# Patent

## Title
Formulations of recombinant furin

## Abstract

The present application provides stabilized formulations of furin (e.g., rfurin) containing a sugar, sugar alcohol, and/or non-ionic surfactant. As compared to non-stabilized compositions, the furin formulations disclosed herein retain greater amounts of furin activity and monomeric furin content, while reducing furin aggregation when stored and/or subjected to mechanical stress. Also provided are methods for stably diluting furin (e.g., rfurin) compositions.

## Background
CROSS-REFERENCES TO RELATED APPLICATIONS
The present application is a Continuation of U.S. patent application Ser. No. 13/488,324, filed Jun. 4, 2012, which claims the benefit of U.S. Provisional Application No. 61/492,712, filed Jun. 2, 2011, the contents of which are expressly incorporated herein by reference in their entireties for all purposes.


FIELD OF THE APPLICATION
This application generally relates to the field of protein formulations.
BACKGROUND OF THE INVENTION
The family of the proteolytic mammalian subtilisin-like proprotein convertases (SPC or PC) is homologous with bacterial subtilisins and yeast Kex2p. To date seven distinct members of the SPC family have been identified, including furin, PC1 (also known as PC3), PC2, PACE4, PC4, PC5 (also known as PC6), PC7 (LPC, PC8, or SPC7), each of which exhibits unique tissue distribution.
Furin, also termed PACE (paired basic amino acid cleavage enzyme) is ubiquitously expressed in all mammalian tissues and cell lines and is capable of processing a wide range of bioactive precursor proteins in the secretory pathway, including also hormones, growth factors, receptors, viral and bacterial proteins, and plasma proteins. It is a calcium-dependent serine endoprotease structurally arranged into several domains, namely a signal peptide, propeptide, catalytic domain, middle domain, (also termed homo-B or P-domain), the C-terminally located cysteine-rich domain, transmembrane domain and the cytoplasmic tail. The furin protease cleavage site comprises a recognition sequence which is characterized by the amino acid sequence Arg-X-Lys/Arg-Arg (Hosaka et al., J Biol Chem. 1991; 266:12127-30).
Furin belongs to the family of the pro-protein convertases and is dependent on calcium (Ca2+). Furin specifically cleaves the C-terminal peptide bond of arginine within a specific sequence, containing arginine at positions −1 and −4. This sequence can be found in numerous human proteins, showing that furin plays a major role in the maturation of a number of human pro-proteins. Accordingly, furin is a proprotein convertase that processes latent precursor proteins into their biologically active products. Furin is a calcium-dependent serine endoprotease that cleaves precursor proteins at their paired basic amino acid processing sites. One substrate for furin is von Willebrand Factor (vWF).
Furin (e.g., rfurin) is capable of converting pro-VWF (pro-von Willebrand factor) into mature VWF by cleaving the Arg741-Ser742 peptide bond of VWF. This maturation step is part of, for example, a rVWF production process which provides a therapy for von Willebrand Disease Type B. The production of activated recombinant proteins is of high clinical and diagnostic importance. For example, active or mature proteins, like mature VWF, may be used to control blood coagulation.
Furin formulations, particularly recombinant furin (rfurin) formulations, often have low shelf life (less than 6 months) due to a loss of rfurin activity from protein degradation (often from self-cleavage of the furin protein). In addition, when used in certain production processes, such as production of mature vWF, rfurin formulations are diluted, often by 200-fold, and the diluted rfurin generally shows a disproportionate loss of activity. Accordingly, there is a need in the field for methods and formulations that stabilize furin (e.g., rfurin) compositions, as well as methods for diluting concentrated furin (e.g., rfurin) compositions without losing a substantial fraction of furin activity.
BRIEF SUMMARY OF THE INVENTION
The present disclosure fulfills these and other needs in the field of furin (e.g., rfurin) formulation, storage, and manipulation, by providing methods and formulations for stabilizing furin (e.g., rfurin compositions). These needs are met, at least in part, by the discovery that the addition of sugars, sugar alcohols, and/or non-ionic surfactants significantly increases the stability of furin (e.g., rfurin) compositions during storage and mechanical stress.
Accordingly, the present invention provides highly stabilized furin formulations, particularly highly stabilized rfurin formulations that show increased stability under conditions of high temperature, agitation, and varying pH as compared to the starting (control) furin formulations.
In one aspect, the present disclosure provides a stabilized aqueous composition of recombinant furin (rfurin), the composition comprising: from 8,000 U/mL to 500,000 U/mL rfurin; from 100 mM to 300 mM of a pharmaceutically acceptable salt; from 0.5 mM to 2 mM calcium; from 2% to 20% sugar or sugar alcohol; from 10 to 200 ppm non-ionic surfactant; from 10 to 200 mM buffering agent; and a pH from 5.5 to 7.5.
In one embodiment, the compositions provided above include 150 mM to 250 mM of a pharmaceutically acceptable salt. In another embodiment, the compositions provided above include 190±10 mM of a pharmaceutically acceptable salt. In another embodiment, the compositions provided above include 190 mM of a pharmaceutically acceptable salt.
In one embodiment of the compositions provided above, the pharmaceutically acceptable salt is sodium chloride. In another embodiment of the compositions provided above, the pharmaceutically acceptable salt is potassium chloride.
In one embodiment, the compositions provided above include 0.9±0.2 mM calcium. In another embodiment, the compositions provided above include 0.92 mM calcium.
In one embodiment, the compositions provided above include from 5% to 15% sugar or sugar alcohol. In another embodiment, the compositions provided above include 10±2% sugar or sugar alcohol. In another embodiment, the compositions provided above include 10% sugar or sugar alcohol.
In one embodiment of the compositions provided above, the sugar or sugar alcohol is selected from the group consisting of sucrose, trehalose, mannitol, and a combination thereof. In another embodiment of the compositions provided above, the sugar or sugar alcohol is trehalose. In another embodiment of the compositions provided above, the sugar or sugar alcohol is sucrose.
In one embodiment, the compositions provided above include from 10 ppm to 100 ppm non-ionic surfactant. In another embodiment, the compositions provided above include from 50 ppm to 100 ppm non-ionic surfactant. In another embodiment, the compositions provided above include 75 ppm non-ionic surfactant.
In one embodiment of the compositions provided above, the non-ionic surfactant is polysorbate-80.
In one embodiment, the compositions provided above include from 50 mM to 150 mM buffering agent. In another embodiment, the compositions provided above include 90±10 mM buffering agent. In another embodiment, the compositions provided above include 91 mM buffering agent.
In one embodiment of the compositions provided above, the buffering agent comprises acetate. In another embodiment of the compositions provided above, the buffering agent comprises HEPES. In another embodiment of the compositions provided above, the buffering agent comprises MES. In another embodiment of the compositions provided above, the buffering agent comprises acetate and HEPES.
In one embodiment, the compositions provided above include from 25 mM to 75 mM acetate and from 25 to 75 mM HEPES. In another embodiment, the compositions provided above include 45±5 mM acetate and 45±5 mM HEPES. In another embodiment, the compositions provided above include 45 mM acetate and 46 mM HEPES.
In one embodiment, the compositions provided above have a pH from 5.5 to 7.0. In another embodiment, the compositions provided above have a pH from 5.5 to 6.5. In another embodiment, the compositions provided above have a pH of 6.0±0.2. In another embodiment, the compositions provided above have a pH of 6.0.
In one embodiment of the compositions provided above, the composition has increased stability when stored at 37° C. as compared to a rfurin composition that does not contain a sugar or sugar alcohol.
In one embodiment of the compositions provided above, the composition maintains a higher percentage of rfurin activity when stored at 37° C. as compared to a rfurin composition that does not contain a sugar or sugar alcohol.
In one embodiment of the compositions provided above, the composition maintains a higher percentage of rfurin monomer content when stored at 37° C. as compared to a rfurin composition that does not contain a sugar or sugar alcohol.
In one embodiment of the compositions provided above, the composition has increased stability when agitated as compared to a rfurin composition that does not contain a non-ionic surfactant.
In one embodiment of the compositions provided above, the composition maintains a higher percentage of rfurin activity when agitated as compared to a rfurin composition that does not contain a non-ionic surfactant.
In one embodiment of the compositions provided above, the composition maintains a higher percentage of rfurin monomer content when agitated as compared to a rfurin composition that does not contain a non-ionic surfactant.
In one embodiment of the compositions provided above, the composition has increased stability when diluted as compared to a rfurin composition that does not contain a non-ionic surfactant.
In one embodiment of the compositions provided above, the composition maintains a higher percentage of rfurin activity when diluted as compared to a rfurin composition that does not contain a non-ionic surfactant.
In one embodiment of the compositions provided above, the composition maintains a higher percentage of rfurin monomer content when diluted as compared to a rfurin composition that does not contain a non-ionic surfactant.
In one aspect, the present disclosure provides a stabilized aqueous composition of recombinant furin (rfurin), the composition comprising: from 8,000 U/mL to 500,000 U/mL rfurin; 190 mM sodium chloride; 0.92 mM calcium; 10% trehalose; 75 ppm polysorbate 80; 45 mM acetic acid; 46 mM HEPES; and a pH from 5.5 to 7.5.
In one embodiment, the compositions provided above have a pH from 5.5 to 7.0. In another embodiment, the compositions provided above have a pH from 5.5 to 6.5. In another embodiment, the compositions provided above have a pH of 6.0±0.2. In another embodiment, the compositions provided above have a pH of 6.0.
In one aspect, the present disclosure provides a method for diluting an aqueous rfurin composition, the method comprising adding a dilution buffer comprising from 10 ppm to 200 ppm of a non-ionic surfactant to the aqueous rfurin composition, thereby forming a diluted rfurin composition.
In one embodiment of the methods provided above, the dilution buffer comprises from 10 ppm to 100 ppm of a non-ionic surfactant. In another embodiment of the methods provided above, the dilution buffer comprises from 50 ppm to 100 ppm of a non-ionic surfactant. In another embodiment of the methods provided above, the dilution buffer comprises 75 ppm of a non-ionic surfactant.
In one embodiment of the methods provided above, the dilution buffer has a pH of from 5.5 to 7.5. In another embodiment of the methods provided above, the dilution buffer has a pH of from 5.5 to 6.5. In another embodiment of the methods provided above, the dilution buffer has a pH of 6.0±0.2. In another embodiment of the methods provided above, the dilution buffer has a pH of 6.0.
In one embodiment of the methods provided above, the diluted rfurin composition comprises at least 50% of rfurin activity present in the aqueous rfurin composition prior to dilution.
In one embodiment of the methods provided above, the diluted rfurin composition comprises at least 75% of rfurin activity present in the aqueous rfurin composition prior to dilution.
In one aspect, the present disclosure provides a highly stabilized formulation of recombinant furin (rfurin), said formulation comprising: 8,000-52,000 U/mL rfurin; 1 mM to 300 mM of a pharmaceutically acceptable salt; 0.1 mM to 2 mM calcium chloride; 1% to 20% sugar and/or sugar dihydrate; 50 to 100 ppm of a surfactant; and 1 mM to 100 mM of a carboxylic acid, a buffering agent in sufficient concentration for maintaining a pH of 4.0-7.0, or both said carboxylic acid and said buffering agent.
In one embodiment, the formulations provided above include 180-200 mM salt.
In one embodiment of the formulations provided above, the salt is sodium chloride.
In one embodiment, the formulations provided above include 0.5-1.0 mM calcium.
In one embodiment, the formulations provided above include 5-15% sugar or sugar dihydrate.
In one embodiment of the formulations provided above, the sugar or sugar dihydrate is selected from the group consisting of sucrose, trehalose, mannitol, and a combination thereof.
In one embodiment of the formulations provided above, the surfactant is polysorbate 80.
In one embodiment of the formulations provided above, the surfactant is present at a concentration of about 65 to about 85 ppm.
In one embodiment of the formulations provided above, the carboxylic acid is acetic acid.
In one embodiment of the formulations provided above, the carboxylic acid is in a concentration of 35-55 mM.
In one embodiment of the formulations provided above, the buffering agent is HEPES.
In one embodiment of the formulations provided above, the buffering agent is in a concentration of about 20 to about 100 mM.
In one embodiment of the formulations provided above, the formulation shows improved stability after storage at 37° C. as compared to a formulation that does not contain a sugar or sugar dihydrate.
In one embodiment of the formulations provided above, the formulation is prepared by adding rfurin to a composition comprising 1% polysorbate 80, 500 mM HEPES, 400 mM acetic acid, 1 mM calcium chloride, and trehalose dihydrate powder, wherein said composition has a pH of 6.0 prior to addition to said rfurin.
In one aspect, the present disclosure provides a highly stabilized formulation of recombinant furin (rfurin), said formulation comprising: 8,000-57,000 U/mL rfurin; 190 mM sodium chloride; 0.92 mM calcium chloride; 10% w/w trehalose dihydrate; 75 ppm polysorbate 80; 45 mM acetic acid; and 46 mM HEPES.
In one aspect, the present disclosure provides a highly stabilized formulation of recombinant furin (rfurin) that includes: (a) 8,000-52,000 U/mL rfurin; (b) 1 mM to 300 mM of a pharmaceutically acceptable salt; (c) 0.1 mM to 2 mM calcium chloride; (d) 1% to 20% sugar and/or sugar dihydrate; (e) 50 to 100 ppm of a surfactant; and (f) 1 mM to 100 mM of a carboxylic acid or a buffering agent in sufficient concentration for maintaining a pH of 4.0-7.0 or both the carboxylic acid and the buffering agent.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1. rFurin in the control formulation—a temperature study analyzed using the furin activity assay. rFurin samples in the control formulation were incubated at: room temperature, 37° C., or 45° C. for up to seven days. The activities of rfurin samples are plotted against the number of days of incubation. Error bars are ±1 standard deviation; n=4.
FIG. 2. rFurin in the control formulation—a temperature study analyzed using SEC. The same samples as in FIG. 1 were analyzed using SEC. Relative rfurin peak heights are plotted against the number of days of incubation. The relative peak heights were calculated as the percentages of the rfurin peak height at time zero.
FIG. 3. rFurin in the control formulation incubated at 37° C. analyzed using SEC. rFurin samples in the control formulation were incubated at 37° C. for seven days. FIG. 3 shows an overlay of SEC absorption profiles at 280 nm of six time points. The same study as in FIG. 1.
FIG. 4. rFurin in the control formulation incubated at room temperature and 37° C. analyzed using Western Blotting. The same study as in FIG. 1. Lane: (1) MWM marker; (2) T=0; (3) rfurin incubated at room temperature for 1 day; (4) rfurin incubated at room temperature for 2 days; (5) rfurin incubated at room temperature for 3 days; (6) rfurin incubated at room temperature for 7 days; (7) MWM marker; (8) rfurin incubated at 37° C. for 1 day; (9) rfurin incubated at 37° C. for 2 days; (10) rfurin incubated at 37° C. for 3 days; and (11) rfurin incubated at 37° C. for 7 days.
FIG. 5. rFurin in the control formulation—a freeze/thaw study analyzed using the furin activity assay. rFurin activity is plotted against the number of freeze/thaw cycles. Error bars are ±1 standard deviation; n=4.
FIG. 6. rFurin in the control formulation—a freeze/thaw study analyzed using SEC. rFurin peak heights are plotted against the number of freeze/thaw cycles.
FIG. 7. rFurin in the control formulation—an agitation study analyzed using the activity assay. rFurin activity is plotted against the time of agitation. Error bars are ±1 standard deviation; n=4.
FIG. 8. rFurin in the control formulation—an agitation study analyzed using SEC. rFurin peak heights are plotted against the time of agitation.
FIG. 9. Comparison of different lots of rfurin using Western Blotting. Various lots of rfurin were analyzed using Western Blotting. Production order is denoted alphabetically. Lane: (1) MWM marker; (2) furin reference; (3) 03-04-09 (lot 018342, 2 mL vial); (4) 03-08-09 (lot 018365, 2 mL vial); (5) 03-15-09 (lot 018366, 2 mL vial); (6) 03-01-09 (lot 018315, 2 mL vial); (7) 07-26-10 (lot X572, 200 mL bottle); (8) 03-06-09 (lot 018365, 200 mL bottle); (9) 08-01-10 (lot X576, 200 mL bottle); (10) 03-09-09 (lot 018366, 200 mL bottle); (11) MWM marker; (12) furin reference; (13) 08-03-10 (lot X577, 200 mL bottle); (14) 07-24-10 (lot X573, 200 mL bottle); (15) 07-30-10 (lot X575, 200 mL bottle); (16) 018346 (QC Stability at −80° C.); (17) 018346 (QC Stability at −25° C.); (18) 018315 (QC Stability at −80° C.); and (19) 018315 (QC Stability at −25° C.).
FIG. 10. Impact of thawing method on rfurin activity analyzed using the furin activity assay. rFurin sample activities are shown as bars for various lots of rfurin thawed at either: 4° C. (left) or room temperature (RT; right). Error bars are ±1 standard deviation; n=4.
FIG. 11. Effect of pH 5 and 5.5 on rfurin activity analyzed using the furin activity assay. rFurin samples were incubated at 37° C. at either pH: 5.0, 5.5, or 6.0 (control formulation) for up to seven days. The activities of rfurin samples are plotted against the number of days of incubation. Error bars are ±1 standard deviation; n=4.
FIG. 12. Effect of pH 5 and 5.5 on rfurin activity analyzed using SEC. rFurin samples were incubated at 37° C. at either pH: 5.0, 5.5 or 6.0 (control formulation). Relative peak heights of rfurin are plotted against the number of days of incubation. The relative peak heights were calculated as the percentage of the rfurin peak height at time zero.
FIG. 13. rFurin stability at 37° C. in the control formulation analyzed using SEC. A rfurin sample in the control formulation was incubated for four days at 37° C. The overlay of 280 nm absorption profiles shows three time points: T=0, 1 day, and 4 days of incubation.
FIG. 14. rFurin stability in MES pH 6.0 analyzed using SEC. A rfurin sample was spiked with MES (final C=100 mM, pH 6.0) and incubated for four days at 37° C. An overlay of 280 nm absorption profiles shows three time points: T=0, 1 day, and 4 days of incubation.
FIG. 15. rFurin stability in HEPES pH 7.0 analyzed using SEC. A rfurin sample was spiked with HEPES (final C=100 mM, pH 7.0) and incubated for four days at 37° C. The overlay of 280 nm absorption profiles shows three time points: T=0, 1 day, and 4 days of incubation.
FIG. 16. rFurin stability in HEPES pH 8.0 analyzed using SEC. A rfurin sample was spiked with HEPES (final C=100 mM, pH 8.0) and incubated for four days at 37° C. The overlay of 280 nm absorption profiles shows three time points: T=0, 1 day, and 4 days of incubation.
FIG. 17. Effects of sucrose and polysorbate 80 additives on rfurin stability at 37° C. analyzed using SEC. The samples were incubated at 37° C. for four days. Relative peak heights of rfurin are plotted against the time of incubation. The relative peak heights were calculated as the percentages of the rfurin peak height at time zero.
FIG. 18. Effects of sucrose and polysorbate 80 on rfurin stability in the control formulation at 37° C. analyzed using the furin activity assay. The relative activities of rfurin samples are plotted against the number of days of incubation to compare the following samples: control formulation without additives, control formulation plus 10% sucrose, and control formulation plus 10% sucrose plus 25 ppm polysorbate 80. The relative activity values were calculated as the percentages of the furin activity at time zero. Error bars are ±1 standard deviation=4.
FIG. 19. Effects of sucrose and polysorbate 80 on rfurin stability in MES buffer at 37° C. analyzed using the furin activity assay. The relative activities of rfurin samples are plotted against the number of days of incubation to compare the following samples: control formulation without additives, spiked with MES (final C=100 mM, pH 6.0), spiked with MES (final C=100 mM, pH 6. plus 10% sucrose, and spiked with MES (final C=100 mM, pH 6.0) plus 10% sucrose plus 25 ppm polysorbate 80. The relative activity values were calculated as the percentages of the furin activity at time zero. Error bars are ±1 standard deviation; n=4.
FIG. 20. Comparison of rfurin stability at 37° C. in the control formulation to the formulation in MES buffer analyzed using the furin activity assay. The relative activities of rfurin samples are plotted against the number of days of incubation to compare the following samples: control formulation without additives, control formulation plus 10% sucrose, and spiked with MES (final C=100 mM, pH 6.0) plus 10% sucrose. The relative activity values were calculated as the percentages of the furin activity at time zero. Error bars are ±1 standard deviation; n=4.
FIG. 21. Comparison of rfurin stability at 37° C. in sucrose to mannitol analyzed using the furin activity assay. All samples were spiked with HEPES/acetic acid, pH 6.0. The relative activities of rfurin samples are plotted against the number of days of incubation to compare the following samples: no additives, 10% sucrose, 10% mannitol, 10% sucrose plus 25 ppm of polysorbate 80, and 10% mannitol plus 25 ppm of polysorbate 80. The relative activity values were calculated as the percentages of the furin activity at time zero. Error bars are ±1 standard deviation; n=4.
FIG. 22. rFurin Stability at 37° C.: sucrose vs. mannitol analyzed using SEC. The same samples as in FIG. 21 were analyzed using SEC. Relative rfurin peak heights are plotted against the time of incubation. The relative peak heights were calculated as the percentages of the rfurin peak height at time zero.
FIG. 23. rFurin stability in trehalose after 5 days of incubation at 37° C. analyzed by the furin activity assay. The effects of varied amounts of trehalose and polysorbate 80 on rfurin stability at 37° C. are examined using the furin activity assay. First, all samples were spiked with HEPES and acetic acid (final concentrations: HEPES—50 mM, acetic acid—50 mM, pH 6.0) then, either 10% sucrose or various amounts of trehalose and/or polysorbate 80 were added. A rfurin sample in the control formulation is also included. The data are sorted from the highest to lowest activity values after 5 days of incubation at 37° C. (right bar in each pair). The left bar in each pair depicts furin activity values at time zero. Error bars are ±1 standard deviation; n=4.
FIG. 24. rFurin activity in trehalose after 5 days of incubation at 37° C. The same samples as in FIG. 23 are displayed after recalculation to show what percentage of each sample's initial activity remained after five days of incubation at 37° C. A rfurin sample in the control formulation is also included. Error bars are ±1 standard deviation; n=4.
FIG. 25. rFurin stability in trehalose after 5 days of incubation at 37° C. analyzed using SEC. The same samples as in FIG. 24. The graph shows what percentage of each sample's initial rfurin peak remained after five days of incubation at 37° C. A rfurin sample in the control formulation is also included.
FIG. 26. rFurin stability in trehalose after 5 days of incubation at 35° C. analyzed using Western Blotting. The same samples as in FIG. 24. The samples are paired: first sample—time zero, second sample—incubated at 37° C. for 5 days. Lanes: (1) MWM marker; (2) furin reference, (3) control; (4) control formulation, T=0; (5) control formulation, 5 days; (6) 10% sucrose, T=0; (7) 10% sucrose, 5 days; (8) 2% trehalose, T=0; (9) 2% trehalose, 5 days; (10) 10% trehalose, T=0; (11) 10% trehalose, 5 days; (12) MWM marker; (13) furin reference; (14) control; (15) 5% trehalose, T=0; (16) 5% trehalose, 5 days; (17) 5% trehalose, +10 ppm Tween80, T=0; (18) 5% trehalose, +10 ppm Tween80, 5 days; (19) 5% trehalose, +25 ppm Tween80, T=0; (20) 5% trehalose, +25 ppm Tween80, 5 days; (21) 5% trehalose, +100 ppm Tween80, T=0; and (22) 5% trehalose, +100 ppm Tween80, 5 days.
FIG. 27. Effect of the trehalose content on rfurin activity tested after 5 days of incubation at 37° C. The same experiment as in FIG. 24. First, all samples were spiked with HEPES and acetic acid (final concentrations: HEPES—50 mM, acetic acid—50 mM, pH 6.0) then, various amounts of trehalose were added. furin activity after five days of incubation at 37° C. is plotted against the percentage of trehalose added to the samples. Error bars are ±1 standard deviation; n=4.
FIG. 28. Effect of the trehalose content on rfurin stability at 37° C. analyzed using SEC after 5 days of incubation. The same experiment as in FIG. 27. rFurin peak heights after five days of incubation at 37° C. are plotted against the percentage of trehalose added to the samples.
FIG. 29. Effect of polysorbate 80 content on rfurin activity tested after 5 days of incubation at 37° C. The same experiment as in FIG. 24. First, all samples were spiked with HEPES, acetic acid (final concentrations: HEPES—50 mM, acetic acid—50 mM, pH 6.0), and 5% trehalose then, various amounts of polysorbate 80 were added. furin activity after five days of incubation at 37° C. is plotted against the amount of polysorbate 80 added to the samples. Error bars are ±1 standard deviation; n=4.
FIG. 30. Effect of Polysorbate 80 content on rfurin stability at 37° C. analyzed using SEC after 5 days of incubation. The same samples as in FIG. 29. rFurin peak heights after five days of incubation at 37° C. are plotted against the amount of polysorbate 80 added to the samples.
FIG. 31. rFurin in the highly stabilized formulation—agitation study analyzed using the furin activity assay. The rfurin samples in the highly stabilized formulation were spiked with various amount of polysorbate 80. The graph shows furin activity values before and after three hours of agitation. The data are sorted from the highest to the lowest activity. Error bars are ±1 standard deviation; n=4. Left bars in each pair show activity at time 0, right bars in each pair show activity after agitation.
FIG. 32. rFurin in the highly stabilized formulation—agitation study analyzed using SEC. The same experiment as in FIG. 31. The graph shows rfurin peak heights before and after three hours of agitation. The data are sorted from the highest to the lowest. Left bars in each pair show activity at time 0, right bars in each pair show activity after agitation.
FIG. 33. rFurin in the highly stabilized formulation—effects of polysorbate content in an agitation study analyzed using the furin activity assay. The same samples as in FIG. 32. furin activity after three hours of agitation is plotted against the content of polysorbate 80. Error bars are ±1 standard deviation; n=4.
FIG. 34. rFurin in the highly stabilized formulation—effect of polysorbate 80 in agitation study analyzed using SEC. The same samples as in FIG. 32. rFurin peak heights after three hours of agitation are plotted against the content of polysorbate 80.
FIG. 35. rFurin in the control formulation—agitation study analyzed using SEC. The chromatogram shows an overlay of SEC absorption profiles at 280 nm comparing rfurin samples in the control formulation before and after three hours of agitation. The rfurin peak of the agitated sample is significantly smaller than the control sample. The absence of high molecular mass peaks in the agitated sample is observed (see paragraph 3.5.1 for explanation).
FIG. 36. UV spectra of rfurin in the control formulation (the same samples as in FIG. 35). The UV spectrum of the rfurin sample after three hours of agitation shows a slanted and extremely elevated profile compared to the sample before agitation, which indicates the presence of a significant level of aggregates in the agitated sample.
FIG. 37. UV spectra of the rfurin samples in the highly stabilized formulation without polysorbate 80. The UV spectrum of the rfurin sample after three hours of agitation shows an elevated profile compared to the sample before agitation, which indicates the presence of significant level of aggregates in the agitated sample.
FIG. 38. rFurin in the highly stabilized Formulation without polysorbate 80—an agitation study analyzed using SEC. The chromatogram shows an overlay of SEC absorption profiles at 280 nm comparing rfurin samples in the highly stabilized formulation without polysorbate 80 before and after three hours of agitation. The rfurin peak of the agitated sample is significantly smaller than the control sample. The absence of high molecular mass peaks is observed.
FIG. 39. UV spectra of the rfurin samples in the highly stabilized formulation containing 10 ppm polysorbate 80. The UV spectrum of the rfurin sample after three hours of agitation shows a slightly elevated profile compared to the sample before agitation, which indicates the presence of aggregates in the agitated sample. The ‘agitated’ spectra is closer to the ‘before agitated’ spectra, relative to those seen in samples without polysorbate (FIGS. 36 and 37), which indicated that that the level of aggregation is less than that seen in samples without polysorbate.
FIG. 40. UV spectra of the rfurin samples in the highly stabilized formulation containing 25 ppm polysorbate 80. The UV spectrum of the rfurin sample after three hours of agitation shows a slightly elevated profile compared to the sample before agitation, which indicates presence of a small amount of aggregates in the agitated sample.
FIG. 41. UV spectra of the rfurin samples in the highly stabilized formulation containing 50 ppm polysorbate 80. The UV spectrum of the rfurin sample after three hours of agitation shows a very similar profile to the sample before agitation. Neither spectra is slanted or elevated, which indicates that neither sample contains significant amounts of large aggregates.
FIG. 42. UV spectra of the rfurin samples in the highly stabilized formulation containing 100 ppm polysorbate 80. The UV spectrum of the rfurin sample after three hours of agitation shows a very similar profile to the sample before agitation. Neither spectra is slanted or elevated, which indicates that neither sample contains significant amounts of large aggregates.
FIG. 43. rFurin in the highly stabilized Formulation—a freeze/thaw study analyzed using the furin activity assay. rFurin samples in the highly stabilized formulation were spiked with different amounts of polysorbate 80. The data show rfurin activity in the samples before and after 5 freeze/thaw cycles. The data are sorted from the highest to the lowest. Error bars are ±1 standard deviation; n=4.
FIG. 44. rFurin in the highly stabilized formulation—a freeze/thaw study analyzed using SEC. The same samples as in FIG. 43. The data show the rfurin peak heights before and after 5 freeze/thaw cycles. The data are sorted from the highest to the lowest.
FIG. 45. rFurin in the highly stabilized Formulation—a freeze/thaw study analyzed using SEC. The same samples as in FIG. 44. The relative peak heights of rfurin are plotted against the number of freeze/thaw cycles. The relative peak heights were calculated as the percentages of the rfurin peak heights before the first freeze/thaw cycle.
FIG. 46. rFurin stability in the highly stabilized formulation at 37° C. analyzed using the furin activity assay. rFurin samples in either the control formulation or in the highly stabilized formulation were incubated at 37° C. for 4 days. Error bars are ±1 standard deviation; n=4.
FIG. 47. rFurin stability in the highly stabilized formulation at 37° C. analyzed using the furin activity assay. The same samples as in FIG. 46 recalculated. The relative activity of each sample was calculated as the percentage of its initial activity. Error bars are ±1 standard deviation; n=4.
FIG. 48. rFurin stability in the highly stabilized formulation at 37° C. analyzed using SEC. The same samples as in FIG. 46. The relative peak height of rfurin for every sample was calculated as the percentage of its initial peak height. Error bars are ±1 standard deviation; n=4.
FIG. 49A and FIG. 49B. Effect of temperature on the pH (B) and conductivity (A) of the highly stabilized formulation stock buffer (500 mM HEPES, 400 mM acetic acid, 1 mM CaCl2, pH 6.0).



