Office Action 1: Non-Final Rejection (2024-03-27)
Rejection types: 112 - Written Description/Enablement, Double Patenting

CLAIMS:
CLAIMS

What is claimed is:

1\. A method of cleaning a separation matrix in a matrix retaining device during purifying a protein expressed in a non-native soluble form in a non-mammalian expression system, the method comprising:
(a) lysing a non-mammalian cell in which the protein is expressed in a non-native soluble form to generate a cell lysate;
(b) contacting the cell lysate with a separation matrix under conditions suitable for the protein to associate with the separation matrix;
(c) washing the separation matrix with a cleaning agent; and
(d) eluting the protein from the separation matrix.

2\. The method of claim 1, wherein the separation matrix is an affinity resin.

3\. The method of claim 1, wherein the separation matrix is a non-affinity resin.

4\. The method of claim 1, wherein the separation matrix is an affinity resin selected from the group consisting of: Protein A, Protein G, and a synthetic mimetic affinity resin.

5\. The method of claim 1, wherein the separation matrix is a non-affinity resin selected from the group consisting of: ion exchange, mixed mode, and a hydrophobic interaction resin.

6\. The method of claim 1, further comprising: repeating (a) to (d) with the same separation matrix.

7\. The method of claim 1, further comprising: repeating (a) to (d) at least 100 times with the same separation matrix.
8\. The method of claim 1, further comprising: repeating (a) to (d) at least 150 times with the same separation matrix.

9\. The method of claim 1, wherein the protein is a complex protein.

10\. The method of claim 1, wherein the cleaning agent is at least one cleaning agent selected from the group consisting of: caustic, detergent, a denaturant, and a reductant.

11\. The method of claim 10, wherein the caustic is sodium or potassium hydroxide.

12\. The method of claim 10, wherein the detergent is SDS or Triton X-100.

13\. The method of claim 10, wherein the denaturant is urea or a guanidine-derivative.

14\. The method of claim 10, wherein the denaturant is DTT or a thioglycolate.

15\. The method of claim 1, further comprising regenerating the separation matrix after the washing.

16\. A method of purifying a protein expressed in a non-native limited solubility form in a non-mammalian expression system comprising:
(a) solubilizing the expressed protein in a solubilization solution comprising one or more of the following:
(i) a denaturant;
(ii) a reductant; and
(iii) a surfactant;
(b) forming a refold solution comprising the solubilization solution and a refold buffer, the refold buffer comprising one or more of the following:
(i) a denaturant;
(ii) an aggregation suppressor;
(iii) a protein stabilizer; and
(iv) a redox component; (c) applying the refold solution to a separation matrix under conditions suitable for the protein to associate with the matrix;
(d) washing the separation matrix; and
(e) eluting the protein from the separation matrix.

17\. A method of purifying a protein expressed in a non-native soluble form in a non- mammalian expression system, the method comprising:
(a) lysing a non-mammalian cell in which the protein is expressed in a non-native soluble form to generate a cell lysate;
(b) filtering the cell lysate;
(c) contacting the filtered cell lysate with a separation matrix under conditions suitable for the protein to associate with the separation matrix;
(d) washing the separation matrix;
(e) eluting the protein from the separation matrix; and
(f) isolating the protein after elution. 40 41 42 5 10 15 20

