Patent Publication Number: US-2023151346-A1

Title: Ph20 polypeptide variants, formulations and uses thereof

Description:
RELATED APPLICATIONS 
     This application is a divisional of U.S. application Ser. No. 17/327,568, entitled “PH20 POLYPEPTIDE VARIANTS, FORMULATIONS AND USES THEREOF,” and filed May 21, 2021, to Ge Wei, H. Michael Shepard, Qiping Zhao and Robert James Connor, which is a continuation of U.S. application Ser. No. 16/912,590, now issued on Jul. 20, 2021, as U.S. Pat. No. 11,066,656, entitled “PH20 POLYPEPTIDE VARIANTS, FORMULATIONS AND USES THEREOF,” and filed Jun. 25, 2020, to Ge Wei, H. Michael Shepard, Qiping Zhao and Robert James Connor, which is a continuation of U.S. application Ser. No. 15/226,489, now issued on Dec. 15, 2020, as U.S. Pat. No. 10,865,400, entitled “PH20 POLYPEPTIDE VARIANTS, FORMULATIONS AND USES THEREOF,” and filed on Aug. 2, 2016, to Ge Wei, H. Michael Shepard, Qiping Zhao and Robert James Connor, which is a divisional of U.S. application Ser. No. 13/694,731, now issued on Sep. 20, 2016, as U.S. Pat. No. 9,447,401, and filed on Dec. 28, 2012, which claims the benefit of priority to U.S. Provisional Application Nos. 61/631,313 and 61/796,208, filed on Dec. 30, 2011, and Nov. 1, 2012, respectively, and each entitled “PH20 POLYPEPTIDE VARIANTS, FORMULATIONS AND USES THEREOF.” 
     U.S. application Ser. No. 17/327,568 also is a continuation of U.S. application Ser. No. 16/824,572, now issued on Jun. 22, 2021, as U.S. Pat. No. 11,041,149, entitled “PH20 POLYPEPTIDE VARIANTS, FORMULATIONS AND USES THEREOF,” and filed Mar. 19, 2020, to Ge Wei, H. Michael Shepard, Qiping Zhao and Robert James Connor, which is a continuation of U.S. application Ser. No. 15/226,489, now issued on Dec. 15, 2020, as U.S. Pat. No. 10,865,400, and filed on Aug. 2, 2016, which is a divisional of U.S. application Ser. No. 13/694,731, now issued on Sep. 20, 2016, as U.S. Pat. No. 9,447,401, filed on Dec. 28, 2012, which claims the benefit of priority to U.S. Provisional Application Nos. 61/631,313 and 61/796,208, filed on Dec. 30, 2011, and Nov. 1, 2012, respectively, and each entitled “PH20 POLYPEPTIDE VARIANTS, FORMULATIONS AND USES THEREOF.” 
     U.S. application Ser. No. 17/327,568 also a continuation of U.S. application Ser. No. 15/226,489, now issued on Dec. 15, 2020, as U.S. Pat. No. 10,865,400, entitled “PH20 POLYPEPTIDE VARIANTS, FORMULATIONS AND USES THEREOF,” and filed on Aug. 2, 2016, to Ge Wei, H. Michael Shepard, Qiping Zhao and Robert James Connor, which is a divisional of U.S. application Ser. No. 13/694,731, now issued on Sep. 20, 2016, as U.S. Pat. No. 9,447,401, and filed on Dec. 28, 2012, which claims the benefit of priority to U.S. Provisional Application Nos. 61/631,313 and 61/796,208, filed on Dec. 30, 2011, and Nov. 1, 2012, respectively, and each entitled “PH20 POLYPEPTIDE VARIANTS, FORMULATIONS AND USES THEREOF.” 
     U.S. application Ser. No. 17/327,568 also is a continuation of U.S. application Ser. No. 13/694,731, now issued on Sep. 20, 2016, as U.S. Pat. No. 9,447,401, entitled “PH20 POLYPEPTIDE VARIANTS, FORMULATIONS AND USES THEREOF,” and filed on Dec. 28, 2012, to Ge Wei, H. Michael Shepard, Qiping Zhao and Robert James Connor, which claims the benefit of priority to U.S. Provisional Application Nos. 61/631,313 and 61/796,208, filed on Dec. 30, 2011, and Nov. 1, 2012, respectively, and each entitled “PH20 POLYPEPTIDE VARIANTS, FORMULATIONS AND USES THEREOF.” 
     This application also is a divisional of U.S. application Ser. No. 17/327,586, entitled “PH20 POLYPEPTIDE VARIANTS, FORMULATIONS AND USES THEREOF,” and filed May 21, 2021, to Ge Wei, H. Michael Shepard, Qiping Zhao and Robert James Connor, which is a continuation of U.S. application Ser. No. 16/912,590, now issued on Jul. 20, 2021, as U.S. Pat. No. 11,066,656, and filed Jun. 25, 2020, which is a continuation of U.S. application Ser. No. 15/226,489, now issued on Dec. 15, 2020, as U.S. Pat. No. 10,865,400, and filed on Aug. 2, 2016, which is a divisional of U.S. application Ser. No. 13/694,731, now issued on Sep. 20, 2016, as U.S. Pat. No. 9,447,401, and filed on Dec. 28, 2012, which claims the benefit of priority to U.S. Provisional Application Nos. 61/631,313 and 61/796,208, filed on Dec. 30, 2011, and Nov. 1, 2012, respectively, and each entitled “PH20 POLYPEPTIDE VARIANTS, FORMULATIONS AND USES THEREOF.” 
     U.S. application Ser. No. 17/327,586 also is a continuation of U.S. application Ser. No. 16/824,572, now issued on Jun. 22, 2021, as U.S. Pat. No. 11,041,149, and filed Mar. 19, 2020, which is a continuation of U.S. application Ser. No. 15/226,489, now issued on Dec. 15, 2020, as U.S. Pat. No. 10,865,400, and filed on Aug. 2, 2016, which is a divisional of U.S. application Ser. No. 13/694,731, now issued on Sep. 20, 2016, as U.S. Pat. No. 9,447,401, filed on Dec. 28, 2012, which claims the benefit of priority to U.S. Provisional Application Nos. 61/631,313 and 61/796,208, filed on Dec. 30, 2011, and Nov. 1, 2012, respectively, and each entitled “PH20 POLYPEPTIDE VARIANTS, FORMULATIONS AND USES THEREOF.” U.S. application Ser. No. 17/327,586 also a continuation of U.S. application Ser. No. 15/226,489, now issued on Dec. 15, 2020, as U.S. Pat. No. 10,865,400, and filed on Aug. 2, 2016, which is a divisional of U.S. application Ser. No. 13/694,731, now issued on Sep. 20, 2016, as U.S. Pat. No. 9,447,401, and filed on Dec. 28, 2012, which claims the benefit of priority to U.S. Provisional Application Nos. 61/631,313 and 61/796,208, filed on Dec. 30, 2011, and Nov. 1, 2012, respectively, and each entitled “PH20 POLYPEPTIDE VARIANTS, FORMULATIONS AND USES THEREOF.” 
     U.S. application Ser. No. 17/327,586 also is a continuation of U.S. application Ser. No. 13/694,731, now issued on Sep. 20, 2016, as U.S. Pat. No. 9,447,401, and filed on Dec. 28, 2012, which claims the benefit of priority to U.S. Provisional Application Nos. 61/631,313 and 61/796,208, filed on Dec. 30, 2011, and Nov. 1, 2012, respectively, and each entitled “PH20 POLYPEPTIDE VARIANTS, FORMULATIONS AND USES THEREOF.” 
     This application also is a divisional of U.S. application Ser. No. 16/912,590, now issued on Jul. 20, 2021, as U.S. Pat. No. 11,066,656, and filed Jun. 25, 2020, which is a continuation of U.S. application Ser. No. 15/226,489, now issued on Dec. 15, 2020, as U.S. Pat. No. 10,865,400, and filed on Aug. 2, 2016, which is a divisional of U.S. application Ser. No. 13/694,731, now issued on Sep. 20, 2016, as U.S. Pat. No. 9,447,401, and filed on Dec. 28, 2012, which claims the benefit of priority to U.S. Provisional Application Nos. 61/631,313 and 61/796,208, filed on Dec. 30, 2011, and Nov. 1, 2012, respectively, and each entitled “PH20 POLYPEPTIDE VARIANTS, FORMULATIONS AND USES THEREOF.” 
     U.S. application Ser. No. 16/912,590, now issued on Jul. 20, 2021, as U.S. Pat. No. 11,066,656, filed Jun. 25, 2020, also is a divisional of U.S. application Ser. No. 13/694,731, now issued on Sep. 20, 2016, as U.S. Pat. No. 9,447,401, and filed on Dec. 28, 2012, which claims the benefit of priority to U.S. Provisional Application Nos. 61/631,313 and 61/796,208, filed on Dec. 30, 2011, and Nov. 1, 2012, respectively, each entitled “PH20 POLYPEPTIDE VARIANTS, FORMULATIONS AND USES THEREOF,” and each to Ge Wei, H. Michael Shepard, Qiping Zhao and Robert James Connor. 
     This application also is a divisional of U.S. application Ser. No. 16/824,572, now issued on Jun. 22, 2021, as U.S. Pat. No. 11,041,149, and filed Mar. 19, 2020, which is a continuation of U.S. application Ser. No. 15/226,489, now issued on Dec. 15, 2020, as U.S. Pat. No. 10,865,400, and filed on Aug. 2, 2016, which is a divisional of U.S. application Ser. No. 13/694,731, now issued on Sep. 20, 2016, as U.S. Pat. No. 9,447,401, filed on Dec. 28, 2012, which claims the benefit of priority to U.S. Provisional Application Nos. 61/631,313 and 61/796,208, filed on Dec. 30, 2011, and Nov. 1, 2012, respectively, and each entitled “PH20 POLYPEPTIDE VARIANTS, FORMULATIONS AND USES THEREOF.” 
     U.S. application Ser. No. 16/824,572, now issued on Jun. 22, 2021, as U.S. Pat. No. 11,041,149, and filed Mar. 19, 2020, also is a divisional of U.S. application Ser. No. 13/694,731, now issued on Sep. 20, 2016, as U.S. Pat. No. 9,447,401, and filed on Dec. 28, 2012, which claims the benefit of priority to U.S. Provisional Application Nos. 61/631,313 and 61/796,208, filed on Dec. 30, 2011, and Nov. 1, 2012, respectively, and each entitled “PH20 POLYPEPTIDE VARIANTS, FORMULATIONS AND USES THEREOF,” and each to Ge Wei, H. Michael Shepard, Qiping Zhao and Robert James Connor. 
     This application is a continuation of U.S. application Ser. No. 15/226,489, now issued on Dec. 15, 2020, as U.S. Pat. No. 10,865,400, entitled “PH20 POLYPEPTIDE VARIANTS, FORMULATIONS AND USES THEREOF,” and filed on Aug. 2, 2016, to Ge Wei, H. Michael Shepard, Qiping Zhao and Robert James Connor, which is a divisional of U.S. application Ser. No. 13/694,731, now issued on Sep. 20, 2016, as U.S. Pat. No. 9,447,401, and filed on Dec. 28, 2012, which claims the benefit of priority to U.S. Provisional Application Nos. 61/631,313 and 61/796,208, filed on Dec. 30, 2011, and Nov. 1, 2012, respectively, and each entitled “PH20 POLYPEPTIDE VARIANTS, FORMULATIONS AND USES THEREOF.” 
     This application also is a divisional of U.S. application Ser. No. 13/694,731, now issued on Sep. 20, 2016, as U.S. Pat. No. 9,447,401, entitled “PH20 POLYPEPTIDE VARIANTS, FORMULATIONS AND USES THEREOF,” and filed on Dec. 28, 2012, to Ge Wei, H. Michael Shepard, Qiping Zhao and Robert James Connor, which claims the benefit of priority to U.S. Provisional Application Nos. 61/631,313 and 61/796,208, filed on Dec. 30, 2011, and Nov. 1, 2012, respectively, and each entitled “PH20 POLYPEPTIDE VARIANTS, FORMULATIONS AND USES THEREOF.” 
     This application also is related to International PCT Application Serial No. PCT/US2012/072182, filed Dec. 28, 2012, entitled “PH20 POLYPEPTIDE VARIANTS, FORMULATIONS AND USES THEREOF,” which also claims priority to U.S. Provisional Application Nos. 61/631,313 and 61/796,208, filed on Dec. 30, 2011, and Nov. 1, 2012, respectively. 
     The subject matter of each of the above-noted applications and patents is incorporated by reference in its entirety. 
    
    
     INCORPORATION BY REFERENCE OF SEQUENCE LISTING PROVIDED ELECTRONICALLY 
     An electronic version of the Sequence Listing is filed herewith, the contents of which are incorporated by reference in their entirety. The electronic file was created on Dec. 15, 2022, is 1,632 kilobytes in size, and is titled 3087Hseq001.xml. 
     FIELD OF THE INVENTION 
     Modified PH20 hyaluronidase polypeptides, including modified polypeptides that exhibit increased stability and/or increased activity, are provided. Also provided are compositions and formulations and uses thereof. 
     BACKGROUND 
     Hyaluronan (hyaluronic acid; HA) is a polypeptide that is found in the extracellular matrix of many cells, especially in soft connective tissues. HA also is found predominantly in skin, cartilage, and in synovial fluid in mammals. Hyaluronan also is the main constituent of the vitreous of the eye. HA has a role in various physiological processes, such as in water and plasma protein homeostasis (Laurent T C et al. (1992) FASEB J 6: 2397-2404)). Certain diseases are associated with expression and/or production of hyaluronan. Hyaluronan-degrading enzymes, such as hyaluronidases, are enzymes that degrade hyaluronan. By catalyzing HA degradation, hyaluronan-degrading enzymes (e.g., hyaluronidases) can be used to treat diseases or disorders associated with accumulation of HA or other glycosaminoglycans. Also, since HA is a major component of the interstitial barrier, hyaluronan-degrading enzymes (e.g., hyaluronidase) increase tissue permeability and therefore can be used to increase the dispersion and delivery of therapeutic agents. Various hyaluronidases have been used therapeutically (e.g., hyaluronidase sold under the trademarks Hydase® (bovine testicular hyaluronidase), Vitrase® (ovine hyaluronidase), and Wydase® (bovine hyaluronidase)), typically as dispersing and spreading agents in combination with other therapeutic agents. Many of these are ovine or bovine forms, which can be immunogenic for treatment of humans. Improved hyaluronan-degrading enzymes, such as hyaluronidases, and compositions thereof that can be used for treatment are needed. 
     SUMMARY 
     Provided are modified PH20 polypeptides that have an altered property or properties compared to the PH20 polypeptide that do not have the modification(s). The modifications include amino acid replacement, deletion and/or insertions. Detailed structure/function of virtually each amino acid in a PH20 polypeptide is provided herein, as well as the identification of residues and loci that contribute to alteration of a property, such as stability in particular conditions, is provided. Hence, provided are modified PH20 polypeptides that contain one or more amino acid replacements that result in a PH20 polypeptide that retains activity and/or exhibits increased or altered stability under a variety of conditions. Activity retained can be, for example, hyaluronidase activity that is as least about 40% or more of the PH20 polypeptide that does not include the replacement. Exemplary modifications are amino acid replacements. For purposes herein, amino acid replacements are denoted by the single amino acid letter followed by the corresponding amino acid position in SEQ ID NO:3 in which the replacement occurs. Single amino acid abbreviations for amino acid residues are well known to a skilled artisan (see e.g. Table 1), and are used herein throughout the description and examples. For example, replacement with P at a position corresponding to position 204 in a PH20 polypeptide with reference to amino acid residue positions set forth in SEQ ID NO:3 means that the replacement encompasses F204P in a PH20 polypeptide set forth in SEQ ID NO:3, or the same replacement at the corresponding position in another PH20 polypeptide. 
     Provided are modified PH20 polypeptides that contain at least one amino acid replacement in a PH20 polypeptide, whereby the modified PH20 polypeptide exhibits increased stability compared to the PH20 polypeptide not containing the amino acid replacement. Increased stability can be manifested as increased resistance to one or more protein conditions that are denaturing to proteins. The stability of modified and unmodified PH20 is compared under the same conditions. Exemplary protein denaturation (or denaturing, used interchangeably herein) conditions include, but are not limited to, elevated temperature greater than 30° C. or about 30° C., agitation, low salt, including essentially or substantially or no salt, and presence of excipients that tend to denature proteins. Exemplary of such excipients are antiadherent(s), binder(s), coating(s), filler(s) and diluent(s), flavor(s), color(s), lubricant(s), glidant(s), preservative(s), detergent(s), sorbent(s) and combinations thereof. 
     The modified PH20 polypeptide can be one in which the unmodified form thereof has at least about 68% sequence identity to SEQ ID NO: 3 and further contains modifications that alter stability and/or can be a PH20 polypeptide that includes as many as about up to 100, 110, 120, 130, 150 amino acid differences from PH20 but retains enzymatic activity, particularly, at least about 40% of the activity of the unmodified PH20 polypeptide and exhibits increased stability, such as stability under denaturing conditions. Thus, included are modified PH20 polypeptides that have at least 68% or about 68% amino acid sequence identity to the sequence of amino acids set forth in SEQ ID NO:3. Included are modified PH20 polypeptides that have at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to the sequence of amino acids set forth in SEQ ID NO:3. Exemplary of such modified PH20 polypeptides are polypeptides that contain amino acid replacement(s) in a PH20 polypeptide that contains the sequence of amino acid residues as set forth in any of SEQ ID NOs: 3, 7, 10, 12, 14, 24, 32-66, 69, 72, 857, 859, 861, 870 or a sequence of amino acids that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to any of SEQ ID NOs: 3, 7, 10, 12, 14, 24, 32-66, 69, 72, 857, 859, 861, or 870. 
     For example, provided herein is a modified PH20 polypeptide that exhibits increased stability containing an amino acid replacement in a PH20 polypeptide that confers the increased stability, wherein increased stability is manifested as increased resistance to denaturation in the presence of one or more protein denaturation conditions, stability is increased compared to the PH20 polypeptide not containing the amino acid replacement, and the unmodified PH20 polypeptide consists of the sequence of amino acids set forth in SEQ ID NO: 7 or is a C-terminal truncated fragment thereof that is a soluble PH20 polypeptide or has at least 85% sequence identity thereto. As above, the modified PH20 polypeptide that exhibits increased stability exhibits increased stability to a denaturation condition that is temperature greater than or about 30° C.; agitation; low or no a salt; or presence of an excipient or a denaturing agent, such as an antiadherent(s), binder(s), coating(s), filler(s) and diluent(s), flavor(s), color(s), lubricant(s), glidant(s), preservative(s), detergent(s), sorbent(s) or sweetener(s) and a combination thereof, and in particular a preservative. In some examples of such modified PH20 polypeptides that exhibit increased stability, the denaturation condition is temperature greater than 30° C., and the modified PH20 polypeptide exhibits greater hyaluronidase activity at the temperature compared to the unmodified PH20 polypeptide not containing the amino acid replacement(s) where the activities are compared under the same conditions. In other examples, the protein denaturation condition is the presence of low concentrations of salt of less than 100 mM, and the modified PH20 polypeptide exhibits increased hyaluronidase activity in the presence of low concentrations of salt compared to the unmodified PH20 polypeptide not containing the amino acid replacement(s) where the activities are compared under the same conditions. 
     In any of the above examples of a modified PH20 polypeptide that exhibits increased stability, stability can be assessed based on a variety of parameters including hyaluronidase activity, solubility, aggregation and/or crystallization. Stability can be assessed in the presence of a denaturing condition. When stability of two or more polypeptides is compared, stability is assessed under the same conditions. In some instances, among the PH20 polypeptides provided herein, the modified PH20 polypeptide exhibits at least 120%, 130%, 135%, 140%, 145%, 150%, 160%, 170%, 180%, 200%, 250%, 300%, 350%, 400%, 500%, 1500%, 2000%, 3000%, 4000%, 5000% or more of the hyaluronidase activity of the PH20 polypeptide not containing the amino acid replacement(s). 
     In any of the above examples of a modified PH20 polypeptide that exhibits increased stability, denaturing conditions include the presence of excipients that denature proteins. Exemplary of such conditions is the presence of a preservative, such as a phenolic preservative. Provided are modified PH20 polypeptides that exhibit increased stability in the presence of an anti-microbial effective amount of one or more phenolic preservatives. An anti-microbial effective amount is the total amount of one or more phenolic preservative agents, which can be expressed as a percentage (%) of mass concentration (w/v) that is or is between (or at least about or at about) 0.05% to 0.6%, 0.1% to 0.4%, 0.1% to 0.3%, 0.15% to 0.325%, 0.15% to 0.25%, 0.1% to 0.2%, 0.2% to 0.3% or 0.3% to 0.4%, inclusive. Exemplary phenolic preservatives include, but are not limited to, phenol, metacresol (m-cresol), benzyl alcohol, and a paraben, such as methylparaben propylparaben, m-cresol, phenol or m-cresol and phenol. Exemplary of the stability achieved by provided modified PH20 polypeptides are those that exhibit at least 15% or about 15% of the hyaluronidase activity for at least 4 hours in the presence of preservative(s) compared to the modified PH20 polypeptide in absence of preservative. Activity is compared under the same conditions except for the presence of preservative(s). For example, provided are modified PH20 polypeptides that exhibit at least (or at least about) 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more of the hyaluronidase activity in the presence of a phenolic preservative(s) compared to absence of the same preservative(s). Thus, provided, among the modified PH20 polypeptides provided herein, are PH20 polypeptides that, by virtue of amino acid replacement(s), are phenophilic compared to PH20 polypeptides without such replacement. Included are modified PH20 polypeptides where the hyaluronidase activity is exhibited after at least 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 3 weeks, 4 weeks or more in the presence of the preservative(s) compared to the hyaluronidase activity of the modified PH20 polypeptide in the absence of preservative for the same time period and under the same conditions except for the presence of preservative(s). 
     In examples of a modified PH20 polypeptide that exhibits increased stability to a phenolic preservative, increased stability in a phenolic preservative can be exhibited under temperature conditions that include any temperature between, for example, 0° C. and 40° C., such as between or about between 0° C. to 40° C., 2° C. to 6° C., 24° C. to 32° C. and 35° C. to 40° C. Exemplary polypeptides exhibit increased stability at temperatures of between or about between 30° C. to 45° C., 35° C. to 45° C., 30° C. to 37° C., 35° C. to 37° C. or 37° C. to 42° C., each inclusive. The particular modified PH20 polypeptide and conditions depend upon the intended formulation, conditions to which the formulation will be exposed and/or intended application. 
     Particular and exemplary modified PH20 polypeptides that exhibit increased stability, such as increased stability to a phenolic preservative, include those that contain a single amino acid modification, such as a replacement, and combinations of modifications, such as at least or 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100 and more modifications. These include modified PH20 polypeptides that contain one or more amino acid replacements, where at least one replacement is at an amino acid position corresponding (i.e., by alignment) to a position selected from among 10, 12, 20, 22, 26, 34, 36, 46, 50, 52, 58, 68, 70, 74, 82, 83, 84, 86, 97, 127, 131, 138, 142, 143, 144, 166, 169, 174, 193, 195, 196, 204, 205, 206, 213, 219, 234, 237, 238, 240, 249, 261, 267, 277, 279, 291, 309, 310, 314, 315, 317, 318, 347, 367, 375, 376, 399, 401, 407, 416, 419, 421, 431, 433, 439, 440, 443 or 445 with reference to amino acid positions set forth in SEQ ID NO:3, wherein corresponding amino acid positions are identified by alignment of the PH20 polypeptide with the polypeptide set forth in SEQ ID NO:3. Exemplary of such modifications are at least one amino acid replacement selected from among replacement with: glycine (G) at a position corresponding to position 10; K at a position corresponding to position 12; S at a position corresponding to position 20; T at a position corresponding to position 22; M at a position corresponding to position 26; W at a position corresponding to position 34; N at a position corresponding to position 36; L at a position corresponding to position 46; M at a position corresponding to position 50; T at a position corresponding to position 52; S at a position corresponding to position 52; C at a position corresponding to position 58; K at a position corresponding to position 58; Rat a position corresponding to position 58; N at a position corresponding to position 58; Y at a position corresponding to position 58; P at a position corresponding to position 58; H at a position corresponding to position 58; P at a position corresponding to position 68; V at a position corresponding to position 70; E at a position corresponding to position 74; L at a position corresponding to position 82; N at a position corresponding to position 82; V at a position corresponding to position 83; Q at a position corresponding to position 83; S at a position corresponding to position 83; G at a position corresponding to position 83; N at a position corresponding to position 84; A at a position corresponding to position 86; K at a position corresponding to position 86; E at a position corresponding to position 97; L at a position corresponding to position 97; R at a position corresponding to position 127; Rat a position corresponding to position 131; L at a position corresponding to position 138; K at a position corresponding to position 142; N at a position corresponding to position 142; P at a position corresponding to position 142; S at a position corresponding to position 142; T at a position corresponding to position 142; G at a position corresponding to position 143; K at a position corresponding to position 143; T at a position corresponding to position 144; Q at a position corresponding to position 166; T at a position corresponding to position 166; L at a position corresponding to position 169; G at a position corresponding to position 174; N at a position corresponding to position 174; Q at a position corresponding to position 193; T at a position corresponding to position 195; N at a position corresponding to position 195; E at a position corresponding to position 196; Rat a position corresponding to position 196; P at a position corresponding to position 204; A at a position corresponding to position 205; E at a position corresponding to position 205; I at a position corresponding to position 206; A at a position corresponding to position 213; I at a position corresponding to position 219; M at a position corresponding to position 234; T at a position corresponding to position 237; H at a position corresponding to position 238; Q at a position corresponding to position 240; V at a position corresponding to position 249; A at a position corresponding to position 261; K at a position corresponding to position 261; Tat a position corresponding to position 267; K at a position corresponding to position 277; H at a position corresponding to position 279; V at a position corresponding to position 279; V at a position corresponding to position 291; E at a position corresponding to position 309; Q at a position corresponding to position 310; Y at a position corresponding to position 314; Y at a position corresponding to position 315; N at a position corresponding to position 317; W at a position corresponding to position 317; D at a position corresponding to position 318; G at a position corresponding to position 347; A at a position corresponding to position 367; R at a position corresponding to position 375; Rat a position corresponding to position 376; V at a position corresponding to position 399; E at a position corresponding to position 401; A at a position corresponding to position 407; L at a position corresponding to position 416; K at a position corresponding to position 419; H at a position corresponding to position 421; E at a position corresponding to position 431; Tat a position corresponding to position 433; V at a position corresponding to position 433; C at a position corresponding to position 439; P at a position corresponding to position 440; G at a position corresponding to position 443; N at a position corresponding to position 445, with reference to amino acid residue positions set forth in SEQ ID NO:3. For example, the modified PH20 polypeptide can contain at least one amino acid replacement selected from among replacement with: T at a position corresponding to position 52, K at a position corresponding to position 58, Rat a position corresponding to position 58, P at a position corresponding to position 68, V at a position corresponding to position 83, P at a position corresponding to position 204, A at a position corresponding to position 261, Tat a position corresponding to position 267, K at a position corresponding to position 277 and H at a position corresponding to position 421, with reference to amino acid residue positions set forth in SEQ ID NO:3. An exemplary modified PH20 polypeptide is one that includes P (or a conservative amino acid thereto) at a position corresponding to position 204 in a PH20 polypeptide with reference to amino acid residue positions set forth in SEQ ID NO:3. 
     Thus, provided herein are modified PH20 polypeptides that exhibit increased stability in the presence of a phenolic preservative containing an amino acid replacement in a PH20 polypeptide that confers the increased stability, wherein stability is increased compared to the unmodified polypeptide without the amino acid replacement, and the unmodified PH20 polypeptide has the sequence of amino acids set forth in SEQ ID NO: 7 or is a C-terminal truncated fragment thereof that is a soluble PH20 polypeptide or has at least 85% sequence identity thereto. For example, the unmodified PH20 polypeptide is a soluble PH20 polypeptide that has the sequence of amino acids set forth in any of SEQ ID NOs: 3 or 32-66. In particular examples, the modified PH20 polypeptide has at least 85% sequence identity to SEQ ID NO:3. In any of such examples of a modified PH20 polypeptide, the polypeptide contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 or more amino acid replacements. In examples herein, the modified PH20 polypeptide is a human PH20. The modified PH20 polypeptide exhibits stability in the presence of phenolic preservatives if it exhibits at least 15% of the hyaluronidase activity in the presence of a preservative(s) for at least 4 hours compared to the hyaluronidase activity in the absence of the phenolic preservative(s), wherein the activity is compared under the same conditions except for the presence of the phenolic preservative(s). In any of the above examples, the modified PH20 polypeptide is stable in the presence of an of an anti-microbial effective amount of one or more phenolic preservatives, such as a total amount of one or more phenolic preservative agents as a percentage (%) of mass concentration (w/v) that is from or from about 0.05% to 0.6%, 0.1% to 0.4%, 0.1% to 0.3%, 0.15% to 0.325%, 0.15% to 0.25%, 0.1% to 0.2%, 0.2% to 0.3% or 0.3% to 0.4%, inclusive. The phenolic preservative can be a phenol, metacresol (m-cresol), benzyl alcohol or a paraben, such as m-cresol, phenol, or m-cresol and phenol. The amino acid replacement can be at amino acid residue 204, 58, 10, 12, 20, 22, 26, 34, 36, 46, 50, 52, 68, 70, 74, 82, 83, 84, 86, 97, 127, 131, 138, 142, 143, 144, 166, 169, 174, 193, 195, 196, 205, 206, 213, 219, 234, 237, 238, 240, 249, 261, 267, 277, 279, 291, 309, 310, 314, 315, 317, 318, 347, 367, 375, 376, 399, 401, 407, 416, 419, 421, 431, 433, 439, 440, 443 or 445 with reference to amino acid positions set forth in SEQ ID NO:3, wherein corresponding amino acid positions are identified by alignment of the PH20 polypeptide with the polypeptide set forth in SEQ ID NO:3. For example, the amino acid replacement is G at a position corresponding to position 10; K at a position corresponding to position 12; S at a position corresponding to position 20; T at a position corresponding to position 22; M at a position corresponding to position 26; W at a position corresponding to position 34; N at a position corresponding to position 36; L at a position corresponding to position 46; M at a position corresponding to position 50; T at a position corresponding to position 52; S at a position corresponding to position 52; C at a position corresponding to position 58; K at a position corresponding to position 58; Rat a position corresponding to position 58; N at a position corresponding to position 58; Y at a position corresponding to position 58; P at a position corresponding to position 58; H at a position corresponding to position 58; P at a position corresponding to position 68; V at a position corresponding to position 70; E at a position corresponding to position 74; L at a position corresponding to position 82; N at a position corresponding to position 82; V at a position corresponding to position 83; Q at a position corresponding to position 83; S at a position corresponding to position 83; G at a position corresponding to position 83; N at a position corresponding to position 84; A at a position corresponding to position 86; K at a position corresponding to position 86; E at a position corresponding to position 97; L at a position corresponding to position 97; R at a position corresponding to position 127; Rat a position corresponding to position 131; L at a position corresponding to position 138; K at a position corresponding to position 142; N at a position corresponding to position 142; P at a position corresponding to position 142; S at a position corresponding to position 142; T at a position corresponding to position 142; G at a position corresponding to position 143; K at a position corresponding to position 143; T at a position corresponding to position 144; Q at a position corresponding to position 166; T at a position corresponding to position 166; L at a position corresponding to position 169; G at a position corresponding to position 174; N at a position corresponding to position 174; Q at a position corresponding to position 193; T at a position corresponding to position 195; N at a position corresponding to position 195; E at a position corresponding to position 196; Rat a position corresponding to position 196; P at a position corresponding to position 204; A at a position corresponding to position 205; E at a position corresponding to position 205; I at a position corresponding to position 206; A at a position corresponding to position 213; I at a position corresponding to position 219; M at a position corresponding to position 234; T at a position corresponding to position 237; H at a position corresponding to position 238; Q at a position corresponding to position 240; V at a position corresponding to position 249; A at a position corresponding to position 261; K at a position corresponding to position 261; T at a position corresponding to position 267; K at a position corresponding to position 277; H at a position corresponding to position 279; V at a position corresponding to position 279; V at a position corresponding to position 291; E at a position corresponding to position 309; Q at a position corresponding to position 310; Y at a position corresponding to position 314; Y at a position corresponding to position 315; N at a position corresponding to position 317; W at a position corresponding to position 317; D at a position corresponding to position 318; G at a position corresponding to position 347; A at a position corresponding to position 367; R at a position corresponding to position 375; Rat a position corresponding to position 376; V at a position corresponding to position 399; E at a position corresponding to position 401; A at a position corresponding to position 407; L at a position corresponding to position 416; K at a position corresponding to position 419; H at a position corresponding to position 421; E at a position corresponding to position 431; T at a position corresponding to position 433; V at a position corresponding to position 433; C at a position corresponding to position 439; P at a position corresponding to position 440; G at a position corresponding to position 443; or N at a position corresponding to position 445, with reference to amino acid residue positions set forth in SEQ ID NO:3. In particular, the amino acid replacement is T at a position corresponding to position 52, K at a position corresponding to position 58, Rat a position corresponding to position 58, P at a position corresponding to position 68, V at a position corresponding to position 83, P at a position corresponding to position 204, A at a position corresponding to position 261, T at a position corresponding to position 267, K at a position corresponding to position 277 or H at a position corresponding to position 421, with reference to amino acid residue positions set forth in SEQ ID NO:3, such as replacement with P at a position corresponding to position 204 or Rat a position corresponding to position 58. The modified PH20 polypeptide that exhibits increased stability to phenolic preservatives can be substantially purified or isolated. The modified PH20 polypeptide that exhibits increased stability to phenolic preservatives can be modified by glycosylation, sialation, albumination, farnysylation, carboxylation, hydroxylation and phosphorylation, and generally is glycosylated, whereby the polypeptide contains at least an N-acetylglucosamine moiety linked to each of at least three asparagine (N) residues, such as at amino acid residues corresponding to amino acid residues 200, 333 and 358 of SEQ ID NO:3. The modified PH20 polypeptide that exhibits increased stability to phenolic preservatives can be conjugated to a polymer, such as PEG or dextran and/or can be conjugated to a moiety that is a multimerization domain, a toxin, a detectable label or a drug. 
     Among modified PH20 polypeptides provided herein that exhibit increased stability are those that exhibit increased hyaluronidase activity at the elevated temperature compared to the PH20 polypeptide not containing the amino acid replacement(s), such as at least 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 300%, 400%, 500% or more hyaluronidase activity for at least 4 hours compared to the PH20 polypeptide not containing the amino acid replacement(s). Also among the polypeptides are those that exhibit activity, but also typically exhibit increased stability or other property at elevated temperatures, such as a modified PH20 polypeptide that exhibits at least 95%, 96%, 97%, 98%, 99%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 300%, 400%, 500% of the hyaluronidase activity for at least 4 hours at a temperature of between or about between 32° C. to 37° C. compared to the hyaluronidase activity of the modified PH20 polypeptide at a temperature of between or about between 2° C. to 8° C., where activity is compared under the same conditions except for the differences in temperature. The hyaluronidase activity can be exhibited after at least 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 3 weeks, 4 weeks or more at elevated temperatures of between or about between 32° C. to 37° C. compared to the hyaluronidase activity of the modified PH20 polypeptide at a temperature between or about between 2° C. to 8° C., where activity is compared for the same time period and under the same conditions except for the difference in temperature. Exemplary of such modified polypeptides are those that contain at least one amino acid replacement at an amino acid position corresponding to a position selected from among 1, 11, 12, 14, 20, 26, 29, 34, 50, 58, 70, 82, 83, 84, 86, 87, 140, 142, 143, 147, 152, 166, 167, 172, 174, 178, 193, 195, 206, 212, 213, 219, 233, 237, 240, 267, 277, 291, 292, 309, 313, 314, 317, 318, 347, 367, 368, 371, 374, 389, 392, 395, 396, 406, 419, 421, 439 and 443 with reference to amino acid positions set forth in SEQ ID NO:3, wherein corresponding amino acid positions are identified by alignment of the PH20 polypeptide with the polypeptide set forth in SEQ ID NO:3. Exemplary mutations include, for example, replacement with Rat a position corresponding to position 1; S at a position corresponding to position 11; I at a position corresponding to position 12; V at a position corresponding to position 14; S at a position corresponding to position 20; M at a position corresponding to position 26; with R at a position corresponding to position 29; W at a position corresponding to position 34; M at a position corresponding to position 50; K at a position corresponding to position 58; Q at a position corresponding to position 58; Q at a position corresponding to position 58; V at a position corresponding to position 70; L at a position corresponding to position 82; Q at a position corresponding to position 83; Rat a position corresponding to position 84; A at a position corresponding to position 86; S at a position corresponding to position 87; K at a position corresponding to position 140; S at a position corresponding to position 142; T at a position corresponding to position 142; K at a position corresponding to position 143; S at a position corresponding to position 147; T at a position corresponding to position 152; T at a position corresponding to position 166; D at a position corresponding to position 167; A at a position corresponding to position 172; G at a position corresponding to position 174; N at a position corresponding to position 174; Rat a position corresponding to position 178; Q at a position corresponding to position 193; T at a position corresponding to position 195; I at a position corresponding to position 206; S at a position corresponding to position 212; A at a position corresponding to position 213; I at a position corresponding to position 219; G at a position corresponding to position 233; Tat a position corresponding to position 237; A at a position corresponding to position 240; Q at a position corresponding to position 240; T at a position corresponding to position 267; E at a position corresponding to position 277; S at a position corresponding to position 291; H at a position corresponding to position 292; V at a position corresponding to position 292; S at a position corresponding to position 309; H at a position corresponding to position 313; S at a position corresponding to position 314; I at a position corresponding to position 317; T at a position corresponding to position 317; W at a position corresponding to position 317; Rat a position corresponding to position 318; G at a position corresponding to position 347; A at a position corresponding to position 367; Rat a position corresponding to position 368; S at a position corresponding to position 371; P at a position corresponding to position 374; A at a position corresponding to position 389; V at a position corresponding to position 392; A at a position corresponding to position 395; H at a position corresponding to position 396; N at a position corresponding to position 406; H at a position corresponding to position 419; K at a position corresponding to position 419; R at a position corresponding to position 421; S at a position corresponding to position 421; A at a position corresponding to position 439; C at a position corresponding to position 439; and G at a position corresponding to position 443, with reference to amino acid positions set forth in SEQ ID NO:3. In particular examples provided herein, any of such modified PH20 polypeptides contain a single amino acid modification, such as a replacement, and combinations of modifications, such as at least or 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100 and more modifications. The modification, such as replacement, can be in an unmodified PH20 polypeptide that has the sequence of amino acids set forth in SEQ ID NO: 7 or is a C-terminal truncated fragment thereof that is a soluble PH20 polypeptide, such as is set forth in any of SEQ ID NOs: 3 or 32-66, or has at least 85% sequence identity thereto. For example, any of such modified PH20 polypeptides has at least 85% sequence identity to SEQ ID NO:3. 
     Also provided are modified PH20 polypeptides that exhibit increased stability in low salt conditions, such as, for example, concentrations of NaCl of less than 100 mM, such as, but not limited to concentrations of NaCl less than 90 mM, 80 mM, 70 mM, 60 mM, 50 mM, 40 mM, 30 mM, 25 mM, 20 mM, 15 mM, 10 mM, 5 mM or less. Among the modified PH20 polypeptides are those that exhibit increased hyaluronidase activity at lower concentrations of salt compared to the PH20 polypeptide not containing the amino acid replacement(s). Such activity includes, for example, at least more than 100%, or at least 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 300%, 400%, 500% or more hyaluronidase activity compared to the PH20 polypeptide not containing the amino acid replacement(s). Exemplary of such modified PH20 polypeptides are those that exhibit at least 60% of the hyaluronidase activity in low concentrations of salt, such as between or about between 10 mM NaCl and 100 mM NaCl, inclusive (or comparable concentrations of other salts or mixtures of salts), compared to the hyaluronidase activity of the modified PH20 polypeptide in 150 mM NaCl, where activities are compared under the same conditions except for the difference in salt concentration. In particular examples provided herein, any of such modified PH20 polypeptides contain a single amino acid modification, such as a replacement, and combinations of modifications, such as at least or 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100 and more modifications. The modification, such as replacement, can be in an the unmodified PH20 polypeptide that has the sequence of amino acids set forth in SEQ ID NO: 7 or is a C-terminal truncated fragment thereof that is a soluble PH20 polypeptide, such as is set forth in any of SEQ ID NOs: 3 or 32-66, or has at least 85% sequence identity thereto. For example, any of such modified PH20 polypeptides has at least 85% sequence identity to SEQ ID NO:3. 
     Also provided are modified PH20 polypeptides that contain at least one amino acid replacement in a PH20 polypeptide, where the modified PH20 polypeptide exhibits increased hyaluronidase activity compared to the PH20 polypeptide not containing the amino acid replacement. When comparing activity among polypeptides, activity is compared under the same conditions. Among these are polypeptides, where the unmodified PH20 exhibits at least 68%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to the sequence of amino acids set forth in SEQ ID NO:3, or the resulting modified PH20 exhibits such sequence identity to the sequence of amino acids set forth in SEQ ID NO:3. Exemplary of such modified PH20 polypeptides are any that contain an amino acid replacement(s) in the sequence of amino acids set forth in any of SEQ ID NOs: 3, 7, 10, 12, 14, 24, 32-66, 69, or 72, or a sequence of amino acids that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to any of SEQ ID NOs: 3, 7, 10, 12, 14, 24, 32-66, 69, or 72. The amino acid replacement(s) also can be made in the sequence of amino acids set forth in any of SEQ ID NOs: 857, 859, 861 or 870, or a sequence of amino acids that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to any of SEQ ID NOs: 857, 859, 861 or 870. In particular, provided are modified PH20 polypeptides that contain an amino acid replacement in the sequence of amino acids set forth in SEQ ID NOs: 3, 7, 32-66, 69 or 72. Among the modified PH20 polypeptides are those that that exhibit at least 120%, 130%, 135%, 140%, 145%, 150%, 160%, 170%, 180%, 200%, 250%, 300%, 350%, 400%, 500%, 1500%, 2000%, 3000%, 4000%, 5000% or more of the hyaluronidase activity of the PH20 polypeptide not containing the amino acid replacement. Activity can be assessed at any temperature, in particular such activity is present when the hyaluronidase is exposed to a temperature that is at a temperature between or about between 2° C. to 8° C. These modified PH20 polypeptides contain at least one amino acid replacement at an amino acid position corresponding to a position selected from among 1, 12, 15, 24, 26, 27, 29, 30, 31, 32, 33, 37, 39, 46, 48, 52, 58, 63, 67, 68, 69, 70, 71, 72, 73, 74, 75, 84, 86, 87, 92, 93, 94, 97, 118, 120, 127, 131, 135, 141, 142, 147, 148, 150, 151, 152, 155, 156, 163, 164, 165, 166, 169, 170, 174, 198, 206, 209, 212, 213, 215, 219, 233, 234, 236, 238, 247, 257, 259, 260, 261, 263, 269, 271, 272, 276, 277, 278, 282, 291, 293, 305, 308, 309, 310, 313, 315, 317, 318, 324, 325, 326, 328, 347, 353, 359, 371, 377, 380, 389, 392, 395, 399, 405, 407, 409, 410, 418, 419, 421, 425, 431, 433, 436, 437, 438, 439, 440, 441, 442, 443, 445, 446 and 447 with reference to amino acid positions set forth in SEQ ID NO:3, wherein corresponding amino acid positions are identified by alignment of the PH20 polypeptide with the polypeptide set forth in SEQ ID NO:3. Exemplary modifications include at least one amino acid replacement selected from among replacement with: histidine (H) at a position corresponding to position 1; Q at a position corresponding to position 1; E at a position corresponding to position 12; T at a position corresponding to position 12; V at a position corresponding to position 15; E at a position corresponding to position 24; H at a position corresponding to position 24; E at a position corresponding to position 26; K at a position corresponding to position 26; K at a position corresponding to position 27; R at a position corresponding to position 27; E at a position corresponding to position 29; I at a position corresponding to position 29; L at a position corresponding to position 29; M at a position corresponding to position 29; P at a position corresponding to position 29; S at a position corresponding to position 29; V at a position corresponding to position 29; G at a position corresponding to position 30; H at a position corresponding to position 30; K at a position corresponding to position 30; M at a position corresponding to position 30; R at a position corresponding to position 30; S at a position corresponding to position 30; A at a position corresponding to position 31; C at a position corresponding to position 31; H at a position corresponding to position 31; I at a position corresponding to position 31; K at a position corresponding to position 31; L at a position corresponding to position 31; P at a position corresponding to position 31; R at a position corresponding to position 31; S at a position corresponding to position 31; T at a position corresponding to position 31; V at a position corresponding to position 31; F at a position corresponding to position 32; G at a position corresponding to position 32; H at a position corresponding to position 32; W at a position corresponding to position 33; F at a position corresponding to position 37; N at a position corresponding to position 39; T at a position corresponding to position 39; R at a position corresponding to position 46; F at a position corresponding to position 48; H at a position corresponding to position 48; N at a position corresponding to position 48; Q at a position corresponding to position 52; K at a position corresponding to position 58; Q at a position corresponding to position 58; W at a position corresponding to position 63; V at a position corresponding to position 67; H at a position corresponding to position 68; Q at a position corresponding to position 68; A at a position corresponding to position 69; C at a position corresponding to position 69; F at a position corresponding to position 69; G at a position corresponding to position 69; I at a position corresponding to position 69; L at a position corresponding to position 69; M at a position corresponding to position 69; P at a position corresponding to position 69; R at a position corresponding to position 69; W at a position corresponding to position 69; Y at a position corresponding to position 69; A at a position corresponding to position 70; C at a position corresponding to position 70; F at a position corresponding to position 70; G at a position corresponding to position 70; H at a position corresponding to position 70; K at a position corresponding to position 70; L at a position corresponding to position 70; N at a position corresponding to position 70; P at a position corresponding to position 70; R at a position corresponding to position 70; S at a position corresponding to position 70; T at a position corresponding to position 70; V at a position corresponding to position 70; Rat a position corresponding to position 71; S at a position corresponding to position 71; M at a position corresponding to position 72; Q at a position corresponding to position 72; H at a position corresponding to position 73; L at a position corresponding to position 73; W at a position corresponding to position 73; A at a position corresponding to position 74; C at a position corresponding to position 74; G at a position corresponding to position 74; N at a position corresponding to position 74; P at a position corresponding to position 74; R at a position corresponding to position 74; S at a position corresponding to position 74; V at a position corresponding to position 74; W at a position corresponding to position 74; F at a position corresponding to position 75; L at a position corresponding to position 75; Rat a position corresponding to position 75; T at a position corresponding to position 75; G at a position corresponding to position 84; Rat a position corresponding to position 84; A at a position corresponding to position 86; C at a position corresponding to position 87; T at a position corresponding to position 87; Y at a position corresponding to position 87; C at a position corresponding to position 92; I at a position corresponding to position 93; L at a position corresponding to position 93; Rat a position corresponding to position 93; T at a position corresponding to position 93; Rat a position corresponding to position 94; G at a position corresponding to position 97; Q at a position corresponding to position 118; F at a position corresponding to position 120; V at a position corresponding to position 120; Y at a position corresponding to position 120; H at a position corresponding to position 127; N at a position corresponding to position 127; G at a position corresponding to position 131; Rat a position corresponding to position 131; V at a position corresponding to position 131; D at a position corresponding to position 135; G at a position corresponding to position 135; Rat a position corresponding to position 135, with H at a position corresponding to position 141; Y at a position corresponding to position 141; Rat a position corresponding to position 142; R at a position corresponding to position 147; V at a position corresponding to position 147; K at a position corresponding to position 148; G at a position corresponding to position 150; K at a position corresponding to position 151; L at a position corresponding to position 151; M at a position corresponding to position 151; Q at a position corresponding to position 151; R at a position corresponding to position 151; R at a position corresponding to position 152; G at a position corresponding to position 155; K at a position corresponding to position 155; D at a position corresponding to position 156; A at a position corresponding to position 163; E at a position corresponding to position 163; K at a position corresponding to position 163; Rat a position corresponding to position 163; M at a position corresponding to position 164; D at a position corresponding to position 165; N at a position corresponding to position 165; A at a position corresponding to position 166; F at a position corresponding to position 166; H at a position corresponding to position 166; L at a position corresponding to position 166; Q at a position corresponding to position 166; Rat a position corresponding to position 166; T at a position corresponding to position 166; Y at a position corresponding to position 166; L at a position corresponding to position 169; Rat a position corresponding to position 170; K at a position corresponding to position 174; D at a position corresponding to position 198; K at a position corresponding to position 206; L at a position corresponding to position 206; N at a position corresponding to position 212; M at a position corresponding to position 213; N at a position corresponding to position 213; M at a position corresponding to position 215; S at a position corresponding to position 219; K at a position corresponding to position 233; Rat a position corresponding to position 233; M at a position corresponding to position 234; R at a position corresponding to position 236; E at a position corresponding to position 237; S at a position corresponding to position 238; I at a position corresponding to position 247; T at a position corresponding to position 257; P at a position corresponding to position 259; Y at a position corresponding to position 260; K at a position corresponding to position 261; N at a position corresponding to position 261; K at a position corresponding to position 263; Rat a position corresponding to position 263; A at a position corresponding to position 269; L at a position corresponding to position 271; M at a position corresponding to position 271; T at a position corresponding to position 272; D at a position corresponding to position 276; S at a position corresponding to position 276; Y at a position corresponding to position 276; K at a position corresponding to position 277; R at a position corresponding to position 277; T at a position corresponding to position 277; H at a position corresponding to position 278; K at a position corresponding to position 278; N at a position corresponding to position 278; R at a position corresponding to position 278; S at a position corresponding to position 278; T at a position corresponding to position 278; Y at a position corresponding to position 278; M at a position corresponding to position 282; V at a position corresponding to position 291; A at a position corresponding to position 293; C at a position corresponding to position 293; F at a position corresponding to position 293; M at a position corresponding to position 293; P at a position corresponding to position 293; Q at a position corresponding to position 293; V at a position corresponding to position 293; E at a position corresponding to position 305; G at a position corresponding to position 308; N at a position corresponding to position 308; E at a position corresponding to position 309; L at a position corresponding to position 309; N at a position corresponding to position 309; Q at a position corresponding to position 309; R at a position corresponding to position 309; T at a position corresponding to position 309; A at a position corresponding to position 310; G at a position corresponding to position 310; K at a position corresponding to position 313; Rat a position corresponding to position 313; H at a position corresponding to position 315; I at a position corresponding to position 317; K at a position corresponding to position 317; Rat a position corresponding to position 317; M at a position corresponding to position 318; H at a position corresponding to position 320; K at a position corresponding to position 320; Rat a position corresponding to position 320; R at a position corresponding to position 324; A at a position corresponding to position 325; D at a position corresponding to position 325; E at a position corresponding to position 325; G at a position corresponding to position 325; H at a position corresponding to position 325; K at a position corresponding to position 325; M at a position corresponding to position 325; N at a position corresponding to position 325; Q at a position corresponding to position 325; S at a position corresponding to position 325; V at a position corresponding to position 326; I at a position corresponding to position 328; K at a position corresponding to position 328; L at a position corresponding to position 328; S at a position corresponding to position 328; Y at a position corresponding to position 328; G at a position corresponding to position 347; S at a position corresponding to position 347; V at a position corresponding to position 353; with T at a position corresponding to position 359; R at a position corresponding to position 371; P at a position corresponding to position 377; Tat a position corresponding to position 377; W at a position corresponding to position 380; Y at a position corresponding to position 380; K at a position corresponding to position 389; M at a position corresponding to position 392; Rat a position corresponding to position 395; M at a position corresponding to position 399; T at a position corresponding to position 399; W at a position corresponding to position 399; G at a position corresponding to position 405; D at a position corresponding to position 407; Q at a position corresponding to position 407; A at a position corresponding to position 409; Q at a position corresponding to position 409; T at a position corresponding to position 410; P at a position corresponding to position 418; F at a position corresponding to position 419; I at a position corresponding to position 419; K at a position corresponding to position 419; Rat a position corresponding to position 419; S at a position corresponding to position 419; H at a position corresponding to position 421; K at a position corresponding to position 421; N at a position corresponding to position 421; Q at a position corresponding to position 421; R at a position corresponding to position 421; S at a position corresponding to position 421; K at a position corresponding to position 425; A at a position corresponding to position 431; H at a position corresponding to position 431; K at a position corresponding to position 431; Q at a position corresponding to position 431; R at a position corresponding to position 431; S at a position corresponding to position 431; V at a position corresponding to position 431; L at a position corresponding to position 433; Rat a position corresponding to position 433; T at a position corresponding to position 433; V at a position corresponding to position 433; K at a position corresponding to position 436; I at a position corresponding to position 437; M at a position corresponding to position 437; T at a position corresponding to position 438; V at a position corresponding to position 439; H at a position corresponding to position 440; R at a position corresponding to position 440; F at a position corresponding to position 441; Rat a position corresponding to position 442; A at a position corresponding to position 443; M at a position corresponding to position 443; M at a position corresponding to position 445; P at a position corresponding to position 445; A at a position corresponding to position 446; D at a position corresponding to position 447; N at a position corresponding to position 447; and/or with Q at a position corresponding to position 447, with reference to amino acid positions set forth in SEQ ID NO:3. 
     Among the polypeptides that exhibit increased hyaluronidase activity are those that exhibit at least 2.0-fold of the hyaluronidase activity of the PH20 polypeptide not containing the amino acid replacement. For example, among these are modified PH20 polypeptides that contain at least one amino acid replacement at an amino acid position corresponding to a position selected from among 24, 29, 31, 48, 58, 69, 70, 75, 84, 97, 165, 166, 271, 278, 317, 320, 325 and 326 with reference to positions set forth in SEQ ID NO:3, wherein corresponding amino acid positions are identified by alignment of the PH20 polypeptide with the polypeptide set forth in SEQ ID NO:3, such as modified PH20 polypeptides that contain at least one amino acid replacement selected from among replacement with: E at a position corresponding to position 24; E at a position corresponding to position 29; V at a position corresponding to position 31; N at a position corresponding to position 48; K at a position corresponding to position 58; Q at a position corresponding to position 58; A at a position corresponding to position 69; F at a position corresponding to position 69; G at a position corresponding to position 69; P at a position corresponding to position 69; R at a position corresponding to position 69; A at a position corresponding to position 70; F at a position corresponding to position 70; G at a position corresponding to position 70; H at a position corresponding to position 70; H at a position corresponding to position 70; N at a position corresponding to position 70; R at a position corresponding to position 70; T at a position corresponding to position 70; V at a position corresponding to position 70; L at a position corresponding to position 75; T at a position corresponding to position 75; G at a position corresponding to position 84; G at a position corresponding to position 97; D at a position corresponding to position 165; L at a position corresponding to position 166; Rat a position corresponding to position 166; T at a position corresponding to position 166; L at a position corresponding to position 271; H at a position corresponding to position 278; Rat a position corresponding to position 278; K at a position corresponding to position 317; K at a position corresponding to position 320; E at a position corresponding to position 325, with G at a position corresponding to position 325; K at a position corresponding to position 325; N at a position corresponding to position 325; Q at a position corresponding to position 325; and V at a position corresponding to position 326; with reference to amino acid positions set forth in SEQ ID NO:3. 
     Among any of the polypeptides provided herein that exhibit increased hyaluronidase activity, any of such modified PH20 polypeptides contain a single amino acid modification, such as a replacement, and combinations of modifications, such as at least or 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100 and more modifications. The modification, such as replacement, can be in an unmodified PH20 polypeptide that has the sequence of amino acids set forth in SEQ ID NO: 7 or is a C-terminal truncated fragment thereof that is a soluble PH20 polypeptide, such as is set forth in any of SEQ ID NOs: 3 or 32-66, or has at least 85% sequence identity thereto. For example, any of such modified PH20 polypeptides has at least 85% sequence identity to SEQ ID NO:3. 
     Also provided are modified PH20 polypeptides that contain at least one amino acid replacement in the PH20 polypeptide whose sequence is set forth in SEQ ID NO:7, a C-terminally truncated fragment thereof, a soluble fragment thereof, or in a PH20 polypeptide that has a sequence of amino acids that is at least 91% identical to the sequence of amino acids set forth in SEQ ID NO:7, where at least one amino replacement(s) is at an amino acid position corresponding to a position selected from among 1, 2, 3, 4, 5, 6, 8, 9, 10, 11, 12, 13, 14, 15, 20, 22, 23, 24, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 54, 58, 59, 60, 61, 63, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 77, 79, 81, 82, 83, 84, 85, 86, 87, 89, 90, 91, 92, 93, 94, 96, 97, 98, 99, 102, 103, 104, 105, 106, 107, 108, 110, 114, 117, 118, 119, 120, 122, 124, 125, 127, 128, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 186, 192, 193, 195, 196, 197, 198, 200, 202, 204, 205, 206, 208, 209, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 224, 226, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 242, 245, 247, 248, 251, 253, 255, 256, 257, 258, 259, 260, 261, 263, 264, 265, 266, 267, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 297, 298, 300, 301, 302, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 320, 321, 323, 324, 325, 326, 327, 328, 331, 334, 335, 338, 339, 342, 343, 347, 348, 349, 351, 353, 356, 357, 358, 359, 360, 361, 367, 368, 369, 371, 373, 374, 375, 376, 377, 378, 379, 380, 381, 383, 385, 387, 388, 389, 391, 392, 393, 394, 395, 396, 397, 398, 399, 401, 403, 404, 405, 406, 407, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 425, 426, 427, 428, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446 and 447 with reference to amino acid positions set forth in SEQ ID NO:3 or 7, where corresponding amino acid positions are identified by alignment of the PH20 polypeptide with the polypeptide set forth in SEQ ID NO:3; and provided that if the modified PH20 polypeptide contains an amino acid replacement at a position corresponding to position 13, 47, 131, or 219 the replacement is not replacement with an Alanine (A). Among these modified PH20 polypeptides are those that exhibit at least 40% of the hyaluronidase activity of the PH20 polypeptide not containing the amino acid replacement, where, as in all instances herein activity is compared under the same conditions. 
     Included among these polypeptides are those that contain an amino acid replacement in the sequence of amino acids set forth in any of SEQ ID NOs: 3, 7, 32-66, 69 and 72, or in a sequence of amino acids that exhibits at least 91% sequence identity to any of SEQ ID NOs: 3, 7, 32-66, 69, or 72. In particular, the modified PH20 polypeptide contains amino acid replacements in SEQ ID NO: 3, 7, 32-66, 69, or 72, which are polypeptides that are a C-terminally truncated fragment of SEQ ID NO:7, or a PH20 polypeptide that has a sequence of amino acids that is at least 91% identical to the sequence of amino acids set forth in SEQ ID NO:7. In particular, among any of such modified PH20 polypeptides provided herein are any including those in which the amino acid replacement is an amino acid replacement set forth in Table 3 below. For example, such modified PH20 polypeptides include those that have at least one amino acid replacement at an amino acid position corresponding to a position selected from among 1, 6, 8, 9, 10, 11, 12, 14, 15, 20, 22, 24, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 46, 47, 48, 49, 50, 52, 58, 59, 63, 67, 68, 69, 70, 71, 72, 73, 74, 75, 79, 82, 83, 84, 86, 87, 89, 90, 92, 93, 94, 97, 102, 104, 107, 114, 118, 120, 127, 128, 130, 131, 132, 135, 138, 139, 140, 141, 142, 143, 144, 146, 147, 148, 149, 150, 151, 152, 155, 156, 158, 160, 162, 163, 164, 165, 166, 167, 169, 170, 172, 173, 174, 175, 178, 179, 193, 195, 196, 198, 204, 205, 206, 209, 212, 213, 215, 219, 220, 221, 222, 232, 233, 234, 235, 236, 237, 238, 240, 247, 248, 249, 257, 258, 259, 260, 261, 263, 267, 269, 271, 272, 273, 274, 276, 277, 278, 279, 282, 283, 285, 287, 289, 291, 292, 293, 298, 305, 307, 308, 309, 310, 313, 314, 315, 317, 318, 320, 321, 324, 325, 326, 328, 335, 347, 349, 351, 353, 356, 359, 367, 368, 369, 371, 373, 374, 375, 376, 377, 380, 381, 383, 385, 389, 392, 393, 395, 396, 399, 401, 404, 405, 406, 407, 409, 410, 412, 416, 418, 419, 421, 425, 427, 428, 431, 433, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446 or 447 with reference to amino acid positions set forth in SEQ ID NO:3. Exemplary of such replacements are those that contain at least one amino acid replacement selected from among replacement with: histidine (H) at a position corresponding to position 1; A at a position corresponding to position 1; E at a position corresponding to position 1; G at a position corresponding to position 1; K at a position corresponding to position 1; Q at a position corresponding to position 1; R at a position corresponding to position 1; A at a position corresponding to position 6; M at a position corresponding to position 8; Q at a position corresponding to position 9; G at a position corresponding to position 10; H at a position corresponding to position 10; S at a position corresponding to position 11; E at a position corresponding to position 12; I at a position corresponding to position 12; K at a position corresponding to position 12; T at a position corresponding to position 12; V at a position corresponding to position 14; V at a position corresponding to position 15; M at a position corresponding to position 15; S at a position corresponding to position 20; T at a position corresponding to position 22; E at a position corresponding to position 24; H at a position corresponding to position 24; R at a position corresponding to position 24; A at a position corresponding to position 26; E at a position corresponding to position 26; K at a position corresponding to position 26; M at a position corresponding to position 26; Q at a position corresponding to position 26; R at a position corresponding to position 26; D at a position corresponding to position 27; K at a position corresponding to position 27; Rat a position corresponding to position 27; Rat a position corresponding to position 28; E at a position corresponding to position 29; I at a position corresponding to position 29; K at a position corresponding to position 29; L at a position corresponding to position 29; M at a position corresponding to position 29; P at a position corresponding to position 29; R at a position corresponding to position 29; S at a position corresponding to position 29; T at a position corresponding to position 29; V at a position corresponding to position 29; G at a position corresponding to position 30; H at a position corresponding to position 30; K at a position corresponding to position 30; L at a position corresponding to position 30; M at a position corresponding to position 30; Rat a position corresponding to position 30; S at a position corresponding to position 30; A at a position corresponding to position 31; C at a position corresponding to position 31; G at a position corresponding to position 31; H at a position corresponding to position 31; I at a position corresponding to position 31; K at a position corresponding to position 31; L at a position corresponding to position 31; P at a position corresponding to position 31; R at a position corresponding to position 31; S at a position corresponding to position 31; Tat a position corresponding to position 31; V at a position corresponding to position 31; W at a position corresponding to position 31; C at a position corresponding to position 32; F at a position corresponding to position 32; G at a position corresponding to position 32; H at a position corresponding to position 32; W at a position corresponding to position 33; G at a position corresponding to position 33; W at a position corresponding to position 34; Q at a position corresponding to position 35; V at a position corresponding to position 35; H at a position corresponding to position 36; N at a position corresponding to position 36; F at a position corresponding to position 37; M at a position corresponding to position 37; Y at a position corresponding to position 38; A at a position corresponding to position 39; L at a position corresponding to position 39; N at a position corresponding to position 39; T at a position corresponding to position 39; L at a position corresponding to position 40; Tat a position corresponding to position 41; L at a position corresponding to position 46; R at a position corresponding to position 46; D at a position corresponding to position 47; F at a position corresponding to position 47; T at a position corresponding to position 47; W at a position corresponding to position 47, with F at a position corresponding to position 48; H at a position corresponding to position 48; K at a position corresponding to position 48; N at a position corresponding to position 48; R at a position corresponding to position 49; D at a position corresponding to position 50; S at a position corresponding to position 50; M at a position corresponding to position 50; N at a position corresponding to position 52; Q at a position corresponding to position 52; R at a position corresponding to position 52; S at a position corresponding to position 52; T at a position corresponding to position 52; C at a position corresponding to position 58; K at a position corresponding to position 58; L at a position corresponding to position 58; P at a position corresponding to position 58; Q at a position corresponding to position 58; Rat a position corresponding to position 58; H at a position corresponding to position 58; N at a position corresponding to position 58; Y at a position corresponding to position 58; N at a position corresponding to position 59; K at a position corresponding to position 63; L at a position corresponding to position 63; M at a position corresponding to position 63; Rat a position corresponding to position 63; W at a position corresponding to position 63; V at a position corresponding to position 67; H at a position corresponding to position 68; P at a position corresponding to position 68; Q at a position corresponding to position 68; A at a position corresponding to position 69; C at a position corresponding to position 69; E at a position corresponding to position 69; F at a position corresponding to position 69; G at a position corresponding to position 69; I at a position corresponding to position 69; L at a position corresponding to position 69; M at a position corresponding to position 69; P at a position corresponding to position 69; R at a position corresponding to position 69; T at a position corresponding to position 69; W at a position corresponding to position 69; Y at a position corresponding to position 69; A at a position corresponding to position 70; C at a position corresponding to position 70; F at a position corresponding to position 70; G at a position corresponding to position 70; H at a position corresponding to position 70; K at a position corresponding to position 70; L at a position corresponding to position 70; N at a position corresponding to position 70; P at a position corresponding to position 70; R at a position corresponding to position 70; S at a position corresponding to position 70; T at a position corresponding to position 70; V at a position corresponding to position 70; Y at a position corresponding to position 70; G at a position corresponding to position 71; N at a position corresponding to position 71; R at a position corresponding to position 71; S at a position corresponding to position 71; K at a position corresponding to position 72; M at a position corresponding to position 72; Q at a position corresponding to position 72; A at a position corresponding to position 73; H at a position corresponding to position 73; K at a position corresponding to position 73; L at a position corresponding to position 73; Q at a position corresponding to position 73; Rat a position corresponding to position 73; T at a position corresponding to position 73; W at a position corresponding to position 73; A at a position corresponding to position 74; C at a position corresponding to position 74; E at a position corresponding to position 74; F at a position corresponding to position 74; G at a position corresponding to position 74; H at a position corresponding to position 74; K at a position corresponding to position 74; L at a position corresponding to position 74; M at a position corresponding to position 74; N at a position corresponding to position 74; P at a position corresponding to position 74; R at a position corresponding to position 74; S at a position corresponding to position 74; V at a position corresponding to position 74; W at a position corresponding to position 74; F at a position corresponding to position 75; L at a position corresponding to position 75; M at position corresponding to position 75; Rat a position corresponding to position 75; T at a position corresponding to position 75; L at a position corresponding to position 79; L at a position corresponding to position 82; N at a position corresponding to position 82; V at a position corresponding to position 83; Q at a position corresponding to position 83; S at a position corresponding to position 83; G at a position corresponding to position 83; E at a position corresponding to position 84; F at a position corresponding to position 84; G at a position corresponding to position 84; N at a position corresponding to position 84; R at a position corresponding to position 84; A at a position corresponding to position 86; H at a position corresponding to position 86; K at a position corresponding to position 86; N at a position corresponding to position 86; S at a position corresponding to position 86; T at a position corresponding to position 86; W at a position corresponding to position 86; C at a position corresponding to position 87; G at a position corresponding to position 87; L at a position corresponding to position 87; M at a position corresponding to position 87; R at a position corresponding to position 87; S at a position corresponding to position 87; T at a position corresponding to position 87; V at a position corresponding to position 87; Y at a position corresponding to position 87; C at a position corresponding to position 89; A at a position corresponding to position 90; E at a position corresponding to position 90; H at a position corresponding to position 90; K at a position corresponding to position 90; N at a position corresponding to position 90; R at a position corresponding to position 90; C at a position corresponding to position 92; L at a position corresponding to position 92; I at a position corresponding to position 93; L at a position corresponding to position 93; Q at a position corresponding to position 93; Rat a position corresponding to position 93; S at a position corresponding to position 93; T at a position corresponding to position 93; D at a position corresponding to position 94; Q at a position corresponding to position 94; R at a position corresponding to position 94; A at a position corresponding to position 97; C at an amino acid residue corresponding to position 97; D at a position corresponding to position 97; E at a position corresponding to position 97; G at a position corresponding to position 97; L at a position corresponding to position 97; S at a position corresponding to position 97; S at a position corresponding to position 102; T at a position corresponding to position 102; Rat a position corresponding to position 104; L at a position corresponding to position 107; A at a position corresponding to position 114; Q at a position corresponding to position 118; H at a position corresponding to position 120; F at a position corresponding to position 120; I at a position corresponding to position 120; S at a position corresponding to position 120; V at a position corresponding to position 120; Y at a position corresponding to position 120; E at a position corresponding to position 127; H at a position corresponding to position 127; N at a position corresponding to position 127; Q at a position corresponding to position 127; Rat a position corresponding to position 127; I at a position corresponding to position 128; Rat a position corresponding to position 130; G at a position corresponding to position 131; I at a position corresponding to position 131; M at a position corresponding to position 131; Q at a position corresponding to position 131; Rat a position corresponding to position 131; V at a position corresponding to position 131; N at a position corresponding to position 132; L at a position corresponding to position 132; D at a position corresponding to position 135; G at a position corresponding to position 135; Rat a position corresponding to position 135, with L at a position corresponding to position 138; T at a position corresponding to position 139; K at a position corresponding to position 140; H at a position corresponding to position 141; Rat a position corresponding to position 141; S at a position corresponding to position 141; W at a position corresponding to position 141; Y at a position corresponding to position 141; D at a position corresponding to position 142; G at a position corresponding to position 142; K at a position corresponding to position 142; N at a position corresponding to position 142; P at a position corresponding to position 142; Q at a position corresponding to position 142; R at a position corresponding to position 142; S at a position corresponding to position 142; T at a position corresponding to position 142; G at a position corresponding to position 143; K at a position corresponding to position 143; Rat a position corresponding to position 144; T at a position corresponding to position 144; P at a position corresponding to position 146; R at a position corresponding to position 146; A at a position corresponding to position 147; F at a position corresponding to position 147; L at a position corresponding to position 147; R at a position corresponding to position 147; S at a position corresponding to position 147; V at a position corresponding to position 147; H at a position corresponding to position 148; K at a position corresponding to position 148; Q at a position corresponding to position 148; T at a position corresponding to position 149; V at a position corresponding to position 149; A at a position corresponding to position 150; D at a position corresponding to position 150; G at a position corresponding to position 150; N at a position corresponding to position 150; S at a position corresponding to position 150; W at a position corresponding to position 150; Y at a position corresponding to position 150; A at a position corresponding to position 151; H at a position corresponding to position 151; K at a position corresponding to position 151; L at a position corresponding to position 151; M at a position corresponding to position 151; Q at a position corresponding to position 151; Rat a position corresponding to position 151; S at a position corresponding to position 151; Tat a position corresponding to position 151; V at a position corresponding to position 151; W at a position corresponding to position 151; Y at a position corresponding to position 151; Rat a position corresponding to position 152; T at a position corresponding to position 152; W at a position corresponding to position 152; D at a position corresponding to position 155; G at a position corresponding to position 155; K at a position corresponding to position 155; Rat a position corresponding to position 155; D at a position corresponding to position 156; Q at a position corresponding to position 158; S at a position corresponding to position 158; S at a position corresponding to position 160; E at a position corresponding to position 162; A at a position corresponding to position 163; E at a position corresponding to position 163; K at a position corresponding to position 163; Q at a position corresponding to position 163; Rat a position corresponding to position 163; S at a position corresponding to position 163; M at a position corresponding to position 164; V at a position corresponding to position 164; D at a position corresponding to position 165; F at a position corresponding to position 165; N at a position corresponding to position 165; S at a position corresponding to position 165; V at a position corresponding to position 165; A at a position corresponding to position 166; E at a position corresponding to position 166; F at a position corresponding to position 166; H at a position corresponding to position 166; L at a position corresponding to position 166; Q at a position corresponding to position 166; Rat a position corresponding to position 166; Tat a position corresponding to position 166; W at a position corresponding to position 166; Y at a position corresponding to position 166; D at a position corresponding to position 167; L at a position corresponding to position 169; Rat a position corresponding to position 170; A at a position corresponding to position 172; Rat a position corresponding to position 173; G at a position corresponding to position 174; K at a position corresponding to position 174; N at a position corresponding to position 174; Rat a position corresponding to position 174; T at a position corresponding to position 174; T at a position corresponding to position 175; K at a position corresponding to position 178; Rat a position corresponding to position 178; K at a position corresponding to position 179; Q at a position corresponding to position 193; T at a position corresponding to position 195; N at a position corresponding to position 195; with E at a position corresponding to position 196; Rat a position corresponding to position 196; with D at a position corresponding to position 198; P at a position corresponding to position 204; A at a position corresponding to position 205; E at a position corresponding to position 205; L at a position corresponding to position 205; T at a position corresponding to position 205; I at a position corresponding to position 206; K at a position corresponding to position 206; L at a position corresponding to position 206; R at a position corresponding to position 206; R at a position corresponding to position 209; N at a position corresponding to position 212; S at a position corresponding to position 212; A at a position corresponding to position 213; M at a position corresponding to position 213; N at a position corresponding to position 213; H at a position corresponding to position 215; M at a position corresponding to position 215; I at a position corresponding to position 219; K at a position corresponding to position 219; S at a position corresponding to position 219; H at a position corresponding to position 220; I at a position corresponding to position 220; L at a position corresponding to position 220; V at a position corresponding to position 220; Q at a position corresponding to position 221; G at a position corresponding to position 222; F at a position corresponding to position 232; G at a position corresponding to position 233; K at a position corresponding to position 233; Rat a position corresponding to position 233; M at a position corresponding to position 234; A at a position corresponding to position 235; R at a position corresponding to position 236; C at a position corresponding to position 237; E at a position corresponding to position 237; H at a position corresponding to position 237; Q at a position corresponding to position 237; T at a position corresponding to position 237; E at a position corresponding to position 238; H at a position corresponding to amino acid position 238; S at a position corresponding to position 238; A at a position corresponding to position 240; Q at a position corresponding to position 240; I at a position corresponding to position 247; A at a position corresponding to position 248; V at a position corresponding to position 249; G at a position corresponding to position 257; T at a position corresponding to position 257; Rat a position corresponding to position 257; N at a position corresponding to position 258; S at a position corresponding to position 258; P at a position corresponding to position 259; M at a position corresponding to position 260; Y at a position corresponding to position 260; A at a position corresponding to position 261; K at a position corresponding to position 261; N at a position corresponding to position 261; K at a position corresponding to position 263; Rat a position corresponding to position 263; T at a position corresponding to position 267; A at a position corresponding to position 269; L at a position corresponding to position 271; M at a position corresponding to position 271; D at a position corresponding to position 272; Tat a position corresponding to position 272; H at a position corresponding to position 273; Y at a position corresponding to position 273; F at a position corresponding to position 274; D at a position corresponding to position 276; H at a position corresponding to position 276; M at a position corresponding to position 276; R at a position corresponding to position 276; S at a position corresponding to position 276; Y at a position corresponding to position 276; A at a position corresponding to position 277; E at a position corresponding to position 277; H at a position corresponding to position 277; K at a position corresponding to position 277; M at a position corresponding to position 277; N at a position corresponding to position 277; Q at a position corresponding to position 277; R at a position corresponding to position 277; S at a position corresponding to position 277; T at a position corresponding to position 277; E at a position corresponding to position 278; F at a position corresponding to position 278; G at a position corresponding to position 278; H at a position corresponding to position 278; K at a position corresponding to position 278; N at a position corresponding to position 278; Rat a position corresponding to position 278; S at a position corresponding to position 278; T at a position corresponding to position 278; Y at a position corresponding to position 278; H at a position corresponding to position 279; M at a position corresponding to position 282; S at a position corresponding to position 283; H at a position corresponding to position 285; T at a position corresponding to position 287; S at a position corresponding to position 289; S at a position corresponding to position 291; V at a position corresponding to position 291; C at a position corresponding to position 292; F at a position corresponding to position 292; H at a position corresponding to position 292; K at a position corresponding to position 292; R at a position corresponding to position 292; V at a position corresponding to position 292; A at a position corresponding to position 293; C at a position corresponding to position 293; D at a position corresponding to position 293; F at a position corresponding to position 293; K at a position corresponding to position 293; M at a position corresponding to position 293; P at a position corresponding to position 293; Q at a position corresponding to position 293; V at a position corresponding to position 293; Y at a position corresponding to position 293; G at a position corresponding to position 298; E at a position corresponding to position 305; G at a position corresponding to position 307; D at a position corresponding to position 308; G at a position corresponding to position 308; K at a position corresponding to position 308; N at a position corresponding to position 308; Rat a position corresponding to position 308; E at a position corresponding to position 309; G at a position corresponding to position 309; H at a position corresponding to position 309; L at a position corresponding to position 309; M at a position corresponding to position 309; N at a position corresponding to position 309; Q at a position corresponding to position 309; Rat a position corresponding to position 309; S at a position corresponding to position 309; T at a position corresponding to position 309; V at a position corresponding to position 309; A at a position corresponding to position 310; G at a position corresponding to position 310; Q at a position corresponding to position 310; S at a position corresponding to position 310; A at a position corresponding to position 313; G at a position corresponding to position 313; H at a position corresponding to position 313; K at a position corresponding to position 313; P at a position corresponding to position 313; Rat a position corresponding to position 313; Tat a position corresponding to position 313; Y at a position corresponding to position 313; with S at a position corresponding to position 314; Y at a position corresponding to position 314; A at a position corresponding to position 315; H at a position corresponding to position 315; Y at a position corresponding to position 315; A at a position corresponding to position 317; I at a position corresponding to position 317; K at a position corresponding to position 317; N at a position corresponding to position 317; Q at a position corresponding to position 317; Rat a position corresponding to position 317; S at a position corresponding to position 317; T at a position corresponding to position 317; W at a position corresponding to position 317; D at a position corresponding to position 318; H at a position corresponding to position 318; K at a position corresponding to position 318; M at a position corresponding to position 318; R at a position corresponding to position 318; H at a position corresponding to position 320; K at a position corresponding to position 320; R at a position corresponding to position 320; Rat a position corresponding to position 321; S at a position corresponding to position 321; N at a position corresponding to position 324; Rat a position corresponding to position 324; A at a position corresponding to position 325; D at a position corresponding to position 325; E at a position corresponding to position 325; G at a position corresponding to position 325; H at a position corresponding to position 325; K at a position corresponding to position 325; M at a position corresponding to position 325; N at a position corresponding to position 325; Q at a position corresponding to position 325; S at a position corresponding to position 325; V at a position corresponding to position 325; L at a position corresponding to position 326; V at a position corresponding to position 326; C at a position corresponding to position 328; G at a position corresponding to position 328; I at a position corresponding to position 328; K at a position corresponding to position 328; L at a position corresponding to position 328; S at a position corresponding to position 328; Y at a position corresponding to position 328; S at a position corresponding to position 335; A at a position corresponding to position 347; G at a position corresponding to position 347; S at a position corresponding to position 347; M at a position corresponding to position 349; R at a position corresponding to position 349; S at a position corresponding to position 351; V at a position corresponding to position 353; with H at a position corresponding to position 356; S at a position corresponding to position 356; E at a position corresponding to position 359; H at a position corresponding to position 359; T at a position corresponding to position 359; A at a position corresponding to position 367; G at a position corresponding to position 367; K at a position corresponding to position 367; S at a position corresponding to position 367; A at a position corresponding to position 368; E at a position corresponding to position 368; K at a position corresponding to position 368; L at a position corresponding to amino acid position 368; M at a position corresponding to amino acid position 368; R at a position corresponding to position 368; T at a position corresponding to amino acid position 368; H at a position corresponding to position 369; R at a position corresponding to position 369; F at a position corresponding to position 371; H at a position corresponding to position 371; K at a position corresponding to position 371; L at a position corresponding to position 371; Rat a position corresponding to position 371; S at a position corresponding to position 371; M at a position corresponding to position 373; H at a position corresponding to position 374; P at a position corresponding to position 374; A at a position corresponding to position 375; G at a position corresponding to position 375; K at a position corresponding to position 375; Rat a position corresponding to position 375; D at a position corresponding to position 376; E at a position corresponding to position 376; Q at a position corresponding to position 376; Rat a position corresponding to position 376; T at a position corresponding to position 376; V at a position corresponding to position 376; Y at a position corresponding to position 376; D at a position corresponding to position 377; E at a position corresponding to position 377; H at a position corresponding to position 377; K at a position corresponding to position 377; P at a position corresponding to position 377; Rat a position corresponding to position 377; S at a position corresponding to position 377; T at a position corresponding to position 377; W at a position corresponding to position 380; Y at a position corresponding to position 380; S at a position corresponding to position 381; I at a position corresponding to position 383; K at a position corresponding to position 383; L at a position corresponding to position 383; S at a position corresponding to position 383; A at a position corresponding to position 385; Q at a position corresponding to position 385; V at a position corresponding to position 385; A at a position corresponding to position 389; G at a position corresponding to position 389; L at a position corresponding to position 389; K at a position corresponding to position 389; Q at a position corresponding to position 389; S at a position corresponding to position 389; A at a position corresponding to position 392; F at a position corresponding to position 392; M at a position corresponding to position 392; Q at a position corresponding to position 392; R at a position corresponding to position 392; V at a position corresponding to position 392; F at a position corresponding to position 393; M at a position corresponding to position 393; A at a position corresponding to position 395; H at a position corresponding to position 395; Rat a position corresponding to position 395; A at a position corresponding to position 396; H at a position corresponding to position 396; Q at a position corresponding to position 396; S at a position corresponding to position 396; K at a position corresponding to position 399; M at a position corresponding to position 399; T at a position corresponding to position 399; V at a position corresponding to position 399; W at a position corresponding to position 399; A at a position corresponding to position 401; E at a position corresponding to position 401; A at a position corresponding to position 404; G at a position corresponding to position 405; F at a position corresponding to position 406; N at a position corresponding to position 406; A at a position corresponding to position 407; D at a position corresponding to position 407; E at a position corresponding to position 407; F at a position corresponding to position 407; H at a position corresponding to position 407; Q at a position corresponding to position 407; P at a position corresponding to position 407; A at a position corresponding to position 409; Q at a position corresponding to position 409; Tat a position corresponding to position 410; Q at a position corresponding to position 412; Rat a position corresponding to position 412; V at a position corresponding to position 412; L at a position corresponding to position 416; E at a position corresponding to position 418; L at a position corresponding to position 418; P at a position corresponding to position 418; Rat a position corresponding to position 418; V at a position corresponding to position 418; F at a position corresponding to position 419; H at a position corresponding to position 419; I at a position corresponding to position 419; K at a position corresponding to position 419; Rat a position corresponding to position 419; S at a position corresponding to position 419; Y at a position corresponding to position 419; A at a position corresponding to position 421; H at a position corresponding to position 421; K at a position corresponding to position 421; N at a position corresponding to position 421; Q at a position corresponding to position 421; R at a position corresponding to position 421; S at a position corresponding to position 421; G at a position corresponding to position 425; K at a position corresponding to position 425; Q at a position corresponding to position 427; T at a position corresponding to position 427; L at a position corresponding to position 428; A at a position corresponding to position 431; G at a position corresponding to position 431; E at a position corresponding to position 431; H at a position corresponding to position 431; K at a position corresponding to position 431; L at a position corresponding to position 431; N at a position corresponding to position 431; Q at a position corresponding to position 431; Rat a position corresponding to position 431; S at a position corresponding to position 431; V at a position corresponding to position 431; A at a position corresponding to position 433; H at a position corresponding to position 433; I at a position corresponding to position 433; K at a position corresponding to position 433; L at a position corresponding to position 433; Rat a position corresponding to position 433; T at a position corresponding to position 433; V at a position corresponding to position 433; W at a position corresponding to position 433; K at a position corresponding to position 436; I at a position corresponding to position 437; M at a position corresponding to position 437; A at a position corresponding to position 438; D at a position corresponding to position 438; Eat a position corresponding to position 438; L at a position corresponding to position 438; N at a position corresponding to position 438; T at a position corresponding to position 438; A at a position corresponding to position 439; C at a position corresponding to position 439; K at a position corresponding to position 439; P at a position corresponding to position 439; Q at a position corresponding to position 439; T at a position corresponding to position 439; V at a position corresponding to position 439; D at a position corresponding to position 440; H at a position corresponding to position 440; M at a position corresponding to position 440; P at a position corresponding to position 440; R at a position corresponding to position 440; S at a position corresponding to position 440; A at a position corresponding to position 441; F at a position corresponding to position 441; C at a position corresponding to position 442; G at a position corresponding to position 442; R at a position corresponding to position 442; A at a position corresponding to position 443; E at a position corresponding to position 443; F at a position corresponding to position 443; G at a position corresponding to position 443; M at a position corresponding to position 443; N at a position corresponding to position 443; E at a position corresponding to position 444; H at a position corresponding to position 444; V at a position corresponding to position 444; H at a position corresponding to position 445; M at a position corresponding to position 445; N at a position corresponding to position 445; P at a position corresponding to position 445; Q at a position corresponding to position 445; S at a position corresponding to position 445; T at a position corresponding to position 445; V at a position corresponding to position 445; W at a position corresponding to position 445; A at a position corresponding to position 446; M at a position corresponding to position 446; W at a position corresponding to position 446; D at a position corresponding to position 447; E at a position corresponding to position 447; G at a position corresponding to position 447; I at a position corresponding to position 447; N at a position corresponding to position 447; P at a position corresponding to position 447; Q at a position corresponding to position 447; T at a position corresponding to position 447, and/or replacement with V at a position corresponding to position 447, each with reference to amino acid positions set forth in SEQ ID NO:3. Among these modified PH20 polypeptides are those that exhibit at least 40% of the activity of the PH20 that does not contain the particular amino acid replacement. Activity can vary between, for example, 40% to 5000%, 40% to 2000%, 40% to 1000%, 40% to 500%, 40% to 100%, 80% to 2000%, 80% to 600%, 80% to 200%, 80% to 300%, of the hyaluronidase activity of the PH20 polypeptide not containing the amino acid replacement. Such activity includes at least 50%, 60%, 70%, 80%, 90%, 100%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, 2000%, 3000% or more of the hyaluronidase activity of the PH20 polypeptide not containing the amino acid replacement, where, as in all instances herein, the activities are compared under the same conditions. 
     In particular, provided are modified PH20 polypeptides that contain at least one amino acid replacement in a PH20 polypeptide set forth in SEQ ID NO:7, a C-terminally truncated fragment thereof, or in a PH20 polypeptide that has a sequence of amino acids that is at least 91% identical to the sequence of amino acids set forth in SEQ ID NO:7 or a corresponding truncated fragment, where: the modified PH20 polypeptides exhibit less than 20% of the hyaluronidase activity of the PH20 polypeptide not containing the amino acid replacement, where activities are compared under the same conditions; the amino acid replacement(s) is at an amino acid position corresponding to a position selected from among 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 25, 27, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 94, 95, 96, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 143, 144, 145, 149, 150, 152, 153, 154, 155, 156, 157, 158, 159, 161, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 197, 198, 199, 200, 201, 202, 203, 204, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 278, 279, 280, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 331, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 408, 410, 411, 412, 413, 414, 415, 416, 417, 419, 420, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 434, 437, 438, 439, 440, 441, 442, 443, 444, or 447 with reference to amino acid positions set forth in SEQ ID NO:3 or 7; 
     corresponding amino acid positions are identified by alignment of the PH20 polypeptide with the polypeptide set forth in SEQ ID NO:3; and provided that: 
     (i) if the modified PH20 polypeptide contains an amino acid replacement at a position corresponding to position 200, 333, 358 or 393 the replacement is not replacement with an Alanine (A). 
     (ii) if the modified PH20 polypeptide contains an amino acid replacement at a position corresponding to position 111 or 249 the replacement is not replacement with an asparagine (N); 
     (iii) if the modified PH20 polypeptide contains an amino acid replacement at a position corresponding to position 113 the replacement is not replacement with a glutamine (Q); 
     (iv) if the modified PH20 polypeptide contains an amino acid replacement at a position corresponding to position 176 the replacement is not replacement with a glycine (G); and 
     (v) if the modified PH20 polypeptide contains an amino acid replacement at a position corresponding to position 252 the replacement is not replacement with a threonine (T). 
     Exemplary of such modified PH20 polypeptides are any that contain amino acid replacement(s) in a PH20 polypeptide that has the sequence of amino acids set forth in any of SEQ ID NOs: 3, 7, 32-66, 69, or 72, or in a sequence of amino acids that exhibits at least 91% sequence identity to any of SEQ ID NOs: 3, 7, 32-66, 69, or 72. For example, the modified PH20 polypeptide contains amino acid replacement(s) in SEQ ID NOs: 3, 7, 32-66, 69, or 72, which are polypeptides that are a C-terminally truncated fragment of SEQ ID NO:7, or a PH20 polypeptide that has a sequence of amino acids that is at least 91% identical to the sequence of amino acids set forth in SEQ ID NO:7. In examples of such modified PH20 polypeptides provided herein, the modified PH20 polypeptides can exhibit similar or the same activity as the PH20 without the modification, or can exhibit increased activity or activity that is less than 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.05% or less of the hyaluronidase activity of the PH20 polypeptide not containing the amino acid replacement. Exemplary of such modified PH20 polypeptides are any set forth in Table 5. 
     Among any and all of the modified PH20 polypeptides provided herein and above, the modified PH20 polypeptide is one that does not consist of the sequence of amino acids set forth in any of SEQ ID NOs: 3, 6-66, 69-72, 856-861, 869 or 870. In particular, among any of the modified PH20 polypeptides provided herein above or elsewhere herein are any that contain an amino acid replacement(s) in a PH20 polypeptide having the sequence of amino acids set forth any of SEQ ID NO: 3, 7, 69 or 72 provided that: (i) where the modified PH20 polypeptide includes only a single amino acid replacement the replacement does not corresponds to amino acid replacements V12A, N47A, D111N, E113Q, N131A, R176G, N200A, N219A, E249Q, R252T, N333A or N358A, with reference to amino acid positions set forth in SEQ ID NO:3; (ii) where the modified PH20 polypeptide includes only two amino acid replacements the replacements do not correspond to amino acid replacements P 13A/L464W, N47A/N131A, N47A/N219A, N131A/N219A or N333A/N358A with reference to positions set forth in SEQ ID NO:3; and (iii) where the modified PH20 polypeptide includes only three amino acid replacements the replacements do not correspond to amino acid replacements N47A/N131A/N219A, with reference to amino acid positions set forth in SEQ ID NO:3. 
     Any of the above modified PH20 polypeptides and any provided herein and described above and below can contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, or more of the amino acid replacements. The modified PH20 polypeptides can include a signal sequence, including the native sequence or a heterologous sequence or a modified sequence, and also include a mature PH20 polypeptide that lacks the signal sequence. 
     Among any of the modified PH20 polypeptides provided herein above or described below are modified PH20 polypeptides that contain or have the sequence of amino acids set forth in any of SEQ ID NOs: 73-855 or a sequence of amino acids that exhibits at least 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a sequence of amino acids set forth in any of SEQ ID NOs: 73-855 and that contains at least one amino acid replacement, such as any described above or elsewhere herein, with reference to positions compared to the sequence of amino acids set forth in SEQ ID NO:3. In any of the examples of the modified PH20 polypeptides provided herein, the modified PH20 polypeptide does not have or contain the sequence of amino acids set forth in any of SEQ ID NOs: 8-31, 69-72, 856-861, 869 or 870. 
     The modified PH20 polypeptides provided herein can be substantially purified or isolated, can exhibit catalytic activity at neutral pH, can be secreted upon expression from cells and are soluble in the supernatant, and/or can include modified amino acids, such as a modification selected from among glycosylation, sialation, albumination, farnysylation, carboxylation, hydroxylation, conjugation to a polymer, such as PEGylation or conjugation to dextran, conjugation to another moiety, such as a multimerization domain, toxin, detectable label or drug, and phosphorylation. The modified PH20 polypeptide can be glycosylated, such as by containing at least an N-acetylglucosamine moiety linked to each of at least three asparagine (N) residues, where, for example, the three asparagine residues correspond to amino acid residues 200, 333 and 358 of SEQ ID NO:3. Multimerization domains include Fc domains. 
     Also provided are nucleic acid molecules that encode any of the modified PH20 polypeptides provided herein. Vectors, eukaryotic and prokaryotic, that contain the nucleic acid molecules are provided. The vectors include expression vectors and include mammalian vectors, including viral vectors. Viral vectors include adenovirus vectors, retrovirus vectors, vaccinia virus vectors, herpes simplex virus and cytomegalovirus vectors, and other such viral vectors. Of interest are oncolytic vectors that accumulate in or are targeted to tumors. Also provided are cells that contain the nucleic acid molecules and cells that contain the vectors. The cells can be prokaryotic or eukaryotic, particularly mammalian cells, such as Chinese Hamster Ovary (CHO) cells. 
     Also provided herein is a modified PH20 polypeptide that is produced by any of the provided cells. Thus, provided herein are methods of producing a modified PH20 polypeptide by culturing any of the cells provided herein under conditions whereby an encoded modified PH20 polypeptide is produced and secreted by the cell, and recovering the expressed polypeptide. Also provided herein is a method of producing a modified PH20 polypeptide by introducing any of the nucleic acids provided herein or any of the vectors provided herein into a cell capable of incorporating N-linked sugar moieties into the polypeptide, culturing the cell under conditions whereby an encoded modified PH20 polypeptide is produced and secreted by the cell, and recovering the expressed polypeptide. In such examples, the nucleic acid is operably linked to a promoter. The cultured cell can be a eukaryotic cell, such as a mammalian cell, for example, a Chinese hamster ovary (CHO) cell. 
     Also provided are pharmaceutical compositions that contain any of the modified PH20 polypeptides provided herein or any of the nucleic acids or vectors provided herein. The compositions can be formulated with other agents and/or with other components, such as preservatives. The compositions can be formulated so that the components, particularly the PH20 and any other active agent, remain active or are stable under preselected conditions. In addition, as described herein, the PH20 polypeptides are modified so that they exhibit increased stability under various conditions. For example, provided are compositions in which the modified PH20 polypeptide is stable (i.e., retains activity as described herein) at a temperature from or from about 2° C. to 8° C., inclusive, for at least 1 month or is stable at a temperature from or from about 30° C. to 42° C., inclusive, for at least 3 days. Provided are compositions in which the modified PH20 polypeptide in the composition is stable at a temperature from or from about 2° C. to 8° C., inclusive, for at least 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 24 months, 25 months, 26 months, 27 months, 28 months, 29 months or 30 months. Also provided are compositions in which the modified PH20 polypeptide in the composition is stable at a temperature from or from about 30° C. to 42° C., inclusive, for at least 3 days, at least 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 35 days, 40 days, 45 days, 50 days, 60 days or more. The pharmaceutical compositions can contain a pharmaceutically acceptable excipient. 
     The conditions, formulations, components, and modified PH20 polypeptide are chosen to achieve a desired stability. The pharmaceutical compositions can be formulated for direct administration or can require dilution. They can be formulated for multiple or single dosage administration. Exemplary compositions include concentrations of modified PH20 between or about between 0.1 μg/mL to 100 μg/mL, 1 μg/mL to 50 μg/mL or 1 μg/mL to 20 μg/mL, or 10 U/mL to 5000 U/mL, 50 U/mL to 4000 U/mL, 100 U/mL to 2000 U/mL, 300 U/mL to 2000 U/mL, 600 U/mL to 2000 U/mL, or 100 U/mL to 1000 U/mL. Exemplary salts include NaCl at a concentration, for example, of less than or about or 200 mM, 180 mM, 150 mM, 140 mM, 130 mM, 120 mM, 110 mM, 100 mM, 90 mM, 80 mM, 70 mM, 60 mM, 50 mM, 40 mM, 30 mM, 25 mM, 20 mM, 15 mM, 10 mM, 5 mM or less, or between or about between 0.1 mM to 200 mM, 0.1 mM to 100 mM, 120 mM to 200 mM, 10 mM to 50 mM, 10 mM to 90 mM, 80 mM to 200 mM, 80 mM to 140 mM, 50 mM to 100 mM, 80 mM to 100 mM, 50 mM to 80 mM, 100 mM to 140 mM or 120 mM to 140 mM. 
     The pharmaceutical compositions can contain an anti-microbially effective amount of a preservative or mixture of preservatives, such as one, two, three, four or more of a phenolic preservative(s), a non-phenolic preservative(s) or a phenolic preservative(s) and a non-phenolic preservative(s), such as, but not limited to, phenol, m-cresol, methylparaben, benzyl alcohol, thimerosal, benzalkonium chloride, 4-chloro-1-butanol, chlorhexidine dihydrochloride, chlorhexidine digluconate, L-phenylalanine, EDTA, bronopol, phenylmercuric acetate, glycerol, imidurea, chlorhexidine, sodium dehydroacetate, o-cresol, p-cresol, chlorocresol, cetrimide, benzethonium chloride, ethyl paraben, propylparaben, butylparaben and any combinations thereof. Phenols include, for example, phenol, metacresol (m-cresol), benzyl alcohol, and parabens, such as methylparaben or propylparaben. Anti-microbial effective concentrations of one or more preservative agents (as a percentage (%) of mass concentration (w/v)) can be between 0.05% to 0.6%, 0.1% to 0.4%, 0.1% to 0.3%, 0.15% to 0.325%, 0.15% to 0.25%, 0.1% to 0.2%, 0.2% to 0.3% or 0.3% to 0.4% inclusive. Examples thereof are pharmaceutical compositions where the preservatives are phenol, m-cresol or phenol and m-cresol and the amount as a % of mass concentration (w/v) in the formulation is between or about between 0.1% to 0.25% phenol and between or about between 0.05% to 0.2% m-cresol, is between or about between 0.10% to 0.2% phenol and between or about between 0.6% to 01.8% m-cresol, between or about between 0.1% to 0.15% phenol and 0.8% to 0.15% m-cresol, is between or about between 0.10% to 0.15% phenol and between or about between 0.06 to 0.09% m-cresol or is between or about between 0.12% to 0.18% phenol and between or about between 0.14 to 0.22% m-cresol. 
     The pharmaceutical compositions can contain a further therapeutically active agent. The active agent can be formulated in the composition or provided as a combination with the PH20-containing composition, but in a separate composition for administration separately, sequentially, intermittently, simultaneously or together. Therapeutically active agents include, for example, an agent selected from among a chemotherapeutic agent, an analgesic agent, an anti-inflammatory agent, an antimicrobial agent, an amoebicidal agent, a trichomonacidal agent, an anti-Parkinson agent, an anti-malarial agent, an anticonvulsant agent, an anti-depressant agent, and antiarthritics agent, an anti-fungal agent, an antihypertensive agent, an antipyretic agent, an anti-parasite agent, an antihistamine agent, an alpha-adrenergic agonist agent, an alpha blocker agent, an anesthetic agent, a bronchial dilator agent, a biocide agent, a bactericide agent, a bacteriostat agent, a beta adrenergic blocker agent, a calcium channel blocker agent, a cardiovascular drug agent, a contraceptive agent, a decongestant agent, a diuretic agent, a depressant agent, a diagnostic agent, a electrolyte agent, a hypnotic agent, a hormone agent, a hyperglycemic agent, a muscle relaxant agent, a muscle contractant agent, an ophthalmic agent, a parasympathomimetic agent, a psychic energizer agent, a sedative agent, a sympathomimetic agent, a tranquilizer agent, an urinary agent, a vaginal agent, a viricide agent, a vitamin agent, a non-steroidal anti-inflammatory agent, an angiotensin converting enzyme inhibitor agent, a polypeptide, a protein, a nucleic acid, a drug, an organic molecule and a sleep inducer. Exemplary of such agents are antibodies, particularly monoclonal antibodies, an Immune Globulin preparation, a bisphosphonate, a cytokine, a chemotherapeutic agent, a coagulation factor and an insulin. Insulins include, for example, basal insulins and fast-acting insulin, such as regular insulin, particularly recombinant human insulin, and insulin analogs, such as insulin lispro, insulin aspart or insulin glulisine. Particular fast-acting insulins are those with an A chain having a sequence of amino acids set forth in SEQ ID NO:862 and a B chain having a sequence of amino acids set forth in SEQ ID NO:863 or an insulin with an A chain with a sequence of amino acids set forth as amino acid residue positions 88-108 of SEQ ID NO:864 and a B chain with a sequence of amino acids set forth as amino acid residue positions 25-54 of SEQ ID NO:864 or an insulin analog that is selected from among an insulin having an A chain with a sequence of amino acids set forth in SEQ ID NO:862 and a B chain having a sequence of amino acids set forth in any of SEQ NOs:865-867. The amount of fast-acting insulin in the compositions can be empirically determined, but typically can be 10 U/mL to 1000 U/mL, 50 U/mL to 500 U/mL, 100 U/mL to 1000 U/mL or 500 U/mL to 1000 U/mL, inclusive. 
     In particular examples, provided herein is a pharmaceutical composition containing any of the modified PH20 polypeptides provided herein that exhibit increased stability to a phenolic preservative and an insulin, such as a fast-acting insulin. The modified PH20 polypeptides and insulin can be provided in therapeutically effective amounts. For example, provided herein is a pharmaceutical composition that contains any of the modified PH20 polypeptides provided herein that exhibits increased stability to a phenolic preservative in an amount that is from or from about 100 U/mL to 1000 U/mL and a fast-acting insulin in an amount that is from or from about 10 U/mL to 1000 U/mL. For example, the fast-acting insulin can be an insulin analog, such as insulin lispro, insulin aspart or insulin glulisine or other analog. Any of such pharmaceutical compositions can be formulated at a pH that is from or from about 7.0 to 7.6. Any of such pharmaceutical compositions also can be formulated to contain salt, such as NaCl, at a concentration that is from or from about 0.1 mM to 200 mM and/or an anti-microbial effective amount of at least one preservative where the composition generally contains at least one phenolic preservative. The anti-microbial effective amount is a total amount of one or more preservative agents as a percentage (%) of mass concentration (w/v) that is or is between 0.05% and 0.6%. The phenolic preservative(s) can be a phenol, metacresol (m-cresol), benzyl alcohol, or a paraben. In any of the above examples of a pharmaceutical composition, the composition also can contain a surfactant, such as a polypropylene glycol, polyethylene glycol, glycerin, sorbitol, poloxamer or polysorbate, in an amount as a % of mass concentration (w/v) in the formulation that is at least or at least about 0.001%; a buffering agent that is a non-metal binding agent or is a metal binding agent, such as Tris, histidine, phosphate or citrate, wherein the concentration of the buffering agent is between or between about 1 mM to 100 mM; glycerin in a concentration less than 60 mM; an antioxidant, such as cysteine, tryptophan or methionine, at a concentration between or from about between 2 mM to 50 mM, inclusive; and/or zinc at a concentration of between or about between 0.001 to 0.1 mg per 100 units of insulin (mg/100U). Also provided herein are closed loop systems, insulin pumps including continuous subcutaneous infusion insulin (CSII) pumps and insulin pens that contain any of the pharmaceutical compositions. The pharmaceutical compositions can be used in methods or uses for treating diabetes, such as type 1 diabetes mellitus, type 2 diabetes mellitus or gestational diabetes. 
     Other therapeutic agents in any of the pharmaceutical compositions provided herein include, but are not limited to Adalimumabs, Agalsidase Betas, Alefacepts, Ampicillins, Anakinras, Antipoliomyelitic Vaccines, Anti-Thymocytes, Azithromycins, Becaplermins, Caspofungins, Cefazolins, Cefepimes, Cefotetans, Ceftazidimes, Ceftriaxones, Cetuximabs, Cilastatins, Clavulanic Acids, Clindamycins, Darbepoetin Alfas, Daclizumabs, Diphtheria, Diphtheria antitoxins, Diphtheria Toxoids, Efalizumabs, Epinephrines, Erythropoietin Alphas, Etanercepts, Filgrastims, Fluconazoles, Follicle-Stimulating Hormones, Follitropin Alphas, Follitropin Betas, Fosphenytoins, Gadodiamides, Gadopentetates, Gatifloxacins, Glatiramers, GM-CSF&#39;s, Goserelins, Goserelin acetates, Granisetrons,  Haemophilus  Influenza B&#39;s, Haloperidols, Hepatitis vaccines, Hepatitis A Vaccines, Hepatitis B Vaccines, Ibritumomab Tiuxetans, Ibritumomabs, Tiuxetans, Immunoglobulins, Hemophilus influenza vaccines, Influenza Virus Vaccines, Infliximabs, Insulin lispro, 75% neutral protamine lispro (NPL)/25% insulin lispro, 50% neutral protamine Hagedorn (NPH)/50% regular insulin, 70% NPH/30% regular insulin; Regular insulin, NPH insulin, Ultra insulin, Ultralente insulin, and Insulin Glargines, Interferons, Interferon alpha, Interferon Betas, Interferon Gammas, Interferon alpha-2a, Interferon alpha 2-b, Interferon Alphacon, Interferon alpha-n, Interferon Betas, Interferon Beta-1a&#39;s, Interferon Gammas, Interferon alpha-con, Iodixanols, Iohexols, Iopamidols, Ioversols, Ketorolacs, Laronidases, Levofloxacins, Lidocaines, Linezolids, Lorazepams, Measles Vaccines, Measles virus, Mumps viruses, Measles-Mumps-Rubella Virus Vaccines, Rubella vaccines, Medroxyprogesterones, Meropenems, Methylprednisolones, Midazolams, Morphines, Octreotides, Omalizumabs, Ondansetrons, Palivizumabs, Pantoprazoles, Pegaspargases, Pegfilgrastims, Peg-Interferon Alpha-2a&#39;s, Peg-Interferon Alpha-2b&#39;s, Pegvisomants, Pertussis vaccines, Piperacillins, Pneumococcal Vaccines and Pneumococcal Conjugate Vaccines, Promethazines, Reteplases, Somatropins, Sulbactams, Sumatriptans, Tazobactams, Tenecteplases, Tetanus Purified Toxoids, Ticarcillins, Tositumomabs, Triamcinolones, Triamcinolone Acetonides, Triamcinolone hexacetonides, Vancomycins, Varicella Zoster immunoglobulins, Varicella vaccines, other vaccines, Alemtuzumabs, Alitretinoins, Allopurinols, Altretamines, Amifostines, Anastrozoles, Arsenics, Arsenic Trioxides, Asparaginases,  Bacillus  Calmette-Guerin (BCG) vaccines, BCG Live, Bexarotenes, Bleomycins, Busulfans, Busulfan intravenous, Busulfan orals, Calusterones, Capecitabines, Carboplatins, Carmustines, Carmustines with Polifeprosans, Celecoxibs, Chlorambucils, Cisplatins, Cladribines, Cyclophosphamides, Cytarabines, Cytarabine liposomals, Dacarbazines, Dactinomycins, Daunorubicin liposomals, Daunorubicins, Daunomycins, Denileukin Diftitoxes, Dexrazoxanes, Docetaxels, Doxorubicins, Doxorubicin liposomals, Dromostanolone propionates, Elliott&#39;s B Solutions, Epirubicins, Epoetin alfas, Estramustines, Etoposides, Etoposide phosphates, Etoposide VP-16s, Exemestanes, Floxuridines, Fludarabines, Fluorouracils, 5-Fluorouracils, Fulvestrants, Gemcitabines, Gemtuzumabs, Ozogamicins, Gemtuzumab ozogamicins, Hydroxyureas, Idarubicins, Ifosfamides, Imatinib mesylates, Irinotecans, Letrozoles, Leucovorins, Levamisoles, Lomustines, CCNUs, Mechlorethamines, Nitrogen mustards, Megestrols, Megestrol acetates, Melphalans, L-PAMs, Mercaptopurines, 6-Mercaptopurines, Mesnas, Methotrexates, Methoxsalens, Mitomycins, Mitomycin C&#39;s, Mitotanes, Mitoxantrones, Nandrolones, Nandrolone Phenpropionates, Nofetumomabs, Oprelvekins, Oxaliplatins, Paclitaxels, Pamidronates, Pegademases, Pentostatins, Pipobromans, Plicamycins, Mithramycins, Porfimers, Porfimer sodiums, Procarbazines, Quinacrines, Rasburicases, Rituximabs, Sargramostims, Streptozocins, Talcs, Tamoxifens, Temozolomides, Teniposides, Testolactones, Thioguanines, 6-Thioguanines, Triethylenethiophosphoramides (Thiotepas), Topotecans, Toremifenes, Trastuzumabs, Tretinoins, Uracil Mustards, Valrubicins, Vinblastines, Vincristines, Vinorelbines, Zoledronates, Acivicins, Aclarubicins, Acodazoles, Acronines, Adozelesins, Aldesleukins, Retinoic Acids, Alitretinoins, 9-Cis-Retinoic Acids, Alvocidibs, Ambazones, Ambomycins, Ametantrones, Aminoglutethimides, Amsacrines, Anaxirones, Ancitabines, Anthramycins, Apaziquones, Argimesnas, Asperlins, Atrimustines, Azacitidines, Azetepas, Azotomycins, Banoxantrones, Batabulins, Batimastats, Benaxibines, Bendamustines, Benzodepas, Bicalutamides, Bietaserpines, Biricodars, Bisantrenes, Bisnafide Dimesylates, Bizelesins, Bortezomibs, Brequinars, Bropirimines, Budotitanes, Cactinomycins, Canertinibs, Caracemides, Carbetimers, Carboquones, Carmofurs, Carubicins, Carzelesins, Cedefingols, Cemadotins, Chlorambucils, Cioteronels, Cirolemycins, Clanfenurs, Clofarabines, Crisnatols, Decitabines, Dexniguldipines, Dexormaplatins, Dezaguanines, Diaziquones, Dibrospidiums, Dienogests, Dinalins, Disermolides, Dofequidars, Doxifluridines, Droloxifenes, Duazomycins, Ecomustines, Edatrexates, Edotecarins, Eflornithines, Elacridars, Elinafides, Elsamitrucins, Emitefurs, Enloplatins, Enpromates, Enzastaurins, Epipropidines, Eptaloprosts, Erbulozoles, Esorubicins, Etanidazoles, Etoglucids, Etoprines, Exisulinds, Fadrozoles, Fazarabines, Fenretinides, Fluoxymesterones, Flurocitabines, Fosquidones, Fostriecins, Fotretamines, Galarubicins, Galocitabines, Geroquinols, Gimatecans, Gimeracils, Gloxazones, Glufosfamides, Ilmofosines, Ilomastats, Imexons, Improsulfans, Indisulams, Inproquones, Interleukins, Interleukin-2s, recombinant Interleukins, Intoplicines, Iobenguanes, Iproplatins, Irsogladines, Ixabepilones, Ketotrexates, L-Alanosines, Lanreotides, Lapatinibs, Ledoxantrones, Leuprolides, Leuprorelins, Lexacalcitols, Liarozoles, Lobaplatins, Lometrexols, Lonafarnibs, Losoxantrones, Lurtotecans, Mafosfamides, Mannosulfans, Marimastats, Masoprocols, Maytansines, Mechlorethamines, Melengestrols, Melphalans, Menogarils, Mepitiostanes, Metesinds, Metomidates, Metoprines, Meturedepas, Miboplatins, Miproxifenes, Misonidazoles, Mitindomides, Mitocarcins, Mitocromins, Mitoflaxones, Mitogillins, Mitoguazones, Mitomalcins, Mitonafides, Mitoquidones, Mitospers, Mitozolomides, Mivobulins, Mizoribines, Mofarotenes, Mopidamols, Mubritinibs, Mycophenolic Acids, Nedaplatins, Neizarabines, Nemorubicins, Nitracrines, Nocodazoles, Nogalamycins, Nolatrexeds, Nortopixantrones, Ormaplatins, Ortataxels, Oteracils, Oxisurans, Oxophenarsines, Patupilones, Peldesines, Peliomycins, Pelitrexols, Pemetrexeds, Pentamustines, Peplomycins, Perfosfamides, Perifosines, Picoplatins, Pinafides, Piposulfans, Pirfenidones, Piroxantrones, Pixantrones, Plevitrexeds, Plomestanes, Porfiromycins, Prednimustines, Propamidines, Prospidiums, Pumitepas, Puromycins, Pyrazofurins, Ranimustines, Riboprines, Ritrosulfans, Rogletimides, Roquinimexs, Rufocromomycins, Sabarubicins, Safingols, Satraplatins, Sebriplatins, Semustines, Simtrazenes, Sizofirans, Sobuzoxanes, Sorafenibs, Sparfosates, Sparfosic Acids, Sparsomycins, Spirogermaniums, Spiromustines, Spiroplatins, Squalamines, Streptonigrins, Streptovarycins, Sufosfamides, Sulofenurs, Tacedinalines, Talisomycins, Tallimustines, Tariquidars, Tauromustines, Tecogalans, Tegafurs, Teloxantrones, Temoporfins, Teroxirones, Thiamiprines, Tiamiprines, Tiazofurins, Tilomisoles, Tilorones, Timcodars, Timonacics, Tirapazamines, Topixantrones, Trabectedins, Ecteinascidin 743, Trestolones, Triciribines, Trilostanes, Trimetrexates, Triplatin Tetranitrates, Triptorelins, Trofosfamides, Tubulozoles, Ubenimexs, Uredepas, Valspodars, Vapreotides, Verteporfins, Vinblastines, Vindesines, Vinepidines, Vinflunines, Vinformides, Vinglycinates, Vinleucinols, Vinleurosines, Vinrosidines, Vintriptols, Vinzolidines, Vorozoles, Xanthomycin A&#39;s, Guamecyclines, Zeniplatins, Zilascorbs [2-H], Zinostatins, Zorubicins, Zosuquidars, Acetazolamides, Acyclovirs, Adipiodones, Alatrofloxacins, Alfentanils, Allergenic extracts, Alpha 1-proteinase inhibitors, Alprostadils, Amikacins, Amino acids, Aminocaproic acids, Aminophyllines, Amitriptylines, Amobarbitals, Amrinones, Analgesics, Anti-poliomyelitic vaccines, Anti-rabic serums, Anti-tetanus immunoglobulins, tetanus vaccines, Antithrombin IIIs, Antivenom serums, Argatrobans, Arginines, Ascorbic acids, Atenolols, Atracuriums, Atropines, Aurothioglucoses, Azathioprines, Aztreonams, Bacitracins, Baclofens, Basiliximabs, Benzoic acids, Benztropines, Betamethasones, Biotins, Bivalirudins, Botulism antitoxins, Bretyliums, Bumetanides, Bupivacaines, Buprenorphines, Butorphanols, Calcitonins, Calcitriols, Calciums, Capreomycins, Carboprosts, Carnitines, Cefamandoles, Cefoperazones, Cefotaximes, Cefoxitins, Ceftizoximes, Cefuroximes, Chloramphenicols, Chloroprocaines, Chloroquines, Chlorothiazides, Chlorpromazines, Chondroitinsulfuric acids, Choriogonadotropin alfas, Chromiums, Cidofovirs, Cimetidines, Ciprofloxacins, Cisatracuriums, Clonidines, Codeines, Colchicines, Colistins, Collagens, Corticorelin ovine triflutates, Corticotrophins, Cosyntropins, Cyanocobalamins, Cyclosporines, Cysteines, Dacliximabs, Dalfopristins, Dalteparins, Danaparoids, Dantrolenes, Deferoxamines, Desmopressins, Dexamethasones, Dexmedetomidines, Dexpanthenols, Dextrans, Iron dextrans, Diatrizoic acids, Diazepams, Diazoxides, Dicyclomines, Digibinds, Digoxins, Dihydroergotamines, Diltiazems, Diphenhydramines, Dipyridamoles, Dobutamines, Dopamines, Doxacuriums, Doxaprams, Doxercalciferols, Doxycyclines, Droperidols, Dyphyllines, Edetic acids, Edrophoniums, Enalaprilats, Ephedrines, Epoprostenols, Ergocalciferols, Ergonovines, Ertapenems, Erythromycins, Esmolols, Estradiols, Estrogenics, Ethacrynic acids, Ethanolamines, Ethanols, Ethiodized oils, Etidronic acids, Etomidates, Famotidines, Fenoldopams, Fentanyls, Flumazenils, Fluoresceins, Fluphenazines, Folic acids, Fomepizoles, Fomivirsens, Fondaparinuxs, Foscarnets, Fosphenytoins, Furosemides, Gadoteridols, Gadoversetamides, Ganciclovirs, Gentamicins, Glucagons, Glucoses, Glycines, Glycopyrrolates, Gonadorelins, Gonadotropin chorionics,  Haemophilus  B polysaccharides, Hemins, Herbals, Histamines, Hydralazines, Hydrocortisones, Hydromorphones, Hydroxocobalamins, Hydroxyzines, Hyoscyamines, Ibutilides, Imiglucerases, Indigo carmines, Indomethacins, Iodides, Iopromides, lothalamic acids, loxaglic acids, Ioxilans, Isoniazids, Isoproterenols, Japanese encephalitis vaccines, Kanamycins, Ketamines, Labetalols, Lepirudins, Levobupivacaines, Levothyroxines, Lincomycins, Liothyronines, Luteinizing hormones, Lyme disease vaccines, Mangafodipirs, Manthtols, Meningococcal polysaccharide vaccines, Meperidines, Mepivacaines, Mesoridazines, Metaraminols, Methadones, Methocarbamols, Methohexitals, Methyldopates, Methylergonovines, Metoclopramides, Metoprolols, Metronidazoles, Minocyclines, Mivacuriums, Morrhuic acids, Moxifloxacins, Muromonab-CD3s, Mycophenolate mofetils, Nafcillins, Nalbuphines, Nalmefenes, Naloxones, Neostigmines, Niacinamides, Nicardipines, Nitroglycerins, Nitroprussides, Norepinephrines, Orphenadrines, Oxacillins, Oxymorphones, Oxytetracyclines, Oxytocins, Pancuroniums, Panthenols, Pantothenic acids, Papaverines, Peginterferon alpha 2As, Penicillin Gs, Pentamidines, Pentazocines, Pentobarbitals, Perflutrens, Perphenazines, Phenobarbitals, Phentolamines, Phenylephrines, Phenytoins, Physostigmines, Phytonadiones, Polymyxin, Pralidoximes, Prilocaines, Procainamides, Procaines, Prochlorperazines, Progesterones, Propranolols, Pyridostigmine hydroxides, Pyridoxines, Quinidines, Quinupristins, Rabies immunoglobulins, Rabies vaccines, Ranitidines, Remifentanils, Riboflavins, Rifampins, Ropivacaines, Samariums, Scopolamines, Seleniums, Sermorelins, Sincalides, Somatrems, Spectinomycins, Streptokinases, Streptomycins, Succinylcholines, Sufentanils, Sulfamethoxazoles, Tacrolimuses, Terbutalines, Teriparatides, Testosterones, Tetanus antitoxins, Tetracaines, Tetradecyl sulfates, Theophyllines, Thiamines, Thiethylperazines, Thiopentals, Thyroid stimulating hormones, Tinzaparins, Tirofibans, Tobramycins, Tolazolines, Tolbutamides, Torsemides, Tranexamic acids, Treprostinils, Trifluoperazines, Trimethobenzamides, Trimethoprims, Tromethamines, Tuberculins, Typhoid vaccines, Urofollitropins, Urokinases, Valproic acids, Vasopressins, Vecuroniums, Verapamils, Voriconazoles, Warfarins, Yellow fever vaccines, Zidovudines, Zincs, Ziprasidone hydrochlorides, Aclacinomycins, Actinomycins, Adriamycins, Azaserines, 6-Azauridines, Carzinophilins, Chromomycins, Denopterins, 6 Diazo 5 Oxo-L-Norleucines, Enocitabines, Floxuridines, Olivomycins, Pirarubicins, Piritrexims, Pteropterins, Tegafurs, Tubercidins, Alteplases, Arcitumomabs, bevacizumabs, Botulinum Toxin Type A&#39;s, Botulinum Toxin Type B&#39;s, Capromab Pendetides, Daclizumabs, Dornase alphas, Drotrecogin alphas, Imciromab Pentetates, Iodine-131&#39;s, an antibiotic agent; an angiogenesis inhibitor; anti-cataract and anti-diabetic retinopathy substances; carbonic anhydrase inhibitors; mydriatics; photodynamic therapy agents; prostaglandin analogs; growth factor; anti-neoplastics; anti-metabolites; anti-viral; amebicides and anti-protozoals; anti-tuberculosis and anti-leprotic; antitoxins and antivenins; antihemophilic factor, anti-inhibitor coagulant complex, antithrombin III, coagulations Factor V, coagulation Factor IX, plasma protein fraction, von Willebrand factor; antiplatelet agent a colony stimulating factor (CSF); an erythropoiesis stimulator; hemostatics and albumins; Immune Globulins; thrombin inhibitors; anticoagulants; a steroidal anti-inflammatory drug selected from among alclometasones, algestones, beclomethasones, betamethasones, budesonides, clobetasols, clobetasones, clocortolones, cloprednols, corticosterones, cortisones, cortivazols, deflazacorts, desonides, desoximetasones, dexamethasones, diflorasones, diflucortolones, difluprednates, enoxolones, fluazacorts, flucloronides, flumethasones, flunisolides, fluocinolones, fluocinonides, fluocortins, fluocortolones, fluorometholones, fluperolones, fluprednidenes, fluprednisolones, flurandrenolides, fluticasones, formocortals, halcinonides, halobetasols, halometasones, halopredones, hydrocortamates, hydrocortisones, loteprednol etabonate, mazipredones, medrysones, meprednisones, methylprednisolones, mometasone furoate, paramethasones, prednicarbates, prednisolones, prednisones, prednivals, prednylidenes, rimexolones, tixocortols and triamcinolones; Docosanols, prostaglandins, prostaglandin analogs, antiprostaglandins and prostaglandin precursors; miotics, cholinergics and anti-cholinesterase; and anti-allergenics. 
     The compositions and modified PH20 polypeptides can be used to treat any condition normally treated by the PH20 polypeptide or the therapeutically active agent. These include, for example, conditions in which hyaluronan plays a role or is associated with the etiology of the disease due to, for example, accumulation or overproduction of hyaluronan. Hence provided are methods, uses of the compositions and modified PH20 polypeptides for treating a hyaluronan-associated disease or condition by administering any of the modified PH20 polypeptides or compositions provided herein. Hyaluronan-associated diseases and conditions include, for example, inflammatory disease and tumors or cancers, including a late-stage cancer, metastatic cancers and undifferentiated cancers, such as ovarian cancer, in situ carcinoma (ISC), squamous cell carcinoma (SCC), prostate cancer, pancreatic cancer, non-small cell lung cancer, breast cancer and colon cancer. The PH20 polypeptide can be modified to exhibit increased half-life for such treatments. For example, the PH20 polypeptide can be modified with a polymer such as a PEG moiety for such treatments. 
     Also provided are methods for increasing delivery of a therapeutic agent to a subject by: administering to a subject any of the modified PH20 polypeptides or compositions provided herein, and administering the therapeutic agent. The therapeutic agent can be administered in the same composition or separately, and can be administered before or after, simultaneously, or intermittently, with administration of the PH20 polypeptide(s). Administration includes any route, including intravenous and subcutaneous administration, such as simultaneously with, intermittently with, or subsequent to administration of the therapeutic agent. The therapeutic agents include any of those set forth above, elsewhere herein and/or known to those of skill in the art. 
     Also provided are methods for treating an excess of glycosaminoglycans; for treating a tumor; for treating glycosaminoglycan accumulation in the brain; for treating a cardiovascular disorder; for treating an ophthalmic disorder; for treating pulmonary disease; for increasing penetration of chemotherapeutic agents into solid tumors; for treating cellulite; for treating a proliferative disorder; or for increasing bioavailability of drugs and other therapeutic agents by administering the modified PH20 polypeptides or compositions provided herein. 
     Also provided are pharmaceutical compositions for use in treating a hyaluronan-associated disease or disorder; for use in delivering a therapeutic agent to a subject; for treating an excess of glycosaminoglycans; for treating a tumor; for treating glycosaminoglycan accumulation in the brain; for treating a cardiovascular disorder; for treating an ophthalmic disorder; for treating pulmonary disease; for increasing penetration of chemotherapeutic agents into solid tumors; for treating cellulite; for treating a proliferative disorder; or for increasing bioavailability of drugs and other therapeutic agents; and for any other use of compositions containing PH20 polypeptides. 
     Provided herein is a method for identifying or selecting a modified hyaluronan-degrading enzyme that exhibits stability under a denaturation condition that includes the steps of: a) testing the activity of a modified hyaluronan-degrading enzyme in a composition containing a denaturing agent and/or under a denaturing condition; b) testing the activity of the modified hyaluronan-degrading enzyme in the same composition and/or under the same conditions as a) except absent the denaturing agent or condition; and c) selecting or identifying a modified hyaluronan-degrading enzyme that exhibits activity in a) that is at least 5% of the activity in b). In such an example, the activity is hyaluronidase activity. In some examples of the methods, a modified hyaluronan-degrading enzyme is selected or identified if the activity in a) is at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more of the activity in b), for example, a modified hyaluronan-degrading enzyme is selected or identified if the activity in a) is at least 40% or more of the activity in b). The method also can include steps of: d) comparing the activity of the modified hyaluronan-degrading enzyme in a) to the activity of the unmodified hyaluronan-degrading enzyme tested under the same conditions; and e) identifying or selecting a modified hyaluronan-degrading enzyme that exhibits at least 120%, 130%, 135%, 140%, 145%, 150%, 160%, 170%, 180%, 200%, 250%, 300%, 350%, 400%, 500%, 1500%, 2000%, 3000%, 4000%, 5000% or more of the hyaluronidase activity compared to the unmodified hyaluronan-degrading enzyme. 
     Also provided herein is a method for identifying or selecting a modified hyaluronan-degrading enzyme that exhibits stability, such as increased stability, under a denaturation condition, that includes the steps of: a) testing the activity of a modified hyaluronan-degrading enzyme in a composition containing a denaturing agent and/or under a denaturing condition; b) testing the activity of the corresponding unmodified hyaluronan-degrading enzyme in a composition containing the same denaturing agent and/or under the same denaturing condition as a), whereby the activity is tested under the same conditions as a); and c) selecting or identifying a modified hyaluronan-degrading enzyme that exhibits greater activity than the unmodified hyaluronan-degrading enzyme, thereby identifying or selecting a modified hyaluronan-degrading enzyme that exhibits increased stability under a denaturation condition. In such an example, the activity can be a hyaluronidase activity. In examples of the method, a modified hyaluronan-degrading enzyme is selected or identified if the activity is at least 120%, 130%, 135%, 140%, 145%, 150%, 160%, 170%, 180%, 200%, 250%, 300%, 350%, 400%, 500%, 1500%, 2000%, 3000%, 4000%, 5000% or more of the activity compared to the unmodified hyaluronan-degrading enzyme. In such an example, the method also can include additional steps of: d) testing the activity of the selected or identified modified hyaluronan-degrading enzyme in a composition containing a denaturing agent and/or under a denaturing condition; e) testing the activity of the same selected or identified modified hyaluronan-degrading enzyme in the same composition and/or under the same conditions as d) except absent the denaturing agent or condition; and f) selecting or identifying a modified hyaluronan-degrading enzyme that exhibits activity in d) that is at least 5% of the activity in e). In such an example, the activity is hyaluronidase activity. In some examples of the methods, a modified hyaluronan-degrading enzyme is selected or identified if the activity in d) is at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more of the activity in e), for example, a modified hyaluronan-degrading enzyme is selected or identified if the activity in d) is at least 40% or more of the activity in e). 
     In any of the methods provided herein for identifying or selecting a modified hyaluronan-degrading enzyme, the denaturing agent or condition is caused by temperature, agitation, no or low salt or the presence of an excipient. For example, the denaturing agent or condition is caused by elevated temperature that is from or from about 30° C. to 42° C., such as greater than or greater than about 30° C., 31° C., 32° C., 33° C., 34° C., 35° C., 36° C., 37° C., 38° C., 39° C., 40° C., 41° C. or 42° C. In other examples, the denaturing agent or condition is the absence of salt or low salt less than 100 mM, such as low salt less than 90 mM, 80 mM, 70 mM, 60 mM, 50 mM, 40 mM, 30 mM, 25 mM, 20 mM, 15 mM, 10 mM, 5 mM. In further examples, the denaturing agent or condition is a denaturing excipient selected from among an antiadherents, binders, coatings, fillers and diluents, flavors, colors, lubricants, glidants, preservatives, sorbents and sweeteners. 
     In particular examples of any of the methods provided herein for identifying or selecting a modified hyaluronan-degrading enzyme, the denaturing agent or condition is a preservative(s), for example, a phenolic preservative(s). The phenolic preservative(s) can be a phenol, metacresol (m-cresol), benzyl alcohol, or a paraben. For example, the denaturing agent or condition is a preservative(s) that is phenol and/or m-cresol. In such examples, the total amount of phenolic preservative in the composition, as a percentage (%) of mass concentration (w/v), is from or from about 0.05% to 0.6%, 0.1% to 0.4%, 0.1% to 0.3%, 0.15% to 0.325%, 0.15% to 0.25%, 0.1% to 0.2%, 0.2% to 0.3% or 0.3% to 0.4% inclusive. 
     In any of the methods provided herein for identifying or selecting a modified hyaluronan-degrading enzyme, prior to testing the activity of a hyaluronan-degrading enzyme composition in a) and/or b), the hyaluronan-degrading enzyme is exposed to the denaturation condition or denaturing agent for a predetermined time. The predetermined time is a time period that is user selected depending on the particular hyaluronan-degrading enzyme that is being evolved or selected, the particular denaturation condition or denaturing agent, the amount or extent of the denaturation condition or denaturing agent, the application or use of the hyaluronan-degrading enzyme and other similar factors. For example, the predetermined time can be from or from about 1 minute to 1 month, 1 minute to 3 weeks, 1 minute to 2 weeks, 1 minute to 1 week, 1 minute to 24 hours, 1 minute to 12 hours, 30 minutes to 6 hours or 1 hour to 4 hours, such as at least or about at least 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 24 hours, two days, three days, four days, five days, six days, 7 days, two weeks or one month. 
     In any of the methods provided herein for identifying or selecting a modified hyaluronan-degrading enzyme, the modified hyaluronan-degrading enzyme is one that contains an amino acid replacement, insertion or deletion of amino acids compared to an unmodified hyaluronan-degrading enzyme. For example, the modified hyaluronan-degrading enzyme contains an amino acid replacement, such as a single amino acid replacement or two, three, four, five, six, seven, eight, nine or more amino acid replacements compared to an unmodified form of the hyaluronan-degrading enzyme. In particular aspects of the method, a library or collection of modified hyaluronan-degrading enzymes are screened in order to evolve or identify or select a modified hyaluronan-degrading enzyme that exhibits stability, such as increased stability, under a denaturation condition. Thus, in examples of the methods herein, a plurality of modified hyaluronan-degrading enzymes are tested in a) and/or b). In such examples, the plurality of modified hyaluronan-degrading enzymes are modified compared to the corresponding unmodified hyaluronan-degrading enzyme to generate a collection of modified hyaluronan-degrading enzymes, whereby each modified protein in the collection is tested in each of a) and/or b). In the collection or library, each modified hyaluronan-degrading enzyme contains a single amino acid replacement compared to the unmodified form of the hyaluronan-degrading enzyme, such that the plurality of modified enzymes are such that the amino acid at each modified position is replaced by up to 1-19 other amino acids other than the original amino acid at the position, whereby each modified hyaluronan-degrading enzyme contains a different amino acid replacement, and every amino acid along the length of the hyaluronan-degrading enzyme, or a selected portion thereof, is replaced. 
     In any of the methods provided herein, the modified hyaluronan-degrading enzyme is modified compared to an unmodified hyaluronan-degrading enzyme by insertion, deletion or replacement of an amino acid(s). The unmodified hyaluronan-degrading enzyme can be a chondroitinase or can be a hyaluronidase. In examples herein, the unmodified hyaluronidase is a PH20 hyaluronidase or truncated form thereof lacking a C-terminal glycosylphosphatidylinositol (GPI) anchor attachment site or a portion of the GPI anchor attachment site, whereby the truncated form exhibits hyaluronidase activity. PH20 hyaluronidase can be a human, monkey, bovine, ovine, rat, fox, mouse or guinea pig PH20. In particular examples, the PH20 hyaluronidase is a human PH20 or a C-terminal truncated form thereof. For example, the unmodified hyaluronan-degrading enzyme is one that has the sequence of amino acids set forth in any of SEQ ID NOs: 3, 7, 10, 12, 14, 24, 32-66, 69, 72, 857, 859, 861, 870 or a sequence of amino acids that is at least 80% sequence identity to any of SEQ ID NOs: 3, 7, 10, 12, 14, 24, 32-66, 69, 72, 857, 859, 861, 870, such as at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to any of SEQ ID NOs: 3, 7, 10, 12, 14, 24, 32-66, 69, 72, 857, 859, 861, or 870. In particular examples, the unmodified hyaluronan-degrading enzyme is a PH20 hyaluronidase having the sequence of amino acids set forth in any of SEQ ID NOs: 3, 7, 32-66, 69 or 72, or a sequence of amino acids that exhibits at least 85% sequence identity to any of SEQ ID NOs: 3, 7, 32-66, 69 or 72, such as a sequence of amino acids that exhibits at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NOs: 3, 7, 32-66, 69 or 72. 
     In any of the methods provided herein for identifying or selecting a modified hyaluronan-degrading enzyme that exhibits stability, the method is performed in vitro. Also provided are any of the methods that are iterative, whereby the steps of the method are repeated a plurality of times, wherein in each repetition, further modified hyaluronan-degrading enzymes of a selected modified hyaluronan-degrading enzyme are generated and tested, whereby the modified hyaluronan-degrading enzyme is evolved to exhibit increased stability under a denaturation condition. Also provided herein is a modified hyaluronan-degrading enzyme identified by any of the methods provided herein. 
    
    
     
       BRIEF DESCRIPTION OF THE FIGURES 
         FIG.  1    depicts the amino acid sequence of full-length human PH20 (set forth in SEQ ID NO:7) and soluble C-terminal truncated variants thereof. The C-terminal amino acid residue of exemplary C-terminal truncated variants of full-length PH20 are indicated by bold font. The complete amino acid sequences of exemplary C-terminal truncated variants of full-length PH20 also are provided in SEQ ID NOs: 3 and 32-66. The C-terminal amino acid residue of an exemplary soluble PH20, whose complete sequence is set forth in SEQ ID NO:3, also is indicated by underline. Exemplary, non-limiting, positions for amino acid replacements are indicated by highlighting. Corresponding positions can be identified by alignment of a sequence of interest with any of SEQ ID NOs: 3, 7 or 32-66, and in particular with SEQ ID NO:3. 
         FIG.  2    (A-L) depicts exemplary alignments of human soluble PH20 set forth in SEQ ID NO:3 with other PH20 polypeptides. A “*” means that the aligned residues are identical, a “:” means that aligned residues are not identical, but are similar and contain conservative amino acids residues at the aligned position, and a “.” means that the aligned residues are similar and contain semi-conservative amino acid residues at the aligned position. Exemplary, non-limiting, corresponding positions for amino acid replacements are indicated by highlighting. For example,  FIG.  2 A  depicts the alignment of a human soluble PH20 set forth in SEQ ID NO:3 with chimpanzee PH20 set forth in SEQ ID NO:10.  FIG.  2 B  depicts the alignment of a human soluble PH20 set forth in SEQ ID NO:3 with Rhesus monkey PH20 set forth in SEQ ID NO:12.  FIG.  2 C  depicts the alignment of a human soluble PH20 set forth in SEQ ID NO:3 with Cynomolgus monkey PH20 set forth in SEQ ID NO:14.  FIG.  2 D  depicts the alignment of human soluble PH20 set forth in SEQ ID NO:3 with bovine PH20 set forth in SEQ ID NO:16.  FIG.  2 E  depicts the alignment of a human soluble PH20 set forth in SEQ ID NO:3 with mouse PH20 set forth in SEQ ID NO:20.  FIG.  2 F  depicts the alignment of a human soluble PH20 set forth in SEQ ID NO:3 with rat PH20 set forth in SEQ ID NO:22.  FIG.  2 G  depicts the alignment of a human soluble PH20 set forth in SEQ ID NO:3 with rabbit PH20 set forth in SEQ ID NO:24.  FIG.  2 H  depicts the alignment of a human soluble PH20 set forth in SEQ ID NO:3 with guinea pig PH20 set forth in SEQ ID NO:29.  FIG.  2 I  depicts the alignment of a human soluble PH20 set forth in SEQ ID NO:3 with Fox PH20 set forth in SEQ ID NO:31.  FIG.  2 J  depicts the alignment of a human soluble PH20 set forth in SEQ ID NO:3 with Gibbon PH20 set forth in SEQ ID NO:857. 
         FIG.  2 K  depicts the alignment of a human soluble PH20 set forth in SEQ ID NO:3 with Marmoset PH20 set forth in SEQ ID NO:859.  FIG.  2 L  depicts the alignment of a human soluble PH20 set forth in SEQ ID NO:3 with Orangutan PH20 set forth in SEQ ID NO:861. 
     
    
    
     DETAILED DESCRIPTION 
     Outline 
     
         
         
           
             A. DEFINITIONS 
             B. PH20 HYALURONIDASE
           1. Structure   2. Function   3. Soluble PH20 Polypeptides   
         
             C. MODIFIED PH20 POLYPEPTIDES
           1. Active Mutants
               a. Increased Activity   b. Increased Stability
                   i. Phenophiles   ii. Thermophiles   iii. Absence of Salt   
                   
               2. Inactive Mutants   3. Additional Modifications and Conjugates
               a. Decreased Immunogenicity   b. Conjugation to Polymers   
               
         
             D. METHODS FOR IDENTIFYING MODIFIED HYALURONAN-DEGRADING ENZYMES WITH ALTERED PROPERTIES OR ACTIVITIES
           1. Hyaluronan-Degrading Enzymes and Libraries of Modified Hyaluronan-Degrading Enzymes   2. Screening or Testing for a Desired Activity or Property   3. Selection or Identification   4. Iterative Methods   
         
             E. PRODUCTION OF MODIFIED POLYPEPTIDES AND ENCODING NUCLEIC ACID MOLECULES
           1. Isolation or Preparation of Nucleic Acids Encoding PH20 Polypeptides   2. Generation of Mutant or Modified Nucleic Acid and Encoding Polypeptides   3. Vectors and Cells   4. Expression
               a. Prokaryotic Cells   b. Yeast Cells   c. Insects and Insect Cells   d. Mammalian expression   e. Plants and plant cells   
               5. Purification   6. Modification of Polypeptides by PEGylation   
         
             F. PHARMACEUTICAL COMPOSITIONS AND FORMULATIONS, DOSAGES AND ADMINISTRATION
           1. Formulations—liquids, injectables, solutions and emulsions
               a. Lyophilized Powders   b. Exemplary Formulations
                   i. Salt (e.g. NaCl)   ii. pH and Buffer   iii. Preservative(s)   iv. Stabilizers   
                   
               2. Compositions for Other Routes of Administration   3. Dosages and Administration   4. Exemplary PH20-Insulin Co-Formulations   5. Packaging, Articles of Manufacture and Kits   
         
             G. METHODS OF ASSESSING PH20 ACTIVITY AND STABILITY
           1. Hyaluronidase Activity   2. Solubility   3. Purity, Crystallization or Aggregation   4. Pharmacodynamics/Pharmacokinetics   
         
             H. METHODS OF TREATMENT AND COMBINATION THERAPY
           1. Methods of Delivering Therapeutic Agents
               Delivery of Insulin   
               2. Methods of Treating Hyaluronan-Associated Disease and Conditions (eg., Tumors)   3. Other Uses   4. Contraception   
         
             I. EXAMPLES 
           
         
       
    
     A. DEFINITIONS 
     Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the invention(s) belong. All patents, patent applications, published applications and publications, GenBank sequences, databases, websites and other published materials referred to throughout the entire disclosure herein, unless noted otherwise, are incorporated by reference in their entirety. In the event that there are a plurality of definitions for terms herein, those in this section prevail. Where reference is made to a URL or other such identifier or address, it understood that such identifiers can change and particular information on the internet can come and go, but equivalent information can be found by searching the internet. Reference thereto evidences the availability and public dissemination of such information. 
     As used herein, a hyaluronan-degrading enzyme refers to an enzyme that catalyzes the cleavage of a hyaluronan polymer (also referred to as hyaluronic acid or HA) into smaller molecular weight fragments. Exemplary hyaluronan-degrading enzymes are hyaluronidases, and particular chondroitinases and lyases that have the ability to depolymerize hyaluronan. Exemplary chondroitinases that are hyaluronan-degrading enzymes include, but are not limited to, chondroitin ABC lyase (also known as chondroitinase ABC), chondroitin AC lyase (also known as chondroitin sulfate lyase or chondroitin sulfate eliminase) and chondroitin C lyase. Chondroitin ABC lyase contains two enzymes, chondroitin-sulfate-ABC endolyase (EC 4.2.2.20) and chondroitin-sulfate-ABC exolyase (EC 4.2.2.21). An exemplary chondroitin-sulfate-ABC endolyases and chondroitin-sulfate-ABC exolyases include, but are not limited to, those from  Proteus vulgaris  and  Pedobacter heparinus  (the  Proteus vulgaris  chondroitin-sulfate-ABC endolyase is set forth in SEQ ID NO:922; Sato et al. (1994)  Appl. Microbiol. Biotechnol.  41(1):39-46). Exemplary chondroitinase AC enzymes from bacteria include, but are not limited to, those from  Pedobacter heparinus , set forth in SEQ ID NO: 923 , Victivallis vadensis , set forth in SEQ ID NO:924, and  Arthrobacter aurescens  (Tkalec et al. (2000)  Applied and Environmental Microbiology  66(1):29-35; Ernst et al. (1995)  Critical Reviews in Biochemistry and Molecular Biology  30(5):387-444). Exemplary chondroitinase C enzymes from bacteria include, but are not limited to, those from  Streptococcus  and  Flavobacterium  (Hibi et al. (1989)  FEMS - Microbiol - Lett.  48(2): 121-4; Michelacci et al. (1976)  J. Biol. Chem.  251:1154-8; Tsuda et al. (1999)  Eur.  1  Biochem.  262:127-133). 
     As used herein, hyaluronidase refers to a class of enzymes that degrade hyaluronan. Hyaluronidases include, but are not limited to, bacterial hyaluronidases (EC 4.2.2.1 or EC 4.2.99.1), hyaluronidases from leeches, other parasites and crustaceans (EC 3.2.1.36), and mammalian-type hyaluronidases (EC 3.2.1.35). Hyaluronidases include any of non-human origin including, but not limited to, murine, canine, feline, leporine, avian, bovine, ovine, porcine, equine, piscine, ranine, bacterial, and any from leeches, other parasites, and crustaceans. Exemplary human hyaluronidases include HYAL1, HYAL2, HYAL3, HYAL4, and PH20. Also included amongst hyaluronidases are soluble hyaluronidases, including, ovine and bovine PH20, and soluble PH20. Exemplary hyaluronidases include any set forth in SEQ ID NOs: 6, 7-31, 69, 70, 71, 72, 856-861, 869-921, mature forms thereof (lacking the signal sequence), or allelic or species variants thereof. Hyaluronidases also include truncated forms thereof that exhibit hyaluronidase activity, including C-terminal truncated variants that are soluble. 
     As used herein, PH20 refers to a type of hyaluronidase that occurs in sperm and is neutral-active. PH-20 occurs on the sperm surface, and in the lysosome-derived acrosome, where it is bound to the inner acrosomal membrane. PH20 includes those of any origin including, but not limited to, human, chimpanzee, Cynomolgus monkey, Rhesus monkey, murine, bovine, ovine, guinea pig, rabbit and rat origin. Exemplary PH20 polypeptides, including precursor and mature forms, include those from human (SEQ ID NOs:6 and 7), chimpanzee (SEQ ID NOs:8, 9, 10, 869 and 870), Rhesus monkey (SEQ ID NOs:11 and 12), Cynomolgus monkey (SEQ ID NOs:13 and 14), cow (e.g., SEQ ID NOs:15-18); mouse (SEQ ID NOs:19 and 20); rat (SEQ ID NOs:21 and 22); rabbit (SEQ ID NOs:23 and 24); sheep (SEQ ID NOs:25-27), guinea pig (SEQ ID NOs:28 and 29); fox (SEQ ID NOs: 30 and 31); Gibbon (SEQ ID NOs:856 and 857), Marmoset (SEQ ID NOs:858 and 859) and orangutan (SEQ ID NOs:860 and 861). Reference to PH20 includes precursor PH20 polypeptides and mature PH20 polypeptides (such as those in which a signal sequence has been removed), truncated forms thereof that have activity, and includes allelic variants and species variants, variants encoded by splice variants, and other variants, including polypeptides that have at least 40%, 45%, 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the precursor polypeptides set forth in SEQ ID NO:7, or the mature forms thereof. PH20 polypeptides also include those that contain chemical or posttranslational modifications and those that do not contain chemical or posttranslational modifications. Such modifications include, but are not limited to, PEGylation, albumination, glycosylation, farnysylation, carboxylation, hydroxylation, phosphorylation, and other polypeptide modifications known in the art. Examples of commercially available bovine or ovine soluble hyaluronidases are Vitrase® hyaluronidase (ovine hyaluronidase) and Amphadase® hyaluronidase (bovine hyaluronidase). 
     As used herein, a soluble PH20 refers to a polypeptide characterized by its solubility under physiological conditions. Generally, a soluble PH20 lacks all or a portion of a glycophosphatidyl anchor (GPI) attachment sequence, or does not otherwise sufficiently anchor to the cell membrane. For example, a soluble PH20 can be a C-terminally truncated variant of a PH20 lacking a contiguous sequence of amino acids that corresponds to all or a portion of a glycophosphatidyl anchor (GPI) attachment sequence. Hence, upon expression from a cell, a soluble PH20 is secreted into the medium. Soluble PH20 proteins can be distinguished, for example, by its partitioning into the aqueous phase of a Triton® X-114 detergent solution warmed to 37° C. (Bordier et al., (1981)  J. Biol. Chem.,  256:1604-7). Membrane-anchored, such as lipid anchored hyaluronidases, will partition into the detergent rich phase, but will partition into the detergent-poor or aqueous phase following treatment with Phospholipase-C. Included among soluble PH20 hyaluronidases are membrane anchored hyaluronidases in which one or more regions associated with anchoring of the hyaluronidase to the membrane has been removed or modified, where the soluble form retains hyaluronidase activity. Soluble hyaluronidases include recombinant soluble hyaluronidases and those contained in or purified from natural sources, such as, for example, testes extracts from sheep or cows. Exemplary of such soluble hyaluronidases are soluble human PH20 (SEQ ID NOs: 3 or 32-66). Other soluble hyaluronidases include ovine (SEQ ID NOs:25-27) and bovine (SEQ ID NO:16 or 18) PH20. 
     As used herein, soluble human PH20 (sHuPH20) includes human PH20 polypeptides that lack a contiguous sequence of amino acids from the C-terminus of human PH20 that includes all or a portion of the glycosylphosphatidylinositol (GPI) anchor sequence (C-terminally truncated PH20 polypeptides) such that upon expression, the polypeptides are soluble under physiological conditions. For example, soluble human PH20 polypeptides are C-terminally truncated polypeptides of human PH20 set forth as SEQ ID NO:6 in its precursor form or in SEQ ID NO:7 in its mature form lacking the signal sequence, or allelic variants thereof (e.g. set forth in any of SEQ ID NOs: 68-72). Solubility can be assessed by any suitable method that demonstrates solubility under physiologic conditions. Exemplary of such methods is the Triton® X-114 assay, that assesses partitioning into the aqueous phase and that is described above. In addition, a soluble human PH20 polypeptide is, if produced in CHO cells, such as CHO-S cells, a polypeptide that is expressed and is secreted into the cell culture medium. Soluble human PH20 polypeptides, however, are not limited to those produced in CHO cells, but can be produced in any cell or by any method, including recombinant expression and polypeptide synthesis. Reference to secretion in CHO cells is definitional. Hence, if a polypeptide could be expressed and secreted in CHO cells and is soluble in the media, i.e., partitions into the aqueous phase when extracted with Triton® X-114 detergent, it is a soluble PH20 polypeptide whether or not it is so-produced. The precursor polypeptides for sHuPH20 polypeptides can include a signal sequence, such as a heterologous or non-heterologous (i.e., native) signal sequence. Exemplary of the precursors are those that include a signal sequence, such as the native 35 amino acid signal sequence at amino acid positions 1-35 (see, e.g., amino acids 1-35 of SEQ ID NO:6). 
     As used herein, “native” or “wildtype” with reference to a PH20 polypeptide refers to a PH20 polypeptide encoded by a native or naturally occurring PH20 gene, including allelic variants, that is present in an organism, including a human and other animals, in nature. Reference to wild-type PH20 without reference to a species is intended to encompass any species of a wild-type PH20. Included among wild-type PH20 polypeptides are the encoded precursor polypeptide, fragments thereof, and processed forms thereof, such as a mature form lacking the signal peptide as well as any pre- or post-translationally processed or modified forms thereof. Also included among native PH20 polypeptides are those that are post-translationally modified, including, but not limited to, those that are modified by glycosylation, carboxylation and/or hydroxylation. The amino acid sequences of exemplary wild-type human PH20 are set forth in SEQ ID NOs: 6 and 7 and those of allelic variants, including mature forms thereof, are set forth in SEQ ID NOs:68-72. Other animals produce native PH20, including, but not limited to, native or wildtype sequences set forth in any of SEQ ID NOs: 8-31, 856-861, 869 or 870. 
     As used herein, modification is in reference to modification of a sequence of amino acids of a polypeptide or a sequence of nucleotides in a nucleic acid molecule and includes deletions, insertions, and replacements of amino acids and nucleotides, respectively. Modifications also can include post-translational modifications or other changes to the molecule that can occur due to conjugation or linkage, directly or indirectly, to another moiety. Methods of modifying a polypeptide are routine to those of skill in the art, such as by using recombinant DNA methodologies. 
     As used herein, a “modified hyaluronan-degrading enzyme” refers to a hyaluronan-degrading enzyme that contains a modification compared to a reference or unmodified hyaluronan-degrading enzyme. The modification can be an amino acid replacement (substitution), insertion (addition) or deletion of one or more amino acid residues. The amino acid residue can be a natural or non-natural amino acid. In some cases, the modification can be a post-translational modification. A modified hyaluronan-degrading enzyme can have up to 150 amino acid differences compared to a reference or unmodified hyaluronan-degrading enzyme, so long as the resulting modified hyaluronan-degrading enzyme exhibits hyaluronidase activity. Typically, a modified hyaluronan-degrading enzyme contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acid modifications. 
     As used herein, an unmodified hyaluronan-degrading enzyme refers to a starting polypeptide that is selected for modification as provided herein. The starting polypeptide can be a naturally-occurring, wild-type form of a polypeptide. In addition, the starting polypeptide can be altered or mutated, such that it differs from a native wild type isoform but is nonetheless referred to herein as a starting unmodified polypeptide relative to the subsequently modified polypeptides produced herein. Thus, existing proteins known in the art that have been modified to have a desired increase or decrease in a particular activity or property compared to an unmodified reference protein can be selected and used as the starting unmodified polypeptide. For example, a protein that has been modified from its native form by one or more single amino acid changes and possesses either an increase or decrease in a desired property, such as a change in an amino acid residue or residues to alter glycosylation, can be selected for modification, and hence referred to herein as unmodified, for further modification. An unmodified hyaluronan-degrading enzyme includes human and non-human hyaluronan-degrading enzymes, including hyaluronan-degrading enzymes from non-human mammals and bacteria. Exemplary unmodified hyaluronan-degrading enzyme are any set forth in SEQ ID NOs: 2, 3, 6, 7-66, 68-72, 856-861, 869-924 or mature, C-terminally truncated forms thereof that exhibit hyaluronidase activity, or a hyaluronan-degrading enzyme that exhibits at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs: 2, 3, 6, 7-66, 68-72, 856-861, 869-924. It is understood that an unmodified hyaluronan-degrading enzyme generally is one that does not contain the modification(s), such as amino acid replacement(s) of a modified hyaluronan-degrading enzyme. 
     As used herein, “modified PH20 polypeptide” or “variant PH20 polypeptide” refers to a PH20 polypeptide that contains at least one amino acid modification, such as at least one amino acid replacement as described herein, in its sequence of amino acids compared to a reference unmodified PH20 polypeptide. A modified PH20 polypeptide can have up to 150 amino acid replacements, so long as the resulting modified PH20 polypeptide exhibits hyaluronidase activity. Typically, a modified PH20 polypeptide contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acid replacements. It is understood that a modified PH20 polypeptide also can include any one or more other modifications, in addition to at least one amino acid replacement as described herein. 
     As used herein, an unmodified PH20 polypeptide refers to a starting PH20 polypeptide that is selected for modification as provided herein. The starting polypeptide can be a naturally-occurring, wild-type form of a polypeptide. In addition, the starting polypeptide can be altered or mutated, such that it differs from a native wild type isoform but is nonetheless referred to herein as a starting unmodified polypeptide relative to the subsequently modified polypeptides produced herein. Thus, existing proteins known in the art that have been modified to have a desired increase or decrease in a particular activity or property compared to an unmodified reference protein can be selected and used as the starting unmodified polypeptide. For example, a protein that has been modified from its native form by one or more single amino acid changes and possesses either an increase or decrease in a desired property, such as a change in an amino acid residue or residues to alter glycosylation, can be selected for modification, and hence referred to herein as unmodified, for further modification. Exemplary unmodified PH20 polypeptides is a human PH20 polypeptide or allelic or species variants thereof or other variants, including mature and precursor polypeptides. For example, exemplary reference PH20 polypeptides is a mature full length PH20 polypeptide set forth in SEQ ID NOs: 7, 69 or 72, or in C-terminally truncated forms thereof such as set forth in any of SEQ ID NOs: 3 and 32-66, or in a PH20 polypeptide that exhibits at least 68%, 69%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs: 3, 7, 32-66, 69 or 72. A reference PH20 polypeptide also can include the corresponding precursor form such as set forth in any of SEQ ID NOs: 2, 6, 68, 70, 71 or other precursor forms, or in a PH20 polypeptide that exhibits at least 68%, 69%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs: 2, 6, 68, 70, 71. It is understood that an unmodified hyaluronan-degrading enzyme generally is one that does not contain the modification(s), such as amino acid replacement(s) of a modified hyaluronan-degrading enzyme. 
     As used herein, an N-linked moiety refers to an asparagine (N) amino acid residue of a polypeptide that is capable of being glycosylated by post-translational modification of a polypeptide. Exemplary N-linked moieties of human PH20 include amino acids N47, N131, N200, N219, N333, N358 and N365 of the sequence of amino acids set forth in SEQ ID NO: 3 or 7 (corresponding to amino acid residues N82, N166, N235, N254, N368, N393 and N490 of human PH20 set forth in SEQ ID NO: 6). 
     As used herein, an N-glycosylated polypeptide refers to a PH20 polypeptide containing oligosaccharide linkage of at least three N-linked amino acid residues, for example, N-linked moieties corresponding to amino acid residues N200, N333 and N358 of SEQ ID NO:3 or 7. An N-glycosylated polypeptide can include a polypeptide where three, four, five and up to all of the N-linked moieties are linked to an oligosaccharide. The N-linked oligosaccharides can include oligomannose, complex, hybrid or sulfated oligosaccharides, or other oligosaccharides and monosaccharides. 
     As used herein, an N-partially glycosylated polypeptide refers to a polypeptide that minimally contains an N-acetylglucosamine glycan linked to at least three N-linked moieties. A partially glycosylated polypeptide can include various glycan forms, including monosaccharides, oligosaccharides, and branched sugar forms, including those formed by treatment of a polypeptide with EndoH, EndoF1, EndoF2 and/or EndoF3. 
     As used herein, “conditions” refers to any parameter that can influence the activity or properties of a protein or agent. For purposes herein, conditions generally refer to the presence, including amount, of excipients, carriers or other components in a formulation other than the active agent (e.g., modified PH20 hyaluronidase); temperature; time (e.g., time of storage or exposure); storage vessel; properties of storage (e.g., agitation) and/or other conditions associated with exposure or use. 
     As used herein, “denaturation” or “denaturing” or grammatical variations thereof with reference to a protein refers to a biochemical change in a protein so that a property or activity of the protein is diminished or eliminated. The biochemical change can be a change in the tertiary structure of the protein to unfold. The property or activity can be completely abolished or can be reduced by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more. 
     As used herein, property refers to a physical or structural property, such as the three-dimensional structure, pI, half-life, conformation and other such physical characteristics. For example, a change in a property can be manifested as the solubility, aggregation or crystallization of a protein. 
     As used herein, activity refers to a functional activity or activities of a polypeptide or portion thereof associated with a full-length (complete) protein. Functional activities include, but are not limited to, biological activity, catalytic or enzymatic activity, antigenicity (ability to bind or compete with a polypeptide for binding to an anti-polypeptide antibody), immunogenicity, ability to form multimers, and the ability to specifically bind to a receptor or ligand for the polypeptide. 
     As used herein, hyaluronidase activity refers to the ability to enzymatically catalyze the cleavage of hyaluronic acid. The United States Pharmacopeia (USP) XXII assay for hyaluronidase determines hyaluronidase activity indirectly by measuring the amount of higher molecular weight hyaluronic acid, or hyaluronan, (HA) substrate remaining after the enzyme is allowed to react with the HA for 30 min at 37° C. (USP XXII-NF XVII (1990) 644-645 United States Pharmacopeia Convention, Inc, Rockville, Md.). A Reference Standard solution can be used in an assay to ascertain the relative activity, in units, of any hyaluronidase. In vitro assays to determine the hyaluronidase activity of hyaluronidases, such as PH20, including modified PH20 polypeptides, are known in the art and described herein. Exemplary assays include the microturbidity assay described herein that measures cleavage of hyaluronic acid by hyaluronidase indirectly by detecting the insoluble precipitate formed when the uncleaved hyaluronic acid binds with serum albumin. Reference Standards can be used, for example, to generate a standard curve to determine the activity in Units of the hyaluronidase being tested. 
     As used herein, neutral active refers to the ability of a PH20 polypeptide to enzymatically catalyze the cleavage of hyaluronic acid at neutral pH, such as at a pH between or about between pH 6.0 to pH 7.8. 
     As used herein, “increased activity” with reference to a modified PH20 hyaluronidase means that, when tested under the same conditions, the modified PH20 hyaluronidase exhibits greater hyaluronidase activity compared to an unmodified PH20 hyaluronidase not containing the amino acid replacement(s). For example, a modified PH20 hyaluronidase exhibits at least or about at least 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 250%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more of the activity of the unmodified or reference PH20 hyaluronidase. 
     As used herein, “solubility” with reference to a protein refers to a protein that is homogenous in an aqueous solution, whereby protein molecules diffuse and do not sediment spontaneously. Hence a soluble protein solution is one in which there is an absence of a visible or discrete particle in a solution containing the protein, such that the particles cannot be easily filtered. Generally, a protein is soluble if there are no visible or discrete particles in the solution. For example, a protein is soluble if it contains no or few particles that can be removed by a filter with a pore size of 0.22 μm. 
     As used herein, aggregation or crystallization with reference to a protein refers to the presence of visible or discrete particles in a solution containing the protein. Typically, the particles are greater than 10 μm in size, such as greater than 15 μm, 20 μm, 25 μm, 30 μm, 40 μm, 50 μm or greater. Aggregation or crystallization can arise due to reduced solubility, increased denaturation of a protein or the formation of covalent bonds. 
     As used herein, “denaturing condition” or “denaturation condition” refers to any condition or agent that, when exposed to a protein, affects or influences the degradation or denaturation of the protein, generally as a result of a loss or partial loss of the tertiary or secondary structure of the protein. Denaturing conditions can result in effects such as loss or reduction in activity, loss or reduction of solubility, aggregation and/or crystallization. The denaturing condition need not be one that is completely deadly to the protein, but nevertheless is one that leads to a reduction in the activity of the protein over time. Thus, a condition is denaturing if the activity of the protein is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more in the presence of the condition than in its absence. A denaturing condition can be due to an external stress or physical condition (e.g., agitation, temperature, time of storage, absence of a stabilizer) or can be due to the presence of a denaturing agent. For example, the denaturing condition can be caused by heat, acid or a chemical denaturant. Exemplary denaturing conditions include, but are not limited to, the presence of a strong acid or base, a concentrated inorganic salt, an organic solvent (e.g., alcohol or chloroform), urea, high or low pH (extremes of pH), elevated temperature (e.g., heat), the presence of excipients that can be denaturing (e.g., phenolic preservatives or detergent), and low or substantially no stabilizing agent that otherwise is required for stability of the protein (e.g., NaCl). 
     As used herein, “denaturing agent” or “denaturant” refers to any substance, molecule or compound that causes denaturation. For example, a denaturing agent can include a strong acid or base, a concentrated inorganic salt, an organic solvent (e.g., alcohol or chloroform), a preservative, detergent or other excipient. 
     As used herein, “resistance to a denaturation condition” refers to any amount of decreased reduction or elimination of a property or activity of the protein associated with or caused by denaturation. For example, denaturation is associated with or causes increased crystallization or aggregation, reduced solubility or decreased activity. Hence, resistance to denaturation means that the protein exhibits decreased aggregation or crystallization, increased solubility or increased or greater activity (e.g., hyaluronidase activity) when exposed to a denaturing condition compared to a reference protein (e.g. unmodified enzyme). The resistance to a denaturation condition need not be absolute or permanent, but can be achieved because the denaturation of the modified hyaluronan-degrading enzyme occurs more slowly than the unmodified enzyme in the denaturation condition such that an activity or property of the modified hyaluronan-degrading enzyme is achieved for longer. For example, a modified hyaluronan-degrading enzyme, such as a modified PH20 hyaluronidase, exhibits resistance to a denaturation condition if it exhibits, for example, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, . . . 20%, . . . 30%, . . . 40%, . . . 50%, . . . 60%, . . . , 70%, . . . 80%, . . . 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% more resistance to denaturation in the presence of a denaturation condition or denaturing agent than an unmodified polypeptide. In some instances, a modified polypeptide exhibits 105%, 110%, 120%, 130%, 140%, 150%, 200%, 300%, 400%, 500%, or more increased resistance to denaturation compared to an unmodified polypeptide. 
     As used herein, stability of a modified PH20 hyaluronidase means that it exhibits resistance to denaturation caused by a denaturation condition or denaturing agent. A modified PH20 polypeptide exhibits stability if it retains some activity in the presence of a denaturation condition or denaturing agent, such as at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the original or initial hyaluronidase activity prior to exposure to the denaturing condition(s). Generally, a modified PH20 hyaluronidase is stable if it retains at least 50% or more of the hyaluronidase activity under a denaturation condition compared to the absence of the denaturation condition. Assays to assess hyaluronidase activity are known to one of skill in the art and described herein. It is understood that the stability of the enzyme need not be permanent or long term, but is manifested for a duration of time in which activity is desired. For example, a modified PH20 hyaluronidase is stable if it exhibits an activity for at least 2 hours, 3 hours, 4 hours, 6 hours, 12 hours, 24 hours, one day, two days, three days, four days, five days, six days, one week, one month, six months or one year upon exposure, or during exposure, to one or more denaturing condition(s) or agent(s) (e.g., presence of a denaturing excipient such as a preservative). For example, a modified PH20 hyaluronidase is stable if it exhibits an activity upon or during exposure to one or more denaturing condition(s) or agent(s) (e.g., presence of a denaturing excipient such as a preservative) for at least 1 month at temperatures from or from about 2° C. to 8° C., inclusive or for at least 3 days at a temperature from or from about 30° C. to 42° C., inclusive. 
     Hence, “stable” or “stability,” with reference to a formulation or a co-formulation provided herein, refers to one in which a modified hyaluronan-degrading enzyme, such as a modified PH20 hyaluronidase, therein is stable upon exposure to one or more denaturing condition(s) or agent(s) therein (e.g., presence of a denaturing excipient such as a preservative) for at least 1 month at temperatures from or from about 2° C. to 8° C., inclusive or for at least 3 days at a temperature from or from about 30° C. to 42° C., inclusive. 
     As used herein, “increased stability” with reference to a modified PH20 hyaluronidase means that, in the presence of the same denaturing or denaturation condition(s) (e.g., presence of a denaturing excipient such as a preservative), the modified PH20 hyaluronidase exhibits greater hyaluronidase activity compared to an unmodified PH20 hyaluronidase not containing the amino acid replacement(s). For example, a modified PH20 hyaluronidase exhibits increased stability if it exhibits at least or about at least 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 250%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more of the activity of the unmodified or reference PH20 hyaluronidase in the presence of a denaturing or denaturation condition(s) (e.g., in the presence of a denaturing excipient such as a preservative). 
     As used herein, “elevated temperatures” refers to temperatures that are greater than room temperature or ambient temperature. Generally, an elevated temperature is a temperature that is at least, greater than, or about 30° C., such as 30° C. to 42° C., and generally 32° C. to 37° C. or 35° C. to 37° C., inclusive. 
     As used herein, room temperature refers to a range generally from about or at to 18° C. to about or at 32° C. Those of skill in the art appreciate that room temperature varies by location and prevailing conditions. For example, room temperatures can be higher in warmer climates such as Italy or Texas. 
     As used herein, recitation that proteins are “compared under the same conditions” means that different proteins are treated identically or substantially identically such that any one or more conditions that can influence the activity or properties of a protein or agent are not varied or not substantially varied between the test agents. For example, when the hyaluronidase activity of a modified PH20 polypeptide is compared to an unmodified PH20 polypeptide any one or more conditions such as the amount or concentration of the polypeptide; presence, including amount, of excipients, carriers or other components in a formulation other than the active agent (e.g., modified PH20 hyaluronidase); temperature; time of storage; storage vessel; properties of storage (e.g., agitation) and/or other conditions associated with exposure or use are identical or substantially identical between and among the compared polypeptides. 
     As used herein, “predetermined time” refers to a time that is established or decided in advance. For example, the predetermined time can be a time chosen in advance that is associated with the desired duration of activity of a hyaluronan-degrading enzyme depending on the desired application or use of the protein. A predetermined time can be hours, days, months or years. For example, a predetermined time can be at least about or about 2 hours, 3 hours, 4 hours, five hours, six hours, 12 hours, 24 hours, 2 days, three days, four days, five days, six days, one week, two weeks, three weeks, one month, six months, one year or more. 
     As used herein, “storage” means that a formulation is not immediately administered to a subject once prepared, but is kept for a period of time under particular conditions (e.g., particular temperature; time, and/or form (e.g., liquid or lyophilized form)) prior to use. For example, a liquid formulation can be kept for days, weeks, months or years, prior to administration to a subject under varied temperatures such as refrigerated (0° C. to 10° C., such as 2° to 8° C.), room temperature (e.g., temperature up to 32° C., such as 18° C. to about or at 32° C.), or elevated temperature (e.g., 30° C. to 42° C., such as 32° C. to 37° C. or 35° C. to 37° C.). 
     As used herein, an “excipient” refers to a compound in a formulation of an active agent that does not provide the biological effect of the active agent when administered in the absence of the active agent. Exemplary excipients include, but are not limited to, salts, buffers, stabilizers, tonicity modifiers, metals, polymers, surfactants, preservatives, amino acids and sugars. 
     As used herein, a stabilizing agent refers to compound added to the formulation to protect the modified PH20 polypeptide or other active agent from degradation, if necessary, such as due to denaturation conditions to which a formulation herein is exposed when handled, stored or used. Thus, included are agents that prevent proteins from degradation from other components in the compositions. Exemplary of such agents are amino acids, amino acid derivatives, amines, sugars, polyols, salts and buffers, surfactants, inhibitors or substrates and other agents as described herein. 
     As used herein, an antimicrobial effectiveness test or preservative effectiveness test (PET) demonstrates the effectiveness of the preservative system in a product. A product is inoculated with a controlled quantity of specific organisms. The test then compares the level of microorganisms found on a control sample versus the test sample over a period of 28 days. Generally, target markets have differing PET requirements. For example, the PET requirements of the United States Pharmacopoeia (USP) and the European Pharmacopoeia (EP) differ. Parameters for performing an antimicrobial effectiveness test, including in different markets, are known to one of skill in the art as described herein. 
     As used herein, an anti-microbially or anti-microbial effective amount of a preservative refers to an amount of the preservative that kills or inhibits the propagation of microbial organisms in a sample that may be introduced from storage or use. For example, for multiple-dose containers, an anti-microbially effective amount of a preservative inhibits the growth of microorganisms that may be introduced from repeatedly withdrawing individual doses. USP and EP (EPA and EPB) have anti-microbial requirements that determine preservative effectiveness, and that vary in stringency. For example, an anti-microbial effective amount of a preservative is an amount such that at least a 1.0 log 10  unit reduction in bacterial organisms occurs at 7 days following inoculation in an antimicrobial preservative effectiveness test (APET). In a particular example, an anti-microbial effective amount of a preservative is an amount such that at least a 1.0 log 10  unit reduction in bacterial organisms occurs at 7 days following inoculation, at least a 3.0 log 10  unit reduction of bacterial organisms occurs at 14 days following inoculation, at least no further increase in bacterial organisms occurs after 28 days following inoculation, and at least no increase in fungal organisms occurs after 7 days following inoculation. In a further example, an anti-microbial effective amount of a preservative is an amount such that at least a 1.0 log 10  unit reduction of bacterial organisms occurs at 24 hours following inoculation, at least a 3.0 log 10  unit reduction of bacterial organisms occurs at 7 days following inoculation, no further increase in bacterial organisms occurs after 28 days following inoculation, at least a 1.0 log 10  unit reduction of fungal organisms occurs at 14 days following inoculation, and at least no further increase in fungal organisms occurs after 28 days following inoculation. In an additional example, an anti-microbial effective amount of a preservative is an amount such that at least a 2.0 log 10  unit reduction of bacterial organisms occurs at 6 hours following inoculation, at least a 3.0 log 10  unit reduction of bacterial organisms occurs at 24 hours following inoculation, no recovery of bacterial organisms occurs after 28 days following inoculation of the composition with the microbial inoculum, at least a 2.0 log 10  unit reduction of fungal organisms occurs at 7 days following inoculation, and at least no further increase in fungal organisms occurs after 28 days following inoculation. 
     As used herein, “preservative” refers to a naturally occurring or synthetically or recombinantly produced substance that, when added to a molecule or protein composition, prevents microbial growth, including bacterial or fungal growth, in the composition. 
     As used herein, a “phenolic preservative” refers to a preservative that contains one hydroxyl group attached to an aromatic carbon ring, such as a benzene ring. Exemplary phenolic preservatives, include but are not limited to, phenol, m-cresol, p-hydroxybenzoic acid, methylparaben, ethylparaben, and propylparaben. For example, cresols, including meta-cresol (m-cresol), has a methyl group substituted onto the benzene ring of a phenol molecule. 
     As used herein, a “phenophile” refers to a protein, such as a modified PH20 polypeptide, that exhibits stability in the presence of an anti-microbially effective amount of a preservative(s). The term “phenolphile” can be used interchangeably herein with “phenophile” and has the same meaning. For example, a modified PH20 polypeptide that is a phenophile or phenolphile typically exhibits increased stability compared to an unmodified PH20 hyaluronidase not containing the amino acid replacement(s) when tested under the same denaturing condition(s) containing a phenolic preservative(s). For example, a modified PH20 hyaluronidase exhibits at least or about at least 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 250%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more of the activity of the unmodified or reference PH20 hyaluronidase in the presence of a phenolic preservative(s). 
     As used herein, a “thermophile” refers to a protein, such as a modified PH20 polypeptide, that exhibits stability under elevated temperatures greater than or about 30° C., such as 30° C. to 42° C., and generally 32° C. to 37° C. or 35° C. to 37° C. For example, a modified PH20 polypeptide that is a thermophile typically exhibits increased stability compared to an unmodified PH20 hyaluronidase not containing the amino acid replacement(s) when tested under the same elevated temperature denaturing condition(s). For example, a modified PH20 hyaluronidase exhibits at least or about at least 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 250%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more of the activity of the unmodified or reference PH20 hyaluronidase under elevated temperatures. 
     As used herein, the term “detergent” is used interchangeably with the term “surfactant” or “surface acting agent.” Surfactants are typically organic compounds that are amphiphilic, i.e., containing both hydrophobic groups (“tails”) and hydrophilic groups (“heads”), which render surfactants soluble in both organic solvents and water. A surfactant can be classified by the presence of formally charged groups in its head. A non-ionic surfactant has no charge groups in its head, whereas an ionic surfactant carries a net charge in its head. A zwitterionic surfactant contains a head with two oppositely charged groups. Some examples of common surfactants include: Anionic (based on sulfate, sulfonate or carboxylate anions): perfluorooctanoate (PFOA or PFO), perfluorooctane sulfonate (PFOS), sodium dodecyl sulfate (SDS), ammonium lauryl sulfate, and other alkyl sulfate salts, sodium laureth sulfate (also known as sodium lauryl ether sulfate, or SLES), alkyl benzene sulfonate; cationic (based on quaternary ammonium cations): cetyl trimethylammonium bromide (CTAB) a.k.a. hexadecyl trimethyl ammonium bromide, and other alkyltrimethylammonium salts, cetylpyridinium chloride (CPC), polyethoxylated tallow amine (POEA), benzalkonium chloride (BAC), benzethonium chloride (BZT); Zwitterionic (amphoteric): dodecyl betaine; cocamidopropyl betaine; coco ampho glycinate; nonionic: alkyl poly(ethylene oxide), alkylphenol poly(ethylene oxide), copolymers of poly(ethylene oxide) and poly(propylene oxide) (commercially known as Poloxamers or Poloxamines), alkyl polyglucosides, including octyl glucoside, decyl maltoside, fatty alcohols (e.g., cetyl alcohol and oleyl alcohol), cocamide MEA, cocamide DEA, polysorbates (Tween 20, Tween 80, etc.), Triton® detergents, and dodecyl dimethylamine oxide. 
     As used herein, a “buffer” refers to a substance, generally a solution, that can keep its pH constant, despite the addition of strong acids or strong bases and external influences of temperature, pressure, volume or redox potential. A buffer prevents change in the concentration of another chemical substance, e.g., proton donor and acceptor systems that prevent marked changes in hydrogen ion concentration (pH). The pH values of all buffers are temperature and concentration dependent. The choice of buffer to maintain a pH value or range can be empirically determined by one of skill in the art based on the known buffering capacity of known buffers. Exemplary buffers include but are not limited to, bicarbonate buffer, cacodylate buffer, phosphate buffer or Tris buffer. For example, Tris buffer (tromethamine) is an amine based buffer that has a pKa of 8.06 and has an effective pH range between 7.9 and 9.2. For Tris buffers, pH increases about 0.03 unit per ° C. temperature decrease, and decreases 0.03 to 0.05 unit per ten-fold dilution. 
     As used herein, the residues of naturally occurring α-amino acids are the residues of those 20 α-amino acids found in nature which are incorporated into protein by the specific recognition of the charged tRNA molecule with its cognate mRNA codon in humans. 
     As used herein, nucleic acids include DNA, RNA and analogs thereof, including peptide nucleic acids (PNA) and mixtures thereof. Nucleic acids can be single or double-stranded. When referring to probes or primers, which are optionally labeled, such as with a detectable label, such as a fluorescent or radiolabel, single-stranded molecules are contemplated. Such molecules are typically of a length such that their target is statistically unique or of low copy number (typically less than 5, generally less than 3) for probing or priming a library. Generally a probe or primer contains at least 14, 16 or 30 contiguous nucleotides of sequence complementary to or identical to a gene of interest. Probes and primers can be 10, 20, 30, 50, 100 or more nucleic acids long. 
     As used herein, a peptide refers to a polypeptide that is from 2 to 40 amino acids in length. 
     As used herein, the amino acids which occur in the various sequences of amino acids provided herein are identified according to their known, three-letter or one-letter abbreviations (Table 1). The nucleotides which occur in the various nucleic acid fragments are designated with the standard single-letter designations used routinely in the art. 
     As used herein, an “amino acid” is an organic compound containing an amino group and a carboxylic acid group. A polypeptide contains two or more amino acids. For purposes herein, amino acids include the twenty naturally-occurring amino acids, non-natural amino acids and amino acid analogs (i.e., amino acids wherein the α-carbon has a side chain). As used herein, “amino acid residue” refers to an amino acid formed upon chemical digestion (hydrolysis) of a polypeptide at its peptide linkages. The amino acid residues described herein are presumed to be in the “L” isomeric form. Residues in the “D” isomeric form, which are so designated, can be substituted for any L-amino acid residue as long as the desired functional property is retained by the polypeptide. NH 2  refers to the free amino group present at the amino terminus of a polypeptide. COOH refers to the free carboxy group present at the carboxyl terminus of a polypeptide. In keeping with standard polypeptide nomenclature described in  J. Biol. Chem.,  243: 3557-3559 (1968), and adopted 37 C.F.R. §§ 1.821-1.822, abbreviations for amino acid residues are shown in Table 1: 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Table of Correspondence 
               
            
           
           
               
               
            
               
                 SYMBOL 
                   
               
            
           
           
               
               
               
            
               
                 1-Letter 
                 3-Letter 
                 AMINO ACID 
               
               
                   
               
               
                 Y 
                 Tyr 
                 Tyrosine 
               
               
                 G 
                 Gly 
                 Glycine 
               
               
                 F 
                 Phe 
                 Phenylalanine 
               
               
                 M 
                 Met 
                 Methionine 
               
               
                 A 
                 Ala 
                 Alanine 
               
               
                 S 
                 Ser 
                 Serine 
               
               
                 I 
                 Ile 
                 Isoleucine 
               
               
                 L 
                 Leu 
                 Leucine 
               
               
                 T 
                 Thr 
                 Threonine 
               
               
                 V 
                 Val 
                 Valine 
               
               
                 P 
                 Pro 
                 Proline 
               
               
                 K 
                 Lys 
                 Lysine 
               
               
                 H 
                 His 
                 Histidine 
               
               
                 Q 
                 Gln 
                 Glutamine 
               
               
                 E 
                 Glu 
                 Glutamic Acid 
               
               
                 Z 
                 Glx 
                 Glu and/or Gln 
               
               
                 W 
                 Trp 
                 Tryptophan 
               
               
                 R 
                 Arg 
                 Arginine 
               
               
                 D 
                 Asp 
                 Aspartic Acid 
               
               
                 N 
                 Asn 
                 Asparagine 
               
               
                 B 
                 Asx 
                 Asn and/or Asp 
               
               
                 C 
                 Cys 
                 Cysteine 
               
               
                 X 
                 Xaa 
                 Unknown or Other 
               
               
                   
               
            
           
         
       
     
     It should be noted that all amino acid residue sequences represented herein by formulae have a left to right orientation in the conventional direction of amino-terminus to carboxyl-terminus. In addition, the phrase “amino acid residue” is broadly defined to include the amino acids listed in the Table of Correspondence (Table 1) and modified and unusual amino acids, such as those referred to in 37 C.F.R. §§ 1.821-1.822, and incorporated herein by reference. Furthermore, it should be noted that a dash at the beginning or end of an amino acid residue sequence indicates a peptide bond to a further sequence of one or more amino acid residues, to an amino-terminal group such as NH 2  or to a carboxyl-terminal group such as COOH. 
     As used herein, “naturally occurring amino acids” refer to the 20 L-amino acids that occur in polypeptides. 
     As used herein, “non-natural amino acid” refers to an organic compound that has a structure similar to a natural amino acid but has been modified structurally to mimic the structure and reactivity of a natural amino acid. Non-naturally occurring amino acids thus include, for example, amino acids or analogs of amino acids other than the 20 naturally-occurring amino acids and include, but are not limited to, the D-stereoisomers of amino acids. Exemplary non-natural amino acids are described herein and are known to those of skill in the art. 
     As used herein, an isokinetic mixture is one in which the molar ratios of amino acids has been adjusted based on their reported reaction rates (see, e.g., Ostresh et al., (1994) Biopolymers 34:1681). 
     As used herein, suitable conservative substitutions of amino acids are known to those of skill in this art and can be made generally without altering the biological activity of the resulting molecule. Those of skill in the art recognize that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson et al. Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin/Cummings Pub. co., p. 224). Such substitutions can be made in accordance with those set forth in TABLE 2 as follows: 
                                 TABLE 2                       Original    Exemplary conservative            residue   substitution                          Ala (A)   Gly; Ser           Arg (R)   Lys           Asn (N)   Gln; His           Cys (C)   Ser           Gln (Q)   Asn           Glu (E)   Asp           Gly (G)   Ala; Pro           His (H)   Asn; Gln           Ile (I)   Leu; Val           Leu (L)   Ile; Val           Lys (K)   Arg; Gln; Glu           Met (M)   Leu; Tyr; Ile           Phe (F)   Met; Leu; Tyr           Ser (S)   Thr           Thr (T)   Ser           Trp (W)   Tyr           Tyr (Y)   Trp; Phe           Val (V)   Ile; Leu                        
Other substitutions also are permissible and can be determined empirically or in accord with known conservative substitutions.
 