DETAILED DESCRIPTION OF THE INVENTION
I. Introduction
The present disclosure is based in part on the discovery that aqueous furin compositions can be stabilized against various chemical and mechanical stresses by the addition of sugars, sugar alcohols, and non-ionic surfactants. The aqueous furin compositions described herein are significantly more stable when stored at or above room temperature, as well as when subjected to mechanical stress, as compared to similar furin compositions lacking sugars, sugar alcohols, and non-ionic surfactants. The present disclosure is also based in part on the discovery that the addition of non-ionic surfactants to furin dilution buffers enables a greater recovery of furin activity when the enzyme is diluted to lower concentrations used during the manufacture of various recombinant biologics, such as VWF.
Advantageously, the studies described herein demonstrate that the addition of sugar or sugar alcohols to a furin composition enhances the stability of the composition when stored over a period of time. For example, it is shown herein that the inclusion of as little as 2% sugar or sugar alcohol in an aqueous furin formulation can increase the stability of the composition by greater than 10%. It is further shown that inclusion of 10% sugar or sugar alcohol in an aqueous furin formulation can increase the stability of the composition by greater than 75%.
Advantageously, the studies described herein also demonstrate that the addition of non-ionic surfactant to a furin composition enhances the stability of the composition when subjected to mechanical stress. For example, it is shown herein that the inclusion of as little as 10 ppm of non-ionic surfactant in an aqueous furin formulation can increase the stability of the composition by greater than 75%.
Advantageously, the studies described herein also demonstrate that the inclusion of non-ionic surfactant to a furin dilution buffer increases the recovery of furin activity after dilution. For example, it is shown herein that the inclusion of 75 ppm of non-ionic surfactant in a furin dilution buffer increases the recovery of furin activity after dilution by 3- to 4-fold.
Accordingly, the present disclosure provides compositions of highly stabilized furin formulations. Although the majority of the discussion herein is in terms of highly stabilized formulations of recombinant furin (rfurin), it will be appreciated that any furin protein, including furin isolated from a subject or any derivatives or mutants of furin, can be included in the formulations of the present invention.
In one aspect, the present disclosure provides highly stabilized furin formulations that show improved stability over control formulations when assayed after subjecting the formulation to one or more stressors, including without limitation exposure of the formulations to a range of temperatures, to several freeze/thaw cycles, and/or to agitation. Improved stability is generally assessed by level of furin activity—for example, after storage at higher than ambient room temperature, a highly stabilized furin formulation will show a higher level of activity than a control formulation stored under identical conditions as compared to the activity of each prior to storage (i.e., at time=0). Methods for assessing the stability of a furin formulation are described herein.
In further aspects, the present disclosure provides methods for forming highly stabilized furin formulations of the present invention. Such methods include methods for stabilizing a formulation of furin for storage, including frozen storage, storage at ambient room temperature or above, or lyophilization.
In yet other aspects, the present disclosure provides methods for diluting concentrated furin solutions that result in increased retention of furin activity after dilution. As compared to furin compositions diluted with solutions lacking non-ionic surfactant, aqueous compositions of furin diluted according to the methods provided herein retain 3- to 4-times more enzymatic activity.
II. Definitions
As used herein, the term “furin” refers to any protein or polypeptide with furin activity, particularly the ability to cleave the peptide bond between residues Arg-763 and Ser-764 of the pro-von Willebrand Factor (pro-VWF) polypeptide. In an exemplary embodiment, furin refers to a polypeptide comprising an amino acid sequence identical or highly identical to that of NP 002560.1 (human furin preproprotein). In an exemplary embodiment, furin refers to a polypeptide comprising an amino acid sequence identical or highly identical to amino acids 25-794 of NP 002560.1 (human furin proprotein). In an exemplary embodiment, furin refers to a polypeptide comprising an amino acid sequence identical or highly identical to amino acids 108-794 of NP 002560.1 (human mature furin protein). As used herein, furin polypeptides also include natural variants of furin with VWF cleaving activity, as well as modified furin constructs with VWF cleaving activity. As used herein, furin encompasses any natural variants, alternative sequences, isoforms or mutant proteins that retain some basal activity (for example, at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or more activity as compared to the activity of wild-type furin). Examples of furin mutations found in the human population include, without limitation, A43E, A43G, A43V, R50W, Y64C, L77P, R81C, E97A, E97V, V109M, V109L, R130W, A139V, P169T, N245S, E271K, Q339R, N407S, E457Q, R464W, K469R, S524Y, T536S, L570F, D624N, A642T, S685P, V728I, V735I, R745Q, P772L, and A793T. Furin polypeptides also include polypeptides containing post-translational modification. For example, it has been shown that furin is phosphorylated at residues S773 and S775 and predicted that furin is glycosylated at residues N387, N440, and N553.
In the context of the present disclosure, furin proteins include recombinant furin polypeptides as well as native furin polypeptides isolated from a source material (e.g., tissue or blood). Furin polypeptides (recombinant and source purified) may be derived from any suitable organism, for example, a mammal such as a primate, human, monkey, rabbit, pig, rodent, mouse, rat, hamster, gerbil, canine, feline, and biologically active derivatives thereof. Mutant and variant furin polypeptides having VWF cleaving activity are also embraced, as are functional fragments, and fusion proteins comprising furin polypeptides. Furthermore, the furin polypeptides described herein may further comprise tags that facilitate purification, detection, or both. The furin polypeptides described herein may further be modified with a therapeutic moiety or a moiety suitable for imaging in vitro or in vivo.
Proteolytically active recombinant furin may be prepared by expression in cell culture (e.g., mammalian cell culture). Non-limiting examples of expression and purification methods for preparing recombinant furin are described in WO 1991/06314, WO 1992/09698, U.S. Pat. Nos. 6,210,929 and 6,596,526, as well as in U.S. Patent Application Publication Nos. 2009/0181423 and 2009/0304669, the contents of which are hereby incorporated by reference in their entireties for all purposes.
As used herein, “activity” refers to a functional activity or activities of furin or portion thereof associated with a full-length (complete) protein. Functional activities include, but are not limited to, biological activity, including participation in the proteolytic maturation of proprotein substrates and cleavage of test substrates such as Boc-Arg-Val-Arg-Arg-AMC (SEQ ID NO:1; AMC=7-amino-4-methoxy coumarin). Substrates for furin include von Willebrand factor, proparathyroid hormone, transforming growth factor beta 1 precursor, proalbumin, pro-beta-secretase, membrane type-1 matrix metalloproteinase, and the beta subunit of pro-nerve growth factor. In one embodiment, one Unit (U) of furin activity is defined as the amount of furin that releases 1 pmol of AMC from Boc-Arg-Val-Arg-Arg-AMC (SEQ ID NO:1) per minute.
As used herein, the term “stability” (such as furin stability or furin formulation stability) is used in a structural context, e.g., relating to the structural integrity of a protein, or in a functional context, e.g., relating to a protein's ability to retain its function and/or activity over time. As will be appreciated, the protein under discussion may be contained within a formulation in accordance with the methods and compositions described herein, and the stability of that protein refers to its stability in that formulation. In one embodiment, the stability of a furin composition is determined by measuring the furin activity of the composition. For example, by using a detectable furin substrate such as Boc-Arg-Val-Arg-Arg-AMC (SEQ ID NO:1; e.g., ALX-260-040-M001 sold by Enzo Life Sciences), for example, in an assay as described in Malloy S S, et al., J Biol Chem. 1992 Aug. 15; 267(23):16396-402, the content of which is hereby incorporated herein by reference in its entirety for all purposes. In one embodiment, the stability of furin composition formulated with sugar, sugar alcohol, and/or non-ionic surfactant, as described herein, is compared to a furin composition formulated without the sugar, sugar alcohol, and/or non-ionic surfactant.
As used herein, a “storage stable” aqueous furin composition refers to a furin polypeptide solution (e.g., a rfurin polypeptide solution) that has been formulated to increase the stability of the protein in solution, for example by at least 10%, over a given storage time. In the context of the present disclosure, a furin polypeptide solution (e.g., a rfurin polypeptide solution) can be made “storage stable” by the addition of a sugar, sugar alcohol, or non-ionic surfactant as a stabilizing agent. In some embodiments, the stability of the furin polypeptide in any given formulation can be measured, for example, by monitoring the formation of aggregates, loss of bulk enzymatic activity, or formation of degradation products, over a period of time. The absolute stability of a formulation, and the stabilizing effects of the sugar, sugar alcohol, or non-ionic surfactant, will vary dependent upon the particular composition being stabilized. In one embodiment, the stability of a furin composition is determined by measuring the furin activity of the composition. For example, by using a detectable furin substrate such as Boc-Arg-Val-Arg-Arg-AMC (SEQ ID NO:1; e.g., ALX-260-040-M001 sold by Enzo Life Sciences), in an assay, for example, as described in Malloy S S, et al., J Biol Chem. 1992 Aug. 15; 267(23):16396-402, the content of which is hereby incorporated by reference in its entirety for all purposes. In one embodiment, the stability of furin composition formulated with sugar, sugar alcohol, and/or non-ionic surfactant, as described herein, is compared to a furin composition formulated without the sugar, sugar alcohol, and/or non-ionic surfactant.
As used herein, “shelf-life” refers to the period of time a formulation maintains a predetermined level of stability at a predetermined temperature. In particular embodiments, the predetermined temperature refers to frozen (e.g., −80° C., −25° C., 0° C.), refrigerated (e.g., 0° to 10° C.), or room temperature (e.g., 18° C. to 32° C.) storage.
As used herein, the term “time of stability” refers to the length of time a formulation is considered stable. For example, the time of stability for a formulation may refer to the length of time for which the level of protein aggregation and/or degradation in the formulation remains below a certain threshold (e.g., 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, etc.), and/or the length of time a formulation maintains biological activity above a certain threshold (e.g., 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, etc.) of the amount of activity present in the formulation at the start of the storage period.
In the context of the present disclosure, a storage stable aqueous composition of a furin polypeptide (e.g., rfurin polypeptide) formulated with a sugar, sugar alcohol, and/or non-ionic surfactant will have a longer time of stability than a composition of the same furin polypeptide formulated without the sugar, sugar alcohol, and/or non-ionic surfactant. In some embodiments, a storage stable aqueous composition of a furin polypeptide, will have a time of stability that is, for example, at least 10% greater than the time of stability for the same composition formulated in the absence of the sugar, sugar alcohol, and/or non-ionic surfactant, or at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190% greater, or at least 2 times greater, or at least 2.5 times, 3.0 times, 3.5 times, 4.0 times, 4.5 times, 5.0 times, 5.5 times, 6.0 times, 6.5 times, 7.0 times, 7.5 times, 8.0 times, 8.5 times, 9.0 times, 9.5 times, 10 times, or more times greater than the time of stability for the same composition formulated in the absence of the sugar, sugar alcohol, and/or non-ionic surfactant.
As used herein, “storage” means that a formulation is not immediately administered to a subject or utilized in a production process once prepared, but is kept for a period of time under particular conditions (e.g. particular temperature) prior to use. For example, a furin formulation can be kept for days, weeks, months or years, prior to administration to a subject under varied temperatures such as frozen (e.g., −80° C., −25° C., 0° C.), refrigerated (e.g., 0° to 10° C.), or room temperature (e.g., 18° C. to 32° C.). As will be appreciated, such formulations may be liquid or lyophilized formulations.
As used herein, the term “about” denotes an approximate range of plus or minus 10% from a specified value. For instance, the language “about 20%” encompasses a range of 18-22%.
As used herein, when referring to a concentration of an individual component of a composition, the phrases “no more than X” and “from 0 to X” are equivalent and refer to any concentration between and including 0 and X. For example, the phrases “a concentration of no more than 2%” and “a concentration of from 0% to 2%” are equivalent and include 0%, 1%, and 2%.
As used herein, when referring to a concentration of an individual component of a composition, the phrases “no less than X” refers to any concentration X or higher. For example, the phrase “a concentration of no less than 98%” includes 98%, 99%, and 100%.
As used herein, when referring to a concentration of an individual component of a composition, the phrases “between X and Y” and “from X to X” are equivalent and refer to any concentration between and including X and Y. For example, the phrases “a concentration of between 49% and 51%” and “a concentration of from 49% to 51%” are equivalent and include 49%, 50%, and 51%.
As used herein, a “sugar” refers to monosaccharides having the general formula CxH2yOy (linear) or CxH(2y-1)Oy (cyclic), and disaccharides consisting of two monosaccharide units formed through a dehydration reaction. Monosaccharides can be classified by the number of carbon atoms they contain: diose (2), triose (3), tetrose (4), pentose (5), hexose (6), heptose (7), etc. Accordingly, as used herein, a C(X) sugar refers to a sugar containing X-number of carbon molecules. For example, a C(5) sugar refers to a pentose sugar, while a C(6) sugar refers to a hexose sugar. Non-limiting examples of sugars that may be used in the formulations provided herein include: diose sugar glycolaldehyde, triose sugars glyceraldehyde and dihydroxyacetone; tetrose sugars erythrose, threose, and erythrulose; pentose sugars arabinose, lyxose, ribose, xylose, ribulose, and xylulose; hexose sugars allose, altrose, glucose, mannose, gulose, idose, galactose, talose, psicose, fructose, sorbose, and tagatose; heptose sugars sedoheptulose, mannoheptulose, and L-glycero-D-manno-heptose; and all possible combinations of disaccharide sugars formed thereform, including without limitation sucrose, lactulose, lactose, maltose, trehalose, cellobiose, kojibiose, nigerose, isomaltose, β,β-trehalose, α,β-trehalose, sophorose, laminaribiose, gentiobiose, turanose, maltulose, palatinose, gentiobiulose, mannobiose, melibiose, melibiulose, rutinose, rutinulose, and xylobiose.
As used herein, a “sugar alcohol” refers to a hydrogenated form of a mono- or disaccharide, whose carbonyl group has been reduced to form a primary or secondary hydroxyl. In one embodiment, the sugar alcohol has between about 4 and about 8 carbon atoms. Non-limiting examples of sugar alcohols that may be used in formulations provided herein include glycol, glycerol, erythritol, threitol, ribitol, fucitol, iditol, volmitol, isomalt, maltitol, lactitol, mannitol, sorbitol, inositol, galactitol, dulcitol, xylitol, and arabitol.
As used herein, a “pharmaceutically acceptable salt” refers to a salt that is safe for administration to a subject (e.g., a human) in a drug formulation. The selection and use of pharmaceutically acceptable salts is well known in the art, for example, see Stahl and Wermuth, Pharmaceutical Salts: Properties, Selection, and Use, 2nd Revised edition, Wiley, Hoboken, N.J. In certain embodiments, the pharmaceutically acceptable salt is sodium chloride, potassium chloride, or a combination thereof.
As used herein, the term “non-ionic surfactant” refers to a surface active agent that is non-ionized under physiologically relevant conditions. Non-limiting examples of non-ionic surfactants useful for the stabilized aqueous furin compositions provided herein include: non-ionic water soluble mono-, di-, and tri-glycerides (e.g., propylene glycol dicarpylate/dicaprate (e.g. MIGLYOL® 840), medium chain mono- and diglycerides (e.g. CAPMUL® and IMWITOR® 72), medium-chain triglycerides (e.g. caprylic and capric triglycerides such as LAVRAFAC, MIGLYOL® 810 or 812, CRODAMOL® GTCC-PN, and SOFTISON 378), long chain monoglycerides (e.g. glyceryl monooleates such as PECEOL, and glyceryl monolinoleates such as MAISINE®), polyoxyl castor oil (e.g. macrogolglycerol ricinoleate, macrogolglycerol hydroxystearate, macrogol cetostearyl ether)); non-ionic water soluble mono- and di-fatty acid esters of polyethyelene glycol; non-ionic water soluble sorbitan fatty acid esters (e.g., sorbitan monolaurates such as polyoxyethylene (20) sorbitan monolaurate (TWEEN 20) and sorbitan monolaurate (SPAN 20); sorbitan monopalmitates such as polyoxyethylene (20) sorbitan monopalmitates (TWEEN 40) and sorbitan monopalmitate (SPAN 40); sorbitan monostearates such as polyoxyethylene (20) sorbitan monostearate (TWEEN 60) and sorbitan monostearate (SPAN 60); sorbitan monooleates such as polyoxyethylene (20) sorbitan monooleate (TWEEN 80) and sorbitan monooleate (SPAN 80); sorbitane trioleates such as sorbitane trioleate (SPAN 85); and sorbitan tristearates such as sorbitan tristearate (SPAN65)); polyglycolyzed glycerides (e.g., lauroyl macrogol-6 glycerides (Labrafil® M2130CS); d-α-tocopheryl polyethyleneglycol 1000 succinate (TPGS), poyethyleneglycol 660 12-hydroxystearate (SOLUTOL® HS 15), polyoxyl oleate and stearate (e.g. PEG 400 monostearate and PEG 1750 monostearate)); non-ionic water soluble triblock copolymers (e.g. poly(ethyleneoxide)/poly-(propyleneoxide)/poly(ethyleneoxide) triblock copolymers such as methyl-oxirane polymer with oxirane BHT (PLURONIC® F-127)).
In one embodiment, storage stable compositions of furin (e.g., rfurin) are provided which contain a non-ionic surfactant selected from a non-ionic water soluble monoglyceride, a non-ionic water soluble diglyceride, a non-ionic water soluble triglyceride, a non-ionic water soluble monofatty acid esters of polyethyelene glycol, a non-ionic water soluble difatty acid esters of polyethyelene glycol, a non-ionic water soluble sorbitan fatty acid ester, a non-ionic polyglycolyzed glyceride, a non-ionic water soluble triblock copolymer, and a combination thereof.
As used herein, the term “biologically active derivative,” when used in the context of furin polypeptide, also embraces polypeptides obtained via recombinant DNA technology. This may include any method known in the art for (i) the production of recombinant DNA by genetic engineering, e.g., via reverse transcription of RNA and/or amplification of DNA, (ii) introducing recombinant DNA into prokaryotic or eukaryotic cells by transfection, e.g., via electroporation or microinjection, (iii) cultivating said transformed cells, e.g., in a continuous or batch-wise manner, (iv) expressing furin protein, e.g., constitutively or upon induction, and (v) isolating said furin protein, e.g., from the culture medium or by harvesting the transformed cells, in order to (vi) obtain substantially purified recombinant furin protein, e.g., via ion exchange chromatography, size exclusion chromatography, affinity chromatography, hydrophobic interaction chromatography, and the like. The term “biologically active derivative” includes also chimeric molecules such as e.g., a furin protein, or functional fragment thereof, in combination with a second polypeptide, e.g., an immunoglobulin Fc domain or an albumin domain, in order to improve the biological/pharmacological properties such as e.g., half life of the furin protein in the circulation system of a mammal, particularly a human.
As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a buffering agent” refers to one agent or mixtures of such agents, and reference to “the method” includes reference to equivalent steps and methods known to those skilled in the art, and so forth.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications mentioned herein are incorporated herein by reference for the purpose of describing and disclosing devices, compositions, formulations and methodologies which are described in the publication and which might be used in connection with the presently described invention.
Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either both of those included limits are also included in the invention.
As used herein, “BDS” refers to “Bulk Drug Substance.”
III. Stabilized Aqueous Compositions of Recombinant Furin
In one aspect, the present disclosure provides stabilized aqueous formulations of furin, e.g., rfurin. The following embodiments are based in part on the discovery that inclusion of a sugar, sugar alcohol, and/or non-ionic surfactant stabilizes aqueous furin compositions, as compared to compositions lacking the sugar, sugar alcohol, and/or non-ionic surfactant.
As will be recognized by one of skill in the art, furin compositions (e.g., rfurin compositions) formulated according to the embodiments provided herein may contain, in addition to the components explicitly disclosed, counter ions contributed by the inclusion of solution components or pH modifying agents, for example, sodium or potassium contributed from an acetate salt, sodium hydroxide, or potassium hydroxide or chloride contributed by calcium chloride or hydrochloric acid. In the context of the present disclosure, a storage stable furin composition (e.g., rfurin) consisting of or consisting essentially of a given formulation may further comprise one or more counter ion, as necessitated by the formulation process at a particular pH.
In one embodiment, a storage stable furin composition (e.g., rfurin composition) provided herein will be stabilized at room temperature (i.e., between 18° C. and 32° C.) for a period of time. For example, in one embodiment, a storage stable, aqueous immunoglobulin composition will be stable when stored at room temperature for at least 4 days. In other embodiments, the composition will be stabile at room temperature for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 21, 28, or more days. In other embodiments, the composition will be stable for at least 1 month. In yet other embodiments, the composition will be stable for at least 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, or more months. In certain embodiments, room temperature refers to between 20° C. and 30° C., between 21° C. and 29° C., between 22° C. and 28° C., between 23° C. and 27° C., between 24° C. and 26° C., or about 25° C. In a specific embodiment, the composition will be stable for an extended period of time when stored at a temperature between 20° C. and 25° C.
In one embodiment, a storage stable furin composition (e.g., rfurin composition) provided herein will be stabilized at refrigerated temperature (i.e., between 2° C. and 10° C.) for a period of time. For example, in one embodiment, a storage stable, aqueous immunoglobulin composition will be stable when stored at refrigerated temperature for at least 4 days. In other embodiments, the composition will be stabile at refrigerated temperature for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 21, 28, or more days. In other embodiments, the composition will be stable for at least 1 month. In yet other embodiments, the composition will be stable for at least 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, or more months. In a specific embodiment, the composition will be stable for an extended period of time when stored at a temperature between 2° C. and 8° C.
In one embodiment, a storage stable furin composition (e.g., rfurin composition) provided herein will be stabilized at elevated temperature (i.e., between 32° C. and 42° C.) for a period of time. For example, in one embodiment, a storage stable, aqueous immunoglobulin composition will be stable when stored at elevated temperature for at least 4 days. In other embodiments, the composition will be stabile at elevated temperature for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 21, 28, or more days. In other embodiments, the composition will be stable for at least 1 month. In yet other embodiments, the composition will be stable for at least 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, or more months. In a specific embodiment, the composition will be stable for an extended period of time when stored at a temperature between 35° C. and 40° C.
In one embodiment, a stored furin composition is considered storage stable as long as the composition maintains at least 40% of the furin activity present at the start of the storage period (e.g., at time=0). In another embodiment, a stored composition is considered stable as long as the composition maintains at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more of the furin activity present at the start of the storage period (e.g., at time=0). In one embodiment, furin activity is measures in an assay as described in Malloy S S, et al., J Biol Chem. 1992 Aug. 15; 267(23):16396-402.
In one embodiment, a furin composition is considered to have been stabilized by the addition of a stabilizing agent (e.g., a sugar, sugar alcohol, or non-ionic surfactant) when the composition contains at least 10% more furin activity after storage for a period of time, as compared to a furin composition not containing the stabilizing agent or containing a lower amount of the stabilizing agent. In other embodiments, a furin composition is considered to have been stabilized by the addition of a stabilizing agent (e.g., a sugar, sugar alcohol, or non-ionic surfactant) when the composition contains at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a greater percentage more furin activity after storage for a period of time, as compared to a furin composition not containing the stabilizing agent or containing a lower amount of the stabilizing agent.
In one embodiment, a stored furin composition is considered stable as long as the percentage of furin present in an aggregated state remains no more than 50%. In other embodiments, a stored furin composition is considered stable as long as the percentage of furin present in an aggregated state remains no more than 45%, 40%, 35%, 30%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less.
In one embodiment, a furin composition is considered to have been stabilized by the addition of a stabilizing agent (e.g., a sugar, sugar alcohol, or non-ionic surfactant) when the composition contains at least 10% less furin present in an aggregated state after storage for a period of time, as compared to a furin composition not containing the stabilizing agent or containing a lower amount of the stabilizing agent. In other embodiments, a furin composition is considered to have been stabilized by the addition of a stabilizing agent (e.g., a sugar, sugar alcohol, or non-ionic surfactant) when the composition contains at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a greater percentage less furin present in an aggregated state after storage for a period of time, as compared to a furin composition not containing the stabilizing agent or containing a lower amount of the stabilizing agent.
In one embodiment, a stored furin composition is considered stable as long as the composition maintains at least 40% of the starting furin activity (e.g., at time=0) after being subjected to mechanical stress. In another embodiment, a stored composition is considered stable as long as the composition maintains 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more of the starting furin activity (e.g., at time=0) after being subjected to mechanical stress. In a specific embodiment, the mechanical stress is agitation (e.g., shaking).
In one embodiment, a furin composition is considered to have been stabilized by the addition of a stabilizing agent (e.g., a sugar, sugar alcohol, or non-ionic surfactant) when the composition contains at least 10% more furin activity after being subjected to mechanical stress, as compared to a furin composition not containing the stabilizing agent or containing a lower amount of the stabilizing agent. In other embodiments, a furin composition is considered to have been stabilized by the addition of a stabilizing agent (e.g., a sugar, sugar alcohol, or non-ionic surfactant) when the composition contains at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a greater percentage more furin activity after being subjected to mechanical stress, as compared to a furin composition not containing the stabilizing agent or containing a lower amount of the stabilizing agent. In a specific embodiment, the mechanical stress is agitation (e.g., shaking).
In one embodiment, a stored furin composition is considered stable as long as the percentage of furin present in an aggregated state remains no more than 50% after being subjected to mechanical stress. In other embodiments, a stored furin composition is considered stable as long as the percentage of furin present in an aggregated state remains no more than 45%, 40%, 35%, 30%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less after being subjected to mechanical stress. In a specific embodiment, the mechanical stress is agitation (e.g., shaking).
In one embodiment, a furin composition is considered to have been stabilized by the addition of a stabilizing agent (e.g., a sugar, sugar alcohol, or non-ionic surfactant) when the composition contains at least 10% less furin present in an aggregated state after being subjected to mechanical stress, as compared to a furin composition not containing the stabilizing agent or containing a lower amount of the stabilizing agent. In other embodiments, a furin composition is considered to have been stabilized by the addition of a stabilizing agent (e.g., a sugar, sugar alcohol, or non-ionic surfactant) when the composition contains at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a greater percentage less furin present in an aggregated state after being subjected to mechanical stress, as compared to a furin composition not containing the stabilizing agent or containing a lower amount of the stabilizing agent. In a specific embodiment, the mechanical stress is agitation (e.g., shaking).
While the furin (e.g., rfurin) formulations described in this application can be lyophilized and reconstituted in the indicated concentrations, it will be appreciated that these preparations can also be reconstituted in more dilute form. For example, a preparation according the present disclosure which is lyophilized and/or normally reconstituted in 2 mL of solution can also be reconstituted in a larger volume of diluent, such as 5 mL. Likewise, lyophilized furin (e.g., rfurin) formulations can also be reconstituted in more concentrated form. For example, a preparation according the present disclosure which is lyophilized and/or normally reconstituted in 2 ml of solution can also be reconstituted in a smaller volume, such as 1 mL.
Advantageously, in one aspect, the highly stabilized furin (e.g., rfurin) formulations of the present invention are combined with a diluent that confers increased furin recovery when the resultant composition is used in a production method, for example, in the maturation of rVWF (also referred to herein as an “rVWF maturation method”). Maturation of pro-von Willebrand factor (vWF) to its active form requires proteolytic processing after a pair of dibasic amino acids (-Lys-Arg-) at residue 763. It has been shown that vWF is preferentially processed by the paired dibasic amino acid-cleaving enzyme furin. Production processes for vWF thus include the use of furin, preferably in a highly stabilized formulation. In a further aspect, the highly stabilized formulation in this diluent increases furin activity recovery in the rVWF maturation step by three to four times compared to control formulations placed in control diluents.
In certain aspects, the highly stabilized formulations of the invention have a shelf life of at least 6 months. As will be appreciated, this shelf life may be at frozen temperatures (i.e., −80° C., −25° C., 0° C.), refrigerated (0° C. to 10° C.), or room temperature (20° C. to 32° C.) in liquid or lyophilized form. In further aspects, the highly stabilized formulations of the invention have a shelf life of at least 12, 18, 24, 30, 36, 42, 48, 54, or 60 months.
In further aspects and in accordance with the above, shelf life is determined by a percent activity remaining after storage at any of the above temperatures for any of the above periods of time. In certain embodiments, shelf life means that the formulation retains at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% of furin activity as measured by any of the assays described herein or known in the art as compared to activity prior to storage for any of the above amounts of time at any of the above temperatures.
In one aspect, a highly stabilized formulation of furin (e.g., rfurin) in accordance with the present disclosure includes: (a) 8,000 U/mL-57,000 U/mL furin (e.g., rfurin); (b) 190 mM sodium chloride; (c) 0.92 mM calcium chloride; (d) 10% w/w trehalose dihydrate; (e) 75 ppm polysorbate 80; (f) 45 mM acetic acid; and (g) 46 mM HEPES.
In certain aspects, highly stabilized formulations of the present disclosure include the following components: 47 mM HEPES, 46 mM acetic acid, 195 mM sodium chloride, 0.094 mM calcium chloride, 0.0075% polysorbate 80, 10% w/w trehalose dihydrate, pH 6.0.
In further aspects, highly stabilized formulations of furin, including rfurin, in accordance with the present disclosure include (a) about 5,500 U/mL to 55,000 U/mL; 6,000 U/mL to 50,000 U/mL; 6,500 U/mL to 45,000 U/mL; 7,000 U/mL to 40,000 U/mL; 7,500 U/mL to 35,000 U/mL; 8,000 U/mL to 30,000 U/mL; 8,500 U/mL to 25,000 U/mL; 9,000 U/mL to 20,000 U/mL; 9,500 U/mL to 15,000 U/mL; or 10,000 U/mL furin; (b) about 100 mM to 300 mM, 110 mM to 280 mM, 120 mM to 260 mM, 130 mM to 240 mM, 140 mM to 220 mM, 150 mM to 200 mM, or 160 mM to 180 mM sodium chloride; (c) about 0.5 mM to 9 mM, 1 mM to 8 mM, 1.5 mM to 7 mM, 2 mM to 6 mM, 2.5 mM to 5 mM, or 3 mM to 4.5 mM calcium chloride; (d) about 0.5% to 19%, 1% to 18%, 1.5% to 17%, 2.0% to 16%, 2.5% to 15%, 3.0% to 14%, 3.5% to 13%, 4.0% to 12%, 4.5% to 11%, 5.0% to 10%, 5.5% to 9%, or 6.0% to 8% trehalose dihydrate; (e) about 0.5 ppm to 140 ppm, 1.0 ppm to 130 ppm, 10 ppm to 120 ppm, 20 ppm to 110 ppm, 30 ppm to 100 ppm, 40 ppm to 95 ppm, 50 ppm to 90 ppm, 55 ppm to 85 ppm, 60 ppm to 80 ppm, or 70 ppm to 75 ppm polysorbate 80; (f) about 25 mM to 90 mM, 30 mM to 80 mM, 35 mM to 70 mM, 40 mM to 60 mM, or 45 mM to 50 mM acetic acid, and (g) 15 mM to 95 mM, 20 mM to 90 mM, 25 mM to 85 mM, 30 mM to 80 mM, 35 mM to 75 mM, 40 mM to 70 mM, 45 mM to 65 mM, or 50 mM to 60 mM HEPES.
In one aspect, the present disclosure provides a stabilized aqueous furin composition (e.g., rfurin) comprising: furin (e.g., rfurin), from 2% to 20% sugar or sugar alcohol, from 10 ppm to 200 ppm non-ionic surfactant, from 50 mM to 500 mM of a pharmaceutically acceptable salt, from 0.5 mM to 10 mM calcium, a buffering agent, and a pH from 5.5 to 7.5. In a specific embodiment, the sugar is a pentose or hexose sugar.
In one embodiment, a stabilized aqueous furin composition (e.g., rfurin) comprises: furin (e.g., rfurin), from 100 mM to 300 mM of a pharmaceutically acceptable salt; from 0.5 mM to 2 mM calcium; from 2% to 20% of a sugar or sugar alcohol; from 100 to 200 ppm of a non-ionic surfactant; from 10 to 200 mM buffering agent; and a pH from 5.5 to 7.5. In a specific embodiment, the sugar is a pentose or hexose sugar.
In a specific embodiment, highly stabilized formulations of the invention include the following components: 47 mM HEPES, 46 mM acetic acid, 195 mM sodium chloride, 0.094 mM calcium chloride, 0.0075% polysorbate 80, 10% w/w trehalose dihydrate, pH 6.0.
In another specific embodiment, a highly stabilized formulation of furin (e.g., rfurin) in accordance with the present invention includes: (a) 8,000-57,000 U/mL rfurin; (b) 190 mM sodium chloride; (c) 0.92 mM calcium chloride; (d) 10% w/w trehalose dihydrate; (e) 75 ppm polysorbate 80; (f) 45 mM acetic acid; and (g) 46 mM HEPES.
A. Stabilizing Agents
Advantageously, it was found that the inclusion of sugars, sugar alcohols, and non-ionic surfactants stabilizes aqueous furin (e.g., rfurin) compositions. These effects are demonstrated in the examples provided herein. For example, the addition of these agents increases furin activity retention upon liquid storage, reduces aggregation of furin polypeptides upon liquid storage, reduces the degradation of furin polypeptides upon liquid storage, reduces the loss of furin activity upon agitation of an aqueous furin composition, and reduces aggregation caused by agitation of an aqueous furin composition.
Accordingly, in one embodiment, the present disclosure provides an aqueous furin composition (e.g., a rfurin composition) comprising from 2% to 20% sugar or sugar alcohol and from 10 ppm to 200 ppm non-ionic surfactant. In another embodiment, the composition comprises from 2% to 10% sugar or sugar alcohol and from 10 ppm to 100 ppm non-ionic surfactant. In another embodiment, the composition comprises 10±2% sugar or sugar alcohol and 75±25 ppm non-ionic surfactant. In a specific embodiment, the composition comprises 10% sugar or sugar alcohol and 75 ppm non-ionic surfactant. In yet other embodiments, the composition comprises a combination of sugar or sugar alcohol and non-ionic surfactant selected from variations 1 to 6035 found in Table 1 to Table 9.







TABLE 1







Exemplary embodiments for the combination of sugar or sugar alcohol and non-ionic


surfactant concentrations useful for the stabilization of furin (e.g., rfurin) compositions.









Sugar or Sugar Alcohol (%)
















2%-20%
2%-17.5%
2%-15%
2%-12.5%
2%-10%
2%-9%
2%-8%
2%-7%




















Non-Ionic
 10-200
Var. 1
Var. 86
Var. 171
Var. 256
Var. 341
Var. 426
Var. 511
Var. 596


Surfactant
 10-175
Var. 2
Var. 87
Var. 172
Var. 257
Var. 342
Var. 427
Var. 512
Var. 597


(ppm)
 10-150
Var. 3
Var. 88
Var. 173
Var. 258
Var. 343
Var. 428
Var. 513
Var. 598



 10-125
Var. 4
Var. 89
Var. 174
Var. 259
Var. 344
Var. 429
Var. 514
Var. 599



 10-100
Var. 5
Var. 90
Var. 175
Var. 260
Var. 345
Var. 430
Var. 515
Var. 600



10-90
Var. 6
Var. 91
Var. 176
Var. 261
Var. 346
Var. 431
Var. 516
Var. 601



10-80
Var. 7
Var. 92
Var. 177
Var. 262
Var. 347
Var. 432
Var. 517
Var. 602



10-75
Var. 8
Var. 93
Var. 178
Var. 263
Var. 348
Var. 433
Var. 518
Var. 603



10-70
Var. 9
Var. 94
Var. 179
Var. 264
Var. 349
Var. 434
Var. 519
Var. 604



10-60
Var. 10
Var. 95
Var. 180
Var. 265
Var. 350
Var. 435
Var. 520
Var. 605



10-50
Var. 11
Var. 96
Var. 181
Var. 266
Var. 351
Var. 436
Var. 521
Var. 606



10-25
Var. 12
Var. 97
Var. 182
Var. 267
Var. 352
Var. 437
Var. 522
Var. 607



 25-200
Var. 13
Var. 98
Var. 183
Var. 268
Var. 353
Var. 438
Var. 523
Var. 608



 25-175
Var. 14
Var. 99
Var. 184
Var. 269
Var. 354
Var. 439
Var. 524
Var. 609



 25-150
Var. 15
Var. 100
Var. 185
Var. 270
Var. 355
Var. 440
Var. 525
Var. 610



 25-125
Var. 16
Var. 101
Var. 186
Var. 271
Var. 356
Var. 441
Var. 526
Var. 611



 25-100
Var. 17
Var. 102
Var. 187
Var. 272
Var. 357
Var. 442
Var. 527
Var. 612



25-90
Var. 18
Var. 103
Var. 188
Var. 273
Var. 358
Var. 443
Var. 528
Var. 613



25-80
Var. 19
Var. 104
Var. 189
Var. 274
Var. 359
Var. 444
Var. 529
Var. 614



25-70
Var. 20
Var. 105
Var. 190
Var. 275
Var. 360
Var. 445
Var. 530
Var. 615



25-60
Var. 21
Var. 106
Var. 191
Var. 276
Var. 361
Var. 446
Var. 531
Var. 616



25-50
Var. 22
Var. 107
Var. 192
Var. 277
Var. 362
Var. 447
Var. 532
Var. 617



 50-200
Var. 23
Var. 108
Var. 193
Var. 278
Var. 363
Var. 448
Var. 533
Var. 618



 50-175
Var. 24
Var. 109
Var. 194
Var. 279
Var. 364
Var. 449
Var. 534
Var. 619



 50-150
Var. 25
Var. 110
Var. 195
Var. 280
Var. 365
Var. 450
Var. 535
Var. 620



 50-125
Var. 26
Var. 111
Var. 196
Var. 281
Var. 366
Var. 451
Var. 536
Var. 621



50-90
Var. 27
Var. 112
Var. 197
Var. 282
Var. 367
Var. 452
Var. 537
Var. 622



50-80
Var. 28
Var. 113
Var. 198
Var. 283
Var. 368
Var. 453
Var. 538
Var. 623



 75-200
Var. 29
Var. 114
Var. 199
Var. 284
Var. 369
Var. 454
Var. 539
Var. 624



 75-175
Var. 30
Var. 115
Var. 200
Var. 285
Var. 370
Var. 455
Var. 540
Var. 625



 75-150
Var. 31
Var. 116
Var. 201
Var. 286
Var. 371
Var. 456
Var. 541
Var. 626



100-200
Var. 32
Var. 117
Var. 202
Var. 287
Var. 372
Var. 457
Var. 542
Var. 627



100-175
Var. 33
Var. 118
Var. 203
Var. 288
Var. 373
Var. 458
Var. 543
Var. 628



50 ± 25
Var. 34
Var. 119
Var. 204
Var. 289
Var. 374
Var. 459
Var. 544
Var. 629



60 ± 25
Var. 35
Var. 120
Var. 205
Var. 290
Var. 375
Var. 460
Var. 545
Var. 630



70 ± 25
Var. 36
Var. 121
Var. 206
Var. 291
Var. 376
Var. 461
Var. 546
Var. 631



75 ± 25
Var. 37
Var. 122
Var. 207
Var. 292
Var. 377
Var. 462
Var. 547
Var. 632



80 ± 25
Var. 38
Var. 123
Var. 208
Var. 293
Var. 378
Var. 463
Var. 548
Var. 633



90 ± 25
Var. 39
Var. 124
Var. 209
Var. 294
Var. 379
Var. 464
Var. 549
Var. 634



100 ± 25 
Var. 40
Var. 125
Var. 210
Var. 295
Var. 380
Var. 465
Var. 550
Var. 635



125 ± 25 
Var. 41
Var. 126
Var. 211
Var. 296
Var. 381
Var. 466
Var. 551
Var. 636



150 ± 25 
Var. 42
Var. 127
Var. 212
Var. 297
Var. 382
Var. 467
Var. 552
Var. 637



175 ± 25 
Var. 43
Var. 128
Var. 213
Var. 298
Var. 383
Var. 468
Var. 553
Var. 638



30 ± 10
Var. 44
Var. 129
Var. 214
Var. 299
Var. 384
Var. 469
Var. 554
Var. 639



40 ± 10
Var. 45
Var. 130
Var. 215
Var. 300
Var. 385
Var. 470
Var. 555
Var. 640



50 ± 10
Var. 46
Var. 131
Var. 216
Var. 301
Var. 386
Var. 471
Var. 556
Var. 641



60 ± 10
Var. 47
Var. 132
Var. 217
Var. 302
Var. 387
Var. 472
Var. 557
Var. 642



70 ± 10
Var. 48
Var. 133
Var. 218
Var. 303
Var. 388
Var. 473
Var. 558
Var. 643



75 ± 10
Var. 49
Var. 134
Var. 219
Var. 304
Var. 389
Var. 474
Var. 559
Var. 644



80 ± 10
Var. 50
Var. 135
Var. 220
Var. 305
Var. 390
Var. 475
Var. 560
Var. 645



90 ± 10
Var. 51
Var. 136
Var. 221
Var. 306
Var. 391
Var. 476
Var. 561
Var. 646



100 ± 10 
Var. 52
Var. 137
Var. 222
Var. 307
Var. 392
Var. 477
Var. 562
Var. 647



110 ± 10 
Var. 53
Var. 138
Var. 223
Var. 308
Var. 393
Var. 478
Var. 563
Var. 648



120 ± 10 
Var. 54
Var. 139
Var. 224
Var. 309
Var. 394
Var. 479
Var. 564
Var. 649



125 ± 10 
Var. 55
Var. 140
Var. 225
Var. 310
Var. 395
Var. 480
Var. 565
Var. 650



130 ± 10 
Var. 56
Var. 141
Var. 226
Var. 311
Var. 396
Var. 481
Var. 566
Var. 651



140 ± 10 
Var. 57
Var. 142
Var. 227
Var. 312
Var. 397
Var. 482
Var. 567
Var. 652



150 ± 10 
Var. 58
Var. 143
Var. 228
Var. 313
Var. 398
Var. 483
Var. 568
Var. 653



160 ± 10 
Var. 59
Var. 144
Var. 229
Var. 314
Var. 399
Var. 484
Var. 569
Var. 654



170 ± 10 
Var. 60
Var. 145
Var. 230
Var. 315
Var. 400
Var. 485
Var. 570
Var. 655



175 ± 10 
Var. 61
Var. 146
Var. 231
Var. 316
Var. 401
Var. 486
Var. 571
Var. 656



180 ± 10 
Var. 62
Var. 147
Var. 232
Var. 317
Var. 402
Var. 487
Var. 572
Var. 657



190 ± 10 
Var. 63
Var. 148
Var. 233
Var. 318
Var. 403
Var. 488
Var. 573
Var. 658



25
Var. 64
Var. 149
Var. 234
Var. 319
Var. 404
Var. 489
Var. 574
Var. 659



30
Var. 65
Var. 150
Var. 235
Var. 320
Var. 405
Var. 490
Var. 575
Var. 660



40
Var. 66
Var. 151
Var. 236
Var. 321
Var. 406
Var. 491
Var. 576
Var. 661



50
Var. 67
Var. 152
Var. 237
Var. 322
Var. 407
Var. 492
Var. 577
Var. 662



60
Var. 68
Var. 153
Var. 238
Var. 323
Var. 408
Var. 493
Var. 578
Var. 663



70
Var. 69
Var. 154
Var. 239
Var. 324
Var. 409
Var. 494
Var. 579
Var. 664



75
Var. 70
Var. 155
Var. 240
Var. 325
Var. 410
Var. 495
Var. 580
Var. 665



80
Var. 71
Var. 156
Var. 241
Var. 326
Var. 411
Var. 496
Var. 581
Var. 666



90
Var. 72
Var. 157
Var. 242
Var. 327
Var. 412
Var. 497
Var. 582
Var. 667



100
Var. 73
Var. 158
Var. 243
Var. 328
Var. 413
Var. 498
Var. 583
Var. 668



110
Var. 74
Var. 159
Var. 244
Var. 329
Var. 414
Var. 499
Var. 584
Var. 669



120
Var. 75
Var. 160
Var. 245
Var. 330
Var. 415
Var. 500
Var. 585
Var. 670



125
Var. 76
Var. 161
Var. 246
Var. 331
Var. 416
Var. 501
Var. 586
Var. 671



130
Var. 77
Var. 162
Var. 247
Var. 332
Var. 417
Var. 502
Var. 587
Var. 672



140
Var. 78
Var. 163
Var. 248
Var. 333
Var. 418
Var. 503
Var. 588
Var. 673



150
Var. 79
Var. 164
Var. 249
Var. 334
Var. 419
Var. 504
Var. 589
Var. 674



160
Var. 80
Var. 165
Var. 250
Var. 335
Var. 420
Var. 505
Var. 590
Var. 675



170
Var. 81
Var. 166
Var. 251
Var. 336
Var. 421
Var. 506
Var. 591
Var. 676



175
Var. 82
Var. 167
Var. 252
Var. 337
Var. 422
Var. 507
Var. 592
Var. 677



180
Var. 83
Var. 168
Var. 253
Var. 338
Var. 423
Var. 508
Var. 593
Var. 678



190
Var. 84
Var. 169
Var. 254
Var. 339
Var. 424
Var. 509
Var. 594
Var. 679



200
Var. 85
Var. 170
Var. 255
Var. 340
Var. 425
Var. 510
Var. 595
Var. 680





Var. = Variation













TABLE 2







Exemplary embodiments for the combination of sugar or sugar alcohol and non-ionic


surfactant concentrations useful for the stabilization of furin (e.g., rfurin) compositions.









