Antibody purification

• US 7,863,426 B2
• Filed: 04/04/2007
• Issued: 01/04/2011
• Est. Priority Date: 04/05/2006
• Status: Active Grant

• Current Assignee: AbbVie Biotechnology Ltd. (Puerto Rico) (Abbvie Incorporated)
• Original Assignee: Abbott Biotechnology Limited (Abbott Laboratories Incorporated)
• Inventors: Avgerinos, George, Wan, Min, Zarbis-Papastoitsis, Gregory
• Primary Examiner(s): Saunders; David A
• Application Number:
  US11/732,918
• Publication Number:
  US 20070292442A1
• Time in Patent Office: 1,371 Days
• Field of Search: None
• US Class Current: 530/390.1
• CPC Class Codes: A61K 2039/505 comprising antibodies
• CPC Class Codes: A61K 2039/54 characterised by the route of administration
• CPC Class Codes: A61K 2039/545 characterised by the dose, timing or administration schedule
• CPC Class Codes: A61K 31/519 ortho- or peri-condensed with heterocyclic rings
• CPC Class Codes: A61K 39/3955 against proteinaceous materials, e.g. enzymes, hormones, lymphokines
• CPC Class Codes: A61K 39/39591 Stabilisation, fragmentation
• CPC Class Codes: A61K 47/22 Heterocyclic compounds, e.g. ascorbic acid, tocopherol or pyrrolidones
• CPC Class Codes: A61K 47/26 Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
• CPC Class Codes: A61P 1/00 Drugs for disorders of the alimentary tract or the digestive system
• CPC Class Codes: A61P 1/04 for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
• CPC Class Codes: A61P 11/00 Drugs for disorders of the respiratory system
• CPC Class Codes: A61P 13/12 of the kidneys
• CPC Class Codes: A61P 17/00 Drugs for dermatological disorders
• CPC Class Codes: A61P 17/06 Antipsoriatics
• CPC Class Codes: A61P 19/02 for joint disorders, e.g. arthritis, arthrosis
• CPC Class Codes: A61P 19/06 Antigout agents, e.g. antihyperuricemic or uricosuric agents
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 27/02 Ophthalmic agents
• 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 29/02 without antiinflammatory effect
• CPC Class Codes: A61P 3/00 : Drugs for disorders of the metabolism of the blood or the extracellular fluid A61P7/00
• CPC Class Codes: A61P 3/04 : Anorexiants; Antiobesity agents
• CPC Class Codes: A61P 3/10 : for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 31/00 : Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• CPC Class Codes: A61P 31/14 : for RNA viruses
• CPC Class Codes: A61P 35/00 : Antineoplastic agents
• CPC Class Codes: A61P 37/00 : Drugs for immunological or allergic disorders
• CPC Class Codes: A61P 37/06 : Immunosuppressants, e.g. drugs for graft rejection
• CPC Class Codes: A61P 37/08 : Antiallergic agents antiasthmatic agents A61P11/06; ophthalmic antiallergics A61P27/14
• CPC Class Codes: A61P 43/00 : Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 9/00 : Drugs for disorders of the cardiovascular system
• CPC Class Codes: A61P 9/10 : for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• 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 1/36 : by a combination of two or more processes of different types
• CPC Class Codes: C07K 16/065 : Purification, fragmentation
• CPC Class Codes: C07K 16/241 : Tumor Necrosis Factors
• 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 2317/94 : Stability, e.g. half-life, pH, temperature or enzyme-resistance

Abstract

The invention provides a method for producing a host cell protein-(HCP) reduced antibody preparation from a mixture comprising an antibody and at least one HCP, comprising an ion exchange separation step wherein the mixture is subjected to a first ion exchange material, such that the HCP-reduced antibody preparation is obtained.

Claims

1. A method for producing a host cell protein (HCP)-reduced antibody preparation from a mixture comprising an antibody and at least one HCP, the method comprising:

applying the mixture to a cation exchange resin equilibrated with an equilibration buffer, wherein greater than 30 grams of antibody per liter of cation exchange resin are applied;

washing HCP from the cation exchange resin with a plurality of wash steps comprising a first wash and a second wash, wherein conductivity of the first wash is equivalent to conductivity of the equilibration buffer and there is an increase in conductivity with each successive wash in the plurality of wash steps;

eluting the antibody from the cation exchange resin with an elution buffer to form a first eluate;

applying the first eluate to an anion exchange resin, wherein prior to applying the first eluate to the anion exchange resin, pH and conductivity of the first eluate are adjusted to be substantially similar to pH and conductivity of the anion exchange resin;

obtaining a first flowthrough comprising the antibody, and

obtaining the HCP-reduced antibody preparation therefrom.