OFFICE ACTION:
DETAILED ACTION
Notice of Pre-AIA  or AIA  Status
The present application is being examined under the pre-AIA  first to invent provisions.
Claims 1-17 are pending and are currently under examination.
Information Disclosure Statement
The information disclosure statements filed on 12/12/2022 have been considered.  Signed copies are enclosed.
Claim Objections
Claims 12 and 14 are objected to because of the following informalities: claims 12 and 14 contain the acronyms SDS and DTT.  While acronyms are permissible shorthand in the claims, the first recitation of the term should include the full recitation followed by the acronym in parentheses.  Appropriate correction is required.
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(B)  CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 1-15 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor, or for pre-AIA  the applicant regards as the invention.
Claims 1-15 are rendered indefinite by the phrase “washing the separation matrix with a cleaning agent.”  It is not clear what limitations are engendered by the phrase “cleaning agent”.  In addition, it is not clear what is meant by the term “washing the separation matrix”.  Generally, in chromatography methods, a “wash” step is used to wash excess and unbound components from the reaction surface.  The instant specification refers to the wash step this way, stating “the protein of interest associates with the matrix in the presence of the components of refold buffer, impurities are washed away and the protein is eluted.”  The specification further states “after the protein of interest has been associated with the separation matrix by contacting the cell lysate containing the protein with the separation matrix, thereby allowing the protein to associate with the adsorbent component of the separation matrix, the separation matrix is washed to remove unbound lysate and impurities. The wash buffer can be of any composition, as long as the composition and pH of the wash buffer is compatible with both the protein and the matrix, and maintains the interaction between the protein and the matrix. Examples of suitable wash buffers that can be employed include solutions containing glycine, Tris, citrate, or phosphate; typically at levels of 5-100 mM (e.g., 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75 or 100 mM). These solutions can also contain an appropriate salt ion, such as chloride, sulfate or acetate at levels of 5-500 mM (e.g., 5, 10, 12, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450 or 500 mM). The resin can be washed once or any number of times. The exact composition of a wash buffer will vary with the protein being purified.  It is of note that this step occurs before the protein is eluted.  Therefore, the wash cannot be something that causes elution of the protein.  The specification also refers to cleaning of the column.  This is the context in which a “cleaning agent” is discussed.  The specification states that the cleaning agent include caustic (e.g. sodium or potassium hydroxide), detergents (e.g. SDS or Triton X-100), denaturants (e.g. urea or guanidine-derivatives), and reductants (e.g. DTT, or thioglycolates).  All of these cleaning agents would be unsuitable for a wash prior to eluting the protein but would be suitable for cleaning (and regenerating) the column after eluting the protein, which is how the use of these agents is described in the specification.  Therefore, while a washing step prior to eluting the protein makes sense, but only as the standard wash that is generally used in chromatography, it does not make sense using any of the cleaning agents recited in the specification.  As such, it is not clear what applicant intends to be the cleaning agent and, in claims where specific agents are specified, these would not work in the method as claimed because they would disrupt binding of the protein and the matrix.
Claim 12 is rendered indefinite by the recitation of the trademark TRITON.  If a trademark or trade name is used in a claim as a limitation to identify or describe a particular material or product, the claim does not comply with the requirements of the 35 U.S.C. 112, second paragraph. Ex parte Simpson, 218 USPQ 1020 (Bd. App. 1982). The claim scope is uncertain since the trademark or trade name cannot be used properly to identify any particular material or product.
Double Patenting
A rejection based on double patenting of the “same invention” type finds its support in the language of 35 U.S.C. 101 which states that “whoever invents or discovers any new and useful process... may obtain a patent therefor...” (Emphasis added). Thus, the term “same invention,” in this context, means an invention drawn to identical subject matter. See Miller v. Eagle Mfg. Co., 151 U.S. 186 (1894); In re Vogel, 422 F.2d 438, 164 USPQ 619 (CCPA 1970); In re Ockert, 245 F.2d 467, 114 USPQ 330 (CCPA 1957).
A statutory type (35 U.S.C. 101) double patenting rejection can be overcome by canceling or amending the claims that are directed to the same invention so they are no longer coextensive in scope. The filing of a terminal disclaimer cannot overcome a double patenting rejection based upon 35 U.S.C. 101.
Claim 16 is rejected under 35 U.S.C. 101 as claiming the same invention as that of claim 9 of prior U.S. Patent No. 9,643,997. This is a statutory double patenting rejection.
Claim 17 is rejected under 35 U.S.C. 101 as claiming the same invention as that of claim 1 of prior U.S. Patent No. 11,407,784. This is a statutory double patenting rejection.