Sugar or Sugar Alcohol (%)
















5%-20%
5%-17.5%
5%-15%
5%-12.5%
5%-10%
7.5%-20%
7.5%-17.5%
7.5%-15%




















Non-Ionic Surfactant
 10-200
Var. 681
Var. 766
Var. 851
Var. 936
Var. 1021
Var. 1106
Var. 1191
Var. 1276


(ppm)
 10-175
Var. 682
Var. 767
Var. 852
Var. 937
Var. 1022
Var. 1107
Var. 1192
Var. 1277



 10-150
Var. 683
Var. 768
Var. 853
Var. 938
Var. 1023
Var. 1108
Var. 1193
Var. 1278



 10-125
Var. 684
Var. 769
Var. 854
Var. 939
Var. 1024
Var. 1109
Var. 1194
Var. 1279



 10-100
Var. 685
Var. 770
Var. 855
Var. 940
Var. 1025
Var. 1110
Var. 1195
Var. 1280



10-90
Var. 686
Var. 771
Var. 856
Var. 941
Var. 1026
Var. 1111
Var. 1196
Var. 1281



10-80
Var. 687
Var. 772
Var. 857
Var. 942
Var. 1027
Var. 1112
Var. 1197
Var. 1282



10-75
Var. 688
Var. 773
Var. 858
Var. 943
Var. 1028
Var. 1113
Var. 1198
Var. 1283



10-70
Var. 689
Var. 774
Var. 859
Var. 944
Var. 1029
Var. 1114
Var. 1199
Var. 1284



10-60
Var. 690
Var. 775
Var. 860
Var. 945
Var. 1030
Var. 1115
Var. 1200
Var. 1285



10-50
Var. 691
Var. 776
Var. 861
Var. 946
Var. 1031
Var. 1116
Var. 1201
Var. 1286



10-25
Var. 692
Var. 777
Var. 862
Var. 947
Var. 1032
Var. 1117
Var. 1202
Var. 1287



 25-200
Var. 693
Var. 778
Var. 863
Var. 948
Var. 1033
Var. 1118
Var. 1203
Var. 1288



 25-175
Var. 694
Var. 779
Var. 864
Var. 949
Var. 1034
Var. 1119
Var. 1204
Var. 1289



 25-150
Var. 695
Var. 780
Var. 865
Var. 950
Var. 1035
Var. 1120
Var. 1205
Var. 1290



 25-125
Var. 696
Var. 781
Var. 866
Var. 951
Var. 1036
Var. 1121
Var. 1206
Var. 1291



 25-100
Var. 697
Var. 782
Var. 867
Var. 952
Var. 1037
Var. 1122
Var. 1207
Var. 1292



25-90
Var. 698
Var. 783
Var. 868
Var. 953
Var. 1038
Var. 1123
Var. 1208
Var. 1293



25-80
Var. 699
Var. 784
Var. 869
Var. 954
Var. 1039
Var. 1124
Var. 1209
Var. 1294



25-70
Var. 700
Var. 785
Var. 870
Var. 955
Var. 1040
Var. 1125
Var. 1210
Var. 1295



25-60
Var. 701
Var. 786
Var. 871
Var. 956
Var. 1041
Var. 1126
Var. 1211
Var. 1296



25-50
Var. 702
Var. 787
Var. 872
Var. 957
Var. 1042
Var. 1127
Var. 1212
Var. 1297



 50-200
Var. 703
Var. 788
Var. 873
Var. 958
Var. 1043
Var. 1128
Var. 1213
Var. 1298



 50-175
Var. 704
Var. 789
Var. 874
Var. 959
Var. 1044
Var. 1129
Var. 1214
Var. 1299



 50-150
Var. 705
Var. 790
Var. 875
Var. 960
Var. 1045
Var. 1130
Var. 1215
Var. 1300



 50-125
Var. 706
Var. 791
Var. 876
Var. 961
Var. 1046
Var. 1131
Var. 1216
Var. 1301



50-90
Var. 707
Var. 792
Var. 877
Var. 962
Var. 1047
Var. 1132
Var. 1217
Var. 1302



50-80
Var. 708
Var. 793
Var. 878
Var. 963
Var. 1048
Var. 1133
Var. 1218
Var. 1303



 75-200
Var. 709
Var. 794
Var. 879
Var. 964
Var. 1049
Var. 1134
Var. 1219
Var. 1304



 75-175
Var. 710
Var. 795
Var. 880
Var. 965
Var. 1050
Var. 1135
Var. 1220
Var. 1305



 75-150
Var. 711
Var. 796
Var. 881
Var. 966
Var. 1051
Var. 1136
Var. 1221
Var. 1306



100-200
Var. 712
Var. 797
Var. 882
Var. 967
Var. 1052
Var. 1137
Var. 1222
Var. 1307



100-175
Var. 713
Var. 798
Var. 883
Var. 968
Var. 1053
Var. 1138
Var. 1223
Var. 1308



50 ± 25
Var. 714
Var. 799
Var. 884
Var. 969
Var. 1054
Var. 1139
Var. 1224
Var. 1309



60 ± 25
Var. 715
Var. 800
Var. 885
Var. 970
Var. 1055
Var. 1140
Var. 1225
Var. 1310



70 ± 25
Var. 716
Var. 801
Var. 886
Var. 971
Var. 1056
Var. 1141
Var. 1226
Var. 1311



75 ± 25
Var. 717
Var. 802
Var. 887
Var. 972
Var. 1057
Var. 1142
Var. 1227
Var. 1312



80 ± 25
Var. 718
Var. 803
Var. 888
Var. 973
Var. 1058
Var. 1143
Var. 1228
Var. 1313



90 ± 25
Var. 719
Var. 804
Var. 889
Var. 974
Var. 1059
Var. 1144
Var. 1229
Var. 1314



100 ± 25 
Var. 720
Var. 805
Var. 890
Var. 975
Var. 1060
Var. 1145
Var. 1230
Var. 1315



125 ± 25 
Var. 721
Var. 806
Var. 891
Var. 976
Var. 1061
Var. 1146
Var. 1231
Var. 1316



150 ± 25 
Var. 722
Var. 807
Var. 892
Var. 977
Var. 1062
Var. 1147
Var. 1232
Var. 1317



175 ± 25 
Var. 723
Var. 808
Var. 893
Var. 978
Var. 1063
Var. 1148
Var. 1233
Var. 1318



30 ± 10
Var. 724
Var. 809
Var. 894
Var. 979
Var. 1064
Var. 1149
Var. 1234
Var. 1319



40 ± 10
Var. 725
Var. 810
Var. 895
Var. 980
Var. 1065
Var. 1150
Var. 1235
Var. 1320



50 ± 10
Var. 726
Var. 811
Var. 896
Var. 981
Var. 1066
Var. 1151
Var. 1236
Var. 1321



60 ± 10
Var. 727
Var. 812
Var. 897
Var. 982
Var. 1067
Var. 1152
Var. 1237
Var. 1322



70 ± 10
Var. 728
Var. 813
Var. 898
Var. 983
Var. 1068
Var. 1153
Var. 1238
Var. 1323



75 ± 10
Var. 729
Var. 814
Var. 899
Var. 984
Var. 1069
Var. 1154
Var. 1239
Var. 1324



80 ± 10
Var. 730
Var. 815
Var. 900
Var. 985
Var. 1070
Var. 1155
Var. 1240
Var. 1325



90 ± 10
Var. 731
Var. 816
Var. 901
Var. 986
Var. 1071
Var. 1156
Var. 1241
Var. 1326



100 ± 10 
Var. 732
Var. 817
Var. 902
Var. 987
Var. 1072
Var. 1157
Var. 1242
Var. 1327



110 ± 10 
Var. 733
Var. 818
Var. 903
Var. 988
Var. 1073
Var. 1158
Var. 1243
Var. 1328



120 ± 10 
Var. 734
Var. 819
Var. 904
Var. 989
Var. 1074
Var. 1159
Var. 1244
Var. 1329



125 ± 10 
Var. 735
Var. 820
Var. 905
Var. 990
Var. 1075
Var. 1160
Var. 1245
Var. 1330



130 ± 10 
Var. 736
Var. 821
Var. 906
Var. 991
Var. 1076
Var. 1161
Var. 1246
Var. 1331



140 ± 10 
Var. 737
Var. 822
Var. 907
Var. 992
Var. 1077
Var. 1162
Var. 1247
Var. 1332



150 ± 10 
Var. 738
Var. 823
Var. 908
Var. 993
Var. 1078
Var. 1163
Var. 1248
Var. 1333



160 ± 10 
Var. 739
Var. 824
Var. 909
Var. 994
Var. 1079
Var. 1164
Var. 1249
Var. 1334



170 ± 10 
Var. 740
Var. 825
Var. 910
Var. 995
Var. 1080
Var. 1165
Var. 1250
Var. 1335



175 ± 10 
Var. 741
Var. 826
Var. 911
Var. 996
Var. 1081
Var. 1166
Var. 1251
Var. 1336



180 ± 10 
Var. 742
Var. 827
Var. 912
Var. 997
Var. 1082
Var. 1167
Var. 1252
Var. 1337



190 ± 10 
Var. 743
Var. 828
Var. 913
Var. 998
Var. 1083
Var. 1168
Var. 1253
Var. 1338



25
Var. 744
Var. 829
Var. 914
Var. 999
Var. 1084
Var. 1169
Var. 1254
Var. 1339



30
Var. 745
Var. 830
Var. 915
Var. 1000
Var. 1085
Var. 1170
Var. 1255
Var. 1340



40
Var. 746
Var. 831
Var. 916
Var. 1001
Var. 1086
Var. 1171
Var. 1256
Var. 1341



50
Var. 747
Var. 832
Var. 917
Var. 1002
Var. 1087
Var. 1172
Var. 1257
Var. 1342



60
Var. 748
Var. 833
Var. 918
Var. 1003
Var. 1088
Var. 1173
Var. 1258
Var. 1343



70
Var. 749
Var. 834
Var. 919
Var. 1004
Var. 1089
Var. 1174
Var. 1259
Var. 1344



75
Var. 750
Var. 835
Var. 920
Var. 1005
Var. 1090
Var. 1175
Var. 1260
Var. 1345



80
Var. 751
Var. 836
Var. 921
Var. 1006
Var. 1091
Var. 1176
Var. 1261
Var. 1346



90
Var. 752
Var. 837
Var. 922
Var. 1007
Var. 1092
Var. 1177
Var. 1262
Var. 1347



100
Var. 753
Var. 838
Var. 923
Var. 1008
Var. 1093
Var. 1178
Var. 1263
Var. 1348



110
Var. 754
Var. 839
Var. 924
Var. 1009
Var. 1094
Var. 1179
Var. 1264
Var. 1349



120
Var. 755
Var. 840
Var. 925
Var. 1010
Var. 1095
Var. 1180
Var. 1265
Var. 1350



125
Var. 756
Var. 841
Var. 926
Var. 1011
Var. 1096
Var. 1181
Var. 1266
Var. 1351



130
Var. 757
Var. 842
Var. 927
Var. 1012
Var. 1097
Var. 1182
Var. 1267
Var. 1352



140
Var. 758
Var. 843
Var. 928
Var. 1013
Var. 1098
Var. 1183
Var. 1268
Var. 1353



150
Var. 759
Var. 844
Var. 929
Var. 1014
Var. 1099
Var. 1184
Var. 1269
Var. 1354



160
Var. 760
Var. 845
Var. 930
Var. 1015
Var. 1100
Var. 1185
Var. 1270
Var. 1355



170
Var. 761
Var. 846
Var. 931
Var. 1016
Var. 1101
Var. 1186
Var. 1271
Var. 1356



175
Var. 762
Var. 847
Var. 932
Var. 1017
Var. 1102
Var. 1187
Var. 1272
Var. 1357



180
Var. 763
Var. 848
Var. 933
Var. 1018
Var. 1103
Var. 1188
Var. 1273
Var. 1358



190
Var. 764
Var. 849
Var. 934
Var. 1019
Var. 1104
Var. 1189
Var. 1274
Var. 1359



200
Var. 765
Var. 850
Var. 935
Var. 1020
Var. 1105
Var. 1190
Var. 1275
Var. 1360





Var. = Variation













TABLE 3







Exemplary embodiments for the combination of sugar or sugar alcohol and non-ionic


surfactant concentrations useful for the stabilization of furin (e.g., rfurin) compositions.









Sugar or Sugar Alcohol (%)
















7.5%-12.5%
10%-20%
10%-17.5%
10%-15%
4 ± 2%
5 ± 2%
6 ± 2%
7 ± 2%




















Non-Ionic Surfactant
 10-200
Var. 1361
Var. 1446
Var. 1531
Var. 1616
Var. 1701
Var. 1786
Var. 1871
Var. 1956


(ppm)
 10-175
Var. 1362
Var. 1447
Var. 1532
Var. 1617
Var. 1702
Var. 1787
Var. 1872
Var. 1957



 10-150
Var. 1363
Var. 1448
Var. 1533
Var. 1618
Var. 1703
Var. 1788
Var. 1873
Var. 1958



 10-125
Var. 1364
Var. 1449
Var. 1534
Var. 1619
Var. 1704
Var. 1789
Var. 1874
Var. 1959



 10-100
Var. 1365
Var. 1450
Var. 1535
Var. 1620
Var. 1705
Var. 1790
Var. 1875
Var. 1960



10-90
Var. 1366
Var. 1451
Var. 1536
Var. 1621
Var. 1706
Var. 1791
Var. 1876
Var. 1961



10-80
Var. 1367
Var. 1452
Var. 1537
Var. 1622
Var. 1707
Var. 1792
Var. 1877
Var. 1962



10-75
Var. 1368
Var. 1453
Var. 1538
Var. 1623
Var. 1708
Var. 1793
Var. 1878
Var. 1963



10-70
Var. 1369
Var. 1454
Var. 1539
Var. 1624
Var. 1709
Var. 1794
Var. 1879
Var. 1964



10-60
Var. 1370
Var. 1455
Var. 1540
Var. 1625
Var. 1710
Var. 1795
Var. 1880
Var. 1965



10-50
Var. 1371
Var. 1456
Var. 1541
Var. 1626
Var. 1711
Var. 1796
Var. 1881
Var. 1966



10-25
Var. 1372
Var. 1457
Var. 1542
Var. 1627
Var. 1712
Var. 1797
Var. 1882
Var. 1967



 25-200
Var. 1373
Var. 1458
Var. 1543
Var. 1628
Var. 1713
Var. 1798
Var. 1883
Var. 1968



 25-175
Var. 1374
Var. 1459
Var. 1544
Var. 1629
Var. 1714
Var. 1799
Var. 1884
Var. 1969



 25-150
Var. 1375
Var. 1460
Var. 1545
Var. 1630
Var. 1715
Var. 1800
Var. 1885
Var. 1970



 25-125
Var. 1376
Var. 1461
Var. 1546
Var. 1631
Var. 1716
Var. 1801
Var. 1886
Var. 1971



 25-100
Var. 1377
Var. 1462
Var. 1547
Var. 1632
Var. 1717
Var. 1802
Var. 1887
Var. 1972



25-90
Var. 1378
Var. 1463
Var. 1548
Var. 1633
Var. 1718
Var. 1803
Var. 1888
Var. 1973



25-80
Var. 1379
Var. 1464
Var. 1549
Var. 1634
Var. 1719
Var. 1804
Var. 1889
Var. 1974



25-70
Var. 1380
Var. 1465
Var. 1550
Var. 1635
Var. 1720
Var. 1805
Var. 1890
Var. 1975



25-60
Var. 1381
Var. 1466
Var. 1551
Var. 1636
Var. 1721
Var. 1806
Var. 1891
Var. 1976



25-50
Var. 1382
Var. 1467
Var. 1552
Var. 1637
Var. 1722
Var. 1807
Var. 1892
Var. 1977



 50-200
Var. 1383
Var. 1468
Var. 1553
Var. 1638
Var. 1723
Var. 1808
Var. 1893
Var. 1978



 50-175
Var. 1384
Var. 1469
Var. 1554
Var. 1639
Var. 1724
Var. 1809
Var. 1894
Var. 1979



 50-150
Var. 1385
Var. 1470
Var. 1555
Var. 1640
Var. 1725
Var. 1810
Var. 1895
Var. 1980



 50-125
Var. 1386
Var. 1471
Var. 1556
Var. 1641
Var. 1726
Var. 1811
Var. 1896
Var. 1981



50-90
Var. 1387
Var. 1472
Var. 1557
Var. 1642
Var. 1727
Var. 1812
Var. 1897
Var. 1982



50-80
Var. 1388
Var. 1473
Var. 1558
Var. 1643
Var. 1728
Var. 1813
Var. 1898
Var. 1983



 75-200
Var. 1389
Var. 1474
Var. 1559
Var. 1644
Var. 1729
Var. 1814
Var. 1899
Var. 1984



 75-175
Var. 1390
Var. 1475
Var. 1560
Var. 1645
Var. 1730
Var. 1815
Var. 1900
Var. 1985



 75-150
Var. 1391
Var. 1476
Var. 1561
Var. 1646
Var. 1731
Var. 1816
Var. 1901
Var. 1986



100-200
Var. 1392
Var. 1477
Var. 1562
Var. 1647
Var. 1732
Var. 1817
Var. 1902
Var. 1987



100-175
Var. 1393
Var. 1478
Var. 1563
Var. 1648
Var. 1733
Var. 1818
Var. 1903
Var. 1988



50 ± 25
Var. 1394
Var. 1479
Var. 1564
Var. 1649
Var. 1734
Var. 1819
Var. 1904
Var. 1989



60 ± 25
Var. 1395
Var. 1480
Var. 1565
Var. 1650
Var. 1735
Var. 1820
Var. 1905
Var. 1990



70 ± 25
Var. 1396
Var. 1481
Var. 1566
Var. 1651
Var. 1736
Var. 1821
Var. 1906
Var. 1991



75 ± 25
Var. 1397
Var. 1482
Var. 1567
Var. 1652
Var. 1737
Var. 1822
Var. 1907
Var. 1992



80 ± 25
Var. 1398
Var. 1483
Var. 1568
Var. 1653
Var. 1738
Var. 1823
Var. 1908
Var. 1993



90 ± 25
Var. 1399
Var. 1484
Var. 1569
Var. 1654
Var. 1739
Var. 1824
Var. 1909
Var. 1994



100 ± 25 
Var. 1400
Var. 1485
Var. 1570
Var. 1655
Var. 1740
Var. 1825
Var. 1910
Var. 1995



125 ± 25 
Var. 1401
Var. 1486
Var. 1571
Var. 1656
Var. 1741
Var. 1826
Var. 1911
Var. 1996



150 ± 25 
Var. 1402
Var. 1487
Var. 1572
Var. 1657
Var. 1742
Var. 1827
Var. 1912
Var. 1997



175 ± 25 
Var. 1403
Var. 1488
Var. 1573
Var. 1658
Var. 1743
Var. 1828
Var. 1913
Var. 1998



30 ± 10
Var. 1404
Var. 1489
Var. 1574
Var. 1659
Var. 1744
Var. 1829
Var. 1914
Var. 1999



40 ± 10
Var. 1405
Var. 1490
Var. 1575
Var. 1660
Var. 1745
Var. 1830
Var. 1915
Var. 2000



50 ± 10
Var. 1406
Var. 1491
Var. 1576
Var. 1661
Var. 1746
Var. 1831
Var. 1916
Var. 2001



60 ± 10
Var. 1407
Var. 1492
Var. 1577
Var. 1662
Var. 1747
Var. 1832
Var. 1917
Var. 2002



70 ± 10
Var. 1408
Var. 1493
Var. 1578
Var. 1663
Var. 1748
Var. 1833
Var. 1918
Var. 2003



75 ± 10
Var. 1409
Var. 1494
Var. 1579
Var. 1664
Var. 1749
Var. 1834
Var. 1919
Var. 2004



80 ± 10
Var. 1410
Var. 1495
Var. 1580
Var. 1665
Var. 1750
Var. 1835
Var. 1920
Var. 2005



90 ± 10
Var. 1411
Var. 1496
Var. 1581
Var. 1666
Var. 1751
Var. 1836
Var. 1921
Var. 2006



100 ± 10 
Var. 1412
Var. 1497
Var. 1582
Var. 1667
Var. 1752
Var. 1837
Var. 1922
Var. 2007



110 ± 10 
Var. 1413
Var. 1498
Var. 1583
Var. 1668
Var. 1753
Var. 1838
Var. 1923
Var. 2008



120 ± 10 
Var. 1414
Var. 1499
Var. 1584
Var. 1669
Var. 1754
Var. 1839
Var. 1924
Var. 2009



125 ± 10 
Var. 1415
Var. 1500
Var. 1585
Var. 1670
Var. 1755
Var. 1840
Var. 1925
Var. 2010



130 ± 10 
Var. 1416
Var. 1501
Var. 1586
Var. 1671
Var. 1756
Var. 1841
Var. 1926
Var. 2011



140 ± 10 
Var. 1417
Var. 1502
Var. 1587
Var. 1672
Var. 1757
Var. 1842
Var. 1927
Var. 2012



150 ± 10 
Var. 1418
Var. 1503
Var. 1588
Var. 1673
Var. 1758
Var. 1843
Var. 1928
Var. 2013



160 ± 10 
Var. 1419
Var. 1504
Var. 1589
Var. 1674
Var. 1759
Var. 1844
Var. 1929
Var. 2014



170 ± 10 
Var. 1420
Var. 1505
Var. 1590
Var. 1675
Var. 1760
Var. 1845
Var. 1930
Var. 2015



175 ± 10 
Var. 1421
Var. 1506
Var. 1591
Var. 1676
Var. 1761
Var. 1846
Var. 1931
Var. 2016



180 ± 10 
Var. 1422
Var. 1507
Var. 1592
Var. 1677
Var. 1762
Var. 1847
Var. 1932
Var. 2017



190 ± 10 
Var. 1423
Var. 1508
Var. 1593
Var. 1678
Var. 1763
Var. 1848
Var. 1933
Var. 2018



25
Var. 1424
Var. 1509
Var. 1594
Var. 1679
Var. 1764
Var. 1849
Var. 1934
Var. 2019



30
Var. 1425
Var. 1510
Var. 1595
Var. 1680
Var. 1765
Var. 1850
Var. 1935
Var. 2020



40
Var. 1426
Var. 1511
Var. 1596
Var. 1681
Var. 1766
Var. 1851
Var. 1936
Var. 2021



50
Var. 1427
Var. 1512
Var. 1597
Var. 1682
Var. 1767
Var. 1852
Var. 1937
Var. 2022



60
Var. 1428
Var. 1513
Var. 1598
Var. 1683
Var. 1768
Var. 1853
Var. 1938
Var. 2023



70
Var. 1429
Var. 1514
Var. 1599
Var. 1684
Var. 1769
Var. 1854
Var. 1939
Var. 2024



75
Var. 1430
Var. 1515
Var. 1600
Var. 1685
Var. 1770
Var. 1855
Var. 1940
Var. 2025



80
Var. 1431
Var. 1516
Var. 1601
Var. 1686
Var. 1771
Var. 1856
Var. 1941
Var. 2026



90
Var. 1432
Var. 1517
Var. 1602
Var. 1687
Var. 1772
Var. 1857
Var. 1942
Var. 2027



100
Var. 1433
Var. 1518
Var. 1603
Var. 1688
Var. 1773
Var. 1858
Var. 1943
Var. 2028



110
Var. 1434
Var. 1519
Var. 1604
Var. 1689
Var. 1774
Var. 1859
Var. 1944
Var. 2029



120
Var. 1435
Var. 1520
Var. 1605
Var. 1690
Var. 1775
Var. 1860
Var. 1945
Var. 2030



125
Var. 1436
Var. 1521
Var. 1606
Var. 1691
Var. 1776
Var. 1861
Var. 1946
Var. 2031



130
Var. 1437
Var. 1522
Var. 1607
Var. 1692
Var. 1777
Var. 1862
Var. 1947
Var. 2032



140
Var. 1438
Var. 1523
Var. 1608
Var. 1693
Var. 1778
Var. 1863
Var. 1948
Var. 2033



150
Var. 1439
Var. 1524
Var. 1609
Var. 1694
Var. 1779
Var. 1864
Var. 1949
Var. 2034



160
Var. 1440
Var. 1525
Var. 1610
Var. 1695
Var. 1780
Var. 1865
Var. 1950
Var. 2035



170
Var. 1441
Var. 1526
Var. 1611
Var. 1696
Var. 1781
Var. 1866
Var. 1951
Var. 2036



175
Var. 1442
Var. 1527
Var. 1612
Var. 1697
Var. 1782
Var. 1867
Var. 1952
Var. 2037



180
Var. 1443
Var. 1528
Var. 1613
Var. 1698
Var. 1783
Var. 1868
Var. 1953
Var. 2038



190
Var. 1444
Var. 1529
Var. 1614
Var. 1699
Var. 1784
Var. 1869
Var. 1954
Var. 2039



200
Var. 1445
Var. 1530
Var. 1615
Var. 1700
Var. 1785
Var. 1870
Var. 1955
Var. 2040





Var. = Variation













TABLE 4







Exemplary embodiments for the combination of sugar or sugar alcohol and non-ionic


surfactant concentrations useful for the stabilization of furin (e.g., rfurin) compositions.









Sugar or Sugar Alcohol (%)
















8 ± 2%
9 ± 2%
10 ± 2%
11 ± 2%
12 ± 2%
13 ± 2%
14 ± 2%
15 ± 2%




















Non-Ionic
10-200
Var. 2041
Var. 2126
Var. 2211
Var. 2296
Var. 2381
Var. 2466
Var. 2551
Var. 2636


Surfactant
10-175
Var. 2042
Var. 2127
Var. 2212
Var. 2297
Var. 2382
Var. 2467
Var. 2552
Var. 2637


(ppm)
10-150
Var. 2043
Var. 2128
Var. 2213
Var. 2298
Var. 2383
Var. 2468
Var. 2553
Var. 2638



10-125
Var. 2044
Var. 2129
Var. 2214
Var. 2299
Var. 2384
Var. 2469
Var. 2554
Var. 2639



10-100
Var. 2045
Var. 2130
Var. 2215
Var. 2300
Var. 2385
Var. 2470
Var. 2555
Var. 2640



10-90 
Var. 2046
Var. 2131
Var. 2216
Var. 2301
Var. 2386
Var. 2471
Var. 2556
Var. 2641



10-80 
Var. 2047
Var. 2132
Var. 2217
Var. 2302
Var. 2387
Var. 2472
Var. 2557
Var. 2642



10-75 
Var. 2048
Var. 2133
Var. 2218
Var. 2303
Var. 2388
Var. 2473
Var. 2558
Var. 2643



10-70 
Var. 2049
Var. 2134
Var. 2219
Var. 2304
Var. 2389
Var. 2474
Var. 2559
Var. 2644



10-60 
Var. 2050
Var. 2135
Var. 2220
Var. 2305
Var. 2390
Var. 2475
Var. 2560
Var. 2645



10-50 
Var. 2051
Var. 2136
Var. 2221
Var. 2306
Var. 2391
Var. 2476
Var. 2561
Var. 2646



10-25 
Var. 2052
Var. 2137
Var. 2222
Var. 2307
Var. 2392
Var. 2477
Var. 2562
Var. 2647



25-200
Var. 2053
Var. 2138
Var. 2223
Var. 2308
Var. 2393
Var. 2478
Var. 2563
Var. 2648



25-175
Var. 2054
Var. 2139
Var. 2224
Var. 2309
Var. 2394
Var. 2479
Var. 2564
Var. 2649



25-150
Var. 2055
Var. 2140
Var. 2225
Var. 2310
Var. 2395
Var. 2480
Var. 2565
Var. 2650



25-125
Var. 2056
Var. 2141
Var. 2226
Var. 2311
Var. 2396
Var. 2481
Var. 2566
Var. 2651



25-100
Var. 2057
Var. 2142
Var. 2227
Var. 2312
Var. 2397
Var. 2482
Var. 2567
Var. 2652



25-90 
Var. 2058
Var. 2143
Var. 2228
Var. 2313
Var. 2398
Var. 2483
Var. 2568
Var. 2653



25-80 
Var. 2059
Var. 2144
Var. 2229
Var. 2314
Var. 2399
Var. 2484
Var. 2569
Var. 2654



25-70 
Var. 2060
Var. 2145
Var. 2230
Var. 2315
Var. 2400
Var. 2485
Var. 2570
Var. 2655



25-60 
Var. 2061
Var. 2146
Var. 2231
Var. 2316
Var. 2401
Var. 2486
Var. 2571
Var. 2656



25-50 
Var. 2062
Var. 2147
Var. 2232
Var. 2317
Var. 2402
Var. 2487
Var. 2572
Var. 2657



50-200
Var. 2063
Var. 2148
Var. 2233
Var. 2318
Var. 2403
Var. 2488
Var. 2573
Var. 2658



50-175
Var. 2064
Var. 2149
Var. 2234
Var. 2319
Var. 2404
Var. 2489
Var. 2574
Var. 2659



50-150
Var. 2065
Var. 2150
Var. 2235
Var. 2320
Var. 2405
Var. 2490
Var. 2575
Var. 2660



50-125
Var. 2066
Var. 2151
Var. 2236
Var. 2321
Var. 2406
Var. 2491
Var. 2576
Var. 2661



50-90 
Var. 2067
Var. 2152
Var. 2237
Var. 2322
Var. 2407
Var. 2492
Var. 2577
Var. 2662



50-80 
Var. 2068
Var. 2153
Var. 2238
Var. 2323
Var. 2408
Var. 2493
Var. 2578
Var. 2663



75-200
Var. 2069
Var. 2154
Var. 2239
Var. 2324
Var. 2409
Var. 2494
Var. 2579
Var. 2664



75-175
Var. 2070
Var. 2155
Var. 2240
Var. 2325
Var. 2410
Var. 2495
Var. 2580
Var. 2665



75-150
Var. 2071
Var. 2156
Var. 2241
Var. 2326
Var. 2411
Var. 2496
Var. 2581
Var. 2666



100-200 
Var. 2072
Var. 2157
Var. 2242
Var. 2327
Var. 2412
Var. 2497
Var. 2582
Var. 2667



100-175 
Var. 2073
Var. 2158
Var. 2243
Var. 2328
Var. 2413
Var. 2498
Var. 2583
Var. 2668



 50 ± 25
Var. 2074
Var. 2159
Var. 2244
Var. 2329
Var. 2414
Var. 2499
Var. 2584
Var. 2669



 60 ± 25
Var. 2075
Var. 2160
Var. 2245
Var. 2330
Var. 2415
Var. 2500
Var. 2585
Var. 2670



 70 ± 25
Var. 2076
Var. 2161
Var. 2246
Var. 2331
Var. 2416
Var. 2501
Var. 2586
Var. 2671



 75 ± 25
Var. 2077
Var. 2162
Var. 2247
Var. 2332
Var. 2417
Var. 2502
Var. 2587
Var. 2672



 80 ± 25
Var. 2078
Var. 2163
Var. 2248
Var. 2333
Var. 2418
Var. 2503
Var. 2588
Var. 2673



 90 ± 25
Var. 2079
Var. 2164
Var. 2249
Var. 2334
Var. 2419
Var. 2504
Var. 2589
Var. 2674



100 ± 25
Var. 2080
Var. 2165
Var. 2250
Var. 2335
Var. 2420
Var. 2505
Var. 2590
Var. 2675



125 ± 25
Var. 2081
Var. 2166
Var. 2251
Var. 2336
Var. 2421
Var. 2506
Var. 2591
Var. 2676



150 ± 25
Var. 2082
Var. 2167
Var. 2252
Var. 2337
Var. 2422
Var. 2507
Var. 2592
Var. 2677



175 ± 25
Var. 2083
Var. 2168
Var. 2253
Var. 2338
Var. 2423
Var. 2508
Var. 2593
Var. 2678



 30 ± 10
Var. 2084
Var. 2169
Var. 2254
Var. 2339
Var. 2424
Var. 2509
Var. 2594
Var. 2679



 40 ± 10
Var. 2085
Var. 2170
Var. 2255
Var. 2340
Var. 2425
Var. 2510
Var. 2595
Var. 2680



 50 ± 10
Var. 2086
Var. 2171
Var. 2256
Var. 2341
Var. 2426
Var. 2511
Var. 2596
Var. 2681



 60 ± 10
Var. 2087
Var. 2172
Var. 2257
Var. 2342
Var. 2427
Var. 2512
Var. 2597
Var. 2682



 70 ± 10
Var. 2088
Var. 2173
Var. 2258
Var. 2343
Var. 2428
Var. 2513
Var. 2598
Var. 2683



 75 ± 10
Var. 2089
Var. 2174
Var. 2259
Var. 2344
Var. 2429
Var. 2514
Var. 2599
Var. 2684



 80 ± 10
Var. 2090
Var. 2175
Var. 2260
Var. 2345
Var. 2430
Var. 2515
Var. 2600
Var. 2685



 90 ± 10
Var. 2091
Var. 2176
Var. 2261
Var. 2346
Var. 2431
Var. 2516
Var. 2601
Var. 2686



100 ± 10
Var. 2092
Var. 2177
Var. 2262
Var. 2347
Var. 2432
Var. 2517
Var. 2602
Var. 2687



110 ± 10
Var. 2093
Var. 2178
Var. 2263
Var. 2348
Var. 2433
Var. 2518
Var. 2603
Var. 2688



120 ± 10
Var. 2094
Var. 2179
Var. 2264
Var. 2349
Var. 2434
Var. 2519
Var. 2604
Var. 2689



125 ± 10
Var. 2095
Var. 2180
Var. 2265
Var. 2350
Var. 2435
Var. 2520
Var. 2605
Var. 2690



130 ± 10
Var. 2096
Var. 2181
Var. 2266
Var. 2351
Var. 2436
Var. 2521
Var. 2606
Var. 2691



140 ± 10
Var. 2097
Var. 2182
Var. 2267
Var. 2352
Var. 2437
Var. 2522
Var. 2607
Var. 2692



150 ± 10
Var. 2098
Var. 2183
Var. 2268
Var. 2353
Var. 2438
Var. 2523
Var. 2608
Var. 2693



160 ± 10
Var. 2099
Var. 2184
Var. 2269
Var. 2354
Var. 2439
Var. 2524
Var. 2609
Var. 2694



170 ± 10
Var. 2100
Var. 2185
Var. 2270
Var. 2355
Var. 2440
Var. 2525
Var. 2610
Var. 2695



175 ± 10
Var. 2101
Var. 2186
Var. 2271
Var. 2356
Var. 2441
Var. 2526
Var. 2611
Var. 2696



180 ± 10
Var. 2102
Var. 2187
Var. 2272
Var. 2357
Var. 2442
Var. 2527
Var. 2612
Var. 2697



190 ± 10
Var. 2103
Var. 2188
Var. 2273
Var. 2358
Var. 2443
Var. 2528
Var. 2613
Var. 2698



25
Var. 2104
Var. 2189
Var. 2274
Var. 2359
Var. 2444
Var. 2529
Var. 2614
Var. 2699



30
Var. 2105
Var. 2190
Var. 2275
Var. 2360
Var. 2445
Var. 2530
Var. 2615
Var. 2700



40
Var. 2106
Var. 2191
Var. 2276
Var. 2361
Var. 2446
Var. 2531
Var. 2616
Var. 2701



50
Var. 2107
Var. 2192
Var. 2277
Var. 2362
Var. 2447
Var. 2532
Var. 2617
Var. 2702



60
Var. 2108
Var. 2193
Var. 2278
Var. 2363
Var. 2448
Var. 2533
Var. 2618
Var. 2703



70
Var. 2109
Var. 2194
Var. 2279
Var. 2364
Var. 2449
Var. 2534
Var. 2619
Var. 2704



75
Var. 2110
Var. 2195
Var. 2280
Var. 2365
Var. 2450
Var. 2535
Var. 2620
Var. 2705



80
Var. 2111
Var. 2196
Var. 2281
Var. 2366
Var. 2451
Var. 2536
Var. 2621
Var. 2706



90
Var. 2112
Var. 2197
Var. 2282
Var. 2367
Var. 2452
Var. 2537
Var. 2622
Var. 2707



100
Var. 2113
Var. 2198
Var. 2283
Var. 2368
Var. 2453
Var. 2538
Var. 2623
Var. 2708



110
Var. 2114
Var. 2199
Var. 2284
Var. 2369
Var. 2454
Var. 2539
Var. 2624
Var. 2709



120
Var. 2115
Var. 2200
Var. 2285
Var. 2370
Var. 2455
Var. 2540
Var. 2625
Var. 2710



125
Var. 2116
Var. 2201
Var. 2286
Var. 2371
Var. 2456
Var. 2541
Var. 2626
Var. 2711



130
Var. 2117
Var. 2202
Var. 2287
Var. 2372
Var. 2457
Var. 2542
Var. 2627
Var. 2712



140
Var. 2118
Var. 2203
Var. 2288
Var. 2373
Var. 2458
Var. 2543
Var. 2628
Var. 2713



150
Var. 2119
Var. 2204
Var. 2289
Var. 2374
Var. 2459
Var. 2544
Var. 2629
Var. 2714



160
Var. 2120
Var. 2205
Var. 2290
Var. 2375
Var. 2460
Var. 2545
Var. 2630
Var. 2715



170
Var. 2121
Var. 2206
Var. 2291
Var. 2376
Var. 2461
Var. 2546
Var. 2631
Var. 2716



175
Var. 2122
Var. 2207
Var. 2292
Var. 2377
Var. 2462
Var. 2547
Var. 2632
Var. 2717



180
Var. 2123
Var. 2208
Var. 2293
Var. 2378
Var. 2463
Var. 2548
Var. 2633
Var. 2718



190
Var. 2124
Var. 2209
Var. 2294
Var. 2379
Var. 2464
Var. 2549
Var. 2634
Var. 2719



200
Var. 2125
Var. 2210
Var. 2295
Var. 2380
Var. 2465
Var. 2550
Var. 2635
Var. 2720





Var. = Variation













TABLE 5







Exemplary embodiments for the combination of sugar or sugar alcohol and non-ionic


surfactant concentrations useful for the stabilization of furin (e.g., rfurin) compositions.









