Enhanced protein purification through a modified protein A elution

Patent metadata

Field	Value
Publication number	US-9428548-B2
Application number	US-201013393525-A
Country	US
Kind code	B2
Filing date	Sep 1, 2010
Priority date	Sep 1, 2009
Publication date	Aug 30, 2016
Grant date	Aug 30, 2016

Abstract

The present invention provides methods for purifying a polypeptide comprising a CH2/CH3 region, comprising binding the polypeptide to Protein A and eluting with a pH gradient starting at a low pH.

First claim

What is claimed is:

1. A method for purifying a polypeptide comprising a C H 2/C H 3 region, comprising the steps of:
(a) binding the polypeptide to Protein A; and
(b) eluting the polypeptide with a pH gradient starting at or below 5.0 using an elution buffer, wherein the elution buffer contains a high pH buffer and a low pH buffer and wherein the pH gradient is formed by adjusting a percentage of each pH buffer in the elution buffer,
wherein the high pH buffer is at about 5.0 and wherein the low pH buffer is at about 2.7, wherein the pH gradient ends at about 3.7, and
wherein an aggregate, a host cell impurity, a virus filter foulant, a virus particle and a virus-like particle are removed from the desired polypeptide.

2. The method of claim 1 , wherein the percentage of low pH buffer starts at about 35%.

3. The method of claim 2 , wherein the elution buffer containing the low pH buffer at about 35% comprises about 16.25 mM acetate and about 8.75 mM formate.

4. The method of claim 1 , wherein the percentage of low pH buffer starts at about 25%.

5. The method of claim 4 , wherein the elution buffer containing the low pH buffer at about 25% comprises about 18.75 mM acetate and 6.25 mM formate.

6. The method of claim 1 , wherein the percentage of low pH buffer starts at about 40%.

7. The method of claim 6 , wherein the elution buffer containing the low pH buffer at about 40% comprises about 15 mM acetate and 10 mM formate.

8. The method of claim 1 , wherein the polypeptide is loaded with a loading density starting at about 14 g/L.

9. The method of claim 1 , wherein the Protein A is a Protein A column chromatography resin or a Protein A chromatography sorbent.

10. The method of claim 9 , wherein the Protein A chromatography sorbent is a membrane or a monolith.

11. The method of claim 9 , wherein the Protein A is a Protein A column chromatography resin and wherein the polypeptide has an elution flow rate ranging from about 5 column volume/hour to about 25 column volume/hour.

12. The method of claim 9 , wherein the Protein A is a Protein A column chromatography resin and wherein a purified fraction of the polypeptide contains about or fewer than about 12 Protein A column volumes.

13. The method of claim 1 , wherein the pH gradient starts at about pH 4.2.

14. The method of claim 1 , wherein the pH gradient starts at about pH 4.3.

15. The method of claim 14 , wherein the polypeptide is an anti-VEGF antibody, an anti-CD20 antibody, an anti-MUC16 antibody, an anti-MET antibody or an anti-CD4 antibody.

16. The method of claim 15 , wherein the antibody is an anti-VEGF antibody.

17. The method of claim 16 , wherein the anti-VEGF antibody is bevacizumab.

18. The method of claim 15 , wherein the antibody is an anti-CD20 antibody.

19. The method of claim 18 , wherein the anti-CD20 antibody is rituximab.

20. The method of claim 1 , wherein the pH gradient starts at about pH 4.6.

21. The method of claim 1 , wherein the host cell impurity is Chinese Hamster Ovary Protein (CHOP).

22. The method of claim 1 , wherein a basic polypeptide variant is separated from the polypeptide.

23. The method of claim 22 , wherein the polypeptide is an anti-VEGF antibody, an anti-CD20 antibody, an anti-MUC16 antibody, an anti-MET antibody or an anti-CD4 antibody.