     As used herein, a DNA construct is a single or double stranded, linear or circular DNA molecule that contains segments of DNA combined and juxtaposed in a manner not found in nature. DNA constructs exist as a result of human manipulation, and include clones and other copies of manipulated molecules. 
     As used herein, a DNA segment is a portion of a larger DNA molecule having specified attributes. For example, a DNA segment encoding a specified polypeptide is a portion of a longer DNA molecule, such as a plasmid or plasmid fragment, which, when read from the 5′ to 3′ direction, encodes the sequence of amino acids of the specified polypeptide. 
     As used herein, the term polynucleotide means a single- or double-stranded polymer of deoxyribonucleotides or ribonucleotide bases read from the 5′ to the 3′ end. Polynucleotides include RNA and DNA, and can be isolated from natural sources, synthesized in vitro, or prepared from a combination of natural and synthetic molecules. The length of a polynucleotide molecule is given herein in terms of nucleotides (abbreviated “nt”) or base pairs (abbreviated “bp”). The term nucleotides is used for single- and double-stranded molecules where the context permits. When the term is applied to double-stranded molecules it is used to denote overall length and will be understood to be equivalent to the term base pairs. It will be recognized by those skilled in the art that the two strands of a double-stranded polynucleotide can differ slightly in length and that the ends thereof can be staggered; thus all nucleotides within a double-stranded polynucleotide molecule cannot be paired. Such unpaired ends will, in general, not exceed 20 nucleotides in length. 
     As used herein, “at a position corresponding to” or recitation that nucleotides or amino acid positions “correspond to” nucleotides or amino acid positions in a disclosed sequence, such as set forth in the Sequence listing, refers to nucleotides or amino acid positions identified upon alignment with the disclosed sequence to maximize identity using a standard alignment algorithm, such as the GAP algorithm. For purposes herein, alignment of a PH20 sequence is to the amino acid sequence set forth in any of SEQ ID NOs: 3, 7 or 32-66, and in particular SEQ ID NO:3. Hence, reference herein that a position or amino acid replacement corresponds to positions with reference to SEQ ID NO:3 also means that the position or amino acid replacement corresponds to positions with reference to any of SEQ ID NOs: 7 or 32-66, since the sequences therein are identical to the corresponding residues as set forth in SEQ ID NO:3. By aligning the sequences, one skilled in the art can identify corresponding residues, for example, using conserved and identical amino acid residues as guides. In general, to identify corresponding positions, the sequences of amino acids are aligned so that the highest order match is obtained (see, e.g.:  Computational Molecular Biology , Lesk, A. M., ed., Oxford University Press, New York, 1988 ; Biocomputing: Informatics and Genome Projects , Smith, D. W., ed., Academic Press, New York, 1993 ; Computer Analysis of Sequence Data, Part I , Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994 ; Sequence Analysis in Molecular Biology , von Heinje, G., Academic Press, 1987; and  Sequence Analysis Primer , Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; Carrillo et al. (1988)  SIAM J Applied Math  48:1073).  FIG.  2    (A-L) exemplifies exemplary alignments and identification of exemplary corresponding residues for replacement. 
     As used herein, “sequence identity” refers to the number of identical or similar amino acids or nucleotide bases in a comparison between a test and a reference polypeptide or polynucleotide. Sequence identity can be determined by sequence alignment of nucleic acid or protein sequences to identify regions of similarity or identity. For purposes herein, sequence identity is generally determined by alignment to identify identical residues. Alignment can be local or global, but for purposes herein alignment is generally a global alignment where the full-length of each sequence is compared. Matches, mismatches and gaps can be identified between compared sequences. Gaps are null amino acids or nucleotides inserted between the residues of aligned sequences so that identical or similar characters are aligned. Generally, there can be internal and terminal gaps. Sequence identity can be determined by taking into account gaps as the number of identical residues/length of the shortest sequence ×100. When using gap penalties, sequence identity can be determined with no penalty for end gaps (e.g., terminal gaps are not penalized). Alternatively, sequence identity can be determined without taking into account gaps as the number of identical positions/length of the total aligned sequence ×100. 
     As used herein, a “global alignment” is an alignment that aligns two sequences from beginning to end, aligning each letter in each sequence only once. An alignment is produced, regardless of whether or not there is similarity or identity between the sequences. For example, 50% sequence identity based on “global alignment” means that in an alignment of the full sequence of two compared sequences each of 100 nucleotides in length, 50% of the residues are the same. It is understood that global alignment also can be used in determining sequence identity even when the length of the aligned sequences is not the same. The differences in the terminal ends of the sequences will be taken into account in determining sequence identity, unless the “no penalty for end gaps” is selected. Generally, a global alignment is used on sequences that share significant similarity over most of their length. Exemplary algorithms for performing global alignment include the Needleman-Wunsch algorithm (Needleman et al.  J. Mol. Biol.  48: 443 (1970). Exemplary programs for performing global alignment are publicly available and include the Global Sequence Alignment Tool available at the National Center for Biotechnology Information (NCBI) website (ncbi.nlm.nih.gov/), and the program available at deepc2.psi.iastate.edu/aat/align/align.html. 
     As used herein, a “local alignment” is an alignment that aligns two sequence, but only aligns those portions of the sequences that share similarity or identity. Hence, a local alignment determines if sub-segments of one sequence are present in another sequence. If there is no similarity, no alignment will be returned. Local alignment algorithms include BLAST or Smith-Waterman algorithm ( Adv. Appl. Math.  2: 482 (1981)). For example, 50% sequence identity based on “local alignment” means that in an alignment of the full sequence of two compared sequences of any length, a region of similarity or identity of 100 nucleotides in length has 50% of the residues that are the same in the region of similarity or identity. 
     For purposes herein, sequence identity can be determined by standard alignment algorithm programs used with default gap penalties established by each supplier. Default parameters for the GAP program can include: (1) a unary comparison matrix (containing a value of 1 for identities and 0 for non identities) and the weighted comparison matrix of Gribskov et al.  Nucl. Acids Res.  14: 6745 (1986), as described by Schwartz and Dayhoff, eds.,  Atlas of Protein Sequence and Structure , National Biomedical Research Foundation, pp. 353-358 (1979); (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap; and (3) no penalty for end gaps. Whether any two nucleic acid molecules have nucleotide sequences or any two polypeptides have amino acid sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% “identical,” or other similar variations reciting a percent identity, can be determined using known computer algorithms based on local or global alignment (see e.g., wikipedia.org/wiki/Sequence_alignment_software, providing links to dozens of known and publicly available alignment databases and programs). Generally, for purposes herein sequence identity is determined using computer algorithms based on global alignment, such as the Needleman-Wunsch Global Sequence Alignment tool available from NCBI/BLAST (blast.ncbi.nlm.nih.gov/Blast.cgi?CMD=Web&amp;Page_TYPE=BlastHome); LAlign (William Pearson implementing the Huang and Miller algorithm ( Adv. Appl. Math . (1991) 12:337-357)); and program from Xiaoqui Huang available at deepc2.psi.iastate.edu/aat/align/align.html. Generally, when comparing nucleotide sequences herein, an alignment with penalty for end gaps is used. Local alignment also can be used when the sequences being compared are substantially the same length. 
     Therefore, as used herein, the term “identity” represents a comparison or alignment between a test and a reference polypeptide or polynucleotide. In one non-limiting example, “at least 90% identical to” refers to percent identities from 90 to 100% relative to the reference polypeptide or polynucleotide. Identity at a level of 90% or more is indicative of the fact that, assuming for exemplification purposes a test and reference polypeptide or polynucleotide length of 100 amino acids or nucleotides are compared, no more than 10% (i.e., 10 out of 100) of amino acids or nucleotides in the test polypeptide or polynucleotide differs from that of the reference polypeptides. Similar comparisons can be made between a test and reference polynucleotides. Such differences can be represented as point mutations randomly distributed over the entire length of an amino acid sequence or they can be clustered in one or more locations of varying length up to the maximum allowable, e.g., 10/100 amino acid difference (approximately 90% identity). Differences also can be due to deletions or truncations of amino acid residues. Differences are defined as nucleic acid or amino acid substitutions, insertions or deletions. Depending on the length of the compared sequences, at the level of homologies or identities above about 85-90%, the result can be independent of the program and gap parameters set; such high levels of identity can be assessed readily, often without relying on software. 
     As used herein, an allelic variant or allelic variation references any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation, and can result in phenotypic polymorphism within populations. Gene mutations can be silent (no change in the encoded polypeptide) or can encode polypeptides having altered amino acid sequence. The term “allelic variant” also is used herein to denote a protein encoded by an allelic variant of a gene. Typically the reference form of the gene encodes a wildtype form and/or predominant form of a polypeptide from a population or single reference member of a species. Typically, allelic variants, which include variants between and among species typically have at least 80%, 90% or greater amino acid identity with a wildtype and/or predominant form from the same species; the degree of identity depends upon the gene and whether comparison is interspecies or intraspecies. Generally, intraspecies allelic variants have at least about 80%, 85%, 90% or 95% identity or greater with a wildtype and/or predominant form, including 96%, 97%, 98%, 99% or greater identity with a wildtype and/or predominant form of a polypeptide. Reference to an allelic variant herein generally refers to variations in proteins among members of the same species. 
     As used herein, “allele,” which is used interchangeably herein with “allelic variant” refers to alternative forms of a gene or portions thereof. Alleles occupy the same locus or position on homologous chromosomes. When a subject has two identical alleles of a gene, the subject is said to be homozygous for that gene or allele. When a subject has two different alleles of a gene, the subject is said to be heterozygous for the gene. Alleles of a specific gene can differ from each other in a single nucleotide or several nucleotides, and can include modifications such as substitutions, deletions and insertions of nucleotides. An allele of a gene also can be a form of a gene containing a mutation. 
     As used herein, species variants refer to variants in polypeptides among different species, including different mammalian species, such as mouse and human. Exemplary of species variants provided herein are primate PH20, such as, but not limited to, human, chimpanzee, macaque, cynomolgus monkey, gibbon, orangutan, or marmoset. Generally, species variants have 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% sequence identity. Corresponding residues between and among species variants can be determined by comparing and aligning sequences to maximize the number of matching nucleotides or residues, for example, such that identity between the sequences is equal to or greater than 95%, equal to or greater than 96%, equal to or greater than 97%, equal to or greater than 98% or equal to greater than 99%. The position of interest is then given the number assigned in the reference nucleic acid molecule. Alignment can be effected manually or by eye, particularly where sequence identity is greater than 80%. 
     As used herein, substantially pure means sufficiently homogeneous to appear free of readily detectable impurities, as determined by standard methods of analysis, such as thin layer chromatography (TLC), gel electrophoresis and high performance liquid chromatography (HPLC), used by those of skill in the art to assess such purity, or sufficiently pure such that further purification would not detectably alter the physical and chemical properties, such as enzymatic and biological activities, of the substance. Methods for purification of the compounds to produce substantially chemically pure compounds are known to those of skill in the art. A substantially chemically pure compound can, however, be a mixture of stereoisomers or isomers. In such instances, further purification might increase the specific activity of the compound. 
     As used herein, isolated or purified polypeptide or protein or biologically-active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue from which the protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized. Preparations can be determined to be substantially free if they appear free of readily detectable impurities as determined by standard methods of analysis, such as thin layer chromatography (TLC), gel electrophoresis and high performance liquid chromatography (HPLC), used by those of skill in the art to assess such purity, or sufficiently pure such that further purification would not detectably alter the physical and chemical properties, such as enzymatic and biological activities, of the substance. Methods for purification of the compounds to produce substantially chemically pure compounds are known to those of skill in the art. A substantially chemically pure compound, however, can be a mixture of stereoisomers. In such instances, further purification might increase the specific activity of the compound. 
     Hence, reference to a substantially purified polypeptide, such as a substantially purified PH20 polypeptide refers to preparations of PH20 proteins that are substantially free of cellular material, includes preparations of proteins in which the protein is separated from cellular components of the cells from which it is isolated or recombinantly-produced. In one embodiment, the term substantially free of cellular material includes preparations of enzyme proteins having less than about 30% (by dry weight) of non-enzyme proteins (also referred to herein as contaminating proteins), generally less than about 20% of non-enzyme proteins or 10% of non-enzyme proteins or less than about 5% of non-enzyme proteins. When the enzyme protein is recombinantly produced, it also is substantially free of culture medium, i.e., culture medium represents less than about or at 20%, 10% or 5% of the volume of the enzyme protein preparation. 
     As used herein, the term substantially free of chemical precursors or other chemicals includes preparations of enzyme proteins in which the protein is separated from chemical precursors or other chemicals that are involved in the synthesis of the protein. The term includes preparations of enzyme proteins having less than about 30% (by dry weight), 20%, 10%, 5% or less of chemical precursors or non-enzyme chemicals or components. 
     As used herein, synthetic, with reference to, for example, a synthetic nucleic acid molecule or a synthetic gene or a synthetic peptide refers to a nucleic acid molecule or polypeptide molecule that is produced by recombinant methods and/or by chemical synthesis methods. 
     As used herein, production by recombinant means or using recombinant DNA methods means the use of the well known methods of molecular biology for expressing proteins encoded by cloned DNA. 
     As used herein, vector (or plasmid) refers to discrete elements that are used to introduce a heterologous nucleic acid into cells for either expression or replication thereof. The vectors typically remain episomal, but can be designed to effect integration of a gene or portion thereof into a chromosome of the genome. Also contemplated are vectors that are artificial chromosomes, such as yeast artificial chromosomes and mammalian artificial chromosomes. Selection and use of such vehicles are well known to those of skill in the art. 
     As used herein, an expression vector includes vectors capable of expressing DNA that is operatively linked with regulatory sequences, such as promoter regions, that are capable of effecting expression of such DNA fragments. Such additional segments can include promoter and terminator sequences, and optionally can include one or more origins of replication, one or more selectable markers, an enhancer, a polyadenylation signal, and the like. Expression vectors are generally derived from plasmid or viral DNA, or can contain elements of both. Thus, an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, a phage, recombinant virus or other vector that, upon introduction into an appropriate host cell, results in expression of the cloned DNA. Appropriate expression vectors are well known to those of skill in the art and include those that are replicable in eukaryotic cells and/or prokaryotic cells and those that remain episomal or those which integrate into the host cell genome. 
     As used herein, vector also includes “virus vectors” or “viral vectors.” Viral vectors are engineered viruses that are operatively linked to exogenous genes to transfer (as vehicles or shuttles) the exogenous genes into cells. Viral vectors include, but are not limited to, adenoviral vectors, retroviral vectors and vaccinia virus vectors. 
     As used herein, “operably” or “operatively linked” when referring to DNA segments means that the segments are arranged so that they function in concert for their intended purposes, e.g., transcription initiates downstream of the promoter and upstream of any transcribed sequences. The promoter is usually the domain to which the transcriptional machinery binds to initiate transcription and proceeds through the coding segment to the terminator. 
     As used herein, a conjugate refers to a modified PH20 polypeptide linked directly or indirectly to one or more other polypeptides or chemical moieties. Such conjugates include fusion proteins, those produced by chemical conjugates and those produced by any other method whereby at least one modified PH20 polypeptide is linked, directly or indirectly to another polypeptide or chemical moiety so long as the conjugate retains hyaluronidase activity. Exemplary of conjugates provided herein include PH20 polypeptides linked directly or indirectly to a multimerization domain (e.g. an Fc moiety), a toxin, a label or a drug. 
     As used herein, a fusion protein refers to a polypeptide encoded by a nucleic acid sequence containing a coding sequence from one nucleic acid molecule and the coding sequence from another nucleic acid molecule in which the coding sequences are in the same reading frame such that when the fusion construct is transcribed and translated in a host cell, the protein is produced containing the two proteins. The two molecules can be adjacent in the construct or separated by a linker polypeptide that contains, 1, 2, 3, or more, but typically fewer than 10, 9, 8, 7, or 6 amino acids. The protein product encoded by a fusion construct is referred to as a fusion polypeptide. Examples of fusion polypeptides include Fc fusions. 
     As used herein, a polymer that is conjugated to a modified PH20 polypeptide refers to any polymer that is covalently or otherwise stably linked, directly or via a linker, to such polypeptide. Such polymers, typically increase serum half-life, and include, but are not limited to, sialic moieties, polyethylene glycol (PEG) moieties, dextran, and sugar and other moieties, such as for glycosylation. 
     As used herein, the term assessing or determining is intended to include quantitative and qualitative determination in the sense of obtaining an absolute value for the activity of a product, and also of obtaining an index, ratio, percentage, visual or other value indicative of the level of the activity. Assessment can be direct or indirect. 
     As used herein, a “composition” refers to any mixture of two or more products or compounds. It can be a solution, a suspension, liquid, powder, a paste, aqueous, non-aqueous, or any combination thereof. 
     As used herein, a formulation refers to a composition containing at least one active pharmaceutical or therapeutic agent and one or more excipients. 
     As used herein, a co-formulation refers to a composition containing two or more active or pharmaceutical or therapeutic agents and one or more excipients. For example, a co-formulation of a fast-acting insulin and a hyaluronan degrading enzyme contains a fast-acting insulin, a hyaluronan degrading enzyme, and one or more excipients. 
     As used herein, “a combination” refers to any association between two or among more items or elements. Exemplary combinations include, but are not limited to, two or more pharmaceutical compositions, a composition containing two or more active ingredients, such as two modified PH20 polypeptides; a modified PH20 polypeptide and an anticancer agent, such as a chemotherapeutic compound; a modified PH20 polypeptide and a therapeutic agent (e.g. an insulin); a modified PH20 polypeptide and a plurality therapeutic and/or imaging agents, or any association thereof. Such combinations can be packaged as kits. 
     As used herein, a kit is a packaged combination, optionally, including instructions for use of the combination and/or other reactions and components for such use. 
     As used herein, “disease or disorder” refers to a pathological condition in an organism resulting from cause or condition including, but not limited to, infections, acquired conditions, genetic conditions, and characterized by identifiable symptoms. 
     As used herein, a hyaluronan-associated disease, disorder or condition refers to any disease or condition in which hyaluronan levels are elevated as cause, consequence or otherwise observed in the disease or condition. Hyaluronan-associated diseases and conditions are associated with elevated hyaluronan expression in a tissue or cell, increased interstitial fluid pressure, decreased vascular volume, and/or increased water content in a tissue. Hyaluronan-associated diseases, disorders or conditions can be treated by administration of a composition containing a hyaluronan degrading enzyme, such as a hyaluronidase, for example, a soluble hyaluronidase, either alone or in combination with or in addition to another treatment and/or agent. Exemplary diseases and conditions, include, but are not limited to, hyaluronan-rich cancers, for example, tumors, including solid tumors such as late-stage cancers, metastatic cancers, undifferentiated cancers, ovarian cancer, in situ carcinoma (ISC), squamous cell carcinoma (SCC), prostate cancer, pancreatic cancer, non-small cell lung cancer, breast cancer, colon cancer and other cancers. Exemplary hyaluronan-associated diseases and conditions also are diseases that are associated with elevated interstitial fluid pressure, such as diseases associated with disc pressure, and edema, for example, edema caused by organ transplant, stroke, brain trauma or other injury. Exemplary hyaluronan-associated diseases and conditions include diseases and conditions associated with elevated interstitial fluid pressure, decreased vascular volume, and/or increased water content in a tissue, including cancers, disc pressure and edema. In one example, treatment of the hyaluronan-associated condition, disease or disorder includes amelioration, reduction, or other beneficial effect on one or more of increased interstitial fluid pressure (IFP), decreased vascular volume, and increased water content in a tissue. 
     As used herein, “treating” a subject with a disease or condition means that the subject&#39;s symptoms are partially or totally alleviated, or remain static following treatment. Hence treatment encompasses prophylaxis, therapy and/or cure. Prophylaxis refers to prevention of a potential disease and/or a prevention of worsening of symptoms or progression of a disease. Treatment also encompasses any pharmaceutical use of a modified interferon and compositions provided herein. 
     As used herein, a pharmaceutically effective agent or therapeutic agent includes any bioactive agent that can exhibit a therapeutic effect to treat a disease or disorder. Exemplary therapeutic agents are described herein. Therapeutic agents include, but are not limited to, anesthetics, vasoconstrictors, dispersing agents, conventional therapeutic drugs, including small molecule drugs, including, but not limited to, bisphosphonates, and therapeutic proteins, including, but not limited to, insulin, IgG molecules, antibodies, cytokines and coagulation factors. 
     As used herein, “insulin” refers to a hormone, precursor or a synthetic or recombinant analog thereof that acts to increase glucose uptake and storage and/or decrease endogenous glucose production. Insulin and analogs thereof are well known to one of skill in the art, including in human and allelic and species variants thereof. Insulin is translated as a precursor polypeptide designated preproinsulin (110 amino acid for human insulin), containing a signal peptide that directs the protein to the endoplasmic reticulum (ER) wherein the signal sequence is cleaved, resulting in proinsulin. Proinsulin is processed further to release a C- or connecting chain peptide (a 31 amino acid C-chain in human insulin). The resulting insulin contains an A-chain (21 amino acid in length in human insulin; set forth in SEQ ID NO:862) and a B-chain (30 amino acid in length in human insulin; set forth in SEQ ID NO:863) which are cross-linked by disulfide bonds. A fully cross-linked human insulin contains three disulfide bridges: one between position 7 of the A-chain and position 7 of the B-chain, a second between position 20 of the A-chain and position 19 of the B-chain, and a third between positions 6 and 11 of the A-chain. Reference to an insulin includes monomeric and multimeric insulins, including hexameric insulins, as well as humanized insulins. Exemplary insulin polypeptides are those of mammalian, including human, origin. Reference to insulin includes preproinsulin, proinsulin and insulin polypeptides in single-chain or two-chain forms, truncated forms thereof that have activity, and includes allelic variants and species variants of human insulin, variants encoded by splice variants, and other variants, such as insulin analogs. An exemplary insulin is human insulin having a sequence of amino acids of the A- and B-chains of human insulin are set forth in SEQ ID NOs: 862 and 863, respectively, and variants or analogs thereof that exhibit at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto to one or both of the A-chain or B-chain and that acts to increase glucose uptake and storage and/or decrease endogenous glucose production. A further exemplary insulin is porcine insulin having a sequence of amino acids for the preproinsulin as set forth in SEQ ID NO:864, whereby the A chain corresponds to amino acid residue positions 88-108 and the B-chain correspond to amino acid, and variants or analogs thereof that exhibit at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto to one or both of the A-chain or B-chain and that acts to increase glucose uptake and storage and/or decrease endogenous glucose production. 
     As used herein, “fast-acting insulin” refers to any insulin that exhibits peak insulin levels at or about not more than four hours following subcutaneous administration to a subject. Fast-acting insulins include any insulin or any fast-acting insulin composition for acute administration to a diabetic subject in response to an actual, perceived, or anticipated hyperglycemic condition in the subject arising at the time of, or within about four hours following, administration of the fast-acting insulin (such as a prandial hyperglycemic condition resulting or anticipated to result from, consumption of a meal), whereby the fast-acting insulin is able to prevent, control or ameliorate the acute hyperglycemic condition. Fast-acting insulins include recombinant insulins and isolated insulins (also referred to as “regular” insulins) such as the insulin sold as human insulin, porcine insulins and bovine insulins, as well as rapid acting insulin analogs (also termed fast-acting insulin analogs herein) designed to be rapid acting by virtue of amino acid changes. Exemplary regular insulin preparations include, but are not limited to, human regular insulins, such as those sold under the trademarks Humulin® R, Novolin® R and Velosulin®, Insulin Human, USP and Insulin Human Injection, USP, as well as acid formulations of insulin, such as, for example, Toronto Insulin, Old Insulin, and Clear Insulin, and regular pig insulins, such as Iletin II® insulin (porcine insulin). Regular insulins typically have an onset of action of between 30 minutes to an hour, and a peak insulin level of 2-5 hours post administration. 
     As used herein, rapid acting insulin analogs (also called fast-acting insulin analogs) are insulins that have a rapid onset of action. Rapid insulins typically are insulin analogs that have been engineered, such as by the introduction of one or more amino acid substitutions, to be more rapid acting than regular insulins. Rapid acting insulin analogs typically have an onset of action of 10-30 minutes post injection, with peak insulin levels observed 30-90 minutes post injection. Exemplary rapid acting insulin analogs are analogs of human insulin containing one or more amino acid changes in the A-chain and/or B-chain of human insulin set forth in SEQ ID NO:862 or 863, respectively, and that exhibit an onset of action 10-30 minutes post injection with peak insulin levels observed 30-90 minutes post injection. Exemplary rapid acting insulin analogs include, but are not limited to, for example, insulin lispro (e.g., Humalog® insulin), insulin aspart (e.g., NovoLog® insulin), and insulin glulisine (e.g., Apidra® insulin) the fast-acting insulin composition sold as VIAject® and VIAtab® (see, e.g., U.S. Pat. No. 7,279,457). The amino acid sequence of exemplary rapid acting insulin analogs have an A chain with a sequence of amino acids set forth in SEQ ID NO:862 and a B chain having a sequence of amino acids set forth in any of SEQ ID NOs:865-867. Also included are any other insulins that have an onset of action of 30 minutes or less and a peak level before 90 minutes, typically 30-90 minutes, post injection. 
     As used herein, a human insulin refers to an insulin that is synthetic or recombinantly produced based upon the human polypeptide, including allelic variants and analogs thereof. 
     As used herein, fast-acting human insulins or human fast-acting insulin compositions include any human insulin or composition of a human insulin that is fast-acting, but excludes non-human insulins, such as regular pig insulin. 
     As used herein, the terms “basal-acting insulins,” or “basal insulins” refer to insulins administered to maintain a basal insulin level as part of an overall treatment regimen for treating a chronic condition such diabetes. Typically, a basal-acting insulin is formulated to maintain an approximately steady state insulin level by the controlled release of insulin when administered periodically (e.g., once or twice daily). Basal-acting insulins include crystalline insulins (e.g., NPH and Lente®, protamine insulin, surfen insulin), basal insulin analogs (insulin glargine, HOE 901, NovoSol Basal) and other chemical formulations of insulin (e.g., gum arabic, lecithin or oil suspensions) that retard the absorption rate of regular insulin. As used herein, the basal-acting insulins can include insulins that are typically understood as long-acting (typically reaching a relatively low peak concentration, while having a maximum duration of action over about 20-30 hours) or intermediate-acting (typically causing peak insulin concentrations at about 4-12 hours after administration). 
     As used herein, treatment means any manner in which the symptoms of a condition, disorder or disease or other indication, are ameliorated or otherwise beneficially altered. 
     As used herein, therapeutic effect means an effect resulting from treatment of a subject that alters, typically improves or ameliorates the symptoms of a disease or condition or that cures a disease or condition. A therapeutically effective amount refers to the amount of a composition, molecule or compound which results in a therapeutic effect following administration to a subject. 
     As used herein, the term “subject” refers to an animal, including a mammal, such as a human being. 
     As used herein, a patient refers to a human subject exhibiting symptoms of a disease or disorder. 
     As used herein, amelioration of the symptoms of a particular disease or disorder by a treatment, such as by administration of a pharmaceutical composition or other therapeutic, refers to any lessening, whether permanent or temporary, lasting or transient, of the symptoms that can be attributed to or associated with administration of the composition or therapeutic. 
     As used herein, prevention or prophylaxis refers to methods in which the risk of developing a disease or condition is reduced. 
     As used herein, a “therapeutically effective amount” or a “therapeutically effective dose” refers to the quantity of an agent, compound, material, or composition containing a compound that is at least sufficient to produce a therapeutic effect. Hence, it is the quantity necessary for preventing, curing, ameliorating, arresting or partially arresting a symptom of a disease or disorder. 
     As used herein, unit dose form refers to physically discrete units suitable for human and animal subjects and packaged individually as is known in the art. 
     As used herein, a single dosage formulation refers to a formulation containing a single dose of therapeutic agent for direct administration. Single dosage formulations generally do not contain any preservatives. 
     As used herein, a multi-dose formulation refers to a formulation that contains multiple doses of a therapeutic agent and that can be directly administered to provide several single doses of the therapeutic agent. The doses can be administered over the course of minutes, hours, weeks, days or months. Multidose formulations can allow dose adjustment, dose-pooling and/or dose-splitting. Because multi-dose formulations are used over time, they generally contain one or more preservatives to prevent microbial growth. 
     As used herein, an “article of manufacture” is a product that is made and sold. As used throughout this application, the term is intended to encompass a therapeutic agent with a soluble PH20, such as esPH20, or an esPH20 alone, contained in the same or separate articles of packaging. 
     As used herein, fluid refers to any composition that can flow. Fluids thus encompass compositions that are in the form of semi-solids, pastes, solutions, aqueous mixtures, gels, lotions, creams and other such compositions. 
     As used herein, a “control” or “standard” refers to a sample that is substantially identical to the test sample, except that it is not treated with a test parameter, or, if it is a plasma sample, it can be from a normal volunteer not affected with the condition of interest. A control also can be an internal control. For example, a control can be a sample, such as a virus, that has a known property or activity. 
     As used herein, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “an” agent includes one or more agents. 
     As used herein, the term “or” is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive. 
     As used herein, ranges and amounts can be expressed as “about” a particular value or range. About also includes the exact amount. Hence “about 5 bases” means “about 5 bases” and also “5 bases.” 
     As used herein, “optional” or “optionally” means that the subsequently described event or circumstance does or does not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not. For example, an optionally substituted group means that the group is unsubstituted or is substituted. 
     As used herein, the abbreviations for any protective groups, amino acids and other compounds, are, unless indicated otherwise, in accord with their common usage, recognized abbreviations, or the IUPAC-IUB Commission on Biochemical Nomenclature (see, (1972)  Biochem.  11:1726). 
     For clarity of disclosure, and not by way of limitation, the detailed description is divided into the subsections that follow. 
     B. PH20 HYALURONIDASE 
     Provided herein are modified PH20 polypeptides. PH20 (also known as sperm surface protein, sperm adhesion molecule 1 or SPAM1) is a hyaluronidase that hydrolyzes hyaluronan (also called hyaluronic acid, hyaluronate or HA) found in connective tissues such as the extracellular matrix. Hyaluronan polymers are composed of repeating disaccharide units, D-glucuronic acid (GlcA) and N-acetyl-D-glucosamine (GlcNAc), linked together via alternating β-1→4 and β-1→3 glycosidic bonds. Hyaluronan chains can reach about 25,000 disaccharide repeats or more in length, and polymers of hyaluronan can range in size from about 5,000 to 20,000,000 Da in vivo. Hyaluronan, also called hyaluronic acid or hyaluronate, is a non-sulfated glycosaminoglycan that is widely distributed throughout connective, epithelial, and neural tissues. Hyaluronan is an essential component of the extracellular matrix and a major constituent of the interstitial barrier. PH20 is an endo-β-N-acetyl-hexosaminidase that hydrolyzes the 131-4 glycosidic bond of hyaluronic acid into various oligosaccharide lengths such as tetrasaccharides and hexasaccharides. PH20 has both hydrolytic and transglycosidase activities. In addition to degrading hyaluronic acid, PH20 also can degrade chondroitin sulfates, such as C4-S and C6-S. PH20 can exhibit hyaluronidase activity at acidic pH and neutral pH. 
     1. Structure 
     PH20 cDNA has been cloned from numerous mammalian species. Exemplary PH20 precursor polypeptides include, but are not limited to, human (SEQ ID NO:6), bovine (SEQ ID NOs:15 or 17), rabbit (SEQ ID NO:23), Cynomolgus monkey (SEQ ID NO:13), guinea pig (SEQ ID NO:28), rat (SEQ ID NO:21), mouse (SEQ ID NO:19), chimpanzee (SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO:869) Rhesus monkey (SEQ ID NO:11), Fox (SEQ ID NO:30), Gibbon (SEQ ID NO:856), Marmoset (SEQ ID NO:858) or orangutan (SEQ ID NO:860) PH20 polypeptides. The mRNA transcript is typically translated to generate a precursor protein containing a 35 amino acid signal sequence at the N-terminus. Following transport to the ER, the signal peptide is removed to yield a mature PH20 polypeptide. Exemplary mature PH20 polypeptides include, but are not limited to, human (SEQ ID NO:7), bovine (SEQ ID NOs:16 or 18), rabbit (SEQ ID NO:24), Cynomolgus monkey (SEQ ID NO:14), guinea pig (SEQ ID NO:29), rat (SEQ ID NO:22), mouse (SEQ ID NO:20), chimpanzee (SEQ ID NO:10 or SEQ ID NO:870), Rhesus monkey (SEQ ID NO:12), Fox (SEQ ID NO:31), Gibbon (SEQ ID NO:857), Marmoset (SEQ ID NO:859) or orangutan (SEQ ID NO:861) PH20 polypeptides. For example, the human PH20 mRNA transcript is normally translated to generate a 509 amino acid precursor protein (SEQ ID NO:6) containing a 35 amino acid signal sequence at the N-terminus (amino acid residue positions 1-35 of SEQ ID NO:6). Thus, following transport to the ER and removal of the signal peptide, a 474 amino acid mature polypeptide with an amino acid sequence set forth in SEQ ID NO:7 is produced. Sequences of PH20 from ovine are also known (see e.g., SEQ ID NOs: 25-27). 
     In particular, human PH20 has the sequence of amino acids set forth in SEQ ID NO:6. The mature human PH20 lacking a signal sequence is set forth in SEQ ID NO:7. Allelic variants and other variants of PH20 are known. Other sequences of PH20 have been reported. For example, a PH20 variant is known as set forth in the precursor sequence set forth in SEQ ID NO:68 that contains an Ala at position 48 and a Trp at position 499, or the mature sequence thereof set forth in SEQ ID NO:69 containing the corresponding differences at positions 13 and 464, respectively, compared to the sequence set forth in SEQ ID NO:7 (see e.g., Gmachl et al. (1993) FEBS Lett., 336:545-548; GenBank Accession No. AAC60607). Further, a natural variant of PH20 has been identified containing a Glutamine (Gln; Q) at position 5 as compared to the precursor sequence of amino acids set forth in SEQ ID NO:6 (see e.g., SEQ ID NO:70, see also Varela et al. (2011) Nature, 469:539-542). Another natural variant contains an Alanine (Ala; A) at position 47 compared to the sequence of amino acids set forth in SEQ ID NO:6 (as set forth in SEQ ID NO: 71) and corresponding to position 12 compared to the sequence of amino acids set forth in SEQ ID NO: 3 or 7 (as set forth in SEQ ID NO:72). 
     The sequence and structure of PH20 polypeptides is highly conserved. Sequence identity between and among PH20 proteins from various species is about 50% to 90%. The hydrophobic N-terminal signal sequence of 35 amino acids in length is generally conserved among PH20 hyaluronidase polypeptides. PH20 hyaluronidases contain a common core hyaluronidase domain region of about 340 amino acids in length that corresponds to amino acid residues 38-374 of the precursor human PH20 sequence set forth in SEQ ID NO:6. A mature PH20 polypeptide lacking the signal sequence and containing a contiguous sequence of amino acids having a C-terminal amino acid residue corresponding to amino acid residue 464 of SEQ ID NO:6 (e.g., amino acid residues corresponding to positions 36-464 of the amino acid sequence set forth in SEQ ID NO:6) is the minimal sequence required for hyaluronidase activity (see e.g., U.S. patent application Ser. No. 10/795,095, which is issued as U.S. Pat. No. 7,767,429; see also U.S. Publication No. US20100143457). 
     Within the common hyaluronidase domain region, at least 57 amino acids are conserved between and among species (see e.g., Arming et al. (1997)  Eur. I Biochem.,  247:810-814; ten Have et al. (1998)  Reprod. Fertil. Dev.,  10:165-72; Chowpongpang et al. (2004)  Biotechnology Letters,  26:1247-1252). For example, PH20 hyaluronidases contain 12 conserved cysteine residues corresponding to amino acid residue 25, 189, 203, 316, 341, 346, 352, 400, 402, 408, 423 and 429 of the sequence of amino acids of a mature PH20 lacking the signal sequence such as set forth in SEQ ID NO: 3 or 7 (corresponding to amino acid residues 60, 224, 238, 351, 376, 381, 387, 435, 437, 443, 458 or 464 of full-length human PH20 set forth in SEQ ID NO:6). Cysteine residues corresponding to 25 and 316 and cysteine residues corresponding to 189 and 203 form disulfide bridges. The other cysteine residues also form disulfide bridges, are involved in posttranslational protein maturation and/or in activity modulation. For example, further four disulfide bonds are formed between the cysteine residues C376 and C387; between C381 and C435; between C437 and C443; and between C458 and C464 of the polypeptide exemplified in SEQ ID NO:6 (corresponding to positions C341 and C352; between C346 and C400; between C402 and C408; and between C423 and C429 of the mature polypeptide set forth in SEQ ID NO:3 or 7, respectively). 
     Amino acid residues corresponding to amino acid residue D111, E113 and E249 of the sequence of amino acids set forth in SEQ ID NO: 3 or 7 are acidic residues part of the enzyme active site and are conserved between and among PH20 species. Amino acid residues R176, R246, R252 of the sequence of amino acids set forth in SEQ ID NO: 3 or 7 are also conserved between and among species and contribute to substrate binding and/or hyaluronidase activity. Amino acid mutations D111N, E113Q, R176G, E249N and R252T result in enzymes that have no detectable enzymatic activity or residual enzymatic activity (see e.g., Arming et al. (1997)  Eur. I Biochem.,  247:810-814). 
     The results herein confirm the requirement of PH20 amino acid residues corresponding to positions 25, 111, 113, 176, 189, 203, 246, 249, 252, 316, 341, 346, 352, 400, 402, 408, 423 and 429 of the sequence of amino acids set forth in a mature PH20 lacking the signal sequence such as set forth in SEQ ID NO: 3 or 7 for hyaluronidase activity, since mutagenesis of these residues results in an enzyme that is not active (e.g., it is not expressed or is inactive when expressed, see e.g., Tables 5 and 10). The exception is that amino acid replacement corresponding to R176K and C316D resulted in mutants that generated some residual hyaluronidase activity. 
     Glycosylation also is required for PH20 hyaluronidase activity based on the recognition motif N×S or N×T. There are six N-linked oligosaccharides at amino acid residues corresponding to positions N47, N131, N200, N219, N333 and N358 of the sequence of amino acids set forth in SEQ ID NO: 3 or 7 (corresponding to amino acid residues N82, N166, N235, N254, N368 and N393 of human PH20 set forth in SEQ ID NO: 6). In particular, at least N-linked glycosylation sites corresponding to amino acid residues N200, N333 and N358 are required for secretion and/or activity of the enzyme (see e.g., U.S. Publication No. US20100143457). For example, a PH20 polypeptide containing amino acid mutations N200A, N333A, N358A or N333A/N393A result in inactive proteins. Single mutations of glycosylation sites N47A, N131A, N219A, N47A/N131A, N47A/N219A, N131A/N291A retain activity. The N-linked glycosylation site corresponding to amino acid residue N368 of human PH20 set forth in SEQ ID NO:6 is conserved between and among species (see e.g., Chowpongpang et al. (2004)  Biotechnology Letters,  26:1247-1252). PH20 hyaluronidases also contains O-linked glycosylation sites. For example, human PH20 has one O-linked oligosaccharide at the amino acid residue corresponding to amino acid T440 of the sequence of amino acids set forth in SEQ ID NO:3 or 7 (corresponding to amino acid residue T475 in SEQ ID NO:6). 
     In addition to the catalytic sites, PH20 also contains a hyaluronan-binding site. This site is located in the Peptide 2 region, which corresponds to amino acid positions 205-235 of the precursor polypeptide set forth in SEQ ID NO:6 and positions 170-200 of the mature polypeptide set forth in SEQ ID NO:3 or 7. This region is highly conserved among hyaluronidases and is similar to the heparin binding motif. Mutation of the arginine residue at position 176 (corresponding to the mature PH20 polypeptide set forth in SEQ ID NO:3 or 7) to a glycine results in a polypeptide with only about 1% of the hyaluronidase activity of the wild type polypeptide (Arming et al., (1997)  Eur. I Biochem.  247:810-814). 
     PH20 polypeptides contain a glycosyl phosphatidylinositol (GPI) anchor attached to the C-terminus of the protein that anchors the protein to the extracellular leaflet of the plasma membrane of cells. At least human, monkey, mouse and guinea pig PH20 are strongly attached to the plasma membrane via the GPI anchor, which can be released by treating with phosphatidylinositol-specific phospholipase C (PI-PLC; see e.g., Lin et al. (1994)  Journal of  Cell Biology, 125:1157-1163; Lin et al. (1993)  Proc. Natl. Acad. Sci.,  90:10071-10075). Other PH20 enzymes, such as bovine PH20, are loosely attached to the plasma membrane and are not anchored via a phospholipase sensitive anchor. As discussed below, soluble active forms that, when expressed, are not attached to the membrane but are secreted can be generated by removal of all of a portion of the GPI anchor attachment signal site (see also U.S. Pat. No. 7,767,429; U.S. Publication No. US20100143457). These include, for example, soluble PH20 polypeptides set forth in any of SEQ ID NOs: 3 or 32-66, or precursor forms thereof containing a signal sequence. 
     GPI-anchored proteins, for example human PH20, are translated with a cleavable N-terminal signal peptide that directs the protein to the endoplasmic reticulum (ER). At the C-terminus of these proteins is another signal sequence that directs addition of a preformed GPI-anchor to the polypeptide within the lumen of the ER. Addition of the GPI anchor occurs following cleavage of the C-terminal portion at a specific amino acid position, called the w-site (typically located approximately 20-30 amino acids from the C-terminus). Although there appears to be no consensus sequence to identify the location of the w-site, GPI anchored proteins contain a C-terminal GPI-anchor attachment signal sequence or domain that typically contains a predominantly hydrophobic region of 8-20 amino acids, preceded by a hydrophilic spacer region of 8-12 amino acids immediately downstream of the w-site. This hydrophilic spacer region often is rich in charged amino acids and proline (White et al. (2000)  J. Cell Sci.  113(Pt.4):721-727). There is generally a region of approximately 11 amino acids before the ω-1 position that is characterized by a low amount of predicted secondary structure, a region around the cleavage site (w-site), from ω-1 to ω+2 that is characterized by the presence of small side chain residues, the spacer region between positions ω+3 and ω+9, and a hydrophobic tail from ω+10 to the C-terminal end (Pierleoni et al., (2008)  BMC Bioinformatics  9:392). 
     Although there is no GPI-anchor attachment signal consensus sequence, various in silico methods and algorithms have been developed that can be used to identify such sequences in polypeptides (see, e.g., Udenfriend et al. (1995) Methods Enzymol.  250:571-582; Eisenhaber et al. (1999)  J. Mol. Chem.  292: 741-758; Kronegg and Buloz, (1999), “Detection/prediction of GPI cleavage site (GPI-anchor) in a protein (DGPI),” 129.194.185.165/dgpi/; Fankhauser et al. (2005)  Bioinformatics  21:1846-1852; Omaetxebarria et al. (2007)  Proteomics  7:1951-1960; Pierleoni et al. (2008)  BMC Bioinformatics  9:392), including those that are readily available on bioinformatic websites, such as the ExPASy Proteomics tools site (expasy.ch/tools/). Thus, one of skill in the art can determine whether a PH20 polypeptide likely contains a GPI-anchor attachment signal sequence, and, therefore, whether the PH20 polypeptide is a GPI-anchored protein. 
     The covalent attachment of a GPI-anchor to the C-terminus of human PH20 and, therefore, the membrane-bound nature of PH20, has been confirmed using phosphatidylinositol-specific phospholipase C (PI-PLC) hydrolysis studies (see e.g., Lin et al., (1994)  J. Biol. Chem.  125:1157-1163). Phosphatidylinositol-specific phospholipase C (PI-PLC) and D (PI-PLD) hydrolyze the GPI anchor, releasing the PH20 polypeptide from the cell membrane. The prior art literature reports that a ω-site cleavage site of human PH20 is identified between Ser-490 and Ala-491 and for monkey PH20 is identified between Ser491 and Thr492 (Lin et al. (1993)  Proc. Natl. Acad. Sci , (1993) 90:10071-10075). Thus, the literature reports that a GPI-anchor attachment signal sequence of human PH20 is located at amino acid positions 491-509 of the precursor polypeptide set forth in SEQ ID NO:6, and the ω-site is amino acid position 490. Thus, in this modeling of human PH20, amino acids 491-509 are cleaved following transport to the ER and a GPI anchor is covalently attached to the serine residue at position 490. 
     2. Function 
     PH20 is normally expressed in sperm from a single testis-specific gene. PH20 is a sperm-associated protein involved in fertilization. PH20 is normally localized on the sperm surface, and in the lysosome-derived acrosome, where it is bound to the inner acrosomal membrane. PH20 is multifunctional and exhibits hyaluronidase activity, hyaluronan (HA)-mediated cell-signaling activity, and acts as a sperm receptor for the zona pellucida surrounding the oocyte when present on acrosome reacted (AR) sperm. For example, PH20 is naturally involved in sperm-egg adhesion and aids penetration by sperm of the layer of cumulus cells by digesting hyaluronic acid. In addition to being a hyaluronidase, PH20 also appears to be a receptor for HA-induced cell signaling, and a receptor for the zona pellucida surrounding the oocyte. Due to the role of PH20 in fertilization, PH20 can be used as an antigen for immunocontraception. 
     PH20 is a neutral active hyaluronidase, although it can exhibit acid-active activity in some cases. The hyaluronidase activity of PH20 is exhibited by the plasma membrane- and inner acrosomal membrane-associated PH20. The plasma membrane PH20 exhibits hyaluronidase activity only at neutral pH, while the inner acrosomal membrane-associated PH20 exhibits acid-active enzyme activity. The structural basis for these differences is due to the presence of two catalytic sites in PH20. A first catalytic site is designated the Peptide 1 region, corresponding to amino acid residues 142-172 of SEQ ID NO:6, which is involved in enzyme activity of PH20 at neutral pH. A second catalytic site is designated the peptide 3 region, corresponding to amino acid residues 277-297 of SEQ ID NO:6, which is involved in enzyme activity at lower pH. A change in the structure of the inner acrosomal membrane-associated PH20 occurs after the acrosome reaction, whereby PH20 is endoproteolytically cleaved but held together by disulfide bonds. The result of the endoproteolysis is that the peptide 3 region is activated and can thus effect neutral and acid-activity to PH20 (see e.g., Cherr et al. (2001)  Matrix Biology,  20:515-525. Also, after the acrosome reaction, lower molecular weight forms are generated by release from the inner acrosomal membrane (e.g., a 53 kDa soluble form of PH20 is generated in monkey). The lower molecular weight form(s) also is acid active. 
     The hyaluronidase activity of PH20 accounts for the spreading activity observed in animal testes extracts that have been used clinically for decades to increase the dispersion and absorption of drugs (see e.g., Bookbinder et al. (2006)  J. Controlled Release,  114:230-241). For example, pharmaceutical preparations containing hyaluronidase were developed as fractionated extracts from bovine testes for therapeutic use as spreading agents and in other applications (Schwartzman (1951)  J. Pediat.,  39:491-502). Original bovine testicular extract preparations included, for example, extracts sold under the trademarks Wydase®, Hylase®, “Dessau,” Neopermease®, Alidase® and Hyazyme®. It is now known that the spreading activity of testicular extract preparations are due to PH20 hyaluronidase activity. For example, in 2001 a sperm hyaluronidase in bull was identified as the hyaluronidase PH20 (Lalancette et al. (2001)  Biol. Reprod.,  65:628-36). By catalyzing the hydrolysis of hyaluronic acid, PH20 hyaluronidase lowers the viscosity of hyaluronic acid, thereby increasing tissue permeability. Hence, soluble forms of PH20 are used as a spreading or dispersing agent in conjunction with other agents, drug and proteins to enhance their dispersion and delivery, and to improve the pharmacokinetic and pharmacodynamic profile of the coadministered agent, drug or protein (see e.g., U.S. Pat. No. 7,767,429; Bookbinder et al. (2006)  J. Controlled Release,  114:230-241). 
     3. Soluble PH20 Polypeptides 
     PH20 can exist in membrane-bound or membrane-associated form, or can be secreted into the media when expressed from cells, and thereby can exist in soluble form. Soluble PH20 can be detected and discriminated from insoluble, membrane-bound PH20 using methods well known in the art, including, but not limited to, those using a Triton® X-114 detergent assay. In this assay, soluble PH20 hyaluronidases partition into the aqueous phase of a Triton® X-114 detergent solution warmed to 37° C. (Bordier et al., (1981)  J. Biol. Chem.,  256:1604-7) while membrane-anchored PH20 hyaluronidases partition into the detergent rich phase. Thus, in addition to using algorithms to assess whether a PH20 polypeptide is naturally GPI-anchored and hence membrane-bound, solubility experiments also can be performed. 
     Soluble PH20 enzymes include hyaluronidases that contain a GPI-anchor attachment signal sequence, but that are loosely attached to the membrane such that they do not contain a phospholipase sensitive anchor. For example, soluble PH20 polypeptides include ovine or bovine PH20. Various forms of such soluble PH20 hyaluronidases have been prepared and approved for therapeutic use in subjects, including humans. For example, animal-derived hyaluronidase preparations include Vitrase® hyaluronidase (ISTA Pharmaceuticals), which is a purified ovine testicular hyaluronidase, and Amphadase® hyaluronidase (Amphastar Pharmaceuticals), which is a bovine testicular hyaluronidase. Soluble PH20 enzymes also include truncated forms of non-human or human membrane-associated PH20 hyaluronidases that lack one or more amino acid residues of a glycosylphosphatidylinositol (GPI) anchor attachment signal sequence and that retain hyaluronidase activity (see e.g., U.S. Pat. No. 7,767,429; U.S. Publication No. US20100143457). Thus, instead of having a GPI-anchor covalently attached to the C-terminus of the protein in the ER and being anchored to the extracellular leaflet of the plasma membrane, these polypeptides are secreted when expressed from cells and are soluble. In instances where the soluble hyaluronan degrading enzyme retains a portion of the GPI anchor attachment signal sequence, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid residues in the GPI-anchor attachment signal sequence can be retained, provided the polypeptide is soluble (i.e., secreted when expressed from cells) and active. 
     Exemplary soluble hyaluronidases that are C-terminally truncated and lack all or a portion of the GPI anchor attachment signal sequence include, but are not limited to, PH20 polypeptides of primate origin, such as, for example, human and chimpanzee PH20 polypeptides. For example, soluble PH20 polypeptides can be made by C-terminal truncation of a polypeptide set forth in SEQ ID NO:7, 10, 12, 14, 69, 72, 857, 859, 861 or 870 or variants thereof that exhibit at least 80%, 85%, 90%, 95% or more sequence identity to any of SEQ ID NO: 7, 10, 12, 14, 69, 72, 857, 859, 861 or 870, wherein the resulting polypeptide is active, soluble and lacks all or a portion of amino acid residues from the GPI-anchor attachment signal sequence. 
     Exemplary soluble PH20 polypeptides are C-terminal truncated human PH20 polypeptides that are mature (lacking a signal sequence), soluble and exhibit neutral activity, and that contain a contiguous sequence of amino acids set forth in SEQ ID NO:6 or SEQ ID NO:7 that minimally has a C-terminal truncated amino acid residue at or after amino acid residue 464 of the sequence of amino acids set forth in SEQ ID NO:6. For example, soluble PH20 polypeptides include C-terminal truncated polypeptides that minimally contain a contiguous sequence of amino acids 36-464 of SEQ ID NO:6, or includes a sequence of amino acids that has at least 85%, for example at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% sequence identity to a contiguous sequence of amino acids that has a C-terminal amino acid residue after amino acid 464 of SEQ ID NO:6 and retains hyaluronidase activity. Exemplary C-terminally truncated human PH20 polypeptides are mature polypeptides (lacking a signal sequence) that include a contiguous sequence of amino acids set forth in SEQ ID NO:6 with a C-terminal residue after 464 such as after amino acid position 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499 or 500 of the sequence of amino acids set forth in SEQ ID NO:6, or a variant thereof that exhibits at least 85% sequence identity, such as at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% sequence identity thereto and retains hyaluronidase activity. For example, exemplary C-terminal PH20 polypeptides have a sequence of amino acids 36 to 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499 or 500 of the sequence of amino acids set forth in SEQ ID NO:6, or a variant thereof that exhibits at least 85% sequence identity, such as at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% sequence identity thereto and retains hyaluronidase activity. Soluble PH20 polypeptides include any that has the sequence of amino acids set forth in SEQ ID NOs: 3 or 32-66 or a sequence of amino acids that exhibits at least 85% sequence identity, such as at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% sequence identity to the sequence of amino acids set forth in any of SEQ ID NOs: 3 or 32-66. 
     In particular, a soluble human PH20 polypeptide is a polypeptide that is truncated after amino acid 482 of the sequence set forth in SEQ ID NO:6. Such a polypeptide can be generated from a nucleic acid molecule containing a signal sequence and encoding amino acids 36-482, for example, as set forth in SEQ ID NO:1 (containing an IgG kappa signal sequence) or SEQ ID NO:67 (containing the native signal sequence). Post translational processing removes the signal sequence, leaving a 447 amino acid soluble recombinant human PH20 (SEQ ID NO:3). A product produced upon expression of a vector set forth in SEQ ID NO:4 or 5, and containing a nucleic acid molecule set forth in SEQ ID NO:67, results in a secreted product, designated rHuPH20, in the culture medium that exhibits heterogeneity at the C-terminus such that the product includes a mixture of species that can include any one or more of SEQ ID NOs: 3 and 44-48 in various abundance. Typically, rHuPH20 is produced in cells that facilitate correct N-glycosylation to retain activity, such as mammalian cells, for example CHO cells (e.g., DG44 CHO cells). Hylenex® hyaluronidase (Halozyme) is a human recombinant hyaluronidase produced by genetically engineered Chinese Hamster Ovary (CHO) cells containing nucleic acid encoding a truncated human PH20 polypeptide (designated rHuPH20). 
     C. MODIFIED PH20 POLYPEPTIDES 
     Provided herein are modified or variant PH20 polypeptides. The modified PH20 polypeptides provided herein exhibit altered activities or properties compared to a wildtype, native or reference PH20 polypeptide. Included among the modified PH20 polypeptides provided herein are PH20 polypeptide that are active mutants, whereby the polypeptides exhibit at least 40% of the hyaluronidase activity of the corresponding PH20 polypeptide not containing the amino acid modification (e.g., amino acid replacement). In particular, provided herein are PH20 polypeptides that exhibit hyaluronidase activity and that exhibit increased stability compared to the PH20 not containing the amino acid modification. Also provided are modified PH20 polypeptides that are inactive, and that can be used, for example, as antigens in contraception vaccines. 
     The modifications can be a single amino acid modification, such as single amino acid replacements (substitutions), insertions or deletions, or multiple amino acid modifications, such as multiple amino acid replacements, insertions or deletions. Exemplary modifications are amino acid replacements, including single or multiple amino acid replacements. The amino acid replacement can be a conservative substitution, such as set forth in Table 2, or a non-conservative substitution, such as any described herein. Modified PH20 polypeptides provided herein can contain at least or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more modified positions compared to the PH20 polypeptide not containing the modification. 
     The modifications described herein can be in any PH20 polypeptide, including, including precursor, mature, or C-terminal truncated forms, so long as the modified form exhibits hyaluronidase activity. For example, the PH20 polypeptides contain modifications compared to a wildtype, native or reference PH20 polypeptide set forth in any of SEQ ID NOs: 2, 3, 6-66, 68-72, 856-861, 869 or 870, or in a polypeptide that has a sequence of amino acids that is at least 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to any of SEQ ID NOs: 3, 6-66, 68-72, 856-861, 869 or 870. For example, the modifications are made in a human PH20 polypeptide having the sequence of amino acids including or set forth in SEQ ID NO:7, SEQ ID NO:69 or SEQ ID NO:72; a bovine PH20 polypeptide having a sequence of amino acids including or set forth in SEQ ID NOs:16 or 18; a rabbit PH20 polypeptide having a sequence of amino acids including or set forth in SEQ ID NO:24; a Cynomolgus monkey PH20 polypeptide having a sequence of amino acids including or set forth in SEQ ID NO:14; a guinea pig PH20 polypeptide having a sequence of amino acids including or set forth in SEQ ID NO:29; a rat PH20 polypeptide having a sequence of amino acids including or set forth in SEQ ID NO:22; a mouse PH20 polypeptide having a sequence of amino acids including or set forth in SEQ ID NO:20; a chimpanzee PH20 polypeptide having a sequence of amino acids including or set forth in SEQ ID NO:10 or 870; a Rhesus monkey PH20 polypeptide having a sequence of amino acids including or set forth in SEQ ID NO:12; a Fox PH20 polypeptide having a sequence of amino acids including or set forth in SEQ ID NO:31; a Gibbon PH20 polypeptide having a sequence of amino acids including or set forth in SEQ ID NO:857; a Marmoset PH20 polypeptide having a sequence of amino acids including or set forth in SEQ ID NO: 859; an Orangutan PH20 polypeptide having a sequence of amino acids including or set forth in SEQ ID NO:861; or a sheep PH20 polypeptide having a sequence of amino acids including or set forth in any of SEQ ID NOs: 25-27; or in sequence variants or truncated variants that exhibit at least 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NOs: 7, 10, 12, 14, 16, 18, 20, 22, 24-27, 29, 31, 69, 72, 857, 859, 861 or 870. 
     In particular, provided herein are PH20 polypeptides that contain modifications compared to a PH20 polypeptide set forth in SEQ ID NO: 3, 7, 32-66, 69 or 72, or a polypeptide that has a sequence of amino acids that is at least 68%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to any of SEQ ID NOs: 3, 7, 32-66, 69 or 72. For example, the modifications provided herein also can be made in a PH20 polypeptide set forth as SEQ ID NO: 10, 12, 14, 24, 857, 859, 861 or 870. 
     In particular, provided herein are modified soluble PH20 polypeptides that are PH20 polypeptides containing a modification provided herein, and that when expressed from cells are secreted into the media as a soluble protein. For example, the modifications are made in a soluble PH20 polypeptide that is C-terminally truncated within or near the C-terminus portion containing the GPI-anchor signal sequence of a PH20 polypeptide that contains a GPI-anchor signal sequence. The C-terminal truncation can be a truncation or deletion of 8 contiguous amino acids at the C-terminus, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more amino acids at the C-terminus, so long as the resulting C-terminally truncated polypeptide exhibits hyaluronidase activity and is secreted from cells (e.g., into the media) when expressed. In some examples, the modifications provided herein are made in a soluble PH20 polypeptide that is a C-terminally truncated polypeptide of SEQ ID NO:7, 10, 12, 14, 69, 72, 857, 859, 861 or 870 or a variant thereof that exhibits at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NOs: 7, 10, 12, 14, 69, 72, 857, 859, 861 or 870. In particular, the modifications provided herein are made in a soluble or C-terminally truncated human PH20 polypeptide having the sequence of amino acids set forth in SEQ ID NOs: 3 or 32-66 or a sequence of amino acids that exhibits at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% sequence identity to the sequence of amino acids set forth in any of SEQ ID NOs: 3 or 32-66. For example, modified PH20 polypeptides provided herein contain amino acid replacements or substitutions, additions or deletions, truncations or combinations thereof with reference to the PH20 polypeptide set forth in SEQ ID NO:3. 
     Modifications also can be made in the corresponding precursor form containing a signal peptide of any of SEQ ID NOs: 3, 7, 10, 12, 14, 16, 18, 20, 22, 24-27, 29, 31, 32-66, 69, 72, 857, 859, 861 or 870. For example, modifications provided herein can be made in a precursor form set forth in any of SEQ ID NOs: 2, 6, 8, 9, 11, 13, 15, 17, 19, 21, 23, 28, 30, 856, 858, 860 or 869 or in a variant thereof that exhibits at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NOs: 2, 6, 8, 9, 11, 13, 15, 17, 19, 21, 23, 28, 30, 856, 858, 860 or 869. 
     In examples of modified PH20 polypeptides provided herein, the modified PH20 polypeptide does not contain the sequence of amino acids set forth in any of SEQ ID NOs: 3-66, 68-72, 856-861, 869 or 870. Typically, the modified PH20 polypeptide is a human PH20 polypeptide, and does not contain the sequence of amino acids set forth in any of SEQ ID NOs: 8-31, 856-861, 869 or 870. 
     Generally, any modification, such as amino acid replacement, deletion or substitution, can be made in a PH20 polypeptide, with the proviso that the modification is not an amino acid replacement where the only modification is a single amino acid replacement that is V12A, N47A, D111N, E113Q, N131A, R176G, N200A, N219A, E249Q, R252T, N333A or N358A. Also, where the modified PH20 polypeptide contains only two amino acid replacements, the amino acid replacements are not P13A/L464W, N47A/N131A, N47A/N219A, N131A/N219A or N333A/N358A. In a further example, where the modified PH20 polypeptide contains only three amino acid replacements, the amino acid replacements are not N47A/N131A/N219A. Exemplary modifications provided herein are described in detail below. 
     For purposes herein, reference to positions and amino acids for modification herein, including amino acid replacement or replacements, are with reference to the PH20 polypeptide set forth in SEQ ID NO:3. It is within the level of one of skill in the art to make any of the modifications provided herein in another PH20 polypeptide by identifying the corresponding amino acid residue in another PH20 polypeptide, such as any set forth in SEQ ID NOs: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24-27, 28, 29, 30, 31, 32-66, 68-72, 856, 857, 858, 859, 860, 861, 869 or 870 or a variant thereof that exhibits at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NOs: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24-27, 28, 29, 30, 31, 32-66, 68-72, 856, 857, 858, 859, 860, 861, 869 or 870. Corresponding positions in another PH20 polypeptide can be identified by alignment of the PH20 polypeptide with the reference to the PH20 polypeptide set forth in SEQ ID NO:3. For example,  FIG.  2    (A-L) depicts alignment of exemplary PH20 polypeptides with SEQ ID NO:3, and identification of exemplary corresponding positions. Also, since SEQ ID NOs: 3, 7, 32-66, 69 and 72 are all forms of a mature human PH20 with a different C-terminal amino acid residue, the numbering of amino acid residues in any of SEQ ID NOs: 7, 32-66, 69 and 72 is the same as SEQ ID NO:3, and hence the corresponding residues of each are identical to that set forth in SEQ ID NO:3 (see e.g.,  FIG.  1   ). Further, SEQ ID NOS set forth in any of SEQ ID NOs: 2, 6, 70 or 71 are precursor forms thereof that differ by only the presence of a signal sequence. For purposes of modification (e.g., amino acid replacement), the corresponding amino acid residue can be any amino acid residue, and need not be identical to the residue set forth in SEQ ID NO:3. Typically, the corresponding amino acid residue identified by alignment with residues in SEQ ID NO:3 is an amino acid residue that is identical to SEQ ID NO:3, or is a conservative or semi-conservative amino acid residue thereto (see e.g.,  FIGS.  2 A- 2 L ). It is also understood that the exemplary replacements provided herein can be made at the corresponding residue in a PH20 polypeptide, so long as the replacement is different than exists in the unmodified form of the PH20 polypeptide. Based on this description and the description elsewhere herein, it is within the level of one of skill in the art to generate a modified PH20 polypeptide containing any one or more of the described mutation, and test each for a property or activity as described herein. 
     Modifications in a PH20 polypeptide also can be made to a PH20 polypeptide that also contains other modifications, including modifications of the primary sequence and modifications not in the primary sequence of the polypeptide. For example, modifications described herein can be in a PH20 polypeptide that is a fusion polypeptide or chimeric polypeptide. The modified PH20 polypeptides provided herein also include polypeptides that are conjugated to a polymer, such as a PEG reagent. 
     Also provided herein are nucleic acid molecules that encode any of the modified PH20 polypeptides provided herein. In particular examples, the nucleic acid sequence can be codon optimized, for example, to increase expression levels of the encoded sequence. The particular codon usage is dependent on the host organism in which the modified polypeptide is expressed. One of skill in the art is familiar with optimal codons for expression in mammalian or human cells, bacteria or yeast, including for example  E. coli  or  Saccharomyces cerevisiae . For example, codon usage information is available from the Codon Usage Database available at kazusa.or.jp.codon (see Richmond (2000)  Genome Biology,  1:reports241 for a description of the database). See also, Forsburg (1994)  Yeast,  10:1045-1047; Brown et al. (1991)  Nucleic Acids Research,  19:4298; Sharp et al. (1988)  Nucleic Acids Res.,  12:8207-8211; Sharp et al. (1991)  Yeast,  657-78). In some examples, the encoding nucleic acid molecules also can be modified to contain a heterologous signal sequence to alter (e.g., increased) expression and secretion of the polypeptide. Exemplary of a heterologous signal sequence is a nucleic acid encoding the IgG kappa signal sequence (set forth in SEQ ID NO:868). 
     The modified polypeptides and encoding nucleic acid molecules provided herein can be produced by standard recombinant DNA techniques known to one of skill in the art. Any method known in the art to effect mutation of any one or more amino acids in a target protein can be employed. Methods include standard site-directed or random mutagenesis of encoding nucleic acid molecules, or solid phase polypeptide synthesis methods. For example, nucleic acid molecules encoding a PH20 polypeptide can be subjected to mutagenesis, such as random mutagenesis of the encoding nucleic acid, error-prone PCR, site-directed mutagenesis, overlap PCR, gene shuffling, or other recombinant methods. The nucleic acid encoding the polypeptides can then be introduced into a host cell to be expressed heterologously. Hence, also provided herein are nucleic acid molecules encoding any of the modified polypeptides provided herein. In some examples, the modified PH20 polypeptides are produced synthetically, such as using solid phase or solutions phase peptide synthesis. 
     In the subsections below, exemplary modified PH20 polypeptide exhibiting altered properties and activities, and encoding nucleic acid molecules, provided herein are described. 
     1. Active Mutants 
     Provided herein are modified PH20 polypeptides that contain one or more amino acid replacements in a PH20 polypeptide and that exhibit hyaluronidase activity. The modified PH20 polypeptides can exhibit 40% to 5000% of the hyaluronidase activity of a wildtype or reference PH20 polypeptide, such as the polypeptide set forth in SEQ ID NOs: 3 or 7. For example, modified PH20 polypeptides provided herein exhibit at least 40% of the hyaluronidase activity, such as at least 50%, 60%, 70%, 80%, 90%, 100%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, 2000%, 3000% or more of the hyaluronidase activity of a wildtype or reference PH20 polypeptide, such as the corresponding polypeptide not containing the amino acid modification (e.g., amino acid replacement), for example, a polypeptide set forth in SEQ ID NO:3 or 7. For example, exemplary positions that can be modified, for example by amino acid replacement or substitution, include, but are not limited to, any of positions corresponding to position 1, 2, 3, 4, 5, 6, 8, 9, 10, 11, 12, 13, 14, 15, 20, 22, 23, 24, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 54, 58, 59, 60, 61, 63, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 77, 79, 81, 82, 83, 84, 85, 86, 87, 89, 90, 91, 92, 93, 94, 96, 97, 98, 99, 102, 103, 104, 105, 106, 107, 108, 110, 114, 117, 118, 119, 120, 122, 124, 125, 127, 128, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 186, 192, 193, 195, 196, 197, 198, 200, 202, 204, 205, 206, 208, 209, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 224, 226, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 242, 245, 247, 248, 251, 253, 255, 256, 257, 258, 259, 260, 261, 263, 264, 265, 266, 267, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 297, 298, 300, 301, 302, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 320, 321, 323, 324, 325, 326, 327, 328, 331, 334, 335, 338, 339, 342, 343, 347, 348, 349, 351, 353, 356, 357, 358, 359, 360, 361, 367, 368, 369, 371, 373, 374, 375, 376, 377, 378, 379, 380, 381, 383, 385, 387, 388, 389, 391, 392, 393, 394, 395, 396, 397, 398, 399, 401, 403, 404, 405, 406, 407, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 425, 426, 427, 428, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446 or 447 with reference to amino acid positions set forth in SEQ ID NO:3. Typically, the amino acid residue that is modified (e.g., replaced with another amino acid) at the position corresponding to any of the above positions in a PH20 polypeptide is an identical residue, a conservative residue or a semi-conservative amino acid residue to the amino acid residue set forth in SEQ ID NO:3. 
     To retain hyaluronidase activity, modifications typically are not made at those positions that are less tolerant to change or required for hyaluronidase activity. For example, generally modifications are not made at a position corresponding to position 7, 16, 17, 18, 19, 21, 25, 53, 55, 56, 57, 62, 64, 76, 78, 80, 88, 95, 100, 101, 109, 111, 112, 113, 115, 116, 121, 123, 126, 129, 185, 187, 188, 189, 190, 191, 194, 199, 201, 203, 207, 210, 223, 225, 227, 228, 229, 241, 243, 244, 246, 249, 250, 252, 254, 262, 268, 295, 296, 299, 303, 319, 322, 329, 330, 332, 333, 336, 337, 340, 341, 344, 345, 346, 350, 352, 354, 355, 362, 363, 364, 365, 366, 370, 372, 382, 384, 386, 390, 400, 402, 408, 423, 424, 429, 430, with reference to amino acid positions set forth in SEQ ID NO:3. Also, in examples where modifications are made at any of positions 2, 3, 4, 5, 6, 8, 9, 10, 11, 12, 13, 14, 15, 20, 22, 23, 27, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 54, 58, 59, 60, 61, 63, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 77, 79, 81, 82, 83, 84, 85, 86, 87, 89, 90, 91, 92, 94, 96, 98, 99, 102, 103, 104, 105, 106, 107, 108, 110, 114, 117, 118, 119, 122, 124, 125, 127, 128, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 143, 144, 145, 149, 150, 152, 153, 154, 155, 156, 157, 158, 159, 161, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 186, 192, 193, 195, 197, 198, 200, 202, 204, 206, 208, 209, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 224, 226, 230, 231, 232, 233, 234, 235, 236, 238, 239, 240, 242, 245, 247, 248, 251, 253, 255, 256, 257, 258, 260, 261, 263, 264, 265, 266, 267, 269, 270, 271, 272, 273, 274, 275, 276, 278, 279, 280, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 297, 298, 300, 301, 302, 304, 305, 306, 307, 308, 310, 311, 312, 313, 314, 315, 316, 317, 318, 320, 321, 323, 324, 325, 326, 327, 331, 334, 335, 338, 339, 342, 343, 347, 348, 349, 351, 353, 356, 357, 358, 359, 360, 361, 367, 368, 369, 371, 373, 374, 375, 376, 377, 378, 379, 380, 381, 383, 385, 387, 388, 389, 391, 392, 393, 394, 395, 396, 397, 398, 399, 401, 403, 404, 405, 406, 410, 411, 412, 413, 414, 415, 416, 417, 419, 420, 422, 425, 426, 427, 428, 431, 432, 434, 437, 438, 439, 440, 441, 442, 443, 444, or 447 with reference to amino acid positions set forth in SEQ ID NO:3, the modification(s) is/are not the corresponding amino acid replacement(s) set forth in Table 5 or 10 herein, which are amino acid replacements that result in an inactive polypeptide. For example, if the modification is a modification at a position corresponding to position 2 with reference to SEQ ID NO:3, the modification is not replacement to a histidine (H), lysine (K), tryptophan (W) or tyrosine (Y). 
     Exemplary amino acid replacements at any of the above corresponding positions are set forth in Table 3. Reference to the corresponding amino acid position in Table 3 is with reference to positions set forth in SEQ ID NO:3. It is understood that the replacements can be made in the corresponding position in another PH20 polypeptide by alignment therewith with the sequence set forth in SEQ ID NO:3 (see e.g.,  FIGS.  1  and  2   ), whereby the corresponding position is the aligned position. In particular examples, the amino acid replacement(s) can be at the corresponding position in a PH20 polypeptide as set forth in any of SEQ ID NOs: 2, 3, 6-66, 68-72, 856-861, 869 or 870 or a variant thereof having at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 86%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto, so long as the resulting modified PH20 polypeptide exhibits at least 40% of the hyaluronidase activity of the corresponding PH20 polypeptide not containing the amino acid replacement. In particular, the replacement(s) can be in a corresponding position in a human PH20 polypeptide, for example, any set forth in any of SEQ ID NOs: 3, 7, 32-66, 69 or 72, or a variant thereof that exhibits at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NOs: 3, 7, 32-66, 69 or 72. In one example, any one or more of the replacements are in SEQ ID NO:3, so long as the resulting modified PH20 polypeptide exhibits at least 40% of the hyaluronidase activity of the PH20 polypeptide set forth in SEO ID NO:3. 
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Active Mutants 
               
            
           
           
               
               
               
               
               
               
            
               
                 Corresponding 
                   
                 Corresponding 
                   
                 Corresponding 
                   
               
               
                 Position 
                 Replacement 
                 Position 
                 Replacement 
                 Position 
                 Replacement 
               
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 1 
                 A C E F G H K N P 
                 2 
                 A C G I L P Q S T 
                 3 
                 E H L Y 
               
               
                   
                 Q R S T V W 
                   
                 V 
                   
                   
               
               
                 4 
                 A I S T V 
                 5 
                 H 
                 6 
                 A H K L N Q R 
               
               
                 7 
                 M 
                 8 
                 I L M P  
                 9 
                 K L Q R S V 
               
               
                 10 
                 D E G H N Q R S W 
                 11 
                 D G H K S 
                 12 
                 A E I K L N R S T 
               
               
                 13 
                 H S T Y 
                 14 
                 D I M V 
                 15 
                 A M V 
               
               
                 20 
                 S 
                 22 
                 H M T Y 
                 23 
                 D 
               
               
                 24 
                 A E G H I K L M N 
                 26 
                 A E G H I K M P Q 
                 27 
                 A D E F H I K L P 
               
               
                   
                 R T V Y 
                   
                 R S T V W Y 
                   
                 Q R S T W 
               
               
                 28 
                 A D E F I L M N P 
                 29 
                 A E G H I K L M P 
                 30 
                 A F G H K L M P 
               
               
                   
                 R S T V W 
                   
                 R S T V W 
                   
                 Q R S T V W 
               
               
                 31 
                 A C G H I K L P R S 
                 32 
                 A C F G H K L M N 
                 33 
                 G M P Q R S T W 
               
               
                   
                 T V W Y 
                   
                 Q R S T V W Y 
                   
                   
               
               
                 34 
                 A E H K Q R W 
                 35 
                 F H L Q T V Y 
                 36 
                 A D G H K L N R 
               
               
                   
                   
                   
                   
                   
                 T 
               
               
                 37 
                 F I K M P R W V 
                 38 
                 Y 
                 39 
                 A L N Q R T Y 
               
               
                 40 
                 L W 
                 41 
                 A C D E G H N T V 
                 42 
                 A 
               
               
                   
                   
                   
                 W 
                   
                   
               
               
                 43 
                 N T 
                 44 
                 E 
                 45 
                 I K 
               
               
                 46 
                 A C E F H L M N R 
                 47 
                 A D F G H K M Q 
                 48 
                 F G H I K M N Q R 
               
               
                   
                 S T V Y 
                   
                 R S T W Y 
                   
                 S V Y 
               
               
                 49 
                 I K R S V 
                 50 
                 A C D E H L M Q R 
                 51 
                 A N R S 
               
               
                   
                   
                   
                 S V Y 
                   
                   
               
               
                 52 
                 N P Q R S T 
                 54 
                 A F N Q S V 
                 58 
                 C G H I K L N P Q 
               
               
                   
                   
                   
                   
                   
                 R S W Y 
               
               
                 59 
                 Q N 
                 60 
                 K 
                 61 
                 F I M V 
               
               
                 63 
                 A H I K L M N R S 
                 65 
                 R 
                 66 
                 H R 
               
               
                   
                 T V W 
                   
                   
                   
                   
               
               
                 67 
                 F L R V Y 
                 68 
                 E G H K L P Q R S 
                 69 
                 A C E F G I L M P 
               
               
                   
                   
                   
                 T 
                   
                 R T W Y 
               
               
                 70 
                 A C F G H K L N P 
                 71 
                 A D G H L M N Q 
                 72 
                 A D E H K L M Q 
               
               
                   
                 R S T V Y 
                   
                 R S 
                   
                 R S Y 
               
               
                 73 
                 A C D G H K L M Q 
                 74 
                 A C E F G H K L M 
                 75 
                 A C F H L M N Q 
               
               
                   
                 R S T W 
                   
                 N P R S V W 
                   
                 R S T Y 
               
               
                 77 
                 H K 
                   
                   
                   
                   
               
               
                 79 
                 L T V 
                 81 
                 P 
                 82 
                 A E G H I L M N Q 
               
               
                   
                   
                   
                   
                   
                 R S T V 
               
               
                 83 
                 F G H K L N Q R S 
                 84 
                 D E F G H I L M N 
                 85 
                 V 
               
               
                   
                 T V 
                   
                 P Q R T W Y 
                   
                   
               
               
                 86 
                 A D E F G H I K L 
                 87 
                 A C E G H I L M P 
                 89 
                 C K M P R W 
               
               
                   
                 M N P R S T V W 
                   
                 Q R S T V Y 
                   
                   
               
               
                 90 
                 A E G H I K L N Q 
                 91 
                 A Q R 
                 92 
                 C H L M T V 
               
               
                   
                 R S T W 
                   
                   
                   
                   
               
               
                 93 
                 D E F G H I L M N 
                 94 
                 A C D E F H L M N 
                 96 
                 D L V 
               
               
                   
                 P Q R S T V 
                   
                 Q R S T 
                   
                   
               
               
                 97 
                 A C D E F G I L N P 
                 98 
                 A C D E H I L M Q 
                 99 
                 A R S 
               
               
                   
                 Q R S W Y 
                   
                 R S V W 
                   
                   
               
               
                 102 
                 A C E G H K L M N 
                   
                   
                   
                   
               
               
                   
                 Q R S T W 
                   
                   
                   
                   
               
               
                 103 
                 N 
                 104 
                 A C G I K M R S T 
                 105 
                 A C G H I P Q R S 
               
               
                   
                   
                   
                   
                   
                 T W V 
               
               
                 106 
                 V 
                   
                   
                   
                   
               
               
                 107 
                 F I L 
                 108 
                 G 
                 110 
                 V 
               
               
                 114 
                 A G H M S 
                 117 
                 D 
                 118 
                 H K L M N Q V 
               
               
                 119 
                 F P Q Y 
                 120 
                 D F G H I L N P R 
                 122 
                 M 
               
               
                   
                   
                   
                 S T V W Y 
                   
                   
               
               
                 124 
                 H L R 
                 125 
                 A H R S 
                 127 
                 A E G H L M N Q 
               
               
                   
                   
                   
                   
                   
                 R S T V W 
               
               
                 128 
                 A C G I K L Q R S 
                 130 
                 I R 
                 131 
                 C E F G H I L M Q 
               
               
                   
                 W 
                   
                   
                   
                 R S T V Y 
               
               
                 132 
                 A C E F H I K L N 
                 133 
                 I 
                 134 
                 L T V 
               
               
                   
                 Q S T V Y 
                   
                   
                   
                   
               
               
                 135 
                 A C D F G H K L N 
                 136 
                 A C D F H I M N Q 
                 137 
                 A C I T A C H I L 
               
               
                   
                 Q R S W Y 
                   
                 R S T W 
                   
                 M N R S W Y 
               
               
                 139 
                 A C D E F G H K L 
                 140 
                 A C D F G H I K L 
                 141 
                 A D E F G H L M 
               
               
                   
                 M R S T V 
                   
                 M R V W Y 
                   
                 Q R S T V W Y 
               
               
                 142 
                 C D E G H I K L M 
                 143 
                 C E G I K L N V 
                 144 
                 R T W 
               
               
                   
                 N P Q R S T 
                   
                   
                   
                   
               
               
                 145 
                 A C D E G H L M N 
                 146 
                 A C E G H I K N P 
                 147 
                 A C D F G I L M P 
               
               
                   
                 P R 
                   
                 Q R S T V Y 
                   
                 Q R S V W Y 
               
               
                 148 
                 C F G H I K L Q R S 
                 149 
                 C G K L M Q R S T 
                 150 
                 A C D E F G I L N 
               
               
                   
                 T V W Y 
                   
                 V 
                   
                 P R S W Y 
               
               
                 151 
                 A C G H K L M N Q 
                 152 
                 A C F I M R T V W 
                 153 
                 I L S 
               
               
                   
                 R S T V W Y 
                   
                 Y 
                   
                   
               
               
                 154 
                 I R T V 
                 155 
                 A C D F G H K L M 
                 156 
                 A C D G I L M Q R 
               
               
                   
                   
                   
                 R S T V W 
                   
                 S T V W 
               
               
                 157 
                 W 
                 158 
                 A F G H L Q S 
                 159 
                 A D E G H L M N 
               
               
                   
                   
                   
                   
                   
                 Q R S V 
               
               
                 160 
                 C F G H I K L M N 
                 161 
                 A C D E R S V 
                 162 
                 A D E G H L M P 
               
               
                   
                 Q R S W V Y 
                   
                   
                   
                 Q R S V W Y 
               
               
                 163 
                 A E G K L Q R S T 
                 164 
                 L M V W 
                 165 
                 A C D F N R S V 
               
               
                   
                 V W 
                   
                   
                   
                 W Y 
               
               
                 166 
                 A C E F G H L N Q 
                 167 
                 A D G H K M N P 
                 168 
                 H 
               
               
                   
                 R T W Y 
                   
                 R S T Y 
                   
                   
               
               
                 169 
                 L R V 
                 170 
                 A Q N R V 
                 171 
                 I V 
               
               
                 172 
                 A C 
                 173 
                 Q N R 
                 174 
                 A G H K M N Q R 
               
               
                   
                   
                   
                   
                   
                 S T V W Y 
               
               
                 175 
                 E H T V Y 
                 176 
                 K L 
                 177 
                 V 
               
               
                 178 
                 G K M R 
                   
                   
                   
                   
               
               
                 179 
                 A C E G I K L M N 
                 180 
                 F G I K M 
                 181 
                 K M Q 
               
               
                   
                 P R S T V 
                   
                   
                   
                   
               
               
                 182 
                 L 
                 183 
                 E L 
                 184 
                 W 
               
               
                 186 
                 Y 
                   
                   
                   
                   
               
               
                 192 
                 S T 
                 193 
                 F G Q R S Y 
                 195 
                 A G H I L N Q R S 
               
               
                   
                   
                   
                   
                   
                 T W V 
               
               
                 196 
                 E G L N R S T W Y 
                 197 
                 A D E F G H K L M 
                 198 
                 A D E H L N Q R S 
               
               
                   
                   
                   
                 Q R S T W 
                   
                 T W Y 
               
               
                 200 
                 D T 
                 202 
                 M 
                 204 
                 P W 
               
               
                 205 
                 L R S T V W Y 
                 206 
                 H I K L M Q R S T 
                 208 
                 A C K L M Q R S 
               
               
                   
                   
                   
                   
                   
                 T V 
               
               
                 209 
                 A E F G L N R S T 
                 211 
                 L W 
                   
                   
               
               
                 212 
                 N S T 
                 213 
                 A E G H K L M N 
                   
                   
               
               
                   
                   
                   
                 Q R V W Y  
                   
                   
               
               
                 214 
                 Q 
                 215 
                 A D E G H K L M 
                 217 
                 M 
               
               
                   
                   
                   
                 Q R T V W Y  
                   
                   
               
               
                 218 
                 F M V 
                 219 
                 A C D E H I K L M 
                 220 
                 A D H I L M S T V 
               
               
                   
                   
                   
                 R S T W 
                   
                   
               
               
                 221 
                 A C I M Q T V 
                 222 
                 D F G I K L N R S 
                 224 
                 I 
               
               
                   
                   
                   
                 V 
                   
                   
               
               
                 226 
                 W 
                   
                   
                   
                   
               
               
                 230 
                 I 
                 231 
                 T 
                 232 
                 S 
               
               
                 233 
                 A F G K L R Y 
                 234 
                 L M 
                 235 
                 A E G H K T 
               
               
                 236 
                 A G H K R S 
                 237 
                 A C E F H L N Q R 
                 238 
                 D E H K Q R S T 
               
               
                   
                   
                   
                 S T W 
                   
                   
               
               
                 239 
                 N 
                   
                   
                   
                   
               
               
                 240 
                 K A M P Q R S V 
                 242 
                 F 
                 245 
                 H 
               
               
                 247 
                 I L M 
                 248 
                 A H W Y 
                 251 
                 L M Y 
               
               
                 253 
                 I 
                 255 
                 A G N Q R S 
                 256 
                 A H L V 
               
               
                 257 
                 A C G I K L M N Q 
                 258 
                 G H N R S 
                 259 
                 E G I K L N P Q R 
               
               
                   
                 R T V 
                   
                   
                   
                 S T V W Y 
               
               
                 260 
                 A D E G H L M Q R 
                 261 
                 A F K M N Q R T V 
                 263 
                 A H K M R T V 
               
               
                   
                 S Y 
                 W 
                   
                   
                   
               
               
                 264 
                 A H 
                 265 
                 I 
                 266 
                 Y 
               
               
                 267 
                 M T 
                 269 
                 A C D S 
                 270 
                 M N S T 
               
               
                 271 
                 F G L M S V 
                 272 
                 D M R S T 
                 273 
                 H T Y 
               
               
                 274 
                 A F S 
                   
                   
                   
                   
               
               
                 275 
                 L V 
                 276 
                 C D E G H I L M R 
                 277 
                 A C D E G H K M 
               
               
                   
                   
                   
                 S Y 
                   
                 N Q R S T Y 
               
               
                 278 
                 A E F G H I K N R 
                 279 
                 A H Q R T 
                 280 
                 GQ 
               
               
                   
                 S T V Y 
                   
                   
                   
                   
               
               
                 282 
                 D G M Q 
                 283 
                 E P R S T 
                 284 
                 A E G H L M N Q 
               
               
                   
                   
                   
                   
                   
                 S T Y 
               
               
                 285 
                 AFGHMNQY 
                 286 
                 RS W 
                 287 
                 I N T 
               
               
                 288 
                   
                 289 
                 KS 
                 290 
                 I M 
               
               
                 291 
                 C Q R S V 
                 292 
                 A C F G H K N P R 
                 293 
                 A C D F G K L M 
               
               
                   
                   
                   
                 V W 
                   
                 P Q S V Y 
               
               
                 294 
                 M 
                   
                   
                   
                   
               
               
                 297 
                 A 
                 298 
                 G I 
                 300 
                 R 
               
               
                 301 
                 A V 
                 302 
                 I W 
                 303 
                 D V 
               
               
                 304 
                 G I 
                 305 
                 D E N 
                 306 
                 D E S 
               
               
                 307 
                 G K N Q S T V W Y 
                 308 
                 D G H K N P R T 
                 309 
                 D E G H K L M N 
               
               
                   
                   
                   
                   
                   
                 Q R S T V W 
               
               
                 310 
                 A F G Q R S V Y 
                 311 
                 G H K Q S T 
                 312 
                 G K L N T 
               
               
                 313 
                 A E G H K L P R S 
                 314 
                 A D H I N Q R S T 
                 315 
                 A E G H K L M R 
               
               
                   
                 T V Y 
                   
                 Y 
                   
                 T Y 
               
               
                 316 
                 D 
                 317 
                 A D H I K M N Q R 
                 318 
                 D F G H I K M N Q 
               
               
                   
                   
                   
                 S T W 
                   
                 R S T 
               
               
                 320 
                 E G H I K L M N R 
                 321 
                 A D H K R S T Y 
                 323 
                 F I L 
               
               
                   
                 S W V Y 
                   
                   
                   
                   
               
               
                 324 
                 A D H M N R S 
                 325 
                 A D E G H K M N 
                 326 
                 C K L V Y 
               
               
                   
                   
                   
                 Q S V W 
                   
                   
               
               
                 327 
                 M 
                 328 
                 A C G H I K L Q R 
                 331 
                 C E V 
               
               
                   
                   
                   
                 S T V W Y 
                   
                   
               
               
                 334 
                 P T 
                 335 
                 S 
                 338 
                 Q 
               
               
                 339 
                 M 
                 342 
                 A 
                 343 
                 T V 
               
               
                 347 
                 A E G L M R S 
                 348 
                 D G S 
                 349 
                 A E K M N R T 
               
               
                 351 
                 A C I Q S 
                 353 
                 T V 
                 356 
                 A D H S 
               
               
                 357 
                 A C K S T 
                 358 
                 C G L T 
                 359 
                 D E H K M T V 
               
               
                 360 
                 T 
                   
                   
                   
                   
               
               
                 361 
                 H 
                 367 
                 A C G K R S 
                 368 
                 A E G H K L M R 
               
               
                   
                   
                   
                   
                   
                 S T V H R S 
               
               
                 371 
                 E F G H I K L M R 
                 373 
                 A E F K L M R S V 
                 374 
                 A H I M N P R S T 
               
               
                   
                 S V 
                   
                   
                   
                 V W Y 
               
               
                 375 
                 A G I K L M N R S 
                 376 
                 A D E L M Q R S T 
                 377 
                 D E H K P R S T 
               
               
                   
                 T 
                   
                 V Y 
                   
                   
               
               
                 378 
                 K N R 
                 379 
                 G H R S T 
                 380 
                 I L P T V W Y 
               
               
                 381 
                 E H K N Q R S V 
                 383 
                 A E H I K L M N S 
                 385 
                 A G H N Q R S T 
               
               
                   
                   
                   
                 T V 
                   
                 V 
               
               
                 387 
                 S 
                 388 
                 F H I M R T V W Y 
                 389 
                 A G H K L M P Q 
               
               
                   
                   
                   
                   
                   
                 R S T Y 
               
               
                 391 
                 C 
                 392 
                 A F G K L M Q R S 
                 393 
                 A D F H K L M N 
               
               
                   
                   
                   
                 T V W Y 
                   
                 R S T 
               
               
                 394 
                 L W 
                 395 
                 A G H K R T W 
                 396 
                 A D H L Q R S T 
               
               
                 397 
                 R 
                 398 
                 L 
                   
                   
               
               
                 399 
                 A C E K M N Q R S 
                 401 
                 A E G Q N 
                 403 
                 F 
               
               
                   
                 T V W 
                   
                   
                   
                   
               
               
                 404 
                 A P T 
                 405 
                 A F G K M P Q R S 
                 406 
                 A C E F G I N Q S 
               
               
                   
                   
                   
                 W Y 
                   
                 T V Y 
               
               
                 407 
                 A D E F G H L M N 
                 409 
                 A D E G H I P Q R 
                 410 
                 D K M N P Q R S 
               
               
                   
                 P Q R V W 
                   
                 S T V 
                   
                 T V Y 
               
               
                 411 
                 A H N P R S T V 
                 412 
                 D G H I L N Q P R 
                 413 
                 A E H K N Q R S 
               
               
                   
                   
                   
                 S V W Y 
                   
                 T 
               
               
                 414 
                 I K L M 
                 415 
                 G S W V Y 
                 416 
                 F G H I K L N Q R 
               
               
                   
                   
                   
                   
                   
                 T V Y 
               
               
                 417 
                 I 
                 418 
                 A E F G I L M N P 
                 419 
                 E F G H I K L N R 
               
               
                   
                   
                   
                 Q R S V Y 
                   
                 S W Y 
               
               
                 420 
                 I P 
                 421 
                 A E G H I K L M N 
                 422 
                 I T 
               
               
                   
                   
                   
                 Q R S T Y 
                   
                   
               
               
                 425 
                 G I K M N R S Y 
                 426 
                 E G K N P Q S Y 
                 427 
                 H I K Q S T 
               
               
                 428 
                 L M P T 
                 431 
                 A E G H I K L N Q 
                 432 
                 E G H N S V 
               
               
                   
                   
                   
                 R S V W Y 
                   
                   
               
               
                 433 
                 A C D E G H I K L 
                 434 
                 F G I M V 
                 435 
                 A C E G H R S T V 
               
               
                   
                 P R S T V W 
                   
                   
                   
                 Y 
               
               
                 436 
                 C D E G H I K L M 
                 437 
                 A D G H I K L M Q 
                 438 
                 A C D E G L N P Q 
               
               
                   
                 Q R S T W Y 
                   
                 R S Y 
                   
                 R S T V W 
               
               
                 439 
                 A C F G H K L P Q 
                 440 
                 A D E F G H I L M 
                 441 
                 A D F G H K L N 
               
               
                   
                 S T V W 
                   
                 P R S V Y 
                   
                 Q S T V Y 
               
               
                 442 
                 C G H K L P Q R T 
                 443 
                 A E F G H L M N Q 
                 444 
                 D E F G H I K M N 
               
               
                   
                 V W Y 
                   
                 R S T W 
                   
                 R V W Y 
               
               
                 445 
                 A G H L M N P Q R 
                 446 
                 A C D E G H I K L 
                 447 
                 D E F G I L M N P 
               
               
                   
                 S T V W Y 
                   
                 M Q R T V W 
                   
                 Q R T V W 
               
               
                   
               
            
           
         
       
     
     In particular examples, provided herein is a modified PH20 polypeptide containing an amino acid replacement or replacements at a position or positions corresponding to 1, 6, 8, 9, 10, 11, 12, 14, 15, 20, 22, 24, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 46, 47, 48, 49, 50, 52, 58, 59, 63, 67, 68, 69, 70, 71, 72, 73, 74, 75, 79, 82, 83, 84, 86, 87, 89, 90, 92, 93, 94, 97, 102, 104, 107, 114, 118, 120, 127, 128, 130, 131, 132, 135, 138, 139, 140, 141, 142, 143, 144, 146, 147, 148, 149, 150, 151, 152, 155, 156, 158, 160, 162, 163, 164, 165, 166, 167, 169, 170, 172, 173, 174, 175, 178, 179, 193, 195, 196, 198, 204, 205, 206, 209, 212, 213, 215, 219, 220, 221, 222, 232, 233, 234, 235, 236, 237, 238, 240, 247, 248, 249, 257, 258, 259, 260, 261, 263, 267, 269, 271, 272, 273, 274, 276, 277, 278, 279, 282, 283, 285, 287, 289, 291, 292, 293, 298, 305, 307, 308, 309, 310, 313, 314, 315, 317, 318, 320, 321, 324, 325, 326, 328, 335, 347, 349, 351, 353, 356, 359, 367, 368, 369, 371, 373, 374, 375, 376, 377, 380, 381, 383, 385, 389, 392, 393, 395, 396, 399, 401, 404, 405, 406, 407, 409, 410, 412, 416, 418, 419, 421, 425, 427, 428, 431, 433, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446 or 447 with reference to amino acid positions set forth in SEQ ID NO:3. For example, the amino acid positions can be replacements at positions corresponding to replacement of Leucine (L) at position 1 (L1), P6, V8, I9, P10, N11, V12, F14, L15, A20, S22, F24, L26, G27, K28, F29, D30, E31, P32, L33, D34, M35, S36, L37, F38, S39, F40, I41, I46, N47, A48, T49, G50, G52, V58, D59, Y63, I67, D68, S69, I70, T71, G72, V73, T74, V75, I79, K82, I83, S84, G86, D87, L89, D90, A92, K93, K94, T97, V102, N104, M107, E114, T118, A120, D127, V128, K130, N131, R132, E135, Q138, Q139, Q140, N141, V142, Q143, L144, L146, T147, E148, A149, T150, E151, K152, Q155, E156, E158, A160, K162, D163, F164, L165, V166, E167, 1169, K170, G172, K173, L174, L175, N178, H179, H193, K195, K196, G198, F204, N205, V206, K209, D212, D213, S215, N219, E220, S221, T222, T232, Q233, Q234, S235, P236, V237, A238, T240, V247, R248, E249, P257, D258, A259, K260, S261, L263, A267, T269, 1271, V272, F273, T274, Q276, V277, L278, K279, S282, Q283, E285, V287, T289, G291, E292, T293, A298, G305, L307, S308, I309, M310, M313, K314, S315, L317, L318, D320, N321, E324, T325, I326, N328, T335, Q347, Q349, V351, I353, N356, S359, P367, D368, N369, A371, Q373, L374, E375, K376, G377, F380, T381, R383, K385, E389, E392, Q393, S395, E396, Y399, S401, S404, T405, L406, S407, K409, E410, A412, D416, D418, A419, D421, A425, G427, A428, D431, F433, P436, P437, M438, E439, T440, E441, E442, P443, Q444, I445, F446 or Y447 with reference to amino acid positions set forth in SEQ ID NO:3. 
     Exemplary amino acid replacements in the modified PH20 polypeptides provided herein include, but are not limited, replacement with: histidine (H) at a position corresponding to position 1; A at a position corresponding to position 1; E at a position corresponding to position 1; G at a position corresponding to position 1; K at a position corresponding to position 1; Q at a position corresponding to position 1; Rat a position corresponding to position 1; A at a position corresponding to position 6; M at a position corresponding to position 8; Q at a position corresponding to position 9; G at a position corresponding to position 10; H at a position corresponding to position 10; S at a position corresponding to position 11; E at a position corresponding to position 12; I at a position corresponding to position 12; K at a position corresponding to position 12; T at a position corresponding to position 12; V at a position corresponding to position 14; V at a position corresponding to position 15; M at a position corresponding to position 15; S at a position corresponding to position 20; T at a position corresponding to position 22; E at a position corresponding to position 24; H at a position corresponding to position 24; R at a position corresponding to position 24; A at a position corresponding to position 26; E at a position corresponding to position 26; K at a position corresponding to position 26; M at a position corresponding to position 26; Q at a position corresponding to position 26; R at a position corresponding to position 26; D at a position corresponding to position 27; K at a position corresponding to position 27; R at a position corresponding to position 27; R at a position corresponding to position 28; E at a position corresponding to position 29; I at a position corresponding to position 29; K at a position corresponding to position 29; L at a position corresponding to position 29; M at a position corresponding to position 29; P at a position corresponding to position 29; R at a position corresponding to position 29; S at a position corresponding to position 29; T at a position corresponding to position 29; V at a position corresponding to position 29; G at a position corresponding to position 30; H at a position corresponding to position 30; K at a position corresponding to position 30; L at a position corresponding to position 30; M at a position corresponding to position 30; Rat a position corresponding to position 30; S at a position corresponding to position 30; A at a position corresponding to position 31; C at a position corresponding to position 31; G at a position corresponding to position 31; H at a position corresponding to position 31; I at a position corresponding to position 31; K at a position corresponding to position 31; L at a position corresponding to position 31; P at a position corresponding to position 31; R at a position corresponding to position 31; S at a position corresponding to position 31; T at a position corresponding to position 31; V at a position corresponding to position 31; W at a position corresponding to position 31; C at a position corresponding to position 32; F at a position corresponding to position 32; G at a position corresponding to position 32; H at a position corresponding to position 32; W at a position corresponding to position 33; G at a position corresponding to position 33; W at a position corresponding to position 34; Q at a position corresponding to position 35; V at a position corresponding to position 35; H at a position corresponding to position 36; N at a position corresponding to position 36; F at a position corresponding to position 37; M at a position corresponding to position 37; Y at a position corresponding to position 38; A at a position corresponding to position 39; L at a position corresponding to position 39; N at a position corresponding to position 39; T at a position corresponding to position 39; L at a position corresponding to position 40; T at a position corresponding to position 41; L at a position corresponding to position 46; Rat a position corresponding to position 46; D at a position corresponding to position 47; F at a position corresponding to position 47; T at a position corresponding to position 47; W at a position corresponding to position 47, with F at a position corresponding to position 48; H at a position corresponding to position 48; K at a position corresponding to position 48; N at a position corresponding to position 48; R at a position corresponding to position 49; D at a position corresponding to position 50; S at a position corresponding to position 50; M at a position corresponding to position 50; N at a position corresponding to position 52; Q at a position corresponding to position 52; R at a position corresponding to position 52; S at a position corresponding to position 52; T at a position corresponding to position 52; C at a position corresponding to position 58; K at a position corresponding to position 58; L at a position corresponding to position 58; P at a position corresponding to position 58; Q at a position corresponding to position 58; Rat a position corresponding to position 58; H at a position corresponding to position 58; N at a position corresponding to position 58; Y at a position corresponding to position 58; N at a position corresponding to position 59; K at a position corresponding to position 63; L at a position corresponding to position 63; M at a position corresponding to position 63; Rat a position corresponding to position 63; W at a position corresponding to position 63; V at a position corresponding to position 67; H at a position corresponding to position 68; P at a position corresponding to position 68; Q at a position corresponding to position 68; A at a position corresponding to position 69; C at a position corresponding to position 69; E at a position corresponding to position 69; F at a position corresponding to position 69; G at a position corresponding to position 69; I at a position corresponding to position 69; L at a position corresponding to position 69; M at a position corresponding to position 69; P at a position corresponding to position 69; R at a position corresponding to position 69; T at a position corresponding to position 69; W at a position corresponding to position 69; Y at a position corresponding to position 69; A at a position corresponding to position 70; C at a position corresponding to position 70; F at a position corresponding to position 70; G at a position corresponding to position 70; H at a position corresponding to position 70; K at a position corresponding to position 70; L at a position corresponding to position 70; N at a position corresponding to position 70; P at a position corresponding to position 70; R at a position corresponding to position 70; S at a position corresponding to position 70; T at a position corresponding to position 70; V at a position corresponding to position 70; Y at a position corresponding to position 70; G at a position corresponding to position 71; N at a position corresponding to position 71; R at a position corresponding to position 71; S at a position corresponding to position 71; K at a position corresponding to position 72; M at a position corresponding to position 72; Q at a position corresponding to position 72; A at a position corresponding to position 73; H at a position corresponding to position 73; K at a position corresponding to position 73; L at a position corresponding to position 73; Q at a position corresponding to position 73; Rat a position corresponding to position 73; Tat a position corresponding to position 73; W at a position corresponding to position 73; A at a position corresponding to position 74; C at a position corresponding to position 74; E at a position corresponding to position 74; F at a position corresponding to position 74; G at a position corresponding to position 74; H at a position corresponding to position 74; K at a position corresponding to position 74; L at a position corresponding to position 74; M at a position corresponding to position 74; N at a position corresponding to position 74; P at a position corresponding to position 74; R at a position corresponding to position 74; S at a position corresponding to position 74; V at a position corresponding to position 74; W at a position corresponding to position 74; F at a position corresponding to position 75; L at a position corresponding to position 75; M at position corresponding to position 75; Rat a position corresponding to position 75; T at a position corresponding to position 75; L at a position corresponding to position 79; L at a position corresponding to position 82; N at a position corresponding to position 82; V at a position corresponding to position 83; Q at a position corresponding to position 83; S at a position corresponding to position 83; G at a position corresponding to position 83; E at a position corresponding to position 84; F at a position corresponding to position 84; G at a position corresponding to position 84; N at a position corresponding to position 84; R at a position corresponding to position 84; A at a position corresponding to position 86; H at a position corresponding to position 86; K at a position corresponding to position 86; N at a position corresponding to position 86; S at a position corresponding to position 86; T at a position corresponding to position 86; W at a position corresponding to position 86; C at a position corresponding to position 87; G at a position corresponding to position 87; L at a position corresponding to position 87; M at a position corresponding to position 87; R at a position corresponding to position 87; S at a position corresponding to position 87; T at a position corresponding to position 87; V at a position corresponding to position 87; Y at a position corresponding to position 87; C at a position corresponding to position 89; A at a position corresponding to position 90; E at a position corresponding to position 90; H at a position corresponding to position 90; K at a position corresponding to position 90; N at a position corresponding to position 90; R at a position corresponding to position 90; C at a position corresponding to position 92; L at a position corresponding to position 92; I at a position corresponding to position 93; L at a position corresponding to position 93; Q at a position corresponding to position 93; Rat a position corresponding to position 93; S at a position corresponding to position 93; T at a position corresponding to position 93; D at a position corresponding to position 94; Q at a position corresponding to position 94; R at a position corresponding to position 94; A at a position corresponding to position 97; C at an amino acid residue corresponding to position 97; D at a position corresponding to position 97; E at a position corresponding to position 97; G at a position corresponding to position 97; L at a position corresponding to position 97; S at a position corresponding to position 97; S at a position corresponding to position 102; T at a position corresponding to position 102; Rat a position corresponding to position 104; L at a position corresponding to position 107; A at a position corresponding to position 114; Q at a position corresponding to position 118; H at a position corresponding to position 120; F at a position corresponding to position 120; I at a position corresponding to position 120; S at a position corresponding to position 120; V at a position corresponding to position 120; Y at a position corresponding to position 120; E at a position corresponding to position 127; H at a position corresponding to position 127; N at a position corresponding to position 127; Q at a position corresponding to position 127; R at a position corresponding to position 127; I at a position corresponding to position 128; R at a position corresponding to position 130; G at a position corresponding to position 131; I at a position corresponding to position 131; M at a position corresponding to position 131; Q at a position corresponding to position 131; Rat a position corresponding to position 131; V at a position corresponding to position 131; N at a position corresponding to position 132; L at a position corresponding to position 132; D at a position corresponding to position 135; G at a position corresponding to position 135; Rat a position corresponding to position 135, with L at a position corresponding to position 138; T at a position corresponding to position 139; K at a position corresponding to position 140; H at a position corresponding to position 141; Rat a position corresponding to position 141; S at a position corresponding to position 141; W at a position corresponding to position 141; Y at a position corresponding to position 141; D at a position corresponding to position 142; G at a position corresponding to position 142; K at a position corresponding to position 142; N at a position corresponding to position 142; P at a position corresponding to position 142; Q at a position corresponding to position 142; R at a position corresponding to position 142; S at a position corresponding to position 142; T at a position corresponding to position 142; G at a position corresponding to position 143; K at a position corresponding to position 143; Rat a position corresponding to position 144; T at a position corresponding to position 144; P at a position corresponding to position 146; R at a position corresponding to position 146; A at a position corresponding to position 147; F at a position corresponding to position 147; L at a position corresponding to position 147; R at a position corresponding to position 147; S at a position corresponding to position 147; V at a position corresponding to position 147; H at a position corresponding to position 148; K at a position corresponding to position 148; Q at a position corresponding to position 148; T at a position corresponding to position 149; V at a position corresponding to position 149; A at a position corresponding to position 150; D at a position corresponding to position 150; G at a position corresponding to position 150; N at a position corresponding to position 150; S at a position corresponding to position 150; W at a position corresponding to position 150; Y at a position corresponding to position 150; A at a position corresponding to position 151; H at a position corresponding to position 151; K at a position corresponding to position 151; L at a position corresponding to position 151; M at a position corresponding to position 151; Q at a position corresponding to position 151; R at a position corresponding to position 151; S at a position corresponding to position 151; T at a position corresponding to position 151; V at a position corresponding to position 151; W at a position corresponding to position 151; Y at a position corresponding to position 151; Rat a position corresponding to position 152; Tat a position corresponding to position 152; W at a position corresponding to position 152; D at a position corresponding to position 155; G at a position corresponding to position 155; K at a position corresponding to position 155; Rat a position corresponding to position 155; D at a position corresponding to position 156; Q at a position corresponding to position 158; S at a position corresponding to position 158; S at a position corresponding to position 160; E at a position corresponding to position 162; A at a position corresponding to position 163; E at a position corresponding to position 163; K at a position corresponding to position 163; Q at a position corresponding to position 163; Rat a position corresponding to position 163; S at a position corresponding to position 163; M at a position corresponding to position 164; V at a position corresponding to position 164; D at a position corresponding to position 165; F at a position corresponding to position 165; N at a position corresponding to position 165; S at a position corresponding to position 165; V at a position corresponding to position 165; A at a position corresponding to position 166; E at a position corresponding to position 166; F at a position corresponding to position 166; H at a position corresponding to position 166; L at a position corresponding to position 166; Q at a position corresponding to position 166; Rat a position corresponding to position 166; T at a position corresponding to position 166; W at a position corresponding to position 166; Y at a position corresponding to position 166; D at a position corresponding to position 167; L at a position corresponding to position 169; Rat a position corresponding to position 170; A at a position corresponding to position 172; Rat a position corresponding to position 173; G at a position corresponding to position 174; K at a position corresponding to position 174; N at a position corresponding to position 174; Rat a position corresponding to position 174; T at a position corresponding to position 174; T at a position corresponding to position 175; K at a position corresponding to position 178; Rat a position corresponding to position 178; K at a position corresponding to position 179; Q at a position corresponding to position 193; T at a position corresponding to position 195; N at a position corresponding to position 195; with E at a position corresponding to position 196; R at a position corresponding to position 196; with D at a position corresponding to position 198; P at a position corresponding to position 204; A at a position corresponding to position 205; E at a position corresponding to position 205; L at a position corresponding to position 205; T at a position corresponding to position 205; I at a position corresponding to position 206; K at a position corresponding to position 206; L at a position corresponding to position 206; R at a position corresponding to position 206; R at a position corresponding to position 209; N at a position corresponding to position 212; S at a position corresponding to position 212; A at a position corresponding to position 213; M at a position corresponding to position 213; N at a position corresponding to position 213; H at a position corresponding to position 215; M at a position corresponding to position 215; A at a position corresponding to position 219; I at a position corresponding to position 219; K at a position corresponding to position 219; S at a position corresponding to position 219; H at a position corresponding to position 220; I at a position corresponding to position 220; L at a position corresponding to position 220; V at a position corresponding to position 220; Q at a position corresponding to position 221; G at a position corresponding to position 222; F at a position corresponding to position 232; G at a position corresponding to position 233; K at a position corresponding to position 233; Rat a position corresponding to position 233; M at a position corresponding to position 234; A at a position corresponding to position 235; R at a position corresponding to position 236; C at a position corresponding to position 237; E at a position corresponding to position 237; H at a position corresponding to position 237; Q at a position corresponding to position 237; T at a position corresponding to position 237; E at a position corresponding to position 238; H at a position corresponding to amino acid position 238; S at a position corresponding to position 238; A at a position corresponding to position 240; Q at a position corresponding to position 240; I at a position corresponding to position 247; A at a position corresponding to position 248; V at a position corresponding to position 249; G at a position corresponding to position 257; Tat a position corresponding to position 257; Rat a position corresponding to position 257; N at a position corresponding to position 258; S at a position corresponding to position 258; P at a position corresponding to position 259; M at a position corresponding to position 260; Y at a position corresponding to position 260; A at a position corresponding to position 261; K at a position corresponding to position 261; N at a position corresponding to position 261; K at a position corresponding to position 263; Rat a position corresponding to position 263; T at a position corresponding to position 267; A at a position corresponding to position 269; L at a position corresponding to position 271; M at a position corresponding to position 271; D at a position corresponding to position 272; Tat a position corresponding to position 272; H at a position corresponding to position 273; Y at a position corresponding to position 273; F at a position corresponding to position 274; D at a position corresponding to position 276; H at a position corresponding to position 276; M at a position corresponding to position 276; R at a position corresponding to position 276; S at a position corresponding to position 276; Y at a position corresponding to position 276; A at a position corresponding to position 277; E at a position corresponding to position 277; H at a position corresponding to position 277; K at a position corresponding to position 277; M at a position corresponding to position 277; N at a position corresponding to position 277; Q at a position corresponding to position 277; R at a position corresponding to position 277; S at a position corresponding to position 277; T at a position corresponding to position 277; E at a position corresponding to position 278; F at a position corresponding to position 278; G at a position corresponding to position 278; H at a position corresponding to position 278; K at a position corresponding to position 278; N at a position corresponding to position 278; R at a position corresponding to position 278; S at a position corresponding to position 278; T at a position corresponding to position 278; Y at a position corresponding to position 278; H at a position corresponding to position 279; M at a position corresponding to position 282; S at a position corresponding to position 283; H at a position corresponding to position 285; T at a position corresponding to position 287; S at a position corresponding to position 289; S at a position corresponding to position 291; V at a position corresponding to position 291; C at a position corresponding to position 292; F at a position corresponding to position 292; H at a position corresponding to position 292; K at a position corresponding to position 292; R at a position corresponding to position 292; V at a position corresponding to position 292; A at a position corresponding to position 293; C at a position corresponding to position 293; D at a position corresponding to position 293; F at a position corresponding to position 293; K at a position corresponding to position 293; M at a position corresponding to position 293; P at a position corresponding to position 293; Q at a position corresponding to position 293; V at a position corresponding to position 293; Y at a position corresponding to position 293; G at a position corresponding to position 298; E at a position corresponding to position 305; G at a position corresponding to position 307; D at a position corresponding to position 308; G at a position corresponding to position 308; K at a position corresponding to position 308; N at a position corresponding to position 308; Rat a position corresponding to position 308; E at a position corresponding to position 309; G at a position corresponding to position 309; H at a position corresponding to position 309; L at a position corresponding to position 309; M at a position corresponding to position 309; N at a position corresponding to position 309; Q at a position corresponding to position 309; Rat a position corresponding to position 309; S at a position corresponding to position 309; T at a position corresponding to position 309; V at a position corresponding to position 309; A at a position corresponding to position 310; G at a position corresponding to position 310; Q at a position corresponding to position 310; S at a position corresponding to position 310; A at a position corresponding to position 313; G at a position corresponding to position 313; H at a position corresponding to position 313; K at a position corresponding to position 313; P at a position corresponding to position 313; Rat a position corresponding to position 313; Tat a position corresponding to position 313; Y at a position corresponding to position 313; with S at a position corresponding to position 314; Y at a position corresponding to position 314; A at a position corresponding to position 315; H at a position corresponding to position 315; Y at a position corresponding to position 315; A at a position corresponding to position 317; I at a position corresponding to position 317; K at a position corresponding to position 317; N at a position corresponding to position 317; Q at a position corresponding to position 317; Rat a position corresponding to position 317; S at a position corresponding to position 317; T at a position corresponding to position 317; W at a position corresponding to position 317; D at a position corresponding to position 318; H at a position corresponding to position 318; K at a position corresponding to position 318; M at a position corresponding to position 318; R at a position corresponding to position 318; H at a position corresponding to position 320; K at a position corresponding to position 320; R at a position corresponding to position 320; Rat a position corresponding to position 321; S at a position corresponding to position 321; N at a position corresponding to position 324; Rat a position corresponding to position 324; A at a position corresponding to position 325; D at a position corresponding to position 325; E at a position corresponding to position 325; G at a position corresponding to position 325; H at a position corresponding to position 325; K at a position corresponding to position 325; M at a position corresponding to position 325; N at a position corresponding to position 325; Q at a position corresponding to position 325; S at a position corresponding to position 325; V at a position corresponding to position 325; L at a position corresponding to position 326; V at a position corresponding to position 326; C at a position corresponding to position 328; G at a position corresponding to position 328; I at a position corresponding to position 328; K at a position corresponding to position 328; L at a position corresponding to position 328; S at a position corresponding to position 328; Y at a position corresponding to position 328; S at a position corresponding to position 335; A at a position corresponding to position 347; G at a position corresponding to position 347; S at a position corresponding to position 347; M at a position corresponding to position 349; R at a position corresponding to position 349; S at a position corresponding to position 351; V at a position corresponding to position 353; with H at a position corresponding to position 356; S at a position corresponding to position 356; E at a position corresponding to position 359; H at a position corresponding to position 359; T at a position corresponding to position 359; A at a position corresponding to position 367; G at a position corresponding to position 367; K at a position corresponding to position 367; S at a position corresponding to position 367; A at a position corresponding to position 368; E at a position corresponding to position 368; K at a position corresponding to position 368; L at a position corresponding to amino acid position 368; M at a position corresponding to amino acid position 368; R at a position corresponding to position 368; Tat a position corresponding to amino acid position 368; H at a position corresponding to position 369; R at a position corresponding to position 369; F at a position corresponding to position 371; H at a position corresponding to position 371; K at a position corresponding to position 371; L at a position corresponding to position 371; Rat a position corresponding to position 371; S at a position corresponding to position 371; M at a position corresponding to position 373; H at a position corresponding to position 374; P at a position corresponding to position 374; A at a position corresponding to position 375; G at a position corresponding to position 375; K at a position corresponding to position 375; Rat a position corresponding to position 375; D at a position corresponding to position 376; E at a position corresponding to position 376; Q at a position corresponding to position 376; R at a position corresponding to position 376; T at a position corresponding to position 376; V at a position corresponding to position 376; Y at a position corresponding to position 376; D at a position corresponding to position 377; E at a position corresponding to position 377; H at a position corresponding to position 377; K at a position corresponding to position 377; P at a position corresponding to position 377; R at a position corresponding to position 377; S at a position corresponding to position 377; T at a position corresponding to position 377; W at a position corresponding to position 380; Y at a position corresponding to position 380; S at a position corresponding to position 381; I at a position corresponding to position 383; K at a position corresponding to position 383; L at a position corresponding to position 383; S at a position corresponding to position 383; A at a position corresponding to position 385; Q at a position corresponding to position 385; V at a position corresponding to position 385; A at a position corresponding to position 389; G at a position corresponding to position 389; L at a position corresponding to position 389; K at a position corresponding to position 389; Q at a position corresponding to position 389; S at a position corresponding to position 389; A at a position corresponding to position 392; F at a position corresponding to position 392; M at a position corresponding to position 392; Q at a position corresponding to position 392; Rat a position corresponding to position 392; V at a position corresponding to position 392; F at a position corresponding to position 393; M at a position corresponding to position 393; A at a position corresponding to position 395; H at a position corresponding to position 395; Rat a position corresponding to position 395; A at a position corresponding to position 396; H at a position corresponding to position 396; Q at a position corresponding to position 396; S at a position corresponding to position 396; K at a position corresponding to position 399; M at a position corresponding to position 399; T at a position corresponding to position 399; V at a position corresponding to position 399; W at a position corresponding to position 399; A at a position corresponding to position 401; E at a position corresponding to position 401; A at a position corresponding to position 404; G at a position corresponding to position 405; F at a position corresponding to position 406; N at a position corresponding to position 406; A at a position corresponding to position 407; D at a position corresponding to position 407; E at a position corresponding to position 407; F at a position corresponding to position 407; H at a position corresponding to position 407; Q at a position corresponding to position 407; P at a position corresponding to position 407; A at a position corresponding to position 409; Q at a position corresponding to position 409; Tat a position corresponding to position 410; Q at a position corresponding to position 412; Rat a position corresponding to position 412; V at a position corresponding to position 412; L at a position corresponding to position 416; E at a position corresponding to position 418; L at a position corresponding to position 418; P at a position corresponding to position 418; Rat a position corresponding to position 418; V at a position corresponding to position 418; F at a position corresponding to position 419; H at a position corresponding to position 419; I at a position corresponding to position 419; K at a position corresponding to position 419; Rat a position corresponding to position 419; S at a position corresponding to position 419; Y at a position corresponding to position 419; A at a position corresponding to position 421; H at a position corresponding to position 421; K at a position corresponding to position 421; N at a position corresponding to position 421; Q at a position corresponding to position 421; Rat a position corresponding to position 421; S at a position corresponding to position 421; G at a position corresponding to position 425; K at a position corresponding to position 425; Q at a position corresponding to position 427; T at a position corresponding to position 427; L at a position corresponding to position 428; A at a position corresponding to position 431; G at a position corresponding to position 431; E at a position corresponding to position 431; H at a position corresponding to position 431; K at a position corresponding to position 431; L at a position corresponding to position 431; N at a position corresponding to position 431; Q at a position corresponding to position 431; Rat a position corresponding to position 431; S at a position corresponding to position 431; V at a position corresponding to position 431; A at a position corresponding to position 433; H at a position corresponding to position 433; I at a position corresponding to position 433; K at a position corresponding to position 433; L at a position corresponding to position 433; Rat a position corresponding to position 433; T at a position corresponding to position 433; V at a position corresponding to position 433; W at a position corresponding to position 433; K at a position corresponding to position 436; I at a position corresponding to position 437; M at a position corresponding to position 437; A at a position corresponding to position 438; D at a position corresponding to position 438; E at a position corresponding to position 438; L at a position corresponding to position 438; N at a position corresponding to position 438; T at a position corresponding to position 438; A at a position corresponding to position 439; C at a position corresponding to position 439; K at a position corresponding to position 439; P at a position corresponding to position 439; Q at a position corresponding to position 439; T at a position corresponding to position 439; V at a position corresponding to position 439; D at a position corresponding to position 440; H at a position corresponding to position 440; M at a position corresponding to position 440; P at a position corresponding to position 440; R at a position corresponding to position 440; S at a position corresponding to position 440; A at a position corresponding to position 441; F at a position corresponding to position 441; C at a position corresponding to position 442; G at a position corresponding to position 442; R at a position corresponding to position 442; A at a position corresponding to position 443; E at a position corresponding to position 443; F at a position corresponding to position 443; G at a position corresponding to position 443; M at a position corresponding to position 443; N at a position corresponding to position 443; E at a position corresponding to position 444; H at a position corresponding to position 444; V at a position corresponding to position 444; H at a position corresponding to position 445; M at a position corresponding to position 445; N at a position corresponding to position 445; P at a position corresponding to position 445; Q at a position corresponding to position 445; S at a position corresponding to position 445; T at a position corresponding to position 445; V at a position corresponding to position 445; W at a position corresponding to position 445; A at a position corresponding to position 446; M at a position corresponding to position 446; W at a position corresponding to position 446; D at a position corresponding to position 447; E at a position corresponding to position 447; G at a position corresponding to position 447; I at a position corresponding to position 447; N at a position corresponding to position 447; P at a position corresponding to position 447; Q at a position corresponding to position 447; T at a position corresponding to position 447, and/or replacement with V at a position corresponding to position 447, each with reference to amino acid positions set forth in SEQ ID NO:3. 
     Exemplary of such modified PH20 polypeptides are any having the sequence of amino acids set forth in any of SEQ ID NOs: 74-855, or having a sequence of amino acids that exhibits at least 68%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NOs:74-855 and contains the amino acid replacement and exhibits hyaluronidase activity. 
     Any of the above modified PH20 polypeptides provided herein can exhibit altered, such as improved or increased, properties or activities compared to the corresponding PH20 polypeptide not containing the amino acid modification (e.g., amino acid replacement). For example, the altered activities or properties can be an increased catalytic activity and/or an increased stability under denaturing conditions. 
     a. In Creased Activity 
     Provided herein are modified or variant PH20 polypeptides that contain one or more amino acid replacements in a PH20 polypeptide and that exhibit increased hyaluronidase activity compared to the corresponding PH20 polypeptide not containing the amino acid replacement(s), for example, the PH20 polypeptide set forth in any of SEQ ID NOs: 2, 3, 6-66, 68-72, 856-861, 869 or 870 or a variant thereof having at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 86%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto. In particular, the modified or variant PH20 polypeptides provided herein exhibit increased hyaluronidase activity compared to the corresponding PH20 polypeptide not containing the amino acid replacement, for example, the PH20 polypeptide set forth in any of SEQ ID NOs: 3, 7, 32-66, 69 or 72 and in particular the PH20 polypeptide set forth in SEQ ID NO:3. 
     The modified PH20 polypeptide can exhibit hyaluronidase activity that is at least or about at least or 120%, 130%, 135%, 140%, 145%, 150%, 160%, 170%, 180%, 200%, 250%, 300%, 350%, 400%, 500%, 1500%, 2000%, 3000%, 4000%, 5000% of the hyaluronidase activity of the corresponding PH20 polypeptide not containing the amino acid replacement(s), for example the PH20 polypeptide set forth in any of any of SEQ ID NOs: 2, 3, 6-66, 68-72, 856-861, 869 or 870 or a variant thereof, under the same conditions. For example, the hyaluronidase activity is increased at least or about at least 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 200-fold, 300-fold, 400-fold or more. 
     In particular examples, the modified PH20 polypeptides contain an amino acid replacement at one or more amino acid positions identified as being associated with increased hyaluronidase activity. As described herein, such positions have been identified using mutagenesis and selection or screening methods to identify those positions that result in increased hyaluronidase activity. The PH20 polypeptide also can contain other modifications, such as other amino acid replacements, that alone are not associated with increased activity so long as the resulting modified PH20 polypeptide exhibits increased hyaluronidase activity compared to the PH20 not containing the amino acid modification(s), such as amino acid replacement(s). The modified PH20 polypeptide provided herein can contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, or more amino acid replacements. Additional modifications, such as insertions or deletions, also can be included. The amino acid replacement can be in a PH20 polypeptide as set forth in any of SEQ ID NOs: 2,3, 6-66, 68-72, 856-861, 869 or 870 or a variant thereof having at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 86%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto. For example, the replacement(s) can be in a human PH20 polypeptide, for example, any set forth in any of SEQ ID NOs: 3, 7, 32-66, 69 or 72 or a variant thereof. 
     For example, the modified PH20 polypeptides provided herein contain an amino acid replacement (substitution) at one or more amino acid positions corresponding to positions 1, 12, 15, 24, 26, 27, 29, 30, 31, 32, 33, 37, 39, 46, 48, 52, 58, 63, 67, 68, 69, 70, 71, 72, 73, 74, 75, 84, 86, 87, 92, 93, 94, 97, 118, 120, 127, 131, 135, 141, 142, 147, 148, 150, 151, 152, 155, 156, 163, 164, 165, 166, 169, 170, 174, 198, 206, 209, 212, 213, 215, 219, 233, 234, 236, 238, 247, 257, 259, 260, 261, 263, 269, 271, 272, 276, 277, 278, 282, 291, 293, 305, 308, 309, 310, 313, 315, 317, 318, 320, 324, 325, 326, 328, 347, 353, 359, 371, 377, 380, 389, 392, 395, 399, 405, 407, 409, 410, 418, 419, 421, 425, 431, 433, 436, 437, 438, 439, 440, 441, 442, 443, 445, 446 or 447 with reference to amino acid positions set forth in SEQ ID NO:3. For example, the amino acid positions can be replacements at positions corresponding to replacement of Leucine (L) at position 1 (L1), V12, L15, F24, L26, G27, F29, D30, E31, P32, L33, L37, S39, I46, A48, G52, V58, Y63, I67, D68, S69, I70, T71, G72, V73, T74, V75, S84, G86, D87, A92, K93, K94, T97, T118, A120, D127, N131, E135, N141, V142, T147, E148, T150, E151, K152, Q155, E156, D163, F164, L165, V166, 1169, K170, L174, G198, V206, K209, D212, D213, S215, N219, Q233, Q234, P236, A238, V247, P257, A259, K260, S261, L263, T269, 1271, V272, Q276, V277, L278, S282, G291, T293, G305, S308, 1309, M310, M313, S315, L317, L318, D320, E324, T325, 1326, N328, Q347, 1353, S359, A371, G377, F380, E389, E392, S395, Y399, T405, S407, K409, E410, D418, A419, D421, A425, D431, F433, P436, P437, M438, E439, T440, E441, E442, P443, 1445, F446 or Y447 with reference to amino acid positions set forth in SEQ ID NO:3. Exemplary of such modified PH20 polypeptides are polypeptides that exhibit at least 1.5-fold or more the activity of the corresponding PH20 polypeptide not containing the amino acid replacement. 
     Exemplary of amino acid replacements in the modified PH20 polypeptides provided herein include, but are not limited, replacement: with histidine (H) at a position corresponding to position 1; Q at a position corresponding to position 1; E at a position corresponding to position 12; T at a position corresponding to position 12; V at a position corresponding to position 15; E at a position corresponding to position 24; H at a position corresponding to position 24; E at a position corresponding to position 26; K at a position corresponding to position 26; K at a position corresponding to position 27; R at a position corresponding to position 27; E at a position corresponding to position 29; I at a position corresponding to position 29; L at a position corresponding to position 29; M at a position corresponding to position 29; P at a position corresponding to position 29; S at a position corresponding to position 29; V at a position corresponding to position 29; G at a position corresponding to position 30; H at a position corresponding to position 30; K at a position corresponding to position 30; M at a position corresponding to position 30; Rat a position corresponding to position 30; S at a position corresponding to position 30; A at a position corresponding to position 31; C at a position corresponding to position 31; H at a position corresponding to position 31; I at a position corresponding to position 31; K at a position corresponding to position 31; L at a position corresponding to position 31; P at a position corresponding to position 31; R at a position corresponding to position 31; S at a position corresponding to position 31; T at a position corresponding to position 31; V at a position corresponding to position 31; F at a position corresponding to position 32; G at a position corresponding to position 32; H at a position corresponding to position 32; W at a position corresponding to position 33; F at a position corresponding to position 37; N at a position corresponding to position 39; T at a position corresponding to position 39; R at a position corresponding to position 46; F at a position corresponding to position 48; H at a position corresponding to position 48; N at a position corresponding to position 48; Q at a position corresponding to position 52; K at a position corresponding to position 58; Q at a position corresponding to position 58; W at a position corresponding to position 63; V at a position corresponding to position 67; H at a position corresponding to position 68; Q at a position corresponding to position 68; A at a position corresponding to position 69; C at a position corresponding to position 69; F at a position corresponding to position 69; G at a position corresponding to position 69; I at a position corresponding to position 69; L at a position corresponding to position 69; M at a position corresponding to position 69; P at a position corresponding to position 69; R at a position corresponding to position 69; W at a position corresponding to position 69; Y at a position corresponding to position 69; A at a position corresponding to position 70; C at a position corresponding to position 70; F at a position corresponding to position 70; G at a position corresponding to position 70; H at a position corresponding to position 70; K at a position corresponding to position 70; L at a position corresponding to position 70; N at a position corresponding to position 70; P at a position corresponding to position 70; R at a position corresponding to position 70; S at a position corresponding to position 70; T at a position corresponding to position 70; V at a position corresponding to position 70; Rat a position corresponding to position 71; S at a position corresponding to position 71; M at a position corresponding to position 72; Q at a position corresponding to position 72; H at a position corresponding to position 73; L at a position corresponding to position 73; W at a position corresponding to position 73; A at a position corresponding to position 74; C at a position corresponding to position 74; G at a position corresponding to position 74; N at a position corresponding to position 74; P at a position corresponding to position 74; R at a position corresponding to position 74; S at a position corresponding to position 74; V at a position corresponding to position 74; W at a position corresponding to position 74; F at a position corresponding to position 75; L at a position corresponding to position 75; Rat a position corresponding to position 75; T at a position corresponding to position 75; G at a position corresponding to position 84; Rat a position corresponding to position 84; A at a position corresponding to position 86; C at a position corresponding to position 87; T at a position corresponding to position 87; Y at a position corresponding to position 87; C at a position corresponding to position 92; I at a position corresponding to position 93; L at a position corresponding to position 93; Rat a position corresponding to position 93; T at a position corresponding to position 93; Rat a position corresponding to position 94; G at a position corresponding to position 97; Q at a position corresponding to position 118; F at a position corresponding to position 120; V at a position corresponding to position 120; Y at a position corresponding to position 120; H at a position corresponding to position 127; N at a position corresponding to position 127; G at a position corresponding to position 131; R at a position corresponding to position 131; V at a position corresponding to position 131; D at a position corresponding to position 135; G at a position corresponding to position 135; Rat a position corresponding to position 135, with H at a position corresponding to position 141; Y at a position corresponding to position 141; Rat a position corresponding to position 142; R at a position corresponding to position 147; V at a position corresponding to position 147; K at a position corresponding to position 148; G at a position corresponding to position 150; K at a position corresponding to position 151; L at a position corresponding to position 151; M at a position corresponding to position 151; Q at a position corresponding to position 151; Rat a position corresponding to position 151; Rat a position corresponding to position 152; G at a position corresponding to position 155; K at a position corresponding to position 155; D at a position corresponding to position 156; A at a position corresponding to position 163; E at a position corresponding to position 163; K at a position corresponding to position 163; Rat a position corresponding to position 163; M at a position corresponding to position 164; D at a position corresponding to position 165; N at a position corresponding to position 165; A at a position corresponding to position 166; F at a position corresponding to position 166; H at a position corresponding to position 166; L at a position corresponding to position 166; Q at a position corresponding to position 166; Rat a position corresponding to position 166; Tat a position corresponding to position 166; Y at a position corresponding to position 166; L at a position corresponding to position 169; Rat a position corresponding to position 170; K at a position corresponding to position 174; D at a position corresponding to position 198; K at a position corresponding to position 206; L at a position corresponding to position 206; N at a position corresponding to position 212; M at a position corresponding to position 213; N at a position corresponding to position 213; M at a position corresponding to position 215; S at a position corresponding to position 219; K at a position corresponding to position 233; Rat a position corresponding to position 233; M at a position corresponding to position 234; R at a position corresponding to position 236; E at a position corresponding to position 237; S at a position corresponding to position 238; I at a position corresponding to position 247; T at a position corresponding to position 257; P at a position corresponding to position 259; Y at a position corresponding to position 260; K at a position corresponding to position 261; N at a position corresponding to position 261; K at a position corresponding to position 263; Rat a position corresponding to position 263; A at a position corresponding to position 269; L at a position corresponding to position 271; M at a position corresponding to position 271; Tat a position corresponding to position 272; D at a position corresponding to position 276; S at a position corresponding to position 276; Y at a position corresponding to position 276; K at a position corresponding to position 277; R at a position corresponding to position 277; T at a position corresponding to position 277; H at a position corresponding to position 278; K at a position corresponding to position 278; N at a position corresponding to position 278; R at a position corresponding to position 278; S at a position corresponding to position 278; T at a position corresponding to position 278; Y at a position corresponding to position 278; M at a position corresponding to position 282; V at a position corresponding to position 291; A at a position corresponding to position 293; C at a position corresponding to position 293; F at a position corresponding to position 293; M at a position corresponding to position 293; P at a position corresponding to position 293; Q at a position corresponding to position 293; V at a position corresponding to position 293; E at a position corresponding to position 305; G at a position corresponding to position 308; N at a position corresponding to position 308; E at a position corresponding to position 309; L at a position corresponding to position 309; N at a position corresponding to position 309; Q at a position corresponding to position 309; R at a position corresponding to position 309; T at a position corresponding to position 309; A at a position corresponding to position 310; G at a position corresponding to position 310; K at a position corresponding to position 313; Rat a position corresponding to position 313; H at a position corresponding to position 315; I at a position corresponding to position 317; K at a position corresponding to position 317; Rat a position corresponding to position 317; M at a position corresponding to position 318; H at a position corresponding to position 320; K at a position corresponding to position 320; R at a position corresponding to position 320; Rat a position corresponding to position 324; A at a position corresponding to position 325; D at a position corresponding to position 325; E at a position corresponding to position 325; G at a position corresponding to position 325; H at a position corresponding to position 325; K at a position corresponding to position 325; M at a position corresponding to position 325; N at a position corresponding to position 325; Q at a position corresponding to position 325; S at a position corresponding to position 325; V at a position corresponding to position 326; I at a position corresponding to position 328; K at a position corresponding to position 328; L at a position corresponding to position 328; S at a position corresponding to position 328; Y at a position corresponding to position 328; G at a position corresponding to position 347; S at a position corresponding to position 347; V at a position corresponding to position 353; with T at a position corresponding to position 359; Rat a position corresponding to position 371; P at a position corresponding to position 377; Tat a position corresponding to position 377; W at a position corresponding to position 380; Y at a position corresponding to position 380; K at a position corresponding to position 389; M at a position corresponding to position 392; Rat a position corresponding to position 395; M at a position corresponding to position 399; T at a position corresponding to position 399; W at a position corresponding to position 399; G at a position corresponding to position 405; D at a position corresponding to position 407; Q at a position corresponding to position 407; A at a position corresponding to position 409; Q at a position corresponding to position 409; T at a position corresponding to position 410; P at a position corresponding to position 418; F at a position corresponding to position 419; I at a position corresponding to position 419; K at a position corresponding to position 419; Rat a position corresponding to position 419; S at a position corresponding to position 419; H at a position corresponding to position 421; K at a position corresponding to position 421; N at a position corresponding to position 421; Q at a position corresponding to position 421; R at a position corresponding to position 421; S at a position corresponding to position 421; K at a position corresponding to position 425; A at a position corresponding to position 431; H at a position corresponding to position 431; K at a position corresponding to position 431; Q at a position corresponding to position 431; R at a position corresponding to position 431; S at a position corresponding to position 431; V at a position corresponding to position 431; L at a position corresponding to position 433; Rat a position corresponding to position 433; T at a position corresponding to position 433; V at a position corresponding to position 433; K at a position corresponding to position 436; I at a position corresponding to position 437; M at a position corresponding to position 437; T at a position corresponding to position 438; V at a position corresponding to position 439; H at a position corresponding to position 440; R at a position corresponding to position 440; F at a position corresponding to position 441; Rat a position corresponding to position 442; A at a position corresponding to position 443; M at a position corresponding to position 443; M at a position corresponding to position 445; P at a position corresponding to position 445; A at a position corresponding to position 446; D at a position corresponding to position 447; N at a position corresponding to position 447; and/or with Q at a position corresponding to position 447, each with reference to amino acid positions set forth in SEQ ID NO:3. The modified PH20 polypeptides can contain any one or more of the recited amino acid substitutions, in any combination, with or without additional modifications, so long at the PH20 polypeptide exhibits hyaluronidase activity, such as increased hyaluronidase activity compared to the PH20 polypeptide not containing the modification(s), for example, at least 1.5-fold increased hyaluronidase activity. 
     In some examples, the modified PH20 polypeptides provided herein contain one or more amino acid replacement(s) at a position(s) corresponding to position(s) 24, 29, 31, 48, 58, 69, 70, 75, 84, 97, 165, 166, 271, 278, 317, 320, 325, and/or 326 with reference to positions set forth in SEQ ID NO:3. For example, exemplary amino acid replacements include, but are not limited to, replacement with: E at a position corresponding to position 24; E at a position corresponding to position 29; V at a position corresponding to position 31; N at a position corresponding to position 48; K at a position corresponding to position 58; Q at a position corresponding to position 58; A at a position corresponding to position 69; F at a position corresponding to position 69; G at a position corresponding to position 69; P at a position corresponding to position 69; R at a position corresponding to position 69; A at a position corresponding to position 70; F at a position corresponding to position 70; G at a position corresponding to position 70; H at a position corresponding to position 70; H at a position corresponding to position 70; N at a position corresponding to position 70; R at a position corresponding to position 70; T at a position corresponding to position 70; V at a position corresponding to position 70; L at a position corresponding to position 75; T at a position corresponding to position 75; G at a position corresponding to position 84; G at a position corresponding to position 97; D at a position corresponding to position 165; L at a position corresponding to position 166; Rat a position corresponding to position 166; T at a position corresponding to position 166; L at a position corresponding to position 271; H at a position corresponding to position 278; R at a position corresponding to position 278; K at a position corresponding to position 317; K at a position corresponding to position 320; E at a position corresponding to position 325, with G at a position corresponding to position 325; K at a position corresponding to position 325; N at a position corresponding to position 325; Q at a position corresponding to position 325; V at a position corresponding to position 326; each with reference to amino acid positions set forth in SEQ ID NO:3. The modified PH20 polypeptides can contain any one or more of the recited amino acid substitutions, in any combination, with or without additional modifications, so long at the PH20 polypeptide exhibits hyaluronidase activity, such as increased hyaluronidase activity compared PH20 polypeptide not containing the modification(s), for example, at least 2.0-fold increased hyaluronidase activity. 
     Exemplary modified PH20 polypeptides that exhibit increased activity compared to the unmodified PH20 polypeptide (e.g., set forth in SEQ ID NO:3) are any having the sequence of amino acids set forth in any of SEQ ID NOs: 73, 78, 86, 89, 91, 95, 96, 99, 100, 105, 106, 108, 109, 111, 112, 113, 115, 117, 118, 119, 120, 123-126, 128-136, 139-141, 149, 154, 155, 159, 164, 165, 167, 173, 178, 181, 191-193, 195-197, 199-205, 207-221, 225, 226, 228, 229, 231, 233, 237-239, 242, 247-254, 256, 257, 267, 269, 270, 277, 283, 293, 295, 296, 298, 300, 303, 308, 316, 318, 321, 322, 324, 325, 330, 334, 335, 338-340, 344, 348, 355, 367, 369, 371, 377, 384-388, 394, 398, 399, 401, 406-408, 410, 412, 414, 416, 419, 421-426, 428, 430, 431, 435, 448, 455, 456, 459, 462, 463, 465, 469, 478-480, 482, 484, 490, 493, 497, 501, 503, 505, 506-508, 510-512, 514, 518, 522, 523, 527, 531, 533, 537-543, 545, 551, 558, 559, 561, 563-566, 569, 572, 574, 576, 579, 581-583, 585, 587, 588, 594, 596, 602, 605, 606, 609, 613, 618-620, 624-634, 637, 640-644, 647, 648, 652, 657, 675, 695, 698, 699, 700, 712, 717, 725, 731, 732, 734, 738, 742, 746, 748-750, 757, 760, 762-765, 768-773, 775, 779, 782, 783, 786-789, 794-797, 799-801, 807, 814, 816, 819, 822, 825, 826, 830, 836, 838, 844, 847, 851, 853 or having a sequence of amino acids that exhibits at least 68%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NOs: 73, 78, 86, 89, 91, 95, 96, 99, 100, 105, 106, 108, 109, 111, 112, 113, 115, 117, 118, 119, 120, 123-126, 128-136, 139-141, 149, 154, 155, 159, 164, 165, 167, 173, 178, 181, 191-193, 195-197, 199-205, 207-221, 225, 226, 228, 229, 231, 233, 237-239, 242, 247-254, 256, 257, 267, 269, 270, 277, 283, 293, 295, 296, 298, 300, 303, 308, 316, 318, 321, 322, 324, 325, 330, 334, 335, 338-340, 344, 348, 355, 367, 369, 371, 377, 384-388, 394, 398, 399, 401, 406-408, 410, 412, 414, 416, 419, 421-426, 428, 430, 431, 435, 448, 455, 456, 459, 462, 463, 465, 469, 478-480, 482, 484, 490, 493, 497, 501, 503, 505, 506-508, 510-512, 514, 518, 522, 523, 527, 531, 533, 537-543, 545, 551, 558, 559, 561, 563-566, 569, 572, 574, 576, 579, 581-583, 585, 587, 588, 594, 596, 602, 605, 606, 609, 613, 618-620, 624-634, 637, 640-644, 647, 648, 652, 657, 675, 695, 698, 699, 700, 712, 717, 725, 731, 732, 734, 738, 742, 746, 748-750, 757, 760, 762-765, 768-773, 775, 779, 782, 783, 786-789, 794-797, 799-801, 807, 814, 816, 819, 822, 825, 826, 830, 836, 838, 844, 847, 851, 853 and contains the amino acid replacement and exhibits increased hyaluronidase activity compared to the corresponding unmodified polypeptide. 
     b. Increased Stability 
     Provided herein are PH20 polypeptides that exhibit increased stability. In particular, the PH20 polypeptides exhibit increased stability in vivo and/or in vitro. For example, the PH20 polypeptides can exhibit increased stability under various storage conditions. The modified PH20 polypeptides provided herein that exhibit increased stability display, among other parameters, increased resistance to denaturation conditions, including but not limited to, denaturation conditions caused by temperature (e.g., elevated temperature such as heat), agitation, no or low salt, and/or presence of excipients. Exemplary excipients include, but are not limited to, antiadherents, binders, coatings, fillers and diluents, flavors, colors, lubricants, glidants, preservatives, sorbents or sweeteners. For example, various excipients, such as preservatives, can act as protein denaturing agents. Modified PH20 polypeptides provided herein that exhibit increased protein stability exhibit reduced aggregation, reduced precipitation and/or increased activity when exposed to a denaturation condition compared to the corresponding PH20 not containing the amino acid replacement. For example, modified PH20 polypeptides provided herein exhibit at least or at least about or 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500% or more increased activity when exposed to a denaturation condition compared to the corresponding PH20 polypeptide not containing the amino acid replacement when exposed to the same denaturation condition. 
     The PH20 polypeptides provided herein that exhibit increased stability are modified or variant PH20 polypeptides that contain an amino acid replacement (substitution), deletion or insertion or other modification. Typically, the PH20 polypeptides provided herein that exhibit increased stability contain one or more amino acid replacements in a PH20 polypeptide compared to the corresponding PH20 polypeptide not containing the amino acid replacement(s), for example, the PH20 polypeptide set forth in any of SEQ ID NOs: 2, 3, 6-66, 68-72, 856-861, 869 or 870 or a variant thereof having at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 86%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto. In particular, the modified or variant PH20 polypeptides provided herein exhibit increased stability compared to the corresponding PH20 polypeptide not containing the amino acid replacement, for example, the PH20 polypeptide set forth in any of SEQ ID NOs: 3, 7, 32-66, 69 or 72 and in particular the PH20 polypeptide set forth in SEQ ID NO:3. 
     In particular examples, the modified PH20 polypeptides contain an amino acid replacement at one or more amino acid positions identified as being associated with increased stability. As described herein, such positions can be identified using mutagenesis and selection or screening methods to identify those positions that result in stability (e.g., increased activity) of the polypeptide compared to the corresponding PH20 not containing the modification upon exposure to one or more denaturation conditions. The PH20 polypeptide also can contain other modifications, such as other amino acid replacements, that alone are not associated with conferring stability, so long as the resulting modified PH20 polypeptide exhibits increased stability under one or more denaturation conditions compared to the PH20 not containing the amino acid modification(s), such as amino acid replacement(s), and exhibits hyaluronidase activity. The modified PH20 polypeptide provided herein can contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, or more amino acid replacements. Additional modifications, such as insertions or deletions, also can be included. The amino acid replacement can be in a PH20 polypeptide as set forth in any of SEQ ID NOs: 2, 3, 6-66, 68-72, 856-861, 869 or 870 or a variant thereof having at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 86%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto. For example, the replacements can be in a human PH20 polypeptide, for example, any set forth in any of SEQ ID NOs: 3, 7, 32-66, 69 or 72 or a variant thereof. 
     Exemplary of modified PH20 polypeptides provided herein are PH20 polypeptides that exhibit increased stability upon exposure to phenol compounds, high temperature (heat), and/or lack of NaCl. 
     i. Phenophiles 
     Provided herein are modified PH20 polypeptides that exhibit increased stability in the presence of phenolic compounds. Multidose formulations must contain antimicrobial preservatives to protect them from microbial contamination. For parenteral drug products, including insulin and other therapeutic agents, the most common preservatives are phenolic compounds, such as phenol, metacresol (m-cresol), benzyl alcohol, and parabens including methylparaben and propylparaben. The preservatives typically must be present at sufficient concentrations to satisfy regulatory rules. For example, regulatory requirements assert that the antimicrobial efficacy of the formulation must satisfy the preservative efficacy test (PET) requirements of the target markets. Currently different regulatory agencies have different pharmacopeial criteria for antimicrobial effectiveness for pharmaceutical products designed for multiple dosing. The PET requirements of the United States Pharmacopoeia (USP) and the European Pharmacopoeia (EP) differ considerably, imposing additional constraints in developing multidose formulations. Table 4 shows the criteria for injectable drugs to meet USP and EP criteria. Typically, formulations that meet EP (EPA or EPB) anti-microbial requirements contain more preservative than those formulated only to meet USP anti-microbial requirements. 
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                 USP and EP requirement for antimicrobial effectiveness testing 
               
            
           
           
               
               
               
               
            
               
                   
                   
                 United 
                 Europe 
               
            
           
           
               
               
               
               
               
            
               
                   
                 Time 
                 States 
                 EPB 
                 EPA 
               
               
                 Requirement 
                 point 
                 USP 
                 (Minimum) 
                 (Preferred) 
               
               
                   
               
               
                 Bacterial Log 
                  6 h 
                   
                   
                 2 
               
               
                 Reduction* 
                 24 h 
                   
                 1 
                 3 
               
               
                   
                  7 d 
                 1.0 
                 3 
                 No recovery 
               
               
                   
                 14 d 
                 3.0 
                 No increase 
                 No recovery 
               
               
                   
                 28 d 
                 No increase 
                 No increase 
                 No recovery 
               
               
                 Fungal Log  
                  7 d 
                 No increase 
                   
                 2 
               
               
                 Reduction* 
                 14 d 
                 No increase 
                 1 
                 No increase 
               
               
                   
                 28 d 
                 No increase 
                 No increase 
                 No increase 
               
               
                   
               
               
                 *Log 10  unit reduction from initial measured inoculum; No increase: not more than 0.5 log 10  unit increase than previously measured value. 
               