Sugar or Sugar Alcohol (%)
















16 ± 2%
17 ± 2%
18 ± 2%
3 ± 1%
4 ± 1%
5 ± 1%
6 ± 1%
7 ± 1%




















Non-Ionic
10-200
Var. 2721
Var. 2806
Var. 2891
Var. 2976
Var. 3061
Var. 3146
Var. 3231
Var. 3316


Surfactant
10-175
Var. 2722
Var. 2807
Var. 2892
Var. 2977
Var. 3062
Var. 3147
Var. 3232
Var. 3317


(ppm)
10-150
Var. 2723
Var. 2808
Var. 2893
Var. 2978
Var. 3063
Var. 3148
Var. 3233
Var. 3318



10-125
Var. 2724
Var. 2809
Var. 2894
Var. 2979
Var. 3064
Var. 3149
Var. 3234
Var. 3319



10-100
Var. 2725
Var. 2810
Var. 2895
Var. 2980
Var. 3065
Var. 3150
Var. 3235
Var. 3320



10-90 
Var. 2726
Var. 2811
Var. 2896
Var. 2981
Var. 3066
Var. 3151
Var. 3236
Var. 3321



10-80 
Var. 2727
Var. 2812
Var. 2897
Var. 2982
Var. 3067
Var. 3152
Var. 3237
Var. 3322



10-75 
Var. 2728
Var. 2813
Var. 2898
Var. 2983
Var. 3068
Var. 3153
Var. 3238
Var. 3323



10-70 
Var. 2729
Var. 2814
Var. 2899
Var. 2984
Var. 3069
Var. 3154
Var. 3239
Var. 3324



10-60 
Var. 2730
Var. 2815
Var. 2900
Var. 2985
Var. 3070
Var. 3155
Var. 3240
Var. 3325



10-50 
Var. 2731
Var. 2816
Var. 2901
Var. 2986
Var. 3071
Var. 3156
Var. 3241
Var. 3326



10-25 
Var. 2732
Var. 2817
Var. 2902
Var. 2987
Var. 3072
Var. 3157
Var. 3242
Var. 3327



25-200
Var. 2733
Var. 2818
Var. 2903
Var. 2988
Var. 3073
Var. 3158
Var. 3243
Var. 3328



25-175
Var. 2734
Var. 2819
Var. 2904
Var. 2989
Var. 3074
Var. 3159
Var. 3244
Var. 3329



25-150
Var. 2735
Var. 2820
Var. 2905
Var. 2990
Var. 3075
Var. 3160
Var. 3245
Var. 3330



25-125
Var. 2736
Var. 2821
Var. 2906
Var. 2991
Var. 3076
Var. 3161
Var. 3246
Var. 3331



25-100
Var. 2737
Var. 2822
Var. 2907
Var. 2992
Var. 3077
Var. 3162
Var. 3247
Var. 3332



25-90 
Var. 2738
Var. 2823
Var. 2908
Var. 2993
Var. 3078
Var. 3163
Var. 3248
Var. 3333



25-80 
Var. 2739
Var. 2824
Var. 2909
Var. 2994
Var. 3079
Var. 3164
Var. 3249
Var. 3334



25-70 
Var. 2740
Var. 2825
Var. 2910
Var. 2995
Var. 3080
Var. 3165
Var. 3250
Var. 3335



25-60 
Var. 2741
Var. 2826
Var. 2911
Var. 2996
Var. 3081
Var. 3166
Var. 3251
Var. 3336



25-50 
Var. 2742
Var. 2827
Var. 2912
Var. 2997
Var. 3082
Var. 3167
Var. 3252
Var. 3337



50-200
Var. 2743
Var. 2828
Var. 2913
Var. 2998
Var. 3083
Var. 3168
Var. 3253
Var. 3338



50-175
Var. 2744
Var. 2829
Var. 2914
Var. 2999
Var. 3084
Var. 3169
Var. 3254
Var. 3339



50-150
Var. 2745
Var. 2830
Var. 2915
Var. 3000
Var. 3085
Var. 3170
Var. 3255
Var. 3340



50-125
Var. 2746
Var. 2831
Var. 2916
Var. 3001
Var. 3086
Var. 3171
Var. 3256
Var. 3341



50-90 
Var. 2747
Var. 2832
Var. 2917
Var. 3002
Var. 3087
Var. 3172
Var. 3257
Var. 3342



50-80 
Var. 2748
Var. 2833
Var. 2918
Var. 3003
Var. 3088
Var. 3173
Var. 3258
Var. 3343



75-200
Var. 2749
Var. 2834
Var. 2919
Var. 3004
Var. 3089
Var. 3174
Var. 3259
Var. 3344



75-175
Var. 2750
Var. 2835
Var. 2920
Var. 3005
Var. 3090
Var. 3175
Var. 3260
Var. 3345



75-150
Var. 2751
Var. 2836
Var. 2921
Var. 3006
Var. 3091
Var. 3176
Var. 3261
Var. 3346



100-200 
Var. 2752
Var. 2837
Var. 2922
Var. 3007
Var. 3092
Var. 3177
Var. 3262
Var. 3347



100-175 
Var. 2753
Var. 2838
Var. 2923
Var. 3008
Var. 3093
Var. 3178
Var. 3263
Var. 3348



 50 ± 25
Var. 2754
Var. 2839
Var. 2924
Var. 3009
Var. 3094
Var. 3179
Var. 3264
Var. 3349



 60 ± 25
Var. 2755
Var. 2840
Var. 2925
Var. 3010
Var. 3095
Var. 3180
Var. 3265
Var. 3350



 70 ± 25
Var. 2756
Var. 2841
Var. 2926
Var. 3011
Var. 3096
Var. 3181
Var. 3266
Var. 3351



 75 ± 25
Var. 2757
Var. 2842
Var. 2927
Var. 3012
Var. 3097
Var. 3182
Var. 3267
Var. 3352



 80 ± 25
Var. 2758
Var. 2843
Var. 2928
Var. 3013
Var. 3098
Var. 3183
Var. 3268
Var. 3353



 90 ± 25
Var. 2759
Var. 2844
Var. 2929
Var. 3014
Var. 3099
Var. 3184
Var. 3269
Var. 3354



100 ± 25
Var. 2760
Var. 2845
Var. 2930
Var. 3015
Var. 3100
Var. 3185
Var. 3270
Var. 3355



125 ± 25
Var. 2761
Var. 2846
Var. 2931
Var. 3016
Var. 3101
Var. 3186
Var. 3271
Var. 3356



150 ± 25
Var. 2762
Var. 2847
Var. 2932
Var. 3017
Var. 3102
Var. 3187
Var. 3272
Var. 3357



175 ± 25
Var. 2763
Var. 2848
Var. 2933
Var. 3018
Var. 3103
Var. 3188
Var. 3273
Var. 3358



 30 ± 10
Var. 2764
Var. 2849
Var. 2934
Var. 3019
Var. 3104
Var. 3189
Var. 3274
Var. 3359



 40 ± 10
Var. 2765
Var. 2850
Var. 2935
Var. 3020
Var. 3105
Var. 3190
Var. 3275
Var. 3360



 50 ± 10
Var. 2766
Var. 2851
Var. 2936
Var. 3021
Var. 3106
Var. 3191
Var. 3276
Var. 3361



 60 ± 10
Var. 2767
Var. 2852
Var. 2937
Var. 3022
Var. 3107
Var. 3192
Var. 3277
Var. 3362



 70 ± 10
Var. 2768
Var. 2853
Var. 2938
Var. 3023
Var. 3108
Var. 3193
Var. 3278
Var. 3363



 75 ± 10
Var. 2769
Var. 2854
Var. 2939
Var. 3024
Var. 3109
Var. 3194
Var. 3279
Var. 3364



 80 ± 10
Var. 2770
Var. 2855
Var. 2940
Var. 3025
Var. 3110
Var. 3195
Var. 3280
Var. 3365



 90 ± 10
Var. 2771
Var. 2856
Var. 2941
Var. 3026
Var. 3111
Var. 3196
Var. 3281
Var. 3366



100 ± 10
Var. 2772
Var. 2857
Var. 2942
Var. 3027
Var. 3112
Var. 3197
Var. 3282
Var. 3367



110 ± 10
Var. 2773
Var. 2858
Var. 2943
Var. 3028
Var. 3113
Var. 3198
Var. 3283
Var. 3368



120 ± 10
Var. 2774
Var. 2859
Var. 2944
Var. 3029
Var. 3114
Var. 3199
Var. 3284
Var. 3369



125 ± 10
Var. 2775
Var. 2860
Var. 2945
Var. 3030
Var. 3115
Var. 3200
Var. 3285
Var. 3370



130 ± 10
Var. 2776
Var. 2861
Var. 2946
Var. 3031
Var. 3116
Var. 3201
Var. 3286
Var. 3371



140 ± 10
Var. 2777
Var. 2862
Var. 2947
Var. 3032
Var. 3117
Var. 3202
Var. 3287
Var. 3372



150 ± 10
Var. 2778
Var. 2863
Var. 2948
Var. 3033
Var. 3118
Var. 3203
Var. 3288
Var. 3373



160 ± 10
Var. 2779
Var. 2864
Var. 2949
Var. 3034
Var. 3119
Var. 3204
Var. 3289
Var. 3374



170 ± 10
Var. 2780
Var. 2865
Var. 2950
Var. 3035
Var. 3120
Var. 3205
Var. 3290
Var. 3375



175 ± 10
Var. 2781
Var. 2866
Var. 2951
Var. 3036
Var. 3121
Var. 3206
Var. 3291
Var. 3376



180 ± 10
Var. 2782
Var. 2867
Var. 2952
Var. 3037
Var. 3122
Var. 3207
Var. 3292
Var. 3377



190 ± 10
Var. 2783
Var. 2868
Var. 2953
Var. 3038
Var. 3123
Var. 3208
Var. 3293
Var. 3378



25
Var. 2784
Var. 2869
Var. 2954
Var. 3039
Var. 3124
Var. 3209
Var. 3294
Var. 3379



30
Var. 2785
Var. 2870
Var. 2955
Var. 3040
Var. 3125
Var. 3210
Var. 3295
Var. 3380



40
Var. 2786
Var. 2871
Var. 2956
Var. 3041
Var. 3126
Var. 3211
Var. 3296
Var. 3381



50
Var. 2787
Var. 2872
Var. 2957
Var. 3042
Var. 3127
Var. 3212
Var. 3297
Var. 3382



60
Var. 2788
Var. 2873
Var. 2958
Var. 3043
Var. 3128
Var. 3213
Var. 3298
Var. 3383



70
Var. 2789
Var. 2874
Var. 2959
Var. 3044
Var. 3129
Var. 3214
Var. 3299
Var. 3384



75
Var. 2790
Var. 2875
Var. 2960
Var. 3045
Var. 3130
Var. 3215
Var. 3300
Var. 3385



80
Var. 2791
Var. 2876
Var. 2961
Var. 3046
Var. 3131
Var. 3216
Var. 3301
Var. 3386



90
Var. 2792
Var. 2877
Var. 2962
Var. 3047
Var. 3132
Var. 3217
Var. 3302
Var. 3387



100
Var. 2793
Var. 2878
Var. 2963
Var. 3048
Var. 3133
Var. 3218
Var. 3303
Var. 3388



110
Var. 2794
Var. 2879
Var. 2964
Var. 3049
Var. 3134
Var. 3219
Var. 3304
Var. 3389



120
Var. 2795
Var. 2880
Var. 2965
Var. 3050
Var. 3135
Var. 3220
Var. 3305
Var. 3390



125
Var. 2796
Var. 2881
Var. 2966
Var. 3051
Var. 3136
Var. 3221
Var. 3306
Var. 3391



130
Var. 2797
Var. 2882
Var. 2967
Var. 3052
Var. 3137
Var. 3222
Var. 3307
Var. 3392



140
Var. 2798
Var. 2883
Var. 2968
Var. 3053
Var. 3138
Var. 3223
Var. 3308
Var. 3393



150
Var. 2799
Var. 2884
Var. 2969
Var. 3054
Var. 3139
Var. 3224
Var. 3309
Var. 3394



160
Var. 2800
Var. 2885
Var. 2970
Var. 3055
Var. 3140
Var. 3225
Var. 3310
Var. 3395



170
Var. 2801
Var. 2886
Var. 2971
Var. 3056
Var. 3141
Var. 3226
Var. 3311
Var. 3396



175
Var. 2802
Var. 2887
Var. 2972
Var. 3057
Var. 3142
Var. 3227
Var. 3312
Var. 3397



180
Var. 2803
Var. 2888
Var. 2973
Var. 3058
Var. 3143
Var. 3228
Var. 3313
Var. 3398



190
Var. 2804
Var. 2889
Var. 2974
Var. 3059
Var. 3144
Var. 3229
Var. 3314
Var. 3399



200
Var. 2805
Var. 2890
Var. 2975
Var. 3060
Var. 3145
Var. 3230
Var. 3315
Var. 3400





Var. = Variation













TABLE 6







Exemplary embodiments for the combination of sugar or sugar alcohol and non-ionic


surfactant concentrations useful for the stabilization of furin (e.g., rfurin) compositions.









Sugar or Sugar Alcohol (%)
















8 ± 1%
9 ± 1%
10 ± 1%
11 ± 1%
12 ± 1%
13 ± 1%
14 ± 1%
15 ± 1%




















Non-Ionic
10-200
Var. 3401
Var. 3486
Var. 3571
Var. 3656
Var. 3741
Var. 3826
Var. 3911
Var. 3996


Surfactant
10-175
Var. 3402
Var. 3487
Var. 3572
Var. 3657
Var. 3742
Var. 3827
Var. 3912
Var. 3997


(ppm)
10-150
Var. 3403
Var. 3488
Var. 3573
Var. 3658
Var. 3743
Var. 3828
Var. 3913
Var. 3998



10-125
Var. 3404
Var. 3489
Var. 3574
Var. 3659
Var. 3744
Var. 3829
Var. 3914
Var. 3999



10-100
Var. 3405
Var. 3490
Var. 3575
Var. 3660
Var. 3745
Var. 3830
Var. 3915
Var. 4000



10-90 
Var. 3406
Var. 3491
Var. 3576
Var. 3661
Var. 3746
Var. 3831
Var. 3916
Var. 4001



10-80 
Var. 3407
Var. 3492
Var. 3577
Var. 3662
Var. 3747
Var. 3832
Var. 3917
Var. 4002



10-75 
Var. 3408
Var. 3493
Var. 3578
Var. 3663
Var. 3748
Var. 3833
Var. 3918
Var. 4003



10-70 
Var. 3409
Var. 3494
Var. 3579
Var. 3664
Var. 3749
Var. 3834
Var. 3919
Var. 4004



10-60 
Var. 3410
Var. 3495
Var. 3580
Var. 3665
Var. 3750
Var. 3835
Var. 3920
Var. 4005



10-50 
Var. 3411
Var. 3496
Var. 3581
Var. 3666
Var. 3751
Var. 3836
Var. 3921
Var. 4006



10-25 
Var. 3412
Var. 3497
Var. 3582
Var. 3667
Var. 3752
Var. 3837
Var. 3922
Var. 4007



25-200
Var. 3413
Var. 3498
Var. 3583
Var. 3668
Var. 3753
Var. 3838
Var. 3923
Var. 4008



25-175
Var. 3414
Var. 3499
Var. 3584
Var. 3669
Var. 3754
Var. 3839
Var. 3924
Var. 4009



25-150
Var. 3415
Var. 3500
Var. 3585
Var. 3670
Var. 3755
Var. 3840
Var. 3925
Var. 4010



25-125
Var. 3416
Var. 3501
Var. 3586
Var. 3671
Var. 3756
Var. 3841
Var. 3926
Var. 4011



25-100
Var. 3417
Var. 3502
Var. 3587
Var. 3672
Var. 3757
Var. 3842
Var. 3927
Var. 4012



25-90 
Var. 3418
Var. 3503
Var. 3588
Var. 3673
Var. 3758
Var. 3843
Var. 3928
Var. 4013



25-80 
Var. 3419
Var. 3504
Var. 3589
Var. 3674
Var. 3759
Var. 3844
Var. 3929
Var. 4014



25-70 
Var. 3420
Var. 3505
Var. 3590
Var. 3675
Var. 3760
Var. 3845
Var. 3930
Var. 4015



25-60 
Var. 3421
Var. 3506
Var. 3591
Var. 3676
Var. 3761
Var. 3846
Var. 3931
Var. 4016



25-50 
Var. 3422
Var. 3507
Var. 3592
Var. 3677
Var. 3762
Var. 3847
Var. 3932
Var. 4017



50-200
Var. 3423
Var. 3508
Var. 3593
Var. 3678
Var. 3763
Var. 3848
Var. 3933
Var. 4018



50-175
Var. 3424
Var. 3509
Var. 3594
Var. 3679
Var. 3764
Var. 3849
Var. 3934
Var. 4019



50-150
Var. 3425
Var. 3510
Var. 3595
Var. 3680
Var. 3765
Var. 3850
Var. 3935
Var. 4020



50-125
Var. 3426
Var. 3511
Var. 3596
Var. 3681
Var. 3766
Var. 3851
Var. 3936
Var. 4021



50-90 
Var. 3427
Var. 3512
Var. 3597
Var. 3682
Var. 3767
Var. 3852
Var. 3937
Var. 4022



50-80 
Var. 3428
Var. 3513
Var. 3598
Var. 3683
Var. 3768
Var. 3853
Var. 3938
Var. 4023



75-200
Var. 3429
Var. 3514
Var. 3599
Var. 3684
Var. 3769
Var. 3854
Var. 3939
Var. 4024



75-175
Var. 3430
Var. 3515
Var. 3600
Var. 3685
Var. 3770
Var. 3855
Var. 3940
Var. 4025



75-150
Var. 3431
Var. 3516
Var. 3601
Var. 3686
Var. 3771
Var. 3856
Var. 3941
Var. 4026



100-200 
Var. 3432
Var. 3517
Var. 3602
Var. 3687
Var. 3772
Var. 3857
Var. 3942
Var. 4027



100-175 
Var. 3433
Var. 3518
Var. 3603
Var. 3688
Var. 3773
Var. 3858
Var. 3943
Var. 4028



 50 ± 25
Var. 3434
Var. 3519
Var. 3604
Var. 3689
Var. 3774
Var. 3859
Var. 3944
Var. 4029



 60 ± 25
Var. 3435
Var. 3520
Var. 3605
Var. 3690
Var. 3775
Var. 3860
Var. 3945
Var. 4030



 70 ± 25
Var. 3436
Var. 3521
Var. 3606
Var. 3691
Var. 3776
Var. 3861
Var. 3946
Var. 4031



 75 ± 25
Var. 3437
Var. 3522
Var. 3607
Var. 3692
Var. 3777
Var. 3862
Var. 3947
Var. 4032



 80 ± 25
Var. 3438
Var. 3523
Var. 3608
Var. 3693
Var. 3778
Var. 3863
Var. 3948
Var. 4033



 90 ± 25
Var. 3439
Var. 3524
Var. 3609
Var. 3694
Var. 3779
Var. 3864
Var. 3949
Var. 4034



100 ± 25
Var. 3440
Var. 3525
Var. 3610
Var. 3695
Var. 3780
Var. 3865
Var. 3950
Var. 4035



125 ± 25
Var. 3441
Var. 3526
Var. 3611
Var. 3696
Var. 3781
Var. 3866
Var. 3951
Var. 4036



150 ± 25
Var. 3442
Var. 3527
Var. 3612
Var. 3697
Var. 3782
Var. 3867
Var. 3952
Var. 4037



175 ± 25
Var. 3443
Var. 3528
Var. 3613
Var. 3698
Var. 3783
Var. 3868
Var. 3953
Var. 4038



 30 ± 10
Var. 3444
Var. 3529
Var. 3614
Var. 3699
Var. 3784
Var. 3869
Var. 3954
Var. 4039



 40 ± 10
Var. 3445
Var. 3530
Var. 3615
Var. 3700
Var. 3785
Var. 3870
Var. 3955
Var. 4040



 50 ± 10
Var. 3446
Var. 3531
Var. 3616
Var. 3701
Var. 3786
Var. 3871
Var. 3956
Var. 4041



 60 ± 10
Var. 3447
Var. 3532
Var. 3617
Var. 3702
Var. 3787
Var. 3872
Var. 3957
Var. 4042



 70 ± 10
Var. 3448
Var. 3533
Var. 3618
Var. 3703
Var. 3788
Var. 3873
Var. 3958
Var. 4043



 75 ± 10
Var. 3449
Var. 3534
Var. 3619
Var. 3704
Var. 3789
Var. 3874
Var. 3959
Var. 4044



 80 ± 10
Var. 3450
Var. 3535
Var. 3620
Var. 3705
Var. 3790
Var. 3875
Var. 3960
Var. 4045



 90 ± 10
Var. 3451
Var. 3536
Var. 3621
Var. 3706
Var. 3791
Var. 3876
Var. 3961
Var. 4046



100 ± 10
Var. 3452
Var. 3537
Var. 3622
Var. 3707
Var. 3792
Var. 3877
Var. 3962
Var. 4047



110 ± 10
Var. 3453
Var. 3538
Var. 3623
Var. 3708
Var. 3793
Var. 3878
Var. 3963
Var. 4048



120 ± 10
Var. 3454
Var. 3539
Var. 3624
Var. 3709
Var. 3794
Var. 3879
Var. 3964
Var. 4049



125 ± 10
Var. 3455
Var. 3540
Var. 3625
Var. 3710
Var. 3795
Var. 3880
Var. 3965
Var. 4050



130 ± 10
Var. 3456
Var. 3541
Var. 3626
Var. 3711
Var. 3796
Var. 3881
Var. 3966
Var. 4051



140 ± 10
Var. 3457
Var. 3542
Var. 3627
Var. 3712
Var. 3797
Var. 3882
Var. 3967
Var. 4052



150 ± 10
Var. 3458
Var. 3543
Var. 3628
Var. 3713
Var. 3798
Var. 3883
Var. 3968
Var. 4053



160 ± 10
Var. 3459
Var. 3544
Var. 3629
Var. 3714
Var. 3799
Var. 3884
Var. 3969
Var. 4054



170 ± 10
Var. 3460
Var. 3545
Var. 3630
Var. 3715
Var. 3800
Var. 3885
Var. 3970
Var. 4055



175 ± 10
Var. 3461
Var. 3546
Var. 3631
Var. 3716
Var. 3801
Var. 3886
Var. 3971
Var. 4056



180 ± 10
Var. 3462
Var. 3547
Var. 3632
Var. 3717
Var. 3802
Var. 3887
Var. 3972
Var. 4057



190 ± 10
Var. 3463
Var. 3548
Var. 3633
Var. 3718
Var. 3803
Var. 3888
Var. 3973
Var. 4058



25
Var. 3464
Var. 3549
Var. 3634
Var. 3719
Var. 3804
Var. 3889
Var. 3974
Var. 4059



30
Var. 3465
Var. 3550
Var. 3635
Var. 3720
Var. 3805
Var. 3890
Var. 3975
Var. 4060



40
Var. 3466
Var. 3551
Var. 3636
Var. 3721
Var. 3806
Var. 3891
Var. 3976
Var. 4061



50
Var. 3467
Var. 3552
Var. 3637
Var. 3722
Var. 3807
Var. 3892
Var. 3977
Var. 4062



60
Var. 3468
Var. 3553
Var. 3638
Var. 3723
Var. 3808
Var. 3893
Var. 3978
Var. 4063



70
Var. 3469
Var. 3554
Var. 3639
Var. 3724
Var. 3809
Var. 3894
Var. 3979
Var. 4064



75
Var. 3470
Var. 3555
Var. 3640
Var. 3725
Var. 3810
Var. 3895
Var. 3980
Var. 4065



80
Var. 3471
Var. 3556
Var. 3641
Var. 3726
Var. 3811
Var. 3896
Var. 3981
Var. 4066



90
Var. 3472
Var. 3557
Var. 3642
Var. 3727
Var. 3812
Var. 3897
Var. 3982
Var. 4067



100
Var. 3473
Var. 3558
Var. 3643
Var. 3728
Var. 3813
Var. 3898
Var. 3983
Var. 4068



110
Var. 3474
Var. 3559
Var. 3644
Var. 3729
Var. 3814
Var. 3899
Var. 3984
Var. 4069



120
Var. 3475
Var. 3560
Var. 3645
Var. 3730
Var. 3815
Var. 3900
Var. 3985
Var. 4070



125
Var. 3476
Var. 3561
Var. 3646
Var. 3731
Var. 3816
Var. 3901
Var. 3986
Var. 4071



130
Var. 3477
Var. 3562
Var. 3647
Var. 3732
Var. 3817
Var. 3902
Var. 3987
Var. 4072



140
Var. 3478
Var. 3563
Var. 3648
Var. 3733
Var. 3818
Var. 3903
Var. 3988
Var. 4073



150
Var. 3479
Var. 3564
Var. 3649
Var. 3734
Var. 3819
Var. 3904
Var. 3989
Var. 4074



160
Var. 3480
Var. 3565
Var. 3650
Var. 3735
Var. 3820
Var. 3905
Var. 3990
Var. 4075



170
Var. 3481
Var. 3566
Var. 3651
Var. 3736
Var. 3821
Var. 3906
Var. 3991
Var. 4076



175
Var. 3482
Var. 3567
Var. 3652
Var. 3737
Var. 3822
Var. 3907
Var. 3992
Var. 4077



180
Var. 3483
Var. 3568
Var. 3653
Var. 3738
Var. 3823
Var. 3908
Var. 3993
Var. 4078



190
Var. 3484
Var. 3569
Var. 3654
Var. 3739
Var. 3824
Var. 3909
Var. 3994
Var. 4079



200
Var. 3485
Var. 3570
Var. 3655
Var. 3740
Var. 3825
Var. 3910
Var. 3995
Var. 4080





Var. = Variation













TABLE 7







Exemplary embodiments for the combination of sugar or sugar alcohol and non-ionic


surfactant concentrations useful for the stabilization of furin (e.g., rfurin) compositions.









Sugar or Sugar Alcohol (%)
















16 ± 1%
17 ± 1%
18 ± 1%
19 ± 1%
2%
3%
4%
5%




















Non-Ionic
10-200
Var. 4081
Var. 4166
Var. 4251
Var. 4336
Var. 4421
Var. 4506
Var. 4591
Var. 4676


Surfactant 
10-175
Var. 4082
Var. 4167
Var. 4252
Var. 4337
Var. 4422
Var. 4507
Var. 4592
Var. 4677


(ppm)
10-150
Var. 4083
Var. 4168
Var. 4253
Var. 4338
Var. 4423
Var. 4508
Var. 4593
Var. 4678



10-125
Var. 4084
Var. 4169
Var. 4254
Var. 4339
Var. 4424
Var. 4509
Var. 4594
Var. 4679



10-100
Var. 4085
Var. 4170
Var. 4255
Var. 4340
Var. 4425
Var. 4510
Var. 4595
Var. 4680



10-90 
Var. 4086
Var. 4171
Var. 4256
Var. 4341
Var. 4426
Var. 4511
Var. 4596
Var. 4681



10-80 
Var. 4087
Var. 4172
Var. 4257
Var. 4342
Var. 4427
Var. 4512
Var. 4597
Var. 4682



10-75 
Var. 4088
Var. 4173
Var. 4258
Var. 4343
Var. 4428
Var. 4513
Var. 4598
Var. 4683



10-70 
Var. 4089
Var. 4174
Var. 4259
Var. 4344
Var. 4429
Var. 4514
Var. 4599
Var. 4684



10-60 
Var. 4090
Var. 4175
Var. 4260
Var. 4345
Var. 4430
Var. 4515
Var. 4600
Var. 4685



10-50 
Var. 4091
Var. 4176
Var. 4261
Var. 4346
Var. 4431
Var. 4516
Var. 4601
Var. 4686



10-25 
Var. 4092
Var. 4177
Var. 4262
Var. 4347
Var. 4432
Var. 4517
Var. 4602
Var. 4687



25-200
Var. 4093
Var. 4178
Var. 4263
Var. 4348
Var. 4433
Var. 4518
Var. 4603
Var. 4688



25-175
Var. 4094
Var. 4179
Var. 4264
Var. 4349
Var. 4434
Var. 4519
Var. 4604
Var. 4689



25-150
Var. 4095
Var. 4180
Var. 4265
Var. 4350
Var. 4435
Var. 4520
Var. 4605
Var. 4690



25-125
Var. 4096
Var. 4181
Var. 4266
Var. 4351
Var. 4436
Var. 4521
Var. 4606
Var. 4691



25-100
Var. 4097
Var. 4182
Var. 4267
Var. 4352
Var. 4437
Var. 4522
Var. 4607
Var. 4692



25-90 
Var. 4098
Var. 4183
Var. 4268
Var. 4353
Var. 4438
Var. 4523
Var. 4608
Var. 4693



25-80 
Var. 4099
Var. 4184
Var. 4269
Var. 4354
Var. 4439
Var. 4524
Var. 4609
Var. 4694



25-70 
Var. 4100
Var. 4185
Var. 4270
Var. 4355
Var. 4440
Var. 4525
Var. 4610
Var. 4695



25-60 
Var. 4101
Var. 4186
Var. 4271
Var. 4356
Var. 4441
Var. 4526
Var. 4611
Var. 4696



25-50 
Var. 4102
Var. 4187
Var. 4272
Var. 4357
Var. 4442
Var. 4527
Var. 4612
Var. 4697



50-200
Var. 4103
Var. 4188
Var. 4273
Var. 4358
Var. 4443
Var. 4528
Var. 4613
Var. 4698



50-175
Var. 4104
Var. 4189
Var. 4274
Var. 4359
Var. 4444
Var. 4529
Var. 4614
Var. 4699



50-150
Var. 4105
Var. 4190
Var. 4275
Var. 4360
Var. 4445
Var. 4530
Var. 4615
Var. 4700



50-125
Var. 4106
Var. 4191
Var. 4276
Var. 4361
Var. 4446
Var. 4531
Var. 4616
Var. 4701



50-90 
Var. 4107
Var. 4192
Var. 4277
Var. 4362
Var. 4447
Var. 4532
Var. 4617
Var. 4702



50-80 
Var. 4108
Var. 4193
Var. 4278
Var. 4363
Var. 4448
Var. 4533
Var. 4618
Var. 4703



75-200
Var. 4109
Var. 4194
Var. 4279
Var. 4364
Var. 4449
Var. 4534
Var. 4619
Var. 4704



75-175
Var. 4110
Var. 4195
Var. 4280
Var. 4365
Var. 4450
Var. 4535
Var. 4620
Var. 4705



75-150
Var. 4111
Var. 4196
Var. 4281
Var. 4366
Var. 4451
Var. 4536
Var. 4621
Var. 4706



100-200 
Var. 4112
Var. 4197
Var. 4282
Var. 4367
Var. 4452
Var. 4537
Var. 4622
Var. 4707



100-175 
Var. 4113
Var. 4198
Var. 4283
Var. 4368
Var. 4453
Var. 4538
Var. 4623
Var. 4708



 50 ± 25
Var. 4114
Var. 4199
Var. 4284
Var. 4369
Var. 4454
Var. 4539
Var. 4624
Var. 4709



 60 ± 25
Var. 4115
Var. 4200
Var. 4285
Var. 4370
Var. 4455
Var. 4540
Var. 4625
Var. 4710



 70 ± 25
Var. 4116
Var. 4201
Var. 4286
Var. 4371
Var. 4456
Var. 4541
Var. 4626
Var. 4711



 75 ± 25
Var. 4117
Var. 4202
Var. 4287
Var. 4372
Var. 4457
Var. 4542
Var. 4627
Var. 4712



 80 ± 25
Var. 4118
Var. 4203
Var. 4288
Var. 4373
Var. 4458
Var. 4543
Var. 4628
Var. 4713



 90 ± 25
Var. 4119
Var. 4204
Var. 4289
Var. 4374
Var. 4459
Var. 4544
Var. 4629
Var. 4714



100 ± 25
Var. 4120
Var. 4205
Var. 4290
Var. 4375
Var. 4460
Var. 4545
Var. 4630
Var. 4715



125 ± 25
Var. 4121
Var. 4206
Var. 4291
Var. 4376
Var. 4461
Var. 4546
Var. 4631
Var. 4716



150 ± 25
Var. 4122
Var. 4207
Var. 4292
Var. 4377
Var. 4462
Var. 4547
Var. 4632
Var. 4717



175 ± 25
Var. 4123
Var. 4208
Var. 4293
Var. 4378
Var. 4463
Var. 4548
Var. 4633
Var. 4718



 30 ± 10
Var. 4124
Var. 4209
Var. 4294
Var. 4379
Var. 4464
Var. 4549
Var. 4634
Var. 4719



 40 ± 10
Var. 4125
Var. 4210
Var. 4295
Var. 4380
Var. 4465
Var. 4550
Var. 4635
Var. 4720



 50 ± 10
Var. 4126
Var. 4211
Var. 4296
Var. 4381
Var. 4466
Var. 4551
Var. 4636
Var. 4721



 60 ± 10
Var. 4127
Var. 4212
Var. 4297
Var. 4382
Var. 4467
Var. 4552
Var. 4637
Var. 4722



 70 ± 10
Var. 4128
Var. 4213
Var. 4298
Var. 4383
Var. 4468
Var. 4553
Var. 4638
Var. 4723



 75 ± 10
Var. 4129
Var. 4214
Var. 4299
Var. 4384
Var. 4469
Var. 4554
Var. 4639
Var. 4724



 80 ± 10
Var. 4130
Var. 4215
Var. 4300
Var. 4385
Var. 4470
Var. 4555
Var. 4640
Var. 4725



 90 ± 10
Var. 4131
Var. 4216
Var. 4301
Var. 4386
Var. 4471
Var. 4556
Var. 4641
Var. 4726



100 ± 10
Var. 4132
Var. 4217
Var. 4302
Var. 4387
Var. 4472
Var. 4557
Var. 4642
Var. 4727



110 ± 10
Var. 4133
Var. 4218
Var. 4303
Var. 4388
Var. 4473
Var. 4558
Var. 4643
Var. 4728



120 ± 10
Var. 4134
Var. 4219
Var. 4304
Var. 4389
Var. 4474
Var. 4559
Var. 4644
Var. 4729



125 ± 10
Var. 4135
Var. 4220
Var. 4305
Var. 4390
Var. 4475
Var. 4560
Var. 4645
Var. 4730



130 ± 10
Var. 4136
Var. 4221
Var. 4306
Var. 4391
Var. 4476
Var. 4561
Var. 4646
Var. 4731



140 ± 10
Var. 4137
Var. 4222
Var. 4307
Var. 4392
Var. 4477
Var. 4562
Var. 4647
Var. 4732



150 ± 10
Var. 4138
Var. 4223
Var. 4308
Var. 4393
Var. 4478
Var. 4563
Var. 4648
Var. 4733



160 ± 10
Var. 4139
Var. 4224
Var. 4309
Var. 4394
Var. 4479
Var. 4564
Var. 4649
Var. 4734



170 ± 10
Var. 4140
Var. 4225
Var. 4310
Var. 4395
Var. 4480
Var. 4565
Var. 4650
Var. 4735



175 ± 10
Var. 4141
Var. 4226
Var. 4311
Var. 4396
Var. 4481
Var. 4566
Var. 4651
Var. 4736



180 ± 10
Var. 4142
Var. 4227
Var. 4312
Var. 4397
Var. 4482
Var. 4567
Var. 4652
Var. 4737



190 ± 10
Var. 4143
Var. 4228
Var. 4313
Var. 4398
Var. 4483
Var. 4568
Var. 4653
Var. 4738



25
Var. 4144
Var. 4229
Var. 4314
Var. 4399
Var. 4484
Var. 4569
Var. 4654
Var. 4739



30
Var. 4145
Var. 4230
Var. 4315
Var. 4400
Var. 4485
Var. 4570
Var. 4655
Var. 4740



40
Var. 4146
Var. 4231
Var. 4316
Var. 4401
Var. 4486
Var. 4571
Var. 4656
Var. 4741



50
Var. 4147
Var. 4232
Var. 4317
Var. 4402
Var. 4487
Var. 4572
Var. 4657
Var. 4742



60
Var. 4148
Var. 4233
Var. 4318
Var. 4403
Var. 4488
Var. 4573
Var. 4658
Var. 4743



70
Var. 4149
Var. 4234
Var. 4319
Var. 4404
Var. 4489
Var. 4574
Var. 4659
Var. 4744



75
Var. 4150
Var. 4235
Var. 4320
Var. 4405
Var. 4490
Var. 4575
Var. 4660
Var. 4745



80
Var. 4151
Var. 4236
Var. 4321
Var. 4406
Var. 4491
Var. 4576
Var. 4661
Var. 4746



90
Var. 4152
Var. 4237
Var. 4322
Var. 4407
Var. 4492
Var. 4577
Var. 4662
Var. 4747



100
Var. 4153
Var. 4238
Var. 4323
Var. 4408
Var. 4493
Var. 4578
Var. 4663
Var. 4748



110
Var. 4154
Var. 4239
Var. 4324
Var. 4409
Var. 4494
Var. 4579
Var. 4664
Var. 4749



120
Var. 4155
Var. 4240
Var. 4325
Var. 4410
Var. 4495
Var. 4580
Var. 4665
Var. 4750



125
Var. 4156
Var. 4241
Var. 4326
Var. 4411
Var. 4496
Var. 4581
Var. 4666
Var. 4751



130
Var. 4157
Var. 4242
Var. 4327
Var. 4412
Var. 4497
Var. 4582
Var. 4667
Var. 4752



140
Var. 4158
Var. 4243
Var. 4328
Var. 4413
Var. 4498
Var. 4583
Var. 4668
Var. 4753



150
Var. 4159
Var. 4244
Var. 4329
Var. 4414
Var. 4499
Var. 4584
Var. 4669
Var. 4754



160
Var. 4160
Var. 4245
Var. 4330
Var. 4415
Var. 4500
Var. 4585
Var. 4670
Var. 4755



170
Var. 4161
Var. 4246
Var. 4331
Var. 4416
Var. 4501
Var. 4586
Var. 4671
Var. 4756



175
Var. 4162
Var. 4247
Var. 4332
Var. 4417
Var. 4502
Var. 4587
Var. 4672
Var. 4757



180
Var. 4163
Var. 4248
Var. 4333
Var. 4418
Var. 4503
Var. 4588
Var. 4673
Var. 4758



190
Var. 4164
Var. 4249
Var. 4334
Var. 4419
Var. 4504
Var. 4589
Var. 4674
Var. 4759



200
Var. 4165
Var. 4250
Var. 4335
Var. 4420
Var. 4505
Var. 4590
Var. 4675
Var. 4760





Var. = Variation













TABLE 8







Exemplary embodiments for the combination of sugar or sugar alcohol and non-ionic


surfactant concentrations useful for the stabilization of furin (e.g., rfurin) compositions.









Sugar or Sugar Alcohol (%)
















6%
7%
8%
9%
10%
11%
12%
13%




















Non-Ionic
10-200
Var. 4761
Var. 4846
Var. 4931
Var. 5016
Var. 5101
Var. 5186
Var. 5271
Var. 5356


Surfactant
10-175
Var. 4762
Var. 4847
Var. 4932
Var. 5017
Var. 5102
Var. 5187
Var. 5272
Var. 5357


(ppm)
10-150
Var. 4763
Var. 4848
Var. 4933
Var. 5018
Var. 5103
Var. 5188
Var. 5273
Var. 5358



10-125
Var. 4764
Var. 4849
Var. 4934
Var. 5019
Var. 5104
Var. 5189
Var. 5274
Var. 5359



10-100
Var. 4765
Var. 4850
Var. 4935
Var. 5020
Var. 5105
Var. 5190
Var. 5275
Var. 5360



10-90 
Var. 4766
Var. 4851
Var. 4936
Var. 5021
Var. 5106
Var. 5191
Var. 5276
Var. 5361



10-80 
Var. 4767
Var. 4852
Var. 4937
Var. 5022
Var. 5107
Var. 5192
Var. 5277
Var. 5362



10-75 
Var. 4768
Var. 4853
Var. 4938
Var. 5023
Var. 5108
Var. 5193
Var. 5278
Var. 5363



10-70 
Var. 4769
Var. 4854
Var. 4939
Var. 5024
Var. 5109
Var. 5194
Var. 5279
Var. 5364



10-60 
Var. 4770
Var. 4855
Var. 4940
Var. 5025
Var. 5110
Var. 5195
Var. 5280
Var. 5365



10-50 
Var. 4771
Var. 4856
Var. 4941
Var. 5026
Var. 5111
Var. 5196
Var. 5281
Var. 5366



10-25 
Var. 4772
Var. 4857
Var. 4942
Var. 5027
Var. 5112
Var. 5197
Var. 5282
Var. 5367



25-200
Var. 4773
Var. 4858
Var. 4943
Var. 5028
Var. 5113
Var. 5198
Var. 5283
Var. 5368



25-175
Var. 4774
Var. 4859
Var. 4944
Var. 5029
Var. 5114
Var. 5199
Var. 5284
Var. 5369



25-150
Var. 4775
Var. 4860
Var. 4945
Var. 5030
Var. 5115
Var. 5200
Var. 5285
Var. 5370



25-125
Var. 4776
Var. 4861
Var. 4946
Var. 5031
Var. 5116
Var. 5201
Var. 5286
Var. 5371



25-100
Var. 4777
Var. 4862
Var. 4947
Var. 5032
Var. 5117
Var. 5202
Var. 5287
Var. 5372



25-90 
Var. 4778
Var. 4863
Var. 4948
Var. 5033
Var. 5118
Var. 5203
Var. 5288
Var. 5373



25-80 
Var. 4779
Var. 4864
Var. 4949
Var. 5034
Var. 5119
Var. 5204
Var. 5289
Var. 5374



25-70 
Var. 4780
Var. 4865
Var. 4950
Var. 5035
Var. 5120
Var. 5205
Var. 5290
Var. 5375



25-60 
Var. 4781
Var. 4866
Var. 4951
Var. 5036
Var. 5121
Var. 5206
Var. 5291
Var. 5376



25-50 
Var. 4782
Var. 4867
Var. 4952
Var. 5037
Var. 5122
Var. 5207
Var. 5292
Var. 5377



50-200
Var. 4783
Var. 4868
Var. 4953
Var. 5038
Var. 5123
Var. 5208
Var. 5293
Var. 5378



50-175
Var. 4784
Var. 4869
Var. 4954
Var. 5039
Var. 5124
Var. 5209
Var. 5294
Var. 5379



50-150
Var. 4785
Var. 4870
Var. 4955
Var. 5040
Var. 5125
Var. 5210
Var. 5295
Var. 5380



50-125
Var. 4786
Var. 4871
Var. 4956
Var. 5041
Var. 5126
Var. 5211
Var. 5296
Var. 5381



50-90 
Var. 4787
Var. 4872
Var. 4957
Var. 5042
Var. 5127
Var. 5212
Var. 5297
Var. 5382



50-80 
Var. 4788
Var. 4873
Var. 4958
Var. 5043
Var. 5128
Var. 5213
Var. 5298
Var. 5383



75-200
Var. 4789
Var. 4874
Var. 4959
Var. 5044
Var. 5129
Var. 5214
Var. 5299
Var. 5384



75-175
Var. 4790
Var. 4875
Var. 4960
Var. 5045
Var. 5130
Var. 5215
Var. 5300
Var. 5385



75-150
Var. 4791
Var. 4876
Var. 4961
Var. 5046
Var. 5131
Var. 5216
Var. 5301
Var. 5386



100-200 
Var. 4792
Var. 4877
Var. 4962
Var. 5047
Var. 5132
Var. 5217
Var. 5302
Var. 5387



100-175 
Var. 4793
Var. 4878
Var. 4963
Var. 5048
Var. 5133
Var. 5218
Var. 5303
Var. 5388



 50 ± 25
Var. 4794
Var. 4879
Var. 4964
Var. 5049
Var. 5134
Var. 5219
Var. 5304
Var. 5389



 60 ± 25
Var. 4795
Var. 4880
Var. 4965
Var. 5050
Var. 5135
Var. 5220
Var. 5305
Var. 5390



 70 ± 25
Var. 4796
Var. 4881
Var. 4966
Var. 5051
Var. 5136
Var. 5221
Var. 5306
Var. 5391



 75 ± 25
Var. 4797
Var. 4882
Var. 4967
Var. 5052
Var. 5137
Var. 5222
Var. 5307
Var. 5392



 80 ± 25
Var. 4798
Var. 4883
Var. 4968
Var. 5053
Var. 5138
Var. 5223
Var. 5308
Var. 5393



 90 ± 25
Var. 4799
Var. 4884
Var. 4969
Var. 5054
Var. 5139
Var. 5224
Var. 5309
Var. 5394



100 ± 25
Var. 4800
Var. 4885
Var. 4970
Var. 5055
Var. 5140
Var. 5225
Var. 5310
Var. 5395



125 ± 25
Var. 4801
Var. 4886
Var. 4971
Var. 5056
Var. 5141
Var. 5226
Var. 5311
Var. 5396



150 ± 25
Var. 4802
Var. 4887
Var. 4972
Var. 5057
Var. 5142
Var. 5227
Var. 5312
Var. 5397



175 ± 25
Var. 4803
Var. 4888
Var. 4973
Var. 5058
Var. 5143
Var. 5228
Var. 5313
Var. 5398



 30 ± 10
Var. 4804
Var. 4889
Var. 4974
Var. 5059
Var. 5144
Var. 5229
Var. 5314
Var. 5399



 40 ± 10
Var. 4805
Var. 4890
Var. 4975
Var. 5060
Var. 5145
Var. 5230
Var. 5315
Var. 5400



 50 ± 10
Var. 4806
Var. 4891
Var. 4976
Var. 5061
Var. 5146
Var. 5231
Var. 5316
Var. 5401



 60 ± 10
Var. 4807
Var. 4892
Var. 4977
Var. 5062
Var. 5147
Var. 5232
Var. 5317
Var. 5402



 70 ± 10
Var. 4808
Var. 4893
Var. 4978
Var. 5063
Var. 5148
Var. 5233
Var. 5318
Var. 5403



 75 ± 10
Var. 4809
Var. 4894
Var. 4979
Var. 5064
Var. 5149
Var. 5234
Var. 5319
Var. 5404



 80 ± 10
Var. 4810
Var. 4895
Var. 4980
Var. 5065
Var. 5150
Var. 5235
Var. 5320
Var. 5405



 90 ± 10
Var. 4811
Var. 4896
Var. 4981
Var. 5066
Var. 5151
Var. 5236
Var. 5321
Var. 5406



100 ± 10
Var. 4812
Var. 4897
Var. 4982
Var. 5067
Var. 5152
Var. 5237
Var. 5322
Var. 5407



110 ± 10
Var. 4813
Var. 4898
Var. 4983
Var. 5068
Var. 5153
Var. 5238
Var. 5323
Var. 5408



120 ± 10
Var. 4814
Var. 4899
Var. 4984
Var. 5069
Var. 5154
Var. 5239
Var. 5324
Var. 5409



125 ± 10
Var. 4815
Var. 4900
Var. 4985
Var. 5070
Var. 5155
Var. 5240
Var. 5325
Var. 5410



130 ± 10
Var. 4816
Var. 4901
Var. 4986
Var. 5071
Var. 5156
Var. 5241
Var. 5326
Var. 5411



140 ± 10
Var. 4817
Var. 4902
Var. 4987
Var. 5072
Var. 5157
Var. 5242
Var. 5327
Var. 5412



150 ± 10
Var. 4818
Var. 4903
Var. 4988
Var. 5073
Var. 5158
Var. 5243
Var. 5328
Var. 5413



160 ± 10
Var. 4819
Var. 4904
Var. 4989
Var. 5074
Var. 5159
Var. 5244
Var. 5329
Var. 5414



170 ± 10
Var. 4820
Var. 4905
Var. 4990
Var. 5075
Var. 5160
Var. 5245
Var. 5330
Var. 5415



175 ± 10
Var. 4821
Var. 4906
Var. 4991
Var. 5076
Var. 5161
Var. 5246
Var. 5331
Var. 5416



180 ± 10
Var. 4822
Var. 4907
Var. 4992
Var. 5077
Var. 5162
Var. 5247
Var. 5332
Var. 5417



190 ± 10
Var. 4823
Var. 4908
Var. 4993
Var. 5078
Var. 5163
Var. 5248
Var. 5333
Var. 5418



25
Var. 4824
Var. 4909
Var. 4994
Var. 5079
Var. 5164
Var. 5249
Var. 5334
Var. 5419



30
Var. 4825
Var. 4910
Var. 4995
Var. 5080
Var. 5165
Var. 5250
Var. 5335
Var. 5420



40
Var. 4826
Var. 4911
Var. 4996
Var. 5081
Var. 5166
Var. 5251
Var. 5336
Var. 5421



50
Var. 4827
Var. 4912
Var. 4997
Var. 5082
Var. 5167
Var. 5252
Var. 5337
Var. 5422



60
Var. 4828
Var. 4913
Var. 4998
Var. 5083
Var. 5168
Var. 5253
Var. 5338
Var. 5423



70
Var. 4829
Var. 4914
Var. 4999
Var. 5084
Var. 5169
Var. 5254
Var. 5339
Var. 5424



75
Var. 4830
Var. 4915
Var. 5000
Var. 5085
Var. 5170
Var. 5255
Var. 5340
Var. 5425



80
Var. 4831
Var. 4916
Var. 5001
Var. 5086
Var. 5171
Var. 5256
Var. 5341
Var. 5426



90
Var. 4832
Var. 4917
Var. 5002
Var. 5087
Var. 5172
Var. 5257
Var. 5342
Var. 5427



100
Var. 4833
Var. 4918
Var. 5003
Var. 5088
Var. 5173
Var. 5258
Var. 5343
Var. 5428



110
Var. 4834
Var. 4919
Var. 5004
Var. 5089
Var. 5174
Var. 5259
Var. 5344
Var. 5429



120
Var. 4835
Var. 4920
Var. 5005
Var. 5090
Var. 5175
Var. 5260
Var. 5345
Var. 5430



125
Var. 4836
Var. 4921
Var. 5006
Var. 5091
Var. 5176
Var. 5261
Var. 5346
Var. 5431



130
Var. 4837
Var. 4922
Var. 5007
Var. 5092
Var. 5177
Var. 5262
Var. 5347
Var. 5432



140
Var. 4838
Var. 4923
Var. 5008
Var. 5093
Var. 5178
Var. 5263
Var. 5348
Var. 5433



150
Var. 4839
Var. 4924
Var. 5009
Var. 5094
Var. 5179
Var. 5264
Var. 5349
Var. 5434



160
Var. 4840
Var. 4925
Var. 5010
Var. 5095
Var. 5180
Var. 5265
Var. 5350
Var. 5435



170
Var. 4841
Var. 4926
Var. 5011
Var. 5096
Var. 5181
Var. 5266
Var. 5351
Var. 5436



175
Var. 4842
Var. 4927
Var. 5012
Var. 5097
Var. 5182
Var. 5267
Var. 5352
Var. 5437



180
Var. 4843
Var. 4928
Var. 5013
Var. 5098
Var. 5183
Var. 5268
Var. 5353
Var. 5438



190
Var. 4844
Var. 4929
Var. 5014
Var. 5099
Var. 5184
Var. 5269
Var. 5354
Var. 5439



200
Var. 4845
Var. 4930
Var. 5015
Var. 5100
Var. 5185
Var. 5270
Var. 5355
Var. 5440





Var. = Variation













TABLE 9







Exemplary embodiments for the combination of sugar or sugar alcohol and non-ionic


surfactant concentrations useful for the stabilization of furin (e.g., rfurin) compositions.









