Methods of purifying Fc region containing proteins

• US 20070072307A1
• Filed: 06/16/2006
• Published: 03/29/2007
• Est. Priority Date: 06/17/2005
• Status: Active Grant

• Current Assignee: Janssen Alzheimer Immunotherapy (Johnson & Johnson), Wyeth LLC (Pfizer Inc.)
• Original Assignee: Janssen Alzheimer Immunotherapy (Johnson & Johnson), Wyeth LLC (Pfizer Inc.)
• Inventors: Godavarti, Ranganathan, Iskra, Timothy
• Granted Patent: US 7,820,799 B2
• Time in Patent Office: Days
• US Class Current: 436/518
• CPC Class Codes: C07K 16/065 Purification, fragmentation
• CPC Class Codes: C07K 16/18 against material from animals or humans
• CPC Class Codes: G01N 33/6854 Immunoglobulins

Abstract

The present application provides methods of purifying polypeptides having a Fc region, for example, antibodies or antibody fusions, by adsorbing the polypeptides to a Fc binding agent, such as, for example, Protein A or Protein G, followed by a wash with a divalent cation salt buffer to remove impurities and subsequent recovery of the adsorbed polypeptides. The present application also features methods of eluting the purified polypeptide as well as the incorporation of the methods within a purification train. Kits comprising components for carrying out the methods and instructions for use are also provided.

Claims

1. A method for purifying a protein having an Fc region from a source liquid comprising the protein and one or more impurities, comprising the steps of:

adsorbing the protein to an Fc binding agent;

washing the Fc binding agent with a buffer solution containing a divalent cation salt to remove one or more impurities; and

recovering the protein from the Fc binding agent in an elution solution.

2. The method of claim 1, wherein said one or more impurities are selected from the group consisting of:

intron read through variant species (IRT), under disulfide bonded species (UDB), and low molecular weight species (LMW).

3. The method of claim 2, wherein said one or more impurities is an IRT.

4. The method of claim 1, wherein the protein is an antibody.

5. The method of claim 4, wherein the antibody is selected from the group consisting of:

an antibody fusion, a murine antibody, a chimeric antibody, a humanized antibody, and a human antibody.

6. The method of claim 4, wherein the antibody is an anti-IL-13 antibody.

7. The method of claim 4, wherein the antibody binds to an antigen selected from the group consisting of:

CD3, CD52, VEGF, EGFR, CD33, CD20, HER-2, TNFα

, CD25, RSV, IgE, gp IIb/IIIa, CD11a and α

4 integrin.

8. The method of claim 1, wherein the protein having an Fc region is recombinantly produced.

9. The method of claim 1, wherein the protein having an Fc region is recombinantly produced in a Chinese Hamster Ovary (CHO) cell.

10. The method of claim 1, wherein the Fc binding agent comprises one or more of Protein A and Protein G.

11. The method of claim 1, wherein the Fc binding agent is immobilized on a solid phase.

12. The method of claim 11, wherein the solid phase comprises one or more of a bead, a gel, a resin, and a particle.

13. The method of claim 1, wherein the divalent cation salt comprises a chaotropic salt.

14. The method of claim 1, wherein the divalent cation salt is selected from the group consisting of:

CaCl₂, MgCl₂, NiCl₂ and mixtures thereof.

15. The method of claim 1, wherein the buffer solution containing the divalent cation salt has a pH value in a range between about 4 to about 8.

16. The method of claim 1, wherein the buffer solution containing the divalent cation salt has a pH value in a range between about 4.5 to about 7.5.

17. The method of claim 1, wherein the buffer solution has a divalent cation salt concentration in a range between about 0.1 M to about 5 M.

18. The method of claim 17, wherein the buffer solution has a divalent cation salt concentration in a range between about 0.5 M to about 3 M.

19. The method of claim 1, wherein the buffer solution containing a divalent cation salt comprises at least about 0.6 M CaCl₂.