2. The method of claim 1, wherein about 35-70 grams of antibody per liter of cation exchange resin are applied.

3. The method of claim 1, wherein the cation exchange resin is at pH 5 and about 70 grams of antibody per liter of resin are applied.

4. The method of claim 1, wherein the mixture comprising an antibody and at least one HCP is not subjected to protein A capture prior to applying the mixture to the cation exchange resin.

5. The method of claim 1, wherein the first wash is with equilibration buffer and the second wash is with a mixture of elution buffer and water.

6. The method of claim 5, wherein the mixture of elution buffer and water comprises about 40-50% elution buffer and about 50-60% water.

7. The method of claim 6, wherein the mixture of elution buffer and water comprises about 45% elution buffer and about 55% water.

8. The method of claim 7, wherein the elution buffer comprises 20 mM sodium phosphate and 150 mM sodium chloride.

9. The method of claim 1, which carried wherein the cation exchange resin is at pH 7.

10. The method of claim 1, wherein the cation exchange resin is at a pH between about pH 5 and about pH 7.

11. The method of claim 1, wherein the cation exchange resin is at a pH of 5.

12. The method of claim 1, wherein the cation exchange resin is formed into a column and the mixture comprising the antibody and at least one HCP is applied to the column.

13. The method of claim 12, wherein the cation exchange resin comprises a synthetic methacrylate based polymeric resin attached to a sulfonate group.

14. The method of claim 1, which further comprises subjecting the first eluate to a viral inactivation step.

15. The method of claim 14, wherein viral inactivation is achieved by pH viral inactivation such that the first eluate is virally inactivated.

16. The method of claim 1, wherein the pH of the anion exchange resin is in a range of about pH 7.7 to about pH 8.3 and the pH of the first eluate is adjusted to be in a range of about pH 7.7 to about pH 8.3.

17. The method of claim 16, wherein the pH of the anion exchange resin is about pH 8.0 and the pH of the first eluate is adjusted to be about pH 8.0.

18. The method of claim 1, wherein the conductivity of the anion exchange resin is in a range of about 3.5 mS/cm to about 5.2 mS/cm and the conductivity of the first eluate is adjusted to be in a range of about 3.5 mS/cm to about 5.2 mS/cm.

19. The method of claim 18, wherein the conductivity of the anion exchange resin is about 5.0 mS/cm and the conductivity of the first eluate is adjusted to be about 5.0 mS/cm.

20. The method of claim 1, wherein the anion exchange resin is a Q sepharose resin.

21. The method of claim 1, wherein the anion exchange resin is formed into a column.

22. The method of claim 1, further comprising applying the first flow through to a hydrophobic interaction column such that a second eluate is obtained.

23. The method of claim 22, wherein the hydrophobic interaction column is a phenyl sepharose column.

24. The method of claim 22, wherein the first flow through comprises about 20 to about 40 grams of antibody per liter of hydrophobic interaction column material.

25. The method of claim 24, wherein the first flow through comprises about 30 to about 36 grams of antibody per liter of hydrophobic interaction column material.

26. The method of claim 22, wherein the second eluate is not subjected to product peak fractionation.

27. The method of claim 1, wherein the HCP comprises procathepsin L such that a procathepsin L-reduced antibody preparation is obtained.

28. The method of claim 27, wherein the first eluate comprises cathepsin L activity ranging from between about 25 to about 60 RFU/s/mg of antibody as measured by a cathepsin L kinetic assay.

29. The method of claim 27, wherein the level of procathepsin L is reproducibly low.

30. The method of any one of claims 1-4, 5, 10, 11 or 14, wherein the antibody is an anti-tumor necrosis factor-alpha (TNFα

) antibody, or antigen-binding portion thereof.

31. The method of claim 30, wherein the anti-TNFα

antibody, or antigen binding portion thereof, is a humanized antibody, a chimeric antibody or a multivalent antibody.

32. The method of claim 30, wherein the anti-TNFα

antibody, or antigen binding portion thereof, is infliximab.

33. The method of claim 30, wherein the anti-TNFα

antibody, or antigen-binding portion thereof, is a human antibody.

34. The method of claim 33, wherein the anti-TNFα

antibody, or antigen-binding portion thereof, is an isolated human antibody that dissociates from human TNFα

with a Kd of 1×

10<sup>−

8</sup> M or less and a K_off rate constant of 1×

10<sup>−

3</sup> s<sup>−

1</sup> or less, both determined by surface plasmon resonance, and neutralizes human TNFα

cytotoxicity in a standard in vitro L929 assay with an IC₅₀ of 1×

10<sup>−

7</sup> M or less.

