Removal of serine proteases by treatment with finely divided silicon dioxide
The present invention provides novel methods for reducing the serine protease and/or serine protease zymogen content of a plasma-derived protein composition. Also provided are methods for manufacturing plasma-derived protein compositions having reduced serine protease and\or serine protease zymogen content. Among yet other aspects, the present invention provides aqueous and lyophilized compositions of plasma-derived proteins having reduced serine protease and/or serine protease zymogen content. Yet other aspects include methods for treating, managing, and/or preventing a disease comprising the administration of a plasma-derived protein composition having a reduced serine protease or serine protease zymogen content.
1. A plasma-derived immunoglobulin G (IgG) composition comprising an IgG concentration of at least 10% and a serine protease concentration that induces hydrolysis of the substrate S-2266 at a rate of less than 5 nmol/ml×min, and wherein said composition is prepared by a method comprising the steps of:
(a) precipitating a cryo-poor plasma fraction, in a first precipitation step, with from about 6% to about 10% alcohol at a pH of from about 7.0 to 7.5 to obtain a first precipitate and a first supernatant;
(b) precipitating IgG from the first supernatant, in a second precipitation step, with from about 23% to about 27% alcohol at a pH of from about 6.7 to about 7.3 to form a second precipitate;
(c) suspending in a buffer the second precipitate to form a first suspension;
(d) contacting the first suspension with finely divided silicon dioxide (SiO 2 ) under a solution condition suitable to bind a serine protease or serine protease zymogen, wherein the solution condition suitable to bind the serine protease or serine protease zymogen comprises a pH between about 4.5 and about 6.0 and a conductivity of between about 0.1 mS/cm and about 3 mS/cm; and
(e) separating the SiO 2 from the first suspension to form a clarified suspension.
2. The composition according to claim 1 , wherein step (b) comprises adjusting the ethanol concentration of the first supernatant formed in step (a) to about 25% (v/v) at a temperature from about −7° C. to about −9° C.
3. The composition according to claim 1 , wherein step (d) comprises the addition of SiO 2 to a final concentration of from about 0.02 grams per gram precipitate formed in step (b) to about 0.06 grams per gram precipitate formed in step (b).
4. The composition according to claim 1 , wherein step (e) comprises the sub-steps of:
(i) separating the SiO 2 from the first suspension using a filter press thereby forming a filtrate, and a filter cake;
(ii) washing the filter cake with at least 3 filter press dead volumes of a wash buffer containing phosphate and acetate, wherein the pH of the wash buffer is adjusted with from about 50 mL to about 200 mL of glacial acetic acid per 1000 L of wash buffer, thereby forming a wash solution; and
(iii) combining the filtrate of sub-step (i) with the wash solution of sub-step (ii), thereby forming the clarified suspension.
5. The composition according to claim 1 , wherein the pH of the solution condition suitable to bind the serine protease or serine protease zymogen is from about 4.9 to about 5.3 and the conductivity of between about 0.5 mS/cm and about 2 mS/cm.
6. The composition according to claim 1 , wherein the method further comprises an anion exchange chromatography enrichment step.
7. The composition according to claim 1 , wherein the method further comprises a cation exchange chromatography enrichment step.
8. The composition according to claim 1 , wherein the method further comprises at least one dedicated viral inactivation or removal step.
9. The composition according to claim 1 , wherein the method comprises a solvent/detergent (S/D) viral inactivation step.
10. The composition according to claim 1 , wherein the method comprises a nanofiltration step.
11. The composition according to claim 1 , wherein the method comprises an incubation step at low pH.
12. The composition according to claim 1 , wherein the IgG composition obtained in step (e) contains at least about 85% of the IgG content found in the cryo-poor plasma fraction of step (a).
13. The composition according to claim 1 , wherein the IgG composition obtained in step (e) contains at least about 90% of the IgG content found in the cryo-poor plasma fraction of step (a).
14. The composition according to claim 1 , wherein the composition further comprises a pharmaceutically acceptable stabilizing agent.
15. The composition according to claim 14 , wherein the pharmaceutically acceptable stabilizing agent is glycine.
16. The composition according to claim 1 , wherein the composition is formulated for intravenous, intramuscular, or subcutaneous administration.
17. The composition according to claim 1 , wherein the composition is aqueous, or lyophilized.
18. A method for treating a disease associated with aberrant activity of a plasma-derived IgG in a subject in need thereof, the method comprising administering a plasma-derived IgG composition according to claim 1 .
19. The composition according to claim 1 , wherein the method after step (e) further comprises:
(f) precipitating IgG from the clarified suspension formed in step (e), in a third precipitation step, with from about 22% to about 28% alcohol at a pH of from about 6.7 to about 7.3 to form a third precipitate;
(g) suspending the third precipitate to form a second suspension; and
(h) separating a soluble fraction from an insoluble fraction in the second suspension formed in step (g), thereby forming a composition comprising a plasma-derived immunoglobulin G (IgG).