Sugar or Sugar Alcohol (%)















14%
15%
16%
17%
18%
19%
20%



















Non-Ionic
 10-200
Var. 5441
Var. 5526
Var. 5611
Var. 5696
Var. 5781
Var. 5866
Var. 5951


Surfactant
 10-175
Var. 5442
Var. 5527
Var. 5612
Var. 5697
Var. 5782
Var. 5867
Var. 5952


(ppm)
 10-150
Var. 5443
Var. 5528
Var. 5613
Var. 5698
Var. 5783
Var. 5868
Var. 5953



 10-125
Var. 5444
Var. 5529
Var. 5614
Var. 5699
Var. 5784
Var. 5869
Var. 5954



 10-100
Var. 5445
Var. 5530
Var. 5615
Var. 5700
Var. 5785
Var. 5870
Var. 5955



10-90
Var. 5446
Var. 5531
Var. 5616
Var. 5701
Var. 5786
Var. 5871
Var. 5956



10-80
Var. 5447
Var. 5532
Var. 5617
Var. 5702
Var. 5787
Var. 5872
Var. 5957



10-75
Var. 5448
Var. 5533
Var. 5618
Var. 5703
Var. 5788
Var. 5873
Var. 5958



10-70
Var. 5449
Var. 5534
Var. 5619
Var. 5704
Var. 5789
Var. 5874
Var. 5959



10-60
Var. 5450
Var. 5535
Var. 5620
Var. 5705
Var. 5790
Var. 5875
Var. 5960



10-50
Var. 5451
Var. 5536
Var. 5621
Var. 5706
Var. 5791
Var. 5876
Var. 5961



10-25
Var. 5452
Var. 5537
Var. 5622
Var. 5707
Var. 5792
Var. 5877
Var. 5962



 25-200
Var. 5453
Var. 5538
Var. 5623
Var. 5708
Var. 5793
Var. 5878
Var. 5963



 25-175
Var. 5454
Var. 5539
Var. 5624
Var. 5709
Var. 5794
Var. 5879
Var. 5964



 25-150
Var. 5455
Var. 5540
Var. 5625
Var. 5710
Var. 5795
Var. 5880
Var. 5965



 25-125
Var. 5456
Var. 5541
Var. 5626
Var. 5711
Var. 5796
Var. 5881
Var. 5966



 25-100
Var. 5457
Var. 5542
Var. 5627
Var. 5712
Var. 5797
Var. 5882
Var. 5967



25-90
Var. 5458
Var. 5543
Var. 5628
Var. 5713
Var. 5798
Var. 5883
Var. 5968



25-80
Var. 5459
Var. 5544
Var. 5629
Var. 5714
Var. 5799
Var. 5884
Var. 5969



25-70
Var. 5460
Var. 5545
Var. 5630
Var. 5715
Var. 5800
Var. 5885
Var. 5970



25-60
Var. 5461
Var. 5546
Var. 5631
Var. 5716
Var. 5801
Var. 5886
Var. 5971



25-50
Var. 5462
Var. 5547
Var. 5632
Var. 5717
Var. 5802
Var. 5887
Var. 5972



 50-200
Var. 5463
Var. 5548
Var. 5633
Var. 5718
Var. 5803
Var. 5888
Var. 5973



 50-175
Var. 5464
Var. 5549
Var. 5634
Var. 5719
Var. 5804
Var. 5889
Var. 5974



 50-150
Var. 5465
Var. 5550
Var. 5635
Var. 5720
Var. 5805
Var. 5890
Var. 5975



 50-125
Var. 5466
Var. 5551
Var. 5636
Var. 5721
Var. 5806
Var. 5891
Var. 5976



50-90
Var. 5467
Var. 5552
Var. 5637
Var. 5722
Var. 5807
Var. 5892
Var. 5977



50-80
Var. 5468
Var. 5553
Var. 5638
Var. 5723
Var. 5808
Var. 5893
Var. 5978



 75-200
Var. 5469
Var. 5554
Var. 5639
Var. 5724
Var. 5809
Var. 5894
Var. 5979



 75-175
Var. 5470
Var. 5555
Var. 5640
Var. 5725
Var. 5810
Var. 5895
Var. 5980



 75-150
Var. 5471
Var. 5556
Var. 5641
Var. 5726
Var. 5811
Var. 5896
Var. 5981



100-200
Var. 5472
Var. 5557
Var. 5642
Var. 5727
Var. 5812
Var. 5897
Var. 5982



100-175
Var. 5473
Var. 5558
Var. 5643
Var. 5728
Var. 5813
Var. 5898
Var. 5983



50 ± 25
Var. 5474
Var. 5559
Var. 5644
Var. 5729
Var. 5814
Var. 5899
Var. 5984



60 ± 25
Var. 5475
Var. 5560
Var. 5645
Var. 5730
Var. 5815
Var. 5900
Var. 5985



70 ± 25
Var. 5476
Var. 5561
Var. 5646
Var. 5731
Var. 5816
Var. 5901
Var. 5986



75 ± 25
Var. 5477
Var. 5562
Var. 5647
Var. 5732
Var. 5817
Var. 5902
Var. 5987



80 ± 25
Var. 5478
Var. 5563
Var. 5648
Var. 5733
Var. 5818
Var. 5903
Var. 5988



90 ± 25
Var. 5479
Var. 5564
Var. 5649
Var. 5734
Var. 5819
Var. 5904
Var. 5989



100 ± 25 
Var. 5480
Var. 5565
Var. 5650
Var. 5735
Var. 5820
Var. 5905
Var. 5990



125 ± 25 
Var. 5481
Var. 5566
Var. 5651
Var. 5736
Var. 5821
Var. 5906
Var. 5991



150 ± 25 
Var. 5482
Var. 5567
Var. 5652
Var. 5737
Var. 5822
Var. 5907
Var. 5992



175 ± 25 
Var. 5483
Var. 5568
Var. 5653
Var. 5738
Var. 5823
Var. 5908
Var. 5993



30 ± 10
Var. 5484
Var. 5569
Var. 5654
Var. 5739
Var. 5824
Var. 5909
Var. 5994



40 ± 10
Var. 5485
Var. 5570
Var. 5655
Var. 5740
Var. 5825
Var. 5910
Var. 5995



50 ± 10
Var. 5486
Var. 5571
Var. 5656
Var. 5741
Var. 5826
Var. 5911
Var. 5996



60 ± 10
Var. 5487
Var. 5572
Var. 5657
Var. 5742
Var. 5827
Var. 5912
Var. 5997



70 ± 10
Var. 5488
Var. 5573
Var. 5658
Var. 5743
Var. 5828
Var. 5913
Var. 5998



75 ± 10
Var. 5489
Var. 5574
Var. 5659
Var. 5744
Var. 5829
Var. 5914
Var. 5999



80 ± 10
Var. 5490
Var. 5575
Var. 5660
Var. 5745
Var. 5830
Var. 5915
Var. 6000



90 ± 10
Var. 5491
Var. 5576
Var. 5661
Var. 5746
Var. 5831
Var. 5916
Var. 6001



100 ± 10 
Var. 5492
Var. 5577
Var. 5662
Var. 5747
Var. 5832
Var. 5917
Var. 6002



110 ± 10 
Var. 5493
Var. 5578
Var. 5663
Var. 5748
Var. 5833
Var. 5918
Var. 6003



120 ± 10 
Var. 5494
Var. 5579
Var. 5664
Var. 5749
Var. 5834
Var. 5919
Var. 6004



125 ± 10 
Var. 5495
Var. 5580
Var. 5665
Var. 5750
Var. 5835
Var. 5920
Var. 6005



130 ± 10 
Var. 5496
Var. 5581
Var. 5666
Var. 5751
Var. 5836
Var. 5921
Var. 6006



140 ± 10 
Var. 5497
Var. 5582
Var. 5667
Var. 5752
Var. 5837
Var. 5922
Var. 6007



150 ± 10 
Var. 5498
Var. 5583
Var. 5668
Var. 5753
Var. 5838
Var. 5923
Var. 6008



160 ± 10 
Var. 5499
Var. 5584
Var. 5669
Var. 5754
Var. 5839
Var. 5924
Var. 6009



170 ± 10 
Var. 5500
Var. 5585
Var. 5670
Var. 5755
Var. 5840
Var. 5925
Var. 6010



175 ± 10 
Var. 5501
Var. 5586
Var. 5671
Var. 5756
Var. 5841
Var. 5926
Var. 6011



180 ± 10 
Var. 5502
Var. 5587
Var. 5672
Var. 5757
Var. 5842
Var. 5927
Var. 6012



190 ± 10 
Var. 5503
Var. 5588
Var. 5673
Var. 5758
Var. 5843
Var. 5928
Var. 6013



25
Var. 5504
Var. 5589
Var. 5674
Var. 5759
Var. 5844
Var. 5929
Var. 6014



30
Var. 5505
Var. 5590
Var. 5675
Var. 5760
Var. 5845
Var. 5930
Var. 6015



40
Var. 5506
Var. 5591
Var. 5676
Var. 5761
Var. 5846
Var. 5931
Var. 6016



50
Var. 5507
Var. 5592
Var. 5677
Var. 5762
Var. 5847
Var. 5932
Var. 6017



60
Var. 5508
Var. 5593
Var. 5678
Var. 5763
Var. 5848
Var. 5933
Var. 6018



70
Var. 5509
Var. 5594
Var. 5679
Var. 5764
Var. 5849
Var. 5934
Var. 6019



75
Var. 5510
Var. 5595
Var. 5680
Var. 5765
Var. 5850
Var. 5935
Var. 6020



80
Var. 5511
Var. 5596
Var. 5681
Var. 5766
Var. 5851
Var. 5936
Var. 6021



90
Var. 5512
Var. 5597
Var. 5682
Var. 5767
Var. 5852
Var. 5937
Var. 6022



100
Var. 5513
Var. 5598
Var. 5683
Var. 5768
Var. 5853
Var. 5938
Var. 6023



110
Var. 5514
Var. 5599
Var. 5684
Var. 5769
Var. 5854
Var. 5939
Var. 6024



120
Var. 5515
Var. 5600
Var. 5685
Var. 5770
Var. 5855
Var. 5940
Var. 6025



125
Var. 5516
Var. 5601
Var. 5686
Var. 5771
Var. 5856
Var. 5941
Var. 6026



130
Var. 5517
Var. 5602
Var. 5687
Var. 5772
Var. 5857
Var. 5942
Var. 6027



140
Var. 5518
Var. 5603
Var. 5688
Var. 5773
Var. 5858
Var. 5943
Var. 6028



150
Var. 5519
Var. 5604
Var. 5689
Var. 5774
Var. 5859
Var. 5944
Var. 6029



160
Var. 5520
Var. 5605
Var. 5690
Var. 5775
Var. 5860
Var. 5945
Var. 6030



170
Var. 5521
Var. 5606
Var. 5691
Var. 5776
Var. 5861
Var. 5946
Var. 6031



175
Var. 5522
Var. 5607
Var. 5692
Var. 5777
Var. 5862
Var. 5947
Var. 6032



180
Var. 5523
Var. 5608
Var. 5693
Var. 5778
Var. 5863
Var. 5948
Var. 6033



190
Var. 5524
Var. 5609
Var. 5694
Var. 5779
Var. 5864
Var. 5949
Var. 6034



200
Var. 5525
Var. 5610
Var. 5695
Var. 5780
Var. 5865
Var. 5950
Var. 6035





Var. = Variation






1. Sugars and Sugar Alcohols
Advantageously, it was found that the addition of sugar and/or sugar alcohol to furin compositions (e.g., rfurin compositions) increases the storage stability of the composition. For example, as shown in FIGS. 17 to 20 and 22 to 25, the inclusion of as little as 2% sugar and/or sugar alcohol can increase the storage stability of a furin composition by at least 20%. And the inclusion of 10% sugar and/or sugar alcohol can increase the storage stability of a furin composition by at least 75%. Accordingly, in one aspect of the present disclosure, a storage stable composition of furin (e.g., rfurin) contains a stabilizing amount of sugar and/or sugar alcohol.
In one embodiment, the storage stable composition of furin (e.g., rfurin) contains monosaccharide sugar. In a particular embodiment, the monosaccharide sugar is selected from the group consisting of a diose sugar, a triose sugar, a tetrose sugar, a pentose sugar, a hexose sugar, a heptose sugar, and an octose sugar. In a particular embodiment, the sugar is a pentose sugar, a hexose sugar, or a combination thereof. In a specific embodiment, the sugar is a hexose sugar.
In another embodiment, the storage stable composition of furin (e.g., rfurin) contains a disaccharide sugar. In a particular embodiment, the disaccharide sugar is selected from disaccharide sugars formed from pentose and/or hexose monosaccharides. In another particular embodiment, the sugar is selected from disaccharide sugars formed from hexose monosaccharides. In one embodiment, the sugar is sucrose, trehalose, or a combination thereof. In one specific embodiment, the sugar is sucrose. In another specific embodiment, the sugar is trehalose. In one embodiment, the sugar is formulated as trehalose dihydrate.
In another embodiment, the storage stable composition of furin (e.g., rfurin) contains a sugar alcohol. In a particular embodiment, the sugar alcohol is selected from glycol, glycerol, erythritol, threitol, ribitol, fucitol, iditol, volmitol, isomalt, maltitol, lactitol, mannitol, sorbitol, inositol, galactitol, dulcitol, xylitol, and arabitol. In another particular embodiment, the sugar alcohol is mannitol.
In yet another embodiment, the stable composition of furin (e.g., rfurin) contains a mixture of sugar and sugar alcohol. In one embodiment, the mixture contains at least two of a monosaccharide, a disaccharide, and a sugar alcohol. In another embodiment, the mixture contains at least two of a pentose sugar, a hexose sugar, a disaccharide formed from pentose and/or hexose monosaccharides, and a sugar alcohol. In another embodiment, the mixture contains at least two of sucrose, trehalose, and mannitol.
In one embodiment, the sugar or sugar alcohol is present at a concentration of from 2% to 20%, 2% to 17.5%, 2% to 15%, 2% to 12.5%, 2% to 10%, 2% to 9%, 2% to 8%, 2% to 7%, 5% to 20%, 5% to 17.5%, 5% to 15%, 5% to 12.5%, 5% to 10%, 7.5% to 20%, 7.5% to 17.5%, 7.5% to 15%, 7.5% to 12.5%, 10% to 20%, 10% to 17.5%, 10% to 15%, 4±2%, 5±2%, 6±2%, 7±2%, 8±2%, 9±2%, 10±2%, 11±2%, 12±2%, 13±2%, 14±2%, 15±2%, 16±2%, 17±2%, 18±2%, 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%, 16±1%, 17±1%, 18±1%, 19±1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%. In one embodiment, the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof. In a specific embodiment, the sugar or sugar alcohol is trehalose.
In one embodiment, a stabilized furin (e.g., rfurin) composition comprises from 50 mM to 500 mM of a pharmaceutically acceptable salt, from 0.5 mM to 5 mM calcium; from 2% to 20% sugar or sugar alcohol, from 10 to 200 mM buffering agent, and a pH from 5.5 to 7.5. In one embodiment, the storage stable furin composition further comprises from 10 ppm to 200 ppm non-ionic surfactant. In a specific embodiment, the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof. In one embodiment, the pH of the composition is from 5.5 to 7.0. In another embodiment, the pH of the composition is from 5.5 to 6.5. In another embodiment, the pH of the composition is 6.0±0.2. In a specific embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.
In one embodiment, a stabilized furin (e.g., rfurin) composition comprises from 50 to 500 mM of a pharmaceutically acceptable salt, from 0.5 mM to 5 mM calcium; from 2% to 20% sugar or sugar alcohol, from 10 ppm to 100 ppm non-ionic surfactant, from 10 mM to 200 mM buffering agent, and a pH from 5.5 to 7.5. In a specific embodiment, the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof. In one embodiment, the pH of the composition is from 5.5 to 7.0. In another embodiment, the pH of the composition is from 5.5 to 6.5. In another embodiment, the pH of the composition is 6.0±0.2. In a specific embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.
In one embodiment, a stabilized furin (e.g., rfurin) composition comprises from 50 to 500 mM of a pharmaceutically acceptable salt, from 0.5 mM to 5 mM calcium; from 2% to 20% trehalose, from 10 ppm to 100 ppm non-ionic surfactant, from 10 mM to 200 mM buffering agent, and a pH from 5.5 to 7.5. In one embodiment, the pH of the composition is from 5.5 to 7.0. In another embodiment, the pH of the composition is from 5.5 to 6.5. In another embodiment, the pH of the composition is 6.0±0.2. In a specific embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.
In one embodiment, a stabilized furin (e.g., rfurin) composition comprises from 50 to 500 mM of a pharmaceutically acceptable salt, from 0.5 mM to 5 mM calcium; a combination of sugar or sugar alcohol and non-ionic surfactant selected from variations 1 to 6035 found in Table 1 to Table 9, from 10 mM to 200 mM buffering agent, and a pH from 5.5 to 7.5. In a specific embodiment, the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof. In one embodiment, the pH of the composition is from 5.5 to 7.0. In another embodiment, the pH of the composition is from 5.5 to 6.5. In another embodiment, the pH of the composition is 6.0±0.2. In a specific embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.
In one embodiment, a stabilized furin (e.g., rfurin) composition comprises from 50 to 500 mM of a pharmaceutically acceptable salt, from 0.5 mM to 5 mM calcium; a combination of trehalose and non-ionic surfactant selected from variations 1 to 6035 found in Table 1 to Table 9, from 10 mM to 200 mM buffering agent, and a pH from 5.5 to 7.5. In one embodiment, the pH of the composition is from 5.5 to 7.0. In another embodiment, the pH of the composition is from 5.5 to 6.5. In another embodiment, the pH of the composition is 6.0±0.2. In a specific embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.
In one embodiment, a stabilized furin (e.g., rfurin) composition comprises from 100 to 300 mM of a pharmaceutically acceptable salt, from 0.5 mM to 2 mM calcium; from 2% to 20% sugar or sugar alcohol, from 10 mM to 200 mM buffering agent, and a pH from 5.5 to 7.5. In one embodiment, the storage stable furin composition further comprises from 10 ppm to 200 ppm non-ionic surfactant. In a specific embodiment, the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof. In one embodiment, the pH of the composition is from 5.5 to 7.0. In another embodiment, the pH of the composition is from 5.5 to 6.5. In another embodiment, the pH of the composition is 6.0±0.2. In a specific embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.
In one embodiment, a stabilized furin (e.g., rfurin) composition comprises from 100 mM to 300 mM of a pharmaceutically acceptable salt, from 0.5 mM to 2 mM calcium; from 2% to 20% sugar or sugar alcohol, from 10 ppm to 100 ppm non-ionic surfactant, from 10 to 200 mM buffering agent, and a pH from 5.5 to 7.5. In a specific embodiment, the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof. In one embodiment, the pH of the composition is from 5.5 to 7.0. In another embodiment, the pH of the composition is from 5.5 to 6.5. In another embodiment, the pH of the composition is 6.0±0.2. In a specific embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.
In one embodiment, a stabilized furin (e.g., rfurin) composition comprises from 100 mM to 300 mM of a pharmaceutically acceptable salt, from 0.5 mM to 2 mM calcium; from 2% to 20% trehalose, from 10 ppm to 100 ppm non-ionic surfactant, from 10 to 200 mM buffering agent, and a pH from 5.5 to 7.5. In one embodiment, the pH of the composition is from 5.5 to 7.0. In another embodiment, the pH of the composition is from 5.5 to 6.5. In another embodiment, the pH of the composition is 6.0±0.2. In a specific embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.
In one embodiment, a stabilized furin (e.g., rfurin) composition comprises from 100 to 300 mM of a pharmaceutically acceptable salt, from 0.5 mM to 2 mM calcium; a combination of sugar or sugar alcohol and non-ionic surfactant selected from variations 1 to 6035 found in Table 1 to Table 9, from 10 mM to 200 mM buffering agent, and a pH from 5.5 to 7.5. In a specific embodiment, the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof. In one embodiment, the pH of the composition is from 5.5 to 7.0. In another embodiment, the pH of the composition is from 5.5 to 6.5. In another embodiment, the pH of the composition is 6.0±0.2. In a specific embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.
In one embodiment, a stabilized furin (e.g., rfurin) composition comprises from 100 to 300 mM of a pharmaceutically acceptable salt, from 0.5 mM to 2 mM calcium; a combination of trehalose and non-ionic surfactant selected from variations 1 to 6035 found in Table 1 to Table 9, from 10 mM to 200 mM buffering agent, and a pH from 5.5 to 7.5. In one embodiment, the pH of the composition is from 5.5 to 7.0. In another embodiment, the pH of the composition is from 5.5 to 6.5. In another embodiment, the pH of the composition is 6.0±0.2. In a specific embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.
In one embodiment, a stabilized furin (e.g., rfurin) composition comprises 190±50 mM of a pharmaceutically acceptable salt, from 0.5 mM to 2 mM calcium; 10±5% sugar or sugar alcohol, 90±25 mM buffering agent, and a pH from 5.5 to 7.5. In one embodiment, the storage stable furin composition further comprises from 10 to 200 ppm non-ionic surfactant. In a specific embodiment, the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof. In one embodiment, the pH of the composition is from 5.5 to 7.0. In another embodiment, the pH of the composition is from 5.5 to 6.5. In another embodiment, the pH of the composition is 6.0±0.2. In a specific embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin. In another specific embodiment, the composition contains 10±2% sugar or sugar alcohol. In another specific embodiment, the composition contains 10±1% sugar or sugar alcohol. In another specific embodiment, the composition contains 10% sugar or sugar alcohol.
In one embodiment, a stabilized furin (e.g., rfurin) composition comprises 190±50 mM of a pharmaceutically acceptable salt, from 0.5 mM to 2 mM calcium; 10±5% sugar or sugar alcohol, from 10 to 100 ppm non-ionic surfactant, 90±25 mM buffering agent, and a pH from 5.5 to 7.5. In a specific embodiment, the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof. In one embodiment, the pH of the composition is from 5.5 to 7.0. In another embodiment, the pH of the composition is from 5.5 to 6.5. In another embodiment, the pH of the composition is 6.0±0.2. In a specific embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin. In another specific embodiment, the composition contains 10±2% sugar or sugar alcohol. In another specific embodiment, the composition contains 10±1% sugar or sugar alcohol. In another specific embodiment, the composition contains 10% sugar or sugar alcohol.
In one embodiment, a stabilized furin (e.g., rfurin) composition comprises 190±50 mM of a pharmaceutically acceptable salt, from 0.5 mM to 2 mM calcium; 10±5% trehalose, from 10 to 100 ppm non-ionic surfactant, 90±25 mM buffering agent, and a pH from 5.5 to 7.5. In one embodiment, the pH of the composition is from 5.5 to 7.0. In another embodiment, the pH of the composition is from 5.5 to 6.5. In another embodiment, the pH of the composition is 6.0±0.2. In a specific embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin. In another specific embodiment, the composition contains 10±2% trehalose. In another specific embodiment, the composition contains 10±1% trehalose. In another specific embodiment, the composition contains 10% trehalose.
In one embodiment, a stabilized furin (e.g., rfurin) composition comprises 190±50 mM of a pharmaceutically acceptable salt, from 0.5 mM to 2 mM calcium; a combination of sugar or sugar alcohol and non-ionic surfactant selected from variations 1 to 6035 found in Table 1 to Table 9, 90±25 mM buffering agent, and a pH from 5.5 to 7.5. In a specific embodiment, the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof. In one embodiment, the pH of the composition is from 5.5 to 7.0. In another embodiment, the pH of the composition is from 5.5 to 6.5. In another embodiment, the pH of the composition is 6.0±0.2. In a specific embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.
In one embodiment, a stabilized furin (e.g., rfurin) composition comprises 190±50 mM of a pharmaceutically acceptable salt, from 0.5 mM to 2 mM calcium; a combination of trehalose and non-ionic surfactant selected from variations 1 to 6035 found in Table 1 to Table 9, from 90±25 mM buffering agent, and a pH from 5.5 to 7.5. In one embodiment, the pH of the composition is from 5.5 to 7.0. In another embodiment, the pH of the composition is from 5.5 to 6.5. In another embodiment, the pH of the composition is 6.0±0.2. In a specific embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.
In one embodiment, a stabilized furin (e.g., rfurin) composition comprises 190 mM of a pharmaceutically acceptable salt, 0.9 mM calcium; 10±5% sugar or sugar alcohol, 91 mM buffering agent, and a pH of 6.0±0.2. In one embodiment, the storage stable furin composition further comprises from 10 to 200 ppm non-ionic surfactant. In a specific embodiment, the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof. In one embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin. In another specific embodiment, the composition contains 10±2% sugar or sugar alcohol. In another specific embodiment, the composition contains 10±1% sugar or sugar alcohol. In another specific embodiment, the composition contains 10% sugar or sugar alcohol.
In one embodiment, a stabilized furin (e.g., rfurin) composition comprises 190 mM of a pharmaceutically acceptable salt, 0.9 mM calcium; 10±5% sugar or sugar alcohol, 75 ppm non-ionic surfactant, 91 mM buffering agent, and a pH from of 6.0±0.2. In a specific embodiment, the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof. In one embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin. In another specific embodiment, the composition contains 10±2% sugar or sugar alcohol. In another specific embodiment, the composition contains 10±1% sugar or sugar alcohol. In another specific embodiment, the composition contains 10% sugar or sugar alcohol.
In one embodiment, a stabilized furin (e.g., rfurin) composition comprises 190 mM of a pharmaceutically acceptable salt, 0.9 mM calcium; 10±5% trehalose, 75 ppm non-ionic surfactant, 91 mM buffering agent, and a pH of 6.0±0.2. In one embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin. In another specific embodiment, the composition contains 10±2% trehalose. In another specific embodiment, the composition contains 10±1% trehalose. In another specific embodiment, the composition contains 10% trehalose.
In one embodiment, a stabilized furin (e.g., rfurin) composition comprises 190 mM of a pharmaceutically acceptable salt, 0.9 mM calcium; a combination of sugar or sugar alcohol and non-ionic surfactant selected from variations 1 to 6035 found in Table 1 to Table 9, 91 mM buffering agent, and a pH of 6.0±0.2. In a specific embodiment, the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof. In one embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.
In one embodiment, a stabilized furin (e.g., rfurin) composition comprises 190 mM of a pharmaceutically acceptable salt, 0.9 mM calcium; a combination of trehalose and non-ionic surfactant selected from variations 1 to 6035 found in Table 1 to Table 9, 91 mM buffering agent, and a pH of 6.0±0.2. In one embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.
In some embodiments, stabilizing agents used in the formulations of the present disclosure are selected from a group that includes, without limitation: sucrose, trehalose, mannitol, raffinose, and arginine. These agents are present in the formulations of the present invention in an amount of from 0.1% to 20%. In certain embodiments, the stabilizing agent is present in an amount of from 5% to 15%, or about 10%. In further embodiments, formulations of the present disclosure include stabilizing agents in an amount of from 0.5% to 19%, 1% to 18%, 1.5% to 17%, 2.0% to 16%, 2.5% to 15%, 3.0% to 14%, 3.5% to 13%, 4.0% to 12%, 4.5% to 11%, 5.0% to 10%, 5.5% to 9%, or 6.0% to 8%. Certain formulations include mannitol, sucrose, and/or trehalose in combination with one or more of any other formulation components disclosed herein.
2. Non Ionic Surfactants
Advantageously, it was found that the addition of sugar non-ionic surfactant to furin compositions (e.g., rfurin compositions) increases the stability of the composition to mechanical stress. For example, as shown in FIGS. 31 to 42, the inclusion of as little as 10 ppm non-ionic surfactant can increase the stability of a furin composition subjected to mechanical stress by at least 25%. And the inclusion of 50 ppm non-ionic surfactant can increase the stability of a furin composition subjected to mechanical stress by at least 40%. Accordingly, in one aspect of the present disclosure, a storage stable composition of furin (e.g., rfurin) contains a stabilizing amount of non-ionic surfactant.
In one embodiment, storage stable compositions of furin (e.g., rfurin) are provided which contain a non-ionic surfactant selected from a non-ionic water soluble monoglyceride, a non-ionic water soluble diglyceride, a non-ionic water soluble triglyceride, a non-ionic water soluble monofatty acid esters of polyethyelene glycol, a non-ionic water soluble difatty acid esters of polyethyelene glycol, a non-ionic water soluble sorbitan fatty acid ester, a non-ionic polyglycolyzed glyceride, a non-ionic water soluble triblock copolymer, and a combination thereof. In one embodiment, the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester. In a specific embodiment, the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate).
In one embodiment, the non-ionic surfactant is present at a concentration of from 10 to 200 ppm, 10 to 175 ppm, 10 to 150 ppm, 10 to 125 ppm, 10 to 100 ppm, 10 to 90 ppm, 10 to 80 ppm, 10 to 75 ppm, 10 to 70 ppm, 10 to 60 ppm, 10 to 50 ppm, 10 to 25 ppm, 25 to 200 ppm, 25 to 175 ppm, 25 to 150 ppm, 25 to 125 ppm, 25 to 100 ppm, 25 to 90 ppm, 25 to 80 ppm, 25 to 70 ppm, 25 to 60 ppm, 25 to 50 ppm, 50 to 200 ppm, 50 to 175 ppm, 50 to 150 ppm, 50 to 125 ppm, 50 to 90 ppm, 50 to 80 ppm, 75 to 200 ppm, 75 to 175 ppm, 75 to 150 ppm, 100 to 200 ppm, 100 to 175 ppm, 50±25 ppm, 60±25 ppm, 70±25 ppm, 75±25 ppm, 80±25 ppm, 90±25 ppm, 100±25 ppm, 125±25 ppm, 150±25 ppm, 175±25 ppm, 30±10 ppm, 40±10 ppm, 50±10 ppm, 60±10 ppm, 70±10 ppm, 75±10 ppm, 80±10 ppm, 90±10 ppm, 100±10 ppm, 110±10 ppm, 120±10 ppm, 125±10 ppm, 130±10 ppm, 140±10 ppm, 150±10 ppm, 160±10 ppm, 170±10 ppm, 175±10 ppm, 180±10 ppm, 190±10 ppm, 25 ppm, 30 ppm, 40 ppm, 50 ppm, 60 ppm, 70 ppm, 75 ppm, 80 ppm, 90 ppm, 100 ppm, 110 ppm, 120 ppm, 125 ppm, 130 ppm, 140 ppm, 150 ppm, 160 ppm, 170 ppm, 175 ppm, 180 ppm, 190 ppm, or 200 ppm. In one embodiment, the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester. In a specific embodiment, the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate).
In one embodiment, a stabilized furin (e.g., rfurin) composition comprises from 50 mM to 500 mM of a pharmaceutically acceptable salt, from 0.5 mM to 5 mM calcium; from 10 ppm to 200 ppm non-ionic surfactant, from 10 to 200 mM buffering agent, and a pH from 5.5 to 7.5. In one embodiment, the storage stable furin composition further comprises from 2% to 20% sugar or sugar alcohol. In one specific embodiment, the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester. In a specific embodiment, the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate). In one embodiment, the pH of the composition is from 5.5 to 7.0. In another embodiment, the pH of the composition is from 5.5 to 6.5. In another embodiment, the pH of the composition is 6.0±0.2. In a specific embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.
In one embodiment, a stabilized furin (e.g., rfurin) composition comprises from 50 to 500 mM of a pharmaceutically acceptable salt, from 0.5 mM to 5 mM calcium; from 2% to 10% sugar or sugar alcohol, from 10 ppm to 200 ppm non-ionic surfactant, from 10 mM to 200 mM buffering agent, and a pH from 5.5 to 7.5. In one specific embodiment, the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester. In a specific embodiment, the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate). In one embodiment, the pH of the composition is from 5.5 to 7.0. In another embodiment, the pH of the composition is from 5.5 to 6.5. In another embodiment, the pH of the composition is 6.0±0.2. In a specific embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.
In one embodiment, a stabilized furin (e.g., rfurin) composition comprises from 50 to 500 mM of a pharmaceutically acceptable salt, from 0.5 mM to 5 mM calcium; from 2% to 10% sugar or sugar alcohol, from 10 ppm to 200 ppm non-ionic water soluble sorbitan fatty acid ester, from 10 mM to 200 mM buffering agent, and a pH from 5.5 to 7.5. In a specific embodiment, the non-ionic water soluble sorbitan fatty acid ester is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate). In one embodiment, the pH of the composition is from 5.5 to 7.0. In another embodiment, the pH of the composition is from 5.5 to 6.5. In another embodiment, the pH of the composition is 6.0±0.2. In a specific embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.
In one embodiment, a stabilized furin (e.g., rfurin) composition comprises from 50 to 500 mM of a pharmaceutically acceptable salt, from 0.5 mM to 5 mM calcium; a combination of sugar or sugar alcohol and non-ionic surfactant selected from variations 1 to 6035 found in Table 1 to Table 9, from 10 mM to 200 mM buffering agent, and a pH from 5.5 to 7.5. In a specific embodiment, the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester. In another specific embodiment, the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof. In yet another specific embodiment, the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester and the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof. In one embodiment, the pH of the composition is from 5.5 to 7.0. In another embodiment, the pH of the composition is from 5.5 to 6.5. In another embodiment, the pH of the composition is 6.0±0.2. In a specific embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.
In one embodiment, a stabilized furin (e.g., rfurin) composition comprises from 50 to 500 mM of a pharmaceutically acceptable salt, from 0.5 mM to 5 mM calcium; a combination of sugar or sugar alcohol and a non-ionic water soluble sorbitan fatty acid ester selected from variations 1 to 6035 found in Table 1 to Table 9, from 10 mM to 200 mM buffering agent, and a pH from 5.5 to 7.5. In a specific embodiment, the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate). In another specific embodiment, the sugar or sugar alcohol is trehalose. In yet another specific embodiment, the non-ionic water soluble sorbitan fatty acid ester is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate) and the sugar or sugar alcohol is trehalose. In one embodiment, the pH of the composition is from 5.5 to 7.0. In another embodiment, the pH of the composition is from 5.5 to 6.5. In another embodiment, the pH of the composition is 6.0±0.2. In a specific embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.
In one embodiment, a stabilized furin (e.g., rfurin) composition comprises from 100 to 300 mM of a pharmaceutically acceptable salt, from 0.5 mM to 2 mM calcium; from 10 ppm to 200 ppm non-ionic surfactant, from 10 mM to 200 mM buffering agent, and a pH from 5.5 to 7.5. In one embodiment, the storage stable furin composition further comprises from 2% to 20% sugar or sugar alcohol. In one embodiment, the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester. In a specific embodiment, the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate). In one embodiment, the pH of the composition is from 5.5 to 7.0. In another embodiment, the pH of the composition is from 5.5 to 6.5. In another embodiment, the pH of the composition is 6.0±0.2. In a specific embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.
In one embodiment, a stabilized furin (e.g., rfurin) composition comprises from 100 mM to 300 mM of a pharmaceutically acceptable salt, from 0.5 mM to 2 mM calcium; from 2% to 10% sugar or sugar alcohol, from 10 ppm to 200 ppm non-ionic surfactant, from 10 to 200 mM buffering agent, and a pH from 5.5 to 7.5. In one embodiment, the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester. In a specific embodiment, the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate). In one embodiment, the pH of the composition is from 5.5 to 7.0. In another embodiment, the pH of the composition is from 5.5 to 6.5. In another embodiment, the pH of the composition is 6.0±0.2. In a specific embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.
In one embodiment, a stabilized furin (e.g., rfurin) composition comprises from 100 mM to 300 mM of a pharmaceutically acceptable salt, from 0.5 mM to 2 mM calcium; from 2% to 10% sugar or sugar alcohol, from 10 ppm to 200 ppm non-ionic water soluble sorbitan fatty acid ester, from 10 to 200 mM buffering agent, and a pH from 5.5 to 7.5. In a specific embodiment, the non-ionic water soluble sorbitan fatty acid ester is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate). In one embodiment, the pH of the composition is from 5.5 to 7.0. In another embodiment, the pH of the composition is from 5.5 to 6.5. In another embodiment, the pH of the composition is 6.0±0.2. In a specific embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.
In one embodiment, a stabilized furin (e.g., rfurin) composition comprises from 100 to 300 mM of a pharmaceutically acceptable salt, from 0.5 mM to 2 mM calcium; a combination of sugar or sugar alcohol and non-ionic surfactant selected from variations 1 to 6035 found in Table 1 to Table 9, from 10 mM to 200 mM buffering agent, and a pH from 5.5 to 7.5. In a specific embodiment, the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester. In another specific embodiment, the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof. In yet another specific embodiment, the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester and the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof. In one embodiment, the pH of the composition is from 5.5 to 7.0. In another embodiment, the pH of the composition is from 5.5 to 6.5. In another embodiment, the pH of the composition is 6.0±0.2. In a specific embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.
In one embodiment, a stabilized furin (e.g., rfurin) composition comprises from 100 to 300 mM of a pharmaceutically acceptable salt, from 0.5 mM to 2 mM calcium; a combination of sugar or sugar alcohol and non-ionic water soluble sorbitan fatty acid ester selected from variations 1 to 6035 found in Table 1 to Table 9, from 10 mM to 200 mM buffering agent, and a pH from 5.5 to 7.5. In a specific embodiment, the non-ionic water soluble sorbitan fatty acid ester is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate). In another specific embodiment, the sugar or sugar alcohol is trehalose. In yet another specific embodiment, the non-ionic water soluble sorbitan fatty acid ester is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate) and the sugar or sugar alcohol is trehalose. In one embodiment, the pH of the composition is from 5.5 to 7.0. In another embodiment, the pH of the composition is from 5.5 to 6.5. In another embodiment, the pH of the composition is 6.0±0.2. In a specific embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.
In one embodiment, a stabilized furin (e.g., rfurin) composition comprises 190±50 mM of a pharmaceutically acceptable salt, from 0.5 mM to 2 mM calcium; 75±25% non-ionic surfactant, 90±25 mM buffering agent, and a pH from 5.5 to 7.5. In one embodiment, the storage stable furin composition further comprises from 2% to 20% sugar or sugar alcohol. In one embodiment, the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester. In a specific embodiment, the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate). In one embodiment, the pH of the composition is from 5.5 to 7.0. In another embodiment, the pH of the composition is from 5.5 to 6.5. In another embodiment, the pH of the composition is 6.0±0.2. In a specific embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin. In one specific embodiment, the composition contains 75±15 ppm non-ionic surfactant. In another specific embodiment, the composition contains 75±5 ppm non-ionic surfactant. In another specific embodiment, the composition contains 75 ppm non-ionic surfactant.
In one embodiment, a stabilized furin (e.g., rfurin) composition comprises 190±50 mM of a pharmaceutically acceptable salt, from 0.5 mM to 2 mM calcium; from 2% to 10% sugar or sugar alcohol, 75±25 ppm non-ionic surfactant, 90±25 mM buffering agent, and a pH from 5.5 to 7.5. In one embodiment, the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester. In a specific embodiment, the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate). In one embodiment, the pH of the composition is from 5.5 to 7.0. In another embodiment, the pH of the composition is from 5.5 to 6.5. In another embodiment, the pH of the composition is 6.0±0.2. In a specific embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin. In one specific embodiment, the composition contains 75±15 ppm non-ionic surfactant. In another specific embodiment, the composition contains 75±5 ppm non-ionic surfactant. In another specific embodiment, the composition contains 75 ppm non-ionic surfactant.
In one embodiment, a stabilized furin (e.g., rfurin) composition comprises 190±50 mM of a pharmaceutically acceptable salt, from 0.5 mM to 2 mM calcium; from 2% to 10% sugar or sugar alcohol, 75±25 ppm non-ionic water soluble sorbitan fatty acid ester, 90±25 mM buffering agent, and a pH from 5.5 to 7.5. In a specific embodiment, the non-ionic water soluble sorbitan fatty acid ester is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate). In another specific embodiment, the sugar or sugar alcohol is trehalose. In yet another specific embodiment, the non-ionic water soluble sorbitan fatty acid ester is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate) and the sugar or sugar alcohol is trehalose. In one embodiment, the pH of the composition is from 5.5 to 7.0. In another embodiment, the pH of the composition is from 5.5 to 6.5. In another embodiment, the pH of the composition is 6.0±0.2. In a specific embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin. In one specific embodiment, the composition contains 75±15 ppm non-ionic water soluble sorbitan fatty acid ester. In another specific embodiment, the composition contains 75±5 ppm non-ionic water soluble sorbitan fatty acid ester. In another specific embodiment, the composition contains 75 ppm non-ionic water soluble sorbitan fatty acid ester.
In one embodiment, a stabilized furin (e.g., rfurin) composition comprises 190±50 mM of a pharmaceutically acceptable salt, from 0.5 mM to 2 mM calcium; a combination of sugar or sugar alcohol and non-ionic surfactant selected from variations 1 to 6035 found in Table 1 to Table 9, 90±25 mM buffering agent, and a pH from 5.5 to 7.5. In a specific embodiment, the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester. In another specific embodiment, the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof. In yet another specific embodiment, the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester and the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof. In one embodiment, the pH of the composition is from 5.5 to 7.0. In another embodiment, the pH of the composition is from 5.5 to 6.5. In another embodiment, the pH of the composition is 6.0±0.2. In a specific embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.
In one embodiment, a stabilized furin (e.g., rfurin) composition comprises 190±50 mM of a pharmaceutically acceptable salt, from 0.5 mM to 2 mM calcium; a combination of sugar or sugar alcohol and non-ionic water soluble sorbitan fatty acid ester selected from variations 1 to 6035 found in Table 1 to Table 9, from 90±25 mM buffering agent, and a pH from 5.5 to 7.5. In a specific embodiment, the non-ionic water soluble sorbitan fatty acid ester is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate). In another specific embodiment, the sugar or sugar alcohol is trehalose. In yet another specific embodiment, the non-ionic water soluble sorbitan fatty acid ester is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate) and the sugar or sugar alcohol is trehalose. In one embodiment, the pH of the composition is from 5.5 to 7.0. In another embodiment, the pH of the composition is from 5.5 to 6.5. In another embodiment, the pH of the composition is 6.0±0.2. In a specific embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.
In one embodiment, a stabilized furin (e.g., rfurin) composition comprises 190 mM of a pharmaceutically acceptable salt, 0.9 mM calcium; 75±25% non-ionic surfactant, 91 mM buffering agent, and a pH of 6.0±0.2. In one embodiment, the storage stable furin composition further comprises from 2% to 10% sugar or sugar alcohol. In one embodiment, the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester. In a specific embodiment, the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate). In one embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin. In one specific embodiment, the composition contains 75±15 ppm non-ionic surfactant. In another specific embodiment, the composition contains 75±5 ppm non-ionic surfactant. In another specific embodiment, the composition contains 75 ppm non-ionic surfactant.
In one embodiment, a stabilized furin (e.g., rfurin) composition comprises 190 mM of a pharmaceutically acceptable salt, 0.9 mM calcium; 10% sugar or sugar alcohol, 75±25 ppm non-ionic surfactant, 91 mM buffering agent, and a pH from of 6.0±0.2. In one embodiment, the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester. In a specific embodiment, the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate). In one embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin. In one specific embodiment, the composition contains 75±15 ppm non-ionic surfactant. In another specific embodiment, the composition contains 75±5 ppm non-ionic surfactant. In another specific embodiment, the composition contains 75 ppm non-ionic surfactant.
In one embodiment, a stabilized furin (e.g., rfurin) composition comprises 190 mM of a pharmaceutically acceptable salt, 0.9 mM calcium; 10% sugar or sugar alcohol, 75±25 ppm non-ionic water soluble sorbitan fatty acid ester, 91 mM buffering agent, and a pH of 6.0±0.2. In one embodiment, the pH of the composition is 6.0. In a specific embodiment, the non-ionic water soluble sorbitan fatty acid ester is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate). In another specific embodiment, the sugar or sugar alcohol is trehalose. In yet another specific embodiment, the non-ionic water soluble sorbitan fatty acid ester is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate) and the sugar or sugar alcohol is trehalose. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin. In one specific embodiment, the composition contains 75±15 ppm non-ionic water soluble sorbitan fatty acid ester. In another specific embodiment, the composition contains 75±5 ppm non-ionic water soluble sorbitan fatty acid ester. In another specific embodiment, the composition contains 75 ppm non-ionic water soluble sorbitan fatty acid ester.
In one embodiment, a stabilized furin (e.g., rfurin) composition comprises 190 mM of a pharmaceutically acceptable salt, 0.9 mM calcium; a combination of sugar or sugar alcohol and non-ionic surfactant selected from variations 1 to 6035 found in Table 1 to Table 9, 91 mM buffering agent, and a pH of 6.0±0.2. In a specific embodiment, the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester. In another specific embodiment, the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof. In yet another specific embodiment, the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester and the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof. In one embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.
In one embodiment, a stabilized furin (e.g., rfurin) composition comprises 190 mM of a pharmaceutically acceptable salt, 0.9 mM calcium; a combination of sugar or sugar alcohol and non-ionic water soluble sorbitan fatty acid ester selected from variations 1 to 6035 found in Table 1 to Table 9, 91 mM buffering agent, and a pH of 6.0±0.2. In a specific embodiment, the non-ionic water soluble sorbitan fatty acid ester is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate). In another specific embodiment, the sugar or sugar alcohol is trehalose. In yet another specific embodiment, the non-ionic water soluble sorbitan fatty acid ester is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate) and the sugar or sugar alcohol is trehalose. In one embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.
The furin (e.g., rfurin) compositions of the present invention also preferably include a surfactant, preferably a non-ionic surfactant, and preferably in an amount from 0.1 ppm to 150 ppm, or from 65 ppm to 80 ppm, or at about 75 ppm. In further embodiments, the furin (e.g., rfurin) compositions include a surfactant in an amount of from 0.5 ppm to 140 ppm, 1.0 ppm to 130 ppm, 10 ppm to 120 ppm, 20 ppm to 110 ppm, 30 ppm to 100 ppm, 40 ppm to 95 ppm, 50 ppm to 90 ppm, 55 ppm to 85 ppm, 60 ppm to 80 ppm, or 70 ppm to 75 ppm. In certain embodiments, the surfactant is chosen from the group consisting of polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, pluronic polyols, glycerol, glucamides (such as Mega 8), tritons, and Brij 35 (polyoxyethylene 23 lauryl ether). Several grades of pluronic polyols (sold under the trade name Pluronic, manufactured by the BASF Wyandotte Corporation) are available. These polyols, of diversified molecular weight (from 1,000 to over 16,000) and physicochemical properties have been used as surfactants. Pluronic F-38, of a molecular weight of 5,000 and Pluronic F-68, molecular weight 9,000, both contain (by weight) 80 percent hydrophilic polyoxyethylene groups and 20 percent hydrophobic polyoxypropylene groups. In one embodiment, the surfactant is polysorbate 80. In a particular embodiment, the polysorbate 80 is vegetable-derived polysorbate 80.
B. Pharmaceutically Acceptable Salts
The stabilized compositions of furin (e.g., rfurin) provided herein generally include a pharmaceutically acceptable salt at a concentration tolerated by the furin polypeptide during storage. In one embodiment, the pharmaceutically acceptable salt is a chloride salt. In a specific embodiment, the pharmaceutically acceptable salt is a monovalent chloride salt. In a more specific embodiment, the pharmaceutically acceptable salt is sodium chloride, potassium chloride, or a combination thereof.
In one embodiment, the concentration of pharmaceutically acceptable salt in a stabilized furin (e.g., rfurin) composition provided herein is from 10 mM to 500 mM. In another embodiment, the concentration of pharmaceutically acceptable salt is from 100 mM to 300 mM. In another embodiment, the concentration of pharmaceutically acceptable salt is from 150 mM to 250 mM. In yet other embodiments, the concentration of pharmaceutically acceptable salt in the stabilized furin (e.g., rfurin) compositions provided herein is selected from variations 6036 to 6180 found in Table 10.