The nonstatutory double patenting rejection is based on a judicially created doctrine grounded in public policy (a policy reflected in the statute) so as to prevent the unjustified or improper timewise extension of the “right to exclude” granted by a patent and to prevent possible harassment by multiple assignees. A nonstatutory double patenting rejection is appropriate where the conflicting claims are not identical, but at least one examined application claim is not patentably distinct from the reference claim(s) because the examined application claim is either anticipated by, or would have been obvious over, the reference claim(s). See, e.g., In re Berg, 140 F.3d 1428, 46 USPQ2d 1226 (Fed. Cir. 1998); In re Goodman, 11 F.3d 1046, 29 USPQ2d 2010 (Fed. Cir. 1993); In re Longi, 759 F.2d 887, 225 USPQ 645 (Fed. Cir. 1985); In re Van Ornum, 686 F.2d 937, 214 USPQ 761 (CCPA 1982); In re Vogel, 422 F.2d 438, 164 USPQ 619 (CCPA 1970); In re Thorington, 418 F.2d 528, 163 USPQ 644 (CCPA 1969).
A timely filed terminal disclaimer in compliance with 37 CFR 1.321(c) or 1.321(d) may be used to overcome an actual or provisional rejection based on nonstatutory double patenting provided the reference application or patent either is shown to be commonly owned with the examined application, or claims an invention made as a result of activities undertaken within the scope of a joint research agreement. See MPEP § 717.02 for applications subject to examination under the first inventor to file provisions of the AIA  as explained in MPEP § 2159. See MPEP § 2146 et seq. for applications not subject to examination under the first inventor to file provisions of the AIA . A terminal disclaimer must be signed in compliance with 37 CFR 1.321(b).
The filing of a terminal disclaimer by itself is not a complete reply to a nonstatutory double patenting (NSDP) rejection. A complete reply requires that the terminal disclaimer be accompanied by a reply requesting reconsideration of the prior Office action. Even where the NSDP rejection is provisional the reply must be complete. See MPEP § 804, subsection I.B.1. For a reply to a non-final Office action, see 37 CFR 1.111(a). For a reply to final Office action, see 37 CFR 1.113(c). A request for reconsideration while not provided for in 37 CFR 1.113(c) may be filed after final for consideration. See MPEP §§ 706.07(e) and 714.13.
The USPTO Internet website contains terminal disclaimer forms which may be used. Please visit www.uspto.gov/patent/patents-forms. The actual filing date of the application in which the form is filed determines what form (e.g., PTO/SB/25, PTO/SB/26, PTO/AIA /25, or PTO/AIA /26) should be used. A web-based eTerminal Disclaimer may be filled out completely online using web-screens. An eTerminal Disclaimer that meets all requirements is auto-processed and approved immediately upon submission. For more information about eTerminal Disclaimers, refer to www.uspto.gov/patents/apply/applying-online/eterminal-disclaimer.
Claims 1-2, 4, 9, and 16 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-25 of U.S. Patent No. 8,940,878. Although the claims at issue are not identical, they are not patentably distinct from each other because both claim sets are drawn to methods of purifying proteins in a separation matrix.  Though the method goal of instant claims 1-2, 4, and 9, differs in that the patented claims do not recite a goal of cleaning the separation matrix, all of the steps of the instant claims are recited.  Though no particular cleaning agent is recited in the patented claims, there is a wash step, which requires a wash buffer of some sort.  This wash buffer is considered to be a cleaning agent.  Otherwise, all of the steps of the claims are recited in the patented claims.
Claims 1-5 and 9 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-30 of U.S. Patent No. 9,643,997. Although the claims at issue are not identical, they are not patentably distinct from each other because both claim sets are drawn to methods of purifying proteins in a separation matrix.  Though the method goal of the instant claims differs in that the patented claims do not recite a goal of cleaning the separation matrix, all of the steps of the instant claims are recited.  Though no particular cleaning agent is recited in the patented claims, there is a wash step, which requires a wash buffer of some sort.  This wash buffer is considered to be a cleaning agent.  Otherwise, all of the steps of the claims are recited in the patented claims.
Claims 1-5, 9, and 16-17 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-23 of U.S. Patent No. 10,577,392. Although the claims at issue are not identical, they are not patentably distinct from each other because both claim sets are drawn to methods of purifying proteins in a separation matrix.  Though the method goal of the instant claims differs in that the patented claims do not recite a goal of cleaning the separation matrix, all of the steps of the instant claims are recited.  Though no particular cleaning agent is recited in the patented claims, there is a wash step, which requires a wash buffer of some sort.  This wash buffer is considered to be a cleaning agent.  Otherwise, all of the steps of the claims are recited in the patented claims.
Claims 1-5,and 9 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-18 of U.S. Patent No. 11,407,784. Although the claims at issue are not identical, they are not patentably distinct from each other because both claim sets are drawn to methods of purifying proteins in a separation matrix.  Though the method goal of the instant claims differs in that the patented claims do not recite a goal of cleaning the separation matrix, all of the steps of the instant claims are recited.  Though no particular cleaning agent is recited in the patented claims, there is a wash step, which requires a wash buffer of some sort.  This wash buffer is considered to be a cleaning agent.  Otherwise, all of the steps of the claims are recited in the patented claims.
Conclusion
No claim is allowed.