35. The method of claim 33, wherein the anti-TNFα

antibody, or antigen-binding portion thereof, is an isolated human antibody with the following characteristics;

a) dissociates from human TNFα

with a K_off rate constant of 1×

10<sup>−

3</sup> s<sup>−

1</sup> or less, as determined by surface plasmon resonance;

b) has a light chain CDR3 domain comprising the amino acid sequence of SEQ ID NO;

3, or modified from SEQ ID NO;

3 by a single alanine substitution at position 1, 4, 5, 7 or 8 or by one to five conservative amino acid substitutions at positions 1, 3, 4, 6, 7, 8 and/or 9;

c) has a heavy chain CDR3 domain comprising the amino acid sequence of SEQ ID NO;

4, or modified from SEQ ID NO;

4 by a single alanine substitution at position 2, 3, 4, 5, 6, 8, 9, 10 or 11 or by one to five conservative amino acid substitutions at positions 2, 3, 4, 5, 6, 8, 9, 10, 11 and/or 12.

36. The method of claim 33, wherein the anti-TNFα

antibody, or antigen binding portion thereof, is an isolated human antibody with a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO;

1 and a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO;

2.

37. The method of claim 33, wherein the anti-TNFα

antibody, or antigen binding portion thereof, is adalimumab.

38. The method of claim 30, wherein the anti-TNFα

antibody, or antigen binding portion thereof, is golimumab.

39. A method for producing a host cell protein (HCP) reduced antibody preparation from a mixture comprising an antibody and at least one HCP, the method comprising:

applying the mixture to a cation exchange resin equilibrated with an equilibration buffer, wherein greater than 30 grams of antibody per liter of resin are applied;

washing HCP from the cation exchange resin with a plurality of wash steps comprising a first wash and a second wash, wherein there is an increase in conductivity from the first wash to the second wash;

eluting the antibody from the cation exchange resin with an elution buffer to form a first eluate;

subjecting the first eluate to a viral inactivation step;

applying the first eluate to an anion exchange resin to obtain a first flow through, wherein prior to applying the first eluate to the anion exchange resin, pH and conductivity of the first eluate are adjusted to be substantially similar to pH and conductivity of the anion exchange resin;

applying the first flow through to a hydrophobic interaction column such that a second eluate is obtained; and

obtaining the HCP-reduced antibody preparation therefrom.

40. The method of claim 39, wherein the cation exchange resin is at pH 7 and about 35 grams of antibody per liter of resin are applied.

41. The method of claim 39, wherein the cation exchange resin is at pH 5 and about 70 grams of antibody per liter of resin are applied.

42. The method of claim 39, wherein the plurality of wash steps comprises washing the resin with a first wash using the equilibration buffer and a second wash using a mixture of the elution buffer and water.

43. The method of claim 42, wherein the mixture of elution buffer and water comprises about 40-50% elution buffer and about 50-60% water.

44. The method of claim 39, wherein the first eluate comprises a range of about 90 to about 100 fold less HCP than the mixture as determined by a HCP ELISA.

45. The method of claim 39, wherein the first flowthrough comprises a range of about 840 to about 850 fold less HCP than the first eluate as determined by a HCP ELISA.

46. The method of claim 39, wherein the second eluate comprises a range of about 3 to about 5 fold less HCP than the first flowthrough as determined by a HCP ELISA.

56. The method of any one of claims 39, 47, or 55, wherein the antibody is an anti-tumor necrosis factor-alpha (TNFα

) antibody, or antigen-binding portion thereof.

57. The method of claim 56, wherein the anti-TNFα

antibody, or antigen binding portion thereof, is a humanized antibody, a chimeric antibody or a multivalent antibody.

58. The method of claim 56, wherein the anti-TNFα

antibody, or antigen binding portion thereof, is infliximab.

59. The method of claim 56, wherein the anti-TNFα

antibody, or antigen-binding portion thereof, is a human antibody.

60. The method of claim 56, wherein the anti-TNFα

antibody, or antigen-binding portion thereof, is an isolated human antibody that dissociates from human TNFα

with a Kd of 1×

10<sup>−

8</sup> M or less and a K_off rate constant of 1×

10<sup>−

3</sup> s<sup>−

1</sup> or less, both determined by surface plasmon resonance, and neutralizes human TNFα

cytotoxicity in a standard in vitro L929 assay with an IC₅₀ of 1×

10<sup>−

7</sup> M or less.