            
           
         
       
     
     Anti-microbial preservatives can interact with proteins resulting in aggregations and negative effects on stability. Thus, although a necessary component, preservatives pose a significant problem in the development of stable, multidose formulations of proteins because they typically induce aggregation of the protein in aqueous solution. In particular, increasing or high amounts of preservatives can negatively impact the stability of a protein, including effects on physical stability (aggregation or precipitation) that can impact protein activity. For example, to meet the EP preservative efficacy requirements, relatively high amounts of phenolic compounds, such as phenol or m-cresol, can be required, which can influence stability of the protein formulation. For example, preservatives such as phenol, m-cresol, and benzyl alcohol have been shown to induce aggregation of human growth hormone (Maa and Hsu (1996)  Int. J. Pharm.  140:155-168), recombinant interleukin-1 receptor (Remmele (1998)  Pharm. Res.  15:200-208), human insulin-like growth factor I (Fransson (1997)  Pharm. Res.  14:606-612), rhIFN-γ (Lam (1997)  Pharm. Res.  14:725-729) and cytochrome c (Singh et al. (2011)  J. Pharm Sci.,  100:1679-89). The destabilizing effect that preservatives have on proteins in solution has been a limiting factor in the development of multidose formulations, and to date, most protein therapeutics have been formulated for single use only. 
     PH20 hyaluronidase, such as rHuPH20, rapidly loses activity in the presence of preservatives, likely due to unfolding of the protein and subsequent aggregate formation. For example, as shown in the Examples herein, preservatives reduce PH20 enzymatic activity, particularly at elevated temperatures (see also U.S. Provisional Appl. No. 61/520,962; and U.S. application Ser. Nos. 13/507,263 and 13/507,262). For example, following incubation with 0.4% m-cresol for 4 hours, PH20 (e.g., rHuPH20) retains only about 10% of its activity (see e.g., Example 5). When incubated in the presence of 0.1% phenol and 0.15% or 0.315% m-cresol for 6 days at 37° C., PH20 (e.g., rHuPH20) retains about 0% to 15% activity, depending on the presence of other excipients or amounts of other excipients in the formulation (see e.g., Examples 9 and 10). For example, the presence of a higher concentration of salt generally increases the stability of PH20. In particular, the melting temperature of PH20, such as rHuPH20, is reduced significantly when phenolic preservatives, such as m-Cresol, are added to the formulation. For example, the unfolding temperature of rHuPH20 is reduced from 44° C. to 24° C. The lower PH20 unfolding temperatures leads to increased PH20 aggregation, especially at elevated temperatures, and reduced enzyme activity. The destabilizing effect is likely due to the hydrophobic nature of the phenolic preservatives. The hydrophobicity of the phenolic compounds can lead to interaction with rHuPH20 through nonspecific binding to the protein, ultimately perturbing the structural integrity of rHuPH20. This translates to a significant loss of rHuPH20 enzymatic activity in the presence of preservatives. 
     The modified PH20 polypeptides provided herein that exhibit increased stability in the presence of phenolic preservatives exhibit more than 15% enzymatic activity in the presence of at least one phenolic preservative for at least 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 3 weeks, 4 weeks or more compared to the enzymatic activity of the modified PH20 polypeptide in the absence of preservative for the same time period and under the same conditions (except for the presence of preservative). In some examples, the modified PH20 polypeptides provided herein exhibit at least 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more enzymatic activity in the presence of a phenolic preservative compared to in the absence of preservative. For example, the phenolic preservative compound can be phenol, metacresol (m-cresol), benzyl alcohol, and/or parabens including methylparaben or propylparaben. 
     In particular examples, the increased stability in the presence of preservative is exhibited under temperature conditions of between or about between 0° C. to 40° C., such as between or about between 2° C. to 6° C., 24° C. to 32° C. or 35° C. to 40° C., and generally at or about at 4° C. or 5° C., 30° C. or 37° C. It is understood that since high temperature also can have a destabilizing effect on PH20 activity (see below), the percentage of enzymatic activity of a modified PH20 polypeptide provided herein in the presence of preservative is greater at lower temperatures than at higher temperatures. 
     Generally, the modified PH20 polypeptides provided herein exhibit increased stability, and the noted enzymatic activities, in the presence of an anti-microbial effective amount of preservative that kills or inhibits the propagation of microbial organisms in a sample of the composition. For example, the modified PH20 polypeptides provided herein exhibit increased stability in the presence of an anti-microbial effective amount of preservative that kills or inhibits the propagation of microbial organisms such that at least a 1.0 log 10  unit reduction in bacterial organisms occurs at 7 days following inoculation. In some examples, the modified PH20 polypeptides provided herein exhibit increased stability in the presence of an anti-microbial effective amount of preservative that kills or inhibits the propagation of microbial organisms such that, when tested in an antimicrobial preservative effectiveness test (APET), following inoculation of the composition with a microbial inoculum there is at least a 1.0 log 10  unit reduction in bacterial organisms at 7 days following inoculation, at least a 3.0 log 10  unit reduction of bacterial organisms at 14 days following inoculation, at least no further increase in bacterial organisms after 28 days following inoculation, and at least no increase in fungal organisms after 7 days following inoculation. In other examples, the modified PH20 polypeptides provided herein exhibit increased stability in the presence of an anti-microbial effective amount of preservative that kills or inhibits the propagation of microbial organisms such that, when tested in an antimicrobial preservative effectiveness test (APET), following inoculation of the composition with a microbial inoculum there is at least a 1.0 log 10  unit reduction of bacterial organisms at 24 hours following inoculation, at least a 3.0 log 10  unit reduction of bacterial organisms at 7 days following inoculation, no further increase in bacterial organisms after 28 days following inoculation, at least a 1.0 log 10  unit reduction of fungal organisms at 14 days following inoculation, and at least no further increase in fungal organisms after 28 days following inoculation. In yet another example, the modified PH20 polypeptides provided herein exhibit increased stability in the presence of an anti-microbial effective amount of the preservative that kills or inhibits the propagation of microbial organisms such that, when tested in an antimicrobial preservative effectiveness test (APET), following inoculation of the composition with a microbial inoculum there is at least a 2.0 log 10  unit reduction of bacterial organisms at 6 hours following inoculation, at least a 3.0 log 10  unit reduction of bacterial organisms at 24 hours following inoculation, no recovery of bacterial organisms after 28 days following inoculation of the composition with the microbial inoculum, at least a 2.0 log 10  unit reduction of fungal organisms at 7 days following inoculation, and at least no further increase in fungal organisms after 28 days following inoculation. 
     For example, the modified PH20 polypeptides provided herein exhibit increased stability, and above recited enzymatic activity, in the presence of a total amount of one or more phenolic preservative agents as a percentage (%) of mass concentration (w/v) that is or is between 0.05% to 0.6%, 0.1% to 0.4%, 0.1% to 0.3%, 0.15% to 0.325%, 0.15% to 0.25%, 0.1% to 0.2%, 0.2% to 0.3% or 0.3% to 0.4% inclusive. 
     Generally, modified PH20 polypeptides provided herein exhibit increased stability in the presence of m-cresol and/or phenol. For example, modified PH20 polypeptides provided herein exhibit increased stability in the presence of m-cresol in an amount as a % of mass concentration (w/v) in a formulation containing the modified PH20 polypeptide of between or about between 0.05% to 0.6%, 0.1% to 0.4%, 0.1% to 0.3%, 0.15% to 0.325%, 0.15% to 0.25%, 0.1% to 0.2%, 0.2% to 0.3% or 0.3% to 0.4%. In other examples, modified PH20 polypeptides provided herein exhibit increased stability in the presence of phenol in an amount at a % of mass concentration (w/v) in a formulation containing the modified PH20 polypeptide of between or about between 0.05% to 0.6%, 0.1% to 0.4%, 0.1% to 0.3%, 0.15% to 0.325%, 0.15% to 0.25%, 0.1% to 0.2%, 0.2% to 0.3% or 0.3% to 0.4% m-cresol. In further examples, modified PH20 polypeptides provided herein exhibit increased stability in the presence of phenol and m-cresol in an amount as a % of mass concentration (w/v) in a formulation containing the modified PH20 polypeptide of between or about between 0.05% to 0.25% phenol and between or about between 0.05% to 0.3% m-cresol, between or about between 0.10% to 0.2% phenol and between or about between 0.6% to 0.18% m-cresol, between or about between 0.1% to 0.15% phenol and 0.8% to 0.15% m-cresol, between or about between 0.10% to 0.15% phenol and between or about between 0.06% to 0.09% m-cresol, or between or about between 0.12% to 0.18% phenol and between or about between 0.14% to 0.22% m-cresol. 
     In examples herein, modified PH20 polypeptides exhibit more than 15%, such as at least 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more enzymatic activity in the presence of at least about between or between 0.3% to 0.4%, inclusive, m-cresol and/or phenol for at least 4 hours at 37° C. compared to the enzymatic activity of the modified PH20 polypeptide in the absence of the preservative for the same time period and under the same conditions (except for the presence of preservative). For example, modified PH20 polypeptides exhibit more than 15%, such as at least 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more enzymatic activity in the presence of about or 0.4% m-cresol for at least 4 hours at 37° C. compared to the enzymatic activity of the modified PH20 polypeptide in the absence of the preservative for the same time period and under the same conditions (except for the presence of preservative). Modified PH20 polypeptides provided herein also exhibit more than 15%, such as at least 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more enzymatic activity in the presence of at least about between or between 0.2% to 0.4%, inclusive, m-cresol and/or phenol for at least 1 day, 2 days, 3 days, 4 days, 5 days or 6 days at 37° C. compared to the enzymatic activity of the modified PH20 polypeptide in the absence of preservative for the same time period and under the same conditions (except for the presence of preservative). For example, modified PH20 polypeptides provided herein exhibit more than 15%, such as at least 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more enzymatic activity in the presence of about or 0.10% phenol and about or 0.15% m-cresol for at least 1 day, 2 days, 3 days, 4 days, 5 days or 6 days at 37° C. compared to the enzymatic activity of the modified PH20 polypeptide in the absence of preservative for the same time period and under the same conditions (except for the presence of preservative). In other examples, modified PH20 polypeptides provided herein exhibit more than 15%, such as at least 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more enzymatic activity in the presence of about or 0.315% m-cresol for at least 1 day, 2 days, 3 days, 4 days, 5 days or 6 days, generally for at least 6 days, at 37° C. compared to the enzymatic activity of the modified PH20 polypeptide in the absence of preservative for the same time period and under the same conditions (except for the presence of preservative). 
     For example, such modified PH20 polypeptides provided herein that exhibit increased stability to phenol compounds contain an amino acid replacement (substitution) at one or more amino acid positions corresponding to positions 10, 12, 20, 22, 26, 34, 36, 46, 50, 52, 58, 68, 70, 74, 82, 83, 84, 86, 97, 127, 131, 138, 142, 143, 144, 166, 169, 174, 193, 195, 196, 204, 205, 206, 213, 219, 234, 237, 238, 240, 249, 261, 267, 277, 279, 291, 309, 310, 314, 315, 317, 318, 347, 367, 375, 376, 399, 401, 407, 416, 419, 421, 431, 433, 439, 440, 443 or 445 with reference to amino acid positions set forth in SEQ ID NO:3. For example, the amino acid positions can be replacements at one or more positions corresponding to replacement of (P) at position 10 (P10), V12, A20, S22, L26, D34, S36, 146, G50, G52, V58, D68, I70, T74, K82, 183, S84, Q86, T97, D127, N131, Q138, V142, Q143, L144, V166, 1169, L174, H193, K195, K196, F204, N205, V206, D213, N219, Q234, V237, A238, T240, E249, S261, A267, V277K279, G291, 1309, M310, K314, S315, L317, Q347, P367, E375, K376, Y399, S401, S407, D416, A419, D421, D431, F433, E439, T440, P443 or 1445 with reference to amino acid positions set forth in SEQ ID NO:3. 
     Exemplary of amino acid replacements in the modified PH20 polypeptides provided herein include, but are not limited to, replacement with: glycine (G) at a position corresponding to position 10; K at a position corresponding to position 12; S at a position corresponding to position 20; T at a position corresponding to position 22; M at a position corresponding to position 26; W at a position corresponding to position 34; N at a position corresponding to position 36; L at a position corresponding to position 46; M at a position corresponding to position 50; T at a position corresponding to position 52; S at a position corresponding to position 52; C at a position corresponding to position 58; K at a position corresponding to position 58; Rat a position corresponding to position 58; N at a position corresponding to position 58; Y at a position corresponding to position 58; P at a position corresponding to position 58; H at a position corresponding to position 58; P at a position corresponding to position 68; V at a position corresponding to position 70; E at a position corresponding to position 74; L at a position corresponding to position 82; N at a position corresponding to position 82; V at a position corresponding to position 83; Q at a position corresponding to position 83; S at a position corresponding to position 83; G at a position corresponding to position 83; N at a position corresponding to position 84; A at a position corresponding to position 86; K at a position corresponding to position 86; E at a position corresponding to position 97; L at a position corresponding to position 97; R at a position corresponding to position 127; Rat a position corresponding to position 131; L at a position corresponding to position 138; K at a position corresponding to position 142; N at a position corresponding to position 142; P at a position corresponding to position 142; S at a position corresponding to position 142; T at a position corresponding to position 142; G at a position corresponding to position 143; K at a position corresponding to position 143; T at a position corresponding to position 144; Q at a position corresponding to position 166; T at a position corresponding to position 166; L at a position corresponding to position 169; G at a position corresponding to position 174; N at a position corresponding to position 174; Q at a position corresponding to position 193; T at a position corresponding to position 195; N at a position corresponding to position 195; E at a position corresponding to position 196; Rat a position corresponding to position 196; P at a position corresponding to position 204; A at a position corresponding to position 205; E at a position corresponding to position 205; I at a position corresponding to position 206; A at a position corresponding to position 213; I at a position corresponding to position 219; M at a position corresponding to position 234; T at a position corresponding to position 237; H at a position corresponding to position 238; Q at a position corresponding to position 240; V at a position corresponding to position 249; A at a position corresponding to position 261; K at a position corresponding to position 261; Tat a position corresponding to position 267; K at a position corresponding to position 277; H at a position corresponding to position 279; V at a position corresponding to position 279; V at a position corresponding to position 291; E at a position corresponding to position 309; Q at a position corresponding to position 310; Y at a position corresponding to position 314; Y at a position corresponding to position 315; N at a position corresponding to position 317; W at a position corresponding to position 317; D at a position corresponding to position 318; G at a position corresponding to position 347; A at a position corresponding to position 367; R at a position corresponding to position 375; Rat a position corresponding to position 376; V at a position corresponding to position 399; E at a position corresponding to position 401; A at a position corresponding to position 407; L at a position corresponding to position 416; K at a position corresponding to position 419; H at a position corresponding to position 421; E at a position corresponding to position 431; Tat a position corresponding to position 433; V at a position corresponding to position 433; C at a position corresponding to position 439; P at a position corresponding to position 440; G at a position corresponding to position 443; N at a position corresponding to position 445, each with reference to amino acid residue positions set forth in SEQ ID NO:3. 
     The amino acid replacement(s) can be in a PH20 polypeptide as set forth in any of SEQ ID NOs: 2, 3, 6-66, 68-72, 856-861, 869 or 870 or a variant thereof having at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 86%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto. For example, the replacement(s) can be in a human PH20 polypeptide, for example, any set forth in any of SEQ ID NOs: 3, 7, 32-66, 69 or 72 or a variant thereof. 
     Exemplary modified PH20 polypeptides that exhibit increased stability to phenol compounds compared to the unmodified PH20 polypeptide (e.g., set forth in SEQ ID NO:3) are any having the sequence of amino acids set forth in any of SEQ ID NOs: 83, 88, 93, 94, 101, 144, 148, 158, 171, 176, 175, 177, 178, 180, 182, 183, 184, 185, 194, 221, 240, 259, 260, 261, 262, 263, 264, 268, 270, 272, 307, 309, 327, 334, 341, 351, 352, 353, 356, 357, 358, 359, 361, 424, 426, 430, 434, 436, 443, 444, 445, 446, 447, 449, 450, 451, 454, 461, 467, 480, 487, 489, 492, 495, 504, 505, 509, 527, 544, 576, 589, 600, 603, 607, 612, 614, 647, 658, 683, 687, 733, 736, 741, 754, 763, 768, 781, 796, 797, 809, 818, 829 or 837 or having a sequence of amino acids that exhibits at least 68%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NOs: 83, 88, 93, 94, 101, 144, 148, 158, 171, 176, 175, 177, 178, 180, 182, 183, 184, 185, 194, 221, 240, 259, 260, 261, 262, 263, 264, 268, 270, 272, 307, 309, 327, 334, 341, 351, 352, 353, 356, 357, 358, 359, 361, 424, 426, 430, 434, 436, 443, 444, 445, 446, 447, 449, 450, 451, 454, 461, 467, 480, 487, 489, 492, 495, 504, 505, 509, 527, 544, 576, 589, 600, 603, 607, 612, 614, 647, 658, 683, 687, 733, 736, 741, 754, 763, 768, 781, 796, 797, 809, 818, 829 or 837 and contains the amino acid replacement, exhibits hyaluronidase activity and exhibits increased stability in the presence phenol compounds compared to the corresponding unmodified polypeptide. 
     In particular, provided herein is a modified PH20 polypeptide that contains an amino acid replacement with P at a position corresponding to amino acid residue 204 with reference to SEQ ID NO:3. Typically, the modified PH20 polypeptide is a human polypeptide. For example, provided herein is a modified PH20 polypeptide that contains an amino acid replacement F204P in a sequence of amino acids set forth in any of SEQ ID NOs: 3, 7, 69, 72 or 32-66, or a sequence of amino acids that exhibits at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NOs:3, 7, 69, 72 or 32-66 so long as the modified polypeptide contains the amino acid replacement corresponding to F204P. In other cases, the modified PH20 polypeptide is a non-human polypeptide. For example, provided herein is a modified PH20 polypeptide that contains an amino acid replacement F204P in a sequence of amino acids set forth in SEQ ID NO:10, 12, 14, 857, 859, 861 or 870 or a sequence that exhibits at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NOs: 10, 12, 14, 857, 859, 861 or 870 so long as the modified polypeptide contains the amino acid replacement corresponding to F204P. In a further example, provided herein is a modified PH20 polypeptide that contains an amino acid replacement F205P in a sequence of amino acids set forth in SEQ ID NO:24 or Y204P in SEQ ID NO:31, or a sequence that exhibits at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:24 or 31. Exemplary of such a modified PH20 polypeptide is a polypeptide having the sequence of amino acids set forth in SEQ ID NO:449, or having a sequence of amino acids that exhibits at least 68%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:449 and contains the amino acid replacement F204P, exhibits increased hyaluronidase activity and exhibits increased stability to phenol compounds compared to the corresponding unmodified polypeptide (e.g., SEQ ID NO:3). In any of the above examples, the modified PH20 polypeptide that contains an amino acid replacement with P at a position corresponding to amino acid residue 204 with reference to SEQ ID NO:3 does not have the sequence of amino acids set forth in SEQ ID NO:15-22, 28 or 29. 
     In another example, provided herein is a modified PH20 polypeptide that contains an amino acid replacement at a position corresponding to amino acid residue 58 with reference to SEQ ID NO:3. Exemplary of amino acid replacements are replacement with lysine (K) or with arginine (R) at a position corresponding to amino acid residue 58 with reference to SEQ ID NO:3. Typically, the modified PH20 polypeptide is a human polypeptide. For example, provided herein is a modified PH20 polypeptide that contains an amino acid replacement V58K or V58R in a sequence of amino acids set forth in any of SEQ ID NOs: 3, 7, 69, 72 or 32-66, or a sequence of amino acids that exhibits at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NOs:3, 7, 69, 72 or 32-66. In other cases, the modified PH20 polypeptide is a non-human polypeptide. For example, provided herein is a modified PH20 polypeptide that contains an amino acid replacement V58K or V58R in a sequence of amino acids set forth in SEQ ID NOs:10, 12, 14, 20, 22, 24, 29, 857, 859, 861 or 870 or a sequence that exhibits at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NOs: 10, 12, 14, 20, 22, 24, 29, 857, 859, 861 or 870. In a further example, provided herein is a modified PH20 polypeptide that contains an amino acid replacement A58R in a sequence of amino acids set forth in SEQ ID NO:16 or 31, or a sequence that exhibits at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:16 or 31. Exemplary of such a modified PH20 polypeptide is a polypeptide having the sequence of amino acids set forth in SEQ ID NO:182, or having a sequence of amino acids that exhibits at least 68%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:182, which contains the amino acid replacement V58R and exhibits increased hyaluronidase activity and exhibits increased stability in the presence of phenol compounds compared to the corresponding unmodified polypeptide (e.g., SEQ ID NO:3). 
     ii. Thermophiles 
     At elevated temperatures, PH20 hyaluronidases can lose activity. 
     Provided herein are modified PH20 polypeptides that exhibit increased stability at elevated temperatures of between or about between 30° C. to 45° C., inclusive, such as between or about between 35° C. to 42° C., in particular at or about 37° C. For example, provided herein are modified PH20 polypeptides that are stable at elevated temperatures greater than 32° C. such as 35° C. to 45° C., 37° C. to 42° C. and in particular at or about 37° C. for at least 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, at least 5 days, at least 6 days or at least 7 days. Modified PH20 polypeptides that exhibit stability at elevated temperatures can be used in applications where temperatures are elevated, can fluctuate or can increase. This can occur, for example, in methods of administration utilizing pumps or other continuous infusion devices. 
     In particular, modified PH20 polypeptides provided herein that exhibit stability at elevated temperatures exhibit increased hyaluronidase activity at elevated temperature compared to the corresponding PH20 polypeptide not containing the modification, e.g., amino acid replacement. The PH20 polypeptides can exhibit increased hyaluronidase activity upon incubation at elevated temperatures greater than 32° C. such as 35° C. to 45° C. or 37° C. to 42° C., in particular at or about 37° C. for at least 4 hours, 5 hours, 6 hours, 12 hours, 1 day, 2 days, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, at least 5 days, at least 6 days or at least 7 days compared to the corresponding PH20 polypeptide not containing the modification incubated under the same conditions. For example, the hyaluronidase activity can be increased at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500% or more compared to the corresponding PH20 polypeptide not containing the modification incubated under the same conditions. For example, the hyaluronidase activity can be increased at least 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold or more compared to the corresponding PH20 polypeptide not containing the modification incubated under the same conditions. 
     In other examples, modified PH20 polypeptides provided herein that exhibit stability at elevated temperatures retain hyaluronidase activity at elevated temperatures compared to the activity of the modified PH20 polypeptide incubated at non-elevated temperatures under the same conditions (except for the differences in temperature). For example, modified PH20 polypeptides exhibit greater than or about 50%, such as greater than or at least 55%, 60%, 65%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the activity at elevated temperatures greater than 32° C. such as 35° C. to 45° C. or 37° C. to 42° C., in particular at or about 37° C. compared to the activity of the PH20 at non-elevated temperatures of between or about between 2° C. to 8° C. In some examples, modified PH20 polypeptides provided herein that exhibit stability at elevated temperatures exhibit increased activity at elevated temperatures compared to the activity of the modified PH20 polypeptide incubated at non-elevated temperatures under the same conditions (except for the difference in temperature). For example, modified PH20 polypeptides exhibit greater than or about 10% increased activity, such as greater than or at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500% or more of activity at elevated temperatures greater than 32° C. such as 35° C. to 45° C. or 37° C. to 42° C., in particular at or about 37° C. compared to the activity of the PH20 at non-elevated temperatures of between or about between 2° C. to 8° C. For example, modified PH20 polypeptides exhibit greater than or at least about 1.1-fold the hyaluronidase activity, such as greater than or at least 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold or more of activity at elevated temperatures greater than 32° C. such as 35° C. to 45° C. or 37° C. to 42° C., in particular at or about 37° C. compared to the activity of the PH20 at non-elevated temperatures of between or about between 2° C. to 8° C. 
     For example, such modified PH20 polypeptides provided herein that exhibit increased stability at elevated temperatures contain an amino acid replacement (substitution) at one or more amino acid positions corresponding to positions 1, 11, 12, 14, 20, 26, 29, 34, 50, 58, 70, 82, 83, 84, 86, 87, 140, 142, 143, 147, 152, 166, 167, 172, 174, 178, 193, 195, 206, 212, 213, 219, 233, 237, 240, 267, 277, 291, 292, 309, 313, 314, 317, 318, 347, 367, 368, 371, 374, 389, 392, 395, 396, 406, 419, 421, 439 or 443 with reference to amino acid positions set forth in SEQ ID NO:3. For example, the amino acid positions can be replacements at one or more positions corresponding to replacement of (L) at position 1 (L1), N11, V12, F14, A20, L26, F29, D34, G50, V58, I70, K82, 183, S84, Q86, D87, Q140, V142, Q143, T147, K152, V166, E167, G172, L174, N178, H193, K195, V206, D212, D213, N219, Q233, V237, T240, A267, V277, G291, E292, 1309, M313, K314, L317, L318, Q347, P367, D368, A371, L374, E389, E392, S395, E396, L406, A419, D421, E439 or P443, with reference to amino acid positions set forth in SEQ ID NO:3. The resulting modified PH20 polypeptide exhibits increased stability at elevated temperatures greater than 32° C. such as 35° C. to 45° C., 37° C. to 42° C. and in particular at or about 37° C. for at least 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, at least 5 days, at least 6 days, at least 7 days or more. 
     Exemplary amino acid replacements in the modified PH20 polypeptides provided herein include, but are not limited, replacement with: R at a position corresponding to position 1; S at a position corresponding to position 11; I at a position corresponding to position 12; V at a position corresponding to position 14; S at a position corresponding to position 20; M at a position corresponding to position 26; with R at a position corresponding to position 29; W at a position corresponding to position 34; M at a position corresponding to position 50; K at a position corresponding to position 58; Q at a position corresponding to position 58; Q at a position corresponding to position 58; V at a position corresponding to position 70; L at a position corresponding to position 82; Q at a position corresponding to position 83; Rat a position corresponding to position 84; A at a position corresponding to position 86; S at a position corresponding to position 87; K at a position corresponding to position 140; S at a position corresponding to position 142; T at a position corresponding to position 142; K at a position corresponding to position 143; S at a position corresponding to position 147; T at a position corresponding to position 152; T at a position corresponding to position 166; D at a position corresponding to position 167; A at a position corresponding to position 172; G at a position corresponding to position 174; N at a position corresponding to position 174; Rat a position corresponding to position 178; Q at a position corresponding to position 193; T at a position corresponding to position 195; I at a position corresponding to position 206; S at a position corresponding to position 212; A at a position corresponding to position 213; I at a position corresponding to position 219; G at a position corresponding to position 233; T at a position corresponding to position 237; A at a position corresponding to position 240; Q at a position corresponding to position 240; T at a position corresponding to position 267; E at a position corresponding to position 277; S at a position corresponding to position 291; H at a position corresponding to position 292; V at a position corresponding to position 292; S at a position corresponding to position 309; H at a position corresponding to position 313; S at a position corresponding to position 314; I at a position corresponding to position 317; Tat a position corresponding to position 317; W at a position corresponding to position 317; Rat a position corresponding to position 318; G at a position corresponding to position 347; A at a position corresponding to position 367; R at a position corresponding to position 368; S at a position corresponding to position 371; P at a position corresponding to position 374; A at a position corresponding to position 389; V at a position corresponding to position 392; A at a position corresponding to position 395; H at a position corresponding to position 396; N at a position corresponding to position 406; H at a position corresponding to position 419; K at a position corresponding to position 419; Rat a position corresponding to position 421; S at a position corresponding to position 421; A at a position corresponding to position 439; C at a position corresponding to position 439; or G at a position corresponding to position 443, each with reference to amino acid residue positions set forth in SEQ ID NO:3. 
     The amino acid replacement(s) can be in a PH20 polypeptide as set forth in any of SEQ ID NOs: 2, 3, 6-66, 68-72, 856-861, 869 or 870 or a variant thereof having at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 86%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto. For example, the replacement(s) can be in a human PH20 polypeptide, for example, any set forth in any of SEQ ID NOs: 3, 7, 32-66, 69 or 72 or a variant thereof. 
     Exemplary modified PH20 polypeptides that exhibit increased stability to phenol compounds compared to the unmodified PH20 polypeptide (e.g., set forth in SEQ ID NO:3) are any having the sequence of amino acids set forth in any of SEQ ID NOs: 79, 85, 87, 90, 93, 101, 114, 144, 171, 178, 181, 221, 259, 262, 269, 270, 282, 343, 356, 357, 359, 368, 395, 426, 429, 432, 434, 436, 441, 443, 444, 454, 460, 461, 467, 477, 487, 491, 492, 509, 525, 550, 554, 557, 584, 593, 599, 605, 611, 612, 617, 647, 658, 667, 676, 679, 709, 720, 723, 727, 740, 761, 763, 772, 773, 808, 809, or 829 or having a sequence of amino acids that exhibits at least 68%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NOs: 79, 85, 87, 90, 93, 101, 114, 144, 171, 178, 181, 221, 259, 262, 269, 270, 282, 343, 356, 357, 359, 368, 395, 426, 429, 432, 434, 436, 441, 443, 444, 454, 460, 461, 467, 477, 487, 491, 492, 509, 525, 550, 554, 557, 584, 593, 599, 605, 611, 612, 617, 647, 658, 667, 676, 679, 709, 720, 723, 727, 740, 761, 763, 772, 773, 808, 809, or 829 and contains the amino acid replacement, exhibits hyaluronidase activity and exhibits increased stability to elevated temperatures compared to the corresponding unmodified polypeptide. 
     iii. Absence of Salt 
     PH20 denatures in the presence of low salt or no salt. Thus, PH20 requires a high salt concentration of between or about between 140 mM to 200 mM to maintain stability. Other therapeutic agents, for example insulin, exhibit decreased solubility and increased crystallization/aggregation in the presence of high salt. Thus, the high salt requirements of PH20 can affect the solubility and/or activity of co-formulated therapeutic agents, while the presence of low salt can decrease the activity of PH20. This can create problems for generating PH20 co-formulations. 
     Provided herein are modified PH20 polypeptides that exhibit increased stability in the presence of low concentrations of salt (e.g. NaCl) less than 100 mM, for example, less than 90 mM, 80 mM, 70 mM, 60 mM, 50 mM, 40 mM, 30 mM, 25 mM, 20 mM, 15 mM, 10 mM, 5 mM or less. Generally, the modified PH20 polypeptides provided herein exhibit stability in the presence of low concentrations of salt, for example, low concentrations of NaCl of between or about between 10 mM NaCl and 100 mM NaCl, such as between or about between 15 mM to 80 mM NaCl. The modified PH20 polypeptides provided herein that exhibit stability at low concentrations of salt, such as low concentrations of NaCl (i.e., less than 100 mM or less), exhibit increased hyaluronidase activity compared to the corresponding PH20 not containing the modification(s) (e.g., amino acid replacements). For example, modified PH20 polypeptides exhibit greater than or about 10% increased activity, such as greater than or at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500% or more of activity at low concentrations of salt, such as low concentrations of NaCl (i.e., less than 100 mM), compared to the activity of the corresponding PH20 not containing the amino acid modification(s) (e.g., amino acid replacement(s) under the same conditions). For example, modified PH20 polypeptides exhibit greater than or at least about 1.1-fold the hyaluronidase activity, such as greater than or at least 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold or more of activity at low concentrations of NaCl less than 100 mM compared to the activity of the corresponding PH20 not containing the amino acid modification(s) (e.g., amino acid replacement(s) under the same conditions. 
     2. Inactive Mutants 
     Provided herein are modified PH20 polypeptides that contain one or more amino acid replacements in a PH20 polypeptide and that are inactive, whereby the polypeptides do not exhibit hyaluronidase activity or exhibit low or diminished hyaluronidase activity. The modified PH20 polypeptides provided herein that are inactive generally exhibit less than 20%, such as less than 10%, of the hyaluronidase activity of a wildtype or reference PH20 polypeptide, such as the polypeptide set forth in SEQ ID NO: 3 or 7. For example, modified PH20 polypeptides provided herein that are inactive exhibit less than 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.05% or less of the hyaluronidase activity of a wildtype or reference PH20 polypeptide, such as the corresponding polypeptide not containing the amino acid modification (e.g., amino acid replacement), for example, a polypeptide set forth in SEQ ID NO:3 or 7. 
     For example, provided herein are PH20 polypeptides that are inactive and that are modified, for example by amino acid replacement or substitution, compared to a wildtype or reference PH20 polypeptide. For example, a modified PH20 polypeptide provided herein that is inactive contains one or more amino acid replacements at position(s) corresponding to position 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 25, 27, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 94, 95, 96, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 143, 144, 145, 149, 150, 152, 153, 154, 155, 156, 157, 158, 159, 161, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 197, 198, 199, 200, 201, 202, 203, 204, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 278, 279, 280, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 331, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 408, 410, 411, 412, 413, 414, 415, 416, 417, 419, 420, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 434, 437, 438, 439, 440, 441, 442, 443, 444, or 447 with reference to amino acid positions set forth in SEQ ID NO:3, so long as the resulting modified PH20 polypeptide is inactive and exhibits less than 20%, and generally less than 10%, of the hyaluronidase activity of the corresponding PH20 polypeptide not containing the amino acid replacement. Typically, the amino acid residue that is modified (e.g., replaced) at the position corresponding to any of the above positions in a PH20 polypeptide is an identical residue, a conservative residue or a semi-conservative amino acid residue to the amino acid residue set forth in SEQ ID NO:3. 
     Exemplary amino acid replacements at any of the above corresponding positions are set forth in Table 5. Reference to corresponding position in Table 5 is with reference to positions set forth in SEQ ID NO:3. It is understood that the replacements can be made in the corresponding position in another PH20 polypeptide by alignment therewith with the sequence set forth in SEQ ID NO:3 (see e.g.,  FIGS.  1  and  2   ), whereby the corresponding position is the aligned position. The amino acid replacement(s) can be at the corresponding position in a PH20 polypeptide as set forth in any of SEQ ID NOs: 2, 3, 6-66, 68-72, 856-861, 869 or 870 or a variant thereof having at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 86%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto, so long as the resulting modified PH20 polypeptide is inactive. For example, the replacement(s) can be in a corresponding position in a human PH20 polypeptide, for example, any set forth in any of SEQ ID NOs: 3, 7, 32-66, 69 or 72, or a variant thereof. In particular, any one or more of the replacements are in SEQ ID NO:3, so long as the resulting modified PH20 polypeptide is inactive and exhibits less than 20%, and generally less than 10%, of the hyaluronidase activity of the PH20 polypeptide set forth in SEQ ID NO:3 
     
       
         
           
               
             
               
                 TABLE 5 
               
             
            
               
                   
               
               
                 Inactive Mutants 
               
            
           
           
               
               
               
               
               
               
            
               
                 Corresponding 
                   
                 Corresponding 
                   
                 Corresponding 
                   
               
               
                 Position 
                 Replacement 
                 Position 
                 Replacement 
                 Position 
                 Replacement 
               
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 2 
                 H K W Y 
                 3 
                 A G K P T V 
                 4 
                 D E F G L P W Y 
               
               
                 5 
                 D G I L M N P Q R 
                 6 
                 E F T V Y 
                 7 
                 C D F G H I K L Q 
               
               
                   
                 T V W Y 
                   
                   
                   
                 R S T W Y 
               
               
                 8 
                 D E G H N R S W 
                 9 
                 C D E G N P 
                 10 
                 F I L M Y 
               
               
                 11 
                 A C F I L P T W Y 
                 12 
                 G H W 
                 13 
                 E G I L M V 
               
               
                 14 
                 A E G H K N P Q W 
                 15 
                 E F G K N P Q R S 
                 16 
                 A C D E F G H K 
               
               
                   
                   
                   
                 Y 
                   
                 M P R S T Y 
               
               
                 17 
                 D E G H I L N P Q 
                 18 
                 C D F G H I L M P 
                 19 
                 A C F G H I L M P 
               
               
                   
                 R S T V W Y 
                   
                 Q S T V Y 
                   
                 Q R S V W Y  
               
               
                 20 
                 D E F H K L N P R 
                 21 
                 A C D E G H I L M 
                 22 
                 C E G K P 
               
               
                   
                 T V Y 
                   
                 R S T V W 
                   
                   
               
               
                 23 
                 A F L M N P R S T 
                 25 
                 D E F G H I K L N 
                 27 
                 C 
               
               
                   
                 V 
                   
                 P R S T V Y 
                   
                   
               
               
                 33 
                 C D H N V Y 
                 34 
                 I L N S T V 
                 35 
                 A D G P R S 
               
               
                 36 
                 C F V W Y 
                 37 
                 C E G N S 
                 38 
                 E G K L N Q R T 
               
               
                   
                   
                   
                   
                   
                 W 
               
               
                 39 
                 C D F W 
                 40 
                 A D E G K N R S T 
                 41 
                 Q 
               
               
                   
                   
                   
                 V 
                   
                   
               
               
                 42 
                 D E H I K L M P Q 
                 43 
                 A E F G I K L Q R 
                 44 
                 A C F G H I L N Q 
               
               
                   
                 R S T V 
                   
                 V 
                   
                 R S T W Y 
               
               
                 45 
                 A D F G P W 
                 46 
                 P W 
                 47 
                 V 
               
               
                 48 
                 P 
                 49 
                 C D G H P 
                 50 
                 V 
               
               
                 51 
                 C F I M P T W Y 
                 52 
                 C E F W Y 
                 53 
                 A C D E G H L N P 
               
               
                   
                   
                   
                   
                   
                 Q R S T W Y 
               
               
                 54 
                 D E G P R Y 
                 55 
                 A D G H N P Q R T 
                 56 
                 A C E G H I K L P 
               
               
                   
                   
                   
                 V Y 
                   
                 R S T V W 
               
               
                 57 
                 A D F G I M P Q R 
                 58 
                 A 
                 59 
                 A E I L M P R T V 
               
               
                   
                 V W 
                   
                   
                   
                 W Y 
               
               
                 60 
                 A D F G H I L N P 
                 61 
                 A E F G H N P Q R 
                 62 
                 A C D F I K L M P 
               
               
                   
                 Q S T V Y 
                   
                 T W Y 
                   
                 Q R S T V Y 
               
               
                 63 
                 C G P 
                 64 
                 A C D E F G H I K 
                 65 
                 A C D G H I K N R 
               
               
                   
                   
                   
                 L P Q R S T V W 
                   
                 S T V W Y 
               
               
                 66 
                 A C D E G I K L N 
                 67 
                 D E G P R T W 
                 68 
                 A C G I L P V Y 
               
               
                   
                 P S T V 
                   
                   
                   
                   
               
               
                 69 
                 N T 
                 70 
                 Q 
                 71 
                 P 
               
               
                 72 
                 C F H I P V W 
                 73 
                 P 
                 75 
                 D G P 
               
               
                 76 
                 A C F G I K L P Q 
                 77 
                 D E L P Q R T V 
                 78 
                 A D I M P T Y 
               
               
                   
                 R S T V W 
                   
                   
                   
                   
               
               
                 79 
                 A D F G H K N P S 
                 80 
                 A D E F G I K L M  
                 81 
                 A C E G H L N P S 
               
               
                   
                 W Y 
                   
                 N R S T V Y 
                   
                 V W Y 
               
               
                 82 
                 Y E K 
                 84 
                 Y 
                 85 
                 A C D E F G H N 
               
               
                   
                   
                   
                   
                   
                 Q S T 
               
               
                 86 
                 C P 
                 87 
                 P 
                 88 
                 A C E F G I K L M 
               
               
                   
                   
                   
                   
                   
                 P R S T V Y 
               
               
                 89 
                 A D E G Q S T W Y 
                 90 
                 C G 
                 91 
                 D E F G H I L T 
               
               
                 92 
                 E F H K P Q R W Y 
                 94 
                 G P 
                 95 
                 A C E F G H K L 
               
               
                   
                   
                   
                   
                   
                 M P Q S V W Y  
               
               
                 96 
                 S V H P R S T W 
                 98 
                 P 
                 99 
                 C E G I N P V W 
               
               
                 100 
                 C E F G N P R S T 
                 101 
                 A C F H I K L M N 
                 102 
                 P 
               
               
                   
                 W Y 
                   
                 Q R S T 
                   
                   
               
               
                 103 
                 A E F G H I L Q R T 
                 104 
                 F P W 
                 105 
                 C M N 
               
               
                   
                 V W Y 
                   
                   
                   
                   
               
               
                 106 
                 A C D F H L M N P 
                 107 
                 A C H K P Q S V W 
                 108 
                 D E F K L M P Q T 
               
               
                   
                 S W Y 
                   
                   
                   
                 V Y 
               
               
                 109 
                 C D E L M R T W 
                 110 
                 F K L M P W 
                 111 
                 H I Q 
               
               
                 112 
                 C E G H L N P S 
                 113 
                 R V 
                 114 
                 I L P T V 
               
               
                 115 
                 A C D F G H I K L 
                 116 
                 A C D E G H I L N 
                 117 
                 D G I K N Q R S V 
               
               
                   
                 M R S V Y 
                   
                 P Q S V W  
                   
                 W 
               
               
                 118 
                 C D E G P R W Y  
                 119 
                 A K I L N P R 
                 121 
                 A C E F G H K L 
               
               
                   
                   
                   
                   
                   
                 M P W Y 
               
               
                 122 
                 A C E F I K Q R S 
                 123 
                 A C D E H L M P Q 
                 124 
                 C D E F N 
               
               
                   
                 T V 
                   
                 R S T V Y 
                   
                   
               
               
                 125 
                 C D G L N W  
                 126 
                 F H I L N P Y 
                 127 
                 K 
               
               
                 128 
                 E P 
                 129 
                 A C D E G H L P Q 
                 130 
                 C D G H L N S T 
               
               
                   
                   
                   
                 S T V W 
                   
                 W Y 
               
               
                 131 
                 P 
                 132 
                 P 
                 133 
                 D E F G H L M N P 
               
               
                   
                   
                   
                   
                   
                 R T V W 
               
               
                 134 
                 A C D F G H K P Q 
                 135 
                 P 
                 136 
                 P 
               
               
                   
                 R S W 
                   
                   
                   
                   
               
               
                 137 
                 F G H N P R W Y 
                 138 
                 V 
                 139 
                 P 
               
               
                 143 
                 C H P R S T  
                 144 
                 A E F I K P Q S V 
                 145 
                 T W 
               
               
                   
                   
                   
                 Y 
                   
                   
               
               
                 149 
                 E 
                 149 
                 P 
                 150 
                 V 
               
               
                 152 
                 L 
                 153 
                 E F M P R T V 
                 154 
                 D E G P S W Y 
               
               
                 155 
                 P Y  
                 156 
                 P 
                 157 
                 A C D E G H I K L 
               
               
                   
                   
                   
                   
                   
                 M P Q R S T V 
               
               
                 158 
                 D K P R Y 
                 159 
                 W Y 
                 161 
                 W 
               
               
                 163 
                 C P 
                 164 
                 A C D E G H N P Q 
                 165 
                 C H P T 
               
               
                   
                   
                   
                 R 
                   
                   
               
               
                 166 
                 D 
                 167 
                 V 
                 168 
                 A C D E F G K L P 
               
               
                   
                   
                   
                   
                   
                 R S V W Y  
               
               
                 169 
                 A D F G H K N P Q 
                 170 
                 C D E G M P W Y 
                 171 
                 C D H M N R S W 
               
               
                   
                 S T Y 
                   
                   
                   
                 Y 
               
               
                 172 
                 D E I L P Q T V W 
                 173 
                 D E G H I L M P S 
                 174 
                 P 
               
               
                   
                 Y 
                   
                 V W Y 
                   
                   
               
               
                 175 
                 C D G K P R S 
                 176 
                 A C E F G H I P Q 
                 177 
                 A C D F G H L M 
               
               
                   
                   
                   
                 S T V W 
                   
                 Q R S T V W 
               
               
                 178 
                 E I L V W 
                 180 
                 A C E P R S 
                 181 
                 A C D E F H I K L 
               
               
                   
                   
                   
                   
                   
                 R S 
               
               
                 182 
                 A C D E H N P Q R 
                 183 
                 C D E G I K N P Q 
                 184 
                 A C D E F G H K L 
               
               
                   
                 S T V Y 
                   
                 R S V 
                   
                 M P R S V 
               
               
                 185 
                 A D E F G I K P R S 
                 186 
                 A D G H I K L N P  
                 187 
                 A F G H I L M N Q 
               
               
                   
                 T V W Y 
                   
                 Q R S V W 
                   
                 R S T V W Y 
               
               
                 188 
                 A C F G H L M N P 
                 189 
                 A E G H K L M N P 
                 190 
                 C E F G H K L N Q 
               
               
                   
                 Q R S T V W 
                   
                 R S T V W Y 
                   
                 R S T V W 
               
               
                 191 
                 A E F G K L M P Q 
                 192 
                 C F G K L M N P Q 
                 193 
                 A D K L M P V 
               
               
                   
                 R S T V W Y 
                   
                 R V W Y  
                   
                   
               
               
                 194 
                 A C I L P S T V 
                 195 
                 S 
                 197 
                 C 
               
               
                 198 
                 V W 
                 199 
                 E G H I K L P R S 
                 200 
                 A F G H K L M P 
               
               
                   
                   
                   
                 W 
                   
                 Q R S W Y 
               
               
                 201 
                 A F L M N P R S T 
                 202 
                 A E F G H K N P Q 
                 203 
                 A D E G H L M N  
               
               
                   
                 V W 
                   
                 R V W Y  
                   
                 Q R S T V 
               
               
                 204 
                 A C E G H I K Q R 
                 206 
                 C D F G P Y  
                 207 
                 A F G M P Q R S T 
               
               
                   
                 S T 
                   
                   
                   
                 V W 
               
               
                 208 
                 D G P W 
                 209 
                 C P 
                 210 
                 A C D E G K M N 
               
               
                   
                   
                   
                   
                   
                 P S T V W Y 
               
               
                 211 
                 C F G H I K M P R 
                 212 
                 A G H I K L M P V 
                 213 
                 P S 
               
               
                   
                 S T V W 
                   
                 W 
                   
                   
               
               
                 214 
                 A C D E G H K N P 
                 215 
                 C P 
                 216 
                 D E G H I K L M N 
               
               
                   
                 R S T Y 
                   
                   
                   
                 P Q R T V 
               
               
                 217 
                 A C G H P Q S T V 
                 218 
                 A I K L P S V 
                 219 
                 P 
               
               
                   
                 W 
                   
                   
                   
                   
               
               
                 220 
                 G K N P R W 
                 221 
                 D E H K P R 
                 222 
                 P Y 
               
               
                 223 
                 C D E G H K L P Q 
                 224 
                 A D E F G M P Q R 
                 225 
                 A D E G H K P Q 
               
               
                   
                 R S T V W Y  
                   
                 S T W Y 
                   
                 R T V W 
               
               
                 226 
                 A C D E F G L N Q 
                 227 
                 A F G H I K L M P 
                 228 
                 A E F G H L M N P 
               
               
                   
                 R S T V W Y 
                   
                 Q R T V W Y  
                   
                 R S T W 
               
               
                 229 
                 E F G K L P Q T V 
                 230 
                 A E G H K M N P R 
                 231 
                 A C D F G H I K L 
               
               
                   
                 W 
                   
                 S T V W Y 
                   
                 P Q R S V 
               
               
                 232 
                 C G H K L N P Q V 
                 233 
                 D I P S T 
                 234 
                 A D E G H N P S T 
               
               
                   
                 Y 
                   
                   
                   
                 V W 
               
               
                 235 
                 F L M R W Y 
                 236 
                 C I L N Q T Y 
                 238 
                 F G L P V W Y 
               
               
                 239 
                 C F G H I L P R S T 
                 240 
                 E F G N W Y  
                 241 
                 A C D E G I P R S 
               
               
                   
                 V W Y 
                   
                   
                   
                 T V W 
               
               
                 242 
                 A C D G I L M P R 
                 243 
                 C D F G H L M P Q 
                 244 
                 A D G I V Y 
               
               
                   
                 S T V W  
                   
                 R S W Y 
                   
                   
               
               
                 245 
                 A C F L P Q R S T 
                 246 
                 A C D E G H I K L 
                 247 
                 A C F H N P Q R S 
               
               
                   
                 V 
                   
                 M P S T V W 
                   
                 T W Y 
               
               
                 248 
                 C D E G I M P T  
                 249 
                 A G H I K M Q S Y 
                 250 
                 C F G H K L M N 
               
               
                   
                   
                   
                   
                   
                 P Q R S T V W 
               
               
                 251 
                 D F G H K P S T W 
                 252 
                 A D E F G H I K L 
                 253 
                 A D E G H L M N 
               
               
                   
                   
                   
                 N P S T Y 
                   
                 Q R S W 
               
               
                 254 
                 C D E G I K L P Q 
                 255 
                 C D L P V W 
                 256 
                 C D E G [ 
               
               
                   
                 R T V W Y 
                   
                   
                   
                   
               
               
                 257 
                 D 
                 258 
                 L P V W 
                 260 
                 C P 
               
               
                 261 
                 P 
                 262 
                 A D E G H I K Q R 
                 263 
                 E F P Q W 
               
               
                   
                   
                   
                 S T V W Y 
                   
                   
               
               
                 264 
                 D E F G L M R T V 
                 265 
                 A D F G H K L M 
                 266 
                 A C G H M P Q R 
               
               
                   
                 W Y 
                   
                 N Q R S 
                   
                 S T V W 
               
               
                 267 
                 D G H I K N R S W 
                 268 
                 A C F G H K L N P 
                 269 
                 E K L M N P Q R 
               
               
                   
                   
                   
                 Q S T V W  
                   
                   
               
               
                 270 
                 A C E F G H I P Y 
                 271 
                 A D E H K T W 
                 272 
                 H L N P W 
               
               
                 273 
                 A C D G I L P Q S 
                 274 
                 C E G H N Q W Y 
                 275 
                 A F G I K L M Q T 
               
               
                   
                 V W 
                   
                   
                   
                 V W 
               
               
                 276 
                 F P W 
                 278 
                 M P 
                 279 
                 A C F G L W Y 
               
               
                 280 
                 D I M N R S T V W 
                 281 
                 A D G H I K N P Q 
                 282 
                 F L V W Y 
               
               
                   
                   
                   
                 R S V W 
                   
                   
               
               
                 283 
                 A C D F W 
                 284 
                 C D F W 
                 284 
                 C I P 
               
               
                 285 
                 K P R T V 
                 286 
                 A C D F H K M P T 
                 287 
                 A C D E G K L N P 
               
               
                   
                   
                   
                 Y 
                   
                 Q R S 
               
               
                 288 
                 D E F G H I K P R T 
                 289 
                 A C E G H L P Q R 
                 290 
                 D Q Y 
               
               
                   
                   
                   
                 S Y 
                   
                   
               
               
                 291 
                 A C D E F M N T W 
                 292 
                 I L T 
                 293 
                 E N 
               
               
                   
                 Y 
                   
                   
                   
                   
               
               
                 294 
                 A E G H K L N P Q 
                 295 
                 C G H I L N P T V 
                 296 
                 C F G I K M Q R S 
               
               
                   
                 R S T W 
                   
                 Y 
                   
                 T V W Y  
               
               
                 297 
                 C E H L N P Q R S 
                 298 
                 C E L M N P Q S T 
                 299 
                 A C D F G H L M 
               
               
                   
                 T Y 
                   
                 W Y 
                   
                 P Q T 
               
               
                 300 
                 A C D E F L M N P 
                 301 
                 E G H K M N P Q R 
                 302 
                 C D E F G H L M P 
               
               
                   
                 Q S T V W  
                   
                 S W Y 
                   
                 R S T Y 
               
               
                 303 
                 A C D E F G K L M 
                 304 
                 A C D G I M N P Q 
                 305 
                 L P Q R S T V Y 
               
               
                   
                 R W Y 
                   
                 S T V Y 
                   
                   
               
               
                 306 
                 A C H I L V W Y 
                 307 
                 C I P 
                 308 
                 C F L M V W Y 
               
               
                 310 
                 C E F K L 
                 311 
                 C E F I L P V W 
                 312 
                 C E M V W 
               
               
                 313 
                 C 
                 314 
                 C L W 
                 315 
                 C I V 
               
               
                 316 
                 E G I K L M P R S T 
                 317 
                 G P 
                 318 
                 C P W 
               
               
                   
                 V W Y 
                   
                   
                   
                   
               
               
                 319 
                 C E F G H I K M P 
                 320 
                 C P V 
                 321 
                 E M P 
               
               
                   
                 Q R S V W Y 
                   
                   
                   
                   
               
               
                 322 
                 C D E G I L N P R S 
                 323 
                 A C E G H K N R S 
                 324 
                 C F P V W Y 
               
               
                   
                 T V W 
                   
                 T V 
                   
                   
               
               
                 325 
                 C R E G H N W 
                 327 
                 A E F G H N Q R S 
                 329 
                 C F G H I K L N Q 
               
               
                   
                   
                   
                 T V W Y  
                   
                 R S T V W Y 
               
               
                 330 
                 A C D E G I L M N 
                 331 
                 A C D E F H K Q R 
                 332 
                 A C D E F G H K L 
               
               
                   
                 P R S V W 
                   
                 S T W Y 
                   
                 N P R S T Y 
               
               
                 333 
                 G H I K P R S T W 
                 334 
                 A C D E G M N R S 
                 335 
                 F G H I K L P V W 
               
               
                   
                 Y 
                   
                   
                   
                 Y 
               
               
                 336 
                 A E F G K N P R S 
                 337 
                 C F G I K L M R T 
                 338 
                 C D E F G H I K L 
               
               
                   
                 T V W Y 
                   
                 W 
                   
                 P R T V 
               
               
                 339 
                 D E F G H L N P S 
                 340 
                 A C D E F G H K P 
                 341 
                 A E G H K L M N 
               
               
                   
                 T V W Y 
                   
                 R S T V W 
                   
                 Q R S T V Y 
               
               
                 342 
                 D E F H K L M P Q 
                 343 
                 C D F I P W 
                 344 
                 F G H L M N P Q 
               
               
                   
                 R T Y 
                   
                   
                   
                 R S T W Y 
               
               
                 345 
                 A C E H K N Q R T 
                 346 
                 A D F G I K L M P 
                 347 
                 C F I P T V W 
               
               
                   
                 V Y 
                   
                 R S T V W 
                   
                   
               
               
                 348 
                 C H I L P Q R T V 
                 349 
                 D F G P V W Y 
                 350 
                 A D E F H K L M 
               
               
                   
                 W Y 
                   
                   
                   
                 N P R S T V Y 
               
               
                 351 
                 C D E F H N R W Y 
                 352 
                 A D E F G K M P Q 
                 353 
                 C F G H K L M Q 
               
               
                   
                   
                   
                 R S T V W Y 
                   
                 R S W 
               
               
                 354 
                 C D E G H I K L M 
                 355 
                 D F G H L M N P Q 
                 356 
                 C G K L P R T V 
               
               
                   
                 P Q S V W Y  
                   
                 R S T V W Y 
                   
                 W 
               
               
                 357 
                 D E F G L M Q R 
                 358 
                 E H I K P Q R W 
                 359 
                 A F G L P W 
               
               
                 360 
                 A C E F G I K L M  
                 361 
                 A C E G M N P Q R 
                 362 
                 A C E G H K L M 
               
               
                   
                 P Q R V 
                   
                 S V W 
                   
                 N P R S T V W 
               
               
                 363 
                 A C D E F G H I P 
                 364 
                 A C D E F G K L M 
                 365 
                 A C D E G M N P 
               
               
                   
                 Q R S T V W  
                   
                 P R S T V Y 
                   
                 Q R S T W Y 
               
               
                 366 
                 A C E F G K M P Q 
                 367 
                 E F I L M Q V 
                 368 
                 C P W 
               
               
                   
                 R T W 
                   
                   
                   
                   
               
               
                 369 
                 C E F I K L P Q V 
                 370 
                 A D E G H K L N P 
                 371 
                 P 
               
               
                   
                 W 
                   
                 Q R S V Y 
                   
                   
               
               
                 372 
                 A D E F G H K L N 
                 373 
                 C P W 
                 374 
                 D E 
               
               
                   
                 P R S T V W 
                   
                   
                   
                   
               
               
                 375 
                 C F P V Y 
                 376 
                 I P W 
                 377 
                 C I L V 
               
               
                 378 
                 D E F I L M Q T W 
                 379 
                 A C E F I L M W  
                 380 
                 C D E G Q R S 
               
               
                   
                 Y 
                   
                   
                   
                   
               
               
                 381 
                 G L P W Y 
                 382 
                 E G H K L M N P Q 
                 383 
                 G P 
               
               
                   
                   
                   
                 R S T W Y  
                   
                   
               
               
                 384 
                 C F M Q S T 
                 385 
                 C L M P W Y 
                 386 
                 A C F G H I L M N 
               
               
                   
                   
                   
                   
                   
                 Q R S T V Y 
               
               
                 387 
                 C E F G H I L M N 
                 388 
                 C G P Q 
                 389 
                 F V 
               
               
                   
                 V W Y 
                   
                   
                   
                   
               
               
                 390 
                 A C E F G H L N P 
                 391 
                 A D G H K N P Q R 
                 392 
                 C P 
               
               
                   
                 R S T V W Y 
                   
                 S T V W Y 
                   
                   
               
               
                 393 
                 C P 
                 394 
                 A D E G I K N P Q 
                 395 
                 C ; , [ 
               
               
                   
                   
                   
                 R S T V 
                   
                   
               
               
                 396 
                 C F G I P Y 
                 397 
                 A C E F G I L M P 
                 398 
                 A C E G H I L N P 
               
               
                   
                   
                   
                 Q T V 
                   
                 R S T V W Y  
               
               
                 399 
                 D P 
                 400 
                 A D E F G I L M P 
                 401 
                 C F H K R W Y 
               
               
                   
                   
                   
                 Q R S T V Y 
                   
                   
               
               
                 402 
                 A D E F L M P Q R 
                 403 
                 A C E G H K L M 
                 404 
                 C D F G H L M N 
               
               
                   
                 S T V W Y  
                   
                 N P Q R T 
                   
                 R V W Y 
               
               
                 405 
                 C I V 
                 406 
                 P R 
                 408 
                 A E F G I K L P R 
               
               
                   
                   
                   
                   
                   
                 S T V W Y 
               
               
                 410 
                 W 
                 411 
                 D E F G 
                 412 
                 E H 
               
               
                 413 
                 H I K L P 
                 414 
                 A D E G H K R S T 
                 415 
                 C D E P 
               
               
                 416 
                 C S 
                 417 
                 A D E F G H K M P 
                 419 
                 D P 
               
               
                   
                   
                   
                 Q R 
                   
                   
               
               
                 420 
                 A D F G H K L N R 
                 422 
                 C D G H L M N Q 
                 423 
                 A D E F G H L M P 
               
               
                   
                 S T W Y 
                   
                 R S Y 
                   
                 Q R S T V W 
               
               
                 424 
                 A C E G H N Q R S 
                 425 
                 E L P W Y 
                 426 
                 C F M R 
               
               
                   
                 W Y 
                   
                   
                   
                   
               
               
                 427 
                 A C F L P V W Y 
                 428 
                 A C D E G H N R S 
                 429 
                 A D K L N P S T V 
               
               
                   
                   
                   
                 Y 
                   
                 W Y 
               
               
                 430 
                 A D E L M N S T V 
                 431 
                 P 
                 432 
                 C F I K L M P Y 
               
               
                 434 
                 H K P Q R W 
                 437 
                 T 
                 438 
                 Y 
               
               
                 439 
                 N R 
                 440 
                 Q 
                 441 
                 R 
               
               
                 442 
                 M N S 
                 443 
                 D 
               
               
                   
               
            
           
         
       
     
     3. Additional Modifications and Conjugates 
     The modified PH20 polypeptides include those that contain chemical or posttranslational modifications. In some examples, modified PH20 polypeptides provided herein do not contain chemical or posttranslational modifications. Chemical and post-translational modifications include, but are not limited to, PEGylation, sialation, albumination, glycosylation, farnysylation, carboxylation, hydroxylation, phosphorylation, and other polypeptide modifications known in the art. 
     Also, in addition to any one or more amino acid modifications, such as amino acid replacements, provided herein, modified PH20 polypeptides provided herein can be conjugated or fused to any moiety using any method known in the art, including chemical and recombinant methods, provided the resulting polypeptide retains hyaluronidase activity. For example, in addition to any one or more amino acid modifications, such as amino acid replacements, provided herein, modified PH20 polypeptides provided herein also can contain other modifications that are or are not in the primary sequence of the polypeptide, including, but not limited to, modification with a carbohydrate moiety, a polyethylene glycol (PEG) moiety, a sialic acid moiety, an Fc domain from immunoglobulin G, or any other domain or moiety. For example, such additional modifications can be made to increase the stability or serum half-life of the protein. 
     In some instances, the domain or other moiety is a targeted agent, including any agent that targets the conjugate to one or more cell types by selectively binding to a cell surface receptor or other cell surface moiety. For example, the domain or other moiety is a targeted agent that targets the conjugate to tumor cells. In such examples, a modified PH20 polypeptide, such as any provided herein, is linked directly or indirectly to a targeted agent. Such targeting agents include, but are not limited to, growth factors, cytokines, chemokines, antibodies, and hormones, or allelic variants, muteins, or fragments thereof so long as the targeting agent is internalized by a cell surface receptor. Exemplary, non-limiting, additional modifications are described below. 
     a. Decreased Immunogenicity 
     The modified PH20 polypeptides provided herein can be made to have decreased immunogenicity. Decreased immunogenicity can be effected by sequence changes that eliminate antigenic epitopes from the polypeptide or by altering post-translational modifications. One of skill in the art is familiar with methods of identifying antigenic epitopes in a polypeptide (see e.g., Liang et al. (2009)  BMC Bioinformatics,  10:302; Yang et al. (2009)  Rev. Med. Vivol.,  19:77-96). In some examples, one or more amino acids can be modified in order to remove or alter an antigenic epitope. 
     In another example, altering the glycosylation of a protein also can effect immunogenicity. For example, altering the glycosylation of the peptide is contemplated, so long as the polypeptides minimally contain at least N-acetylglucosamine at amino acid residues corresponding to amino acid residues set forth as N200, N333 and N358 of SEQ ID NO:3 or 7. 
     For example, the PH20 polypeptides can be modified such that they lack fucose, particularly bifucosylation. In particular, the PH20 polypeptides provided herein are not bifucosylated. This can be achieved by expressing and producing the PH20 polypeptide in host cells that do not effect bifucosylation. Fucose is a deoxyhexose that is present in a wide variety of organisms, including mammals, insects and plants. Fucosylated glycans are synthesized by fucosyl-transferases; see, e.g., Ma et al.,  Glycobiology,  16(12):158R-184R, (2006); Nakayama et al.,  J. Biol. Chem.,  276:16100-16106 (2001); and Sturla et al.,  Glycobiology,  15(10):924-935 (2005). In humans, fucose frequently exists as a terminal modification to glycan structures, and the presence of fucose α1,6-linked to N-acetylglucosamine has been shown to be important in glycoprotein processing and recognition. In insects, N-glycan core structures exhibit bifucosylation with α1,6- and α1,3-linkages. Insect cell core fucosylation with α1,3-linkages generates a carbohydrate epitope that is immunogenic in humans (see, e.g., US Publication No. 20070067855). For example, PH20 polypeptides provided herein can be generated in host cells that are incapable of bifucosylating the polypeptide. Thus, while insect cells or other cells that bifucosylate can be used for expression of the polypeptides, typically mammalian cells, such as CHO cells, are used. 
     In some examples, defucosylated, or fucose-deficient PH20 polypeptides can be generated in insect cells with modified glycosylation pathways, through the use of baculovirus expression vectors containing eukaryotic oligosaccharide processing genes, thereby creating “mammalianized” insect cell expression systems (see, e.g., U.S. Pat. No. 6,461,863). Alternatively, antigenicity can be eliminated by expression of PH20 polypeptides in insect cells lacking α1,3-fucosylatransferase (FT3) (see, e.g., US Publication No. 20070067855). In other examples, defucosylated or fucose-deficient PH20 polypeptides can be generated, for example, in cell lines that produce defucosylated proteins, including Lec13 CHO cells deficient in protein fucosylation (Ripka et al.  Arch. Biochem. Biophys.  249:533-545 (1986); U.S. Pat. Pub. No. 2003/0157108; and WO 2004/056312), and knockout cell lines, such as alpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (Yamane-Ohnuki et al.  Biotech. Bioeng.  87: 614 (2004)). 
     b. Conjugation to Polymers 
     In some examples, the modified PH20 polypeptides provided herein are conjugated to polymers. Exemplary polymers that can be conjugated to the PH20 polypeptides, include natural and synthetic homopolymers, such as polyols (i.e., poly-OH), polyamines (i.e., poly-NH 2 ) and polycarboxylic acids (i.e., poly-COOH), and further heteropolymers, i.e., polymers containing one or more different coupling groups, e.g., hydroxyl groups and amine groups. Examples of suitable polymeric molecules include polymeric molecules selected from among polyalkylene oxides (PAO), such as polyalkylene glycols (PAG), including polyethylene glycols (PEG), methoxypolyethylene glycols (mPEG) and polypropylene glycols, PEG-glycidyl ethers (Epox-PEG), PEG-oxycarbonylimidazole (CDI-PEG), branched polyethylene glycols (PEGs), polyvinyl alcohol (PVA), polycarboxylates, polyvinylpyrrolidone, poly-D,L-amino acids, polyethylene-co-maleic acid anhydride, polystyrene-co-maleic acid anhydride, dextrans including carboxymethyl-dextrans, heparin, homologous albumin, celluloses, including methylcellulose, carboxymethylcellulose, ethylcellulose, hydroxyethylcellulose, carboxyethylcellulose and hydroxypropylcellulose, hydrolysates of chitosan, starches such as hydroxyethyl-starches and hydroxypropyl-starches, glycogen, agaroses and derivatives thereof, guar gum, pullulan, inulin, xanthan gum, carrageenan, pectin, alginic acid hydrolysates and bio-polymers. 
     Typically, the polymers are polyalkylene oxides (PAO), such as polyethylene oxides, such as PEG, typically mPEG, which have few reactive groups capable of cross-linking. Typically, the polymers are non-toxic polymeric molecules such as (methoxy)polyethylene glycol (mPEG) which can be covalently conjugated to the PH20 polypeptides (e.g., to attachment groups on the protein surface) using a relatively simple chemistry. 
     Suitable polymeric molecules for attachment to the PH20 polypeptides include, but are not limited to, polyethylene glycol (PEG) and PEG derivatives such as methoxy-polyethylene glycols (mPEG), PEG-glycidyl ethers (Epox-PEG), PEG-oxycarbonylimidazole (CDI-PEG), branched PEGs, and polyethylene oxide (PEO) (see e.g., Roberts et al.,  Advanced Drug Delivery Review  2002, 54:459-476; Harris and Zalipsky (eds.) “Poly(ethylene glycol), Chemistry and Biological Applications” ACS Symposium Series 680, 1997; Mehvar et al.,  J. Pharm. Pharmaceut. Sci.,  3(1):125-136, 2000; Harris and Chess (2003)  Nat Rev Drug Discov.  2(3):214-21; and Tsubery,  J Biol. Chem  279(37):38118-24, 2004). The polymeric molecule can be of a molecular weight typically ranging from about 3 kDa to about 60 kDa. In some embodiments the polymeric molecule that is conjugated to a PH20 polypeptide provided herein has a molecular weight of 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 or more than 60 kDa. 
     Various methods of modifying polypeptides by covalently attaching (conjugating) a PEG or PEG derivative (i.e., “PEGylation”) are known in the art (see e.g., U.S. 2006/0104968; U.S. Pat. Nos. 5,672,662; 6,737,505; and U.S. 2004/0235734). Techniques for PEGylation include, but are not limited to, specialized linkers and coupling chemistries (see e.g., Roberts,  Adv. Drug Deliv. Rev.  54:459-476, 2002), attachment of multiple PEG moieties to a single conjugation site (such as via use of branched PEGs; see e.g., Guiotto et al.,  Bioorg. Med. Chem. Lett.  12:177-180, 2002), site-specific PEGylation and/or mono-PEGylation (see e.g., Chapman et al., Nature Biotech. 17:780-783, 1999), and site-directed enzymatic PEGylation (see e.g., Sato,  Adv. Drug Deliv. Rev.,  54:487-504, 2002) (see, also, for example, Lu and Felix (1994)  Int. J. Peptide Protein Res.  43:127-138; Lu and Felix (1993)  Peptide Res.  6:140-6, 1993; Felix et al. (1995)  Int. J. Peptide Res.  46:253-64; Benhar et al. (1994)  J. Biol. Chem.  269:13398-404; Brumeanu et al. (1995)  J Immunol.  154:3088-95; see also, Caliceti et al. (2003)  Adv. Drug Deliv. Rev.  55(10):1261-77 and Molineux (2003)  Pharmacotherapy  23 (8 Pt 2):3S-8S). Methods and techniques described in the art can produce proteins having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 PEG or PEG derivatives attached to a single protein molecule (see e.g., U.S. 2006/0104968). 
     Numerous reagents for PEGylation have been described in the art. Such reagents include, but are not limited to, N-hydroxysuccinimidyl (NHS) activated PEG, succinimidyl mPEG, mPEG2-N-hydroxysuccinimide, mPEG succinimidyl alpha-methylbutanoate, mPEG succinimidyl propionate, mPEG succinimidyl butanoate, mPEG carboxymethyl 3-hydroxybutanoic acid succinimidyl ester, homobifunctional PEG-succinimidyl propionate, homobifunctional PEG propionaldehyde, homobifunctional PEG butyraldehyde, PEG maleimide, PEG hydrazide, p-nitrophenyl-carbonate PEG, mPEG-benzotriazole carbonate, propionaldehyde PEG, mPEG butryaldehyde, branched mPEG2 butyraldehyde, mPEG acetyl, mPEG piperidone, mPEG methylketone, mPEG “linkerless” maleimide, mPEG vinyl sulfone, mPEG thiol, mPEG orthopyridylthioester, mPEG orthopyridyl disulfide, Fmoc-PEG-NHS, Boc-PEG-NHS, vinylsulfone PEG-NHS, acrylate PEG-NHS, fluorescein PEG-NHS, and biotin PEG-NHS (see e.g., Monfardini et al.,  Bioconjugate Chem.  6:62-69, 1995; Veronese et al.,  J. Bioactive Compatible Polymers  12:197-207, 1997; U.S. Pat. Nos. 5,672,662; 5,932,462; 6,495,659; 6,737,505; 4,002,531; 4,179,337; 5,122,614; 5,324,844; 5,446,090; 5,612,460; 5,643,575; 5,766,581; 5,795,569; 5,808,096; 5,900,461; 5,919,455; 5,985,263; 5,990,237; 6,113,906; 6,214,966; 6,258,351; 6,340,742; 6,413,507; 6,420,339; U.S. Pat. Nos. 6,437,025; 6,448,369; 6,461,802; 6,828,401; 6,858,736; U.S. 2001/0021763; U.S. 2001/0044526; U.S. 2001/0046481; U.S. 2002/0052430; U.S. 2002/0072573; U.S. 2002/0156047; U.S. 2003/0114647; U.S. 2003/0143596; U.S. 2003/0158333; U.S. 2003/0220447; U.S. 2004/0013637; US 2004/0235734; U.S. 2005/0114037; U.S. 2005/0171328; U.S. 2005/0209416; EP 1064951; EP 0822199; WO 01076640; WO 0002017; WO 0249673; WO 9428024; WO 0187925; and WO 2005000360). 
     D. METHODS FOR IDENTIFYING MODIFIED HYALURONAN-DEGRADING ENZYMES WITH ALTERED PROPERTIES OR ACTIVITIES 
     Provided herein are methods for identifying a modified or variant hyaluronan-degrading enzyme, such as a modified hyaluronidase or modified PH20 polypeptide, that exhibits an altered activity or property compared to an unmodified hyaluronan-degrading enzyme. In particular, the methods provided herein can be used to screen for one or more modified hyaluronan-degrading enzymes, such as one or more modified hyaluronidase or PH20 polypeptide, that exhibits increased activity and/or increased stability in the presence of a denaturation agent or condition. For example, the methods can be used to identify a modified or variant hyaluronan-degrading enzyme, such as a modified or variant hyaluronidase or modified or variant PH20 polypeptide, that exhibits increased stability by virtue of increased resistance to denaturation conditions, including but not limited to, denaturation conditions caused by temperature (e.g., elevated temperature such as heat), agitation, no or low salt, presence of an excipient and/or a denaturing agent. Exemplary denaturing agents or excipients include, but are not limited to, antiadherents, binders, coatings, fillers and diluents, flavors, colors, lubricants, glidants, preservatives, sorbents or sweeteners. For example, various excipients, such as preservatives, can act as protein denaturing agents. In the method, the activity also can be compared to an unmodified hyaluronan-degrading enzyme under the same denaturation condition, and a modified hyaluronan-degrading enzyme identified or selected that exhibits greater activity than the corresponding unmodified hyaluronan-degrading enzyme. 
     In the method, one or more modified hyaluronan-degrading enzymes are provided. In some examples, a library of modified molecules is prepared. Methods of mutagenesis and generation of libraries or collections of variant molecules is described herein and is known to one of skill in the art using standard recombinant DNA techniques. In one example, the enzymes that are tested can be pooled and screened, whereby the method permits selection of only those enzymes that exhibit a desired activity. In another example, the tested enzymes can be physically separated and screened individually, such as by formatting in arrays, such as addressable arrays. 
     In one aspect of the method, the modified hyaluronan-degrading enzymes are tested or screened for hyaluronidase activity in the presence and absence of one or more denaturation conditions or denaturing agent. After testing under both sets of conditions, the activities are assessed in order to identify modified hyaluronan-degrading enzymes that exhibit activity in the presence of the denaturation condition. The desired level or amount of activity selected as a cut-off in the methods can be empirically determined by the user, and is dependent on factors such as the particular hyaluronan-degrading enzyme, the desired application or use of the hyaluronan-degrading enzyme, the particular denaturation condition or denaturing agent and other similar factors. Typically, a modified hyaluronan-degrading enzyme is identified that exhibits at least 5% or 10% of the activity in the presence of a denaturing agent or condition compared to in its absence, and generally at least 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more, for example at least 40% of the activity. 
     Additionally or alternatively, the activity of the modified hyaluronan-degrading enzyme in the presence of one or more denaturation conditions or denaturing agents is compared to the activity of the corresponding unmodified hyaluronan-degrading enzyme in the presence of the same denaturation agent(s) or condition(s). In such examples, it is understood that the activity of the modified and unmodified enzyme are tested under the same conditions (e.g., time, temperature, composition), except for the difference in the particular enzyme tested (unmodified versus modified). A modified hyaluronan-degrading enzyme is identified that exhibits greater activity, such as at least 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 250%, 300%, 400%, 500% or more of the activity of the unmodified hyaluronan-degrading enzyme. 
     The method can be performed a plurality of times, whereby the steps of the method are repeated 1, 2, 3, 4, or 5 times. The method provided herein also is iterative. In one example, after the method is performed, any identified modified hyaluronan-degrading enzyme can be modified or further modified to increase or optimize the activity. 
     A description of the steps of the method and components of the method are provided in the subsections that follow. 
     1. Hyaluronan-Degrading Enzymes and Libraries of Modified Hyaluronan-Degrading Enzymes 
     In the methods herein, one or more modified hyaluronan-degrading enzymes, such as a hyaluronidase or a PH20 polypeptide, are tested for a desired activity or property, such as increased stability (e.g., increased resistance to a denaturation condition). The modified hyaluronan-degrading enzyme can be modified compared to an unmodified hyaluronan-degrading enzyme, such as any hyaluronan-degrading enzyme known in the art. Hyaluronan-degrading enzymes are a family of enzymes that degrade hyaluronic acid, which is an essential component of the extracellular matrix and a major constituent of the interstitial barrier. Hyaluronan-degrading enzymes act to degrade hyaluronan by cleaving hyaluronan polymers, which are composed of repeating disaccharides units: D-glucuronic acid (GlcA) and N-acetyl-D-glucosamine (GlcNAc), linked together via alternating β-1→4 and β-1→3 glycosidic bonds. By catalyzing the hydrolysis of hyaluronic acid, a major constituent of the interstitial barrier, hyaluronan-degrading enzymes lower the viscosity of hyaluronic acid, thereby increasing tissue permeability. Accordingly, hyaluronan-degrading enzymes for the uses and methods provided herein include any enzyme having the ability to catalyze the cleavage of a hyaluronan disaccharide chain or polymer. In some examples, the hyaluronan-degrading enzyme cleaves the β-1→4 glycosidic bond in the hyaluronan chain or polymer. In other examples, the hyaluronan-degrading enzyme catalyzes the cleavage of the β-1→3 glycosidic bond in the hyaluronan chain or polymer. 
     Hyaluronan-degrading enzymes include enzymes that are membrane-bound or that are soluble forms that are secreted from cells. Thus, where hyaluronan-degrading enzymes include a glycosylphosphatidylinositol (GPI) anchor signal sequence and/or are otherwise membrane-anchored or insoluble, such hyaluronan-degrading enzymes can be provided in soluble form by C-terminal truncation or deletion of all or a portion of the GPI anchor signal sequence to render the enzyme secreted and soluble. Thus, hyaluronan-degrading enzymes include C-terminally truncated variants, e.g., truncated to remove all or a portion of a GPI anchor signal sequence. Examples of such soluble hyaluronidases are soluble PH20 hyaluronides, such as any set forth in U.S. Pat. No. 7,767,429; U.S. Publication Nos. 2004/0268425 and 2010/0143457. 
     Exemplary hyaluronan-degrading enzymes are non-human animal or human hyaluronidases, bacterial hyaluronidases, hyaluronidases from leeches or chondroitinases that exhibit hyaluronan-degrading activity, including soluble or truncated forms thereof that are active. Exemplary non-human animal hyaluronidases are any set forth in any of SEQ ID NOs: 8-31, 856-861, 869, 870, 871-886, or mature, C-terminally truncated variants that are soluble and active, or active forms thereof. Exemplary human hyaluronidases are any set forth in any of SEQ ID NOs: 2, 3, 6, 7, 32-66, 68-72 or 887-890, or mature, C-terminally truncated variants that are soluble and active, or active forms thereof, and in particular any of SEQ ID NOs: 3, 7, 32-66, 69 or 72. Exemplary bacterial hyaluronidases are any set forth in any of SEQ ID NOs: 891-919 or mature, C-terminally truncated variants that are soluble and active, or active forms thereof. Exemplary hyaluronidases from leeches are set forth in SEQ ID NO:920 or 921, or mature, C-terminally truncated variants that are soluble and active, or active forms thereof. Exemplary chondroitinases that have hyaluronan-degrading enzyme activity are set forth in SEQ ID NO:922-924, or mature, C-terminally truncated variants that are soluble and active, or active forms thereof. 
     For example, one or more modified PH20 polypeptides are tested for a desired activity or property, such as increased stability (e.g., increased resistance to a denaturation condition). The modified PH20 polypeptide can be modified compared to an unmodified PH20 polypeptide, such as any known PH20 polypeptide native, wildtype or reference polypeptide. For example, the modified PH20 polypeptide is modified compared to a full-length, soluble or active form of a PH20 polypeptide, such as any set forth in any of SEQ ID NOs: 3, 7, 32-66, 69 or 72, or a polypeptide that exhibits at least 85%, such as at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NOs: 3, 7, 32-66, 69 or 72. In particular examples of the method herein, the starting or unmodified PH20 polypeptide has the sequence of amino acids set forth in SEQ ID NO:3. 
     Libraries or collections of modified hyaluronan-degrading enzymes can be screened. Hyaluronan-degrading enzymes can be modified by any process known to one of skill in the art that can alter the structure of a protein. Examples of modifications include replacement, addition, and deletion of one or more amino acids of the protein to form libraries or collections of modified hyaluronan-degrading enzymes. It is within the level of one of skill in the art to generate modified or variant proteins for use in the methods herein. Methods of mutagenesis are well known in the art and include, for example, site-directed mutagenesis such as for example QuikChange (Stratagene) or saturation mutagenesis. Mutagenesis methods include, but are not limited to, site-mediated mutagenesis, PCR mutagenesis, cassette mutagenesis, site-directed mutagenesis, random point mutagenesis, mutagenesis using uracil containing templates, oligonucleotide-directed mutagenesis, phosphorothioate-modified DNA mutagenesis, mutagenesis using gapped duplex DNA, point mismatch repair, mutagenesis using repair-deficient host strains, restriction-selection and restriction-purification, deletion mutagenesis, mutagenesis by total gene synthesis, double-strand break repair, and many others known to persons of skill. In the methods herein, mutagenesis can be effected across the full length of a protein or within a region of a protein. The mutations can be made rationally or randomly. 
     In some examples, the methods provided herein are performed such that the identity of each mutant protein is known a priori before the protein is tested. For example, the methods provided herein can be conducive to mutagenesis and screening or testing methods that are addressable. This can permit the ease of comparisons between the activities of tested proteins without the need for sequencing of identified proteins. For example, site-directed mutagenesis methods can be used to individually generate mutant proteins. Mutagenesis can be performed by the replacement of single amino acid residues at specific target positions one-by-one, such that each individual mutant generated is the single product of each single mutagenesis reaction. Mutant DNA molecules can be designed, generated by mutagenesis and cloned individually, such as in addressable arrays, such that they are physically separated from each other and each one is the single product of an independent mutagenesis reaction. The amino acids selected to replace the target positions on the particular protein being optimized can be either all of the remaining 19 amino acids, or a more restricted group containing only selected amino acids. In some methods provided herein, each amino acid that is replaced is independently replaced by 19 of the remaining amino acids or by less than 19 of the remaining amino acids, such as 10, 11, 12, 13, 14, 15, 16, 17 or 18 of the remaining amino acids. 
     2. Screening or Testing For A Desired Activity or Property 
     The hyaluronidase activity or other activity of a composition containing a modified hyaluronan-degrading enzyme is screened or tested under conditions that expose the hyaluronan-degrading enzyme to a denaturation condition or a denaturing agent (presence of denaturation condition or denaturing agent). The denaturing condition or denaturing agent need not be a condition or agent that is completely deadly to the enzyme, but generally is any condition or agent that destabilizes enzyme activity over time. For example, the denaturation condition can be a condition caused by temperature (e.g., elevated temperature such as greater than or about or 30° C., for example, 30° C. to 42° C. such as or about 37° C.), agitation, no or low salt (e.g., NaCl), and/or caused by the presence of a denaturing agent, such as the presence of excipients (e.g., presence of preservatives). 
     For purposes of selecting or identifying a modified hyaluronan-degrading enzyme that exhibits stability or increased stability under the denaturation condition, activity can be compared to activity of the modified hyaluronan-degrading enzyme in the absence of the denaturation condition and/or activity of the corresponding unmodified hyaluronan-degrading enzyme in the presence of the denaturation condition. For example, the modified hyaluronan-degrading enzyme also can be screened or tested under the same conditions, except not including a denaturing condition or denaturing agent (absence of denaturation condition or denaturing agent). If desired, the activity of the corresponding unmodified hyaluronan-degrading enzyme (e.g., the hyaluronan-degrading enzyme not containing the amino acid replacement(s)) can also be tested under the same conditions that expose the hyaluronan-degrading enzyme to the same denaturation condition or a denaturing agent. 
     For example, each member of a library or collection of modified hyaluronan-degrading enzymes is incubated under or exposed to one or more denaturation conditions. The incubation or exposure can occur in vivo or in vitro. Typically, the assay is performed in vitro. The same modified enzyme also is exposed or incubated to a reference or control condition that does not contain the denaturation condition. The activities under both conditions are compared in order to identify modified hyaluronan-degrading enzymes that exhibit stability upon exposure to a denaturation condition or conditions. Further, in screening or identifying the activity of the enzyme under the two different sets of conditions, generally the only conditions that are varied in the assay relate to the presence or absence of one or more denaturation conditions. The other conditions of the assay, including but not limited to, time, temperature and/or other incubation conditions, can be the same for both sets of conditions. 
     For example, exposure can be achieved by incubation of a modified hyaluronan-degrading enzyme in an assay buffer or composition that has been modified or adjusted to contain a denaturing agent such as an excipient or low or no salt. Exemplary denaturing agents or excipients include, but are not limited to, antiadherents, binders, coatings, fillers and diluents, flavors, colors, lubricants, glidants, preservatives, sorbents or sweeteners. The choice of buffer that is used can be empirically determined by one skilled in the art depending on the particular parameter or parameters being modified. Exemplary assay buffers are Good&#39;s buffers (see e.g., Good et al. (1966) Biochemistry, 5:467-477). Examples of such buffers include, but are not limited to ACES, ADA, BES, Bicine, BIS-TRIS, CAPS, HEPES, MES, MOPS, PIPES, TRIS or Trizma® buffers. Further, the amount or concentration of the excipient or salt can be empirically determined by one of skill in the art depending on the choice of excipient or salt and the desired level or activity of the modified hyaluronan-degrading enzyme. 
     In one example, the assay buffer or composition is modified by inclusion of an amount of a denaturing agent or denaturing excipient that is a preservative, for example, a phenolic preservative. The phenolic preservative can be phenol, metacresol (m-cresol), benzyl alcohol, and parabens including methylparaben and propylparaben. In particular, the phenolic preservative is phenol and/or m-cresol. The total amount of one or more phenolic preservative agents as a percentage (%) of mass concentration (w/v) can be between 0.05% to 0.6%, 0.1% to 0.4%, 0.1% to 0.3%, 0.15% to 0.325%, 0.15% to 0.25%, 0.1% to 0.2%, 0.2% to 0.3% or 0.3% to 0.4% inclusive. In such an example, the activity of the modified hyaluronan-degrading enzyme is tested or assessed in the presence of such a total amount (e.g., between or about between 0.05% to 0.6%) of one or more preservatives, for example, one or more phenolic preservatives. In some examples, the modified hyaluronan-degrading enzyme also can be tested or assessed under a control or reference condition in which the assay buffer or composition is not modified to contain a preservative. In certain instances, as a control, the activity of modified hyaluronan-degrading enzymes also can be compared to the corresponding unmodified hyaluronan-degrading enzyme not containing the modification(s) under conditions that contain a preservative agent and/or under conditions that do not contain a preservative agent. 
     In another example, the assay buffer is modified by the presence of a denaturation condition that is low or no salt. As discussed elsewhere herein, hyaluronan-degrading enzymes, such as PH20, generally require salt (e.g., NaCl, Lys-Lys or MgCl 2 ) for activity. Hence, the absence of salt or low salt is denaturing to the enzyme. In one example, the assay buffer is modified by inclusion of an amount of salt that is less than 100 mM, for example, less than 90 mM, 80 mM, 70 mM, 60 mM, 50 mM, 40 mM, 30 mM, 25 mM, 20 mM, 15 mM, 10 mM, 5 mM or less. In such an example, the activity of the modified hyaluronan-degrading enzyme is tested in the absence of salt or in the presence of salt that is less than 100 mM. In some examples, the modified hyaluronan-degrading enzyme also can be tested or assessed under a control or reference condition in which the assay buffer contains a higher salt concentration, generally between or about between 140 mM to 200 mM. In certain instances, as a control, the activity of modified hyaluronan-degrading enzymes also can be compared to the corresponding unmodified hyaluronan-degrading enzyme not containing the modification(s) under conditions that contain low or no salt, such as less than 100 mM and/or under conditions that contain salt in an amount that is between or about between 140 mM to 200 mM. 
     Exposure of a hyaluronan-degrading enzyme to a denaturation condition also can be achieved by incubation of a modified hyaluronan-degrading enzyme under conditions that are known to be denaturing, such as under conditions of elevated temperature such as a temperature greater than or about or 30° C. (e.g., 30° C. to 42° C. such as or about 37° C.) or agitation. For example, the activity of the modified hyaluronan-degrading enzyme is tested at elevated temperatures greater than or about or 30° C. to 42° C. In some examples, the modified hyaluronan-degrading enzyme also can be tested or assessed under a control or reference condition where the temperatures is less than 30° C., such as between or about between 0° C. to 25° C., for example, 0° C. to 5° C. or 18° C. to 25° C. In certain instances, as a control, the activity of modified hyaluronan-degrading enzymes also can be compared to the corresponding unmodified hyaluronan-degrading enzyme not containing the modification(s) under elevated temperatures greater than or about or 30° C. to 42° C. and/or temperatures is less than 30° C., such as between or about between 0° C. to 25° C., for example, 0° C. to 5° C. or 18° C. to 25° C. 
     The modified hyaluronan-degrading enzyme can be exposed to one or more than one of the conditions. The exposure to one condition can occur simultaneously, subsequently, intermittently or periodically to exposure to one or more other conditions. 
     In one example, in the method herein, the modified hyaluronan-degrading enzyme is incubated or exposed to the denaturation condition or denaturing agent prior to performing an assay for hyaluronidase activity. For example, the modified hyaluronan-degrading enzyme is incubated in the presence of a denaturing agent or exposed to one or more denaturation conditions or control conditions, such as one or more of the denaturation conditions or control conditions as described above. The incubation or exposure can be for any desired length of time, and can be empirically determined by one of skill in the art. For example, the modified hyaluronan-degrading enzyme can be incubated or exposed to one or more denaturation conditions, denaturing agents or control conditions for or about for 1 minute to 1 month, such as 1 minute to 3 weeks, 1 minute to 2 weeks, 1 minute to 1 week, 1 minute to 24 hours, 1 minute to 12 hours, such as 30 minutes to 6 hours or 1 hour to 4 hours, and generally at least or about at least 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours or 12 hours. After the time of incubation or exposure, the sample or composition containing the modified hyaluronan-degrading enzyme (or control unmodified enzyme) is assessed for hyaluronidase assay. In another example, the modified hyaluronan-degrading enzyme is exposed or incubated under one or more denaturation conditions and is simultaneously or concurrently assessed for hyaluronidase activity. In any examples where a modified hyaluronan-degrading enzyme is assessed, it is understood that an unmodified hyaluronan-degrading enzyme not containing the modifications(s) also can be assessed under similar assay conditions for comparison. 
     Assays to assess hyaluronidase activity are well known in the art. Examples of such assays are described in Section G. In one example, hyaluronidase activity can be assessed in a microturbidity assay, wherein the amount of undegraded HA is measured by the addition of a reagent that precipitates HA (e.g., Cetylpyridinium chloride (CPC) or acidified serum) after the enzyme is allowed to react with HA. In another example, hyaluronidase activity can be assessed using a microtiter assay in which residual biotinylated hyaluronic acid is measured following incubation with hyaluronidase (see e.g., Frost and Stern (1997)  Anal. Biochem.  251:263-269, U.S. Pat. Publication No. 20050260186). The resulting activities under each of the tested conditions is determined and compared. 
     3. Selection or Identification 
     In the method, after screening modified hyaluronan-degrading enzymes under one or more denaturation conditions, the hyaluronidase activities of the tested enzymes are compared. The method is practiced in order to identify a modified hyaluronan-degrading enzyme that is more resistant to denaturation by a condition or a denaturing agent, whereby the activity of the enzyme is indicative of the stability of the enzyme as a measure of its resistance to denaturation. It is understood that some reduction of enzyme activity, as a result of denaturation, can be tolerated in various applications, and thus the method can be practiced to select for a modified hyaluronan-degrading enzymes that exhibits a requisite activity upon exposure to a denaturation condition to permit its use or application (e.g., therapeutic activity). For example, a modified enzyme can be selected that loses activity more slowly than the corresponding unmodified or reference hyaluronan-degrading enzyme, but whose retained activity is sufficient for a particular application or purpose. 
     In examples of the methods herein, the activity of the modified hyaluronan degrading enzyme is assessed upon exposure to a first denaturation condition and also assessed upon exposure to a second condition that is a control or non-denaturation condition, and the resulting hyaluronidase activities compared. For comparison, in some examples, the activity can be represented as a ratio of activity or a percentage of activity under a denaturation condition compared to under a control or non-denaturation condition. For example, where the parameter that differs between the first and second condition is the presence of preservative (e.g., phenolic preservative), activity can be represented as a ratio of activity or percentage of activity observed in the presence of preservative (e.g., phenolic preservative) versus activity in the absence of preservative (e.g., phenolic preservative). In another example, where the parameter that differs between the first and second condition is temperature, activity can be represented as a ratio of activity or percentage of activity observed in the presence of elevated temperature (e.g., 30° C. to 42° C.) compared to activity in the presence of a lower temperature such as 0° C. to 25° C., for example 0° C. to 5° C. or 18° C. to 25° C. 
     A modified hyaluronan-degrading enzyme is selected or identified that retains or exhibits any desired activity in the presence of the denaturation condition compared to in its absence. The particular cut-off of activity for selection of enzymes herein is dependent on the particular user and/or practice of the method and can be empirically determined depending on factors such as the particular denaturation condition or denaturing agent, the particular modified hyaluronan-degrading enzyme, the desired application of the identified or selected hyaluronan-degrading enzyme and other similar factors. Generally, a selected or identified modified hyaluronan-degrading enzyme exhibits stability if any detectable activity is measured or assessed upon exposure or incubation with a denaturation condition or denaturing agent. For example, a selected or identified modified hyaluronan-degrading enzyme exhibits stability, or resistance to a denaturation condition or denaturing agent, if it exhibits at least 5% or 10% of the activity of the same enzyme in the absence of the denaturation condition or denaturing agent, and generally if the modified hyaluronan-degrading enzyme exhibits an activity that is at least 15% of the initial hyaluronidase activity prior to incubation in the presence of the denaturation condition. For example, a modified hyaluronan-degrading enzyme is selected or identified that exhibits at least (or at least about) 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 200%, 300%, 400%, 500% or more of the initial hyaluronidase activity of the modified hyaluronan-degrading enzyme tested under a control or non-denaturation condition. 
     In other examples of the methods herein, the activity of the modified hyaluronan degrading enzyme is assessed upon exposure to a denaturation condition and the activity of the unmodified or reference hyaluronan-degrading enzyme also is assessed upon exposure to the same denaturation conditions. In such examples, the activities are compared when the enzymes are exposed to the same conditions. For comparison, the activity under a denaturation condition can be represented as a ratio of activity or a percentage of activity of a modified hyaluronan-degrading enzyme compared to an unmodified or reference hyaluronan-degrading enzyme. In such examples, a modified hyaluronan-degrading enzyme is selected that exhibits greater activity under a denaturation condition than the unmodified or reference hyaluronan-degrading enzyme. Thus, the modified hyaluronan-degrading enzyme is one that is more resistant to the condition. For example, where the denaturation condition is the presence of preservative (e.g., phenolic preservative), the activity observed in the presence of preservative (e.g., phenolic preservative) can be represented as a ratio of activity or percentage of activity of the modified hyaluronan-degrading enzyme compared to the unmodified or reference hyaluronan-degrading enzyme. In another example, where the denaturation condition is high temperature, activity observed in the presence of elevated temperature (e.g., 30° C. to 42° C.) can be represented as a ratio of activity or percentage of activity of the modified hyaluronan-degrading enzyme compared to the unmodified or reference hyaluronan-degrading enzyme. 
     In such examples, a modified hyaluronan-degrading enzyme, such as a modified PH20, is identified or selected that exhibits a ratio of activity that is greater than or at least 1.1, such that the enzyme exhibits greater activity than the unmodified or reference hyaluronan-degrading enzyme under the denaturation condition. For example, the ratio is at least or at least about 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, 3.0, 3.5, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0 or greater. A modified hyaluronan-degrading enzyme (e.g., a modified PH20) can be selected if its activity is at least 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 250%, 300%, 400%, 500% or more of the activity of the unmodified or reference hyaluronan-degrading enzyme when tested under the same conditions. Thus, modified hyaluronan-degrading enzymes are identified that exhibit greater or improved stability compared to the unmodified hyaluronan-degrading enzyme or a reference hyaluronan-degrading enzyme as manifested by increased resistance to a denaturation condition or denaturing agent. 
     4. Iterative Methods 
     The method provided herein also is iterative. In one example, after the method is performed, any modified hyaluronan-degrading enzymes identified as exhibiting stability, such as increased stability, under a denaturation condition can be modified or further modified to increase or optimize the stability. A secondary library can be created by introducing additional modifications in a first identified modified hyaluronan-degrading enzyme. For example, modifications that were identified as conferring stability, such as increasing stability, can be combined to generate a combinatorial library. The secondary library can be tested using the assays and methods described herein. 
     In another example of an iterative aspect of the method, modified hyaluronan-degrading enzymes that are identified as not exhibiting stability such as increased stability (e.g., such that they are not active or do not have increased activity under the a denaturation condition), can be further modified and retested for stability under a denaturation condition. The further modifications can be targeted near particular regions (e.g., particular amino acid residues) associated with activity and/or stability of the molecule. For example, residues that are associated with activity and/or stability of the molecule generally are critical residues that are involved in the structural folding or other activities of the molecule. Hence, such residues are required for activity, generally under any condition. Critical residues can be identified because, when mutated, a normal activity of the protein is ablated or reduced. For example, critical residues can be identified that, when mutated in a hyaluronan-degrading enzyme, exhibit reduced or ablated hyaluronidase activity under a normal or control assay condition. A further library of modified proteins can be generated with amino acid mutations targeted at or near to the identified critical amino acid residues, such as adjacent to the identified critical amino acid residues. In some examples, the mutations can be amino acid replacement to any other of up to 19 other amino acid residues. The secondary library can be tested using the assays and methods described herein. 
     E. PRODUCTION OF MODIFIED PH20 POLYPEPTIDES AND ENCODING NUCLEIC ACID MOLECULES 
     Polypeptides of a modified PH20 polypeptide set forth herein can be obtained by methods well known in the art for protein purification and recombinant protein expression. Polypeptides also can be synthesized chemically. Modified or variant, including truncated, forms can be engineered from a wildtype polypeptide using standard recombinant DNA methods. For example, modified PH20 polypeptides can be engineered from a wildtype polypeptide, such as by site-directed mutagenesis. 
     1. Isolation or Preparation of Nucleic Acids Encoding PH20 Polypeptides 
     Polypeptides can be cloned or isolated using any available methods known in the art for cloning and isolating nucleic acid molecules. Such methods include PCR amplification of nucleic acids and screening of libraries, including nucleic acid hybridization screening, antibody-based screening and activity-based screening. For example, when the polypeptides are produced by recombinant means, any method known to those of skill in the art for identification of nucleic acids that encode desired genes can be used. Any method available in the art can be used to obtain a full length or partial (i.e., encompassing the entire coding region) cDNA or genomic DNA clone encoding a PH20, such as from a cell or tissue source. 
     Methods for amplification of nucleic acids can be used to isolate nucleic acid molecules encoding a desired polypeptide, including for example, polymerase chain reaction (PCR) methods. Examples of such methods include use of a Perkin-Elmer Cetus thermal cycler and Taq polymerase (Gene Amp). A nucleic acid containing material can be used as a starting material from which a desired polypeptide-encoding nucleic acid molecule can be isolated. For example, DNA and mRNA preparations, cell extracts, tissue extracts, fluid samples (e.g., blood, serum, saliva), samples from healthy and/or diseased subjects can be used in amplification methods. The source can be from any eukaryotic species including, but not limited to, vertebrate, mammalian, human, porcine, bovine, feline, avian, equine, canine, and other primate sources. Nucleic acid libraries also can be used as a source of starting material. Primers can be designed to amplify a desired polypeptide. For example, primers can be designed based on expressed sequences from which a desired polypeptide is generated. Primers can be designed based on back-translation of a polypeptide amino acid sequence. If desired, degenerate primers can be used for amplification. Oligonucleotide primers that hybridize to sequences at the 3′ and 5′ termini of the desired sequence can be uses as primers to amplify by PCR sequences from a nucleic acid sample. Primers can be used to amplify the entire full-length PH20, or a truncated sequence thereof, such as a nucleic acid encoding any of the soluble PH20 polypeptides provided herein. Nucleic acid molecules generated by amplification can be sequenced and confirmed to encode a desired polypeptide. 
     Additional nucleotide sequences can be joined to a polypeptide-encoding nucleic acid molecule, including linker sequences containing restriction endonuclease sites for the purpose of cloning the synthetic gene into a vector, for example, a protein expression vector or a vector designed for the amplification of the core protein coding DNA sequences. Furthermore, additional nucleotide sequences specifying functional DNA elements can be operatively linked to a polypeptide-encoding nucleic acid molecule. Examples of such sequences include, but are not limited to, promoter sequences designed to facilitate intracellular protein expression, and secretion sequences, for example heterologous signal sequences, designed to facilitate protein secretion. Such sequences are known to those of skill in the art. For example, exemplary heterologous signal sequences include, but are not limited to, human and mouse kappa IgG heterologous signal sequences set forth in SEQ ID NO: 868. Additional nucleotide residue sequences such as sequences of bases specifying protein binding regions also can be linked to enzyme-encoding nucleic acid molecules. Such regions include, but are not limited to, sequences of residues that facilitate or encode proteins that facilitate uptake of an enzyme into specific target cells, or otherwise alter pharmacokinetics of a product of a synthetic gene. 
     In addition, tags or other moieties can be added, for example, to aid in detection or affinity purification of the polypeptide. For example, additional nucleotide residue sequences such as sequences of bases specifying an epitope tag or other detectable marker also can be linked to enzyme-encoding nucleic acid molecules. Examples of such sequences include nucleic acid sequences encoding a His tag or Flag Tag. 
     The identified and isolated nucleic acids can then be inserted into an appropriate cloning vector. A large number of vector-host systems known in the art can be used. Possible vectors include, but are not limited to, plasmids or modified viruses, but the vector system must be compatible with the host cell used. Such vectors include, but are not limited to, bacteriophages such as lambda derivatives, or plasmids such as pCMV4, pBR322 or pUC plasmid derivatives or the Bluescript vector (Stratagene, La Jolla, Calif.). Other expression vectors include the HZ24 expression vector exemplified herein (see e.g., SEQ ID NOs:4 and 5). The insertion into a cloning vector can, for example, be accomplished by ligating the DNA fragment into a cloning vector which has complementary cohesive termini. Insertion can be effected using TOPO cloning vectors (Invitrogen, Carlsbad, Calif.). 
     If the complementary restriction sites used to fragment the DNA are not present in the cloning vector, the ends of the DNA molecules can be enzymatically modified. Alternatively, any site desired can be produced by ligating nucleotide sequences (linkers) onto the DNA termini; these ligated linkers can contain specific chemically synthesized oligonucleotides encoding restriction endonuclease recognition sequences. In an alternative method, the cleaved vector and protein gene can be modified by homopolymeric tailing. 
     Recombinant molecules can be introduced into host cells via, for example, transformation, transfection, infection, electroporation and sonoporation, so that many copies of the gene sequence are generated. In specific embodiments, transformation of host cells with recombinant DNA molecules that incorporate the isolated protein gene, cDNA, or synthesized DNA sequence enables generation of multiple copies of the gene. Thus, the gene can be obtained in large quantities by growing transformants, isolating the recombinant DNA molecules from the transformants and, when necessary, retrieving the inserted gene from the isolated recombinant DNA. 
     In addition to recombinant production, modified PH20 polypeptides provided herein can be produced by direct peptide synthesis using solid-phase techniques (see e.g., Stewart et al. (1969)  Solid - Phase Peptide Synthesis , WH Freeman Co., San Francisco; Merrifield J (1963)  J Am Chem Soc.,  85:2149-2154). In vitro protein synthesis can be performed using manual techniques or by automation. Automated synthesis can be achieved, for example, using Applied Biosystems 431A Peptide Synthesizer (Perkin Elmer, Foster City Calif.) in accordance with the instructions provided by the manufacturer. Various fragments of a polypeptide can be chemically synthesized separately and combined using chemical methods. 
     2. Generation of Mutant or Modified Nucleic Acid and Encoding Polypeptides 
     The modifications provided herein can be made by standard recombinant DNA techniques such as are routine to one of skill in the art. Any method known in the art to effect mutation of any one or more amino acids in a target protein can be employed. Methods include standard site-directed mutagenesis (using e.g., a kit, such as QuikChange available from Stratagene) of encoding nucleic acid molecules, or by solid phase polypeptide synthesis methods. 
     3. Vectors and Cells 
     For recombinant expression of one or more of the desired proteins, such as any modified PH20 polypeptide described herein, the nucleic acid containing all or a portion of the nucleotide sequence encoding the protein can be inserted into an appropriate expression vector, i.e., a vector that contains the necessary elements for the transcription and translation of the inserted protein coding sequence. The necessary transcriptional and translational signals also can be supplied by the native promoter for enzyme genes, and/or their flanking regions. 
     Also provided are vectors that contain a nucleic acid encoding the enzyme. Cells containing the vectors also are provided. The cells include eukaryotic and prokaryotic cells, and the vectors are any suitable for use therein. Generally, the cell is a cell that is capable of effecting glyosylation of the encoded protein. 
     Prokaryotic and eukaryotic cells containing the vectors are provided. Such cells include bacterial cells, yeast cells, fungal cells, Archea, plant cells, insect cells and animal cells. The cells are used to produce a protein thereof by growing the above-described cells under conditions whereby the encoded protein is expressed by the cell, and recovering the expressed protein. For purposes herein, for example, the enzyme can be secreted into the medium. 
     A host cell strain can be chosen for its ability to modulate the expression of the inserted sequences or to process the expressed protein in the desired fashion. Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation and acylation. Post-translational processing can impact the folding and/or function of the polypeptide. Different host cells, such as, but not limited to, CHO (DG44, DXB11, CHO-K1), HeLa, MCDK, 293 and W138 have specific cellular machinery and characteristic mechanisms for such post-translational activities and can be chosen to ensure the correct modification and processing of the introduced protein. Generally, the choice of cell is one that is capable of introducing N-linked glycosylation into the expressed polypeptide. Hence, eukaryotic cells containing the vectors are provided. 
     Exemplary eukaryotic cells are mammalian Chinese Hamster Ovary (CHO) cells. For example, CHO cells deficient in dihydrofolate reductase (e.g., DG44 cells) are used to produce polypeptides provided herein. Note that bacterial expression of an PH20 polypeptide provided herein will not result in a catalytically active polypeptide, but when combined with proper glycosylation machinery, the PH20 can be artificially glycosylated. 
     Provided are vectors that contain a sequence of nucleotides that encodes the modified PH20 polypeptide, coupled to the native or heterologous signal sequence, as well as multiple copies thereof. The vectors can be selected for expression of the enzyme protein in the cell or such that the enzyme protein is expressed as a secreted protein. 
     A variety of host-vector systems can be used to express the protein encoding sequence. These include but are not limited to mammalian cell systems infected with virus (e.g., vaccinia virus, adenovirus and other viruses); insect cell systems infected with virus (e.g., baculovirus); microorganisms such as yeast containing yeast vectors; or bacteria transformed with bacteriophage, DNA, plasmid DNA, or cosmid DNA. The expression elements of vectors vary in their strengths and specificities. Depending on the host-vector system used, any one of a number of suitable transcription and translation elements can be used. 
     Any methods known to those of skill in the art for the insertion of DNA fragments into a vector can be used to construct expression vectors containing a chimeric gene containing appropriate transcriptional/translational control signals and protein coding sequences. These methods can include in vitro recombinant DNA and synthetic techniques and in vivo recombinants (genetic recombination). Expression of nucleic acid sequences encoding protein, or domains, derivatives, fragments or homologs thereof, can be regulated by a second nucleic acid sequence so that the genes or fragments thereof are expressed in a host transformed with the recombinant DNA molecule(s). For example, expression of the proteins can be controlled by any promoter/enhancer known in the art. In a specific embodiment, the promoter is not native to the genes for a desired protein. Promoters which can be used include, but are not limited to, the SV40 early promoter (Bernoist and Chambon,  Nature  290:304-310 (1981)), the promoter contained in the 3′ long terminal repeat of Rous sarcoma virus (Yamamoto et al.  Cell  22:787-797 (1980)), the herpes thymidine kinase promoter (Wagner et al.,  Proc. Natl. Acad. Sci. USA  78:1441-1445 (1981)), the regulatory sequences of the metallothionein gene (Brinster et al.,  Nature  296:39-42 (1982)); prokaryotic expression vector promoters, such as the β-lactamase promoter (Jay et al., (1981)  Proc. Natl. Acad. Sci. USA  78:5543) or the tac promoter (DeBoer et al.,  Proc. Natl. Acad. Sci. USA  80:21-25 (1983); see also Gilbert and Villa-Komaroff, “Useful Proteins from Recombinant Bacteria,” Scientific American 242:74-94 (1980)); plant expression vector promoters, such as the nopaline synthetase promoter (Herrera-Estrella et al.,  Nature  303:209-213 (1984)) or the cauliflower mosaic virus 35S RNA promoter (Gardner et al.,  Nucleic Acids Res.  9:2871 (1981)), and the promoter of the photosynthetic enzyme ribulose bisphosphate carboxylase (Herrera-Estrella et al.,  Nature  310:115-120 (1984)); promoter elements from yeast and other fungi such as the Gal4 promoter, the alcohol dehydrogenase promoter, the phosphoglycerol kinase promoter, the alkaline phosphatase promoter, and the following animal transcriptional control regions that exhibit tissue specificity and have been used in transgenic animals: elastase I gene control region which is active in pancreatic acinar cells (Swift et al.,  Cell  38:639-646 (1984); Ornitz et al.,  Cold Spring Harbor Symp. Quant. Biol.  50:399-409 (1986); MacDonald,  Hepatology  7:425-515 (1987)); insulin gene control region which is active in pancreatic beta cells (Hanahan et al.,  Nature  315:115-122 (1985)), immunoglobulin gene control region which is active in lymphoid cells (Grosschedl et al.,  Cell  38:647-658 (1984); Adams et al.,  Nature  318:533-538 (1985); Alexander et al.,  Mol. Cell Biol.  7:1436-1444 (1987)), mouse mammary tumor virus control region which is active in testicular, breast, lymphoid and mast cells (Leder et al.,  Cell  45:485-495 (1986)), albumin gene control region which is active in liver (Pinkert et al.,  Genes and Devel.  1:268-276 (1987)), alpha-fetoprotein gene control region which is active in liver (Krumlauf et al.,  Mol. Cell. Biol.  5:1639-1648 (1985); Hammer et al.,  Science  235:53-58 1987)), alpha-1 antitrypsin gene control region which is active in liver (Kelsey et al.,  Genes and Devel.  1:161-171 (1987)), beta globin gene control region which is active in myeloid cells (Magram et al.,  Nature  315:338-340 (1985); Kollias et al.,  Cell  46:89-94 (1986)), myelin basic protein gene control region which is active in oligodendrocyte cells of the brain (Readhead et al.,  Cell  48:703-712 (1987)), myosin light chain-2 gene control region which is active in skeletal muscle (Shani,  Nature  314:283-286 (1985)), and gonadotrophic releasing hormone gene control region which is active in gonadotrophs of the hypothalamus (Mason et al.,  Science  234:1372-1378 (1986)). 
     In a specific embodiment, a vector is used that contains a promoter operably linked to nucleic acids encoding a desired protein, or a domain, fragment, derivative or homolog thereof, one or more origins of replication, and optionally, one or more selectable markers (e.g., an antibiotic resistance gene). Depending on the expression system, specific initiation signals also are required for efficient translation of a PH20 sequence. These signals include the ATG initiation codon and adjacent sequences. In cases where the initiation codon and upstream sequences of PH20 or soluble forms thereof are inserted into the appropriate expression vector, no additional translational control signals are needed. In cases where only a coding sequence, or a portion thereof, is inserted, exogenous transcriptional control signals including the ATG initiation codon must be provided. Furthermore, the initiation codon must be in the correct reading frame to ensure transcription of the entire insert. Exogenous transcriptional elements and initiation codons can be of various origins, both natural and synthetic. The efficiency of expression can be enhanced by the inclusion of enhancers appropriate to the cell system in use (Scharf et al. (1994)  Results Probl Cell Differ  20:125-62; Bittner et al. (1987)  Methods in Enzymol,  153:516-544). 
     Exemplary plasmid vectors for transformation of  E. coli  cells include, for example, the pQE expression vectors (available from Qiagen, Valencia, Calif.; see also literature published by Qiagen describing the system). pQE vectors have a phage T5 promoter (recognized by  E. coli  RNA polymerase) and a double lac operator repression module to provide tightly regulated, high-level expression of recombinant proteins in  E. coli , a synthetic ribosomal binding site (RBS II) for efficient translation, a 6×His tag coding sequence, to and T1 transcriptional terminators, ColE1 origin of replication, and a beta-lactamase gene for conferring ampicillin resistance. The pQE vectors enable placement of a 6×His tag at either the N- or C-terminus of the recombinant protein. Such plasmids include pQE 32, pQE 30, and pQE 31 which provide multiple cloning sites for all three reading frames and provide for the expression of N-terminally 6×His-tagged proteins. Other exemplary plasmid vectors for transformation of  E. coli  cells, include, for example, the pET expression vectors (see, U.S. Pat. No. 4,952,496; available from Novagen, Madison, Wis.; see, also literature published by Novagen describing the system). Such plasmids include pET 11a, which contains the T7lac promoter, T7 terminator, the inducible  E. coli  lac operator, and the lac repressor gene; pET 12a-c, which contains the T7 promoter, T7 terminator, and the  E. coli  ompT secretion signal; and pET 15b and pET19b (Novagen, Madison, Wis.), which contain a His-Tag™ leader sequence for use in purification with a His column and a thrombin cleavage site that permits cleavage following purification over the column, the T7-lac promoter region and the T7 terminator. 
     Typically, vectors can be plasmids, viral vectors, or others known in the art, used for expression of the modified PH20 polypeptide in vivo or in vitro. For example, the modified PH20 polypeptide is expressed in mammalian cells, including, for example, Chinese Hamster Ovary (CHO) cells. An exemplary vector for mammalian cell expression is the HZ24 expression vector. The HZ24 expression vector was derived from the pCI vector backbone (Promega). It contains DNA encoding the Beta-lactamase resistance gene (AmpR), an F1 origin of replication, a Cytomegalovirus immediate-early enhancer/promoter region (CMV), and an SV40 late polyadenylation signal (SV40). The expression vector also has an internal ribosome entry site (IRES) from the ECMV virus (Clontech) and the mouse dihydrofolate reductase (DHFR) gene. 
     Viral vectors, such as adenovirus, retrovirus or vaccinia virus vectors, can be employed. In some examples, the vector is a defective or attenuated retroviral or other viral vector (see U.S. Pat. No. 4,980,286). For example, a retroviral vector can be used (see Miller et al., Meth. Enzymol. 217: 581-599 (1993)). These retroviral vectors have been modified to delete retroviral sequences that are not necessary for packaging of the viral genome and integration into host cell DNA. 
     In some examples, viruses armed with a nucleic acid encoding a modified PH20 polypeptide can facilitate their replication and spread within a target tissue for example. The target tissue can be a cancerous tissue whereby the virus is capable of selective replication within the tumor. The virus can also be a non-lytic virus wherein the virus selectively replicates under a tissue specific promoter. As the viruses replicate, the coexpression of the PH20 polypeptide with viral genes will facilitate the spread of the virus in vivo. 
     4. Expression 
     Modified PH20 polypeptides can be produced by any method known to those of skill in the art including in vivo and in vitro methods. Desired proteins can be expressed in any organism suitable to produce the required amounts and forms of the proteins, such as for example, those needed for administration and treatment. Expression hosts include prokaryotic and eukaryotic organisms such as  E. coli , yeast, plants, insect cells, mammalian cells, including human cell lines and transgenic animals. Expression hosts can differ in their protein production levels as well as the types of post-translational modifications that are present on the expressed proteins. The choice of expression host can be made based on these and other factors, such as regulatory and safety considerations, production costs and the need and methods for purification. 
     Many expression vectors are available and known to those of skill in the art and can be used for expression of proteins. The choice of expression vector will be influenced by the choice of host expression system. In general, expression vectors can include transcriptional promoters and optionally enhancers, translational signals, and transcriptional and translational termination signals. Expression vectors that are used for stable transformation typically have a selectable marker which allows selection and maintenance of the transformed cells. In some cases, an origin of replication can be used to amplify the copy number of the vector. 
     Modified PH20 polypeptides also can be utilized or expressed as protein fusions. For example, an enzyme fusion can be generated to add additional functionality to an enzyme. Examples of enzyme fusion proteins include, but are not limited to, fusions of a signal sequence, a tag such as for localization, e.g., a 6×His or His 6  tag or a myc tag, or a tag for purification, for example, a GST fusion, and a sequence for directing protein secretion and/or membrane association. 
     For long-term, high-yield production of recombinant proteins, stable expression is desired. For example, cell lines that stably express a modified PH20 polypeptide can be transformed using expression vectors that contain viral origins of replication or endogenous expression elements and a selectable marker gene. Following the introduction of the vector, cells can be allowed to grow for 1-2 days in an enriched medium before they are switched to selective media. The purpose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells that successfully express the introduced sequences. Resistant cells of stably transformed cells can be proliferated using tissue culture techniques appropriate to the cell types. 
     Any number of selection systems can be used to recover transformed cell lines. These include, but are not limited to, the herpes simplex virus thymidine kinase (Wigler, M et al. (1977) Cell, 11:223-32) and adenine phosphoribosyltransferase (Lowy, I et al. (1980) Cell, 22:817-23) genes, which can be employed in TK- or APRT-cells, respectively. Also, antimetabolite, antibiotic or herbicide resistance can be used as the basis for selection. For example, DHFR, which confers resistance to methotrexate (Wigler, M et al. (1980) Proc. Natl. Acad. Sci, 77:3567-70); npt, which confers resistance to the aminoglycosides neomycin and G-418 (Colbere-Garapin, F et al. (1981) J. Mol. Biol., 150:1-14); and also or pat, which confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively, can be used. Additional selectable genes have been described, for example, trpB, which allows cells to utilize indole in place of typtophan or hisD, which allows cells to utilize histinol in place of histidine (Hartman S C and RC Mulligan (1988) Proc. Natl. Acad. Sci, 85:8047-51). Visible markers, such as but not limited to, anthocyanins, beta glucuronidase and its substrate, GUS, and luciferase and its substrate luciferin, also can be used to identify transformants and also to quantify the amount of transient or stable protein expression attributable to a particular vector system (Rhodes C A et al. (1995) Methods Mol. Biol. 55:121-131). 
     The presence and expression of PH20 polypeptides can be monitored. For example, detection of a functional polypeptide can be determined by testing the conditioned media for hyaluronidase enzyme activity under appropriate conditions. Exemplary assays to assess the solubility and activity of expressed proteins are provided herein. 
     a. Prokaryotic Cells 
     Prokaryotes, especially  E. coli , provide a system for producing large amounts of proteins. Transformation of  E. coli  is a simple and rapid technique well known to those of skill in the art. Expression vectors for  E. coli  can contain inducible promoters. Such promoters are useful for inducing high levels of protein expression and for expressing proteins that exhibit some toxicity to the host cells. Examples of inducible promoters include the lac promoter, the trp promoter, the hybrid tac promoter, the T7 and SP6 RNA promoters and the temperature regulated λPL promoter. 
     Proteins, such as any provided herein, can be expressed in the cytoplasmic environment of  E. coli . The cytoplasm is a reducing environment, and for some molecules, this can result in the formation of insoluble inclusion bodies. Reducing agents such as dithiothreotol and β-mercaptoethanol and denaturants, such as guanidine-HCl and urea can be used to resolubilize the proteins. An alternative approach effects protein expression in the periplasmic space of bacteria which provides an oxidizing environment and chaperonin-like and disulfide isomerases, which can aid in the production of soluble protein. Typically, a leader sequence is fused to the protein to be expressed which directs the protein to the periplasm. The leader is then removed by signal peptidases inside the periplasm. Examples of periplasmic-targeting leader sequences include the pelB leader from the pectate lyase gene and the leader derived from the alkaline phosphatase gene. In some cases, periplasmic expression allows leakage of the expressed protein into the culture medium. The secretion of proteins allows quick and simple purification from the culture supernatant. Proteins that are not secreted can be obtained from the periplasm by osmotic lysis. Similar to cytoplasmic expression, in some cases proteins can become insoluble and denaturants and reducing agents can be used to facilitate solubilization and refolding. Temperature of induction and growth also can influence expression levels and solubility, typically temperatures between 25° C. and 37° C. are used. Typically, bacteria produce aglycosylated proteins. Thus, if proteins require glycosylation for function, glycosylation can be added in vitro after purification from host cells. 
     b. Yeast Cells 
     Yeasts such as  Saccharomyces cerevisae, Schizosaccharomyces pombe, Yarrowia lipolytica, Kluyveromyces lactis  and  Pichia pastoris  are well known yeast expression hosts that can be used for production of proteins, such as any described herein. Yeast can be transformed with episomal replicating vectors or by stable chromosomal integration by homologous recombination. Typically, inducible promoters are used to regulate gene expression. Examples of such promoters include GAL1, GALT and GALS and metallothionein promoters, such as CUP1, AOX1 or other  Pichia  or other yeast promoters. Expression vectors often include a selectable marker such as LEU2, TRP1, HIS3 and URA3 for selection and maintenance of the transformed DNA. Proteins expressed in yeast are often soluble. Co-expression with chaperonins such as Bip and protein disulfide isomerase can improve expression levels and solubility. Additionally, proteins expressed in yeast can be directed for secretion using secretion signal peptide fusions such as the yeast mating type alpha-factor secretion signal from  Saccharomyces cerevisae  and fusions with yeast cell surface proteins such as the Aga2p mating adhesion receptor or the Arxula adeninivorans glucoamylase. A protease cleavage site such as for the Kex-2 protease, can be engineered to remove the fused sequences from the expressed polypeptides as they exit the secretion pathway. Yeast also is capable of glycosylation at Asn-X-Ser/Thr motifs. 
     c. Insects and Insect Cells 
     Insect cells, particularly using baculovirus expression, are useful for expressing polypeptides such as PH20 polypeptides. Insect cells express high levels of protein and are capable of most of the post-translational modifications used by higher eukaryotes. Baculoviruses have a restrictive host range which improves the safety and reduces regulatory concerns of eukaryotic expression. Typical expression vectors use a promoter for high level expression such as the polyhedrin promoter of baculovirus. Commonly used baculovirus systems include a baculovirus, such as the  Autographa californica  nuclear polyhedrosis virus (AcNPV) or the  Bombyx mori  nuclear polyhedrosis virus (BmNPV), and an insect cell line, such as Sf9 derived from  Spodoptera frugiperda, Pseudaletia unipuncta  (A7S) and Danaus plexippus (DpN1). For high-level expression, the nucleotide sequence of the molecule to be expressed is fused immediately downstream of the polyhedrin initiation codon of the virus. Mammalian secretion signals are accurately processed in insect cells and can be used to secrete the expressed protein into the culture medium. In addition, the cell lines Pseudaletia unipuncta (A7S) and Danaus plexippus (DpN1) produce proteins with glycosylation patterns similar to mammalian cell systems. Exemplary insect cells are those that have been altered to reduce immunogenicity, including those with “mammalianized” baculovirus expression vectors and those lacking the enzyme FT3. 
     An alternative expression system in insect cells employs stably transformed cells. Cell lines such as the Schnieder 2 (S2) and Kc cells ( Drosophila melanogaster ) and C7 cells ( Aedes albopictus ) can be used for expression. The  Drosophila  metallothionein promoter can be used to induce high levels of expression in the presence of heavy metal induction with cadmium or copper. Expression vectors are typically maintained by the use of selectable markers such as neomycin and hygromycin. 
     d. Mammalian Expression 
     Mammalian expression systems can be used to express proteins including PH20 polypeptides. Expression constructs can be transferred to mammalian cells by viral infection such as by adenovirus or by direct DNA transfer such as liposomes, calcium phosphate, DEAE-dextran and by physical means such as electroporation and microinjection. Expression vectors for mammalian cells typically include an mRNA cap site, a TATA box, a translational initiation sequence (Kozak consensus sequence) and polyadenylation elements. IRES elements also can be added to permit bicistronic expression with another gene, such as a selectable marker. Such vectors often include transcriptional promoter-enhancers for high-level expression, for example the SV40 promoter-enhancer, the human cytomegalovirus (CMV) promoter and the long terminal repeat of Rous sarcoma virus (RSV). These promoter-enhancers are active in many cell types. Tissue and cell-type promoters and enhancer regions also can be used for expression. Exemplary promoter/enhancer regions include, but are not limited to, those from genes such as elastase I, insulin, immunoglobulin, mouse mammary tumor virus, albumin, alpha fetoprotein, alpha 1 antitrypsin, beta globin, myelin basic protein, myosin light chain 2, and gonadotropic releasing hormone gene control. Selectable markers can be used to select for and maintain cells with the expression construct. Examples of selectable marker genes include, but are not limited to, hygromycin B phosphotransferase, adenosine deaminase, xanthine-guanine phosphoribosyl transferase, aminoglycoside phosphotransferase, dihydrofolate reductase (DHFR) and thymidine kinase. For example, expression can be performed in the presence of methotrexate to select for only those cells expressing the DHFR gene. Fusion with cell surface signaling molecules such as TCR-ζ and Fc ε RI-γ can direct expression of the proteins in an active state on the cell surface. 
     Many cell lines are available for mammalian expression including mouse, rat human, monkey, chicken and hamster cells. Exemplary cell lines include but are not limited to CHO, Balb/3T3, HeLa, MT2, mouse NS0 (nonsecreting) and other myeloma cell lines, hybridoma and heterohybridoma cell lines, lymphocytes, fibroblasts, Sp2/0, COS, NIH3T3, HEK293, 293S, 2B8, and HKB cells. Cell lines also are available adapted to serum-free media which facilitates purification of secreted proteins from the cell culture media. Examples include CHO—S cells (Invitrogen, Carlsbad, Calif., cat #11619-012) and the serum free EBNA-1 cell line (Pham et al., (2003)  Biotechnol. Bioeng.  84:332-42). Cell lines also are available that are adapted to grow in special mediums optimized for maximal expression. For example, DG44 CHO cells are adapted to grow in suspension culture in a chemically defined, animal product-free medium. 
     e. Plants and Plant Cells 
     Transgenic plant cells and plants can be used to express proteins such as any described herein. Expression constructs are typically transferred to plants using direct DNA transfer such as microprojectile bombardment and PEG-mediated transfer into protoplasts, and with  agrobacterium -mediated transformation. Expression vectors can include promoter and enhancer sequences, transcriptional termination elements and translational control elements. Expression vectors and transformation techniques are usually divided between dicot hosts, such as  Arabidopsis  and tobacco, and monocot hosts, such as corn and rice. Examples of plant promoters used for expression include the cauliflower mosaic virus promoter, the nopaline synthase promoter, the ribose bisphosphate carboxylase promoter and the ubiquitin and UBQ3 promoters. Selectable markers such as hygromycin, phosphomannose isomerase and neomycin phosphotransferase are often used to facilitate selection and maintenance of transformed cells. Transformed plant cells can be maintained in culture as cells, aggregates (callus tissue) or regenerated into whole plants. Transgenic plant cells also can include algae engineered to produce hyaluronidase polypeptides. Because plants have different glycosylation patterns than mammalian cells, this can influence the choice of protein produced in these hosts. 
     5. Purification 
     Host cells transformed with a nucleic acid sequence encoding a modified PH20 polypeptide can be cultured under conditions suitable for the expression and recovery of the encoded protein from cell culture. The protein produced by a recombinant cell is generally secreted, but may be contained intracellularly depending on the sequence and/or the vector used. As will be understood by those of skill in the art, expression vectors containing nucleic acid encoding PH20 can be designed with signal sequences that facilitate direct secretion of PH20 through prokaryotic or eukaryotic cell membranes. 
     Thus, methods for purification of polypeptides from host cells will depend on the chosen host cells and expression systems. For secreted molecules, proteins are generally purified from the culture media after removing the cells. For intracellular expression, cells can be lysed and the proteins purified from the extract. When transgenic organisms such as transgenic plants and animals are used for expression, tissues or organs can be used as starting material to make a lysed cell extract. Additionally, transgenic animal production can include the production of polypeptides in milk or eggs, which can be collected, and if necessary, the proteins can be extracted and further purified using standard methods in the art. 
     Proteins, such as modified PH20 polypeptides, can be purified using standard protein purification techniques known in the art including but not limited to, SDS-PAGE, size fractionation and size exclusion chromatography, ammonium sulfate precipitation and ionic exchange chromatography, such as anion exchange chromatography. Affinity purification techniques also can be utilized to improve the efficiency and purity of the preparations. For example, antibodies, receptors and other molecules that bind PH20 hyaluronidase enzymes can be used in affinity purification. For example, soluble PH20 can be purified from conditioned media. 
     Expression constructs also can be engineered to add an affinity tag to a protein such as a myc epitope, GST fusion or His 6  and affinity purified with myc antibody, glutathione resin or Ni-resin, respectively. Such tags can be joined to the nucleotide sequence encoding a soluble PH20 as described elsewhere herein, which can facilitate purification of soluble proteins. For example, a modified PH20 polypeptide can be expressed as a recombinant protein with one or more additional polypeptide domains added to facilitate protein purification. Such purification facilitating domains include, but are not limited to, metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin and the domain utilized in the FLAGS extension/affinity purification system (Immunex Corp., Seattle Wash.). The inclusion of a cleavable linker sequence such as Factor XA or enterokinase (Invitrogen, San Diego, Calif.) between the purification domain and the expressed PH20 polypeptide is useful to facilitate purification. One such expression vector provides for expression of a fusion protein containing a PH20 polypeptide in and an enterokinase cleavage site. The histidine residues facilitate purification on IMIAC (immobilized metal ion affinity chromatography), while the enterokinase cleavage site provides a means for purifying the polypeptide from the fusion protein. 
     Purity can be assessed by any method known in the art including gel electrophoresis, orthogonal HPLC methods, staining and spectrophotometric techniques. The expressed and purified protein can be analyzed using any assay or method known to one of skill in the art, for example, any described in Section G. These include assays based on the physical and/or functional properties of the protein, including, but not limited to, analysis by gel electrophoresis, immunoassay and assays of hyaluronidase activity. 
     Depending on the expression system and host cells used, the resulting polypeptide can be heterogeneous due to peptidases present in the culture medium upon production and purification. For example, culture of soluble PH20 in CHO cells can result in a mixture of heterogeneous polypeptides. 
     6. Modification of Polypeptides by PEGylation 
     Polyethylene glycol (PEG) has been widely used in biomaterials, biotechnology and medicine primarily because PEG is a biocompatible, nontoxic, water-soluble polymer that is typically nonimmunogenic (Zhao and Harris,  ACS Symposium Series  680: 458-72, 1997). In the area of drug delivery, PEG derivatives have been widely used in covalent attachment (i.e., “PEGylation”) to proteins to reduce immunogenicity, proteolysis and kidney clearance and to enhance solubility (Zalipsky, Adv. Drug Del. Rev. 16:157-82, 1995). Similarly, PEG has been attached to low molecular weight, relatively hydrophobic drugs to enhance solubility, reduce toxicity and alter biodistribution. Typically, PEGylated drugs are injected as solutions. 
     A closely related application is synthesis of crosslinked degradable PEG networks or formulations for use in drug delivery since much of the same chemistry used in design of degradable, soluble drug carriers can also be used in design of degradable gels (Sawhney et al.,  Macromolecules  26: 581-87, 1993). It also is known that intermacromolecular complexes can be formed by mixing solutions of two complementary polymers. Such complexes are generally stabilized by electrostatic interactions (polyanion-polycation) and/or hydrogen bonds (polyacid-polybase) between the polymers involved, and/or by hydrophobic interactions between the polymers in an aqueous surrounding (Krupers et al.,  Eur. Polym J.  32:785-790, 1996). For example, mixing solutions of polyacrylic acid (PAAc) and polyethylene oxide (PEO) under the proper conditions results in the formation of complexes based mostly on hydrogen bonding. Dissociation of these complexes at physiologic conditions has been used for delivery of free drugs (i.e., non-PEGylated). In addition, complexes of complementary polymers have been formed from both homopolymers and copolymers. 
     Numerous reagents for PEGylation have been described in the art. Such reagents include, but are not limited to, reaction of the polypeptide with N-hydroxysuccinimidyl (NHS) activated PEG, succinimidyl mPEG, mPEG 2 -N-hydroxysuccinimide, mPEG succinimidyl alpha-methylbutanoate, mPEG succinimidyl propionate, mPEG succinimidyl butanoate, mPEG carboxymethyl 3-hydroxybutanoic acid succinimidyl ester, homobifunctional PEG-succinimidyl propionate, homobifunctional PEG propionaldehyde, homobifunctional PEG butyraldehyde, PEG maleimide, PEG hydrazide, p-nitrophenyl-carbonate PEG, mPEG-benzotriazole carbonate, propionaldehyde PEG, mPEG butryaldehyde, branched mPEG 2  butyraldehyde, mPEG acetyl, mPEG piperidone, mPEG methylketone, mPEG “linkerless” maleimide, mPEG vinyl sulfone, mPEG thiol, mPEG orthopyridylthioester, mPEG orthopyridyl disulfide, Fmoc-PEG-NHS, Boc-PEG-NHS, vinylsulfone PEG-NHS, acrylate PEG-NHS, fluorescein PEG-NHS, and biotin PEG-NHS (see e.g., Monfardini et al.,  Bioconjugate Chem.  6:62-69, 1995; Veronese et al.,  J. Bioactive Compatible Polymers  12:197-207, 1997; U.S. Pat. Nos. 5,672,662; 5,932,462; 6,495,659; 6,737,505; 4,002,531; 4,179,337; 5,122,614; 5,324,844; U.S. 5,446,090; U.S. Pat. Nos. 5,612,460; 5,643,575; 5,766,581; 5,795,569; 5,808,096; 5,900,461; 5,919,455; 5,985,263; 5,990,237; 6,113,906; U.S. 6,214,966; U.S. Pat. Nos. 6,258,351; 6,340,742; 6,413,507; 6,420,339; 6,437,025; 6,448,369; 6,461,802; 6,828,401; 6,858,736; U.S. 2001/0021763; U.S. 2001/0044526; U.S. 2001/0046481; U.S. 2002/0052430; U.S. 2002/0072573; U.S. 2002/0156047; U.S. 2003/0114647; U.S. 2003/0143596; U.S. 2003/0158333; U.S. 2003/0220447; U.S. 2004/0013637; US 2004/0235734; WO05000360; U.S. 2005/0114037; U.S. 2005/0171328; U.S. 2005/0209416; EP 1064951; EP 0822199; WO 01076640; WO 0002017; WO 0249673; WO 9428024; and WO 0187925). 
     In one example, the polyethylene glycol has a molecular weight ranging from about 3 kD to about 50 kD, and typically from about 5 kD to about 30 kD. Covalent attachment of the PEG to the drug (known as “PEGylation”) can be accomplished by known chemical synthesis techniques. For example, the PEGylation of protein can be accomplished by reacting NHS-activated PEG with the protein under suitable reaction conditions. 
     While numerous reactions have been described for PEGylation, those that are most generally applicable confer directionality, utilize mild reaction conditions, and do not necessitate extensive downstream processing to remove toxic catalysts or bi-products. For instance, monomethoxy PEG (mPEG) has only one reactive terminal hydroxyl, and thus its use limits some of the heterogeneity of the resulting PEG-protein product mixture. Activation of the hydroxyl group at the end of the polymer opposite to the terminal methoxy group is generally necessary to accomplish efficient protein PEGylation, with the aim being to make the derivatised PEG more susceptible to nucleophilic attack. The attacking nucleophile is usually the epsilon-amino group of a lysyl residue, but other amines also can react (e.g., the N-terminal alpha-amine or the ring amines of histidine) if local conditions are favorable. A more directed attachment is possible in proteins containing a single lysine or cysteine. The latter residue can be targeted by PEG-maleimide for thiol-specific modification. Alternatively, PEG hydrazide can be reacted with a periodate oxidized hyaluronan-degrading enzyme and reduced in the presence of NaCNBH 3 . More specifically, PEGylated CMP sugars can be reacted with a hyaluronan-degrading enzyme in the presence of appropriate glycosyl-transferases. One technique is the “PEGylation” technique where a number of polymeric molecules are coupled to the polypeptide in question. When using this technique, the immune system has difficulties in recognizing the epitopes on the polypeptide&#39;s surface responsible for the formation of antibodies, thereby reducing the immune response. For polypeptides introduced directly into the circulatory system of the human body to give a particular physiological effect (i.e., pharmaceuticals) the typical potential immune response is an IgG and/or IgM response, while polypeptides which are inhaled through the respiratory system (i.e., industrial polypeptide) potentially can cause an IgE response (i.e., allergic response). One of the theories explaining the reduced immune response is that the polymeric molecule(s) shield(s) epitope(s) on the surface of the polypeptide responsible for the immune response leading to antibody formation. Another theory or at least a partial factor is that the heavier the conjugate is, the more reduced the resulting immune response is. 
     Typically, to make the PEGylated PH20 polypeptide provided herein, PEG moieties are conjugated, via covalent attachment, to the polypeptides. Techniques for PEGylation include, but are not limited to, specialized linkers and coupling chemistries (see e.g., Roberts,  Adv. Drug Deliv. Rev.  54:459-476, 2002), attachment of multiple PEG moieties to a single conjugation site (such as via use of branched PEGs; see e.g., Guiotto et al.,  Bioorg. Med. Chem. Lett.  12:177-180, 2002), site-specific PEGylation and/or mono-PEGylation (see e.g., Chapman et al.,  Nature Biotech.  17:780-783, 1999), and site-directed enzymatic PEGylation (see e.g., Sato,  Adv. Drug  Deliv. Rev., 54:487-504, 2002). Methods and techniques described in the art can produce proteins having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 PEG or PEG derivatives attached to a single protein molecule (see e.g., U.S. 2006/0104968). 
     As an exemplary illustrative method for making a PEGylated PH20 polypeptide, PEG aldehydes, succinimides and carbonates have each been applied to conjugate PEG moieties, typically succinimidyl PEGs, to rHuPH20. For example, rHuPH20 has been conjugated with exemplary succinimidyl monoPEG (mPEG) reagents including mPEG-Succinimidyl Propionates (mPEG-SPA), mPEG-Succinimidyl Butanoates (mPEG-SBA), and (for attaching “branched” PEGs) mPEG2-N-Hydroxylsuccinimide. These PEGylated succinimidyl esters contain different length carbon backbones between the PEG group and the activated cross-linker, and either a single or branched PEG group. These differences can be used, for example, to provide for different reaction kinetics and to potentially restrict sites available for PEG attachment to rHuPH20 during the conjugation process. 
     Succinimidyl PEGs (as above) containing either linear or branched PEGs can be conjugated to PH20. PEGs can used to generate PH20s reproducibly containing molecules having, on the average, between about three to six or three to six PEG molecules per hyaluronidase. Such PEGylated rHuPH20 compositions can be readily purified to yield compositions having specific activities of approximately 25,000 or 30,000 Unit/mg protein hyaluronidase activity, and being substantially free of non-PEGylated PH20 (less than 5% non-PEGylated). 
     Using various PEG reagents, exemplary versions of a PEGylated PH20 polypeptide can be prepared, for example, using mPEG-SBA (30 kD), mPEG-SMB (30 kD), and branched versions based on mPEG2-NHS (40 kD) and mPEG2-NHS (60 kD). PEGylated versions of PH20 can be generated using NHS chemistries, as well as carbonates, and aldehydes, using each of the following reagents: mPEG2-NHS-40K branched, mPEG-NHS-10K branched, mPEG-NHS-20K branched, mPEG2-NHS-60K branched; mPEG-SBA-5K, mPEG-SBA-20K, mPEG-SBA-30K; mPEG-SMB-20K, mPEG-SMB-30K; mPEG-butyrldehyde; mPEG-SPA-20K, mPEG-SPA-30K; and PEG-NHS-5K-biotin. PEGylated PH20 also can be prepared using PEG reagents available from Dowpharma, a division of Dow Chemical Corporation; including PH20 polypeptides PEGylated with Dowpharma&#39;s p-nitrophenyl-carbonate PEG (30 kDa) and with propionaldehyde PEG (30 kDa). 
     In one example, the PEGylation includes conjugation of mPEG-SBA, for example, mPEG-SBA-30K (having a molecular weight of about 30 kDa) or another succinimidyl ester of a PEG butanoic acid derivative, to a PH20 polypeptide. Succinimidyl esters of PEG butanoic acid derivatives, such as mPEG-SBA-30K readily couple to amino groups of proteins. For example, covalent conjugation of m-PEG-SBA-30K and rHuPH20 (which is approximately 60 KDa in size) provides stable amide bonds between rHuPH20 and mPEG, as shown in Scheme 1, below. 
     