NEXT OFFICE ACTION: DETAILED ACTION
Notice of Pre-AIA  or AIA  Status
The present application is being examined under the pre-AIA  first to invent provisions.
Applicant’s amendment and remarks filed on 9/25/2024 are acknowledged.  Claims 1, 12, and 14 are amended.  Claims 16-17 are cancelled.  Claims 1-15 are pending.
Information Disclosure Statement
The information disclosure statements filed on 9/26/2024 and 10/2/2024 have been considered.  Signed copies are enclosed.
Terminal Disclaimer
The terminal disclaimer filed on 9/25/2024 disclaiming the terminal portion of any patent granted on this application which would extend beyond the expiration date of US Patents 8940878, 9643997, 10577392, and 11407784 has been reviewed and is accepted.  The terminal disclaimer has been recorded.
Claim Objections Withdrawn
The objection to claims 12 and 14 because of informalities is withdrawn in light of applicant’s amendment thereto.
Claim Rejections Withdrawn
The rejection of claims 1-15 under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor, or for pre-AIA  the applicant regards as the invention is withdrawn in light of applicant’s amendment thereto.
The rejection of claim 16 under 35 U.S.C. 101 as claiming the same invention as that of claim 9 of prior U.S. Patent No. 9,643,997 is withdrawn.  The cancellation of the claim renders the rejection moot.
The rejection of claim 17 under 35 U.S.C. 101 as claiming the same invention as that of claim 1 of prior U.S. Patent No. 11,407,784 is withdrawn.  The cancellation of the claim renders the rejection moot.
The rejection of claims 1-2, 4, 9, and 16 on the ground of nonstatutory double patenting as being unpatentable over claims 1-25 of U.S. Patent No. 8,940,878 is withdrawn in light of the terminal disclaimer filed on 9/25/2024.
The rejection of claims 1-5 and 9 on the ground of nonstatutory double patenting as being unpatentable over claims 1-30 of U.S. Patent No. 9,643,997 is withdrawn in light of the terminal disclaimer filed on 9/25/2024.
The rejection of claims 1-5, 9, and 16-17 on the ground of nonstatutory double patenting as being unpatentable over claims 1-23 of U.S. Patent No. 10,577,392 is withdrawn in light of the terminal disclaimer filed on 9/25/2024.
The rejection of claims 1-5 and 9 on the ground of nonstatutory double patenting as being unpatentable over claims 1-18 of U.S. Patent No. 11,407,784 is withdrawn in light of the terminal disclaimer filed on 9/25/2024.
Conclusion
Claims 1-15 are allowed.

