Method to produce an immunoglobulin preparation with improved yield
The present invention provides improved methods for the manufacturing of IVIG products. These methods offer various advantages such as reduced loss of IgG during purification and improved quality of final products. In other aspects, the present invention provides aqueous and pharmaceutical compositions suitable for intravenous, subcutaneous, and/or intramuscular administration. In yet other embodiments, the present invention provides methods of treating a disease or condition comprising administration of an IgG composition provided herein.
1. A method for preparing an enriched IgG composition from plasma, the method comprising the steps of:
(a) precipitating a cryo-poor plasma fraction, in a first precipitation step, with from 6% to 10% ethyl alcohol at a pH of from 7.0 to 7.5 to form a first precipitate and a first supernatant;
(b) precipitating IgG from the first supernatant, in a second precipitation step, with from 23% to 27% ethyl alcohol at a pH of from 6.7 to 7.3 to form a second precipitate;
(c) suspending the second precipitate to form a first suspension;
(d) mixing fumed silica with the first suspension formed in (c) for at least 30 minutes; and
(e) separating a solubilized portion of the first suspension mixed with fumed silica in step
(d) from a non-solubilized portion of the first suspension, thereby forming an enriched IgG composition.
2. The method of claim 1 , wherein the second precipitation step is performed at a temperature of from −7° C. to −9° C.
3. The method of claim 1 , wherein the second precipitate is suspended with an extraction buffer at a ratio of 1 part precipitate to from 12 parts to 18 parts of an extraction buffer.
4. The method of claim 3 , wherein the extraction buffer has a pH of from about 4.5 to about 5.0.
5. The method of claim 4 , wherein the extraction buffer comprises 5 mM sodium phosphate and 5 mM acetate.
6. The method of claim 5 , wherein the extraction buffer comprises from 510 mL to 600 mL of glacial acetic acid per 1000 L of buffer.
7. The method of claim 1 , wherein from 0.01 g to 0.07 g fumed silica per g of the second precipitate formed in step (b) is mixed with the first suspension in step (d).
8. The method of claim 1 , wherein from 0.02 g to 0.06 g fumed silica per g of the second precipitate formed in step (b) is mixed with the first suspension in step (d).
9. The method of claim 1 , wherein from 0.03 g to 0.05 g fumed silica per g of the second precipitate formed in step (b) is mixed with the first suspension in step (d).
10. The method of claim 1 , wherein the solubilized portion of the first suspension mixed with fumed silica is separated from the non-solubilized portion of the first suspension by depth filtration.
11. The method of claim 10 , wherein the depth filtration further comprises washing a depth filter used in the depth filtration with at least 3 filter void volumes of buffer.
12. The method of claim 1 , wherein the soluble portion of the first suspension, separated in step (e), contains at least 85% of the IgG content of the cryo-poor plasma fraction used in step (a).
13. The method of claim 1 , wherein the soluble portion of the first suspension, separated in step (e), contains at least 90% of the IgG content of the cryo-poor plasma fraction used in step (a).
14. The method of claim 1 , further comprising the steps of:
(f) precipitating IgG from the solubilized portion of the suspension, in a third precipitation step, with from 22% to 28% ethyl alcohol at a pH of from 6.7 to 7.3 to form a third precipitate;
(g) suspending the third precipitate to form a second suspension;
(h) separating a solubilized portion of the second suspension formed in step (g) from a non-solubilized portion of the second suspension.
15. The method of claim 2 , further comprising the steps of:
(f) precipitating IgG from the solubilized portion of the suspension, in a third precipitation step, with from 22% to 28% ethyl alcohol at a pH of from 6.7 to 7.3 to form a third precipitate;
(g) suspending the third precipitate to form a second suspension;
(h) separating a solubilized portion of the second suspension formed in step (g) from a non-solubilized portion of the second suspension.
16. The method of claim 9 , further comprising the steps of:
(f) precipitating IgG from the solubilized portion of the suspension, in a third precipitation step, with from 22% to 28% ethyl alcohol at a pH of from 6.7 to 7.3 to form a third precipitate;
(g) suspending the third precipitate to form a second suspension;
(h) separating a solubilized portion of the second suspension formed in step (g) from a non-solubilized portion of the second suspension.
17. The method of claim 14 , further comprising treating the solubilized portion of the second suspension separated in step (h) with a solvent and detergent (S/D) treatment step.
18. The method of claim 14 , further comprising the steps of:
binding IgG in the solubilized portion of the second suspension to a cation exchange material;
eluting IgG from the cation exchange material to form a cation exchange eluate.
19. The method of claim 18 , further comprising the steps of:
loading IgG from the cation exchange eluate onto an anion exchange column;
collecting an effluent comprising IgG from the anion exchange column to form an anion exchange flow-through.
20. The method of claim 19 , further comprising the step of:
nanofiltering IgG from the anion exchange flow-through to form a nanofiltrate.
21. The method of claim 20 , wherein the nanofiltration is performed with a nanofilter having a mean pore size of about 35 nm.
22. The method of claim 20 , further comprising the step of:
ultrafiltering and diafiltering IgG from the nanofiltrate to form a filtrate having a protein concentration of at least 11% (w/v), thereby obtaining an enriched IgG composition.
23. The method of claim 22 , wherein the ultrafiltration and diafiltration comprises the sub-steps of:
(1) concentrating IgG from the nanofiltrate to a protein concentration of 5±2% (w/v) to form a first IgG concentrate;
(2) diafiltering the IgG concentrate of (1) against a buffer comprising glycine to form an IgG diafiltrate; and
(3) concentrating the IgG diafiltrate of (2) to a protein concentration of at least 11% (w/v).