       
         
         
             
             
         
       
     
     Typically, the mPEG-SBA-30K or other PEG is added to the PH20 polypeptide at a PEG:polypeptide molar ratio of 10:1 in a suitable buffer, e.g., 130 mM NaCl/10 mM HEPES at pH 6.8 or 70 mM phosphate buffer, pH 7, followed by sterilization, e.g., sterile filtration, and continued conjugation, for example, with stirring, overnight at 4° C. in a cold room. In one example, the conjugated PEG-PH20 is concentrated and buffer-exchanged. 
     Other methods of coupling succinimidyl esters of PEG butanoic acid derivatives, such as mPEG-SBA-30K are known in the art (see e.g., U.S. Pat. Nos. 5,672,662; 6,737,505; and U.S. 2004/0235734). For example, a polypeptide, such as a PH20 polypeptide, can be coupled to an NHS activated PEG derivative by reaction in a borate buffer (0.1 M, pH 8.0) for one hour at 4° C. The resulting PEGylated protein can be purified by ultrafiltration. Another method reacts polypeptide with mPEG-SBA in deionized water to which triethylamine is added to raise the pH to 7.2-9. The resulting mixture is stirred at room temperature for several hours to complete the PEGylation. 
     Methods for PEGylation of PH20 polypeptides, including, for example, animal-derived hyaluronidases and bacterial hyaluronan-degrading enzymes, are known to one of skill in the art. See, for example, European Patent No. EP 0400472, which describes the PEGylation of bovine testes hyaluorindase and chondroitin ABC lyase. Also, U.S. Publication No. 2006014968 describes PEGylation of a human hyaluronidase derived from human PH20. For example, the PEGylated hyaluronan-degrading enzyme generally contains at least 3 PEG moieties per molecule. In some examples, the PH20 polypeptide contains three to six PEG molecules. In other examples, the enzyme can have a PEG to protein molar ratio between 5:1 and 9:1, for example, 7:1. 
     F. PHARMACEUTICAL COMPOSITIONS AND FORMULATIONS, DOSAGES AND ADMINISTRATION 
     Pharmaceutical compositions of any of the modified PH20 polypeptides are provided herein for administration. Pharmaceutically acceptable compositions are prepared in view of approvals for a regulatory agency or other agency prepared in accordance with generally recognized pharmacopeia for use in animals and in humans. Typically, the compounds are formulated into pharmaceutical compositions using techniques and procedures well known in the art (see e.g., Ansel  Introduction to Pharmaceutical Dosage Forms , Fourth Edition, 1985, 126). 
     In particular, provided herein are pharmaceutical compositions that are stable as a liquid formulation for prolonged periods of time for at least 1 month at temperatures from or from about 2° C. to 8° C., inclusive or for at least 3 days at a temperature from or from about 30° C. to 42° C., inclusive. Pharmaceutical compositions, in particular liquid formulations, can be limited by the stability of the active agent, which can be susceptible to effects of storage conditions (time or length of storage, temperature and/or agitation) and/or formulation components contained in the composition. Hence, the stable pharmaceutical compositions generally contain a modified PH20 polypeptide as described in Section C.1.b that exhibits increased stability manifested as an increased resistance to one or more protein denaturation conditions. Such protein denaturation conditions can include, but are not limited to, elevated temperature greater than or equal to or about 30° C., agitation, low or no salt, and presence of excipients. The increased stability is characterized by improved storage time, decreased fragmentation, and/or decreased aggregate formation, while still retaining the activity of the active agent(s), e.g., the PH20 hyaluronidase. Such formulations can be provided as “ready-to use” liquid formulations without further reconstitution and/or without any requirement for further dilution. In some examples, the formulations also can be prepared in a lyophilized or concentrated form. 
     Pharmaceutical compositions containing a modified PH20 polypeptide can be co-administered with another therapeutic agent. In such examples, the modified PH20 polypeptides can be formulated separately as a pharmaceutical composition and administered prior to, simultaneously with, intermittently with, or subsequent to a second composition containing an active therapeutic agent. In other examples, modified PH20 polypeptides can be co-formulated with pharmaceutical formulations of other therapeutic agents. 
     In particular, provided herein are co-formulations containing a modified PH20 polypeptide as described herein and a therapeutic agent that is a chemotherapeutic agent, an analgesic agent, an anti-inflammatory agent, an antimicrobial agent, an amoebicidal agent, a trichomonacidal agent, an anti-Parkinson agent, an anti-malarial agent, an anticonvulsant agent, an anti-depressant agent, and antiarthritics agent, an anti-fungal agent, an antihypertensive agent, an antipyretic agent, an anti-parasite agent, an antihistamine agent, an alpha-adrenergic agonist agent, an alpha blocker agent, an anesthetic agent, a bronchial dilator agent, a biocide agent, a bactericide agent, a bacteriostat agent, a beta adrenergic blocker agent, a calcium channel blocker agent, a cardiovascular drug agent, a contraceptive agent, a decongestant agent, a diuretic agent, a depressant agent, a diagnostic agent, a electrolyte agent, a hypnotic agent, a hormone agent, a hyperglycemic agent, a muscle relaxant agent, a muscle contractant agent, an ophthalmic agent, a parasympathomimetic agent, a psychic energizer agent, a sedative agent, a sympathomimetic agent, a tranquilizer agent, an urinary agent, a vaginal agent, a viricide agent, a vitamin agent, a non-steroidal anti-inflammatory agent, an angiotensin converting enzyme inhibitor agent, a polypeptide, a protein, a nucleic acid, a drug, an organic molecule or a sleep inducer. For example, modified PH20 polypeptides provided herein can be co-formulated with an antibody such as a monoclonal antibody, an Immune Globulin, an antibiotic, a bisphosphonate, a cytokine, a chemotherapeutic agent, a coagulation factor or an insulin. Exemplary therapeutic agents that can be co-formulated with a modified PH20 polypeptide are described in described in Section H. In particular, provided herein are co-formulations containing a modified PH20 polypeptide and an insulin, such as a fast-acting insulin, for example, a regular insulin or a fast-acting (rapid-acting) insulin analog. The co-formulations provided herein include stable co-formulations, whereby the active agents, i.e., the modified PH20 polypeptide and the therapeutic agent, exhibit increased stability and retain activity for prolonged periods as described herein. 
     Formulations containing PH20 provided herein, including separate formulations thereof and co-formulations, are stable for prolonged periods of time, including at varied temperatures and under varied storage or use conditions such as agitation. For example, the formulations provided herein are stable and retain activity of active agent(s) (e.g., PH20 hyaluronidase) at “refrigerator” conditions, for example, at 2° C. to 8° C., such as at or about 4° C., for at least at least 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 24 months, 25 months, 26 months, 27 months, 28 months, 29 months or 30 months or more. In another example, the formulations provided herein are stable and retain activity of active agent(s) (e.g., PH20 hyaluronidase) at room temperature for example at 18° C. to 32° C., generally 20° C. to 32° C., such as 28° C. to 32° C., for at least 2 weeks to 1 year, for example, at least 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, at least 7 months, at least 8 months, at least 9 months, or at least 1 year or more. In a further example, the formulations provided herein are stable and retain activity of active agent(s) (e.g., PH20 hyaluronidase) at elevated temperatures of about or greater than 30° C., generally from or from about 30° C. to 42° C., such as 32° C. to 37° C. or 35° C. to 37° C. or about or 37° C. for at least 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 35 days, 40 days, 45 days, 50 days, 60 days or more. 
     Compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, and sustained release formulations. A composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and other such agents. Topical formulations also are contemplated. The formulation should suit the mode of administration. 
     1. Formulations—Liquids, Injectables and Emulsions 
     The formulation generally is made to suit the route of administration. Parenteral administration, generally characterized by injection or infusion, either subcutaneously, intramuscularly, intravenously or intradermally is contemplated herein. Preparations for parenteral administration include sterile solutions ready for injection, sterile dry soluble products, such as lyophilized powders, ready to be combined with a solvent just prior to use, including hypodermic tablets, sterile suspensions ready for injection, sterile dry insoluble products ready to be combined with a vehicle just prior to use and sterile emulsions. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. For example, the compositions containing a modified PH20 polypeptide, formulated separately or co-formulated with another therapeutic agent, can be provided as a pharmaceutical preparation in liquid form as a solution, syrup or suspension. In liquid form, the pharmaceutical preparations can be provided as a concentrated preparation to be diluted to a therapeutically effective concentration before use. Generally, the preparations are provided in a dosage form that does not require dilution for use. In another example, pharmaceutical preparations can be presented in lyophilized form for reconstitution with water or other suitable vehicle before use. 
     Injectables are designed for local and systemic administration. For purposes herein, local administration is desired for direct administration to the affected interstitium. The solutions can be either aqueous or nonaqueous. If administered intravenously, suitable carriers include physiological saline or phosphate buffered saline (PBS), and solutions containing thickening and solubilizing agents, such as glucose, polyethylene glycol, and polypropylene glycol and mixtures thereof. 
     The concentration of the pharmaceutically active compound is adjusted so that an injection or infusion provides an effective amount to produce the desired pharmacological effect. The exact dose depends on the age, weight and condition of the patient or animal as is known in the art. The unit-dose parenteral preparations can be packaged in, for example, an ampoule, a cartridge, a vial or a syringe with a needle. The volume of liquid solution or reconstituted powder preparation, containing the pharmaceutically active compound, is a function of the disease to be treated and the particular article of manufacture chosen for package. All preparations for parenteral administration must be sterile, as is known and practiced in the art. The percentage of active compound contained in such parenteral compositions is highly dependent on the specific nature thereof, as well as the activity of the compound and the needs of the subject. 
     Pharmaceutical compositions can include carriers or other excipients. For example, pharmaceutical compositions provided herein can contain any one or more of a diluents(s), adjuvant(s), antiadherent(s), binder(s), coating(s), filler(s), flavor(s), color(s), lubricant(s), glidant(s), preservative(s), detergent(s), sorbent(s) or sweetener(s) and a combination thereof or vehicle with which a modified PH20 polypeptide is administered. For example, pharmaceutically acceptable carriers or excipients used in parenteral preparations include aqueous vehicles, nonaqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, emulsifying agents, sequestering or chelating agents and other pharmaceutically acceptable substances. Formulations, including liquid preparations, can be prepared by conventional means with pharmaceutically acceptable additives or excipients. 
     Examples of suitable pharmaceutical carriers are described in “Remington&#39;s Pharmaceutical Sciences” by E. W. Martin. Such compositions will contain a therapeutically effective amount of the compound, generally in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. Such pharmaceutical carriers can be sterile liquids, such as water or oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, and sesame oil. Water is a typical carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions also can be employed as liquid carriers, particularly for injectable solutions. Examples of aqueous vehicles include Sodium Chloride Injection, Ringers Injection, Isotonic Dextrose Injection, Sterile Water Injection, Dextrose and Lactated Ringers Injection. Nonaqueous parenteral vehicles include fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil and peanut oil. Suspending and dispersing agents include, but are not limited to, sorbitol syrup, cellulose derivatives or hydrogenated edible fats, sodium carboxymethylcellulose, hydroxypropyl methylcellulose and polyvinylpyrrolidone. Emulsifying agents include, but are not limited to, lecithin or acacia. Detergents include, but are not limited to, Polysorbate 80 (TWEEN 80). Non-aqueous vehicles include, but are not limited to, almond oil, oily esters, or fractionated vegetable oils. Anti-microbial agents or preservatives include, but are not limited to, methyl or propyl-p-hydroxybenzoates or sorbic acid, m-cresol, phenol. A diluent includes, but is not limited to, lactose, sucrose, dicalcium phosphate, or carboxymethylcellulose. A lubricant includes, but is not limited to, magnesium stearate, calcium stearate or talc. A binder includes, but is not limited to, starch, natural gums, such as gum acacia, gelatin, glucose, molasses, polyvinylpyrrolidine, celluloses and derivatives thereof, povidone, crospovidones and other such binders known to those of skill in the art. Isotonic agents include, but are not limited to, sodium chloride and dextrose. Buffers include, but are not limited to, phosphate and citrate. Antioxidants include sodium bisulfate. Local anesthetics include procaine hydrochloride. A sequestering or chelating agent of metal ions includes EDTA. Other suitable pharmaceutical excipients include, but are not limited to, starch, glucose, lactose, dextrose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, saline, water, and ethanol. Pharmaceutical carriers also include ethyl alcohol, polyethylene glycol and propylene glycol for water miscible vehicles and sodium hydroxide, hydrochloric acid, citric acid or lactic acid for pH adjustment. A composition, if desired, also can contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, or pH buffering agents, for example, acetate, sodium citrate, cyclodextrin derivatives, sorbitan monolaurate, triethanolamine sodium acetate, triethanolamine oleate, stabilizers, solubility enhancers, and other such agents such as for example, sodium acetate, sodium phosphate, sorbitan monolaurate, triethanolamine oleate and cyclodextrins. 
     In particular, antimicrobial agents (e.g., preservatives) in bacteriostatic or fungistatic concentrations (e.g., an anti-microbial effective amount) can be added to parenteral preparations packaged in multiple-dose containers, which include phenols or cresols, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride and benzethonium chloride. 
     The volume of the formulations, including the separately formulated or co-formulated PH20-containing formulations provided herein, can be any volume suitable for the container in which it is provided. In some examples, the formulations are provided in a vial, syringe, pen, reservoir for a pump or a closed loop system, or any other suitable container. For example, the formulations provided herein are between or about between 0.1 mL to 500 mL, such as 0.1 mL to 100 mL, 1 mL to 100 mL, 0.1 mL to 50 mL, such as at least or about at least or about or 0.1 mL, 1 mL, 2 mL, 3 mL, 4 mL, 5 mL, 10 mL, 15 mL, 20 mL, 30 mL, 40 mL, 50 mL or more. 
     a. Lyophilized Powders 
     Of interest herein are lyophilized powders, which can be reconstituted for administration as solutions, emulsions and other mixtures. They may also be reconstituted and formulated as solids or gels. 
     The sterile, lyophilized powder is prepared by dissolving a compound of enzyme in a buffer solution. The buffer solution may contain an excipient which improves the stability or other pharmacological component of the powder or reconstituted solution, prepared from the powder. Subsequent sterile filtration of the solution followed by lyophilization under standard conditions known to those of skill in the art provides the desired formulation. A liquid formulation as described herein above can be prepared. The resulting mixture is sterile filtered or treated to remove particulates and to insure sterility, and apportioned into vials for lyophilization. For example, the lyophilized powder can be prepared by dissolving an excipient, such as dextrose, sorbitol, fructose, corn syrup, xylitol, glycerin, glucose, sucrose or other suitable agent, in a suitable buffer, such as citrate, sodium or potassium phosphate or other such buffer known to those of skill in the art. Then, a selected enzyme is added to the resulting mixture, and stirred until it dissolves. 
     Each vial is made to contain a single dosage or multiple dosages of the compound. The lyophilized powder can be stored under appropriate conditions, such as at about 4° C. to room temperature. Reconstitution of this lyophilized powder with an appropriate buffer solution provides a formulation for use in parenteral administration. 
     b. Exemplary Formulations 
     Single dose formulations of PH20 are known in the art. For example, Hylenex® recombinant hyaluronidase (hyaluronidase human injection) contains, per mL, 8.5 mg NaCl (145 mM), 1.4 mg dibasic sodium phosphate (9.9 mM), 1.0 mg human albumin, 0.9 mg edetate disodium (2.4 mM), 0.3 mg CaCl 2  (2.7 mM) and NaOH to adjust the pH to 7.4. Other formulations of human soluble hyaluronidase, such as the rHuPH20 formulations described in U.S. Pat. Pub. No. US2011/0053247, include 130 mM NaCl, 10 mM Hepes, pH 7.0; or 10 mM histidine, 130 mM NaCl, pH 6.0. Any of the modified PH20 polypeptides provided herein can be similarly formulated. 
     In addition to a therapeutically effective amount of a modified PH20 polypeptide and/or other therapeutic agent, exemplary pharmaceutical compositions provided herein, including separately formulated- and co-formulated-PH20 containing formulations, can contain a concentration of NaCl and are prepared at a requisite pH to maintain the stability of the active agent(s) (e.g., PH20 hyaluronidase and/or other co-formulated therapeutic agent). For multi-dose formulations and other formulations stored for a prolonged time, the compositions generally also contain one or more preservatives. Further stabilizing agents and other excipients also can be included. Exemplary components are described below. 
     i. Salt (e.g. NaCl) 
     In examples herein, the pharmaceutical compositions provided herein contain a concentration of salt, such as sodium chloride (NaCl), to maintain the stability of the active agent(s) (e.g., PH20 hyaluronidase). Salt, such as NaCl, is generally required to retain PH20 stability and activity. Low salt concentrations of generally less than 120 mM can have deleterious effects on PH20 activity over time and depending on temperature conditions. Hence, the absence of salt (e.g. NaCl) or a low concentration of salt (e.g. NaCl) can result in instability of the protein. In some examples herein, however, modified PH20 polypeptides that exhibit increased stability in the absence of low or no salt, such as low or no NaCl (see e.g., Section C.1.b.iii), are not susceptible to denaturation. Also, the presence of salt (e.g. NaCl) can have differing effects on other therapeutic agents. For example, the solubility of insulin and insulin analogs tends to increase with lower salt concentration (e.g., &lt;140 mM) and high salt concentrations can result in crystallization/aggregation of insulin, especially at lower temperatures (see e.g., U.S. Provisional Appl. No. 61/520,962; U.S. application Ser. Nos. 13/507,263 and 13/507,262; and International PCT Application No. PCT/US2012/042816). Thus, pharmaceutical compositions provided herein are prepared in accordance with the requirements of the active agent(s). It is within the level of one of skill in the art to assess the stability of the active agent(s) in the formulation and under various storage conditions (see e.g., Section G). In particular examples herein, the pharmaceutical compositions, including the separately formulated or co-formulated PH20-containing formulations provided herein, contain NaCl at a concentration of between or about between 10 mM to 200 mM, such as 10 mM to 50 mM, 50 mM to 200 mM, 50 mM to 120 mM, 50 mM to 100 mM, 50 mM to 90 mM, 120 mM to 160 mM, 130 mM to 150 mM, 80 mM to 140 mM, 80 mM to 120 mM, 80 mM to 100 mM, 80 mM to 160 mM, 100 mM to 140 mM, 120 mM to 120 mM or 140 mM to 180 mM. 
     ii. pH and Buffer 
     In examples herein, the pharmaceutical compositions provided herein are prepared at a pH to maintain the stability of the active agent(s) (e.g., PH20 hyaluronidase). For example, the pharmaceutical compositions provided herein are prepared at a pH of between or about between 6.5 to 7.8 such as between or about between 6.5 to 7.2, 7.0 to 7.8, 7.0 to 7.6 or 7.2 to 7.4. Reference to pH herein is based on measurement of pH at room temperature. It is understood that the pH can change during storage over time, but typically will remain between or between about pH 6.5 to or to about 7.8. For example, the pH can vary by ±0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.2, 1.3, 1.4, 1.5 or more. Exemplary co-formulations provided herein have a pH of or of about 7.0±0.2, 7.1±0.2, 7.2±0.2, 7.3±0.2, 7.4±0.2, 7.5±0.2 or 7.6±0.2 when prepared. If necessary, pH can be adjusted using acidifying agents to lower the pH or alkalizing agents to increase the pH. Exemplary acidifying agents include, but are not limited to, acetic acid, citric acid, sulfuric acid, hydrochloric acid, monobasic sodium phosphate solution, and phosphoric acid. Exemplary alkalizing agents include, but are not limited to, dibasic sodium phosphate solution, sodium carbonate, or sodium hydroxide. 
     The compositions are generally prepared using a buffering agent that maintains the pH range. Any buffer can be used in formulations provided herein so long as it does not adversely affect the stability of the active agent(s) (e.g., PH20 hyaluronidase), and supports the requisite pH range required. Examples of particularly suitable buffers include Tris, succinate, acetate, phosphate buffers, citrate, aconitate, malate and carbonate. Those of skill in the art, however, will recognize that formulations provided herein are not limited to a particular buffer, so long as the buffer provides an acceptable degree of pH stability, or “buffer capacity” in the range indicated. Generally, a buffer has an adequate buffer capacity within about 1 pH unit of its pK (Lachman et al. In: The Theory and Practice of Industrial Pharmacy 3rd Edn. (Lachman, L., Lieberman, H A. and Kanig, J. L., Eds.), Lea and Febiger, Philadelphia, p. 458-460, 1986). Buffer suitability can be estimated based on published pK tabulations or can be determined empirically by methods well known in the art. The pH of the solution can be adjusted to the desired endpoint within the range as described above, for example, using any acceptable acid or base. 
     Buffers that can be included in the co-formulations provided herein include, but are not limited to, Tris (Tromethamine), histidine, phosphate buffers, such as dibasic sodium phosphate, and citrate buffers. Such buffering agents can be present in the co-formulations at concentrations between or about between 1 mM to 100 mM, such as 10 mM to 50 mM or 20 mM to 40 mM, such as at or about 30 mM. For example, such buffering agents can be present in the co-formulations in a concentration of or about 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, or more. 
     iii. Preservative(s) 
     In examples herein, multi-dose formulations or formulations stored for prolonged periods contain an anti-microbially effective amount of preservative or mixture of preservatives in an amount to have a bacteriostatic or fungistatic effect. In particular examples, the preservatives are present in a sufficient concentration to provide the anti-microbial requirements of, for example, the United States Pharmacopoeia (USP) and the European Pharmacopoeia (EP), including the EP anti-microbial requirements (EPA) and the preferred EP anti-microbial requirements (EPB) (see Table 4). Since the presence of preservatives, and in particular phenolic preservatives, can have deleterious effects on the stability of PH20, such formulations typically contain a modified PH20 polypeptide that exhibits increased stability in the presence of preservatives, such as any described in Section C.1.b.i herein. Generally, the amount maintains the stability of the active agent(s) (e.g., PH20 hyaluronidase). 
     An anti-microbial effective amount of preservative is an amount that exhibits anti-microbial activity by killing or inhibiting the propagation of microbial organisms in a sample of the composition as assessed in an antimicrobial preservative effectiveness test (APET). One of skill in the art is familiar with the antimicrobial preservative effectiveness test and standards to be meet under the USP and EPA or EPB in order to meet minimum requirements. In general, the antimicrobial preservative effectiveness test involves challenging a composition with prescribed inoculums of suitable microorganisms, i.e., bacteria, yeast and fungi, storing the inoculated preparation at a prescribed temperature, withdrawing samples at specified intervals of time and counting the organisms in the sample (see, Sutton and Porter, (2002)  PDA Journal of Pharmaceutical Science and Technology  56(4):300-311; The United States Pharmacopeial Convention, Inc., (effective Jan. 1, 2002),  The United States Pharmacopeia  25 th    Revision , Rockville, Md., Chapter &lt;51&gt; Antimicrobial Effectiveness Testing; and European Pharmacopoeia, Chapter 5.1.3, Efficacy of Antimicrobial Preservation). The microorganisms used in the challenge generally include three strains of bacteria, namely  E. coli  (ATCC No. 8739),  Pseudomonas aeruginosa  (ATCC No. 9027) and  Staphylococcus aureus  (ATCC No. 6538), yeast ( Candida albicans  ATCC No. 10231) and fungus ( Aspergillus niger  ATCC No. 16404), all of which are added such that the inoculated composition contains 10 5  or 10 6  colony forming units (cfu) of microorganism per mL of composition. The preservative properties of the composition are deemed adequate if, under the conditions of the test, there is a significant fall or no increase, as specified in Table 3 in the number of microorganisms in the inoculated composition after the times and at the temperatures prescribed. The criteria for evaluation are given in terms of the log reduction in the number of viable microorganism as compared to the initial sample or the previous time point. 
     Non-limiting examples of preservatives that can be included in the co-formulations provided herein include, but are not limited to, phenol, meta-cresol (m-cresol), methylparaben, benzyl alcohol, thimerosal, benzalkonium chloride, 4-chloro-1-butanol, chlorhexidine dihydrochloride, chlorhexidine digluconate, L-phenylalanine, EDTA, bronopol (2-bromo-2-nitropropane-1,3-diol), phenylmercuric acetate, glycerol (glycerin), imidurea, chlorhexidine, sodium dehydroacetate, ortho-cresol (o-cresol), para-cresol (p-cresol), chlorocresol, cetrimide, benzethonium chloride, ethylparaben, propylparaben or butylparaben and any combination thereof. For example, formulations provided herein can contain a single preservative. In other examples, the formulations contain at least two different preservatives or at least three different preservatives. For example, formulations provided herein can contain two preservatives such as L-phenylalanine and m-cresol, L-phenylalanine and methylparaben, L-phenylalanine and phenol, m-cresol and methylparaben, phenol and methylparaben, m-cresol and phenol or other similar combinations. In one example, the preservative in the formulation contains at least one phenolic preservative. For example, the formulation contains phenol, m-cresol or phenol and m-cresol. 
     In the formulations provided herein, the total amount of the one or more preservative agents as a percentage (%) of mass concentration (w/v) in the formulation can be, for example, between from or between about from 0.1% to 0.4%, such as 0.1% to 0.3%, 0.15% to 0.325%, 0.15% to 0.25%, 0.1% to 0.2%, 0.2% to 0.3%, or 0.3% to 0.4%. Generally, the formulations contain less than 0.4% (w/v) preservative. For example, the co-formulations provided herein contain at least or about at least 0.1%, 0.12%, 0.125%, 0.13%, 0.14%, 0.15%, 0.16% 0.17%, 0.175%, 0.18%, 0.19%, 0.2%, 0.25%, 0.3%, 0.325%, 0.35% but less than 0.4% total preservative. 
     In some examples, the formulations provided herein contain between or between about 0.1% to 0.25% phenol and between or about between 0.05% to 0.2% m-cresol, such as between or about between 0.10% to 0.2% phenol and between or about between 0.06% to 0.18% m-cresol, or between or about between 0.1% to 0.15% phenol and between or about between 0.08% to 0.15% m-cresol. For example, formulations provided herein contain or contain about 0.1% phenol and 0.075% m-cresol; 0.1% phenol and 0.15% m-cresol; 0.125% phenol and 0.075% m-cresol; 0.13% phenol and 0.075% m-cresol; 0.13% phenol and 0.08% m-cresol; 0.15% phenol and 0.175% m-cresol; or 0.17% phenol and 0.13% m-cresol. 
     iv. Stabilizers 
     In examples herein, the pharmaceutical compositions provided herein optionally can contain one or more other stabilizing agent to maintain the stability of the active agent(s) (e.g., PH20 hyaluronidase). Included among the types of stabilizers that can be contained in the formulations provided herein are amino acids, amino acid derivatives, amines, sugars, polyols, salts and buffers, surfactants, and other agents. The formulations provided herein contain at least one stabilizer. For example, the formulations provided herein contain at least one, two, three, four, five, six or more stabilizers. Hence, any one or more of an amino acids, amino acid derivatives, amines, sugars, polyols, salts and buffers, surfactants, and other agents can be included in the formulations herein. Generally, the formulations herein contain at least contain a surfactant and an appropriate buffer. Optionally, the formulations provided herein can contain other additional stabilizers. Other components include, for example, one or more tonicity modifiers, one or more anti-oxidation agents, or other stabilizer. 
     Exemplary amino acid stabilizers, amino acid derivatives or amines include, but are not limited to, L-Arginine, Glutamine, Glycine, Lysine, Methionine, Proline, Lys-Lys, Gly-Gly, Trimethylamine oxide (TMAO) or betaine. Exemplary sugars and polyols include, but are not limited to, glycerol, sorbitol, mannitol, inositol, sucrose or trehalose. Exemplary salts and buffers include, but are not limited to, magnesium chloride, sodium sulfate, Tris such as Tris (100 mM), or sodium Benzoate. Exemplary surfactants include, but are not limited to, poloxamer 188 (e.g., Pluronic® F68), polysorbate 80 (PS80), polysorbate 20 (PS20). Other stabilizers include, but are not limited to, hyaluronic acid (HA), human serum albumin (HSA), phenyl butyric acid, taurocholic acid, polyvinylpyrolidone (PVP) or zinc. 
     In particular examples herein, the formulations contain one or more detergents, such as surfactants, to maintain the stability of the active agent(s) (e.g., PH20 hyaluronidase). For example, surfactants can inhibit aggregation of PH20 and minimize absorptive loss. The surfactants generally are non-ionic surfactants. Surfactants that can be included in the formulations herein include, but are not limited to, partial and fatty acid esters and ethers of polyhydric alcohols such as of glycerol, or sorbitol, poloxamers and polysorbates. For example, exemplary surfactants in the —formulations herein include any one or more of poloxamer 188 (PLURONICS® poloxamer such as PLURONIC® F68 poloxamer), TETRONICS® surfactant, polysorbate 20, polysorbate 80, PEG 400, PEG 3000, Tween® surfactant (e.g., Tween® 20 surfactant or Tween® 80 surfactant), Triton® X-100 surfactant, SPAN® surfactant, MYRJ® surfactant, BRIJ® surfactant, CREMOPHOR® surfactant, polypropylene glycols or polyethylene glycols. In some examples, the formulations herein contain poloxamer 188, polysorbate 20, polysorbate 80, generally poloxamer 188 (pluronic F68). The formulations provided herein generally contain at least one surfactant, such as 1, 2 or 3 surfactants. 
     In the formulations provided herein, the total amount of the one or more surfactants as a percentage (%) of mass concentration (w/v) in the formulation can be, for example, between from or between about from 0.005% to 1.0%, such as between from or between about from 0.01% to 0.5%, such as 0.01% to 0.1% or 0.01% to 0.02%. Generally, the formulations contain at least 0.01% surfactant and contain less than 1.0%, such as less than 0.5% or less than 0.1% surfactant. For example, the formulations provided herein can contain at or about 0.001%, 0.005%, 0.01%, 0.015%, 0.02%, 0.025%, 0.03%, 0.035%, 0.04%, 0.045%, 0.05%, 0.055%, 0.06%, 0.065%, 0.07%, 0.08%, or 0.09% surfactant. In particular examples, the formulations provided herein contain or contain about 0.01% to or to about 0.05% surfactant. 
     Tonicity modifiers can be included in the formulation provided herein to produce a solution with the desired osmolality. The formulations provided herein have an osmolality of between or about between 245 mOsm/kg to 305 mOsm/kg. For example, the osmolality is or is about 245 mOsm/kg, 250 mOsm/kg, 255 mOsm/kg, 260 mOsm/kg, 265 mOsm/kg, 270 mOsm/kg, 275 mOsm/kg, 280 mOsm/kg, 285 mOsm/kg, 290 mOsm/kg, 295 mOsm/kg, 300 mOsm/kg or 305 mOsm/kg. In some examples, the formulations have an osmolality of or of about 275 mOsm/kg. Tonicity modifiers include, but are not limited to, glycerin, NaCl, amino acids, polyalcohols, trehalose, and other salts and/or sugars. The particular amount can be empirically determined in order to retain enzyme activity, and/or tonicity. 
     In other instances, glycerin (glycerol) is included in the formulations. For example, formulations provided herein typically contain less than 60 mM glycerin, such as less than 55 mM, less than 50 mM, less than 45 mM, less than 40 mM, less than 35 mM, less than 30 mM, less than 25 mM, less than 20 mM, less than 15 mM, 10 mM or less. The amount of glycerin typically depends on the amount of NaCl present: the more NaCl present in the formulation, the less glycerin is required to achieve the desired osmolality or osmolarity. Thus, for example, in formulations containing higher NaCl concentrations, little or no glycerin need be included in the formulation. In contrast, in formulations containing slightly lower NaCl concentrations, glycerin can be included. For example, formulations provided herein can contain glycerin at a concentration of 40 mM to 60 mM, such as less than 50 mM, such as 20 mM to 50 mM, for example at or about 50 mM. 
     The formulations provided herein also can contain antioxidants to reduce or prevent oxidation, in particular oxidation of the PH20 polypeptide. For example, oxidation can be effected by high concentrations of surfactant or hyaluronan oligomers. Exemplary antioxidants include, but are not limited to, cysteine, tryptophan and methionine. In particular examples, the anti-oxidant is methionine. The formulations provided herein can include an antioxidant at a concentration from between or from about between 5 mM to or to about 50 mM, such as 5 mM to 40 mM, 5 mM to 20 mM or 10 mM to 20 mM. For example, methionine can be provided in the formulations herein at a concentration from between or from about between 5 mM to or to about 50 mM, such as 5 mM to 40 mM, 5 mM to 20 mM or 10 mM to 20 mM. For example, an antioxidant, for example methionine, can be included at a concentration that is or is about 5 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM, 21 mM, 22 mM, 23 mM, 24 mM, 25 mM, 26 mM, 27 mM, 28 mM, 29 mM, 30 mM, 35 mM, 40 mM, 45 mM or 50 mM. In some examples, the formulations contain 10 mM to 20 mM methionine, such as or about 10 mM or 20 mM methionine. 
     The formulations provided herein also can contain an amino acid stabilizer, which contributes to the stability of the preparation. The stabilizer can be a non-polar or basic amino acid. Exemplary non-polar and basic amino acids include, but are not limited to, alanine, histidine, arginine, lysine, ornithine, isoleucine, valine, methionine, glycine and proline. For example, the amino acid stabilizer is glycine or proline, typically glycine. The stabilizer can be a single amino acid or it can be a combination of 2 or more such amino acids. The amino acid stabilizers can be natural amino acids, amino acid analogues, modified amino acids or amino acid equivalents. Generally, the amino acid is an L-amino acid. For example, when proline is used as the stabilizer, it is generally L-proline. It is also possible to use amino acid equivalents, for example, proline analogues. The concentration of amino acid stabilizer, for example glycine, included in the formulation ranges from 0.1 M to 1 M amino acid, typically 0.1 M to 0.75 M, generally 0.2 M to 0.5 M, for example, at least at or about 0.1 M, 0.15 M, 0.2 M, 0.25 M, 0.3 M, 0.35 M, 0.4 M, 0.45 M, 0.5 M, 0.6 M, 0.7 M, 0.75 M or more amino acid. The amino acid, for example glycine, can be used in a form of a pharmaceutically acceptable salt, such as hydrochloride, hydrobromide, sulfate, acetate, etc. The purity of the amino acid, for example glycine, should be at least 98%, at least 99%, or at least 99.5% or more. 
     In examples herein, if necessary, hyaluronidase inhibitors are included in a formulation to stabilize PH20, in particular to reduce the effects of otherwise destabilizing agents and conditions, such as, for example, low salt, high pH, the presence of preservatives and elevated temperatures, present in the formulation. Such a component generally is not required for pharmaceutical compositions containing a modified PH20 polypeptide as provided herein that exhibits increased stability under such conditions. When provided, the hyaluronidase inhibitor is provided at least at its equilibrium concentration. One of skill in the art is familiar with various classes of hyaluronidase inhibitors (see e.g., Girish et al. (2009)  Current Medicinal Chemistry,  16:2261-2288, and references cited therein). One of skill in the art knows or can determine by standard methods in the art the equilibrium concentration of a hyaluronidase inhibitor in a reaction or stable composition herein. 
     An exemplary hyaluronidase inhibitor for use in the compositions herein is hyaluronan (HA). Hyaluronic acid (HA, also known as hyaluronan and hyaluronate) is the natural substrate for PH20. HA is a non-sulfated glycosaminoglycan that is widely distributed throughout connective, epithelial, and neural tissues. It is a polymer of up to 25,000 disaccharide units, themselves composed of D-glucuronic acid and D-N-acetylglucosamine. The molecular weight of HA ranges from about 5 kDa to 200,000 kDa. Any size HA can be used in the compositions as a stabilizer. In some examples, the HA is a disaccharide, composed of D-glucuronic acid and D-N-acetylglucosamine. In other examples, the HA is an oligosaccharide, such as a tetrasaccharide, containing 2 repeating disaccharide units, or alternatively, the HA used in the co-formulations provided herein can contain multiple repeating disaccharide units, such as 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 or more disaccharide units. In another example, the HA used in the formulations provided herein has a molecular weight that is from or from about 5 kDa to or to about 5,000 kDa; from or from about 5 kDa to or to about 1,000 kDa; from or from about 5 kDa to or to about 500 kDa; or from or from about 5 kDa to or to about 200 kDa. Exemplary HA oligosaccharides for use in the formulations herein have a molecular weight of or of about 6.4 kDa, 74.0 kDa. or 234.4 kDa. The formulations can contain 1 mg/mL to 20 mg/mL HA, 8 mg/mL to 12 mg/mL, such as at least or about 1 mg/mL, 2 mg/mL, 3 mg/mL, 4 mg/mL, 5 mg/mL, 6 mg/mL, 7 mg/mL, 8 mg/mL, 9 mg/mL, 10 mg/mL, 11 mg/mL, 12 mg/mL, 13 mg/mL, 14 mg/mL, 15 mg/mL, 16 mg/mL, 17 mg/mL, 18 mg/mL, 19 mg/mL or 20 mg/mL or more HA. In some examples, the molar ratio of HA to PH20 is or is about 100,000:1, 95,000:1, 90,000:1, 85,000:1, 80,000:1, 75,000:1, 70,000:1, 65,000:1, 60,000:1, 55,000:1, 50,000:1, 45,000:1, 40,000:1, 35,000:1, 30,000:1, 25,000:1, 20,000:1, 15,000:1, 10,000:1, 5,000:1, 1,000:1, 900:1, 800:1, 700:1, 600:1, 500:1, 400:1, 300:1, 200:1, or 100:1 or less. 
     In some examples, a nicotinic compound is used as a stabilizing agent. Nicotinic compounds include, but are not limited to, nicotinamide, nicotinic acid, niacin, niacinamide, vitamin B3 and/or salts thereof and/or any combination thereof. In particular applications, the stabilizing agent can include a nicotinic compound an amino acid or amino acids (see e.g., International Publication No. WO2010149772). For example, the amino acid can be arginine, glutamic acid and/or salts thereof or combinations thereof. 
     2. Compositions for Other Routes of Administration 
     Depending upon the condition treated other routes of administration, such as topical application, transdermal patches, oral and rectal administration are also contemplated herein. 
     For example, pharmaceutical dosage forms for rectal administration are rectal suppositories, capsules and tablets for systemic effect. Rectal suppositories include solid bodies for insertion into the rectum which melt or soften at body temperature releasing one or more pharmacologically or therapeutically active ingredients. Pharmaceutically acceptable substances utilized in rectal suppositories are bases or vehicles and agents to raise the melting point. Examples of bases include cocoa butter (theobroma oil), glycerin-gelatin, carbowax (polyoxyethylene glycol) and appropriate mixtures of mono-, di- and triglycerides of fatty acids. Combinations of the various bases may be used. Agents to raise the melting point of suppositories include spermaceti and wax. Rectal suppositories may be prepared either by the compressed method or by molding. The typical weight of a rectal suppository is about 2 to 3 gm. Tablets and capsules for rectal administration are manufactured using the same pharmaceutically acceptable substance and by the same methods as for formulations for oral administration. Formulations suitable for rectal administration can be provided as unit dose suppositories. These can be prepared by admixing the active compound with one or more conventional solid carriers, for example, cocoa butter, and then shaping the resulting mixture. 
     For oral administration, pharmaceutical compositions can take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinized maize starch, polyvinyl pyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate). The tablets can be coated by methods well-known in the art. 
     Formulations suitable for buccal (sublingual) administration include, for example, lozenges containing the active compound in a flavored base, usually sucrose and acacia or tragacanth; and pastilles containing the compound in an inert base such as gelatin and glycerin or sucrose and acacia. 
     Topical mixtures are prepared as described for the local and systemic administration. The resulting mixtures can be solutions, suspensions, emulsion or the like and are formulated as creams, gels, ointments, emulsions, solutions, elixirs, lotions, suspensions, tinctures, pastes, foams, aerosols, irrigations, sprays, suppositories, bandages, dermal patches or any other formulations suitable for topical administration. 
     The compounds or pharmaceutically acceptable derivatives thereof may be formulated as aerosols for topical application, such as by inhalation (see, e.g., U.S. Pat. Nos. 4,044,126, 4,414,209, and 4,364,923, which describe aerosols for delivery of a steroid useful for treatment of inflammatory diseases, particularly asthma). These formulations, for administration to the respiratory tract, can be in the form of an aerosol or solution for a nebulizer, or as a microfine powder for insufflation, alone or in combination with an inert carrier such as lactose. In such a case, the particles of the formulation will typically have diameters of less than 50 microns, or less than 10 microns. 
     The compounds can be formulated for local or topical application, such as for topical application to the skin and mucous membranes, such as in the eye, in the form of gels, creams, and lotions and for application to the eye or for intracisternal or intraspinal application. Topical administration is contemplated for transdermal delivery and also for administration to the eyes or mucosa, or for inhalation therapies. Nasal solutions of the active compound alone or in combination with other pharmaceutically acceptable excipients also can be administered. 
     Formulations suitable for transdermal administration are provided. They can be provided in any suitable format, such as discrete patches adapted to remain in intimate contact with the epidermis of the recipient for a prolonged period of time. Such patches contain the active compound in an optionally buffered aqueous solution of, for example, 0.1 to 0.2 M concentration with respect to the active compound. Formulations suitable for transdermal administration also can be delivered by iontophoresis (see, e.g., Tyle, P,  Pharmaceutical Research  3(6):318-326 (1986)) and typically take the form of an optionally buffered aqueous solution of the active compound. 
     Pharmaceutical compositions also can be administered by controlled release formulations and/or delivery devices (see e.g., in U.S. Pat. Nos. 3,536,809; 3,598,123; 3,630,200; 3,845,770; 3,916,899; 4,008,719; 4,769,027; 5,059,595; 5,073,543; 5,120,548; 5,591,767; 5,639,476; 5,674,533 and 5,733,566). 
     3. Dosages and Administration 
     The modified PH20 polypeptides provided herein can be formulated as pharmaceutical compositions for single dosage or multiple dosage administration. The PH20 polypeptide is included in an amount sufficient to exert a therapeutically useful effect in the absence of undesirable side effects on the patient treated. The therapeutically effective concentration can be determined empirically by testing the polypeptides in known in vitro and in vivo systems such as by using the assays provided herein or known in the art (see e.g., Taliani et al., (1996)  Anal. Biochem.,  240: 60-67; Filocamo et al., (1997)  J Virology,  71: 1417-1427; Sudo, (1996)  Antiviral Res.  32: 9-18; Bouffard et al., (1995)  Virology,  209:52-59; Bianchi et al., (1996)  Anal. Biochem.,  237: 239-244; Hamatake et al., (1996)  Intervirology  39:249-258; Steinkuhler et al., (1998)  Biochem.,  37:8899-8905; D&#39;Souza et al., (1995)  J Gen. Virol.,  76:1729-1736; Takeshita et al., (1997)  Anal. Biochem.,  247:242-246; see also e.g., Shimizu et al., (1994)  J. Virol.  68:8406-8408; Mizutani et al., (1996)  J. Virol.  70:7219-7223; Mizutani et al., (1996)  Biochem. Biophys. Res. Commun.,  227:822-826; Lu et al. (1996) Proc.  Natl. Acad. Sci  (USA), 93:1412-1417; Hahm et al., (1996)  Virology,  226:318-326; Ito et al. (1996)  J. Gen. Virol.,  77:1043-1054; Mizutani et al. (1995)  Biochem. Biophys. Res. Commun.,  212:906-911; Cho et al., (1997)  J. Virol. Meth.  65:201-207) and then extrapolated therefrom for dosages for humans. 
     The amount of a modified PH20 to be administered for the treatment of a disease or condition can be determined by standard clinical techniques. In addition, in vitro assays and animal models can be employed to help identify optimal dosage ranges. The precise dosage, which can be determined empirically, can depend on the particular enzyme, the route of administration, the type of disease to be treated and the seriousness of the disease. 
     Hence, it is understood that the precise dosage and duration of treatment is a function of the disease being treated and can be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro test data. It is to be noted that concentrations and dosage values also can vary with the severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that the concentration ranges set forth herein are exemplary only and are not intended to limit the scope or use of compositions and combinations containing them. The compositions can be administered hourly, daily, weekly, monthly, yearly or once. Generally, dosage regimens are chosen to limit toxicity. It should be noted that the attending physician would know how to and when to terminate, interrupt or adjust therapy to lower dosage due to toxicity, or bone marrow, liver or kidney or other tissue dysfunctions. Conversely, the attending physician would also know how to and when to adjust treatment to higher levels if the clinical response is not adequate (precluding toxic side effects). 
     Typically, a therapeutically effective dose of a modified PH20 enzyme is at or about 10 Unit (U) to 500,000 Units, 100 Units to 100,000 Units, 500 Units to 50,000 Units, 1000 Units to 10,000 Units, 5000 Units to 7500 Units, 5000 Units to 50,000 Units, or 1,000 Units to 10,000 Units, generally 1,000 to 50,000 Units, in a stabilized solution or suspension or a lyophilized form. For example, a PH20 polypeptide, can be administered at a dose of at least or about at least or 10 U, 20 U, 30 U, 40 U, 50 U, 100 U, 150 U, 200 U, 250 U, 300 U, 350 U, 400 U, 450 U, 500 U, 600 U, 700 U, 800 U, 900 U, 1000 U, 2,000 U, 3,000 U, 4,000 Units, 5,000 U or more. The formulations can be provided in unit-dose forms such as, but not limited to, ampoules, syringes and individually packaged tablets or capsules. 
     The PH20 enzyme can be administered alone, or with other pharmacologically effective agent(s) or therapeutic agent(s), in a total volume of 0.1-100 mL, 1-50 mL, 10-50 mL, 10-30 mL, 1-20 mL, or 1-10 mL, typically 10-50 mL. Typically, volumes of injections or infusions of a PH20-containing composition are at least or at least about 0.01 mL, 0.05 mL, 0.1 mL, 0.2 mL, 0.3 mL, 0.4 mL, 0.5 mL, 1 mL, 2 mL, 3 mL, 4 mL, 5 mL, 6 mL, 7 mL, 8 mL, 9 mL, 10 mL, 20 mL, 30 mL, 40 mL, 50 mL or more. The formulations provided herein contain a modified PH20 polypeptide in an amount between or about between 30 Units/mL to 3000 U/mL, 300 U/mL to 2000 U/mL or 600 U/mL to 2000 U/mL or 600 U/mL to 1000 U/mL, such as at least or about at least 30 U/mL, 35 U/mL, 40 U/mL, 50 U/mL, 100 U/mL, 200 U/mL, 300 U/mL, 400 U/mL, 500 U/mL, 600 U/mL, 700 U/mL, 800 U/mL, 900 U/mL, 1000 U/mL, 2000 U/mL or 3000 U/mL. For example, the formulations provided herein contain a PH20 that is in an amount that is at least 100 U/mL to 1000 U/mL, for example at least or about at least or about or 600 U/mL. 
     The PH20 polypeptide can be provided as a solution in an amount that is at least or about or is 100 U/mL, 150 U/mL, 200 U/mL, 300 U/mL, 400 U/mL, 500 U/mL, 600 U/mL, 800 U/mL or 1000 U/mL, or can be provided in a more concentrated form, for example in an amount that is at least or about or is 2000 U/mL, 3000 Units/mL, 4000 U/mL, 5000 U/mL, 8000 U/mL, 10,000 U/mL or 20,000 U/mL for use directly or for dilution to the effective concentration prior to use. The PH20 polypeptide compositions can be provided as a liquid or lyophilized formulation. 
     When the PH20 is co-formulated with a therapeutic agent, dosages can be provided as a ratio of the amount of a PH20 polypeptide to the amount of therapeutic agent administered. For example, a PH20 polypeptide can be administered at 1 hyaluronidase U/therapeutic agent U (1:1) to 50:1 or more, for example, at or about 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1 or more. 
     The formulations provided herein, including co-formulations and/or stable formulations, can be prepared for single dose administration, multiple dose administration or continuous infusion administrations. Implantation of a slow-release or sustained-release system, such that a constant level of dosage is maintained (see e.g., U.S. Pat. No. 3,710,795), is also contemplated herein. 
     For example, formulations of pharmaceutically therapeutically active compounds and derivatives thereof are provided for administration to humans and animals in unit dosage forms or multiple dosage forms. For example, compounds can be formulated as tablets, capsules, pills, powders, granules, sterile parenteral solutions or suspensions, oral solutions or suspensions, or oil-water emulsions containing suitable quantities of the compounds or pharmaceutically acceptable derivatives thereof. Each unit dose contains a predetermined quantity of therapeutically active compound(s) sufficient to produce the desired therapeutic effect, in association with the required pharmaceutical carrier, vehicle or diluent. Examples of unit dose forms include ampoules and syringes and individually packaged tablets or capsules. Unit dose forms can be administered in fractions or multiples thereof. A multiple dose form is a plurality of identical unit dosage forms packaged in a single container to be administered in segregated unit dose forms. Examples of multiple dose forms include vials, bottles of tablets or capsules or bottles of pints or gallons. Hence, multiple dose form is a multiple of unit doses that are not segregated in packaging. Generally, dosage forms or compositions containing active ingredient in the range of 0.005% to 100% with the balance made up from non-toxic carrier can be prepared. 
     Compositions provided herein typically are formulated for administration by subcutaneous route, although other routes of administration are contemplated, such as any route known to those of skill in the art including intramuscular, intraperitoneal, intravenous, intradermal, intralesional, intraperitoneal injection, epidural, vaginal, rectal, local, otic, transdermal administration or any route of administration. Formulations suited for such routes are known to one of skill in the art. Administration can be local, topical or systemic depending upon the locus of treatment. Local administration to an area in need of treatment can be achieved by, for example, but not limited to, local infusion during surgery, topical application, e.g., in conjunction with a wound dressing after surgery, by injection, by means of a catheter, by means of a suppository, or by means of an implant. Compositions also can be administered with other biologically active agents, either sequentially, intermittently or in the same composition. 
     The most suitable route in any given case depends on a variety of factors, such as the nature of the disease, the tolerance of the subject to a particular administration route, the severity of the disease, and the particular composition that is used. Typically, the compositions provided herein are administered parenterally. In some examples, modified PH20 polypeptide compositions are administered so that they reach the interstitium of skin or tissues, thereby degrading the interstitial space for subsequent delivery of a therapeutic agent. Thus, in some examples, direct administration under the skin, such as by subcutaneous administration methods, is contemplated. Thus, in one example, local administration can be achieved by injection, such as from a syringe or other article of manufacture containing an injection device such as a needle. In another example, local administration can be achieved by infusion, which can be facilitated by the use of a pump or other similar device. Other modes of administration also are contemplated. For example, modified PH20 polypeptides, included conjugated forms with increased half-life such as PEGylated forms thereof, can be administered intravenously. Pharmaceutical compositions can be formulated in dosage forms appropriate for each route of administration. 
     Administration methods can be employed to decrease the exposure of selected modified PH20 polypeptides to degradative processes, such as proteolytic degradation and immunological intervention via antigenic and immunogenic responses. Examples of such methods include local administration at the site of treatment. PEGylation of therapeutics increases resistance to proteolysis, increases plasma half-life, and decreases antigenicity and immunogenicity. Examples of PEGylation methodologies are known in the art (see for example, Lu and Felix,  Int. J. Peptide Protein Res.,  43: 127-138, 1994; Lu and Felix,  Peptide Res.,  6: 140-6, 1993; Felix et al.,  Int. J. Peptide Res.,  46: 253-64, 1995; Benhar et al.,  J. Biol. Chem.,  269: 13398-404, 1994; Brumeanu et al.,  J. Immunol.,  154: 3088-95, 1995; see also, Caliceti et al. (2003)  Adv. Drug Deliv. Rev.  55(10):1261-77 and Molineux (2003)  Pharmacotherapy  23 (8 Pt 2):3S-8S). PEGylation also can be used in the delivery of nucleic acid molecules in vivo. For example, PEGylation of adenovirus can increase stability and gene transfer (see, e.g., Cheng e et al., (2003)  Pharm. Res.  20(9): 1444-51). 
     Various other delivery systems are known and can be used to administer selected PH20 polypeptides, such as but not limited to, encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compound, receptor mediated endocytosis, and delivery of nucleic acid molecules encoding selected PH20 polypeptides such as retrovirus delivery systems. 
     Hence, in certain embodiments, liposomes and/or nanoparticles also can be employed with administration of soluble PH20 polypeptides. Liposomes are formed from phospholipids that are dispersed in an aqueous medium and spontaneously form multilamellar concentric bilayer vesicles (also termed multilamellar vesicles (MLVs)). MLVs generally have diameters of from 25 nm to 4 μm. Sonication of MLVs results in the formation of small unilamellar vesicles (SUVs) with diameters in the range of 200 to 500 angstroms containing an aqueous solution in the core. 
     Phospholipids can form a variety of structures other than liposomes when dispersed in water, depending on the molar ratio of lipid to water. At low ratios of lipid to water, liposomes form. Physical characteristics of liposomes depend on the pH, ionic strength and the presence of divalent cations. Liposomes can show low permeability to ionic and polar substances, but at elevated temperatures undergo a phase transition which markedly alters their permeability. The phase transition involves a change from a closely packed, ordered structure, known as the gel state, to a loosely packed, less-ordered structure, known as the fluid state. This occurs at a characteristic phase-transition temperature and results in an increase in permeability to ions, sugars and drugs. 
     Liposomes interact with cells via different mechanisms: endocytosis by phagocytic cells of the reticuloendothelial system such as macrophages and neutrophils; adsorption to the cell surface, either by nonspecific weak hydrophobic or electrostatic forces, or by specific interactions with cell-surface components; fusion with the plasma cell membrane by insertion of the lipid bilayer of the liposome into the plasma membrane, with simultaneous release of liposomal contents into the cytoplasm; and by transfer of liposomal lipids to cellular or subcellular membranes, or vice versa, without any association of the liposome contents. Varying the liposome formulation can alter which mechanism is operative, although more than one can operate at the same time. Nanocapsules can generally entrap compounds in a stable and reproducible way. To avoid side effects due to intracellular polymeric overloading, such ultrafine particles (sized around 0.1 μm) should be designed using polymers able to be degraded in vivo. Biodegradable polyalkyl-cyanoacrylate nanoparticles that meet these requirements are contemplated for use herein, and such particles can be easily made. 
     4. Exemplary PH20-Insulin Co-Formulations Provided herein are stable co-formulations of a fast acting insulin, such as a rapid acting (fast-acting) insulin analog, and a modified PH20 polypeptide. Any of the modified PH20 polypeptides provided herein can be included in a co-formulation with insulin, such as any of the co-formulations described in U.S. application Ser. No. 13/507,263 or 13/507,262 or in International PCT Application Serial No. PCT/US2012/042816. 
     In particular, the modified PH20 polypeptide is a modified PH20 polypeptide that exhibits increased stability under denaturation conditions, such as any set forth in Sections C.1.b. In particular, the PH20 polypeptide is a modified PH20 polypeptide that exhibits increased stability to one or more phenolic preservatives, such as any set forth in Section C.1.b.i. For example, the PH20 polypeptide is a modified PH20 polypeptide that contains an amino acid replacement with P at a position corresponding to position 204 with reference to amino acid positions set forth in SEQ ID NO:3, such as F204P with reference to any of SEQ ID NOs: 3, 7 or 32-66. In other examples, the PH20 polypeptide is a modified PH20 polypeptide that contains an amino acid replacement with R at a position corresponding to position 58 with reference to amino acid positions set forth in SEQ ID NO:3, such as V58R with reference to any of SEQ ID NOs: 3, 7 or 32-66. 
     The fast acting insulin can be a regular insulin or a rapid acting (fast-acting) insulin analog. Insulin is a polypeptide that when processed is composed of 51 amino acids containing an A- and B-chain. Generally, insulin contains an A-chain of about 21 amino acids and a B-chain of about 30 amino acids. The A- and B-chains are linked by disulfide bridges. Exemplary regular insulins include, for example, a human insulin (with an A chain having a sequence of amino acids set forth in SEQ ID NO:862 and a B chain having a sequence of amino acids set forth in SEQ ID NO:863) or a porcine insulin (with an A chain having a sequence of amino acids set forth as amino acid residue positions 88-108 of SEQ ID NO:864 and a B chain having a sequence of amino acids set forth as amino acid residue positions 25-54 of SEQ ID NO:864). Exemplary fast-acting insulin analogs are insulin variants that contain one or more amino acid modifications compared to a human insulin set forth in SEQ ID NO: 862 and 863 (A and B chains). For example, exemplary insulin analogs are known to one of skill in the art, and include, but are not limited to, glulisine having an A-chain set forth in SEQ ID NO:862 and a B-chain that is a variant of SEQ ID NO:863 (B-chain; LysB3, GluB29), HMR-1 153 having an A-chain set forth in SEQ ID NO:862 and a B-chain that is a variant of SEQ ID NO:863 (B-chain; LysB3, IleB28), insulin aspart having an A-chain set forth in SEQ ID NO:862 and a B-chain that is a variant of SEQ ID NO:863 (B-chain; AspB28), and insulin lispro having an A-chain set forth in SEQ ID NO:862 and a B-chain that is a variant of SEQ ID NO:863 (B-chain; LysB28, ProB29). In every instance above, the nomenclature of the analogs is based on a description of the amino acid substitution at specific positions on the A or B chain of insulin, numbered from the N-terminus of the chain, in which the remainder of the sequence is that of natural human insulin. Exemplary of such analog forms, are set forth in SEQ ID NO:862 (A-chain) and having a B-chain set forth in any of SEQ ID NOs: 865-867. 
     The co-formulations are stable as a liquid formulation for prolonged periods of time for at least 1 month at temperatures from or from about 2° C. to 8° C., inclusive, or for at least 3 days at a temperature from or from about 30° C. to 42° C., inclusive. For example, the co-formulations are stable and retain activity of the PH20 hyaluronidase and insulin at “refrigerator” conditions, for example, at 2° C. to 8° C., such as at or about 4° C., for at least at least 2 months, 3 months, 4 months, 5 months, 6 months, or 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 24 months, 25 months, 26 months, 27 months, 28 months, 29 months or 30 months or more. In another example, the formulations provided herein are stable and retain activity of the PH20 hyaluronidase and insulin at room temperature for example at 18° C. to 32° C., generally 20° C. to 32° C., such as 28° C. to 32° C., for at least 2 weeks to 1 year, for example, at least 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, at least 7 months, at least 8 months, at least 9 months, or at least 1 year or more. In a further example, the formulations provided herein are stable and retain activity of the PH20 hyaluronidase and insulin at elevated temperatures of about or greater than 30° C., generally from or from about 30° C. to 42° C., such as 32° C. to 37° C. or 35° C. to 37° C. or about or 37° C. for at least 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 35 days, 40 days, 45 days, 50 days, 60 days or more. 
     Assays to assess stability of active agents are well-known to one of skill in the art. Section G provides exemplary assays to assess stability of PH20 hyaluronidase. The stability of insulin can be assessed using similar methods well-known to one of skill in the art. For example, insulin stability and solubility can be assessed by visual assessment (e.g., including changes in color, clarity, presence of aggregates or clumping and material adhesion, or frosting), acid clarification, optical microscopy, reversed phase high performance liquid chromatography (RP-HPLC), in vitro or in vivo bioassays and denaturing and non-denaturing size exclusion chromatography (SEC). In vitro or in vivo bioassays for insulin activity include, but are not limited to, a competitive binding assay using cells expressing insulin receptors (e.g., human placental cell membranes) and a radiolabeled insulin (see e.g., Weiss et al., (2001)  J. Biol. Chem.  276:40018-40024; Duttaroy et al., (2005)  Diabetes  54:251-258); insulin-stimulated glucose uptake (Louveau et al., (2004)  J. Endocrin.  181:271-280, Duttaroy et al., (2005)  Diabetes  54:251-258); assays to assess glucose production in the presence of insulin (Wang et al., (2000)  J. Biochem.,  275:14717-14721, Duttaroy et al., (2005)  Diabetes  54:251-258); and studies using diabetic and/or healthy animal models (Atkinson et al., (1999)  Nature Med.  5:601-604; Nagoya-Shibata-Yasuda (NSY) mice, Zucker diabetic fatty (ZDF) rats and Gato-Katazaki (GK) rats (Cefalu (2006)  ILAR Journal  47:186-198). 
     Examples of such formulations contain 100 U/mL to 1000 U/mL of a modified PH20 polypeptide, and in particular at or about or at least 600 U/mL; 10 U/mL to 1000 U/mL of a fast-acting insulin, and in particular at or at least or about 100 U/mL; NaCl at a concentration of between or about between 80-140 mM; a pH of between or about between 7.0 to 7.8; a buffering agent that maintains the pH range of between or about between 7.0 to 7.8; 0.1% to 0.4% preservative as a mass concentration (w/v). Optionally, a further stabilizing agent can be included. For example, the co-formulations provided herein contain 1 mM to 100 mM of a buffering agent. For example, the co-formulations provided herein contain 0.005% to 0.5% surfactant. Exemplary co-formulations provided herein also can contain less than 60 mM glycerin (glycerol) and 2 mM to or to about 50 mM of an antioxidant. 
     The following stable formulations are exemplary only and provide a platform from which minor adjustments can be made. It is understood that very small changes in the concentrations of the various excipients and other components (e.g., ±15% of the stated concentrations), or small changes in pH, can be made while retaining some if not all of the insulin solubility and stability and PH20 stability. Further changes also can be made by adding or removing excipients. For example, the type of stabilizing surfactant can be changed. 
     For example, the exemplary co-formulations herein contain 100 U/mL to 1000 U/mL of a modified PH20 polypeptide, and in particular at least or about at least or about 600 U/mL of a modified PH20 polypeptide; 10 U/mL to 1000 U/mL of a fast-acting insulin, and in particular at least or about at least or about 100 U/mL of a fast-acting insulin; from or from about 10 mM to or to about 50 mM Tris (e.g., from or from about 20 mM to 40 mM Tris, such as or as about 20 mM, 25 mM, 30 mM, 35 mM or 40 mM Tris); from or from about 80 mM to or to about 160 mM NaCl (e.g., at or about 80 mM, 90 mM, 100 mM, 110 mM 120 mM, 130 mM, 140 mM, 150 mM or 160 mM NaCl); from or from about 2 mM to or to about 50 mM methionine (e.g., at or about 5 mM, 10 mM, 20 mM, 30 mM, 40 mM or 50 mM methionine); from or from about 0 mM to or to about 50 mM glycerin (e.g., at or about 5 mM, 10 mM, 20 mM, 30 mM, 40 mM or 50 mM glycerin); from or from about 0.005% to or to about 0.5% poloxamer 188, such as 0.01% to 0.05% (e.g., at or about 0.01%, 0.02%, 0.03%, 0.04% or 0.05% poloxamer 188); from or from about 0.05% to or to about 0.25% phenol (e.g., at or about 0.1%, 0.12%, 0.125%, 0.13%, 0.14%, 0.15%, 0.16% or 0.17% phenol); and from or from about 0.05% to or to about 0.4% m-cresol (e.g., at or about 0.075%, 0.08%, 0.09%, 0.1%, 0.12%, 0.13%, 0.14%, 0.15%, 0.16% or 0.17% m-cresol). The formulations are prepared with a pH from or from about 7.0 to or to about 7.6 (e.g., at or about pH 7.0, 7.1, 7.2, 7.3, 7.4, 7.5 or 7.6). In further examples, zinc is included at a concentration of or about 0.017 mg/100 U, 0.018 mg/100 U, 0.02 mg/100 U, 0.022 mg/100 U or 0.024 mg/100 U insulin. 
     In particular examples, the fast acting insulin is insulin aspart, insulin lispro or insulin glulisine. Exemplary co-formulations provided herein that contain a modified PH20 polypeptide and insulin lispro are those that contain from or about 25 mM to or to about 35 mM Tris (e.g., at or about 30 mM Tris); from or from about 70 mM to or to about 100 mM NaCl (e.g., at or about 80 mM or 100 mM NaCl); from or from about 10 mM to or to about 30 mM methionine (e.g., at or about 10 mM or 20 mM methionine); from or from about 40 mM to or to about 60 mM glycerin (e.g., at or about 50 mM glycerin); from or from about 0.005% to or to about 0.05% poloxamer 188 (e.g., at or about 0.01% poloxamer 188); from or from about 0.017 mg zinc/100 U insulin to or to about 0.024 mg zinc/100 U insulin (e.g., 0.017 mg zinc/100 U insulin, 0.018 mg/100 U, 0.02 mg/100 U, 0.022 mg/100 U or 0.024 mg zinc/100 U insulin); from or from about 0.08% to or to about 0.17% phenol (e.g., 0.1%, 0.125% or 0.13% phenol); and from or from about 0.07% to or to about 0.17% m-cresol (e.g., 0.075%, 0.08%, 0.13% or 0.15% m-cresol). For example, the co-formulations can contain at or about 0.1% phenol and 0.015% m-cresol; at or about 0.125% phenol and 0.075% m-cresol; at or about 0.13% phenol and 0.075% m-cresol; at or about 0.13% phenol and 0.08% m-cresol; or at or about 0.17% phenol and 0.13% m-cresol. Such formulations of insulin lispro and a modified PH20 polypeptide are prepared with a pH of or about 7.0 to or to about 7.5 (typically a pH of or about pH 7.2). 
     Exemplary co-formulations provided herein that contain a modified PH20 polypeptide and insulin aspart are those that contain from or from about 25 mM to or to about 35 mM Tris (e.g., at or about 30 mM Tris); from or from about 70 mM to or to about 120 mM NaCl (e.g., at or about 80 mM or 100 mM NaCl); from or from about 2 mM to or to about 30 mM methionine, such as 2 mM to 10 mM or 5 mM to 30 mM methionine (e.g., at or about 5 mM, 10 mM or 20 mM methionine); from or from about 0.005% to or to about 0.05% poloxamer 188 (e.g., at or about 0.01% poloxamer 188); from or from about 0.08% to or to about 0.17% phenol (e.g., 0.1%, 0.125% or 0.13% phenol); and from or from about 0.07% to or to about 0.17% m-cresol (e.g., 0.075%, 0.08%, 0.13% or 0.15% m-cresol). For example, the co-formulations can contain at or about 0.1% phenol and 0.015% m-cresol; at or about 0.125% phenol and 0.075% m-cresol; at or about 0.13% phenol and 0.075% m-cresol; at or about 0.13% phenol and 0.08% m-cresol; or at or about 0.17% phenol and 0.13% m-cresol. Such formulations of insulin aspart and a modified PH20 polypeptide are prepared with a pH of or about 7.0 to or to about 7.6 (typically a pH of or about pH 7.4 or 7.3). 
     Further exemplary formulations provided herein that contain a modified PH20 polypeptide and insulin aspart are those that do not contain phenol. Such exemplary formulations contain from or from about 25 mM to or to about 35 mM Tris (e.g., at or about 30 mM Tris); from or from about 70 mM to or to about 120 mM NaCl (e.g., at or about 80 mM or 100 mM NaCl); from or from about 2 mM to or to about 30 mM methionine, such as 2 mM to 10 mM or 5 mM to 30 mM methionine (e.g., at or about 5 mM, 10 mM or 20 mM methionine); from or from about 0.005% to or to about 0.05% poloxamer 188 (e.g., at or about 0.01% poloxamer 188); and from or from about 0.07% to or to about 0.4% m-cresol, such as from or from about 0.2% to 0.4% m-cresol (e.g., 0.3%, 0.315%, 0.35%, 0.4% m-cresol). Such formulations of insulin aspart and a modified PH20 polypeptide are prepared with a pH of or about 7.0 to or to about 7.6 (typically a pH of or about pH 7.4 or 7.3). 
     Exemplary co-formulations provided herein that contain a modified PH20 polypeptide and insulin glulisine are those that contain from or from about 25 mM to or to about 35 mM Tris (e.g., at or about 30 mM Tris); from or from about 100 mM to or to about 150 mM NaCl (e.g., at or about 100 mM or 140 mM NaCl); from or from about 10 mM to or to about 30 mM methionine (e.g., at or about 10 mM or 20 mM methionine); from or from about 40 mM to or to about 60 mM glycerin (e.g., at or about 50 mM glycerin); from or from about 0.005% to or to about 0.05% poloxamer 188 (e.g., at or about 0.01% poloxamer 188); from or from about 0.08% to or to about 0.17% phenol (e.g., 0.1%, 0.125% or 0.13% phenol); and from or from about 0.07% to or to about 0.17% m-cresol (e.g., 0.075%, 0.08%, 0.13% or 0.15% m-cresol). For example, the co-formulations can contain at or about 0.1% phenol and 0.015% m-cresol; at or about 0.125% phenol and 0.075% m-cresol; at or about 0.13% phenol and 0.075% m-cresol; at or about 0.13% phenol and 0.08% m-cresol; or at or about 0.17% phenol and 0.13% m-cresol. Such formulations of insulin glulisine and a modified PH20 polypeptide are prepared with a pH of or about 7.0 to or to about 7.6 (typically a pH of or about pH 7.4). 
     5. Packaging, Articles of Manufacture and Kits 
     Pharmaceutical compounds of modified PH20 polypeptides, or nucleic acids encoding such polypeptides, or derivatives or variants thereof can be packaged as articles of manufacture containing packaging material, a pharmaceutical composition which is effective for treating a disease or disorder, and a label that indicates that the pharmaceutical composition or therapeutic molecule is to be used for treating the disease or disorder. Combinations of a selected modified PH20 polypeptide, or a derivative or variant thereof and an therapeutic agent also can be packaged in an article of manufacture. 
     The articles of manufacture provided herein contain packaging materials. Packaging materials for use in packaging pharmaceutical products are well known to those of skill in the art. See, for example, U.S. Pat. Nos. 5,323,907, 5,052,558 and 5,033,252, each of which is incorporated herein in its entirety. Examples of pharmaceutical packaging materials include, but are not limited to, blister packs, bottles, tubes, inhalers, pumps, bags, vials, containers, syringes, bottles, and any packaging material suitable for a selected formulation and intended mode of administration and treatment. The articles of manufacture can include a needle or other injection device so as to facilitate administration (e.g., sub-epidermal administration) for local injection purposes. A wide array of formulations of the compounds and compositions provided herein are contemplated including a modified PH20 polypeptide and a therapeutic agent, such as a fast-acting insulin, known to treat a particular disease or disorder. The choice of package depends on the PH20 and/or therapeutic agent, and whether such compositions will be packaged together or separately. In one example, the PH20 can be packaged as a mixture with the therapeutic agent. In another example, the components can be packaged as separate compositions 
     Modified PH20 polypeptides, therapeutic agents and/or articles of manufacture thereof also can be provided as kits. Kits can include a pharmaceutical composition described herein and an item for administration provided as an article of manufacture. For example a PH20 polypeptide can be supplied with a device for administration, such as a syringe, an inhaler, a dosage cup, a dropper, or an applicator. The compositions can be contained in the item for administration or can be provided separately to be added later. The kit can, optionally, include instructions for application including dosages, dosing regimens and instructions for modes of administration. Kits also can include a pharmaceutical composition described herein and an item for diagnosis. For example, such kits can include an item for measuring the concentration, amount or activity of the selected protease in a subject. 
     G. METHODS OF ASSESSING PH20 ACTIVITY AND STABILITY 
     Assays can be used to assess the stability and activity of the PH20 polypeptides provided herein. The assays can be used to assess the hyaluronidase activity of the PH20 polypeptide under particular conditions, temperature, and/or over time. Such assays can be used, for example, to determine the stability of the PH20 polypeptide under specific denaturation conditions, including, but not limited to, elevated temperatures greater than or about or 30° C. (e.g., 30° C. to 42° C. such as or about 37° C.), agitation, presence of excipients (e.g., preservative), or low or no NaCl (salt). For example, stability under specific conditions can be monitored by assessing activity, solubility, and stability (e.g., formation of aggregates, etc.) in the absence of exposure to the denaturation condition and then at various time points thereafter in the presence of the condition. Hence, stability can be assessed over time. Stability also can be assessed by comparing any one or more of activity, solubility or aggregation in the presence of one or more denaturation conditions compared to a native, wildtype or reference PH20 polypeptide. The assays also can be used make minor adjustments to the formulations provided herein while retaining the stability of both active agents. 
     1. Hyaluronidase Activity 
     The activity of a modified PH20 polypeptide can be assessed using methods well known in the art. For example, the USP XXII assay for hyaluronidase determines activity indirectly by measuring the amount of undegraded hyaluronic acid, or hyaluronan, (HA) substrate remaining after the enzyme is allowed to react with the HA for 30 min at 37° C. (USP XXII-NF XVII (1990) 644-645 United States Pharmacopeia Convention, Inc, Rockville, Md.). A Hyaluronidase Reference Standard (USP) or National Formulary (NF) Standard Hyaluronidase solution can be used in an assay to ascertain the activity, in units, of any hyaluronidase. In one example, activity is measured using a microturbidity assay. This is based on the formation of an insoluble precipitate when hyaluronic acid binds with a reagent that precipitates it, such as acidified serum or cetylpyridinium chloride (CPC). The activity is measured by incubating hyaluronidase with sodium hyaluronate (hyaluronic acid) for a set period of time (e.g., 10 minutes) and then precipitating the undigested sodium hyaluronate with the addition of acidified serum or CPC. The turbidity of the resulting sample is measured at 640 nm after an additional development period. The decrease in turbidity resulting from hyaluronidase activity on the sodium hyaluronate substrate is a measure of hyaluronidase enzymatic activity. 
     In another example, hyaluronidase activity is measured using a microtiter assay in which residual biotinylated hyaluronic acid is measured following incubation with hyaluronidase (see e.g., Frost and Stern (1997)  Anal. Biochem.  251:263-269, U.S. Pat. Publication No. 20050260186). The free carboxyl groups on the glucuronic acid residues of hyaluronic acid are biotinylated, and the biotinylated hyaluronic acid substrate is covalently coupled to a microtiter plate. Following incubation with hyaluronidase, the residual biotinylated hyaluronic acid substrate is detected using an avidin-peroxidase reaction, and compared to that obtained following reaction with hyaluronidase standards of known activity. 
     Other assays to measure hyaluronidase activity also are known in the art and can be used in the methods herein (see e.g., Delpech et al., (1995)  Anal. Biochem.  229:35-41; Takahashi et al., (2003)  Anal. Biochem.  322:257-263). 
     Many hyaluronidase assays have been based upon the measurement of the generation of new reducing N-acetylamino groups (Bonner and Cantey,  Clin. Chim. Acta  13:746-752, 1966), or loss of viscosity (De Salegui et al.,  Arch. Biochem. Biophys.  121:548-554, 1967) or turbidity (Doifnian and Ott,  J. Biol. Chem.  172:367, 1948). With purified substrates all of these methods suffice for determination of the presence or absence of endoglycosidase activity. 
     Substantially purified glycosaminoglycan substrates can also be used in a Gel Shift Assay. Glycosaminoglycans are mixed with recombinant PH20, such as a soluble PH20, to test for endoglycosidase activity that results in a shift in substrate mobility within the gel. Examples of such substrates include, but are not limited to, chondroitin-4 and 6 sulfate, dermatan sulfate, heparan-sulfate, which can be obtained from Sigma Chemical. Human umbilical cord Hyaluronan can be obtained from ICN. For example, each test substrate can be diluted to at or about 0.1 mg/mL in a buffer range from pH 3.5-7.5. In such an exemplary assay, at or about 10 μl samples of purified soluble PH20 or conditioned media from PH20 expressing cells can be mixed with at or about 90 μl of test substrate in desired buffer and incubated for 3 hours at 37° C. Following incubation, samples are neutralized with sample buffer (Tris EDTA pH 8.0, Bromophenol Blue and glycerol) followed by electrophoresis. Glycosaminoglycans can be detected using any method known in the art, for example, glycosaminoglycans can be detected by staining the gels using 0.5% Alcian Blue in 3% Glacial Acetic Acid overnight followed by destaining in 7% Glacial Acetic Acid. Degradation is determined by comparison of substrate mobility in the presence and absence of enzyme. 
     Hyaluronidase activity can also be detected by substrate gel zymography (Guentenhoner et al., (1992)  Matrix  12:388-396). In this assay, a sample is applied to an SDS-PAGE gel containing hyaluronic acid and the proteins in the sample separated by electrophoresis. The gel is then incubated in an enzyme assay buffer and subsequently stained to detect the hyaluronic acid in the gel. Hyaluronidase activity is visualized as a cleared zone in the substrate gel. 
     The ability of a PH20 polypeptide, including a modified PH20 polypeptide provided herein, to act as a spreading or diffusing agent also can be assessed. For example, trypan blue dye can be injected subcutaneously with or without a PH20 polypeptide into the lateral skin on each side of nude mice. The dye area is then measured, such as with a microcaliper, to determine the ability of the PH20 polypeptide to act as a spreading agent (U.S. Pat. Pub. No. 20060104968). 
     The functional activity of a PH20 polypeptide can be compared and/or normalized to a reference standard using any of these assays. This can be done to determine what a functionally equivalent amount of a PH20 polypeptide is. For example, the ability of a PH20 polypeptide to act as a spreading or diffusing agent can be assessed by injecting it into the lateral skin of mice with trypan blue, and the amount required to achieve the same amount of diffusion as, for example, 100 units of a Hyaluronidase Reference Standard, can be determined. The amount of PH20 polypeptide required is, therefore, functionally equivalent to 100 hyaluronidase units. 
     2. Solubility 
     The solubility of a PH20 polypeptide can be determined by any method known to one of the skill in the art. One method for determining solubility is detergent partitioning. For example, a soluble PH20 polypeptide can be distinguished, for example, by its partitioning into the aqueous phase of a Triton® X-114 detergent solution at 37° C. (Bordier et al., (1981)  J. Biol. Chem.,  256:1604-1607). Membrane-anchored polypeptides, such as lipid-anchored hyaluronidases, including GPI-anchored hyaluronidases, will partition into the detergent-rich phase, but will partition into the detergent-poor or aqueous phase following treatment with Phospholipase C. Phospholipase C is an enzyme that cleaves the phospho-glycerol bond found in GPI-anchored proteins. Treatment with PLC will cause release of GPI-linked proteins from the outer cell membrane. 
     3. Purity, Crystallization or Aggregation 
     The stability of a PH20 polypeptide provided herein also can be assessed using other methods and assays known in the art. In addition to assessing stability based on hyaluronidase activity, stability can be assessed by visual inspection, percent recovery, protein purity and apparent melting temperature. 
     For example, protein purity can be measured by reversed phase high performance liquid chromatography (RP-HPLC). Protein purity, as determined by RP-HPLC, is the percent of the main PH20 protein peak present, as compared to all of the protein species present. Thus, RP-HPLC, and similar methods known to one of skill in the art, can assess degradation of the enzyme. Protein purity can be assessed over time. Protein purity also can be assessed in the presence of one or more denaturation conditions and in varying amounts thereof. Percent recovery also can be determined as the relative percentage of the polypeptide under various conditions (denaturation conditions, time of storage, mode of storage such as vessel or container, or other similar parameters that can be altered) as compared to a reference sample. PH20 polypeptide stability also can be determined by measuring the oxidation of the hyaluronidase by RP-HPLC. Percent oxidation is a measure of sum of the peak areas of the major (ox-1) and minor (ox-2) peaks. 
     In one example, the melting temperature of a PH20 polypeptide, such as a modified PH20 polypeptide, can be determined by measuring the hydrodynamic radius of particles by dynamic light scattering under various conditions (e.g., denaturation conditions or other storage conditions). An increase in particle size and a decrease in the melting temperature indicates denaturation and subsequent aggregation of the hyaluronidase. 
     Other methods known to one of skill in the art that can be used to determine the stability of the hyaluronidase in the co-formulations provided herein, include polyacrylamide gel electrophoresis (PAGE), immunoblotting, nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry, circular dichroism (CD) and dye-based fluorescence assays. 
     4. Pharmacodynamics/Pharmacokinetics 
     The effect of administration of a PH20 polypeptide, such as a modified PH20 polypeptide, alone or in combination with another therapeutic agent, on the pharmacokinetic and pharmacodynamic properties of any administered agent also can be assessed in vivo using animal models and/or human subjects, such as in the setting of a clinical trial. Pharmacokinetic or pharmacodynamic studies can be performed using animal models or can be performed during studies with patients administered with a PH20 polypeptide or modified PH20 polypeptide. 
     Animal models include, but are not limited to, mice, rats, rabbits, dogs, guinea pigs and non-human primate models, such as cynomolgus monkeys or rhesus macaques. In some instances, pharmacokinetic or pharmacodynamic studies are performed using healthy animals. In other examples, the studies are performed using animal models of a disease for which therapy with hyaluronan is considered, such as animal models of any hyaluronan-associated disease or disorder, for example a tumor model. 
     The pharmacokinetic properties of a PH20 polypeptide, such as a modified PH20 polypeptide, can be assessed by measuring such parameters as the maximum (peak) concentration (C max ), the peak time (i.e., when maximum concentration occurs; T max ), the minimum concentration (i.e., the minimum concentration between doses; C max ), the elimination half-life (T 1/2 ) and area under the curve (i.e., the area under the curve generated by plotting time versus concentration; AUC), following administration. The absolute bioavailability of the hyaluronidase can be determined by comparing the area under the curve of hyaluronidase following subcutaneous delivery (AUCsc) with the AUC of hyaluronidase following intravenous delivery (AUC iv ). Absolute bioavailability (F), can be calculated using the formula: F=([AUC] sc ×dose sc )/([AUC] iv ×dose iv ). A range of doses and different dosing frequency of dosing can be administered in the pharmacokinetic studies to assess the effect of increasing or decreasing concentrations enzyme, such as modified PH20 polypeptide, in the dose. 
     H. METHODS OF TREATMENT AND COMBINATION THERAPY 
     Provided herein are methods and uses of any of the modified PH20 polypeptides provided herein that exhibit hyaluronidase activity based on its ability to degrade glycosaminoglycan(s) such as hyaluronan. Due to such activity, the modified PH20 polypeptides can be used as a spreading factor to increase the delivery and/or bioavailability of subcutaneously administered therapeutic agents. Delivery of any therapeutic agent, including but not limited to, peptides, proteins, small molecule drugs, nucleic acids, or viruses can be facilitated or enhanced by co-administration with a modified PH20 polypeptide provided herein. For example, modified PH20 polypeptides can be used to increase the delivery of therapeutic agents such as antibodies (e.g., monoclonal antibodies), cytokines, Immune Globulin, an Insulin, or coagulation factors, to a desired locus, such as by increasing penetration of chemotherapeutic agents into solid tumors. The modified PH20 polypeptides also can be used to treat a hyaluronan-disease or disorder that is characterized by an excess or accumulation of hyaluronan. For example, modified PH20 polypeptides provided herein can be used to for treating a tumor; for treating glycosaminoglycan accumulation in the brain; for treating a cardiovascular disorder; for treating an ophthalmic disorder; for treating pulmonary disease; for treating cellulite; and/or for treating a proliferative disorder. 
     Other methods and uses of a modified PH20 polypeptide include any that are known to one of skill in the art. For example, various forms of PH20 hyaluronidases have been prepared and approved for therapeutic use in humans. For example, animal-derived hyaluronidase preparations include Vitrase® hyaluronidase (ISTA Pharmaceuticals), a purified ovine testicular hyaluronidase, and Amphadase® hyaluronidase (Amphastar Pharmaceuticals), a bovine testicular hyaluronidase. Hylenex® hyaluronidase (Halozyme Therapeutics) is a human recombinant hyaluronidase produced by genetically engineered Chinese Hamster Ovary (CHO) cells containing nucleic acid encoding for soluble rHuPH20 (see e.g., U.S. Pat. No. 7,767,429). Approved therapeutic uses for hyaluronidases include use as an adjuvant to increase the absorption and dispersion of other therapeutic agents for hypodermoclysis (subcutaneous fluid administration), and as an adjunct in subcutaneous urography for improving resorption of radiopaque agents. In addition to these indications, hyaluronidases can be used as a therapeutic or cosmetic agent for the treatment of additional diseases and conditions. For example, hyaluronidase is commonly used, for example, for peribulbar block in local anesthesia prior ophthalmic surgery. The presence of the enzyme prevents the need for additional blocks and reduces the time to the onset of akinesia (loss of eye movement). Peribulbar and sub-Tenon&#39;s block are the most common applications of hyaluronidase for ophthalmic procedures. Hyaluronidase also can promote akinesia in cosmetic surgery, such as blepharoplasties and face lifts. It is understood that soluble PH20 hyaluronidases provided herein, including esPH20 hyaluronidases, can be used in any method of treatment or combination therapy for which a PH20 hyaluronidase is used (see e.g., U.S. Publication Nos. US20040268425; US20050260186; US20060104968; and U.S. application Ser. No. 12/381,844, published as U.S. Publication No. US20100074885; 12/386,249, published as U.S. Publication No. US20090311237; 12/387,225, published as U.S. Publication No. US20090304665; and Ser. No. 12/386,222, published as U.S. Publication No. US2010003238, each incorporated by reference in their entirety). 
     Exemplary, non-limiting, methods and uses are described in the following subsections. 
     1. Methods of Delivering Therapeutic Agents 
     As noted above, hyaluronidase is a spreading or diffusing substance that modifies the permeability of connective tissue through the hydrolysis of hyaluronic acid, a polysaccharide found in the intercellular ground substance of connective tissue, and of certain specialized tissues, such as the umbilical cord and vitreous humor. When no spreading factor is present, materials injected subcutaneously, such as drugs, proteins, peptides and nucleic acid, spread very slowly. Co-injection with hyaluronidase, however, can cause rapid spreading. The rate of diffusion is proportional to the amount of enzyme, and the extent of diffusion is proportional to the volume of solution. 
     Modified PH20 polypeptides provided herein can be used to promote or enhance the delivery agents and molecules to any of a variety of mammalian tissues in vivo. It can be used to facilitate the diffusion and, therefore, promote the delivery, of small molecule pharmacologic agents as well as larger molecule pharmacologic agents, such as proteins, nucleic acids and ribonucleic acids, and macromolecular compositions than can contain a combination of components including, but not limited to, nucleic acids, proteins, carbohydrates, lipids, lipid-based molecules and drugs (see e.g., U.S. Publication Nos. US20040268425; US20050260186; and US20060104968). Modified PH20 polypeptides can be co-administered and/or co-formulated with a therapeutic agent to improve the bioavailability as well as pharmacokinetic (PK) and/or pharmacodynamic (PD) characteristics of co-formulated or co-administered agents. PK/PD parameters that can be improved by using soluble PH20, such as esPH20, include such measures as C max  (the maximal concentration of agent achieved following absorption in, e.g., the bloodstream), T max  (the time required to achieve maximal concentration), T 1/2  (the time required for the concentration to fall by half), C min  (the minimal concentration of agent following metabolism and excretion), AUC (area under the curve of concentration versus time, a measure of the overall amount of bioavailability), concentrations in various tissues of interest (including, e.g., the rate of achieving desired concentrations, the overall levels, and the duration of maintaining desired levels), and E. (the maximal effect achieved). 
     The methods of treatment provided herein include combination therapies with a therapeutic agent for the treatment of a disease or disorder for which the therapeutic agent threats. Any therapeutic agent that ameliorates and or otherwise lessens the severity of a disease or condition can be combined with a modified PH20 polypeptide provided herein in order to increase the bioavailability of such therapeutic agent. In particular, modified PH20 polypeptides provided herein can be used in each and all of the combinations described in applications see e.g., U.S. Publication Nos. US20040268425; US20050260186; US20060104968 and U.S. Appl. Serial Nos. 12/381,844, published as U.S. Publication No. US20100074885; 12/386,249, published as U.S. Publication No. US20090311237; 12/387,225, published as U.S. Publication No. US20090304665; and Ser. No. 12/386,222, published as U.S. Publication No. US2010003238 in place of the disclosed hyaluronidase or hyaluronidase degrading enzyme. 
     Modified PH20 polypeptides can be administered prior to, subsequent to, intermittently with or simultaneously with the therapeutic agent preparation. Generally, the modified PH20 polypeptide is administered prior to or simultaneously with administration of the therapeutic agent preparation to permit the PH20 to degrade the hyaluronic acid in the interstitial space. The PH20 can be administered at a site different from the site of administration of the therapeutic molecule or the soluble PH20 can be administered at a site the same as the site of administration of the therapeutic molecule. 
     Examples of pharmaceutical, therapeutic and cosmetic agents and molecules that can be administered with hyaluronidase include, but are not limited to, a chemotherapeutic or anticancer agent, an analgesic agent, an antibiotic agent, an anti-inflammatory agent, an antimicrobial agent, an amoebicidal agent, a trichomonacidal agent, an anti-Parkinson agent, an anti-malarial agent, an anticonvulsant agent, an anti-depressant agent, an anti-arthritic agent, an anti-fungal agent, an antihypertensive agent, an antipyretic agent, an anti-parasitic agent, an antihistamine agent, an alpha-adrenergic agonist agent, an alpha blocker agent, an anesthetic agent, a bronchial dilator agent, a biocide agent, a bactericide agent, a bacteriostatic agent, a beta adrenergic blocker agent, a calcium channel blocker agent, a cardiovascular drug agent, a contraceptive agent, a cosmetic or esthetic agent, a decongestant agent, a diuretic agent, a depressant agent, a diagnostic agent, an electrolyte agent, a hypnotic agent, a hormone agent, a hyperglycemic agent, a muscle relaxant agent, a muscle contractant agent, an ophthalmic agent, a parasympathomimetic agent, a psychic energizer agent, a sedative agent, a sleep inducer, a sympathomimetic agent, a tranquilizer agent, a urinary agent, a vaginal agent, a viricide agent, a vitamin agent, a non-steroidal anti-inflammatory agent, or an angiotensin converting enzyme inhibitor agent, and any combination thereof. In particular, therapeutic agents include antibodies, including monoclonal antibodies, bisphosphonates, insulins, coagulation factors, cytokines and Immun Globulins. 
     For example, modified PH20 polypeptides provided herein can be used to increase the delivery of chemotherapeutic agents. Hyaluronidases have also been used to enhance the activity of chemotherapeutics and/or the accessibility of tumors to chemotherapeutics (Schuller et al., 1991 , Proc. Amer. Assoc. Cancer Res.  32:173, abstract no. 1034; Czejka et al., 1990 , Pharmazie  45:H.9; Baumgartner et al., (1988)  Reg. Cancer Treat.  1:55-58; Zanker et al., (1986)  Proc. Amer. Assoc. Cancer Res.  27:390). Combination chemotherapy with hyaluronidase is effective in the treatment of a variety of cancers including urinary bladder cancer (Horn et al., (1985)  J. Surg. Oncol.  28:304-307), squamous cell carcinoma (Kohno et al., (1994)  J. Cancer Res. Oncol.  120:293-297), breast cancer (Beckenlehner et al., (1992)  J. Cancer Res. Oncol.  118:591-596), and gastrointestinal cancer (Scheithauer et al., (1988)  Anticancer Res.  8:391-396). In this example, the modified PH20 hyaluronidase enhances penetration of chemotherapeutic or other anti-cancer agents into solid tumors, thereby treating the disease. 
     Compositions containing soluble PH20 can be injected intratumorally with anti-cancer agents or intravenously for disseminated cancers or hard to reach tumors. The anticancer agent can be a chemotherapeutic, an antibody, a peptide, or a gene therapy vector, virus or DNA. Additionally, hyaluronidase can be used to recruit tumor cells into the cycling pool for sensitization in previously chemorefractory tumors that have acquired multiple drug resistance (St Croix et al., (1998)  Cancer Lett  September 131(1): 35-44). 
     Exemplary anti-cancer agents that can be administered after, coincident with or before administration of a soluble PH20, such as an esPH20, include, but are not limited to Acivicins; Aclarubicins; Acodazoles; Acronines; Adozelesins; Aldesleukins; Alemtuzumabs; Alitretinoins (9-Cis-Retinoic Acids); Allopurinols; Altretamines; Alvocidibs; Ambazones; Ambomycins; Ametantrones; Amifostines; Aminoglutethimides; Amsacrines; Anastrozoles; Anaxirones; Ancitabines; Anthramycins; Apaziquones; Argimesnas; Arsenic Trioxides; Asparaginases; Asperlins; Atrimustines; Azacitidines; Azetepas; Azotomycins; Banoxantrones; Batabulins; Batimastats; BCG Live; Benaxibines; Bendamustines; Benzodepas; Bexarotenes; Bevacizumab; Bicalutamides; Bietaserpines; Biricodars; Bisantrenes; Bisantrenes; Bisnafide Dimesylates; Bizelesins; Bleomycins; Bortezomibs; Brequinars; Bropirimines; Budotitanes; Busulfans; Cactinomycins; Calusterones; Canertinibs; Capecitabines; Caracemides; Carbetimers; Carboplatins; Carboquones; Carmofurs; Carmustines with Polifeprosans; Carmustines; Carubicins; Carzelesins; Cedefingols; Celecoxibs; Cemadotins; Chlorambucils; Cioteronels; Ciplactin; Cirolemycins; Cisplatins; Cladribines; Clanfenurs; Clofarabines; Crisnatols; Cyclophosphamides; Cytarabine liposomals; Cytarabines; Dacarbazines; Dactinomycins; Darbepoetin Alfas; Daunorubicin liposomals; Daunorubicins/Daunomycins; Daunorubicins; Decitabines; Denileukin Diftitoxes; Dexniguldipines; Dexonas; Dexrazoxanes; Dezaguanines; Diaziquones; Dibrospidiums; Dienogests; Dinalins; Disermolides; Docetaxels; Dofequidars; Doxifluridines; Doxorubicin liposomals; Doxorubicin HCL; Doxorubicin HCL liposome injection; Doxorubicins; Droloxifenes; Dromostanolone Propionates; Duazomycins; Ecomustines; Edatrexates; Edotecarins; Eflornithines; Elacridars; Elinafides; Elliott&#39;s B Solutions; Elsamitrucins; Emitefurs; Enloplatins; Enpromates; Enzastaurins; Epipropidines; Epirubicins; Epoetin alfas; Eptaloprosts; Erbulozoles; Esorubicins; Estramustines; Etanidazoles; Etoglucids; Etoposide phosphates; Etoposide VP-16s; Etoposides; Etoprines; Exemestanes; Exisulinds; Fadrozoles; Fazarabines; Fenretinides; Filgrastims; Floxuridines; Fludarabines; Fluorouracils; 5-fluorouracils; Fluoxymesterones; Flurocitabines; Fosquidones; Fostriecins; Fostriecins; Fotretamines; Fulvestrants; Galarubicins; Galocitabines; Gemcitabines; Gemtuzumabs/Ozogamicins; Geroquinols; Gimatecans; Gimeracils; Gloxazones; Glufosfamides; Goserelin acetates; Hydroxyureas; Ibritumomabs/Tiuxetans; Idarubicins; Ifosfamides; Ilmofosines; Ilomastats; Imatinib mesylates; Imexons; Improsulfans; Indisulams; Inproquones; Interferon alfa-2as; Interferon alfa-2bs; Interferon Alfas; Interferon Betas; Interferon Gammas; Interferons; Interleukin-2s and other Interleukins (including recombinant Interleukins); Intoplicines; Iobenguanes [131-I]; Iproplatins; Irinotecans; Irsogladines; Ixabepilones; Ketotrexates; L-Alanosines; Lanreotides; Lapatinibs; Ledoxantrones; Letrozoles; Leucovorins; Leuprolides; Leuprorelins (Leuprolides); Levamisoles; Lexacalcitols; Liarozoles; Lobaplatins; Lometrexols; Lomustines/CCNUs; Lomustines; Lonafarnibs; Losoxantrones; Lurtotecans; Mafosfamides; Mannosulfans; Marimastats; Masoprocols; Maytansines; Mechlorethamines; Mechlorethamines/Nitrogen mustards; Megestrol acetates; Megestrols; Melengestrols; Melphalans; Melphalan L-PAMs; Menogarils; Mepitiostanes; Mercaptopurines; 6-Mecaptopurine; Mesnas; Metesinds; Methotrexates; Methoxsalens; Metomidates; Metoprines; Meturedepas; Miboplatins; Miproxifenes; Misonidazoles; Mitindomides; Mitocarcins; Mitocromins; Mitoflaxones; Mitogillins; Mitoguazones; Mitomalcins; Mitomycin Cs; Mitomycins; Mitonafides; Mitoquidones; Mitospers; Mitotanes; Mitoxantrones; Mitozolomides; Mivobulins; Mizoribines; Mofarotenes; Mopidamols; Mubritinibs; Mycophenolic Acids; Nandrolone Phenpropionates; Nedaplatins; Nelarabines; Nemorubicins; Nitracrines; Nocodazoles; Nofetumomabs; Nogalamycins; Nolatrexeds; Nortopixantrones; Octreotides; Oprelvekins; Ormaplatins; Ortataxels; Oteracils; Oxaliplatins; Oxisurans; Oxophenarsines; Paclitaxels; Pamidronates; Patupilones; Pegademases; Pegaspargases; Pegfilgrastims; Peldesines; Peliomycins; Pelitrexols; Pemetrexeds; Pentamustines; Pentostatins; Peplomycins; Perfosfamides; Perifosines; Picoplatins; Pinafides; Pipobromans; Piposulfans; Pirfenidones; Piroxantrones; Pixantrones; Plevitrexeds; Plicamycin Mithramycins; Plicamycins; Plomestanes; Plomestanes; Porfimer sodiums; Porfimers; Porfiromycins; Prednimustines; Procarbazines; Propamidines; Prospidiums; Pumitepas; Puromycins; Pyrazofurins; Quinacrines; Ranimustines; Rasburicases; Riboprines; Ritrosulfans; Rituximabs; Rogletimides; Roquinimexs; Rufocromomycins; Sabarubicins; Safingols; Sargramostims; Satraplatins; Sebriplatins; Semustines; Simtrazenes; Sizofirans; Sobuzoxanes; Sorafenibs; Sparfosates; Sparfosic Acids; Sparsomycins; Spirogermaniums; Spiromustines; Spiroplatins; Spiroplatins; Squalamines; Streptonigrins; Streptovarycins; Streptozocins; Sufosfamides; Sulofenurs; Sunitinib Malate; 6-TG; Tacedinalines; Talcs; Talisomycins; Tallimustines; Tamoxifens; Tariquidars; Tauromustines; Tecogalans; Tegafurs; Teloxantrones; Temoporfins; Temozolomides; Teniposides/VM-26s; Teniposides; Teroxirones; Testolactones; Thiamiprines; Thioguanines; Thiotepas; Tiamiprines; Tiazofurins; Tilomisoles; Tilorones; Timcodars; Timonacics; Tirapazamines; Topixantrones; Topotecans; Toremifenes; Tositumomabs; Trabectedins (Ecteinascidin 743); Trastuzumabs; Trestolones; Tretinoins/ATRA; Triciribines; Trilostanes; Trimetrexates; Triplatin Tetranitrates; Triptorelins; Trofosfamides; Tubulozoles; Ubenimexs; Uracil Mustards; Uredepas; Valrubicins; Valspodars; Vapreotides; Verteporfins; Vinblastines; Vincristines; Vindesines; Vinepidines; Vinflunines; Vinformides; Vinglycinates; Vinleucinols; Vinleurosines; Vinorelbines; Vinrosidines; Vintriptols; Vinzolidines; Vorozoles; Xanthomycin A&#39;s (Guamecyclines); Zeniplatins; Zilascorbs [2-H]; Zinostatins; Zoledronate; Zorubicins; and Zosuquidars, for example: 
     Aldesleukins (e.g., PROLEUKIN®); Alemtuzumabs (e.g., CAMPATH®); Alitretinoins (e.g., PANRETIN®); Allopurinols (e.g., ZYLOPRIM®); Altretamines (e.g., HEXALEN®); Amifostines (e.g., ETHYOL®); Anastrozoles (e.g., ARIMIDEX®); Arsenic Trioxides (e.g., TRISENOX®); Asparaginases (e.g., ELSPAR®); BCG Live (e.g., TICE® BCG); Bexarotenes (e.g., TARGRETIN®); Bevacizumab (AVASTIN®); Bleomycins (e.g., BLENOXANE R); Busulfan intravenous (e.g., BUSULFEX R); Busulfan orals (e.g., MYLERAN™); Calusterones (e.g., METHOSARB®); Capecitabines (e.g., XELODA®); Carboplatins (e.g., PARAPLATIN®); Carmustines (e.g., BCNU®, BiCNU®); Carmustines with Polifeprosans (e.g., GLIADEL® Wafer); Celecoxibs (e.g., CELEBREX®); Chlorambucils (e.g., LEUKERAN®)); Cisplatins (e.g., PLATINOL®); Cladribines (e.g., LEUSTATIN®, 2-CdA®); Cyclophosphamides (e.g., CYTOXAN®, NEOSAR®); Cytarabines (e.g., CYTOSAR-U®); Cytarabine liposomals (e.g., DepoCyt®); Dacarbazines (e.g., DTIC-Domeu): Dactinomycins (e.g., COSMEGEN®); Darbepoetin Alfas (e.g., ARANESP®); Daunorubicin liposomals (e. g. DAUNOXOME®); Daunorubicins/Daunomycins (e.g., CERUBIDINE®); Denileukin Diftitoxes (e.g., ONTAK®); Dexrazoxanes (e.g., ZINECARD®); Docetaxels (e.g., TAXOTERE R); Doxorubicins (e.g., ADRIAMYCIN®, RUBEX®); Doxorubicin liposomals, including Doxorubicin HCL liposome injections (e.g., DOXIL R); Dromostanolone propionates (e.g., DROMOSTANOLONE® and MASTERONE R Injection); Elliott&#39;s B Solutions (e.g., Elliott&#39;s B Solution®); Epirubicins (e.g., ELLENCE®); Epoetin alfas (e.g., EPOGEN®); Estramustines (e.g., EMCYT®); Etoposide phosphates (e.g., ETOPOPHOS®); Etoposide VP-16s (e.g., VEPESID®); Exemestanes (e.g., AROMASIN®); Filgrastims (e.g., NEUPOGEN®)); Floxuridines (e.g., FUDR®); Fludarabines (e.g., FLUDARA®); Fluorouracils incl. 5-FUs (e.g., ADRUCIL®); Fulvestrants (e.g., FASLODEX®); Gemcitabines (e.g., GEMZAR®); Gemtuzumabs/Ozogamicins (e.g., MYLOTARG®); Goserelin acetates (e.g., ZOLADEX®); Hydroxyureas (e.g., HYDREA®); Ibritumomabs/Tiuxetans (e.g., ZEVALIN®); Idarubicins (e.g., IDAMYCIN®); Ifosfamides (e.g., IFEX®); Imatinib mesylates (e.g., GLEEVEC®); Interferon alfa-2as (e.g., ROFERON-A®); Interferon alfa-2bs (e.g., INTRON A®); Irinotecans (e.g., CAMPTOSAR®); Letrozoles (e.g., FEMARA®); Leucovorins (e.g., WELLCOVORIN®, LEUCOVORIN®); Levamisoles (e.g., ERGAMISOL®); Lomustines/CCNUs (e.g., CeeNU®); Mechlorethamines/Nitrogen mustards (e.g., MUSTARGEN®); Megestrol acetates (e.g., MEGACE®); Melphalans/L-PAMs (e.g., ALKERAN®); Mercaptopurine incl. 6-MPs (e.g., PURINETHOL®); Mesnas (e.g., MESNEX®); Methotrexates; Methoxsalens (e.g., UVADEX®); Mitomycin Cs (e.g., MUTAMYCINED, MITOZYTREX®); Mitotanes (e.g., LYSODREN®); Mitoxantrones (e.g., NOVANTRONE®); Nandrolone Phenpropionates (e.g., DURABOLIN-50®); Nofetumomabs (e.g., VERLUMA®); Oprelvekins (e.g., NEUMEGA®); Oxaliplatins (e.g., ELOXATIN®); Paclitaxels (e.g., PAXENE®, TAXOL®); Pamidronates (e.g., AREDIA®); Pegademases (e.g., ADAGEN®); Pegaspargases (e.g., ONCASPAR®); Pegfilgrastims (e.g., NEULASTA®)); Pentostatins (e.g., NIPENT®); Pipobromans (e.g., VERCYTE®); Plicamycin/Mithramycins (e.g., MITHRACIN®); Porfimer sodiums (e.g., PHOTOFRIN®); Procarbazines (e.g., MATULANE®); Quinacrines (e.g., ATABRINE®); Rasburicases (e.g., ELITEK®); Rituximabs (e.g., RITUXAN®)); Sargramostims (e.g., PROKINE®); Streptozocins (e.g., ZANOSAR®); Sunitinib Malates (e.g., SUTENT®); Talcs (e.g., SCLEROSOL®); Tamoxifens (e.g., NOLVADEX®); Temozolomides (e.g., TEMODAR®); Teniposides/VM-26s (e.g., VUMON®); Testolactones (e.g., TESLAC®); Thioguanines incl. 6-TG; Thiotepas (e.g., THEOPLEX®); Topotecans (e.g., HYCAMTIN®); Toremifenes (e.g., FARESTON®); Tositumomabs (e.g., BEXXAR®); Trastuzumabs (e.g., HERCEPTIN®); Tretinoins/ATRA (e.g., VESANOID R); Uracil Mustards; Valrubicins (e.g., VALSTAR0); Vinblastines (e.g., VELBAN®); Vincristines (e.g., ONCOVIN®); Vinorelbines (e.g., NAVELBINE®); and Zoledronates (e.g., ZOMETA®). 
     For example, exemplary antibiotic agents include, but are not limited to, Aminoglycosides; Amphenicols; Ansamycins; Carbacephems; Carbapenems; Cephalosporins or Cephems; Cephamycins; Clavams; Cyclic lipopeptides; Diaminopyrimidines; Ketolides; Lincosamides; Macrolides; Monobactams; Nitrofurans; Oxacephems; Oxazolidinones; Penems, thienamycins and miscellaneous beta-lactams; Penicillins; Polypeptides antibiotics; Quinolones; Sulfonamides; Sulfones; Tetracyclines; and other antibiotics (such as Clofoctols, Fusidic acids, Hexedines, Methenamines, Nitrofurantoins Nitroxolines, Ritipenems, Taurolidines, Xibomols). 
     Also included among exemplary therapeutic agents are coagulation factors or other blood modifiers such as antihemophilic factors, anti-inhibitor coagulant complexes, antithrombin III, coagulation Factor V, coagulation Factor VIII, coagulation Factor IX, plasma protein fractions, von Willebrand factors; antiplatelet agents (including, for example, abciximabs, anagrelides, cilostazols, clopidogrel bisulfates, dipyridamoles, epoprostenols, eptifibatides, tirofibans; colony stimulating factors (CSFs) (including, for example, Granulocyte CSFs and Granulocyte Macrophage CSFs); erythropoiesis stimulators (including, for example, erythropoietins such as darbepoetin alfas) and epoetin alfas; hemostatics and albumins (including, for example, aprotinins, combinations of antihemophilic factors and plasma, Desmopressin Acetates, and albumins); immune globulins, as well as hepatitis B immune globulins; thrombin inhibitors (including for example direct thrombin inhibitors and lepirudin), and drotrecogin alfas; anticoagulants (including, for example, dalteparins, enoxaparins and other heparins, and warfarins). 
     Exemplary antibodies or other therapeutic agents include, but are not limited to, Cetuximab (e.g., IMC-C225; Erbitux®); Trastuzumab (e.g., Herceptin®); Rituximab (e.g., Rituxant; MabTherat); Bevacizumab (e.g., Avastint); Alemtuzumab (e.g., Campatht; Campath-1H®; Mabcampath®); Panitumumab (e.g., ABX-EGF; Vectibix®); Ranibizumab (e.g., Lucentis®); Ibritumomab; Ibritumomab tiuxetan (e.g., Zevalin R); Tositumomab; Iodine I 131 Tositumomab (e.g., BEXXAR®); Catumaxomab (e.g., Removab®); Gemtuzumab; Gemtuzumab ozogamicin (e.g., Mylotarg R); Abatacept (e.g., CTLA4-Ig; Orencia®); Belatacept (L104EA29YIg; LEA29Y; LEA); Ipilimumab (e.g., MDX-010; MDX-101); Tremelimumab (e.g., ticilimumab; CP-675,206); PRS-010 (see e.g., US20090042785); PRS-050 (see e.g., U.S. Pat. No. 7,585,940; US20090305982); Aflibercept (VEGF Trap, AVE005; Holash et al., (2002)  PNAS  99:11393-11398); Volociximab (M200); F200 (Chimeric (human/murine) IgG4 Fab fragment of Volociximab (M200)); MORAb-009 Mouse/human chimeric IgG1(US20050054048); Soluble fusion protein:Anti-mesothelin Fv linked to a truncated  Pseudomonas  exotoxin A (SS1P (CAT-5001); US20070189962); Cixutumumab (IMC-A12); Nimotuzumab (h-R3) (Spicer (2005)  Curr Opin Mol Ther  7:182-191); Zalutumumab (HuMax-EGFR; Lammerts van Bueren et al. (2008)  PNAS  105:6109-14); Necitumumab IMC-11F8 (Li et al. (2008)  Structure  16:216-227); Sym004 (Pedersen et al. 2010  Cancer Res  70:588-597); and mAb-425. 
     In particular, therapeutic agents include, but are not limited to, immunoglobulins, Interferon beta, Interferon alpha-2as, Interferon alpha-1s, Interferon alpha-n3s, Interferon beta-1, Interferon beta-1as, Interferon gamma-1bs, Peg-interferon alpha-2 and Peginterferon alpha-2bs, insulin, a bisphosphate (e.g., Pamidronates or Zoledronates), Docetaxels, Doxorubicins, Doxorubicin liposomals and bevacizumabs. 
     Other exemplary therapeutic agents that can be combined by co-administration and/or co-formulation with a modified PH20 polypeptide provided herein, include, but are not limited to, Adalimumabs, Agalsidase Betas, Alefacepts, Ampicillins, Anakinras, Antipoliomyelitic Vaccines, Anti-Thymocytes, Azithromycins, Becaplermins, Caspofungins, Cefazolins, Cefepimes, Cefotetans, Ceftazidimes, Ceftriaxones, Cetuximabs, Cilastatins, Clavulanic Acids, Clindamycins, Darbepoetin Alfas, Daclizumabs, Diphtheria, Diphtheria antitoxins, Diphtheria Toxoids, Efalizumabs, Epinephrines, Erythropoietin Alphas, Etanercepts, Filgrastims, Fluconazoles, Follicle-Stimulating Hormones, Follitropin Alphas, Follitropin Betas, Fosphenytoins, Gadodiamides, Gadopentetates, Gatifloxacins, Glatiramers, GM-CSF&#39;s, Goserelins, Goserelin acetates, Granisetrons,  Haemophilus  Influenza B&#39;s, Haloperidols, Hepatitis vaccines, Hepatitis A Vaccines, Hepatitis B Vaccines, Ibritumomab Tiuxetans, Ibritumomabs, Tiuxetans, Immunoglobulins, Hemophilus influenza vaccines, Influenza Virus Vaccines, Infliximabs, Insulins, Insulin Glargines, Interferons, Interferon alphas, Interferon Betas, Interferon Gammas, Interferon alpha-2a&#39;s, Interferon alpha-2b&#39;s, Interferon alpha-1&#39;s, Interferon alpha-n3&#39;s, Interferon Betas, Interferon Beta-1a&#39;s, Interferon Gammas, Interferon alpha-consensus, Iodixanols, Iohexols, Iopamidols, Ioversols, Ketorolacs, Laronidases, Levofloxacins, Lidocaines, Linezolids, Lorazepams, Measles Vaccines, Measles virus, Mumps viruses, Measles-Mumps-Rubella Virus Vaccines, Rubella vaccines, Medroxyprogesterones, Meropenems, Methylprednisolones, Midazolams, Morphines, Octreotides, Omalizumabs, Ondansetrons, Palivizumabs, Pantoprazoles, Pegaspargases, Pegfilgrastims, Peg-Interferon Alfa-2a&#39;s, Peg-Interferon Alfa-2b&#39;s, Pegvisomants, Pertussis vaccines, Piperacillins, Pneumococcal Vaccines and Pneumococcal Conjugate Vaccines, Promethazines, Reteplases, Somatropins, Sulbactams, Sumatriptans, Tazobactams, Tenecteplases, Tetanus Purified Toxoids, Ticarcillins, Tositumomabs, Triamcinolones, Triamcinolone Acetonides, Triamcinolone hexacetonides, Vancomycins, Varicella Zoster immunoglobulins, Varicella vaccines, other vaccines, Alemtuzumabs, Alitretinoins, Allopurinols, Altretamines, Amifostines, Anastrozoles, Arsenics, Arsenic Trioxides, Asparaginases,  Bacillus  Calmette-Guerin (BCG) vaccines, BCG Live, Bexarotenes, Bleomycins, Busulfans, Busulfan intravenous, Busulfan orals, Calusterones, Capecitabines, Carboplatins, Carmustines, Carmustines with Polifeprosans, Celecoxibs, Chlorambucils, Cisplatins, Cladribines, Cyclophosphamides, Cytarabines, Cytarabine liposomals, Dacarbazines, Dactinomycins, Daunorubicin liposomals, Daunorubicins, Daunomycins, Denileukin Diftitoxes, Dexrazoxanes, Docetaxels, Doxorubicins, Doxorubicin liposomals, Dromostanolone propionates, Elliott&#39;s B Solutions, Epirubicins, Epoetin alfas, Estramustines, Etoposides, Etoposide phosphates, Etoposide VP-16s, Exemestanes, Floxuridines, Fludarabines, Fluorouracils, 5-Fluorouracils, Fulvestrants, Gemcitabines, Gemtuzumabs, Ozogamicins, Gemtuzumab ozogamicins, Hydroxyureas, Idarubicins, Ifosfamides, Imatinib mesylates, Irinotecans, Letrozoles, Leucovorins, Levamisoles, Lomustines, CCNUs, Mechlorethamines, Nitrogen mustards, Megestrols, Megestrol acetates, Melphalans, L-PAMs, Mercaptopurines, 6-Mercaptopurines, Mesnas, Methotrexates, Methoxsalens, Mitomycins, Mitomycin C&#39;s, Mitotanes, Mitoxantrones, Nandrolones, Nandrolone Phenpropionates, Nofetumomabs, Oprelvekins, Oxaliplatins, Paclitaxels, Pamidronates, Pegademases, Pentostatins, Pipobromans, Plicamycins, Mithramycins, Porfimers, Porfimer sodiums, Procarbazines, Quinacrines, Rasburicases, Rituximabs, Sargramostims, Streptozocins, Talcs, Tamoxifens, Temozolomides, Teniposides, Testolactones, Thioguanines, 6-Thioguanines, Triethylenethiophosphoramides (Thiotepas), Topotecans, Toremifenes, Trastuzumabs, Tretinoins, Uracil Mustards, Valrubicins, Vinblastines, Vincristines, Vinorelbines, Zoledronates, Acivicins, Aclarubicins, Acodazoles, Acronines, Adozelesins, Aldesleukins, Retinoic Acids, Alitretinoins, 9-Cis-Retinoic Acids, Alvocidibs, Ambazones, Ambomycins, Ametantrones, Aminoglutethimides, Amsacrines, Anaxirones, Ancitabines, Anthramycins, Apaziquones, Argimesnas, Asperlins, Atrimustines, Azacitidines, Azetepas, Azotomycins, Banoxantrones, Batabulins, Batimastats, Benaxibines, Bendamustines, Benzodepas, Bicalutamides, Bietaserpines, Biricodars, Bisantrenes, Bisnafide Dimesylates, Bizelesins, Bortezomibs, Brequinars, Bropirimines, Budotitanes, Cactinomycins, Canertinibs, Caracemides, Carbetimers, Carboquones, Carmofurs, Carubicins, Carzelesins, Cedefingols, Cemadotins, Chlorambucils, Cioteronels, Cirolemycins, Clanfenurs, Clofarabines, Crisnatols, Decitabines, Dexniguldipines, Dexormaplatins, Dezaguanines, Diaziquones, Dibrospidiums, Dienogests, Dinalins, Disermolides, Dofequidars, Doxifluridines, Droloxifenes, Duazomycins, Ecomustines, Edatrexates, Edotecarins, Eflomithines, Elacridars, Elinafides, Elsamitrucins, Emitefurs, Enloplatins, Enpromates, Enzastaurins, Epipropidines, Eptaloprosts, Erbulozoles, Esorubicins, Etanidazoles, Etoglucids, Etoprines, Exisulinds, Fadrozoles, Fazarabines, Fenretinides, Fluoxymesterones, Flurocitabines, Fosquidones, Fostriecins, Fotretamines, Galarubicins, Galocitabines, Geroquinols, Gimatecans, Gimeracils, Gloxazones, Glufosfamides, Ilmofosines, Ilomastats, Imexons, Improsulfans, Indisulams, Inproquones, Interleukins, Interleukin-2s, recombinant Interleukins, Intoplicines, Iobenguanes, Iproplatins, Irsogladines, Ixabepilones, Ketotrexates, L-Alanosines, Lanreotides, Lapatinibs, Ledoxantrones, Leuprolides, Leuprorelins, Lexacalcitols, Liarozoles, Lobaplatins, Lometrexols, Lonafarnibs, Losoxantrones, Lurtotecans, Mafosfamides, Mannosulfans, Marimastats, Masoprocols, Maytansines, Mechlorethamines, Melengestrols, Melphalans, Menogarils, Mepitiostanes, Metesinds, Metomidates, Metoprines, Meturedepas, Miboplatins, Miproxifenes, Misonidazoles, Mitindomides, Mitocarcins, Mitocromins, Mitoflaxones, Mitogillins, Mitoguazones, Mitomalcins, Mitonafides, Mitoquidones, Mitospers, Mitozolomides, Mivobulins, Mizoribines, Mofarotenes, Mopidamols, Mubritinibs, Mycophenolic Acids, Nedaplatins, Neizarabines, Nemorubicins, Nitracrines, Nocodazoles, Nogalamycins, Nolatrexeds, Nortopixantrones, Ormaplatins, Ortataxels, Oteracils, Oxisurans, Oxophenarsines, Patupilones, Peldesines, Peliomycins, Pelitrexols, Pemetrexeds, Pentamustines, Peplomycins, Perfosfamides, Perifosines, Picoplatins, Pinafides, Piposulfans, Pirfenidones, Piroxantrones, Pixantrones, Plevitrexeds, Plomestanes, Porfiromycins, Prednimustines, Propamidines, Prospidiums, Pumitepas, Puromycins, Pyrazofurins, Ranimustines, Riboprines, Ritrosulfans, Rogletimides, Roquinimexs, Rufocromomycins, Sabarubicins, Safingols, Satraplatins, Sebriplatins, Semustines, Simtrazenes, Sizofirans, Sobuzoxanes, Sorafenibs, Sparfosates, Sparfosic Acids, Sparsomycins, Spirogermaniums, Spiromustines, Spiroplatins, Squalamines, Streptonigrins, Streptovarycins, Sufosfamides, Sulofenurs, Tacedinalines, Talisomycins, Tallimustines, Tariquidars, Tauromustines, Tecogalans, Tegafurs, Teloxantrones, Temoporfins, Teroxirones, Thiamiprines, Tiamiprines, Tiazofurins, Tilomisoles, Tilorones, Timcodars, Timonacics, Tirapazamines, Topixantrones, Trabectedins, Ecteinascidin 743, Trestolones, Triciribines, Trilostanes, Trimetrexates, Triplatin Tetranitrates, Triptorelins, Trofosfamides, Tubulozoles, Ubenimexs, Uredepas, Valspodars, Vapreotides, Verteporfins, Vinblastines, Vindesines, Vinepidines, Vinflunines, Vinformides, Vinglycinates, Vinleucinols, Vinleurosines, Vinrosidines, Vintriptols, Vinzolidines, Vorozoles, Xanthomycin A&#39;s, Guamecyclines, Zeniplatins, Zilascorbs [2-H], Zinostatins, Zorubicins, Zosuquidars, Acetazolamides, Acyclovirs, Adipiodones, Alatrofloxacins, Alfentanils, Allergenic extracts, Alpha 1-proteinase inhibitors, Alprostadils, Amikacins, Amino acids, Aminocaproic acids, Aminophyllines, Amitriptylines, Amobarbitals, Amrinones, Analgesics, Anti-poliomyelitic vaccines, Anti-rabic serums, Anti-tetanus immunoglobulins, tetanus vaccines, Antithrombin IIIs, Antivenom serums, Argatrobans, Arginines, Ascorbic acids, Atenolols, Atracuriums, Atropines, Aurothioglucoses, Azathioprines, Aztreonams, Bacitracins, Baclofens, Basiliximabs, Benzoic acids, Benztropines, Betamethasones, Biotins, Bivalirudins, Botulism antitoxins, Bretyliums, Bumetanides, Bupivacaines, Buprenorphines, Butorphanols, Calcitonins, Calcitriols, Calciums, Capreomycins, Carboprosts, Carnitines, Cefamandoles, Cefoperazones, Cefotaximes, Cefoxitins, Ceftizoximes, Cefuroximes, Chloramphenicols, Chloroprocaines, Chloroquines, Chlorothiazides, Chlorpromazines, Chondroitinsulfuric acids, Choriogonadotropin alfas, Chromiums, Cidofovirs, Cimetidines, Ciprofloxacins, Cisatracuriums, Clonidines, Codeines, Colchicines, Colistins, Collagens, Corticorelin ovine triflutates, Corticotrophins, Cosyntropins, Cyanocobalamins, Cyclosporines, Cysteines, Dacliximabs, Dalfopristins, Dalteparins, Danaparoids, Dantrolenes, Deferoxamines, Desmopressins, Dexamethasones, Dexmedetomidines, Dexpanthenols, Dextrans, Iron dextrans, Diatrizoic acids, Diazepams, Diazoxides, Dicyclomines, Digibinds, Digoxins, Dihydroergotamines, Diltiazems, Diphenhydramines, Dipyridamoles, Dobutamines, Dopamines, Doxacuriums, Doxaprams, Doxercalciferols, Doxycyclines, Droperidols, Dyphyllines, Edetic acids, Edrophoniums, Enalaprilats, Ephedrines, Epoprostenols, Ergocalciferols, Ergonovines, Ertapenems, Erythromycins, Esmolols, Estradiols, Estrogenics, Ethacrynic acids, Ethanolamines, Ethanols, Ethiodized oils, Etidronic acids, Etomidates, Factor VIIIs, Famotidines, Fenoldopams, Fentanyls, Flumazenils, Fluoresceins, Fluphenazines, Folic acids, Fomepizoles, Fomivirsens, Fondaparinuxs, Foscarnets, Fosphenytoins, Furosemides, Gadoteridols, Gadoversetamides, Ganciclovirs, Gentamicins, Glucagons, Glucoses, Glycines, Glycopyrrolates, Gonadorelins, Gonadotropin chorionics,  Haemophilus  B polysaccharides, Hemins, Herbals, Histamines, Hydralazines, Hydrocortisones, Hydromorphones, Hydroxocobalamins, Hydroxyzines, Hyoscyamines, Ibutilides, Imiglucerases, Indigo carmines, Indomethacins, Iodides, Iopromides, lothalamic acids, loxaglic acids, Ioxilans, Isoniazids, Isoproterenols, Japanese encephalitis vaccines, Kanamycins, Ketamines, Labetalols, Lepirudins, Levobupivacaines, Levothyroxines, Lincomycins, Liothyronines, Luteinizing hormones, Lyme disease vaccines, Mangafodipirs, Manthtols, Meningococcal polysaccharide vaccines, Meperidines, Mepivacaines, Mesoridazines, Metaraminols, Methadones, Methocarbamols, Methohexitals, Methyldopates, Methylergonovines, Metoclopramides, Metoprolols, Metronidazoles, Minocyclines, Mivacuriums, Morrhuic acids, Moxifloxacins, Muromonab-CD3s, Mycophenolate mofetils, Nafcillins, Nalbuphines, Nalmefenes, Naloxones, Neostigmines, Niacinamides, Nicardipines, Nitroglycerins, Nitroprussides, Norepinephrines, Orphenadrines, Oxacillins, Oxymorphones, Oxytetracyclines, Oxytocins, Pancuroniums, Panthenols, Pantothenic acids, Papaverines, Peginterferon-alpha (e.g., interferon alpha 2a or 2b), Penicillin Gs, Pentamidines, Pentazocines, Pentobarbitals, Perflutrens, Perphenazines, Phenobarbitals, Phentolamines, Phenylephrines, Phenytoins, Physostigmines, Phytonadiones, Polymyxin bs, Pralidoximes, Prilocaines, Procainamides, Procaines, Prochlorperazines, Progesterones, Propranolols, Pyridostigmine hydroxides, Pyridoxines, Quinidines, Quinupristins, Rabies immunoglobulins, Rabies vaccines, Ranitidines, Remifentanils, Riboflavins, Rifampins, Ropivacaines, Samariums, Scopolamines, Seleniums, Sermorelins, Sincalides, Somatrems, Spectinomycins, Streptokinases, Streptomycins, Succinylcholines, Sufentanils, Sulfamethoxazoles, Tacrolimuses, Terbutalines, Teriparatides, Testosterones, Tetanus antitoxins, Tetracaines, Tetradecyl sulfates, Theophyllines, Thiamines, Thiethylperazines, Thiopentals, Thyroid stimulating hormones, Tinzaparins, Tirofibans, Tobramycins, Tolazolines, Tolbutamides, Torsemides, Tranexamic acids, Treprostinils, Trifluoperazines, Trimethobenzamides, Trimethoprims, Tromethamines, Tuberculins, Typhoid vaccines, Urofollitropins, Urokinases, Valproic acids, Vasopressins, Vecuroniums, Verapamils, Voriconazoles, Warfarins, Yellow fever vaccines, Zidovudines, Zincs, Ziprasidone hydrochlorides, Aclacinomycins, Actinomycins, Adriamycins, Azaserines, 6-Azauridines, Carzinophilins, Chromomycins, Denopterins, 6-Diazo-5-Oxo-L-Norleucines, Enocitabines, Floxuridines, Olivomycins, Pirarubicins, Piritrexims, Pteropterins, Tegafurs, Tubercidins, Alteplases, Arcitumomabs, bevacizumabs, Botulinum Toxin Type A&#39;s, Botulinum Toxin Type B&#39;s, Capromab Pendetides, Daclizumabs, Dornase alfas, Drotrecogin alfas, Imciromab Pentetates, and Iodine-131&#39;s. 
     Delivery of Insulin 
     Methods provided herein include methods of co-administering a modified PH20 polypeptide and an insulin to increase subcutaneous delivery of the insulin, such as a fast-acting insulin (see e.g., U.S. Pat. Nos. 7,767,429; 7,846,431; U.S. Publication No. US20090304665; and U.S. application Ser. Nos. 13/507,263; 13/507,262 and 13/507,261). Such methods include methods of direct administration, and pump and continuous infusion methods, including open and closed pump systems. For example, exemplary insulins that can be administered with a modified PH20 hyaluronidase provided herein are fast-acting insulins or insulin analogs. For example, a co-administered insulin includes a regular insulin, insulin aspart, insulin lispro, insulin glulisine or other similar analog variants. Exemplary insulins are insulins that contain an A chain set forth in SEQ ID NO:862 and a B chain set forth in SEQ ID NO:863 or variants that contain one or more amino acid modifications compared to a human insulin set forth in SEQ ID NO: 862 and 863 (A and B chains) For example, exemplary insulin analogs are known to one of skill in the art, and include, but are not limited to, those set forth in SEQ ID NO:862 (A-chain) and having a B-chain set forth in any of SEQ ID NOs: 865-867. 
     The co-formulations can be administered subcutaneously to treat any condition that is amenable to treatment with insulin. Therapeutic uses include, but are not limited to, treatment for type 1 diabetes mellitus, type 2 diabetes mellitus, gestational diabetes, and for glycemic control in critically ill patients. For example, the co-formulations of a fast acting insulin and hyaluronan degrading enzyme can be administered subcutaneously in discrete doses, such as via a syringe or insulin pen, prior to a meal as prandial insulin therapy in subjects with diabetes to achieve glycemic control. The co-formulations also can be administered subcutaneously or intraperitoneally using an insulin pump or in the context of a closed loop system to continuously control blood glucose levels throughout the day and night and/or to control post-prandial glycemic excursions. It is within the skill of a treating physician to identify such diseases or conditions. 
     For any disease or condition, including all those exemplified above, for which a fast-acting insulin is indicated or has been used and for which other agents and treatments are available, the co-formulations can be used in combination therewith. Depending on the disease or condition to be treated, exemplary combinations include, but are not limited to, combinations with anti-diabetic drugs, including, but not limited to, sulfonylureas, biguanides, meglitinides, thiazolidinediones, alpha-glucosidase inhibitors, peptide analogs, including glucagon-like peptide (GLP) analogs and, gastric inhibitory peptide (GIP) analogs and DPP-4 inhibitors. In another example, the co-formulations of a fast acting insulin and modified PH20 polypeptide described herein can be administered in combination with, prior to, intermittently with, or subsequent to, one or more other insulins, including fast-acting insulin, and basal-acting insulins. 
     2. Methods of Hyaluronan-Associated Diseases and Conditions (e.g., Tumors) 
     In particular, PH20 hyaluronidase can be used to treat hyaluronan-associated diseases or conditions. Typically, hyaluronan-associated diseases and conditions are associated with elevated hyaluronan expression in a tissue, cell, or body fluid (e.g., tumor tissue or tumor-associated tissue, blood, or interstitial space) compared to a control, e.g., another tissue, cell or body fluid. The elevated hyaluronan expression can be elevated compared to a normal tissue, cell or body fluid, for example, a tissue, cell or body fluid that is analogous to the sample being tested, but isolated from a different subject, such as a subject that is normal (i.e., does not have a disease or condition, or does not have the type of disease or condition that the subject being tested has), for example, a subject that does not have a hyaluronan-associated disease or condition. The elevated hyaluronan expression can be elevated compared to an analogous tissue from another subject that has a similar disease or condition, but whose disease is not as severe and/or is not hyaluronan-associated or expresses relatively less hyaluronan and thus is hyaluronan-associated to a lesser degree. For example, the subject being tested can be a subject with a hyaluronan-associated cancer, where the HA amounts in the tissue, cell or fluid are relatively elevated compared to a subject having a less severe cancer, such as an early stage, differentiated or other type of cancer. In another example, the cell, tissue or fluid contains elevated levels of hyaluronan compared to a control sample, such as a fluid, tissue, extract (e.g., cellular or nuclear extract), nucleic acid or peptide preparation, cell line, biopsy, standard or other sample, with a known amount or relative amount of HA, such as a sample, for example a tumor cell line, known to express relatively low levels of HA, such as exemplary tumor cell lines described herein that express low levels of HA, for example, the HCT 116 cell line, the HT29 cell line, the NCI H460 cell line, the DU145 cell line, the Capan-1 cell line, and tumors from tumor models generated using such cell lines. 
     Hyaluronan-associated diseases and conditions include those associated with high interstitial fluid pressure, such as disc pressure, proliferative disorders, such as cancer and benign prostatic hyperplasia, and edema. Edema can result from or be manifested in, for example, organ transplant, stroke or brain trauma. Proliferative disorders include, but are not limited to, cancer, smooth muscle cell proliferation, systemic sclerosis, cirrhosis of the liver, adult respiratory distress syndrome, idiopathic cardiomyopathy, lupus erythematosus, retinopathy, e.g., diabetic retinopathy or other retinopathies, cardiac hyperplasia, reproductive system associated disorders, such as benign prostatic hyperplasia (BPH) and ovarian cysts, pulmonary fibrosis, endometriosis, fibromatosis, hamartomas, lymphangiomatosis, sarcoidosis, desmoid tumors. Cancers include solid and lymphatic/blood tumors and metastatic disease, and undifferentiated tumors. The tumors amenable to treatment typically exhibit cellular and/or stromal expression of a hyaluronan, compared to a non-cancerous tissue of the same tissue type or compared to a non-metastatic tumor of the same tumor-type. Cancers include any one or more of ovarian cancer, in situ carcinoma (ISC), squamous cell carcinoma (SCC), prostate cancer, pancreatic cancer, other gastric cancers, non-small cell lung cancer, breast cancer, brain cancer and colon cancer. 
     Modified PH20 polypeptides provided herein, such as PEGylated forms thereof, can be used to treat tumors. Thus, in addition to its indirect anticancer effects, hyaluronidases also have direct anticarcinogenic effects. Hyaluronidase prevents growth of tumors transplanted into mice (De Maeyer et al., (1992)  Int. J. Cancer  51:657-660) and inhibits tumor formation upon exposure to carcinogens (Pawlowski et al., (1979)  Int. J. Cancer  23:105-109; Haberman et al., (1981) Proceedings of the 17th Annual Meeting of the American Society of Clinical Oncology, Washington, D.C., 22:105, abstract no. 415). PH20 hyaluronidase has been shown to treat various tumors (see e.g., U.S. Publication No. US2010/0003238 and U.S. application Ser. No. 13/135,817, published as U.S. Publication No. US20120020951). 
     The hyaluronan-rich cancer can be a cancer in which the cancer cells produce HALOS, cancers that have elevated expression of hyaluronan (as determined by immunostaining, e.g., histological staining of sections from the tumor), cancers that have elevated HAS2 (Hyaluronan synthase 2), cancers that do not produce hyaluronidase (HYAL1) in vitro. Hyaluronan-rich cancers can be identified by any method for assessing hyaluronan expression, and other known methods for assaying protein/mRNA expression. 
     Several hyaluronan-rich cancers have been identified. In some cases, hyaluronan expression correlates with poor prognosis, for example, decreased survival rate and/or recurrence-free survival rate, metastases, angiogenesis, cancer cell invasion into other tissues/areas, and other indicators of poor prognosis. Such correlation has been observed, for example, in hyaluronan-rich tumors including ovarian cancer, SCC, ISC, prostate cancer, lung cancer, including non-small-cell lung cancer (NSCLC), breast cancer, colon cancer and pancreatic cancer (see, for example, Anttila et al.,  Cancer Research,  60:150-155 (2000); Karvinen et al.,  British Journal of Dermatology,  148:86-94 (2003); Lipponen et al.,  Eur. Journal of Cancer,  849-856 (2001); Pirinen et al.,  Int. J. Cancer:  95: 12-17 (2001); Auvinen et al.,  American Journal of Pathology,  156(2):529-536 (2000); Ropponen et al.,  Cancer Research,  58: 342-347 (1998)). Thus, hyaluronan-rich cancers can be treated by administration of a hyaluronidase, such as a soluble PH20, to treat one or more symptoms of the cancer. Hyaluronan-rich tumors include, but are not limited to those of the prostate, breast, colon, ovarian, stomach, head and neck and other tumors and cancers. 
     Other hyaluronan-associated diseases or conditions that are associated with excess glycosaminoglycans and that can be treated with a modified PH20 polypeptide provided herein include, but are not limited to, cardiovascular disease (e.g., following ischemia reperfusion; in arteriosclerosis); vitrectomy and ophthalmic disorders and conditions (e.g., in methods to liquefy the vitreous humor of the eye; reduce postoperative pressure; other ocular surgical procedures such as glaucoma, vitreous and retina surgery and in corneal transplantation); in hypodermoclysis (i.e., infusion of fluids and electrolytes into the hypodermis of the skin); cosmetic applications (e.g., in the treatment of cellulite, “pigskin” edema or “orange peel” edema); organ transplantation (e.g., associated with interstitial edemas in connection with grafting of an organ); pulmonary disease. 
     3. Other Uses 
     In further examples of its therapeutic use, modified PH20 polypeptides provided herein, can be used for such purposes as an antidote to local necrosis from paravenous injection of necrotic substances such as  vinca  alkaloids (Few et al. (1987)  Amer. J. Matern. Child Nurs.  12, 23-26), treatment of ganglion cysts (Paul et al. (1997)  J Hand Surg.  22 (2): 219-21) and treatment of tissue necrosis due to venous insufficiency (Elder et al. (1980)  Lancet  648-649). Modified PH20 polypeptides also can be used to treat ganglion cysts (also known as a wrist cyst, Bible cyst, or dorsal tendon cyst), which are the most common soft tissue mass of the hand and are fluid filled sacs that can be felt below the skin. 
     Modified PH20 polypeptides can be used in the treatment of spinal cord injury by degrading chondroitin sulfate proteoglycans (CSPGs). Following spinal cord injury, glial scars containing CSPGs are produced by astrocytes. CSPGs play a crucial role in the inhibition of axon growth. In addition, the expression of CSPG has been shown to increase following injury of the central nervous system (CNS). Soluble PH20 also can be utilized for the treatment of herniated disks in a process known as chemonucleolysis. Chondroitinase ABC, an enzyme cleaving similar substrates as hyaluronidase, can induce the reduction of intradiscal pressure in the lumbar spine. There are three types of disk injuries. A protruded disk is one that is intact but bulging. In an extruded disk, the fibrous wrapper has torn and the NP has oozed out, but is still connected to the disk. In a sequestered disk, a fragment of the NP has broken loose from the disk and is free in the spinal canal. Chemonucleolysis is typically effective on protruded and extruded disks, but not on sequestered disk injuries. 
     4. Contraception 
     Modified PH20 polypeptides provided herein can be used as vaccines in contraceptive applications. PH20 is present in the male reproductive tract, and is expressed in both the testis and epididymis and is present in sperm. PH20 plays a role in fertilization by facilitating entry of the sperm through the cumulus layer surrounding the unfertilized egg. PH20 also is able to bind to hyaluronic acid (HA) on the zona pellucida during early phases of fertilization. This binding also initiates intracellular signaling that aids in the acrosome reaction. Immunization with PH20 has been shown to be an effective contraceptive in male guinea pigs (Primakoff et al. (1988)  Nature  335:543-546, Tung et al. (1997)  Biol. Reprod.  56:1133-1141). It also has been shown to be an effective contraceptive in female guinea pigs due to the generation of anti-PH20 antibodies that prevent sperm and egg binding. In examples herein, the modified PH20 polypeptides can be inactive enzymes, such as any described in Sections C.2. The polypeptides can be administered directly or can be administered as a recombinant virus to deliver the antigen. 
     I. EXAMPLES 
     The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention. 
     Example 1 
     Generation of Recombinant Human pH20 Hyaluronidase (rHuPH20)
 
A. Generation of a Soluble rHuPH20-Expressing Cell Line
 
     A recombinant human PH20 hyaluronidase designated rHuPH20 was generated as described in published U.S. Publication No. US20110053247. Briefly, the pCI-PH20-IRES-DHFR-SV40pa (HZ24) plasmid (set forth in SEQ ID NO:5) was used to transfect Chinese Hamster Ovary (CHO cells) (see e.g., U.S. Pat. Nos. 7,767,429 and 7,781,607 and U.S. Publication No. 2006-0104968). The HZ24 plasmid vector for expression of soluble rHuPH20 contains a pCI vector backbone (Promega), DNA encoding amino acids 1-482 of human PH20 hyaluronidase (SEQ ID NO:2), an internal ribosomal entry site (IRES) from the ECMV virus (Clontech), and the mouse dihydrofolate reductase (DHFR) gene. The pCI vector backbone also includes DNA encoding the Beta-lactamase resistance gene (AmpR), an f1 origin of replication, a Cytomegalovirus immediate-early enhancer/promoter region (CMV), a chimeric intron, and an SV40 late polyadenylation signal (SV40). The DNA encoding the soluble rHuPH20 construct contains an NheI site and a Kozak consensus sequence prior to the DNA encoding the methionine at amino acid position 1 of the native 35 amino acid signal sequence of human PH20, and a stop codon following the DNA encoding the tyrosine corresponding to amino acid position 482 of the human PH20 hyaluronidase set forth in SEQ ID NO:2, followed by a BamHI restriction site. 
     Non-transfected DG44 CHO cells growing in GIBCO Modified CD-CHO media for DHFR(−) cells, supplemented with 4 mM Glutamine and 18 mL/L Plurionic F68/L (Gibco), were seeded at 0.5×10 6  cells/mL in a shaker flask in preparation for transfection. Cells were grown at 37° C. in 5% CO 2  in a humidified incubator, shaking at 120 rpm. Exponentially growing non-transfected DG44 CHO cells were tested for viability prior to transfection. 
     Sixty million viable cells of the non-transfected DG44 CHO cell culture were pelleted and resuspended to a density of 2×10 7  cells in 0.7 mL of 2× transfection buffer (2× HeBS: 40 mM Hepes, pH 7.0, 274 mM NaCl, 10 mM KCl, 1.4 mM Na 2 HPO 4 , 12 mM dextrose). To each aliquot of resuspended cells, 0.09 mL (250 μg) of the linear HZ24 plasmid (linearized by overnight digestion with Cla I (New England Biolabs) was added, and the cell/DNA solutions were transferred into 0.4 cm gap BTX (Gentronics) electroporation cuvettes at room temperature. A negative control electroporation was performed with no plasmid DNA mixed with the cells. The cell/plasmid mixes were electroporated with a capacitor discharge of 330 V and 960 μF or at 350 V and 960 μF. 
     The cells were removed from the cuvettes after electroporation and transferred into 5 mL of Modified CD-CHO media for DHFR(−) cells, supplemented with 4 mM Glutamine and 18 mL/L Plurionic F68/L (Gibco), and allowed to grow in a well of a 6-well tissue culture plate without selection for 2 days at 37° C. in 5% CO 2  in a humidified incubator. 
     Two days post-electroporation, 0.5 mL of tissue culture media was removed from each well and tested for the presence of hyaluronidase activity, using the microturbidity assay described in Example 8. The results are set forth in Table 6. 
     
       
         
           
               
             
               
                 TABLE 6 
               
             
            
               
                   
               
               
                 Initial Hyaluronidase Activity of HZ24 Transfected  
               
               
                 DG44 CHO cells at 40 hours post-transfection 
               
            
           
           
               
               
               
               
            
               
                   
                   
                   
                 Activity  
               
               
                   
                   
                 Dilution 
                 (Units/mL) 
               
               
                   
                   
               
            
           
           
               
               
               
               
            
               
                   
                 Transfection 1 330 V 
                 1 to 10 
                 0.25 
               
               
                   
                 Transfection 2 350 V 
                 1 to 10 
                 0.52 
               
               
                   
                 Negative Control 
                 1 to 10 
                 0.015 
               
               
                   
                   
               
            
           
         
       
     
     Cells from Transfection 2 (350V) were collected from the tissue culture well, counted and diluted to 1×10 4  to 2×10 4  viable cells per mL. A 0.1 mL aliquot of the cell suspension was transferred to each well of five, 96 well round bottom tissue culture plates. One hundred microliters of CD-CHO media (GIBCO) containing 4 mM GlutaMAX™-1 supplement (GIBCO™, Invitrogen Corporation) and without hypoxanthine and thymidine supplements were added to the wells containing cells (final volume 0.2 mL). Ten clones were identified from the 5 plates grown without methotrexate (Table 7). 
     
       
         
           
               
             
               
                 TABLE 7 
               
             
            
               
                   
               
               
                 Hyaluronidase activity of identified clones 
               
            
           
           
               
               
               
            
               
                   
                 Plate/Well ID 
                 Relative Hyaluronidase 
               
               
                   
                   
               
               
                   
                 1C3 
                 261 
               
               
                   
                 2C2 
                 261 
               
               
                   
                 3D3 
                 261 
               
               
                   
                 3E5 
                 243 
               
               
                   
                 3C6 
                 174 
               
               
                   
                 2G8 
                 103 
               
               
                   
                 1B9 
                 304 
               
               
                   
                 2D9 
                 273 
               
               
                   
                 4D10 
                 302 
               
               
                   
                   
               
            
           
         
       
     
     Six HZ24 clones were expanded in culture and transferred into shaker flasks as single cell suspensions. Clones 3D3, 3E5, 2G8, 2D9, 1E11, and 4D10 were plated into 96-well round bottom tissue culture plates using a two-dimensional infinite dilution strategy in which cells were diluted 1:2 down the plate, and 1:3 across the plate, starting at 5000 cells in the top left hand well. Diluted clones were grown in a background of 500 non-transfected DG44 CHO cells per well, to provide necessary growth factors for the initial days in culture. Ten plates were made per subclone, with 5 plates containing 50 nM methotrexate and 5 plates without methotrexate. 
     Clone 3D3 produced 24 visual subclones (13 from the no methotrexate treatment, and 11 from the 50 nM methotrexate treatment). Significant hyaluronidase activity was measured in the supernatants from 8 of the 24 subclones (&gt;50 Units/mL), and these 8 subclones were expanded into T-25 tissue culture flasks. Clones isolated from the methotrexate treatment protocol were expanded in the presence of 50 nM methotrexate. Clone 3D35M was further expanded in 500 nM methotrexate giving rise to clones producing hyaluronidase activity in excess of 1,000 Units/mL in shaker flasks (clone 3D35M; or Gen′ 3D35M). A master cell bank (MCB) of the 3D35M cells was then prepared. 
     B. Production Gen2 Cells Containing Soluble human PH20 (rHuPH20) 
     The Gen′ 3D35M cell line described in Example 1.A was adapted to higher methotrexate levels to produce generation 2 (Gen2) clones. 3D35M cells were seeded from established methotrexate-containing cultures into CD CHO medium containing 4 mM GlutaMAX-1™ and 1.0 μM methotrexate. The cells were adapted to a higher methotrexate level by growing and passaging them 9 times over a period of 46 days in a 37° C., 7% CO 2  humidified incubator. The amplified population of cells was cloned out by limiting dilution in 96-well tissue culture plates containing medium with 2.0 μM methotrexate. After approximately 4 weeks, clones were identified and clone 3E10B was selected for expansion. 3E10B cells were grown in CD CHO medium containing 4 mM GlutaMAX-1™ and 2.0 μM methotrexate for 20 passages. A master cell bank (MCB) of the 3E10B cell line was created and frozen and used for subsequent studies. 
     Amplification of the cell line continued by culturing 3E10B cells in CD CHO medium containing 4 mM GlutaMAX-1™ and 4.0 μM methotrexate. After the 12th passage, cells were frozen in vials as a research cell bank (RCB). One vial of the RCB was thawed and cultured in medium containing 8.0 μM methotrexate. After 5 days, the methotrexate concentration in the medium was increased to 16.0 μM, then 20.0 μM 18 days later. Cells from the 8th passage in medium containing 20.0 μM methotrexate were cloned out by limiting dilution in 96-well tissue culture plates containing CD CHO medium containing 4 mM GlutaMAX-1™ and 20.0 μM methotrexate. Clones were identified 5-6 weeks later and clone 2B2 was selected for expansion in medium containing 20.0 μM methotrexate. After the 11th passage, 2B2 cells were frozen in vials as a research cell bank (RCB). 
     The resultant 2B2 cells are dihydrofolate reductase deficient (dhfr-) DG44 CHO cells that express soluble recombinant human PH20 (rHuPH20). The soluble PH20 is present in 2B2 cells at a copy number of approximately 206 copies/cell. Southern blot analysis of Spe I-, Xba I- and BamH I/Hind III-digested genomic 2B2 cell DNA using a rHuPH20-specific probe revealed the following restriction digest profile: one major hybridizing band of  18  7.7 kb and four minor hybridizing bands (˜13.9, ˜6.6, ˜5.7 and ˜4.6 kb) with DNA digested with Spe I; one major hybridizing band of ˜5.0 kb and two minor hybridizing bands (˜13.9 and ˜6.5 kb) with DNA digested with Xba I; and one single hybridizing band of ˜1.4 kb observed using 2B2 DNA digested with BamH I/Hind III. 
     C. Production of Gen2 soluble rHuPH20 in 300 L Bioreactor Cell Culture 
     A vial of HZ24-2B2 was thawed and expanded from shaker flasks through 36 L spinner flasks in CD-CHO media (Invitrogen, Carlsbad, Calif.) supplemented with 20 μM methotrexate and GlutaMAX-1™ (Invitrogen). Briefly, the vial of cells was thawed in a 37° C. water bath, medium was added and the cells were centrifuged. The cells were re-suspended in a 125 mL shake flask with 20 mL of fresh medium and placed in a 37° C., 7% CO 2  incubator. The cells were expanded up to 40 mL in the 125 mL shake flask. When the cell density reached greater than 1.5×10 6  cells/mL, the culture was expanded into a 125 mL spinner flask in a 100 mL culture volume. The flask was incubated at 37° C., 7% CO 2 . When the cell density reached greater than 1.5×10 6  cells/mL, the culture was expanded into a 250 mL spinner flask in 200 mL culture volume, and the flask was incubated at 37° C., 7% CO 2 . When the cell density reached greater than 1.5×10 6  cells/mL, the culture was expanded into a 1 L spinner flask in 800 mL culture volume and incubated at 37° C., 7% CO 2 . When the cell density reached greater than 1.5×10 6  cells/mL the culture was expanded into a 6 L spinner flask in 5000 mL culture volume and incubated at 37° C., 7% CO 2 . When the cell density reached greater than 1.5×10 6  cells/mL the culture was expanded into a 36 L spinner flask in 32 L culture volume and incubated at 37° C., 7% CO 2 . 
     A 400 L reactor was sterilized and 230 mL of CD-CHO media were added. Before use, the reactor was checked for contamination. Approximately 30 L cells were transferred from the 36 L spinner flasks to the 400 L bioreactor (Braun) at an inoculation density of 4.0×10 5  viable cells per mL and a total volume of 260 L. Parameters were: temperature setpoint, 37° C.; Impeller Speed 40-55 RPM; Vessel Pressure: 3 psi; Air Sparge 0.5-1.5 L/Min.; Air Overlay: 3 L/min. The reactor was sampled daily for cell counts, pH verification, media analysis, protein production and retention. Also, during the run nutrient feeds were added. At 120 hrs (day 5), 10.4L of Feed #1 Medium (4×CD-CHO+33 g/L Glucose+160 mL/L Glutamax-1™+83 mL/L Yeastolate+33 mg/L rHulnsulin) was added. At 168 hours (day 7), 10.8 L of Feed #2 (2×CD-CHO+33 g/L Glucose+80 mL/L Glutamax-1™+167 mL/L Yeastolate+0.92 g/L Sodium Butyrate) was added, and culture temperature was changed to 36.5° C. At 216 hours (day 9), 10.8 L of Feed #3 (1× CD-CHO+50 g/L Glucose+50 mL/L Glutamax-1™+250 mL/L Yeastolate+1.80 g/L Sodium Butyrate) was added, and culture temperature was changed to 36° C. At 264 hours (day 11), 10.8 L of Feed #4 (1×CD-CHO+33 g/L Glucose+33 mL/L Glutamax-1™+250 mL/L Yeastolate+0.92 g/L Sodium Butyrate) was added, and culture temperature was changed to 35.5° C. The addition of the feed media was observed to dramatically enhance the production of soluble rHuPH20 in the final stages of production. The reactor was harvested at 14 or 15 days or when the viability of the cells dropped below 40%. The process resulted in a final productivity of 17,000 Units per mL with a maximal cell density of 12 million cells/mL. At harvest, the culture was sampled for mycoplasma, bioburden, endotoxin and virus in vitro and in vivo, by Transmission Electron Microscopy (TEM) and enzyme activity. 
     The culture was pumped by a peristaltic pump through four Millistak filtration system modules (Millipore) in parallel, each containing a layer of diatomaceous earth graded to 4-8 μm and a layer of diatomaceous earth graded to 1.4-1.1 μm, followed by a cellulose membrane, then through a second single Millistak filtration system (Millipore) containing a layer of diatomaceous earth graded to 0.4-0.11 μm and a layer of diatomaceous earth graded to &lt;0.1 μm, followed by a cellulose membrane, and then through a 0.22 μm final filter into a sterile single use flexible bag with a 350 L capacity. The harvested cell culture fluid was supplemented with 10 mM EDTA and 10 mM Tris to a pH of 7.5. The culture was concentrated 10× with a tangential flow filtration (TFF) apparatus using four Sartoslice TFF 30 kDa molecular weight cut-off (MWCO) polyether sulfone (PES) filter (Sartorious), followed by a 10× buffer exchange with 10 mM Tris, 20 mM Na 2 SO 4 , pH 7.5 into a 0.22 μm final filter into a 50 L sterile storage bag. 
     The concentrated, diafiltered harvest was inactivated for virus. Prior to viral inactivation, a solution of 10% Triton® X-100 detergent, and 3% tri (n-butyl) phosphate (TNBP) was prepared. The concentrated, diafiltered harvest was exposed to 1% Triton® X-100 detergent, and 0.3% TNBP for 1 hour in a 36 L glass reaction vessel immediately prior to purification on the Q column. 
     D. Purification of Gen2 soluble rHuPH20 
     A Q Sepharose (Pharmacia) ion exchange column (9 L resin, H=29 cm, D=20 cm) was prepared. Wash samples were collected for a determination of pH, conductivity and endotoxin (LAL assay). The column was equilibrated with 5 column volumes of 10 mM Tris, 20 mM Na 2 SO 4 , pH 7.5. Following viral inactivation, the concentrated, diafiltered harvest was loaded onto the Q column at a flow rate of 100 cm/hr. The column was washed with 5 column volumes of 10 mM Tris, 20 mM Na 2 SO 4 , pH 7.5 and 10 mM Hepes, 50 mM NaCl, pH7.0. The protein was eluted with 10 mM Hepes, 400 mM NaCl, pH 7.0 into a 0.22 μm final filter into sterile bag. The eluate sample was tested for bioburden, protein concentration and hyaluronidase activity. A 280  absorbance readings were taken at the beginning and end of the exchange. 
     Phenyl-Sepharose (Pharmacia) hydrophobic interaction chromatography was next performed. A Phenyl-Sepharose (PS) column (19-21 L resin, H=29 cm, D=30 cm) was prepared. The wash was collected and sampled for pH, conductivity and endotoxin (LAL assay). The column was equilibrated with 5 column volumes of 5 mM potassium phosphate, 0.5 M ammonium sulfate, and 0.1 mM CaCl 2 , pH 7.0. The protein eluate from the Q sepharose column was supplemented with 2M ammonium sulfate, 1 M potassium phosphate and 1 M CaCl 2  stock solutions to yield final concentrations of 5 mM, 0.5 M and 0.1 mM, respectively. The protein was loaded onto the PS column at a flow rate of 100 cm/hr and the column flow thru collected. The column was washed with 5 mM potassium phosphate, 0.5 M ammonium sulfate and 0.1 mM CaCl 2  pH 7.0 at 100 cm/hr and the wash was added to the collected flow thru. Combined with the column wash, the flow through was passed through a 0.22 μm final filter into a sterile bag. The flow through was sampled for bioburden, protein concentration and enzyme activity. 
     An aminophenyl boronate column (Prometics) was prepared. The wash was collected and sampled for pH, conductivity and endotoxin (LAL assay). The column was equilibrated with 5 column volumes of 5 mM potassium phosphate, 0.5 M ammonium sulfate. The PS flow through containing purified protein was loaded onto the aminophenyl boronate column at a flow rate of 100 cm/hr. The column was washed with 5 mM potassium phosphate, 0.5 M ammonium sulfate, pH 7.0. The column was washed with 20 mM bicine, 0.5 M ammonium sulfate, pH 9.0. The column was washed with 20 mM bicine, 100 mM sodium chloride, pH 9.0. The protein was eluted with 50 mM Hepes, 100 mM NaCl, pH 6.9 and passed through a sterile filter into a sterile bag. The eluted sample was tested for bioburden, protein concentration and enzyme activity. 
     The hydroxyapatite (HAP) column (Biorad) was prepared. The wash was collected and tested for pH, conductivity and endotoxin (LAL assay). The column was equilibrated with 5 mM potassium phosphate, 100 mM NaCl, 0.1 mM CaCl 2 , pH 7.0. The aminophenyl boronate purified protein was supplemented to final concentrations of 5 mM potassium phosphate and 0.1 mM CaCl 2  and loaded onto the HAP column at a flow rate of 100 cm/hr. The column was washed with 5 mM potassium phosphate, pH 7, 100 mM NaCl, 0.1 mM CaCl 2 . The column was next washed with 10 mM potassium phosphate, pH 7, 100 mM NaCl, 0.1 mM CaCl 2 . The protein was eluted with 70 mM potassium phosphate, pH 7.0 and passed through a 0.22 μm sterile filter into a sterile bag. The eluted sample was tested for bioburden, protein concentration and enzyme activity. 
     The HAP purified protein was then passed through a virus removal filter. The sterilized Viosart filter (Sartorius) was first prepared by washing with 2 L of 70 mM potassium phosphate, pH 7.0. Before use, the filtered buffer was sampled for pH and conductivity. The HAP purified protein was pumped via a peristaltic pump through the 20 nM virus removal filter. The filtered protein in 70 mM potassium phosphate, pH 7.0 was passed through a 0.22 μm final filter into a sterile bag. The filtered sample was tested for protein concentration, enzyme activity, oligosaccharide, monosaccharide and sialic acid profiling. The sample also was tested for process related impurities. 
     The protein in the filtrate was then concentrated to 10 mg/mL using a 10 kDa molecular weight cut off (MWCO) Sartocon Slice tangential flow filtration (TFF) system (Sartorius). The filter was first prepared by washing with 10 mM histidine, 130 mM NaCl, pH 6.0 and the permeate was sampled for pH and conductivity. Following concentration, the concentrated protein was sampled and tested for protein concentration and enzyme activity. A 6× buffer exchange was performed on the concentrated protein into the final buffer: 10 mM histidine, 130 mM NaCl, pH 6.0. Following buffer exchange, the concentrated protein was passed though a 0.22 μm filter into a 20 L sterile storage bag. The protein was sampled and tested for protein concentration, enzyme activity, free sulfydryl groups, oligosaccharide profiling and osmolality. Lot number WRS2 was used as a standard in the assays described below, the results showed that the test description for appearance was clear and colorless; the pH was 7.4; the endotoxin level was &lt;0.01 EU/mL; the osmolality was 308 mOsm/Kg; the density was 1.005 g/mL; the rHuPH20 content was 1.3 ppm; and the hyaluronidase activity was 145 USP U/mL. 
     The sterile filtered bulk protein was then asceptically dispensed at 20 mL into 30 mL sterile Teflon vials (Nalgene). The vials were then flash frozen and stored at −20±5° C. 
     Example 2 
     Generation of PH20 Mutant Library 
     A. Cloning and Mutagenesis 
     In this example, a human hyaluronidase PH20 library was created by cloning DNA encoding human PH20 into a plasmid followed by transfection and protein expression. 
     The library was created by mutagenesis of a PH20 template that is a codon optimized version of PH20 with an Ig Kappa leader sequence. Specifically, for generating the library of variants, the HZ24-PH20(OHO)-IRES-SEAP expression vector (set forth in SEQ ID NO:4) was used as a template, which contains the sequence of nucleotides encoding PH20 set forth in SEQ ID NO:1, which encodes a precursor PH20 set forth in SEQ ID NO:2 or a mature PH20 set forth in SEQ ID NO:3 lacking residues 1-22 corresponding to the IgK signal sequence. The backbone of the vector was derived from the original HZ24 vector containing the DHFR selection marker (see Example 1 and SEQ ID NO:5) with the addition of an IgK leader sequence and codon optimization. The expression vector also was modified to contain the gene for secreted alkaline phosphatase (SEAP). Hence, in addition to sequence encoding PH20, the HZ24-PH20(OHO)-IRES-SEAP expression vector also contains an internal ribosome entry site (EMCV IRES) that is linked to the coding sequence for the gene for secreted alkaline phosphatase (SEAP), and a single CMV promoter that drives expression of PH20 and SEAP in the construct. It also contains a gene for ampilcillin resistance. With reference to the sequence of nucleotides set forth in SEQ ID NO:4, the sequence of nucleotides encoding PH20 corresponds to nucleotides 1058-2464 (including the IgK leader sequence), the sequence of nucleotides encoding SEAP corresponds to nucleotides 2970-4529, and the ampicillin resistance gene corresponds to nucleotides 5778-6635. 
     The first library was made to generate encoded variant proteins wherein each of residues 23-469 of SEQ ID NO:2 (corresponding to residues 1-447 of SEQ ID NO:3 or residues 36-482 of SEQ ID NO:6) was changed to one of about 15 amino acid residues, such that each member contained a single amino change. The resulting library contained 6753 variant members, each containing a single amino acid mutation compared to residues 23-469 of SEQ ID NO:2 (corresponding to residues 1-447 of SEQ ID NO:3 or residues 36-482 of SEQ ID NO:6). Glycerol stocks of the resulting library were prepared and stored at −80° C. The amino acid replacements (mut) in each member are listed in Table 8 below, and correspond to amino acid replacements with reference to the sequence of amino acids of PH20 set forth in SEQ ID NO:3 (and SEQ ID NOs: 7 or 32-66, which are the mature sequence of PH20 or other C-terminally truncated fragments thereof). The corresponding mutated codons (cod) of each PH20 variant in the library are also listed in Table 8, and correspond to nucleotide residue changes in the corresponding encoding nucleotide for PH20 set forth as 1058-2464 of SEQ ID NO:4. Each member was expressed and screened for hyaluoridase activity as described below. 
     
