ISOLATION AND PURIFICATION OF ANTIBODIES USING PROTEIN A AFFINITY CHROMATOGRAPHY

• US 20140255423A1
• Filed: 03/26/2014
• Published: 09/11/2014
• Est. Priority Date: 10/20/2008
• Status: Active Grant

• Current Assignee: Abbvie Incorporated
• Original Assignee: Abbvie Incorporated
• Inventors: Hickman, Robert K., Weed, Cheryl L., Ennis, Scott T., Perilli-Palmer, Barbara, Wan, Min, Huang, Qing
• Granted Patent: US 9,018,361 B2
• Time in Patent Office: Days
• US Class Current: 424/158.1
• CPC Class Codes: A61P 1/00 Drugs for disorders of the alimentary tract or the digestive system
• CPC Class Codes: A61P 17/06 Antipsoriatics
• CPC Class Codes: A61P 19/02 for joint disorders, e.g. arthritis, arthrosis
• CPC Class Codes: A61P 29/00 Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 37/02 Immunomodulators
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C07K 1/165 mixed-mode chromatography
• CPC Class Codes: C07K 1/18 Ion-exchange chromatography
• CPC Class Codes: C07K 1/20 Partition-, reverse-phase or hydrophobic interaction chromatography
• CPC Class Codes: C07K 1/22 Affinity chromatography or related techniques based upon selective absorption processes
• CPC Class Codes: C07K 1/36 by a combination of two or more processes of different types
• CPC Class Codes: C07K 16/00 Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies antibodies with enzymatic activity, e.g. abzymes C12N9/0002
• CPC Class Codes: C07K 16/241 Tumor Necrosis Factors
• CPC Class Codes: C07K 16/244 Interleukins [IL]
• CPC Class Codes: C07K 2317/14 Specific host cells or culture conditions, e.g. components, pH or temperature
• CPC Class Codes: C07K 2317/21 from primates, e.g. man
• CPC Class Codes: C07K 2317/22 from camelids, e.g. camel, llama or dromedary
• CPC Class Codes: C07K 2317/24 containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• CPC Class Codes: Y10S 436/824 Immunological separation techniques

Abstract

Disclosed herein are methods for the isolation and purification of antibodies wherein the use of an affinity chromatographic step results in an antibody composition sufficiently pure for pharmaceutical uses. The methods described herein comprise pH viral reduction/inactivation, ultrafiltration/diafiltration, affinity chromatography, preferably Protein A affinity, ion exchange chromatography, and hydrophobic chromatography. Further, the present invention is directed toward pharmaceutical compositions comprising one or more antibodies of the present invention.

Claims

1-27. -27. (canceled)

28. A process for purifying an anti-TNFα

antibody that comprises a heavy chain variable region amino acid sequence of SEQ ID NO;

5 and a light chain variable region amino acid sequence of SEQ ID NO;

6 from a Chinese Hamster Ovary (CHO) cell culture expressing said anti-TNFα

antibody, said process comprising;

binding the anti-TNFα

antibody from said cell culture to a Protein A resin, and

eluting the bound anti-TNFα

antibody with an acidic elution buffer, wherein said process further comprises incubating the anti-TNFα

antibody at a pH of between 2 and 5 for 0.5 to 2 hours.

29. The process of claim 28, wherein said eluting is at an elution pH range from 2.5 to 4.

30. The process of claim 28, wherein said incubating is at a pH of between 2 and 5 for 0.5 to 1.5 hours.

31. The process of claim 28, wherein said incubating is at a pH of between 3 and 4 for from 0.5 to 2 hours.

32. The process of claim 28, wherein said incubating is at a pH of 3.5 for 1 hour.

33. The process of claim 28, further comprising subjecting the anti-TNFα

antibody to an ion exchange chromatography.

34. The process of claim 33, wherein said ion exchange chromatography is cation exchange chromatography or anion exchange chromatography.

35. The process of claim 28, further comprising subjecting the anti-TNFα

antibody to a hydrophobic interaction chromatography.

36. The process of claim 34, further comprising subjecting the anti-TNFα

antibody to a hydrophobic interaction chromatography.

37. The process of claim 28, further comprising purifying the anti-TNFα

using ionic interaction, hydrophobic interaction, or a combination thereof.

38. The process of claim 28, wherein the purified anti-TNFα

is pharmaceutical grade.

39. The process of claim 28, wherein said process produces greater than 90% monomers of said antibody.

40. The process of claim 28, wherein said process using Protein A resin produces a greater clearance of host cell protein as compared to a similar process that employs a Fractogel™

resin.

41. The process of claim 40, wherein said process using Protein A resin removes greater than 93% of the host cell proteins present in an anti-TNFα

antibody preparation applied to said Protein A resin.

42. A process for purifying adalimumab from a Chinese Hamster Ovary (CHO) cell culture expressing said adalimumab, said process comprising:

binding the adalimumab from said cell culture to a Protein A resin, and

eluting the bound adalimumab with an acidic elution buffer,

wherein said process further comprises incubating adalimumab at a pH of between 2 and 5 for 0.5 to 2 hours.

43. The process of claim 42, wherein said eluting is at an elution pH range from 2.5 to 4.

44. The process of claim 42, wherein said incubating is at a pH of between 2 and 5 for 0.5 to 1.5 hours.

45. The process of claim 42, wherein said incubating is at a pH of between 3 and 4 for 0.5 to 2 hours.

46. The process of claim 42, wherein said incubating is at a pH of 3.5 for 1 hour.

47. The process of claim 42, further comprising subjecting adalimumab to an ion exchange chromatography.

48. The process of claim 47, wherein said ion exchange chromatography is cation exchange chromatography or anion exchange chromatography.

49. The process of claim 48, further comprising subjecting adalimumab to a hydrophobic interaction chromatography.

50. The process of claim 42, further comprising subjecting adalimumab to a hydrophobic interaction chromatography.

51. The process of claim 42, further comprising purifying adalimumab using ionic interaction, hydrophobic interaction, or a combination thereof.

52. The process of claim 42, wherein the purified adalimumab is pharmaceutical grade.

53. The process of claim 42, wherein said process produces greater than 90% monomers of said adalimumab.

54. The process of claim 42, wherein said eluting is at an elution pH of 3.2.

55. A process for purifying adalimumab from a Chinese Hamster Ovary (CHO) cell culture expressing said adalimumab, said process comprising:

binding the adalimumab from said cell culture to a Protein A resin, and

eluting the bound adalimumab with an acidic elution buffer,

wherein said process further comprises incubating adalimumab at a pH of between 2 and 5 for 0.5 to 2 hours;

and wherein said process further comprises subjecting adalimumab to one or more further chromatographic separations to produce a host cell protein-reduced adalimumab preparation, wherein said one or more further chromatographic separations comprise an ion exchange chromatography, a hydrophobic interactive chromatography or a combination thereof, wherein said process produces greater than 90% monomers of said adalimumab.

56. The process of claim 55 wherein said process using Protein A resin produces a greater clearance of host cell protein as compared to a similar process that employs a Fractogel™

resin.

57. The process of claim 56, wherein said process using Protein A resin removes greater than 93% of the host cell proteins present in an adalimumab preparation applied to said Protein A resin.