CITED REFERENCE SUMMARIES: Ref US9643997: Claims:
1. A method of purifying a protein expressed in a non-native soluble form in a non-mammalian expression system comprising:
(a) lysing a non-mammalian cell in which the protein is expressed in a nonnative soluble form to generate a cell lysate;
(b) contacting the cell lysate with a separation matrix under conditions suitable for the protein to associate with the separation matrix;
(c) washing the separation matrix; and
(d) eluting the protein from the separation matrix.
2. The method of claim 1, wherein the protein is a complex protein.
3. The method of claim 2, wherein the complex protein is selected from the group consisting of a multimeric protein, an antibody and an Fc fusion protein.
4. The method of claim 1, wherein the non-mammalian expression system comprises bacteria or yeast cells.
5. The method of claim 1, wherein the separation matrix is an affinity resin.
6. The method of claim 1, wherein the separation matrix is a non-affinity resin selected from the group consisting of ion exchange, mixed mode, and a hydrophobic interaction resin.
7. The method of claim 1, wherein the cell lysate is filtered before it is contacted with the separation matrix.
8. The method of claim 1, further comprising refolding the protein to its native form after it is eluted.
9. A method of purifying a protein expressed in a non-native limited solubility form in a non-mammalian expression system comprising:
(a) solubilizing the expressed protein in a solubilization solution comprising one or more of the following:
(i) a denaturant;
(ii) a reductant; and
(iii) a surfactant;