       
         
           
               
             
               
                 TABLE 8 
               
             
            
               
                   
               
               
                 PH20 Variants 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                 mut 
                 cod 
                 mut 
                 cod 
                 mut 
                 cod 
                 mut 
                 cod 
                 mut 
                 cod 
                 mut 
                 cod 
               
               
                   
               
               
                 L001A 
                 GCG 
                 Y066S 
                 AGT 
                 R132N 
                 AAT 
                 G198T 
                 ACT 
                 V265G 
                 GGT 
                 I331K 
                 AAG 
               
               
                 L001C 
                 TGT 
                 Y066T 
                 ACG 
                 R132P 
                 CCT 
                 G198V 
                 GTT 
                 V265H 
                 CAT 
                 I331L 
                 CTG 
               
               
                 L001D 
                 GAT 
                 Y066V 
                 GTG 
                 R132Q 
                 CAG 
                 G198W 
                 TGG 
                 V265I 
                 ATT 
                 I331Q 
                 CAG 
               
               
                 L001E 
                 GAG 
                 I067C 
                 TGT 
                 R132S 
                 AGT 
                 G198Y 
                 TAT 
                 V265K 
                 AAG 
                 I331R 
                 CGT 
               
               
                 L001F 
                 TTT 
                 I067D 
                 GAT 
                 R132T 
                 ACT 
                 Y199A 
                 GCG 
                 V265L 
                 CTG 
                 I331S 
                 AGT 
               
               
                 L001G 
                 GGT 
                 I067E 
                 GAG 
                 R132V 
                 GTG 
                 Y199C 
                 TGT 
                 V265M 
                 ATG 
                 I331T 
                 ACT 
               
               
                 L001H 
                 CAT 
                 I067F 
                 TTT 
                 R132Y 
                 TAT 
                 Y199E 
                 GAG 
                 V265N 
                 AAT 
                 I331W 
                 TGG 
               
               
                 L001K 
                 AAG 
                 I067G 
                 GGG 
                 S133A 
                 GCT 
                 Y199G 
                 GGG 
                 V265P 
                 CCT 
                 I331Y 
                 TAT 
               
               
                 L001N 
                 AAT 
                 I067H 
                 CAT 
                 S133D 
                 GAT 
                 Y199H 
                 CAT 
                 V265Q 
                 CAG 
                 I332A 
                 GCT 
               
               
                 L001P 
                 CCG 
                 I067L 
                 TTG 
                 S133E 
                 GAG 
                 Y199I 
                 ATT 
                 V265R 
                 AGG 
                 I332C 
                 TGT 
               
               
                 L001Q 
                 CAG 
                 I067N 
                 AAT 
                 S133F 
                 TTT 
                 Y199K 
                 AAG 
                 V265S 
                 TCT 
                 I332D 
                 GAT 
               
               
                 L001R 
                 CGG 
                 I067P 
                 CCG 
                 S133G 
                 GGG 
                 Y199L 
                 CTT 
                 V265W 
                 TGG 
                 I332E 
                 GAG 
               
               
                 L001S 
                 TCT 
                 I067Q 
                 CAG 
                 S133H 
                 CAT 
                 Y199N 
                 AAT 
                 V265Y 
                 TAT 
                 I332F 
                 TTT 
               
               
                 L001T 
                 ACG 
                 I067R 
                 CGG 
                 S133I 
                 ATT 
                 Y199P 
                 CCT 
                 F266A 
                 GCG 
                 I332G 
                 GGT 
               
               
                 L001V 
                 GTG 
                 I067T 
                 ACG 
                 S133L 
                 CTG 
                 Y199Q 
                 CAG 
                 F266C 
                 TGT 
                 I332H 
                 CAT 
               
               
                 L001W 
                 TGG 
                 I067V 
                 GTT 
                 S133M 
                 ATG 
                 Y199R 
                 AGG 
                 F266D 
                 GAT 
                 I332K 
                 AAG 
               
               
                 N002A 
                 GCT 
                 I067W 
                 TGG 
                 S133N 
                 AAT 
                 Y199S 
                 TCG 
                 F266G 
                 GGG 
                 I332L 
                 CTG 
               
               
                 N002C 
                 TGT 
                 I067Y 
                 TAT 
                 S133P 
                 CCT 
                 Y199T 
                 ACG 
                 F266H 
                 CAT 
                 I332N 
                 AAT 
               
               
                 N002F 
                 TTT 
                 D068A 
                 GCT 
                 S133R 
                 CGG 
                 Y199W 
                 TGG 
                 F266L 
                 CTT 
                 I332P 
                 CCT 
               
               
                 N002G 
                 GGG 
                 D068C 
                 TGT 
                 S133T 
                 ACT 
                 N200A 
                 GCT 
                 F266M 
                 CCG 
                 I332R 
                 AGG 
               
               
                 N002H 
                 CAT 
                 D068E 
                 GAG 
                 S133V 
                 GTT 
                 N200D 
                 GAT 
                 F266P 
                 ATG 
                 I332S 
                 AGT 
               
               
                 N002I 
                 ATT 
                 D068G 
                 GGG 
                 S133W 
                 TGG 
                 N200F 
                 CAG 
                 F266Q 
                 CAG 
                 I332T 
                 ACT 
               
               
                 N002K 
                 AAG 
                 D068H 
                 CAC 
                 I134A 
                 GCT 
                 N200G 
                 GGT 
                 F266R 
                 CGG 
                 I332Y 
                 TAT 
               
               
                 N002L 
                 TTG 
                 D068I 
                 ATT 
                 I134C 
                 TGT 
                 N200H 
                 CAT 
                 F266S 
                 TCG 
                 N333A 
                 GCT 
               
               
                 N002P 
                 CCG 
                 D068K 
                 AAG 
                 I134D 
                 GAT 
                 N200K 
                 AAG 
                 F266T 
                 ACG 
                 N333E 
                 GAG 
               
               
                 N002Q 
                 CAG 
                 D068L 
                 TTG 
                 I134F 
                 TTT 
                 N200L 
                 CTG 
                 F266V 
                 GTG 
                 N333G 
                 GGT 
               
               
                 N002S 
                 AGT 
                 D068P 
                 CCT 
                 I134G 
                 GGG 
                 N200M 
                 ATG 
                 F266W 
                 TGG 
                 N333H 
                 CAT 
               
               
                 N002T 
                 ACG 
                 D068Q 
                 CAG 
                 I134H 
                 CAT 
                 N200P 
                 CCT 
                 F266Y 
                 TAT 
                 N333I 
                 ATT 
               
               
                 N002V 
                 GTT 
                 D068R 
                 CGG 
                 I134K 
                 AAG 
                 N200Q 
                 CAG 
                 A267D 
                 GAT 
                 N333K 
                 AAG 
               
               
                 N002W 
                 TGG 
                 D068S 
                 TCG 
                 I134L 
                 TTG 
                 N200R 
                 AGG 
                 A267E 
                 GAG 
                 N333L 
                 CTG 
               
               
                 N002Y 
                 TAT 
                 D068T 
                 ACT 
                 I134P 
                 CCT 
                 N200S 
                 TCT 
                 A267G 
                 GGT 
                 N333M 
                 ATG 
               
               
                 F003A 
                 GCT 
                 D068V 
                 GTG 
                 I134Q 
                 CAG 
                 N200T 
                 ACT 
                 A267H 
                 CAT 
                 N333P 
                 CCT 
               
               
                 F003E 
                 GAG 
                 D068Y 
                 TAT 
                 I134R 
                 CGT 
                 N200V 
                 GTG 
                 A267I 
                 ATT 
                 N333R 
                 CGG 
               
               
                 F003G 
                 GGG 
                 S069A 
                 GCT 
                 I134S 
                 TCG 
                 N200W 
                 TGG 
                 A267K 
                 AAG 
                 N333S 
                 AGT 
               
               
                 F003H 
                 CAT 
                 S069C 
                 TGT 
                 I134T 
                 ACT 
                 N200Y 
                 TAT 
                 A267L 
                 CTT 
                 N333T 
                 ACT 
               
               
                 F003I 
                 ATT 
                 S069E 
                 GAG 
                 I134V 
                 GTG 
                 G201A 
                 GCG 
                 A267M 
                 ATG 
                 N333V 
                 GTT 
               
               
                 F003K 
                 AAG 
                 S069F 
                 TTT 
                 I134W 
                 TGG 
                 G201E 
                 GAG 
                 A267N 
                 AAT 
                 N333W 
                 TGG 
               
               
                 F003L 
                 TTG 
                 S069G 
                 GGG 
                 E135A 
                 GCT 
                 G201F 
                 TTT 
                 A267P 
                 CCG 
                 N333Y 
                 TAT 
               
               
                 F003M 
                 ATG 
                 S069I 
                 ATT 
                 E135C 
                 TGT 
                 G201H 
                 CAT 
                 A267R 
                 AGG 
                 V334A 
                 GCT 
               
               
                 F003N 
                 AAT 
                 S069L 
                 CTT 
                 E135D 
                 GAT 
                 G201K 
                 AAG 
                 A267S 
                 TCT 
                 V334C 
                 TGT 
               
               
                 F003P 
                 CCT 
                 S069M 
                 ATG 
                 E135F 
                 TTT 
                 G201L 
                 CTT 
                 A267T 
                 GTG 
                 V334D 
                 GAT 
               
               
                 F003R 
                 CGT 
                 S069N 
                 AAT 
                 E135G 
                 GGG 
                 G201M 
                 ATG 
                 A267V 
                 ACT 
                 V334E 
                 GAG 
               
               
                 F003S 
                 TCG 
                 S069P 
                 CCT 
                 E135H 
                 CAT 
                 G201N 
                 AAT 
                 A267W 
                 TGG 
                 V334G 
                 GGG 
               
               
                 F003T 
                 ACT 
                 S069R 
                 CGT 
                 E135K 
                 AAG 
                 G201P 
                 CCT 
                 Y268A 
                 GCT 
                 V334H 
                 CAT 
               
               
                 F003V 
                 GTG 
                 S069T 
                 ACG 
                 E135L 
                 TTG 
                 G201Q 
                 CAG 
                 Y268C 
                 TGT 
                 V334L 
                 TTG 
               
               
                 F003Y 
                 TAT 
                 S069V 
                 GTT 
                 E135N 
                 AAT 
                 G201R 
                 CGT 
                 Y268F 
                 TTT 
                 V334M 
                 ATG 
               
               
                 R004A 
                 GCG 
                 S069W 
                 TGG 
                 E135P 
                 CCT 
                 G201S 
                 TCG 
                 Y268G 
                 GGG 
                 V334N 
                 AAT 
               
               
                 R004D 
                 GAT 
                 S069Y 
                 TAT 
                 E135Q 
                 CAG 
                 G201T 
                 ACG 
                 Y268H 
                 CAT 
                 V334P 
                 CCT 
               
               
                 R004E 
                 GAG 
                 I070A 
                 GCT 
                 E135R 
                 CGG 
                 G201V 
                 GTG 
                 Y268K 
                 AAG 
                 V334Q 
                 CAG 
               
               
                 R004F 
                 TTT 
                 I070C 
                 TGT 
                 E135S 
                 TCT 
                 G201W 
                 TGG 
                 Y268L 
                 CTT 
                 V334R 
                 AGG 
               
               
                 R004G 
                 GGG 
                 I070F 
                 TTT 
                 E135W 
                 TGG 
                 S202A 
                 GCG 
                 Y268N 
                 AAT 
                 V334S 
                 TCT 
               
               
                 R004I 
                 ATT 
                 I070G 
                 GGG 
                 E135Y 
                 TAT 
                 S202E 
                 GAG 
                 Y268P 
                 CCT 
                 V334T 
                 ACT 
               
               
                 R004L 
                 TTG 
                 I070H 
                 CAT 
                 L136A 
                 GCT 
                 S202F 
                 TTT 
                 Y268Q 
                 CAG 
                 V334Y 
                 TAT 
               
               
                 R004M 
                 ATG 
                 I070K 
                 AAG 
                 L136C 
                 TGT 
                 S202G 
                 GGT 
                 Y268R 
                 CGT 
                 T335A 
                 GCT 
               
               
                 R004N 
                 AAT 
                 I070L 
                 TTG 
                 L136D 
                 GAT 
                 S202H 
                 CAT 
                 Y268S 
                 TCG 
                 T335C 
                 TGT 
               
               
                 R004P 
                 CCT 
                 I070N 
                 AAT 
                 L136F 
                 TTT 
                 S202K 
                 AAG 
                 Y268T 
                 ACT 
                 T335F 
                 TTT 
               
               
                 R004S 
                 TCT 
                 I070P 
                 CCG 
                 L136G 
                 GGT 
                 S202M 
                 ATG 
                 Y268V 
                 GTG 
                 T335G 
                 GGT 
               
               
                 R004T 
                 ACG 
                 I070Q 
                 CAG 
                 L136H 
                 CAT 
                 S202N 
                 AAT 
                 Y268W 
                 TGG 
                 T335H 
                 CAT 
               
               
                 R004V 
                 GTG 
                 I070R 
                 CGT 
                 L136I 
                 ATT 
                 S202P 
                 CCT 
                 T269A 
                 GCT 
                 T335I 
                 ATT 
               
               
                 R004W 
                 TGG 
                 I070S 
                 TCT 
                 L136M 
                 ATG 
                 S202Q 
                 CAG 
                 T269C 
                 TGT 
                 T335K 
                 AAG 
               
               
                 R004Y 
                 TAT 
                 I070T 
                 ACT 
                 L136N 
                 AAT 
                 S202R 
                 CGT 
                 T269D 
                 GAT 
                 T335L 
                 TTG 
               
               
                 A005D 
                 GAT 
                 I070V 
                 GTT 
                 L136P 
                 CCT 
                 S202T 
                 ACG 
                 T269E 
                 GAG 
                 T335N 
                 AAT 
               
               
                 A005G 
                 GGG 
                 I070Y 
                 TAT 
                 L136Q 
                 CAG 
                 S202V 
                 GTT 
                 T269G 
                 GGT 
                 T335P 
                 CCT 
               
               
                 A005H 
                 CAT 
                 T071A 
                 GCT 
                 L136R 
                 CGT 
                 S202W 
                 TGG 
                 T269K 
                 AAG 
                 T335Q 
                 CAG 
               
               
                 A005I 
                 ATT 
                 T071C 
                 TGT 
                 L136S 
                 TCG 
                 S202Y 
                 TAT 
                 T269L 
                 CTG 
                 T335S 
                 TCT 
               
               
                 A005L 
                 CTT 
                 T071D 
                 GAT 
                 L136T 
                 ACT 
                 C203A 
                 GCG 
                 T269M 
                 ATG 
                 T335V 
                 GTG 
               
               
                 A005M 
                 ATG 
                 T071E 
                 GAG 
                 L136W 
                 TGG 
                 C203D 
                 GAT 
                 T269N 
                 AAT 
                 T335W 
                 TGG 
               
               
                 A005N 
                 AAT 
                 T071G 
                 GGG 
                 V137A 
                 GCT 
                 C203E 
                 GAG 
                 T269P 
                 CCG 
                 T335Y 
                 TAT 
               
               
                 A005P 
                 CCG 
                 T071H 
                 CAT 
                 V137C 
                 TGT 
                 C203G 
                 GGG 
                 T269Q 
                 CAG 
                 L336A 
                 GCT 
               
               
                 A005Q 
                 CAG 
                 T071L 
                 TTG 
                 V137E 
                 GAG 
                 C203H 
                 CAT 
                 T269R 
                 AGG 
                 L336E 
                 GAG 
               
               
                 A005R 
                 AGG 
                 T071M 
                 ATG 
                 V137F 
                 TTT 
                 C203L 
                 CTT 
                 T269S 
                 TCG 
                 L336F 
                 TTT 
               
               
                 A005S 
                 TCG 
                 T071N 
                 AAT 
                 V137G 
                 GGG 
                 C203M 
                 ATG 
                 T269V 
                 GTG 
                 L336G 
                 GGG 
               
               
                 A005T 
                 ACG 
                 T071P 
                 CCT 
                 V137H 
                 CAT 
                 C203N 
                 AAT 
                 T269Y 
                 TAT 
                 L336H 
                 CAT 
               
               
                 A005V 
                 GTG 
                 T071Q 
                 CAG 
                 V137I 
                 ATT 
                 C203P 
                 CCG 
                 R270A 
                 GCT 
                 L336K 
                 AAG 
               
               
                 A005W 
                 TGG 
                 T071R 
                 CGG 
                 V137L 
                 TTG 
                 C203Q 
                 CAG 
                 R270C 
                 TGT 
                 L336M 
                 ATG 
               
               
                 A005Y 
                 TAT 
                 T071S 
                 TCG 
                 V137N 
                 AAT 
                 C203R 
                 AGG 
                 R270D 
                 GAT 
                 L336N 
                 AAT 
               
               
                 P006A 
                 GCG 
                 T071V 
                 GTG 
                 V137P 
                 CCT 
                 C203S 
                 AGT 
                 R270E 
                 GAG 
                 L336P 
                 CCT 
               
               
                 P006D 
                 GAT 
                 T071Y 
                 TAT 
                 V137Q 
                 CAG 
                 C203T 
                 ACT 
                 R270F 
                 TTT 
                 L336R 
                 AGG 
               
               
                 P006E 
                 GAG 
                 G072A 
                 GCT 
                 V137R 
                 CGT 
                 C203V 
                 GTG 
                 R270G 
                 GGG 
                 L336S 
                 TCT 
               
               
                 P006F 
                 TTT 
                 G072C 
                 TGT 
                 V137S 
                 TCT 
                 C203W 
                 TGG 
                 R270H 
                 CAT 
                 L336T 
                 ACT 
               
               
                 P006G 
                 GGG 
                 G072D 
                 GAT 
                 V137T 
                 ACT 
                 F204A 
                 GCG 
                 R270I 
                 ATT 
                 L336V 
                 GTG 
               
               
                 P006H 
                 CAT 
                 G072E 
                 GAG 
                 V137W 
                 TGG 
                 F204C 
                 TGT 
                 R270M 
                 ATG 
                 L336W 
                 TGG 
               
               
                 P006K 
                 AAG 
                 G072F 
                 TTT 
                 V137Y 
                 TAT 
                 F204E 
                 GAG 
                 R270N 
                 AAT 
                 L336Y 
                 TAT 
               
               
                 P006L 
                 CTT 
                 G072H 
                 CAT 
                 Q138A 
                 GCT 
                 F204G 
                 GGG 
                 R270P 
                 CCT 
                 A337C 
                 TGT 
               
               
                 P006N 
                 AAT 
                 G072I 
                 ATT 
                 Q138C 
                 TGT 
                 F204H 
                 CAT 
                 R270Q 
                 CAG 
                 A337F 
                 TTT 
               
               
                 P006Q 
                 CAG 
                 G072K 
                 AAG 
                 Q138E 
                 GAG 
                 F204I 
                 ATT 
                 R270S 
                 TCG 
                 A337G 
                 GGG 
               
               
                 P006R 
                 AGG 
                 G072L 
                 TTG 
                 Q138F 
                 TTT 
                 F204K 
                 AAG 
                 R270T 
                 ACT 
                 A337H 
                 CAT 
               
               
                 P006S 
                 AGT 
                 G072M 
                 ATG 
                 Q138G 
                 GGG 
                 F204L 
                 CTT 
                 R270V 
                 GTG 
                 A337I 
                 ATT 
               
               
                 P006T 
                 ACG 
                 G072P 
                 CCT 
                 Q138H 
                 CAT 
                 F204M 
                 ATG 
                 R270Y 
                 TAT 
                 A337K 
                 AAG 
               
               
                 P006V 
                 GTG 
                 G072Q 
                 CAG 
                 Q138I 
                 ATT 
                 F204P 
                 CCT 
                 I271A 
                 GCT 
                 A337L 
                 TTG 
               
               
                 P006W 
                 TGG 
                 G072R 
                 CGG 
                 Q138L 
                 TTG 
                 F204Q 
                 CAG 
                 I271D 
                 GAT 
                 A337M 
                 ATG 
               
               
                 P006Y 
                 TAT 
                 G072S 
                 TCT 
                 Q138M 
                 ATG 
                 F204R 
                 AGG 
                 I271E 
                 GAG 
                 A337N 
                 AAT 
               
               
                 P007A 
                 GCT 
                 G072T 
                 ACT 
                 Q138N 
                 AAT 
                 F204S 
                 AGT 
                 I271F 
                 TTT 
                 A337P 
                 CCT 
               
               
                 P007C 
                 TGT 
                 G072V 
                 GTG 
                 Q138R 
                 CGT 
                 F204T 
                 ACT 
                 I271G 
                 GGG 
                 A337R 
                 CGG 
               
               
                 P007D 
                 GAT 
                 G072W 
                 TGG 
                 Q138S 
                 AGT 
                 F204V 
                 GTG 
                 I271H 
                 CAT 
                 A337S 
                 TCT 
               
               
                 P007F 
                 TTT 
                 G072Y 
                 TAT 
                 Q138V 
                 GTT 
                 F204W 
                 TGG 
                 I271K 
                 AAG 
                 A337T 
                 ACT 
               
               
                 P007G 
                 GGT 
                 V073A 
                 GCG 
                 Q138W 
                 TGG 
                 N205A 
                 GCG 
                 I271L 
                 CTT 
                 A337V 
                 GTT 
               
               
                 P007H 
                 CAT 
                 V073C 
                 TGT 
                 Q138Y 
                 TAT 
                 N205D 
                 GAT 
                 I271M 
                 ATG 
                 A337W 
                 TGG 
               
               
                 P007I 
                 ATT 
                 V073D 
                 GAT 
                 Q139A 
                 GCT 
                 N205E 
                 GAG 
                 I271P 
                 CCT 
                 A338C 
                 TGT 
               
               
                 P007K 
                 AAG 
                 V073F 
                 TTT 
                 Q139C 
                 TGT 
                 N205F 
                 TTT 
                 I271R 
                 AGG 
                 A338D 
                 GAT 
               
               
                 P007L 
                 TTG 
                 V073G 
                 GGG 
                 Q139D 
                 GAT 
                 N205G 
                 GGG 
                 I271S 
                 AGT 
                 A338E 
                 GAG 
               
               
                 P007M 
                 ATG 
                 V073H 
                 CAT 
                 Q139E 
                 GAG 
                 N205K 
                 AAG 
                 I271T 
                 ACT 
                 A338F 
                 TTT 
               
               
                 P007Q 
                 CAG 
                 V073K 
                 AAG 
                 Q139F 
                 TTT 
                 N205L 
                 CTG 
                 I271V 
                 GTT 
                 A338G 
                 GGG 
               
               
                 P007R 
                 CGG 
                 V073L 
                 CTT 
                 Q139G 
                 GGG 
                 N205M 
                 ATG 
                 I271W 
                 TGG 
                 A338H 
                 CAT 
               
               
                 P007S 
                 AGT 
                 V073M 
                 ATG 
                 Q139H 
                 CAT 
                 N205P 
                 CCT 
                 V272A 
                 GCT 
                 A338I 
                 ATT 
               
               
                 P007T 
                 ACT 
                 V073P 
                 CCG 
                 Q139K 
                 AAG 
                 N205R 
                 AGG 
                 V272C 
                 TGT 
                 A338K 
                 AAG 
               
               
                 P007V 
                 GTG 
                 V073Q 
                 CAG 
                 Q139L 
                 CTG 
                 N205S 
                 TCG 
                 V272D 
                 GAT 
                 A338L 
                 CTT 
               
               
                 P007W 
                 TGG 
                 V073R 
                 TGG 
                 Q139M 
                 ATG 
                 N205T 
                 ACG 
                 V272E 
                 GAG 
                 A338P 
                 CCT 
               
               
                 P007Y 
                 TAT 
                 V073S 
                 TCG 
                 Q139P 
                 CCT 
                 N205V 
                 GTG 
                 V272G 
                 GGG 
                 A338Q 
                 CAG 
               
               
                 V008A 
                 GCT 
                 V073T 
                 ACG 
                 Q139R 
                 CGT 
                 N205W 
                 TGG 
                 V272H 
                 CAT 
                 A338R 
                 CGT 
               
               
                 V008D 
                 GAT 
                 V073W 
                 CGG 
                 Q139S 
                 TCT 
                 N205Y 
                 TAT 
                 V272K 
                 AAG 
                 A338S 
                 TCG 
               
               
                 V008E 
                 GAG 
                 T074A 
                 GCT 
                 Q139T 
                 ACT 
                 V206C 
                 TGT 
                 V272L 
                 TTG 
                 A338T 
                 ACT 
               
               
                 V008G 
                 GGT 
                 T074C 
                 TGT 
                 Q139V 
                 GTG 
                 V206D 
                 GAT 
                 V272M 
                 ATG 
                 A338V 
                 GTG 
               
               
                 V008H 
                 CAT 
                 T074E 
                 GAG 
                 Q140A 
                 GCT 
                 V206F 
                 TTT 
                 V272N 
                 AAT 
                 K339D 
                 GAT 
               
               
                 V008I 
                 ATT 
                 T074F 
                 TTT 
                 Q140C 
                 TGT 
                 V206G 
                 GGG 
                 V272P 
                 CCT 
                 K339E 
                 GAG 
               
               
                 V008L 
                 TTG 
                 T074G 
                 GGT 
                 Q140D 
                 GAT 
                 V206H 
                 CAT 
                 V272R 
                 AGG 
                 K339F 
                 TTT 
               
               
                 V008M 
                 ATG 
                 T074H 
                 CAT 
                 Q140F 
                 TTT 
                 V206I 
                 ATT 
                 V272S 
                 TCG 
                 K339G 
                 GGG 
               
               
                 V008N 
                 AAT 
                 T074K 
                 AAG 
                 Q140G 
                 GGG 
                 V206K 
                 AAG 
                 V272T 
                 ACT 
                 K339H 
                 CAT 
               
               
                 V008P 
                 CCT 
                 T074L 
                 TTG 
                 Q140H 
                 CAT 
                 V206L 
                 CTT 
                 V272W 
                 TGG 
                 K339L 
                 CTG 
               
               
                 V008Q 
                 CAG 
                 T074M 
                 ATG 
                 Q140I 
                 ATT 
                 V206M 
                 ATG 
                 F273A 
                 GCT 
                 K339M 
                 ATG 
               
               
                 V008R 
                 CGG 
                 T074N 
                 AAT 
                 Q140K 
                 AAG 
                 V206P 
                 CCG 
                 F273C 
                 TGT 
                 K339N 
                 AAT 
               
               
                 V008S 
                 TCT 
                 T074P 
                 CCG 
                 Q140L 
                 TTG 
                 V206Q 
                 CAG 
                 F273D 
                 GAT 
                 K339P 
                 CCT 
               
               
                 V008T 
                 ACT 
                 T074R 
                 CGG 
                 Q140M 
                 ATG 
                 V206R 
                 CGG 
                 F273G 
                 GGG 
                 K339R 
                 CGG 
               
               
                 V008W 
                 TGG 
                 T074S 
                 TCG 
                 Q140R 
                 CGG 
                 V206S 
                 TCT 
                 F273H 
                 CAT 
                 K339S 
                 AGT 
               
               
                 I009A 
                 GCT 
                 T074V 
                 GTG 
                 Q140S 
                 AGT 
                 V206T 
                 ACG 
                 F273I 
                 ATT 
                 K339T 
                 ACT 
               
               
                 I009C 
                 TGT 
                 T074W 
                 TGG 
                 Q140V 
                 GTG 
                 V206Y 
                 TAT 
                 F273L 
                 CTG 
                 K339V 
                 GTT 
               
               
                 I009D 
                 GAT 
                 V075A 
                 GCG 
                 Q140W 
                 TGG 
                 E207A 
                 GCT 
                 F273P 
                 CCT 
                 K339W 
                 TGG 
               
               
                 I009E 
                 GAG 
                 V075C 
                 TGT 
                 Q140Y 
                 TAT 
                 E207F 
                 TTT 
                 F273Q 
                 CAG 
                 K339Y 
                 TAT 
               
               
                 I009G 
                 GGG 
                 V075D 
                 GAT 
                 N141A 
                 GCT 
                 E207G 
                 GGG 
                 F273R 
                 CGG 
                 M340A 
                 GCT 
               
               
                 I009H 
                 CAT 
                 V075F 
                 TTT 
                 N141D 
                 GAT 
                 E207H 
                 CAT 
                 F273S 
                 TCG 
                 M340C 
                 TGT 
               
               
                 I009K 
                 AAG 
                 V075G 
                 GGG 
                 N141E 
                 GAG 
                 E207I 
                 ATT 
                 F273T 
                 ACG 
                 M340D 
                 GAT 
               
               
                 I009L 
                 CTT 
                 V075H 
                 CAT 
                 N141F 
                 TTT 
                 E207K 
                 AAG 
                 F273V 
                 GTT 
                 M340E 
                 GAG 
               
               
                 I009N 
                 AAT 
                 V075L 
                 CTT 
                 N141G 
                 GGT 
                 E207L 
                 TTG 
                 F273W 
                 TGG 
                 M340F 
                 TTT 
               
               
                 I009P 
                 CCT 
                 V075M 
                 ATG 
                 N141H 
                 CAT 
                 E207M 
                 ATG 
                 F273Y 
                 TAT 
                 M340G 
                 GGG 
               
               
                 I009Q 
                 CAG 
                 V075N 
                 AAT 
                 N141L 
                 TTG 
                 E207P 
                 CCG 
                 T274A 
                 GCG 
                 M340H 
                 CAT 
               
               
                 I009R 
                 CGG 
                 V075P 
                 CCG 
                 N141M 
                 ATG 
                 E207Q 
                 CAG 
                 T274C 
                 TGT 
                 M340K 
                 AAG 
               
               
                 I009S 
                 AGT 
                 V075Q 
                 CAG 
                 N141P 
                 CCT 
                 E207R 
                 AGG 
                 T274E 
                 GAG 
                 M340L 
                 CTG 
               
               
                 I009T 
                 ACG 
                 V075R 
                 CGT 
                 N141Q 
                 CAG 
                 E207S 
                 TCT 
                 T274F 
                 ATG 
                 M340P 
                 CCT 
               
               
                 I009V 
                 GTT 
                 V075S 
                 TCT 
                 N141R 
                 CGT 
                 E207T 
                 ACG 
                 T274G 
                 GGG 
                 M340R 
                 CGG 
               
               
                 P010D 
                 GAT 
                 V075T 
                 ACT 
                 N141S 
                 TCT 
                 E207V 
                 GTT 
                 T274H 
                 CAT 
                 M340S 
                 TCG 
               
               
                 P010E 
                 GAG 
                 V075W 
                 TGG 
                 N141T 
                 ACT 
                 E207W 
                 TGG 
                 T274L 
                 CTG 
                 M340T 
                 ACT 
               
               
                 P010F 
                 TTT 
                 V075Y 
                 TAT 
                 N141V 
                 GTT 
                 I208A 
                 GCT 
                 T274N 
                 AAT 
                 M340V 
                 GTG 
               
               
                 P010G 
                 GGT 
                 N076A 
                 GCT 
                 N141W 
                 TGG 
                 I208C 
                 TGT 
                 T274P 
                 CCT 
                 M340W 
                 TGG 
               
               
                 P010H 
                 CAT 
                 N076C 
                 TGT 
                 N141Y 
                 TAT 
                 I208D 
                 GAT 
                 T274Q 
                 CAG 
                 C341A 
                 GCT 
               
               
                 P010I 
                 ATT 
                 N076D 
                 GAT 
                 V142C 
                 TGT 
                 I208E 
                 GAG 
                 T274R 
                 CGT 
                 C341E 
                 GAG 
               
               
                 P010L 
                 CTT 
                 N076F 
                 TTT 
                 V142D 
                 GAT 
                 I208G 
                 GGG 
                 T274S 
                 AGT 
                 C341G 
                 GGG 
               
               
                 P010M 
                 ATG 
                 N076G 
                 GGG 
                 V142E 
                 GAG 
                 I208K 
                 AAG 
                 T274V 
                 GTT 
                 C341H 
                 CAT 
               
               
                 P010N 
                 AAT 
                 N076I 
                 ATT 
                 V142G 
                 GGG 
                 I208L 
                 TTG 
                 T274W 
                 TGG 
                 C341K 
                 AAG 
               
               
                 P010Q 
                 CAG 
                 N076K 
                 AAG 
                 V142H 
                 CAT 
                 I208M 
                 ATG 
                 T274Y 
                 TAT 
                 C341L 
                 TTG 
               
               
                 P010R 
                 CGG 
                 N076L 
                 CTG 
                 V142I 
                 ATT 
                 I208P 
                 CCG 
                 D275A 
                 GCT 
                 C341M 
                 ATG 
               
               
                 P010S 
                 TCG 
                 N076P 
                 CCT 
                 V142K 
                 AAG 
                 I208Q 
                 CAG 
                 D275C 
                 TGT 
                 C341N 
                 AAT 
               
               
                 P010T 
                 ACT 
                 N076Q 
                 CAG 
                 V142L 
                 TTG 
                 I208R 
                 CGT 
                 D275E 
                 GAG 
                 C341Q 
                 CAG 
               
               
                 P010W 
                 TGG 
                 N076R 
                 CGT 
                 V142M 
                 ATG 
                 I208S 
                 AGT 
                 D275F 
                 TTT 
                 C341R 
                 AGG 
               
               
                 P010Y 
                 TAT 
                 N076S 
                 AGT 
                 V142N 
                 AAT 
                 I208T 
                 ACG 
                 D275G 
                 GGG 
                 C341S 
                 TCT 
               
               
                 N011A 
                 GCG 
                 N076T 
                 ACT 
                 V142P 
                 CCT 
                 I208V 
                 GTG 
                 D275I 
                 ATT 
                 C341T 
                 ACT 
               
               
                 N011C 
                 TGT 
                 N076V 
                 GTT 
                 V142Q 
                 CAG 
                 I208W 
                 TGG 
                 D275K 
                 AAG 
                 C341V 
                 GTT 
               
               
                 N011D 
                 GAT 
                 N076W 
                 TGG 
                 V142R 
                 CGG 
                 K209A 
                 GCG 
                 D275L 
                 CTT 
                 C341W 
                 TGG 
               
               
                 N011E 
                 GAG 
                 G077D 
                 GAT 
                 V142S 
                 AGT 
                 K209C 
                 TGT 
                 D275M 
                 ATG 
                 C341Y 
                 TAT 
               
               
                 N011F 
                 TTT 
                 G077E 
                 GAG 
                 V142T 
                 ACT 
                 K209D 
                 GAT 
                 D275Q 
                 CAG 
                 S342A 
                 GCT 
               
               
                 N011G 
                 GGG 
                 G077F 
                 TTT 
                 Q143C 
                 TGT 
                 K209E 
                 GAG 
                 D275R 
                 CGT 
                 S342D 
                 GAT 
               
               
                 N011H 
                 CAT 
                 G077H 
                 CAT 
                 Q143E 
                 GAG 
                 K209F 
                 TTT 
                 D275S 
                 TCG 
                 S342E 
                 GAG 
               
               
                 N011I 
                 ATT 
                 G077K 
                 AAG 
                 Q143F 
                 TTT 
                 K209G 
                 GGT 
                 D275T 
                 ACT 
                 S342F 
                 TTT 
               
               
                 N011K 
                 AAG 
                 G077L 
                 TTG 
                 Q143G 
                 GGG 
                 K209L 
                 CTG 
                 D275V 
                 GTG 
                 S342G 
                 GGG 
               
               
                 N011L 
                 CTG 
                 G077M 
                 ATG 
                 Q143H 
                 CAT 
                 K209N 
                 AAT 
                 D275W 
                 TGG 
                 S342H 
                 CAT 
               
               
                 N011P 
                 CCG 
                 G077N 
                 AAT 
                 Q143I 
                 ATT 
                 K209P 
                 CCG 
                 Q276C 
                 TGT 
                 S342I 
                 ATT 
               
               
                 N011S 
                 TCG 
                 G077P 
                 CCG 
                 Q143K 
                 AAG 
                 K209R 
                 CGG 
                 Q276D 
                 GAT 
                 S342K 
                 AAG 
               
               
                 N011T 
                 ACG 
                 G077Q 
                 CAG 
                 Q143L 
                 TTG 
                 K209S 
                 AGT 
                 Q276E 
                 GAG 
                 S342L 
                 TTG 
               
               
                 N011W 
                 TGG 
                 G077R 
                 CGT 
                 Q143M 
                 ATG 
                 K209T 
                 ACT 
                 Q276F 
                 TTT 
                 S342M 
                 ATG 
               
               
                 N011Y 
                 TAT 
                 G077S 
                 TCG 
                 Q143N 
                 AAT 
                 K209V 
                 GTT 
                 Q276G 
                 GGG 
                 S342P 
                 CCT 
               
               
                 V012A 
                 GCT 
                 G077T 
                 ACG 
                 Q143P 
                 CCT 
                 K209W 
                 TGG 
                 Q276H 
                 CAT 
                 S342Q 
                 CAG 
               
               
                 V012D 
                 GAT 
                 G077V 
                 GTG 
                 Q143R 
                 CGG 
                 K209Y 
                 TAT 
                 Q276I 
                 ATT 
                 S342R 
                 CGG 
               
               
                 V012E 
                 GAG 
                 G077Y 
                 TAT 
                 Q143S 
                 TCG 
                 R210A 
                 GCG 
                 Q276L 
                 CTT 
                 S342T 
                 ACT 
               
               
                 V012G 
                 GGG 
                 G078A 
                 GCG 
                 Q143T 
                 ACT 
                 R210C 
                 TGT 
                 Q276M 
                 ATG 
                 S342Y 
                 TAT 
               
               
                 V012H 
                 CAT 
                 G078C 
                 TGT 
                 Q143V 
                 GTG 
                 R210D 
                 GAT 
                 Q276P 
                 CCT 
                 Q343C 
                 TGT 
               
               
                 V012I 
                 ATT 
                 G078D 
                 GAT 
                 Q143Y 
                 TAT 
                 R210E 
                 GAG 
                 Q276R 
                 CGT 
                 Q343D 
                 GAT 
               
               
                 V012K 
                 AAG 
                 G078H 
                 CAT 
                 L144A 
                 GCT 
                 R210G 
                 GGT 
                 Q276S 
                 AGT 
                 Q343E 
                 GAG 
               
               
                 V012L 
                 CTT 
                 G078I 
                 ATT 
                 L144E 
                 GAG 
                 R210K 
                 AAG 
                 Q276V 
                 GTT 
                 Q343F 
                 TTT 
               
               
                 V012M 
                 ATG 
                 G078K 
                 AAG 
                 L144F 
                 TTT 
                 R210L 
                 CTG 
                 Q276W 
                 TGG 
                 Q343G 
                 GGG 
               
               
                 V012N 
                 AAT 
                 G078L 
                 TTG 
                 L144G 
                 GGG 
                 R210M 
                 ATG 
                 Q276Y 
                 TAT 
                 Q343I 
                 ATT 
               
               
                 V012P 
                 CCG 
                 G078M 
                 ATG 
                 L144I 
                 ATT 
                 R210N 
                 AAT 
                 V277A 
                 GCT 
                 Q343L 
                 CTT 
               
               
                 V012R 
                 AGG 
                 G078P 
                 CCG 
                 L144K 
                 AAG 
                 R210P 
                 CCT 
                 V277C 
                 TGT 
                 Q343M 
                 ATG 
               
               
                 V012S 
                 TCG 
                 G078Q 
                 CAG 
                 L144N 
                 AAT 
                 R210S 
                 TCG 
                 V277D 
                 GAT 
                 Q343P 
                 CCT 
               
               
                 V012T 
                 ACT 
                 G078R 
                 AGG 
                 L144P 
                 CCT 
                 R210T 
                 ACT 
                 V277E 
                 GAG 
                 Q343R 
                 AGG 
               
               
                 V012W 
                 TGG 
                 G078S 
                 TCG 
                 L144Q 
                 CAG 
                 R210V 
                 GTG 
                 V277G 
                 GGG 
                 Q343S 
                 AGT 
               
               
                 P013A 
                 GCT 
                 G078T 
                 ACT 
                 L144R 
                 CGT 
                 R210W 
                 TGG 
                 V277H 
                 CAT 
                 Q343T 
                 ACT 
               
               
                 P013E 
                 GAG 
                 G078V 
                 GTG 
                 L144S 
                 TCT 
                 R210Y 
                 TAT 
                 V277K 
                 AAG 
                 Q343V 
                 GTG 
               
               
                 P013F 
                 TTT 
                 G078Y 
                 TAT 
                 L144T 
                 ACT 
                 N211A 
                 GCG 
                 V277L 
                 TTG 
                 Q343W 
                 TGG 
               
               
                 P013G 
                 GGG 
                 I079A 
                 GCT 
                 L144V 
                 GTT 
                 N211C 
                 TGT 
                 V277M 
                 ATG 
                 Q343Y 
                 TAT 
               
               
                 P013H 
                 CAT 
                 I079D 
                 GAT 
                 L144W 
                 TGG 
                 N211F 
                 TTT 
                 V277N 
                 AAT 
                 V344E 
                 GAG 
               
               
                 P013I 
                 ATT 
                 I079F 
                 TTT 
                 L144Y 
                 TAT 
                 N211G 
                 GGG 
                 V277Q 
                 CAG 
                 V344F 
                 TTT 
               
               
                 P013L 
                 CTT 
                 I079G 
                 GGG 
                 S145A 
                 GCT 
                 N211H 
                 CAT 
                 V277R 
                 AGG 
                 V344G 
                 GGG 
               
               
                 P013M 
                 ATG 
                 I079H 
                 CAT 
                 S145C 
                 TGT 
                 N211I 
                 ATT 
                 V277S 
                 TCT 
                 V344H 
                 CAT 
               
               
                 P013Q 
                 CAG 
                 I079K 
                 AAG 
                 S145D 
                 GAT 
                 N211K 
                 AAG 
                 V277T 
                 ACT 
                 V344I 
                 ATT 
               
               
                 P013R 
                 CGT 
                 I079L 
                 TTG 
                 S145E 
                 GAG 
                 N211L 
                 CTG 
                 V277Y 
                 TAT 
                 V344L 
                 CTG 
               
               
                 P013S 
                 TCG 
                 I079N 
                 AAT 
                 S145F 
                 TTT 
                 N211M 
                 ATG 
                 L278A 
                 GCT 
                 V344M 
                 ATG 
               
               
                 P013T 
                 ACT 
                 I079P 
                 CCG 
                 S145G 
                 GGG 
                 N211P 
                 CCT 
                 L278E 
                 GAG 
                 V344N 
                 AAT 
               
               
                 P013V 
                 GTG 
                 I079R 
                 CGT 
                 S145H 
                 CAT 
                 N211R 
                 CGG 
                 L278F 
                 TTT 
                 V344P 
                 CCT 
               
               
                 P013W 
                 TGG 
                 I079S 
                 AGT 
                 S145L 
                 TTG 
                 N211S 
                 AGT 
                 L278G 
                 GGG 
                 V344Q 
                 CAG 
               
               
                 P013Y 
                 TAT 
                 I079T 
                 ACT 
                 S145M 
                 ATG 
                 N211T 
                 ACT 
                 L278H 
                 CAT 
                 V344R 
                 CGT 
               
               
                 F014A 
                 GCG 
                 I079V 
                 GTT 
                 S145N 
                 AAT 
                 N211V 
                 GTT 
                 L278I 
                 ATT 
                 V344S 
                 TCG 
               
               
                 F014D 
                 GAT 
                 I079W 
                 TGG 
                 S145P 
                 CCT 
                 N211W 
                 TGG 
                 L278K 
                 AAG 
                 V344T 
                 ACT 
               
               
                 F014E 
                 GAG 
                 I079Y 
                 TAT 
                 S145R 
                 CGT 
                 D212A 
                 GCT 
                 L278M 
                 TTT 
                 V344W 
                 TGG 
               
               
                 F014G 
                 GGT 
                 P080A 
                 GCG 
                 S145T 
                 ACT 
                 D212E 
                 GAG 
                 L278N 
                 AAT 
                 V344Y 
                 TAT 
               
               
                 F014H 
                 CAT 
                 P080D 
                 GAT 
                 S145V 
                 GTT 
                 D212G 
                 GGG 
                 L278P 
                 CCG 
                 L345A 
                 GCT 
               
               
                 F014I 
                 ATT 
                 P080E 
                 GAG 
                 S145W 
                 TGG 
                 D212H 
                 CAT 
                 L278R 
                 CGT 
                 L345C 
                 TGT 
               
               
                 F014K 
                 AAG 
                 P080F 
                 TTT 
                 L146A 
                 GCT 
                 D212I 
                 ATT 
                 L278S 
                 TCT 
                 L345D 
                 GAT 
               
               
                 F014M 
                 ATG 
                 P080G 
                 GGG 
                 L146C 
                 TGT 
                 D212K 
                 AAG 
                 L278T 
                 ACT 
                 L345E 
                 GAG 
               
               
                 F014N 
                 AAT 
                 P080I 
                 ATT 
                 L146E 
                 GAG 
                 D212L 
                 CTG 
                 L278V 
                 GTT 
                 L345G 
                 GGG 
               
               
                 F014P 
                 CCT 
                 P080K 
                 AAG 
                 L146G 
                 GGG 
                 D212M 
                 ATG 
                 L278Y 
                 TAT 
                 L345H 
                 CAT 
               
               
                 F014Q 
                 CAG 
                 P080L 
                 CTT 
                 L146H 
                 CAT 
                 D212N 
                 AAT 
                 K279A 
                 GCG 
                 L345K 
                 AAG 
               
               
                 F014R 
                 CGG 
                 P080M 
                 ATG 
                 L146I 
                 ATT 
                 D212P 
                 CCT 
                 K279C 
                 TGT 
                 L345N 
                 AAT 
               
               
                 F014T 
                 ACT 
                 P080N 
                 AAT 
                 L146K 
                 AAG 
                 D212Q 
                 CAG 
                 K279D 
                 GAT 
                 L345P 
                 CCT 
               
               
                 F014V 
                 GTG 
                 P080R 
                 AGG 
                 L146N 
                 AAT 
                 D212S 
                 TCG 
                 K279F 
                 TTT 
                 L345Q 
                 CAG 
               
               
                 F014W 
                 TGG 
                 P080S 
                 TCT 
                 L146P 
                 CCT 
                 D212T 
                 ACT 
                 K279G 
                 GGG 
                 L345R 
                 CGT 
               
               
                 L015A 
                 GCG 
                 P080T 
                 ACG 
                 L146Q 
                 CAG 
                 D212V 
                 GTG 
                 K279H 
                 CAT 
                 L345T 
                 ACT 
               
               
                 L015E 
                 GAG 
                 P080V 
                 GTG 
                 L146R 
                 CGG 
                 D212W 
                 TGG 
                 K279L 
                 CTG 
                 L345V 
                 GTT 
               
               
                 L015F 
                 TTT 
                 P080Y 
                 TAT 
                 L146S 
                 TCG 
                 D213A 
                 GCT 
                 K279P 
                 CCT 
                 L345W 
                 TGG 
               
               
                 L015G 
                 GGG 
                 Q081A 
                 GCT 
                 L146T 
                 ACT 
                 D213E 
                 GAG 
                 K279Q 
                 CAG 
                 L345Y 
                 TAT 
               
               
                 L015K 
                 AAG 
                 Q081C 
                 TGT 
                 L146V 
                 GTT 
                 D213G 
                 GGG 
                 K279R 
                 AGG 
                 C346A 
                 GCT 
               
               
                 L015M 
                 ATG 
                 Q081E 
                 GAG 
                 L146Y 
                 TAT 
                 D213H 
                 CAT 
                 K279S 
                 TCT 
                 C346D 
                 GAT 
               
               
                 L015N 
                 AAT 
                 Q081F 
                 TTT 
                 T147A 
                 GCT 
                 D213K 
                 AAG 
                 K279T 
                 ACG 
                 C346F 
                 TTT 
               
               
                 L015P 
                 CCG 
                 Q081G 
                 GGG 
                 T147C 
                 TGT 
                 D213L 
                 CTG 
                 K279V 
                 GTG 
                 C346G 
                 GGG 
               
               
                 L015Q 
                 CAG 
                 Q081H 
                 CAT 
                 T147D 
                 GAT 
                 D213M 
                 ATG 
                 K279W 
                 TGG 
                 C346I 
                 ATT 
               
               
                 L015R 
                 CGG 
                 Q081L 
                 CTG 
                 T147F 
                 TTT 
                 D213N 
                 AAT 
                 K279Y 
                 TAT 
                 C346K 
                 AAG 
               
               
                 L015S 
                 TCG 
                 Q081M 
                 ATG 
                 T147G 
                 GGT 
                 D213P 
                 CCT 
                 F280D 
                 GAT 
                 C346L 
                 CTT 
               
               
                 L015T 
                 ACT 
                 Q081N 
                 AAT 
                 T147I 
                 ATT 
                 D213Q 
                 CAG 
                 F280E 
                 GAG 
                 C346M 
                 ATG 
               
               
                 L015V 
                 GTT 
                 Q081P 
                 CCG 
                 T147L 
                 CTT 
                 D213R 
                 CGT 
                 F280G 
                 GGG 
                 C346P 
                 CCT 
               
               
                 L015W 
                 TGG 
                 Q081R 
                 AGG 
                 T147M 
                 ATG 
                 D213S 
                 TCG 
                 F280H 
                 CAT 
                 C346Q 
                 CAG 
               
               
                 L015Y 
                 TAT 
                 Q081S 
                 TCT 
                 T147P 
                 CCT 
                 D213V 
                 GTG 
                 F280I 
                 ATT 
                 C346R 
                 CGG 
               
               
                 W016A 
                 GCG 
                 Q081V 
                 GTT 
                 T147Q 
                 CAG 
                 D213W 
                 TGG 
                 F280L 
                 TTG 
                 C346S 
                 TCT 
               
               
                 W016C 
                 TGT 
                 Q081W 
                 TGG 
                 T147R 
                 CGT 
                 D213Y 
                 TAT 
                 F280M 
                 ATG 
                 C346T 
                 ACT 
               
               
                 W016D 
                 GAT 
                 Q081Y 
                 TAT 
                 T147S 
                 AGT 
                 L214A 
                 GCG 
                 F280N 
                 AAT 
                 C346V 
                 GTG 
               
               
                 W016E 
                 GAG 
                 K082A 
                 GCT 
                 T147V 
                 GTT 
                 L214C 
                 TGT 
                 F280P 
                 CCT 
                 C346W 
                 TGG 
               
               
                 W016F 
                 TTT 
                 K082E 
                 GAG 
                 T147W 
                 TGG 
                 L214D 
                 GAT 
                 F280Q 
                 CAG 
                 Q347A 
                 GCT 
               
               
                 W016G 
                 GGT 
                 K082G 
                 GGT 
                 T147Y 
                 TAT 
                 L214E 
                 GAG 
                 F280R 
                 CGT 
                 Q347C 
                 TGT 
               
               
                 W016H 
                 CAT 
                 K082H 
                 CAT 
                 E148C 
                 TGT 
                 L214G 
                 GGG 
                 F280S 
                 TCG 
                 Q347E 
                 GAG 
               
               
                 W016K 
                 AAG 
                 K082I 
                 ATT 
                 E148F 
                 TTT 
                 L214H 
                 CAT 
                 F280T 
                 ACT 
                 Q347F 
                 TTT 
               
               
                 W016L 
                 CTT 
                 K082L 
                 CTT 
                 E148G 
                 GGG 
                 L214K 
                 AAG 
                 F280V 
                 GTG 
                 Q347G 
                 GGT 
               
               
                 W016M 
                 ATG 
                 K082M 
                 ATG 
                 E148H 
                 CAT 
                 L214N 
                 AAT 
                 F280W 
                 TGG 
                 Q347I 
                 ATT 
               
               
                 W016P 
                 CCT 
                 K082N 
                 AAT 
                 E148I 
                 ATT 
                 L214P 
                 CCG 
                 L281A 
                 GCG 
                 Q347L 
                 TTG 
               
               
                 W016R 
                 CGT 
                 K082P 
                 CCT 
                 E148K 
                 AAG 
                 L214Q 
                 CAG 
                 L281D 
                 GAT 
                 Q347M 
                 ATG 
               
               
                 W016S 
                 TCG 
                 K082Q 
                 CAG 
                 E148L 
                 CTG 
                 L214R 
                 CGG 
                 L281F 
                 TTT 
                 Q347P 
                 CCT 
               
               
                 W016T 
                 ACT 
                 K082R 
                 CGT 
                 E148P 
                 CCT 
                 L214S 
                 TCG 
                 L281G 
                 GGT 
                 Q347R 
                 AGG 
               
               
                 W016Y 
                 TAT 
                 K082S 
                 AGT 
                 E148Q 
                 CAG 
                 L214T 
                 ACG 
                 L281H 
                 CAT 
                 Q347S 
                 TCT 
               
               
                 A017D 
                 GAT 
                 K082T 
                 ACT 
                 E148R 
                 CGG 
                 L214V 
                 GTG 
                 L281I 
                 ATT 
                 Q347T 
                 ACT 
               
               
                 A017E 
                 GAG 
                 K082V 
                 GTG 
                 E148S 
                 TCT 
                 L214Y 
                 TAT 
                 L281K 
                 AAG 
                 Q347V 
                 GTG 
               
               
                 A017G 
                 GGG 
                 K082W 
                 TGG 
                 E148T 
                 ACT 
                 S215A 
                 GCT 
                 L281N 
                 AAT 
                 Q347W 
                 TGG 
               
               
                 A017H 
                 CAT 
                 K082Y 
                 TAT 
                 E148V 
                 GTG 
                 S215C 
                 TGT 
                 L281P 
                 CCG 
                 Q347Y 
                 TAT 
               
               
                 A017I 
                 ATT 
                 I083E 
                 GAG 
                 E148W 
                 TGG 
                 S215D 
                 GAT 
                 L281Q 
                 CAG 
                 E348C 
                 TGT 
               
               
                 A017L 
                 CTT 
                 I083F 
                 TTT 
                 E148Y 
                 TAT 
                 S215E 
                 GAG 
                 L281R 
                 CGG 
                 E348D 
                 GAT 
               
               
                 A017N 
                 AAT 
                 I083G 
                 GGT 
                 A149C 
                 TGT 
                 S215G 
                 GGG 
                 L281S 
                 AGT 
                 E348G 
                 GGT 
               
               
                 A017P 
                 CCG 
                 I083H 
                 CAT 
                 A149E 
                 GAG 
                 S215H 
                 CAT 
                 L281V 
                 GTT 
                 E348H 
                 CAT 
               
               
                 A017Q 
                 CAG 
                 I083K 
                 AAG 
                 A149F 
                 TTT 
                 S215K 
                 AAG 
                 L281W 
                 TGG 
                 E348I 
                 ATT 
               
               
                 A017R 
                 AGG 
                 I083L 
                 CTG 
                 A149G 
                 GGT 
                 S215L 
                 TTG 
                 L281Y 
                 TAT 
                 E348L 
                 TTG 
               
               
                 A017S 
                 TCG 
                 I083N 
                 AAT 
                 A149K 
                 AAG 
                 S215M 
                 ATG 
                 S282A 
                 GCG 
                 E348M 
                 ATG 
               
               
                 A017T 
                 ACG 
                 I083P 
                 CCT 
                 A149L 
                 TTG 
                 S215P 
                 CCG 
                 S282C 
                 TGT 
                 E348P 
                 CCT 
               
               
                 A017V 
                 GTG 
                 I083Q 
                 CAA 
                 A149M 
                 ATG 
                 S215Q 
                 CAG 
                 S282D 
                 GAT 
                 E348Q 
                 CAG 
               
               
                 A017W 
                 TGG 
                 I083R 
                 CGT 
                 A149P 
                 CCT 
                 S215R 
                 CGG 
                 S282E 
                 GAG 
                 E348R 
                 CGG 
               
               
                 A017Y 
                 TAT 
                 I083S 
                 TCG 
                 A149Q 
                 CAG 
                 S215T 
                 ACT 
                 S282F 
                 TTT 
                 E348S 
                 TCT 
               
               
                 W018C 
                 TGT 
                 I083T 
                 ACT 
                 A149R 
                 CGG 
                 S215V 
                 GTG 
                 S282G 
                 GGT 
                 E348T 
                 ACT 
               
               
                 W018D 
                 GAT 
                 I083V 
                 GTT 
                 A149S 
                 TCT 
                 S215W 
                 TGG 
                 S282L 
                 CTT 
                 E348V 
                 GTT 
               
               
                 W018F 
                 TTT 
                 I083Y 
                 TAT 
                 A149T 
                 ACT 
                 W216D 
                 GAT 
                 S282M 
                 ATG 
                 E348W 
                 TGG 
               
               
                 W018G 
                 GGG 
                 S084D 
                 GAT 
                 A149V 
                 GTT 
                 W216E 
                 GAG 
                 S282P 
                 CCT 
                 E348Y 
                 TAT 
               
               
                 W018H 
                 CAT 
                 S084E 
                 GAG 
                 A149W 
                 TGG 
                 W216G 
                 GGT 
                 S282Q 
                 CAG 
                 Q349A 
                 GCT 
               
               
                 W018I 
                 ATT 
                 S084F 
                 TTT 
                 A149Y 
                 TAT 
                 W216H 
                 CAT 
                 S282R 
                 CGT 
                 Q349D 
                 GAT 
               
               
                 W018L 
                 CTG 
                 S084G 
                 GGT 
                 T150A 
                 GCT 
                 W216I 
                 ATT 
                 S282T 
                 ACT 
                 Q349E 
                 GAG 
               
               
                 W018M 
                 ATG 
                 S084H 
                 CAT 
                 T150C 
                 TGT 
                 W216K 
                 AAG 
                 S282V 
                 GTT 
                 Q349F 
                 TTT 
               
               
                 W018P 
                 CCG 
                 S084I 
                 ATT 
                 T150D 
                 GAT 
                 W216L 
                 CTG 
                 S282W 
                 TGG 
                 Q349G 
                 GGT 
               
               
                 W018Q 
                 CAG 
                 S084L 
                 CTT 
                 T150E 
                 GAG 
                 W216M 
                 ATG 
                 S282Y 
                 TAT 
                 Q349H 
                 CAT 
               
               
                 W018R 
                 CGG 
                 S084M 
                 ATG 
                 T150F 
                 TTT 
                 W216N 
                 AAT 
                 Q283A 
                 GCG 
                 Q349K 
                 AAG 
               
               
                 W018S 
                 AGT 
                 S084N 
                 AAT 
                 T150G 
                 GGG 
                 W216P 
                 CCT 
                 Q283C 
                 TGT 
                 Q349L 
                 CTG 
               
               
                 W018T 
                 ACG 
                 S084P 
                 CCT 
                 T150I 
                 ATT 
                 W216Q 
                 CAG 
                 Q283D 
                 GAT 
                 Q349M 
                 ATG 
               
               
                 W018V 
                 GTG 
                 S084Q 
                 CAG 
                 T150L 
                 TTG 
                 W216R 
                 CGG 
                 Q283E 
                 GAG 
                 Q349N 
                 AAT 
               
               
                 W018Y 
                 TAT 
                 S084R 
                 CGG 
                 T150N 
                 AAT 
                 W216T 
                 ACG 
                 Q283F 
                 TTT 
                 Q349P 
                 CCT 
               
               
                 N019A 
                 GCG 
                 S084T 
                 ACT 
                 T150P 
                 CCT 
                 W216V 
                 GTG 
                 Q283G 
                 GGG 
                 Q349R 
                 CGT 
               
               
                 N019C 
                 TGT 
                 S084W 
                 TGG 
                 T150R 
                 AGG 
                 W216Y 
                 TAT 
                 Q283H 
                 CAT 
                 Q349S 
                 TCG 
               
               
                 N019F 
                 TTT 
                 S084Y 
                 TAT 
                 T150S 
                 TCT 
                 L217A 
                 GCG 
                 Q283L 
                 CTT 
                 Q349T 
                 ACT 
               
               
                 N019G 
                 GGG 
                 L085A 
                 GCT 
                 T150V 
                 GTG 
                 L217C 
                 TGT 
                 Q283N 
                 AAT 
                 Q349V 
                 GTG 
               
               
                 N019H 
                 CAT 
                 L085C 
                 TGT 
                 T150W 
                 TGG 
                 L217E 
                 GAG 
                 Q283P 
                 CCG 
                 Q349W 
                 TGG 
               
               
                 N019I 
                 ATT 
                 L085D 
                 GAT 
                 T150Y 
                 TAT 
                 L217G 
                 GGT 
                 Q283R 
                 CGT 
                 Q349Y 
                 TAT 
               
               
                 N019L 
                 CTG 
                 L085E 
                 GAG 
                 E151A 
                 GCT 
                 L217H 
                 CAT 
                 Q283S 
                 TCT 
                 G350A 
                 GCT 
               
               
                 N019M 
                 ATG 
                 L085F 
                 TTT 
                 E151C 
                 TGT 
                 L217I 
                 ATT 
                 Q283T 
                 ACT 
                 G350D 
                 GAT 
               
               
                 N019P 
                 CCG 
                 L085G 
                 GGG 
                 E151G 
                 GGT 
                 L217M 
                 ATG 
                 Q283W 
                 TGG 
                 G350E 
                 GAG 
               
               
                 N019Q 
                 CAG 
                 L085H 
                 CAT 
                 E151H 
                 CAT 
                 L217P 
                 CCG 
                 Q283Y 
                 TAT 
                 G350F 
                 TTT 
               
               
                 N019R 
                 CGT 
                 L085K 
                 AAG 
                 E151K 
                 AAG 
                 L217Q 
                 CAG 
                 D284A 
                 GCT 
                 G350H 
                 CAT 
               
               
                 N019S 
                 TCG 
                 L085N 
                 AAT 
                 E151L 
                 TTG 
                 L217R 
                 AGG 
                 D284C 
                 TGT 
                 G350K 
                 AAG 
               
               
                 N019V 
                 GTT 
                 L085P 
                 CCT 
                 E151M 
                 ATG 
                 L217S 
                 TCT 
                 D284E 
                 GAG 
                 G350L 
                 CTG 
               
               
                 N019W 
                 TGG 
                 L085Q 
                 CAG 
                 E151N 
                 AAT 
                 L217T 
                 ACG 
                 D284G 
                 GGT 
                 G350M 
                 ATG 
               
               
                 N019Y 
                 TAT 
                 L085R 
                 CGT 
                 E151Q 
                 CAG 
                 L217V 
                 GTG 
                 D284H 
                 CAT 
                 G350N 
                 AAT 
               
               
                 A020D 
                 GAT 
                 L085S 
                 TCG 
                 E151R 
                 AGG 
                 L217W 
                 TGG 
                 D284I 
                 ATT 
                 G350P 
                 CCT 
               
               
                 A020E 
                 GAG 
                 L085T 
                 ACT 
                 E151S 
                 TCG 
                 L217Y 
                 TAT 
                 D284L 
                 TTG 
                 G350R 
                 CGT 
               
               
                 A020F 
                 TTT 
                 L085V 
                 GTT 
                 E151T 
                 ACT 
                 W218A 
                 GCT 
                 D284M 
                 ATG 
                 G350S 
                 TCT 
               
               
                 A020G 
                 GGG 
                 Q086A 
                 GCT 
                 E151V 
                 GTT 
                 W218D 
                 GAT 
                 D284N 
                 AAT 
                 G350T 
                 ACT 
               
               
                 A020H 
                 CAT 
                 Q086C 
                 TGT 
                 E151W 
                 TGG 
                 W218F 
                 TTT 
                 D284P 
                 CCG 
                 G350V 
                 GTG 
               
               
                 A020K 
                 AAG 
                 Q086D 
                 GAT 
                 E151Y 
                 TAT 
                 W218G 
                 GGT 
                 D284Q 
                 CAG 
                 G350Y 
                 TAT 
               
               
                 A020L 
                 CTG 
                 Q086E 
                 GAG 
                 K152A 
                 GCT 
                 W218H 
                 CAT 
                 D284S 
                 TCT 
                 V351A 
                 GCT 
               
               
                 A020N 
                 AAT 
                 Q086F 
                 TTT 
                 K152C 
                 TGT 
                 W218I 
                 ATT 
                 D284T 
                 ACG 
                 V351C 
                 TGT 
               
               
                 A020P 
                 CCG 
                 Q086G 
                 GGT 
                 K152F 
                 TTT 
                 W218K 
                 AAG 
                 D284V 
                 GTT 
                 V351D 
                 GAT 
               
               
                 A020Q 
                 CAG 
                 Q086H 
                 CAT 
                 K152G 
                 GGT 
                 W218L 
                 CTT 
                 D284Y 
                 TAT 
                 V351E 
                 GAG 
               
               
                 A020R 
                 CGT 
                 Q086I 
                 ATT 
                 K152I 
                 ATT 
                 W218M 
                 ATG 
                 E285A 
                 GCG 
                 V351F 
                 TTT 
               
               
                 A020S 
                 TCT 
                 Q086K 
                 AAG 
                 K152L 
                 TTG 
                 W218P 
                 CCT 
                 E285F 
                 TTT 
                 V351G 
                 GGT 
               
               
                 A020T 
                 ACT 
                 Q086L 
                 CTG 
                 K152M 
                 ATG 
                 W218Q 
                 CAG 
                 E285G 
                 GGG 
                 V351H 
                 CAT 
               
               
                 A020V 
                 GTT 
                 Q086M 
                 ATG 
                 K152N 
                 AAT 
                 W218R 
                 CGG 
                 E285H 
                 CAT 
                 V351I 
                 ATT 
               
               
                 A020Y 
                 TAT 
                 Q086N 
                 AAT 
                 K152P 
                 CCT 
                 W218S 
                 TCG 
                 E285K 
                 AAG 
                 V351L 
                 TTG 
               
               
                 P021A 
                 GCG 
                 Q086P 
                 CCT 
                 K152R 
                 AGG 
                 W218T 
                 ACT 
                 E285M 
                 ATG 
                 V351N 
                 AAT 
               
               
                 P021C 
                 TGT 
                 Q086R 
                 CGG 
                 K152S 
                 TCT 
                 W218V 
                 GTG 
                 E285N 
                 AAT 
                 V351Q 
                 CAG 
               
               
                 P021D 
                 GAT 
                 Q086S 
                 TCT 
                 K152T 
                 ACT 
                 N219A 
                 GCG 
                 E285P 
                 CCT 
                 V351R 
                 AGG 
               
               
                 P021E 
                 GAG 
                 Q086T 
                 ACT 
                 K152V 
                 GTG 
                 N219C 
                 TGT 
                 E285Q 
                 CAG 
                 V351S 
                 TCT 
               
               
                 P021G 
                 GGG 
                 Q086V 
                 GTG 
                 K152W 
                 TGG 
                 N219D 
                 GAT 
                 E285R 
                 CGT 
                 V351W 
                 TGG 
               
               
                 P021H 
                 CAT 
                 Q086W 
                 TGG 
                 K152Y 
                 TAT 
                 N219E 
                 GAG 
                 E285S 
                 AGT 
                 V351Y 
                 TAT 
               
               
                 P021I 
                 ATT 
                 D087A 
                 GCT 
                 A153C 
                 TGT 
                 N219G 
                 GGG 
                 E285T 
                 ACG 
                 C352A 
                 GCT 
               
               
                 P021K 
                 AAG 
                 D087C 
                 TGT 
                 A153E 
                 GAG 
                 N219H 
                 CAT 
                 E285V 
                 GTG 
                 C352D 
                 GAT 
               
               
                 P021L 
                 CTT 
                 D087E 
                 GAG 
                 A153F 
                 TTT 
                 N219I 
                 ATT 
                 E285W 
                 TGG 
                 C352E 
                 GAG 
               
               
                 P021M 
                 ATG 
                 D087G 
                 GGG 
                 A153G 
                 GGT 
                 N219K 
                 AAG 
                 E285Y 
                 TAT 
                 C352F 
                 TTT 
               
               
                 P021R 
                 CGT 
                 D087H 
                 CAT 
                 A153H 
                 CAT 
                 N219L 
                 CTT 
                 L286A 
                 GCG 
                 C352G 
                 GGG 
               
               
                 P021S 
                 TCT 
                 D087I 
                 ATT 
                 A153I 
                 ATT 
                 N219M 
                 ATG 
                 L286C 
                 TGT 
                 C352K 
                 AAG 
               
               
                 P021T 
                 ACG 
                 D087L 
                 CTG 
                 A153K 
                 AAG 
                 N219P 
                 CCT 
                 L286D 
                 GAT 
                 C352M 
                 ATG 
               
               
                 P021V 
                 GTT 
                 D087M 
                 ATG 
                 A153L 
                 CTG 
                 N219R 
                 CGT 
                 L286E 
                 GAG 
                 C352P 
                 CCT 
               
               
                 P021W 
                 TGG 
                 D087P 
                 CCT 
                 A153M 
                 ATG 
                 N219S 
                 TCG 
                 L286F 
                 TTT 
                 C352Q 
                 CAG 
               
               
                 S022A 
                 GCT 
                 D087Q 
                 CAG 
                 A153P 
                 CCT 
                 N219T 
                 ACT 
                 L286G 
                 GGT 
                 C352R 
                 CGT 
               
               
                 S022C 
                 TGT 
                 D087R 
                 AGG 
                 A153Q 
                 CAG 
                 N219W 
                 TGG 
                 L286H 
                 CAT 
                 C352S 
                 AGT 
               
               
                 S022D 
                 GAT 
                 D087S 
                 TCG 
                 A153R 
                 CGT 
                 E220A 
                 GCG 
                 L286K 
                 AAG 
                 C352T 
                 ACT 
               
               
                 S022E 
                 GAG 
                 D087T 
                 ACT 
                 A153S 
                 AGT 
                 E220D 
                 GAT 
                 L286M 
                 ATG 
                 C352V 
                 GTG 
               
               
                 S022G 
                 GGG 
                 D087V 
                 GTT 
                 A153T 
                 ACT 
                 E220G 
                 GGG 
                 L286P 
                 CCT 
                 C352W 
                 TGG 
               
               
                 S022H 
                 CAT 
                 D087Y 
                 TAT 
                 A153V 
                 GTG 
                 E220H 
                 CAT 
                 L286R 
                 AGG 
                 C352Y 
                 TAT 
               
               
                 S022K 
                 AAG 
                 H088A 
                 GCT 
                 A153W 
                 TGG 
                 E220I 
                 ATT 
                 L286S 
                 AGT 
                 I353A 
                 GCT 
               
               
                 S022L 
                 CTG 
                 H088C 
                 TGT 
                 K154A 
                 GCT 
                 E220K 
                 AAG 
                 L286T 
                 ACG 
                 I353C 
                 TGT 
               
               
                 S022M 
                 ATG 
                 H088E 
                 GAG 
                 K154C 
                 TGT 
                 E220L 
                 TTG 
                 L286W 
                 TGG 
                 I353E 
                 GAG 
               
               
                 S022N 
                 AAT 
                 H088F 
                 TTT 
                 K154D 
                 GAT 
                 E220M 
                 ATG 
                 L286Y 
                 TAT 
                 I353F 
                 TTT 
               
               
                 S022P 
                 CCG 
                 H088G 
                 GGG 
                 K154E 
                 GAG 
                 E220N 
                 AAT 
                 V287A 
                 GCT 
                 I353G 
                 GGG 
               
               
                 S022R 
                 CGG 
                 H088I 
                 ATT 
                 K154G 
                 GGT 
                 E220P 
                 CCG 
                 V287C 
                 TGT 
                 I353H 
                 CAT 
               
               
                 S022T 
                 ACT 
                 H088K 
                 AAG 
                 K154H 
                 CAT 
                 E220R 
                 CGG 
                 V287D 
                 GAT 
                 I353K 
                 AAG 
               
               
                 S022V 
                 GTG 
                 H088L 
                 TTG 
                 K154I 
                 ATT 
                 E220S 
                 TCT 
                 V287E 
                 GAG 
                 I353L 
                 CTT 
               
               
                 S022Y 
                 TAT 
                 H088M 
                 ATG 
                 K154L 
                 CTG 
                 E220T 
                 ACG 
                 V287F 
                 TTT 
                 I353M 
                 ATG 
               
               
                 E023A 
                 GCT 
                 H088P 
                 CCT 
                 K154P 
                 CCT 
                 E220V 
                 GTG 
                 V287G 
                 GGG 
                 I353Q 
                 CAG 
               
               
                 E023D 
                 GAT 
                 H088R 
                 CGT 
                 K154R 
                 CGG 
                 E220W 
                 TGG 
                 V287I 
                 ATT 
                 I353R 
                 CGT 
               
               
                 E023F 
                 TTT 
                 H088S 
                 AGT 
                 K154S 
                 AGT 
                 S221A 
                 GCG 
                 V287K 
                 AAG 
                 I353S 
                 TCG 
               
               
                 E023G 
                 GGG 
                 H088T 
                 ACT 
                 K154T 
                 ACT 
                 S221C 
                 TGT 
                 V287L 
                 CTT 
                 I353T 
                 ACT 
               
               
                 E023H 
                 CAT 
                 H088V 
                 GTT 
                 K154V 
                 GTG 
                 S221D 
                 GAT 
                 V287N 
                 AAT 
                 I353V 
                 GTG 
               
               
                 E023L 
                 CTT 
                 H088Y 
                 TAT 
                 K154W 
                 TGG 
                 S221E 
                 GAG 
                 V287P 
                 CCT 
                 I353W 
                 TGG 
               
               
                 E023M 
                 ATG 
                 L089A 
                 GCT 
                 K154Y 
                 TAT 
                 S221G 
                 GGG 
                 V287Q 
                 CAG 
                 R354C 
                 TGT 
               
               
                 E023N 
                 AAT 
                 L089C 
                 TGT 
                 Q155A 
                 GCT 
                 S221H 
                 CAT 
                 V287R 
                 CGG 
                 R354D 
                 GAT 
               
               
                 E023P 
                 CCT 
                 L089D 
                 GAT 
                 Q155C 
                 TGT 
                 S221I 
                 ATT 
                 V287S 
                 TCT 
                 R354E 
                 GAG 
               
               
                 E023Q 
                 CAG 
                 L089E 
                 GAG 
                 Q155D 
                 GAT 
                 S221K 
                 AAG 
                 V287T 
                 ACT 
                 R354G 
                 GGT 
               
               
                 E023R 
                 CGG 
                 L089G 
                 GGG 
                 Q155F 
                 TTT 
                 S221L 
                 TTG 
                 Y288D 
                 GAC 
                 R354H 
                 CAT 
               
               
                 E023S 
                 TCT 
                 L089K 
                 AAG 
                 Q155G 
                 GGG 
                 S221M 
                 ATG 
                 Y288E 
                 GAG 
                 R354I 
                 ATT 
               
               
                 E023T 
                 ACG 
                 L089M 
                 ATG 
                 Q155H 
                 CAT 
                 S221P 
                 CCG 
                 Y288F 
                 TTT 
                 R354K 
                 AAG 
               
               
                 E023V 
                 GTG 
                 L089N 
                 AAT 
                 Q155K 
                 AAG 
                 S221Q 
                 CAG 
                 Y288G 
                 GGG 
                 R354L 
                 CTT 
               
               
                 E023W 
                 TGG 
                 L089P 
                 CCT 
                 Q155L 
                 CTT 
                 S221R 
                 CGG 
                 Y288H 
                 CAT 
                 R354M 
                 ATG 
               
               
                 F024A 
                 GCG 
                 L089Q 
                 CAG 
                 Q155M 
                 ATG 
                 S221T 
                 ACT 
                 Y288I 
                 ATT 
                 R354P 
                 CCT 
               
               
                 F024C 
                 TGT 
                 L089R 
                 AGG 
                 Q155P 
                 CCT 
                 S221V 
                 GTG 
                 Y288K 
                 AAG 
                 R354Q 
                 CAG 
               
               
                 F024E 
                 GAG 
                 L089S 
                 TCG 
                 Q155R 
                 CGG 
                 T222A 
                 GCG 
                 Y288L 
                 CTG 
                 R354S 
                 TCT 
               
               
                 F024G 
                 GGG 
                 L089T 
                 ACT 
                 Q155S 
                 AGT 
                 T222D 
                 GAT 
                 Y288P 
                 CCT 
                 R354V 
                 GTG 
               
               
                 F024H 
                 CAT 
                 L089W 
                 TGG 
                 Q155T 
                 ACT 
                 T222E 
                 GAG 
                 Y288Q 
                 CAG 
                 R354W 
                 TGG 
               
               
                 F024I 
                 ATT 
                 L089Y 
                 TAT 
                 Q155V 
                 GTT 
                 T222F 
                 TTT 
                 Y288R 
                 CGT 
                 R354Y 
                 TAT 
               
               
                 F024K 
                 AAG 
                 D090A 
                 GCT 
                 Q155W 
                 TGG 
                 T222G 
                 GGG 
                 Y288S 
                 TCT 
                 K355D 
                 GAT 
               
               
                 F024L 
                 TTG 
                 D090C 
                 TGT 
                 Q155Y 
                 TAT 
                 T222I 
                 ATT 
                 Y288T 
                 ACT 
                 K355F 
                 TTT 
               
               
                 F024M 
                 ATG 
                 D090E 
                 GAG 
                 E156A 
                 GCT 
                 T222K 
                 AAA 
                 Y288V 
                 GTG 
                 K355G 
                 GGG 
               
               
                 F024N 
                 AAT 
                 D090G 
                 GGG 
                 E156C 
                 TGT 
                 T222L 
                 TTG 
                 Y288W 
                 TGG 
                 K355H 
                 CAT 
               
               
                 F024P 
                 CCT 
                 D090H 
                 CAT 
                 E156D 
                 GAT 
                 T222N 
                 AAT 
                 T289A 
                 GCT 
                 K355L 
                 CTG 
               
               
                 F024R 
                 CGT 
                 D090I 
                 ATT 
                 E156G 
                 GGT 
                 T222P 
                 CCG 
                 T289C 
                 TGT 
                 K355M 
                 ATG 
               
               
                 F024T 
                 ACG 
                 D090K 
                 AAG 
                 E156I 
                 ATT 
                 T222R 
                 CGG 
                 T289E 
                 GAG 
                 K355N 
                 AAT 
               
               
                 F024V 
                 GTT 
                 D090L 
                 CTT 
                 E156K 
                 AAG 
                 T222S 
                 AGT 
                 T289G 
                 GGT 
                 K355P 
                 CCT 
               
               
                 F024Y 
                 TAT 
                 D090N 
                 AAT 
                 E156L 
                 CTG 
                 T222V 
                 GTT 
                 T289H 
                 CAT 
                 K355Q 
                 CAG 
               
               
                 C025D 
                 GAT 
                 D090P 
                 CCT 
                 E156M 
                 ATG 
                 T222W 
                 TGG 
                 T289K 
                 AAG 
                 K355R 
                 CGT 
               
               
                 C025E 
                 GAG 
                 D090Q 
                 CAG 
                 E156P 
                 CCT 
                 T222Y 
                 TAT 
                 T289L 
                 CTT 
                 K355S 
                 TCT 
               
               
                 C025F 
                 TTT 
                 D090R 
                 AGG 
                 E156Q 
                 CAG 
                 A223C 
                 TGT 
                 T289M 
                 ATG 
                 K355T 
                 ACT 
               
               
                 C025G 
                 GGG 
                 D090S 
                 AGT 
                 E156R 
                 CGG 
                 A223D 
                 GAT 
                 T289N 
                 AAT 
                 K355V 
                 GTG 
               
               
                 C025H 
                 CAT 
                 D090T 
                 ACT 
                 E156S 
                 TCT 
                 A223E 
                 GAG 
                 T289P 
                 CCT 
                 K355W 
                 TGG 
               
               
                 C025I 
                 ATT 
                 D090W 
                 TGG 
                 E156T 
                 ACT 
                 A223G 
                 GGG 
                 T289Q 
                 CAG 
                 K355Y 
                 TAT 
               
               
                 C025K 
                 AAG 
                 K091A 
                 GCT 
                 E156V 
                 GTT 
                 A223H 
                 CAT 
                 T289R 
                 AGG 
                 N356A 
                 GCT 
               
               
                 C025L 
                 TTG 
                 K091D 
                 GAT 
                 E156W 
                 TGG 
                 A223K 
                 AAG 
                 T289S 
                 TCG 
                 N356C 
                 TGT 
               
               
                 C025N 
                 AAT 
                 K091E 
                 GAG 
                 F157A 
                 GCT 
                 A223L 
                 CTG 
                 T289V 
                 GTG 
                 N356D 
                 GAT 
               
               
                 C025P 
                 CCT 
                 K091F 
                 TTT 
                 F157C 
                 TGT 
                 A223P 
                 CCT 
                 T289Y 
                 TAT 
                 N356F 
                 TTT 
               
               
                 C025R 
                 CGT 
                 K091G 
                 GGG 
                 F157D 
                 GAT 
                 A223Q 
                 CAG 
                 F290A 
                 GCT 
                 N356G 
                 GGG 
               
               
                 C025S 
                 TCT 
                 K091H 
                 CAT 
                 F157E 
                 GAG 
                 A223R 
                 AGG 
                 F290C 
                 TGT 
                 N356H 
                 CAT 
               
               
                 C025T 
                 ACT 
                 K091I 
                 ATT 
                 F157G 
                 GGT 
                 A223S 
                 TCT 
                 F290D 
                 GAT 
                 N356K 
                 AAG 
               
               
                 C025V 
                 GTG 
                 K091L 
                 TTG 
                 F157H 
                 CAT 
                 A223T 
                 ACG 
                 F290G 
                 GGG 
                 N356L 
                 CTG 
               
               
                 C025Y 
                 TAT 
                 K091N 
                 AAT 
                 F157I 
                 ATT 
                 A223V 
                 GTG 
                 F290H 
                 CAT 
                 N356P 
                 CCT 
               
               
                 L026A 
                 GCT 
                 K091Q 
                 CAG 
                 F157K 
                 AAG 
                 A223W 
                 TGG 
                 F290I 
                 ATT 
                 N356Q 
                 CAG 
               
               
                 L026E 
                 GAG 
                 K091R 
                 CGT 
                 F157L 
                 TTG 
                 A223Y 
                 TAT 
                 F290K 
                 AAG 
                 N356R 
                 CGG 
               
               
                 L026G 
                 GGT 
                 K091S 
                 TCT 
                 F157M 
                 ATG 
                 L224A 
                 GCT 
                 F290L 
                 TTG 
                 N356S 
                 AGT 
               
               
                 L026H 
                 CAT 
                 K091T 
                 ACT 
                 F157P 
                 CCT 
                 L224D 
                 GAT 
                 F290M 
                 ATG 
                 N356T 
                 ACT 
               
               
                 L026I 
                 ATT 
                 K091Y 
                 TAT 
                 F157Q 
                 CAG 
                 L224E 
                 GAG 
                 F290Q 
                 CAG 
                 N356V 
                 GTG 
               
               
                 L026K 
                 AAG 
                 A092C 
                 TGT 
                 F157R 
                 CGG 
                 L224F 
                 TTT 
                 F290R 
                 AGG 
                 N356W 
                 TGG 
               
               
                 L026M 
                 ATG 
                 A092E 
                 GAG 
                 F157S 
                 TCG 
                 L224G 
                 GGG 
                 F290S 
                 TCG 
                 W357A 
                 GCT 
               
               
                 L026P 
                 CCG 
                 A092F 
                 TTT 
                 F157T 
                 ACT 
                 L224I 
                 ATT 
                 F290T 
                 ACT 
                 W357C 
                 TGT 
               
               
                 L026Q 
                 CAG 
                 A092G 
                 GGT 
                 F157V 
                 GTG 
                 L224M 
                 ATG 
                 F290V 
                 GTT 
                 W357D 
                 GAT 
               
               
                 L026R 
                 CGG 
                 A092H 
                 CAT 
                 F157W 
                 TGG 
                 L224P 
                 CCG 
                 F290Y 
                 TAT 
                 W357E 
                 GAG 
               
               
                 L026S 
                 TCT 
                 A092K 
                 AAG 
                 E158A 
                 GCT 
                 L224Q 
                 CAG 
                 G291A 
                 GCT 
                 W357F 
                 TTT 
               
               
                 L026T 
                 ACT 
                 A092L 
                 CTG 
                 E158C 
                 TGT 
                 L224R 
                 AGG 
                 G291C 
                 TGT 
                 W357G 
                 GGG 
               
               
                 L026V 
                 GTT 
                 A092M 
                 ATG 
                 E158D 
                 GAT 
                 L224S 
                 AGT 
                 G291D 
                 GAT 
                 W357K 
                 AAG 
               
               
                 L026W 
                 TGG 
                 A092P 
                 CCT 
                 E158F 
                 TTT 
                 L224T 
                 ACT 
                 G291E 
                 GAG 
                 W357L 
                 TTG 
               
               
                 L026Y 
                 TAT 
                 A092Q 
                 CAG 
                 E158G 
                 GGG 
                 L224V 
                 GTT 
                 G291F 
                 TTT 
                 W357M 
                 ATG 
               
               
                 G027A 
                 GCT 
                 A092R 
                 CGT 
                 E158H 
                 CAT 
                 L224W 
                 TGG 
                 G291H 
                 CAT 
                 W357P 
                 CCT 
               
               
                 G027C 
                 TGT 
                 A092T 
                 ACT 
                 E158K 
                 AAG 
                 L224Y 
                 TAT 
                 G291L 
                 CTG 
                 W357Q 
                 CAG 
               
               
                 G027D 
                 GAT 
                 A092V 
                 GTT 
                 E158L 
                 CTG 
                 Y225A 
                 GCG 
                 G291M 
                 ATG 
                 W357R 
                 CGT 
               
               
                 G027E 
                 GAG 
                 A092W 
                 TGG 
                 E158N 
                 AAT 
                 Y225D 
                 GAT 
                 G291N 
                 AAT 
                 W357S 
                 AGT 
               
               
                 G027F 
                 TTT 
                 A092Y 
                 TAT 
                 E158P 
                 CCT 
                 Y225E 
                 GAG 
                 G291P 
                 CCT 
                 W357T 
                 ACT 
               
               
                 G027H 
                 CAT 
                 K093D 
                 GAT 
                 E158Q 
                 CAG 
                 Y225G 
                 GGT 
                 G291Q 
                 CAG 
                 W357V 
                 GTG 
               
               
                 G027I 
                 ATT 
                 K093E 
                 GAG 
                 E158R 
                 CGG 
                 Y225H 
                 CAT 
                 G291R 
                 CGG 
                 N358C 
                 TGT 
               
               
                 G027K 
                 AAG 
                 K093F 
                 TTT 
                 E158S 
                 TCG 
                 Y225K 
                 AAG 
                 G291S 
                 TCT 
                 N358D 
                 GAT 
               
               
                 G027L 
                 CTG 
                 K093G 
                 GGT 
                 E158V 
                 GTG 
                 Y225L 
                 CTG 
                 G291T 
                 ACT 
                 N358E 
                 GAG 
               
               
                 G027P 
                 CCT 
                 K093H 
                 CAT 
                 E158Y 
                 TAT 
                 Y225P 
                 CCG 
                 G291V 
                 GTG 
                 N358G 
                 GGG 
               
               
                 G027Q 
                 CAG 
                 K093I 
                 ATT 
                 K159A 
                 GCT 
                 Y225Q 
                 CAG 
                 G291W 
                 TGG 
                 N358H 
                 CAT 
               
               
                 G027R 
                 CGG 
                 K093L 
                 CTG 
                 K159D 
                 GAT 
                 Y225R 
                 AGG 
                 G291Y 
                 TAT 
                 N358I 
                 ATT 
               
               
                 G027S 
                 TCG 
                 K093M 
                 ATG 
                 K159E 
                 GAG 
                 Y225S 
                 TCT 
                 E292A 
                 GCT 
                 N358K 
                 AAG 
               
               
                 G027T 
                 ACT 
                 K093N 
                 AAT 
                 K159F 
                 TTT 
                 Y225T 
                 ACG 
                 E292C 
                 TGT 
                 N358L 
                 CTG 
               
               
                 G027W 
                 TGG 
                 K093P 
                 CCT 
                 K159G 
                 GGT 
                 Y225V 
                 GTG 
                 E292F 
                 TTT 
                 N358P 
                 CCT 
               
               
                 K028A 
                 GCG 
                 K093Q 
                 CAG 
                 K159H 
                 CAT 
                 Y225W 
                 TGG 
                 E292G 
                 GGT 
                 N358Q 
                 CAG 
               
               
                 K028D 
                 GAT 
                 K093R 
                 CGG 
                 K159L 
                 CTT 
                 P226A 
                 GCG 
                 E292H 
                 CAT 
                 N358R 
                 CGT 
               
               
                 K028E 
                 GAG 
                 K093S 
                 AGT 
                 K159M 
                 ATG 
                 P226C 
                 TGT 
                 E292I 
                 ATT 
                 N358S 
                 TCT 
               
               
                 K028F 
                 TTT 
                 K093T 
                 ACT 
                 K159N 
                 AAT 
                 P226D 
                 GAT 
                 E292K 
                 AAG 
                 N358T 
                 ACT 
               
               
                 K028G 
                 GGG 
                 K093V 
                 GTT 
                 K159Q 
                 CAG 
                 P226E 
                 GAG 
                 E292L 
                 TTG 
                 N358V 
                 GTG 
               
               
                 K028I 
                 ATT 
                 K094A 
                 GCT 
                 K159R 
                 CGG 
                 P226F 
                 TTT 
                 E292N 
                 AAT 
                 N358W 
                 TGG 
               
               
                 K028L 
                 TTG 
                 K094C 
                 TGT 
                 K159S 
                 TCT 
                 P226G 
                 GGT 
                 E292P 
                 CCT 
                 S359A 
                 GCT 
               
               
                 K028M 
                 ATG 
                 K094D 
                 GAT 
                 K159V 
                 GTG 
                 P226L 
                 CTT 
                 E292Q 
                 CAG 
                 S359C 
                 TGT 
               
               
                 K028N 
                 AAT 
                 K094E 
                 GAG 
                 K159W 
                 TGG 
                 P226N 
                 AAT 
                 E292R 
                 CGG 
                 S359D 
                 GAT 
               
               
                 K028P 
                 CCT 
                 K094F 
                 TTT 
                 K159Y 
                 TAT 
                 P226Q 
                 CAG 
                 E292T 
                 ACT 
                 S359E 
                 GAG 
               
               
                 K028R 
                 CGG 
                 K094G 
                 GGG 
                 A160C 
                 TGT 
                 P226R 
                 AGG 
                 E292V 
                 GTT 
                 S359F 
                 TTT 
               
               
                 K028S 
                 AGT 
                 K094H 
                 CAT 
                 A160F 
                 TTT 
                 P226S 
                 TCT 
                 E292W 
                 TGG 
                 S359G 
                 GGG 
               
               
                 K028T 
                 ACT 
                 K094L 
                 TTG 
                 A160G 
                 GGG 
                 P226T 
                 ACG 
                 T293A 
                 GCT 
                 S359H 
                 CAT 
               
               
                 K028V 
                 GTT 
                 K094M 
                 ATG 
                 A160H 
                 CAT 
                 P226V 
                 GTT 
                 T293C 
                 TGT 
                 S359K 
                 AAG 
               
               
                 K028W 
                 TGG 
                 K094N 
                 AAT 
                 A160I 
                 ATT 
                 P226W 
                 TGG 
                 T293D 
                 GAT 
                 S359L 
                 TTG 
               
               
                 F029A 
                 GCT 
                 K094P 
                 CCT 
                 A160K 
                 AAG 
                 P226Y 
                 TAT 
                 T293E 
                 GAG 
                 S359M 
                 ATG 
               
               
                 F029C 
                 TGT 
                 K094Q 
                 CAG 
                 A160L 
                 CTG 
                 S227A 
                 GCT 
                 T293F 
                 TTT 
                 S359P 
                 CCT 
               
               
                 F029E 
                 GAG 
                 K094R 
                 AGG 
                 A160M 
                 ATG 
                 S227F 
                 TTT 
                 T293G 
                 GGT 
                 S359R 
                 CGG 
               
               
                 F029G 
                 GGG 
                 K094S 
                 TCT 
                 A160N 
                 AAT 
                 S227G 
                 GGG 
                 T293K 
                 AAG 
                 S359T 
                 ACT 
               
               
                 F029H 
                 CAT 
                 K094T 
                 ACT 
                 A160Q 
                 CAG 
                 S227H 
                 CAT 
                 T293L 
                 CTT 
                 S359V 
                 GTT 
               
               
                 F029I 
                 ATT 
                 D095A 
                 GCT 
                 A160R 
                 AGG 
                 S227I 
                 ATT 
                 T293M 
                 ATG 
                 S359W 
                 TGG 
               
               
                 F029K 
                 AAG 
                 D095C 
                 TGT 
                 A160S 
                 AGT 
                 S227K 
                 AAG 
                 T293N 
                 AAT 
                 S360A 
                 GCT 
               
               
                 F029L 
                 CTT 
                 D095E 
                 GAG 
                 A160V 
                 GTG 
                 S227L 
                 TTG 
                 T293P 
                 CCT 
                 S360C 
                 TGT 
               
               
                 F029M 
                 ATG 
                 D095F 
                 TTT 
                 A160W 
                 TGG 
                 S227M 
                 ATG 
                 T293Q 
                 CAG 
                 S360E 
                 GAG 
               
               
                 F029P 
                 CCG 
                 D095G 
                 GGG 
                 A160Y 
                 TAT 
                 S227P 
                 CCT 
                 T293S 
                 TCT 
                 S360F 
                 TTT 
               
               
                 F029R 
                 CGG 
                 D095H 
                 CAT 
                 G161A 
                 GCT 
                 S227Q 
                 CAG 
                 T293V 
                 GTG 
                 S360G 
                 GGG 
               
               
                 F029S 
                 TCG 
                 D095K 
                 AAG 
                 G161C 
                 TGT 
                 S227R 
                 CGG 
                 T293Y 
                 TAT 
                 S360I 
                 ATT 
               
               
                 F029T 
                 ACG 
                 D095L 
                 TTG 
                 G161D 
                 GAT 
                 S227T 
                 ACG 
                 V294A 
                 GCT 
                 S360K 
                 AAG 
               
               
                 F029V 
                 GTG 
                 D095M 
                 ATG 
                 G161E 
                 GAG 
                 S227V 
                 GTG 
                 V294C 
                 TGT 
                 S360L 
                 CTG 
               
               
                 F029W 
                 TGG 
                 D095P 
                 CCT 
                 G161H 
                 CAT 
                 S227W 
                 TGG 
                 V294E 
                 GAG 
                 S360M 
                 ATG 
               
               
                 D030A 
                 GCG 
                 D095Q 
                 CAG 
                 G161I 
                 ATT 
                 S227Y 
                 TAT 
                 V294G 
                 GGG 
                 S360N 
                 AAT 
               
               
                 D030E 
                 GAG 
                 D095S 
                 TCT 
                 G161K 
                 AAG 
                 I228A 
                 GCG 
                 V294H 
                 CAT 
                 S360P 
                 CCT 
               
               
                 D030F 
                 TTT 
                 D095V 
                 GTG 
                 G161L 
                 CTT 
                 I228E 
                 GAG 
                 V294K 
                 AAG 
                 S360Q 
                 CAG 
               
               
                 D030G 
                 GGG 
                 D095W 
                 TGG 
                 G161M 
                 ATG 
                 I228F 
                 TTT 
                 V294L 
                 TTG 
                 S360R 
                 AGG 
               
               
                 D030H 
                 CAT 
                 D095Y 
                 TAT 
                 G161Q 
                 CAG 
                 I228G 
                 GGG 
                 V294M 
                 ATG 
                 S360T 
                 ACT 
               
               
                 D030K 
                 AAG 
                 I096A 
                 GCT 
                 G161R 
                 CGT 
                 I228H 
                 CAT 
                 V294N 
                 AAT 
                 S360V 
                 GTT 
               
               
                 D030L 
                 TTG 
                 I096C 
                 TGT 
                 G161S 
                 AGT 
                 I228K 
                 AAG 
                 V294P 
                 CCT 
                 D361A 
                 GCT 
               
               
                 D030M 
                 ATG 
                 I096D 
                 GAT 
                 G161T 
                 ACT 
                 I228L 
                 TTG 
                 V294Q 
                 CAG 
                 D361C 
                 TGT 
               
               
                 D030P 
                 CCT 
                 I096E 
                 GAG 
                 G161V 
                 GTG 
                 I228M 
                 ATG 
                 V294R 
                 AGG 
                 D361E 
                 GAG 
               
               
                 D030Q 
                 CAG 
                 I096F 
                 TTT 
                 G161W 
                 TGG 
                 I228N 
                 AAT 
                 V294S 
                 AGT 
                 D361G 
                 GGG 
               
               
                 D030R 
                 CGG 
                 I096G 
                 GGG 
                 K162A 
                 GCT 
                 I228P 
                 CCG 
                 V294T 
                 ACT 
                 D361H 
                 CAT 
               
               
                 D030S 
                 TCG 
                 I096H 
                 CAT 
                 K162D 
                 GAT 
                 I228Q 
                 CAG 
                 V294W 
                 TGG 
                 D361L 
                 TTG 
               
               
                 D030T 
                 ACT 
                 I096L 
                 TTG 
                 K162E 
                 GAG 
                 I228R 
                 CGT 
                 A295C 
                 TGT 
                 D361M 
                 ATG 
               
               
                 D030V 
                 GTT 
                 I096N 
                 AAT 
                 K162F 
                 TTT 
                 I228S 
                 TCT 
                 A295D 
                 GAT 
                 D361N 
                 AAT 
               
               
                 D030W 
                 TGG 
                 I096P 
                 CCT 
                 K162G 
                 GGG 
                 I228T 
                 ACT 
                 A295E 
                 GAG 
                 D361P 
                 CCT 
               
               
                 E031A 
                 GCG 
                 I096R 
                 CGT 
                 K162H 
                 CAT 
                 I228W 
                 TGG 
                 A295F 
                 TTT 
                 D361Q 
                 CAG 
               
               
                 E031C 
                 TGT 
                 I096S 
                 AGT 
                 K162L 
                 TTG 
                 Y229E 
                 GAG 
                 A295G 
                 GGG 
                 D361R 
                 AGG 
               
               
                 E031G 
                 GGG 
                 I096T 
                 ACT 
                 K162M 
                 ATG 
                 Y229F 
                 TTT 
                 A295H 
                 CAT 
                 D361S 
                 TCG 
               
               
                 E031H 
                 CAT 
                 I096V 
                 GTG 
                 K162P 
                 CCT 
                 Y229G 
                 GGT 
                 A295I 
                 ATT 
                 D361V 
                 GTT 
               
               
                 E031I 
                 ATT 
                 I096W 
                 TGG 
                 K162Q 
                 CAG 
                 Y229H 
                 CAT 
                 A295L 
                 CTG 
                 D361W 
                 TGG 
               
               
                 E031K 
                 AAG 
                 T097A 
                 GCT 
                 K162R 
                 CGG 
                 Y229I 
                 ATT 
                 A295N 
                 AAT 
                 D361Y 
                 TAT 
               
               
                 E031L 
                 CTG 
                 T097C 
                 TGT 
                 K162S 
                 TCG 
                 Y229K 
                 AAG 
                 A295P 
                 CCT 
                 Y362A 
                 GCT 
               
               
                 E031N 
                 AAC 
                 T097D 
                 GAT 
                 K162V 
                 GTG 
                 Y229L 
                 TTG 
                 A295Q 
                 CAG 
                 Y362C 
                 TGT 
               
               
                 E031P 
                 CCG 
                 T097E 
                 GAG 
                 K162W 
                 TGG 
                 Y229N 
                 AAT 
                 A295S 
                 AGT 
                 Y362E 
                 GAG 
               
               
                 E031R 
                 CGG 
                 T097F 
                 TTT 
                 K162Y 
                 TAT 
                 Y229P 
                 CCT 
                 A295T 
                 ACT 
                 Y362G 
                 GGG 
               
               
                 E031S 
                 TCT 
                 T097G 
                 GGG 
                 D163A 
                 GCT 
                 Y229Q 
                 CAG 
                 A295V 
                 GTT 
                 Y362H 
                 CAT 
               
               
                 E031T 
                 ACG 
                 T097I 
                 ATT 
                 D163C 
                 TGT 
                 Y229R 
                 CGT 
                 A295Y 
                 TAT 
                 Y362K 
                 AAG 
               
               
                 E031V 
                 GTG 
                 T097L 
                 CTT 
                 D163E 
                 GAG 
                 Y229S 
                 TCG 
                 L296A 
                 GCT 
                 Y362L 
                 CTT 
               
               
                 E031W 
                 TGG 
                 T097N 
                 AAT 
                 D163F 
                 TTT 
                 Y229T 
                 ACT 
                 L296C 
                 TGT 
                 Y362M 
                 ATG 
               
               
                 E031Y 
                 TAT 
                 T097P 
                 CCT 
                 D163G 
                 GGG 
                 Y229V 
                 GTG 
                 L296F 
                 TTT 
                 Y362N 
                 AAT 
               
               
                 P032A 
                 GCG 
                 T097Q 
                 CAG 
                 D163H 
                 CAC 
                 Y229W 
                 TGG 
                 L296G 
                 GGT 
                 Y362P 
                 CCT 
               
               
                 P032C 
                 TGT 
                 T097R 
                 CGG 
                 D163K 
                 AAG 
                 L230A 
                 GCG 
                 L296I 
                 ATT 
                 Y362R 
                 CGG 
               
               
                 P032F 
                 TTT 
                 T097S 
                 TCG 
                 D163L 
                 CTT 
                 L230E 
                 GAG 
                 L296K 
                 AAG 
                 Y362S 
                 AGT 
               
               
                 P032G 
                 GGG 
                 T097W 
                 TGG 
                 D163P 
                 CCT 
                 L230G 
                 GGG 
                 L296M 
                 ATG 
                 Y362T 
                 ACT 
               
               
                 P032H 
                 CAT 
                 T097Y 
                 TAT 
                 D163Q 
                 CAG 
                 L230H 
                 CAT 
                 L296P 
                 CCT 
                 Y362V 
                 GTG 
               
               
                 P032K 
                 AAG 
                 F098A 
                 GCT 
                 D163R 
                 AGG 
                 L230I 
                 ATT 
                 L296Q 
                 CAG 
                 Y362W 
                 TGG 
               
               
                 P032L 
                 CTG 
                 F098C 
                 TGT 
                 D163S 
                 TCG 
                 L230K 
                 AAG 
                 L296R 
                 CGT 
                 L363A 
                 GCT 
               
               
                 P032M 
                 ATG 
                 F098D 
                 GAT 
                 D163T 
                 ACT 
                 L230M 
                 ATG 
                 L296S 
                 TCG 
                 L363C 
                 TGT 
               
               
                 P032N 
                 AAT 
                 F098E 
                 GAG 
                 D163V 
                 GTG 
                 L230N 
                 AAT 
                 L296T 
                 ACT 
                 L363D 
                 GAT 
               
               
                 P032Q 
                 CAG 
                 F098G 
                 GGG 
                 D163W 
                 TGG 
                 L230P 
                 CCT 
                 L296V 
                 GTT 
                 L363E 
                 GAG 
               
               
                 P032R 
                 CGG 
                 F098H 
                 CAT 
                 F164A 
                 GCT 
                 L230R 
                 CGT 
                 L296W 
                 TGG 
                 L363F 
                 TTT 
               
               
                 P032S 
                 TCG 
                 F098I 
                 ATT 
                 F164C 
                 TGT 
                 L230S 
                 AGT 
                 L296Y 
                 TAT 
                 L363G 
                 GGG 
               
               
                 P032T 
                 ACT 
                 F098L 
                 TTG 
                 F164D 
                 GAT 
                 L230T 
                 ACT 
                 G297A 
                 GCT 
                 L363H 
                 CAT 
               
               
                 P032V 
                 GTG 
                 F098M 
                 ATG 
                 F164E 
                 GAG 
                 L230V 
                 GTT 
                 G297C 
                 TGT 
                 L363I 
                 ATT 
               
               
                 P032W 
                 TGG 
                 F098P 
                 CCT 
                 F164G 
                 GGG 
                 L230W 
                 TGG 
                 G297E 
                 GAG 
                 L363P 
                 CCT 
               
               
                 P032Y 
                 TAT 
                 F098Q 
                 CAG 
                 F164H 
                 CAT 
                 L230Y 
                 TAT 
                 G297H 
                 CAT 
                 L363Q 
                 CAG 
               
               
                 L033C 
                 TGT 
                 F098R 
                 CGT 
                 F164L 
                 TTG 
                 N231A 
                 GCT 
                 G297I 
                 ATT 
                 L363R 
                 CGG 
               
               
                 L033D 
                 GAT 
                 F098S 
                 TCG 
                 F164M 
                 ATG 
                 N231C 
                 TGT 
                 G297L 
                 CTT 
                 L363S 
                 TCG 
               
               
                 L033G 
                 GGG 
                 F098V 
                 GTT 
                 F164N 
                 AAT 
                 N231D 
                 GAT 
                 G297N 
                 AAT 
                 L363T 
                 ACT 
               
               
                 L033H 
                 CAT 
                 F098W 
                 TGG 
                 F164P 
                 CCT 
                 N231F 
                 TTT 
                 G297P 
                 CCT 
                 L363V 
                 GTG 
               
               
                 L033I 
                 ATT 
                 Y099A 
                 GCT 
                 F164Q 
                 CAG 
                 N231G 
                 GGG 
                 G297Q 
                 CAG 
                 L363W 
                 TGG 
               
               
                 L033M 
                 ATG 
                 Y099C 
                 TGT 
                 F164R 
                 CGG 
                 N231H 
                 CAT 
                 G297R 
                 CGG 
                 H364A 
                 GCT 
               
               
                 L033N 
                 AAT 
                 Y099E 
                 GAG 
                 F164S 
                 AGT 
                 N231I 
                 ATT 
                 G297S 
                 AGT 
                 H364C 
                 TGT 
               
               
                 L033P 
                 CCG 
                 Y099F 
                 TTT 
                 F164V 
                 GTT 
                 N231K 
                 AAG 
                 G297T 
                 ACT 
                 H364D 
                 GAT 
               
               
                 L033Q 
                 CAG 
                 Y099G 
                 GGT 
                 F164W 
                 TGG 
                 N231L 
                 CTT 
                 G297V 
                 GTG 
                 H364E 
                 GAG 
               
               
                 L033R 
                 AGG 
                 Y099I 
                 ATT 
                 L165A 
                 GCT 
                 N231P 
                 CCT 
                 G297W 
                 TGG 
                 H364F 
                 TTT 
               
               
                 L033S 
                 TCG 
                 Y099L 
                 TTG 
                 L165C 
                 TGT 
                 N231Q 
                 CAG 
                 G297Y 
                 TAT 
                 H364G 
                 GGG 
               
               
                 L033T 
                 ACT 
                 Y099N 
                 AAT 
                 L165D 
                 GAT 
                 N231R 
                 CGT 
                 A298C 
                 TGT 
                 H364K 
                 AAG 
               
               
                 L033V 
                 GTT 
                 Y099P 
                 CCT 
                 L165F 
                 TTT 
                 N231S 
                 TCT 
                 A298E 
                 GAG 
                 H364L 
                 CTG 
               
               
                 L033W 
                 TGG 
                 Y099Q 
                 CAG 
                 L165G 
                 GGG 
                 N231T 
                 ACG 
                 A298G 
                 GGG 
                 H364M 
                 ATG 
               
               
                 L033Y 
                 TAT 
                 Y099R 
                 AGG 
                 L165H 
                 CAT 
                 N231V 
                 GTG 
                 A298I 
                 ATT 
                 H364P 
                 CCT 
               
               
                 D034A 
                 GCT 
                 Y099S 
                 TCG 
                 L165N 
                 AAT 
                 T232A 
                 GCG 
                 A298L 
                 TTG 
                 H364R 
                 CGG 
               
               
                 D034E 
                 GAG 
                 Y099T 
                 ACT 
                 L165P 
                 CCT 
                 T232C 
                 TGT 
                 A298M 
                 ATG 
                 H364S 
                 TCT 
               
               
                 D034G 
                 GGT 
                 Y099V 
                 GTT 
                 L165Q 
                 CAG 
                 T232F 
                 TTT 
                 A298N 
                 AAT 
                 H364T 
                 ACT 
               
               
                 D034H 
                 CAT 
                 Y099W 
                 TGG 
                 L165R 
                 CGG 
                 T232G 
                 GGG 
                 A298P 
                 CCT 
                 H364V 
                 GTG 
               
               
                 D034I 
                 ATT 
                 M100C 
                 TGT 
                 L165S 
                 TCG 
                 T232H 
                 CAT 
                 A298Q 
                 CAG 
                 H364Y 
                 TAT 
               
               
                 D034K 
                 AAG 
                 M100E 
                 GAG 
                 L165T 
                 ACT 
                 T232K 
                 AAG 
                 A298R 
                 CGT 
                 L365A 
                 GCT 
               
               
                 D034L 
                 CTT 
                 M100F 
                 TTT 
                 L165V 
                 GTG 
                 T232L 
                 CTT 
                 A298S 
                 TCG 
                 L365C 
                 TGT 
               
               
                 D034N 
                 AAT 
                 M100G 
                 GGT 
                 L165W 
                 TGG 
                 T232M 
                 ATG 
                 A298T 
                 ACT 
                 L365D 
                 GAT 
               
               
                 D034P 
                 CCT 
                 M100K 
                 AAG 
                 L165Y 
                 TAT 
                 T232N 
                 AAT 
                 A298V 
                 GTG 
                 L365E 
                 GAG 
               
               
                 D034Q 
                 CAG 
                 M100L 
                 CTG 
                 V166A 
                 GCT 
                 T232P 
                 CCG 
                 A298W 
                 TGG 
                 L365G 
                 GGG 
               
               
                 D034R 
                 CGT 
                 M100N 
                 AAT 
                 V166C 
                 TGT 
                 T232Q 
                 CAG 
                 A298Y 
                 TAT 
                 L365I 
                 ATT 
               
               
                 D034S 
                 AGT 
                 M100P 
                 CCT 
                 V166D 
                 GAT 
                 T232R 
                 AGG 
                 S299A 
                 GCT 
                 L365M 
                 ATG 
               
               
                 D034T 
                 ACG 
                 M100Q 
                 CAG 
                 V166E 
                 GAG 
                 T232S 
                 AGT 
                 S299C 
                 TGT 
                 L365N 
                 AAT 
               
               
                 D034V 
                 GTT 
                 M100R 
                 CGG 
                 V166F 
                 TTT 
                 T232V 
                 GTG 
                 S299D 
                 GAT 
                 L365P 
                 CCT 
               
               
                 D034W 
                 TGG 
                 M100S 
                 TCT 
                 V166G 
                 GGT 
                 T232Y 
                 TAT 
                 S299E 
                 GAG 
                 L365Q 
                 CAG 
               
               
                 M035A 
                 GCG 
                 M100T 
                 ACT 
                 V166H 
                 CAT 
                 Q233A 
                 GCG 
                 S299F 
                 TTT 
                 L365R 
                 CGG 
               
               
                 M035D 
                 GAT 
                 M100V 
                 GTT 
                 V166L 
                 CTT 
                 Q233C 
                 TGT 
                 S299G 
                 GGG 
                 L365S 
                 AGT 
               
               
                 M035F 
                 TTT 
                 M100W 
                 TGG 
                 V166N 
                 AAT 
                 Q233D 
                 GAT 
                 S299H 
                 CAT 
                 L365T 
                 ACT 
               
               
                 M035G 
                 GGG 
                 M100Y 
                 TAT 
                 V166P 
                 CCT 
                 Q233F 
                 TTT 
                 S299I 
                 ATT 
                 L365V 
                 GTG 
               
               
                 M035H 
                 CAT 
                 P101A 
                 GCT 
                 V166Q 
                 CAG 
                 Q233G 
                 GGG 
                 S299L 
                 CTT 
                 L365W 
                 TGG 
               
               
                 M035I 
                 ATT 
                 P101C 
                 TGT 
                 V166R 
                 CGG 
                 Q233I 
                 ATT 
                 S299M 
                 ATG 
                 L365Y 
                 TAT 
               
               
                 M035L 
                 TTG 
                 P101F 
                 TTT 
                 V166T 
                 ACT 
                 Q233K 
                 AAG 
                 S299P 
                 CCT 
                 N366A 
                 GCT 
               
               
                 M035N 
                 AAT 
                 P101G 
                 GGG 
                 V166W 
                 TGG 
                 Q233L 
                 CTG 
                 S299Q 
                 CAG 
                 N366C 
                 TGT 
               
               
                 M035P 
                 CCG 
                 P101H 
                 CAT 
                 V166Y 
                 TAT 
                 Q233P 
                 CCG 
                 S299R 
                 AGG 
                 N366E 
                 GAG 
               
               
                 M035Q 
                 CAG 
                 P101I 
                 ATT 
                 E167A 
                 GCT 
                 Q233R 
                 AGG 
                 S299T 
                 ACT 
                 N366F 
                 TTT 
               
               
                 M035R 
                 CGT 
                 P101K 
                 AAG 
                 E167D 
                 GAT 
                 Q233S 
                 TCG 
                 S299Y 
                 TAT 
                 N366G 
                 GGG 
               
               
                 M035S 
                 TCT 
                 P101L 
                 CTT 
                 E167F 
                 TTT 
                 Q233T 
                 ACG 
                 G300A 
                 GCT 
                 N366K 
                 AAG 
               
               
                 M035T 
                 ACT 
                 P101M 
                 ATG 
                 E167G 
                 GGT 
                 Q233V 
                 GTG 
                 G300C 
                 TGT 
                 N366L 
                 TTG 
               
               
                 M035V 
                 GTT 
                 P101N 
                 AAT 
                 E167H 
                 CAT 
                 Q233W 
                 TGG 
                 G300D 
                 GAT 
                 N366M 
                 ATG 
               
               
                 M035Y 
                 TAT 
                 P101Q 
                 CAG 
                 E167K 
                 AAG 
                 Q233Y 
                 TAT 
                 G300E 
                 GAG 
                 N366P 
                 CCT 
               
               
                 S036A 
                 GCG 
                 P101R 
                 AGG 
                 E167L 
                 TTG 
                 Q234A 
                 GCT 
                 G300F 
                 TTT 
                 N366Q 
                 CAG 
               
               
                 S036C 
                 TGT 
                 P101S 
                 TCT 
                 E167M 
                 ATG 
                 Q234C 
                 TGT 
                 G300L 
                 CTT 
                 N366R 
                 AGG 
               
               
                 S036D 
                 GAT 
                 P101T 
                 ACT 
                 E167N 
                 AAT 
                 Q234D 
                 GAT 
                 G300M 
                 ATG 
                 N366S 
                 TCT 
               
               
                 S036F 
                 TTT 
                 P101Y 
                 TAT 
                 E167P 
                 CCT 
                 Q234E 
                 GAG 
                 G300N 
                 AAT 
                 N366T 
                 ACT 
               
               
                 S036G 
                 GGT 
                 V102A 
                 GCT 
                 E167R 
                 AGG 
                 Q234G 
                 GGT 
                 G300P 
                 CCT 
                 N366V 
                 GTT 
               
               
                 S036H 
                 CAT 
                 V102C 
                 TGT 
                 E167S 
                 TCG 
                 Q234H 
                 CAT 
                 G300Q 
                 CAG 
                 N366W 
                 TGG 
               
               
                 S036K 
                 AAG 
                 V102E 
                 GAG 
                 E167T 
                 ACT 
                 Q234L 
                 CTT 
                 G300R 
                 AGG 
                 P367A 
                 GCT 
               
               
                 S036L 
                 TTG 
                 V102G 
                 GGT 
                 E167V 
                 GTT 
                 Q234M 
                 ATG 
                 G300S 
                 TCG 
                 P367C 
                 TGT 
               
               
                 S036N 
                 AAT 
                 V102H 
                 CAT 
                 E167Y 
                 TAT 
                 Q234N 
                 AAT 
                 G300T 
                 ACT 
                 P367E 
                 GAG 
               
               
                 S036P 
                 CCG 
                 V102K 
                 AAG 
                 T168A 
                 GCT 
                 Q234P 
                 CCG 
                 G300V 
                 GTT 
                 P367F 
                 TTT 
               
               
                 S036R 
                 CGG 
                 V102L 
                 TTG 
                 T168C 
                 TGT 
                 Q234R 
                 CGG 
                 G300W 
                 TGG 
                 P367G 
                 GGT 
               
               
                 S036T 
                 ACG 
                 V102M 
                 ATG 
                 T168D 
                 GAT 
                 Q234S 
                 AGT 
                 I301A 
                 GCT 
                 P367H 
                 CAT 
               
               
                 S036V 
                 GTT 
                 V102N 
                 AAT 
                 T168E 
                 GAG 
                 Q234T 
                 ACT 
                 I301E 
                 GAG 
                 P367I 
                 ATT 
               
               
                 S036W 
                 TGG 
                 V102P 
                 CCT 
                 T168F 
                 TTT 
                 Q234V 
                 GTG 
                 I301G 
                 GGG 
                 P367K 
                 AAG 
               
               
                 S036Y 
                 TAT 
                 V102Q 
                 CAG 
                 T168G 
                 GGG 
                 Q234W 
                 TGG 
                 I301H 
                 CAT 
                 P367L 
                 CTG 
               
               
                 L037A 
                 GCG 
                 V102R 
                 AGG 
                 T168H 
                 CAT 
                 S235A 
                 GCG 
                 I301K 
                 AAG 
                 P367M 
                 ATG 
               
               
                 L037C 
                 TGT 
                 V102S 
                 TCT 
                 T168K 
                 AAG 
                 S235E 
                 GAG 
                 I301L 
                 CTG 
                 P367Q 
                 CAG 
               
               
                 L037E 
                 GAG 
                 V102T 
                 ACT 
                 T168L 
                 CTG 
                 S235F 
                 TTT 
                 I301M 
                 ATG 
                 P367R 
                 CGT 
               
               
                 L037F 
                 TTT 
                 V102W 
                 TGG 
                 T168P 
                 CCT 
                 S235G 
                 GGG 
                 I301N 
                 AAT 
                 P367S 
                 TCG 
               
               
                 L037G 
                 GGG 
                 D103A 
                 GCT 
                 T168R 
                 CGG 
                 S235H 
                 CAT 
                 I301P 
                 CCT 
                 P367V 
                 GTT 
               
               
                 L037I 
                 ATT 
                 D103E 
                 GAG 
                 T168S 
                 TCT 
                 S235K 
                 AAG 
                 I301Q 
                 CAG 
                 P367W 
                 TGG 
               
               
                 L037K 
                 AAG 
                 D103F 
                 TTT 
                 T168V 
                 GTG 
                 S235L 
                 CTT 
                 I301R 
                 CGG 
                 D368A 
                 GCT 
               
               
                 L037M 
                 ATG 
                 D103G 
                 GGG 
                 T168W 
                 TGG 
                 S235M 
                 ATG 
                 I301S 
                 AGT 
                 D368C 
                 TGT 
               
               
                 L037N 
                 AAT 
                 D103H 
                 CAT 
                 T168Y 
                 TAT 
                 S235P 
                 CCT 
                 I301V 
                 GTT 
                 D368E 
                 GAG 
               
               
                 L037P 
                 CCT 
                 D103I 
                 ATT 
                 I169A 
                 GCT 
                 S235Q 
                 CAG 
                 I301W 
                 TGG 
                 D368G 
                 GGT 
               
               
                 L037R 
                 AGG 
                 D103L 
                 CTT 
                 I169D 
                 GAT 
                 S235R 
                 CGG 
                 I301Y 
                 TAT 
                 D368H 
                 CAT 
               
               
                 L037S 
                 TCT 
                 D103N 
                 AAT 
                 I169F 
                 TTT 
                 S235T 
                 ACG 
                 V302C 
                 TGT 
                 D368K 
                 AAG 
               
               
                 L037T 
                 ACG 
                 D103Q 
                 CAG 
                 I169G 
                 GGG 
                 S235V 
                 GTG 
                 V302D 
                 GAT 
                 D368L 
                 CTT 
               
               
                 L037V 
                 GTG 
                 D103R 
                 AGG 
                 I169H 
                 CAT 
                 S235W 
                 TGG 
                 V302E 
                 GAG 
                 D368M 
                 ATG 
               
               
                 L037W 
                 TGG 
                 D103S 
                 TCG 
                 I169K 
                 AAG 
                 S235Y 
                 TAT 
                 V302F 
                 TTT 
                 D368P 
                 CCT 
               
               
                 F038A 
                 GCG 
                 D103T 
                 ACT 
                 I169L 
                 TTG 
                 P236A 
                 GCT 
                 V302G 
                 GGT 
                 D368R 
                 CGT 
               
               
                 F038C 
                 TGT 
                 D103V 
                 GTT 
                 I169N 
                 AAT 
                 P236C 
                 TGT 
                 V302H 
                 CAT 
                 D368S 
                 AGT 
               
               
                 F038E 
                 GAG 
                 D103W 
                 TGG 
                 I169P 
                 CCT 
                 P236E 
                 GAG 
                 V302I 
                 ATT 
                 D368T 
                 ACT 
               
               
                 F038G 
                 GGG 
                 D103Y 
                 TAT 
                 I169Q 
                 CAG 
                 P236G 
                 GGG 
                 V302L 
                 TTG 
                 D368V 
                 GTT 
               
               
                 F038K 
                 AAG 
                 N104A 
                 GCT 
                 I169R 
                 CGG 
                 P236H 
                 CAT 
                 V302M 
                 ATG 
                 D368W 
                 TGG 
               
               
                 F038L 
                 CTT 
                 N104C 
                 TGT 
                 I169S 
                 TCG 
                 P236I 
                 ATT 
                 V302P 
                 CCT 
                 D368Y 
                 TAT 
               
               
                 F038M 
                 ATG 
                 N104F 
                 TTT 
                 I169T 
                 ACT 
                 P236K 
                 AAG 
                 V302R 
                 AGG 
                 N369A 
                 GCT 
               
               
                 F038N 
                 AAT 
                 N104G 
                 GGG 
                 I169V 
                 GTT 
                 P236L 
                 CTG 
                 V302S 
                 TCG 
                 N369C 
                 TGT 
               
               
                 F038P 
                 CCT 
                 N104H 
                 CAT 
                 I169Y 
                 TAT 
                 P236N 
                 AAT 
                 V302T 
                 ACT 
                 N369E 
                 GAG 
               
               
                 F038Q 
                 CAG 
                 N104I 
                 ATT 
                 K170A 
                 GCT 
                 P236Q 
                 CAG 
                 V302W 
                 TGG 
                 N369F 
                 TTT 
               
               
                 F038R 
                 AGG 
                 N104K 
                 AAG 
                 K170C 
                 TGT 
                 P236R 
                 CGT 
                 V302Y 
                 TAT 
                 N369H 
                 CAT 
               
               
                 F038S 
                 TCT 
                 N104L 
                 CTG 
                 K170D 
                 GAT 
                 P236S 
                 AGT 
                 I303A 
                 GCT 
                 N369I 
                 ATT 
               
               
                 F038T 
                 ACT 
                 N104M 
                 ATG 
                 K170E 
                 GAG 
                 P236T 
                 ACT 
                 I303C 
                 TGT 
                 N369K 
                 AAG 
               
               
                 F038W 
                 TGG 
                 N104P 
                 CCT 
                 K170G 
                 GGG 
                 P236W 
                 TGG 
                 I303D 
                 GAT 
                 N369L 
                 CTT 
               
               
                 F038Y 
                 TAT 
                 N104R 
                 AGG 
                 K170I 
                 ATT 
                 P236Y 
                 TAT 
                 I303E 
                 GAG 
                 N369P 
                 CCT 
               
               
                 S039A 
                 GCG 
                 N104S 
                 TCT 
                 K170L 
                 TTG 
                 V237A 
                 GCG 
                 I303F 
                 TTT 
                 N369Q 
                 CAG 
               
               
                 S039C 
                 TGT 
                 N104T 
                 ACT 
                 K170M 
                 ATG 
                 V237C 
                 TGT 
                 I303G 
                 GGT 
                 N369R 
                 CGG 
               
               
                 S039D 
                 GAT 
                 N104V 
                 GTT 
                 K170N 
                 AAT 
                 V237E 
                 GAG 
                 I303K 
                 AAG 
                 N369S 
                 TCG 
               
               
                 S039F 
                 TTT 
                 N104W 
                 TGG 
                 K170P 
                 CCT 
                 V237F 
                 TTT 
                 I303L 
                 TTG 
                 N369T 
                 ACT 
               
               
                 S039G 
                 GGT 
                 L105A 
                 GCT 
                 K170Q 
                 CAG 
                 V237G 
                 GGT 
                 I303M 
                 ATG 
                 N369V 
                 GTG 
               
               
                 S039L 
                 TTG 
                 L105C 
                 TGT 
                 K170R 
                 CGT 
                 V237H 
                 CAT 
                 I303P 
                 CCT 
                 N369W 
                 TGG 
               
               
                 S039M 
                 ATG 
                 L105D 
                 GAT 
                 K170V 
                 GTT 
                 V237L 
                 TTG 
                 I303R 
                 CGT 
                 F370A 
                 GCT 
               
               
                 S039N 
                 AAT 
                 L105E 
                 GAG 
                 K170W 
                 TGG 
                 V237N 
                 AAT 
                 I303S 
                 AGT 
                 F370D 
                 GAT 
               
               
                 S039P 
                 CCG 
                 L105G 
                 GGT 
                 K170Y 
                 TAT 
                 V237P 
                 CCT 
                 I303V 
                 GTG 
                 F370E 
                 GAG 
               
               
                 S039Q 
                 CAG 
                 L105H 
                 CAT 
                 L171A 
                 GCT 
                 V237Q 
                 CAG 
                 I303W 
                 TGG 
                 F370G 
                 GGG 
               
               
                 S039R 
                 CGT 
                 L105I 
                 ATT 
                 L171C 
                 TGT 
                 V237R 
                 CGG 
                 I303Y 
                 TAT 
                 F370H 
                 CAT 
               
               
                 S039T 
                 ACT 
                 L105M 
                 ATG 
                 L171D 
                 GAT 
                 V237S 
                 TCG 
                 W304A 
                 GCT 
                 F370I 
                 ATT 
               
               
                 S039V 
                 GTT 
                 L105N 
                 AAT 
                 L171G 
                 GGG 
                 V237T 
                 ACG 
                 W304C 
                 TGT 
                 F370K 
                 AAG 
               
               
                 S039W 
                 TGG 
                 L105P 
                 CCT 
                 L171H 
                 CAT 
                 V237W 
                 TGG 
                 W304D 
                 GAT 
                 F370L 
                 CTG 
               
               
                 S039Y 
                 TAT 
                 L105Q 
                 CAG 
                 L171I 
                 ATT 
                 V237Y 
                 TAT 
                 W304G 
                 GGT 
                 F370N 
                 AAT 
               
               
                 F040A 
                 GCG 
                 L105R 
                 CGG 
                 L171M 
                 ATG 
                 A238D 
                 GAT 
                 W304I 
                 ATT 
                 F370P 
                 CCT 
               
               
                 F040D 
                 GAT 
                 L105S 
                 TCT 
                 L171N 
                 AAT 
                 A238E 
                 GAG 
                 W304L 
                 CTG 
                 F370Q 
                 CAG 
               
               
                 F040E 
                 GAG 
                 L105T 
                 ACT 
                 L171P 
                 CCT 
                 A238F 
                 TTT 
                 W304M 
                 ATG 
                 F370R 
                 AGG 
               
               
                 F040G 
                 GGT 
                 L105V 
                 GTT 
                 L171Q 
                 CAG 
                 A238G 
                 GGT 
                 W304N 
                 AAT 
                 F370S 
                 TCT 
               
               
                 F040I 
                 ATT 
                 L105W 
                 TGG 
                 L171R 
                 CGT 
                 A238H 
                 CAT 
                 W304P 
                 CCT 
                 F370V 
                 GTG 
               
               
                 F040K 
                 AAG 
                 G106A 
                 GCT 
                 L171S 
                 AGT 
                 A238K 
                 AAG 
                 W304Q 
                 CAG 
                 F370Y 
                 TAT 
               
               
                 F040L 
                 CTG 
                 G106C 
                 TGT 
                 L171V 
                 GTG 
                 A238L 
                 CTT 
                 W304R 
                 CGG 
                 A371C 
                 TGT 
               
               
                 F040N 
                 AAT 
                 G106D 
                 GAT 
                 L171W 
                 TGG 
                 A238P 
                 CCG 
                 W304S 
                 AGT 
                 A371E 
                 GAG 
               
               
                 F040Q 
                 CAG 
                 G106E 
                 GAG 
                 L171Y 
                 TAT 
                 A238Q 
                 CAG 
                 W304T 
                 ACT 
                 A371F 
                 TTT 
               
               
                 F040R 
                 CGG 
                 G106F 
                 TTT 
                 G172A 
                 GCT 
                 A238R 
                 AGG 
                 W304V 
                 GTG 
                 A371G 
                 GGG 
               
               
                 F040S 
                 TCT 
                 G106H 
                 CAT 
                 G172C 
                 TGT 
                 A238S 
                 AGT 
                 W304Y 
                 TAT 
                 A371H 
                 CAT 
               
               
                 F040T 
                 ACT 
                 G106I 
                 ATT 
                 G172D 
                 GAT 
                 A238T 
                 ACG 
                 G305C 
                 TGT 
                 A371I 
                 ATT 
               
               
                 F040V 
                 GTT 
                 G106L 
                 CTG 
                 G172E 
                 GAG 
                 A238V 
                 GTG 
                 G305D 
                 GAT 
                 A371K 
                 AAG 
               
               
                 F040W 
                 TGG 
                 G106M 
                 ATG 
                 G172I 
                 ATT 
                 A238W 
                 TGG 
                 G305E 
                 GAG 
                 A371L 
                 CTT 
               
               
                 F040Y 
                 TAT 
                 G106N 
                 AAT 
                 G172L 
                 CTT 
                 A238Y 
                 TAT 
                 G305F 
                 TTT 
                 A371M 
                 ATG 
               
               
                 I041A 
                 GCG 
                 G106P 
                 CCT 
                 G172M 
                 ATG 
                 A239C 
                 TGT 
                 G305H 
                 CAT 
                 A371P 
                 CCT 
               
               
                 I041C 
                 TGT 
                 G106S 
                 AGT 
                 G172P 
                 CCT 
                 A239F 
                 TTT 
                 G305K 
                 AAG 
                 A371R 
                 CGT 
               
               
                 I041D 
                 GAT 
                 G106V 
                 GTG 
                 G172Q 
                 CAG 
                 A239G 
                 GGT 
                 G305L 
                 CTT 
                 A371S 
                 TCG 
               
               
                 I041E 
                 GAG 
                 G106W 
                 TGG 
                 G172R 
                 CGT 
                 A239H 
                 CAT 
                 G305N 
                 AAT 
                 A371T 
                 ACT 
               
               
                 I041F 
                 TTT 
                 G106Y 
                 TAT 
                 G172S 
                 TCT 
                 A239I 
                 ATT 
                 G305P 
                 CCT 
                 A371V 
                 GTG 
               
               
                 I041G 
                 GGG 
                 M107A 
                 GCT 
                 G172T 
                 ACT 
                 A239K 
                 AAG 
                 G305Q 
                 CAG 
                 A371W 
                 TGG 
               
               
                 I041H 
                 CAT 
                 M107C 
                 TGT 
                 G172V 
                 GTT 
                 T240K 
                 AAG 
                 G305R 
                 CGT 
                 I372A 
                 GCT 
               
               
                 I041N 
                 AAT 
                 M107D 
                 GAT 
                 G172W 
                 TGG 
                 A239L 
                 TTG 
                 G305S 
                 TCG 
                 I372D 
                 GAT 
               
               
                 I041P 
                 CCG 
                 M107F 
                 TTT 
                 G172Y 
                 TAT 
                 A239N 
                 AAT 
                 G305T 
                 ACT 
                 I372E 
                 GAG 
               
               
                 I041Q 
                 CAG 
                 M107G 
                 GGG 
                 K173D 
                 GAT 
                 A239P 
                 CCT 
                 G305V 
                 GTG 
                 I372F 
                 TTT 
               
               
                 I041R 
                 AGG 
                 M107H 
                 CAT 
                 K173E 
                 GAG 
                 A239R 
                 AGG 
                 G305Y 
                 TAT 
                 I372G 
                 GGT 
               
               
                 I041S 
                 TCT 
                 M107I 
                 ATT 
                 K173G 
                 GGG 
                 A239S 
                 TCT 
                 T306A 
                 GCT 
                 I372H 
                 CAT 
               
               
                 I041T 
                 ACG 
                 M107K 
                 AAG 
                 K173H 
                 CAT 
                 A239T 
                 ACT 
                 T306C 
                 TGT 
                 I372K 
                 AAG 
               
               
                 I041V 
                 GTT 
                 M107L 
                 CTT 
                 K173I 
                 ATT 
                 A239V 
                 GTT 
                 T306D 
                 GAT 
                 I372L 
                 CTG 
               
               
                 I041W 
                 TGG 
                 M107P 
                 CCT 
                 K173L 
                 CTT 
                 A239W 
                 TGG 
                 T306E 
                 GAG 
                 I372N 
                 AAT 
               
               
                 G042A 
                 GCT 
                 M107Q 
                 CAG 
                 K173M 
                 ATG 
                 A239Y 
                 TAT 
                 T306F 
                 TTT 
                 I372P 
                 CCT 
               
               
                 G042C 
                 TGT 
                 M107R 
                 CGT 
                 K173N 
                 AAT 
                 T240A 
                 GCG 
                 T306G 
                 GGT 
                 I372R 
                 CGG 
               
               
                 G042D 
                 GAT 
                 M107S 
                 TCT 
                 K173P 
                 CCT 
                 T240E 
                 GAG 
                 T306H 
                 CAT 
                 I372S 
                 TCT 
               
               
                 G042E 
                 GAG 
                 M107V 
                 GTT 
                 K173Q 
                 CAG 
                 T240F 
                 TTT 
                 T306I 
                 ATT 
                 I372T 
                 ACT 
               
               
                 G042H 
                 CAT 
                 M107W 
                 TGG 
                 K173R 
                 CGG 
                 T240G 
                 GGG 
                 T306L 
                 CTG 
                 I372V 
                 GTG 
               
               
                 G042I 
                 ATT 
                 A108D 
                 GAT 
                 K173S 
                 TCG 
                 T240L 
                 CTT 
                 T306P 
                 CCT 
                 I372W 
                 TGG 
               
               
                 G042K 
                 AAG 
                 A108E 
                 GAG 
                 K173V 
                 GTG 
                 T240M 
                 ATG 
                 T306R 
                 AGG 
                 Q373A 
                 GCT 
               
               
                 G042L 
                 CTG 
                 A108F 
                 TTT 
                 K173W 
                 TGG 
                 T240N 
                 AAT 
                 T306S 
                 AGT 
                 Q373C 
                 TGT 
               
               
                 G042M 
                 ATG 
                 A108G 
                 GGT 
                 K173Y 
                 TAT 
                 T240P 
                 CCT 
                 T306V 
                 GTG 
                 Q373E 
                 GAG 
               
               
                 G042P 
                 CCT 
                 A108H 
                 CAT 
                 L174A 
                 GCT 
                 T240Q 
                 CAG 
                 T306W 
                 TGG 
                 Q373F 
                 TTT 
               
               
                 G042Q 
                 CAG 
                 A108K 
                 AAG 
                 L174C 
                 TGT 
                 T240R 
                 CGT 
                 T306Y 
                 TAT 
                 Q373G 
                 GGT 
               
               
                 G042R 
                 CGG 
                 A108L 
                 TTG 
                 L174G 
                 GGG 
                 T240S 
                 AGT 
                 L307C 
                 TGT 
                 Q373H 
                 CAT 
               
               
                 G042S 
                 TCT 
                 A108M 
                 ATG 
                 L174H 
                 CAT 
                 T240V 
                 GTG 
                 L307E 
                 GAG 
                 Q373K 
                 AAG 
               
               
                 G042T 
                 ACT 
                 A108N 
                 AAT 
                 L174K 
                 AAG 
                 T240W 
                 TGG 
                 L307F 
                 TTT 
                 Q373L 
                 CTG 
               
               
                 G042V 
                 GTT 
                 A108P 
                 CCT 
                 L174M 
                 ATG 
                 T240Y 
                 TAT 
                 L307G 
                 GGG 
                 Q373M 
                 ATG 
               
               
                 S043A 
                 GCG 
                 A108Q 
                 CAG 
                 L174N 
                 AAT 
                 L241A 
                 GCG 
                 L307I 
                 ATT 
                 Q373N 
                 AAT 
               
               
                 S043D 
                 GAT 
                 A108R 
                 CGG 
                 L174P 
                 CCT 
                 L241C 
                 TGT 
                 L307K 
                 AAG 
                 Q373P 
                 CCT 
               
               
                 S043E 
                 GAG 
                 A108S 
                 TCT 
                 L174Q 
                 CAG 
                 L241D 
                 GAT 
                 L307N 
                 AAT 
                 Q373R 
                 CGT 
               
               
                 S043F 
                 TTT 
                 A108T 
                 ACT 
                 L174R 
                 CGT 
                 L241E 
                 GAG 
                 L307P 
                 CCT 
                 Q373S 
                 TCT 
               
               
                 S043G 
                 GGT 
                 A108V 
                 GTG 
                 L174S 
                 TCG 
                 L241F 
                 TTT 
                 L307Q 
                 CAG 
                 Q373T 
                 ACT 
               
               
                 S043H 
                 CAT 
                 A108Y 
                 TAT 
                 L174T 
                 ACT 
                 L241G 
                 GGG 
                 L307R 
                 AGG 
                 Q373V 
                 GTT 
               
               
                 S043I 
                 ATT 
                 V109A 
                 GCT 
                 L174V 
                 GTT 
                 L241I 
                 ATT 
                 L307S 
                 AGT 
                 Q373W 
                 TGG 
               
               
                 S043K 
                 AAG 
                 V109C 
                 TGT 
                 L174W 
                 TGG 
                 L241K 
                 AAG 
                 L307T 
                 ACT 
                 L374A 
                 GCT 
               
               
                 S043L 
                 CTT 
                 V109D 
                 GAT 
                 L174Y 
                 TAT 
                 L241P 
                 CCT 
                 L307V 
                 GTG 
                 L374D 
                 GAT 
               
               
                 S043N 
                 AAT 
                 V109E 
                 GAG 
                 L175C 
                 TGT 
                 L241Q 
                 CAG 
                 L307W 
                 TGG 
                 L374E 
                 GAG 
               
               
                 S043P 
                 CCT 
                 V109F 
                 TTT 
                 L175D 
                 GAT 
                 L241R 
                 CGG 
                 L307Y 
                 TAT 
                 L374G 
                 GGT 
               
               
                 S043Q 
                 CAG 
                 V109G 
                 GGG 
                 L175E 
                 GAG 
                 L241S 
                 TCT 
                 S308C 
                 TGT 
                 L374H 
                 CAT 
               
               
                 S043R 
                 CGG 
                 V109H 
                 CAT 
                 L175F 
                 TTT 
                 L241T 
                 ACG 
                 S308D 
                 GAT 
                 L374I 
                 ATT 
               
               
                 S043T 
                 ACT 
                 V109L 
                 TTG 
                 L175G 
                 GGG 
                 L241V 
                 GTT 
                 S308F 
                 TTT 
                 L374M 
                 ATG 
               
               
                 S043V 
                 GTG 
                 V109M 
                 ATG 
                 L175H 
                 CAT 
                 L241W 
                 TGG 
                 S308G 
                 GGT 
                 L374N 
                 AAT 
               
               
                 P044A 
                 GCT 
                 V109P 
                 CCT 
                 L175K 
                 AAG 
                 Y242A 
                 GCG 
                 S308H 
                 CAT 
                 L374P 
                 CCT 
               
               
                 P044C 
                 TGT 
                 V109Q 
                 CAG 
                 L175N 
                 AAT 
                 Y242C 
                 TGT 
                 S308K 
                 AAG 
                 L374R 
                 AGG 
               
               
                 P044E 
                 GAG 
                 V109R 
                 AGG 
                 L175P 
                 CCT 
                 Y242D 
                 GAT 
                 S308L 
                 CTG 
                 L374S 
                 AGT 
               
               
                 P044F 
                 TTT 
                 V109T 
                 ACT 
                 L175R 
                 CGT 
                 Y242F 
                 TTT 
                 S308M 
                 ATG 
                 L374T 
                 ACT 
               
               
                 P044G 
                 GGG 
                 V109W 
                 TGG 
                 L175S 
                 TCT 
                 Y242G 
                 GGT 
                 S308N 
                 AAT 
                 L374V 
                 GTG 
               
               
                 P044H 
                 CAT 
                 V109Y 
                 TAT 
                 L175T 
                 ACT 
                 Y242I 
                 ATT 
                 S308P 
                 CCT 
                 L374W 
                 TGG 
               
               
                 P044I 
                 ATT 
                 I110A 
                 GCT 
                 L175V 
                 GTG 
                 Y242K 
                 AAG 
                 S308R 
                 CGG 
                 L374Y 
                 TAT 
               
               
                 P044L 
                 CTT 
                 I110C 
                 TGT 
                 L175W 
                 TGG 
                 Y242L 
                 CTT 
                 S308T 
                 ACT 
                 E375A 
                 GCT 
               
               
                 P044N 
                 AAT 
                 I110D 
                 GAT 
                 L175Y 
                 TAT 
                 Y242M 
                 ATG 
                 S308V 
                 GTT 
                 E375C 
                 TGT 
               
               
                 P044Q 
                 CAG 
                 I110F 
                 TTT 
                 R176A 
                 GCT 
                 Y242P 
                 CCG 
                 S308W 
                 TGG 
                 E375F 
                 TTT 
               
               
                 P044R 
                 CGT 
                 I110G 
                 GGG 
                 R176C 
                 TGT 
                 Y242R 
                 CGG 
                 S308Y 
                 TAT 
                 E375G 
                 GGT 
               
               
                 P044S 
                 TCT 
                 I110H 
                 CAT 
                 R176E 
                 GAG 
                 Y242S 
                 TCT 
                 I309D 
                 GAT 
                 E375I 
                 ATT 
               
               
                 P044T 
                 ACT 
                 I110K 
                 AAG 
                 R176F 
                 TTT 
                 Y242T 
                 ACG 
                 I309E 
                 GAG 
                 E375K 
                 AAG 
               
               
                 P044W 
                 TGG 
                 I110L 
                 CTG 
                 R176G 
                 GGG 
                 Y242V 
                 GTT 
                 I309G 
                 GGT 
                 E375L 
                 CTT 
               
               
                 P044Y 
                 ACG 
                 I110M 
                 ATG 
                 R176H 
                 CAT 
                 Y242W 
                 TGG 
                 I309H 
                 CAT 
                 E375M 
                 ATG 
               
               
                 R045A 
                 GCG 
                 I110N 
                 AAT 
                 R176I 
                 ATT 
                 V243A 
                 GCG 
                 I309K 
                 AAG 
                 E375N 
                 AAT 
               
               
                 R045D 
                 GAT 
                 I110P 
                 CCT 
                 R176K 
                 AAG 
                 V243C 
                 TGT 
                 I309L 
                 CTG 
                 E375P 
                 CCT 
               
               
                 R045F 
                 TTT 
                 I110R 
                 CGT 
                 R176L 
                 CTT 
                 V243D 
                 GAT 
                 I309M 
                 ATG 
                 E375R 
                 CGT 
               
               
                 R045G 
                 GGG 
                 I110S 
                 AGT 
                 R176P 
                 CCT 
                 V243F 
                 TTT 
                 I309N 
                 AAT 
                 E375S 
                 TCT 
               
               
                 R045H 
                 CAT 
                 I110V 
                 GTT 
                 R176Q 
                 CAG 
                 V243G 
                 GGG 
                 I309Q 
                 CAG 
                 E375T 
                 ACT 
               
               
                 R045I 
                 ATT 
                 I110W 
                 TGG 
                 R176S 
                 AGT 
                 V243H 
                 CAT 
                 I309R 
                 CGT 
                 E375V 
                 GTT 
               
               
                 R045K 
                 AAG 
                 D111C 
                 TGT 
                 R176T 
                 ACT 
                 V243L 
                 CTT 
                 I309S 
                 AGT 
                 E375Y 
                 TAT 
               
               
                 R045M 
                 ATG 
                 D111E 
                 GAG 
                 R176V 
                 GTG 
                 V243M 
                 ATG 
                 I309T 
                 ACT 
                 K376A 
                 GCT 
               
               
                 R045P 
                 CCT 
                 D111G 
                 GGT 
                 R176W 
                 TGG 
                 V243P 
                 CCT 
                 I309V 
                 GTG 
                 K376D 
                 GAT 
               
               
                 R045Q 
                 CAG 
                 D111H 
                 CAT 
                 P177A 
                 GCT 
                 V243Q 
                 CAG 
                 I309W 
                 TGG 
                 K376E 
                 GAG 
               
               
                 R045S 
                 TCG 
                 D111I 
                 ATT 
                 P177C 
                 TGT 
                 V243R 
                 AGG 
                 I309Y 
                 TAT 
                 K376G 
                 GGG 
               
               
                 R045T 
                 ACG 
                 D111K 
                 AAG 
                 P177D 
                 GAT 
                 V243S 
                 AGT 
                 M310A 
                 GCT 
                 K376I 
                 ATT 
               
               
                 R045V 
                 GTG 
                 D111L 
                 TTG 
                 P177F 
                 TTT 
                 V243T 
                 ACG 
                 M310C 
                 TGT 
                 K376L 
                 TTG 
               
               
                 R045W 
                 TGG 
                 D111M 
                 ATG 
                 P177G 
                 GGG 
                 V243W 
                 TGG 
                 M310E 
                 GAG 
                 K376M 
                 ATG 
               
               
                 R045Y 
                 TAT 
                 D111P 
                 ACT 
                 P177H 
                 CAT 
                 V243Y 
                 TAT 
                 M310F 
                 TTT 
                 K376P 
                 CCT 
               
               
                 I046A 
                 GCG 
                 D111Q 
                 CAG 
                 P177L 
                 CTT 
                 R244A 
                 GCG 
                 M310G 
                 GGG 
                 K376Q 
                 CAG 
               
               
                 I046C 
                 TGT 
                 D111R 
                 CGG 
                 P177M 
                 ATG 
                 R244D 
                 GAT 
                 M310K 
                 AAG 
                 K376R 
                 CGT 
               
               
                 I046E 
                 GAG 
                 D111S 
                 AGT 
                 P177Q 
                 CAG 
                 R244G 
                 GGG 
                 M310L 
                 CTG 
                 K376S 
                 AGT 
               
               
                 I046F 
                 TTT 
                 D111T 
                 ACT 
                 P177R 
                 CGG 
                 R244H 
                 CAT 
                 M310N 
                 AAT 
                 K376T 
                 ACT 
               
               
                 I046H 
                 CAT 
                 D111V 
                 GTT 
                 P177S 
                 TCT 
                 R244I 
                 ATT 
                 M310P 
                 CCT 
                 K376V 
                 GTG 
               
               
                 I046L 
                 CTT 
                 D111W 
                 TGG 
                 P177T 
                 ACT 
                 R244K 
                 AAG 
                 M310Q 
                 CAG 
                 K376W 
                 TGG 
               
               
                 I046M 
                 ATG 
                 D111Y 
                 TAT 
                 P177V 
                 GTT 
                 R244M 
                 ATG 
                 M310R 
                 CGG 
                 K376Y 
                 TAT 
               
               
                 I046N 
                 AAT 
                 W112C 
                 TGT 
                 P177W 
                 TGG 
                 R244N 
                 AAT 
                 M310S 
                 AGT 
                 G377C 
                 TGT 
               
               
                 I046P 
                 CCT 
                 W112D 
                 GAT 
                 P177Y 
                 TAT 
                 R244P 
                 CCT 
                 M310V 
                 GTG 
                 G377D 
                 GAT 
               
               
                 I046R 
                 CGT 
                 W112E 
                 GAG 
                 N178A 
                 GCT 
                 R244Q 
                 CAG 
                 M310W 
                 TGG 
                 G377E 
                 GAG 
               
               
                 I046S 
                 TCT 
                 W112F 
                 TTT 
                 N178D 
                 GAT 
                 R244S 
                 TCT 
                 M310Y 
                 TAT 
                 G377F 
                 TTT 
               
               
                 I046T 
                 ACT 
                 W112G 
                 GGG 
                 N178E 
                 GAG 
                 R244T 
                 ACG 
                 R311A 
                 GCT 
                 G377H 
                 CAT 
               
               
                 I046V 
                 GTT 
                 W112H 
                 CAT 
                 N178G 
                 GGG 
                 R244V 
                 GTG 
                 R311C 
                 TGT 
                 G377I 
                 ATT 
               
               
                 I046W 
                 TGG 
                 W112I 
                 ATT 
                 N178I 
                 ATT 
                 R244W 
                 TGG 
                 R311E 
                 GAG 
                 G377K 
                 AAG 
               
               
                 I046Y 
                 TAT 
                 W112L 
                 CTT 
                 N178K 
                 AAG 
                 R244Y 
                 TAT 
                 R311F 
                 TTT 
                 G377L 
                 CTT 
               
               
                 N047A 
                 GCT 
                 W112N 
                 AAT 
                 N178L 
                 TTG 
                 N245A 
                 GCG 
                 R311G 
                 GGT 
                 G377M 
                 ATG 
               
               
                 N047D 
                 GAT 
                 W112P 
                 CCT 
                 N178M 
                 ATG 
                 N245C 
                 TGT 
                 R311H 
                 CAT 
                 G377P 
                 CCT 
               
               
                 N047F 
                 TTT 
                 W112Q 
                 CAG 
                 N178P 
                 CCT 
                 N245F 
                 TTT 
                 R311I 
                 ATT 
                 G377R 
                 AGG 
               
               
                 N047G 
                 GGG 
                 W112R 
                 CGT 
                 N178R 
                 CGG 
                 N245G 
                 GGG 
                 R311K 
                 AAG 
                 G377S 
                 TCG 
               
               
                 N047H 
                 CAT 
                 W112S 
                 TCT 
                 N178S 
                 AGT 
                 N245H 
                 CAT 
                 R311L 
                 TTG 
                 G377T 
                 ACT 
               
               
                 N047I 
                 ATT 
                 W112V 
                 GTT 
                 N178T 
                 ACT 
                 N245I 
                 ATT 
                 R311P 
                 CCT 
                 G377V 
                 GTG 
               
               
                 N047K 
                 AAG 
                 W112Y 
                 TAT 
                 N178V 
                 GTG 
                 N245K 
                 AAG 
                 R311Q 
                 CAG 
                 G377Y 
                 TAT 
               
               
                 N047L 
                 CTT 
                 E113A 
                 GCT 
                 N178W 
                 TGG 
                 N245L 
                 CTG 
                 R311S 
                 TCT 
                 G378D 
                 GAT 
               
               
                 N047M 
                 ATG 
                 E113C 
                 TGT 
                 N178Y 
                 TAT 
                 N245P 
                 CCG 
                 R311T 
                 ACT 
                 G378E 
                 GAG 
               
               
                 N047P 
                 CCT 
                 E113D 
                 GAT 
                 H179A 
                 GCT 
                 N245Q 
                 CAG 
                 R311V 
                 GTG 
                 G378F 
                 TTT 
               
               
                 N047Q 
                 CAG 
                 E113F 
                 TTT 
                 H179C 
                 TGT 
                 N245R 
                 CGG 
                 R311W 
                 TGG 
                 G378I 
                 ATT 
               
               
                 N047R 
                 CGG 
                 E113G 
                 GGG 
                 H179E 
                 GAG 
                 N245S 
                 TCG 
                 S312A 
                 GCT 
                 G378K 
                 AAG 
               
               
                 N047S 
                 TCT 
                 E113H 
                 CAT 
                 H179G 
                 GGG 
                 N245T 
                 ACG 
                 S312C 
                 TGT 
                 G378L 
                 CTG 
               
               
                 N047T 
                 ACG 
                 E113L 
                 CTT 
                 H179I 
                 ATT 
                 N245V 
                 GTG 
                 S312E 
                 GAG 
                 G378M 
                 ATG 
               
               
                 N047V 
                 GTG 
                 E113P 
                 CCT 
                 H179K 
                 AAG 
                 N245W 
                 TGG 
                 S312F 
                 TTT 
                 G378N 
                 AAT 
               
               
                 N047W 
                 TGG 
                 E113Q 
                 CAG 
                 H179L 
                 CTG 
                 R246A 
                 GCG 
                 S312G 
                 GGG 
                 G378Q 
                 CAG 
               
               
                 N047Y 
                 TAT 
                 E113R 
                 CGT 
                 H179M 
                 ATG 
                 R246C 
                 TGT 
                 S312H 
                 CAT 
                 G378R 
                 AGG 
               
               
                 A048C 
                 TGT 
                 E113S 
                 TCT 
                 H179N 
                 AAT 
                 R246D 
                 GAT 
                 S312K 
                 AAG 
                 G378S 
                 TCT 
               
               
                 A048E 
                 GAG 
                 E113T 
                 ACT 
                 H179P 
                 CCT 
                 R246E 
                 GAG 
                 S312L 
                 CTG 
                 G378T 
                 ACT 
               
               
                 A048F 
                 TTT 
                 E113V 
                 GTT 
                 H179R 
                 AGG 
                 R246G 
                 GGG 
                 S312M 
                 ATG 
                 G378V 
                 GTG 
               
               
                 A048G 
                 GGT 
                 E113W 
                 TGG 
                 H179S 
                 AGT 
                 R246H 
                 CAT 
                 S312N 
                 AAT 
                 G378W 
                 TGG 
               
               
                 A048H 
                 CAT 
                 E113Y 
                 CAT 
                 H179T 
                 ACT 
                 R246I 
                 ATT 
                 S312P 
                 CCT 
                 G378Y 
                 TAT 
               
               
                 A048I 
                 ATT 
                 E114A 
                 GCT 
                 H179V 
                 GTG 
                 R246K 
                 AAG 
                 S312Q 
                 CAG 
                 K379A 
                 GCT 
               
               
                 A048K 
                 AAG 
                 E114C 
                 TGT 
                 H179W 
                 TGG 
                 R246L 
                 TTG 
                 S312R 
                 CGG 
                 K379C 
                 TGT 
               
               
                 A048L 
                 CTG 
                 E114D 
                 GAT 
                 L180A 
                 GCT 
                 R246M 
                 ATG 
                 S312T 
                 ACT 
                 K379E 
                 GAG 
               
               
                 A048M 
                 ATG 
                 E114G 
                 GGG 
                 L180C 
                 TGT 
                 R246P 
                 CCT 
                 S312V 
                 GTT 
                 K379F 
                 TTT 
               
               
                 A048N 
                 AAT 
                 E114H 
                 CAT 
                 L180E 
                 GAG 
                 R246S 
                 AGT 
                 S312W 
                 TGG 
                 K379G 
                 GGG 
               
               
                 A048P 
                 CCT 
                 E114I 
                 ATT 
                 L180F 
                 TTT 
                 R246T 
                 ACG 
                 M313A 
                 GCT 
                 K379H 
                 CAT 
               
               
                 A048Q 
                 CAG 
                 E114L 
                 CTG 
                 L180G 
                 GGT 
                 R246V 
                 GTT 
                 M313C 
                 TGT 
                 K379I 
                 ATT 
               
               
                 A048R 
                 CGG 
                 E114M 
                 ATG 
                 L180H 
                 CAT 
                 R246W 
                 TGG 
                 M313D 
                 GAT 
                 K379L 
                 CTT 
               
               
                 A048S 
                 TCT 
                 E114P 
                 CCT 
                 L180I 
                 ATT 
                 V247A 
                 GCG 
                 M313E 
                 GAG 
                 K379M 
                 ATG 
               
               
                 A048V 
                 GTT 
                 E114R 
                 CGG 
                 L180K 
                 AAG 
                 V247C 
                 TGT 
                 M313F 
                 TTT 
                 K379N 
                 AAT 
               
               
                 A048W 
                 TGG 
                 E114S 
                 TCT 
                 L180M 
                 ATG 
                 V247F 
                 TTT 
                 M313G 
                 GGG 
                 K379R 
                 CGT 
               
               
                 A048Y 
                 TAT 
                 E114T 
                 ACT 
                 L180N 
                 AAT 
                 V247H 
                 CAT 
                 M313H 
                 CAT 
                 K379S 
                 TCT 
               
               
                 T049A 
                 GCG 
                 E114V 
                 GTG 
                 L180P 
                 CCT 
                 V247I 
                 ATT 
                 M313K 
                 AAG 
                 K379T 
                 ACT 
               
               
                 T049C 
                 TGT 
                 E114W 
                 TGG 
                 L180R 
                 AGG 
                 V247L 
                 CTG 
                 M313L 
                 CTT 
                 K379V 
                 GTT 
               
               
                 T049D 
                 GAT 
                 E114Y 
                 TAT 
                 L180S 
                 TCG 
                 V247M 
                 ATG 
                 M313P 
                 CCT 
                 K379W 
                 TGG 
               
               
                 T049F 
                 TTT 
                 W115A 
                 GCT 
                 L180T 
                 ACT 
                 V247N 
                 AAT 
                 M313R 
                 CGT 
                 F380A 
                 GCT 
               
               
                 T049G 
                 GGG 
                 W115C 
                 TGT 
                 L180W 
                 TGG 
                 V247P 
                 CCT 
                 M313S 
                 TCG 
                 F380C 
                 TGT 
               
               
                 T049H 
                 CAT 
                 W115D 
                 GAT 
                 W181A 
                 GCT 
                 V247Q 
                 CAG 
                 M313T 
                 ACT 
                 F380D 
                 GAT 
               
               
                 T049I 
                 ATT 
                 W115F 
                 TTT 
                 W181C 
                 TGT 
                 V247R 
                 CGT 
                 M313V 
                 GTT 
                 F380E 
                 GAG 
               
               
                 T049K 
                 AAG 
                 W115G 
                 GGT 
                 W181D 
                 GAT 
                 V247S 
                 TCT 
                 M313Y 
                 TAT 
                 F380G 
                 GGG 
               
               
                 T049L 
                 TTG 
                 W115H 
                 CAT 
                 W181E 
                 GAG 
                 V247T 
                 ACT 
                 K314A 
                 GCT 
                 F380I 
                 ATT 
               
               
                 T049N 
                 AAT 
                 W115I 
                 ATT 
                 W181F 
                 TTT 
                 V247W 
                 TGG 
                 K314C 
                 TGT 
                 F380L 
                 CTT 
               
               
                 T049P 
                 CCG 
                 W115K 
                 AAG 
                 W181H 
                 CAT 
                 V247Y 
                 TAT 
                 K314D 
                 GAT 
                 F380P 
                 CCT 
               
               
                 T049R 
                 AGG 
                 W115L 
                 CTT 
                 W181I 
                 ATT 
                 R248A 
                 GCT 
                 K314H 
                 CAT 
                 F380Q 
                 CAG 
               
               
                 T049S 
                 TCG 
                 W115M 
                 ATG 
                 W181K 
                 AAG 
                 R248C 
                 TGT 
                 K314I 
                 ATT 
                 F380R 
                 CGG 
               
               
                 T049V 
                 GTT 
                 W115P 
                 CCT 
                 W181L 
                 CTG 
                 R248D 
                 GAT 
                 K314L 
                 TTG 
                 F380S 
                 AGT 
               
               
                 T049W 
                 TGG 
                 W115R 
                 CGG 
                 W181M 
                 ATG 
                 R248E 
                 GAG 
                 K314N 
                 AAT 
                 F380T 
                 ACT 
               
               
                 G050A 
                 GCG 
                 W115S 
                 AGT 
                 W181N 
                 AAT 
                 R248G 
                 GGG 
                 K314P 
                 CCT 
                 F380V 
                 GTG 
               
               
                 G050C 
                 TGT 
                 W115V 
                 GTG 
                 W181Q 
                 CAG 
                 R248H 
                 CAT 
                 K314Q 
                 CAG 
                 F380W 
                 TGG 
               
               
                 G050D 
                 GAT 
                 W115Y 
                 TAT 
                 W181R 
                 CGT 
                 R248I 
                 ATT 
                 K314R 
                 CGG 
                 F380Y 
                 TAT 
               
               
                 G050E 
                 GAG 
                 R116A 
                 GCT 
                 W181S 
                 TCT 
                 R248L 
                 CTT 
                 K314S 
                 TCG 
                 T381A 
                 AGC 
               
               
                 G050F 
                 TTT 
                 R116C 
                 TGT 
                 W181V 
                 GTG 
                 R248M 
                 ATG 
                 K314T 
                 ACT 
                 T381E 
                 GAG 
               
               
                 G050H 
                 CAT 
                 R116D 
                 GAT 
                 G182A 
                 GCT 
                 R248P 
                 CCG 
                 K314V 
                 GTT 
                 T381F 
                 TTT 
               
               
                 G050L 
                 CTT 
                 R116E 
                 GAG 
                 G182C 
                 TGT 
                 R248S 
                 TCG 
                 K314W 
                 TGG 
                 T381G 
                 GGT 
               
               
                 G050M 
                 ATG 
                 R116G 
                 GGG 
                 G182D 
                 GAT 
                 R248T 
                 ACG 
                 K314Y 
                 TAT 
                 T381H 
                 CAT 
               
               
                 G050P 
                 CCT 
                 R116H 
                 CAT 
                 G182E 
                 GAG 
                 R248V 
                 GTG 
                 S315A 
                 GCT 
                 T381K 
                 AAG 
               
               
                 G050Q 
                 CAG 
                 R116I 
                 ATT 
                 G182H 
                 CAT 
                 R248W 
                 TGG 
                 S315C 
                 TGT 
                 T381L 
                 TTG 
               
               
                 G050R 
                 CGG 
                 R116L 
                 CTG 
                 G182L 
                 CTT 
                 R248Y 
                 TAT 
                 S315E 
                 GAG 
                 T381N 
                 AAT 
               
               
                 G050S 
                 AGT 
                 R116N 
                 AAT 
                 G182M 
                 ATG 
                 E249A 
                 GCT 
                 S315G 
                 GGT 
                 T381P 
                 CCT 
               
               
                 G050V 
                 GTT 
                 R116P 
                 CCT 
                 G182N 
                 AAT 
                 E249G 
                 GGG 
                 S315H 
                 CAT 
                 T381Q 
                 CAG 
               
               
                 G050W 
                 TGG 
                 R116Q 
                 CAG 
                 G182P 
                 CCT 
                 E249H 
                 CAT 
                 S315I 
                 ATT 
                 T381R 
                 CGT 
               
               
                 G050Y 
                 TAT 
                 R116S 
                 TCT 
                 G182Q 
                 CAG 
                 E249I 
                 ATT 
                 S315K 
                 AAG 
                 T381S 
                 AGT 
               
               
                 Q051A 
                 GCG 
                 R116T 
                 ACT 
                 G182R 
                 CGT 
                 E249K 
                 AAG 
                 S315L 
                 CTG 
                 T381V 
                 GTG 
               
               
                 Q051C 
                 TGT 
                 R116V 
                 GTG 
                 G182S 
                 AGT 
                 E249L 
                 CTG 
                 S315M 
                 ATG 
                 T381W 
                 TGG 
               
               
                 Q051D 
                 GAT 
                 R116W 
                 TGG 
                 G182T 
                 ACT 
                 E249M 
                 ATG 
                 S315P 
                 CCT 
                 T381Y 
                 TAT 
               
               
                 Q051F 
                 TTT 
                 P117D 
                 GAT 
                 G182V 
                 GTT 
                 E249P 
                 CCT 
                 S315R 
                 CGG 
                 V382E 
                 GAG 
               
               
                 Q051H 
                 CAT 
                 P117E 
                 GAG 
                 G182Y 
                 TAT 
                 E249Q 
                 CAG 
                 S315T 
                 ACT 
                 V382G 
                 GGG 
               
               
                 Q051I 
                 ATT 
                 P117F 
                 TTT 
                 Y183A 
                 GCT 
                 E249R 
                 CGG 
                 S315V 
                 GTT 
                 V382H 
                 CAT 
               
               
                 Q051K 
                 AAG 
                 P117G 
                 GGT 
                 Y183C 
                 TGT 
                 E249S 
                 TCT 
                 S315W 
                 TGG 
                 V382I 
                 ATT 
               
               
                 Q051M 
                 ATG 
                 P117H 
                 CAT 
                 Y183D 
                 GAT 
                 E249T 
                 ACT 
                 S315Y 
                 TAT 
                 V382K 
                 AAG 
               
               
                 Q051N 
                 AAT 
                 P117I 
                 ATT 
                 Y183E 
                 GAG 
                 E249V 
                 GTG 
                 C316A 
                 GCT 
                 V382L 
                 TTG 
               
               
                 Q051P 
                 CCT 
                 P117K 
                 AAG 
                 Y183G 
                 GGG 
                 E249W 
                 TGG 
                 C316D 
                 GAT 
                 V382M 
                 ATG 
               
               
                 Q051R 
                 CGG 
                 P117N 
                 AAT 
                 Y183I 
                 ATT 
                 E249Y 
                 TAT 
                 C316E 
                 GAG 
                 V382N 
                 AAT 
               
               
                 Q051S 
                 TCT 
                 P117Q 
                 CAG 
                 Y183K 
                 AAG 
                 A250C 
                 TGT 
                 C316G 
                 GGG 
                 V382P 
                 CCT 
               
               
                 Q051T 
                 ACG 
                 P117R 
                 AGG 
                 Y183L 
                 TTG 
                 A250F 
                 TTT 
                 C316I 
                 ATT 
                 V382Q 
                 CAG 
               
               
                 Q051W 
                 TGG 
                 P117S 
                 TCG 
                 Y183N 
                 AAT 
                 A250G 
                 GGT 
                 C316K 
                 AAG 
                 V382R 
                 CGG 
               
               
                 Q051Y 
                 TAT 
                 P117T 
                 ACT 
                 Y183P 
                 CCT 
                 A250H 
                 CAT 
                 C316L 
                 CTG 
                 V382S 
                 TCG 
               
               
                 G052A 
                 GCT 
                 P117V 
                 GTT 
                 Y183Q 
                 CAG 
                 A250K 
                 AAG 
                 C316M 
                 ATG 
                 V382T 
                 ACT 
               
               
                 G052C 
                 TGT 
                 P117W 
                 TGG 
                 Y183R 
                 CGT 
                 A250L 
                 CTG 
                 C316P 
                 CCT 
                 V382W 
                 TGG 
               
               
                 G052E 
                 GAG 
                 P117Y 
                 TAT 
                 Y183S 
                 TCT 
                 A250M 
                 ATG 
                 C316R 
                 AGG 
                 V382Y 
                 TAT 
               
               
                 G052F 
                 TTT 
                 T118C 
                 TGT 
                 Y183V 
                 GTT 
                 A250N 
                 AAT 
                 C316S 
                 TCT 
                 R383A 
                 GCT 
               
               
                 G052H 
                 CAT 
                 T118D 
                 GAT 
                 Y183W 
                 TGG 
                 A250P 
                 CCT 
                 C316T 
                 ACT 
                 R383E 
                 GAG 
               
               
                 G052K 
                 AAG 
                 T118E 
                 GAG 
                 Y184A 
                 GCT 
                 A250Q 
                 CAG 
                 C316V 
                 GTT 
                 R383F 
                 TTT 
               
               
                 G052L 
                 CTT 
                 T118G 
                 GGG 
                 Y184C 
                 TGT 
                 A250R 
                 AGG 
                 C316W 
                 TGG 
                 R383G 
                 GGG 
               