61. The method of claim 56, wherein the anti-TNFα

antibody, or antigen-binding portion thereof, is an isolated human antibody with the following characteristics;

a) dissociates from human TNFα

with a K_off rate constant of 1×

10<sup>−

3</sup> s<sup>−

1</sup> or less, as determined by surface plasmon resonance;

b) has a light chain CDR3 domain comprising the amino acid sequence of SEQ ID NO;

3, or modified from SEQ ID NO;

3 by a single alanine substitution at position 1, 4, 5, 7 or 8 or by one to five conservative amino acid substitutions at positions 1, 3, 4, 6, 7, 8 and/or 9;

c) has a heavy chain CDR3 domain comprising the amino acid sequence of SEQ ID NO;

4, or modified from SEQ ID NO;

4 by a single alanine substitution at position 2, 3, 4, 5, 6, 8, 9, 10 or 11 or by one to five conservative amino acid substitutions at positions 2, 3, 4, 5, 6, 8, 9, 10, 11 and/or 12.

62. The method of claim 56, wherein the anti-TNFα

antibody, or antigen binding portion thereof, is an isolated human antibody with a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO;

1 and a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO;

2.

63. The method of claim 56, wherein the anti-TNFα

antibody, or antigen binding portion thereof, is golimumab.

64. The method of claim 56, wherein the anti-TNFα

antibody, or antigen binding portion thereof, is adalimumab.

47. A method for producing a host cell protein (HCP)-reduced antibody preparation from a mixture comprising an antibody and at least one HCP, the method comprising:

applying the mixture to a cation exchange resin equilibrated with an equilibration buffer, wherein the cation exchange resin is at pH 7 and about 35 grams of antibody per liter of resin are applied, or the cation exchange resin is at a pH in a range of pH 5 to pH 7 and about 35 to about 70 grams of antibody per liter of resin are applied;

washing HCP from the cation exchange resin with wash steps comprising a first wash using the equilibration buffer and a second wash using a mixture of an elution buffer and water;

eluting the antibody from the cation exchange resin with the elution buffer to form a first eluate;

subjecting the first eluate to a viral inactivation step, wherein viral inactivation is achieved by pH viral inactivation such that the first eluate is virally inactivated;

applying the first eluate to an anion exchange resin, wherein, prior to applying the first eluate to the anion ion exchange resin, pH and conductivity of the first eluate are adjusted to be substantially similar to pH and conductivity of the anion exchange resin, such that a first flow through is obtained;

applying the first flow through to a hydrophobic interaction column such that a second eluate is obtained; and

obtaining the HCP-reduced antibody preparation therefrom.

48. The method of claim 47, wherein the antibody mixture has not been subjected to protein A capture prior to applying to the cation exchange resin.

49. The method of claim 47, wherein the mixture of elution buffer and water comprises about 40-50% elution buffer and about 50-60% water.

50. The method of claim 47, wherein the first eluate comprises a range of about 90 to about 100 fold less HCP than the mixture as determined by a HCP ELISA.

51. The method of claim 47, wherein the first flowthrough comprises a range of about 840 to about 850 fold less HCP than the first eluate as determined by a HCP ELISA.

52. The method of claim 47, wherein the second eluate comprises a range of about 3 to about 5 fold less HCP than the first flowthrough as determined by a HCP ELISA.

53. The method of claim 47 wherein the first flowthrough comprises cathepsin L activity ranging from between about 0.4 to about 4 RFU/s/mg of antibody as measured by a cathepsin L kinetic assay.

54. The method of claim 47 wherein the second eluate comprises cathepsin L activity ranging from between about 0.5 to about 1.5 RFU/s/mg of antibody as measured by a cathepsin L kinetic assay.

55. A method for producing a host cell protein (HCP)-reduced antibody preparation from a mixture comprising an antibody and at least one HCP, the method comprising:

applying the mixture to a cation exchange resin equilibrated with an equilibration buffer, wherein greater than about 30 grams of antibody per liter of resin are applied;

washing HCP from the cation exchange resin with a plurality of wash steps comprising a first wash and a second wash, wherein the first wash is with equilibration buffer and the second wash is a mixture of an elution buffer and water;

eluting the antibody from the cation exchange resin with the elution buffer to form a first eluate;

applying the first eluate to an anion exchange resin, wherein prior to applying the first eluate to the anion exchange resin, pH and conductivity of the first eluate are adjusted to be substantially similar to pH and conductivity of the anion exchange resin;

obtaining a first flow through comprising the antibody; and

obtaining the HCP-reduced antibody preparation therefrom.