(b) forming a refold solution comprising the solubilization solution and a refold buffer, the refold buffer comprising one or more of the following:
(i) a denaturant;
(ii) an aggregation suppressor;
(iii) a protein stabilizer; and
(iv) a redox component;

(c) applying the refold solution to a separation matrix under conditions suitable for the protein to associate with the matrix;
(d) washing the separation matrix; and
(e) eluting the protein from the separation matrix.
10. The method of claim 9, wherein the non-native limited solubility form is a component of an inclusion body.
11. The method of claim 9, wherein the protein is a complex protein.
12. The method of claim 10, wherein the complex protein is selected from the group consisting of a multimeric protein, an antibody, a peptibody, and an Fc fusion protein.
13. The method of any one of claims 9-12, wherein the non-mammalian expression system comprises bacteria or yeast cells.
14. The method of any one of claims 9-12, wherein the denaturant of the solubilization solution or the refold buffer comprises one or more of urea, guanidinium salts, dimethyl urea, methylurea and ethylurea.
15. The method of claim 9, wherein the reductant comprises one or more of cysteine, dithiothreitol (DTT), beta-mercaptoethanol and glutathione.
16. The method of claim 9, wherein the surfactant comprises one or more of sarcosyl and sodium dodecylsulfate.
17. The method of claim 9, wherein the aggregation suppressor is selected from the group consisting of arginine, proline, polyethylene glycols, nonionic surfactants, ionic surfactants, polyhydric alcohols, glycerol, sucrose, sorbitol, glucose, Tris, sodium sulfate, potassium sulfate and osmolytes.
18. The method of claim 9, wherein the protein stabilizer comprises one or more of arginine, proline, polyethylene glycols, non-ionic surfactants, ionic surfactants, polyhydric alcohols, glycerol, sucrose, sorbitol, glucose, tris, sodium sulfate, potassium sulfate and osmolytes.
19. The method of claim 9, wherein the redox component comprises one or more of glutathione-reduced, glutathione-oxidized, cysteine, cystine, cysteamine, cystamine and beta-mercaptoethanol.
20. The method of claim 9, wherein the separation matrix is:
(i) an affinity resin, selected from the group consisting of Protein A, Protein G, and synthetic mimetic affinity resin; or
(ii) a non-affinity resin selected from the group consisting of ion exchange, mixed mode, and a hydrophobic interaction resin.
21. The method of any one of claim 1 or 9-12, wherein the protein is isolated after elution from the separation matrix.
22. The method of claim 8, wherein the protein is isolated after refolding.
23. The method of claim 14, wherein the reductant comprises one or more of cysteine, dithiothreitol (DTT), beta-mercaptoethanol and glutathione.
24. The method of claim 15, wherein the surfactant comprises one or more of sarcosyl and sodium dodecylsulfate.
25. The method of claim 16, wherein the aggregation suppressor is selected from the group consisting of arginine, proline, polyethylene glycols, nonionic surfactants, ionic surfactants, polyhydric alcohols, glycerol, sucrose, sorbitol, glucose, Tris, sodium sulfate, potassium sulfate and osmolytes.
26. The method of claim 17, wherein the protein stabilizer comprises one or more of arginine, proline, polyethylene glycols, non-ionic surfactants, ionic surfactants, polyhydric alcohols, glycerol, sucrose, sorbitol, glucose, tris, sodium sulfate, potassium sulfate and osmolytes.
27. The method of claim 18, wherein the redox component comprises one or more of glutathione-reduced, glutathione-oxidized, cysteine, cystine, cysteamine, cystamine and beta-mercaptoethanol.
28. The method of claim 19, wherein the separation matrix is:
(i) an affinity resin, selected from the group consisting of Protein A, Protein G, and synthetic mimetic affinity resin; or
(ii) a non-affinity resin selected from the group consisting of ion exchange, mixed mode, and a hydrophobic interaction resin.
29. The method of claim 13, wherein the protein is isolated after elution from the separation matrix.
30. The method of claim 20, wherein the protein is isolated after elution from the separation matrix.
Ref US11407784: Claims:
1. A method of purifying a protein expressed in a non-native soluble form in a non-mammalian expression system, the method comprising:
(a) lysing a non-mammalian cell in which the protein is expressed in a nonnative soluble form to generate a cell lysate;
(b) filtering the cell lysate;
(c) contacting the filtered cell lysate with a separation matrix under conditions suitable for the protein to associate with the separation matrix;
(d) washing the separation matrix;
(e) eluting the protein from the separation matrix; and
(f) isolating the protein after elution.
2. The method of claim 1, wherein the non-mammalian expression system comprises bacteria cells.
3. The method of claim 2, further comprising refolding the isolated protein to its native form.
4. The method of claim 2, wherein the separation matrix is a nonaffinity resin selected from the group consisting of: ion exchange, mixed mode, and a hydrophobic interaction resin.
5. The method of claim 2, wherein the separation matrix is an affinity resin selected from the group consisting of: Protein A, Protein G, and a synthetic mimetic affinity resin.
6. The method of claim 2, wherein the protein is a complex protein.
7. The method of claim 6, wherein the complex protein is selected from the group consisting of: a multimeric protein, an antibody, and an Fc fusion protein.
8. The method of claim 1, wherein the non-mammalian expression system comprises yeast cells.
9. The method of claim 8, further comprising refolding the isolated protein to its native form.
10. The method of claim 8, wherein the separation matrix is a nonaffinity resin selected from the group consisting of: ion exchange, mixed mode, and a hydrophobic interaction resin.
11. The method of claim 8, wherein the separation matrix is an affinity resin selected from the group consisting of: Protein A, Protein G, and a synthetic mimetic affinity resin.
12. The method of claim 8, wherein the protein is a complex protein.
13. The method of claim 12, wherein the complex protein is selected from the group consisting of: a multimeric protein, an antibody, and an Fc fusion protein.
14. The method of claim 1, further comprising refolding the isolated protein to its native form.
15. The method of claim 14, wherein the protein is a complex protein.
16. The method of claim 15, wherein the complex protein is selected from the group consisting of: a multimeric protein, an antibody, and an Fc fusion protein.
17. The method of claim 14, wherein the separation matrix is a nonaffinity resin selected from the group consisting of: ion exchange, mixed mode, and a hydrophobic interaction resin.
18. The method of claim 14, wherein the separation matrix is an affinity resin selected from the group consisting of: Protein A, Protein G, and a synthetic mimetic affinity resin.
Ref US8940878: Claims:
1. A method of purifying a protein expressed in a non-native soluble form in a non-mammalian expression system comprising:
(a) lysing a non-mammalian cell in which the protein is expressed in a non-native soluble form to generate a cell lysate;
(b) contacting the cell lysate with a separation matrix under conditions suitable for the protein to associate with the separation matrix;
(c) washing the separation matrix; and
(d) eluting the protein from the separation matrix, wherein the separation matrix is an affinity resin selected from the group consisting of Protein A, Protein G and a synthetic mimetic affinity resin.
2. The method of claim 1, wherein the protein is a complex protein.
3. The method of claim 2, wherein the complex protein is selected from the group consisting of a multimeric protein, an antibody and an Fc fusion protein.
4. The method of claim 1, wherein the non-mammalian expression system comprises bacteria or yeast cells.
5. The method of claim 1, wherein the cell lysate is filtered before it is contacted with the separation matrix.
6. The method of claim 1, further comprising refolding the protein to its native form after it is eluted.
7. A method of purifying a protein expressed in a non-native limited solubility form in a non-mammalian expression system comprising:
(a) expressing a protein in a non-native limited solubility form in a non-mammalian cell;
(b) lysing a non-mammalian cell;
(c) solubilizing the expressed protein in a solubilization solution comprising one or more of the following:
(i) a denaturant;
(ii) a reductant; and
(iii) a surfactant;