               
                 G052N 
                 AAT 
                 T118H 
                 CAT 
                 Y184D 
                 GAT 
                 A250S 
                 TCT 
                 C316Y 
                 TAT 
                 R383H 
                 CAT 
               
               
                 G052P 
                 CCT 
                 T118K 
                 AAG 
                 Y184E 
                 GAG 
                 A250T 
                 ACG 
                 L317A 
                 GCT 
                 R383I 
                 ATT 
               
               
                 G052Q 
                 CAG 
                 T118L 
                 CTG 
                 Y184F 
                 TTT 
                 A250V 
                 GTG 
                 L317C 
                 TGT 
                 R383K 
                 AAG 
               
               
                 G052R 
                 CGG 
                 T118M 
                 ATG 
                 Y184G 
                 GGT 
                 A250W 
                 TGG 
                 L317D 
                 GAT 
                 R383L 
                 CTG 
               
               
                 G052S 
                 AGT 
                 T118N 
                 AAT 
                 Y184H 
                 CAT 
                 I251C 
                 TGT 
                 L317G 
                 GGG 
                 R383M 
                 ATG 
               
               
                 G052T 
                 ACT 
                 T118P 
                 CCT 
                 Y184K 
                 AAG 
                 I251D 
                 GAT 
                 L317H 
                 CAT 
                 R383N 
                 AAT 
               
               
                 G052W 
                 TGG 
                 T118Q 
                 CAG 
                 Y184L 
                 CTT 
                 I251F 
                 TTT 
                 L317I 
                 ATT 
                 R383P 
                 CCT 
               
               
                 G052Y 
                 TAT 
                 T118R 
                 CGT 
                 Y184M 
                 ATG 
                 I251G 
                 GGG 
                 L317K 
                 AAG 
                 R383S 
                 TCG 
               
               
                 V053A 
                 GCG 
                 T118V 
                 GTT 
                 Y184P 
                 CCT 
                 I251H 
                 CAT 
                 L317M 
                 ATG 
                 R383T 
                 ACT 
               
               
                 V053C 
                 TGT 
                 T118W 
                 TGG 
                 Y184R 
                 AGG 
                 I251K 
                 AAG 
                 L317N 
                 AAT 
                 R383V 
                 GTG 
               
               
                 V053D 
                 GAT 
                 T118Y 
                 TAT 
                 Y184S 
                 TCG 
                 I251L 
                 CTT 
                 L317P 
                 CCT 
                 R383W 
                 TGG 
               
               
                 V053E 
                 GAG 
                 W119A 
                 GCT 
                 Y184V 
                 GTG 
                 I251M 
                 ATG 
                 L317Q 
                 CAG 
                 G384A 
                 GCT 
               
               
                 V053G 
                 GGG 
                 W119D 
                 GAT 
                 Y184W 
                 TGG 
                 I251P 
                 CCG 
                 L317R 
                 AGG 
                 G384C 
                 TGT 
               
               
                 V053H 
                 CAT 
                 W119E 
                 GAG 
                 L185A 
                 GCT 
                 I251Q 
                 CAG 
                 L317S 
                 TCG 
                 G384D 
                 GAT 
               
               
                 V053L 
                 CTG 
                 W119F 
                 TTT 
                 L185D 
                 GAT 
                 I251S 
                 AGT 
                 L317T 
                 ACT 
                 G384E 
                 GAG 
               
               
                 V053N 
                 AAT 
                 W119G 
                 GGT 
                 L185E 
                 GAG 
                 I251T 
                 ACT 
                 L317W 
                 TGG 
                 G384F 
                 TTT 
               
               
                 V053P 
                 CCG 
                 W119I 
                 ATT 
                 L185F 
                 TTT 
                 I251V 
                 GTG 
                 L318C 
                 TGT 
                 G384H 
                 CAT 
               
               
                 V053Q 
                 CAG 
                 W119K 
                 AAG 
                 L185G 
                 GGG 
                 I251W 
                 TGG 
                 L318D 
                 GAT 
                 G384I 
                 ATT 
               
               
                 V053R 
                 CGG 
                 W119L 
                 CTG 
                 L185I 
                 ATT 
                 I251Y 
                 TAT 
                 L318F 
                 TTT 
                 G384K 
                 AAG 
               
               
                 V053S 
                 AGT 
                 W119N 
                 AAT 
                 L185K 
                 AAG 
                 R252A 
                 GCT 
                 L318G 
                 GGG 
                 G384L 
                 CTT 
               
               
                 V053T 
                 ACT 
                 W119P 
                 CCT 
                 L185N 
                 AAT 
                 R252D 
                 GAT 
                 L318H 
                 CAT 
                 G384M 
                 ATG 
               
               
                 V053W 
                 TGG 
                 W119Q 
                 CAG 
                 L185P 
                 CCT 
                 R252E 
                 GAG 
                 L318I 
                 ATT 
                 G384P 
                 CCT 
               
               
                 V053Y 
                 TAT 
                 W119R 
                 CGG 
                 L185R 
                 CGG 
                 R252F 
                 TTT 
                 L318K 
                 AAG 
                 G384Q 
                 CAG 
               
               
                 T054A 
                 GCG 
                 W119S 
                 TCT 
                 L185S 
                 TCG 
                 R252G 
                 GGT 
                 L318M 
                 ATG 
                 G384R 
                 AGG 
               
               
                 T054D 
                 GAT 
                 W119V 
                 GTT 
                 L185T 
                 ACT 
                 R252H 
                 CAT 
                 L318N 
                 AAT 
                 G384S 
                 TCG 
               
               
                 T054E 
                 GAG 
                 W119Y 
                 TAT 
                 L185V 
                 GTG 
                 R252I 
                 ATT 
                 L318P 
                 CCT 
                 G384T 
                 ACT 
               
               
                 T054F 
                 TTT 
                 A120C 
                 TGT 
                 L185W 
                 TGG 
                 R252K 
                 AAG 
                 L318Q 
                 CAG 
                 K385A 
                 GCT 
               
               
                 T054G 
                 GGG 
                 A120D 
                 GAT 
                 L185Y 
                 TAT 
                 R252L 
                 CTG 
                 L318R 
                 CGG 
                 K385C 
                 TGT 
               
               
                 T054H 
                 CAT 
                 A120F 
                 TTT 
                 F186A 
                 GCT 
                 R252N 
                 AAT 
                 L318S 
                 AGT 
                 K385G 
                 GGG 
               
               
                 T054I 
                 ATT 
                 A120G 
                 GGG 
                 F186D 
                 GAT 
                 R252P 
                 CCT 
                 L318T 
                 ACT 
                 K385H 
                 CAT 
               
               
                 T054M 
                 ATG 
                 A120H 
                 CAT 
                 F186G 
                 GGT 
                 R252S 
                 TCG 
                 L318W 
                 TGG 
                 K385L 
                 CTT 
               
               
                 T054N 
                 AAT 
                 A120I 
                 ATT 
                 F186H 
                 CAT 
                 R252T 
                 ACT 
                 L319C 
                 TGT 
                 K385M 
                 ATG 
               
               
                 T054P 
                 CCG 
                 A120L 
                 CTT 
                 F186I 
                 ATT 
                 R252V 
                 GTG 
                 L319E 
                 GAG 
                 K385N 
                 AAT 
               
               
                 T054Q 
                 CAG 
                 A120N 
                 AAT 
                 F186K 
                 AAG 
                 R252Y 
                 TAT 
                 L319F 
                 TTT 
                 K385P 
                 CCG 
               
               
                 T054R 
                 CGT 
                 A120P 
                 CCT 
                 F186L 
                 CTT 
                 V253A 
                 GCG 
                 L319G 
                 GGG 
                 K385Q 
                 CAG 
               
               
                 T054S 
                 AGT 
                 A120R 
                 CGT 
                 F186N 
                 AAT 
                 V253D 
                 GAT 
                 L319H 
                 CAT 
                 K385R 
                 CGT 
               
               
                 T054V 
                 GTT 
                 A120S 
                 TCT 
                 F186P 
                 CCT 
                 V253E 
                 GAG 
                 L319I 
                 ATT 
                 K385S 
                 TCT 
               
               
                 T054Y 
                 TAT 
                 A120T 
                 ACT 
                 F186Q 
                 CAG 
                 V253G 
                 GGG 
                 L319K 
                 AAG 
                 K385T 
                 ACG 
               
               
                 I055A 
                 GCT 
                 A120V 
                 GTG 
                 F186R 
                 AGG 
                 V253H 
                 CAT 
                 L319M 
                 ATG 
                 K385V 
                 GTT 
               
               
                 I055C 
                 TGT 
                 A120W 
                 TGG 
                 F186S 
                 TCT 
                 V253I 
                 ATT 
                 L319P 
                 CCT 
                 K385W 
                 TGG 
               
               
                 I055D 
                 GAT 
                 A120Y 
                 TAT 
                 F186V 
                 GTT 
                 V253L 
                 CTG 
                 L319Q 
                 CAG 
                 K385Y 
                 TAT 
               
               
                 I055F 
                 TTT 
                 R121A 
                 GCT 
                 F186W 
                 TGG 
                 V253M 
                 ATG 
                 L319R 
                 AGG 
                 P386A 
                 GCG 
               
               
                 I055G 
                 GGG 
                 R121C 
                 TGT 
                 F186Y 
                 TAT 
                 V253N 
                 AAT 
                 L319S 
                 TCG 
                 P386C 
                 TGT 
               
               
                 I055H 
                 CAT 
                 R121D 
                 GAT 
                 P187A 
                 GCT 
                 V253P 
                 CCT 
                 L319V 
                 GTT 
                 P386F 
                 TTT 
               
               
                 I055L 
                 CTG 
                 R121E 
                 GAG 
                 P187F 
                 TTT 
                 V253Q 
                 CAG 
                 L319W 
                 TGG 
                 P386G 
                 GGG 
               
               
                 I055N 
                 AAT 
                 R121F 
                 TTT 
                 P187G 
                 GGG 
                 V253R 
                 CGG 
                 L319Y 
                 TAT 
                 P386H 
                 CAT 
               
               
                 I055P 
                 CCT 
                 R121G 
                 GGT 
                 P187H 
                 CAT 
                 V253S 
                 TCG 
                 D320C 
                 TGT 
                 P386I 
                 ATT 
               
               
                 I055Q 
                 CAG 
                 R121H 
                 CAT 
                 P187I 
                 ATT 
                 V253T 
                 ACG 
                 D320E 
                 GAG 
                 P386L 
                 CTT 
               
               
                 I055R 
                 CGT 
                 R121K 
                 AAG 
                 P187L 
                 CTT 
                 V253W 
                 TGG 
                 D320F 
                 TTT 
                 P386M 
                 ATG 
               
               
                 I055S 
                 TCG 
                 R121L 
                 CTG 
                 P187M 
                 ATG 
                 S254C 
                 TGT 
                 D320G 
                 GGG 
                 P386N 
                 AAT 
               
               
                 I055T 
                 ACT 
                 R121M 
                 ATG 
                 P187N 
                 AAT 
                 S254D 
                 GAT 
                 D320H 
                 CAT 
                 P386Q 
                 CAG 
               
               
                 I055V 
                 GTT 
                 R121P 
                 CCT 
                 P187Q 
                 CAG 
                 S254E 
                 GAG 
                 D320I 
                 ATT 
                 P386R 
                 CGT 
               
               
                 I055Y 
                 TAT 
                 R121S 
                 TCG 
                 P187R 
                 AGG 
                 S254G 
                 GGG 
                 D320K 
                 AAG 
                 P386S 
                 AGT 
               
               
                 F056A 
                 GCG 
                 R121T 
                 ACT 
                 P187S 
                 TCG 
                 S254I 
                 ATT 
                 D320L 
                 TTG 
                 P386T 
                 ACG 
               
               
                 F056C 
                 TGT 
                 R121V 
                 GTT 
                 P187T 
                 ACT 
                 S254K 
                 AAG 
                 D320M 
                 ATG 
                 P386V 
                 GTT 
               
               
                 F056E 
                 GAG 
                 R121W 
                 TGG 
                 P187V 
                 GTT 
                 S254L 
                 TTG 
                 D320N 
                 AAT 
                 P386Y 
                 TAT 
               
               
                 F056G 
                 GGG 
                 R121Y 
                 TAT 
                 P187W 
                 TGG 
                 S254N 
                 AAT 
                 D320P 
                 CCT 
                 T387C 
                 TGT 
               
               
                 F056H 
                 CAT 
                 N122A 
                 GCT 
                 P187Y 
                 TAT 
                 S254P 
                 CCT 
                 D320R 
                 AGG 
                 T387E 
                 GAG 
               
               
                 F056I 
                 ATT 
                 N122C 
                 TGT 
                 D188A 
                 GCT 
                 S254Q 
                 CAG 
                 D320S 
                 AGT 
                 T387F 
                 TTT 
               
               
                 F056K 
                 AAG 
                 N122E 
                 GAG 
                 D188C 
                 TGT 
                 S254R 
                 CGG 
                 D320V 
                 GTG 
                 T387G 
                 GGG 
               
               
                 F056L 
                 TTG 
                 N122F 
                 TTT 
                 D188F 
                 TTT 
                 S254T 
                 ACT 
                 D320W 
                 TGG 
                 T387H 
                 CAT 
               
               
                 F056N 
                 AAT 
                 N122I 
                 ATT 
                 D188G 
                 GGG 
                 S254V 
                 GTG 
                 D320Y 
                 TAT 
                 T387I 
                 ATT 
               
               
                 F056P 
                 CCG 
                 N122K 
                 AAG 
                 D188H 
                 CAT 
                 S254W 
                 TGG 
                 N321A 
                 GCT 
                 T387K 
                 AAG 
               
               
                 F056R 
                 CGT 
                 N122L 
                 CTG 
                 D188L 
                 CTT 
                 S254Y 
                 TAT 
                 N321D 
                 GAT 
                 T387L 
                 CTG 
               
               
                 F056S 
                 TCT 
                 N122M 
                 ATG 
                 D188M 
                 ATG 
                 K255A 
                 GCG 
                 N321E 
                 GAG 
                 T387M 
                 ATG 
               
               
                 F056T 
                 ACT 
                 N122P 
                 CCT 
                 D188N 
                 AAT 
                 K255C 
                 TGT 
                 N321G 
                 GGT 
                 T387N 
                 AAT 
               
               
                 F056V 
                 GTT 
                 N122Q 
                 CAG 
                 D188P 
                 CCT 
                 K255D 
                 GAT 
                 N321H 
                 CAT 
                 T387Q 
                 CAG 
               
               
                 F056W 
                 TGG 
                 N122R 
                 CGG 
                 D188Q 
                 CAG 
                 K255G 
                 GGT 
                 N321I 
                 ATT 
                 T387S 
                 TCG 
               
               
                 Y057A 
                 GCT 
                 N122S 
                 TCT 
                 D188R 
                 AGG 
                 K255H 
                 CAT 
                 N321K 
                 AAG 
                 T387V 
                 GTT 
               
               
                 Y057D 
                 GAT 
                 N122T 
                 ACT 
                 D188S 
                 AGT 
                 K255L 
                 TTG 
                 N321L 
                 CTG 
                 T387W 
                 TGG 
               
               
                 Y057E 
                 GAG 
                 N122V 
                 GTT 
                 D188T 
                 ACT 
                 K255N 
                 AAT 
                 N321M 
                 ATG 
                 T387Y 
                 TAT 
               
               
                 Y057F 
                 TTT 
                 N122W 
                 TGG 
                 D188V 
                 GTG 
                 K255P 
                 CCG 
                 N321P 
                 CCT 
                 L388A 
                 GCG 
               
               
                 Y057G 
                 GGG 
                 W123A 
                 GCT 
                 D188W 
                 TGG 
                 K255Q 
                 CAG 
                 N321R 
                 CGG 
                 L388C 
                 TGT 
               
               
                 Y057I 
                 ATT 
                 W123C 
                 TGT 
                 C189A 
                 GCT 
                 K255R 
                 CGG 
                 N321S 
                 TCT 
                 L388F 
                 TTT 
               
               
                 Y057L 
                 TTG 
                 W123D 
                 GAT 
                 C189E 
                 GAG 
                 K255S 
                 TCG 
                 N321T 
                 ACT 
                 L388G 
                 GGG 
               
               
                 Y057M 
                 ATG 
                 W123E 
                 GAG 
                 C189G 
                 GGT 
                 K255T 
                 ACT 
                 N321V 
                 GTG 
                 L388H 
                 CAT 
               
               
                 Y057P 
                 CCG 
                 W123G 
                 GGG 
                 C189H 
                 CAT 
                 K255V 
                 GTT 
                 N321Y 
                 TAT 
                 L388I 
                 ATT 
               
               
                 Y057Q 
                 CAG 
                 W123H 
                 CAT 
                 C189K 
                 AAG 
                 K255W 
                 TGG 
                 Y322C 
                 TGT 
                 L388M 
                 ATG 
               
               
                 Y057R 
                 CGG 
                 W123L 
                 CTT 
                 C189L 
                 TTG 
                 K255Y 
                 TAT 
                 Y322D 
                 GAT 
                 L388P 
                 CCT 
               
               
                 Y057S 
                 AGT 
                 W123M 
                 ATG 
                 C189M 
                 ATG 
                 I256A 
                 GCT 
                 Y322E 
                 GAG 
                 L388Q 
                 CAG 
               
               
                 Y057T 
                 ACG 
                 W123P 
                 CCT 
                 C189N 
                 ACT 
                 I256C 
                 TGT 
                 Y322F 
                 TTT 
                 L388R 
                 CGT 
               
               
                 Y057V 
                 GTG 
                 W123Q 
                 CAG 
                 C189P 
                 CCT 
                 I256D 
                 GAT 
                 Y322G 
                 GGT 
                 L388S 
                 TCG 
               
               
                 Y057W 
                 TGG 
                 W123R 
                 AGG 
                 C189R 
                 AGG 
                 I256E 
                 GAG 
                 Y322H 
                 CAT 
                 L388T 
                 ACG 
               
               
                 V058A 
                 GCT 
                 W123S 
                 AGT 
                 C189S 
                 TCG 
                 I256G 
                 GGG 
                 Y322I 
                 ATT 
                 L388V 
                 GTT 
               
               
                 V058C 
                 TGT 
                 W123T 
                 ACT 
                 C189T 
                 ACT 
                 I256H 
                 CAT 
                 Y322L 
                 CTG 
                 L388W 
                 TGG 
               
               
                 V058D 
                 GAT 
                 W123V 
                 GTT 
                 C189V 
                 GTG 
                 I256L 
                 CTT 
                 Y322N 
                 AAT 
                 L388Y 
                 TAT 
               
               
                 V058G 
                 GGT 
                 W123Y 
                 TAT 
                 C189W 
                 TGG 
                 I256M 
                 ATG 
                 Y322P 
                 CCT 
                 E389A 
                 GCT 
               
               
                 V058H 
                 CAT 
                 K124A 
                 GCT 
                 C189Y 
                 TAT 
                 I256N 
                 AAT 
                 Y322R 
                 CGT 
                 E389F 
                 TTT 
               
               
                 V058I 
                 ATT 
                 K124C 
                 TGT 
                 Y190C 
                 TGT 
                 I256P 
                 CCG 
                 Y322S 
                 TCT 
                 E389G 
                 GGT 
               
               
                 V058K 
                 AAG 
                 K124D 
                 GAT 
                 Y190E 
                 GAG 
                 I256Q 
                 CAG 
                 Y322T 
                 ACT 
                 E389H 
                 CAT 
               
               
                 V058L 
                 CTT 
                 K124E 
                 GAG 
                 Y190F 
                 TTT 
                 I256R 
                 AGG 
                 Y322V 
                 GTG 
                 E389I 
                 ATT 
               
               
                 V058N 
                 AAT 
                 K124F 
                 TTT 
                 Y190G 
                 GGG 
                 I256T 
                 ACG 
                 Y322W 
                 TGG 
                 E389K 
                 AAG 
               
               
                 V058P 
                 CCT 
                 K124G 
                 GGG 
                 Y190H 
                 CAT 
                 I256V 
                 GTT 
                 M323A 
                 GCT 
                 E389L 
                 CTG 
               
               
                 V058Q 
                 CAG 
                 K124H 
                 CAT 
                 Y190K 
                 AAG 
                 I256W 
                 TGG 
                 M323C 
                 TGT 
                 E389M 
                 ATG 
               
               
                 V058R 
                 CGG 
                 K124I 
                 ATT 
                 Y190L 
                 CTT 
                 P257A 
                 GCG 
                 M323E 
                 GAG 
                 E389P 
                 CCT 
               
               
                 V058S 
                 TCG 
                 K124L 
                 CTT 
                 Y190N 
                 AAT 
                 P257C 
                 TGT 
                 M323F 
                 TTT 
                 E389Q 
                 CAG 
               
               
                 V058W 
                 TGG 
                 K124N 
                 AAT 
                 Y190P 
                 CCT 
                 P257D 
                 GAT 
                 M323G 
                 GGG 
                 E389R 
                 CGG 
               
               
                 V058Y 
                 TAT 
                 K124P 
                 CCT 
                 Y190Q 
                 CAG 
                 P257G 
                 GGG 
                 M323H 
                 CAT 
                 E389S 
                 TCG 
               
               
                 D059A 
                 GCT 
                 K124R 
                 CGG 
                 Y190R 
                 CGT 
                 P257I 
                 ATT 
                 M323I 
                 ATT 
                 E389T 
                 ACT 
               
               
                 D059E 
                 GAG 
                 K124S 
                 TCT 
                 Y190S 
                 TCT 
                 P257K 
                 AAG 
                 M323K 
                 AAG 
                 E389V 
                 GTT 
               
               
                 D059G 
                 GGG 
                 K124T 
                 ACT 
                 Y190T 
                 ACT 
                 P257L 
                 CTT 
                 M323L 
                 TTG 
                 E389Y 
                 TAT 
               
               
                 D059H 
                 CAT 
                 K124V 
                 GTG 
                 Y190V 
                 GTG 
                 P257M 
                 ATG 
                 M323N 
                 AAT 
                 D390A 
                 GCG 
               
               
                 D059I 
                 ATT 
                 K124W 
                 TGG 
                 Y190W 
                 TGG 
                 P257N 
                 AAT 
                 M323P 
                 CCT 
                 D390C 
                 TGT 
               
               
                 D059L 
                 CTT 
                 P125A 
                 GCT 
                 N191A 
                 GCT 
                 P257Q 
                 CAG 
                 M323R 
                 CGG 
                 D390E 
                 GAG 
               
               
                 D059M 
                 ATG 
                 P125C 
                 TGT 
                 N191E 
                 GAG 
                 P257R 
                 CGT 
                 M323S 
                 AGT 
                 D390F 
                 TTT 
               
               
                 D059N 
                 AAT 
                 P125D 
                 GAT 
                 N191F 
                 TTT 
                 P257S 
                 TCG 
                 M323T 
                 ACT 
                 D390G 
                 GGG 
               
               
                 D059P 
                 CCT 
                 P125G 
                 GGG 
                 N191G 
                 GGG 
                 P257T 
                 ACG 
                 M323V 
                 GTT 
                 D390H 
                 CAT 
               
               
                 D059Q 
                 CAG 
                 P125H 
                 CAT 
                 N191K 
                 AAG 
                 P257V 
                 GTG 
                 E324A 
                 GCT 
                 D390L 
                 CTT 
               
               
                 D059R 
                 CGT 
                 P125I 
                 ATT 
                 N191L 
                 TTG 
                 P257W 
                 TGG 
                 E324C 
                 TGT 
                 D390N 
                 AAT 
               
               
                 D059T 
                 ACG 
                 P125L 
                 CTT 
                 N191M 
                 ATG 
                 D258A 
                 GCG 
                 E324D 
                 GAT 
                 D390P 
                 CCG 
               
               
                 D059V 
                 GTG 
                 P125N 
                 AAT 
                 N191P 
                 CCT 
                 D258E 
                 GAG 
                 E324F 
                 TTT 
                 D390R 
                 CGG 
               
               
                 D059W 
                 TGG 
                 P125Q 
                 CAG 
                 N191Q 
                 CAG 
                 D258G 
                 GGG 
                 E324G 
                 GGG 
                 D390S 
                 AGT 
               
               
                 D059Y 
                 TAT 
                 P125R 
                 CGT 
                 N191R 
                 CGG 
                 D258H 
                 CAT 
                 E324H 
                 CAT 
                 D390T 
                 ACT 
               
               
                 R060A 
                 GCG 
                 P125S 
                 TCG 
                 N191S 
                 TCG 
                 D258I 
                 ATT 
                 E324L 
                 TTG 
                 D390V 
                 GTG 
               
               
                 R060D 
                 GAT 
                 P125T 
                 ACT 
                 N191T 
                 ACT 
                 D258L 
                 CTT 
                 E324M 
                 ATG 
                 D390W 
                 TGG 
               
               
                 R060F 
                 TTT 
                 P125V 
                 GTG 
                 N191V 
                 GTT 
                 D258N 
                 AAT 
                 E324N 
                 AAT 
                 D390Y 
                 TAT 
               
               
                 R060G 
                 GGT 
                 P125W 
                 TGG 
                 N191W 
                 TGG 
                 D258P 
                 CCG 
                 E324P 
                 CCT 
                 L391A 
                 GCT 
               
               
                 R060H 
                 CAT 
                 P125Y 
                 TAT 
                 N191Y 
                 TAT 
                 D258Q 
                 CAG 
                 E324R 
                 CGG 
                 L391C 
                 TGT 
               
               
                 R060I 
                 ATT 
                 K126A 
                 GCT 
                 H192C 
                 TGT 
                 D258R 
                 CGT 
                 E324S 
                 AGT 
                 L391D 
                 GAT 
               
               
                 R060K 
                 AAG 
                 K126D 
                 GAT 
                 H192F 
                 TTT 
                 D258S 
                 AGT 
                 E324V 
                 GTG 
                 L391G 
                 GGG 
               
               
                 R060L 
                 CTT 
                 K126E 
                 GAG 
                 H192G 
                 GGT 
                 D258T 
                 ACG 
                 E324W 
                 TGG 
                 L391H 
                 CAT 
               
               
                 R060N 
                 AAT 
                 K126F 
                 TTT 
                 H192K 
                 AAG 
                 D258V 
                 GTG 
                 E324Y 
                 TAT 
                 L391K 
                 AAG 
               
               
                 R060P 
                 CCG 
                 K126G 
                 GGT 
                 H192L 
                 CTT 
                 D258W 
                 TGG 
                 T325A 
                 GCT 
                 L391N 
                 AAT 
               
               
                 R060Q 
                 CAG 
                 K126H 
                 CAT 
                 H192M 
                 ATG 
                 D258Y 
                 TAT 
                 T325C 
                 TGT 
                 L391P 
                 CCT 
               
               
                 R060S 
                 TCG 
                 K126I 
                 ATT 
                 H192N 
                 AAT 
                 A259E 
                 GAG 
                 T325D 
                 GAT 
                 L391Q 
                 CAG 
               
               
                 R060T 
                 ACG 
                 K126L 
                 CTG 
                 H192P 
                 CCT 
                 A259G 
                 GGG 
                 T325E 
                 GAG 
                 L391R 
                 CGG 
               
               
                 R060V 
                 GTT 
                 K126M 
                 ATG 
                 H192Q 
                 CAG 
                 A259I 
                 ATT 
                 T325G 
                 GGT 
                 L391S 
                 TCT 
               
               
                 R060Y 
                 TAT 
                 K126N 
                 AAT 
                 H192R 
                 CGT 
                 A259K 
                 AAG 
                 T325H 
                 CAT 
                 L391T 
                 ACT 
               
               
                 L061A 
                 GCT 
                 K126P 
                 CCT 
                 H192S 
                 TCG 
                 A259L 
                 TTG 
                 T325I 
                 ATT 
                 L391V 
                 GTG 
               
               
                 L061E 
                 GAG 
                 K126Q 
                 CAG 
                 H192T 
                 ACT 
                 A259M 
                 ATG 
                 T325K 
                 AAG 
                 L391W 
                 TGG 
               
               
                 L061F 
                 TTT 
                 K126R 
                 AGG 
                 H192V 
                 GTT 
                 A259N 
                 AAT 
                 T325M 
                 ATG 
                 L391Y 
                 TAT 
               
               
                 L061G 
                 GGG 
                 K126S 
                 TCT 
                 H192W 
                 TGG 
                 A259P 
                 CCT 
                 T325N 
                 AAT 
                 E392A 
                 GCT 
               
               
                 L061H 
                 CAT 
                 K126T 
                 ACT 
                 H192Y 
                 TAT 
                 A259Q 
                 CAG 
                 T325Q 
                 CAG 
                 E392C 
                 TGT 
               
               
                 L061I 
                 ATT 
                 K126V 
                 GTG 
                 H193A 
                 GCT 
                 A259R 
                 CGT 
                 T325R 
                 CGG 
                 E392F 
                 TTT 
               
               
                 L061M 
                 ATG 
                 K126W 
                 TGG 
                 H193C 
                 TGT 
                 A259S 
                 AGT 
                 T325S 
                 TCG 
                 E392G 
                 GGG 
               
               
                 L061N 
                 AAT 
                 K126Y 
                 TAT 
                 H193D 
                 GAT 
                 A259T 
                 ACT 
                 T325V 
                 GTG 
                 E392K 
                 AAG 
               
               
                 L061P 
                 CCT 
                 D127A 
                 GCT 
                 H193F 
                 TTT 
                 A259V 
                 GTG 
                 T325W 
                 TGG 
                 E392L 
                 CTG 
               
               
                 L061Q 
                 CAG 
                 D127E 
                 GAG 
                 H193G 
                 GGG 
                 A259W 
                 TGG 
                 I326A 
                 GCT 
                 E392M 
                 ATG 
               
               
                 L061R 
                 AGG 
                 D127F 
                 TTT 
                 H193K 
                 AAG 
                 A259Y 
                 TAT 
                 I326C 
                 TGT 
                 E392P 
                 CCT 
               
               
                 L061T 
                 ACT 
                 D127G 
                 GGT 
                 H193L 
                 TTG 
                 K260A 
                 GCG 
                 I326D 
                 GAT 
                 E392Q 
                 CAG 
               
               
                 L061V 
                 GTT 
                 D127H 
                 CAT 
                 H193M 
                 ATG 
                 K260C 
                 TGT 
                 I326E 
                 GAG 
                 E392R 
                 AGG 
               
               
                 L061W 
                 TGG 
                 D127K 
                 AAG 
                 H193P 
                 CCG 
                 K260D 
                 GAT 
                 I326G 
                 GGG 
                 E392S 
                 AGT 
               
               
                 L061Y 
                 TAT 
                 D127L 
                 CTG 
                 H193Q 
                 CAG 
                 K260E 
                 GAG 
                 I326H 
                 CAT 
                 E392T 
                 ACT 
               
               
                 G062A 
                 GCG 
                 D127M 
                 ATG 
                 H193R 
                 AGG 
                 K260G 
                 GGG 
                 I326K 
                 AAG 
                 E392V 
                 GTT 
               
               
                 G062C 
                 TGT 
                 D127N 
                 AAT 
                 H193S 
                 TCT 
                 K260H 
                 CAT 
                 I326L 
                 CTT 
                 E392W 
                 TGG 
               
               
                 G062D 
                 GAT 
                 D127Q 
                 CAG 
                 H193T 
                 ACG 
                 K260L 
                 TTG 
                 I326N 
                 AAT 
                 E392Y 
                 TAT 
               
               
                 G062F 
                 TTT 
                 D127R 
                 CGT 
                 H193V 
                 GTG 
                 K260M 
                 ATG 
                 I326P 
                 CCT 
                 Q393A 
                 GCG 
               
               
                 G062I 
                 ATT 
                 D127S 
                 AGT 
                 H193Y 
                 TAT 
                 K260P 
                 CCG 
                 I326R 
                 CGG 
                 Q393C 
                 TGT 
               
               
                 G062K 
                 AAG 
                 D127T 
                 ACT 
                 Y194A 
                 GCT 
                 K260Q 
                 CAG 
                 I326S 
                 TCT 
                 Q393D 
                 GAT 
               
               
                 G062L 
                 CTT 
                 D127V 
                 GTT 
                 Y194C 
                 TGT 
                 K260R 
                 CGG 
                 I326V 
                 GTG 
                 Q393F 
                 TTT 
               
               
                 G062M 
                 ATG 
                 D127W 
                 TGG 
                 Y194E 
                 GAG 
                 K260S 
                 TCT 
                 I326W 
                 TGG 
                 Q393G 
                 GGT 
               
               
                 G062P 
                 CCT 
                 V128A 
                 GCT 
                 Y194F 
                 TTT 
                 K260V 
                 GTT 
                 I326Y 
                 TAT 
                 Q393H 
                 CAT 
               
               
                 G062Q 
                 CAG 
                 V128C 
                 TGT 
                 Y194G 
                 GGG 
                 K260W 
                 TGG 
                 L327A 
                 GCT 
                 Q393I 
                 ATT 
               
               
                 G062R 
                 CGT 
                 V128E 
                 GAG 
                 Y194I 
                 ATT 
                 K260Y 
                 TAT 
                 L327D 
                 GAT 
                 Q393K 
                 AAG 
               
               
                 G062S 
                 AGT 
                 V128F 
                 TTT 
                 Y194L 
                 TTG 
                 S261A 
                 GCG 
                 L327E 
                 GAG 
                 Q393L 
                 TTG 
               
               
                 G062T 
                 ACT 
                 V128G 
                 GGG 
                 Y194N 
                 AAT 
                 S261E 
                 GAG 
                 L327F 
                 TTT 
                 Q393M 
                 ATG 
               
               
                 G062V 
                 GTG 
                 V128H 
                 CAT 
                 Y194P 
                 CCT 
                 S261F 
                 TTT 
                 L327G 
                 GGG 
                 Q393N 
                 AAT 
               
               
                 G062Y 
                 TAT 
                 V128I 
                 ATT 
                 Y194Q 
                 CAG 
                 S261G 
                 GGG 
                 L327H 
                 CAT 
                 Q393P 
                 CCG 
               
               
                 Y063A 
                 GCG 
                 V128K 
                 AAG 
                 Y194R 
                 AGG 
                 S261I 
                 ATT 
                 L327M 
                 ATG 
                 Q393R 
                 CGT 
               
               
                 Y063C 
                 TGT 
                 V128L 
                 CTG 
                 Y194S 
                 TCG 
                 S261K 
                 AAG 
                 L327N 
                 AAT 
                 Q393S 
                 TCG 
               
               
                 Y063G 
                 GGT 
                 V128P 
                 CCT 
                 Y194T 
                 ACG 
                 S261L 
                 CTT 
                 L327Q 
                 CAG 
                 Q393T 
                 ACG 
               
               
                 Y063H 
                 CAT 
                 V128Q 
                 CAG 
                 Y194V 
                 GTG 
                 S261M 
                 ATG 
                 L327R 
                 CGG 
                 F394A 
                 GCG 
               
               
                 Y063I 
                 ATT 
                 V128R 
                 AGG 
                 Y194W 
                 TGG 
                 S261N 
                 AAT 
                 L327S 
                 AGT 
                 F394D 
                 GAT 
               
               
                 Y063K 
                 AAG 
                 V128S 
                 TCG 
                 K195A 
                 GCG 
                 S261P 
                 CCT 
                 L327T 
                 ACT 
                 F394E 
                 GAG 
               
               
                 Y063L 
                 CTG 
                 V128W 
                 TGG 
                 K195E 
                 GAG 
                 S261Q 
                 CAG 
                 L327V 
                 GTG 
                 F394G 
                 GGG 
               
               
                 Y063M 
                 ATG 
                 V128Y 
                 TAT 
                 K195F 
                 TTT 
                 S261R 
                 CGT 
                 L327W 
                 TGG 
                 F394I 
                 ATT 
               
               
                 Y063N 
                 AAT 
                 Y129A 
                 GCT 
                 K195G 
                 GGT 
                 S261T 
                 ACT 
                 L327Y 
                 TAT 
                 F394K 
                 AAG 
               
               
                 Y063P 
                 CCT 
                 Y129C 
                 TGT 
                 K195H 
                 CAT 
                 S261V 
                 GTT 
                 N328A 
                 GCT 
                 F394L 
                 CTG 
               
               
                 Y063R 
                 AGG 
                 Y129D 
                 GAT 
                 K195I 
                 ATT 
                 S261W 
                 TGG 
                 N328C 
                 TGT 
                 F394N 
                 AAT 
               
               
                 Y063S 
                 TCT 
                 Y129E 
                 GAG 
                 K195L 
                 TTG 
                 P262A 
                 GCG 
                 N328D 
                 GAT 
                 F394P 
                 CCG 
               
               
                 Y063T 
                 ACG 
                 Y129G 
                 GGG 
                 K195N 
                 AAT 
                 P262D 
                 GAT 
                 N328G 
                 GGT 
                 F394Q 
                 CAG 
               
               
                 Y063V 
                 GTG 
                 Y129H 
                 CAT 
                 K195Q 
                 CAG 
                 P262E 
                 GAG 
                 N328H 
                 CAT 
                 F394R 
                 CGT 
               
               
                 Y063W 
                 TGG 
                 Y129L 
                 TTG 
                 K195R 
                 CGT 
                 P262F 
                 TTT 
                 N328I 
                 ATT 
                 F394S 
                 TCG 
               
               
                 Y064A 
                 GCT 
                 Y129M 
                 ATG 
                 K195S 
                 TCT 
                 P262G 
                 GGG 
                 N328K 
                 AAG 
                 F394T 
                 ACT 
               
               
                 Y064C 
                 TGT 
                 Y129P 
                 CCT 
                 K195T 
                 ACT 
                 P262H 
                 CAT 
                 N328L 
                 CTT 
                 F394V 
                 GTT 
               
               
                 Y064D 
                 GAT 
                 Y129Q 
                 CAG 
                 K195V 
                 GTG 
                 P262I 
                 ATT 
                 N328Q 
                 CAG 
                 F394W 
                 TGG 
               
               
                 Y064E 
                 GAG 
                 Y129R 
                 CGG 
                 K195W 
                 TGG 
                 P262K 
                 AAG 
                 N328R 
                 AGG 
                 S395A 
                 GCG 
               
               
                 Y064F 
                 TTT 
                 Y129S 
                 AGT 
                 K195Y 
                 TAT 
                 P262Q 
                 CAG 
                 N328S 
                 AGT 
                 S395C 
                 TGT 
               
               
                 Y064G 
                 GGT 
                 Y129T 
                 ACT 
                 K196A 
                 GCT 
                 P262R 
                 CGT 
                 N328T 
                 ACT 
                 S395D 
                 GAT 
               
               
                 Y064H 
                 CAT 
                 Y129V 
                 GTT 
                 K196C 
                 TGT 
                 P262S 
                 TCT 
                 N328V 
                 GTG 
                 S395E 
                 GAG 
               
               
                 Y064I 
                 ATT 
                 Y129W 
                 TGG 
                 K196D 
                 GAT 
                 P262T 
                 ACT 
                 N328W 
                 TGG 
                 S395G 
                 GGG 
               
               
                 Y064K 
                 AAG 
                 K130C 
                 TGT 
                 K196E 
                 GAG 
                 P262V 
                 GTG 
                 N328Y 
                 TAT 
                 S395H 
                 CAT 
               
               
                 Y064L 
                 CTT 
                 K130D 
                 GAT 
                 K196G 
                 GGG 
                 P262W 
                 TGG 
                 P329C 
                 TGT 
                 S395K 
                 AAG 
               
               
                 Y064P 
                 CCT 
                 K130E 
                 GAG 
                 K196I 
                 ATT 
                 P262Y 
                 TAT 
                 P329F 
                 TTT 
                 S395L 
                 CTT 
               
               
                 Y064Q 
                 CAG 
                 K130G 
                 GGG 
                 K196L 
                 TTG 
                 L263A 
                 GCT 
                 P329G 
                 GGT 
                 S395M 
                 ATG 
               
               
                 Y064R 
                 CGG 
                 K130H 
                 CAT 
                 K196N 
                 AAT 
                 L263E 
                 GAG 
                 P329H 
                 CAT 
                 S395P 
                 CCT 
               
               
                 Y064S 
                 AGT 
                 K130I 
                 ATT 
                 K196P 
                 CCG 
                 L263F 
                 TTT 
                 P329I 
                 ATT 
                 S395R 
                 CGG 
               
               
                 Y064T 
                 ACT 
                 K130L 
                 TTG 
                 K196R 
                 CGT 
                 L263G 
                 GGG 
                 P329K 
                 AAG 
                 S395T 
                 ACG 
               
               
                 Y064V 
                 GTT 
                 K130N 
                 AAT 
                 K196S 
                 TCG 
                 L263H 
                 CAT 
                 P329L 
                 CTG 
                 S395V 
                 GTT 
               
               
                 Y064W 
                 TGG 
                 K130Q 
                 CAG 
                 K196T 
                 ACT 
                 L263K 
                 AAG 
                 P329N 
                 AAT 
                 S395W 
                 TGG 
               
               
                 P065A 
                 GCT 
                 K130R 
                 AGG 
                 K196V 
                 GTG 
                 L263M 
                 ATG 
                 P329Q 
                 CAG 
                 S395Y 
                 TAT 
               
               
                 P065C 
                 TGT 
                 K130S 
                 TCT 
                 K196W 
                 TGG 
                 L263N 
                 AAT 
                 P329R 
                 CGT 
                 E396A 
                 GCG 
               
               
                 P065D 
                 GAT 
                 K130T 
                 ACT 
                 K196Y 
                 TAT 
                 L263P 
                 CCG 
                 P329S 
                 AGT 
                 E396C 
                 TGT 
               
               
                 P065F 
                 TTT 
                 K130V 
                 GTG 
                 P197A 
                 GCT 
                 L263Q 
                 CAG 
                 P329T 
                 ACT 
                 E396D 
                 GAT 
               
               
                 P065G 
                 GGG 
                 K130W 
                 TGG 
                 P197C 
                 TGT 
                 L263R 
                 CGG 
                 P329V 
                 GTT 
                 E396F 
                 TTT 
               
               
                 P065H 
                 CAT 
                 K130Y 
                 TAT 
                 P197D 
                 GAT 
                 L263S 
                 AGT 
                 P329W 
                 TGG 
                 E396G 
                 GGG 
               
               
                 P065I 
                 ATT 
                 N131C 
                 TGT 
                 P197E 
                 GAG 
                 L263T 
                 ACT 
                 P329Y 
                 TAT 
                 E396H 
                 CAT 
               
               
                 P065K 
                 AAG 
                 N131E 
                 GAG 
                 P197F 
                 TTT 
                 L263V 
                 GTT 
                 Y330A 
                 GCT 
                 E396I 
                 ATT 
               
               
                 P065N 
                 AAT 
                 N131F 
                 TTT 
                 P197G 
                 GGT 
                 L263W 
                 TGG 
                 Y330C 
                 TGT 
                 E396L 
                 CTT 
               
               
                 P065R 
                 CGG 
                 N131G 
                 GGG 
                 P197H 
                 CAT 
                 P264A 
                 GCG 
                 Y330D 
                 GAT 
                 E396P 
                 CCG 
               
               
                 P065S 
                 TCG 
                 N131H 
                 CAT 
                 P197K 
                 AAG 
                 P264D 
                 GAT 
                 Y330E 
                 GAG 
                 E396Q 
                 CAG 
               
               
                 P065T 
                 ACG 
                 N131I 
                 ATT 
                 P197L 
                 TTG 
                 P264E 
                 GAG 
                 Y330F 
                 TTT 
                 E396R 
                 AGG 
               
               
                 P065V 
                 GTT 
                 N131L 
                 CTT 
                 P197M 
                 ATG 
                 P264F 
                 TTT 
                 Y330G 
                 GGT 
                 E396S 
                 TCT 
               
               
                 P065W 
                 TGG 
                 N131M 
                 ATG 
                 P197Q 
                 CAG 
                 P264G 
                 GGT 
                 Y330I 
                 ATT 
                 E396T 
                 ACT 
               
               
                 P065Y 
                 TAT 
                 N131P 
                 CCT 
                 P197R 
                 CGT 
                 P264H 
                 CAT 
                 Y330L 
                 CTG 
                 E396V 
                 GTG 
               
               
                 Y066A 
                 GCG 
                 N131Q 
                 CAG 
                 P197S 
                 AGT 
                 P264L 
                 CTT 
                 Y330M 
                 ATG 
                 E396Y 
                 TAT 
               
               
                 Y066C 
                 TGT 
                 N131R 
                 CGG 
                 P197T 
                 ACT 
                 P264M 
                 ATG 
                 Y330N 
                 AAT 
                 K397A 
                 GCT 
               
               
                 Y066D 
                 GAT 
                 N131S 
                 AGT 
                 P197W 
                 TGG 
                 P264N 
                 AAT 
                 Y330P 
                 CCT 
                 K397C 
                 TGT 
               
               
                 Y066E 
                 GAG 
                 N131T 
                 ACT 
                 G198A 
                 GCT 
                 P264R 
                 CGG 
                 Y330R 
                 AGG 
                 K397E 
                 GAG 
               
               
                 Y066G 
                 GGT 
                 N131V 
                 GTG 
                 G198C 
                 TGT 
                 P264S 
                 AGT 
                 Y330S 
                 AGT 
                 K397F 
                 TTT 
               
               
                 Y066H 
                 CAT 
                 N131Y 
                 TAT 
                 G198D 
                 GAT 
                 P264T 
                 ACT 
                 Y330V 
                 GTT 
                 K397G 
                 GGT 
               
               
                 Y066I 
                 ATT 
                 R132A 
                 GCT 
                 G198E 
                 GAG 
                 P264V 
                 GTT 
                 I331V 
                 GTG 
                 K397I 
                 ATT 
               
               
                 Y066K 
                 AAG 
                 R132C 
                 TGT 
                 G198H 
                 CAT 
                 P264W 
                 TGG 
                 Y330W 
                 TGG 
                 K397L 
                 TTG 
               
               
                 Y066L 
                 CTG 
                 R132E 
                 GAG 
                 G198L 
                 CTG 
                 P264Y 
                 TAT 
                 I331A 
                 GCT 
                 K397M 
                 ATG 
               
               
                 Y066N 
                 AAT 
                 R132F 
                 TTT 
                 G198N 
                 AAT 
                 V265A 
                 GCG 
                 I331C 
                 TGT 
                 K397N 
                 AAT 
               
               
                 Y066P 
                 CCT 
                 R132H 
                 CAT 
                 G198P 
                 CCG 
                 V265C 
                 TGT 
                 I331D 
                 GAT 
                 K397P 
                 CCG 
               
               
                 Y066R 
                 CGG 
                 R132I 
                 ATT 
                 G198Q 
                 CAG 
                 V265D 
                 GAT 
                 I331E 
                 GAG 
                 K397Q 
                 CAG 
               
               
                 K397T 
                 ACT 
                 R132K 
                 AAG 
                 G198R 
                 AGG 
                 V265E 
                 GAG 
                 I331F 
                 TTT 
                 K397R 
                 AGG 
               
               
                 K397V 
                 GTT 
                 R132L 
                 TTG 
                 G198S 
                 TCT 
                 V265F 
                 TTT 
                 I331H 
                 CAT 
                 K397S 
                 TCG 
               
               
                 F398A 
                 GCT 
                 L406P 
                 CCT 
                 K415G 
                 GGT 
                 C423T 
                 ACT 
                 A432L 
                 TTG 
                 E441D 
                 GAT 
               
               
                 F398C 
                 TGT 
                 L406Q 
                 CAG 
                 K415L 
                 CTG 
                 C423V 
                 GTG 
                 A432M 
                 ATG 
                 E441F 
                 TTT 
               
               
                 F398E 
                 GAG 
                 L406R 
                 CGG 
                 K415M 
                 ATG 
                 C423W 
                 TGG 
                 A432N 
                 AAT 
                 E441G 
                 GGG 
               
               
                 F398G 
                 GGT 
                 L406S 
                 AGT 
                 K415P 
                 CCG 
                 I424A 
                 GCT 
                 A432P 
                 CCT 
                 E441H 
                 CAT 
               
               
                 F398H 
                 CAT 
                 L406T 
                 ACG 
                 K415Q 
                 CAG 
                 I424C 
                 TGT 
                 A432R 
                 AGG 
                 E441K 
                 AAG 
               
               
                 F398I 
                 ATT 
                 L406V 
                 GTT 
                 K415R 
                 CGG 
                 I424E 
                 GAG 
                 A432S 
                 TCT 
                 E441L 
                 CTT 
               
               
                 F398L 
                 CTT 
                 L406Y 
                 TAT 
                 K415S 
                 TCT 
                 I424G 
                 GGG 
                 A432V 
                 GTG 
                 E441N 
                 AAT 
               
               
                 F398N 
                 AAT 
                 S407A 
                 GCG 
                 K415T 
                 ACT 
                 I424H 
                 CAT 
                 A432Y 
                 TAT 
                 E441Q 
                 CAG 
               
               
                 F398P 
                 CCT 
                 S407D 
                 GAT 
                 K415V 
                 GTG 
                 I424K 
                 AAG 
                 F433A 
                 GCT 
                 E441R 
                 CGG 
               
               
                 F398R 
                 AGG 
                 S407E 
                 GAG 
                 K415W 
                 TGG 
                 I424L 
                 CTT 
                 F433C 
                 TGT 
                 E441S 
                 AGT 
               
               
                 F398S 
                 TCT 
                 S407F 
                 TTT 
                 K415Y 
                 TAT 
                 I424N 
                 AAT 
                 F433D 
                 GAT 
                 E441T 
                 ACT 
               
               
                 F398T 
                 ACT 
                 S407G 
                 GGT 
                 D416C 
                 TGT 
                 I424Q 
                 CAG 
                 F433E 
                 GAG 
                 E441V 
                 GTG 
               
               
                 F398V 
                 GTT 
                 S407H 
                 CAT 
                 D416F 
                 TTT 
                 I424R 
                 CGG 
                 F433G 
                 GGG 
                 E441Y 
                 TAT 
               
               
                 F398W 
                 TGG 
                 S407L 
                 CTG 
                 D416G 
                 GGT 
                 I424S 
                 TCG 
                 F433H 
                 CAT 
                 E442C 
                 TGT 
               
               
                 F398Y 
                 TAT 
                 S407M 
                 ATG 
                 D416H 
                 CAT 
                 I424T 
                 ACT 
                 F433I 
                 ATT 
                 E442G 
                 GGG 
               
               
                 Y399A 
                 GCG 
                 S407N 
                 AAT 
                 D416I 
                 ATT 
                 I424V 
                 GTT 
                 F433K 
                 AAG 
                 E442H 
                 CAT 
               
               
                 Y399C 
                 TGT 
                 S407P 
                 CCT 
                 D416K 
                 AAG 
                 I424W 
                 TGG 
                 F433L 
                 TTG 
                 E442K 
                 AAG 
               
               
                 Y399D 
                 GAT 
                 S407Q 
                 CAG 
                 D416L 
                 CTT 
                 I424Y 
                 TAT 
                 F433P 
                 CCT 
                 E442L 
                 CTT 
               
               
                 Y399E 
                 GAG 
                 S407R 
                 CGG 
                 D416N 
                 AAT 
                 A425C 
                 TGT 
                 F433R 
                 CGG 
                 E442M 
                 ATG 
               
               
                 Y399G 
                 GGG 
                 S407T 
                 ACG 
                 D416Q 
                 CAG 
                 A425D 
                 GAT 
                 F433S 
                 AGT 
                 E442N 
                 AAT 
               
               
                 Y399K 
                 AAG 
                 S407V 
                 GTG 
                 D416R 
                 CGG 
                 A425E 
                 GAG 
                 F433T 
                 ACT 
                 E442P 
                 CCT 
               
               
                 Y399M 
                 ATG 
                 S407W 
                 TGG 
                 D416S 
                 TCT 
                 A425G 
                 GGT 
                 F433V 
                 GTG 
                 E442Q 
                 CAG 
               
               
                 Y399N 
                 AAT 
                 C408A 
                 GCG 
                 D416T 
                 ACG 
                 A425I 
                 ATT 
                 F433W 
                 TGG 
                 E442R 
                 CGG 
               
               
                 Y399P 
                 CCT 
                 C408E 
                 GAG 
                 D416V 
                 GTG 
                 A425K 
                 AAG 
                 L434F 
                 TTT 
                 E442S 
                 AGT 
               
               
                 Y399Q 
                 CAG 
                 C408F 
                 TTT 
                 D416W 
                 TGG 
                 A425L 
                 TTG 
                 L434G 
                 GGT 
                 E442T 
                 ACT 
               
               
                 Y399R 
                 CGG 
                 C408G 
                 GGG 
                 D416Y 
                 TAT 
                 A425M 
                 ATG 
                 L434H 
                 CAT 
                 E442V 
                 GTG 
               
               
                 Y399S 
                 TCG 
                 C408I 
                 ATT 
                 T417A 
                 GCT 
                 A425N 
                 AAT 
                 L434I 
                 ATT 
                 E442W 
                 TGG 
               
               
                 Y399T 
                 ACG 
                 C408K 
                 AAG 
                 T417D 
                 GAT 
                 A425P 
                 CCT 
                 L434K 
                 AAG 
                 E442Y 
                 TAT 
               
               
                 Y399V 
                 GTT 
                 C408L 
                 CTT 
                 T417E 
                 GAG 
                 A425R 
                 AGG 
                 L434M 
                 ATG 
                 P443A 
                 GCT 
               
               
                 Y399W 
                 TGG 
                 C408N 
                 AAT 
                 T417F 
                 TTT 
                 A425S 
                 AGT 
                 L434N 
                 AAT 
                 P443D 
                 GAT 
               
               
                 C400A 
                 GCG 
                 C408P 
                 CCT 
                 T417G 
                 GGG 
                 A425V 
                 GTG 
                 L434P 
                 CCT 
                 P443E 
                 GAG 
               
               
                 C400D 
                 GAT 
                 C408R 
                 CGT 
                 T417H 
                 CAT 
                 A425W 
                 TGG 
                 L434Q 
                 CAG 
                 P443F 
                 TTT 
               
               
                 C400E 
                 GAG 
                 C408S 
                 TCG 
                 T417I 
                 ATT 
                 A425Y 
                 TAT 
                 L434R 
                 CGG 
                 P443G 
                 GGG 
               
               
                 C400F 
                 TTT 
                 C408T 
                 ACT 
                 T417K 
                 AAG 
                 D426A 
                 GCT 
                 L434S 
                 AGT 
                 P443H 
                 CAT 
               
               
                 C400G 
                 GGG 
                 C408V 
                 GTT 
                 T417L 
                 TTG 
                 D426C 
                 TGT 
                 L434T 
                 ACT 
                 P443I 
                 ATT 
               
               
                 C400I 
                 ATT 
                 C408W 
                 TGG 
                 T417M 
                 ATG 
                 D426E 
                 GAG 
                 L434V 
                 GTT 
                 P443L 
                 CTT 
               
               
                 C400L 
                 CTG 
                 C408Y 
                 TAT 
                 T417P 
                 CCT 
                 D426F 
                 TTT 
                 L434W 
                 TGG 
                 P443M 
                 ATG 
               
               
                 C400M 
                 ATG 
                 K409A 
                 GCG 
                 T417Q 
                 CAG 
                 D426G 
                 GGG 
                 L434Y 
                 TAT 
                 P443N 
                 AAT 
               
               
                 C400P 
                 CCG 
                 K409C 
                 TGT 
                 T417R 
                 CGT 
                 D426I 
                 ATT 
                 K435A 
                 GCT 
                 P443Q 
                 CAG 
               
               
                 C400Q 
                 CAG 
                 K409D 
                 GAT 
                 T417S 
                 TCG 
                 D426K 
                 AAG 
                 K435C 
                 TGT 
                 P443R 
                 AGG 
               
               
                 C400R 
                 CGG 
                 K409E 
                 GAG 
                 T417W 
                 TGG 
                 D426L 
                 CTG 
                 K435E 
                 GAG 
                 P443S 
                 TCT 
               
               
                 C400S 
                 AGT 
                 K409G 
                 GGT 
                 D418A 
                 GCT 
                 D426M 
                 ATG 
                 K435F 
                 TTT 
                 P443T 
                 ACT 
               
               
                 C400T 
                 ACG 
                 K409H 
                 CAT 
                 D418C 
                 TGT 
                 D426N 
                 AAT 
                 K435G 
                 GGT 
                 P443W 
                 TGG 
               
               
                 C400V 
                 GTG 
                 K409I 
                 ATT 
                 D418E 
                 GAG 
                 D426P 
                 CCT 
                 K435H 
                 CAT 
                 Q444C 
                 TGT 
               
               
                 C400Y 
                 TAT 
                 K409L 
                 CTG 
                 D418F 
                 TTT 
                 D426Q 
                 CAG 
                 K435I 
                 ATT 
                 Q444D 
                 GAT 
               
               
                 S401A 
                 GCT 
                 K409P 
                 CCG 
                 D418G 
                 GGT 
                 D426R 
                 CGT 
                 K435L 
                 CTG 
                 Q444E 
                 GAG 
               
               
                 S401C 
                 TGT 
                 K409Q 
                 CAG 
                 D418I 
                 ATT 
                 D426S 
                 TCG 
                 K435P 
                 CCT 
                 Q444F 
                 TTT 
               
               
                 S401D 
                 GAT 
                 K409R 
                 AGG 
                 D418L 
                 TTG 
                 D426Y 
                 TAT 
                 K435R 
                 AGG 
                 Q444G 
                 GGG 
               
               
                 S401E 
                 GAG 
                 K409S 
                 TCG 
                 D418M 
                 ATG 
                 G427A 
                 GCT 
                 K435S 
                 TCT 
                 Q444H 
                 CAT 
               
               
                 S401F 
                 TTT 
                 K409T 
                 ACG 
                 D418N 
                 AAT 
                 G427C 
                 TGT 
                 K435T 
                 ACT 
                 Q444I 
                 ATT 
               
               
                 S401G 
                 GGG 
                 K409V 
                 GTG 
                 D418P 
                 CCT 
                 G427F 
                 TTT 
                 K435V 
                 GTT 
                 Q444K 
                 AAG 
               
               
                 S401H 
                 CAT 
                 K409W 
                 TGG 
                 D418Q 
                 CAG 
                 G427H 
                 CAT 
                 K435W 
                 TGG 
                 Q444L 
                 CTG 
               
               
                 S401K 
                 AAG 
                 A412Y 
                 TAT 
                 D418R 
                 CGG 
                 G427I 
                 ATT 
                 K435Y 
                 TAT 
                 Q444M 
                 ATG 
               
               
                 S401L 
                 CTT 
                 E410D 
                 GAT 
                 D418S 
                 TCG 
                 G427K 
                 AAG 
                 P436C 
                 TGT 
                 Q444N 
                 AAT 
               
               
                 S401N 
                 AAT 
                 E410G 
                 GGG 
                 D418V 
                 GTG 
                 G427L 
                 CTG 
                 P436D 
                 GAT 
                 Q444R 
                 CGG 
               
               
                 S401Q 
                 CAG 
                 E410I 
                 ATT 
                 D418Y 
                 TAT 
                 G427P 
                 CCT 
                 P436E 
                 GAG 
                 Q444V 
                 GTT 
               
               
                 S401R 
                 CGT 
                 E410K 
                 AAG 
                 A419D 
                 GAT 
                 G427Q 
                 CAG 
                 P436G 
                 GGG 
                 Q444W 
                 TGG 
               
               
                 S401T 
                 ACT 
                 E410L 
                 CTT 
                 A419E 
                 GAG 
                 G427R 
                 CGT 
                 P436H 
                 CAT 
                 Q444Y 
                 TAT 
               
               
                 S401W 
                 TGG 
                 E410M 
                 ATG 
                 A419F 
                 TTT 
                 G427S 
                 AGT 
                 P436I 
                 ATT 
                 I445A 
                 GCT 
               
               
                 S401Y 
                 TAT 
                 E410N 
                 AAT 
                 A419G 
                 GGG 
                 G427T 
                 ACT 
                 P436K 
                 AAG 
                 I445C 
                 TGT 
               
               
                 C402A 
                 GCT 
                 E410P 
                 CCG 
                 A419H 
                 CAT 
                 G427V 
                 GTG 
                 P436L 
                 CTG 
                 I445D 
                 GAT 
               
               
                 C402D 
                 GAT 
                 E410Q 
                 CAG 
                 A419I 
                 ATT 
                 G427W 
                 TGG 
                 P436M 
                 ATG 
                 I445G 
                 GGG 
               
               
                 C402E 
                 GAG 
                 E410R 
                 CGT 
                 A419K 
                 AAG 
                 G427Y 
                 TAT 
                 P436Q 
                 CAG 
                 I445H 
                 CAT 
               
               
                 C402F 
                 TTT 
                 E410S 
                 TCG 
                 A419L 
                 CTT 
                 V428A 
                 GCT 
                 P436R 
                 CGG 
                 I445K 
                 AAG 
               
               
                 C402G 
                 GGG 
                 E410T 
                 ACG 
                 A419N 
                 AAT 
                 V428C 
                 TGT 
                 P436S 
                 TCT 
                 I445L 
                 CTT 
               
               
                 C402L 
                 TTG 
                 E410V 
                 GTG 
                 A419P 
                 CCT 
                 V428D 
                 GAT 
                 P436T 
                 ACT 
                 I445M 
                 ATG 
               
               
                 C402M 
                 ATG 
                 E410W 
                 TGG 
                 A419R 
                 CGG 
                 V428E 
                 GAG 
                 P436W 
                 TGG 
                 I445N 
                 AAT 
               
               
                 C402P 
                 CCT 
                 E410Y 
                 TAT 
                 A419S 
                 TCT 
                 V428F 
                 TTT 
                 P436Y 
                 TAT 
                 I445P 
                 CCT 
               
               
                 C402Q 
                 CAG 
                 K411A 
                 GCT 
                 A419T 
                 ACT 
                 V428G 
                 GGT 
                 P437A 
                 GCT 
                 I445Q 
                 CAG 
               
               
                 C402R 
                 CGG 
                 K411D 
                 GAT 
                 A419W 
                 TGG 
                 V428H 
                 CAT 
                 P437D 
                 GAT 
                 I445R 
                 AGG 
               
               
                 C402S 
                 TCT 
                 K411E 
                 GAG 
                 A419Y 
                 TAT 
                 V428L 
                 CTT 
                 P437F 
                 TTT 
                 I445S 
                 AGT 
               
               
                 C402T 
                 ACG 
                 K411F 
                 TTT 
                 V420A 
                 GCT 
                 V428M 
                 ATG 
                 P437G 
                 GGT 
                 I445T 
                 ACT 
               
               
                 C402V 
                 GTT 
                 K411G 
                 GGG 
                 V420D 
                 GAT 
                 V428N 
                 AAT 
                 P437H 
                 CAT 
                 I445V 
                 GTG 
               
               
                 C402W 
                 TGG 
                 K411H 
                 CAT 
                 V420F 
                 TTT 
                 V428P 
                 CCT 
                 P437I 
                 ATT 
                 I445W 
                 TGG 
               
               
                 C402Y 
                 TAT 
                 K411I 
                 ATT 
                 V420G 
                 GGT 
                 V428R 
                 CGG 
                 P437K 
                 AAG 
                 I445Y 
                 TAT 
               
               
                 Y403A 
                 GCT 
                 K411L 
                 CTG 
                 V420H 
                 CAT 
                 V428S 
                 TCG 
                 P437L 
                 CTG 
                 F446A 
                 GCT 
               
               
                 Y403C 
                 TGT 
                 K411N 
                 AAT 
                 V420I 
                 ATT 
                 V428T 
                 ACT 
                 P437M 
                 ATG 
                 F446C 
                 TGT 
               
               
                 Y403E 
                 GAG 
                 K411P 
                 CCT 
                 V420K 
                 AAG 
                 V428Y 
                 TAT 
                 P437Q 
                 CAG 
                 F446D 
                 GAT 
               
               
                 Y403F 
                 TTT 
                 K411R 
                 AGG 
                 V420L 
                 CTT 
                 C429A 
                 GCT 
                 P437R 
                 CGT 
                 F446E 
                 GAG 
               
               
                 Y403G 
                 GGT 
                 K411S 
                 TCG 
                 V420N 
                 AAT 
                 C429D 
                 GAT 
                 P437S 
                 TCT 
                 F446G 
                 GGG 
               
               
                 Y403H 
                 CAT 
                 K411T 
                 ACT 
                 V420P 
                 CCT 
                 C429G 
                 GGT 
                 P437T 
                 ACT 
                 F446H 
                 CAT 
               
               
                 Y403K 
                 AAG 
                 K411V 
                 GTT 
                 V420R 
                 AGG 
                 C429I 
                 ATT 
                 P437W 
                 TGG 
                 F446I 
                 ATT 
               
               
                 Y403L 
                 TTG 
                 K411W 
                 TGG 
                 V420S 
                 TCT 
                 C429K 
                 AAG 
                 P437Y 
                 TAT 
                 F446K 
                 AAG 
               
               
                 Y403M 
                 ATG 
                 A412D 
                 GAT 
                 V420T 
                 ACT 
                 C429L 
                 TTG 
                 M438A 
                 GCT 
                 F446L 
                 TTG 
               
               
                 Y403N 
                 AAT 
                 A412E 
                 GAG 
                 V420W 
                 TGG 
                 C429M 
                 ATG 
                 M438C 
                 TGT 
                 F446M 
                 ATG 
               
               
                 Y403P 
                 CCG 
                 A412G 
                 GGG 
                 V420Y 
                 TAT 
                 C429N 
                 AAT 
                 M438D 
                 GAT 
                 F446Q 
                 CAG 
               
               
                 Y403Q 
                 CAG 
                 A412H 
                 CAT 
                 D421A 
                 GCT 
                 C429P 
                 CCT 
                 M438E 
                 GAG 
                 F446R 
                 CGG 
               
               
                 Y403R 
                 CGG 
                 A412I 
                 ATT 
                 D421E 
                 GAG 
                 C429R 
                 CGG 
                 M438G 
                 GGG 
                 F446T 
                 ACT 
               
               
                 Y403S 
                 TCT 
                 A412L 
                 CTG 
                 D421G 
                 GGT 
                 C429S 
                 TCG 
                 M438L 
                 TTG 
                 F446V 
                 GTT 
               
               
                 Y403T 
                 ACG 
                 A412N 
                 AAT 
                 D421H 
                 CAT 
                 C429T 
                 ACT 
                 M438N 
                 AAT 
                 F446W 
                 TGG 
               
               
                 S404A 
                 GCT 
                 A412P 
                 CCT 
                 D421I 
                 ATT 
                 C429V 
                 GTT 
                 M438P 
                 CCT 
                 Y447D 
                 GAT 
               
               
                 S404C 
                 TGT 
                 A412Q 
                 CAG 
                 D421K 
                 AAG 
                 C429W 
                 TGG 
                 M438Q 
                 CAG 
                 Y447E 
                 GAG 
               
               
                 S404D 
                 GAT 
                 A412R 
                 CGG 
                 D421L 
                 TTG 
                 C429Y 
                 TAT 
                 M438R 
                 AGG 
                 Y447F 
                 TTT 
               
               
                 S404F 
                 TTT 
                 A412S 
                 AGT 
                 D421M 
                 ATG 
                 I430A 
                 GCT 
                 M438S 
                 TCG 
                 Y447G 
                 GGT 
               
               
                 S404G 
                 GGT 
                 A412V 
                 GTT 
                 D421N 
                 AAT 
                 I430D 
                 GAT 
                 M438T 
                 ACT 
                 Y447I 
                 ATT 
               
               
                 S404H 
                 CAT 
                 A412W 
                 TGG 
                 D421Q 
                 CAG 
                 I430E 
                 GAG 
                 M438V 
                 GTG 
                 Y447K 
                 AAG 
               
               
                 S404L 
                 CTT 
                 D413A 
                 GCG 
                 D421R 
                 CGG 
                 I430G 
                 GGG 
                 M438W 
                 TGG 
                 Y447L 
                 CTT 
               
               
                 S404M 
                 ATG 
                 D413E 
                 GAG 
                 D421S 
                 TCG 
                 I430H 
                 CAT 
                 M438Y 
                 TAT 
                 Y447M 
                 ATG 
               
               
                 S404N 
                 AAT 
                 D413F 
                 TTT 
                 D421T 
                 ACT 
                 I430K 
                 AAG 
                 E439A 
                 GCT 
                 Y447N 
                 AAT 
               
               
                 S404P 
                 CCT 
                 D413G 
                 GGT 
                 D421W 
                 TGG 
                 I430L 
                 TTG 
                 E439C 
                 TGT 
                 Y447P 
                 CCT 
               
               
                 S404R 
                 AGG 
                 D413H 
                 CAT 
                 D421Y 
                 TAT 
                 I430M 
                 ATG 
                 E439F 
                 TTT 
                 Y447Q 
                 CAG 
               
               
                 S404T 
                 ACG 
                 D413I 
                 ATT 
                 V422A 
                 GCT 
                 I430N 
                 AAT 
                 E439G 
                 GGG 
                 Y447R 
                 AGG 
               
               
                 S404V 
                 GTG 
                 D413K 
                 AAG 
                 V422C 
                 TGT 
                 I430P 
                 CCT 
                 E439H 
                 CAT 
                 Y447T 
                 ACT 
               
               
                 S404W 
                 TGG 
                 D413L 
                 CTG 
                 V422D 
                 GAT 
                 I430R 
                 AGG 
                 E439K 
                 AAG 
                 Y447V 
                 GTT 
               
               
                 S404Y 
                 TAT 
                 D413N 
                 AAT 
                 V422E 
                 GAG 
                 I430S 
                 TCT 
                 E439L 
                 CTT 
                 Y447W 
                 TGG 
               
               
                 T405A 
                 GCG 
                 D413P 
                 CCG 
                 V422G 
                 GGG 
                 I430T 
                 ACT 
                 E439N 
                 AAT 
                   
                   
               
               
                 T405C 
                 TGT 
                 D413Q 
                 CAG 
                 V422H 
                 CAT 
                 I430V 
                 GTT 
                 E439P 
                 CCT 
                   
                   
               
               
                 T405F 
                 TTT 
                 D413R 
                 CGT 
                 V422I 
                 ATT 
                 I430W 
                 TGG 
                 E439Q 
                 CAG 
                   
                   
               
               
                 T405G 
                 GGG 
                 D413S 
                 TCG 
                 V422L 
                 CTG 
                 D431A 
                 GCT 
                 E439R 
                 CGG 
                   
                   
               
               
                 T405I 
                 ATT 
                 D413T 
                 ACT 
                 V422M 
                 ATG 
                 D431E 
                 GAG 
                 E439S 
                 TCG 
                   
                   
               
               
                 T405K 
                 AAG 
                 D413W 
                 TGG 
                 V422N 
                 AAT 
                 D431G 
                 GGT 
                 E439T 
                 ACT 
                   
                   
               
               
                 T405L 
                 TTG 
                 V414A 
                 GCG 
                 V422P 
                 CCT 
                 D431H 
                 CAT 
                 E439V 
                 GTT 
                   
                   
               
               
                 T405M 
                 ATG 
                 V414D 
                 GAT 
                 V422Q 
                 CAG 
                 D431I 
                 ATT 
                 E439W 
                 TGG 
                   
                   
               
               
                 T405P 
                 CCG 
                 V414E 
                 GAG 
                 V422R 
                 CGT 
                 D431K 
                 AAG 
                 T440A 
                 GCT 
                   
                   
               
               
                 T405Q 
                 CAG 
                 V414F 
                 TTT 
                 V422S 
                 TCG 
                 D431L 
                 CTT 
                 T440D 
                 GAT 
                   
                   
               
               
                 T405R 
                 CGT 
                 V414G 
                 GGT 
                 V422T 
                 ACT 
                 D431N 
                 AAT 
                 T440E 
                 GAG 
                   
                   
               
               
                 T405S 
                 TCT 
                 V414H 
                 CAT 
                 V422W 
                 TGG 
                 D431P 
                 CCT 
                 T440F 
                 TTT 
                   
                   
               
               
                 T405V 
                 GTG 
                 V414I 
                 ATT 
                 V422Y 
                 TAT 
                 D431Q 
                 CAG 
                 T440G 
                 GGG 
                   
                   
               
               
                 T405W 
                 TGG 
                 V414K 
                 AAG 
                 C423A 
                 GCT 
                 D431R 
                 CGT 
                 T440H 
                 CAT 
                   
                   
               
               
                 T405Y 
                 TAT 
                 V414L 
                 TTG 
                 C423D 
                 GAT 
                 D431S 
                 TCT 
                 T440I 
                 ATT 
                   
                   
               
               
                 L406A 
                 GCT 
                 V414M 
                 ATG 
                 C423E 
                 GAG 
                 D431V 
                 GTT 
                 T440L 
                 CTT 
                   
                   
               
               
                 L406C 
                 TGT 
                 V414Q 
                 CAG 
                 C423F 
                 TTT 
                 D431W 
                 TGG 
                 T440M 
                 ATG 
                   
                   
               
               
                 L406D 
                 GAT 
                 V414R 
                 AGG 
                 C423G 
                 GGG 
                 D431Y 
                 TAT 
                 T440P 
                 CCT 
                   
                   
               
               
                 L406E 
                 GAG 
                 V414S 
                 TCG 
                 C423H 
                 CAT 
                 A432C 
                 TGT 
                 T440Q 
                 CAG 
                   
                   
               
               
                 L406F 
                 TTT 
                 V414T 
                 ACT 
                 C423L 
                 CTG 
                 A432E 
                 GAG 
                 T440R 
                 AGG 
                   
                   
               
               
                 L406G 
                 GGT 
                 V414Y 
                 TAT 
                 C423M 
                 ATG 
                 A432F 
                 TTT 
                 T440S 
                 AGT 
                   
                   
               
               
                 L406I 
                 ATT 
                 K415A 
                 GCG 
                 C423P 
                 CCT 
                 A432G 
                 GGG 
                 T440V 
                 GTG 
                   
                   
               
               
                 L406N 
                 AAT 
                 K415C 
                 TGT 
                 C423Q 
                 CAG 
                 A432H 
                 CAT 
                 T440Y 
                 TAT 
                   
                   
               
               
                   
                   
                 K415D 
                 GAT 
                 C423R 
                 AGG 
                 A432I 
                 ATT 
                 E441A 
                 GCT 
                   
                   
               
               
                   
                   
                 K415E 
                 GAG 
                 C423S 
                 TCG 
                 A432K 
                 AAG 
                 E441C 
                 TGT 
               
               
                   
               
            
           
         
       
     
     2. Expression 
     For expression of each mutant, HZ24-PH20-IRES-SEAP plasmid DNA containing cDNA encoding one of the variant PH20 or encoding wildtype PH20 was transfected into monolayer CHO-S cells (Invitrogen, Cat. No. 11619-012) using Lipofectamine 2000 (Invitrogen, Cat. No. 11668-027) according to the protocol suggested by the manufacturer. CHO-S cells were seeded the night before transfection and grown in DMEM with 10% FBS to be 80% confluent the next day. Then, the medium of the CHO-S cells was replaced with Opti-MEM. A mixture of plasmid DNA and lipofectamine was made (0.2 μg DNA and 0.5 μL Lipofetamine). The Lipofectamine/DNA mixture was added to CHO-S cells and incubated overnight. The next day, the cells were supplemented with CD-CHO serum free medium (Invitrogen, Cat. No. 10743-029). Supernatant from transfected cells was collected at various time points after transfection, and generally 96 hours after transfection. The supernatant, containing the variant PH20 protein or wildtype PH20 having a sequence of amino acids set forth in SEQ ID NO:3, was stored at −20° C. Activities of the supernatants were screened as described in the following examples. 
     Example 3 
     Screening of Library with a Hyaluronidase Activity Assay to Identify Activity Mutants 
     In this example, supernatants of expressed PH20 variants generated in Example 2 were screened using a hyaluronidase activity assay to assess activity of each mutant. In addition, activity of the secreted alkaline phosphatase (SEAP) was also measured to allow for normalizing PH20 activity of the expressed mutants to the PH20 wildtype. Active and inactive mutants were identified. 
     1. Generation of Biotinylated HA (bHA) Substrate 
     A 1.2-MDa HA (Lifecore) was biotinylated for use as a substrate in the hyaluronidase activity assay. First, 1.2 grams (g) of 1.2 MDa HA was dissolved at 4° C. in 600 mL ddH 2 0 for a week at a concentration of 2 mg/mL with stirring. Next, 645.71 mg Biotin Hydrazide was dissolved in 100 mL DMSO to a concentration of 25 mM (6.458 mg/mL, 247.8 mg in 38.37 mL DMSO). The biotin solution was warmed briefly at 37° C. until the solution was clear. Also, 368.61 mg Sulfo-NHS in 20 mL ddH 2 O was dissolved to make a 100× solution (18.4 mg/mL Sulfo-NHS). A 30 mM (1000×) water-soluble carbodiimide EDC solution was made by dissolving 17.63 mg EDC in 3 mL ddH20 at a concentration of 5.7513 mg/mL right before the reaction was started. 
     To four (4) 1000-mL sterile capped bottles, the following components were added at room temperature (RT) and in the following order with stirring: 1) 200 mL of 2 mg/mL HA solution; 2) 80 mL of 0.5M MES, pH 5.0 with gentle mixing; and 3) 91.6 mL of ddH 2 O with gentle mixing. Next, 24 mL of 25 mM Biotin-Hydrazide and 4 mL of 100× Sulfo-NHS solution were added sequentially, immediately followed by the addition of 500 μL EDC. After the addition of each component, the solution was mixed by inverting three times and stirring. After the addition of the last component, the solution was mixed by stirring overnight at 4° C. Then, Guanidine hydrochloride was added to a final concentration of 4 M by adding 38.2 g per 100 mL and was allowed to dissolve completely before adjusting the solution volume to 600 mL with ddH 2 O. 
     For dialysis, 200 mL from each batch of the conjugated HA guanidine hydrochloride solution was transferred into dialysis membranes. Over the course of three days, the solution was dialyzed against ddH 2 O with a change in ddH 2 O at least six times. The resulting volume of about 840 mL was adjusted to a final volume of 1000 mL with ddH 2 O. The final concentration of the biotinylated hyaluronan (bHA) was 0.4 mg/mL. 
     2. Hyaluronidase Activity Assay 
     The enzyme assay was a modification of the method described by Frost et al. (1997) (A Microtiter-Based Assay for Hyaluronidase Activity Not Requiring Specialized Reagents. Analytical Biochemistry (1997) 251:263-269) that provides a measure of PH20 hyaluronidase activity. 
     First, biotinylated HA (bHA) substrate was bound to plastic microtiter plates to generate assay plates. Briefly, 100 μl of b-HA at 1 mg/mL in 0.5 M carbonate buffer (pH 9.6) was dispensed into each well of a high bind microplate (Immunolon 4 HBX extra high binding; Thermo Scientific). The plate was covered with a plate sealer and stored between 2-8° C. for 24-48 hours. 
     Then, the assay plate was washed with 1× phosphate buffered saline (PBS) wash buffer containing 0.05% (v/v) Tween 20 (PBST). PBST was generated from 1× PBS (generated from Catalog No. P5368, Sigma (10 mM Phosphate Buffer, 2.7 mM Potassium Chloride, 137 mM Sodium Chloride, pH 7.4) by placing the contents of one packet of PBS into a 1-L graduated cylinder with 800 mL deionized water, dissolved by stirring or shaking and adding sufficient quantity of water to 1 L) by adding 500 μl Tween 20 (Catalog No. 6505; EMD Bioscience) to 900 mL of 1× PBS and adding sufficient quantity of water to 1 L. Washing was done using the BioTek ELx405 Select CW plate washer (BioTek) by washing five (5) times with 300 μl PBST wash buffer per well for each wash. At the end of each wash, the plate was tapped on a paper towel to remove excess liquid from each well. Prior to incubation with samples, 200 μl Blocking Buffer (1.0% w/v Bovine Serum Albumin (BSA) in PBS) was added to each well and the assay plate was incubated at 37° C. for approximately 1 hour prior. The Blocking buffer was generated by adding 2.5 g of BSA (Catalog No. 001-000-162; Jackson Immuno Research) to 200 mL 1× PBS, stirring, adding a sufficient quantity of 1× PBS to 250 mL and filtering through an 0.2 μM PES filter unit. 
     Transfected variant or wildtype PH20 supernatants generated as described in Example 1 were diluted in duplicate 1:25 in assay diluent buffer (pH 7.4 HEPES buffer; 10 mM HEPES, 50 mM NaCl, 1 mM CaCl 2 , 1 mg/mL BSA, pH 7.4, 0.05% Tween-20) in uncoated 4×HB high bound microplates. For the standard curve, 1:3 serial dilutions of rHuPH20 (generated as described in Example 1 with a specific activity of 145 U/mL) were made in assay diluent buffer in duplicate starting from 3 U/mL for standards as follows: 3 U/mL, 1 U/mL, ⅓ U/mL, 1/9 U/mL, 1/27 U/mL, 1/81 U/mL, and 1/243 U/mL. One hundred microliters (100 up of each standard and sample were transferred to the assay plates and incubated for approximately 1.5 hours at 37° C. 
     After the incubation, the plate was washed with PBST using the BioTek ELx405 Select CW plate washer by washing five (5) times with 300 μl PBST wash buffer per well for each wash. At the end of each wash, the plate was tapped on a paper towel to remove excess liquid from each well. Then, 100 μl of 1:5000 diluted Streptavidin-HRP (SA-HRP) was added to each well of the plate and incubated at ambient temperature for approximately 1 hour. For the dilution, a 1 mg/mL stock of Streptavidin-HRP conjugate (Catalog No. 21126; Thermo Scientific) was diluted 1:5000 into dilution buffer (1 mg/mL BSA, 0.025% Tween20, 137 mM NaCl, 20 mM Tris pH 7.5). After the incubation, the plate was washed with PBST using the BioTek ELx405 Select CW plate washer by washing five (5) times with 300 μl PBST wash buffer per well for each wash. At the end of each wash, the plate was tapped on a paper towel to remove excess liquid from each well. Then, 100 μl of TMB solution (Catalog No. 52-00-03, KPL; ambient temperature and protected from light) was added to each well for approximately five (5) minutes at room temperature or until an optimal color development was yielded. To stop the reaction, 100 μl 1.0 N Sulfuric Acid or TMB Stop solution (Catalog No. 50-85-06) were added to each well and the plates tapped to mix. Optical density was measured at 450 nm within 30 minutes of adding the stop solution. Since more PH20 in a standard or sample would lead to less bHA available to bind SA-HRP, the optical density (450 nm) value was inversely proportional to the concentration of hyaluronidase activity in each specimen. 
     3. SEAP Activity 
     Activity of secreted alkaline phosphatase (SEAP) in the cell culture supernatant also was measured using a colorimetric assay of placental alkaline phosphatase using pNPP as a phosphatase substrate (Anaspec SensoLyte pNPP SEAP kit; Catalog No. 72144, Anaspec) according to the manufacturer&#39;s instructions. The absorbance signal was measured at optical density (OD) of 405 nm. 
     The criteria for the high throughput (HTP) screening were that the transfected supernatant resulted in a SEAP signal of ≥0.1 and the signal for the rHuPH20 wildtype control produced a signal of ≥1 U/mL. Also, the criteria for each screen were that the standard curves had a signal to noise ratio (S/N) for the 0 U/mL standard versus the 3 u/mL standard at 0D405 of ≥5, had less than three β) standards with a coefficient of variation (CV) ≥10%, and at least four (4) of the standards were in the linear range. 
     Example 4 
     Selected pH20 Variants with Altered Hyaluronidase Activity 
     Each generated variant was screened for hyaluonidase activity as described in Example 3. The SEAP expression was used to normalize PH20 activity of each variant to the PH20 wildtype. Mutants were identified that exhibited altered hyaluronidase activity compared to wildtype. 
     1. Active Mutants 
     Active mutants were selected whereby at least one duplicate sample exhibited greater than 40% of wildtype activity when normalized to SEAP activity. The identified active mutants are set forth in Table 9. The Table sets forth the amino acid replacement compared to the sequence of amino acids of PH20 set forth in SEQ ID NO:3. The amino acid sequence of exemplary mutants also is set forth by reference to a SEQ ID NO. The Table also sets forth the average hyaluronidase activity of tested duplicates normalized by SEAP values compared to average of wildtype PH20 activities in each plate, which were also normalized by their own SEAP values. For example, a value of 0.40 indicates that the variant exhibits 40% of the hyaluronidase activity of wildtype PH20, a value of 1 indicates that the variant exhibits a similar hyaluronidase activity of wildtype and a value of 3.00 indicates that the variant exhibits 300% of the hyaluronidase activity of wildtype PH20 or 3-fold increased activity compared to wildtype. 
     The results in Table 9 show that over 600 tested mutants exhibit activity that is increased compared to wildtype. For example, about 536 mutants exhibit 120% or greater than 120% of the hyaluronidase activity of wildtype PH20 and about 75 of the mutants exhibit 300% or greater than 300% of the hyaluronidase activity of wildtype PH20. In particular, the results in Table 9 show that that hyaluronidase activity compared to wildtype of mutant S69A is about 22-fold; mutant S69R is about 14-fold; mutant 170A is about 27-fold; mutant 170K is about 14-fold; mutant 170R is about 14-fold; and mutant I271L is about 10-fold. 
     
       
         
           
               
             
               
                 TABLE 9 
               
             
            
               
                   
               
               
                 ACTIVE MUTANTS 
               
            
           
           
               
               
               
               
            
               
                   
                   
                 SEQ 
                   
               
               
                   
                   
                 ID 
                 AvgNorm 
               
               
                   
                 mutant 
                 NO 
                 Act. 
               
               
                   
                   
               
            
           
           
               
               
               
               
            
               
                   
                 L001A 
                 74 
                 0.95 
               
               
                   
                 L001C 
                   
                 0.89 
               
               
                   
                 L001E 
                 75 
                 0.55 
               
               
                   
                 L001F 
                   
                 0.41 
               
               
                   
                 L001G 
                 76 
                 0.62 
               
               
                   
                 L001H 
                 73 
                 1.90 
               
               
                   
                 L001K 
                 77 
                 1.39 
               
               
                   
                 L001N 
                   
                 0.87 
               
               
                   
                 L001P 
                   
                 0.92 
               
               
                   
                 L001Q 
                 78 
                 3.27 
               
               
                   
                 L001R 
                 79 
                 0.72 
               
               
                   
                 L001S 
                   
                 0.74 
               
               
                   
                 L001T 
                   
                 0.99 
               
               
                   
                 L001V 
                   
                 1.00 
               
               
                   
                 L001W 
                   
                 0.88 
               
               
                   
                 N002A 
                   
                 0.61 
               
               
                   
                 N002C 
                   
                 0.4 
               
               
                   
                 G291C 
                   
                 0.27 
               
               
                   
                 N002G 
                   
                 0.44 
               
               
                   
                 N002L 
                   
                 0.46 
               
               
                   
                 N002P 
                   
                 0.54 
               
               
                   
                 N002Q 
                   
                 0.84 
               
               
                   
                 N002S 
                   
                 0.78 
               
               
                   
                 N002T 
                   
                 1.05 
               
               
                   
                 N002V 
                   
                 0.65 
               
               
                   
                 F003E 
                   
                 0.42 
               
               
                   
                 F003H 
                   
                 0.68 
               
               
                   
                 F003L 
                   
                 0.59 
               
               
                   
                 F003Y 
                   
                 0.50 
               
               
                   
                 R004A 
                   
                 0.73 
               
               
                   
                 R004I 
                   
                 0.54 
               
               
                   
                 R004S 
                   
                 0.60 
               
               
                   
                 R004T 
                   
                 0.66 
               
               
                   
                 R004V 
                   
                 1.09 
               
               
                   
                 A005H 
                   
                 0.44 
               
               
                   
                 P006A 
                 80 
                 0.78 
               
               
                   
                 P006H 
                   
                 0.58 
               
               
                   
                 P006K 
                   
                 0.80 
               
               
                   
                 P006L 
                   
                 0.76 
               
               
                   
                 P006N 
                   
                 0.40 
               
               
                   
                 P006Q 
                   
                 0.89 
               
               
                   
                 P006R 
                   
                 0.56 
               
               
                   
                 P007M 
                   
                 0.57 
               
               
                   
                 V008I 
                   
                 1.17 
               
               
                   
                 V008L 
                   
                 0.53 
               
               
                   
                 V008M 
                 81 
                 0.47 
               
               
                   
                 V008P 
                   
                 0.33 
               
               
                   
                 I009K 
                   
                 0.69 
               
               
                   
                 I009L 
                   
                 1.08 
               
               
                   
                 I009R 
                   
                 0.53 
               
               
                   
                 I009S 
                   
                 0.98 
               
               
                   
                 I009V 
                   
                 0.84 
               
               
                   
                 P010D 
                   
                 0.62 
               
               
                   
                 P010E 
                   
                 0.66 
               
               
                   
                 P010G 
                 83 
                 0.55 
               
               
                   
                 P010H 
                 84 
                 0.43 
               
               
                   
                 P010N 
                   
                 0.55 
               
               
                   
                 P010Q 
                   
                 0.89 
               
               
                   
                 P010R 
                   
                 0.73 
               
               
                   
                 P010S 
                   
                 0.55 
               
               
                   
                 P010W 
                   
                 0.59 
               
               
                   
                 N011D 
                   
                 0.54 
               
               
                   
                 N011G 
                   
                 0.45 
               
               
                   
                 N011H 
                   
                 0.69 
               
               
                   
                 N011K 
                   
                 0.58 
               
               
                   
                 N011S 
                 85 
                 0.39 
               
               
                   
                 M310F 
                   
                 0.30 
               
               
                   
                 V012A 
                   
                 0.56 
               
               
                   
                 V012E 
                 86 
                 1.86 
               
               
                   
                 V012I 
                 87 
                 0.68 
               
               
                   
                 V012K 
                 88 
                 0.65 
               
               
                   
                 V012L 
                   
                 0.44 
               
               
                   
                 V012N 
                   
                 0.46 
               
               
                   
                 V012R 
                   
                 0.50 
               
               
                   
                 V012S 
                   
                 0.75 
               
               
                   
                 V012T 
                 89 
                 1.50 
               
               
                   
                 P013H 
                   
                 0.46 
               
               
                   
                 P013S 
                   
                 0.68 
               
               
                   
                 P013T 
                   
                 0.90 
               
               
                   
                 P013Y 
                   
                 0.51 
               
               
                   
                 F014D 
                   
                 0.64 
               
               
                   
                 F014I 
                   
                 0.42 
               
               
                   
                 F014M 
                   
                 0.47 
               
               
                   
                 F014V 
                 90 
                 0.46 
               
               
                   
                 L015A 
                   
                 0.65 
               
               
                   
                 L015M 
                 92 
                 0.45 
               
               
                   
                 L015V 
                 91 
                 2.20 
               
               
                   
                 A020S 
                 93 
                 0.50 
               
               
                   
                 S022H 
                   
                 0.57 
               
               
                   
                 S022M 
                   
                 0.49 
               
               
                   
                 S022T 
                 94 
                 0.48 
               
               
                   
                 S022Y 
                   
                 0.45 
               
               
                   
                 E023D 
                   
                 0.97 
               
               
                   
                 F024A 
                   
                 0.69 
               
               
                   
                 F024E 
                 95 
                 3.99 
               
               
                   
                 F024G 
                   
                 0.75 
               
               
                   
                 F024H 
                 96 
                 2.07 
               
               
                   
                 F024I 
                   
                 0.70 
               
               
                   
                 F024K 
                   
                 0.96 
               
               
                   
                 F024L 
                   
                 0.62 
               
               
                   
                 F024M 
                   
                 0.85 
               
               
                   
                 F024N 
                   
                 0.60 
               
               
                   
                 F024R 
                 97 
                 1.22 
               
               
                   
                 F024T 
                   
                 1.18 
               
               
                   
                 F024V 
                   
                 1.15 
               
               
                   
                 F024Y 
                   
                 0.90 
               
               
                   
                 L026A 
                 98 
                 1.30 
               
               
                   
                 L026E 
                 99 
                 3.22 
               
               
                   
                 L026G 
                   
                 0.81 
               
               
                   
                 L026H 
                   
                 0.97 
               
               
                   
                 L026I 
                   
                 0.51 
               
               
                   
                 L026K 
                 100 
                 1.88 
               
               
                   
                 L026M 
                 101 
                 1.43 
               
               
                   
                 L026P 
                   
                 0.55 
               
               
                   
                 L026Q 
                 102 
                 1.44 
               
               
                   
                 L026R 
                 103 
                 1.43 
               
               
                   
                 L026S 
                   
                 0.78 
               
               
                   
                 L026T 
                   
                 0.87 
               
               
                   
                 L026V 
                   
                 0.52 
               
               
                   
                 L026W 
                   
                 0.53 
               
               
                   
                 L026Y 
                   
                 0.52 
               
               
                   
                 G027A 
                   
                 0.79 
               
               
                   
                 G027D 
                 104 
                 1.22 
               
               
                   
                 G027E 
                   
                 1.18 
               
               
                   
                 G027F 
                   
                 0.61 
               
               
                   
                 G027H 
                   
                 1.11 
               
               
                   
                 G027I 
                   
                 0.41 
               
               
                   
                 G027K 
                 105 
                 2.71 
               
               
                   
                 G027L 
                   
                 0.76 
               
               
                   
                 G027P 
                   
                 0.46 
               
               
                   
                 G027Q 
                   
                 1.12 
               
               
                   
                 G027R 
                 106 
                 1.88 
               
               
                   
                 G027S 
                   
                 0.94 
               
               
                   
                 G027T 
                   
                 0.61 
               
               
                   
                 G027W 
                   
                 0.76 
               
               
                   
                 K028A 
                   
                 0.78 
               
               
                   
                 K028D 
                   
                 0.62 
               
               
                   
                 K028E 
                   
                 0.54 
               
               
                   
                 K028F 
                   
                 0.75 
               
               
                   
                 K028I 
                   
                 0.55 
               
               
                   
                 K028L 
                   
                 0.51 
               
               
                   
                 K028M 
                   
                 0.67 
               
               
                   
                 K028N 
                   
                 0.58 
               
               
                   
                 K028P 
                   
                 0.40 
               
               
                   
                 K028R 
                 107 
                 0.71 
               
               
                   
                 K028S 
                   
                 0.46 
               
               
                   
                 K028T 
                   
                 0.68 
               
               
                   
                 K028V 
                   
                 0.76 
               
               
                   
                 K028W 
                   
                 0.51 
               
               
                   
                 F029A 
                   
                 0.90 
               
               
                   
                 F029E 
                 108 
                 4.03 
               
               
                   
                 F029G 
                   
                 1.05 
               
               
                   
                 F029H 
                   
                 0.82 
               
               
                   
                 F029I 
                 109 
                 1.53 
               
               
                   
                 F029K 
                 110 
                 1.34 
               
               
                   
                 F029L 
                 111 
                 2.36 
               
               
                   
                 F029M 
                 112 
                 2.08 
               
               
                   
                 F029P 
                 113 
                 3.79 
               
               
                   
                 F029R 
                 114 
                 1.24 
               
               
                   
                 F029S 
                 115 
                 2.21 
               
               
                   
                 F029T 
                 116 
                 0.85 
               
               
                   
                 F029V 
                 117 
                 1.65 
               
               
                   
                 F029W 
                   
                 0.48 
               
               
                   
                 D030A 
                   
                 1.12 
               
               
                   
                 D030F 
                   
                 0.84 
               
               
                   
                 D030G 
                 118 
                 2.02 
               
               
                   
                 D030H 
                 119 
                 1.69 
               
               
                   
                 D030K 
                 120 
                 2.63 
               
               
                   
                 D030L 
                 121 
                 1.32 
               
               
                   
                 D030M 
                 122 
                 1.85 
               
               
                   
                 D030P 
                   
                 1.19 
               
               
                   
                 D030Q 
                   
                 0.84 
               
               
                   
                 D030R 
                 123 
                 1.82 
               
               
                   
                 D030S 
                 124 
                 1.62 
               
               
                   
                 D030T 
                   
                 0.57 
               
               
                   
                 D030V 
                   
                 0.46 
               
               
                   
                 D030W 
                   
                 0.62 
               
               
                   
                 E031A 
                 125 
                 2.05 
               
               
                   
                 E031C 
                 126 
                 2.95 
               
               
                   
                 E031G 
                 127 
                 1.27 
               
               
                   
                 E031H 
                 128 
                 2.74 
               
               
                   
                 E031I 
                 129 
                 3.89 
               
               
                   
                 E031K 
                 130 
                 3.13 
               
               
                   
                 E031L 
                 131 
                 2.62 
               
               
                   
                 E031P 
                 132 
                 1.51 
               
               
                   
                 E031R 
                 133 
                 2.27 
               
               
                   
                 E031S 
                 134 
                 1.70 
               
               
                   
                 E031T 
                 135 
                 3.96 
               
               
                   
                 E031V 
                 136 
                 4.57 
               
               
                   
                 E031W 
                 137 
                 1.26 
               
               
                   
                 E031Y 
                   
                 1.13 
               
               
                   
                 P032A 
                   
                 0.92 
               
               
                   
                 P032C 
                 138 
                 0.40 
               
               
                   
                 P032F 
                 139 
                 2.71 
               
               
                   
                 I326C 
                   
                 0.39 
               
               
                   
                 I331C 
                   
                 0.27 
               
               
                   
                 P032G 
                 140 
                 1.60 
               
               
                   
                 P032H 
                 141 
                 2.08 
               
               
                   
                 P032K 
                   
                 1.04 
               
               
                   
                 P032L 
                   
                 0.82 
               
               
                   
                 P032M 
                   
                 0.67 
               
               
                   
                 P032N 
                   
                 0.70 
               
               
                   
                 P032Q 
                   
                 1.11 
               
               
                   
                 P032R 
                   
                 1.17 
               
               
                   
                 P032S 
                   
                 1.01 
               
               
                   
                 P032T 
                   
                 0.77 
               
               
                   
                 P032V 
                   
                 0.81 
               
               
                   
                 P032W 
                   
                 0.54 
               
               
                   
                 P032Y 
                   
                 1.01 
               
               
                   
                 L033G 
                 143 
                 0.57 
               
               
                   
                 L033M 
                   
                 0.69 
               
               
                   
                 L033P 
                   
                 0.87 
               
               
                   
                 L033Q 
                   
                 0.45 
               
               
                   
                 L033R 
                   
                 0.61 
               
               
                   
                 L033S 
                   
                 0.48 
               
               
                   
                 L033T 
                   
                 0.45 
               
               
                   
                 L033W 
                 142 
                 1.58 
               
               
                   
                 D034A 
                   
                 0.38 
               
               
                   
                 D034E 
                   
                 0.58 
               
               
                   
                 D034H 
                   
                 0.41 
               
               
                   
                 D034K 
                   
                 0.54 
               
               
                   
                 D034Q 
                   
                 0.59 
               
               
                   
                 D034R 
                   
                 1.17 
               
               
                   
                 D034W 
                 144 
                 0.46 
               
               
                   
                 M035F 
                   
                 0.87 
               
               
                   
                 M035H 
                   
                 0.60 
               
               
                   
                 M035L 
                   
                 0.52 
               
               
                   
                 M035T 
                   
                 0.83 
               
               
                   
                 M035Y 
                   
                 0.78 
               
               
                   
                 S036A 
                   
                 0.45 
               
               
                   
                 S036D 
                   
                 0.32 
               
               
                   
                 S036G 
                   
                 0.64 
               
               
                   
                 S036H 
                 147 
                 0.54 
               
               
                   
                 S036K 
                   
                 0.83 
               
               
                   
                 S036L 
                   
                 0.71 
               
               
                   
                 S036R 
                   
                 1.09 
               
               
                   
                 Q347L 
                   
                 0.39 
               
               
                   
                 V351Q 
                   
                 0.34 
               
               
                   
                 S036T 
                   
                 0.51 
               
               
                   
                 L037F 
                 149 
                 3.33 
               
               
                   
                 L037I 
                   
                 0.62 
               
               
                   
                 L037K 
                   
                 0.43 
               
               
                   
                 L037M 
                 150 
                 1.46 
               
               
                   
                 L037P 
                   
                 0.63 
               
               
                   
                 L037R 
                   
                 0.51 
               
               
                   
                 L037V 
                   
                 0.57 
               
               
                   
                 F038Y 
                 151 
                 1.29 
               
               
                   
                 S039A 
                 152 
                 1.06 
               
               
                   
                 S039L 
                 153 
                 0.80 
               
               
                   
                 S039N 
                 154 
                 2.32 
               
               
                   
                 S039Q 
                   
                 1.10 
               
               
                   
                 S039R 
                   
                 0.56 
               
               
                   
                 S039T 
                 155 
                 1.57 
               
               
                   
                 S039Y 
                   
                 0.56 
               
               
                   
                 F040L 
                 156 
                 0.92 
               
               
                   
                 F040W 
                   
                 1.11 
               
               
                   
                 I041A 
                   
                 0.67 
               
               
                   
                 I041C 
                   
                 0.53 
               
               
                   
                 I041D 
                   
                 0.78 
               
               
                   
                 I041E 
                   
                 0.51 
               
               
                   
                 I041G 
                   
                 0.76 
               
               
                   
                 I041H 
                   
                 0.77 
               
               
                   
                 I041N 
                   
                 0.40 
               
               
                   
                 I041T 
                 157 
                 1.47 
               
               
                   
                 I041V 
                   
                 0.73 
               
               
                   
                 I041W 
                   
                 0.66 
               
               
                   
                 G042A 
                   
                 0.64 
               
               
                   
                 S043T 
                   
                 0.43 
               
               
                   
                 P044E 
                   
                 0.59 
               
               
                   
                 R045I 
                   
                 0.45 
               
               
                   
                 R045K 
                   
                 0.53 
               
               
                   
                 I046A 
                   
                 1.04 
               
               
                   
                 I046C 
                   
                 0.37 
               
               
                   
                 I046E 
                   
                 0.43 
               
               
                   
                 I046F 
                   
                 0.73 
               
               
                   
                 I046H 
                   
                 0.82 
               
               
                   
                 I046L 
                 158 
                 1.08 
               
               
                   
                 I046M 
                   
                 1.00 
               
               
                   
                 I046N 
                   
                 0.66 
               
               
                   
                 I046R 
                 159 
                 2.29 
               
               
                   
                 I046S 
                   
                 0.64 
               
               
                   
                 I046T 
                   
                 0.55 
               
               
                   
                 I046V 
                   
                 1.01 
               
               
                   
                 I046Y 
                   
                 0.76 
               
               
                   
                 N047A 
                   
                 0.48 
               
               
                   
                 N047D 
                 160 
                 0.82 
               
               
                   
                 N047F 
                 161 
                 1.32 
               
               
                   
                 N047G 
                   
                 0.82 
               
               
                   
                 N047H 
                   
                 1.16 
               
               
                   
                 N047K 
                   
                 0.67 
               
               
                   
                 N047M 
                   
                 0.77 
               
               
                   
                 N047Q 
                   
                 0.69 
               
               
                   
                 N047R 
                   
                 0.84 
               
               
                   
                 N047S 
                   
                 0.85 
               
               
                   
                 N047T 
                 162 
                 1.49 
               
               
                   
                 N047W 
                 163 
                 0.63 
               
               
                   
                 N047Y 
                   
                 0.45 
               
               
                   
                 A048F 
                 164 
                 2.51 
               
               
                   
                 A048G 
                   
                 0.83 
               
               
                   
                 A048H 
                 165 
                 1.99 
               
               
                   
                 A048I 
                   
                 0.64 
               
               
                   
                 A048K 
                 166 
                 1.28 
               
               
                   
                 A048M 
                   
                 0.76 
               
               
                   
                 A048N 
                 167 
                 4.25 
               
               
                   
                 A048Q 
                   
                 1.05 
               
               
                   
                 A048R 
                   
                 0.66 
               
               
                   
                 A048S 
                   
                 1.06 
               
               
                   
                 A048V 
                   
                 0.60 
               
               
                   
                 A048Y 
                   
                 0.81 
               
               
                   
                 T049I 
                   
                 0.42 
               
               
                   
                 T049K 
                   
                 0.85 
               
               
                   
                 T049R 
                 168 
                 1.41 
               
               
                   
                 T049S 
                   
                 0.92 
               
               
                   
                 T049V 
                   
                 0.45 
               
               
                   
                 G050A 
                   
                 0.93 
               
               
                   
                 G050C 
                   
                 0.41 
               
               
                   
                 G050D 
                 169 
                 1.37 
               
               
                   
                 G050E 
                   
                 0.78 
               
               
                   
                 G050H 
                   
                 0.74 
               
               
                   
                 G050L 
                   
                 0.43 
               
               
                   
                 G050M 
                 171 
                 0.47 
               
               
                   
                 G050Q 
                   
                 0.86 
               
               
                   
                 G050R 
                   
                 0.86 
               
               
                   
                 G050S 
                 170 
                 1.24 
               
               
                   
                 G050V 
                   
                 0.3 
               
               
                   
                 G050Y 
                   
                 0.58 
               
               
                   
                 Q051N 
                   
                 0.60 
               
               
                   
                 Q051S 
                   
                 0.46 
               
               
                   
                 G052N 
                 172 
                 0.89 
               
               
                   
                 G052P 
                   
                 0.43 
               
               
                   
                 G052Q 
                 173 
                 3.71 
               
               
                   
                 G052R 
                 174 
                 0.53 
               
               
                   
                 G052S 
                 175 
                 1.32 
               
               
                   
                 E375I 
                   
                 0.36 
               
               
                   
                 F380V 
                   
                 0.39 
               
               
                   
                 G052T 
                 176 
                 0.49 
               
               
                   
                 T054A 
                   
                 0.43 
               
               
                   
                 T054F 
                   
                 0.56 
               
               
                   
                 T054N 
                   
                 0.48 
               
               
                   
                 T054Q 
                   
                 0.91 
               
               
                   
                 T054S 
                   
                 0.70 
               
               
                   
                 T054V 
                   
                 0.66 
               
               
                   
                 V058C 
                 177 
                 0.55 
               
               
                   
                 V058G 
                   
                 0.54 
               
               
                   
                 V058H 
                 183 
                 1.09 
               
               
                   
                 V058I 
                   
                 0.57 
               
               
                   
                 V058K 
                 178 
                 4.08 
               
               
                   
                 V058L 
                 179 
                 1.54 
               
               
                   
                 V058N 
                 184 
                 0.49 
               
               
                   
                 V058P 
                 180 
                 0.90 
               
               
                   
                 V058Q 
                 181 
                 4.54 
               
               
                   
                 V058R 
                 182 
                 1.92 
               
               
                   
                 V058S 
                   
                 0.83 
               
               
                   
                 V058W 
                   
                 0.65 
               
               
                   
                 V058Y 
                 185 
                 1.07 
               
               
                   
                 D059Q 
                   
                 0.40 
               
               
                   
                 D059N 
                 186 
                 1.27 
               
               
                   
                 R060K 
                   
                 0.69 
               
               
                   
                 L061I 
                   
                 0.42 
               
               
                   
                 L061M 
                   
                 0.73 
               
               
                   
                 L061V 
                   
                 0.59 
               
               
                   
                 Y063A 
                   
                 0.63 
               
               
                   
                 Y063H 
                   
                 1.07 
               
               
                   
                 Y063I 
                   
                 1.03 
               
               
                   
                 Y063K 
                 187 
                 1.36 
               
               
                   
                 Y063L 
                 188 
                 1.33 
               
               
                   
                 Y063M 
                 189 
                 1.32 
               
               
                   
                 Y063N 
                   
                 0.96 
               
               
                   
                 Y063R 
                 190 
                 1.40 
               
               
                   
                 Y063S 
                   
                 1.00 
               
               
                   
                 Y063T 
                   
                 1.07 
               
               
                   
                 Y063V 
                   
                 0.43 
               
               
                   
                 Y063W 
                 191 
                 1.53 
               
               
                   
                 P065R 
                   
                 0.57 
               
               
                   
                 Y066H 
                   
                 0.47 
               
               
                   
                 Y066R 
                   
                 0.51 
               
               
                   
                 I067F 
                   
                 1.00 
               
               
                   
                 I067L 
                   
                 0.45 
               
               
                   
                 I067R 
                   
                 0.24 
               
               
                   
                 I067V 
                 192 
                 1.80 
               
               
                   
                 I067Y 
                   
                 0.55 
               
               
                   
                 D068E 
                   
                 0.72 
               
               
                   
                 D068H 
                 193 
                 2.06 
               
               
                   
                 D068K 
                   
                 1.08 
               
               
                   
                 D068L 
                   
                 0.43 
               
               
                   
                 D068P 
                 194 
                 0.50 
               
               
                   
                 D068Q 
                 195 
                 1.67 
               
               
                   
                 D068R 
                   
                 0.70 
               
               
                   
                 D068S 
                   
                 0.81 
               
               
                   
                 D068T 
                   
                 0.75 
               
               
                   
                 S069A 
                 196 
                 22.06 
               
               
                   
                 S069C 
                 197 
                 1.97 
               
               
                   
                 S069E 
                 198 
                 1.48 
               
               
                   
                 S069F 
                 199 
                 8.75 
               
               
                   
                 S069G 
                 200 
                 6.06 
               
               
                   
                 S069I 
                 201 
                 3.12 
               
               
                   
                 S069L 
                 202 
                 3.44 
               
               
                   
                 S069M 
                 203 
                 2.67 
               
               
                   
                 S069P 
                 204 
                 8.14 
               
               
                   
                 S069R 
                 205 
                 14.06 
               
               
                   
                 S069T 
                 206 
                 0.58 
               
               
                   
                 S069W 
                 207 
                 2.18 
               
               
                   
                 S069Y 
                 208 
                 2.71 
               
               
                   
                 I070A 
                 209 
                 27.00 
               
               
                   
                 I070C 
                 210 
                 2.57 
               
               
                   
                 I070F 
                 211 
                 5.69 
               
               
                   
                 I070G 
                 212 
                 6.22 
               
               
                   
                 I070H 
                 213 
                 9.09 
               
               
                   
                 I070K 
                 214 
                 14.64 
               
               
                   
                 I070L 
                 215 
                 3.05 
               
               
                   
                 I070N 
                 216 
                 6.19 
               
               
                   
                 I070P 
                 217 
                 3.03 
               
               
                   
                 I070R 
                 218 
                 13.95 
               
               
                   
                 I070S 
                 219 
                 3.63 
               
               
                   
                 I070T 
                 220 
                 5.43 
               
               
                   
                 I070V 
                 221 
                 6.34 
               
               
                   
                 I070Y 
                 222 
                 1.26 
               
               
                   
                 T071A 
                   
                 0.86 
               
               
                   
                 T071D 
                   
                 0.50 
               
               
                   
                 T071G 
                 223 
                 1.41 
               
               
                   
                 T071H 
                   
                 0.93 
               
               
                   
                 T071L 
                   
                 1.09 
               
               
                   
                 T071M 
                   
                 0.89 
               
               
                   
                 T071N 
                 224 
                 1.21 
               
               
                   
                 T071Q 
                   
                 0.68 
               
               
                   
                 T071R 
                 225 
                 2.17 
               
               
                   
                 T071S 
                 226 
                 1.54 
               
               
                   
                 G072A 
                   
                 0.45 
               
               
                   
                 G072D 
                   
                 0.60 
               
               
                   
                 S395W 
                   
                 0.4 
               
               
                   
                 G072E 
                   
                 0.69 
               
               
                   
                 G072H 
                   
                 0.46 
               
               
                   
                 G072K 
                 227 
                 1.39 
               
               
                   
                 G072L 
                   
                 0.43 
               
               
                   
                 G072M 
                 228 
                 3.11 
               
               
                   
                 G072Q 
                 229 
                 2.33 
               
               
                   
                 G072R 
                   
                 0.65 
               
               
                   
                 G072S 
                   
                 0.51 
               
               
                   
                 V073A 
                 230 
                 1.38 
               
               
                   
                 V073C 
                   
                 0.84 
               
               
                   
                 V073D 
                   
                 0.94 
               
               
                   
                 V073G 
                   
                 1.17 
               
               
                   
                 V073H 
                 231 
                 1.54 
               
               
                   
                 V073K 
                 232 
                 1.42 
               
               
                   
                 V073L 
                 233 
                 1.59 
               
               
                   
                 V073M 
                   
                 0.68 
               
               
                   
                 V073Q 
                 234 
                 0.96 
               
               
                   
                 V073R 
                 235 
                 0.72 
               
               
                   
                 V073S 
                   
                 0.86 
               
               
                   
                 K297R 
                   
                 0.34 
               
               
                   
                 S401Q 
                   
                 0.39 
               
               
                   
                 V073T 
                 236 
                 1.34 
               
               
                   
                 V073W 
                 237 
                 1.91 
               
               
                   
                 T074A 
                 238 
                 2.28 
               
               
                   
                 T074C 
                 239 
                 2.18 
               
               
                   
                 T074E 
                 240 
                 1.38 
               
               
                   
                 T074F 
                 241 
                 1.43 
               
               
                   
                 T074G 
                 242 
                 2.75 
               
               
                   
                 T074H 
                 243 
                 1.40 
               
               
                   
                 T074K 
                 244 
                 1.29 
               
               
                   
                 T074L 
                 245 
                 1.43 
               
               
                   
                 T074M 
                 246 
                 0.52 
               
               
                   
                 T074N 
                 247 
                 2.12 
               
               
                   
                 T074P 
                 248 
                 2.45 
               
               
                   
                 T074R 
                 249 
                 2.22 
               
               
                   
                 T074S 
                 250 
                 1.80 
               
               
                   
                 T074V 
                 251 
                 2.27 
               
               
                   
                 T074W 
                 252 
                 2.13 
               
               
                   
                 V075A 
                   
                 0.71 
               
               
                   
                 V075C 
                   
                 0.46 
               
               
                   
                 V075F 
                 253 
                 2.00 
               
               
                   
                 V075H 
                   
                 0.62 
               
               
                   
                 V075L 
                 254 
                 5.22 
               
               
                   
                 V075M 
                 255 
                 1.16 
               
               
                   
                 V075N 
                   
                 0.81 
               
               
                   
                 V075Q 
                   
                 1.51 
               
               
                   
                 V075R 
                 256 
                 3.02 
               
               
                   
                 V075S 
                   
                 0.76 
               
               
                   
                 V075T 
                 257 
                 4.34 
               
               
                   
                 V075Y 
                   
                 0.63 
               
               
                   
                 G077H 
                   
                 0.32 
               
               
                   
                 I079L 
                 258 
                 1.44 
               
               
                   
                 I079T 
                   
                 0.79 
               
               
                   
                 I079V 
                   
                 1.01 
               
               
                   
                 Q081P 
                   
                 0.60 
               
               
                   
                 K082A 
                   
                 0.94 
               
               
                   
                 K082E 
                   
                 0.50 
               
               
                   
                 K082G 
                   
                 0.64 
               
               
                   
                 K082H 
                   
                 0.44 
               
               
                   
                 K082I 
                   
                 1.01 
               
               
                   
                 K082L 
                 259 
                 0.87 
               
               
                   
                 K082M 
                   
                 0.58 
               
               
                   
                 K082N 
                 260 
                 0.96 
               
               
                   
                 K082Q 
                   
                 0.76 
               
               
                   
                 K082R 
                   
                 0.85 
               
               
                   
                 K082S 
                   
                 0.62 
               
               
                   
                 K082T 
                   
                 0.56 
               
               
                   
                 K082Y 
                   
                 0.32 
               
               
                   
                 K082V 
                   
                 0.57 
               
               
                   
                 I083F 
                   
                 0.57 
               
               
                   
                 I083G 
                 264 
                 1.05 
               
               
                   
                 I083L 
                   
                 0.93 
               
               
                   
                 I083N 
                   
                 0.82 
               
               
                   
                 I083Q 
                 262 
                 1.07 
               
               
                   
                 I083R 
                   
                 0.45 
               
               
                   
                 I083S 
                 263 
                 0.79 
               
               
                   
                 I083T 
                   
                 0.95 
               
               
                   
                 I083V 
                 261 
                 0.99 
               
               
                   
                 S084D 
                   
                 0.98 
               
               
                   
                 S084E 
                 265 
                 0.52 
               
               
                   
                 S084F 
                 266 
                 0.72 
               
               
                   
                 S084G 
                 267 
                 8.68 
               
               
                   
                 S084H 
                   
                 0.96 
               
               
                   
                 S084I 
                   
                 0.90 
               
               
                   
                 S084L 
                   
                 0.92 
               
               
                   
                 S084M 
                   
                 0.77 
               
               
                   
                 S084N 
                 268 
                 0.89 
               
               
                   
                 S084P 
                   
                 0.57 
               
               
                   
                 S084Q 
                   
                 0.86 
               
               
                   
                 S084R 
                 269 
                 1.89 
               
               
                   
                 S084T 
                   
                 0.82 
               
               
                   
                 S084W 
                   
                 0.86 
               
               
                   
                 S084Y 
                   
                 0.30 
               
               
                   
                 S221I 
                   
                 0.35 
               
               
                   
                 L085V 
                   
                 0.42 
               
               
                   
                 Q086A 
                 270 
                 2.70 
               
               
                   
                 Q086D 
                   
                 0.88 
               
               
                   
                 Q086E 
                   
                 1.18 
               
               
                   
                 Q086F 
                   
                 0.54 
               
               
                   
                 Q086G 
                   
                 1.02 
               
               
                   
                 Q086H 
                 271 
                 1.70 
               
               
                   
                 Q086I 
                   
                 0.65 
               
               
                   
                 Q086K 
                 272 
                 0.97 
               
               
                   
                 Q086L 
                   
                 0.92 
               
               
                   
                 Q086M 
                   
                 1.06 
               
               
                   
                 Q086N 
                 273 
                 1.28 
               
               
                   
                 Q086P 
                   
                 0.42 
               
               
                   
                 Q086R 
                   
                 0.93 
               
               
                   
                 Q086S 
                 274 
                 0.85 
               
               
                   
                 Q086T 
                 275 
                 0.58 
               
               
                   
                 Q086V 
                   
                 0.97 
               
               
                   
                 Q086W 
                 276 
                 1.21 
               
               
                   
                 D087A 
                   
                 1.00 
               
               
                   
                 D087C 
                 277 
                 1.77 
               
               
                   
                 D087E 
                   
                 0.86 
               
               
                   
                 D087G 
                 278 
                 1.00 
               
               
                   
                 D087H 
                   
                 0.72 
               
               
                   
                 D087I 
                   
                 0.53 
               
               
                   
                 D087L 
                 279 
                 0.55 
               
               
                   
                 D087M 
                 280 
                 0.58 
               
               
                   
                 D087P 
                   
                 0.31 
               
               
                   
                 D087Q 
                   
                 1.05 
               
               
                   
                 D087R 
                 281 
                 1.28 
               
               
                   
                 D087S 
                 282 
                 0.99 
               
               
                   
                 D087T 
                 283 
                 1.70 
               
               
                   
                 A412H 
                   
                 0.39 
               
               
                   
                 D413H 
                   
                 0.31 
               
               
                   
                 V414K 
                   
                 0.3 
               
               
                   
                 K415V 
                   
                 0.39 
               
               
                   
                 D087V 
                 284 
                 0.66 
               
               
                   
                 D087Y 
                 285 
                 2.72 
               
               
                   
                 L089C 
                 286 
                 1.46 
               
               
                   
                 L089R 
                   
                 0.34 
               
               
                   
                 L089K 
                   
                 0.45 
               
               
                   
                 L089M 
                   
                 0.63 
               
               
                   
                 D090A 
                 287 
                 1.48 
               
               
                   
                 D090E 
                 288 
                 1.15 
               
               
                   
                 D090G 
                   
                 0.41 
               
               
                   
                 D090H 
                 289 
                 1.24 
               
               
                   
                 D090I 
                   
                 1.10 
               
               
                   
                 D090K 
                 290 
                 1.36 
               
               
                   
                 D090L 
                   
                 1.15 
               
               
                   
                 D090N 
                 291 
                 1.18 
               
               
                   
                 D090Q 
                   
                 1.11 
               
               
                   
                 D090R 
                 292 
                 1.49 
               
               
                   
                 D090S 
                   
                 1.15 
               
               
                   
                 D090T 
                   
                 1.02 
               
               
                   
                 D090W 
                   
                 0.81 
               
               
                   
                 K091A 
                   
                 0.89 
               
               
                   
                 K091Q 
                   
                 0.43 
               
               
                   
                 K091R 
                   
                 0.67 
               
               
                   
                 A092C 
                 293 
                 1.97 
               
               
                   
                 A092H 
                   
                 0.22 
               
               
                   
                 A092L 
                 294 
                 1.29 
               
               
                   
                 A092M 
                   
                 0.86 
               
               
                   
                 A092T 
                   
                 0.70 
               
               
                   
                 A092V 
                   
                 1.09 
               
               
                   
                 K093D 
                   
                 0.71 
               
               
                   
                 K093E 
                   
                 0.83 
               
               
                   
                 K093F 
                   
                 0.50 
               
               
                   
                 K093G 
                   
                 0.97 
               
               
                   
                 K093H 
                   
                 0.61 
               
               
                   
                 K093I 
                 295 
                 3.25 
               
               
                   
                 R248H 
                   
                 0.4 
               
               
                   
                 K093L 
                 296 
                 1.53 
               
               
                   
                 K093M 
                   
                 0.70 
               
               
                   
                 K093N 
                   
                 0.71 
               
               
                   
                 K093Q 
                 297 
                 0.84 
               
               
                   
                 K093R 
                 298 
                 1.52 
               
               
                   
                 K093S 
                 299 
                 1.25 
               
               
                   
                 K093T 
                 300 
                 3.93 
               
               
                   
                 K093V 
                   
                 0.24 
               
               
                   
                 K094A 
                   
                 0.64 
               
               
                   
                 K094D 
                 301 
                 0.93 
               
               
                   
                 K094E 
                   
                 0.79 
               
               
                   
                 K094F 
                   
                 0.59 
               
               
                   
                 K094H 
                   
                 0.72 
               
               
                   
                 K094L 
                   
                 0.52 
               
               
                   
                 K094M 
                   
                 0.66 
               
               
                   
                 K094N 
                   
                 0.99 
               
               
                   
                 K094Q 
                 302 
                 1.22 
               
               
                   
                 K094R 
                 303 
                 3.94 
               
               
                   
                 K094S 
                   
                 0.94 
               
               
                   
                 K094T 
                   
                 1.14 
               
               
                   
                 I096D 
                   
                 0.69 
               
               
                   
                 I096L 
                   
                 0.46 
               
               
                   
                 I096V 
                   
                 0.68 
               
               
                   
                 T097A 
                 304 
                 1.25 
               
               
                   
                 T097C 
                 305 
                 0.53 
               
               
                   
                 T097D 
                 306 
                 1.31 
               
               
                   
                 T097E 
                 307 
                 1.19 
               
               
                   
                 T097F 
                   
                 0.75 
               
               
                   
                 P257C 
                   
                 0.36 
               
               
                   
                 D426K 
                   
                 0.26 
               
               
                   
                 G427H 
                   
                 0.35 
               
               
                   
                 T097G 
                 308 
                 4.84 
               
               
                   
                 T097I 
                   
                 0.85 
               
               
                   
                 T097L 
                 309 
                 1.22 
               
               
                   
                 T097N 
                   
                 1.10 
               
               
                   
                 T097P 
                   
                 0.62 
               
               
                   
                 T097Q 
                   
                 1.17 
               
               
                   
                 T097R 
                   
                 0.95 
               
               
                   
                 T097S 
                 310 
                 1.21 
               
               
                   
                 T097W 
                   
                 0.53 
               
               
                   
                 T097Y 
                   
                 0.74 
               
               
                   
                 F098A 
                   
                 0.60 
               
               
                   
                 F098C 
                   
                 0.58 
               
               
                   
                 F098D 
                   
                 0.47 
               
               
                   
                 F098E 
                   
                 0.44 
               
               
                   
                 F098H 
                   
                 1.06 
               
               
                   
                 F098I 
                   
                 0.52 
               
               
                   
                 F098L 
                   
                 0.58 
               
               
                   
                 F098M 
                   
                 0.87 
               
               
                   
                 F098Q 
                   
                 0.65 
               
               
                   
                 P436C 
                   
                 0.39 
               
               
                   
                 F098R 
                   
                 0.72 
               
               
                   
                 F098S 
                   
                 0.56 
               
               
                   
                 F098V 
                   
                 0.46 
               
               
                   
                 F098W 
                   
                 0.81 
               
               
                   
                 Y099A 
                   
                 0.33 
               
               
                   
                 Y099R 
                   
                 0.53 
               
               
                   
                 Y099S 
                   
                 0.43 
               
               
                   
                 V102A 
                   
                 0.83 
               
               
                   
                 V102C 
                   
                 0.69 
               
               
                   
                 V102E 
                   
                 0.90 
               
               
                   
                 V102G 
                   
                 0.67 
               
               
                   
                 V102H 
                   
                 0.88 
               
               
                   
                 V102K 
                   
                 1.03 
               
               
                   
                 V102L 
                   
                 0.71 
               
               
                   
                 V102M 
                   
                 0.77 
               
               
                   
                 V102N 
                   
                 1.02 
               
               
                   
                 V102Q 
                   
                 1.03 
               
               
                   
                 V102R 
                   
                 0.94 
               
               
                   
                 V102S 
                 311 
                 1.41 
               
               
                   
                 V102T 
                 312 
                 1.26 
               
               
                   
                 V102W 
                   
                 0.76 
               
               
                   
                 D103N 
                   
                 0.39 
               
               
                   
                 N104A 
                   
                 0.69 
               
               
                   
                 N104C 
                   
                 0.41 
               
               
                   
                 N104G 
                   
                 0.48 
               
               
                   
                 N104K 
                   
                 0.88 
               
               
                   
                 N104M 
                   
                 0.61 
               
               
                   
                 N104R 
                 313 
                 1.25 
               
               
                   
                 N104S 
                   
                 1.03 
               
               
                   
                 N104T 
                   
                 0.71 
               
               
                   
                 L105A 
                   
                 0.54 
               
               
                   
                 L105G 
                   
                 0.51 
               
               
                   
                 L105I 
                   
                 0.94 
               
               
                   
                 L105P 
                   
                 0.84 
               
               
                   
                 L105Q 
                   
                 0.90 
               
               
                   
                 L105R 
                   
                 0.65 
               
               
                   
                 L105S 
                   
                 0.61 
               
               
                   
                 L105T 
                   
                 0.51 
               
               
                   
                 L105W 
                   
                 0.34 
               
               
                   
                 L105V 
                   
                 0.99 
               
               
                   
                 G106V 
                   
                 0.43 
               
               
                   
                 M107F 
                   
                 0.91 
               
               
                   
                 M107I 
                   
                 0.67 
               
               
                   
                 M107L 
                 314 
                 1.32 
               
               
                   
                 A108G 
                   
                 0.47 
               
               
                   
                 I110V 
                   
                 0.51 
               
               
                   
                 E114A 
                 315 
                 1.44 
               
               
                   
                 E114G 
                   
                 0.73 
               
               
                   
                 E114H 
                   
                 0.75 
               
               
                   
                 E114M 
                   
                 0.44 
               
               
                   
                 E114S 
                   
                 0.69 
               
               
                   
                 P117D 
                   
                 0.56 
               
               
                   
                 T118H 
                   
                 0.47 
               
               
                   
                 T118K 
                   
                 0.53 
               
               
                   
                 T118L 
                   
                 1.09 
               
               
                   
                 T118M 
                   
                 0.53 
               
               
                   
                 T118N 
                   
                 0.67 
               
               
                   
                 T118Q 
                 316 
                 3.37 
               
               
                   
                 T118V 
                   
                 0.79 
               
               
                   
                 W119F 
                   
                 0.53 
               
               
                   
                 W119P 
                   
                 0.36 
               
               
                   
                 W119Y 
                   
                 1.08 
               
               
                   
                 A120D 
                   
                 0.76 
               
               
                   
                 A120F 
                 318 
                 2.62 
               
               
                   
                 A120G 
                   
                 1.03 
               
               
                   
                 A120H 
                 317 
                 1.11 
               
               
                   
                 A120I 
                 319 
                 1.33 
               
               
                   
                 A120L 
                   
                 1.25 
               
               
                   
                 A120N 
                   
                 0.81 
               
               
                   
                 A120P 
                   
                 0.42 
               
               
                   
                 A120R 
                   
                 0.82 
               
               
                   
                 A120S 
                 320 
                 1.21 
               
               
                   
                 A120T 
                   
                 0.62 
               
               
                   
                 A120V 
                 321 
                 1.53 
               
               
                   
                 A120W 
                   
                 0.59 
               
               
                   
                 A120Y 
                 322 
                 1.95 
               
               
                   
                 N122M 
                   
                 0.56 
               
               
                   
                 K124L 
                   
                 0.34 
               
               
                   
                 K124R 
                   
                 0.62 
               
               
                   
                 P125H 
                   
                 0.43 
               
               
                   
                 P125R 
                   
                 0.63 
               
               
                   
                 P125S 
                   
                 0.54 
               
               
                   
                 D127A 
                   
                 0.89 
               
               
                   
                 D127E 
                 323 
                 1.31 
               
               
                   
                 D127G 
                   
                 0.97 
               
               
                   
                 D127H 
                 324 
                 2.33 
               
               
                   
                 D127L 
                   
                 0.84 
               
               
                   
                 D127M 
                   
                 0.4 
               
               
                   
                 D127N 
                 325 
                 1.69 
               
               
                   
                 D127Q 
                 326 
                 1.21 
               
               
                   
                 D127R 
                 327 
                 0.51 
               
               
                   
                 D127S 
                   
                 0.77 
               
               
                   
                 D127T 
                   
                 1.11 
               
               
                   
                 D127V 
                   
                 0.56 
               
               
                   
                 D127W 
                   
                 0.44 
               
               
                   
                 V128A 
                   
                 0.53 
               
               
                   
                 V128C 
                   
                 0.68 
               
               
                   
                 V128G 
                   
                 0.49 
               
               
                   
                 V128I 
                 328 
                 1.25 
               
               
                   
                 V128K 
                   
                 1.16 
               
               
                   
                 V128L 
                   
                 0.95 
               
               
                   
                 V128Q 
                   
                 0.55 
               
               
                   
                 V128R 
                   
                 0.74 
               
               
                   
                 V128S 
                   
                 0.53 
               
               
                   
                 V128W 
                   
                 0.50 
               
               
                   
                 K130I 
                   
                 0.50 
               
               
                   
                 K130R 
                 329 
                 1.42 
               
               
                   
                 N131C 
                   
                 0.60 
               
               
                   
                 N131E 
                   
                 0.44 
               
               
                   
                 N131F 
                   
                 0.63 
               
               
                   
                 N131G 
                 330 
                 2.47 
               
               
                   
                 N131H 
                   
                 0.80 
               
               
                   
                 N131I 
                 331 
                 1.40 
               
               
                   
                 N131L 
                   
                 0.82 
               
               
                   
                 N131M 
                 332 
                 0.99 
               
               
                   
                 N131Q 
                 333 
                 1.24 
               
               
                   
                 N131R 
                 334 
                 2.81 
               
               
                   
                 N131S 
                   
                 0.76 
               
               
                   
                 N131T 
                   
                 1.02 
               
               
                   
                 N131V 
                 335 
                 2.08 
               
               
                   
                 N131Y 
                   
                 0.85 
               
               
                   
                 R132A 
                   
                 0.68 
               
               
                   
                 R132C 
                   
                 0.58 
               
               
                   
                 R132E 
                   
                 0.70 
               
               
                   
                 R132F 
                   
                 0.60 
               
               
                   
                 R132H 
                   
                 0.66 
               
               
                   
                 K279A 
                   
                 0.27 
               
               
                   
                 E285A 
                   
                 0.34 
               
               
                   
                 R132I 
                   
                 0.56 
               
               
                   
                 R132K 
                   
                 1.05 
               
               
                   
                 R132L 
                 337 
                 0.76 
               
               
                   
                 R132N 
                 336 
                 1.28 
               
               
                   
                 R132Q 
                   
                 0.69 
               
               
                   
                 R132S 
                   
                 0.79 
               
               
                   
                 R132T 
                   
                 0.61 
               
               
                   
                 R132V 
                   
                 0.73 
               
               
                   
                 R132Y 
                   
                 0.78 
               
               
                   
                 S133I 
                   
                 0.54 
               
               
                   
                 I134L 
                   
                 1.04 
               
               
                   
                 I134T 
                   
                 0.60 
               
               
                   
                 I134V 
                   
                 1.08 
               
               
                   
                 E135A 
                   
                 0.99 
               
               
                   
                 E135C 
                   
                 0.77 
               
               
                   
                 E135D 
                 338 
                 2.68 
               
               
                   
                 E135F 
                   
                 0.73 
               
               
                   
                 E442L 
                   
                 0.4 
               
               
                   
                 E135G 
                 339 
                 2.79 
               
               
                   
                 E135H 
                   
                 0.79 
               
               
                   
                 E135K 
                   
                 1.15 
               
               
                   
                 E135L 
                   
                 0.82 
               
               
                   
                 E135N 
                   
                 0.56 
               
               
                   
                 E135Q 
                   
                 1.59 
               
               
                   
                 E135R 
                 340 
                 2.08 
               
               
                   
                 E135S 
                   
                 1.13 
               
               
                   
                 E135W 
                   
                 0.63 
               
               
                   
                 E135Y 
                   
                 0.50 
               
               
                   
                 L136A 
                   
                 0.73 
               
               
                   
                 L136C 
                   
                 0.56 
               
               
                   
                 L136D 
                   
                 0.47 
               
               
                   
                 L136F 
                   
                 0.96 
               
               
                   
                 L136H 
                   
                 1.00 
               
               
                   
                 L136I 
                   
                 0.65 
               
               
                   
                 L136M 
                   
                 1.05 
               
               
                   
                 L136N 
                   
                 0.48 
               
               
                   
                 L136Q 
                   
                 0.61 
               
               
                   
                 L136R 
                   
                 0.74 
               
               
                   
                 L136S 
                   
                 0.80 
               
               
                   
                 L136T 
                   
                 0.72 
               
               
                   
                 L136W 
                   
                 1.11 
               
               
                   
                 V137A 
                   
                 0.48 
               
               
                   
                 V137I 
                   
                 1.01 
               
               
                   
                 V137T 
                   
                 0.51 
               
               
                   
                 Q138A 
                   
                 0.69 
               
               
                   
                 Q138C 
                   
                 0.65 
               
               
                   
                 Q138H 
                   
                 0.71 
               
               
                   
                 Q138I 
                   
                 0.54 
               
               
                   
                 Q138L 
                 341 
                 0.59 
               
               
                   
                 Q138M 
                   
                 0.68 
               
               
                   
                 Q138N 
                   
                 0.61 
               
               
                   
                 Q138R 
                   
                 0.53 
               
               
                   
                 Q138S 
                   
                 0.48 
               
               
                   
                 Q138W 
                   
                 0.41 
               
               
                   
                 Q138Y 
                   
                 0.60 
               
               
                   
                 Q139A 
                   
                 0.92 
               
               
                   
                 Q139C 
                   
                 0.44 
               
               
                   
                 Q139D 
                   
                 0.48 
               
               
                   
                 Q139E 
                   
                 0.94 
               
               
                   
                 Q139F 
                   
                 0.53 
               
               
                   
                 Q139G 
                   
                 0.65 
               
               
                   
                 Q139H 
                   
                 0.56 
               
               
                   
                 Q139K 
                   
                 0.73 
               
               
                   
                 Q139L 
                   
                 0.70 
               
               
                   
                 Q139M 
                   
                 0.95 
               
               
                   
                 Q139R 
                   
                 0.79 
               
               
                   
                 Q139S 
                   
                 0.81 
               
               
                   
                 Q139T 
                 342 
                 1.31 
               
               
                   
                 Q139V 
                   
                 0.77 
               
               
                   
                 Q140A 
                   
                 0.96 
               
               
                   
                 Q140C 
                   
                 0.50 
               
               
                   
                 Q140D 
                   
                 0.59 
               
               
                   
                 Q140F 
                   
                 0.66 
               
               
                   
                 Q140G 
                   
                 0.73 
               
               
                   
                 Q140H 
                   
                 0.84 
               
               
                   
                 Q140I 
                   
                 0.75 
               
               
                   
                 Q140K 
                 343 
                 0.93 
               
               
                   
                 Q140L 
                   
                 0.51 
               
               
                   
                 Q140M 
                   
                 0.80 
               
               
                   
                 Q140R 
                   
                 0.85 
               
               
                   
                 Q140V 
                   
                 0.61 
               
               
                   
                 Q140W 
                   
                 0.59 
               
               
                   
                 Q140Y 
                   
                 0.41 
               
               
                   
                 N141A 
                   
                 1.12 
               
               
                   
                 N141D 
                   
                 1.09 
               
               
                   
                 N141E 
                   
                 0.67 
               
               
                   
                 N141F 
                   
                 0.81 
               
               
                   
                 N141G 
                   
                 1.15 
               
               
                   
                 N141H 
                 344 
                 2.03 
               
               
                   
                 N002I 
                   
                 0.37 
               
               
                   
                 G297A 
                   
                 0.57 
               
               
                   
                 N141L 
                   
                 0.61 
               
               
                   
                 N141M 
                   
                 0.48 
               
               
                   
                 N141Q 
                   
                 1.16 
               
               
                   
                 N141R 
                 345 
                 1.40 
               
               
                   
                 N141S 
                 346 
                 0.72 
               
               
                   
                 N141T 
                   
                 0.45 
               
               
                   
                 N141V 
                   
                 0.50 
               
               
                   
                 N141W 
                 347 
                 0.83 
               
               
                   
                 N141Y 
                 348 
                 1.55 
               
               
                   
                 V142C 
                   
                 0.61 
               
               
                   
                 V142D 
                 349 
                 0.71 
               
               
                   
                 V142E 
                   
                 0.87 
               
               
                   
                 V142G 
                 350 
                 0.98 
               
               
                   
                 V142H 
                   
                 1.11 
               
               
                   
                 V142I 
                   
                 0.81 
               
               
                   
                 V142K 
                 351 
                 1.40 
               
               
                   
                 V142L 
                   
                 0.75 
               
               
                   
                 V142M 
                   
                 0.76 
               
               
                   
                 V142N 
                 352 
                 0.98 
               
               
                   
                 V142P 
                 353 
                 0.88 
               
               
                   
                 V142Q 
                 354 
                 1.04 
               
               
                   
                 V142R 
                 355 
                 1.53 
               
               
                   
                 V142S 
                 356 
                 0.93 
               
               
                   
                 V142T 
                 357 
                 1.19 
               
               
                   
                 Q143E 
                   
                 0.77 
               
               
                   
                 Q143G 
                 358 
                 0.62 
               
               
                   
                 Q143I 
                   
                 0.44 
               
               
                   
                 Q143K 
                 359 
                 1.30 
               
               
                   
                 I009Q 
                 82 
                 0.4 
               
               
                   
                 Q143L 
                   
                 0.56 
               
               
                   
                 Q143N 
                   
                 0.73 
               
               
                   
                 Q143V 
                   
                 0.57 
               
               
                   
                 L144T 
                 361 
                 1.02 
               
               
                   
                 L144W 
                   
                 0.79 
               
               
                   
                 S145A 
                   
                 0.58 
               
               
                   
                 S145C 
                   
                 0.44 
               
               
                   
                 S145D 
                   
                 0.48 
               
               
                   
                 S145E 
                   
                 0.56 
               
               
                   
                 S145G 
                   
                 0.94 
               
               
                   
                 S145H 
                   
                 0.56 
               
               
                   
                 S145L 
                   
                 0.44 
               
               
                   
                 S145M 
                   
                 0.56 
               
               
                   
                 S145N 
                   
                 0.58 
               
               
                   
                 S145P 
                   
                 1.04 
               
               
                   
                 S145R 
                   
                 0.97 
               
               
                   
                 L146A 
                   
                 0.52 
               
               
                   
                 L146C 
                   
                 0.42 
               
               
                   
                 G305N 
                   
                 0.36 
               
               
                   
                 M310Y 
                   
                 0.38 
               
               
                   
                 L146E 
                   
                 0.50 
               
               
                   
                 L146G 
                   
                 0.62 
               
               
                   
                 L146H 
                   
                 0.78 
               
               
                   
                 L146I 
                   
                 0.82 
               
               
                   
                 L146K 
                   
                 0.84 
               
               
                   
                 L146N 
                   
                 0.57 
               
               
                   
                 L146P 
                 362 
                 0.93 
               
               
                   
                 L146Q 
                   
                 0.84 
               
               
                   
                 L146R 
                 363 
                 1.47 
               
               
                   
                 L146S 
                   
                 0.71 
               
               
                   
                 L146T 
                   
                 0.74 
               
               
                   
                 L146V 
                   
                 0.84 
               
               
                   
                 L146Y 
                   
                 0.80 
               
               
                   
                 S312K 
                   
                 0.38 
               
               
                   
                 T147A 
                 364 
                 1.20 
               
               
                   
                 T147C 
                   
                 0.47 
               
               
                   
                 T147D 
                   
                 0.71 
               
               
                   
                 T147F 
                 365 
                 1.24 
               
               
                   
                 T147G 
                   
                 1.05 
               
               
                   
                 T147I 
                   
                 0.85 
               
               
                   
                 T147L 
                 366 
                 1.30 
               
               
                   
                 T147M 
                   
                 0.79 
               
               
                   
                 T147P 
                   
                 1.09 
               
               
                   
                 T147Q 
                   
                 1.29 
               
               
                   
                 T147R 
                 367 
                 2.11 
               
               
                   
                 T147S 
                 368 
                 1.27 
               
               
                   
                 T147V 
                 369 
                 2.04 
               
               
                   
                 T147W 
                   
                 0.97 
               
               
                   
                 T147Y 
                   
                 1.04 
               
               
                   
                 E148C 
                   
                 0.66 
               
               
                   
                 E148F 
                   
                 0.42 
               
               
                   
                 E148G 
                   
                 1.05 
               
               
                   
                 E148H 
                 370 
                 1.24 
               
               
                   
                 E148I 
                   
                 0.73 
               
               
                   
                 E148K 
                 371 
                 1.63 
               
               
                   
                 E148L 
                   
                 0.85 
               
               
                   
                 E148Q 
                 372 
                 1.44 
               
               
                   
                 E148R 
                   
                 0.97 
               
               
                   
                 E148S 
                   
                 1.15 
               
               
                   
                 E148T 
                   
                 0.82 
               
               
                   
                 E148V 
                   
                 0.99 
               
               
                   
                 E148W 
                   
                 0.43 
               
               
                   
                 E148Y 
                   
                 0.95 
               
               
                   
                 A149C 
                   
                 1.15 
               
               
                   
                 A149G 
                   
                 0.52 
               
               
                   
                 A149K 
                   
                 0.51 
               
               
                   
                 A149L 
                   
                 0.88 
               
               
                   
                 A149M 
                   
                 0.88 
               
               
                   
                 A149Q 
                   
                 1.15 
               
               
                   
                 A149R 
                   
                 1.02 
               
               
                   
                 A149S 
                   
                 1.08 
               
               
                   
                 A149T 
                 373 
                 1.24 
               
               
                   
                 A149V 
                 374 
                 1.34 
               
               
                   
                 T150A 
                 375 
                 1.21 
               
               
                   
                 T150C 
                   
                 0.70 
               
               
                   
                 T150D 
                 376 
                 1.24 
               
               
                   
                 T150E 
                   
                 1.05 
               
               
                   
                 T150F 
                   
                 0.71 
               
               
                   
                 T150G 
                 377 
                 2.19 
               
               
                   
                 T150I 
                   
                 0.52 
               
               
                   
                 T150L 
                   
                 0.70 
               
               
                   
                 T150N 
                 378 
                 0.91 
               
               
                   
                 T150P 
                   
                 0.88 
               
               
                   
                 T150R 
                   
                 0.90 
               
               
                   
                 T150S 
                 379 
                 0.92 
               
               
                   
                 T150W 
                 380 
                 1.25 
               
               
                   
                 T150Y 
                 381 
                 1.36 
               
               
                   
                 E151A 
                 382 
                 1.27 
               
               
                   
                 E151C 
                   
                 1.00 
               
               
                   
                 E151G 
                   
                 1.06 
               
               
                   
                 E151H 
                 383 
                 1.34 
               
               
                   
                 E151K 
                 384 
                 2.05 
               
               
                   
                 E151L 
                 385 
                 1.03 
               
               
                   
                 E151M 
                 386 
                 1.26 
               
               
                   
                 E151N 
                   
                 0.95 
               
               
                   
                 E151Q 
                 387 
                 2.01 
               
               
                   
                 D320L 
                   
                 0.37 
               
               
                   
                 E151R 
                 388 
                 1.61 
               
               
                   
                 E151S 
                 389 
                 1.28 
               
               
                   
                 E151T 
                 390 
                 1.21 
               
               
                   
                 E151V 
                 391 
                 1.38 
               
               
                   
                 E151W 
                 392 
                 1.31 
               
               
                   
                 E151Y 
                 393 
                 1.31 
               
               
                   
                 K152A 
                   
                 0.51 
               
               
                   
                 K152C 
                   
                 0.52 
               
               
                   
                 K152F 
                   
                 0.61 
               
               
                   
                 K152I 
                   
                 0.65 
               
               
                   
                 K152M 
                   
                 0.75 
               
               
                   
                 K152R 
                 394 
                 1.85 
               
               
                   
                 K152T 
                 395 
                 1.20 
               
               
                   
                 K152V 
                   
                 0.82 
               
               
                   
                 K152Y 
                   
                 0.67 
               
               
                   
                 A153I 
                   
                 0.93 
               
               
                   
                 A153L 
                   
                 0.51 
               
               
                   
                 K154R 
                   
                 0.86 
               
               
                   
                 K154T 
                   
                 0.83 
               
               
                   
                 K154V 
                   
                 0.46 
               
               
                   
                 Q155A 
                   
                 0.91 
               
               
                   
                 Q155C 
                   
                 0.60 
               
               
                   
                 Q155D 
                 397 
                 1.49 
               
               
                   
                 Q155F 
                   
                 0.70 
               
               
                   
                 Q155G 
                 398 
                 1.61 
               
               
                   
                 Q155H 
                   
                 1.03 
               
               
                   
                 Q155K 
                 399 
                 1.57 
               
               
                   
                 Q155L 
                   
                 0.86 
               
               
                   
                 Q155M 
                   
                 0.97 
               
               
                   
                 Q155R 
                 400 
                 1.27 
               
               
                   
                 Q155S 
                   
                 0.77 
               
               
                   
                 Q155T 
                   
                 0.76 
               
               
                   
                 Q155V 
                   
                 0.73 
               
               
                   
                 Q155W 
                   
                 0.91 
               
               
                   
                 E156A 
                   
                 0.79 
               
               
                   
                 E156D 
                 401 
                 1.95 
               
               
                   
                 E156G 
                   
                 0.49 
               
               
                   
                 E156I 
                   
                 0.51 
               
               
                   
                 E156L 
                   
                 0.43 
               
               
                   
                 E156M 
                   
                 0.87 
               
               
                   
                 E156Q 
                   
                 0.84 
               
               
                   
                 E156R 
                   
                 0.43 
               
               
                   
                 E156S 
                   
                 0.62 
               
               
                   
                 E156T 
                   
                 0.69 
               
               
                   
                 E156V 
                   
                 0.45 
               
               
                   
                 E156W 
                   
                 0.49 
               
               
                   
                 F157W 
                   
                 0.61 
               
               
                   
                 E158A 
                   
                 0.56 
               
               
                   
                 E158F 
                   
                 0.51 
               
               
                   
                 E158H 
                   
                 0.54 
               
               
                   
                 I326S 
                   
                 0.95 
               
               
                   
                 I331E 
                   
                 0.34 
               
               
                   
                 E158L 
                   
                 0.44 
               
               
                   
                 E158Q 
                 402 
                 1.25 
               
               
                   
                 E158S 
                 403 
                 0.95 
               
               
                   
                 K159A 
                   
                 0.64 
               
               
                   
                 K159D 
                   
                 0.52 
               
               
                   
                 K159E 
                   
                 0.49 
               
               
                   
                 K159H 
                   
                 0.74 
               
               
                   
                 K159L 
                   
                 0.62 
               
               
                   
                 K159M 
                   
                 0.66 
               
               
                   
                 K159N 
                   
                 0.73 
               
               
                   
                 K159Q 
                   
                 0.92 
               
               
                   
                 K159R 
                   
                 0.88 
               
               
                   
                 K159S 
                   
                 0.67 
               
               
                   
                 K159V 
                   
                 0.41 
               
               
                   
                 A160C 
                   
                 0.61 
               
               
                   
                 A160F 
                   
                 0.79 
               
               
                   
                 A160G 
                   
                 0.75 
               
               
                   
                 A160H 
                   
                 0.47 
               
               
                   
                 A160I 
                   
                 0.43 
               
               
                   
                 A160K 
                   
                 0.91 
               
               
                   
                 A160L 
                   
                 0.67 
               
               
                   
                 M035Q 
                   
                 0.37 
               
               
                   
                 A160M 
                   
                 0.77 
               
               
                   
                 A160N 
                   
                 0.56 
               
               
                   
                 A160Q 
                   
                 0.65 
               
               
                   
                 A160R 
                   
                 0.89 
               
               
                   
                 A160S 
                 404 
                 1.35 
               
               
                   
                 A160V 
                   
                 0.73 
               
               
                   
                 A160Y 
                   
                 1.07 
               
               
                   
                 G161A 
                   
                 0.99 
               
               
                   
                 G161C 
                   
                 0.44 
               
               
                   
                 G161D 
                   
                 0.86 
               
               
                   
                 G161E 
                   
                 0.49 
               
               
                   
                 G161R 
                   
                 0.48 
               
               
                   
                 S036N 
                 148 
                 0.38 
               
               
                   
                 G161S 
                   
                 0.77 
               
               
                   
                 G161V 
                   
                 0.42 
               
               
                   
                 K162A 
                   
                 0.50 
               
               
                   
                 K162D 
                   
                 0.77 
               
               
                   
                 K162E 
                 405 
                 0.51 
               
               
                   
                 V351C 
                   
                 0.35 
               
               
                   
                 W357K 
                   
                 0.36 
               
               
                   
                 K162G 
                   
                 0.56 
               
               
                   
                 K162H 
                   
                 0.62 
               
               
                   
                 K162L 
                   
                 0.54 
               
               
                   
                 K162M 
                   
                 1.04 
               
               
                   
                 K162P 
                   
                 0.64 
               
               
                   
                 K162Q 
                   
                 0.58 
               
               
                   
                 K162R 
                   
                 0.52 
               
               
                   
                 K162S 
                   
                 0.47 
               
               
                   
                 K162V 
                   
                 0.52 
               
               
                   
                 K162W 
                   
                 1.01 
               
               
                   
                 K162Y 
                   
                 0.72 
               
               
                   
                 D163A 
                 406 
                 1.52 
               
               
                   
                 D163E 
                 407 
                 1.63 
               
               
                   
                 D163G 
                   
                 1.15 
               
               
                   
                 D163K 
                 408 
                 1.90 
               
               
                   
                 D163L 
                   
                 1.18 
               
               
                   
                 D163Q 
                 409 
                 1.40 
               
               
                   
                 D163R 
                 410 
                 1.80 
               
               
                   
                 D163S 
                 411 
                 1.34 
               
               
                   
                 D163T 
                   
                 1.13 
               
               
                   
                 D163V 
                   
                 0.76 
               
               
                   
                 F164L 
                   
                 1.13 
               
               
                   
                 F164M 
                 412 
                 1.66 
               
               
                   
                 F164V 
                 413 
                 1.23 
               
               
                   
                 S043N 
                   
                 0.34 
               
               
                   
                 F164W 
                   
                 0.72 
               
               
                   
                 L165A 
                   
                 0.48 
               
               
                   
                 L165D 
                 414 
                 5.79 
               
               
                   
                 L165F 
                 415 
                 1.23 
               
               
                   
                 L165N 
                 416 
                 2.19 
               
               
                   
                 L165R 
                   
                 0.59 
               
               
                   
                 L165S 
                 417 
                 1.31 
               
               
                   
                 L165V 
                 418 
                 1.22 
               
               
                   
                 L165W 
                   
                 1.14 
               
               
                   
                 A371G 
                   
                 0.38 
               
               
                   
                 L165Y 
                   
                 0.66 
               
               
                   
                 V166A 
                 419 
                 2.85 
               
               
                   
                 V166C 
                   
                 1.16 
               
               
                   
                 V166E 
                 420 
                 1.28 
               
               
                   
                 V166F 
                 421 
                 1.67 
               
               
                   
                 V166G 
                   
                 1.11 
               
               
                   
                 V166H 
                 422 
                 1.74 
               
               
                   
                 V166L 
                 423 
                 4.38 
               
               
                   
                 V166Q 
                 424 
                 3.61 
               
               
                   
                 V166R 
                 425 
                 5.56 
               
               
                   
                 V166T 
                 426 
                 4.26 
               
               
                   
                 V166W 
                 427 
                 1.26 
               
               
                   
                 V166Y 
                 428 
                 2.08 
               
               
                   
                 E167A 
                   
                 0.84 
               
               
                   
                 E167D 
                 429 
                 0.69 
               
               
                   
                 E167G 
                   
                 0.60 
               
               
                   
                 E167H 
                   
                 0.89 
               
               
                   
                 E167K 
                   
                 0.91 
               
               
                   
                 E167M 
                   
                 0.87 
               
               
                   
                 E167N 
                   
                 0.83 
               
               
                   
                 E167P 
                   
                 0.58 
               
               
                   
                 E167R 
                   
                 1.02 
               
               
                   
                 E167S 
                   
                 1.17 
               
               
                   
                 E167T 
                   
                 0.59 
               
               
                   
                 E167Y 
                   
                 0.55 
               
               
                   
                 T168H 
                   
                 0.46 
               
               
                   
                 I169L 
                 430 
                 2.08 
               
               
                   
                 I169R 
                   
                 0.54 
               
               
                   
                 I169V 
                   
                 0.74 
               
               
                   
                 K170N 
                   
                 0.72 
               
               
                   
                 K170R 
                 431 
                 2.58 
               
               
                   
                 K170V 
                   
                 0.58 
               
               
                   
                 L171I 
                   
                 0.73 
               
               
                   
                 L171V 
                   
                 0.64 
               
               
                   
                 G172A 
                 432 
                 1.20 
               
               
                   
                 G172C 
                   
                 1.03 
               
               
                   
                 K173N 
                   
                 0.44 
               
               
                   
                 K173R 
                 433 
                 0.82 
               
               
                   
                 L174A 
                   
                 1.20 
               
               
                   
                 L174G 
                 434 
                 0.40 
               
               
                   
                 L174K 
                 435 
                 2.39 
               
               
                   
                 L174M 
                   
                 0.79 
               
               
                   
                 L174N 
                 436 
                 1.36 
               
               
                   
                 L174Q 
                   
                 0.99 
               
               
                   
                 L174R 
                 437 
                 1.50 
               
               
                   
                 L174S 
                   
                 0.85 
               
               
                   
                 L174T 
                 438 
                 1.12 
               
               
                   
                 L174V 
                   
                 0.62 
               
               
                   
                 L174W 
                   
                 0.78 
               
               
                   
                 L174Y 
                   
                 1.06 
               
               
                   
                 L175E 
                   
                 0.43 
               
               
                   
                 Q051A 
                   
                 0.34 
               
               
                   
                 L175H 
                   
                 0.57 
               
               
                   
                 L175T 
                 439 
                 1.43 
               
               
                   
                 L175V 
                   
                 0.94 
               
               
                   
                 L175Y 
                   
                 0.66 
               
               
                   
                 R176K 
                   
                 0.67 
               
               
                   
                 N178G 
                   
                 0.85 
               
               
                   
                 N178K 
                 440 
                 0.85 
               
               
                   
                 N178M 
                   
                 0.88 
               
               
                   
                 K376L 
                   
                 0.37 
               
               
                   
                 F380T 
                   
                 0.39 
               
               
                   
                 N178R 
                 441 
                 1.10 
               
               
                   
                 H179A 
                   
                 1.06 
               
               
                   
                 H179C 
                   
                 0.94 
               
               
                   
                 H179E 
                   
                 0.62 
               
               
                   
                 H179G 
                   
                 0.86 
               
               
                   
                 H179I 
                   
                 0.90 
               
               
                   
                 H179K 
                 442 
                 1.39 
               
               
                   
                 H179L 
                   
                 0.73 
               
               
                   
                 H179M 
                   
                 0.63 
               
               
                   
                 H179N 
                   
                 0.96 
               
               
                   
                 H179P 
                   
                 0.44 
               
               
                   
                 H179R 
                   
                 0.96 
               
               
                   
                 H179S 
                   
                 0.51 
               
               
                   
                 H179T 
                   
                 0.43 
               
               
                   
                 H179V 
                   
                 0.42 
               
               
                   
                 L180F 
                   
                 0.59 
               
               
                   
                 L180G 
                   
                 0.62 
               
               
                   
                 L180K 
                   
                 0.44 
               
               
                   
                 L180M 
                   
                 0.64 
               
               
                   
                 W181M 
                   
                 0.88 
               
               
                   
                 L061F 
                   
                 0.3 
               
               
                   
                 W181Q 
                   
                 0.88 
               
               
                   
                 G182L 
                   
                 0.90 
               
               
                   
                 Y183L 
                   
                 0.70 
               
               
                   
                 F186Y 
                   
                 0.59 
               
               
                   
                 H192S 
                   
                 0.49 
               
               
                   
                 H192T 
                   
                 0.50 
               
               
                   
                 H193G 
                   
                 0.68 
               
               
                   
                 H193Q 
                 443 
                 0.82 
               
               
                   
                 H193S 
                   
                 0.42 
               
               
                   
                 H193Y 
                   
                 0.58 
               
               
                   
                 K195A 
                   
                 0.51 
               
               
                   
                 K195G 
                   
                 0.45 
               
               
                   
                 K195H 
                   
                 0.45 
               
               
                   
                 K195I 
                   
                 0.50 
               
               
                   
                 K195L 
                   
                 0.45 
               
               
                   
                 K195N 
                 445 
                 0.74 
               
               
                   
                 K195Q 
                   
                 0.71 
               
               
                   
                 K195R 
                   
                 0.85 
               
               
                   
                 K195S 
                   
                 0.42 
               
               
                   
                 K195T 
                 444 
                 0.58 
               
               
                   
                 K195W 
                   
                 0.49 
               
               
                   
                 K196E 
                 446 
                 0.43 
               
               
                   
                 D068G 
                   
                 0.37 
               
               
                   
                 K196G 
                   
                 0.41 
               
               
                   
                 K196L 
                   
                 0.65 
               
               
                   
                 K196R 
                 447 
                 0.58 
               
               
                   
                 K196S 
                   
                 0.68 
               
               
                   
                 K196T 
                   
                 1.18 
               
               
                   
                 K196W 
                   
                 0.55 
               
               
                   
                 P197A 
                   
                 0.81 
               
               
                   
                 P197D 
                   
                 0.58 
               
               
                   
                 P197E 
                   
                 0.52 
               
               
                   
                 P197F 
                   
                 0.48 
               
               
                   
                 P197G 
                   
                 0.75 
               
               
                   
                 P197H 
                   
                 0.62 
               
               
                   
                 P197K 
                   
                 0.99 
               
               
                   
                 P197L 
                   
                 0.56 
               
               
                   
                 P197M 
                   
                 1.03 
               
               
                   