24. The method of claim 23 , wherein the antibody is an anti-VEGF antibody.

25. The method of claim 24 , wherein the anti-VEGF antibody is bevacizumab.

26. The method of claim 23 , wherein the antibody is an anti-CD20 antibody.

27. The method of claim 26 , wherein the anti-CD20 antibody is rituximab.

28. The method of claim 1 , wherein the C H 2/C H 3 region comprises a Fc region of an immunoglobulin.

29. The method of claim 1 , wherein the polypeptide is an antibody.

30. The method of claim 29 , wherein the antibody is a monoclonal antibody, a polyclonal antibody, a multi-specific antibody, or an antibody fragment.

31. The method of claim 1 , wherein the polypeptide is an immunoadhesion.

32. The method of claim 1 , wherein the purified polypeptide has a purity of at least about 98% monomer.

33. The method of claim 32 , wherein the polypeptide is an anti-VEGF antibody, an anti-CD20 antibody, an anti-MUC16 antibody, an anti-MET antibody or an anti-CD4 antibody.

34. The method of claim 33 , wherein the antibody is an anti-VEGF antibody.

35. The method of claim 34 , wherein the anti-VEGF antibody is bevacizumab.

36. The method of claim 33 , wherein the antibody is an anti-CD20 antibody.

37. The method of claim 36 , wherein the anti-CD20 antibody is rituximab.

38. The method of claim 1 , wherein the purified polypeptide has a purity of at least about 99% monomer.

39. The method of claim 38 , wherein the polypeptide is an anti-VEGF antibody, an anti-CD20 antibody, an anti-MUC16 antibody, an anti-MET antibody or an anti-CD4 antibody.

40. The method of claim 39 , wherein the antibody is an anti-VEGF antibody.

41. The method of claim 40 , wherein the anti-VEGF antibody is bevacizumab.

42. The method of claim 39 , wherein the antibody is an anti-CD20 antibody.

43. The method of claim 42 wherein the anti-CD20 antibody is rituximab.

44. The method of claim 1 , wherein a ratio of a host cell impurity to the purified polypeptide is at least about 20% lower than the ratio in a polypeptide purified by a step elution method, wherein the step elution method comprises binding the polypeptide to Protein A and eluting with a pH starting at or below 3.6.

45. The method of claim 44 , wherein the polypeptide is an anti-VEGF antibody, an anti-CD20 antibody, an anti-MUC16 antibody, an anti-MET antibody or an anti-CD4 antibody.

46. The method of claim 45 , wherein the antibody is an anti-VEGF antibody.

47. The method of claim 46 , wherein the anti-VEGF antibody is bevacizumab.

48. The method of claim 45 , wherein the antibody is an anti-CD20 antibody.

49. The method of claim 48 , wherein the anti-CD20 antibody is rituximab.

50. The method of claim 1 , wherein a ratio of a host cell impurity to the purified polypeptide is at least about 60% lower than the ratio in a polypeptide purified by a step elution method, wherein the step elution method comprises binding the polypeptide to Protein A and eluting with a pH starting at about 3.6.

51. The method of claim 50 , wherein the polypeptide is an anti-VEGF antibody, an anti-CD20 antibody, an anti-MUC16 antibody, an anti-MET antibody or an anti-CD4 antibody.

52. The method of claim 51 , wherein the antibody is an anti-VEGF antibody.

53. The method of claim 52 , wherein the anti-VEGF antibody is bevacizumab.

54. The method of claim 51 , wherein the antibody is an anti-CD20 antibody.

55. The method of claim 54 , wherein the anti-CD20 antibody is rituximab.

56. The method of claim 44 or 50 , wherein the purified polypeptide is a polypeptide monomer.

Assignees

• Brown Arick
• Dowd Christopher J
• Radhamohan Asha Nandini
• Genentech Inc

Inventors

• Brown Arick
• Dowd Christopher J
• Radhamohan Asha Nandini

Classifications

• C07K1/22 Primary

  Affinity chromatography or related techniques based upon selective absorption processes · CPC title

• C12P21/005

  Glycopeptides, glycoproteins · CPC title

• C07K16/46

  Hybrid immunoglobulins (hybrids of an immunoglobulin with a peptide not being an immunoglobulin C07K19/00) · CPC title

• C07K1/18 Primary

  Ion-exchange chromatography · CPC title