TABLE 10





Exemplary pharmaceutically acceptable salt concentrations useful


for the stabilization of furin (e.g., rfurin) compositions.




















 10 mM to 500 mM
Var. 6036
140 ± 5 mM
Var. 6085
179 mM
Var. 6134


100 mM to 300 mM
Var. 6037
145 ± 5 mM
Var. 6086
180 mM
Var. 6135


150 mM to 250 mM
Var. 6038
150 ± 5 mM
Var. 6087
181 mM
Var. 6136


 50 mM to 500 mM
Var. 6039
155 ± 5 mM
Var. 6088
182 mM
Var. 6137


 50 mM to 450 mM
Var. 6040
160 ± 5 mM
Var. 6089
183 mM
Var. 6138


 50 mM to 400 mM
Var. 6041
165 ± 5 mM
Var. 6090
184 mM
Var. 6139


   100 to 400 mM
Var. 6042
170 ± 5 mM
Var. 6091
185 mM
Var. 6140


100 mM to 350 mM
Var. 6043
175 ± 5 mM
Var. 6092
186 mM
Var. 6141


100 mM to 250 mM
Var. 6044
180 ± 5 mM
Var. 6093
187 mM
Var. 6142


100 mM to 200 mM
Var. 6045
185 ± 5 mM
Var. 6094
188 mM
Var. 6143


150 mM to 400 mM
Var. 6046
186 ± 5 mM
Var. 6095
189 mM
Var. 6144


150 mM to 350 mM
Var. 6047
187 ± 5 mM
Var. 6096
190 mM
Var. 6145


150 mM to 300 mM
Var. 6048
188 ± 5 mM
Var. 6097
191 mM
Var. 6146


150 mM to 250 mM
Var. 6049
189 ± 5 mM
Var. 6098
192 mM
Var. 6147


150 mM to 200 mM
Var. 6050
190 ± 5 mM
Var. 6099
193 mM
Var. 6148


175 mM to 225 mM
Var. 6051
191 ± 5 mM
Var. 6100
194 mM
Var. 6149


175 mM to 200 mM
Var. 6052
192 ± 5 mM
Var. 6101
195 mM
Var. 6150


100 ± 25 mM
Var. 6053
193 ± 5 mM
Var. 6102
196 mM
Var. 6151


150 ± 25 mM
Var. 6054
194 ± 5 mM
Var. 6103
197 mM
Var. 6152


200 ± 25 mM
Var. 6055
195 ± 5 mM
Var. 6104
198 mM
Var. 6153


250 ± 25 mM
Var. 6056
196 ± 5 mM
Var. 6105
199 mM
Var. 6154


300 ± 25 mM
Var. 6057
197 ± 5 mM
Var. 6106
200 mM
Var. 6155


350 ± 25 mM
Var. 6058
198 ± 5 mM
Var. 6107
201 mM
Var. 6156


400 ± 25 mM
Var. 6059
199 ± 5 mM
Var. 6108
202 mM
Var. 6157


450 ± 25 mM
Var. 6060
200 ± 5 mM
Var. 6109
203 mM
Var. 6158


190 ± 150 mM
Var. 6061
205 ± 5 mM
Var. 6110
204 mM
Var. 6159


190 ± 125 mM
Var. 6062
210 ± 5 mM
Var. 6111
205 mM
Var. 6160


190 ± 100 mM
Var. 6063
215 ± 5 mM
Var. 6112
206 mM
Var. 6161


190 ± 90 mM
Var. 6064
220 ± 5 mM
Var. 6113
207 mM
Var. 6162


190 ± 80 mM
Var. 6065
225 ± 5 mM
Var. 6114
208 mM
Var. 6163


190 ± 70 mM
Var. 6066
230 ± 5 mM
Var. 6115
209 mM
Var. 6164


190 ± 60 mM
Var. 6067
235 ± 5 mM
Var. 6116
210 mM
Var. 6165


190 ± 50 mM
Var. 6068
240 ± 5 mM
Var. 6117
211 mM
Var. 6166


190 ± 40 mM
Var. 6069
245 ± 5 mM
Var. 6118
212 mM
Var. 6167


190 ± 30 mM
Var. 6070
250 ± 5 mM
Var. 6119
213 mM
Var. 6168


190 ± 25 mM
Var. 6071
255 ± 5 mM
Var. 6120
214 mM
Var. 6169


190 ± 20 mM
Var. 6072
260 ± 5 mM
Var. 6121
215 mM
Var. 6170


190 ± 15 mM
Var. 6073
265 ± 5 mM
Var. 6122
216 mM
Var. 6171


190 ± 10 mM
Var. 6074
270 ± 5 mM
Var. 6123
217 mM
Var. 6172


190 ± 5 mM
Var. 6075
275 ± 5 mM
Var. 6124
218 mM
Var. 6173


190 ± 2 mM
Var. 6076
280 ± 5 mM
Var. 6125
219 mM
Var. 6174


100 ± 5 mM
Var. 6077
285 ± 5 mM
Var. 6126
220 mM
Var. 6175


105 ± 5 mM
Var. 6078
290 ± 5 mM
Var. 6127
221 mM
Var. 6176


110 ± 5 mM
Var. 6079
295 ± 5 mM
Var. 6128
222 mM
Var. 6177


115 ± 5 mM
Var. 6080
300 ± 5 mM
Var. 6129
223 mM
Var. 6178


120 ± 5 mM
Var. 6081
175 mM
Var. 6130
224 mM
Var. 6179


125 ± 5 mM
Var. 6082
176 mM
Var. 6131
225 mM
Var. 6180


130 ± 5 mM
Var. 6083
177 mM
Var. 6132


135 ± 5 mM
Var. 6084
178 mM
Var. 6133





Var. = Variation






In one embodiment, a stabilized furin (e.g., rfurin) composition comprises a pharmaceutically acceptable salt at a concentration selected from variations 6036 to 6180 found in Table 10, from 0.5 mM to 5 mM calcium, from 10 ppm to 200 ppm non-ionic surfactant, from 10 to 200 mM buffering agent, and a pH from 5.5 to 7.5. In one embodiment, the storage stable furin composition further comprises from 2% to 20% sugar or sugar alcohol. In one embodiment, the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester. In a specific embodiment, the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate). In one embodiment, the pharmaceutically acceptable salt is sodium chloride, potassium chloride, or a combination thereof. In a specific embodiment, pharmaceutically acceptable salt is sodium chloride. In another specific embodiment, pharmaceutically acceptable salt is potassium chloride. In one embodiment, the pH of the composition is from 5.5 to 7.0. In another embodiment, the pH of the composition is from 5.5 to 6.5. In another embodiment, the pH of the composition is 6.0±0.2. In a specific embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.
In one embodiment, a stabilized furin (e.g., rfurin) composition comprises a pharmaceutically acceptable salt at a concentration selected from variations 6036 to 6180 found in Table 10, from 0.5 mM to 5 mM calcium, from 2% to 20% sugar or sugar alcohol, from 10 to 200 mM buffering agent, and a pH from 5.5 to 7.5. In one embodiment, the storage stable furin composition further comprises from 10 ppm to 200 ppm non-ionic surfactant. In one embodiment, the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof. In a specific embodiment, the sugar or sugar alcohol is trehalose. In one embodiment, the pharmaceutically acceptable salt is sodium chloride, potassium chloride, or a combination thereof. In a specific embodiment, pharmaceutically acceptable salt is sodium chloride. In another specific embodiment, pharmaceutically acceptable salt is potassium chloride. In one embodiment, the pH of the composition is from 5.5 to 7.0. In another embodiment, the pH of the composition is from 5.5 to 6.5. In another embodiment, the pH of the composition is 6.0±0.2. In a specific embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.
In one embodiment, a stabilized furin (e.g., rfurin) composition comprises a pharmaceutically acceptable salt at a concentration selected from variations 6036 to 6180 found in Table 10, from 0.5 mM to 5 mM calcium, from 2% to 20% sugar or sugar alcohol, from 10 ppm to 200 ppm non-ionic surfactant, from 10 to 200 mM buffering agent, and a pH from 5.5 to 7.5. In one embodiment, the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester. In a specific embodiment, the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate). In one embodiment, the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof. In a specific embodiment, the sugar or sugar alcohol is trehalose. In a specific embodiment, the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate) and the sugar or sugar alcohol is trehalose. In one embodiment, the pharmaceutically acceptable salt is sodium chloride, potassium chloride, or a combination thereof. In a specific embodiment, pharmaceutically acceptable salt is sodium chloride. In another specific embodiment, pharmaceutically acceptable salt is potassium chloride. In one embodiment, the pH of the composition is from 5.5 to 7.0. In another embodiment, the pH of the composition is from 5.5 to 6.5. In another embodiment, the pH of the composition is 6.0±0.2. In a specific embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.
In one embodiment, a stabilized furin (e.g., rfurin) composition comprises a pharmaceutically acceptable salt at a concentration selected from variations 6036 to 6180 found in Table 10, from 0.5 mM to 5 mM calcium, from 2% to 10% sugar or sugar alcohol, from 10 ppm to 100 ppm non-ionic surfactant, from 10 to 200 mM buffering agent, and a pH from 5.5 to 7.5. In one embodiment, the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester. In a specific embodiment, the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate). In one embodiment, the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof. In a specific embodiment, the sugar or sugar alcohol is trehalose. In a specific embodiment, the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate) and the sugar or sugar alcohol is trehalose. In one embodiment, the pharmaceutically acceptable salt is sodium chloride, potassium chloride, or a combination thereof. In a specific embodiment, pharmaceutically acceptable salt is sodium chloride. In another specific embodiment, pharmaceutically acceptable salt is potassium chloride. In one embodiment, the pH of the composition is from 5.5 to 7.0. In another embodiment, the pH of the composition is from 5.5 to 6.5. In another embodiment, the pH of the composition is 6.0±0.2. In a specific embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.
In one embodiment, a stabilized furin (e.g., rfurin) composition comprises a pharmaceutically acceptable salt at a concentration selected from variations 6036 to 6180 found in Table 10, from 0.5 mM to 2 mM calcium, 10±5% sugar or sugar alcohol, 75±25 ppm non-ionic surfactant, from 10 to 200 mM buffering agent, and a pH from 5.5 to 7.5. In one embodiment, the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester. In a specific embodiment, the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate). In one embodiment, the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof. In a specific embodiment, the sugar or sugar alcohol is trehalose. In a specific embodiment, the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate) and the sugar or sugar alcohol is trehalose. In one embodiment, the pharmaceutically acceptable salt is sodium chloride, potassium chloride, or a combination thereof. In a specific embodiment, pharmaceutically acceptable salt is sodium chloride. In another specific embodiment, pharmaceutically acceptable salt is potassium chloride. In one embodiment, the concentration of the buffering agent is 90±25 mM. In one embodiment, the pH of the composition is from 5.5 to 7.0. In another embodiment, the pH of the composition is from 5.5 to 6.5. In another embodiment, the pH of the composition is 6.0±0.2. In a specific embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.
In one embodiment, a stabilized furin (e.g., rfurin) composition comprises a pharmaceutically acceptable salt at a concentration selected from variations 6036 to 6180 found in Table 10, from 0.9 mM calcium, 10% sugar or sugar alcohol, 75 ppm non-ionic surfactant, from 91 mM buffering agent, and a pH of 6.0±0.2. In one embodiment, the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester. In a specific embodiment, the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate). In one embodiment, the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof. In a specific embodiment, the sugar or sugar alcohol is trehalose. In a specific embodiment, the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate) and the sugar or sugar alcohol is trehalose. In one embodiment, the pharmaceutically acceptable salt is sodium chloride, potassium chloride, or a combination thereof. In a specific embodiment, pharmaceutically acceptable salt is sodium chloride. In another specific embodiment, pharmaceutically acceptable salt is potassium chloride. In a specific embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.
In some embodiments, monovalent chloride salts, such as sodium chloride and potassium chloride, are used in formulations of the present disclosure. Although the formulation component comprising monovalent chloride salts is described primarily in terms of sodium chloride, it will be appreciated that any monovalent chloride salt, including potassium chloride, may be used in accordance with the descriptions herein for sodium chloride. In certain embodiments, sodium chloride is included in the present formulations at an amount of from 50 to 500 mM. In other embodiments, sodium chloride is included in the formulations at an amount of from 100 to 300 mM, from 150 to 250 mM, or at about 190 mM. In further embodiments, sodium chloride is included in the present formulations in an amount of from 100 to 300 mM, 110 to 280 mM, 120 to 260 mM, 130 to 240 mM, 140 to 220 mM, 150 to 200 mM, or 160 to 180 mM. In a specific embodiment, the formulation includes 190 mM sodium chloride in combination with one or more of any other formulation components disclosed herein.
C. Buffering Agents
Advantageously, it was found that furin (e.g., rfurin) compositions are stabilized at pH 6.0 to 7.0. For example, it is shown in FIGS. 11 and 12 that furin compositions formulated at pH 5.5 are stabilized as compared to furin compositions formulated at pH 5.0. Also shown is that furin compositions formulated at pH 6.0 are further stabilized as compared to compositions formulated at pH 5.5. Furthermore, as shown in FIG. 17, furin (e.g., rfurin) compositions formulated at pH 7.0 have similar stability to compositions formulated at pH 6.0.
Accordingly, in certain embodiments, the stabilized furin (e.g., rfurin) compositions provided herein are formulated at a pH from 5.5 to 7.5. In a specific embodiment, a stabilized furin (e.g., rfurin) composition is formulated at a pH from 6.0 to 7.0. In another specific embodiment, a stabilized furin composition is formulated at pH 6.0±0.2. In a more specific embodiment, a stabilized furin composition is formulated at pH 6.0. In yet other embodiments, a stabilized furin composition is formulated at pH selected from variations 6181 to 6403 found in Table 11.







TABLE 11





Exemplary pH ranges useful for the stabilization of furin (e.g., rfurin) compositions.






















5.5-8.0
Var. 6181
5.7-6.3
Var. 6237
6.0-6.7
Var. 6293
5.9
Var. 6349


5.5-7.9
Var. 6182
5.7-6.2
Var. 6238
6.0-6.6
Var. 6294
6
Var. 6350


5.5-7.8
Var. 6183
5.7-6.1
Var. 6239
6.0-6.5
Var. 6295
6.1
Var. 6351


5.5-7.6
Var. 6184
5.7-6.0
Var. 6240
6.0-6.4
Var. 6296
6.2
Var. 6352


5.5-7.5
Var. 6185
5.8-8.0
Var. 6241
6.0-6.3
Var. 6297
6.3
Var. 6353


5.5-7.4
Var. 6186
5.8-7.9
Var. 6242
6.0-6.2
Var. 6298
6.4
Var. 6354


5.5-7.3
Var. 6187
5.8-7.8
Var. 6243
6.0-6.1
Var. 6299
6.5
Var. 6355


5.5-7.2
Var. 6188
5.8-7.6
Var. 6244
5.7 ± 0.2
Var. 6300
6.6
Var. 6356


5.5-7.1
Var. 6189
5.8-7.5
Var. 6245
5.8 ± 0.2
Var. 6301
6.7
Var. 6357


5.5-7.0
Var. 6190
5.8-7.4
Var. 6246
5.9 ± 0.2
Var. 6302
6.8
Var. 6358


5.5-6.9
Var. 6191
5.8-7.3
Var. 6247
6.0 ± 0.2
Var. 6303
6.9
Var. 6359


5.5-6.8
Var. 6192
5.8-7.2
Var. 6248
6.1 ± 0.2
Var. 6304
7
Var. 6360


5.5-6.7
Var. 6193
5.8-7.1
Var. 6249
6.2 ± 0.2
Var. 6305
7.1
Var. 6361


5.5-6.6
Var. 6194
5.8-7.0
Var. 6250
6.3 ± 0.2
Var. 6306
7.2
Var. 6362


5.5-6.5
Var. 6195
5.8-6.9
Var. 6251
6.4 ± 0.2
Var. 6307
7.3
Var. 6363


5.5-6.4
Var. 6196
5.8-6.8
Var. 6252
6.5 ± 0.2
Var. 6308
7.4
Var. 6364


5.5-6.3
Var. 6197
5.8-6.7
Var. 6253
6.6 ± 0.2
Var. 6309
7.5
Var. 6365


5.5-6.2
Var. 6198
5.8-6.6
Var. 6254
6.7 ± 0.2
Var. 6310
7.6
Var. 6366


5.5-6.1
Var. 6199
5.8-6.5
Var. 6255
6.8 ± 0.2
Var. 6311
7.7
Var. 6367


5.5-6.0
Var. 6200
5.8-6.4
Var. 6256
6.9 ± 0.2
Var. 6312
7.8
Var. 6368


5.6-8.0
Var. 6201
5.8-6.3
Var. 6257
7.0 ± 0.2
Var. 6313
7.9
Var. 6369


5.6-7.9
Var. 6202
5.8-6.2
Var. 6258
7.1 ± 0.2
Var. 6314
8
Var. 6370


5.6-7.8
Var. 6203
5.8-6.1
Var. 6259
7.2 ± 0.2
Var. 6315
6.0 ± 0.3
Var. 6371


5.6-7.6
Var. 6204
5.8-6.0
Var. 6260
7.3 ± 0.2
Var. 6316
6.0 ± 0.4
Var. 6372


5.6-7.5
Var. 6205
5.9-8.0
Var. 6261
7.4 ± 0.2
Var. 6317
6.0 ± 0.5
Var. 6373


5.6-7.4
Var. 6206
5.9-7.9
Var. 6262
7.5 ± 0.2
Var. 6318
6.1 ± 0.3
Var. 6374


5.6-7.3
Var. 6207
5.9-7.8
Var. 6263
7.6 ± 0.2
Var. 6319
6.1 ± 0.4
Var. 6375


5.6-7.2
Var. 6208
5.9-7.6
Var. 6264
7.7 ± 0.2
Var. 6320
6.1 ± 0.5
Var. 6376


5.6-7.1
Var. 6209
5.9-7.5
Var. 6265
7.8 ± 0.2
Var. 6321
6.2 ± 0.3
Var. 6377


5.6-7.0
Var. 6210
5.9-7.4
Var. 6266
5.6 ± 0.1
Var. 6322
6.2 ± 0.4
Var. 6378


5.6-6.9
Var. 6211
5.9-7.3
Var. 6267
5.7 ± 0.1
Var. 6323
6.2 ± 0.5
Var. 6379


5.6-6.8
Var. 6212
5.9-7.2
Var. 6268
5.8 ± 0.1
Var. 6324
6.3 ± 0.3
Var. 6380


5.6-6.7
Var. 6213
5.9-7.1
Var. 6269
5.9 ± 0.1
Var. 6325
6.3 ± 0.4
Var. 6381


5.6-6.6
Var. 6214
5.9-7.0
Var. 6270
6.0 ± 0.1
Var. 6326
6.3 ± 0.5
Var. 6382


5.6-6.5
Var. 6215
5.9-6.9
Var. 6271
6.1 ± 0.1
Var. 6327
6.4 ± 0.3
Var. 6383


5.6-6.4
Var. 6216
5.9-6.8
Var. 6272
6.2 ± 0.1
Var. 6328
6.4 ± 0.4
Var. 6384


5.6-6.3
Var. 6217
5.9-6.7
Var. 6273
6.3 ± 0.1
Var. 6329
6.4 ± 0.5
Var. 6385


5.6-6.2
Var. 6218
5.9-6.6
Var. 6274
6.4 ± 0.1
Var. 6330
6.5 ± 0.3
Var. 6386


5.6-6.1
Var. 6219
5.9-6.5
Var. 6275
6.5 ± 0.1
Var. 6331
6.5 ± 0.4
Var. 6387


5.6-6.0
Var. 6220
5.9-6.4
Var. 6276
6.6 ± 0.1
Var. 6332
6.5 ± 0.5
Var. 6388


5.7-8.0
Var. 6221
5.9-6.3
Var. 6277
6.7 ± 0.1
Var. 6333
6.6 ± 0.3
Var. 6389


5.7-7.9
Var. 6222
5.9-6.2
Var. 6278
6.8 ± 0.1
Var. 6334
6.6 ± 0.4
Var. 6390


5.7-7.8
Var. 6223
5.9-6.1
Var. 6279
6.9 ± 0.1
Var. 6335
6.6 ± 0.5
Var. 6391


5.7-7.6
Var. 6224
5.9-6.0
Var. 6280
7.0 ± 0.1
Var. 6336
6.7 ± 0.3
Var. 6392


5.7-7.5
Var. 6225
6.0-8.0
Var. 6281
7.1 ± 0.1
Var. 6337
6.7 ± 0.4
Var. 6393


5.7-7.4
Var. 6226
6.0-7.9
Var. 6282
7.2 ± 0.1
Var. 6338
6.7 ± 0.5
Var. 6394


5.7-7.3
Var. 6227
6.0-7.8
Var. 6283
7.3 ± 0.1
Var. 6339
6.8 ± 0.3
Var. 6395


5.7-7.2
Var. 6228
6.0-7.6
Var. 6284
7.4 ± 0.1
Var. 6340
6.8 ± 0.4
Var. 6396


5.7-7.1
Var. 6229
6.0-7.5
Var. 6285
7.5 ± 0.1
Var. 6341
6.8 ± 0.5
Var. 6397


5.7-7.0
Var. 6230
6.0-7.4
Var. 6286
7.6 ± 0.1
Var. 6342
6.9 ± 0.3
Var. 6398


5.7-6.9
Var. 6231
6.0-7.3
Var. 6287
7.7 ± 0.1
Var. 6343
6.9 ± 0.4
Var. 6399


5.7-6.8
Var. 6232
6.0-7.2
Var. 6288
7.8 ± 0.1
Var. 6344
6.9 ± 0.5
Var. 6400


5.7-6.7
Var. 6233
6.0-7.1
Var. 6289
5.5
Var. 6345
7.0 ± 0.3
Var. 6401


5.7-6.6
Var. 6234
6.0-7.0
Var. 6290
5.6
Var. 6346
7.0 ± 0.4
Var. 6402


5.7-6.5
Var. 6235
6.0-6.9
Var. 6291
5.7
Var. 6347
7.0 ± 0.5
Var. 6403


5.7-6.4
Var. 6236
6.0-6.8
Var. 6292
5.8
Var. 6348





Var. = Variation






In one embodiment, a stabilized furin (e.g., rfurin) composition comprises from 50 to 500 mM of a pharmaceutically acceptable salt, from 0.5 mM to 5 mM calcium, from 10 ppm to 200 ppm non-ionic surfactant, from 10 to 200 mM buffering agent, and a pH selected from variations 6181 to 6403 found in Table 11. In one embodiment, the storage stable furin composition further comprises from 2% to 20% sugar or sugar alcohol. In one embodiment, the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester. In a specific embodiment, the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate). In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.
In one embodiment, a stabilized furin (e.g., rfurin) composition comprises from 50 to 500 mM of a pharmaceutically acceptable salt, from 0.5 mM to 5 mM calcium, from 2% to 20% sugar or sugar alcohol, from 10 to 200 mM buffering agent, and a pH selected from variations 6181 to 6403 found in Table 11. In one embodiment, the storage stable furin composition further comprises from 10 ppm to 200 ppm non-ionic surfactant. In one embodiment, the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof. In a specific embodiment, the sugar or sugar alcohol is trehalose. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.
In one embodiment, a stabilized furin (e.g., rfurin) composition comprises from 50 to 500 mM of a pharmaceutically acceptable salt, from 0.5 mM to 5 mM calcium, from 2% to 20% sugar or sugar alcohol, from 10 ppm to 200 ppm non-ionic surfactant, from 10 to 200 mM buffering agent, and a pH selected from variations 6181 to 6403 found in Table 11. In one embodiment, the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester. In a specific embodiment, the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate). In one embodiment, the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof. In a specific embodiment, the sugar or sugar alcohol is trehalose. In a specific embodiment, the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate) and the sugar or sugar alcohol is trehalose. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.
In one embodiment, a stabilized furin (e.g., rfurin) composition comprises from 50 to 500 mM of a pharmaceutically acceptable salt, from 0.5 mM to 5 mM calcium, from 2% to 10% sugar or sugar alcohol, from 10 ppm to 100 ppm non-ionic surfactant, from 10 to 200 mM buffering agent, and a pH selected from variations 6181 to 6403 found in Table 11. In one embodiment, the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester. In a specific embodiment, the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate). In one embodiment, the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof. In a specific embodiment, the sugar or sugar alcohol is trehalose. In a specific embodiment, the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate) and the sugar or sugar alcohol is trehalose. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.
In one embodiment, a stabilized furin (e.g., rfurin) composition comprises from 50 to 300 mM of a pharmaceutically acceptable salt, from 0.5 mM to 2 mM calcium, from 2% to 10% sugar or sugar alcohol, from 10 ppm to 100 ppm non-ionic surfactant, from 10 to 200 mM buffering agent, and a pH selected from variations 6181 to 6403 found in Table 11. In one embodiment, the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester. In a specific embodiment, the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate). In one embodiment, the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof. In a specific embodiment, the sugar or sugar alcohol is trehalose. In a specific embodiment, the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate) and the sugar or sugar alcohol is trehalose. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.
In one embodiment, a stabilized furin (e.g., rfurin) composition comprises from 50 to 300 mM of a pharmaceutically acceptable salt, from 0.5 mM to 2 mM calcium, 10±5% sugar or sugar alcohol, 75±25 ppm non-ionic surfactant, from 10 to 200 mM buffering agent, and a pH selected from variations 6181 to 6403 found in Table 11. In one embodiment, the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester. In a specific embodiment, the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate). In one embodiment, the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof. In a specific embodiment, the sugar or sugar alcohol is trehalose. In a specific embodiment, the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate) and the sugar or sugar alcohol is trehalose. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.
In one embodiment, a stabilized furin (e.g., rfurin) composition comprises 190±50 mM of a pharmaceutically acceptable salt, from 0.5 mM to 2 mM calcium, 10±5% sugar or sugar alcohol, 75±25 ppm non-ionic surfactant, from 90±25 mM buffering agent, and a pH selected from variations 6181 to 6403 found in Table 11. In one embodiment, the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester. In a specific embodiment, the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate). In one embodiment, the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof. In a specific embodiment, the sugar or sugar alcohol is trehalose. In a specific embodiment, the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate) and the sugar or sugar alcohol is trehalose. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.
In one embodiment, a stabilized furin (e.g., rfurin) composition comprises 191 mM of a pharmaceutically acceptable salt, 0.9 mM calcium, 10% sugar or sugar alcohol, 75 ppm non-ionic surfactant, 91 mM buffering agent, and a pH selected from variations 6181 to 6403 found in Table 11. In one embodiment, the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester. In a specific embodiment, the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate). In one embodiment, the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof. In a specific embodiment, the sugar or sugar alcohol is trehalose. In a specific embodiment, the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate) and the sugar or sugar alcohol is trehalose. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.
In one embodiment, the stabilized furin (e.g., rfurin) compositions provided herein are formulated at an appropriate pH using one or more buffering agents. In one embodiment, the one or more buffering agent is selected from histidine, imidazole, phosphate, citrate, Tris, acetate (e.g., acetic acid), BIS-Tris Propane, PIPES, MOPS, HEPES, MES, ACES, and a combination thereof. In a particular embodiment, the buffering agent is acetate, HEPES, or a combination thereof. In a specific embodiment, the buffering agent is a combination of acetate and HEPES.
Buffering agent is present in the stabilized furin (e.g., rfurin) compositions at a concentration suitable to maintain the pH of the composition during storage over a period of time (e.g., weeks, months, or years). In one embodiment, the concentration of buffering agent in the formulation is from 10 mM to 300 mM. In another embodiment, the concentration of buffering agent in the formulation is between 10 mM to 200 mM. In a specific embodiment, the concentration of buffering agent in the formulation is 90±25 mM. In yet other embodiments, the concentration of buffering agent in the composition is selected from variations 6404 to 6469 found in Table 12.