                 P197Q 
                   
                 0.69 
               
               
                   
                 P197R 
                   
                 0.58 
               
               
                   
                 P197S 
                   
                 0.70 
               
               
                   
                 P197T 
                   
                 0.41 
               
               
                   
                 G198A 
                   
                 0.80 
               
               
                   
                 G198D 
                 448 
                 1.99 
               
               
                   
                 G198E 
                   
                 0.49 
               
               
                   
                 G198H 
                   
                 0.84 
               
               
                   
                 G198L 
                   
                 0.48 
               
               
                   
                 G198N 
                   
                 0.80 
               
               
                   
                 G198Q 
                   
                 0.55 
               
               
                   
                 G198R 
                   
                 0.58 
               
               
                   
                 G198S 
                   
                 0.76 
               
               
                   
                 G198T 
                   
                 0.41 
               
               
                   
                 G198Y 
                   
                 0.81 
               
               
                   
                 N200D 
                   
                 0.46 
               
               
                   
                 S202M 
                   
                 0.40 
               
               
                   
                 F204P 
                 449 
                 0.63 
               
               
                   
                 N205A 
                 450 
                 1.30 
               
               
                   
                 N205D 
                   
                 0.85 
               
               
                   
                 N205E 
                 451 
                 1.94 
               
               
                   
                 N205F 
                   
                 0.52 
               
               
                   
                 N205G 
                   
                 0.79 
               
               
                   
                 N205K 
                   
                 0.76 
               
               
                   
                 N205M 
                   
                 0.58 
               
               
                   
                 N205P 
                   
                 0.75 
               
               
                   
                 N205R 
                   
                 0.54 
               
               
                   
                 N205S 
                   
                 0.80 
               
               
                   
                 N205T 
                 453 
                 0.85 
               
               
                   
                 N205V 
                   
                 0.49 
               
               
                   
                 N205W 
                   
                 0.41 
               
               
                   
                 V206H 
                   
                 0.50 
               
               
                   
                 V206I 
                 454 
                 0.94 
               
               
                   
                 V206K 
                 455 
                 1.75 
               
               
                   
                 V206L 
                 456 
                 1.57 
               
               
                   
                 S395T 
                   
                 0.39 
               
               
                   
                 V206M 
                   
                 0.43 
               
               
                   
                 V206R 
                 457 
                 1.30 
               
               
                   
                 V206S 
                   
                 0.72 
               
               
                   
                 G072Y 
                   
                 0.35 
               
               
                   
                 V206T 
                   
                 0.59 
               
               
                   
                 I208A 
                   
                 0.62 
               
               
                   
                 I208C 
                   
                 0.48 
               
               
                   
                 I208K 
                   
                 0.91 
               
               
                   
                 I208L 
                   
                 0.84 
               
               
                   
                 I208M 
                   
                 0.88 
               
               
                   
                 I208Q 
                   
                 0.77 
               
               
                   
                 I208R 
                   
                 1.14 
               
               
                   
                 I208S 
                   
                 0.62 
               
               
                   
                 I208T 
                   
                 1.01 
               
               
                   
                 I208V 
                   
                 1.07 
               
               
                   
                 K209A 
                   
                 0.53 
               
               
                   
                 K209E 
                   
                 0.46 
               
               
                   
                 K209G 
                   
                 0.44 
               
               
                   
                 K209N 
                   
                 0.50 
               
               
                   
                 F398L 
                   
                 0.35 
               
               
                   
                 S404T 
                   
                 0.37 
               
               
                   
                 K209R 
                 458 
                 0.68 
               
               
                   
                 K209S 
                   
                 0.50 
               
               
                   
                 K209T 
                   
                 0.50 
               
               
                   
                 D212N 
                 459 
                 1.52 
               
               
                   
                 D212S 
                 460 
                 0.93 
               
               
                   
                 D212T 
                   
                 0.76 
               
               
                   
                 D213A 
                 461 
                 0.85 
               
               
                   
                 D213E 
                   
                 0.79 
               
               
                   
                 D213G 
                   
                 0.81 
               
               
                   
                 D213H 
                   
                 0.75 
               
               
                   
                 D213K 
                   
                 0.82 
               
               
                   
                 D213L 
                   
                 0.56 
               
               
                   
                 D213M 
                 462 
                 1.56 
               
               
                   
                 D213N 
                 463 
                 1.53 
               
               
                   
                 D213Q 
                   
                 1.04 
               
               
                   
                 D213R 
                   
                 0.92 
               
               
                   
                 D213V 
                   
                 0.47 
               
               
                   
                 D213W 
                   
                 0.49 
               
               
                   
                 D213Y 
                   
                 0.49 
               
               
                   
                 L214Q 
                   
                 0.57 
               
               
                   
                 S215A 
                   
                 0.74 
               
               
                   
                 S215D 
                   
                 0.62 
               
               
                   
                 S215E 
                   
                 0.74 
               
               
                   
                 S215G 
                   
                 0.88 
               
               
                   
                 S215H 
                 464 
                 0.91 
               
               
                   
                 S215K 
                   
                 0.99 
               
               
                   
                 S215L 
                   
                 0.60 
               
               
                   
                 S215M 
                 465 
                 1.77 
               
               
                   
                 S215Q 
                   
                 0.79 
               
               
                   
                 G077K 
                   
                 0.32 
               
               
                   
                 S215R 
                   
                 0.71 
               
               
                   
                 S215T 
                   
                 0.80 
               
               
                   
                 S215V 
                   
                 0.69 
               
               
                   
                 S215W 
                   
                 0.52 
               
               
                   
                 W216Y 
                   
                 0.48 
               
               
                   
                 L217M 
                   
                 0.51 
               
               
                   
                 W218F 
                   
                 0.57 
               
               
                   
                 N219A 
                 466 
                 1.29 
               
               
                   
                 N219C 
                   
                 0.43 
               
               
                   
                 N219D 
                   
                 0.75 
               
               
                   
                 N219E 
                   
                 0.95 
               
               
                   
                 N219H 
                   
                 0.97 
               
               
                   
                 N219I 
                 467 
                 0.60 
               
               
                   
                 N219K 
                 468 
                 1.45 
               
               
                   
                 N219L 
                   
                 0.72 
               
               
                   
                 N219M 
                   
                 1.02 
               
               
                   
                 I083H 
                   
                 0.4 
               
               
                   
                 N219R 
                   
                 1.10 
               
               
                   
                 N219S 
                 469 
                 2.48 
               
               
                   
                 N219T 
                   
                 0.82 
               
               
                   
                 N219W 
                   
                 0.48 
               
               
                   
                 E220A 
                   
                 0.75 
               
               
                   
                 E220H 
                 470 
                 1.40 
               
               
                   
                 E220I 
                 471 
                 1.34 
               
               
                   
                 E220L 
                 472 
                 1.45 
               
               
                   
                 E220S 
                   
                 0.62 
               
               
                   
                 E220T 
                   
                 0.91 
               
               
                   
                 E220V 
                 473 
                 1.35 
               
               
                   
                 S221A 
                   
                 0.72 
               
               
                   
                 S221C 
                   
                 0.59 
               
               
                   
                 S221M 
                   
                 0.46 
               
               
                   
                 S221Q 
                 474 
                 1.37 
               
               
                   
                 S221T 
                   
                 0.94 
               
               
                   
                 S221V 
                   
                 1.04 
               
               
                   
                 T222D 
                   
                 0.43 
               
               
                   
                 T222F 
                   
                 0.43 
               
               
                   
                 T222G 
                 475 
                 0.49 
               
               
                   
                 T222K 
                   
                 0.75 
               
               
                   
                 T222L 
                   
                 0.64 
               
               
                   
                 T222N 
                   
                 0.80 
               
               
                   
                 T222R 
                   
                 0.75 
               
               
                   
                 E220D 
                   
                 0.39 
               
               
                   
                 T222I 
                   
                 0.4 
               
               
                   
                 T222S 
                   
                 0.63 
               
               
                   
                 T222V 
                   
                 0.79 
               
               
                   
                 L224I 
                   
                 0.61 
               
               
                   
                 L230I 
                   
                 0.87 
               
               
                   
                 N231T 
                   
                 1.10 
               
               
                   
                 T232F 
                 476 
                 0.73 
               
               
                   
                 T232S 
                   
                 0.76 
               
               
                   
                 Q233A 
                   
                 0.71 
               
               
                   
                 Q233F 
                   
                 0.53 
               
               
                   
                 Q233G 
                 477 
                 0.46 
               
               
                   
                 Q233K 
                 478 
                 1.69 
               
               
                   
                 Q233L 
                   
                 0.69 
               
               
                   
                 Q233R 
                 479 
                 1.50 
               
               
                   
                 Q233Y 
                   
                 0.50 
               
               
                   
                 Q234M 
                 480 
                 1.65 
               
               
                   
                 S235A 
                 481 
                 0.47 
               
               
                   
                 S235E 
                   
                 1.00 
               
               
                   
                 S235G 
                   
                 0.95 
               
               
                   
                 S235H 
                   
                 0.44 
               
               
                   
                 S235K 
                   
                 0.53 
               
               
                   
                 S235T 
                   
                 0.66 
               
               
                   
                 P236A 
                   
                 1.07 
               
               
                   
                 P236G 
                   
                 1.09 
               
               
                   
                 P236H 
                   
                 0.46 
               
               
                   
                 P236K 
                   
                 0.71 
               
               
                   
                 P236R 
                 482 
                 3.09 
               
               
                   
                 Q234L 
                   
                 0.40 
               
               
                   
                 P236S 
                   
                 0.91 
               
               
                   
                 V237A 
                   
                 0.90 
               
               
                   
                 V237E 
                 484 
                 1.93 
               
               
                   
                 V237F 
                   
                 0.41 
               
               
                   
                 A412Q 
                 751 
                 0.35 
               
               
                   
                 A413Q 
                   
                 0.38 
               
               
                   
                 V414L 
                   
                 0.36 
               
               
                   
                 D418G 
                   
                 0.45 
               
               
                   
                 V237H 
                 485 
                 0.75 
               
               
                   
                 V237L 
                   
                 1.12 
               
               
                   
                 V237N 
                   
                 0.67 
               
               
                   
                 L089W 
                   
                 0.26 
               
               
                   
                 V237Q 
                 486 
                 1.46 
               
               
                   
                 V237R 
                   
                 0.71 
               
               
                   
                 V237S 
                   
                 1.03 
               
               
                   
                 V237T 
                 487 
                 1.01 
               
               
                   
                 V237W 
                   
                 0.52 
               
               
                   
                 A238D 
                   
                 0.75 
               
               
                   
                 A238E 
                 488 
                 0.59 
               
               
                   
                 A238H 
                 489 
                 0.60 
               
               
                   
                 A238K 
                   
                 0.60 
               
               
                   
                 A238Q 
                   
                 1.02 
               
               
                   
                 A238R 
                   
                 0.49 
               
               
                   
                 A238S 
                 490 
                 2.62 
               
               
                   
                 A238T 
                   
                 0.44 
               
               
                   
                 T240K 
                   
                 1.13 
               
               
                   
                 T240A 
                 491 
                 0.48 
               
               
                   
                 T240M 
                   
                 0.48 
               
               
                   
                 T240P 
                   
                 0.56 
               
               
                   
                 T240Q 
                 492 
                 0.75 
               
               
                   
                 T240R 
                   
                 0.91 
               
               
                   
                 A239N 
                   
                 0.32 
               
               
                   
                 T240S 
                   
                 0.74 
               
               
                   
                 T240V 
                   
                 0.77 
               
               
                   
                 Y242F 
                   
                 1.08 
               
               
                   
                 N245H 
                   
                 0.50 
               
               
                   
                 V247I 
                 493 
                 2.01 
               
               
                   
                 V247L 
                   
                 0.83 
               
               
                   
                 V247M 
                   
                 0.52 
               
               
                   
                 R248A 
                 494 
                 0.43 
               
               
                   
                 R248W 
                   
                 0.52 
               
               
                   
                 R248Y 
                   
                 0.67 
               
               
                   
                 I251Y 
                   
                 0.37 
               
               
                   
                 I251L 
                   
                 0.58 
               
               
                   
                 I251M 
                   
                 0.43 
               
               
                   
                 V253I 
                   
                 0.76 
               
               
                   
                 K255A 
                   
                 0.40 
               
               
                   
                 K255N 
                   
                 0.52 
               
               
                   
                 K255Q 
                   
                 0.91 
               
               
                   
                 K255R 
                   
                 0.71 
               
               
                   
                 K093P 
                   
                 0.38 
               
               
                   
                 K255S 
                   
                 0.43 
               
               
                   
                 I256A 
                   
                 0.42 
               
               
                   
                 I256H 
                   
                 0.51 
               
               
                   
                 I256L 
                   
                 0.64 
               
               
                   
                 I256V 
                   
                 0.51 
               
               
                   
                 P257A 
                   
                 0.82 
               
               
                   
                 P257G 
                 496 
                 0.51 
               
               
                   
                 P257I 
                   
                 1.07 
               
               
                   
                 P257K 
                   
                 0.92 
               
               
                   
                 P257L 
                   
                 0.69 
               
               
                   
                 P257M 
                   
                 0.90 
               
               
                   
                 P257N 
                   
                 0.69 
               
               
                   
                 P257Q 
                   
                 0.61 
               
               
                   
                 P257R 
                 498 
                 1.38 
               
               
                   
                 P257T 
                 497 
                 2.04 
               
               
                   
                 P257V 
                   
                 0.88 
               
               
                   
                 D258H 
                   
                 0.84 
               
               
                   
                 D258N 
                 499 
                 1.44 
               
               
                   
                 D258R 
                   
                 0.45 
               
               
                   
                 D258S 
                 500 
                 1.44 
               
               
                   
                 D258G 
                   
                 0.39 
               
               
                   
                 D426S 
                   
                 0.36 
               
               
                   
                 G427I 
                   
                 0.54 
               
               
                   
                 A259E 
                   
                 0.85 
               
               
                   
                 A259G 
                   
                 0.68 
               
               
                   
                 A259I 
                   
                 0.46 
               
               
                   
                 A259K 
                   
                 0.76 
               
               
                   
                 A259L 
                   
                 0.53 
               
               
                   
                 A259N 
                   
                 0.49 
               
               
                   
                 A259P 
                 501 
                 1.54 
               
               
                   
                 A259Q 
                   
                 0.70 
               
               
                   
                 A259R 
                   
                 0.72 
               
               
                   
                 A259S 
                   
                 0.63 
               
               
                   
                 A259T 
                   
                 0.51 
               
               
                   
                 A259V 
                   
                 0.41 
               
               
                   
                 A259W 
                   
                 0.55 
               
               
                   
                 A259Y 
                   
                 0.51 
               
               
                   
                 K260A 
                   
                 0.66 
               
               
                   
                 K260D 
                   
                 0.41 
               
               
                   
                 K260E 
                   
                 0.58 
               
               
                   
                 K260H 
                   
                 0.87 
               
               
                   
                 K260L 
                   
                 0.60 
               
               
                   
                 E249V 
                 495 
                   
               
               
                   
                 K260M 
                 502 
                 0.85 
               
               
                   
                 K260Q 
                   
                 0.58 
               
               
                   
                 K260R 
                   
                 0.83 
               
               
                   
                 K260S 
                   
                 0.66 
               
               
                   
                 K260G 
                   
                 0.37 
               
               
                   
                 K260Y 
                 503 
                 1.73 
               
               
                   
                 S261A 
                 504 
                 0.74 
               
               
                   
                 S261F 
                   
                 0.73 
               
               
                   
                 S261K 
                 505 
                 2.54 
               
               
                   
                 S261M 
                   
                 0.56 
               
               
                   
                 S261N 
                 506 
                 1.98 
               
               
                   
                 S261Q 
                   
                 0.76 
               
               
                   
                 S261R 
                   
                 1.19 
               
               
                   
                 S261T 
                   
                 0.66 
               
               
                   
                 S261V 
                   
                 0.48 
               
               
                   
                 S261W 
                   
                 0.44 
               
               
                   
                 L263A 
                   
                 0.76 
               
               
                   
                 L263K 
                 507 
                 2.73 
               
               
                   
                 L263M 
                   
                 0.89 
               
               
                   
                 L263R 
                 508 
                 1.63 
               
               
                   
                 L263T 
                   
                 0.49 
               
               
                   
                 N104I 
                   
                 0.35 
               
               
                   
                 L263V 
                   
                 0.75 
               
               
                   
                 P264A 
                   
                 0.43 
               
               
                   
                 P264H 
                   
                 0.60 
               
               
                   
                 V265I 
                   
                 0.58 
               
               
                   
                 F266Y 
                   
                 0.58 
               
               
                   
                 A267M 
                   
                 0.45 
               
               
                   
                 A267T 
                 509 
                 1.34 
               
               
                   
                 T269A 
                 510 
                 1.63 
               
               
                   
                 T269C 
                   
                 0.75 
               
               
                   
                 T269D 
                   
                 0.76 
               
               
                   
                 T269S 
                   
                 1.01 
               
               
                   
                 R270M 
                   
                 0.46 
               
               
                   
                 R270N 
                   
                 0.52 
               
               
                   
                 R270S 
                   
                 0.69 
               
               
                   
                 I271F 
                   
                 0.72 
               
               
                   
                 I271G 
                   
                 1.29 
               
               
                   
                 L105C 
                   
                 0.33 
               
               
                   
                 I271L 
                 511 
                 10.62 
               
               
                   
                 V272E 
                   
                 0.39 
               
               
                   
                 I271M 
                 512 
                 3.24 
               
               
                   
                 I271S 
                   
                 0.42 
               
               
                   
                 I271V 
                   
                 1.05 
               
               
                   
                 V272D 
                 513 
                 1.36 
               
               
                   
                 V272R 
                   
                 0.74 
               
               
                   
                 V272S 
                   
                 0.96 
               
               
                   
                 V272T 
                 514 
                 1.61 
               
               
                   
                 F273H 
                 515 
                 1.41 
               
               
                   
                 F273T 
                   
                 0.48 
               
               
                   
                 F273Y 
                 516 
                 0.90 
               
               
                   
                 T274A 
                   
                 0.51 
               
               
                   
                 1274F 
                 517 
                 1.28 
               
               
                   
                 T274S 
                   
                 0.62 
               
               
                   
                 Q276C 
                   
                 0.88 
               
               
                   
                 Q276D 
                 518 
                 1.69 
               
               
                   
                 Q276E 
                   
                 1.05 
               
               
                   
                 Q276H 
                 519 
                 1.20 
               
               
                   
                 Q276I 
                   
                 0.51 
               
               
                   
                 Q276L 
                   
                 0.48 
               
               
                   
                 W119Q 
                   
                 0.72 
               
               
                   
                 Q276M 
                 520 
                 1.14 
               
               
                   
                 Q276R 
                 521 
                 1.30 
               
               
                   
                 Q276S 
                 522 
                 1.63 
               
               
                   
                 Q276Y 
                 523 
                 1.94 
               
               
                   
                 V277A 
                 524 
                 0.65 
               
               
                   
                 V277C 
                   
                 0.41 
               
               
                   
                 V277D 
                   
                 0.79 
               
               
                   
                 V277E 
                 525 
                 1.02 
               
               
                   
                 V277G 
                   
                 1.18 
               
               
                   
                 V277H 
                 526 
                 1.09 
               
               
                   
                 V277K 
                 527 
                 1.51 
               
               
                   
                 V277M 
                 528 
                 0.94 
               
               
                   
                 V277N 
                 529 
                 1.15 
               
               
                   
                 V277Q 
                 530 
                 0.82 
               
               
                   
                 V277R 
                 531 
                 1.63 
               
               
                   
                 V277S 
                 532 
                 0.83 
               
               
                   
                 K124H 
                   
                 0.35 
               
               
                   
                 V277T 
                 533 
                 1.94 
               
               
                   
                 V277Y 
                   
                 0.66 
               
               
                   
                 L278A 
                   
                 1.13 
               
               
                   
                 L278E 
                 534 
                 1.03 
               
               
                   
                 L278F 
                 535 
                 1.26 
               
               
                   
                 L278G 
                 536 
                 1.33 
               
               
                   
                 L278H 
                 537 
                 4.50 
               
               
                   
                 L278I 
                   
                 0.93 
               
               
                   
                 L278K 
                 538 
                 1.75 
               
               
                   
                 D275V 
                   
                 0.4 
               
               
                   
                 L278N 
                 539 
                 1.74 
               
               
                   
                 L278R 
                 540 
                 5.87 
               
               
                   
                 L278S 
                 541 
                 1.67 
               
               
                   
                 L278T 
                 542 
                 1.66 
               
               
                   
                 L278V 
                   
                 0.44 
               
               
                   
                 L278Y 
                 543 
                 1.51 
               
               
                   
                 K279H 
                 544 
                 0.44 
               
               
                   
                 K279Q 
                   
                 0.84 
               
               
                   
                 K279R 
                   
                 1.10 
               
               
                   
                 K279T 
                   
                 0.86 
               
               
                   
                 F280G 
                   
                 0.47 
               
               
                   
                 F280Q 
                   
                 0.43 
               
               
                   
                 S282D 
                   
                 0.41 
               
               
                   
                 S282G 
                   
                 0.54 
               
               
                   
                 S282M 
                 545 
                 2.64 
               
               
                   
                 S282Q 
                   
                 0.41 
               
               
                   
                 Q283E 
                   
                 0.63 
               
               
                   
                 Q283P 
                   
                 1.18 
               
               
                   
                 Q283R 
                   
                 0.59 
               
               
                   
                 Q283S 
                 546 
                 1.73 
               
               
                   
                 Q283T 
                   
                 0.65 
               
               
                   
                 D284A 
                   
                 0.58 
               
               
                   
                 D284E 
                   
                 1.21 
               
               
                   
                 D284G 
                   
                 0.60 
               
               
                   
                 D284H 
                   
                 0.51 
               
               
                   
                 D284L 
                   
                 0.50 
               
               
                   
                 D284M 
                   
                 0.56 
               
               
                   
                 D284N 
                   
                 0.40 
               
               
                   
                 D284Q 
                   
                 0.95 
               
               
                   
                 D284S 
                   
                 0.99 
               
               
                   
                 E285F 
                   
                 0.47 
               
               
                   
                 E285G 
                   
                 0.52 
               
               
                   
                 E285H 
                 547 
                 1.30 
               
               
                   
                 E285M 
                   
                 0.43 
               
               
                   
                 E285N 
                   
                 0.40 
               
               
                   
                 E285Q 
                   
                 0.59 
               
               
                   
                 E285Y 
                   
                 0.99 
               
               
                   
                 L286S 
                   
                 0.46 
               
               
                   
                 D284T 
                   
                 0.39 
               
               
                   
                 L286R 
                   
                 0.53 
               
               
                   
                 V287I 
                   
                 0.51 
               
               
                   
                 V287T 
                 548 
                 0.50 
               
               
                   
                 Y288L 
                   
                 0.79 
               
               
                   
                 Y288W 
                   
                 0.49 
               
               
                   
                 T289K 
                   
                 0.75 
               
               
                   
                 T289S 
                 549 
                 0.48 
               
               
                   
                 F290I 
                   
                 0.41 
               
               
                   
                 F290M 
                   
                 1.03 
               
               
                   
                 G291Q 
                   
                 0.80 
               
               
                   
                 G291R 
                   
                 0.45 
               
               
                   
                 G291S 
                 550 
                 0.41 
               
               
                   
                 G291V 
                 551 
                 1.63 
               
               
                   
                 E292A 
                   
                 0.66 
               
               
                   
                 E292C 
                 552 
                 0.71 
               
               
                   
                 E292F 
                 553 
                 0.90 
               
               
                   
                 E292G 
                   
                 0.41 
               
               
                   
                 E292H 
                 554 
                 1.26 
               
               
                   
                 E442W 
                   
                 0.38 
               
               
                   
                 E292K 
                 555 
                 1.27 
               
               
                   
                 E292N 
                   
                 0.99 
               
               
                   
                 E292P 
                   
                 1.05 
               
               
                   
                 E292R 
                 556 
                 0.42 
               
               
                   
                 E292V 
                 557 
                 1.28 
               
               
                   
                 E292W 
                   
                 0.83 
               
               
                   
                 T293A 
                 558 
                 1.90 
               
               
                   
                 T293C 
                 559 
                 1.67 
               
               
                   
                 T293D 
                 560 
                 1.46 
               
               
                   
                 V137C 
                   
                 0.37 
               
               
                   
                 V137S 
                   
                 0.36 
               
               
                   
                 V137L 
                   
                 0.21 
               
               
                   
                 Q143C 
                   
                 0.28 
               
               
                   
                 L144R 
                 360 
                 0.26 
               
               
                   
                 K152W 
                 396 
                 0.37 
               
               
                   
                 A153S 
                   
                 0.34 
               
               
                   
                 K154I 
                   
                 0.38 
               
               
                   
                 E156C 
                   
                 0.35 
               
               
                   
                 E158G 
                   
                 0.37 
               
               
                   
                 K159G 
                   
                 0.38 
               
               
                   
                 A160W 
                   
                 0.39 
               
               
                   
                 G161V 
                   
                 0.42 
               
               
                   
                 D163W 
                   
                 0.38 
               
               
                   
                 D163F 
                   
                 0.39 
               
               
                   
                 L165C 
                   
                 0.27 
               
               
                   
                 V166N 
                   
                 0.47 
               
               
                   
                 E167F 
                   
                 0.31 
               
               
                   
                 K170A 
                   
                 0.40 
               
               
                   
                 K170Q 
                   
                 0.40 
               
               
                   
                 K173Q 
                   
                 0.32 
               
               
                   
                 L174H 
                   
                 0.38 
               
               
                   
                 R176L 
                   
                 0.40 
               
               
                   
                 P177V 
                   
                 0.36 
               
               
                   
                 L180I 
                   
                 0.38 
               
               
                   
                 W181K 
                   
                 0.29 
               
               
                   
                 Y183E 
                   
                 0.32 
               
               
                   
                 Y184W 
                   
                 0.39 
               
               
                   
                 H193R 
                   
                 0.33 
               
               
                   
                 H193F 
                   
                 0.38 
               
               
                   
                 K195V 
                   
                 0.36 
               
               
                   
                 K196N 
                   
                 0.39 
               
               
                   
                 K196Y 
                   
                 0.39 
               
               
                   
                 P197W 
                   
                 0.39 
               
               
                   
                 G198W 
                   
                 0.29 
               
               
                   
                 N200T 
                   
                 0.37 
               
               
                   
                 F204W 
                   
                 0.39 
               
               
                   
                 N205L 
                 452 
                 0.39 
               
               
                   
                 N205Y 
                   
                 0.4 
               
               
                   
                 V206Q 
                   
                 0.33 
               
               
                   
                 K209F 
                   
                 0.4 
               
               
                   
                 K209L 
                   
                 0.38 
               
               
                   
                 N211L 
                   
                 0.41 
               
               
                   
                 N211W 
                   
                 0.51 
               
               
                   
                 W218M 
                   
                 0.38 
               
               
                   
                 W218V 
                   
                 0.28 
               
               
                   
                 T293F 
                 561 
                 1.94 
               
               
                   
                 T293G 
                   
                 1.00 
               
               
                   
                 T293K 
                 562 
                 1.35 
               
               
                   
                 T293L 
                   
                 1.00 
               
               
                   
                 T293M 
                 563 
                 2.29 
               
               
                   
                 T293P 
                 564 
                 1.64 
               
               
                   
                 T293Q 
                 565 
                 1.83 
               
               
                   
                 T293S 
                   
                 0.89 
               
               
                   
                 T293V 
                 566 
                 2.15 
               
               
                   
                 T293Y 
                 567 
                 1.49 
               
               
                   
                 V294M 
                   
                 0.41 
               
               
                   
                 A298G 
                 568 
                 0.43 
               
               
                   
                 A298I 
                   
                 0.41 
               
               
                   
                 G300R 
                   
                 0.42 
               
               
                   
                 I301A 
                   
                 0.88 
               
               
                   
                 I301V 
                   
                 0.88 
               
               
                   
                 V287N 
                   
                 0.35 
               
               
                   
                 V302W 
                   
                 0.46 
               
               
                   
                 V302I 
                   
                 0.45 
               
               
                   
                 I303V 
                   
                 0.47 
               
               
                   
                 W304G 
                   
                 1.13 
               
               
                   
                 W304I 
                   
                 1.17 
               
               
                   
                 G305D 
                   
                 1.00 
               
               
                   
                 G305E 
                 569 
                 1.62 
               
               
                   
                 T306D 
                   
                 0.76 
               
               
                   
                 T306E 
                   
                 0.52 
               
               
                   
                 T306S 
                   
                 1.02 
               
               
                   
                 L307K 
                   
                 0.43 
               
               
                   
                 L307N 
                   
                 0.76 
               
               
                   
                 L307Q 
                   
                 0.61 
               
               
                   
                 L307S 
                   
                 0.86 
               
               
                   
                 L307T 
                   
                 1.08 
               
               
                   
                 L307V 
                   
                 0.48 
               
               
                   
                 L307W 
                   
                 0.64 
               
               
                   
                 L307Y 
                   
                 0.60 
               
               
                   
                 S308D 
                 571 
                 0.92 
               
               
                   
                 S308G 
                 572 
                 1.73 
               
               
                   
                 S308H 
                   
                 1.15 
               
               
                   
                 S308K 
                 573 
                 1.33 
               
               
                   
                 S308N 
                 574 
                 2.33 
               
               
                   
                 S308P 
                   
                 0.65 
               
               
                   
                 S308R 
                 575 
                 1.34 
               
               
                   
                 S308T 
                   
                 0.72 
               
               
                   
                 I309D 
                   
                 0.72 
               
               
                   
                 I309E 
                 576 
                 1.99 
               
               
                   
                 I309G 
                 577 
                 1.44 
               
               
                   
                 I303D 
                   
                 0.34 
               
               
                   
                 I309H 
                 578 
                 1.30 
               
               
                   
                 I309K 
                   
                 0.98 
               
               
                   
                 I309L 
                 579 
                 1.72 
               
               
                   
                 I309M 
                 580 
                 1.47 
               
               
                   
                 I309N 
                 581 
                 3.11 
               
               
                   
                 I309Q 
                 582 
                 1.64 
               
               
                   
                 I309R 
                 583 
                 2.27 
               
               
                   
                 I309S 
                 584 
                 1.16 
               
               
                   
                 I309T 
                 585 
                 2.09 
               
               
                   
                 I309V 
                 586 
                 0.60 
               
               
                   
                 I309W 
                   
                 0.88 
               
               
                   
                 M310A 
                 587 
                 1.50 
               
               
                   
                 M310G 
                 588 
                 2.73 
               
               
                   
                 M310Q 
                 589 
                 0.59 
               
               
                   
                 M310R 
                   
                 0.50 
               
               
                   
                 M310S 
                 590 
                 1.61 
               
               
                   
                 M310V 
                   
                 0.70 
               
               
                   
                 R311G 
                   
                 0.53 
               
               
                   
                 L307G 
                 570 
                 0.32 
               
               
                   
                 R311G 
                   
                 0.54 
               
               
                   
                 R311H 
                   
                 0.48 
               
               
                   
                 R311K 
                   
                 0.72 
               
               
                   
                 R311Q 
                   
                 0.43 
               
               
                   
                 R311S 
                   
                 0.84 
               
               
                   
                 R311T 
                   
                 0.52 
               
               
                   
                 S312G 
                   
                 0.49 
               
               
                   
                 S312N 
                   
                 1.26 
               
               
                   
                 S312T 
                   
                 0.75 
               
               
                   
                 M313A 
                 591 
                 1.34 
               
               
                   
                 M313E 
                   
                 0.63 
               
               
                   
                 M313G 
                 592 
                 0.56 
               
               
                   
                 M313H 
                 593 
                 1.23 
               
               
                   
                 M313K 
                 594 
                 2.85 
               
               
                   
                 S312L 
                   
                 0.38 
               
               
                   
                 M313L 
                   
                 1.05 
               
               
                   
                 M313P 
                 595 
                 1.11 
               
               
                   
                 M313R 
                 596 
                 2.30 
               
               
                   
                 M313S 
                   
                 0.88 
               
               
                   
                 M313T 
                 597 
                 0.67 
               
               
                   
                 M313V 
                   
                 0.99 
               
               
                   
                 M313Y 
                 598 
                 1.12 
               
               
                   
                 K314A 
                   
                 0.82 
               
               
                   
                 K314D 
                   
                 0.53 
               
               
                   
                 K314H 
                   
                 1.10 
               
               
                   
                 K314I 
                   
                 0.54 
               
               
                   
                 K314N 
                   
                 0.57 
               
               
                   
                 K314Q 
                   
                 0.62 
               
               
                   
                 K314R 
                   
                 0.95 
               
               
                   
                 K314S 
                 599 
                 0.61 
               
               
                   
                 K314T 
                   
                 0.61 
               
               
                   
                 K314Y 
                 600 
                 0.45 
               
               
                   
                 S315A 
                 601 
                 0.85 
               
               
                   
                 S315E 
                   
                 0.41 
               
               
                   
                 S315G 
                   
                 0.72 
               
               
                   
                 S315H 
                 602 
                 2.04 
               
               
                   
                 S315K 
                   
                 0.62 
               
               
                   
                 S315L 
                   
                 0.42 
               
               
                   
                 S315M 
                   
                 0.63 
               
               
                   
                 S315R 
                   
                 1.04 
               
               
                   
                 S315T 
                   
                 0.97 
               
               
                   
                 S315Y 
                 603 
                 0.50 
               
               
                   
                 C316D 
                   
                 0.41 
               
               
                   
                 L317A 
                 604 
                 1.27 
               
               
                   
                 L317D 
                   
                 0.61 
               
               
                   
                 L317H 
                   
                 1.05 
               
               
                   
                 L317I 
                 605 
                 1.76 
               
               
                   
                 L317K 
                 606 
                 5.11 
               
               
                   
                 L317M 
                   
                 1.20 
               
               
                   
                 L317N 
                 607 
                 0.73 
               
               
                   
                 L317Q 
                 608 
                 1.67 
               
               
                   
                 L317R 
                 609 
                 2.41 
               
               
                   
                 L317S 
                 610 
                 1.03 
               
               
                   
                 L317T 
                 611 
                 0.93 
               
               
                   
                 L317W 
                 612 
                 0.84 
               
               
                   
                 L318D 
                 614 
                 0.46 
               
               
                   
                 L318F 
                   
                 0.51 
               
               
                   
                 L318G 
                   
                 0.49 
               
               
                   
                 L318H 
                 615 
                 0.45 
               
               
                   
                 L318I 
                   
                 0.70 
               
               
                   
                 L318K 
                 616 
                 1.36 
               
               
                   
                 L318M 
                 613 
                 1.68 
               
               
                   
                 L318N 
                   
                 0.52 
               
               
                   
                 L318Q 
                   
                 0.71 
               
               
                   
                 L318R 
                 617 
                 1.34 
               
               
                   
                 L318S 
                   
                 0.71 
               
               
                   
                 L318T 
                   
                 0.63 
               
               
                   
                 D320E 
                   
                 0.78 
               
               
                   
                 D320G 
                   
                 0.83 
               
               
                   
                 D320H 
                 618 
                 1.75 
               
               
                   
                 D320I 
                   
                 1.00 
               
               
                   
                 D320K 
                 619 
                 6.42 
               
               
                   
                 D320M 
                   
                 0.79 
               
               
                   
                 D320N 
                   
                 0.52 
               
               
                   
                 D320R 
                 620 
                 3.19 
               
               
                   
                 D320S 
                   
                 1.19 
               
               
                   
                 D320W 
                   
                 0.40 
               
               
                   
                 D320V 
                   
                 0.35 
               
               
                   
                 D320Y 
                   
                 0.86 
               
               
                   
                 N321A 
                   
                 1.01 
               
               
                   
                 N321D 
                   
                 1.25 
               
               
                   
                 N321H 
                   
                 0.92 
               
               
                   
                 N321K 
                   
                 1.29 
               
               
                   
                 N321R 
                 621 
                 1.23 
               
               
                   
                 N321S 
                 622 
                 1.26 
               
               
                   
                 N321T 
                   
                 0.64 
               
               
                   
                 N321Y 
                   
                 0.40 
               
               
                   
                 M323F 
                   
                 0.64 
               
               
                   
                 M323I 
                   
                 0.55 
               
               
                   
                 M323L 
                   
                 0.55 
               
               
                   
                 E324A 
                   
                 0.59 
               
               
                   
                 E324D 
                   
                 1.15 
               
               
                   
                 E324H 
                   
                 0.79 
               
               
                   
                 E324M 
                   
                 0.50 
               
               
                   
                 E324N 
                 623 
                 1.01 
               
               
                   
                 E324R 
                 624 
                 2.28 
               
               
                   
                 E324S 
                   
                 0.62 
               
               
                   
                 T325A 
                 625 
                 1.87 
               
               
                   
                 T325D 
                 626 
                 1.78 
               
               
                   
                 T325E 
                 627 
                 4.03 
               
               
                   
                 T325G 
                 628 
                 4.21 
               
               
                   
                 T325H 
                 629 
                 3.45 
               
               
                   
                 T325K 
                 630 
                 4.37 
               
               
                   
                 T325M 
                 631 
                 2.11 
               
               
                   
                 T325N 
                 632 
                 4.64 
               
               
                   
                 T325Q 
                 633 
                 5.08 
               
               
                   
                 T325S 
                 634 
                 3.19 
               
               
                   
                 T325V 
                 635 
                 1.24 
               
               
                   
                 T325W 
                   
                 0.62 
               
               
                   
                 I326K 
                   
                 0.95 
               
               
                   
                 I326L 
                 636 
                 1.50 
               
               
                   
                 I326V 
                 637 
                 6.29 
               
               
                   
                 I326Y 
                   
                 0.77 
               
               
                   
                 L327M 
                   
                 0.52 
               
               
                   
                 N328A 
                   
                 0.67 
               
               
                   
                 N328C 
                 638 
                 1.25 
               
               
                   
                 N328G 
                 639 
                 0.56 
               
               
                   
                 N328H 
                   
                 0.88 
               
               
                   
                 N328I 
                 642 
                 1.85 
               
               
                   
                 N328K 
                 640 
                 2.12 
               
               
                   
                 N328L 
                 641 
                 2.01 
               
               
                   
                 N328Q 
                   
                 1.13 
               
               
                   
                 N328R 
                   
                 0.68 
               
               
                   
                 N328S 
                 643 
                 2.22 
               
               
                   
                 N328T 
                   
                 0.59 
               
               
                   
                 N328V 
                   
                 1.16 
               
               
                   
                 N328Y 
                 644 
                 1.66 
               
               
                   
                 I331V 
                   
                 0.94 
               
               
                   
                 N328W 
                   
                 0.33 
               
               
                   
                 V334T 
                   
                 0.39 
               
               
                   
                 V334P 
                   
                 0.46 
               
               
                   
                 T335S 
                 645 
                 0.47 
               
               
                   
                 A338Q 
                   
                 0.63 
               
               
                   
                 K339M 
                   
                 0.61 
               
               
                   
                 S342A 
                   
                 0.68 
               
               
                   
                 Q343T 
                   
                 0.49 
               
               
                   
                 Q343V 
                   
                 0.51 
               
               
                   
                 Q347A 
                 646 
                 0.78 
               
               
                   
                 Q347E 
                   
                 0.78 
               
               
                   
                 Q347G 
                 647 
                 2.68 
               
               
                   
                 Q347M 
                   
                 0.61 
               
               
                   
                 Q347R 
                   
                 0.55 
               
               
                   
                 Q347S 
                 648 
                 2.38 
               
               
                   
                 E348D 
                   
                 0.67 
               
               
                   
                 E348G 
                   
                 0.55 
               
               
                   
                 E348S 
                   
                 0.44 
               
               
                   
                 Q349A 
                   
                 0.47 
               
               
                   
                 Q349E 
                   
                 0.83 
               
               
                   
                 Q349K 
                   
                 0.93 
               
               
                   
                 Q349M 
                 649 
                 0.70 
               
               
                   
                 Q349N 
                   
                 0.44 
               
               
                   
                 M035V 
                 146 
                 0.37 
               
               
                   
                 Q349R 
                 650 
                 0.73 
               
               
                   
                 Q349T 
                   
                 0.49 
               
               
                   
                 V351A 
                   
                 1.14 
               
               
                   
                 V351S 
                 651 
                 0.92 
               
               
                   
                 I353T 
                   
                 0.42 
               
               
                   
                 I353V 
                 652 
                 1.61 
               
               
                   
                 N356A 
                   
                 0.41 
               
               
                   
                 N356D 
                   
                 0.79 
               
               
                   
                 N356H 
                 653 
                 0.82 
               
               
                   
                 N356S 
                 654 
                 0.46 
               
               
                   
                 W357A 
                   
                 0.80 
               
               
                   
                 W357C 
                   
                 0.67 
               
               
                   
                 L037W 
                   
                 0.36 
               
               
                   
                 W357S 
                   
                 0.41 
               
               
                   
                 W357T 
                   
                 0.62 
               
               
                   
                 N358C 
                   
                 0.66 
               
               
                   
                 N358G 
                   
                 0.41 
               
               
                   
                 N358T 
                   
                 0.58 
               
               
                   
                 V351I 
                   
                 0.36 
               
               
                   
                 N358L 
                   
                 0.38 
               
               
                   
                 S359D 
                   
                 0.45 
               
               
                   
                 S359E 
                 655 
                 1.05 
               
               
                   
                 S359H 
                 656 
                 0.44 
               
               
                   
                 S359K 
                   
                 0.66 
               
               
                   
                 S359M 
                   
                 0.63 
               
               
                   
                 S359T 
                 657 
                 2.11 
               
               
                   
                 S359V 
                   
                 0.65 
               
               
                   
                 S360T 
                   
                 0.50 
               
               
                   
                 P367A 
                 658 
                 0.55 
               
               
                   
                 P367C 
                   
                 0.83 
               
               
                   
                 P367G 
                 659 
                 0.47 
               
               
                   
                 P367K 
                 660 
                 0.57 
               
               
                   
                 P367R 
                   
                 0.46 
               
               
                   
                 P367S 
                 661 
                 0.52 
               
               
                   
                 D368A 
                 662 
                 1.34 
               
               
                   
                 D368E 
                 663 
                 1.28 
               
               
                   
                 D368G 
                   
                 0.49 
               
               
                   
                 D368H 
                   
                 0.96 
               
               
                   
                 D368K 
                 664 
                 1.31 
               
               
                   
                 D368L 
                 665 
                 0.64 
               
               
                   
                 D368M 
                 666 
                 0.78 
               
               
                   
                 D368R 
                 667 
                 1.31 
               
               
                   
                 D368S 
                   
                 0.93 
               
               
                   
                 D368T 
                 668 
                 0.80 
               
               
                   
                 D361H 
                   
                 0.37 
               
               
                   
                 D368V 
                   
                 0.41 
               
               
                   
                 N369H 
                 669 
                 1.33 
               
               
                   
                 N369R 
                 670 
                 0.55 
               
               
                   
                 N369S 
                   
                 0.54 
               
               
                   
                 A371E 
                   
                 1.05 
               
               
                   
                 A371F 
                 671 
                 0.52 
               
               
                   
                 A371H 
                 672 
                 1.20 
               
               
                   
                 A371I 
                   
                 0.50 
               
               
                   
                 A371K 
                 673 
                 1.76 
               
               
                   
                 L374W 
                   
                 0.34 
               
               
                   
                 A371L 
                 674 
                 0.57 
               
               
                   
                 A371M 
                   
                 0.57 
               
               
                   
                 A371R 
                 675 
                 1.51 
               
               
                   
                 A371S 
                 676 
                 1.45 
               
               
                   
                 A371V 
                   
                 0.94 
               
               
                   
                 Q373A 
                   
                 0.65 
               
               
                   
                 Q373E 
                   
                 0.81 
               
               
                   
                 Q373F 
                   
                 0.62 
               
               
                   
                 Q373K 
                   
                 0.73 
               
               
                   
                 Q373L 
                   
                 0.84 
               
               
                   
                 Q373M 
                 677 
                 1.43 
               
               
                   
                 Q373R 
                   
                 0.68 
               
               
                   
                 Q373S 
                   
                 0.87 
               
               
                   
                 Q373V 
                   
                 1.05 
               
               
                   
                 L374A 
                   
                 0.60 
               
               
                   
                 L374H 
                 678 
                 1.42 
               
               
                   
                 L374I 
                   
                 0.80 
               
               
                   
                 L374M 
                   
                 1.11 
               
               
                   
                 L374N 
                   
                 0.43 
               
               
                   
                 L374P 
                 679 
                 0.43 
               
               
                   
                 L374R 
                   
                 0.83 
               
               
                   
                 L374S 
                   
                 0.58 
               
               
                   
                 L374T 
                   
                 0.47 
               
               
                   
                 L374V 
                   
                 0.56 
               
               
                   
                 L374Y 
                   
                 0.66 
               
               
                   
                 E375A 
                 680 
                 0.42 
               
               
                   
                 E375G 
                 681 
                 0.90 
               
               
                   
                 E375K 
                 682 
                 1.49 
               
               
                   
                 E375L 
                   
                 0.46 
               
               
                   
                 E375M 
                   
                 0.54 
               
               
                   
                 E375N 
                   
                 0.81 
               
               
                   
                 E375R 
                 683 
                 0.43 
               
               
                   
                 E375S 
                   
                 0.77 
               
               
                   
                 E375T 
                   
                 1.17 
               
               
                   
                 K376A 
                   
                 0.95 
               
               
                   
                 K376D 
                 684 
                 0.78 
               
               
                   
                 K376E 
                 685 
                 0.88 
               
               
                   
                 K376M 
                   
                 0.46 
               
               
                   
                 K376Q 
                 686 
                 0.69 
               
               
                   
                 K376R 
                 687 
                 0.67 
               
               
                   
                 K376S 
                   
                 0.80 
               
               
                   
                 K376T 
                 688 
                 0.53 
               
               
                   
                 K376V 
                 689 
                 0.58 
               
               
                   
                 K376Y 
                 690 
                 0.42 
               
               
                   
                 G377D 
                 691 
                 1.35 
               
               
                   
                 G377E 
                 692 
                 0.59 
               
               
                   
                 G377H 
                 693 
                 1.49 
               
               
                   
                 G377K 
                 694 
                 1.50 
               
               
                   
                 G377P 
                 695 
                 2.30 
               
               
                   
                 G377R 
                 696 
                 1.28 
               
               
                   
                 G377S 
                 697 
                 1.80 
               
               
                   
                 Q051R 
                   
                 0.36 
               
               
                   
                 G377T 
                 698 
                 3.83 
               
               
                   
                 G378K 
                   
                 1.22 
               
               
                   
                 G378N 
                   
                 0.64 
               
               
                   
                 G378R 
                   
                 1.03 
               
               
                   
                 K379G 
                   
                 0.52 
               
               
                   
                 K379H 
                   
                 0.57 
               
               
                   
                 K379R 
                   
                 0.74 
               
               
                   
                 K379S 
                   
                 0.46 
               
               
                   
                 K379T 
                   
                 0.4 
               
               
                   
                 M035Q 
                 145 
                 0.37 
               
               
                   
                 F380I 
                   
                 0.56 
               
               
                   
                 F380L 
                   
                 0.67 
               
               
                   
                 F380P 
                   
                 0.47 
               
               
                   
                 F380W 
                 699 
                 2.15 
               
               
                   
                 F380Y 
                 700 
                 1.50 
               
               
                   
                 T381H 
                   
                 0.48 
               
               
                   
                 T381K 
                   
                 1.06 
               
               
                   
                 T381N 
                   
                 0.51 
               
               
                   
                 T381Q 
                   
                 0.84 
               
               
                   
                 T381R 
                   
                 0.87 
               
               
                   
                 T381S 
                 701 
                 0.87 
               
               
                   
                 T381V 
                   
                 0.89 
               
               
                   
                 R383A 
                   
                 0.51 
               
               
                   
                 R383E 
                   
                 0.51 
               
               
                   
                 R383H 
                   
                 0.71 
               
               
                   
                 R383I 
                 702 
                 0.71 
               
               
                   
                 R383K 
                 703 
                 1.30 
               
               
                   
                 R383L 
                 704 
                 1.31 
               
               
                   
                 R383M 
                   
                 0.61 
               
               
                   
                 R383N 
                   
                 0.77 
               
               
                   
                 T381E 
                   
                 0.35 
               
               
                   
                 R383S 
                 705 
                 0.87 
               
               
                   
                 R383T 
                   
                 0.98 
               
               
                   
                 R383V 
                   
                 1.05 
               
               
                   
                 K385A 
                 706 
                 1.12 
               
               
                   
                 K385G 
                   
                 0.62 
               
               
                   
                 K385H 
                   
                 0.50 
               
               
                   
                 K385N 
                   
                 0.41 
               
               
                   
                 K385Q 
                 707 
                 0.73 
               
               
                   
                 K385R 
                   
                 0.94 
               
               
                   
                 K385S 
                   
                 1.05 
               
               
                   
                 K385T 
                   
                 0.46 
               
               
                   
                 K385V 
                 708 
                 0.43 
               
               
                   
                 T387S 
                   
                 0.93 
               
               
                   
                 L388F 
                   
                 0.92 
               
               
                   
                 L388H 
                   
                 0.47 
               
               
                   
                 L388I 
                   
                 0.98 
               
               
                   
                 L388M 
                   
                 0.79 
               
               
                   
                 L388R 
                   
                 0.60 
               
               
                   
                 L388T 
                   
                 0.51 
               
               
                   
                 L388V 
                   
                 0.78 
               
               
                   
                 L388W 
                   
                 0.77 
               
               
                   
                 L388Y 
                   
                 1.18 
               
               
                   
                 E392W 
                   
                 0.31 
               
               
                   
                 E389A 
                 709 
                 1.14 
               
               
                   
                 E389G 
                 710 
                 0.91 
               
               
                   
                 E389H 
                   
                 1.17 
               
               
                   
                 E389K 
                 712 
                 1.91 
               
               
                   
                 E389L 
                 711 
                 0.65 
               
               
                   
                 E389M 
                   
                 0.60 
               
               
                   
                 E389P 
                   
                 0.75 
               
               
                   
                 E389Q 
                 713 
                 0.69 
               
               
                   
                 E389R 
                   
                 0.94 
               
               
                   
                 E389S 
                 714 
                 1.08 
               
               
                   
                 E389T 
                   
                 0.70 
               
               
                   
                 E389Y 
                   
                 0.77 
               
               
                   
                 L391C 
                   
                 0.90 
               
               
                   
                 E392A 
                 715 
                 0.58 
               
               
                   
                 E392F 
                 716 
                 0.54 
               
               
                   
                 E392G 
                   
                 1.00 
               
               
                   
                 E392K 
                   
                 0.66 
               
               
                   
                 E392L 
                   
                 0.80 
               
               
                   
                 E392M 
                 717 
                 1.54 
               
               
                   
                 E392Q 
                 718 
                 1.01 
               
               
                   
                 E392R 
                 719 
                 0.66 
               
               
                   
                 E392S 
                   
                 0.52 
               
               
                   
                 E392T 
                   
                 0.72 
               
               
                   
                 E392V 
                 720 
                 1.27 
               
               
                   
                 E392Y 
                   
                 0.92 
               
               
                   
                 Q393A 
                   
                 1.26 
               
               
                   
                 Q393D 
                   
                 0.45 
               
               
                   
                 Q393F 
                 721 
                 1.23 
               
               
                   
                 Q393H 
                   
                 1.05 
               
               
                   
                 Q393K 
                   
                 0.80 
               
               
                   
                 Q393L 
                   
                 0.91 
               
               
                   
                 Q393M 
                 722 
                 0.80 
               
               
                   
                 Q393N 
                   
                 0.72 
               
               
                   
                 Q393R 
                   
                 0.74 
               
               
                   
                 Q393S 
                   
                 1.15 
               
               
                   
                 Q393T 
                   
                 0.41 
               
               
                   
                 F394L 
                   
                 0.56 
               
               
                   
                 F394W 
                   
                 0.41 
               
               
                   
                 S395A 
                 723 
                 1.10 
               
               
                   
                 S395G 
                   
                 0.77 
               
               
                   
                 S395H 
                 724 
                 0.56 
               
               
                   
                 S395K 
                   
                 0.96 
               
               
                   
                 S395R 
                 725 
                 1.98 
               
               
                   
                 E396A 
                 726 
                 0.52 
               
               
                   
                 E396D 
                   
                 0.64 
               
               
                   
                 E396H 
                 727 
                 0.47 
               
               
                   
                 E396Q 
                 728 
                 0.73 
               
               
                   
                 E396R 
                   
                 0.61 
               
               
                   
                 E396S 
                 729 
                 0.61 
               
               
                   
                 E396T 
                   
                 0.89 
               
               
                   
                 E396L 
                   
                 0.39 
               
               
                   
                 Y399A 
                   
                 1.01 
               
               
                   
                 Y399C 
                   
                 0.46 
               
               
                   
                 Y399E 
                   
                 1.49 
               
               
                   
                 S407L 
                   
                 0.4 
               
               
                   
                 Y399K 
                 730 
                 1.94 
               
               
                   
                 Y399M 
                 731 
                 2.70 
               
               
                   
                 Y399N 
                   
                 0.52 
               
               
                   
                 Y399Q 
                   
                 1.18 
               
               
                   
                 Y399R 
                   
                 1.20 
               
               
                   
                 Y399S 
                   
                 1.01 
               
               
                   
                 Y399T 
                 732 
                 2.40 
               
               
                   
                 Y399V 
                 733 
                 1.44 
               
               
                   
                 Y399W 
                 734 
                 1.92 
               
               
                   
                 S401A 
                 735 
                 0.82 
               
               
                   
                 S401E 
                 736 
                 0.46 
               
               
                   
                 S401N 
                   
                 0.42 
               
               
                   
                 Y403F 
                   
                 0.62 
               
               
                   
                 S404A 
                 737 
                 0.63 
               
               
                   
                 S404P 
                   
                 0.64 
               
               
                   
                 S401G 
                   
                 0.38 
               
               
                   
                 T405F 
                   
                 0.36 
               
               
                   
                 T405A 
                   
                 0.56 
               
               
                   
                 T405G 
                 738 
                 2.32 
               
               
                   
                 T405K 
                   
                 0.74 
               
               
                   
                 T405M 
                   
                 0.48 
               
               
                   
                 T405P 
                   
                 0.64 
               
               
                   
                 T405Q 
                   
                 0.75 
               
               
                   
                 T405R 
                   
                 0.60 
               
               
                   
                 T405S 
                   
                 0.94 
               
               
                   
                 T405W 
                   
                 0.73 
               
               
                   
                 T405Y 
                   
                 0.44 
               
               
                   
                 L406A 
                   
                 0.70 
               
               
                   
                 L406C 
                   
                 0.98 
               
               
                   
                 L406E 
                   
                 0.73 
               
               
                   
                 L406F 
                 739 
                 1.42 
               
               
                   
                 L406G 
                   
                 1.00 
               
               
                   
                 L406I 
                   
                 0.61 
               
               
                   
                 L406N 
                 740 
                 0.76 
               
               
                   
                 L406Q 
                   
                 0.93 
               
               
                   
                 L406S 
                   
                 0.47 
               
               
                   
                 L406T 
                   
                 0.83 
               
               
                   
                 L406V 
                   
                 0.87 
               
               
                   
                 L406Y 
                   
                 0.74 
               
               
                   
                 S407A 
                 741 
                 1.16 
               
               
                   
                 S407D 
                 742 
                 1.52 
               
               
                   
                 S407E 
                 743 
                 1.38 
               
               
                   
                 S407F 
                 744 
                 1.42 
               
               
                   
                 S407G 
                   
                 0.75 
               
               
                   
                 S407H 
                 745 
                 1.34 
               
               
                   
                 S407M 
                   
                 0.74 
               
               
                   
                 K411H 
                   
                 0.33 
               
               
                   
                 S407N 
                   
                 0.72 
               
               
                   
                 S407P 
                 747 
                 0.94 
               
               
                   
                 S407Q 
                 746 
                 1.71 
               
               
                   
                 S407R 
                   
                 1.04 
               
               
                   
                 S407V 
                   
                 0.56 
               
               
                   
                 S407W 
                   
                 0.41 
               
               
                   
                 K409A 
                 748 
                 2.18 
               
               
                   
                 K409D 
                   
                 0.65 
               
               
                   
                 K409E 
                   
                 0.62 
               
               
                   
                 K409G 
                   
                 0.50 
               
               
                   
                 K409H 
                   
                 0.64 
               
               
                   
                 K409I 
                   
                 0.51 
               
               
                   
                 K409P 
                   
                 0.48 
               
               
                   
                 K409Q 
                 749 
                 3.33 
               
               
                   
                 K409R 
                   
                 0.84 
               
               
                   
                 K409S 
                   
                 0.72 
               
               
                   
                 I208K 
                   
                 0.30 
               
               
                   
                 K409T 
                   
                 0.63 
               
               
                   
                 K409V 
                   
                 0.48 
               
               
                   
                 A412Y 
                   
                 0.66 
               
               
                   
                 E410D 
                   
                 0.47 
               
               
                   
                 E410K 
                   
                 0.70 
               
               
                   
                 E410M 
                   
                 0.42 
               
               
                   
                 E410N 
                   
                 0.67 
               
               
                   
                 E410P 
                   
                 0.73 
               
               
                   
                 E410Q 
                   
                 0.85 
               
               
                   
                 E410R 
                   
                 0.61 
               
               
                   
                 E410S 
                   
                 0.81 
               
               
                   
                 E410T 
                 750 
                 1.54 
               
               
                   
                 E410V 
                   
                 0.65 
               
               
                   
                 E410Y 
                   
                 0.62 
               
               
                   
                 K411A 
                   
                 0.48 
               
               
                   
                 K411N 
                   
                 1.02 
               
               
                   
                 K411P 
                   
                 0.42 
               
               
                   
                 K411R 
                   
                 0.97 
               
               
                   
                 K411S 
                   
                 1.21 
               
               
                   
                 K411T 
                   
                 0.63 
               
               
                   
                 K411V 
                   
                 0.99 
               
               
                   
                 A412D 
                   
                 0.74 
               
               
                   
                 A412G 
                   
                 0.80 
               
               
                   
                 A412I 
                   
                 0.81 
               
               
                   
                 E220M 
                   
                 0.36 
               
               
                   
                 P226W 
                   
                 0.51 
               
               
                   
                 A412L 
                   
                 0.65 
               
               
                   
                 A412N 
                   
                 0.86 
               
               
                   
                 A412P 
                   
                 0.77 
               
               
                   
                 A412R 
                 752 
                 0.66 
               
               
                   
                 A412S 
                   
                 0.86 
               
               
                   
                 A412V 
                 753 
                 0.53 
               
               
                   
                 A412W 
                   
                 0.54 
               
               
                   
                 D413E 
                   
                 0.52 
               
               
                   
                 D413K 
                   
                 0.42 
               
               
                   
                 D413N 
                   
                 0.94 
               
               
                   
                 D413R 
                   
                 0.50 
               
               
                   
                 D413T 
                   
                 0.41 
               
               
                   
                 V414I 
                   
                 1.12 
               
               
                   
                 V414M 
                   
                 0.53 
               
               
                   
                 K415G 
                   
                 0.40 
               
               
                   
                 K415S 
                   
                 0.42 
               
               
                   
                 K415W 
                   
                 0.42 
               
               
                   
                 D416F 
                   
                 0.41 
               
               
                   
                 D416G 
                   
                 0.67 
               
               
                   
                 D416H 
                   
                 0.57 
               
               
                   
                 D416I 
                   
                 0.63 
               
               
                   
                 D416K 
                   
                 0.76 
               
               
                   
                 D416L 
                 754 
                 0.75 
               
               
                   
                 D416N 
                   
                 0.73 
               
               
                   
                 D416Q 
                   
                 0.83 
               
               
                   
                 D416R 
                   
                 0.46 
               
               
                   
                 V237C 
                 483 
                 0.35 
               
               
                   
                 D416T 
                   
                 0.85 
               
               
                   
                 D416V 
                   
                 0.59 
               
               
                   
                 D416Y 
                   
                 0.40 
               
               
                   
                 T417I 
                   
                 1.22 
               
               
                   
                 D413A 
                   
                 0.38 
               
               
                   
                 D413S 
                   
                 0.39 
               
               
                   
                 K415Y 
                   
                 0.39 
               
               
                   
                 D418A 
                   
                 0.92 
               
               
                   
                 D418E 
                 755 
                 1.31 
               
               
                   
                 D418F 
                   
                 0.81 
               
               
                   
                 L089P 
                   
                 0.38 
               
               
                   
                 D418G 
                   
                 0.45 
               
               
                   
                 D418I 
                   
                 0.99 
               
               
                   
                 D418L 
                 756 
                 1.28 
               
               
                   
                 D418M 
                   
                 1.09 
               
               
                   
                 D418N 
                   
                 0.91 
               
               
                   
                 D418P 
                 757 
                 2.11 
               
               
                   
                 D418Q 
                   
                 1.05 
               
               
                   
                 D418R 
                 758 
                 1.18 
               
               
                   
                 D418S 
                   
                 0.78 
               
               
                   
                 D418V 
                 759 
                 1.43 
               
               
                   
                 D418Y 
                   
                 0.97 
               
               
                   
                 A419E 
                   
                 0.45 
               
               
                   
                 A419F 
                 760 
                 2.17 
               
               
                   
                 A419G 
                   
                 0.42 
               
               
                   
                 A419H 
                 761 
                 1.21 
               
               
                   
                 A419I 
                 762 
                 1.64 
               
               
                   
                 A419K 
                 763 
                 1.88 
               
               
                   
                 A419L 
                   
                 0.56 
               
               
                   
                 A419N 
                   
                 0.53 
               
               
                   
                 V421I 
                   
                 0.39 
               
               
                   
                 A419R 
                 764 
                 1.81 
               
               
                   
                 A419S 
                 765 
                 2.65 
               
               
                   
                 A419W 
                   
                 0.69 
               
               
                   
                 A419Y 
                 766 
                 1.44 
               
               
                   
                 V420I 
                   
                 1.04 
               
               
                   
                 V420P 
                   
                 0.48 
               
               
                   
                 D421A 
                 767 
                 1.28 
               
               
                   
                 D421E 
                   
                 0.81 
               
               
                   
                 D421G 
                   
                 0.62 
               
               
                   
                 D421H 
                 768 
                 1.98 
               
               
                   
                 K255G 
                   
                 0.39 
               
               
                   
                 D421K 
                 769 
                 2.42 
               
               
                   
                 D421L 
                   
                 0.73 
               
               
                   
                 D421M 
                   
                 0.94 
               
               
                   
                 D421N 
                 770 
                 1.89 
               
               
                   
                 D421Q 
                 771 
                 1.54 
               
               
                   
                 D421R 
                 772 
                 2.21 
               
               
                   
                 D421S 
                 773 
                 2.12 
               
               
                   
                 K094C 
                   
                 0.33 
               
               
                   
                 D421T 
                   
                 0.80 
               
               
                   
                 D421Y 
                   
                 0.66 
               
               
                   
                 V422I 
                   
                 0.42 
               
               
                   
                 V422T 
                   
                 0.49 
               
               
                   
                 A425G 
                 774 
                 1.20 
               
               
                   
                 A425I 
                   
                 0.44 
               
               
                   
                 A425K 
                 775 
                 1.75 
               
               
                   
                 A425M 
                   
                 0.70 
               
               
                   
                 A425N 
                   
                 0.46 
               
               
                   
                 A425R 
                   
                 0.49 
               
               
                   
                 A425S 
                   
                 0.47 
               
               
                   
                 D426E 
                   
                 0.62 
               
               
                   
                 D426G 
                   
                 0.85 
               
               
                   
                 D426N 
                   
                 0.61 
               
               
                   
                 D426P 
                   
                 1.03 
               
               
                   
                 D426Q 
                   
                 0.42 
               
               
                   
                 D426Y 
                   
                 0.43 
               
               
                   
                 G427K 
                   
                 0.52 
               
               
                   
                 G427S 
                   
                 0.42 
               
               
                   
                 V428L 
                 778 
                 1.25 
               
               
                   
                 A425Y 
                   
                 0.39 
               
               
                   
                 G427T 
                 777 
                 0.35 
               
               
                   
                 G427Q 
                 776 
                 0.39 
               
               
                   
                 V428M 
                   
                 0.42 
               
               
                   
                 V428P 
                   
                 0.82 
               
               
                   
                 V428T 
                   
                 0.62 
               
               
                   
                 D431A 
                 779 
                 2.42 
               
               
                   
                 D431E 
                 781 
                 1.27 
               
               
                   
                 D431G 
                 780 
                 0.55 
               
               
                   
                 D431H 
                 782 
                 3.13 
               
               
                   
                 D431I 
                   
                 1.05 
               
               
                   
                 D431K 
                 783 
                 1.83 
               
               
                   
                 D431L 
                 784 
                 0.62 
               
               
                   
                 D431N 
                 785 
                 1.30 
               
               
                   
                 D431Q 
                 786 
                 2.16 
               
               
                   
                 D431R 
                 787 
                 2.20 
               
               
                   
                 D431S 
                 788 
                 1.91 
               
               
                   
                 D431V 
                 789 
                 1.52 
               
               
                   
                 D431W 
                   
                 0.56 
               
               
                   
                 D431Y 
                   
                 0.85 
               
               
                   
                 A432E 
                   
                 0.60 
               
               
                   
                 A432G 
                   
                 0.52 
               
               
                   
                 A432H 
                   
                 0.34 
               
               
                   
                 A432N 
                   
                 0.51 
               
               
                   
                 A432S 
                   
                 0.61 
               
               
                   
                 A432V 
                   
                 0.56 
               
               
                   
                 F433A 
                 790 
                 0.97 
               
               
                   
                 R270T 
                   
                 0.40 
               
               
                   
                 F433C 
                   
                 0.69 
               
               
                   
                 F433D 
                   
                 0.95 
               
               
                   
                 F433E 
                   
                 0.82 
               
               
                   
                 F433G 
                   
                 0.54 
               
               
                   
                 F433H 
                 791 
                 0.83 
               
               
                   
                 F433I 
                 792 
                 1.06 
               
               
                   
                 F433K 
                 793 
                 1.36 
               
               
                   
                 F433L 
                 794 
                 1.87 
               
               
                   
                 F433P 
                   
                 0.95 
               
               
                   
                 F433R 
                 795 
                 1.63 
               
               
                   
                 F433S 
                   
                 0.86 
               
               
                   
                 F433T 
                 796 
                 1.86 
               
               
                   
                 F433V 
                 797 
                 1.63 
               
               
                   
                 F433W 
                 798 
                 1.28 
               
               
                   
                 L434F 
                   
                 0.41 
               
               
                   
                 L434G 
                   
                 0.47 
               
               
                   
                 L263H 
                   
                 0.36 
               
               
                   
                 L434I 
                   
                 0.89 
               
               
                   
                 L434M 
                   
                 0.60 
               
               
                   
                 L434V 
                   
                 0.46 
               
               
                   
                 K435A 
                   
                 1.08 
               
               
                   
                 K435C 
                   
                 0.53 
               
               
                   
                 K435E 
                   
                 0.78 
               
               
                   
                 K435G 
                   
                 0.64 
               
               
                   
                 K435H 
                   
                 1.05 
               
               
                   
                 K435R 
                   
                 1.01 
               
               
                   
                 K435S 
                   
                 1.03 
               
               
                   
                 K435T 
                   
                 0.73 
               
               
                   
                 K435V 
                   
                 0.44 
               
               
                   
                 K435Y 
                   
                 0.50 
               
               
                   
                 P436D 
                   
                 1.19 
               
               
                   
                 P436E 
                   
                 0.74 
               
               
                   
                 P436G 
                   
                 1.19 
               
               
                   
                 L105H 
                   
                 0.36 
               
               
                   
                 P436H 
                   
                 0.72 
               
               
                   
                 V272M 
                   
                 0.31 
               
               
                   
                 P436I 
                   
                 0.84 
               
               
                   
                 P436K 
                 799 
                 2.05 
               
               
                   
                 P436L 
                   
                 0.63 
               
               
                   
                 P436M 
                   
                 0.61 
               
               
                   
                 P436Q 
                   
                 0.86 
               
               
                   
                 P436R 
                   
                 1.00 
               
               
                   
                 P436S 
                   
                 0.92 
               
               
                   
                 P436T 
                   
                 0.59 
               
               
                   
                 P436W 
                   
                 0.43 
               
               
                   
                 P436Y 
                   
                 0.49 
               
               
                   
                 P437A 
                   
                 0.56 
               
               
                   
                 P437D 
                   
                 0.62 
               
               
                   
                 P437G 
                   
                 0.50 
               
               
                   
                 P437H 
                   
                 1.11 
               
               
                   
                 P437I 
                 800 
                 2.46 
               
               
                   
                 P437K 
                   
                 0.83 
               
               
                   
                 P437L 
                   
                 0.51 
               
               
                   
                 P437M 
                 801 
                 2.55 
               
               
                   
                 P437Q 
                   
                 0.96 
               
               
                   
                 D275L 
                   
                 0.24 
               
               
                   
                 P437R 
                   
                 0.85 
               
               
                   
                 P437S 
                   
                 0.57 
               
               
                   
                 P437Y 
                   
                 0.42 
               
               
                   
                 M438A 
                 802 
                 0.75 
               
               
                   
                 M438C 
                   
                 0.63 
               
               
                   
                 M438D 
                 803 
                 0.87 
               
               
                   
                 M438E 
                 804 
                 0.72 
               
               
                   
                 M438G 
                   
                 0.83 
               
               
                   
                 M438L 
                 805 
                 0.86 
               
               
                   
                 M438N 
                 806 
                 1.08 
               
               
                   
                 M438P 
                   
                 0.81 
               
               
                   
                 M438Q 
                   
                 0.85 
               
               
                   
                 M438R 
                   
                 0.99 
               
               
                   
                 M438S 
                   
                 0.83 
               
               
                   
                 M438T 
                 807 
                 3.99 
               
               
                   
                 M438V 
                   
                 0.85 
               
               
                   
                 P125A 
                   
                 0.36 
               
               
                   
                 M438W 
                   
                 0.57 
               
               
                   
                 E439A 
                 808 
                 1.20 
               
               
                   
                 E439C 
                 809 
                 0.58 
               
               
                   
                 E439F 
                   
                 1.00 
               
               
                   
                 E439G 
                   
                 1.22 
               
               
                   
                 E439H 
                   
                 0.74 
               
               
                   
                 E439K 
                 810 
                 1.20 
               
               
                   
                 E439L 
                   
                 0.88 
               
               
                   
                 E439P 
                 811 
                 1.16 
               
               
                   
                 Q276G 
                   
                 0.36 
               
               
                   
                 E439Q 
                 812 
                 1.32 
               
               
                   
                 E439S 
                   
                 1.02 
               
               
                   
                 E439T 
                 813 
                 1.15 
               
               
                   
                 E439V 
                 814 
                 1.57 
               
               
                   
                 E439W 
                   
                 0.62 
               
               
                   
                 T440A 
                   
                 1.22 
               
               
                   
                 T440D 
                 815 
                 1.03 
               
               
                   
                 T440E 
                   
                 1.00 
               
               
                   
                 T440F 
                   
                 0.85 
               
               
                   
                 T440G 
                   
                 0.86 
               
               
                   
                 T440H 
                 816 
                 3.00 
               
               
                   
                 T440I 
                   
                 1.04 
               
               
                   
                 T440L 
                   
                 0.97 
               
               
                   
                 T440M 
                 817 
                 1.08 
               
               
                   
                 T440P 
                 818 
                 0.88 
               
               
                   
                 T440R 
                 819 
                 1.77 
               
               
                   
                 T440S 
                 820 
                 1.17 
               
               
                   
                 T440V 
                   
                 1.02 
               
               
                   
                 T440Y 
                   
                 1.11 
               
               
                   
                 E441A 
                 821 
                 1.47 
               
               
                   
                 E441D 
                   
                 0.67 
               
               
                   
                 E441F 
                 822 
                 3.91 
               
               
                   
                 E441G 
                   
                 0.87 
               
               
                   
                 E441H 
                   
                 0.65 
               
               
                   
                 E441K 
                   
                 0.80 
               
               
                   
                 E441L 
                   
                 0.82 
               
               
                   
                 E441N 
                   
                 0.82 
               
               
                   
                 E441Q 
                   
                 0.81 
               
               
                   
                 E441S 
                   
                 0.79 
               
               
                   
                 E441T 
                   
                 0.66 
               
               
                   
                 E441V 
                   
                 0.54 
               
               
                   
                 E441Y 
                   
                 0.51 
               
               
                   
                 E442C 
                 823 
                 1.38 
               
               
                   
                 E442G 
                 824 
                 0.51 
               
               
                   
                 E442H 
                   
                 0.76 
               
               
                   
                 E442K 
                   
                 0.73 
               
               
                   
                 E442P 
                   
                 0.91 
               
               
                   
                 E442Q 
                   
                 0.74 
               
               
                   
                 D284Y 
                   
                 0.37 
               
               
                   
                 L286W 
                   
                 0.38 
               
               
                   
                 E442R 
                 825 
                 3.94 
               
               
                   
                 E442T 
                   
                 0.61 
               
               
                   
                 E442V 
                   
                 0.65 
               
               
                   
                 E442Y 
                   
                 0.60 
               
               
                   
                 P443A 
                 826 
                 1.63 
               
               
                   
                 P443E 
                 827 
                 1.07 
               
               
                   
                 P443F 
                 828 
                 0.70 
               
               
                   
                 P443G 
                 829 
                 1.12 
               
               
                   
                 P443H 
                   
                 1.08 
               
               
                   
                 P443L 
                   
                 1.19 
               
               
                   
                 P443M 
                 830 
                 1.99 
               
               
                   
                 P443N 
                 831 
                 1.25 
               
               
                   
                 P443Q 
                   
                 0.96 
               
               
                   
                 P443R 
                   
                 1.04 
               
               
                   
                 P443S 
                   
                 0.99 
               
               
                   
                 P443T 
                   
                 0.87 
               
               
                   
                 P443W 
                   
                 0.64 
               
               
                   
                 Q444M 
                   
                 0.37 
               
               
                   
                 Q444D 
                   
                 0.97 
               
               
                   
                 Q444E 
                 832 
                 1.19 
               
               
                   
                 Q444F 
                   
                 0.66 
               
               
                   
                 Q444G 
                   
                 0.93 
               
               
                   
                 Q444H 
                 833 
                 0.97 
               
               
                   
                 Q444I 
                   
                 0.58 
               
               
                   
                 Q444K 
                   
                 1.03 
               
               
                   
                 Q444N 
                   
                 1.01 
               
               
                   
                 Q444R 
                   
                 0.85 
               
               
                   
                 Q444V 
                 834 
                 1.12 
               
               
                   
                 Q444W 
                   
                 0.64 
               
               
                   
                 Q444Y 
                   
                 0.67 
               
               
                   
                 I445A 
                   
                 0.97 
               
               
                   
                 I445G 
                   
                 0.98 
               
               
                   
                 I445H 
                 835 
                 1.35 
               
               
                   
                 I445L 
                   
                 1.06 
               
               
                   
                 I445M 
                 836 
                 1.57 
               
               
                   
                 I445N 
                 837 
                 1.24 
               
               
                   
                 I445P 
                 838 
                 1.67 
               
               
                   
                 I445Q 
                 839 
                 1.26 
               
               
                   
                 I445R 
                   
                 1.08 
               
               
                   
                 I445S 
                 840 
                 1.21 
               
               
                   
                 I445T 
                 841 
                 1.38 
               
               
                   
                 I445V 
                 842 
                 1.25 
               
               
                   
                 I445W 
                 843 
                 0.69 
               
               
                   
                 I445Y 
                   
                 0.53 
               
               
                   
                 F446A 
                 844 
                 1.58 
               
               
                   
                 F446C 
                   
                 0.75 
               
               
                   
                 F446D 
                   
                 1.18 
               
               
                   
                 F446E 
                   
                 1.10 
               
               
                   
                 F446G 
                   
                 1.12 
               
               
                   
                 F446H 
                   
                 1.28 
               
               
                   
                 F446I 
                   
                 1.06 
               
               
                   
                 F446K 
                   
                 0.94 
               
               
                   
                 F446L 
                   
                 0.93 
               
               
                   
                 F446M 
                 845 
                 1.31 
               
               
                   
                 F446Q 
                   
                 0.72 
               
               
                   
                 F446R 
                   
                 0.89 
               
               
                   
                 F446T 
                   
                 0.89 
               
               
                   
                 F446V 
                   
                 0.91 
               
               
                   
                 F446W 
                 846 
                 1.40 
               
               
                   
                 Y447D 
                 847 
                 3.25 
               
               
                   
                 Y447E 
                 848 
                 1.36 
               
               
                   
                 Y447F 
                   
                 1.41 
               
               
                   
                 Y447G 
                 849 
                 0.92 
               
               
                   
                 Y447I 
                 850 
                 1.36 
               
               
                   
                 Y447L 
                   
                 1.09 
               
               
                   
                 Y447M 
                   
                 0.90 
               
               
                   
                 Y447N 
                 851 
                 1.58 
               
               
                   
                 Y447P 
                 852 
                 1.46 
               
               
                   
                 Y447Q 
                 853 
                 2.37 
               
               
                   
                 Y447R 
                   
                 1.12 
               
               
                   
                 Y447T 
                 854 
                 1.90 
               
               
                   
                 Y447V 
                 855 
                 1.38 
               
               
                   
                 Y447W 
                   
                 1.07 
               
               
                   
                   
               
            
           
         
       
     
     2. Inactive Mutants 
     The other mutants that exhibited less than 20% hyaluronidase activity of wildtype PH20, in at least one of the duplicates, were rescreened to confirm that the dead mutants are inactive. To confirm the inactive mutants, the hyaluronidase activity assay described in Example 3 was modified to incorporate an overnight 37° C. substrate-sample incubation step prior to measurement of enzymatic activity. The modified assay is intended to detect PH20 activities below 0.2 U/mL. 
     The preparation of the bHA coated plates and blocking of the plates prior to addition of the transfected variant supernatants or wildtype PH20 was the same as described in Example 3. The assay was modified as follows. First, transfected variant supernatants or wildypte PH20 not containing a mutation generated as described in Example 2 were diluted in duplicate 1:25 in assay diluent. For the standard curve, 1:3 serial dilutions of rHuPH20 (generated as described in Example 1) were made in assay diluent in duplicate starting from 0.1 U/mL down to 0.00014 U/mL. A blank well also was included. Then, 100 μl of the diluted samples or standard were added to pre-designated wells of the bHA-coated and blocked plate and allowed to incubate at 37° C. overnight. After the incubation, the plates were washed and binding to bHA detected as described above in Example 3. Optical density was measured at 450 nm within 30 minutes of adding the stop solution. 
     The identified reconfirmed inactive mutants are set forth in Table 10. The Table sets forth the amino acid replacement compared to the sequence of amino acids of PH20 set forth in SEQ ID NO:3. 
     
       
         
           
               
             
               
                 TABLE 10 
               
               
                   
               
               
                 Inactive Mutants 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 N002H 
                 R060V 
                 R121W 
                 C189P 
                 P236I 
                 V287N 
                 L336W 
                 G377V 
               
               
                 N002K 
                 R060Y 
                 R121Y 
                 C189R 
                 P236L 
                 V287P 
                 L336Y 
                 G378D 
               
               
                 N002W 
                 L061A 
                 N122A 
                 C189S 
                 P236N 
                 V287Q 
                 A337C 
                 G378E 
               
               
                 N002Y 
                 L061E 
                 N122C 
                 C189T 
                 P236Q 
                 V287R 
                 A337F 
                 G378F 
               
               
                 F003A 
                 L061F 
                 N122E 
                 C189V 
                 P236T 
                 V287S 
                 A337G 
                 G378I 
               
               
                 F003G 
                 L061G 
                 N122F 
                 C189W 
                 P236Y 
                 Y288D 
                 A337I 
                 G378L 
               
               
                 F003K 
                 L061H 
                 N122I 
                 C189Y 
                 A238F 
                 Y288E 
                 A337K 
                 G378M 
               
               
                 F003P 
                 L061N 
                 N122K 
                 Y190C 
                 A238G 
                 Y288F 
                 A337L 
                 G378Q 
               
               
                 F003T 
                 L061P 
                 N122Q 
                 Y190E 
                 A238L 
                 Y288G 
                 A337M 
                 G378T 
               
               
                 F003V 
                 L061Q 
                 N122R 
                 Y190F 
                 A238P 
                 Y288H 
                 A337R 
                 G378W 
               
               
                 R004D 
                 L061R 
                 N122S 
                 Y190G 
                 A238V 
                 Y288I 
                 A337T 
                 G378Y 
               
               
                 R004E 
                 L061T 
                 N122T 
                 Y190H 
                 A238W 
                 Y288K 
                 A337W 
                 K379A 
               
               
                 R004F 
                 L061W 
                 N122V 
                 Y190K 
                 A238Y 
                 Y288P 
                 A338C 
                 K379C 
               
               
                 R004G 
                 L061Y 
                 W123A 
                 Y190L 
                 A239C 
                 Y288R 
                 A338D 
                 K379E 
               
               
                 R004L 
                 G062A 
                 W123C 
                 Y190N 
                 A239F 
                 Y288T 
                 A338E 
                 K379F 
               
               
                 R004P 
                 G062C 
                 W123D 
                 Y190Q 
                 A239G 
                 T289A 
                 A338F 
                 K379I 
               
               
                 R004W 
                 G062D 
                 W123E 
                 Y190R 
                 A239H 
                 T289C 
                 A338G 
                 K379L 
               
               
                 R004Y 
                 G062F 
                 W123H 
                 Y190S 
                 A239I 
                 T289E 
                 A338H 
                 K379M 
               
               
                 A005D 
                 G062I 
                 W123L 
                 Y190T 
                 A239L 
                 T289G 
                 A338I 
                 K379W 
               
               
                 A005G 
                 G062K 
                 W123M 
                 Y190V 
                 A239P 
                 T289H 
                 A338K 
                 F380C 
               
               
                 A005I 
                 G062L 
                 W123P 
                 Y190W 
                 A239R 
                 T289L 
                 A338L 
                 F380D 
               
               
                 A005L 
                 G062M 
                 W123Q 
                 N191A 
                 A239S 
                 T289P 
                 A338P 
                 F380E 
               
               
                 A005M 
                 G062P 
                 W123R 
                 N191E 
                 A239T 
                 T289Q 
                 A338R 
                 F380G 
               
               
                 A005N 
                 G062Q 
                 W123S 
                 N191F 
                 A239V 
                 T289R 
                 A338T 
                 F380Q 
               
               
                 A005P 
                 G062R 
                 W123T 
                 N191G 
                 A239W 
                 T289S 
                 A338V 
                 F380R 
               
               
                 A005Q 
                 G062S 
                 W123V 
                 N191K 
                 A239Y 
                 T289Y 
                 K339D 
                 F380S 
               
               
                 A005R 
                 G062T 
                 W123Y 
                 N191L 
                 T240E 
                 F290D 
                 K339E 
                 T381G 
               
               
                 A005T 
                 G062V 
                 K124C 
                 N191M 
                 T240F 
                 F290Q 
                 K339F 
                 T381L 
               
               
                 A005V 
                 G062Y 
                 K124D 
                 N191P 
                 T240G 
                 F290Y 
                 K339G 
                 T381P 
               
               
                 A005W 
                 Y063C 
                 K124E 
                 N191Q 
                 T240N 
                 G291A 
                 K339H 
                 T381W 
               
               
                 A005Y 
                 Y063G 
                 K124F 
                 N191R 
                 T240W 
                 G291C 
                 K339L 
                 T381Y 
               
               
                 P006E 
                 Y063P 
                 K124N 
                 N191S 
                 T240Y 
                 G291D 
                 K339N 
                 V382E 
               
               
                 P006F 
                 Y064A 
                 P125C 
                 N191T 
                 L241A 
                 G291E 
                 K339P 
                 V382G 
               
               
                 P006T 
                 Y064C 
                 P125D 
                 N191V 
                 L241C 
                 G291F 
                 K339S 
                 V382H 
               
               
                 P006V 
                 Y064D 
                 P125G 
                 N191W 
                 L241D 
                 G291M 
                 K339T 
                 V382K 
               
               
                 P006Y 
                 Y064E 
                 P125L 
                 N191Y 
                 L241E 
                 G291N 
                 K339V 
                 V382L 
               
               
                 P007C 
                 Y064F 
                 P125N 
                 H192C 
                 L241G 
                 G291T 
                 K339W 
                 V382M 
               
               
                 P007D 
                 Y064G 
                 P125W 
                 H192F 
                 L241I 
                 G291W 
                 K339Y 
                 V382N 
               
               
                 P007F 
                 Y064H 
                 K126F 
                 H192G 
                 L241P 
                 G291Y 
                 M340A 
                 V382P 
               
               
                 P007G 
                 Y064I 
                 K126H 
                 H192K 
                 L241R 
                 E292I 
                 M340C 
                 V382Q 
               
               
                 P007H 
                 Y064K 
                 K126I 
                 H192L 
                 L241S 
                 E292L 
                 M340D 
                 V382R 
               
               
                 P007I 
                 Y064L 
                 K126L 
                 H192M 
                 L241T 
                 E292T 
                 M340E 
                 V382S 
               
               
                 P007K 
                 Y064P 
                 K126N 
                 H192N 
                 L241V 
                 T293E 
                 M340F 
                 V382T 
               
               
                 P007L 
                 Y064Q 
                 K126P 
                 H192P 
                 L241W 
                 T293N 
                 M340G 
                 V382W 
               
               
                 P007Q 
                 Y064R 
                 K126Y 
                 H192Q 
                 Y242A 
                 V294A 
                 M340H 
                 V382Y 
               
               
                 P007R 
                 Y064S 
                 D127K 
                 H192R 
                 Y242C 
                 V294E 
                 M340K 
                 R383G 
               
               
                 P007S 
                 Y064T 
                 V128E 
                 H192V 
                 Y242D 
                 V294G 
                 M340P 
                 R383P 
               
               
                 P007T 
                 Y064V 
                 V128P 
                 H192W 
                 Y242G 
                 V294H 
                 M340R 
                 G384C 
               
               
                 P007W 
                 Y064W 
                 Y129A 
                 H192Y 
                 Y242I 
                 V294K 
                 M340S 
                 G384F 
               
               
                 P007Y 
                 P065A 
                 Y129C 
                 H193A 
                 Y242L 
                 V294L 
                 M340T 
                 G384M 
               
               
                 V008D 
                 P065C 
                 Y129D 
                 H193D 
                 Y242M 
                 V294N 
                 M340V 
                 G384Q 
               
               
                 V008E 
                 P065D 
                 Y129E 
                 H193K 
                 Y242P 
                 V294P 
                 M340W 
                 G384S 
               
               
                 V008G 
                 P065G 
                 Y129G 
                 H193L 
                 Y242R 
                 V294Q 
                 C341A 
                 G384T 
               
               
                 V008H 
                 P065H 
                 Y129H 
                 H193M 
                 Y242S 
                 V294R 
                 C341E 
                 K385C 
               
               
                 V008N 
                 P065I 
                 Y129L 
                 H193P 
                 Y242T 
                 V294S 
                 C341G 
                 K385L 
               
               
                 V008R 
                 P065K 
                 Y129P 
                 H193V 
                 Y242V 
                 V294T 
                 C341H 
                 K385M 
               
               
                 V008S 
                 P065N 
                 Y129Q 
                 Y194A 
                 Y242W 
                 V294W 
                 C341K 
                 K385P 
               
               
                 V008W 
                 P065R 
                 Y129S 
                 Y194C 
                 V243C 
                 A295C 
                 C341L 
                 K385W 
               
               
                 I009C 
                 P065S 
                 Y129T 
                 Y194I 
                 V243D 
                 A295G 
                 C341M 
                 K385Y 
               
               
                 I009D 
                 P065T 
                 Y129V 
                 Y194L 
                 V243F 
                 A295H 
                 C341N 
                 P386A 
               
               
                 I009E 
                 P065V 
                 Y129W 
                 Y194P 
                 V243G 
                 A295I 
                 C341Q 
                 P386C 
               
               
                 I009G 
                 P065W 
                 K130C 
                 Y194S 
                 V243H 
                 A295L 
                 C341R 
                 P386F 
               
               
                 I009N 
                 P065Y 
                 K130D 
                 Y194T 
                 V243L 
                 A295N 
                 C341S 
                 P386G 
               
               
                 I009P 
                 Y066A 
                 K130G 
                 Y194V 
                 V243M 
                 A295P 
                 C341T 
                 P386H 
               
               
                 P010F 
                 Y066C 
                 K130H 
                 K195S 
                 V243P 
                 A295T 
                 C341V 
                 P386I 
               
               
                 P010I 
                 Y066D 
                 K130L 
                 P197C 
                 V243Q 
                 A295V 
                 C341Y 
                 P386L 
               
               
                 P010L 
                 Y066E 
                 K130N 
                 G198V 
                 V243R 
                 A295Y 
                 S342D 
                 P386M 
               
               
                 P010M 
                 Y066G 
                 K130S 
                 G198W 
                 V243S 
                 L296C 
                 S342E 
                 P386N 
               
               
                 P010Y 
                 Y066I 
                 K130T 
                 Y199E 
                 V243W 
                 L296F 
                 S342F 
                 P386Q 
               
               
                 N011A 
                 Y066K 
                 K130W 
                 Y199G 
                 V243Y 
                 L296G 
                 S342H 
                 P386R 
               
               
                 N011C 
                 Y066L 
                 K130Y 
                 Y199H 
                 R244A 
                 L296I 
                 S342K 
                 P386S 
               
               
                 N011F 
                 Y066N 
                 N131P 
                 Y199I 
                 R244D 
                 L296K 
                 S342L 
                 P386T 
               
               
                 N011I 
                 Y066P 
                 R132P 
                 Y199K 
                 R244G 
                 L296M 
                 S342M 
                 P386V 
               
               
                 N011L 
                 Y066S 
                 S133D 
                 Y199L 
                 R244I 
                 L296Q 
                 S342P 
                 P386Y 
               
               
                 N011P 
                 Y066T 
                 S133E 
                 Y199P 
                 R244V 
                 L296R 
                 S342Q 
                 T387C 
               
               
                 N011T 
                 Y066V 
                 S133F 
                 Y199R 
                 R244Y 
                 L296S 
                 S342R 
                 T387E 
               
               
                 N011W 
                 I067D 
                 S133G 
                 Y199S 
                 N245A 
                 L296T 
                 S342T 
                 T387F 
               
               
                 N011Y 
                 I067E 
                 S133H 
                 Y199W 
                 N245C 
                 L296V 
                 S342Y 
                 T387G 
               
               
                 V012G 
                 I067G 
                 S133L 
                 N200A 
                 N245F 
                 L296W 
                 Q343C 
                 T387H 
               
               
                 V012H 
                 I067P 
                 S133M 
                 N200F 
                 N245L 
                 L296Y 
                 Q343D 
                 T387I 
               
               
                 V012W 
                 I067R 
                 S133N 
                 N200G 
                 N245P 
                 G297C 
                 Q343F 
                 T387L 
               
               
                 P013E 
                 I067T 
                 S133P 
                 N200H 
                 N245Q 
                 G297E 
                 Q343I 
                 T387M 
               
               
                 P013G 
                 I067W 
                 S133R 
                 N200K 
                 N245R 
                 G297H 
                 Q343P 
                 T387N 
               
               
                 P013I 
                 D068A 
                 S133T 
                 N200L 
                 N245S 
                 G297L 
                 Q343W 
                 T387V 
               
               
                 P013L 
                 D068C 
                 S133V 
                 N200M 
                 N245T 
                 G297N 
                 V344F 
                 T387W 
               
               
                 P013M 
                 D068G 
                 S133W 
                 N200P 
                 N245V 
                 G297P 
                 V344G 
                 T387Y 
               
               
                 P013V 
                 D068I 
                 I134A 
                 N200Q 
                 R246A 
                 G297Q 
                 V344H 
                 L388C 
               
               
                 F014A 
                 D068L 
                 I134C 
                 N200R 
                 R246C 
                 G297R 
                 V344L 
                 L388G 
               
               
                 F014E 
                 D068P 
                 I134D 
                 N200S 
                 R246D 
                 G297S 
                 V344M 
                 L388P 
               
               
                 F014G 
                 D068V 
                 I134F 
                 N200W 
                 R246E 
                 G297T 
                 V344N 
                 L388Q 
               
               
                 F014H 
                 D068Y 
                 I134G 
                 N200Y 
                 R246G 
                 G297Y 
                 V344P 
                 L388S 
               
               
                 F014K 
                 S069N 
                 I134H 
                 G201A 
                 R246H 
                 A298C 
                 V344Q 
                 E389F 
               
               
                 F014N 
                 S069T 
                 I134K 
                 G201F 
                 R246I 
                 A298E 
                 V344R 
                 E389V 
               
               
                 F014P 
                 I070Q 
                 I134P 
                 G201L 
                 R246K 
                 A298L 
                 V344S 
                 D390A 
               
               
                 F014Q 
                 T071P 
                 I134Q 
                 G201M 
                 R246L 
                 A298M 
                 V344T 
                 D390C 
               
               
                 F014W 
                 G072C 
                 I134R 
                 G201N 
                 R246M 
                 A298N 
                 V344W 
                 D390E 
               
               
                 L015E 
                 G072F 
                 I134S 
                 G201P 
                 R246P 
                 A298P 
                 V344Y 
                 D390F 
               
               
                 L015F 
                 G072H 
                 I134W 
                 G201R 
                 R246S 
                 A298Q 
                 L345A 
                 D390G 
               
               
                 L015G 
                 G072I 
                 E135P 
                 G201S 
                 R246T 
                 A298S 
                 L345C 
                 D390H 
               
               
                 L015K 
                 G072P 
                 L136P 
                 G201T 
                 R246V 
                 A298T 
                 L345E 
                 D390L 
               
               
                 L015N 
                 G072V 
                 V137F 
                 G201V 
                 R246W 
                 A298W 
                 L345H 
                 D390N 
               
               
                 L015P 
                 G072W 
                 V137G 
                 G201W 
                 V247A 
                 A298Y 
                 L345K 
                 D390P 
               
               
                 L015Q 
                 V073P 
                 V137H 
                 S202A 
                 V247C 
                 S299A 
                 L345N 
                 D390R 
               
               
                 L015R 
                 V075D 
                 V137N 
                 S202E 
                 V247F 
                 S299C 
                 L345Q 
                 D390S 
               
               
                 L015S 
                 V075G 
                 V137P 
                 S202F 
                 V247H 
                 S299D 
                 L345R 
                 D390T 
               
               
                 L015Y 
                 V075P 
                 V137R 
                 S202G 
                 V247N 
                 S299F 
                 L345T 
                 D390V 
               
               
                 W016A 
                 N076A 
                 V137W 
                 S202H 
                 V247P 
                 S299G 
                 L345V 
                 D390W 
               
               
                 W016C 
                 N076C 
                 V137Y 
                 S202K 
                 V247Q 
                 S299H 
                 L345Y 
                 D390Y 
               
               
                 W016D 
                 N076F 
                 Q138V 
                 S202N 
                 V247R 
                 S299L 
                 C346A 
                 L391A 
               
               
                 W016E 
                 N076G 
                 Q139P 
                 S202P 
                 V247S 
                 S299M 
                 C346D 
                 L391D 
               
               
                 W016F 
                 N076I 
                 Q143C 
                 S202Q 
                 V247T 
                 S299P 
                 C346F 
                 L391G 
               
               
                 W016G 
                 N076K 
                 Q143H 
                 S202R 
                 V247W 
                 S299Q 
                 C346G 
                 L391H 
               
               
                 W016H 
                 N076L 
                 Q143P 
                 S202V 
                 V247Y 
                 S299T 
                 C346I 
                 L391K 
               
               
                 W016K 
                 N076P 
                 Q143R 
                 S202W 
                 R248C 
                 G300A 
                 C346K 
                 L391N 
               
               
                 W016M 
                 N076Q 
                 Q143S 
                 S202Y 
                 R248D 
                 G300C 
                 C346L 
                 L391P 
               
               
                 W016P 
                 N076R 
                 Q143T 
                 C203A 
                 R248E 
                 G300D 
                 C346M 
                 L391Q 
               
               
                 W016R 
                 N076S 
                 L144A 
                 C203D 
                 R248G 
                 G300E 
                 C346P 
                 L391R 
               
               
                 W016S 
                 N076T 
                 L144E 
                 C203E 
                 R248I 
                 G300F 
                 C346R 
                 L391S 
               
               
                 W016T 
                 N076V 
                 L144F 
                 C203G 
                 R248M 
                 G300L 
                 C346S 
                 L391T 
               
               
                 W016Y 
                 N076W 
                 L144I 
                 C203H 
                 R248P 
                 G300M 
                 C346T 
                 L391V 
               
               
                 A017D 
                 G077D 
                 L144K 
                 C203L 
                 R248T 
                 G300N 
                 C346V 
                 L391W 
               
               
                 A017E 
                 G077E 
                 L144P 
                 C203M 
                 E249A 
                 G300P 
                 C346W 
                 L391Y 
               
               
                 A017G 
                 G077L 
                 L144Q 
                 C203N 
                 E249G 
                 G300Q 
                 Q347C 
                 E392C 
               
               
                 A017H 
                 G077P 
                 L144S 
                 C203Q 
                 E249H 
                 G300S 
                 Q347F 
                 E392P 
               
               
                 A017I 
                 G077Q 
                 L144V 
                 C203R 
                 E249I 
                 G300T 
                 Q347I 
                 Q393C 
               
               
                 A017L 
                 G077R 
                 L144Y 
                 C203S 
                 E249K 
                 G300V 
                 Q347P 
                 Q393P 
               
               
                 A017N 
                 G077T 
                 S145T 
                 C203T 
                 E249M 
                 G300W 
                 Q347T 
                 F394A 
               
               
                 A017P 
                 G077V 
                 S145W 
                 C203V 
                 E249Q 
                 I301E 
                 Q347V 
                 F394D 
               
               
                 A017Q 
                 G078A 
                 A149E 
                 F204A 
                 E249S 
                 I301G 
                 Q347W 
                 F394E 
               
               
                 A017R 
                 G078D 
                 A149P 
                 F204C 
                 E249Y 
                 I301H 
                 E348C 
                 F394G 
               
               
                 A017S 
                 G078I 
                 T150V 
                 F204E 
                 A250C 
                 I301K 
                 E348H 
                 F394I 
               
               
                 A017T 
                 G078M 
                 K152L 
                 F204G 
                 A250F 
                 I301M 
                 E348I 
                 F394K 
               
               
                 A017V 
                 G078P 
                 A153E 
                 F204H 
                 A250G 
                 I301N 
                 E348L 
                 F394N 
               
               
                 A017W 
                 G078T 
                 A153F 
                 F204I 
                 A250H 
                 I301P 
                 E348P 
                 F394P 
               
               
                 A017Y 
                 G078Y 
                 A153M 
                 F204K 
                 A250K 
                 I301Q 
                 E348Q 
                 F394Q 
               
               
                 W018C 
                 I079A 
                 A153P 
                 F204Q 
                 A250L 
                 I301R 
                 E348R 
                 F394R 
               
               
                 W018D 
                 I079D 
                 A153R 
                 F204R 
                 A250M 
                 I301S 
                 E348T 
                 F394S 
               
               
                 W018F 
                 I079F 
                 A153T 
                 F204S 
                 A250N 
                 I301W 
                 E348V 
                 F394T 
               
               
                 W018G 
                 I079G 
                 A153V 
                 F204T 
                 A250P 
                 I301Y 
                 E348W 
                 F394V 
               
               
                 W018H 
                 I079H 
                 K154D 
                 V206C 
                 A250Q 
                 V302C 
                 E348Y 
                 S395C 
               
               
                 W018I 
                 I079K 
                 K154E 
                 V206D 
                 A250R 
                 V302D 
                 Q349D 
                 S395L 
               
               
                 W018L 
                 I079N 
                 K154G 
                 V206F 
                 A250S 
                 V302E 
                 Q349F 
                 S395M 
               
               
                 W018M 
                 I079P 
                 K154P 
                 V206G 
                 A250T 
                 V302F 
                 Q349G 
                 S395P 
               
               
                 W018P 
                 I079S 
                 K154S 
                 V206P 
                 A250V 
                 V302G 
                 Q349P 
                 E396C 
               
               
                 W018Q 
                 I079W 
                 K154W 
                 V206Y 
                 A250W 
                 V302H 
                 Q349V 
                 E396F 
               
               
                 W018S 
                 I079Y 
                 K154Y 
                 E207A 
                 I251D 
                 V302L 
                 Q349W 
                 E396G 
               
               
                 W018T 
                 P080A 
                 Q155P 
                 E207F 
                 I251F 
                 V302M 
                 Q349Y 
                 E396I 
               
               
                 W018V 
                 P080D 
                 Q155Y 
                 E207G 
                 I251G 
                 V302P 
                 G350A 
                 E396P 
               
               
                 W018Y 
                 P080E 
                 E156P 
                 E207M 
                 I251H 
                 V302R 
                 G350D 
                 E396Y 
               
               
                 N019A 
                 P080F 
                 F157A 
                 E207P 
                 I251K 
                 V302S 
                 G350E 
                 K397A 
               
               
                 N019C 
                 P080G 
                 F157C 
                 E207Q 
                 I251P 
                 V302T 
                 G350F 
                 K397C 
               
               
                 N019F 
                 P080I 
                 F157D 
                 E207R 
                 I251S 
                 V302Y 
                 G350H 
                 K397E 
               
               
                 N019G 
                 P080K 
                 F157E 
                 E207S 
                 I251T 
                 I303A 
                 G350K 
                 K397F 
               
               
                 N019H 
                 P080L 
                 F157G 
                 E207T 
                 I251W 
                 I303C 
                 G350L 
                 K397G 
               
               
                 N019I 
                 P080M 
                 F157H 
                 E207V 
                 R252A 
                 I303D 
                 G350M 
                 K397I 
               
               
                 N019L 
                 P080N 
                 F157I 
                 E207W 
                 R252D 
                 I303E 
                 G350N 
                 K397L 
               
               
                 N019M 
                 P080R 
                 F157K 
                 I208D 
                 R252E 
                 I303F 
                 G350P 
                 K397M 
               
               
                 N019P 
                 P080S 
                 F157L 
                 I208G 
                 R252F 
                 I303G 
                 G350R 
                 K397P 
               
               
                 N019Q 
                 P080T 
                 F157M 
                 I208P 
                 R252G 
                 I303K 
                 G350S 
                 K397Q 
               
               
                 N019R 
                 P080V 
                 F157P 
                 I208W 
                 R252H 
                 I303L 
                 G350T 
                 K397T 
               
               
                 N019S 
                 P080Y 
                 F157Q 
                 K209C 
                 R252I 
                 I303M 
                 G350V 
                 K397V 
               
               
                 N019V 
                 Q081A 
                 F157R 
                 K209P 
                 R252K 
                 I303R 
                 G350Y 
                 F398A 
               
               
                 N019W 
                 Q081C 
                 F157S 
                 R210A 
                 R252L 
                 I303W 
                 V351C 
                 F398C 
               
               
                 N019Y 
                 Q081E 
                 F157T 
                 R210C 
                 R252N 
                 I303Y 
                 V351D 
                 F398E 
               
               
                 A020D 
                 Q081G 
                 F157V 
                 R210D 
                 R252P 
                 W304A 
                 V351E 
                 F398G 
               
               
                 A020E 
                 Q081H 
                 E158D 
                 R210E 
                 R252S 
                 W304C 
                 V351F 
                 F398H 
               
               
                 A020F 
                 Q081L 
                 E158K 
                 R210G 
                 R252T 
                 W304D 
                 V351H 
                 F398I 
               
               
                 A020H 
                 Q081N 
                 E158P 
                 R210K 
                 R252Y 
                 W304G 
                 V351N 
                 F398L 
               
               
                 A020K 
                 Q081P 
                 E158R 
                 R210M 
                 V253A 
                 W304I 
                 V351R 
                 F398N 
               
               
                 A020L 
                 Q081S 
                 E158Y 
                 R210N 
                 V253D 
                 W304M 
                 V351W 
                 F398P 
               
               
                 A020N 
                 Q081V 
                 K159W 
                 R210P 
                 V253E 
                 W304N 
                 V351Y 
                 F398R 
               
               
                 A020P 
                 Q081W 
                 K159Y 
                 R210S 
                 V253G 
                 W304P 
                 C352A 
                 F398S 
               
               
                 A020R 
                 Q081Y 
                 G161W 
                 R210T 
                 V253H 
                 W304Q 
                 C352D 
                 F398T 
               
               
                 A020T 
                 K082W 
                 D163C 
                 R210V 
                 V253L 
                 W304S 
                 C352E 
                 F398V 
               
               
                 A020V 
                 K082Y 
                 D163P 
                 R210W 
                 V253M 
                 W304T 
                 C352F 
                 F398W 
               
               
                 A020Y 
                 I083E 
                 F164A 
                 R210Y 
                 V253N 
                 W304V 
                 C352G 
                 F398Y 
               
               
                 P021A 
                 I083K 
                 F164C 
                 N211C 
                 V253Q 
                 W304Y 
                 C352K 
                 Y399D 
               
               
                 P021C 
                 S084Y 
                 F164D 
                 N211F 
                 V253R 
                 G305L 
                 C352M 
                 Y399P 
               
               
                 P021D 
                 L085A 
                 F164E 
                 N211G 
                 V253S 
                 G305P 
                 C352P 
                 C400A 
               
               
                 P021E 
                 L085C 
                 F164G 
                 N211H 
                 V253W 
                 G305Q 
                 C352Q 
                 C400D 
               
               
                 P021G 
                 L085D 
                 F164H 
                 N211I 
                 S254C 
                 G305R 
                 C352R 
                 C400E 
               
               
                 P021H 
                 L085E 
                 F164N 
                 N211K 
                 S254D 
                 G305S 
                 C352S 
                 C400F 
               
               
                 P021I 
                 L085F 
                 F164P 
                 N211M 
                 S254E 
                 G305T 
                 C352T 
                 C400G 
               
               
                 P021L 
                 L085G 
                 F164Q 
                 N211P 
                 S254G 
                 G305V 
                 C352V 
                 C400I 
               
               
                 P021M 
                 L085H 
                 F164R 
                 N211R 
                 S254I 
                 G305Y 
                 C352W 
                 C400L 
               
               
                 P021R 
                 L085N 
                 L165C 
                 N211S 
                 S254K 
                 T306A 
                 C352Y 
                 C400M 
               
               
                 P021S 
                 L085Q 
                 L165H 
                 N211T 
                 S254L 
                 T306C 
                 I353C 
                 C400P 
               
               
                 P021T 
                 L085S 
                 L165P 
                 N211V 
                 S254P 
                 T306H 
                 I353F 
                 C400Q 
               
               
                 P021V 
                 L085T 
                 L165T 
                 N211W 
                 S254Q 
                 T306I 
                 I353G 
                 C400R 
               
               
                 P021W 
                 Q086C 
                 V166D 
                 D212A 
                 S254R 
                 T306L 
                 I353H 
                 C400S 
               
               
                 S022C 
                 Q086P 
                 E167V 
                 D212G 
                 S254T 
                 T306V 
                 I353K 
                 C400T 
               
               
                 S022E 
                 D087P 
                 T168A 
                 D212H 
                 S254V 
                 T306W 
                 I353L 
                 C400V 
               
               
                 S022G 
                 H088A 
                 T168C 
                 D212I 
                 S254W 
                 T306Y 
                 I353M 
                 C400Y 
               
               
                 S022K 
                 H088C 
                 T168D 
                 D212K 
                 S254Y 
                 L307C 
                 I353Q 
                 S401C 
               
               
                 S022P 
                 H088E 
                 T168E 
                 D212L 
                 K255C 
                   
                 I353R 
                 S401F 
               
               
                 E023A 
                 H088F 
                 T168F 
                 D212M 
                 K255D 
                 L307I 
                 I353S 
                 S401H 
               
               
                 E023F 
                 H088G 
                 T168G 
                 D212P 
                 K255L 
                 L307P 
                 I353W 
                 S401K 
               
               
                 E023L 
                 H088I 
                 T168K 
                 D212V 
                 K255P 
                 S308C 
                 R354C 
                 S401R 
               
               
                 E023M 
                 H088K 
                 T168L 
                 D212W 
                 K255V 
                 S308F 
                 R354D 
                 S401W 
               
               
                 E023N 
                 H088L 
                 T168P 
                 D213P 
                 K255W 
                 S308L 
                 R354E 
                 S401Y 
               
               
                 E023P 
                 H088M 
                 T168R 
                 D213S  
                 I256C 
                 S308M 
                 R354G 
                 C402A 
               
               
                 E023R 
                 H088P 
                 T168S 
                 L214A 
                 I256D 
                 S308V 
                 R354H 
                 C402D 
               
               
                 E023S 
                 H088R 
                 T168V 
                 L214C 
                 I256E 
                 S308W 
                 R354I 
                 C402E 
               
               
                 E023T 
                 H088S 
                 T168W 
                 L214D 
                 I256G 
                 S308Y 
                 R354K 
                 C402F 
               
               
                 E023V 
                 H088T 
                 T168Y 
                 L214E 
                 I256P 
                 M310C 
                 R354L 
                 C402L 
               
               
                 C025D 
                 H088V 
                 I169A 
                 L214G 
                 P257D 
                 M310E 
                 R354M 
                 C402M 
               
               
                 C025E 
                 H088Y 
                 I169D 
                 L214H 
                 D258L 
                 M310F 
                 R354P 
                 C402P 
               
               
                 C025F 
                 L089A 
                 I169F 
                 L214K 
                 D258P 
                 M310K 
                 R354Q 
                 C402Q 
               
               
                 C025G 
                 L089D 
                 I169G 
                 L214N 
                 D258V 
                 M310L 
                 R354S 
                 C402R 
               
               
                 C025H 
                 L089E 
                 I169H 
                 L214P 
                 D258W 
                 R311C 
                 R354V 
                 C402S 
               
               
                 C025I 
                 L089G 
                 I169K 
                 L214R 
                 K260C 
                 R311E 
                 R354W 
                 C402T 
               
               
                 C025K 
                 L089Q 
                 I169N 
                 L214S 
                 K260P 
                 R311F 
                 R354Y 
                 C402V 
               
               
                 C025L 
                 L089S 
                 I169P 
                 L214T 
                 S261P 
                 R311I 
                 K355D 
                 C402W 
               
               
                 C025N 
                 L089T 
                 I169Q 
                 L214Y 
                 P262A 
                 R311L 
                 K355F 
                 C402Y 
               
               
                 C025P 
                 L089W 
                 I169S 
                 S215C 
                 P262D 
                 R311P 
                 K355G 
                 Y403A 
               
               
                 C025R 
                 L089Y 
                 I169T 
                 S215P 
                 P262E 
                 R311V 
                 K355H 
                 Y403C 
               
               
                 C025S 
                 D090C 
                 I169Y 
                 W216D 
                 P262F 
                 R311W 
                 K355L 
                 Y403E 
               
               
                 C025T 
                 D090G 
                 K170C 
                 W216E 
                 P262G 
                 S312C 
                 K355M 
                 Y403G 
               
               
                 C025V 
                 K091D 
                 K170D 
                 W216G 
                 P262H 
                 S312E 
                 K355N 
                 Y403H 
               
               
                 C025Y 
                 K091E 
                 K170E 
                 W216H 
                 P262I 
                 S312M 
                 K355P 
                 Y403K 
               
               
                 G027C 
                 K091F 
                 K170G 
                 W216I 
                 P262K 
                 S312V 
                 K355Q 
                 Y403L 
               
               
                 L033C 
                 K091G 
                 K170M 
                 W216K 
                 P262Q 
                 S312W 
                 K355R 
                 Y403M 
               
               
                 L033D 
                 K091H 
                 K170P 
                 W216L 
                 P262R 
                 M313C 
                 K355S 
                 Y403N 
               
               
                 L033H 
                 K091I 
                 K170W 
                 W216M 
                 P262S 
                 K314C 
                 K355T 
                 Y403P 
               
               
                 L033N 
                 K091L 
                 K170Y 
                 W216N 
                 P262T 
                 K314L 
                 K355V 
                 Y403Q 
               
               
                 L033V 
                 K091N 
                 L171C 
                 W216P 
                 P262V 
                 K314W 
                 K355W 
                 Y403R 
               
               
                 L033Y 
                 K091T 
                 L171D 
                 W216Q 
                 P262W 
                 S315C 
                 K355Y 
                 Y403T 
               
               
                 D034I 
                 A092E 
                 L171H 
                 W216R 
                 P262Y 
                 S315I 
                 N356C 
                 S404C 
               
               
                 D034L 
                 A092F 
                 L171M 
                 W216T 
                 L263E 
                 S315V 
                 N356G 
                 S404D 
               
               
                 D034N 
                 A092H 
                 L171N 
                 W216V 
                 L263F 
                 C316E 
                 N356K 
                 S404F 
               
               
                 D034S 
                 A092K 
                 L171R 
                 L217A 
                 L263P 
                 C316G 
                 N356L 
                 S404G 
               
               
                 D034T 
                 A092P 
                 L171S 
                 L217C 
                 L263Q 
                 C316I 
                 N356P 
                 S404H 
               
               
                 D034V 
                 A092Q 
                 L171W 
                 L217G 
                 L263W 
                 C316K 
                 N356R 
                 S404L 
               
               
                 M035A 
                 A092R 
                 L171Y 
                 L217H 
                 P264D 
                 C316L 
                 N356T 
                 S404M 
               
               
                 M035D 
                 A092W 
                 G172D 
                 L217P 
                 P264E 
                 C316M 
                 N356V 
                 S404N 
               
               
                 M035G 
                 A092Y 
                 G172E 
                 L217Q 
                 P264F 
                 C316P 
                 N356W 
                 S404R 
               
               
                 M035P 
                 K094G 
                 G172I 
                 L217 S  
                 P264G 
                 C316R 
                 W357D 
                 S404V 
               
               
                 M035R 
                 K094P 
                 G172L 
                 L217T 
                 P264L 
                 C316S 
                 W357E 
                 S404W 
               
               
                 M035S 
                 D095A 
                 G172P 
                 L217V 
                 P264M 
                 C316T 
                 W357F 
                 S404Y 
               
               
                 S036C 
                 D095C 
                 G172Q 
                 L217W 
                 P264R 
                 C316V 
                 W357G 
                 T405C 
               
               
                 S036F 
                 D095E 
                 G172T 
                 W218A 
                 P264T 
                 C316W 
                 W357L 
                 T405I 
               
               
                 S036V 
                 D095F 
                 G172V 
                 W218I 
                 P264V 
                 C316Y 
                 W357M 
                 T405V 
               
               
                 S036W 
                 D095G 
                 G172W 
                 W218K 
                 P264W 
                 L317G 
                 W357Q 
                 L406P 
               
               
                 S036Y 
                 D095H 
                 G172Y 
                 W218L 
                 P264Y 
                 L317P 
                 W357R 
                 L406R 
               
               
                 L037C 
                 D095K 
                 K173D 
                 W218P 
                 V265A 
                 L318C 
                 N358E 
                 C408A 
               
               
                 L037E 
                 D095L 
                 K173E 
                 W218S 
                 V265D 
                 L318P 
                 N358H 
                 C408E 
               
               
                 L037G 
                 D095M 
                 K173G 
                 W218V 
                 V265F 
                 L318W 
                 N358I 
                 C408F 
               
               
                 L037N 
                 D095P 
                 K173H 
                 N219P 
                 V265G 
                 L319C 
                 N358K 
                 C408G 
               
               
                 L037S 
                 D095Q 
                 K173I 
                 E220G 
                 V265H 
                 L319E 
                 N358P 
                 C408I 
               
               
                 F038E 
                 D095S 
                 K173L 
                 E220K 
                 V265K 
                 L319F 
                 N358Q 
                 C408K 
               
               
                 F038G 
                 D095V 
                 K173M 
                 E220N 
                 V265L 
                 L319G 
                 N358R 
                 C408L 
               
               
                 F038K 
                 D095W 
                 K173P 
                 E220P 
                 V265M 
                 L319H 
                 N358W 
                 C408P 
               
               
                 F038L 
                 D095Y 
                 K173S 
                 E220R 
                 V265N 
                 L319I 
                 S359A 
                 C408R 
               
               
                 F038N 
                 I096A 
                 K173V 
                 E220W 
                 V265Q 
                 L319K 
                 S359F 
                 C408S 
               
               
                 F038Q 
                 I096C 
                 K173W 
                 S221D 
                 V265R 
                 L319M 
                 S359G 
                 C408T 
               
               
                 F038R 
                 I096G 
                 K173Y 
                 S221E 
                 V265S 
                 L319P 
                 S359L 
                 C408V 
               
               
                 F038T 
                 I096H 
                 L174P 
                 S221H 
                 F266A 
                 L319Q 
                 S359P 
                 C408W 
               
               
                 F038W 
                 I096P 
                 L175C 
                 S221K 
                 F266C 
                 L319R 
                 S359W 
                 C408Y 
               
               
                 S039C 
                 I096R 
                 L175D 
                 S221P 
                 F266G 
                 L319S 
                 S360A 
                 E410W 
               
               
                 S039D 
                 I096S 
                 L175G 
                 S221R 
                 F266H 
                 L319V 
                 S360C 
                 K411D 
               
               
                 S039F 
                 I096T 
                 L175K 
                 T222P 
                 F266M 
                 L319W 
                 S360E 
                 K411E 
               
               
                 S039W 
                 I096W 
                 L175P 
                 T222Y 
                 F266P 
                 L319Y 
                 S360F 
                 K411F 
               
               
                 F040A 
                 F098P 
                 L175R 
                 A223C 
                 F266Q 
                 D320C 
                 S360G 
                 K411G 
               
               
                 F040D 
                 Y099C 
                 L175S 
                 A223D 
                 F266R 
                 D320P 
                 S360I 
                 A412E 
               
               
                 F040E 
                 Y099E 
                 R176A 
                 A223E 
                 F266S 
                 D320V 
                 S360K 
                 A412H 
               
               
                 F040G 
                 Y099G 
                 R176C 
                 A223G 
                 F266T 
                 N321E 
                 S360L 
                 D413H 
               
               
                 F040K 
                 Y099I 
                 R176E 
                 A223H 
                 F266V 
                 N321M 
                 S360M 
                 D413I 
               
               
                 F040N 
                 Y099N 
                 R176F 
                 A223K 
                 F266W 
                 N321P 
                 S360P 
                 D413K 
               
               
                 F040R 
                 Y099P 
                 R176G 
                 A223L 
                 A267D 
                 Y322C 
                 S360Q 
                 D413L 
               
               
                 F040S 
                 Y099V 
                 R176H 
                 A223P 
                 A267G 
                 Y322D 
                 S360R 
                 D413P 
               
               
                 F040T 
                 Y099W 
                 R176I 
                 A223Q 
                 A267H 
                 Y322E 
                 S360V 
                 V414A 
               
               
                 F040V 
                 M100C 
                 R176P 
                 A223R 
                 A267I 
                 Y322G 
                 D361A 
                 V414D 
               
               
                 I041Q 
                 M100E 
                 R176Q 
                 A223S  
                 A267K 
                 Y322I 
                 D361C 
                 V414E 
               
               
                 G042D 
                 M100F 
                 R176S 
                 A223T 
                 A267N 
                 Y322L 
                 D361E 
                 V414G 
               
               
                 G042E 
                 M100G 
                 R176T 
                 A223V 
                 A267R 
                 Y322N 
                 D361G 
                 V414H 
               
               
                 G042H 
                 M100N 
                 R176V 
                 A223W 
                 A267S 
                 Y322P 
                 D361M 
                 V414K 
               
               
                 G042I 
                 M100P 
                 R176W 
                 A223Y 
                 A267W 
                 Y322R 
                 D361N 
                 V414R 
               
               
                 G042K 
                 M100R 
                 P177A 
                 L224A 
                 Y268A 
                 Y322S 
                 D361P 
                 V414S 
               
               
                 G042L 
                 M100S 
                 P177C 
                 L224D 
                 Y268C 
                 Y322T 
                 D361Q 
                 V414T 
               
               
                 G042M 
                 M100T 
                 P177D 
                 L224E 
                 Y268F 
                 Y322V 
                 D361R 
                 K415C 
               
               
                 G042P 
                 M100W 
                 P177F 
                 L224F 
                 Y268G 
                 Y322W 
                 D361S 
                 K415D 
               
               
                 G042Q 
                 M100Y 
                 P177G 
                 L224G 
                 Y268H 
                 M323A 
                 D361V 
                 K415E 
               
               
                 G042R 
                 P101A 
                 P177H 
                 L224M 
                 Y268K 
                 M323C 
                 D361W 
                 K415P 
               
               
                 G042S 
                 P101C 
                 P177L 
                 L224P 
                 Y268L 
                 M323E 
                 Y362A 
                 D416C 
               
               
                 G042T 
                 P101F 
                 P177M 
                 L224Q 
                 Y268N 
                 M323G 
                 Y362C 
                 D416S 
               
               
                 G042V 
                 P101H 
                 P177Q 
                 L224R 
                 Y268P 
                 M323H 
                 Y362E 
                 T417A 
               
               
                 S043A 
                 P101I 
                 P177R 
                 L224S 
                 Y268Q 
                 M323K 
                 Y362G 
                 T417D 
               
               
                 S043E 
                 P101K 
                 P177S 
                 L224T 
                 Y268S 
                 M323N 
                 Y362H 
                 T417E 
               
               
                 S043F 
                 P101L 
                 P177T 
                 L224W 
                 Y268T 
                 M323R 
                 Y362K 
                 T417F 
               
               
                 S043G 
                 P101M 
                 P177V 
                 L224Y 
                 Y268V 
                 M323S 
                 Y362L 
                 T417G 
               
               
                 S043I 
                 P101N 
                 P177W 
                 Y225A 
                 Y268W 
                 M323T 
                 Y362M 
                 T417H 
               
               
                 S043K 
                 P101Q 
                 N178E 
                 Y225D 
                 T269E 
                 M323V 
                 Y362N 
                 T417K 
               
               
                 S043L 
                 P101R 
                 N178I 
                 Y225E 
                 T269K 
                 E324C 
                 Y362P 
                 T417M 
               
               
                 S043Q 
                 P101S 
                 N178L 
                 Y225G 
                 T269L 
                 E324F 
                 Y362R 
                 T417P 
               
               
                 S043R 
                 P101T 
                 N178V 
                 Y225H 
                 T269M 
                 E324P 
                 Y362S 
                 T417Q 
               
               
                 S043V 
                 V102P 
                 N178W 
                 Y225K 
                 T269N 
                 E324V 
                 Y362T 
                 T417R 
               
               
                 P044A 
                 D 103A 
                 N178Y 
                 Y225P 
                 T269P 
                 E324W 
                 Y362V 
                 A419D 
               
               
                 P044C 
                 D103E 
                 H179W 
                 Y225Q 
                 T269Q 
                 E324Y 
                 Y362W 
                 A419P 
               
               
                 P044F 
                 D103F 
                 L180A 
                 Y225R 
                 T269R 
                 T325C 
                 L363A 
                 V420A 
               
               
                 P044G 
                 D103G 
                 L180C 
                 Y225T 
                 R270A 
                 T325R 
                 L363C 
                 V420D 
               
               
                 P044H 
                 D103H 
                 L180E 
                 Y225V 
                 R270C 
                 I326E 
                 L363D 
                 V420F 
               
               
                 P044I 
                 D103I 
                 L180P 
                 Y225W 
                 R270E 
                 I326G 
                 L363E 
                 V420G 
               
               
                 P044L 
                 D103L 
                 L180R 
                 P226A 
                 R270F 
                 I326H 
                 L363F 
                 V420H 
               
               
                 P044N 
                 D103Q 
                 L180S 
                 P226C 
                 R270G 
                 I326N 
                 L363G 
                 V420K 
               
               
                 P044Q 
                 D103R 
                 W181A 
                 P226D 
                 R270H 
                 I326W 
                 L363H 
                 V420L 
               
               
                 P044R 
                 D103T 
                 W181C 
                 P226E 
                 R270I 
                 L327A 
                 L363I 
                 V420N 
               
               
                 P044S 
                 D103V 
                 W181D 
                 P226F 
                 R270P 
                 L327E 
                 L363P 
                 V420R 
               
               
                 P044T 
                 D103W 
                 W181E 
                 P226G 
                 R270Y 
                 L327F 
                 L363Q 
                 V420S 
               
               
                 P044W 
                 D103Y 
                 W181F 
                 P226L 
                 I271A 
                 L327G 
                 L363R 
                 V420T 
               
               
                 P044Y 
                 N104F 
                 W181H 
                 P226N 
                 I271D 
                 L327H 
                 L363S 
                 V420W 
               
               
                 R045A 
                 N104P 
                 W181I 
                 P226Q 
                 I271E 
                 L327N 
                 L363T 
                 V420Y 
               
               
                 R045D 
                 N104W 
                 W181K 
                 P226R 
                 I271H 
                 L327Q 
                 L363V 
                 V422C 
               
               
                 R045F 
                 L105C 
                 W181L 
                 P226S 
                 I271K 
                 L327R 
                 L363W 
                 V422D 
               
               
                 R045G 
                 L105M 
                 W181R 
                 P226T 
                 I271T 
                 L327S 
                 H364A 
                 V422G 
               
               
                 R045P 
                 L105N 
                 W181S 
                 P226V 
                 I271W 
                 L327T 
                 H364C 
                 V422H 
               
               
                 R045W 
                 G106A 
                 W181V 
                 P226W 
                 V272A 
                 L327V 
                 H364D 
                 V422L 
               
               
                 I046P 
                 G106C 
                 G182A 
                 P226Y 
                 V272H 
                 L327W 
                 H364E 
                 V422M 
               
               
                 I046W 
                 G106D 
                 G182C 
                 S227A 
                 V272L 
                 L327Y 
                 H364F 
                 V422N 
               
               
                 N047V 
                 G106F 
                 G182D 
                 S227F 
                 V272N 
                 P329C 
                 H364G 
                 V422Q 
               
               
                 A048P 
                 G106H 
                 G182E 
                 S227G 
                 V272P 
                 P329F 
                 H364K 
                 V422R 
               
               
                 T049C 
                 G106L 
                 G182H 
                 S227H 
                 V272W 
                 P329G 
                 H364L 
                 V422S 
               
               
                 T049D 
                 G106M 
                 G182N 
                 S227I 
                 F273A 
                 P329H 
                 H364M 
                 V422Y 
               
               
                 T049G 
                 G106N 
                 G182P 
                 S227K 
                 F273C 
                 P329I 
                 H364P 
                 C423A 
               
               
                 T049H 
                 G106P 
                 G182Q 
                 S227L 
                 F273D 
                 P329K 
                 H364R 
                 C423D 
               
               
                 T049P 
                 G106S 
                 G182R 
                 S227M 
                 F273G 
                 P329L 
                 H364S 
                 C423E 
               
               
                   
                 G106W 
                 G182S 
                 S227P 
                 F273I 
                 P329N 
                 H364T 
                 C423F 
               
               
                 Q051C 
                 G106Y 
                 G182T 
                 S227Q 
                 F273L 
                 P329Q 
                 H364V 
                 C423G 
               
               
                 Q051F 
                 M107A 
                 G182V 
                 S227R 
                 F273P 
                 P329R 
                 H364Y 
                 C423H 
               
               
                 Q051I 
                 M107C 
                 G182Y 
                 S227T 
                 F273Q 
                 P329S 
                 L365A 
                 C423L 
               
               
                 Q051M 
                 M107H 
                 Y183C 
                 S227V 
                 F273S 
                 P329T 
                 L365C 
                 C423M 
               
               
                 Q051P 
                 M107K 
                 Y183D 
                 S227W 
                 F273V 
                 P329V 
                 L365D 
                 C423P 
               
               
                 Q051T 
                 M107P 
                 Y183E 
                 S227Y 
                 F273W 
                 P329W 
                 L365E 
                 C423Q 
               
               
                 Q051W 
                 M107Q 
                 Y183G 
                 I228A 
                 T274C 
                 P329Y 
                 L365G 
                 C423R 
               
               
                 Q051Y 
                 M107S 
                 Y183I 
                 I228E 
                 T274E 
                 Y330A 
                 L365M 
                 C423S 
               
               
                 G052C 
                 M107V 
                 Y183K 
                 I228F 
                 T274G 
                 Y330C 
                 L365N 
                 C423T 
               
               
                 G052E 
                 M107W 
                 Y183N 
                 I228G 
                 T274H 
                 Y330D 
                 L365P 
                 C423V 
               
               
                 G052F 
                 A108D 
                 Y183P 
                 I228H 
                 T274N 
                 Y330E 
                 L365Q 
                 C423W 
               
               
                 G052W 
                 A108E 
                 Y183Q 
                 I228L 
                 T274Q 
                 Y330G 
                 L365R 
                 I424A 
               
               
                 G052Y 
                 A108F 
                 Y183R 
                 I228M 
                 T274W 
                 Y330I 
                 L365S 
                 I424C 
               
               
                 V053A 
                 A108K 
                 Y183S 
                 I228N 
                 T274Y 
                 Y330L 
                 L365T 
                 I424E 
               
               
                 V053C 
                 A108L 
                 Y183V 
                 I228P 
                 D275A 
                 Y330M 
                 L365W 
                 I424G 
               
               
                 V053D 
                 A108M 
                 Y184A 
                 I228R 
                 D275F 
                 Y330N 
                 L365Y 
                 I424H 
               
               
                 V053E 
                 A108P 
                 Y184C 
                 I228S 
                 D275G 
                 Y330P 
                 N366A 
                 I424N 
               
               
                 V053G 
                 A108Q 
                 Y184D 
                 I228T 
                 D275I 
                 Y330R 
                 N366C 
                 I424Q 
               
               
                 V053H 
                 A108T 
                 Y184E 
                 I228W 
                 D275K 
                 Y330S 
                 N366E 
                 I424R 
               
               
                 V053L 
                 A108V 
                 Y184F 
                 Y229E 
                 D275L 
                 Y330V 
                 N366F 
                 I424S 
               
               
                 V053N 
                 A108Y 
                 Y184G 
                 Y229F 
                 D275M 
                 Y330W 
                 N366G 
                 I424W 
               
               
                 V053P 
                 V109C 
                 Y184H 
                 Y229G 
                 D275Q 
                 I331A 
                 N366K 
                 I424Y 
               
               
                 V053Q 
                 V109D 
                 Y184K 
                 Y229K 
                 D275T 
                 I331C 
                 N366M 
                 A425E 
               
               
                 V053R 
                 V109E 
                 Y184L 
                 Y229L 
                 D275V 
                 I331D 
                 N366P 
                 A425L 
               
               
                 V053S 
                 V109L 
                 Y184M 
                 Y229P 
                 D275W 
                 I331E 
                 N366Q 
                 A425P 
               
               
                 V053T 
                 V109M 
                 Y184P 
                 Y229Q 
                 Q276F 
                 I331F  
                 N366R 
                 A425W 
               
               
                 V053W 
                 V109R 
                 Y184R 
                 Y229T 
                 Q276P 
                 I331H 
                 N366T 
                 A425Y 
               
               
                 V053Y 
                 V109T 
                 Y184S 
                 Y229V 
                 Q276W 
                 I331K 
                 N366W 
                 D426C 
               
               
                 T054D 
                 V109W 
                 Y184V 
                 Y229W 
                 L278M 
                 I331Q 
                 P367E 
                 D426F 
               
               
                 T054E 
                 I110F 
                 L185A 
                 L230A 
                 L278P 
                 I331R 
                 P367F 
                 D426M 
               
               
                 T054G 
                 I110K 
                 L185D 
                 L230E 
                 K279A 
                 I331S 
                 P367I 
                 D426R 
               
               
                 T054P 
                 I110L 
                 L185E 
                 L230G 
                 K279C 
                 I331T  
                 P367L 
                 G427A 
               
               
                 T054R 
                 I110M 
                 L185F 
                 L230H 
                 K279F 
                 I331W 
                 P367M 
                 G427C 
               
               
                 T054Y 
                 I110P 
                 L185G 
                 L230K 
                 K279G 
                 I331Y 
                 P367Q 
                 G427F 
               
               
                 I055A 
                 I110W 
                 L185I 
                 L230M 
                 K279L 
                 I332A 
                 P367V 
                 G427L 
               
               
                 I055D 
                 D111H 
                 L185K 
                 L230N 
                 K279W 
                 I332C 
                 D368C 
                 G427P 
               
               
                 I055G 
                 D111I 
                 L185P 
                 L230P 
                 K279Y 
                 I332D 
                 D368P 
                   
               
               
                 I055H 
                 D111Q 
                 L185R 
                 L230R 
                 F280D 
                 I332E 
                 D368W 
                 G427V 
               
               
                 I055N 
                 W112C 
                 L185S 
                 L230S 
                 F280I 
                 I332F 
                 N369C 
                 G427W 
               
               
                 I055P 
                 W112E 
                 L185T 
                 L230T 
                 F280L 
                 I332G 
                 N369E 
                 G427Y 
               
               
                 I055Q 
                 W112G 
                 L185V 
                 L230V 
                 F280M 
                 I332H 
                 N369F 
                 V428A 
               
               
                 I055R 
                 W112H 
                 L185W 
                 L230W 
                 F280N 
                 I332K 
                 N369I 
                 V428C 
               
               
                 I055T 
                 W112L 
                 L185Y 
                 L230Y 
                 F280R 
                 I332L 
                 N369K 
                 V428D 
               
               
                 I055V 
                 W112N 
                 F186A 
                 N231A 
                 F280S 
                 I332N 
                 N369L 
                 V428E 
               
               
                 I055Y 
                 W112P 
                 F186D 
                 N231C 
                 F280T 
                 I332P 
                 N369P 
                 V428G 
               
               
                 F056A 
                 W112S 
                 F186G 
                 N231D 
                 F280V 
                 I332R 
                 N369Q 
                 V428H 
               
               
                 F056C 
                 E113R 
                 F186H 
                 N231F 
                 F280W 
                 I332S 
                 N369V 
                 V428N 
               
               
                 F056E 
                 E113V 
                 F186I 
                 N231G 
                 L281A 
                 I332T 
                 N369W 
                 V428R 
               
               
                 F056G 
                 E114I 
                 F186K 
                 N231H 
                 L281D 
                 I332Y 
                 F370A 
                 V428S 
               
               
                 F056H 
                 E114L 
                 F186L 
                 N231I 
                 L281G 
                 N333G 
                 F370D 
                 V428Y 
               
               
                 F056I 
                 E114P 
                 F186N 
                 N231K 
                 L281H 
                 N333H 
                 F370E 
                 C429A 
               
               
                 F056K 
                 E114T 
                 F186P 
                 N231L 
                 L281I 
                 N333I 
                 F370G 
                 C429D 
               
               
                 F056L 
                 E114V 
                 F186Q 
                 N231P 
                 L281K 
                 N333K 
                 F370H 
                 C429K 
               
               
                 F056P 
                 W115A 
                 F186R 
                 N231Q 
                 L281N 
                 N333P 
                 F370K 
                 C429L 
               
               
                 F056R 
                 W115C 
                 F186S 
                 N231R 
                 L281P 
                 N333R 
                 F370L 
                 C429N 
               
               
                 F056S 
                 W115D 
                 F186V 
                 N231S 
                 L281Q 
                 N333S  
                 F370N 
                 C429P 
               
               
                 F056T 
                 W115F 
                 F186W 
                 N231V 
                 L281R 
                 N333T 
                 F370P 
                 C429S 
               
               
                 F056V 
                 W115G 
                 P187A 
                 T232C 
                 L281S 
                 N333W 
                 F370Q 
                 C429T 
               
               
                 F056W 
                 W115H 
                 P187F 
                 T232G 
                 L281V 
                 N333Y 
                 F370R 
                 C429V 
               
               
                 Y057A 
                 W115I 
                 P187G 
                 T232H 
                 L281W 
                 V334A 
                 F370S 
                 C429W 
               
               
                 Y057D 
                 W115K 
                 P187H 
                 T232K 
                 S282F 
                 V334C 
                 F370V 
                 C429Y 
               
               
                 Y057F 
                 W115L 
                 P187I 
                 T232L 
                 S282L 
                 V334D 
                 F370Y 
                 I430A 
               
               
                 Y057G 
                 W115M 
                 P187L 
                 T232N 
                 S282V 
                 V334E 
                 A371P 
                 I430D 
               
               
                 Y057I 
                 W115R 
                 P187M 
                 T232P 
                 S282W 
                 V334G 
                 A371W 
                 I430E 
               
               
                 Y057L 
                 W115S 
                 P187N 
                 T232Q 
                 S282Y 
                 V334M 
                 I372A 
                 I430L 
               
               
                 Y057M 
                 W115V 
                 P187Q 
                 T232V 
                 Q283A 
                 V334N 
                 I372D 
                 I430M 
               
               
                 Y057P 
                 W115Y 
                 P187R 
                 T232Y 
                 Q283C 
                 V334R 
                 I372E 
                 I430N 
               
               
                 Y057Q 
                 R116A 
                 P187S 
                 Q233D 
                 Q283D 
                 V334S 
                 I372F 
                 I430S 
               
               
                 Y057R 
                 R116C 
                 P187T 
                 Q233I 
                 Q283F 
                 T335F 
                 I372G 
                 I430T 
               
               
                 Y057V 
                 R116D 
                 P187V 
                 Q233P 
                 Q283W 
                 T335G 
                 I372H 
                 I430V 
               
               
                 Y057W 
                 R116E 
                 P187W 
                 Q233S 
                 D284C 
                 T335H 
                 I372K 
                 D431P 
               
               
                 V058A 
                 R116G 
                 P187Y 
                 Q233T 
                 D284I 
                 T335I 
                 I372L 
                 A432C 
               
               
                 D059A 
                 R116H 
                 D188A 
                 Q234A 
                 D284P 
                 T335K 
                 I372N 
                 A432F 
               
               
                 D059E 
                 R1161 
                 D188C 
                 Q234D 
                 E285K 
                 T335L 
                 I372P 
                 A432I 
               
               
                 D0591 
                 R116L 
                 D188F 
                 Q234E 
                 E285P 
                 T335P 
                 I372R 
                 A432K 
               
               
                 D059L 
                 R116N 
                 D188G 
                 Q234G 
                 E285R 
                 T335V 
                 I372S 
                 A432L 
               
               
                 D059M 
                 R116P 
                 D188H 
                 Q234H 
                 E285T 
                 T335W 
                 I372T 
                 A432M 
               
               
                 D059P 
                 R116Q 
                 D188L 
                 Q234N 
                 E285V 
                 T335Y 
                 I372V 
                 A432P 
               
               
                 D059R 
                 R116S 
                 D188M 
                 Q234P 
                 L286A 
                 L336A 
                 I372W 
                 A432Y 
               
               
                 D059T 
                 R116V 
                 D188N 
                 Q234S 
                 L286C 
                 L336E 
                 Q373C 
                 L434H 
               
               
                 D059V 
                 R116W 
                 D188P 
                 Q234T 
                 L286D 
                 L336F 
                 Q373P 
                 L434K 
               
               
                 D059W 
                 P117D 
                 D188Q 
                 Q234V 
                 L286F 
                 L336G 
                 Q373W 
                 L434P 
               
               
                 D059Y 
                 P117G 
                 D188R 
                 Q234W 
                 L286H 
                 L336K 
                 L374D 
                 L434Q 
               
               
                 R060A 
                 P117I 
                 D188S 
                 S235F 
                 L286K 
                 L336N 
                 L374E 
                 L434R 
               
               
                 R060D 
                 P117K 
                 D188T 
                 S235L 
                 L286M 
                 L336P 
                 E375C 
                 L434W 
               
               
                 R060F 
                 P117N 
                 D188V 
                 S235M 
                 L286P 
                 L336R 
                 E375F 
                 P437T 
               
               
                 R060G 
                 P117Q 
                 D188W 
                 S235R 
                 L286T 
                 L336S 
                 E375P 
                 M438Y 
               
               
                 R060H 
                 P117R 
                 C189A 
                 S235W 
                 L286Y 
                 L336T 
                 E375V 
                 E439N 
               
               
                 R060I 
                 P117S 
                 C189E 
                 S235Y 
                 V287A 
                 L336V 
                 E375Y 
                 E439R 
               
               
                 R060L 
                 P117V 
                 C189G 
                 P236C 
                 V287C 
                 R121G 
                 K376I 
                 T440Q 
               
               
                 R060N 
                 P117W 
                 C189H 
                 W119L 
                 V287D 
                 R121H 
                 K376P 
                 E441R 
               
               
                 R060P 
                 T118C 
                 C189K 
                 W119N 
                 V287E 
                 R121K 
                 K376W 
                 E442M 
               
               
                 R060Q 
                 T118D 
                 C189L 
                 W119P 
                 V287G 
                 R121L 
                 G377C 
                 E442N 
               
               
                 R060S 
                 T118E 
                 C189M 
                 W119R 
                 V287K 
                 R121M 
                 G377I 
                 E442S 
               
               
                 R060T 
                 T118G 
                 C189N 
                 R121A 
                 V287L 
                 R121P 
                 G377L 
                 P443D 
               
               
                 T118R 
                 T118P 
                 T118W 
                 R121C 
                 R121F 
                 G378D 
                 G377V 
                 G378E 
               
               
                 T118Y 
                 W119I 
                 W119A 
                 W119K 
                 R121E 
                 G378F 
                 G378I 
               
               
                   
               
            
           
         
       
     
     Example 5 
     Assay for Hyaluronidase Activity Under Temperature and Phenophilic Conditions 
     Supernatants from PH20 activity variants set forth in Table 9, as identified in Example 4, were tested for stability under thermophilic and/or phenophilic conditions. The assay to measure hyaluronidase activity under temperature and phenophile conditions using biotinylated-HA (bHA) as substrate for measuring hyaluronidase activity was modified from the original assay described in Example 3 in that it incorporated a 4-hour 37° C. incubation of samples with or without m-cresol prior to measurement of enzymatic activity. The assay was used to identify PH20 mutants with thermophilic properties (activity greater at 37° C. condition than at 4° C.) and/or with phenolphilic properties (greater activity in the presence of m-cresol than wildtype PH20). 
     1. Primary Screen 
     Prior to incubating samples with bHA, variant PH20 samples were diluted into designated wells of an uncoated 4×HB plate for pre-incubation at 37° C. for 4 hours under the following conditions: 1) pre-incubation at 37° C. with 0.4% m-cresol; and 2) pre-incubation at 37° C. without 0.4% m-cresol. For the preincubation at 37° C. with 0.4% m-cresol, a 1% m-cresol intermediate stock was prepared from 50% (v/v) m-cresol stock solution. Briefly, in a 2 mL Wheaton glass vial a 50% stock of m-cresol (Fluka, Catalog No. 65996; Spectrum, Catalog No. C2773) was made in methanol based on the density (D=1.034 g/L). The vial was sealed and stored at −20° C. with protection from light in small aliquotes. Then, the 1% intermediate stock was generated by dilution in HEPES assay buffer (10 mM HEPES, 50 mM NaCl, 1 mM CaCl 2 , 1 mg/mL BSA, pH 7.4, 0.05% Tween-20) daily immediately prior to use in a fume hood with vortexing. 
     Then, duplicates of transfected variant supernatant samples set forth in Table 9, generated as described above in Example 2, were each separately subjected to a 1:2.5 dilution of 1% m-cresol in HEPES assay buffer/transfected supernatant to obtain 0.4% final concentration of m-cresol. For the preincubation at 37° C. without 0.4% m-cresol, transfected variant supernatant samples were subjected to a 1:2.5 dilution in HEPES assay buffer/transfected supernatant. In addition, for each condition, an internal killing control was also tested by spiking in 3 U/mL of rHuPH20 in pH 7.4 HEPES buffer (generated as described in Example 1) that was diluted the same as described above for the transfected samples. The plates were sealed with plate sealers and incubated at 37° C. for 4 hours. 
     The preparation of the bHA coated plates and blocking of the plates prior to addition of the transfected variant supernatants or wildtype PH20 was the same as described in Example 3. The assay was further modified as follows. First, samples were diluted in duplicate 1:10 in HEPES assay buffer in 4×HB plates. For each variant, the samples that were tested were 1) non-preincubated transfected variant supernatant (no incubation; 4° C.); 2) preincubated transfected variant supernatants preincubated at 37° C. for 4 hours with 0.4% m-cresol (Cresol); or 3) preincubated transfected variant supernatant preincubated at 37° C. for 4 hours without 0.4% m-cresol (no cresol; 37° C.). In addition, the spiked-in samples also were tested. A standard curve using rHuPH20 was made as described in Example 3 without m-cresol. One hundred microliters (1000) of each standard and sample were transferred to pre-designated wells of the bHA-coated and blocked plate and incubated for approximately 1.5 hours at 37° C. Thus, each sample of each variant was tested in quadruplicate due to the preincubation of duplicate samples of each transfected variant supernatants in the pre-incubation step and the further duplicate of each sample in the bHA assay. 
     After the incubation, the plates were washed and binding to bHA detected as described above in Example 3. Optical density was measured at 450 nm within 30 minutes of adding the stop solution. 
     The U/mL activity was calculated from the standard curve and compared. The results were depicted as the percent (%) activity remaining under each of the following parameters: ratio of activity at 1) 37° C. preincubation without m-cresol/4° C.; 2) 37° C. after preincubation with m-cresol/4° C.; and 3) 37° C. after preincubation with m-cresol/after preincubation at 37° C. without m-cresol. Initial phenophile hits for reconfirmation were identified as those that in a duplicate assay exhibited a percentage of remaining activity under condition 3) of ≥20% of the original activity at 37° C. 
     Initial Hits were rescreened using a 6-well plate rescreen assay. For the rescreen, plasmid DNA corresponding to the potential Hit was transformed into  E. coli  bacteria and plasmid DNA prepared and purified using MaxiPrep according to the manufacturers instructions. The DNA sequence was confirmed. 
     The plasmid DNA was transfected into monolayer CHO-S cells (Invitrogen, Cat. No. 11619-012) grown on 6-well plates at a density of about 50-80% confluency using Lipofectamine 2000 (Invitrogen, Cat. No. 11668-027) according to the protocol suggested by the manufacturer. Transfections were performed in duplicate. The cells were incubated at 37° C. in a CO 2  incubator for 96 hours post-transfection before collecting the supernatant for the assay. As controls, cells also were transfected with the HZ24-PH20(OHO)-IRES-SEAP expression vector (SEQ ID NO:4) that contains a codon-optimized wildtype PH20 sequence (OHO). Mock cells also were included as controls. 
     Ninety-Six (96) hours post-transfections, supernatant was collected from each sample, including the OHO and mock controls, and assayed for hyaluronidase activity under various conditions as described above: 1) non-preincubated transfected variant supernatant (no incubation; 4° C.); 2) preincubated transfected variant supernatants preincubated at 37° C. for 4 hours with 0.4% m-cresol (Cresol; 37° C.); or 3) preincubated transfected variant supernatant preincubated at 37° C. for 4 hours without 0.4% m-cresol (no cresol; 37° C.). Hyaluronidase activity was determined as described above using the bHA assay. 
     The results were assessed as described above. Absolute hyaluronidase activity (U/mL) was generated from the standard curve. In addition, percent activity was determined as a ratio of activity at 37° C./4° C., 37° C. plus m-cresol/4° C., and 37° C. plus m-cresol/37° C. The results are set forth in Tables 11 and 12 below. 
     