(d) forming a refold solution comprising the solubilization solution and a refold buffer, the refold buffer comprising one or more of the following:
(i) a denaturant;
(ii) an aggregation suppressor;
(iii) a protein stabilizer; and
(iv) a redox component;

(e) directly applying the refold solution to a separation matrix under conditions suitable for the protein to associate with the matrix;
(f) washing the separation matrix; and
(g) eluting the protein from the separation matrix, wherein the separation matrix is a non-affinity resin selected from the group consisting of ion exchange, mixed mode, and a hydrophobic interaction resin.
8. The method of claim 7, wherein the non-native limited solubility form is a component of an inclusion body.
9. The method of claim 7, wherein the protein is a complex protein.
10. The method of claim 7, wherein the complex protein is selected from the group consisting of a multimeric protein, an antibody, a peptibody, and an Fc fusion protein.
11. The method of claim 7, wherein the non-mammalian expression system is bacteria or yeast cells.
12. The method of claim 7, wherein the denaturant of the solubilization solution or the refold buffer comprises one or more of urea, guanidinium salts, dimethyl urea, methylurea and ethylurea.
13. The method of claim 7, wherein the reductant comprises one or more of cysteine, dithiothreitol (DTT), beta-mercaptoethanol and glutathione.
14. The method of claim 7, wherein the surfactant comprises one or more of sarcosyl and sodium dodecylsulfate.
15. The method of claim 7, wherein the aggregation suppressor is selected from the group consisting of arginine, proline, polyethylene glycols, non-ionic surfactants, ionic surfactants, polyhydric alcohols, glycerol, sucrose, sorbitol, glucose, Tris, sodium sulfate, potassium sulfate and osmolytes.
16. The method of claim 7, wherein the protein stabilizer comprises one or more of arginine, proline, polyethylene glycols, non-ionic surfactants, ionic surfactants, polyhydric alcohols, glycerol, sucrose, sorbitol, glucose, tris, sodium sulfate, potassium sulfate and osmolytes.
17. The method of claim 7, wherein the redox component comprises one or more of glutathione-reduced, glutathione-oxidized, cysteine, cystine, cysteamine, cystamine and beta-mercaptoethanol.
18. The method of claim 1 or 7, further comprising the step of washing the separation matrix with a regeneration reagent.
19. The method of claim 18, wherein the regeneration reagent is one of a strong base or a strong acid.
20. The method of claim 19, wherein the strong acid is phosphoric acid.
21. The method of claim 19, wherein the strong base is sodium hydroxide.
22. The method of claim 18, wherein the regenerating comprises washing the separation matrix with a solution comprising one or both of a chaotrope present at a concentration of 4-6 M and a reductant.
23. The method of claim 22, wherein the chaotrope is one of urea, dimethyl urea, methylurea, ethylurea, and guanidinium.
24. The method of claim 22, wherein the reductant is one of cysteine, dithiothreitol (DTT), beta-mercaptoethanol and glutathione.
25. The method of claim 18, wherein the regenerating comprises washing the separation matrix with a solution comprising 50 mM Tris, 10 mM citrate, 6 M urea, 50 mM dithiothreitol (DTT) at pH 7.4.
Ref US10577392: Claims:
1. A method of purifying a protein expressed in a non-native soluble form in a non-mammalian expression system comprising:
(a) expressing a protein in a non-native soluble form in E. coli;  
(b) lysing the E. coli to generate a cell lysate;
(c) contacting the cell lysate with a separation matrix under conditions suitable for the protein to associate with the separation matrix;
(d) washing the separation matrix; and
(e) eluting the protein from the separation matrix.
2. The method of claim 1, wherein the protein is a complex protein.
3. The method of claim 2, wherein the complex protein is selected from the group consisting of: a multimeric protein, an antibody, and an Fc fusion protein.
4. The method of claim 1, wherein the separation matrix is an affinity resin selected from the group consisting of: Protein A, Protein G, and a synthetic mimetic affinity resin.
5. The method of claim 1, wherein the separation matrix is a non-affinity resin selected from the group consisting of: ion exchange, mixed mode, and a hydrophobic interaction resin.
6. The method of claim 1, wherein the cell lysate is filtered before it is contacted with the separation matrix.
7. The method of claim 1, further comprising:
(f) refolding the protein to its native form.
8. A method of purifying a protein expressed in a non-native limited solubility form in a non-mammalian expression system comprising:
(a) expressing a protein in a non-native limited solubility form in a non-mammalian cell;
(b) lysing the non-mammalian cell;
(c) solubilizing the expressed protein in a solubilization solution comprising one or more of the following:
(i) a denaturant;
(ii) a reductant; and
(iii) a surfactant;