TABLE 12





Exemplary buffering agent concentrations (mM) useful for


the stabilization of furin (e.g., rfurin) compositions.
















10-300
Var. 6404


10-275
Var. 6405


10-250
Var. 6406


10-225
Var. 6407


10-200
Var. 6408


10-175
Var. 6409


10-150
Var. 6410


10-125
Var. 6411


10-100
Var. 6412


25-300
Var. 6413


25-275
Var. 6414


25-250
Var. 6415


25-225
Var. 6416


25-200
Var. 6417


25-175
Var. 6418


25-150
Var. 6419


25-125
Var. 6420


25-100
Var. 6421


50-300
Var. 6422


50-275
Var. 6423


50-250
Var. 6424


50-225
Var. 6425


50-200
Var. 6426


50-175
Var. 6427


50-150
Var. 6428


50-125
Var. 6429


50-100
Var. 6430


75-300
Var. 6431


75-275
Var. 6432


75-250
Var. 6433


75-225
Var. 6434


75-200
Var. 6435


75-175
Var. 6436


75-150
Var. 6437


75-125
Var. 6438


75-100
Var. 6439


90 ± 75
Var. 6440


90 ± 50
Var. 6441


90 ± 25
Var. 6442


90 ± 10
Var. 6443


90 ± 5 
Var. 6444


90 ± 2 
Var. 6445


91 ± 10
Var. 6446


91 ± 5 
Var. 6447


91 ± 2 
Var. 6448


80
Var. 6449


81
Var. 6450


82
Var. 6451


83
Var. 6452


84
Var. 6453


85
Var. 6454


86
Var. 6455


87
Var. 6456


88
Var. 6457


89
Var. 6458


90
Var. 6459


91
Var. 6460


92
Var. 6461


93
Var. 6462


94
Var. 6463


95
Var. 6464


96
Var. 6465


97
Var. 6466


98
Var. 6467


99
Var. 6468


100 
Var. 6469





Var. = Variation






In one embodiment, a stabilized furin (e.g., rfurin) composition comprises from 50 to 500 mM of a pharmaceutically acceptable salt, from 0.5 mM to 5 mM calcium, from 10 ppm to 200 ppm non-ionic surfactant, buffering agent at a concentration selected from variations 6404 to 6469 found in Table 12, and a pH from 5.5 to 7.5. In one embodiment, the storage stable furin composition further comprises from 2% to 20% sugar or sugar alcohol. In one embodiment, the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester. In a specific embodiment, the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate). In one embodiment, the buffering agent is acetate, HEPES, or a combination thereof. In a specific embodiment, the buffering agent is a combination of acetate and HEPES. In one embodiment, the pH of the composition is from 5.5 to 7.0. In another embodiment, the pH of the composition is from 5.5 to 6.5. In another embodiment, the pH of the composition is 6.0±0.2. In a specific embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.
In one embodiment, a stabilized furin (e.g., rfurin) composition comprises from 50 to 500 mM of a pharmaceutically acceptable salt, from 0.5 mM to 5 mM calcium, from 2% to 20% sugar or sugar alcohol, buffering agent at a concentration selected from variations 6404 to 6469 found in Table 12, and a pH from 5.5 to 7.5. In one embodiment, the storage stable furin composition further comprises from 10 ppm to 200 ppm non-ionic surfactant. In one embodiment, the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof. In a specific embodiment, the sugar or sugar alcohol is trehalose. In one embodiment, the buffering agent is acetate, HEPES, or a combination thereof. In a specific embodiment, the buffering agent is a combination of acetate and HEPES. In one embodiment, the pH of the composition is from 5.5 to 7.0. In another embodiment, the pH of the composition is from 5.5 to 6.5. In another embodiment, the pH of the composition is 6.0±0.2. In a specific embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.
In one embodiment, a stabilized furin (e.g., rfurin) composition comprises from 50 to 500 mM of a pharmaceutically acceptable salt, from 0.5 mM to 5 mM calcium, from 2% to 20% sugar or sugar alcohol, from 10 ppm to 200 ppm non-ionic surfactant, buffering agent at a concentration selected from variations 6404 to 6469 found in Table 12, and a pH from 5.5 to 7.5. In one embodiment, the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester. In a specific embodiment, the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate). In one embodiment, the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof. In a specific embodiment, the sugar or sugar alcohol is trehalose. In a specific embodiment, the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate) and the sugar or sugar alcohol is trehalose. In one embodiment, the buffering agent is acetate, HEPES, or a combination thereof. In a specific embodiment, the buffering agent is a combination of acetate and HEPES. In one embodiment, the pH of the composition is from 5.5 to 7.0. In another embodiment, the pH of the composition is from 5.5 to 6.5. In another embodiment, the pH of the composition is 6.0±0.2. In a specific embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.
In one embodiment, a stabilized furin (e.g., rfurin) composition comprises from 50 to 500 mM of a pharmaceutically acceptable salt, from 0.5 mM to 5 mM calcium, from 2% to 10% sugar or sugar alcohol, from 10 ppm to 100 ppm non-ionic surfactant, buffering agent at a concentration selected from variations 6404 to 6469 found in Table 12, and a pH from 5.5 to 7.5. In one embodiment, the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester. In a specific embodiment, the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate). In one embodiment, the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof. In a specific embodiment, the sugar or sugar alcohol is trehalose. In a specific embodiment, the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate) and the sugar or sugar alcohol is trehalose. In one embodiment, the buffering agent is acetate, HEPES, or a combination thereof. In a specific embodiment, the buffering agent is a combination of acetate and HEPES. In one embodiment, the pH of the composition is from 5.5 to 7.0. In another embodiment, the pH of the composition is from 5.5 to 6.5. In another embodiment, the pH of the composition is 6.0±0.2. In a specific embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.
In one embodiment, a stabilized furin (e.g., rfurin) composition comprises from 50 to 300 mM of a pharmaceutically acceptable salt, from 0.5 mM to 2 mM calcium, from 2% to 10% sugar or sugar alcohol, from 10 ppm to 100 ppm non-ionic surfactant, buffering agent at a concentration selected from variations 6404 to 6469 found in Table 12, and a pH from 5.5 to 7.5. In one embodiment, the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester. In a specific embodiment, the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate). In one embodiment, the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof. In a specific embodiment, the sugar or sugar alcohol is trehalose. In a specific embodiment, the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate) and the sugar or sugar alcohol is trehalose. In one embodiment, the buffering agent is acetate, HEPES, or a combination thereof. In a specific embodiment, the buffering agent is a combination of acetate and HEPES. In one embodiment, the pH of the composition is from 5.5 to 7.0. In another embodiment, the pH of the composition is from 5.5 to 6.5. In another embodiment, the pH of the composition is 6.0±0.2. In a specific embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.
In one embodiment, a stabilized furin (e.g., rfurin) composition comprises from 50 to 300 mM of a pharmaceutically acceptable salt, from 0.5 mM to 2 mM calcium, 10±5% sugar or sugar alcohol, 75±25 ppm non-ionic surfactant, buffering agent at a concentration selected from variations 6404 to 6469 found in Table 12, and a pH from 5.5 to 7.5. In one embodiment, the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester. In a specific embodiment, the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate). In one embodiment, the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof. In a specific embodiment, the sugar or sugar alcohol is trehalose. In a specific embodiment, the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate) and the sugar or sugar alcohol is trehalose. In one embodiment, the buffering agent is acetate, HEPES, or a combination thereof. In a specific embodiment, the buffering agent is a combination of acetate and HEPES. In one embodiment, the pH of the composition is from 5.5 to 7.0. In another embodiment, the pH of the composition is from 5.5 to 6.5. In another embodiment, the pH of the composition is 6.0±0.2. In a specific embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.
In one embodiment, a stabilized furin (e.g., rfurin) composition comprises 190±50 mM of a pharmaceutically acceptable salt, from 0.5 mM to 2 mM calcium, 10±5% sugar or sugar alcohol, 75±25 ppm non-ionic surfactant, buffering agent at a concentration selected from variations 6404 to 6469 found in Table 12, and a pH from 5.5 to 7.5. In one embodiment, the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester. In a specific embodiment, the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate). In one embodiment, the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof. In a specific embodiment, the sugar or sugar alcohol is trehalose. In a specific embodiment, the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate) and the sugar or sugar alcohol is trehalose. In one embodiment, the buffering agent is acetate, HEPES, or a combination thereof. In a specific embodiment, the buffering agent is a combination of acetate and HEPES. In one embodiment, the pH of the composition is from 5.5 to 7.0. In another embodiment, the pH of the composition is from 5.5 to 6.5. In another embodiment, the pH of the composition is 6.0±0.2. In a specific embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.
In one embodiment, a stabilized furin (e.g., rfurin) composition comprises 191 mM of a pharmaceutically acceptable salt, 0.9 mM calcium, 10% sugar or sugar alcohol, 75 ppm non-ionic surfactant, buffering agent at a concentration selected from variations 6404 to 6469 found in Table 12, and a pH from 5.5 to 7.5. In one embodiment, the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester. In a specific embodiment, the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate). In one embodiment, the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof. In a specific embodiment, the sugar or sugar alcohol is trehalose. In a specific embodiment, the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate) and the sugar or sugar alcohol is trehalose. In one embodiment, the buffering agent is acetate, HEPES, or a combination thereof. In a specific embodiment, the buffering agent is a combination of acetate and HEPES. In one embodiment, the pH of the composition is from 5.5 to 7.0. In another embodiment, the pH of the composition is from 5.5 to 6.5. In another embodiment, the pH of the composition is 6.0±0.2. In a specific embodiment, the pH of the composition is 6.0. In a specific embodiment, the composition contains from 8,000 U/mL to 500,000 U/mL rfurin.
Buffers may also be present in formulations of the invention in combination with one or more of any other formulation components described herein. As shown in the Examples, the highly stabilized formulations of the present invention show increased stability at pH 6.0. In certain embodiments, the pH of the highly stabilized formulations should preferably be maintained in the range of from 6.0 to 8.0, or at a pH of about 6.0. The buffering agent can be any physiologically acceptable chemical entity or combination of chemical entities which have the capacity to act as buffers, including, without limitation: histidine, imidazole, phosphate, citrate, Tris, Acetate, BIS-Tris Propane, PIPES, MOPS, HEPES, MES, and ACES. The full chemical designations of many of these buffering agents are listed in Table 1 below. In certain embodiments, if calcium is present in the formulation at a concentration above about 5 mM, phosphate is not used as a buffering agent. In some embodiments, the buffering agent is included in a concentration of from 10 mM to 200 mM, or 10 to 100 mM, or 30-60 mM, or about 46 mM. In further embodiments, an individual buffering agent is included in a concentration of from 15 to 95 mM, 20 to 90 mM, 25 to 85 mM, 30 to 80 mM, 35 to 75 mM, 40 to 70 mM, 45 to 65 mM, or 50 to 60 mM. In certain embodiments, the formulation contains two buffering agents.







TABLE 13





Exemplary Buffering Agents
















Tris
tris-(hydroxymethyl)-aminomethane


BIS-Tris Propane
1,3-bis-[tris-(hydroxy-methyl)methylamino]-propane


PIPES
piperazine-N,N′-bis-(2-ethanesulfonic acid)


MOPS
3-(N-morpholino) propanesulfonic acid


HEPES
N-2-hydroxyethyl-piperazine-N′-2-ethanesulfonic acid


MES
2-(N-morpholino) ethanesulfonic acid


ACES
N-2-acetamido-2-aminoethanesulfonic acid









In certain aspects the present formulations include a carboxylic acid in combination with one or more of the formulation components described herein. In further aspects, the carboxylic acid is preferably acetic acid (e.g., acetate). In certain embodiments, the formulation includes from 20 mM to 100 mM acetic acid, or from 30 mM to 50 mM, or about 45 mM acetic acid or any other carboxylic acid. In further embodiments, the formulation includes from 25 mM to 90 mM, 30 mM to 80 mM, 35 mM to 70 mM, 40 mM to 60 mM, or 45 mM to 50 mM acetic acid or any other carboxylic acid.
D. Additional Formulation Components
The highly stabilized formulations of the present disclosure include furin, e.g., rfurin, and one or more of stabilizing agents, buffering agents, sodium chloride, salts, and other excipients. Such components are described in further detail below. As will be appreciated, any of the formulation components described herein can be used singly or in any combination.
The highly stabilized Furin formulations of the present disclosure show increased stability toward shear (agitation), lyophilization, and freeze/thaw stress as well as resistance to product loss or denaturation at container surfaces as compared to control or starting formulations.
Furin is included in formulations of the present disclosure in concentrations from 5,000 to 500,000 U/mL. In certain embodiments, Furin is included in concentrations from 5,500 to 55,000, from 6,000 to 50,000, from 6,500 to 45,000, from 7,000 to 40,000, from 7,500 to 35,000, from 8,000 to 30,000, from 8,500 to 25,000, from 9,000 to 20,000, from 9,500 to 15,000, and about 10,000 U/mL. In yet other embodiments, furin is included in the formulations of the present disclosure at a concentration selected from variations 6470 to 6533 found in Table 14, reported in thousands of Units of furin activity per mL. In specific embodiments, the furin contained in the highly stabilized formulations of the present disclosure is rfurin.







TABLE 14





Exemplary furin (e.g., rfurin) concentrations


(expressed in thousands of Units furin activity per mL)


used in the stabilized compositions provided herein.
















  1-1,000
Var. 6470


1-900
Var. 6471


1-800
Var. 6472


1-700
Var. 6473


1-600
Var. 6474


1-500
Var. 6475


1-450
Var. 6476


1-400
Var. 6477


1-350
Var. 6478


1-300
Var. 6479


1-250
Var. 6480


1-200
Var. 6481


1-150
Var. 6482


1-100
Var. 6483


1-52 
Var. 6484


1-50 
Var. 6485


  5-1,000
Var. 6486


5-900
Var. 6487


5-800
Var. 6488


5-700
Var. 6489


5-600
Var. 6490


5-500
Var. 6491


5-450
Var. 6492


5-400
Var. 6493


5-350
Var. 6494


5-300
Var. 6495


5-250
Var. 6496


5-200
Var. 6497


5-150
Var. 6498


5-100
Var. 6499


5-52 
Var. 6500


5-50 
Var. 6501


  8-1,000
Var. 6502


8-900
Var. 6503


8-800
Var. 6504


8-700
Var. 6505


8-600
Var. 6506


8-500
Var. 6507


8-450
Var. 6508


8-400
Var. 6509


8-350
Var. 6510


8-300
Var. 6511


8-250
Var. 6512


8-200
Var. 6513


8-150
Var. 6514


8-100
Var. 6515


8-52 
Var. 6516


8-50 
Var. 6517


 10-1,000
Var. 6518


10-900 
Var. 6519


10-800 
Var. 6520


10-700 
Var. 6521


10-600 
Var. 6522


10-500 
Var. 6523


10-450 
Var. 6524


10-400 
Var. 6525


10-350 
Var. 6526


10-300 
Var. 6527


10-250 
Var. 6528


10-200 
Var. 6529


10-150 
Var. 6530


10-100 
Var. 6531


10-52 
Var. 6532


10-50 
Var. 6533





Var. = Variation






In another embodiment, the furin (e.g., rfurin) concentration of a stabilized composition provided herein can be expressed as the mass of furin present in the composition. In one embodiment, furin (e.g., rfurin) is present in a stabilized composition as described herein at a concentration from 100 ng/mL to 100 mg/mL. In another embodiment, furin (e.g., rfurin) is present in a stabilized composition as described herein at a concentration from 1 μg/mL to 10 mg/mL. In another embodiment, furin (e.g., rfurin) is present in a stabilized composition as described herein at a concentration from 1 μg/mL to 1 mg/mL. In yet other embodiments, furin (e.g., rfurin) is present in a stabilized composition as described herein at a concentration selected from variations 6534 to 6638 found in Table 15. In specific embodiments, the furin contained in the highly stabilized formulations of the present disclosure is rfurin.







TABLE 15





Exemplary furin (e.g., rfurin) concentrations used in


the stabilized compositions provided herein.
















100 ng/mL to 100 mg/mL
Var. 6534


100 ng/mL to 50 mg/mL 
Var. 6535


100 ng/mL to 25 mg/mL 
Var. 6536


100 ng/mL to 10 mg/mL 
Var. 6537


100 ng/mL to 5 mg/mL 
Var. 6538


100 ng/mL to 1 mg/mL 
Var. 6539


100 ng/mL to 500 μg/mL
Var. 6540


100 ng/mL to 250 μg/mL
Var. 6541


100 ng/mL to 100 μg/mL
Var. 6542


100 ng/mL to 50 μg/mL 
Var. 6543


100 ng/mL to 25 μg/mL 
Var. 6544


100 ng/mL to 10 μg/mL 
Var. 6545


100 ng/mL to 5 μg/mL 
Var. 6546


100 ng/mL to 1 μg/mL 
Var. 6547


 1 μg/mL to 100 mg/mL
Var. 6548


 1 μg/mL to 50 mg/mL
Var. 6549


 1 μg/mL to 25 mg/mL
Var. 6550


 1 μg/mL to 10 mg/mL
Var. 6551


1 μg/mL to 5 mg/mL
Var. 6552


1 μg/mL to 1 mg/mL
Var. 6553


 1 μg/mL to 500 μg/mL
Var. 6554


 1 μg/mL to 250 μg/mL
Var. 6555


 1 μg/mL to 100 μg/mL
Var. 6556


 1 μg/mL to 50 μg/mL
Var. 6557


 1 μg/mL to 25 μg/mL
Var. 6558


 1 μg/mL to 10 μg/mL
Var. 6559


1 μg/mL to 5 μg/mL
Var. 6560


 5 μg/mL to 100 mg/mL
Var. 6561


 5 μg/mL to 50 mg/mL
Var. 6562


 5 μg/mL to 25 mg/mL
Var. 6563


 5 μg/mL to 10 mg/mL
Var. 6564


5 μg/mL to 5 mg/mL
Var. 6565


5 μg/mL to 1 mg/mL
Var. 6566


 5 μg/mL to 500 μg/mL
Var. 6567


 5 μg/mL to 250 μg/mL
Var. 6568


 5 μg/mL to 100 μg/mL
Var. 6569


 5 μg/mL to 50 μg/mL
Var. 6570


 5 μg/mL to 25 μg/mL
Var. 6571


 5 μg/mL to 10 μg/mL
Var. 6572


 10 μg/mL to 100 mg/mL
Var. 6573


10 μg/mL to 50 mg/mL
Var. 6574


10 μg/mL to 25 mg/mL
Var. 6575


10 μg/mL to 10 mg/mL
Var. 6576


10 μg/mL to 5 mg/mL 
Var. 6577


10 μg/mL to 1 mg/mL 
Var. 6578


 10 μg/mL to 500 μg/mL
Var. 6579


 10 μg/mL to 250 μg/mL
Var. 6580


 10 μg/mL to 100 μg/mL
Var. 6581


10 μg/mL to 50 μg/mL
Var. 6582


10 μg/mL to 25 μg/mL
Var. 6583


 25 μg/mL to 100 mg/mL
Var. 6584


25 μg/mL to 50 mg/mL
Var. 6585


25 μg/mL to 25 mg/mL
Var. 6586


25 μg/mL to 10 mg/mL
Var. 6587


25 μg/mL to 5 mg/mL 
Var. 6588


25 μg/mL to 1 mg/mL 
Var. 6589


 25 μg/mL to 500 μg/mL
Var. 6590


 25 μg/mL to 250 μg/mL
Var. 6591


 25 μg/mL to 100 μg/mL
Var. 6592


25 μg/mL to 50 μg/mL
Var. 6593


 50 μg/mL to 100 mg/mL
Var. 6594


50 μg/mL to 50 mg/mL
Var. 6595


50 μg/mL to 25 mg/mL
Var. 6596


50 μg/mL to 10 mg/mL
Var. 6597


50 μg/mL to 5 mg/mL 
Var. 6598


50 μg/mL to 1 mg/mL 
Var. 6599


 50 μg/mL to 500 μg/mL
Var. 6600


 50 μg/mL to 250 μg/mL
Var. 6601


 50 μg/mL to 100 μg/mL
Var. 6602


100 μg/mL to 100 mg/mL
Var. 6603


100 μg/mL to 50 mg/mL 
Var. 6604


100 μg/mL to 25 mg/mL 
Var. 6605


100 μg/mL to 10 mg/mL 
Var. 6606


100 μg/mL to 5 mg/mL 
Var. 6607


100 μg/mL to 1 mg/mL 
Var. 6608


100 μg/mL to 500 μg/mL
Var. 6609


100 μg/mL to 250 μg/mL
Var. 6610


250 μg/mL to 100 mg/mL
Var. 6611


250 μg/mL to 50 mg/mL 
Var. 6612


250 μg/mL to 25 mg/mL 
Var. 6613


250 μg/mL to 10 mg/mL 
Var. 6614


250 μg/mL to 5 mg/mL 
Var. 6615


250 μg/mL to 1 mg/mL 
Var. 6616


250 μg/mL to 500 μg/mL
Var. 6617


500 μg/mL to 100 mg/mL
Var. 6618


500 μg/mL to 50 mg/mL 
Var. 6619


500 μg/mL to 25 mg/mL 
Var. 6620


500 μg/mL to 10 mg/mL 
Var. 6621


500 μg/mL to 5 mg/mL 
Var. 6622


500 μg/mL to 1 mg/mL 
Var. 6623


  1 mg/mL to 100 mg/mL
Var. 6624


 1 mg/mL to 50 mg/mL
Var. 6625


 1 mg/mL to 25 mg/mL
Var. 6626


 1 mg/mL to 10 mg/mL
Var. 6627


1 mg/mL to 5 mg/mL 
Var. 6628


  5 mg/mL to 100 mg/mL
Var. 6629


 5 mg/mL to 50 mg/mL
Var. 6630


 5 mg/mL to 25 mg/mL
Var. 6631


 5 mg/mL to 10 mg/mL
Var. 6632


 10 mg/mL to 100 mg/mL
Var. 6633


10 mg/mL to 50 mg/mL 
Var. 6634


10 mg/mL to 25 mg/mL 
Var. 6635


 25 mg/mL to 100 mg/mL
Var. 6636


25 mg/mL to 50 mg/mL 
Var. 6637


 50 mg/mL to 100 mg/mL
Var. 6638





Var. = Variation






In some aspects of the present disclosure, furin (e.g., rfurin) formulations include calcium or another divalent metal cations. In further aspects, the divalent cation is present as a salt, preferably a chloride salt. In certain embodiments, from 0.1 mM to 10 mM of a divalent cation salt can be used, or from 0.5 mM to 2 mM, or about 0.92 mM. In still further embodiments, from 0.5 mM to 9 mM, from 1 mM to 8 mM, from 1.5 mM to 7 mM, from 2 mM to 6 mM, from 2.5 mM to 5 mM, or from 3 mM to 4.5 mM divalent cation salt is used in formulations of the invention. When calcium salt is used, it is preferably calcium chloride, but can also be other calcium salts such as calcium gluconate, calcium glubionate, or calcium gluceptate. Divalent cations, including calcium, can be included in combination with one or more other formulation components disclosed herein. Non-limiting examples of divalent metal cations useful in the formulations provided herein include calcium, barium, manganese, magnesium, cobalt, copper, nickel, and zinc.
In some embodiments, the furin (e.g., rfurin) formulations include an antioxidant. The addition of antioxidants to aqueous and lyophilized formulations has been found to improve the stability of these formulations, and thus extend their shelf lives. The antioxidants used must be compatible for use with a pharmaceutical preparation, and in addition are preferably water soluble. When adding antioxidants to a formulation, it is preferable to add such antioxidants as late in the process prior to lyophilization as possible, in order to avoid spontaneous oxidation of the antioxidant. Table 2 below lists suitable antioxidants, which are available commercially through companies such as Calbiochem and Sigma.







TABLE 16





Exemplary Antioxidants

















N-Acetyl-L-Cysteine/Homocysteine



Glutathione



6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox)



Lipoic acid



Methionine



Sodium Thiosulfate



Platinum



Glycme-glycine-histidme (tripeptide)



Butylatedhydroxytoluene (BHT)










Concentrations in the range of about 0.05 mg/ml to more than 1.0 mg/ml of antioxidants can be used, and it is believed that higher concentrations would also be useful (up to the point of any toxic effects or adverse manufacturing effects, such as a depression of the glass transition temperature of the lyophilized product). Accordingly, in one embodiment, the furin (e.g., rfurin) composition comprises from 0.05 mg/mL to 1.0 mg/mL antioxidant. In other embodiments, the furin (e.g., rfurin) composition comprises from 0.05 to 0.5 mg/mL, 0.1 mg/mL to 0.9 mg/mL, 0.1 mg/mL to 0.8 mg/mL, 0.1 mg/mL to 0.7 mg/mL, 0.1 mg/mL to 0.6 mg/mL, 0.1 mg/mL to 0.5 mg/mL, 0.1 mg/mL to 0.4 mg/mL, 0.1 mg/mL to 0.3 mg/mL, 0.1 mg/mL to 0.2 mg/mL, 0.2 mg/mL to 0.9 mg/mL, 0.2 mg/mL to 0.8 mg/mL, 0.2 mg/mL to 0.7 mg/mL, 0.2 mg/mL to 0.6 mg/mL, 0.2 mg/mL to 0.5 mg/mL, 0.2 mg/mL to 0.4 mg/mL, 0.2 mg/mL to 0.3 mg/mL, 0.3 mg/mL to 0.7 mg/mL, 0.4 mg/mL to 0.6 mg/mL antioxidant.
E. Formulation Development
In certain aspects of the present disclosure, highly stabilized formulations of furin (e.g., rfurin) are prepared by providing a starting (control) furin formulation and adding components to achieve a desired level of component concentrations. For example, the addition of one part 750 ppm non-ionic surfactant to 9 parts furin composition lacking non-ionic surfactant to provide a final formulation comprising 75 ppm non-ionic surfactant. This process is also called “spiking in” to a starting formulation.
In certain embodiments, to produce the highly stabilized furin (e.g., rfurin) formulations of the present disclosure, furin is contained in a starting formulation and a buffer composition is spiked into the starting formulation. In other embodiments, the highly stabilized furin (e.g., rfurin) formulation is formed using dialysis according to methods known in the art. In some embodiments, the methods of formulation involve spiking a concentrated buffer composition into a starting formulation of furin. In one embodiment, polysorbate 80 is added to the starting formulation prior to the addition of other components, particularly prior to the addition of trehalose, in order to protect Furin against agitation during mixing.
In an exemplary embodiment, furin (e.g., rfurin) bulk drug substance (BDS) is contained in a starting formulation of 10 mM sodium acetate, 230 mM sodium chloride, 1 mM calcium chloride, pH 6.0. In one embodiment, the following components are spiked into the starting formulation: 1% polysorbate 80, 500 mM HEPES, 400 mM acetic acid, 1 mM CaCl2, pH 6.0 and trehalose dihydrate powder to attain the following composition: 46 mM HEPES, 45 mM acetic acid, 190 mM NaCl, 0.92 mM CaCl2, 75 ppm polysorbate 80, 10% w/w trehalose dihydrate, pH 6.0. The increase in acetic acid and the addition of HEPES results in greater stability of the pH. Although at pH 6.0 both acetic acid and HEPES are outside of their highest buffering capacity, the high concentration of these chemicals greatly increases the buffering capacity of the highly stabilized formulation. In certain embodiments, the polysorbate 80 is added to the starting formulation prior to the other components to protect furin from aggregation and adsorption to container surfaces during mixing when the other reagents are added. In certain embodiments, the furin in the starting formulation is mixed with polysorbate 80 and/or any other components except for trehalose. Trehalose and any remaining components are added after polysorbate 80 is added to protect furin from aggregation and adsorption during mixing.
In certain embodiments, the stabilized formulation is combined with a diluent so that the formulation can be used in methods such as an rVWF maturation process. In further embodiments, a stabilized diluent is prepared by spiking a starting diluent (50 mM HEPES, 150 mM NaCl, 1 mM CaCl2, pH 7.0) with polysorbate 80 to 75 ppm. The combination of the highly stabilized furin (e.g., rfurin) formulation of the present disclosure with this stabilized diluent increases furin recovery in the rVWF maturation step by three to four times compared to the use of the starting furin and diluent formulations.
In certain embodiments, the highly stabilized furin formulations of the present disclosure are lyophilized. During lyophilization, the formulation is converted from being in an aqueous phase to being in an amorphous solid phase, which is thought to protect the protein from chemical and/or conformational instability. In further embodiments, the lyophilized preparation not only contains an amorphous phase, but also includes a component which crystallizes during lyophilization.
One or more components of highly stabilized formulations may in some embodiments be dispersed in the amorphous phase of the lyophilized cake. In addition, the apparent glass transition temperature (Tg′) of the amorphous phase is preferably relatively high during freeze-drying, and the glass transition temperature (Tg) of the solid is likewise preferably high during storage.
IV. Methods for Diluting Aqueous Compositions of Recombinant Furin
In one aspect, the present disclosure provides methods for diluting aqueous compositions of furin (e.g., rfurin). The following embodiments are based in part on the discovery that inclusion of a non-ionic surfactant in a buffer used to dilute furin (e.g., rfurin) results in the recovery of 3-4 times more furin activity as compared to dilution with a buffer lacking the non-ionic surfactant.
In one embodiment, the method includes adding a dilution buffer containing non-ionic surfactant to a furin (e.g., rfurin) composition, to form a diluted furin (e.g., rfurin) composition. In some embodiments, the dilution buffer is added at a ratio of from 1:1 (dilution buffer:furin composition) to 1,000:1 (dilution buffer:furin composition). In another embodiment, the dilution buffer is added at a ratio of from 1:1 (dilution buffer:furin composition) to 500:1 (dilution buffer:furin composition). In another embodiment, the dilution buffer is added at a ratio of from 1:1 (dilution buffer:furin composition) to 250:1 (dilution buffer:furin composition). In another embodiment, the dilution buffer is added at a ratio of from 1:1 (dilution buffer:furin composition) to 200:1 (dilution buffer:furin composition). In another embodiment, the dilution buffer is added at a ratio of from 1:1 (dilution buffer:furin composition) to 100:1 (dilution buffer:furin composition). In another embodiment, the dilution buffer is added at a ratio of from 1:1 (dilution buffer:furin composition) to 50:1 (dilution buffer:furin composition).
In one embodiment, the method comprises a first step of adding a non-ionic surfactant to a furin (e.g., rfurin) composition and a second step of adding a dilution buffer to the furin (e.g., rfurin) composition containing the non-ionic surfactant, to form a diluted furin composition. In one embodiment, the surfactant will be added to the furin composition at a final concentration of X-fold a desired concentration in the diluted furin composition, where X is the dilution factor. For example, if a final concentration of 10 ppm non-ionic surfactant is desired in a furin composition to be diluted 100-fold, the non-ionic surfactant is added to the starting furin composition at a final concentration of 1,000 ppm (10 ppm×100-fold dilution), and the composition is subsequently diluted by adding 99 parts dilution buffer per 1 part starting solution (accounting for volume added during addition of the non-ionic surfactant).
In certain embodiments, the furin (e.g., rfurin) composition is diluted from 1-fold to 1,000-fold, from 1-fold to 500-fold, from 1-fold to 250-fold, from 1-fold to 200-fold, from 1-fold to 100-fold, from 1-fold to 50-fold, from 1-fold to 10-fold, from 10-fold to 1,000-fold, from 10-fold to 500-fold, from 10-fold to 250-fold, from 10-fold to 200-fold, from 10-fold to 100-fold, from 10-fold to 50-fold, from 50-fold to 1,000-fold, from 50-fold to 500-fold, from 50-fold to 250-fold, from 50-fold to 200-fold, from 50-fold to 100-fold, from 100-fold to 1,000-fold, from 100-fold to 500-fold, from 100-fold to 250-fold, from 100-fold to 200-fold, from 200-fold to 1,000-fold, from 200-fold to 500-fold, or from 200-fold to 250-fold.
A. Furin Dilution Buffer
In one embodiment, the furin dilution buffer will include a pharmaceutically acceptable salt, non-ionic surfactant, and a buffering agent. In one embodiment, the dilution buffer further includes calcium. In another embodiment, the dilution buffer further includes a sugar and/or sugar alcohol.
In one embodiment, pharmaceutically acceptable salt is present in the dilution buffer at a concentration of from 10 mM to 500 mM. In another embodiment, the concentration of pharmaceutically acceptable salt is from 100 mM to 300 mM. In another embodiment, the concentration of pharmaceutically acceptable salt is from 150 mM to 250 mM. In yet other embodiments, the concentration of pharmaceutically acceptable salt in the furin dilution buffer is selected from variations 6036 to 6180 found in Table 10. In one embodiment, the pharmaceutically acceptable salt is sodium chloride, potassium chloride, or a combination thereof. In a specific embodiment, pharmaceutically acceptable salt is sodium chloride. In another specific embodiment, pharmaceutically acceptable salt is potassium chloride.
In one embodiment, the dilution buffer has a pH of from 5.5 to 7.5. In one embodiment, the pH of the dilution buffer is from 5.5 to 7.0. In another embodiment, the pH of the dilution buffer is from 5.5 to 6.5. In another embodiment, the pH of the dilution buffer is 6.0±0.2. In a specific embodiment, the pH of the dilution buffer is 6.0. In other embodiments, the dilution buffer has a pH selected from variations 6181 to 6403 found in Table 11.
In one embodiment, buffering agent is present in the dilution buffer at a concentration of from 10 mM to 300 mM. In another embodiment, the concentration of buffering agent in the dilution buffer is between 10 mM to 200 mM. In other embodiments, the concentration of buffering agent in the dilution buffer is selected from variations 6404 to 6469 found in Table 12. In one embodiment, the buffering agent is acetate, HEPES, or a combination thereof. In a specific embodiment, the buffering agent is a combination of acetate and HEPES.
In some embodiments, the furin dilution buffer includes calcium or another divalent metal cation. In one embodiment, the divalent cation is added as a salt, preferably a chloride salt. In certain embodiments, the concentration of divalent cation (e.g., calcium) in the dilution buffer is from 0.1 mM to 10 mM, from 0.5 mM to 2 mM, or about 0.92 mM. In still further embodiments, from 0.5 mM to 9 mM, from 1 mM to 8 mM, from 1.5 mM to 7 mM, from 2 mM to 6 mM, from 2.5 mM to 5 mM, or from 3 mM to 4.5 mM divalent cation is used in the dilution buffer. When calcium salt is used, it is preferably calcium chloride, but can also be other calcium salts such as calcium gluconate, calcium glubionate, or calcium gluceptate. Divalent cations, including calcium, can be included in combination with one or more other formulation components disclosed herein. Non-limiting examples of divalent metal cations useful in the dilution buffers provided herein include calcium, barium, manganese, magnesium, cobalt, copper, nickel, and zinc.
In some embodiments, the furin dilution buffer includes a sugar and/or sugar alcohol. In one embodiment, the dilution buffer contains a monosaccharide sugar. In a particular embodiment, the monosaccharide sugar is selected from the group consisting of a diose sugar, a triose sugar, a tetrose sugar, a pentose sugar, a hexose sugar, a heptose sugar, and an octose sugar. In a particular embodiment, the sugar is a pentose sugar, a hexose sugar, or a combination thereof. In a specific embodiment, the sugar is a hexose sugar.
In another embodiment, the dilution buffer contains a disaccharide sugar. In a particular embodiment, the disaccharide sugar is selected from disaccharide sugars formed from pentose and/or hexose monosaccharides. In another particular embodiment, the sugar is selected from disaccharide sugars formed from hexose monosaccharides. In one embodiment, the sugar is sucrose, trehalose, or a combination thereof. In one specific embodiment, the sugar is sucrose. In another specific embodiment, the sugar is trehalose. In one embodiment, the sugar is formulated as trehalose dihydrate.
In another embodiment, the dilution buffer contains a sugar alcohol. In a particular embodiment, the sugar alcohol is selected from glycol, glycerol, erythritol, threitol, ribitol, fucitol, iditol, volmitol, isomalt, maltitol, lactitol, mannitol, sorbitol, inositol, galactitol, dulcitol, xylitol, and arabitol. In another particular embodiment, the sugar alcohol is mannitol.
In yet another embodiment, the dilution buffer contains a mixture of sugar and sugar alcohol. In one embodiment, the mixture contains at least two of a monosaccharide, a disaccharide, and a sugar alcohol. In another embodiment, the mixture contains at least two of a pentose sugar, a hexose sugar, a disaccharide formed from pentose and/or hexose monosaccharides, and a sugar alcohol. In another embodiment, the mixture contains at least two of sucrose, trehalose, and mannitol.
In one embodiment, the sugar or sugar alcohol is present in the dilution buffer at a concentration of from 2% to 20%, 2% to 17.5%, 2% to 15%, 2% to 12.5%, 2% to 10%, 2% to 9%, 2% to 8%, 2% to 7%, 5% to 20%, 5% to 17.5%, 5% to 15%, 5% to 12.5%, 5% to 10%, 7.5% to 20%, 7.5% to 17.5%, 7.5% to 15%, 7.5% to 12.5%, 10% to 20%, 10% to 17.5%, 10% to 15%, 4±2%, 5±2%, 6±2%, 7±2%, 8±2%, 9±2%, 10±2%, 11±2%, 12±2%, 13±2%, 14±2%, 15±2%, 16±2%, 17±2%, 18±2%, 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%, 16±1%, 17±1%, 18±1%, 19±1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%. In one embodiment, the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, and a combination thereof. In a specific embodiment, the sugar or sugar alcohol is trehalose.
In one embodiment, storage stable compositions of furin (e.g., rfurin) are provided which contain a non-ionic surfactant selected from a non-ionic water soluble monoglyceride, a non-ionic water soluble diglyceride, a non-ionic water soluble triglyceride, a non-ionic water soluble monofatty acid esters of polyethyelene glycol, a non-ionic water soluble difatty acid esters of polyethyelene glycol, a non-ionic water soluble sorbitan fatty acid ester, a non-ionic polyglycolyzed glyceride, a non-ionic water soluble triblock copolymer, and a combination thereof. In one embodiment, the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester. In a specific embodiment, the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate).
1. Non Ionic Surfactants
In one embodiment, the furin dilution buffer, or non-ionic surfactant solution added prior to dilution, contains a non-ionic surfactant selected from a non-ionic water soluble monoglyceride, a non-ionic water soluble diglyceride, a non-ionic water soluble triglyceride, a non-ionic water soluble monofatty acid esters of polyethyelene glycol, a non-ionic water soluble difatty acid esters of polyethyelene glycol, a non-ionic water soluble sorbitan fatty acid ester, a non-ionic polyglycolyzed glyceride, a non-ionic water soluble triblock copolymer, and a combination thereof. In one embodiment, the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester. In a specific embodiment, the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate).
In one embodiment, the non-ionic surfactant is present in the dilution buffer at a concentration of from 10 to 200 ppm, 10 to 175 ppm, 10 to 150 ppm, 10 to 125 ppm, 10 to 100 ppm, 10 to 90 ppm, 10 to 80 ppm, 10 to 75 ppm, 10 to 70 ppm, 10 to 60 ppm, 10 to 50 ppm, 10 to 25 ppm, 25 to 200 ppm, 25 to 175 ppm, 25 to 150 ppm, 25 to 125 ppm, 25 to 100 ppm, 25 to 90 ppm, 25 to 80 ppm, 25 to 70 ppm, 25 to 60 ppm, 25 to 50 ppm, 50 to 200 ppm, 50 to 175 ppm, 50 to 150 ppm, 50 to 125 ppm, 50 to 90 ppm, 50 to 80 ppm, 75 to 200 ppm, 75 to 175 ppm, 75 to 150 ppm, 100 to 200 ppm, 100 to 175 ppm, 50±25 ppm, 60±25 ppm, 70±25 ppm, 75±25 ppm, 80±25 ppm, 90±25 ppm, 100±25 ppm, 125±25 ppm, 150±25 ppm, 175±25 ppm, 30±10 ppm, 40±10 ppm, 50±10 ppm, 60±10 ppm, 70±10 ppm, 75±10 ppm, 80±10 ppm, 90±10 ppm, 100±10 ppm, 110±10 ppm, 120±10 ppm, 125±10 ppm, 130±10 ppm, 140±10 ppm, 150±10 ppm, 160±10 ppm, 170±10 ppm, 175±10 ppm, 180±10 ppm, 190±10 ppm, 25 ppm, 30 ppm, 40 ppm, 50 ppm, 60 ppm, 70 ppm, 75 ppm, 80 ppm, 90 ppm, 100 ppm, 110 ppm, 120 ppm, 125 ppm, 130 ppm, 140 ppm, 150 ppm, 160 ppm, 170 ppm, 175 ppm, 180 ppm, 190 ppm, or 200 ppm. In one embodiment, the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester. In a specific embodiment, the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate).
In one embodiment, the target concentration of non-ionic surfactant after dilution is from 10 to 200 ppm, 10 to 175 ppm, 10 to 150 ppm, 10 to 125 ppm, 10 to 100 ppm, 10 to 90 ppm, 10 to 80 ppm, 10 to 75 ppm, 10 to 70 ppm, 10 to 60 ppm, 10 to 50 ppm, 10 to 25 ppm, 25 to 200 ppm, 25 to 175 ppm, 25 to 150 ppm, 25 to 125 ppm, 25 to 100 ppm, 25 to 90 ppm, 25 to 80 ppm, 25 to 70 ppm, 25 to 60 ppm, 25 to 50 ppm, 50 to 200 ppm, 50 to 175 ppm, 50 to 150 ppm, 50 to 125 ppm, 50 to 90 ppm, 50 to 80 ppm, 75 to 200 ppm, 75 to 175 ppm, 75 to 150 ppm, 100 to 200 ppm, 100 to 175 ppm, 50±25 ppm, 60±25 ppm, 70±25 ppm, 75±25 ppm, 80±25 ppm, 90±25 ppm, 100±25 ppm, 125±25 ppm, 150±25 ppm, 175±25 ppm, 30±10 ppm, 40±10 ppm, 50±10 ppm, 60±10 ppm, 70±10 ppm, 75±10 ppm, 80±10 ppm, 90±10 ppm, 100±10 ppm, 110±10 ppm, 120±10 ppm, 125±10 ppm, 130±10 ppm, 140±10 ppm, 150±10 ppm, 160±10 ppm, 170±10 ppm, 175±10 ppm, 180±10 ppm, 190±10 ppm, 25 ppm, 30 ppm, 40 ppm, 50 ppm, 60 ppm, 70 ppm, 75 ppm, 80 ppm, 90 ppm, 100 ppm, 110 ppm, 120 ppm, 125 ppm, 130 ppm, 140 ppm, 150 ppm, 160 ppm, 170 ppm, 175 ppm, 180 ppm, 190 ppm, or 200 ppm. In one embodiment, the non-ionic surfactant is a non-ionic water soluble sorbitan fatty acid ester. In a specific embodiment, the non-ionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate).
In one embodiment, the inclusion of a non-ionic surfactant in a furin dilution buffer, or addition prior to dilution, increases the furin activity recovered in a diluted furin composition by at least 10%, as compared to the activity recovered in a furin composition diluted without use of the non-ionic surfactant, or with the use of the non-ionic surfactant at a lower concentration. In other embodiments, the furin activity recovered after dilution using a non-ionic surfactant is at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 175%, 2-fold, 3-fold, 4-fold, 5-fold, or more greater than the furin activity recovered after dilution without the non-ionic surfactant, or using a lower concentration of the non-ionic surfactant.
V. Stability Assays
As discussed herein, the highly stabilized furin (e.g., rfurin) formulations of the present disclosure show improved stability as compared to control formulations. In one embodiment, improved stability includes retention of a higher percentage of activity than control formulations in various stability assays. Such assays can be used to determine if a formulation is a highly stabilized formulation. In some embodiments, the highly stabilized formulation has at least 5%, 10%, 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater activity than a control formulation when assessed by any of the stability assays discussed herein or known in the art.
In further aspects, furin (e.g., rfurin) formulations are tested under stressor conditions, such as storage at high temperature, agitation, freeze/thaw cycles, or some combination thereof. After such stressors, the formulations are assayed using any of the methods described herein or known in the art to determine the stability under these conditions.
In one aspect, furin activity assays are used to assess the stability of a formulation. In some embodiments, a furin activity assays involve a measurement of substrate cleavage. In one embodiment, the substrate is Boc-Arg-Val-Arg-Arg-AMC (SEQ ID NO:1; AMC=7-amino-4-methoxy coumarin), which is cleaved by furin, liberating 7-amino-4-methoxy coumarin. The assay is performed, for example, on an ELISA plate. The furin activity in a sample is determined from a standard curve created, for example, from a quality control (QC) furin standard. The activity values are generally calculated as mean values from two dilutions, each in duplicate.
In another aspect, size exclusion chromatography (SEC) is used to evaluate the stability of a furin (e.g., rfurin) formulation. In such assays, stability is indicated by the height of the peak corresponding to monomeric furin. A reduction in monomer peak height means the product is being lost (unstable) and the generation of higher molecular weight peaks indicates that aggregation is occurring and lower molecular weight peaks means the product is degrading. In an exemplary embodiment, after 4 days at 37° C., the relative peak height of a control furin formulation was 58.5%, but in a highly stabilized formulation, the relative peak height was 74.8% (FIG. 48). Thus, the highly stabilized formulation maintained the stability of furin in the composition and retained a larger percentage of the peak height as compared to that seen prior to storage at four days at a higher (37° C.) temperature.
In another aspect, qualitative assays such as Western Blot analyses, are used to evaluate the stability of a Furin formulation. For example, FIG. 26 shows that after 5 days at 35° C., a greater percent of the signal is present as the intact furin molecule rather than the degraded species in the stabilized formulation (lane 11), as compared to a non-stabilized formulation (lane 5).
In still another aspect, qualitative assays such as UV spectra are used to evaluate stability. Such assays are particularly useful when the stressor used is agitation. The UV spectra can indicate the presence of aggregate—the presence of an aggregate is an indication of instability. For example, FIG. 36 shows a major displacement of the spectra upward (indicating aggregation) when the control formulation is agitated. FIGS. 41 and 42 show the protective power of a highly stabilized formulation in that, with agitation, the spectra of the agitated sample is not displaced upward, but overlays the non-agitated sample, which indicates that aggregation was not formed when the formulation was placed under stress through agitation.
The practice of the present invention may employ, unless otherwise indicated, conventional techniques and descriptions of organic chemistry, polymer technology, molecular biology (including recombinant techniques), cell biology, biochemistry, and immunology, which are within the skill of the art. Such conventional techniques include polymer array synthesis, hybridization, ligation, and detection of hybridization using a label. Specific illustrations of suitable techniques can be had by reference to the example herein below. However, other equivalent conventional procedures can, of course, also be used. Such conventional techniques and descriptions can be found in standard laboratory manuals such as Genome Analysis: A Laboratory Manual Series (Vols. I-IV), Using Antibodies: A Laboratory Manual, Cells: A Laboratory Manual, PCR Primer: A Laboratory Manual, and Molecular Cloning: A Laboratory Manual (all from Cold Spring Harbor Laboratory Press), Stryer, L. (1995) Biochemistry (4th Ed.) Freeman, Highly stabilized York, Gait, “Oligonucleotide Synthesis: A Practical Approach” 1984, IRL Press, London, Nelson and Cox (2000), Lehninger, Principles of Biochemistry 3rd Ed., W. H. Freeman Pub., Highly stabilized York, N.Y. and Berg et al. (2002) Biochemistry, 5th Ed., W. H. Freeman Pub., Highly stabilized York, N.Y., all of which are herein incorporated in their entirety by reference for all purposes.
In the following description, numerous specific details are set forth to provide a more thorough understanding of the present invention. However, it will be apparent to one of skill in the art that the present invention may be practiced without one or more of these specific details. In other instances, well-known features and procedures well known to those skilled in the art have not been described in order to avoid obscuring the invention.
Although the present invention is described primarily with reference to specific embodiments, it is also envisioned that other embodiments will become apparent to those skilled in the art upon reading the present disclosure, and it is intended that such embodiments be contained within the present inventive methods.
EXAMPLES
Example 1
Storage Stability of Aqueous Rfurin Formulations
The stability of a control furin formulation was tested at three different temperatures: room temperature (e.g., 25° C.), 37° C., and 45° C. These studies at elevated temperatures were used to study accelerated stability, which is comparable to long term studies of stability at lower temperatures. The samples were tested at time zero, then after 1, 2, 3, 4, and 7 days of incubation. Analyses included the furin activity assay, SEC, and western blotting.
Briefly, several 5 mL polypropylene vials were filled with 3 mL of rfurin and incubated at three different temperatures: ambient (room), 37° C. and 45° C. One vial from each incubation condition was taken out at time zero, then after 1, 2, 3, 4, and 7 days. 200 μL aliquots were made into 0.65 mL polypropylene vials and frozen at −80° C. awaiting analyses.
The stability of several furin formulations was determined under these conditions. Specifics of the various formulations are given in Table 17.







