Claims for Patent: 9,428,548

Summary for Patent: 9,428,548

Title:	Enhanced protein purification through a modified protein A elution
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.
Inventor(s):	Brown; Arick (South San Francisco, CA), Dowd; Christopher J. (South San Francisco, CA), Radhamohan; Asha Nandini (South San Fracisco, CA)
Assignee:	Genentech, Inc. (South San Francisco, CA)
Application Number:	13/393,525
Patent Litigation and PTAB cases:	See patent lawsuits and PTAB cases for patent 9,428,548
Patent Claims:	1. A method for purifying a polypeptide comprising a C.sub.H2/C.sub.H3 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.sub.H2/C.sub.H3 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. 57. The method of claim 1, wherein the Protein A is a modified or a non-modified Protein A ligand. 58. The method of claim 1, wherein the purification is a manufacturing scale process. 59. The method of claim 1, further comprising subjecting the polypeptide to a virus filtration step. 60. The method of claim 1, further comprising subjecting the polypeptide to an ion exchange chromatography step. 61. The method of claim 60, wherein the ion exchange chromatography step runs continuously after step (b). 62. The method of claim 1, wherein the method does not comprise a further purification step to remove an aggregate. 63. The method of claim 1, wherein the method does not comprise a further purification step to remove a virus filter foulant. 64. The method of claim 63, 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. 65. The method of claim 64, wherein the antibody is an anti-VEGF antibody. 66. The method of claim 65, wherein the anti-VEGF antibody is bevacizumab. 67. The method of claim 64, wherein the antibody is an anti-CD20 antibody. 68. The method of claim 67, wherein the anti-CD20 antibody is rituximab. 69. The method of claim 1, 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. 70. The method of claim 69, wherein the antibody is an anti-VEGF antibody. 71. The method of claim 70, wherein the anti-VEGF antibody is bevacizumab. 72. The method of claim 69, wherein the antibody is an anti-CD20 antibody. 73. The method of claim 72, wherein the anti-CD20 antibody is rituximab.

Details for Patent 9,428,548

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Genentech, Inc.	RITUXAN	rituximab	Injection	103705	11/26/1997		2029-09-01
Idec Pharmaceuticals Corp.	RITUXAN	rituximab	Injection	103737	02/19/2002		2029-09-01
Genentech, Inc.	AVASTIN	bevacizumab	Injection	125085	02/26/2004		2029-09-01
Genentech, Inc.	RITUXAN HYCELA	rituximab and hyaluronidase human	Injection	761064	06/22/2017		2029-09-01