20. The method of claim 1, wherein the buffer solution containing a divalent cation salt comprises at least about 2 M MgCl₂.

21. The method of claim 1, wherein the buffer solution containing a divalent cation salt comprises at least about 2 M CaCl₂.

22. The method of claim 1, wherein the steps of adsorbing the protein to an Fc binding agent and washing the Fc binding agent are performed at a temperature in a range between about 2°

C. to about 24°

C.

23. The method of claim 1, wherein the one or more impurities comprise one or more of a host cell protein, a host cell DNA, a cell culture protein, and mixtures thereof.

24. The method of claim 1, wherein the one or more impurities comprise an undesired species of the protein having an Fc region.

25. The method of claim 24, wherein the undesired species of the protein having an Fc region comprises one or more of antibody chains or fragments thereof having intronic read through sequence, one or more antibody chains or fragments thereof having an improper disulfide linkage, a half-antibody or fragment thereof, a light chain dimer or fragment thereof, and a heavy chain dimer or fragment thereof.

26. The method of claim 1, wherein the step of recovering the protein from the Fc binding agent comprises eluting the protein using an elution buffer having a pH in a range from about 2.0 to about 6.5.

27. The method of claim 1, wherein the method further comprises a chromatography step selected from the group consisting of:

anion exchange chromatography, cation exchange chromatography, immobilized metal affinity chromatography and hydrophobic interaction chromatography (HIC).

28. The method of claim 1, wherein the method further comprises a chromatography step selected from the group consisting of:

hydroxyapatite chromatography, dialysis, affinity chromatography, ammonium sulphate precipitation, ethanol precipitation, reverse phase HPLC (RP-HPLC), and chromatofocusing.

29. The method of claim 1, wherein the one or more impurities comprise one or more intron read-through variants of the protein and the elution solution containing the protein has a level of intron read-through variants that is at least 5 fold less than the level of intron read-through variants in the source liquid.

30. The method of claim 29, wherein a solution containing the protein recovered in the elution solution has a level of intron read-through variants that is at least 10 fold less than the level of intron read-through variants in the source liquid.

31. The method of claim 1, wherein the one or more impurities comprise one or more intron read-through variants of the protein and the intron read-through variants comprise less than about 1% of a species of said protein in the elution solution.

32. The method of claim 31, wherein the intron read-through variants comprise less than about 0.8% of a species of said protein in the elution solution.

33. The method of claim 32, wherein the intron read-through variants comprise less than about 0.5% of a species of said protein in the elution solution.

34. The method of claim 33, wherein the intron read-through variants comprise less than about 0.2% of a species of said protein in the elution solution.

35. The method of claim 1, wherein the one or more impurities comprise one or more low molecular weight species of the protein and the low molecular weight species comprise less than about 1% of a species of said protein in the elution solution.

36. The method of claim 35, wherein the low molecular weight species comprise less than about 0.8% of a species of said protein in the elution solution.

37. The method of claim 36, wherein the low molecular weight species comprise less than about 0.5% of a species of said protein in the elution solution.

38. The method of claim 37, wherein the low molecular weight species comprise less than about 0.2% of a species of said protein in the elution solution.

39. The method of claim 1, wherein the one or more impurities comprise one or more under-disulfide bonded variants of the protein and the under-disulfide bonded variants comprise less than about 15% of a species of said protein in the elution solution.

40. The method of claim 39, wherein the under-disulfide bonded variants comprise less than about 10% of a species of said protein in the elution solution.

41. The method of claim 40, wherein the under-disulfide bonded variants comprise less than about 5% of a species of said protein in the elution solution.

42. The method of claim 41, wherein the under-disulfide bonded variants comprise less than about 2% of a species of said protein in the elution.

43. A protein purified according to the method of claim 1.

44. The protein of claim 43, wherein the protein is an antibody.

45. A kit comprising reagents for purifying a protein having an Fc region from impurities, the kit comprising,

a reagent selected from the group consisting of:

a divalent cation salt, and a Fc binding agent, and

instructions for use.