(d) forming a refold solution comprising the solubilized protein and a refold buffer, the refold buffer comprising one or more of the following:
(i) a denaturant;
(ii) an aggregation suppressor;
(iii) a protein stabilizer; and
(iv) a redox component;

(e) applying the refold solution to a separation matrix under conditions suitable for the protein to associate with the separation matrix;
(f) washing the separation matrix; and
(g) eluting the protein from the separation matrix to yield a purified protein.
9. A method of purifying a protein expressed in a non-native soluble form in non-mammalian expression system comprising:
(a) expressing a protein in a non-native soluble form in a non-mammalian cell;
(b) lysing the non-mammalian cell to generate a cell lysate;
(c) contacting the cell lysate with a separation matrix under conditions suitable for the protein to associate with the separation matrix without diluting the protein prior to the contacting;
(d) washing the separation matrix; and
(e) eluting the protein from the separation matrix.
10. A method of purifying a protein expressed in a non-native limited solubility form in E. coli comprising:
(a) expressing a protein in a non-native limited solubility form in E. coli;  
(b) lysing the E. coli;  
(c) solubilizing the expressed protein in a solubilization solution comprising one or more of the following:
(i) a denaturant;
(ii) a reductant; and
(iii) a surfactant;

(d) forming a refold solution comprising the solubilized protein and a refold buffer, the refold buffer comprising one or more of the following:
(i) a denaturant;
(ii) an aggregation suppressor;
(iii) a protein stabilizer; and
(iv) a redox component;

(e) applying the refold solution to a separation matrix under conditions suitable for the protein to associate with the separation matrix and obtaining a purified protein.
11. The method of claim 8 or 10, wherein the non-native limited solubility form is a component of an inclusion body.
12. The method of claim 8 or 10, wherein the protein is a complex protein.
13. The method of claim 12, wherein the complex protein is selected from the group consisting of: a multimeric protein, an antibody, a peptibody, and an Fc fusion protein.
14. The method of claim 8 or 10, wherein the non-native limited solubility form is a component of an inclusion body.
15. The method of claim 8 or 10, wherein the denaturant in the solubilization solution comprises one or more of: urea, guanidinium salts, dimethyl urea, methylurea, and ethylurea.
16. The method of claim 8 or 10, wherein the reductant comprises one or more of: cysteine, dithiothreitol (DTT), beta-mercaptoethanol, and glutathione.
17. The method of claim 8 or 10, wherein the surfactant comprises one or more of: sarcosyl and sodium dodecylsulfate.
18. The method of claim 8 or 10, wherein the aggregation suppressor is selected from the group consisting of: arginine, proline, polyethylene glycols, nonionic surfactants, ionic surfactants, polyhydric alcohols, glycerol, sucrose, sorbitol, glucose, Tris, sodium sulfate, potassium sulfate, and osmolytes.
19. The method of claim 8 or 10, wherein the protein stabilizer comprises one or more of: arginine, proline, polyethylene glycols, non-ionic surfactants, ionic surfactants, polyhydric alcohols, glycerol, sucrose, sorbitol, glucose, tris, sodium sulfate, potassium sulfate, and osmolytes.
20. The method of claim 8 or 10, wherein the redox component comprises one or more of: glutathione-reduced, glutathione-oxidized, cysteine, cystine, cysteamine, cystamine, and beta-mercaptoethanol.
21. The method of claim 8 or 10, wherein the separation matrix is:
an affinity resin selected from the group consisting of: Protein A, Protein G, and synthetic mimetic affinity resin.
22. The method of claim 8 or 10, wherein the separation matrix is: a non-affinity resin selected from the group consisting of: ion exchange, mixed mode, and a hydrophobic interaction resin.
23. The method of claim 8 or 10, wherein the refold solution is directly applied to the separation matrix.