TABLE 17





Furin formulations tested for stability during


storage at elevated temperatures.


















Control Formulation
10 mM Acetate




230 mM sodium chloride




1 mM calcium chloride




pH 6.0



Test Formulation #1
46 mM HEPES




45 mM Acetate




190 mM sodium chloride




0.92 mM calcium chloride




75 ppm polysorbate 80




10% (w/w) trehalose dehydrate




pH 6.0



Test Formulation #2
46 mM HEPES




45 mM Acetate




190 mM sodium chloride




0.92 mM calcium chloride




10% (w/w) trehalose dehydrate




pH 6.0



Test Formulation #3
46 mM HEPES




45 mM Acetate




190 mM sodium chloride




0.92 mM calcium chloride




10 ppm polysorbate 80




10% (w/w) trehalose dehydrate




pH 6.0



Test Formulation #4
46 mM HEPES




45 mM Acetate




190 mM sodium chloride




0.92 mM calcium chloride




25 ppm polysorbate 80




10% (w/w) trehalose dehydrate




pH 6.0



Test Formulation #5
46 mM HEPES




45 mM Acetate




190 mM sodium chloride




0.92 mM calcium chloride




50 ppm polysorbate 80




10% (w/w) trehalose dehydrate




pH 6.0



Test Formulation #6
46 mM HEPES




45 mM Acetate




190 mM sodium chloride




0.92 mM calcium chloride




100 ppm polysorbate 80




10% (w/w) trehalose dehydrate




pH 6.0










One method of assessing stability is through a furin activity assay. In this assay, the activity of furin is determined from the rate of a substrate cleavage. The substrate, Boc-Arg-Val-Arg-Arg-AMC (SEQ ID NO:1; AMC=7-amino-4-methoxy coumarin), is cleaved by furin and a fluorescent AMC is liberated. The assay is performed, for example, on an ELISA plate. The furin activity in a sample is determined from a standard curve created, for example, from a quality control rfurin standard. The activity values were calculated as mean values from two dilutions, each in duplicate.
rFurin Stability at 37° C. in the Highly Stabilized Formulation.
The purpose of this experiment was to test rfurin stability in a stabilized formulation containing 75 ppm polysorbate 80. There was a small change in the composition of this sample because trehalose dihydrate (C12H22O11*2H2O, FW 378.33) was used, rather than, nonhydrated trehalose (C12H22O11, FW 342.30). Use of the trehalose dihydrate created a slightly more dilute sample. Agitation studies suggested that the polysorbate 80 concentration should be kept between 50 ppm and 100 ppm to provide the best protection against rfurin denaturation induced by agitation. In this study, a rfurin sample in test formulation #1 was spiked with polysorbate 80 to 75 ppm and incubated at 37° C. for four days. An rfurin sample formulated with the control formulation served as a control.
Analyses by the Furin activity assay (FIG. 1) show loss of activity in the samples in the control formulation that were incubated at 37° C. and 45° C. Since the 45° C. sample was highly degraded and aggregated after just two days, the sample was not tested after that time. For the samples incubated at room temperature, the data was inconclusive as a result of the amount of variability in the assay, but the data suggested that the rfurin activity was relatively stable, with the activity after 7 days roughly equivalent to T=0.
In contrast to the data in the control formulation, analyses by the furin activity assay (FIG. 46) show that rfurin in test formulation #1 retained more of its activity than in the control formulation. After four days of incubation at 37° C., rfurin retained 76.7% of its original activity, while the control formulation preserved only 55.5% of its original activity (FIG. 47).
Size exclusion chromatograph (SEC) was also used to assess the stability of various furin formulations. For SEC methods, the following equipment and materials were used:

    
    
        Agilent HPLC 1100 Series equipped with a temperature controlled autosampler and a Photo-Diode Array detector
        Autosampler 100 μL polypropylene vials
        Size Exclusion Chromatography column from TOSOH Bioscience, TSKgel
        G3000SWx1, 7.8 mm ID×30 cm, 5 μm

In addition, following conditions were used for the SEC method:

        Mobile Phase: 50 mM MOPS, 200 mM sodium sulfate, 0.02% sodium azide, pH 7.0
        Flow Rate: 0.5 mL/min
        Run Time: 30 min
        Sample Volume: 100 μL
        Autosampler Temperature: 4° C.
        Column Temperature: Ambient
    
    


Single wavelength absorption at 280 nm (Reference off) was used to create chromatographs. After manual integration, the height of the main peak at 19 min, corresponding to the rfurin content of the formulation, was used to evaluate stability. The reason for using the peak height instead of the peak area was that the chromatographic peaks were often not baseline separated, which could lead to large errors. Only one injection of each sample was conducted. Repeated analyses of the rfurin control formulation showed less than 2% variation in the peak height (data not shown).
The SEC results correlated with the furin activity assay results reported above. Analyses by SEC (FIG. 2) show that the height of the rfurin monomer peak (retention time 19 min) of the control formulation decreased with the time of incubation for all temperatures. The rate of the rfurin monomer peak decrease was relatively low in the samples incubated at room temperature, as 82% of the peak height remained after seven days of incubation. However, at 37° C., the height of the rfurin monomer peak dropped to 21.6%, of the original height, after seven days of incubation. At 45° C., after two days of incubation, the main peak decreased to 14.1%. Consistent with the loss of rfurin monomer content, other chromatographic peaks began to appear over time (FIG. 3), corresponding to aggregates (eluting at earlier time points) and degradation products (eluting at later time points). These additional peaks indicate both degradation and aggregation of rfurin in the control formulation. Increased degradation at 37° C. compared to room temperature storage was also confirmed by Western Blotting (FIG. 4). rFurin degradation may be caused by proteases that were co-purified with rfurin, or by the autocatalytic activity of rfurin itself.
In contrast, SEC analysis confirmed the increased stability of test formulation #1 (FIG. 48), containing trehalose, as compared to the control formulation, which does not contain a sugar or sugar alcohol. FIG. 48 shows that test formulation #1 maintained 74.8% of rfurin monomer peak height of the starting solution after incubation at 37° C. for 4 days, as compared to the control formulation, which maintained only 58.5% of rfurin monomer peak height of the starting solution during the same incubation.
Example 2
Storage Stability of Aqueous Rfurin Formulations Upon Freeze/Thaw
The stability of the control formulation and test formulations #2-6 were tested by a freeze/thaw study. Five freeze/thaw cycles were completed and samples were analyzed by the furin activity assay and SEC. Investigating the impact of freeze/thaw is important because rfurin samples in this study and rfurin BDS are stored frozen. Freeze/thaw experiments using the control formulation (n=4) demonstrate a minor loss of furin activity (approximately 7%) after a total of five freeze/thaw cycles. While there appears to be a downward trend, this may be due to true loss of protein or a reflection of assay variability. Regardless, this data supports the ability to freeze and thaw the rFurin, not only once, but multiple times, with little to no loss of activity. Likewise, SEC analysis of the oligomeric state of furin in the control formulation showed no loss in monomeric content over the course of the freeze/thaw experiment (FIG. 6).
Since rFurin is generally stored frozen, the impact of a thawing method was evaluated. Analyses by the activity assay (Figure) demonstrate no significant difference between the two thawing methods: thawing at 4° C. overnight, or at room temperature for 2 hours.
Taken together, these data suggest that rfurin in the control formulation is relatively stable for short periods at room temperature. Furthermore, loss of activity upon storage at elevated temperatures correlates to both rfurin degradation and aggregation. In addition, rfurin in the control formulation retained its activity well even after five freeze/thaw cycles.
Freeze/Thaw Study of rFurin in a Highly Stabilized Formulation.
The freeze/thaw study was carried out using the same set of samples as the previous Freeze/thaw study; the stability of rfurin in test formulations #2 (0 ppm polysorbate 80), #3 (10 ppm polysorbate 80), #4 (25 ppm polysorbate 80), #5 (50 ppm polysorbate 80), and #6 (100 ppm polysorbate 80) was evaluated. Furin in the control formulation served as a control. Consistent with the prior analysis of the control formulation, analysis of furin activity (FIG. 43) demonstrated no significant loss of activity after five freeze/thaw cycles in any of the tested formulations. Analyses of the rfurin monomer content by SEC (FIGS. 44 and 45) demonstrated only a small trend of sample deterioration after freeze/thaw. Even after five freeze/thaw cycles, all samples retained more than 95% of their original activity.
Example 3
Storage Stability of Aqueous Rfurin Formulations Upon Mechanical Stress
Without being limited by theory, one potential mechanism of the agitation effect on rfurin samples is that it induces denaturation of rfurin, exposing hydrophobic surfaces. The hydrophobic surfaces are attracted to other hydrophobic surfaces, such as other denatured rFurin proteins which induces aggregation, or causing adsorption to hydrophobic surfaces on sample tubes and materials.
The agitation study was not designed to mimic the true process of rfurin use, but to overstress the sample. To be considered stabilized, a formulation of rfurin performs at least as well as the control formulation, and a highly stabilized formulation performs better than the control formulation.
The agitation study was carried out by shaking a rfurin sample in a bottle containing Teflon balls. Samples were taken after 2 and 17 hours of agitation and analyzed by furin activity assay and SEC. As shown in the results for both the furin activity assay and SEC analysis (FIGS. 7 and 8, respectively), rfurin in the control formulation is fairly resistant to agitation for a limited period of time. After two hours of vigorous agitation, such as might occur during a diafiltration step, more than 85% of rfurin was still active in the control formulation. However, after 17 hours of agitation, as might occur during shipping of a sample, less than 10% of the rfurin content remained active.
The effect was then tested on furin test formulations #2 to 6, which were created by spiking nine parts of a rFurin sample with one part of 500 mM HEPES, 400 mM acetic acid, 1 mM CaCl2, pH 6 plus 10% w/w trehalose plus varied amounts of polysorbate 80.
This agitation study was carried out by shaking the test rfurin formulations and control formulation containing bottles with Teflon balls for three hours. Test formulations containing the following levels of polysorbate 80 were evaluated: none, 10 ppm, 25 ppm, 50 ppm, or 100 ppm. As shown in FIG. 31, polysorbate 80 in the test formulations provided significant protection to rfurin during agitation. After three hours of agitation, the activity of rFurin in the samples containing polysorbate 80 ranged from 31,499±1,218 U/mL to 36,787±847 U/mL, while the sample in the formulations without polysorbate 80 had 17,835±1,706 U/mL of Furin activity. The sample in the control formulation (10 mM sodium acetate, 230 mM sodium chloride, 1 mM calcium chloride, pH 6.0) had an activity of 24,368±1,135 U/mL.
Analyses by SEC confirmed the beneficial effect of polysorbate 80 in this study (FIG. 32). After three hours of agitation, the peak heights of rfurin in the samples containing polysorbate 80 ranged from 37.5 mAU to 35.9 mAU, while the height of the rFurin peak in the formulation without polysorbate 80 was 22.1 mAU. The height of the rFurin peak in the control formulation was 29.9 mAU. The effect of polysorbate 80 is also shown in FIGS. 33 and 34. The small trend suggests that the addition of 10 ppm and 25 ppm polysorbate 80, while still beneficial, is not as effective as use of 50 ppm or 100 ppm polysorbate 80. The SEC profile for the agitated sample in the control formulation (FIG. 35) does not show any additional peaks compared to the non-agitated sample. This would suggest that surface adsorption was the main reason for the activity loss. However, the UV absorption spectra (FIG. 36), show that the agitated sample has a slanted and elevated profile, which is a sign of light scattering and obscuration caused by the presence of aggregates. Light obscuration is generally caused by vary large aggregates that are more opaque than smaller molecules, which cause an elevation of the UV spectra. This phenomenon cannot be distinguished from absorption by a spectrophotometer because its detector measures the amount of light that passes through a sample. Since less light gets to the detector, it is reported as absorption; hence the absorption profile is elevated.
Light can also be scattered by aggregates, which would also decrease the amount of light getting into a detector. The shorter the wave length, the more light is scattered (the amount of scattered light is inversely proportional to the fourth power on the wavelength). This means that the shorter the wavelength the less light gets to a detector; hence the absorption profile is slanted. It is likely that the aggregates were present in this sample, but the SEC analysis was unable to detect them. These kinds of problems with SEC analyses are not uncommon as protein aggregates are often too large to pass through the column, or are absorbed by the column matrix. The UV absorption spectrum of the rfurin sample in the formulation without polysorbate 80 (FIG. 37) shows a similar elevated profile. Again, no aggregates were detected in the SEC analysis of this sample (FIG. 38). The samples formulated with polysorbate 80 show less aggregation than those without polysorbate 80 when analyzed by the UV absorption spectra (FIGS. 39 to 42). Although a UV spectrum cannot quantify the amount of aggregates, again, a small trend can be observed hinting that the samples with polysorbate 80 at 50 ppm and 100 ppm were better in protecting rFurin from aggregation than 10 ppm or 25 ppm. The data suggest that polysorbate 80 included in the formulation at a level of 75 ppm would be effective, as this concentration is in the middle of a wide plateau (50-10 ppm) and would represent a robust condition.
The UV spectrophotometry analysis was conducted using the following equipment and materials:

    
    
        Agilent 8453 Spectrophotometer
        Quartz cuvette 1 cm light pathway
    
    


The spectrophotometry analysis was conducted by first placing 0.5 mL of an appropriate formulation in a cuvette and measured as a blank. Then, 0.5 mL of a rfurin sample was placed in a cuvette, and the scan was taken from 240 nm to 400 nm.
Example 4
Impact of pH, Sucrose and Polysorbate 80 on rFurin Stability
rFurin samples were spiked with various buffers in order to adjust the pH to 5.0, 5.5, 6.0, 7.0 and 8.0. In addition, sucrose and/or polysorbate 80 were added to the samples at pH 6.0, 7.0 and 8.0. The samples were incubated at 37° C. and tested by the furin activity assay and SEC.
Both the furin activity assay (FIG. 11) and SEC (FIG. 12) showed that at 37° C. the rfurin was more stable at pH 6.0 than at pH 5.0 or 5.5. The samples at pH 6.0, in both the control formulation (FIG. 13) and in MES (FIG. 14), were more stable than those formulated in pH 7.0 or 8.0 (FIGS. 15 and 16) when analyzed by SEC. The addition of sucrose to the samples in pH 6.0 and 7.0 had a beneficial effect on rfurin stability. The SEC analyses (FIG. 17) showed that after four days of incubation at 37° C., the peak heights of the samples containing sucrose were on average at 75% of T=0, as compared to 52% without the sucrose. Analyses by the furin activity assay confirmed the results of the SEC experiments (FIGS. 18 and 19). The addition of sucrose improved rfurin stability in both the control formulation and in MES pH 6.0 (FIGS. 18 and 19), with similar stability seen using either buffer (FIG. 20). The effects of polysorbate 80 on rfurin stability in a sucrose-containing formulation at 37° C. were minimal, if any, as shown in FIGS. 17, 18, and 19.
These data suggest that at 37° C. rfurin is most stable in the pH 6.0 formulations compared to pH 5.0, 5.5, 7.0 and 8.0. The data further show that sucrose had a beneficial effect on rfurin stability at 37° C. In addition, no significant difference was seen in rfurin stability between the control buffer (acetate) or MES at pH 6.0 in the presence of 10% sucrose. Finally, there was no significant effect of polysorbate 80 on rfurin stability in a sucrose formulation at 37° C.
Example 5
Comparison of Furin Formulation Stability in Sucrose and Mannitol
As seen in Example 4, the addition of sucrose slowed down rfurin degradation and/or activity loss at 37° C. While sucrose is not used in downstream applications such as the rVWF process, other sugars and/or sugar alcohols are, such as mannitol. The addition of mannitol to the rfurin formulation, rather than sucrose, would therefore not introduce a new raw material to the vWF process. This study was conducted to determine whether the addition of mannitol would have a beneficial effect on rfurin stability. The test formulation also possessed an improved buffering capacity by addition of HEPES and acetic acid to final concentrations of 50 mM each at pH 6.0. A stabilized formulation was created by spiking a rfurin sample with 10% of 500 mM HEPES, 400 mM acetic acid, 1 mM CaCl2, pH 6, and then, adding either sucrose, mannitol, and/or polysorbate 80. Analyses by both the furin activity assay (FIG. 21) and SEC (FIG. 22) showed that after four days of incubation at 37° C., mannitol and sucrose had similar stabilizing effects on rfurin formulations. Again, the effect of polysorbate 80 on these formulations was not significant. Although the activity data (FIG. 21) show that the sample containing mannitol and polysorbate 80 after 4 days of incubation at 37° C. was better than other samples, not only does this sample not follow the overall pattern, but also the SEC analyses (Figure) did not confirm it. It is likely that this result was an outlier.
Example 6
Comparison of Furin Formulation Stability in Sucrose and Trehalose
Various publications have shown that mannitol accelerates the denaturation of proteins during freezing, which raised some concerns about its inclusion in the rfurin formulation buffer. No such problems have been reported with trehalose, another sugar already part of the rVWF production process. This study was conducted to determine whether addition of trehalose would have a beneficial effect on rfurin stability. In addition, various concentrations of polysorbate 80 were evaluated. Analyses using the furin activity assay (FIGS. 23 and 24) showed that after 5 days of incubation at 37° C., the sample containing 10% sucrose retained 66.2%±2.9% of its original activity while the sample containing 10% trehalose retained 60.1%±4%. The difference between these samples is not significant. Both samples were significantly better in preserving rfurin activity than the control formulation, which retained only 37.5%±4.2% of its original activity.
Analyses by SEC (FIG. 25) confirmed the activity analyses. The rfurin peak for the sample in 10% sucrose retained 78.3% of its height compared to 75.1% for the sample in 10% trehalose and 44.2% for the control formulation. Also, analyses by western blotting (FIG. 26) showed the least degradation in the samples containing 10% sucrose or 10% trehalose. Note that the samples are presented as pairs, with day 0 followed by day 5 for each of the formulations.
Both the furin activity assay (FIG. 27) and SEC (FIG. 28) analyses show that 10% trehalose was better than 5% or 2% in preserving rfurin activity at 37° C. These findings were also confirmed by western blotting (FIG. 26). The impact of polysorbate 80 on rfurin stability at 37° C. was minimal according to the analyses by the furin activity assay and SEC (FIGS. 29 and 30, respectively).
SEC and furin activity assays were conducted as described above. Western blotting was performed using the following equipment and materials:

    
    
        Goat anti-Furin (peptide) primary antibody (Cat# SC-12484) from Santa Cruz Biotechnology, Inc.
        Rabbit anti-goat IgG-HRP conjugate secondary antibody (Cat# A5420) from Sigma
        Pierce Metal Enhanced Substrate Kit containing DAB/Metal Concentrate 10×,
        Pierce (Cat#1856090) and Stable Peroxidase Buffer, Pierce (Cat#185591)
        4-20% Tris-Glycine gel (Cat# EC6025BOX) from Invitrogen
        NuPAGE® Sample Reducing Agent (0.5M DTT, 10×, Cat# NP0004) from Invitrogen
        Non-Reducing Sample Buffer (4×, Cat#84788) from Thermo
        PBS (Cat# P4417) from Sigma
        Tween 20 (Cat#9480) from EMD Chemicals
        BSA (Cat# A7906) from Sigma
        Gel transfer device (iBLOT Cat# IB401001) from Invitrogen
        PVDF (Immobilon-P, Cat# IPVH07850) transfer membrane from Millipore
    
    


A polyclonal antibody was used in this study instead of a monoclonal to increase detection of rFurin degradation products. 5 μl of a sample was combined with 1 μl of 10×DTT, and 4 μl of 4× non-reducing dye. The sample was heated at 70° C. to 75° C. for 10 minutes. 10 μl of the sample was loaded to 4-20% SDS-PAGE gel, and run at 150 V for 60 to 70 minutes. The protein gel was transferred to PVDF membrane using the iBLOT at 20 V for 6 minutes. The PVDF membrane was then blocked with PBS buffer containing 0.1% TWEEN 20 and 3% BSA for 1 hour. The membrane was incubated for one hour in the primary antibody diluted 125 fold with the same buffer. The membrane was washed with PBST buffer for five minutes, three times each. The membrane was incubated for one hour in the secondary antibody diluted 10,000 fold with the same buffer as the primary antibody. The incubation time was 1 hour. The blot was washed using PBST buffer for five minutes, three times each. The blot was developed in 10 mL of premixed chromagenic substrate (1 ml concentrate plus 9 ml of Stable Peroxidase Buffer) followed by the wash with water for 10 minutes.
Example 7
Effects of Stabilizing Agents Upon Dilution of Furin Compositions
The rFurin dilution study was carried out to compare a starting formulation and its diluent to a stabilized formulation and a stabilized diluent. The study mimicked the dilution method that used in the rVWF maturation process. Briefly, rfurin BDS was passed through a filter followed by the addition of a 200-fold volume of the diluent. The main difference between the test dilution method and the starting dilution method is that in the test (highly stabilized) method, 75 ppm polysorbate 80 is present in both the rfurin BDS and the diluent, while in the starting method, there is no polysorbate 80 in either the rfurin BDS or the diluent.
Two dilution experiments were carried out, and both have shown that the combination of the stabilized formulation with its stabilized diluent was superior to the starting furin and diluent formulations. In the first experiment, the test method using the stabilized formulations recovered 83% to 87% of the expected rfurin activity, while the control methods recovered only 21% to 22% (Table 18). In this experiment, the rfurin sample was passed through a filter and followed by a 200-fold volume of a diluent. The filtrate was collected to a bag and then transferred to a graduated cylinder.







TABLE 18







Experimental results of rfurin dilution experiments


performed with and without non-ionic surfactant.













Activity
St.
Dilution
Expected
Recovery


Sample
(IU)
Dev.
Factor
Activity (IU)
(% Activity)
















Starting Material

25,953
1,118
1
N/A
N/A


Control Formulation
From a Bag
27.264
2.815
200
129.8
21.0


After Filtration
From a
28.3
1.56
200
129.8
21.8



Cylinder


New Formulation After
From a Bag
108.137
6.611
200
129.8
83.3


Filtration
From a
113.4
5.21
200
129.8
87.4



Cylinder









In the second experiment, the test method recovered 80% of the expected rfurin activity, while the control method recovered only 27% (Table 19). In this experiment, the rfurin sample was passed through a filter and followed by a 200-fold volume of a diluent. The filtrate was collected to a cylinder. The combination of the stabilized rfurin formulation and the stabilized diluent increased rfurin recovery three to four times compared to the control method. The improved recovery is, without being limited by theory, linked to the presence of polysorbate 80.







TABLE 19







Experimental results of rfurin dilution experiments performed


with and without non-ionic surfactant.
















Expected
Recovery



Activity

Dilution
Activity
(%


Sample
(IU)
St. Dev.
Factor
(IU)
Activity)















Starting
32,379
1,654
N/A
N/A
N/A


Material


Control
44.267
1.607
200
161.9
27.3


Formulation


After


Filtration


New
129.646
11.514
200
161.9
80.1


Formulation


After


Filtration









Example 8
Characteristics of Stabilized Formulation Stock Buffer
The stock buffer for the stabilized rfurin formulation (500 mM HEPES, 400 mM acetic acid, 1 mM CaCl2, pH 6.0) can be made consistently (Table 20). The RSD's for pH and conductivity were <0.3%. The pH was slightly and indirectly correlated with temperature, dropping 0.014 units per degree Celsius, while conductivity was directly correlated with temperature, raising 0.40 mS/cm per degree C. (FIG. 49). The average temperature of the samples when the pH and conductivity were measured was 27.6° C. In manufacturing, the samples are read at 25±0.5° C. Based on the linear regressions in FIG. 48, the expected pH and conductivity at 25° C. were calculated. The relative standard deviation from Table 20 was applied to this value to determine the expected range (±3 sd). pH at 25° C. is expected to be 6.04 (±0.01). Conductivity at 25° C. is expected to be 19.9±0.2 mS/cm. These expected ranges are based on three lots made under limited circumstances. The specification ranges for the buffer need not be set this strictly. The pH specification will be the same as the final BDS pH specification, 5.90-6.10. The conductivity range will be similar to that used for the previous BDS buffer, e.g., with a range of ±20%, for a specification of 15.9-23.9 mS/cm. The requirements of the rVWF process are generally not of concern in setting the buffer specifications because the rfurin will be diluted 200-fold in the rVWF process. Experimental data in Table 21 and Table 22 correspond to the graphs presented in FIG. 49.







TABLE 20







Characteristics of stabilized formulation stock buffer.















Reagent
Buffer 1
Buffer 2
Buffer 3
mean
sd
rsd, %
mean + 3 sd
mean − 3 sd


















Water, L
0.85
0.85
0.85







HEPES, g
119.15
119.15
119.15


CaCl2, g
0.111
0.111
0.111


Acetic acid, mL
23
23
23


10M NaOH, mL
39.4
39.4
39.4


pH
6.011
6.003
6.005
6.006
0.004
0.07
6.019
5.994


Cond., mS/cm
21.23
21.15
21.11
21.16
0.06
0.29
21.35
20.98


Temp., C.
27.5
27.6
27.6
27.6
0.06
0.21
27.7
27.4
















TABLE 21







Dependence of stock buffer conductivity on temperature.










Temp, C.
Cond, mS/cm














18
17.23



18.4
17.42



18.7
17.53



19
17.63



19.3
17.75



19.6
17.86



19.7
17.91



20
18.03



20.2
17.72



20.5
17.83



21.6
17.86



24.4
19.81



27.6
21.1

















TABLE 22







Dependence of stock buffer pH on temperature.










Temp, C.
pH














18
6.135



19.3
6.103



21.6
6.088



27.6
6.005










REFERENCES
All references cited herein are hereby incorporated by reference herein in their entirety and for all purposes to the same extent as if each individual publication or patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.
Many modifications and variations of this application can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. The specific embodiments described herein are offered by way of example only, and the application is to be limited only by the terms of the appended claims, along with the full scope of equivalents to which the claims are entitled.

## Claims

1. A stabilized aqueous composition of recombinant furin (rfurin), the composition comprising:
(a) from 8,000 U/mL to 500,000 U/mL rfurin;
(b) from 175 mM to 260 mM of a pharmaceutically acceptable salt;
(c) from 0.5 mM to 2 mM calcium;
(d) from 2% to 20% sugar or sugar alcohol;
(e) from 10 to 200 ppm non-ionic surfactant;
(f) from 10 to 200 mM buffering agent; and
(g) a pH from 5.5 to 7.5.

2. The composition of claim 1, comprising from 180 mM to 260 mM of a pharmaceutically acceptable salt.

3. The composition of claim 1, wherein the pharmaceutically acceptable salt is selected from the group consisting of sodium chloride and potassium chloride.

4. The composition of claim 1, comprising 0.9±0.2 mM calcium.

5. The composition of claim 1, comprising from 5% to 15% sugar or sugar alcohol.

6. The composition of claim 5, comprising 10% sugar or sugar alcohol.

7. The composition of claim 1, wherein the sugar or sugar alcohol is selected from the group consisting of sucrose, trehalose, mannitol, and a combination thereof.

8. The composition of claim 1, wherein the non-ionic surfactant is polysorbate-80.

9. The composition of claim 1, comprising from 10 ppm to 100 ppm non-ionic surfactant.

10. The composition of claim 9, comprising 75 ppm non-ionic surfactant.

11. The composition of claim 1, comprising from 50 mM to 150 mM buffering agent.

12. The composition of claim 1, comprising 90±10 mM buffering agent.

13. The composition of claim 1, wherein the buffering agent comprises acetate and HEPES.

14. The composition of claim 13, comprising from 25 mM to 75 mM acetate and from 25 to 75 mM HEPES.

15. The composition of claim 13, wherein the buffering agent comprises 45±5 mM acetate and 45±5 mM HEPES.

16. The composition of claim 1, wherein the buffering agent comprises MES.

17. The composition of claim 1, having a pH from 5.5 to 7.0.

18. The composition of claim 1, having a pH from 5.5 to 6.5.

19. The composition of claim 1, having a pH of 6.0.

20. The composition of claim 1, having a pH of 6.0±0.2.

21. The composition of claim 1, wherein the composition has increased stability when stored at 37° C. as compared to a rfurin composition that does not contain a sugar or sugar alcohol.

22. The composition of claim 1, wherein the composition maintains a higher percentage of rfurin activity when stored at 37° C. as compared to a rfurin composition that does not contain a sugar or sugar alcohol.

23. The composition of claim 1, wherein the composition maintains a higher percentage of rfurin monomer content when stored at 37° C. as compared to a rfurin composition that does not contain a sugar or sugar alcohol.

24. The composition of claim 1, wherein the composition has increased stability when agitated as compared to a rfurin composition that does not contain a non-ionic surfactant.

25. The composition of claim 1, wherein the composition maintains a higher percentage of rfurin activity when agitated as compared to a rfurin composition that does not contain a non-ionic surfactant.

26. The composition of claim 1, wherein the composition maintains a higher percentage of rfurin monomer content when agitated as compared to a rfurin composition that does not contain a non-ionic surfactant.

27. The composition of claim 1, wherein the composition has increased stability when diluted as compared to a rfurin composition that does not contain a non-ionic surfactant.

28. The composition of claim 1, wherein the composition maintains a higher percentage of rfurin activity when diluted as compared to a rfurin composition that does not contain a non-ionic surfactant.

29. The composition of claim 1, wherein the composition maintains a higher percentage of rfurin monomer content when diluted as compared to a rfurin composition that does not contain a non-ionic surfactant.