       
         
           
               
             
               
                 TABLE 11 
               
             
            
               
                   
               
               
                 Absolute Hyaluronidase Activity 
               
            
           
           
               
               
               
               
            
               
                   
                 No incubation 
                 37° C. no cresol 
                 37° C. with m-cresol 
               
               
                 Mutant 
                 (4° C.) 
                 (37° C.) 
                 (37° C. plus m-cresol) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 L001A 
                 2.993 
                 2.511 
                 3.529 
                 3.214 
                 0.287 
                 0.295 
               
               
                 L001E 
                 2.669 
                 2.539 
                 2.862 
                 3.179 
                 0.376 
                 0.341 
               
               
                 L001G 
                 0.348 
                 0.583 
                 0.596 
                 0.676 
                 0.055 
                 0.031 
               
               
                 L001Q 
                 5.135 
                 6.443 
                 6.133 
                 5.719 
                 0.621 
                 0.636 
               
               
                 L001R 
                 5.603 
                 4.390 
                 6.576 
                 7.042 
                 0.458 
                 0.396 
               
               
                 P006A 
                 2.965 
                 3.208 
                 4.088 
                 3.495 
                 0.404 
                 0.435 
               
               
                 V008M 
                 1.376 
                 1.401 
                 1.856 
                 1.678 
                 0.000 
                 0.008 
               
               
                 I009Q 
                 0.447 
                 0.381 
                 0.469 
                 0.476 
                 0.031 
                 0.030 
               
               
                 P010G 
                 0.747 
                 0.564 
                 0.820 
                 0.688 
                 0.123 
                 0.114 
               
               
                 P010H 
                 0.473 
                 0.485 
                 0.624 
                 0.548 
                 0.000 
                 0.000 
               
               
                 N011S 
                 0.862 
                 0.962 
                 1.313 
                 1.263 
                 0.094 
                 0.064 
               
               
                 V012E 
                 11.019 
                 5.519 
                 5.312 
                 5.528 
                 0.753 
                 0.934 
               
               
                 V012I 
                 2.804 
                 3.844 
                 3.610 
                 6.566 
                 0.106 
                 0.090 
               
               
                 V012K 
                 1.691 
                 1.963 
                 2.479 
                 2.243 
                 0.330 
                 0.321 
               
               
                 F014V 
                 0.144 
                 0.165 
                 0.222 
                 0.242 
                 0.003 
                 0.000 
               
               
                 L015M 
                 0.902 
                 1.073 
                 1.026 
                 0.901 
                 0.017 
                 0.017 
               
               
                 A020S 
                 1.494 
                 2.205 
                 2.822 
                 2.620 
                 0.413 
                 0.397 
               
               
                 S022T 
                 3.035 
                 3.788 
                 3.375 
                 3.273 
                 0.684 
                 0.748 
               
               
                 L026M 
                 1.482 
                 1.226 
                 2.027 
                 1.704 
                 0.224 
                 0.178 
               
               
                 K028R 
                 0.944 
                 0.845 
                 1.043 
                 0.925 
                 0.112 
                 0.095 
               
               
                 F029R 
                 1.195 
                 1.511 
                 1.848 
                 1.839 
                 0.140 
                 0.140 
               
               
                 F029S 
                 3.019 
                 3.615 
                 3.566 
                 3.521 
                 0.250 
                 0.283 
               
               
                 F029T 
                 1.451 
                 1.712 
                 1.839 
                 2.065 
                 0.220 
                 0.212 
               
               
                 P032C 
                 0.370 
                 0.419 
                 0.476 
                 0.534 
                 0.006 
                 0.040 
               
               
                 L033G 
                 0.566 
                 0.700 
                 0.686 
                 0.627 
                 0.001 
                 0.026 
               
               
                 D034W 
                 0.340 
                 0.321 
                 0.499 
                 0.471 
                 0.076 
                 0.069 
               
               
                 M035V 
                 0.887 
                 0.639 
                 0.721 
                 0.652 
                 0.116 
                 0.023 
               
               
                 S036H 
                 1.109 
                 0.752 
                 1.178 
                 1.135 
                 0.117 
                 0.026 
               
               
                 S036N 
                 0.797 
                 0.933 
                 0.893 
                 0.859 
                 0.171 
                 0.260 
               
               
                 L037M 
                 0.574 
                 0.404 
                 0.455 
                 0.353 
                 0.049 
                 0.032 
               
               
                 F040L 
                 2.603 
                 3.941 
                 3.515 
                 4.148 
                 0.277 
                 0.361 
               
               
                 I046L 
                 3.027 
                 2.959 
                 4.011 
                 3.342 
                 0.513 
                 0.557 
               
               
                 N047D 
                 2.222 
                 2.359 
                 2.573 
                 2.639 
                 0.032 
                 0.021 
               
               
                 N047W 
                 0.404 
                 0.415 
                 0.423 
                 0.456 
                 0.000 
                 0.017 
               
               
                 A048N 
                 12.398 
                 45.971 
                 14.252 
                 23.873 
                 0.797 
                 0.902 
               
               
                 T049R 
                 7.893 
                 13.334 
                 9.685 
                 12.102 
                 0.563 
                 0.649 
               
               
                 G050D 
                 3.287 
                 3.148 
                 3.084 
                 3.020 
                 0.242 
                 0.264 
               
               
                 G050M 
                 1.763 
                 2.333 
                 2.780 
                 3.244 
                 0.250 
                 0.393 
               
               
                 G052N 
                 7.217 
                 9.809 
                 6.939 
                 13.978 
                 1.109 
                 1.083 
               
               
                 G052T 
                 1.542 
                 1.224 
                 1.795 
                 1.433 
                 0.381 
                 0.463 
               
               
                 G052S 
                 2.152 
                 1.999 
                 2.120 
                 1.963 
                 0.498 
                 0.566 
               
               
                 V058C 
                 1.428 
                 1.312 
                 1.321 
                 1.301 
                 0.212 
                 0.210 
               
               
                 V058K 
                 28.000 
                 28.000 
                 61.016 
                 61.016 
                 23.586 
                 23.586 
               
               
                 V058R 
                 5.719 
                 4.688 
                 5.542 
                 4.822 
                 3.134 
                 3.149 
               
               
                 V058N 
                 1.200 
                 1.175 
                 1.550 
                 1.525 
                 0.200 
                 0.175 
               
               
                 V058Y 
                 1.040 
                 0.770 
                 1.071 
                 1.088 
                 0.388 
                 0.454 
               
               
                 V058Q 
                 11.956 
                 15.363 
                 18.458 
                 45.092 
                 1.567 
                 2.166 
               
               
                 V058P 
                 3.360 
                 2.949 
                 2.799 
                 5.121 
                 0.592 
                 0.884 
               
               
                 V058H 
                 3.790 
                 5.074 
                 7.590 
                 9.222 
                 0.826 
                 1.205 
               
               
                 D068P 
                 0.215 
                 0.215 
                 0.213 
                 0.180 
                 0.001 
                 0.184 
               
               
                 S069T 
                 1.927 
                 2.179 
                 2.671 
                 2.671 
                 0.289 
                 0.240 
               
               
                 I070P 
                 1.284 
                 1.593 
                 1.306 
                 1.589 
                 0.010 
                 0.032 
               
               
                 I070V 
                 1.818 
                 2.437 
                 3.099 
                 3.335 
                 0.433 
                 0.363 
               
               
                 V073Q 
                 4.846 
                 5.441 
                 5.880 
                 5.827 
                 0.383 
                 0.477 
               
               
                 V073R 
                 0.522 
                 0.803 
                 0.720 
                 0.804 
                 0.018 
                 0.059 
               
               
                 T074E 
                 2.903 
                 3.834 
                 3.868 
                 3.871 
                 0.666 
                 0.626 
               
               
                 T074M 
                 0.569 
                 0.744 
                 0.656 
                 0.771 
                 0.079 
                 0.083 
               
               
                 T074N 
                 2.792 
                 1.905 
                 2.565 
                 2.995 
                 0.281 
                 0.204 
               
               
                 T074P 
                 2.331 
                 1.593 
                 2.525 
                 2.648 
                 0.309 
                 0.265 
               
               
                 T074R 
                 0.999 
                 0.820 
                 0.806 
                 1.066 
                 0.060 
                 0.023 
               
               
                 T074V 
                 1.186 
                 1.280 
                 1.365 
                 1.460 
                 0.101 
                 0.080 
               
               
                 V075M 
                 0.917 
                 1.087 
                 1.233 
                 1.321 
                 0.003 
                 0.028 
               
               
                 K082L 
                 1.362 
                 1.311 
                 1.563 
                 3.302 
                 0.325 
                 0.354 
               
               
                 K082N 
                 3.202 
                 3.411 
                 3.396 
                 3.244 
                 0.792 
                 0.861 
               
               
                 I083V 
                 3.706 
                 2.633 
                 5.194 
                 3.615 
                 1.552 
                 1.017 
               
               
                 I083Q 
                 2.376 
                 1.946 
                 2.665 
                 3.674 
                 0.720 
                 0.510 
               
               
                 I083S 
                 0.841 
                 1.054 
                 0.880 
                 1.005 
                 0.235 
                 0.268 
               
               
                 I083G 
                 2.276 
                 2.443 
                 2.418 
                 1.866 
                 0.545 
                 0.601 
               
               
                 S084E 
                 1.470 
                 1.484 
                 1.834 
                 1.683 
                 0.115 
                 0.115 
               
               
                 S084F 
                 1.179 
                 1.212 
                 0.982 
                 1.103 
                 0.025 
                 0.000 
               
               
                 S084N 
                 2.255 
                 1.888 
                 3.268 
                 2.476 
                 0.597 
                 0.547 
               
               
                 S084R 
                 8.534 
                 14.779 
                 10.230 
                 30.016 
                 1.117 
                 1.494 
               
               
                 Q086A 
                 2.084 
                 2.120 
                 2.845 
                 3.310 
                 0.405 
                 0.322 
               
               
                 Q086H 
                 1.187 
                 1.000 
                 1.218 
                 1.296 
                 0.087 
                 0.065 
               
               
                 Q086K 
                 0.127 
                 0.110 
                 0.126 
                 0.072 
                 0.032 
                 0.023 
               
               
                 Q086S 
                 2.528 
                 2.082 
                 2.539 
                 2.149 
                 0.173 
                 0.241 
               
               
                 Q086T 
                 3.018 
                 2.542 
                 2.832 
                 4.562 
                 0.290 
                 0.406 
               
               
                 D087G 
                 2.755 
                 2.176 
                 2.252 
                 1.971 
                 0.034 
                 0.122 
               
               
                 D087L 
                 2.070 
                 2.277 
                 2.195 
                 2.311 
                 0.324 
                 0.299 
               
               
                 D087M 
                 2.262 
                 2.325 
                 2.510 
                 2.038 
                 0.191 
                 0.335 
               
               
                 D087S 
                 5.210 
                 10.305 
                 6.983 
                 14.399 
                 0.569 
                 0.928 
               
               
                 D087V 
                 1.361 
                 1.364 
                 1.553 
                 1.187 
                 0.142 
                 0.189 
               
               
                 D090E 
                 8.251 
                 12.299 
                 7.666 
                 19.836 
                 1.093 
                 1.234 
               
               
                 D090N 
                 2.812 
                 2.775 
                 3.123 
                 2.737 
                 0.379 
                 0.290 
               
               
                 K093Q 
                 2.491 
                 2.065 
                 2.267 
                 1.971 
                 0.132 
                 0.131 
               
               
                 K093R 
                 2.986 
                 2.862 
                 3.094 
                 2.842 
                 0.362 
                 0.465 
               
               
                 K094D 
                 2.393 
                 2.088 
                 2.071 
                 2.132 
                 0.135 
                 0.211 
               
               
                 K094R 
                 1.407 
                 1.542 
                 1.764 
                 1.676 
                 0.158 
                 0.166 
               
               
                 T097C 
                 0.330 
                 0.618 
                 0.545 
                 0.505 
                 0.044 
                 0.087 
               
               
                 T097D 
                 0.520 
                 0.565 
                 0.643 
                 0.664 
                 0.055 
                 0.073 
               
               
                 T097E 
                 1.096 
                 1.410 
                 1.394 
                 1.623 
                 0.217 
                 0.262 
               
               
                 T097L 
                 0.899 
                 1.198 
                 1.065 
                 1.241 
                 0.246 
                 0.300 
               
               
                 N104R 
                 2.508 
                 2.356 
                 2.876 
                 2.790 
                 0.279 
                 0.238 
               
               
                 A120H 
                 2.155 
                 2.551 
                 2.028 
                 2.883 
                 0.168 
                 0.199 
               
               
                 D127R 
                 0.264 
                 0.339 
                 0.149 
                 0.199 
                 0.105 
                 0.068 
               
               
                 V128I 
                 3.120 
                 3.313 
                 3.546 
                 3.401 
                 0.389 
                 0.504 
               
               
                 N131M 
                 15.335 
                 20.678 
                 27.143 
                 15.899 
                 0.505 
                 0.447 
               
               
                 N131R 
                 8.195 
                 8.748 
                 7.724 
                 8.392 
                 1.645 
                 1.626 
               
               
                 N131V 
                 1.656 
                 1.870 
                 2.280 
                 1.962 
                 0.233 
                 0.214 
               
               
                 R132L 
                 3.306 
                 3.235 
                 3.259 
                 2.966 
                 0.337 
                 0.430 
               
               
                 Q138L 
                 1.494 
                 1.660 
                 1.611 
                 1.521 
                 0.410 
                 0.347 
               
               
                 Q140K 
                 2.829 
                 4.065 
                 4.996 
                 4.464 
                 0.546 
                 0.559 
               
               
                 N141R 
                 1.290 
                 1.320 
                 1.334 
                 1.527 
                 0.058 
                 0.035 
               
               
                 N141S 
                 2.201 
                 2.708 
                 2.900 
                 2.966 
                 0.135 
                 0.164 
               
               
                 N141W 
                 1.475 
                 1.568 
                 1.927 
                 1.643 
                 0.100 
                 0.105 
               
               
                 V142D 
                 2.552 
                 2.186 
                 2.914 
                 3.193 
                 0.128 
                 0.067 
               
               
                 V142G 
                 1.357 
                 1.796 
                 1.597 
                 1.621 
                 0.211 
                 0.219 
               
               
                 V142K 
                 3.532 
                 2.381 
                 3.867 
                 3.681 
                 0.571 
                 0.575 
               
               
                 V142N 
                 0.432 
                 0.567 
                 0.672 
                 0.589 
                 0.103 
                 0.087 
               
               
                 V142P 
                 4.624 
                 7.213 
                 7.722 
                 7.021 
                 1.074 
                 1.081 
               
               
                 V142Q 
                 5.090 
                 6.900 
                 7.618 
                 6.897 
                 0.678 
                 0.678 
               
               
                 V142R 
                 1.968 
                 2.595 
                 2.941 
                 2.689 
                 0.364 
                 0.330 
               
               
                 V142S 
                 2.789 
                 2.988 
                 4.763 
                 3.497 
                 0.416 
                 0.591 
               
               
                 V142T 
                 1.926 
                 3.260 
                 4.313 
                 4.031 
                 0.495 
                 0.472 
               
               
                 Q143G 
                 3.922 
                 4.903 
                 5.632 
                 4.846 
                 0.782 
                 0.780 
               
               
                 Q143K 
                 3.634 
                 3.671 
                 7.285 
                 5.008 
                 1.043 
                 1.039 
               
               
                 L144R 
                 3.810 
                 4.581 
                 5.191 
                 5.107 
                 0.556 
                 0.520 
               
               
                 L144T 
                 1.496 
                 1.681 
                 1.941 
                 1.831 
                 0.285 
                 0.219 
               
               
                 L146P 
                 0.818 
                 0.782 
                 0.954 
                 0.904 
                 0.011 
                 0.031 
               
               
                 T147S 
                 0.984 
                 1.149 
                 1.399 
                 1.497 
                 0.055 
                 0.039 
               
               
                 T150N 
                 0.442 
                 0.585 
                 0.622 
                 0.684 
                 0.039 
                 0.046 
               
               
                 T150S 
                 1.747 
                 1.400 
                 1.875 
                 1.988 
                 0.120 
                 0.121 
               
               
                 E151A 
                 2.870 
                 2.269 
                 2.965 
                 2.860 
                 0.359 
                 0.337 
               
               
                 E151L 
                 3.365 
                 3.289 
                 4.446 
                 4.007 
                 0.218 
                 0.251 
               
               
                 E151S 
                 5.187 
                 4.591 
                 5.987 
                 6.262 
                 0.371 
                 0.294 
               
               
                 E151T 
                 2.442 
                 3.000 
                 3.134 
                 3.309 
                 0.000 
                 0.000 
               
               
                 E151V 
                 3.998 
                 4.247 
                 4.459 
                 4.232 
                 0.326 
                 0.314 
               
               
                 E151W 
                 7.166 
                 14.248 
                 11.352 
                 13.524 
                 0.131 
                 0.121 
               
               
                 K152T 
                 1.204 
                 1.377 
                 1.796 
                 1.883 
                 0.100 
                 0.067 
               
               
                 K152W 
                 2.084 
                 1.795 
                 2.549 
                 2.406 
                 0.063 
                 0.069 
               
               
                 E158S 
                 0.339 
                 0.397 
                 0.451 
                 0.407 
                 0.000 
                 0.000 
               
               
                 K162E 
                 0.168 
                 0.195 
                 0.114 
                 0.080 
                 0.004 
                 0.024 
               
               
                 L165F 
                 4.775 
                 5.250 
                 5.075 
                 5.075 
                 0.600 
                 0.725 
               
               
                 V166Q 
                 1.883 
                 2.507 
                 2.937 
                 2.958 
                 0.392 
                 0.324 
               
               
                 V166T 
                 0.993 
                 1.315 
                 1.821 
                 1.800 
                 0.231 
                 0.235 
               
               
                 E167D 
                 0.811 
                 0.910 
                 1.109 
                 1.480 
                 0.111 
                 0.056 
               
               
                 I169L 
                 1.812 
                 1.796 
                 2.540 
                 2.196 
                 0.335 
                 0.341 
               
               
                 K170R 
                 1.578 
                 2.054 
                 2.536 
                 1.995 
                 0.209 
                 0.201 
               
               
                 G172A 
                 0.413 
                 0.581 
                 0.692 
                 0.777 
                 0.052 
                 0.056 
               
               
                 K173R 
                 1.654 
                 1.551 
                 1.766 
                 2.083 
                 0.173 
                 0.156 
               
               
                 L174G 
                 0.184 
                 0.087 
                 0.210 
                 0.230 
                 0.026 
                 0.031 
               
               
                 L174N 
                 1.616 
                 2.276 
                 2.494 
                 2.872 
                 0.331 
                 0.543 
               
               
                 L174T 
                 0.552 
                 0.566 
                 0.689 
                 0.820 
                 0.090 
                 0.050 
               
               
                 N178K 
                 2.931 
                 4.375 
                 4.891 
                 4.513 
                 0.258 
                 0.362 
               
               
                 N178R 
                 8.160 
                 13.820 
                 16.287 
                 20.033 
                 0.665 
                 0.790 
               
               
                 H193Q 
                 1.060 
                 1.367 
                 2.264 
                 1.888 
                 0.346 
                 0.346 
               
               
                 K195T 
                 1.227 
                 0.806 
                 1.548 
                 1.911 
                 0.348 
                 0.292 
               
               
                 K195N 
                 1.266 
                 1.437 
                 1.649 
                 1.385 
                 0.369 
                 0.353 
               
               
                 K196E 
                 0.732 
                 0.660 
                 0.663 
                 1.017 
                 0.244 
                 0.239 
               
               
                 K196R 
                 2.246 
                 2.285 
                 2.383 
                 2.174 
                 0.315 
                 0.384 
               
               
                 F204P 
                 3.500 
                 4.550 
                 2.925 
                 3.750 
                 2.475 
                 4.725 
               
               
                 N205A 
                 0.515 
                 0.837 
                 0.717 
                 0.854 
                 0.153 
                 0.160 
               
               
                 N205E 
                 1.011 
                 2.004 
                 1.627 
                 1.870 
                 0.314 
                 0.346 
               
               
                 N205L 
                 1.084 
                 1.029 
                 1.165 
                 0.000 
                 0.123 
                 0.088 
               
               
                 N205T 
                 0.295 
                 0.367 
                 0.428 
                 0.406 
                 0.043 
                 0.053 
               
               
                 V206I 
                 0.317 
                 0.508 
                 0.600 
                 0.565 
                 0.079 
                 0.088 
               
               
                 K209R 
                 2.041 
                 2.453 
                 2.445 
                 1.951 
                 0.291 
                 0.077 
               
               
                 D212N 
                 5.568 
                 4.549 
                 6.271 
                 6.016 
                 0.167 
                 0.322 
               
               
                 D212S 
                 1.987 
                 1.502 
                 2.442 
                 2.222 
                 0.204 
                 0.152 
               
               
                 D213A 
                 0.235 
                 0.283 
                 0.432 
                 0.438 
                 0.116 
                 0.060 
               
               
                 D213M 
                 1.664 
                 2.080 
                 2.650 
                 2.046 
                 0.181 
                 0.142 
               
               
                 S215H 
                 2.448 
                 3.056 
                 2.670 
                 2.414 
                 0.268 
                 0.139 
               
               
                 S215M 
                 1.497 
                 2.175 
                 2.618 
                 1.630 
                 0.110 
                 0.146 
               
               
                 N219I 
                 0.338 
                 0.250 
                 0.860 
                 0.728 
                 0.076 
                 0.082 
               
               
                 E220V 
                 3.783 
                 3.828 
                 4.993 
                 4.349 
                 0.371 
                 0.257 
               
               
                 T222G 
                 3.528 
                 5.262 
                 5.399 
                 5.549 
                 0.033 
                 0.044 
               
               
                 T232F 
                 0.539 
                 1.242 
                 0.716 
                 0.781 
                 0.089 
                 0.153 
               
               
                 Q233G 
                 0.041 
                 0.095 
                 0.115 
                 0.121 
                 0.000 
                 0.000 
               
               
                 Q234M 
                 6.029 
                 6.031 
                 5.764 
                 4.871 
                 1.286 
                 0.988 
               
               
                 S235A 
                 0.550 
                 0.502 
                 0.714 
                 0.607 
                 0.079 
                 0.073 
               
               
                 V237C 
                 0.623 
                 0.708 
                 0.860 
                 0.824 
                 0.000 
                 0.000 
               
               
                 V237H 
                 0.303 
                 0.316 
                 0.370 
                 0.459 
                 0.046 
                 0.034 
               
               
                 V237T 
                 0.152 
                 0.196 
                 0.254 
                 0.247 
                 0.054 
                 0.053 
               
               
                 A238E 
                 2.050 
                 1.800 
                 1.945 
                 2.559 
                 0.159 
                 0.171 
               
               
                 A238H 
                 0.579 
                 0.363 
                 0.345 
                 0.743 
                 0.090 
                 0.062 
               
               
                 T240A 
                 1.107 
                 0.900 
                 1.564 
                 1.302 
                 0.143 
                 0.118 
               
               
                 T240Q 
                 0.333 
                 0.510 
                 0.542 
                 0.617 
                 0.080 
                 0.085 
               
               
                 R248A 
                 2.274 
                 2.499 
                 2.575 
                 3.115 
                 0.027 
                 0.075 
               
               
                 E249V 
                 3.001 
                 3.894 
                 4.284 
                 4.325 
                 0.655 
                 0.712 
               
               
                 P257G 
                 3.981 
                 4.452 
                 4.985 
                 5.022 
                 0.039 
                 0.034 
               
               
                 K260M 
                 0.719 
                 0.960 
                 0.839 
                 0.935 
                 0.072 
                 0.068 
               
               
                 S261A 
                 3.253 
                 3.117 
                 1.872 
                 2.686 
                 1.264 
                 1.451 
               
               
                 S261K 
                 6.089 
                 5.421 
                 9.860 
                 6.297 
                 1.583 
                 1.437 
               
               
                 S261N 
                 14.149 
                 40.257 
                 20.219 
                 14.303 
                 2.115 
                 1.917 
               
               
                 A267T 
                 0.052 
                 0.095 
                 0.102 
                 0.106 
                 0.036 
                 0.041 
               
               
                 F273H 
                 0.340 
                 0.436 
                 0.417 
                 0.519 
                 0.025 
                 0.031 
               
               
                 F273Y 
                 0.558 
                 0.505 
                 0.668 
                 0.519 
                 0.052 
                 0.050 
               
               
                 Q276H 
                 2.706 
                 1.877 
                 2.027 
                 1.997 
                 0.181 
                 0.201 
               
               
                 Q276M 
                 0.775 
                 0.768 
                 0.762 
                 0.806 
                 0.043 
                 0.000 
               
               
                 Q276R 
                 6.080 
                 9.717 
                 7.383 
                 14.593 
                 0.807 
                 1.281 
               
               
                 Q276S 
                 1.353 
                 1.212 
                 1.497 
                 1.681 
                 0.149 
                 0.147 
               
               
                 V277A 
                 1.202 
                 1.643 
                 1.692 
                 2.129 
                 0.118 
                 0.110 
               
               
                 V277E 
                 2.440 
                 2.340 
                 4.289 
                 4.577 
                 0.161 
                 0.239 
               
               
                 V277H 
                 5.548 
                 5.302 
                 7.181 
                 7.300 
                 0.227 
                 0.512 
               
               
                 V277K 
                 8.950 
                 8.996 
                 33.627 
                 33.627 
                 4.442 
                 4.045 
               
               
                 V277M 
                 1.279 
                 1.622 
                 1.754 
                 1.818 
                 0.264 
                 0.270 
               
               
                 V277N 
                 14.351 
                 4.306 
                 12.865 
                 11.772 
                 0.938 
                 0.796 
               
               
                 V277Q 
                 5.459 
                 5.461 
                 6.547 
                 6.343 
                 0.373 
                 0.493 
               
               
                 V277R 
                 18.300 
                 12.038 
                 17.581 
                 20.641 
                 2.737 
                 2.023 
               
               
                 V277S 
                 14.351 
                 10.444 
                 9.509 
                 15.135 
                 0.727 
                 0.716 
               
               
                 V277T 
                 8.412 
                 7.804 
                 8.497 
                 11.184 
                 0.679 
                 0.871 
               
               
                 L278E 
                 4.416 
                 2.795 
                 3.330 
                 2.800 
                 0.170 
                 0.202 
               
               
                 L278G 
                 7.502 
                 7.456 
                 9.173 
                 7.760 
                 0.596 
                 0.612 
               
               
                 K279H 
                 0.888 
                 1.087 
                 1.234 
                 1.339 
                 0.185 
                 0.269 
               
               
                 V287T 
                 0.580 
                 0.667 
                 0.843 
                 0.832 
                 0.139 
                 0.100 
               
               
                 T289S 
                 0.783 
                 1.019 
                 0.819 
                 1.001 
                 0.008 
                 0.007 
               
               
                 G291S 
                 0.227 
                 0.322 
                 0.419 
                 0.385 
                 0.051 
                 0.016 
               
               
                 G291V 
                 3.662 
                 3.707 
                 4.131 
                 5.599 
                 0.821 
                 0.706 
               
               
                 E292C 
                 1.344 
                 1.599 
                 1.711 
                 1.617 
                 0.138 
                 0.144 
               
               
                 E292F 
                 6.106 
                 4.697 
                 8.422 
                 6.216 
                 0.520 
                 0.363 
               
               
                 E292H 
                 2.620 
                 3.316 
                 4.458 
                 3.830 
                 0.389 
                 0.451 
               
               
                 E292R 
                 2.810 
                 2.178 
                 3.155 
                 2.829 
                 0.398 
                 0.339 
               
               
                 E292V 
                 0.891 
                 1.121 
                 1.453 
                 1.494 
                 0.193 
                 0.177 
               
               
                 T293A 
                 1.986 
                 3.110 
                 2.546 
                 1.789 
                 0.086 
                 0.076 
               
               
                 A298G 
                 0.161 
                 0.274 
                 0.342 
                 0.236 
                 0.030 
                 0.022 
               
               
                 L307G 
                 0.616 
                 0.661 
                 0.726 
                 0.605 
                 0.000 
                 0.000 
               
               
                 S308D 
                 0.264 
                 0.325 
                 0.337 
                 0.344 
                 0.014 
                 0.010 
               
               
                 S308K 
                 0.651 
                 0.722 
                 0.826 
                 0.716 
                 0.011 
                 0.000 
               
               
                 S308N 
                 3.995 
                 4.406 
                 6.808 
                 6.128 
                 0.386 
                 0.362 
               
               
                 I309E 
                 3.166 
                 2.819 
                 3.921 
                 3.663 
                 0.637 
                 0.528 
               
               
                 I309G 
                 6.651 
                 5.429 
                 6.824 
                 6.194 
                 0.503 
                 0.400 
               
               
                 I309L 
                 0.326 
                 0.403 
                 0.501 
                 0.431 
                 0.048 
                 0.047 
               
               
                 I309M 
                 2.809 
                 2.473 
                 3.467 
                 3.383 
                 0.278 
                 0.239 
               
               
                 I309N 
                 4.865 
                 5.191 
                 5.444 
                 5.054 
                 0.380 
                 0.327 
               
               
                 I309S 
                 10.719 
                 28.759 
                 18.217 
                 158.604 
                 0.748 
                 1.367 
               
               
                 I309T 
                 3.052 
                 2.509 
                 2.989 
                 3.735 
                 0.228 
                 0.207 
               
               
                 I309V 
                 1.705 
                 1.292 
                 1.929 
                 1.787 
                 0.029 
                 0.062 
               
               
                 M310G 
                 4.514 
                 6.397 
                 7.568 
                 7.084 
                 0.866 
                 0.915 
               
               
                 M310Q 
                 3.648 
                 3.179 
                 3.912 
                 3.380 
                 1.088 
                 0.955 
               
               
                 M313G 
                 0.252 
                 0.325 
                 0.348 
                 0.355 
                 0.034 
                 0.036 
               
               
                 M313H 
                 3.767 
                 5.276 
                 10.243 
                 10.395 
                 0.380 
                 0.404 
               
               
                 M313K 
                 12.689 
                 12.122 
                 15.085 
                 12.984 
                 0.129 
                 0.072 
               
               
                 M313P 
                 4.050 
                 2.951 
                 4.198 
                 3.919 
                 0.209 
                 0.177 
               
               
                 M313R 
                 4.634 
                 10.863 
                 7.288 
                 3.568 
                 0.337 
                 0.296 
               
               
                 M313T 
                 2.903 
                 4.474 
                 4.705 
                 4.467 
                 0.331 
                 0.313 
               
               
                 M313Y 
                 1.063 
                 1.262 
                 1.276 
                 1.300 
                 0.096 
                 0.089 
               
               
                 K314S 
                 2.848 
                 4.450 
                 4.042 
                 5.879 
                 0.391 
                 0.533 
               
               
                 K314Y 
                 0.093 
                 0.131 
                 0.226 
                 0.182 
                 0.013 
                 0.020 
               
               
                 S315A 
                 1.472 
                 1.082 
                 1.345 
                 1.484 
                 0.222 
                 0.148 
               
               
                 S315H 
                 2.412 
                 3.242 
                 3.648 
                 3.414 
                 0.440 
                 0.371 
               
               
                 S315Y 
                 0.279 
                 0.626 
                 0.477 
                 0.362 
                 0.146 
                 0.143 
               
               
                 L317A 
                 3.254 
                 2.845 
                 4.019 
                 3.776 
                 0.280 
                 0.317 
               
               
                 L317I 
                 1.078 
                 1.524 
                 2.021 
                 1.687 
                 0.257 
                 0.180 
               
               
                 L317K 
                 12.129 
                 9.382 
                 11.668 
                 12.591 
                 0.402 
                 0.445 
               
               
                 L317N 
                 2.907 
                 3.066 
                 3.703 
                 3.717 
                 0.445 
                 0.540 
               
               
                 L317R 
                 8.631 
                 15.187 
                 20.585 
                 15.106 
                 0.796 
                 0.857 
               
               
                 L317S 
                 11.586 
                 29.267 
                 10.535 
                 25.114 
                 1.637 
                 1.613 
               
               
                 L317T 
                 1.338 
                 1.073 
                 1.953 
                 1.656 
                 0.136 
                 0.018 
               
               
                 L317W 
                 0.810 
                 1.128 
                 1.326 
                 1.665 
                 0.158 
                 0.171 
               
               
                 L318D 
                 1.750 
                 1.970 
                 1.847 
                 1.930 
                 0.322 
                 0.322 
               
               
                 L318H 
                 1.073 
                 0.806 
                 1.072 
                 1.005 
                 0.046 
                 0.074 
               
               
                 L318R 
                 2.856 
                 3.464 
                 4.583 
                 4.187 
                 0.258 
                 0.260 
               
               
                 N321R 
                 3.069 
                 4.409 
                 5.059 
                 4.946 
                 0.482 
                 0.426 
               
               
                 N321S 
                 0.683 
                 0.710 
                 0.700 
                 0.772 
                 0.058 
                 0.035 
               
               
                 E324N 
                 4.309 
                 2.530 
                 4.508 
                 3.321 
                 0.348 
                 0.303 
               
               
                 T325E 
                 1.071 
                 1.270 
                 1.337 
                 1.352 
                 0.193 
                 0.143 
               
               
                 N328G 
                 0.379 
                 0.504 
                 0.747 
                 0.553 
                 0.031 
                 0.040 
               
               
                 N328Y 
                 2.629 
                 4.543 
                 4.758 
                 4.543 
                 0.490 
                 0.477 
               
               
                 T335S 
                 0.905 
                 0.787 
                 0.977 
                 0.986 
                 0.113 
                 0.062 
               
               
                 Q347A 
                 8.316 
                 11.961 
                 8.432 
                 11.508 
                 0.918 
                 1.266 
               
               
                 Q347G 
                 1.358 
                 1.120 
                 3.021 
                 2.319 
                 0.253 
                 0.209 
               
               
                 Q349M 
                 1.493 
                 1.629 
                 1.486 
                 1.760 
                 0.178 
                 0.217 
               
               
                 Q349R 
                 0.451 
                 0.572 
                 0.663 
                 0.598 
                 0.078 
                 0.079 
               
               
                 V351S 
                 1.379 
                 1.633 
                 1.804 
                 1.647 
                 0.000 
                 0.000 
               
               
                 I353V 
                 2.335 
                 1.954 
                 3.090 
                 2.697 
                 0.323 
                 0.321 
               
               
                 N356H 
                 0.445 
                 0.451 
                 0.445 
                 0.588 
                 0.038 
                 0.023 
               
               
                 N356S 
                 0.262 
                 0.253 
                 0.136 
                 0.318 
                 0.000 
                 0.008 
               
               
                 S359E 
                 2.616 
                 2.635 
                 3.547 
                 3.560 
                 0.382 
                 0.333 
               
               
                 S359H 
                 0.403 
                 0.371 
                 0.445 
                 0.374 
                 0.000 
                 0.000 
               
               
                 P367A 
                 0.643 
                 0.782 
                 1.074 
                 0.996 
                 0.139 
                 0.131 
               
               
                 P367G 
                 0.593 
                 0.530 
                 0.686 
                 0.650 
                 0.000 
                 0.000 
               
               
                 P367K 
                 0.707 
                 0.767 
                 0.890 
                 0.513 
                 0.045 
                 0.052 
               
               
                 P367S 
                 3.967 
                 3.478 
                 2.946 
                 3.073 
                 0.424 
                 0.505 
               
               
                 D368A 
                 1.762 
                 2.321 
                 2.143 
                 1.895 
                 0.031 
                 0.040 
               
               
                 D368E 
                 3.464 
                 4.944 
                 5.772 
                 4.842 
                 0.530 
                 0.555 
               
               
                 D368L 
                 0.557 
                 0.566 
                 0.607 
                 0.619 
                 0.000 
                 0.006 
               
               
                 D368M 
                 0.861 
                 1.065 
                 1.031 
                 1.104 
                 0.028 
                 0.028 
               
               
                 D368R 
                 4.503 
                 5.270 
                 7.418 
                 6.226 
                 0.754 
                 0.735 
               
               
                 D368T 
                 2.345 
                 1.993 
                 2.512 
                 2.525 
                 0.072 
                 0.085 
               
               
                 N369R 
                 1.548 
                 2.719 
                 2.503 
                 2.022 
                 0.160 
                 0.125 
               
               
                 A371F 
                 2.760 
                 5.207 
                 4.974 
                 3.980 
                 0.308 
                 0.222 
               
               
                 A371H 
                 8.101 
                 86.587 
                 77.531 
                 77.531 
                 1.403 
                 1.316 
               
               
                 A371H 
                 3.509 
                 4.058 
                 3.900 
                 3.879 
                 0.000 
                 0.334 
               
               
                 A371K 
                 2.903 
                 3.546 
                 3.963 
                 4.055 
                 0.509 
                 0.505 
               
               
                 A371L 
                 11.018 
                 40.668 
                 76.587 
                 43.516 
                 1.159 
                 0.964 
               
               
                 A371L 
                 3.328 
                 3.445 
                 3.472 
                 2.075 
                 0.000 
                 0.025 
               
               
                 A371R 
                 25.855 
                 25.855 
                 n/a 
                 n/a 
                 2.851 
                 3.634 
               
               
                 A371R 
                 6.592 
                 7.733 
                 7.987 
                 7.576 
                 0.000 
                 0.196 
               
               
                 A371S 
                 3.329 
                 3.505 
                 4.916 
                 4.611 
                 0.412 
                 0.781 
               
               
                 L374P 
                 2.939 
                 7.129 
                 11.522 
                 8.771 
                 0.665 
                 0.646 
               
               
                 E375A 
                 0.627 
                 0.507 
                 0.557 
                 0.683 
                 0.000 
                 0.014 
               
               
                 E375G 
                 1.596 
                 1.299 
                 2.025 
                 1.806 
                 0.209 
                 0.265 
               
               
                 E375R 
                 0.937 
                 1.132 
                 1.529 
                 1.318 
                 0.201 
                 0.260 
               
               
                 K376D 
                 0.458 
                 0.312 
                 0.518 
                 0.515 
                 0.064 
                 0.026 
               
               
                 K376E 
                 1.572 
                 1.094 
                 1.572 
                 1.674 
                 0.213 
                 0.174 
               
               
                 K376Q 
                 0.727 
                 0.940 
                 0.910 
                 0.846 
                 0.116 
                 0.102 
               
               
                 K376R 
                 2.086 
                 1.351 
                 1.704 
                 2.690 
                 0.539 
                 0.279 
               
               
                 K376T 
                 0.847 
                 1.001 
                 1.026 
                 1.135 
                 0.153 
                 0.064 
               
               
                 K376V 
                 0.834 
                 0.861 
                 1.036 
                 1.021 
                 0.033 
                 0.026 
               
               
                 K376Y 
                 1.316 
                 0.777 
                 1.353 
                 0.747 
                 0.125 
                 0.097 
               
               
                 G377D 
                 1.159 
                 1.332 
                 1.285 
                 1.763 
                 0.202 
                 0.186 
               
               
                 G377E 
                 0.877 
                 0.926 
                 1.144 
                 1.189 
                 0.092 
                 0.088 
               
               
                 G377H 
                 3.037 
                 3.432 
                 4.460 
                 3.598 
                 0.372 
                 0.364 
               
               
                 G377K 
                 3.445 
                 4.101 
                 6.405 
                 4.911 
                 0.283 
                 0.245 
               
               
                 G377R 
                 1.096 
                 1.257 
                 1.312 
                 1.191 
                 0.077 
                 0.085 
               
               
                 G377S 
                 0.453 
                 0.452 
                 0.492 
                 0.457 
                 0.034 
                 0.036 
               
               
                 G377T 
                 2.198 
                 2.313 
                 2.474 
                 2.522 
                 0.424 
                 0.461 
               
               
                 F380W 
                 17.497 
                 27.987 
                 25.734 
                 29.353 
                 2.566 
                 2.716 
               
               
                 T381S 
                 2.861 
                 3.161 
                 3.886 
                 3.558 
                 0.521 
                 0.367 
               
               
                 R383I 
                 1.959 
                 6.936 
                 10.340 
                 6.820 
                 0.655 
                 0.513 
               
               
                 R383S 
                 2.429 
                 2.548 
                 3.228 
                 3.044 
                 0.339 
                 0.321 
               
               
                 K385A 
                 0.479 
                 0.669 
                 0.604 
                 0.754 
                 0.028 
                 0.000 
               
               
                 K385Q 
                 1.746 
                 2.089 
                 2.403 
                 2.609 
                 0.217 
                 0.196 
               
               
                 K385V 
                 1.232 
                 1.750 
                 1.387 
                 1.410 
                 0.071 
                 0.042 
               
               
                 E389A 
                 6.872 
                 10.944 
                 21.081 
                 24.610 
                 0.449 
                 0.449 
               
               
                 E389G 
                 0.166 
                 0.203 
                 0.188 
                 0.284 
                 0.004 
                 0.000 
               
               
                 E389L 
                 1.814 
                 2.142 
                 2.598 
                 2.403 
                 0.370 
                 0.303 
               
               
                 E389Q 
                 2.547 
                 3.432 
                 3.459 
                 3.423 
                 0.411 
                 0.437 
               
               
                 E389S 
                 1.847 
                 2.640 
                 3.059 
                 2.456 
                 0.000 
                 0.007 
               
               
                 E392A 
                 1.797 
                 1.370 
                 2.021 
                 2.133 
                 0.147 
                 0.136 
               
               
                 E392F 
                 1.575 
                 1.407 
                 1.821 
                 2.023 
                 0.071 
                 0.079 
               
               
                 E392Q 
                 5.826 
                 4.653 
                 6.583 
                 4.364 
                 0.693 
                 0.729 
               
               
                 E392R 
                 4.555 
                 5.306 
                 5.900 
                 6.548 
                 0.218 
                 0.193 
               
               
                 E392V 
                 3.817 
                 2.936 
                 4.747 
                 4.544 
                 0.367 
                 0.291 
               
               
                 Q393F 
                 1.754 
                 2.186 
                 2.455 
                 2.222 
                 0.260 
                 0.226 
               
               
                 Q393M 
                 1.252 
                 1.826 
                 1.749 
                 1.588 
                 0.028 
                 0.049 
               
               
                 S395A 
                 4.220 
                 6.127 
                 8.788 
                 6.906 
                 1.141 
                 0.856 
               
               
                 S395H 
                 1.609 
                 2.261 
                 2.574 
                 2.564 
                 0.323 
                 0.268 
               
               
                 E396A 
                 1.135 
                 1.184 
                 1.497 
                 1.524 
                 0.126 
                 0.149 
               
               
                 E396H 
                 0.357 
                 0.532 
                 0.751 
                 0.684 
                 0.069 
                 0.022 
               
               
                 E396Q 
                 1.310 
                 1.625 
                 1.611 
                 1.559 
                 0.162 
                 0.160 
               
               
                 E396S 
                 3.375 
                 5.709 
                 5.274 
                 6.380 
                 0.146 
                 0.129 
               
               
                 Y399T 
                 2.538 
                 3.250 
                 3.313 
                 3.989 
                 0.000 
                 0.002 
               
               
                 Y399V 
                 2.738 
                 2.697 
                 3.028 
                 3.129 
                 0.484 
                 0.557 
               
               
                 Y399W 
                 1.400 
                 1.883 
                 1.715 
                 1.946 
                 0.236 
                 0.233 
               
               
                 S401A 
                 2.636 
                 3.171 
                 3.216 
                 3.148 
                 0.447 
                 0.410 
               
               
                 S401E 
                 1.685 
                 1.601 
                 2.110 
                 2.060 
                 0.344 
                 0.309 
               
               
                 S404A 
                 1.288 
                 1.635 
                 1.924 
                 1.724 
                 0.000 
                 0.019 
               
               
                 L406F 
                 0.706 
                 0.490 
                 0.867 
                 0.716 
                 0.000 
                 0.000 
               
               
                 L406N 
                 0.617 
                 0.795 
                 0.943 
                 1.044 
                 0.060 
                 0.070 
               
               
                 S407A 
                 2.428 
                 2.949 
                 3.432 
                 3.255 
                 0.389 
                 0.548 
               
               
                 S407D 
                 2.090 
                 5.790 
                 5.038 
                 5.682 
                 0.569 
                 0.575 
               
               
                 S407P 
                 2.660 
                 2.708 
                 3.812 
                 3.301 
                 0.261 
                 0.366 
               
               
                 A412Q 
                 2.001 
                 2.918 
                 2.925 
                 2.902 
                 0.279 
                 0.247 
               
               
                 A412R 
                 4.562 
                 5.132 
                 6.390 
                 6.347 
                 0.570 
                 0.596 
               
               
                 A412V 
                 2.581 
                 3.451 
                 3.789 
                 3.511 
                 0.189 
                 0.189 
               
               
                 D416L 
                 0.610 
                 0.817 
                 0.737 
                 1.043 
                 0.130 
                 0.160 
               
               
                 D418R 
                 4.541 
                 4.847 
                 5.347 
                 5.438 
                 0.406 
                 0.583 
               
               
                 A419H 
                 10.409 
                 20.311 
                 25.109 
                 38.221 
                 2.214 
                 2.293 
               
               
                 A419K 
                 12.835 
                 10.298 
                 24.536 
                 208.289 
                 2.556 
                 3.173 
               
               
                 D421A 
                 5.968 
                 5.617 
                 6.094 
                 16.940 
                 0.761 
                 0.764 
               
               
                 D421H 
                 48.012 
                 48.012 
                 160.106 
                 32.481 
                 16.300 
                 28.113 
               
               
                 D421K 
                 5.527 
                 5.225 
                 6.864 
                 5.346 
                 0.523 
                 0.725 
               
               
                 D421N 
                 9.060 
                 8.635 
                 10.039 
                 8.645 
                 1.502 
                 1.422 
               
               
                 D421Q 
                 7.529 
                 5.581 
                 7.858 
                 8.016 
                 0.842 
                 0.994 
               
               
                 D421R 
                 6.637 
                 5.463 
                 9.211 
                 7.537 
                 0.815 
                 0.737 
               
               
                 D421S 
                 5.556 
                 5.355 
                 7.899 
                 8.898 
                 0.869 
                 0.762 
               
               
                 A425G 
                 10.421 
                 8.827 
                 7.796 
                 10.676 
                 0.827 
                 1.189 
               
               
                 G427Q 
                 1.008 
                 1.252 
                 1.342 
                 1.230 
                 0.031 
                 0.106 
               
               
                 G427T 
                 1.330 
                 1.380 
                 1.664 
                 1.643 
                 0.080 
                 0.065 
               
               
                 V428L 
                 2.138 
                 2.769 
                 2.930 
                 3.029 
                 0.053 
                 0.030 
               
               
                 D431E 
                 2.810 
                 2.220 
                 1.972 
                 2.112 
                 0.519 
                 0.438 
               
               
                 D431H 
                 2.154 
                 3.185 
                 4.017 
                 3.028 
                 0.294 
                 0.301 
               
               
                 D431K 
                 8.123 
                 16.953 
                 19.563 
                 11.575 
                 2.272 
                 2.339 
               
               
                 D431L 
                 1.211 
                 1.215 
                 1.564 
                 1.448 
                 0.164 
                 0.170 
               
               
                 D431N 
                 11.819 
                 12.063 
                 16.358 
                 15.131 
                 1.601 
                 1.399 
               
               
                 D431Q 
                 6.077 
                 9.828 
                 14.157 
                 10.760 
                 1.533 
                 1.153 
               
               
                 D431S 
                 14.523 
                 10.220 
                 11.338 
                 9.075 
                 0.853 
                 0.829 
               
               
                 F433A 
                 4.035 
                 4.673 
                 5.943 
                 4.649 
                 0.581 
                 0.595 
               
               
                 F433H 
                 1.836 
                 2.397 
                 2.574 
                 2.108 
                 0.347 
                 0.356 
               
               
                 F433I 
                 2.754 
                 2.643 
                 2.990 
                 2.299 
                 0.338 
                 0.382 
               
               
                 F433K 
                 17.815 
                 14.495 
                 16.240 
                 49.615 
                 1.806 
                 1.790 
               
               
                 F433R 
                 8.198 
                 6.719 
                 10.572 
                 8.960 
                 1.113 
                 0.857 
               
               
                 F433T 
                 6.005 
                 5.941 
                 9.716 
                 8.019 
                 1.327 
                 1.542 
               
               
                 F433V 
                 10.645 
                 7.762 
                 150.315 
                 8.696 
                 2.415 
                 1.505 
               
               
                 F433W 
                 0.526 
                 0.795 
                 0.784 
                 0.903 
                 0.082 
                 0.068 
               
               
                 P437I 
                 0.759 
                 0.996 
                 1.130 
                 1.066 
                 0.027 
                 0.019 
               
               
                 M438A 
                 1.996 
                 1.518 
                 2.125 
                 2.060 
                 0.214 
                 0.210 
               
               
                 M438D 
                 2.849 
                 2.522 
                 3.002 
                 2.857 
                 0.305 
                 0.074 
               
               
                 M438E 
                 4.681 
                 4.992 
                 5.386 
                 5.680 
                 0.431 
                 0.518 
               
               
                 M438L 
                 10.127 
                 5.268 
                 6.663 
                 11.324 
                 0.670 
                 0.739 
               
               
                 M438N 
                 6.172 
                 5.531 
                 8.050 
                 5.568 
                 0.649 
                 0.662 
               
               
                 M438T 
                 2.218 
                 2.411 
                 2.308 
                 2.500 
                 0.309 
                 0.304 
               
               
                 E439A 
                 3.557 
                 4.432 
                 4.883 
                 4.235 
                 0.568 
                 0.596 
               
               
                 E439A 
                 1.099 
                 0.998 
                 1.694 
                 1.470 
                 0.080 
                 0.109 
               
               
                 E439C 
                 0.148 
                 0.256 
                 0.286 
                 0.286 
                 0.042 
                 0.045 
               
               
                 E439K 
                 0.466 
                 0.588 
                 0.580 
                 0.616 
                 0.077 
                 0.065 
               
               
                 E439P 
                 2.868 
                 3.736 
                 3.394 
                 3.267 
                 0.529 
                 0.490 
               
               
                 E439Q 
                 1.070 
                 0.848 
                 1.087 
                 1.080 
                 0.116 
                 0.115 
               
               
                 E439T 
                 1.965 
                 1.889 
                 2.179 
                 2.323 
                 0.313 
                 0.263 
               
               
                 T440D 
                 4.148 
                 4.443 
                 4.931 
                 3.533 
                 0.568 
                 0.651 
               
               
                 T440H 
                 2.317 
                 1.982 
                 3.297 
                 2.595 
                 0.147 
                 0.196 
               
               
                 T440M 
                 3.397 
                 3.305 
                 2.878 
                 2.873 
                 0.254 
                 0.367 
               
               
                 T440P 
                 3.562 
                 3.593 
                 3.987 
                 3.277 
                 0.540 
                 0.566 
               
               
                 T440S 
                 2.522 
                 2.207 
                 2.533 
                 2.895 
                 0.283 
                 0.284 
               
               
                 E441F 
                 1.402 
                 1.407 
                 1.813 
                 1.560 
                 0.204 
                 0.178 
               
               
                 E442G 
                 2.871 
                 3.340 
                 3.193 
                 3.347 
                 0.327 
                 0.367 
               
               
                 P443E 
                 0.907 
                 0.710 
                 0.856 
                 0.928 
                 0.044 
                 0.063 
               
               
                 P443F 
                 1.830 
                 2.370 
                 2.683 
                 2.321 
                 0.301 
                 0.286 
               
               
                 P443G 
                 4.077 
                 2.921 
                 9.751 
                 4.614 
                 0.835 
                 0.756 
               
               
                 Q444E 
                 8.293 
                 3.861 
                 6.800 
                 6.213 
                 0.581 
                 0.594 
               
               
                 Q444H 
                 3.823 
                 3.936 
                 5.746 
                 4.710 
                 0.486 
                 0.513 
               
               
                 Q444V 
                 2.193 
                 2.107 
                 2.847 
                 2.583 
                 0.384 
                 0.284 
               
               
                 I445M 
                 5.265 
                 4.438 
                 4.480 
                 4.489 
                 0.773 
                 0.691 
               
               
                 I445N 
                 3.375 
                 4.024 
                 3.592 
                 3.515 
                 0.499 
                 0.455 
               
               
                 I445W 
                 2.289 
                 2.694 
                 2.683 
                 2.695 
                 0.314 
                 0.296 
               
               
                 Y447E 
                 2.373 
                 2.464 
                 2.363 
                 2.685 
                 0.391 
                 0.345 
               
               
                 Y447G 
                 0.945 
                 1.352 
                 1.358 
                 1.401 
                 0.187 
                 0.162 
               
               
                 Y447P 
                 0.991 
                 1.383 
                 1.379 
                 1.490 
                 0.190 
                 0.183 
               
               
                 positive 
                 2.919 
                 2.173 
                 2.773 
                 2.105 
                 0.145 
                 0.178 
               
               
                 control 
                 3.984 
                 4.463 
                 4.215 
                 4.823 
                 0.189 
                 0.253 
               
               
                 (OHO) 
                 3 
                 2.725 
                 3 
                 3.325 
                 0.1 
                 0.125 
               
               
                   
                 2.501 
                 2.883 
                 2.370 
                 3.158 
                 0.452 
                 0.522 
               
               
                   
                 7.629 
                 2.989 
                 10.835 
                 3.914 
                 0.485 
                 0.219 
               
               
                   
                 5.783 
                 5.356 
                 2.609 
                 3.643 
                 0.542 
                 0.402 
               
               
                   
                 5.279 
                 5.422 
                 2.815 
                 4.026 
                 0.618 
                 0.401 
               
               
                   
                 4.775 
                 4.385 
                 2.845 
                 3.327 
                 0.718 
                 0.540 
               
               
                   
                 3.617 
                 4.264 
                 3.322 
                 3.427 
                 0.633 
                 0.479 
               
               
                   
                 5.881 
                 4.511 
                 5.518 
                 4.359 
                 0.743 
                 0.848 
               
               
                   
                 6.754 
                 4.932 
                 3.902 
                 4.120 
                 0.665 
                 0.724 
               
               
                   
                 3.911 
                 3.494 
                 3.911 
                 5.179 
                 0.726 
                 0.841 
               
               
                   
                 5.406 
                 7.559 
                 4.018 
                 4.620 
                 0.735 
                 0.429 
               
               
                   
                 4.015 
                 3.887 
                 3.9400 
                 3.4080 
                 0.3340 
                 0.3410 
               
               
                   
                 2.604 
                 2.339 
                 2.4430 
                 2.3910 
                 0.2350 
                 0.2330 
               
               
                   
                 3.736 
                 3.473 
                 3.6210 
                 3.0560 
                 0.3100 
                 0.2770 
               
               
                   
                 3.759 
                 3.509 
                 3.6330 
                 3.0490 
                 0.3600 
                 0.3030 
               
               
                   
               
               
                 n/a (not available; e.g., beyond detection limit) 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 12 
               
             
            
               
                   
               
               
                 Percent (%) Activity 
               
            
           
           
               
               
               
            
               
                   
                 duplicate 1 
                 duplicate 2 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                 % activity 
                 % activity 
                 % activity 
                 % activity 
                 % activity 
                 % activity 
               
               
                   
                 at 
                 37° C. + m- 
                 37° C. + m- 
                 at 
                 37° C. + m- 
                 37° C. + m- 
               
               
                   
                 37° C./4° C. 
                 cresol/37° C. 
                 cresol/4° C. 
                 37° C./4° C. 
                 cresol/37° C. 
                 cresol/4° C. 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 L001A 
                 117.908 
                 8.13 
                 9.59 
                 127.997 
                 9.179 
                 11.75 
               
               
                 L001E 
                 107.231 
                 13.14 
                 14.09 
                 125.207 
                 10.727 
                 13.43 
               
               
                 L001G 
                 171.264 
                 9.23 
                 15.80 
                 115.952 
                 4.586 
                 5.32 
               
               
                 L001Q 
                 119.435 
                 10.13 
                 12.09 
                 88.763 
                 11.121 
                 9.87 
               
               
                 L001R 
                 117.366 
                 6.96 
                 8.17 
                 160.410 
                 5.623 
                 9.02 
               
               
                 P006A 
                 137.875 
                 9.88 
                 13.63 
                 108.946 
                 12.446 
                 13.56 
               
               
                 V008M 
                 134.884 
                 0.00 
                 0.00 
                 119.772 
                 0.477 
                 0.57 
               
               
                 I009Q 
                 104.922 
                 6.61 
                 6.94 
                 124.934 
                 6.303 
                 7.87 
               
               
                 P010G 
                 109.772 
                 15.00 
                 16.47 
                 121.986 
                 16.570 
                 20.21 
               
               
                 P010H 
                 131.924 
                 0.00 
                 0.00 
                 112.990 
                 0.000 
                 0.00 
               
               
                 N011S 
                 152.320 
                 7.16 
                 10.90 
                 131.289 
                 5.067 
                 6.65 
               
               
                 V012E 
                 48.208 
                 14.18 
                 6.83 
                 100.163 
                 16.896 
                 16.92 
               
               
                 V012I 
                 128.745 
                 2.94 
                 3.78 
                 170.812 
                 1.371 
                 2.34 
               
               
                 V012K 
                 146.600 
                 13.31 
                 19.52 
                 114.264 
                 14.311 
                 16.35 
               
               
                 F014V 
                 154.167 
                 1.35 
                 2.08 
                 146.667 
                 0.000 
                 0.00 
               
               
                 L015M 
                 113.747 
                 1.66 
                 1.88 
                 83.970 
                 1.887 
                 1.58 
               
               
                 A020S 
                 188.889 
                 14.64 
                 27.64 
                 118.821 
                 15.153 
                 18.00 
               
               
                 S022T 
                 111.203 
                 20.27 
                 22.54 
                 86.404 
                 22.854 
                 19.75 
               
               
                 L026M 
                 136.775 
                 11.05 
                 15.11 
                 138.989 
                 10.446 
                 14.52 
               
               
                 K028R 
                 110.487 
                 10.74 
                 11.86 
                 109.467 
                 10.270 
                 11.24 
               
               
                 F029R 
                 154.644 
                 7.58 
                 11.72 
                 121.707 
                 7.613 
                 9.27 
               
               
                 F029S 
                 118.119 
                 7.01 
                 8.28 
                 97.400 
                 8.037 
                 7.83 
               
               
                 F029T 
                 126.740 
                 11.96 
                 15.16 
                 120.619 
                 10.266 
                 12.38 
               
               
                 P032C 
                 128.649 
                 1.26 
                 1.62 
                 127.446 
                 7.491 
                 9.55 
               
               
                 L033G 
                 121.201 
                 0.15 
                 0.18 
                 89.571 
                 4.147 
                 3.71 
               
               
                 D034W 
                 146.765 
                 15.23 
                 22.35 
                 146.729 
                 14.650 
                 21.50 
               
               
                 M035V 
                 81.285 
                 16.09 
                 13.08 
                 102.034 
                 3.528 
                 3.60 
               
               
                 S036H 
                 106.222 
                 9.93 
                 10.55 
                 150.931 
                 2.291 
                 3.46 
               
               
                 S036N 
                 112.045 
                 19.15 
                 21.46 
                 92.069 
                 30.268 
                 27.87 
               
               
                 L037M 
                 79.268 
                 10.77 
                 8.54 
                 87.376 
                 9.065 
                 7.92 
               
               
                 F040L 
                 135.036 
                 7.88 
                 10.64 
                 105.252 
                 8.703 
                 9.16 
               
               
                 I046L 
                 132.507 
                 12.79 
                 16.95 
                 112.944 
                 16.667 
                 18.82 
               
               
                 N047D 
                 115.797 
                 1.24 
                 1.44 
                 111.869 
                 0.796 
                 0.89 
               
               
                 N047W 
                 104.703 
                 0.00 
                 0.00 
                 109.880 
                 3.728 
                 4.10 
               
               
                 A048N 
                 114.954 
                 5.59 
                 6.43 
                 51.931 
                 3.778 
                 1.96 
               
               
                 T049R 
                 122.704 
                 5.81 
                 7.13 
                 90.760 
                 5.363 
                 4.87 
               
               
                 G050D 
                 93.824 
                 7.85 
                 7.36 
                 95.934 
                 8.742 
                 8.39 
               
               
                 G050M 
                 157.686 
                 8.99 
                 14.18 
                 139.048 
                 12.115 
                 16.85 
               
               
                 G052N 
                 96.148 
                 15.98 
                 15.37 
                 142.502 
                 7.748 
                 11.04 
               
               
                 G052T 
                 116.407 
                 21.23 
                 24.71 
                 117.075 
                 32.310 
                 37.83 
               
               
                 G052S 
                 98.513 
                 23.49 
                 23.14 
                 98.199 
                 28.833 
                 28.31 
               
               
                 V058C 
                 92.507 
                 16.05 
                 14.85 
                 99.162 
                 16.141 
                 16.01 
               
               
                 V058K 
                 217.914 
                 38.66 
                 84.24 
                 217.914 
                 38.655 
                 84.24 
               
               
                 V058R 
                 96.905 
                 56.55 
                 54.80 
                 102.858 
                 65.305 
                 67.17 
               
               
                 V058N 
                 129.167 
                 12.90 
                 16.67 
                 129.787 
                 11.475 
                 14.89 
               
               
                 V058Y 
                 102.981 
                 36.23 
                 37.31 
                 141.299 
                 41.728 
                 58.96 
               
               
                 V058Q 
                 154.383 
                 8.49 
                 13.11 
                 293.510 
                 4.804 
                 14.10 
               
               
                 V058P 
                 83.304 
                 21.15 
                 17.62 
                 173.652 
                 17.262 
                 29.98 
               
               
                 V058H 
                 200.264 
                 10.88 
                 21.79 
                 181.750 
                 13.067 
                 23.75 
               
               
                 D068P 
                 99.070 
                 0.47 
                 0.47 
                 83.721 
                 102.222 
                 85.58 
               
               
                 S069T 
                 138.609 
                 10.82 
                 15.00 
                 122.579 
                 8.985 
                 11.01 
               
               
                 I070P 
                 101.713 
                 0.77 
                 0.78 
                 99.749 
                 2.014 
                 2.01 
               
               
                 I070V 
                 170.462 
                 13.97 
                 23.82 
                 136.849 
                 10.885 
                 14.90 
               
               
                 V073Q 
                 121.337 
                 6.51 
                 7.90 
                 107.094 
                 8.186 
                 8.77 
               
               
                 V073R 
                 137.931 
                 2.50 
                 3.45 
                 100.125 
                 7.338 
                 7.35 
               
               
                 T074E 
                 133.241 
                 17.22 
                 22.94 
                 100.965 
                 16.172 
                 16.33 
               
               
                 T074M 
                 115.290 
                 12.04 
                 13.88 
                 103.629 
                 10.765 
                 11.16 
               
               
                 T074N 
                 91.870 
                 10.96 
                 10.06 
                 157.218 
                 6.811 
                 10.71 
               
               
                 T074P 
                 108.323 
                 12.24 
                 13.26 
                 166.227 
                 10.008 
                 16.64 
               
               
                 T074R 
                 80.681 
                 7.44 
                 6.01 
                 130.000 
                 2.158 
                 2.80 
               
               
                 T074V 
                 115.093 
                 7.40 
                 8.52 
                 114.063 
                 5.479 
                 6.25 
               
               
                 V075M 
                 134.460 
                 0.24 
                 0.33 
                 121.527 
                 2.120 
                 2.58 
               
               
                 K082L 
                 114.758 
                 20.79 
                 23.86 
                 251.869 
                 10.721 
                 27.00 
               
               
                 K082N 
                 106.059 
                 23.32 
                 24.73 
                 95.104 
                 26.541 
                 25.24 
               
               
                 I083V 
                 140.151 
                 29.88 
                 41.88 
                 137.296 
                 28.133 
                 38.63 
               
               
                 I083Q 
                 112.163 
                 27.02 
                 30.30 
                 188.798 
                 13.881 
                 26.21 
               
               
                 I083S 
                 104.637 
                 26.70 
                 27.94 
                 95.351 
                 26.667 
                 25.43 
               
               
                 I083G 
                 106.239 
                 22.54 
                 23.95 
                 76.381 
                 32.208 
                 24.60 
               
               
                 S084E 
                 124.762 
                 6.27 
                 7.82 
                 113.410 
                 6.833 
                 7.75 
               
               
                 S084F 
                 83.291 
                 2.55 
                 2.12 
                 91.007 
                 0.000 
                 0.00 
               
               
                 S084N 
                 144.922 
                 18.27 
                 26.47 
                 131.144 
                 22.092 
                 28.97 
               
               
                 S084R 
                 119.873 
                 10.92 
                 13.09 
                 203.099 
                 4.977 
                 10.11 
               
               
                 Q086A 
                 136.516 
                 14.24 
                 19.43 
                 156.132 
                 9.728 
                 15.19 
               
               
                 Q086H 
                 102.612 
                 7.14 
                 7.33 
                 129.600 
                 5.015 
                 6.50 
               
               
                 Q086K 
                 99.213 
                 25.40 
                 25.20 
                 65.455 
                 31.944 
                 20.91 
               
               
                 Q086S 
                 100.435 
                 6.81 
                 6.84 
                 103.218 
                 11.215 
                 11.58 
               
               
                 Q086T 
                 93.837 
                 10.24 
                 9.61 
                 179.465 
                 8.900 
                 15.97 
               
               
                 D087G 
                 81.742 
                 1.51 
                 1.23 
                 90.579 
                 6.190 
                 5.61 
               
               
                 D087L 
                 106.039 
                 14.76 
                 15.65 
                 101.493 
                 12.938 
                 13.13 
               
               
                 D087M 
                 110.964 
                 7.61 
                 8.44 
                 87.656 
                 16.438 
                 14.41 
               
               
                 D087S 
                 134.031 
                 8.15 
                 10.92 
                 139.728 
                 6.445 
                 9.01 
               
               
                 D087V 
                 114.107 
                 9.14 
                 10.43 
                 87.023 
                 15.922 
                 13.86 
               
               
                 D090E 
                 92.910 
                 14.26 
                 13.25 
                 161.281 
                 6.221 
                 10.03 
               
               
                 D090N 
                 111.060 
                 12.14 
                 13.48 
                 98.631 
                 10.596 
                 10.45 
               
               
                 K093Q 
                 91.008 
                 5.82 
                 5.30 
                 95.448 
                 6.646 
                 6.34 
               
               
                 K093R 
                 103.617 
                 11.70 
                 12.12 
                 99.301 
                 16.362 
                 16.25 
               
               
                 K094D 
                 86.544 
                 6.52 
                 5.64 
                 102.107 
                 9.897 
                 10.11 
               
               
                 K094R 
                 125.373 
                 8.96 
                 11.23 
                 108.690 
                 9.905 
                 10.77 
               
               
                 T097C 
                 165.152 
                 8.07 
                 13.33 
                 81.715 
                 17.228 
                 14.08 
               
               
                 T097D 
                 123.654 
                 8.55 
                 10.58 
                 117.522 
                 10.994 
                 12.92 
               
               
                 T097E 
                 127.190 
                 15.57 
                 19.80 
                 115.106 
                 16.143 
                 18.58 
               
               
                 T097L 
                 118.465 
                 23.10 
                 27.36 
                 103.589 
                 24.174 
                 25.04 
               
               
                 N104R 
                 114.673 
                 9.70 
                 11.12 
                 118.421 
                 8.530 
                 10.10 
               
               
                 A120H 
                 94.107 
                 8.28 
                 7.80 
                 113.015 
                 6.903 
                 7.80 
               
               
                 D127R 
                 56.439 
                 70.47 
                 39.77 
                 58.702 
                 34.171 
                 20.06 
               
               
                 V128I 
                 113.654 
                 10.97 
                 12.47 
                 102.656 
                 14.819 
                 15.21 
               
               
                 N131M 
                 177.000 
                 1.86 
                 3.29 
                 76.888 
                 2.811 
                 2.16 
               
               
                 N131R 
                 94.253 
                 21.30 
                 20.07 
                 95.930 
                 19.376 
                 18.59 
               
               
                 N131V 
                 137.681 
                 10.22 
                 14.07 
                 104.920 
                 10.907 
                 11.44 
               
               
                 R132L 
                 98.578 
                 10.34 
                 10.19 
                 91.685 
                 14.498 
                 13.29 
               
               
                 Q138L 
                 107.831 
                 25.45 
                 27.44 
                 91.627 
                 22.814 
                 20.90 
               
               
                 Q140K 
                 176.600 
                 10.93 
                 19.30 
                 109.815 
                 12.522 
                 13.75 
               
               
                 N141R 
                 103.411 
                 4.35 
                 4.50 
                 115.682 
                 2.292 
                 2.65 
               
               
                 N141S 
                 131.758 
                 4.66 
                 6.13 
                 109.527 
                 5.529 
                 6.06 
               
               
                 N141W 
                 130.644 
                 5.19 
                 6.78 
                 104.783 
                 6.391 
                 6.70 
               
               
                 V142D 
                 114.185 
                 4.39 
                 5.02 
                 146.066 
                 2.098 
                 3.06 
               
               
                 V142G 
                 117.686 
                 13.21 
                 15.55 
                 90.256 
                 13.510 
                 12.19 
               
               
                 V142K 
                 109.485 
                 14.77 
                 16.17 
                 154.599 
                 15.621 
                 24.15 
               
               
                 V142N 
                 155.556 
                 15.33 
                 23.84 
                 103.880 
                 14.771 
                 15.34 
               
               
                 V142P 
                 166.998 
                 13.91 
                 23.23 
                 97.338 
                 15.397 
                 14.99 
               
               
                 V142Q 
                 149.666 
                 8.90 
                 13.32 
                 99.957 
                 9.830 
                 9.83 
               
               
                 V142R 
                 149.441 
                 12.38 
                 18.50 
                 103.622 
                 12.272 
                 12.72 
               
               
                 V142S 
                 170.778 
                 8.73 
                 14.92 
                 117.035 
                 16.900 
                 19.78 
               
               
                 V142T 
                 223.936 
                 11.48 
                 25.70 
                 123.650 
                 11.709 
                 14.48 
               
               
                 Q143G 
                 143.600 
                 13.88 
                 19.94 
                 98.837 
                 16.096 
                 15.91 
               
               
                 Q143K 
                 200.468 
                 14.32 
                 28.70 
                 136.421 
                 20.747 
                 28.30 
               
               
                 L144R 
                 136.247 
                 10.71 
                 14.59 
                 111.482 
                 10.182 
                 11.35 
               
               
                 L144T 
                 129.746 
                 14.68 
                 19.05 
                 108.923 
                 11.961 
                 13.03 
               
               
                 L146P 
                 116.626 
                 1.15 
                 1.34 
                 115.601 
                 3.429 
                 3.96 
               
               
                 T147S 
                 142.175 
                 3.93 
                 5.59 
                 130.287 
                 2.605 
                 3.39 
               
               
                 T150N 
                 140.724 
                 6.27 
                 8.82 
                 116.923 
                 6.725 
                 7.86 
               
               
                 T150S 
                 107.327 
                 6.40 
                 6.87 
                 142.000 
                 6.087 
                 8.64 
               
               
                 E151A 
                 103.310 
                 12.11 
                 12.51 
                 126.047 
                 11.783 
                 14.85 
               
               
                 E151L 
                 132.125 
                 4.90 
                 6.48 
                 121.830 
                 6.264 
                 7.63 
               
               
                 E151S 
                 115.423 
                 6.20 
                 7.15 
                 136.397 
                 4.695 
                 6.40 
               
               
                 E151T 
                 128.337 
                 0.00 
                 0.00 
                 110.300 
                 0.000 
                 0.00 
               
               
                 E151V 
                 111.531 
                 7.31 
                 8.15 
                 99.647 
                 7.420 
                 7.39 
               
               
                 E151W 
                 158.415 
                 1.15 
                 1.83 
                 94.919 
                 0.895 
                 0.85 
               
               
                 K152T 
                 149.169 
                 5.57 
                 8.31 
                 136.747 
                 3.558 
                 4.87 
               
               
                 K152W 
                 122.313 
                 2.47 
                 3.02 
                 134.039 
                 2.868 
                 3.84 
               
               
                 E158S 
                 133.038 
                 0.00 
                 0.00 
                 102.519 
                 0.000 
                 0.00 
               
               
                 K162E 
                 67.857 
                 3.51 
                 2.38 
                 41.026 
                 30.000 
                 12.31 
               
               
                 L165F 
                 106.283 
                 11.82 
                 12.57 
                 96.667 
                 14.286 
                 13.81 
               
               
                 V166Q 
                 155.975 
                 13.35 
                 20.82 
                 117.990 
                 10.953 
                 12.92 
               
               
                 V166T 
                 183.384 
                 12.69 
                 23.26 
                 136.882 
                 13.056 
                 17.87 
               
               
                 E167D 
                 136.745 
                 10.01 
                 13.69 
                 162.637 
                 3.784 
                 6.15 
               
               
                 I169L 
                 140.177 
                 13.19 
                 18.49 
                 122.272 
                 15.528 
                 18.99 
               
               
                 K170R 
                 160.710 
                 8.24 
                 13.24 
                 97.128 
                 10.075 
                 9.79 
               
               
                 G172A 
                 167.554 
                 7.51 
                 12.59 
                 133.735 
                 7.207 
                 9.64 
               
               
                 K173R 
                 106.771 
                 9.80 
                 10.46 
                 134.300 
                 7.489 
                 10.06 
               
               
                 L174G 
                 114.130 
                 12.38 
                 14.13 
                 264.368 
                 13.478 
                 35.63 
               
               
                 L174N 
                 154.332 
                 13.27 
                 20.48 
                 126.186 
                 18.907 
                 23.86 
               
               
                 L174T 
                 124.819 
                 13.06 
                 16.30 
                 144.876 
                 6.098 
                 8.83 
               
               
                 N178K 
                 166.871 
                 5.27 
                 8.80 
                 103.154 
                 8.021 
                 8.27 
               
               
                 N178R 
                 199.596 
                 4.08 
                 8.15 
                 144.957 
                 3.943 
                 5.72 
               
               
                 H193Q 
                 213.585 
                 15.28 
                 32.64 
                 138.113 
                 18.326 
                 25.31 
               
               
                 K195T 
                 126.161 
                 22.48 
                 28.36 
                 237.097 
                 15.280 
                 36.23 
               
               
                 K195N 
                 130.253 
                 22.38 
                 29.15 
                 96.381 
                 25.487 
                 24.57 
               
               
                 K196E 
                 90.574 
                 36.80 
                 33.33 
                 154.091 
                 23.500 
                 36.21 
               
               
                 K196R 
                 106.100 
                 13.22 
                 14.02 
                 95.142 
                 17.663 
                 16.81 
               
               
                 F204P 
                 83.571 
                 84.62 
                 70.71 
                 82.418 
                 126.000 
                 103.85 
               
               
                 N205A 
                 139.223 
                 21.34 
                 29.71 
                 102.031 
                 18.735 
                 19.12 
               
               
                 N205E 
                 160.930 
                 19.30 
                 31.06 
                 93.313 
                 18.503 
                 17.27 
               
               
                 N205L 
                 107.472 
                 10.56 
                 11.35 
                 0.000 
                 #DIV/0! 
                 8.55 
               
               
                 N205T 
                 145.085 
                 10.05 
                 14.58 
                 110.627 
                 13.054 
                 14.44 
               
               
                 V206I 
                 189.274 
                 13.17 
                 24.92 
                 111.220 
                 15.575 
                 17.32 
               
               
                 K209R 
                 119.794 
                 11.90 
                 14.26 
                 79.535 
                 3.947 
                 3.14 
               
               
                 D212N 
                 112.626 
                 2.66 
                 3.00 
                 132.249 
                 5.352 
                 7.08 
               
               
                 D212S 
                 122.899 
                 8.35 
                 10.27 
                 147.936 
                 6.841 
                 10.12 
               
               
                 D213A 
                 183.830 
                 26.85 
                 49.36 
                 154.770 
                 13.699 
                 21.20 
               
               
                 D213M 
                 159.255 
                 6.83 
                 10.88 
                 98.365 
                 6.940 
                 6.83 
               
               
                 S215H 
                 109.069 
                 10.04 
                 10.95 
                 78.992 
                 5.758 
                 4.55 
               
               
                 S215M 
                 174.883 
                 4.20 
                 7.35 
                 74.943 
                 8.957 
                 6.71 
               
               
                 N219I 
                 254.438 
                 8.84 
                 22.49 
                 291.200 
                 11.264 
                 32.80 
               
               
                 E220V 
                 131.985 
                 7.43 
                 9.81 
                 113.610 
                 5.909 
                 6.71 
               
               
                 T222G 
                 153.033 
                 0.61 
                 0.94 
                 105.454 
                 0.793 
                 0.84 
               
               
                 T232F 
                 132.839 
                 12.43 
                 16.51 
                 62.882 
                 19.590 
                 12.32 
               
               
                 Q233G 
                 280.488 
                 0.00 
                 0.00 
                 127.368 
                 0.000 
                 0.00 
               
               
                 Q234M 
                 95.605 
                 22.31 
                 21.33 
                 80.766 
                 20.283 
                 16.38 
               
               
                 S235A 
                 129.818 
                 11.06 
                 14.36 
                 120.916 
                 12.026 
                 14.54 
               
               
                 V237C 
                 138.042 
                 0.00 
                 0.00 
                 116.384 
                 0.000 
                 0.00 
               
               
                 V237H 
                 122.112 
                 12.43 
                 15.18 
                 145.253 
                 7.407 
                 10.76 
               
               
                 V237T 
                 167.105 
                 21.26 
                 35.53 
                 126.020 
                 21.457 
                 27.04 
               
               
                 A238E 
                 94.878 
                 8.17 
                 7.76 
                 142.167 
                 6.682 
                 9.50 
               
               
                 A238H 
                 59.585 
                 26.09 
                 15.54 
                 204.683 
                 8.345 
                 17.08 
               
               
                 T240A 
                 141.283 
                 9.14 
                 12.92 
                 144.667 
                 9.063 
                 13.11 
               
               
                 T240Q 
                 162.763 
                 14.76 
                 24.02 
                 120.980 
                 13.776 
                 16.67 
               
               
                 R248A 
                 113.237 
                 1.05 
                 1.19 
                 124.650 
                 2.408 
                 3.00 
               
               
                 E249V 
                 142.752 
                 15.29 
                 21.83 
                 111.068 
                 16.462 
                 18.28 
               
               
                 P257G 
                 125.220 
                 0.78 
                 0.98 
                 112.803 
                 0.677 
                 0.76 
               
               
                 K260M 
                 116.690 
                 8.58 
                 10.01 
                 97.396 
                 7.273 
                 7.08 
               
               
                 S261A 
                 57.547 
                 67.52 
                 38.86 
                 86.173 
                 54.021 
                 46.55 
               
               
                 S261K 
                 161.931 
                 16.05 
                 26.00 
                 116.159 
                 22.820 
                 26.51 
               
               
                 S261N 
                 142.901 
                 10.46 
                 14.95 
                 35.529 
                 13.403 
                 4.76 
               
               
                 A267T 
                 196.154 
                 35.29 
                 69.23 
                 111.579 
                 38.679 
                 43.16 
               
               
                 F273H 
                 122.647 
                 6.00 
                 7.35 
                 119.037 
                 5.973 
                 7.11 
               
               
                 F273Y 
                 119.713 
                 7.78 
                 9.32 
                 102.772 
                 9.634 
                 9.90 
               
               
                 Q276H 
                 74.908 
                 8.93 
                 6.69 
                 106.393 
                 10.065 
                 10.71 
               
               
                 Q276M 
                 98.323 
                 5.64 
                 5.55 
                 104.948 
                 0.000 
                 0.00 
               
               
                 Q276R 
                 121.431 
                 10.93 
                 13.27 
                 150.180 
                 8.778 
                 13.18 
               
               
                 Q276S 
                 110.643 
                 9.95 
                 11.01 
                 138.696 
                 8.745 
                 12.13 
               
               
                 V277A 
                 140.765 
                 6.97 
                 9.82 
                 129.580 
                 5.167 
                 6.70 
               
               
                 V277E 
                 175.779 
                 3.75 
                 6.60 
                 195.598 
                 5.222 
                 10.21 
               
               
                 V277H 
                 129.434 
                 3.16 
                 4.09 
                 137.684 
                 7.014 
                 9.66 
               
               
                 V277K 
                 375.721 
                 13.21 
                 49.63 
                 373.799 
                 12.029 
                 44.96 
               
               
                 V277M 
                 137.138 
                 15.05 
                 20.64 
                 112.084 
                 14.851 
                 16.65 
               
               
                 V277N 
                 89.645 
                 7.29 
                 6.54 
                 273.386 
                 6.762 
                 18.49 
               
               
                 V277Q 
                 119.930 
                 5.70 
                 6.83 
                 116.151 
                 7.772 
                 9.03 
               
               
                 V277R 
                 96.071 
                 15.57 
                 14.96 
                 171.465 
                 9.801 
                 16.81 
               
               
                 V277S 
                 66.260 
                 7.65 
                 5.07 
                 144.916 
                 4.731 
                 6.86 
               
               
                 V277T 
                 101.010 
                 7.99 
                 8.07 
                 143.311 
                 7.788 
                 11.16 
               
               
                 L278E 
                 75.408 
                 5.11 
                 3.85 
                 100.179 
                 7.214 
                 7.23 
               
               
                 L278G 
                 122.274 
                 6.50 
                 7.94 
                 104.077 
                 7.887 
                 8.21 
               
               
                 K279H 
                 138.964 
                 14.99 
                 20.83 
                 123.183 
                 20.090 
                 24.75 
               
               
                 V287T 
                 145.345 
                 16.49 
                 23.97 
                 124.738 
                 12.019 
                 14.99 
               
               
                 T289S 
                 104.598 
                 0.98 
                 1.02 
                 98.234 
                 0.699 
                 0.69 
               
               
                 G291S 
                 184.581 
                 12.17 
                 22.47 
                 119.565 
                 4.156 
                 4.97 
               
               
                 G291V 
                 112.807 
                 19.87 
                 22.42 
                 151.039 
                 12.609 
                 19.05 
               
               
                 E292C 
                 127.307 
                 8.07 
                 10.27 
                 101.126 
                 8.905 
                 9.01 
               
               
                 E292F 
                 137.930 
                 6.17 
                 8.52 
                 132.340 
                 5.840 
                 7.73 
               
               
                 E292H 
                 170.153 
                 8.73 
                 14.85 
                 115.501 
                 11.775 
                 13.60 
               
               
                 E292R 
                 112.278 
                 12.61 
                 14.16 
                 129.890 
                 11.983 
                 15.56 
               
               
                 E292V 
                 163.075 
                 13.28 
                 21.66 
                 133.274 
                 11.847 
                 15.79 
               
               
                 T293A 
                 128.197 
                 3.38 
                 4.33 
                 57.524 
                 4.248 
                 2.44 
               
               
                 A298G 
                 212.422 
                 8.77 
                 18.63 
                 86.131 
                 9.322 
                 8.03 
               
               
                 L307G 
                 117.857 
                 0.00 
                 0.00 
                 91.528 
                 0.000 
                 0.00 
               
               
                 S308D 
                 127.652 
                 4.15 
                 5.30 
                 105.846 
                 2.907 
                 3.08 
               
               
                 S308K 
                 126.882 
                 1.33 
                 1.69 
                 99.169 
                 0.000 
                 0.00 
               
               
                 S308N 
                 170.413 
                 5.67 
                 9.66 
                 139.083 
                 5.907 
                 8.22 
               
               
                 I309E 
                 123.847 
                 16.25 
                 20.12 
                 129.940 
                 14.414 
                 18.73 
               
               
                 I309G 
                 102.601 
                 7.37 
                 7.56 
                 114.091 
                 6.458 
                 7.37 
               
               
                 I309L 
                 153.681 
                 9.58 
                 14.72 
                 106.948 
                 10.905 
                 11.66 
               
               
                 I309M 
                 123.425 
                 8.02 
                 9.90 
                 136.797 
                 7.065 
                 9.66 
               
               
                 I309N 
                 111.901 
                 6.98 
                 7.81 
                 97.361 
                 6.470 
                 6.30 
               
               
                 I309S 
                 169.951 
                 4.11 
                 6.98 
                 551.493 
                 0.862 
                 4.75 
               
               
                 I309T 
                 97.936 
                 7.63 
                 7.47 
                 148.864 
                 5.542 
                 8.25 
               
               
                 I309V 
                 113.138 
                 1.50 
                 1.70 
                 138.313 
                 3.470 
                 4.80 
               
               
                 M310G 
                 167.656 
                 11.44 
                 19.18 
                 110.739 
                 12.916 
                 14.30 
               
               
                 M310Q 
                 107.237 
                 27.81 
                 29.82 
                 106.323 
                 28.254 
                 30.04 
               
               
                 M313G 
                 138.095 
                 9.77 
                 13.49 
                 109.231 
                 10.141 
                 11.08 
               
               
                 M313H 
                 271.914 
                 3.71 
                 10.09 
                 197.024 
                 3.886 
                 7.66 
               
               
                 M313K 
                 118.882 
                 0.86 
                 1.02 
                 107.111 
                 0.555 
                 0.59 
               
               
                 M313P 
                 103.654 
                 4.98 
                 5.16 
                 132.802 
                 4.516 
                 6.00 
               
               
                 M313R 
                 157.272 
                 4.62 
                 7.27 
                 32.845 
                 8.296 
                 2.72 
               
               
                 M313T 
                 162.074 
                 7.04 
                 11.40 
                 99.844 
                 7.007 
                 7.00 
               
               
                 M313Y 
                 120.038 
                 7.52 
                 9.03 
                 103.011 
                 6.846 
                 7.05 
               
               
                 K314S 
                 141.924 
                 9.67 
                 13.73 
                 132.112 
                 9.066 
                 11.98 
               
               
                 K314Y 
                 243.011 
                 5.75 
                 13.98 
                 138.931 
                 10.989 
                 15.27 
               
               
                 S315A 
                 91.372 
                 16.51 
                 15.08 
                 137.153 
                 9.973 
                 13.68 
               
               
                 S315H 
                 151.244 
                 12.06 
                 18.24 
                 105.305 
                 10.867 
                 11.44 
               
               
                 S315Y 
                 170.968 
                 30.61 
                 52.33 
                 57.827 
                 39.503 
                 22.84 
               
               
                 L317A 
                 123.510 
                 6.97 
                 8.60 
                 132.724 
                 8.395 
                 11.14 
               
               
                 L317I 
                 187.477 
                 12.72 
                 23.84 
                 110.696 
                 10.670 
                 11.81 
               
               
                 L317K 
                 96.199 
                 3.45 
                 3.31 
                 134.204 
                 3.534 
                 4.74 
               
               
                 L317N 
                 127.382 
                 12.02 
                 15.31 
                 121.233 
                 14.528 
                 17.61 
               
               
                 L317R 
                 238.501 
                 3.87 
                 9.22 
                 99.467 
                 5.673 
                 5.64 
               
               
                 L317S 
                 90.929 
                 15.54 
                 14.13 
                 85.810 
                 6.423 
                 5.51 
               
               
                 L317T 
                 145.964 
                 6.96 
                 10.16 
                 154.334 
                 1.087 
                 1.68 
               
               
                 L317W 
                 163.704 
                 11.92 
                 19.51 
                 147.606 
                 10.270 
                 15.16 
               
               
                 L318D 
                 105.543 
                 17.43 
                 18.40 
                 97.970 
                 16.684 
                 16.35 
               
               
                 L318H 
                 99.907 
                 4.29 
                 4.29 
                 124.690 
                 7.363 
                 9.18 
               
               
                 L318R 
                 160.469 
                 5.63 
                 9.03 
                 120.872 
                 6.210 
                 7.51 
               
               
                 N321R 
                 164.842 
                 9.53 
                 15.71 
                 112.180 
                 8.613 
                 9.66 
               
               
                 N321S 
                 102.489 
                 8.29 
                 8.49 
                 108.732 
                 4.534 
                 4.93 
               
               
                 E324N 
                 104.618 
                 7.72 
                 8.08 
                 131.265 
                 9.124 
                 11.98 
               
               
                 T325E 
                 124.837 
                 14.44 
                 18.02 
                 106.457 
                 10.577 
                 11.26 
               
               
                 N328G 
                 197.098 
                 4.15 
                 8.18 
                 109.722 
                 7.233 
                 7.94 
               
               
                 N328Y 
                 180.981 
                 10.30 
                 18.64 
                 100.000 
                 10.500 
                 10.50 
               
               
                 T335S 
                 107.956 
                 11.57 
                 12.49 
                 125.286 
                 6.288 
                 7.88 
               
               
                 Q347A 
                 101.395 
                 10.89 
                 11.04 
                 96.213 
                 11.001 
                 10.58 
               
               
                 Q347G 
                 222.459 
                 8.37 
                 18.63 
                 207.054 
                 9.013 
                 18.66 
               
               
                 Q349M 
                 99.531 
                 11.98 
                 11.92 
                 108.042 
                 12.330 
                 13.32 
               
               
                 Q349R 
                 147.007 
                 11.76 
                 17.29 
                 104.545 
                 13.211 
                 13.81 
               
               
                 V351S 
                 130.819 
                 0.00 
                 0.00 
                 100.857 
                 0.000 
                 0.00 
               
               
                 I353V 
                 132.334 
                 10.45 
                 13.83 
                 138.025 
                 11.902 
                 16.43 
               
               
                 N356H 
                 100.000 
                 8.54 
                 8.54 
                 130.377 
                 3.912 
                 5.10 
               
               
                 N356S 
                 51.908 
                 0.00 
                 0.00 
                 125.692 
                 2.516 
                 3.16 
               
               
                 S359E 
                 135.589 
                 10.77 
                 14.60 
                 135.104 
                 9.354 
                 12.64 
               
               
                 S359H 
                 110.422 
                 0.00 
                 0.00 
                 100.809 
                 0.000 
                 0.00 
               
               
                 P367A 
                 167.030 
                 12.94 
                 21.62 
                 127.366 
                 13.153 
                 16.75 
               
               
                 P367G 
                 115.683 
                 0.00 
                 0.00 
                 122.642 
                 0.000 
                 0.00 
               
               
                 P367K 
                 125.884 
                 5.06 
                 6.36 
                 66.884 
                 10.136 
                 6.78 
               
               
                 P367S 
                 74.263 
                 14.39 
                 10.69 
                 88.355 
                 16.433 
                 14.52 
               
               
                 D368A 
                 121.623 
                 1.45 
                 1.76 
                 81.646 
                 2.111 
                 1.72 
               
               
                 D368E 
                 166.628 
                 9.18 
                 15.30 
                 97.937 
                 11.462 
                 11.23 
               
               
                 D368L 
                 108.977 
                 0.00 
                 0.00 
                 109.364 
                 0.969 
                 1.06 
               
               
                 D368M 
                 119.744 
                 2.72 
                 3.25 
                 103.662 
                 2.536 
                 2.63 
               
               
                 D368R 
                 164.735 
                 10.16 
                 16.74 
                 118.140 
                 11.805 
                 13.95 
               
               
                 D368T 
                 107.122 
                 2.87 
                 3.07 
                 126.693 
                 3.366 
                 4.26 
               
               
                 N369R 
                 161.693 
                 6.39 
                 10.34 
                 74.366 
                 6.182 
                 4.60 
               
               
                 A371F 
                 180.217 
                 6.19 
                 11.16 
                 76.436 
                 5.578 
                 4.26 
               
               
                 A371H 
                 957.055 
                 1.81 
                 17.32 
                 89.541 
                 1.697 
                 1.52 
               
               
                 A371H 
                 111.143 
                 0.00 
                 0.00 
                 95.589 
                 8.610 
                 8.23 
               
               
                 A371K 
                 136.514 
                 12.84 
                 17.53 
                 114.354 
                 12.454 
                 14.24 
               
               
                 A371L 
                 695.108 
                 1.51 
                 10.52 
                 107.003 
                 2.215 
                 2.37 
               
               
                 A371L 
                 104.327 
                 0.00 
                 0.00 
                 60.232 
                 1.205 
                 0.73 
               
               
                 A371R 
                 #VALUE! 
                 #VALUE! 
                 11.03 
                 #VALUE! 
                 #VALUE! 
                 14.06 
               
               
                 A371R 
                 121.162 
                 0.00 
                 0.00 
                 97.970 
                 2.587 
                 2.53 
               
               
                 A371S 
                 147.672 
                 8.38 
                 12.38 
                 131.555 
                 16.938 
                 22.28 
               
               
                 L374P 
                 392.038 
                 5.77 
                 22.63 
                 123.033 
                 7.365 
                 9.06 
               
               
                 E375A 
                 88.836 
                 0.00 
                 0.00 
                 134.714 
                 2.050 
                 2.76 
               
               
                 E375G 
                 126.880 
                 10.32 
                 13.10 
                 139.030 
                 14.673 
                 20.40 
               
               
                 E375R 
                 163.180 
                 13.15 
                 21.45 
                 116.431 
                 19.727 
                 22.97 
               
               
                 K376D 
                 113.100 
                 12.36 
                 13.97 
                 165.064 
                 5.049 
                 8.33 
               
               
                 K376E 
                 100.000 
                 13.55 
                 13.55 
                 153.016 
                 10.394 
                 15.90 
               
               
                 K376Q 
                 125.172 
                 12.75 
                 15.96 
                 90.000 
                 12.057 
                 10.85 
               
               
                 K376R 
                 81.687 
                 31.63 
                 25.84 
                 199.112 
                 10.372 
                 20.65 
               
               
                 K376T 
                 121.133 
                 14.91 
                 18.06 
                 113.387 
                 5.639 
                 6.39 
               
               
                 K376V 
                 124.221 
                 3.19 
                 3.96 
                 118.583 
                 2.547 
                 3.02 
               
               
                 K376Y 
                 102.812 
                 9.24 
                 9.50 
                 96.139 
                 12.985 
                 12.48 
               
               
                 G377D 
                 110.871 
                 15.72 
                 17.43 
                 132.357 
                 10.550 
                 13.96 
               
               
                 G377E 
                 130.445 
                 8.04 
                 10.49 
                 128.402 
                 7.401 
                 9.50 
               
               
                 G377H 
                 146.855 
                 8.34 
                 12.25 
                 104.837 
                 10.117 
                 10.61 
               
               
                 G377K 
                 185.922 
                 4.42 
                 8.21 
                 119.751 
                 4.989 
                 5.97 
               
               
                 G377R 
                 119.708 
                 5.87 
                 7.03 
                 94.749 
                 7.137 
                 6.76 
               
               
                 G377S 
                 108.609 
                 6.91 
                 7.51 
                 101.106 
                 7.877 
                 7.96 
               
               
                 G377T 
                 112.557 
                 17.14 
                 19.29 
                 109.036 
                 18.279 
                 19.93 
               
               
                 F380W 
                 147.077 
                 9.97 
                 14.67 
                 104.881 
                 9.253 
                 9.70 
               
               
                 T381S 
                 135.827 
                 13.41 
                 18.21 
                 112.559 
                 10.315 
                 11.61 
               
               
                 R383I 
                 527.820 
                 6.33 
                 33.44 
                 98.328 
                 7.522 
                 7.40 
               
               
                 R383S 
                 132.894 
                 10.50 
                 13.96 
                 119.466 
                 10.545 
                 12.60 
               
               
                 K385A 
                 126.096 
                 4.64 
                 5.85 
                 112.706 
                 0.000 
                 0.00 
               
               
                 K385Q 
                 137.629 
                 9.03 
                 12.43 
                 124.892 
                 7.512 
                 9.38 
               
               
                 K385V 
                 112.581 
                 5.12 
                 5.76 
                 80.571 
                 2.979 
                 2.40 
               
               
                 E389A 
                 306.767 
                 2.13 
                 6.53 
                 224.872 
                 1.824 
                 4.10 
               
               
                 E389G 
                 113.253 
                 2.13 
                 2.41 
                 139.901 
                 0.000 
                 0.00 
               
               
                 E389L 
                 143.219 
                 14.24 
                 20.40 
                 112.185 
                 12.609 
                 14.15 
               
               
                 E389Q 
                 135.807 
                 11.88 
                 16.14 
                 99.738 
                 12.767 
                 12.73 
               
               
                 E389S 
                 165.620 
                 0.00 
                 0.00 
                 93.030 
                 0.285 
                 0.27 
               
               
                 E392A 
                 112.465 
                 7.27 
                 8.18 
                 155.693 
                 6.376 
                 9.93 
               
               
                 E392F 
                 115.619 
                 3.90 
                 4.51 
                 143.781 
                 3.905 
                 5.61 
               
               
                 E392Q 
                 112.993 
                 10.53 
                 11.89 
                 93.789 
                 16.705 
                 15.67 
               
               
                 E392R 
                 129.528 
                 3.69 
                 4.79 
                 123.407 
                 2.947 
                 3.64 
               
               
                 E392V 
                 124.365 
                 7.73 
                 9.61 
                 154.768 
                 6.404 
                 9.91 
               
               
                 Q393F 
                 139.966 
                 10.59 
                 14.82 
                 101.647 
                 10.171 
                 10.34 
               
               
                 Q393M 
                 139.696 
                 1.60 
                 2.24 
                 86.966 
                 3.086 
                 2.68 
               
               
                 S395A 
                 208.246 
                 12.98 
                 27.04 
                 112.714 
                 12.395 
                 13.97 
               
               
                 S395H 
                 159.975 
                 12.55 
                 20.07 
                 113.401 
                 10.452 
                 11.85 
               
               
                 E396A 
                 131.894 
                 8.42 
                 11.10 
                 128.716 
                 9.777 
                 12.58 
               
               
                 E396H 
                 210.364 
                 9.19 
                 19.33 
                 128.571 
                 3.216 
                 4.14 
               
               
                 E396Q 
                 122.977 
                 10.06 
                 12.37 
                 95.938 
                 10.263 
                 9.85 
               
               
                 E396S 
                 156.267 
                 2.77 
                 4.33 
                 111.753 
                 2.022 
                 2.26 
               
               
                 Y399T 
                 130.536 
                 0.00 
                 0.00 
                 122.738 
                 0.050 
                 0.06 
               
               
                 Y399V 
                 110.592 
                 15.98 
                 17.68 
                 116.018 
                 17.801 
                 20.65 
               
               
                 Y399W 
                 122.500 
                 13.76 
                 16.86 
                 103.346 
                 11.973 
                 12.37 
               
               
                 S401A 
                 122.003 
                 13.90 
                 16.96 
                 99.275 
                 13.024 
                 12.93 
               
               
                 S401E 
                 125.223 
                 16.30 
                 20.42 
                 128.670 
                 15.000 
                 19.30 
               
               
                 S404A 
                 149.379 
                 0.00 
                 0.00 
                 105.443 
                 1.102 
                 1.16 
               
               
                 L406F 
                 122.805 
                 0.00 
                 0.00 
                 146.122 
                 0.000 
                 0.00 
               
               
                 L406N 
                 152.836 
                 6.36 
                 9.72 
                 131.321 
                 6.705 
                 8.81 
               
               
                 S407A 
                 141.351 
                 11.33 
                 16.02 
                 110.376 
                 16.836 
                 18.58 
               
               
                 S407D 
                 241.053 
                 11.29 
                 27.22 
                 98.135 
                 10.120 
                 9.93 
               
               
                 S407P 
                 143.308 
                 6.85 
                 9.81 
                 121.898 
                 11.088 
                 13.52 
               
               
                 A412Q 
                 146.177 
                 9.54 
                 13.94 
                 99.452 
                 8.511 
                 8.46 
               
               
                 A412R 
                 140.070 
                 8.92 
                 12.49 
                 123.675 
                 9.390 
                 11.61 
               
               
                 A412V 
                 146.804 
                 4.99 
                 7.32 
                 101.739 
                 5.383 
                 5.48 
               
               
                 D416L 
                 120.820 
                 17.64 
                 21.31 
                 127.662 
                 15.340 
                 19.58 
               
               
                 D418R 
                 117.749 
                 7.59 
                 8.94 
                 112.193 
                 10.721 
                 12.03 
               
               
                 A419H 
                 241.224 
                 8.82 
                 21.27 
                 188.179 
                 5.999 
                 11.29 
               
               
                 A419K 
                 191.165 
                 10.42 
                 19.91 
                 2022.616 
                 1.523 
                 30.81 
               
               
                 D421A 
                 102.111 
                 12.49 
                 12.75 
                 301.584 
                 4.510 
                 13.60 
               
               
                 D421H 
                 333.471 
                 10.18 
                 33.95 
                 67.652 
                 86.552 
                 58.55 
               
               
                 D421K 
                 124.190 
                 7.62 
                 9.46 
                 102.316 
                 13.562 
                 13.88 
               
               
                 D421N 
                 110.806 
                 14.96 
                 16.58 
                 100.116 
                 16.449 
                 16.47 
               
               
                 D421Q 
                 104.370 
                 10.72 
                 11.18 
                 143.630 
                 12.400 
                 17.81 
               
               
                 D421R 
                 138.783 
                 8.85 
                 12.28 
                 137.964 
                 9.778 
                 13.49 
               
               
                 D421S 
                 142.171 
                 11.00 
                 15.64 
                 166.162 
                 8.564 
                 14.23 
               
               
                 A425G 
                 74.810 
                 10.61 
                 7.94 
                 120.947 
                 11.137 
                 13.47 
               
               
                 G427Q 
                 133.135 
                 2.31 
                 3.08 
                 98.243 
                 8.618 
                 8.47 
               
               
                 G427T 
                 125.113 
                 4.81 
                 6.02 
                 119.058 
                 3.956 
                 4.71 
               
               
                 V428L 
                 137.044 
                 1.81 
                 2.48 
                 109.390 
                 0.990 
                 1.08 
               
               
                 D431E 
                 70.178 
                 26.32 
                 18.47 
                 95.135 
                 20.739 
                 19.73 
               
               
                 D431H 
                 186.490 
                 7.32 
                 13.65 
                 95.071 
                 9.941 
                 9.45 
               
               
                 D431K 
                 240.835 
                 11.61 
                 27.97 
                 68.277 
                 20.207 
                 13.80 
               
               
                 D431L 
                 129.149 
                 10.49 
                 13.54 
                 119.177 
                 11.740 
                 13.99 
               
               
                 D431N 
                 138.404 
                 9.79 
                 13.55 
                 125.433 
                 9.246 
                 11.60 
               
               
                 D431Q 
                 232.960 
                 10.83 
                 25.23 
                 109.483 
                 10.716 
                 11.73 
               
               
                 D431S 
                 78.069 
                 7.52 
                 5.87 
                 88.796 
                 9.135 
                 8.11 
               
               
                 F433A 
                 147.286 
                 9.78 
                 14.40 
                 99.486 
                 12.798 
                 12.73 
               
               
                 F433H 
                 140.196 
                 13.48 
                 18.90 
                 87.943 
                 16.888 
                 14.85 
               
               
                 F433I 
                 108.569 
                 11.30 
                 12.27 
                 86.984 
                 16.616 
                 14.45 
               
               
                 F433K 
                 91.159 
                 11.12 
                 10.14 
                 342.290 
                 3.608 
                 12.35 
               
               
                 F433R 
                 128.958 
                 10.53 
                 13.58 
                 133.353 
                 9.565 
                 12.75 
               
               
                 F433T 
                 161.799 
                 13.66 
                 22.10 
                 134.977 
                 19.229 
                 25.96 
               
               
                 F433V 
                 1412.071 
                 1.61 
                 22.69 
                 112.033 
                 17.307 
                 19.39 
               
               
                 F433W 
                 149.049 
                 10.46 
                 15.59 
                 113.585 
                 7.530 
                 8.55 
               
               
                 P437I 
                 148.880 
                 2.39 
                 3.56 
                 107.028 
                 1.782 
                 1.91 
               
               
                 M438A 
                 106.463 
                 10.07 
                 10.72 
                 135.705 
                 10.194 
                 13.83 
               
               
                 M438D 
                 105.370 
                 10.16 
                 10.71 
                 113.283 
                 2.590 
                 2.93 
               
               
                 M438E 
                 115.061 
                 8.00 
                 9.21 
                 113.782 
                 9.120 
                 10.38 
               
               
                 M438L 
                 65.794 
                 10.06 
                 6.62 
                 214.958 
                 6.526 
                 14.03 
               
               
                 M438N 
                 130.428 
                 8.06 
                 10.52 
                 100.669 
                 11.889 
                 11.97 
               
               
                 M438T 
                 104.058 
                 13.39 
                 13.93 
                 103.691 
                 12.160 
                 12.61 
               
               
                 E439A 
                 137.279 
                 11.63 
                 15.97 
                 95.555 
                 14.073 
                 13.45 
               
               
                 E439A 
                 154.140 
                 4.72 
                 7.28 
                 147.295 
                 7.415 
                 10.92 
               
               
                 E439C 
                 193.243 
                 14.69 
                 28.38 
                 111.719 
                 15.734 
                 17.58 
               
               
                 E439K 
                 124.464 
                 13.28 
                 16.52 
                 104.762 
                 10.552 
                 11.05 
               
               
                 E439P 
                 118.340 
                 15.59 
                 18.44 
                 87.446 
                 14.998 
                 13.12 
               
               
                 E439Q 
                 101.589 
                 10.67 
                 10.84 
                 127.358 
                 10.648 
                 13.56 
               
               
                 E439T 
                 110.891 
                 14.36 
                 15.93 
                 122.975 
                 11.322 
                 13.92 
               
               
                 T440D 
                 118.877 
                 11.52 
                 13.69 
                 79.518 
                 18.426 
                 14.65 
               
               
                 T440H 
                 142.296 
                 4.46 
                 6.34 
                 130.928 
                 7.553 
                 9.89 
               
               
                 T440M 
                 84.722 
                 8.83 
                 7.48 
                 86.929 
                 12.774 
                 11.10 
               
               
                 T440P 
                 111.931 
                 13.54 
                 15.16 
                 91.205 
                 17.272 
                 15.75 
               
               
                 T440S 
                 100.436 
                 11.17 
                 11.22 
                 131.174 
                 9.810 
                 12.87 
               
               
                 E441F 
                 129.315 
                 11.25 
                 14.55 
                 110.874 
                 11.410 
                 12.65 
               
               
                 E442G 
                 111.216 
                 10.24 
                 11.39 
                 100.210 
                 10.965 
                 10.99 
               
               
                 P443E 
                 94.377 
                 5.14 
                 4.85 
                 130.704 
                 6.789 
                 8.87 
               
               
                 P443F 
                 146.612 
                 11.22 
                 16.45 
                 97.932 
                 12.322 
                 12.07 
               
               
                 P443G 
                 239.171 
                 8.56 
                 20.48 
                 157.960 
                 16.385 
                 25.88 
               
               
                 Q444E 
                 81.997 
                 8.54 
                 7.01 
                 160.917 
                 9.561 
                 15.38 
               
               
                 Q444H 
                 150.301 
                 8.46 
                 12.71 
                 119.665 
                 10.892 
                 13.03 
               
               
                 Q444V 
                 129.822 
                 13.49 
                 17.51 
                 122.591 
                 10.995 
                 13.48 
               
               
                 I445M 
                 85.090 
                 17.25 
                 14.68 
                 101.149 
                 15.393 
                 15.57 
               
               
                 I445N 
                 106.430 
                 13.89 
                 14.79 
                 87.351 
                 12.945 
                 11.31 
               
               
                 I445W 
                 117.213 
                 11.70 
                 13.72 
                 100.037 
                 10.983 
                 10.99 
               
               
                 Y447E 
                 99.579 
                 16.55 
                 16.48 
                 108.969 
                 12.849 
                 14.00 
               
               
                 Y447G 
                 143.704 
                 13.77 
                 19.79 
                 103.624 
                 11.563 
                 11.98 
               
               
                 Y447P 
                 139.152 
                 13.78 
                 19.17 
                 107.737 
                 12.282 
                 13.23 
               
               
                 positive 
                 94.998 
                 5.23 
                 4.97 
                 96.871 
                 8.456 
                 8.19 
               
               
                 control 
                 105.798 
                 4.48 
                 4.74 
                 108.066 
                 5.246 
                 5.67 
               
               
                 (OHO) 
                 100.000 
                 3.33 
                 3.33 
                 82.7780 
                 3.759 
                 4.59 
               
               
                   
                 94.762 
                 19.07 
                 18.07 
                 109.539 
                 16.529 
                 18.11 
               
               
                   
                 142.024 
                 4.48 
                 6.36 
                 130.947 
                 5.595 
                 7.33 
               
               
                   
                 45.115 
                 20.77 
                 9.37 
                 68.017 
                 11.035 
                 7.51 
               
               
                   
                 53.324 
                 21.95 
                 11.71 
                 74.253 
                 9.960 
                 7.40 
               
               
                   
                 59.581 
                 25.24 
                 15.04 
                 75.872 
                 16.231 
                 12.31 
               
               
                   
                 91.844 
                 19.05 
                 17.50 
                 80.371 
                 13.977 
                 11.23 
               
               
                   
                 93.828 
                 13.47 
                 12.63 
                 96.630 
                 19.454 
                 18.80 
               
               
                   
                 57.773 
                 17.04 
                 9.85 
                 83.536 
                 17.573 
                 14.68 
               
               
                   
                 100.000 
                 18.56 
                 18.56 
                 148.226 
                 16.239 
                 24.07 
               
               
                   
                 74.325 
                 18.29 
                 13.60 
                 61.119 
                 9.286 
                 5.68 
               
               
                   
                 98.132 
                 8.48 
                 8.32 
                 87.677 
                 10.006 
                 8.77 
               
               
                   
                 93.817 
                 9.62 
                 9.02 
                 102.223 
                 9.745 
                 9.96 
               
               
                   
                 96.922 
                 8.56 
                 8.30 
                 87.993 
                 9.064 
                 7.98 
               
               
                   
                 96.648 
                 9.91 
                 9.58 
                 86.891 
                 9.938 
                 8.63 
               
               
                   
               
               
                 n/a (not available; e.g., beyond detection limit) 
               
            
           
         
       
     
     2. Summary of Results for F204P 
     For mutant F204P, the results above of tested supernatant from transient transfection of CHO-S cells incubated in the presence of m-cresol in a bHA enzymatic activity assay showed that the F204P mutant protein was highly resistant to 0.4% m-cresol treatment. The results showed that the activity that remained after 4 hours incubation with 0.4% m-cresol at 37° C. was approximately equal to the activity observed when the enzyme was incubated at either 4° C. or at 37° C. in the absence of m-cresol. The positive control (WT PH20-OHO) showed a reduction in activity of 75% and 83% on the day of the assay (as assayed from two different OHO transfections). This demonstrated that the F204P phenophile was able to retain 60% to 90% or greater of its activity above the residual activity of the wildtype PH20 control enzyme. 
     In order to confirm the stability of F204P upon m-cresol treatment or exposure to increased temperature, a second transfection of F204P was performed in duplicate using CHO-S cells, and clarified supernatant was again tested for its stability at 4° C., at 37° C. for 4 hours with 0.4% m-cresol and at 37° C. for 4 hours without 0.4% m-cresol. The results confirmed that the F204P mutant enzyme retained a high amount of hyaluronidase activity after the 4 hour incubation in m-cresol at 37° C. The results were similar to the results seen in the first screening of the mutant, with F204P retaining anywhere from 57% to greater than 90% of its activity above the residual activity of the wildtype PH20 control enzyme after the 4 hour incubation. 
     A summary of the enzyme activity of F204P compared to the wildtype control is set forth in Table 13. 
     
       
         
           
               
             
               
                 TABLE 13 
               
             
            
               
                   
               
               
                 Summary of Enzyme Activity 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                   
                 Remaining Activity 
                   
               
               
                   
                 Remaining Activity 
                   
                 after 4 h incubation 
                   
               
               
                   
                 after 4 h incubation 
                   
                 (37° C. + m-cre/ 
                   
               
               
                   
                 (37° C. + m-cre/37° C.) 
                 Net % Increase 
                 4° C.) 
                 Net % Increase 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                   
                 WT 
                 in Activity Over 
                   
                 WT 
                 in Activity Over 
               
               
                 Transfection # 
                 F204P 
                 (OHO) 
                 WT (37° C.) 
                 F204P 
                 (OHO) 
                 WT (4° C.) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 1 
                 73.6% 
                 16.4% 
                 57.2% 
                 86.0% 
                 25.3% 
                 60.7% 
               
               
                 2 
                 122.3% 
                 25.2% 
                 97.1% 
                 109.7% 
                 16.6% 
                 93.1% 
               
               
                   
               
            
           
         
       
     
     Example 6 
     Large Scale Expression and Purification of pH20 Hit Variant 1. Expression and Purification 
     HZ24-PH20-IRES-SEAP plasmid DNA containing cDNA encoding one of the variant PH20 was transfected into monolayer CHO-S cells as generally described in Example 2. CHO-S cells were cultured in shaker flasks using CD-CHO media supplemented with GlutaMAX (8 mM). On the day of transfection, 15 flasks were prepared of approximately 300 mL volume containing the CHO-S cells at an approximate density of 1.0×10 6  cells/mL. Each 300 mL flask was transfected using 375 μg of plasmid DNA encoding the F204P mutant combined with 375 μL of Freestyle MAX transfection reagent. The transfected plasmid DNA had a sequence of nucleotides set forth in SEQ ID NO:4 containing a codon change of TTC to CCT at nucleotide positions 1733-1735, thereby encoding the F204P mutant. The transfected cells were then allowed to remain in culture for 96 hours, whereupon the cells and media were harvested and pooled. The cells were pelleted by centrifugation (4000×g, 20′), and the supernatant retained for purification of the F204P protein (approximately 4.5 liters). 
     The crude supernatant was concentrated 10× using a 30 kDa Tangential flow filter (TFF) system (Millipore Pellicon XL, Bimax 30, 200 mL void volume; 50 cm 2  filter surface area) until the volume was approximately 450 mL. The permeate was saved for assay to detect flow through of the F204P protein. A free-flow buffer exchange for the retentate was then performed using 4 liters of buffer (10 mM NaPO 4 ; 25 mM NaCl, pH 7.2). The volume of the retentate was reduced again to approximately 200 mL, and then the remaining permeate in the system was purged (void volume ˜200 mL) and the system was flushed using approximately 50 mL of buffer to yield a final concentrated product of approximately 450 mL. 
     An anti-rHuPH20 affinity column was prepared by coupling antigen affinity purified Rabbit anti-rHuPH20 IgG to CNBr-activated Sepharose 4 Fast Flow (GEHealth catalog No. 17-0981-01). Briefly, 0.7 g of pre-activated Sepharose 4 powder was suspended in 1 mM HCl in a 10 mL glass column for 30 minutes to allow the powder to swell. The solution was drained from the column and washed with 15 gel volumes (about 30 mL) of cold 1 mM HCl by gravity. The column was washed with 5 gel volumens of coupling buffer (0.1M NaHCO 3 , 0.5M NaCl at pH 8.3). Next, 5 mg of Rabbit anti-rHuPH20 IgG at &gt;1.0 mg/mL in coupling buffer was added to the column at a protein/gel ratio of 2-3 mg/mL gel. The column was rotated head to head at 4° C. overnight. The flow-through was collected for coupling efficiency determination. The gel was washed with 2 gel volumes of coupling buffer, and then washed and resuspended in 1 M ethanolaminine pH 9.5 for 2 hours at room temperature to block unused activated sites. The gel was washed 6 times with 5 gel volumes per wash alternating coupling buffer and 0.1 NaAc, 0.5M NaCl, pH 4.5. The gel was then washed with 10 gel volumes of TBS (20 mM Tris-HCl, 0.15 M NaCl, pH 7.5). The coupling efficiency was determined (1-post-coupling protein concentration/pre-coupling protein concentration ×100%). The antibody coupled gel was stored in TBS with 0.02% NaN 3  at 4° C. 
     The concentrated supernatant product was subsequently loaded onto a anti-rHuPH20 affinity column at an approximate rate of 5 mL/min. The elution was performed according to standard procedure using a GE™ AKTA FPLC purification system (GE Healthcare, Product No. 18-1900-26), whereby the protein was eluted via a low pH glycine wash (0.1 M glycine-HCl, pH 2.5) in 1 mL fractions. Each fraction was immediately neutralized by the addition of 100 μL of 1M Tris, pH 7.5. 
     The eluted protein was assayed by resolving protein bands on a 4-20% SDS-PAGE gradient Tris-glycine gel. SeeBlue®Plus2 Pre-stained MW standards (Life Teechnologies; Catalog No. LC5925) were used as molecular weight standards, and 50 ng rHuPH20 (as described in Example 1) was used as a positive control. The polyacrylamide gel was stained with Instant Blue to show total protein from each fraction. To confirm the bands on the gel are PH20, the gel was transferred to a PVDF membrane (Invitrogen), which was subjected to Western Blot using a Rabbit anti-PH20 primary antibody generated by immunizing rabbits with rHuPH20 and an HRP-Goat anti-rabbit secondary antibody (Calbiochem, Cat. No. DC03L). 
     Then, the flow-through from the initial loading of the affinity column was re-loaded onto the column twice due to the low capacity of the affinity column. All fractions containing the protein were then combined resulting in a total volume that was approximately 13 mL. This product was then dialyzed overnight versus four liters of buffer (10 mM NaPO 4 , 140 mM NaCl, pH 7.2) using a Slide-A-Lyzer Dialysis Cassette G2 (20,000 MWCO) with a 15 mL capacity. The buffer was then changed and the product dialyzed against a second fresh four liters of the same buffer. The F204P protein was then concentrated using an Amicon Ultra Centrifugation column (Millipore; 10,000 MWCO) to a final volume of approximately 450 μL (10 minutes at 4000×g). 
     2. Characterization of Protein 
     The purified protein was characterized for its protein concentration, activity, and purity. 
     To determine the protein concentration of the purified protein, a quantification ELISA was performed as described in Example 7. Also, hyaluronidase activity was determined as described in Example 3. The protein concentration after centrifugation was estimated to be approximately 400 μg/mL. The purified protein also was resolved on a 4-20% SDS-PAGE gradient Tris-glycine gel, which was then stained with Instant Blue. The staining results demonstrated that the protein was essentially a single molecular weight protein of approximately 63 kDa, similar to the rHuPH20 control. No appreciable degradative products were detected by this method. Approximate yields of the protein at various timepoints and activity during the purification are described in Table 14. 
     
       
         
           
               
             
               
                 TABLE 14 
               
             
            
               
                   
               
               
                 Characterization of Purification Steps 
               
            
           
           
               
               
               
               
            
               
                   
                   
                 Quant ELISA 
                   
               
               
                   
                 Activity Assay 
                 Assay 
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                   
                   
                 Total 
                 Protein 
                 Total 
                 Specific 
               
               
                 Purification  
                 Volume  
                 Activity 
                 Activity 
                 Conc. 
                 Protein 
                 Activity 
               
               
                 Step 
                 (mL) 
                 (U/mL) 
                 (U) 
                 (μg/mL) 
                 (μg) 
                 (U/μg) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Supernatant 
                 4500 
                 2.66 
                 11,700 
                 0.046 
                 207 
                 56.5 
               
               
                 Conc. after  
                 450 
                 42 
                 18,900 
                 0.4 
                 178 
                 105.9 
               
               
                 TFF &amp; Buffer  
                   
                   
                   
                   
                   
                   
               
               
                 Exchange 
                   
                   
                   
                   
                   
                   
               
               
                 Pooled  
                 0.45 
                 11,741 
                 5283 
                 396 
                 180 
                 35.3 
               
               
                 Fractions 5-7  
                   
                   
                   
                   
                   
                   
               
               
                 after AC,  
                   
                   
                   
                   
                   
                   
               
               
                 Dialysis &amp; 
                   
                   
                   
                   
                   
                   
               
               
                 Conc.—A280 
               
               
                   
               
            
           
         
       
     
     The purity of the purified protein was determined by Reverse Phase HPLC (RP-HPLC). The elution time from the reverse phase column was essentially identical as that observed with the recombinant human hyaluronidase (HUB), and provides a basis for crude estimation of the purity of the sample at approximately 80-90%. 
     Example 7 
     Quantification Using ELISA 
     The quantification of PH20 or variants were performed using an ELISA that captures the protein using a monoclonal anti-rHuPH20 capture antibody. Specifically, one day prior to performing the ELISA, 96-well 4HBX plates were coated with capture antibody (Protein G purified rabbit polyclonal anti-PH20 antibody generated by immunizing rabbits with rHuPH20; 1 mg/mL stock) at 1 μg/mL in 100 mM phosphate (pH 7.2) in a total volume of 100 μL per well. The plates were stored at 4° C. overnight. On the next day, the plates were washed 5× with 1×PBS at 300 μL/well with a plate washer. After each wash, the plated were patted dry on paper towels. Then, the plates were blocked with 200 μL PBS containing Tween 20 (1×PBST) per well at room temperature for 1 hour. 
     The standards and samples were added to the plate. For generation of the standard, a 1 mg/mL stock of rHuPH20 (Example 1) was freshly diluted to 50 μg/mL in HEPES pH 7.4 assay buffer as an intermediate stock. Then, for the standards, the 50 μg/mL stock was diluted in duplicates into 360 μL of 0.5×PBST at 300 ng/mL for the first standard (first row). For the other standard rows, 240 μL 0.5×PBST were added to each well, and 1:3 serial dilutions made. For the transfected supernatant samples, 360 μL per well was added in duplicate into the first row, and each were also serially diluted as described above into 0.5× PBST. For purified samples, 100 μL was added per well. The plates were incubated for 2 hours at room temperature. After incubation, the plates were washed 5× with 1×PBST at 300 μL/well using a plate washer. After each wash, the plates were patted dry on paper towels. 
     An HRP-conjugated anti-PH20 antibody was prepared for detection using an HRP conjugation kit (Pierce, Thermo-Fisher; Catalog No. 31489). 1 mg of a Protein G purified rabbit polyclonal antibody generated by immunizing rabbits with rHuPH20 was diluted in 1 mL PBS and 1 mL of 2× carbonate kit buffer. Next, 100 μL of peroxidase were added to 1 mL of the above antibody solution and incubated at room temperature for 1 hour. Then, 10 μL NaBH4 stock was added in a fume hood, and the sample incubated at room temperature for 20 minutes. To quench the reaction, 20 μL of ethanolamine was added and incubated at room temperature for 15 minutes. To this, 1/25 volume 5% human serum albumin (0.1 mL syringe) was added to give a 2 mg/mL albumin stock reaction. The pH was adjusted to about 7.9 by addition of 250 μL of 1 M Tris pH 7.4. The concentration of the stock was 400 μg/mL. The stock solution was further diluted 1/10 in PBS Tween20 (0.05%) containing 0.5% human serum albumin and preservatives, and then was sterile filtered. The stock was stored at 4° C. or was frozen at −20° C. 
     Antibodies were detecting using the HRP-conjugated anti-PH20 antibody that was diluted 1000× into 0.5× PBST. 100 μL of the diluted antibody was added to all wells of the plate and the plate incubated for a further 2 hours at room temperature. After incubation, the plates were washed 5× with 1×PBST at 300 μL/well using a plate washer. After each wash, the plates were patted dry on paper towels. Then, 100 μL of TMB substrate were added to each well and the reaction was stopped after 5-10 minutes by adding 100 μL of stop solution per well. The plate was read at OD 450 . 
     Example 8 
     Determination of Enzymatic Activity of pH20 
     Enzymatic activity of PH20 in samples such as cell cultures, purification fractions and purified solutions was determined using a turbidimetric assay, which is based on the formation of an insoluble precipitate when hyaluronic acid binds with cetylpyridinium chloride (CPC). The activity is measured by incubating PH20 with hyaluronan for a set period of time (30 minutes) and then precipitating the undigested hyaluronan with the addition of CDC. The turbidity of the resulting sample is measured at 640 nm. The decrease in turbidity resulting from enzyme activity on the hyaluronan substrate is a measure of the PH20 enzymatic activity. The method is run using a calibration curve generated with dilutions of a PH20 assay working reference standard (rHuPH20 standard generated as described in Example 1), and sample activity measurements are made relative to this calibration curve. 
     Dilutions of the sample and standards were prepared in Enzyme Diluent Solution (70 mM NaCl, 0.1% human serum albumin [HSA], 0.67 g/L gelatin hydrolysate in 25 mM PIPES buffer, pH 5.5). The samples were diluted to an appropriate concentration. Hyaluronic acid (HA, average MW of 20-50 kDa) from Lifecore Biomedical (Chaska, Minn.) also was prepared at 1 mg/mL in substrate solution that contains 25 mM PIPES, 70 mM NaCl at pH 5.5. Equal amounts of the above two solutions were mixed to prepare a 1 mL reaction mixture and incubated at 37° C. for 30 min. The reaction was stopped by addition of 4 mL of Cetylpyridinium Chloride Solution (CPC, 5.0 mg/mL). After brief vortexing, the turbidity of the sample mixture was read at 640 nm and the activity was determined by fitting against a standard curve. Specific activity (Units/mg) was calculated by dividing the enzyme activity (U/mL) by the protein concentration (mg/mL). 
     Example 9 
     Stability of F204P-pH20 Variant in Preservative 
     To confirm the screening results, an amount estimated to be about 450 U/mL of the purified F204P protein as described in Example 6 was formulated in 10 mM sodium phosphate, pH 6.5, 120 mM NaCl, 10 mM methionine, 0.01% Pluronic F-68, 0.1% phenol and 0.15% m-cresol. A test article that also contained an amount estimated to be about 450 U/mL wild type rHuPH20 (generated as described in Example 1) in the same formulation was also prepared to serve as a control. Each formulation solution was aliquotted in 0.5 mL and filled into 2 mL USP Type I borosilicate glass with a chlorobutyl rubber stopper and an aluminum seal. The vials were incubated at 5° C., 30° C. or 37° C. Samples were withdrawn from the incubator at various times and enzymatic activity was measured as described in Example 8. 
     The results of the enzymatic activity measurements are shown in Table 15. As can be seen, the rHuPH20 wild type control showed a rapid decrease in activity when incubated at 37° C. in the presence of phenolic preservatives. In contrast, the F204P mutant showed no significant loss in activity throughout the study. The results also show that activity of PH20 is retained after incubation for up to 4 weeks at 5° C. and 30° C. compared to the activity of the rHuPH20 wildtype control not containing the mutation. These results confirm that F204P tolerates EPB level of preservative (0.1% phenol and 0.15% m-cresol) and is stable at 37° C. for at least up to 6 days at at 5° C. and 30° C. for greater than one month. 
     
       
         
           
               
             
               
                 TABLE 15 
               
             
            
               
                   
               
               
                 Stability of rHuPH20 wildtype and F204P mutant incubated  
               
               
                 at with preservative 
               
            
           
           
               
               
               
               
            
               
                   
                 PH20 relative 
                 PH20 relative 
                 PH20 relative  
               
               
                   
                 activity (%) at 
                 activity (%) at 
                 activity 
               
               
                   
                 5° C. 
                 30° C. 
                 (%) at 37° C. 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                 ID 
                 T0 
                 2 w 
                 4 w 
                 6 d 
                 2 w 
                 4 w 
                 2 d 
                 4 d 
                 6 d 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                 F204P 
                 100 
                 — 
                 91.8 
                 84.1 
                 100 
                 96.6 
                 105 
                 91.1 
                 95.9 
               
               
                 wildtype  
                 100 
                 — 
                 81.9 
                 66.7 
                 61.7 
                 60.5 
                 48.6 
                 29.6 
                 15.2 
               
               
                 control 
               
               
                   
               
            
           
         
       
     
     Example 10 
     Stability of F204P-pH20 Variant in Insulin Coformulation 
     The PH20 variant F204P was tested for its stability in a coformulation containing an insulin analog (insulin aspart or insulin lispro). 
     In the tested coformulations, the insulin lispro was a commercial product (Insulin Lispro: Eli Lilly Humalog® (insulin Lispro) 100 U/mL, Lot A572364). 
     In the tested coformulations, the insulin aspart analog was a reprocessed aspart prepared by pooling 12 vials (10 mL each) of a commercial product (Insulin Aspart: Novo Nordisk, NovoRapid® (insulin Aspart), Lot XS60195), which was then concentrated using an Amicon Ultracel-10 K column concentrator until the final concentration was about 5 times the original concentration. The insulin analog was precipitated by addition of 1 M sodium acetate, pH 5.3 and 30 mM zinc chloride (ZnCl 2 , EMD, Cat No. ZX0065-1) at 1/10 of the protein solution volume. The solution was placed on ice for 30 minutes followed by centrifugation at 5600 rpm for 20 minutes in an Avanti J-E Centrifuge with JS-5.3 swinging bucket rotor (Beckman Coulter). The supernatant was decanted and the pellet was resuspended and washed with 20 mM sodium acetate, 2 mM zinc chloride, pH 5.5 solution. The resuspended solution was centrifuged as described above. The washing step was repeated a total of 5 times. A final wash was performed with 20 mM sodium acetate, pH 5.5 to remove all traces of zinc chloride. The resulting protein paste was dissolved with water containing 20 mM HCl. After complete dissolution, 250 mM Tris, pH 10.7 was added to a final Tris concentration of 20 mM. The pH of the resulting solution was adjusted such that the insulin analog was formulated as described below and the protein concentration was adjusted to about 15-20 mg/mL. An insulin analog prepared in this way typically had a yield of about 90%, with a residual preservative concentration at less than 100 times the starting material. 
     Briefly, three β) formulations were generated each containing 600 Units (U) of PH20-F204P or wildtype rHuPH20 (generated as described in Example 1) for a total of 6 formulations as set forth in Table 16: 
     
       
         
           
               
             
               
                 TABLE 16 
               
             
            
               
                   
               
               
                 Summary of Insulin Formulations 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                 Buffer 
                 Tonicity 
                   
                   
                   
                 API 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                   
                 Tris/ 
                 modifier 
                 Anti-Ox 
                   
                 Metal 
                 Surfactant 
                 Preservatives 
                 PH20 
                 Analog 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                 ID 
                 pH 
                 NaPO 4   
                 HCl 
                 NaCl 
                 Methionine 
                 Glycerin 
                 Zn 
                 F68 
                 Phenol 
                 m-Cresol 
                 (U/mL) 
                 (mg/mL) 
               
               
                   
               
               
                 F1. Humalog + 
                 7.0-7.8 
                 13.2 mM 
                   
                   
                   
                 173.7 mM 
                 0.242 mM 
                   
                   
                 0.315% 
                 600 
                 3.5 
               
               
                 F204P 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 F2. Humalog + 
                 7.0-7.8 
                 13.2 mM 
                   
                   
                   
                 173.7 mM 
                 0.242 mM 
                   
                   
                 0.315% 
                 600 
                 3.5 
               
               
                 wt 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 F3. Aspart + 
                 7.3 
                   
                 30 mM 
                 100 mM 
                 5 mM 
                   
                   
                 0.010% 
                 0.100% 
                 0.150% 
                 600 
                 3.5 
               
               
                 F204P 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 F4. Aspart + 
                 7.3 
                   
                 30 mM 
                 100 mM 
                 5 mM 
                   
                   
                 0.010% 
                 0.100% 
                 0.150% 
                 600 
                 3.5 
               
               
                 wt 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 F5. Aspart + 
                 7.3 
                   
                 30 mM 
                 100 mM 
                 5 mM 
                   
                   
                 0.010% 
                   
                 0.315% 
                 600 
                 3.5 
               
               
                 F204P 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 F6. Aspart + 
                 7.3 
                   
                 30 mM 
                 100 mM 
                 5 mM 
                   
                   
                 0.010% 
                   
                 0.315% 
                 600 
                 3.5 
               
               
                 wt 
               
               
                   
               
            
           
         
       
     
     Each formulation solution was dispensed in 0.5 mL aliquots into 2 mL USP Type I borosilicate glass vials with a chlorobutyl rubber stopper and an aluminum seal. The vials were incubated at 5° C., 30° C. and 37° C. Samples were withdrawn from the incubator at scheduled time points for enzymatic activity measurements as described in Example 8. 
     The results of the enzymatic activity measurements for samples incubated at 37° C., 30° C. and 5° C. are shown in Tables 17-19, respectively. At 37° C., the enzymatic activity of samples containing wildtype rHuPH20 (F2, F4 and F6) were almost totally lost within two days of incubation. In contrast, after 6 days incubation at 37° C., formulation F3 and F5, which contains PH20-F204P, lost only about 10% and 30%, respectively. The PH20-F204P formulated in commercial Humalog (F1) lost most of its activity within 2 days at 37° C. most likely due to the lack of NaCl in the formulation. 
     A similar trend for enzymatic activities of ampoules incubated at 30° C. was noted between the PH20-F204P and rHuPH20. For formulations that contain an EPA preservative level, the differences between wild type and F204P were dramatic (Table 17; F1 and F5 vs. F2 and F6). When the preservative concentration was reduced to an EPB level (F3 and F4), the F204P still outperformed wildtype rHuPH20, although there was slightly higher rHuPH20 stability compared to EPA conditions. In both EPA and EPB preservative levels, PH20-F204P was able to maintain its activity up to 14 days at 30° C. when 100 mM of NaCl was included in the formulation. 
     
       
         
           
               
             
               
                 TABLE 17 
               
             
            
               
                   
               
               
                 Enzymatic activity of rHuPH20 wild type and F204P mutant incubated at 37° C. 
               
            
           
           
               
               
            
               
                   
                 PH20 activity U/mL, (% of remaining activity) 
               
            
           
           
               
               
               
               
               
               
            
               
                 ID 
                 Initial Activity 
                 2 d 
                 4 d 
                 6 d 
                 2 w 
               
               
                   
               
               
                 F1. Humalog + F204P 
                 583 (100%) 
                  61 (10%) 
                 15 (3%) 
                 10 (2%) 
                 — 
               
               
                 F2. Humalog + wt 
                 439 (100%) 
                  4 (1%) 
                 — 
                 — 
                 — 
               
               
                 F3. Aspart + F204P 
                 625 (100%) 
                 613 (98%) 
                 496 (79%) 
                 570 (91%) 
                 532 (85%) 
               
               
                 F4. Aspart + wt 
                 566 (100%) 
                  58 (10%) 
                 24 (4%) 
                  4 (1%) 
                 — 
               
               
                 F5. Aspart + F204P 
                 657 (100%) 
                 484 (74%) 
                 462 (70%) 
                 478 (73%) 
                 360 (55%) 
               
               
                 F6. Aspart + wt 
                 596 (100%) 
                 −1 (0%) 
                 — 
                 — 
                 — 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 18 
               
             
            
               
                   
               
               
                 Enzymatic activity of rHuPH20 wild type  
               
               
                 and F204P mutant incubated at 30° C. 
               
            
           
           
               
               
            
               
                   
                 PH20 activity U/mL, (% of remaining activity) 
               
            
           
           
               
               
               
               
               
            
               
                   
                 Initial  
                   
                   
                   
               
               
                 ID 
                 Activity 
                 6 d  
                 2 w 
                 4 w 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 F1. Humalog +  
                 583 (100%) 
                 345 (59%) 
                 250 (43%)  
                 111 (19%) 
               
               
                 F204P 
                   
                   
                   
                   
               
               
                 F2. Humalog + wt 
                 439 (100%) 
                  1 (0%) 
                 16 (4%)  
                 −1 
               
               
                 F3. Aspart + F204P 
                 625 (100%) 
                 601 (96%) 
                 650 (104%) 
                 579 (93%) 
               
               
                 F4. Aspart + wt 
                 566 (100%) 
                 428 (76%) 
                 390 (69%)  
                 277 (49%) 
               
               
                 F5. Aspart + F204P 
                 657 (100%) 
                 632 (96%) 
                 655 (100%) 
                 561 (85%) 
               
               
                 F6. Aspart + wt 
                 596 (100%) 
                 145 (24%) 
                 65 (11%) 
                   9 (1.5%) 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 19 
               
             
            
               
                   
               
               
                 Enzymatic Activity at 5° C. 
               
            
           
           
               
               
               
            
               
                   
                   
                 PH20 activity  
               
               
                   
                   
                 (U/mL) at 5° C. 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                 Initial  
                   
                   
               
               
                   
                 ID 
                 Activity 
                 2 w 
                 4 w 
               
               
                   
                   
               
               
                   
                 F1. Humalog + F204P 
                 583 
                 544 
                 565 
               
               
                   
                 F2. Humalog + wt 
                 439 
                 428 
                 404 
               
               
                   
                 F3. Aspart + F204P 
                 625 
                 647 
                 607 
               
               
                   
                 F4. Aspart + wt 
                 566 
                 580 
                 496 
               
               
                   
                 F5. Aspart + F204P 
                 657 
                 695 
                 574 
               
               
                   
                 F6. Aspart + wt 
                 596 
                 583 
                 519 
               
               
                   
                   
               
            
           
         
       
     
     Example 11 
     Stability of V58R-pH20 in Insulin Coformulation 
     A. Stability of V58R-PH20 
     The PH20 variant V58R was expressed in CHO-S cells as described in Example 2 or Example 6. The transfected plasmid DNA had a sequence of nucleotides set forth in SEQ ID NO:4 containing a codon change of GTG to CGG at nucleotide positions 1295-1297, thereby encoding the V58R mutant. The V58R mutant was tested for its stability in a coformulation containing insulin aspart (insulin aspart analog prepared as described in Example 10) and under EPA or EPB preservative levels. Briefly, four (4) formulations were generated each containing 600 Units (U) of PH20-V58R or wildtype rHuPH20 (generated as described in Example 1) as set forth in Table 20. Formulations F1 and F2 represent the EPB preservative levels while formulations F3 and F4 represent the EPA preservative levels. 
     
       
         
           
               
             
               
                 TABLE 20 
               
             
            
               
                   
               
               
                 Summary of Insulin Formulations 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 Tonicity 
                   
                   
                   
                 API 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                   
                 Buffer 
                 modifier 
                 Anti-Ox 
                   
                 Metal 
                 Surfactant 
                 Preservatives 
                 PH20 
                 Analog 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                 ID 
                 pH 
                 NaPO 4   
                 Tris/HCl 
                 NaCl 
                 Methionine 
                 Glycerin 
                 Zn 
                 F68 
                 Phenol 
                 m-Cresol 
                 (U/mL) 
                 (mg/mL) 
               
               
                   
               
               
                 F1. Aspart + 
                 7.3 
                   
                 30 mM 
                 100 mM 
                 5 mM 
                   
                   
                 0.010% 
                 0.100% 
                 0.150% 
                 600 
                 3.5 
               
               
                 V58R 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 F2. Aspart + 
                 7.3 
                   
                 30 mM 
                 100 mM 
                 5 mM 
                   
                   
                 0.010% 
                 0.100% 
                 0.150% 
                 600 
                 3.5 
               
               
                 rHuPH20 wt 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 F3. Aspart + 
                 7.3 
                   
                 30 mM 
                 100 mM 
                 5 mM 
                   
                   
                 0.010% 
                   
                 0.315% 
                 600 
                 3.5 
               
               
                 V58R 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 F4. Aspart + 
                 7.3 
                   
                 30 mM 
                 100 mM 
                 5 mM 
                   
                   
                 0.010% 
                   
                 0.315% 
                 600 
                 3.5 
               
               
                 rHuPH20 wt 
               
               
                   
               
            
           
         
       
     
     Each formulation solution was dispensed in 0.5 mL aliquots into 2 mL USP Type I borosilicate glass vials with a chlorobutyl rubber stopper and an aluminum seal. The vials were incubated at 30° C. and 37° C. Samples were withdrawn from the incubator at scheduled time points for enzymatic activity measurements as described in Example 8. 
     The results of the enzymatic activity measurements for samples incubated at 37° C. and 30° C. are shown in Table 21 and Table 22. At 37° C., the enzymatic activity of samples containing wildtype rHuPH20 (F2 and F4) were almost totally lost within two days of incubation. In contrast, after 6 days incubation at 37° C., formulations F1 (EPB) and F3 (EPA), containing V58R-PH20, lost only about 25% and 40% activity, respectively. At 30° C., the enzymatic activity of samples containing wildtype rHuPH20 also was dramatically reduced in the presence of EPA or EPB preservatives levels within one month of incubation, although there was a slightly less dramatic loss in activity in the presence of EPB preservative levels. In contrast, for V58R-PH20, there was no loss of enzymatic activity for either tested formulation up to 1 month. 
     
       
         
           
               
             
               
                 TABLE 21 
               
             
            
               
                   
               
               
                 Enzymatic activity of rHuPH20 wild type  
               
               
                 and V58R mutant incubated at 37° C. 
               
            
           
           
               
               
               
            
               
                   
                   
                 PH20 activity U/mL 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                   
                 Initial  
                   
                   
                   
               
               
                   
                 Formulation 
                 Activity 
                 2 d 
                 4 d 
                 6 d 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 F1. Aspart + V58R 
                 1350 
                 1099 
                 1094 
                 1006 
               
               
                   
                 F2. Aspart + rHuPH20 wt 
                 677 
                 53 
                 −3 
                 — 
               
               
                   
                 F3. Aspart + V58R 
                 1189 
                 793 
                 581 
                 464 
               
               
                   
                 F4. Aspart + rHuPH20 wt 
                 744 
                 12 
                 −9 
                 — 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 22 
               
             
            
               
                   
               
               
                 Enzymatic activity of rHuPH20 wild  
               
               
                 type and V58R mutant incubated at 30° C. 
               
            
           
           
               
               
            
               
                   
                 PH20 activity U/mL 
               
            
           
           
               
               
               
               
            
               
                 Formulation 
                 Initial Activity 
                 2 weeks 
                 4 weeks 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 F1. Aspart + V58R 
                 1350 
                 1368 
                 1208 
               
               
                 F2. Aspart + rHuPH20 wt 
                 677 
                 422 
                 256 
               
               
                 F3. Aspart +V58R 
                 1189 
                 1228 
                 1171 
               
               
                 F4. Aspart + rHuPH20 wt 
                 744 
                 21 
                 −5 
               
               
                   
               
            
           
         
       
     
     B. Comparison of Stability of F204P and V58R 
     The PH20 variant V58R-PH20 was compared to F204P for its stability in a coformulation containing insulin aspart (insulin aspart analog prepared as described in Example 10) and under EPA or EPB preservative levels. Briefly, eight (8) formulations were generated as set forth in Table 23. Formulations F1-F4 represent the EPB preservative levels while formulations F5-F8 represent the EPA preservative levels. Formulations F3 and F4 and formulations F7 and F8 were identical and represent the wildtype control formulations formulations used for the EPB or EPA studies, respectively. 
     
       
         
           
               
             
               
                 TABLE 23 
               
             
            
               
                   
               
               
                 Summary of Insulin Formulations 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                 Buffer 
                 Tonicity 
                   
                   
                 Preservatives 
                 API 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
            
               
                   
                 Tris/ 
                 modifier 
                 Anti-Ox 
                   
                 Metal 
                 Surfactant 
                   
                 m- 
                 PH20 
                 Analog 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                 ID 
                 pH 
                 NaPO 4   
                 HCl 
                 NaCl 
                 Methionine 
                 Glycerin 
                 Zn 
                 F68 
                 Phenol 
                 Cresol 
                 U/mL 
                 mg/mL 
               
               
                   
               
               
                 F1. Aspart + 
                 7.3 
                   
                 30 mM 
                 100 mM 
                 5 mM 
                   
                   
                 0.010% 
                 0.100% 
                 0.150% 
                 600 
                 3.5 
               
               
                 V58R 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 F2 Aspart + 
                 7.3 
                   
                 30 mM 
                 100 mM 
                 5 mM 
                   
                   
                 0.010% 
                 0.100% 
                 0.150% 
                 600 
                 3.5 
               
               
                 F204P 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 F3. Aspart + 
                 7.3 
                   
                 30 mM 
                 100 mM 
                 5 mM 
                   
                   
                 0.010% 
                 0.100% 
                 0.150% 
                 600 
                 3.5 
               
               
                 rHuPH20 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 wt(1) 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 F4. Aspart + 
                 7.3 
                   
                 30 mM 
                 100 mM 
                 5 mM 
                   
                   
                 0.010% 
                 0.100% 
                 0.150% 
                 600 
                 3.5 
               
               
                 rHuPH20 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 wt(2) 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 F5. Aspart + 
                 7.3 
                   
                 30 mM 
                 100 mM 
                 5 mM 
                   
                   
                 0.010% 
                   
                 0.315% 
                 600 
                 3.5 
               
               
                 V58R 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 F6 Aspart + 
                 7.3 
                   
                 30 mM 
                 100 mM 
                 5 mM 
                   
                   
                 0.010% 
                   
                 0.315% 
                 600 
                 3.5 
               
               
                 F204P 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 F7. Aspart + 
                 7.3 
                   
                 30 mM 
                 100 mM 
                 5 mM 
                   
                   
                 0.010% 
                   
                 0.315% 
                 600 
                 3.5 
               
               
                 rHuPH20 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 wt(1) 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 F8. Aspart + 
                 7.3 
                   
                 30 mM 
                 100 mM 
                 5 mM 
                   
                   
                 0.010% 
                   
                 0.315% 
                 600 
                 3.5 
               
               
                 rHuPH20 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 wt(2) 
               
               
                   
               
            
           
         
       
     
     Each formulation solution was dispensed in 0.5 mL aliquots into 2 mL USP Type I borosilicate glass vials with a chlorobutyl rubber stopper and an aluminum seal. The vials were incubated at 30° C. and 37° C. Samples were withdrawn from the incubator at scheduled time points for enzymatic activity measures as described in Example 8. 
     The results show that the percentage hyaluronidase activity in the tested formulations after preincubation at 37° C. was slightly greater for both PH20 mutants when formulated in EPB and not EPA preservative levels. While the percent of activity remaining was greater than 80% for both tested mutants after 6 days incubation in formulations containing EPB preservative levels, it was less in the presence of EPA preservative levels. For example, the activity remaining at 6 days in EPA preservative levels was slightly less than 80% after 6 days for F204P-PH20, while it was only about 40% for V58R-PH20. Hence, the results also show that at 37° C., V58R-PH20 is somewhat less stable than the F204P-PH20, in particular in a formulation with EPA preservative levels. After incubation at 30° C. for at least a week, the F204P-PH20 and V58R-PH20 were stable and exhibited almost 100% initial activity in the presence of both EPA and EPB preservative levels. In contrast, rHuPH20 exhibited only about 40% of its initial activity after 4 weeks at 30° C. in the presence of EPB preservative levels, while it exhibited no detectable activity after 4 weeks at 30° C. in the presence of EPA preservative levels. 
     Example 12 
     Expression of F204P-pH20 Using a Lentivirus Expression Vector 
     A lentivirus expression vector, pLV-EF1a-PH20(F204P)-IRES-GFP-Bsd was generated containing a codon-optimized mutant hyaluronidase cDNA encoding F204P-PH20. The sequence of pLV-EF 1a-PH20(F204P)-IRES-GFP-Bsd is set forth in SEQ ID NO:925. The pLV-EF1a-PH20(F204P)-IRES-GFP-Bsd vector contains an ampicillin resistance gene (AmpR) located at nucleotides 8611-9471, an EF1a promoter at residues 1933 to 2327, an IRES at residues 4786-5370, a GFP-Bsd at residues 5394-6527 and nucleotides encoding F204P-PH20 at residues 3369-4781. 
     Lentivirus was produced as described in Bandaranayake et al. ((2011) Nucleic Acids Research, 39:e143). Briefly, 293T cells (ATCC) were plated at 6×10 6  cells onto 10 cm tissue culture plates. After 24 hours, 6 μg of psPAX2 (SEQ ID NO:926; Addgene plasmid No. 12260), 3 μg of PMD2.G (SEQ ID NO:927; Addgene plasmid #12259) and 9 μg lentiviral vector plasmid pLV-EF1a-PH20(F204P)-IRES-GFP-Bsd were mixed in 1.5 mL Opti-MEM (Life Technologies). 45 μL of Lipofectamine 2000 (LF2000; Life Technologies) were diluted into 1.5 mL Opti-MEM (Life Technologies). The DNA and LF2000 were mixed gently, and incubated at room temperature for 20 minutes to allow the DNA and lipid to form complexes. In the meantime, the overnight culture medium was replaced with 5.0 mL DMEM+10% FBS without antibiotics. A volume of 3.0 mL containing the DNA-LF2000 complexes were added to the 293T cells. The medium containing the DNA-LF2000 complexes was replaced with 10 mL complete medium at 12-16 hours post-transfection. The supernatant was collected at 48 hours post-transfection and the medium was transferred to a polypropylene storage tube. The virus-containing medium was spun at 1300 rpm for 5 minutes to pellet any 293T cells that were carried over during collection. The supernatant was carefully transferred to a sterile polypropylene storage tube. 
     CHO-S cells (Invitrogen) were grown in CHO- S media  (Invitrogen) with shaking at 120 rpm at 37° C. and 5% CO 2  in vented 125-mL shake flasks (Nalgene). For transduction, CHO-S cells were added to wells of a six-well plate at 2×10 6  cells per well in 2 ml of CHO-S media containing 4 μg/mL hexadimethrine bromide at a final concentration of 4 μg/mL (Polybrene; SIGMA). Virus was added to each well at a multiplicity of infection (MOI) of 10 and the cells were incubated with shaking (120 rpm) at 37° C. and 5% CO 2  for 6 hours. The cells were then harvested and pelleted by low speed centrifugation (500×g, 5 min). The transduction medium was removed and replaced with 10 mL of fresh CHO-S medium (Invitrogen) supplemented with GlutaMax (50 mL/liter) and transferred to a T-25 flask. Three days post infection, blasticidin (Invitrogen) was added to the growth medium at a concentration of 1 μg/mL. The medium was changed regularly at 3-4 day intervals, and the cells were transferred to a T75 flask for expansion. Two weeks after the initial infection, the cells were expanded to shaker flasks and maintained in culture using medium containing 1 μg/mL blasticidin. F204P-PH20 protein secreted into the CHO-S medium was collected and purified by affinity chromatography using an anti-rHuPH20 affinity column as described in Example 6. The protein was prepared in standard API buffer (10 mM Histidine, 130 mM NaCl, pH 6.5). 
     Example 13 
     Analysis of Secondary Structure and Melting Temperature 
     The secondary structure and melting temperature of the PH20 variant F204P was tested and compared to wild-type rHuPH20 (generated as described in Example 1) to further assess stability of the variant. The secondary structure was tested by circular dichroism. A Jasco J-810-1505 equipped with PTC-4245 was employed for the CD spectral measurement and the CD spectra were collected by Spectra Manager (Version 1.5, Jasco). Procedures for instrumental set up and data collection are described in Table 24. 
     
       
         
           
               
             
               
                 TABLE 24 
               
             
            
               
                   
               
               
                 CD Spectroscopy Operation Conditions 
               
            
           
           
               
               
               
            
               
                   
                 Parameters 
                 Conditions 
               
               
                   
                   
               
               
                   
                 Nitrogen flow rate 
                 25 W/h 
               
               
                   
                 Sample temperature 
                 30-75° C. 
               
               
                   
                 Sample concentration 
                 Approx. 0.1 mg/mL 
               
               
                   
                 Cell pathlength 
                 1 mm 
               
               
                   
                 Wavelength 
                 220 nm  
               
               
                   
                 Data pitch 
                 1° C. 
               
               
                   
                 Delay time 
                 60 seconds 
               
               
                   
                 Temperature slope 
                 1° C./min 
               
               
                   
                 Sensitivity 
                 standard 
               
               
                   
                 Response 
                 4 seconds 
               
               
                   
                 Band width 
                 1 nm 
               
               
                   
                   
               
            
           
         
       
     
     1. Sample Preparation and Measurement Two hundred (200) μL of a 0.1 mg·mL protein sample diluted in Mcllvaine&#39;s buffer (McIlvaine (1921) JBC 49:183) adjusted to pH 6.5 were prepared. A series of samples of the F204P variant were also generated that varied in pH by adjustment using Mcllvaine&#39;s buffer to a pH range from 5.0 to 7.5 as set forth in Table 25. In addition, samples also were generated by adjusting the NaCl concentration to 17.5 mM to 140 mM as set forth in Table 26. Samples were filtered using a 0.2 μm syringe filter prior to measurement. Similar samples were generated for rHuPH20. Then, 200 μL samples were transferred to a rectangular cuvetted having a 1 mm width and seated on Jasco J-810 spectropolarimeter. CD spectra of the samples were collected under the conditions described in Table 20. The melting temperature (T m ) was calculated using Spectra Manager (v 1.5, Jasco) from the CD spectral intensity measured at the temperature range from 30° C. to 75° C. The cuvettes were cleaned by Chromerge® cleaner (C577-12, Fisher scientific) between individual sample loading and after the run. 
     
       
         
           
               
             
               
                 TABLE 25 
               
             
            
               
                   
               
               
                 Sample pH and concentration 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                   
                   
                 F204P 
               
               
                 Target 
                 Actual  
                 F204P 
                 Buffer  
                 concentration 
               
               
                 pH 
                 pH 
                 (μL) 
                 (μL) 
                 (mg/mL) 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 5.0 
                 4.92 
                 25 
                 175 
                 0.1 
               
               
                 5.5 
                 5.38 
                 25 
                 175 
                 0.1 
               
               
                 6.0 
                 5.99 
                 25 
                 175 
                 0.1 
               
               
                 6.5 
                 6.49 
                 25 
                 175 
                 0.1 
               
               
                 7.0 
                 7.00 
                 25 
                 175 
                 0.1 
               
               
                 7.5 
                 7.5 
                 25 
                 175 
                 0.1 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 26 
               
             
            
               
                   
               
               
                 Sodium Concentration in Samples at pH 6.5 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 Target NaCl 
                 NaCl, 
                   
                 Buffer 
                 F204P 
               
               
                   
                 concentration 
                 2.8M 
                 F204P  
                 at pH 6.5  
                 concentration 
               
               
                   
                 (mM) 
                 (μL) 
                 (μL) 
                 (μL) 
                 (mg/mL) 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 17.5 
                 0.00 
                 25 
                 175 
                 0.1 
               
               
                   
                 50.0 
                 2.32 
                 25 
                 172.7 
                 0.1 
               
               
                   
                 75.0 
                 4.11 
                 25 
                 170.9 
                 0.1 
               
               
                   
                 100.0 
                 5.89 
                 25 
                 169.1 
                 0.1 
               
               
                   
                 140.0 
                 8.75 
                 25 
                 166.3 
                 0.1 
               
               
                   
                   
               
            
           
         
       
     
     2. Results 
     The results show that the secondary structure of F204P is similar to rHuPH20. As a function of temperature, circular dichroism showed that a change in the absorption was measured with increasing temperatures. As a function of pH, the T m  distribution was closely comparable for both F204P and rHuPH20 and the highest T m  for each was obtained between pH 5.5 and pH 6.0. The results, however, showed that T m  of the F204P variant was approximately 9° C. higher at all tested ranges than wildtype rHuPH20. This result indicated that the F204P mutant is more stable against thermal stress conditions. As a function of salt, the results show that the F204P and wildtype rHuPH20 both exhibited an increasing T m  with higher salt concentration, showing that both have a proportional inclination toward salt concentration. 
     Example 14 
     Assessment of Enzymatic Activity in an Intradermal Trypan Blue Dispersion Assay 
     Spreading activity of the PH20 variant F204P was assessed using a dye dispersion in vivo assay. Briefly, purified PH20 variant F204P (prepared as described in Example 12) and wild-type rHuPH20 (prepared as described in Example 1) were both formulated in API buffer (10 mM Histidine, 130 mM NaCl, pH 6.5) at a concentration of 10,000 U/mL. The stocks were further diluted to three target concentrations of 1000, 100 and 10 U/mL by serial 1:10 dilutions in API buffer. Purified proteins (either rHuPH20 or F204P-PH20) were diluted 1:1 with 0.4% Trypan Blue (0.4% liquid solution; Catalog No. 15250, Invitrogen) to give a final concentration of 5, 50 and 500 U/mL protein, each containing 0.2% trypan blue. A vehicle control (API buffer) also was prepared. Forty-two (42) female NCr nu/nu homozygous mice were used in the study with six mice used per group as set forth in Table 27. 
     
       
         
           
               
             
               
                 TABLE 27 
               
             
            
               
                   
               
               
                 Summary of Treatment Groups for Dye Dispersion Study 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 No.  
                   
                 Final Dose with 
                   
                 Injection 
               
               
                   
                 of 
                 Test  
                 Trypan Blue 
                 Trypan  
                 Volume 
               
               
                 Group 
                 Mice 
                 Article 
                 (Units/mL) 
                 Blue  
                 (mL) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 1 
                 6 
                 Control 
                 0 
                 0.2% 
                 0.04 
               
               
                 2 
                 6 
                 rHuPH20 
                 5 
                 0.2% 
                 0.04 
               
               
                 3 
                 6 
                 rHuPH20 
                 50 
                 0.2% 
                 0.04 
               
               
                 4 
                 6 
                 rHuPH20 
                 500 
                 0.2% 
                 0.04 
               
               
                 5 
                 6 
                 F204P-PH20 
                 5 
                 0.2% 
                 0.04 
               
               
                 6 
                 6 
                 F204P-PH20 
                 50 
                 0.2% 
                 0.04 
               
               
                 7 
                 6 
                 F204P-PH20 
                 500 
                 0.2% 
                 0.04 
               
               
                   
               
            
           
         
       
     
     Forty (40) μL of samples were administered by a single intradermal injection. The area of dye dispersion was measured at 2.5, 5, 10, 15 and 20 minutes post-injection and was recorded by photographic imaging by photograph of the injection site with a Nikon D90 digital camera with 60 mm prime micro-lens. A laser distance meter (Leica D3) was used to accurately position the camera at a pre-determined distance from the Trypan Blue dye area on the animal. The area of the dye was determined using Image-Pro Analyzer 7.0 (MediaCybernetics, Inc). The calculated areas were expressed as mm 2 . 
     The results are set forth in Table 28. The results showed that the dispersion activity of the PH20 variant F204P was substantially identical to the dispersion activity of rHuPH20. The ability to increase the area of dye dispersion was dose-dependent, with both proteins having greatest activity at 500 U/mL. The results also showed that the area of dye dispersion increased with time post-intradermal injection. The areas of dye dispersion of rHuPH20 and F204P-PH20 were significantly greater than the areas of dye dispersion for the controls (p&lt;0.05) at all time points when formulated at all concentrations (5, 50 and 500 U/mL) with the exception of rHuPH20 at the lowest concentration (5 U/mL). When compared to each other, rHuPH20 and F204P-PH20 showed similar dispersion effects, although there was a significant difference in dispersion between the two groups at 5 U/mL and 500 U/mL but not at 50 U/mL. In sum, the results show that both rHuPH20 and F204P-PH20 provided a statistically significant increase in the area of dye dispersion compared to the vehicle control. 
     
       
         
           
               
             
               
                 TABLE 28 
               
             
            
               
                   
               
               
                 Trypan Blue Dispersion 
               
            
           
           
               
               
            
               
                 Group 
                 Area (mm2) 
               
            
           
           
               
               
               
               
               
               
            
               
                 Avg. (n = 6) 
                 2.5 min 
                 5 min 
                 10 min 
                 15 min 
                 20 min 
               
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 1: Control 
                 37.44 ± 2.81 
                 38.16 ± 3.33 
                 43.71 ± 2.12 
                 45.70 ± 2.38 
                 48.77 ± 2.14 
               
               
                 2: rHuPH20 (5 U/mL) 
                 36.68 ± 2.83 
                 42.31 ± 2.57 
                 45.41 ± 2.75 
                 46.72 ± 3.35 
                 49.61 ± 2.97 
               
               
                 3: rHuPH20 (50 U/mL) 
                 39.24 ± 1.20 
                 44.90 ± 1.44 
                 46.96 ± 1.70 
                 50.08 ± 2.07 
                 53.50 ± 1.59 
               
               
                 4: rHuPH20 (500 U/mL) 
                 44.72 ± 1.35 
                 50.21 ± 1.92 
                 57.47 ± 1.29 
                 59.77 ± 1.25 
                 57.17 ± 3.28 
               
               
                 5: F204P (5 U/mL) 
                 39.65 ± 1.53 
                 46.09 ± 2.73 
                 48.07 ± 1.43 
                 52.54 ± 2.01 
                 54.11 ± 1.01 
               
               
                 6: F204P (50 U/mL) 
                 38.10 ± 1.92 
                 47.07 ± 2.12 
                 51.48 ± 2.14 
                 55.24 ± 1.90 
                 58.34 ± 2.89 
               
               
                 7: F204P (500 U/mL) 
                 46.58 ± 1.67 
                 54.06 ± 2.52 
                 58.96 ± 1.85 
                 64.37 ± 1.72 
                 64.44 ± 2.17 
               
               
                   
               
            
           
         
       
     
     Example 15 
     Assessment of Enzymatic Activity By Dermal Barrier Reconstitution 
     Activity of F204P-PH20 was assessed and compared to rHuPH20 to measure the amount of time required for the dermal barrier to reconstitute itself after intradermal hyaluronidase administration. Dermal reconstitution was evaluated by comparing the duration of the hyaluronidase spreading activity as assessed by monitoring the area of diffusion of 0.4% Trypan Blue over time. The proteins used in the study were purified PH20 variant F204P (prepared as described in Example 12) and wild-type rHuPH20 (prepared as described in Example 1) that were both formulated in API buffer (10 mM Histidine, 130 mM NaCl, pH 6.5). Vehicle (API buffer) was used as a control. Male NCr nu/nu homozygous mice were used in the study with three animals per time point for a total of fifteen mice used per group as set forth in Table 29. 
     
       
         
           
               
             
               
                 TABLE 29 
               
             
            
               
                   
               
               
                 Summary of Treatment Groups for  
               
               
                 Dermal Barrier Reconstitution Study 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                   
                 No.  
                 Time  
                   
                 Final  
                 Injection  
               
               
                   
                   
                 of 
                 Points 
                 Test  
                 Dose 
                 Volume 
               
               
                   
                 Group 
                 Mice 
                 (h) 
                 Article 
                 (Units/mL) 
                 (mL) 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                 1 
                 15 
                 0.5, 1,  
                 Control 
                 0 
                 0.04 
               
               
                   
                   
                   
                 4, 24, 48 
                   
                   
                   
               
               
                   
                 2 
                 15 
                 0.5, 1,  
                 rHuPH20 
                 100 
                 0.04 
               
               
                   
                   
                   
                 4, 24, 48 
                   
                   
                   
               
               
                   
                 3 
                 15 
                 0.5, 1,  
                 F204P 
                 100 
                 0.04 
               
               
                   
                   
                   
                 4, 24, 48 
                   
                   
                   
               
               
                   
                   
               
            
           
         
       
     
     All mice received two intradermal doses of vehicle control or rHuPH20 or F204P-PH20 at 100 U/mL in 0.04 mL at study time 0. The same control or test article was injected on the opposing lateral sides of each animal (right, R; left, L). Injection sites were marked with a permanent marker. Trypan Blue Stain (0.4% liquid solution; 15250, Invitrogen) was administered at a volume of 0.04 mL by intradermal injection at the same injection site at 0.5, 1, 4, 24 and 48 hours post-injection of test article or control. At 5 and 20 minutes post-injection of the Trypan Blue Stain, the area of the dye at the injection site was measured by digital imaging of the region as described in Example 14. 
     The results are set forth in Table 30. The results show that when the area of dye dispersion was measured at various time points after administration of the test article or control, there was a statistically significant increase in the area of dye dispersion at 30 min and 1 hour post-injection of rHuPH20 or F204P-PH20. By 4 hours post-administration of the enzymes, however, there was not a statistically significant increase in the area of dye dispersion compared to control. In addition, no statistically significant differences in the area of dye dispersion was observed between the rHuPH20 and F204P-PH20 treatment groups. Therefore, the duration of the spreading activity of rHuPH20 and F204P were similar and show that rHuPH20 and F204P-PH20 have comparable in vivo performance. 
     
       
         
           
               
             
               
                 TABLE 30 
               
             
            
               
                   
               
               
                 Dermal Reconstitution 
               
            
           
           
               
               
               
               
               
            
               
                   
                 min 
                   
                   
                   
               
               
                 time 
                 post- 
                   
                   
                   
               
               
                 Point 
                 injection 
                 Vehicle 
                 rHuPH20 
                 F204P-PH20 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 30 
                 5 
                 49.96 ± 2.05 
                 80.84 ± 8.03 
                 80.76 ± 4.46 
               
               
                   
                 20 
                 64.42 ± 2.49 
                 94.55 ± 7.09 
                 95.75 ± 5.18 
               
               
                  1 hour 
                 5 
                 58.01 ± 3.21 
                 82.56 ± 6.40 
                 77.11 ± 3.18 
               
               
                   
                 20 
                 65.19 ± 6.21 
                 96.19 ± 6.39 
                 91.45 ± 1.73 
               
               
                  4 hour 
                 5 
                 52.10 ± 3.47 
                 67.19 ± 2.39 
                 67.33 ± 3.93 
               
               
                   
                 20 
                 57.69 ± 3.92 
                 81.15 ± 4.45 
                 82.21 ± 4.14 
               
               
                 24 hour 
                 5 
                 49.87 ± 3.25 
                 59.01 ± 2.15 
                 54.91 ± 3.54 
               
               
                   
                 20 
                 57.15 ± 3.47 
                 67.65 ± 2.27 
                 62.91 ± 3.30 
               
               
                 48 hour 
                 5 
                 53.64 ± 2.99 
                 53.53 ± 4.88 
                 55.64 ± 7.19 
               
               
                   
                 20 
                 61.57 ± 4.02 
                 66.33 ± 4.12 
                 63.11 ± 5.97 
               
               
                   
               
            
           
         
       
     
     Example 16 
     In Vivo Pharmacokinetics of F204P-PH20 Compared to rHuPH20 
     The pharmacokinetics (PK) of rHuPH20 and F204P-PH20 were compared following intravenous tail-vein administration by measuring the plasma hyaluronidase levels over time after administration. The proteins used in the study were purified PH20 variant F204P (prepared as described in Example 12; batch concentration 1.02 mg/mL) and wild-type rHuPH20 (prepared as described in Example 1; batch concentration 0.95 mg/mL) formulated in API buffer (10 mM Histidine, 130 mM NaCl, pH 6.5). The proteins were prepared at a concentration of 0.087 mg/mL in API buffer for a dose volume of about 5 mL. An animal that was not administered with protein was used a control (pre-dose control). Forty two (42) male CD-1 mice (˜20-30 grams) were used in the study with six animals per treatment group as set forth in Table 31. 
     
       
         
           
               
             
               
                 TABLE 31 
               
             
            
               
                   
               
               
                 Pharmacokinetics of Single Intravenous  
               
               
                 Dose of rHuPH20 or F204P-PH20 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                 Dose 
                   
               
               
                   
                 number of 
                 Test  
                 Dose 
                 Volume 
                   
               
               
                 Group 
                 animals (No.) 
                 Article 
                 (mg/kg) 
                 (mL/kg) 
                 Euthanasia 
               
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 1 
                 6 (Nos. 1-6) 
                 no treatment 
                 N/A 
                 N/A 
                 pre-dose 
               
               
                 2 
                 6 (Nos. 7-12) 
                 rHuPH20 
                 0.433 
                 5 
                 1 min 
               
               
                 3 
                 6 (Nos. 13-18) 
                 rHuPH20 
                 0.433 
                 5 
                  5 ± 1 min 
               
               
                 4 
                 6 (Nos. 19-24) 
                 rHuPH20 
                 0.433 
                 5 
                 10 ± 2 min 
               
               
                 5 
                 6 (Nos. 25-30) 
                 F204P-PH20 
                 0.433 
                 5 
                 1 min 
               
               
                 6 
                 6 (Nos. 21-36) 
                 F204P-PH20 
                 0.433 
                 5 
                  5 ± 1 min 
               
               
                 7 
                 6 (Nos. 37-42) 
                 F204P-PH20 
                 0.433 
                 5 
                 10 ± 2 min 
               
               
                   
               
            
           
         
       
     
     Mice were intravenously administered 0.433 mg/kg rHuPH20 or F204P-PH20 by tail vein injection. Blood samples were obtained from animals 1 minute, 5 minutes and 10 minutes post-administration. Blood samples were obtained by terminal bleed (cardiac puncture) and collected into blood collection tubes containing the anti-coagulant EDTA for the preparation of plasma. Blood samples were centrifuged at 500 g for 10 minutes and the plasma removed and frozen at −80° C. until assessment of hyaluronidase activity using the microturbidity assay described in Example 8. 
     The results are set forth in Table 32. The results show that hyaluronidase activity is detected in plasma prior to treatment with the hyaluronidase. Within 1 minute post-treatment with either rHuPH20 or F204P-PH20 hyaluronidase, there is a detectably high amount of hyaluronidase activity present in the plasma, which is similar between both treatment groups. Over time, the hyaluronidase activity rapidly decreases for both treatment groups, although there is detectably hyaluronidase activity present in the plasma 10 minutes post-administration. At the 5 minute and 10 minute post-administration time points, activity in the plasma in animals treated with F204P-PH20 is greater than in animals treated with rHuPH20. This shows that F204P-PH20 exhibits somewhat greater activity for a prolonged time period, and therefore exhibits greater half-life in vivo than rHuPH20. 
     
       
         
           
               
             
               
                 TABLE 32 
               
             
            
               
                   
               
               
                 rHuPH20 and F204P-PH20 Activity (U/mL) in Mouse Plasma K2EDTA 
               
            
           
           
               
               
            
               
                   
                 Time Point (min) 
               
            
           
           
               
               
               
               
               
            
               
                   
                 Predose 
                 1 minute 
                 5 minute 
                 10 minute 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                   
                 Animal 
                   
                 Animal 
                   
                 Animal 
                   
                 Animal 
                   
               
               
                 Protein 
                 No. 
                 U/mL 
                 No. 
                 U/mL 
                 No. 
                 U/mL 
                 No. 
                 U/mL 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 rHuPH20 
                 1 
                 BQL 
                 7 
                     235 a   
                 13 
                 18.3 
                 19 
                 3.76 
               
               
                   
                 2 
                 BQL 
                 8 
                   13.5 
                 14 
                 7.70 
                 20 
                 3.70 
               
               
                   
                 3 
                 BQL 
                 9 
                 278 
                 15 
                 8.85 
                 21 
                 2.64 
               
               
                   
                 4 
                 BQL 
                 10 
                 328 
                 16 
                 10.5 
                 22 
                 2.70 
               
               
                   
                 5 
                 BQL 
                 11 
                 356 
                 17 
                 12.8 
                 23 
                 2.36 
               
               
                   
                 6 
                 BQL 
                 12 
                 287 
                 18 
                 18.0 
                 24 
                 2.80 
               
               
                 F204P-PH20 
                 1 
                 BQL 
                 25 
                 249 
                 31 
                 48.0 
                 37 
                 11.5 
               
               
                   
                 2 
                 BQL 
                 26 
                 223 
                 32 
                 21.6 
                 38 
                 11.4 
               
               
                   
                 3 
                 BQL 
                 27 
                 246 
                 33 
                 38.4 
                 39 
                 10.1 
               
               
                   
                 4 
                 BQL 
                 28 
                 246 
                 34 
                 38.6 
                 40 
                 12.2 
               
               
                   
                 5 
                 BQL 
                 20 
                     0.696 
                 35 
                 38.2 
                 41 
                 10.8 
               
               
                   
                 6 
                 BQL 
                 30 
                 257 
                 36 
                 28.5 
                 42 
                 10.2 
               
               
                   
               
               
                 BQL—Below Quantifiable Limit &lt;0.625 U/mL with minimum required dilution 
               
               
                   a Hemolyzed 
               
            
           
         
       
     
     Since modifications will be apparent to those of skill in this art, it is intended that this invention be limited only by the scope of the appended claims.