Removal of serine proteases by treatment with finely divided silicon dioxide
View Patent ↗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 method for preparing an Immunoglobulin G (IgG) composition having a reduced amount of a serine protease or a serine protease zymogen, the 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 the second precipitate to form a first suspension;
(d) contacting the first suspension with from about 0.02 grams fumed silica per gram precipitate formed in step (b) to about 0.06 grams fumed silica per gram precipitate formed in step (b) at a pH of from about 4 to about 7 at a conductivity of from about 0.1 mS/cm to about 3.0 mS/cm; and
(e) separating the fumed silica from the first suspension to form a clarified suspension.
2. The method of claim 1 , wherein the method further comprises the steps of:
(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 an enriched IgG composition.
3. The method of claim 1 , wherein the method further comprises an anion exchange chromatography enrichment step.
4. The method of claim 1 , wherein the method further comprises a cation exchange chromatography enrichment step.
5. The method of claim 1 , wherein the method further comprises at least one dedicated viral inactivation or removal step.
6. The method of claim 5 , wherein the method comprises a solvent/detergent (S/D) viral inactivation step.
7. The method of claim 5 , wherein the method comprises a nanofiltration step.
8. The method of claim 5 , wherein the method comprises an incubation step at low pH.
9. The method of 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.
10. The method of claim 9 , wherein step (b) is performed at a temperature of about −9° C.
11. The method of claim 2 , wherein step (c) comprises suspending the precipitate formed in PM step (b) with a buffer containing phosphate and acetate, wherein the pH of the buffer is adjusted with from 300 mL to 700 mL of glacial acetic acid per 1000 L of the buffer.
12. The method of claim 1 , wherein the first suspension is contacted with fumed silica at a pH of from 4.9 to 5.3.
13. The method of claim 12 , wherein the first suspension is contacted with fumed silica at a conductivity of from 0.5 mS/cm to 2 mS/cm.
14. The method of claim 1 , wherein step (e) comprises the sub-steps of:
(i) separating the fumed silica from the first suspension using a filter press thereby forming a filtrate;
(ii) washing the filter press 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 50 mL to 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 with the wash solution of sub-step (ii), thereby forming the clarified suspension.
15. The method of claim 14 , further comprising the sub-step of:
(iv) treating the clarified suspension with a detergent.
16. The method of claim 2 , wherein the enriched IgG composition obtained in step (h) contains at least 85% of the IgG content found in the cryo-poor plasma fraction used in step (a).
17. The method of claim 16 , wherein the enriched IgG composition obtained in step (h) contains at least 90% of the IgG content found in the cryo-poor plasma fraction used in step (a).
18. The method of claim 2 , 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.
19. The method of claim 2 , wherein the first suspension is contacted with fumed silica at a pH of from 4.9 to 5.3.
20. The method of claim 19 , wherein the first suspension is contacted with fumed silica at a conductivity of from 0.5 mS/cm to 2 mS/cm.