Claims for Patent: 7,820,799

Summary for Patent: 7,820,799

Title:	Methods of purifying Fc region containing proteins
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.
Inventor(s):	Godavarti; Ranganathan (Burlington, MA), Iskra; Timothy (Derry, NH)
Assignee:	Janssen Alzheimer Immunotherapy (Little Island, County Cork, IE) Wyeth LLC (Madison, NJ)
Application Number:	11/455,203
Patent Litigation and PTAB cases:	See patent lawsuits and PTAB cases for patent 7,820,799
Patent 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 bound to the protein with a buffer solution containing CaCl.sub.2 at a concentration from about 0.5 M to about 3 M to reduce the one or more impurities; and recovering the protein from the Fc binding agent in an elution solution, wherein the protein having an Fc region is purified from the source liquid. 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 that binds to an antigen selected from the group consisting of: CD3, CD52, VEGF, EGFR, CD33, CD20, HER-2, TNF.alpha., CD25, RSV, IgE, gp IIb/IIIa, CD11a and .alpha.4 integrin. 5. The method of claim 1, wherein the protein is an anti-IL-13 antibody. 6. The method of claim 1, wherein the protein having an Fc region is recombinantly produced. 7. The method of claim 1, wherein the protein having an Fc region is recombinantly produced in a Chinese Hamster Ovary (CHO) cell. 8. The method of claim 1, wherein the Fc binding agent comprises one or more of Protein A and Protein G. 9. The method of claim 1, wherein the Fc binding agent is immobilized on a solid phase. 10. The method of claim 9, wherein the solid phase comprises one or more of a bead, a gel, a resin, and a particle. 11. The method of claim 1, wherein the buffer solution containing the CaCl.sub.2 has a pH value in a range between about 4 to about 8. 12. The method of claim 1, wherein the buffer solution containing the CaCl.sub.2 has a pH value in a range between about 4.5 to about 7.5. 13. The method of claim 1, wherein the buffer solution has a CaCl.sub.2 concentration in a range between about 1 M to about 3 M. 14. The method of claim 13, wherein the buffer solution has a CaCl.sub.2 concentration in a range between about 0.6 M to about 2.5 M. 15. The method of claim 1, wherein the buffer solution has a CaCl.sub.2 concentration in a range between about 1.5 M to about 2.5 M. 16. The method of claim 1, wherein the buffer solution containing CaCl.sub.2 comprises about 2 M CaCl.sub.2. 17. 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 the range between about 2.degree. C. to about 24.degree. C. 18. 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. 19. The method of claim 1, wherein the one or more impurities comprise an undesired species of the protein having an Fc region. 20. The method of claim 19, wherein the undesired species of the protein having an Fc region comprises one or more of antibody chains or fragments thereof having an 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 hereof. 21. 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. 22. 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). 23. The method of claim 1, wherein the method further comprises a step selected from the group consisting of: hydroxyapatite chromatography, dialysis, affinity chromatography, ammonium sulphate precipitation, ethanol precipitation, reverse phase HPLC (RP-HPLC), and chromatofocusing. 24. 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. 25. The method of claim 24, 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. 26. 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. 27. The method of claim 26, wherein the intron read-through variants comprise less than about 0.8% of a species of said protein in the elution solution. 28. The method of claim 27, wherein the intron read-through variants comprise less than about 0.5% of the species of said protein in the elution solution. 29. The method of claim 28, wherein the intron read-through variants comprise less than about 0.2% of a species of said protein in the elution solution. 30. 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. 31. The method of claim 30, wherein the low molecular weight species comprise less than about 0.8% of a species of said protein in the elution solution. 32. The method of claim 31, wherein the low molecular weight species comprise less than about 0.5% of a species of said protein in the elution solution. 33. The method of claim 32, wherein the low molecular weight species comprise less than about 0.2% of a species of said protein in the elution solution. 34. 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. 35. The method of claim 34, wherein the under-disulfide bonded variants comprise less than about 10% of a species of said protein in the elution solution. 36. The method of claim 35, wherein the under-disulfide bonded variants comprise less than about 5% of a species of said protein in the elution solution. 37. The method of claim 36, wherein the under-disulfide bonded variants comprise less than about 2% of a species of said protein in the elution solution. 38. The method of claim 1, wherein the Fc binding agent is a protein selected from the group consisting of: Protein A, Protein G, and Protein A/G. 39. The method of claim 1, wherein the Fc binding agent is a moiety that binds selectively or preferentially to the protein through a specific interaction with a binding site of the protein. 40. The method of claim 1, further comprising washing the Fc binding agent bound to the protein having an Fc region with a buffer solution containing NaCl at a concentration of from 0.75 M to 2.0 M after washing with the CaCl.sub.2, wherein said one or more impurities is selected from the group consisting of: intron read through variant species (IRT), under disulfide bonded species (UDB), low molecular weight species (LMW), host cell protein and host cell DNA. 41. The method of claim 1, wherein said one or more impurities is selected from the group consisting of: intron read through variant species (IRT), under disulfide-bonded species (UDB), and low molecular weight species (LMW), and wherein the one or more impurities is reduced to a level of at least about 2-fold lower than that present in the source liquid. 42. A method for purifying a protein having an Fc region from a source liquid comprising the protein and one or more impurities, wherein the one or more impurities comprise one or more intron read through variant species (IRT), the method comprising the steps of: adsorbing the protein having an Fc region to an affinity ligand which is an Fc binding agent; washing the affinity ligand with a buffer solution containing CaCl.sub.2 at a concentration from about 0.5 M to about 3 M to reduce the IRT; and recovering the protein having an Fc region from the affinity ligand in an elution solution, wherein the protein having an Fc region is purified from the source liquid. 43. The method of claim 42, wherein the buffer solution containing the CaCl.sub.2 has a pH value in a range between about 4 to about 8. 44. The method of claim 42, wherein the buffer solution containing the CaCl.sub.2 has a pH value in a range between about 4.5 to about 7.5. 45. The method of claim 42, wherein the buffer solution has a CaCl.sub.2 concentration in a range between about 1 M to about 3 M. 46. The method of claim 45, wherein the buffer solution has a CaCl.sub.2 concentration in a range between about 1.5 M to about 2.5 M. 47. The method of claim 42, wherein the buffer solution containing CaCl.sub.2 comprises about 2 M CaCl.sub.2. 48. The method of claim 42, wherein the protein having an Fc region is recombinantly produced. 49. The method of claim 48, wherein the protein having an Fc region is recombinantly produced in a Chinese Hamster Ovary (CHO) cell. 50. The method of claim 42, wherein the Fc binding agent is a protein selected from the group consisting of: Protein A, Protein G and Protein A/G. 51. The method claim 42, wherein the Fc binding agent is immobilized on a solid phase. 52. The method of claim 42, wherein the steps of adsorbing the protein having an Fc region to an Fc binding agent and washing the Fc binding agent are performed at a temperature in the range between about 2.degree. C. to about 24.degree. C. 53. The method of claim 42, wherein the step of recovering the protein having an Fc region 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. 54. The method of claim 42, 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). 55. The method of claim 42, wherein the method further comprises a step selected from the group consisting of: hydroxyapatite chromatography, dialysis, affinity chromatography, ammonium sulphate precipitation, ethanol precipitation, reverse phase HPLC (RP-HPLC), and chromatofocusing. 56. The method of claim 42, wherein the elution solution containing the protein having an Fc region 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. 57. The method of claim 42, wherein the intron read-through variants comprise less than about 1% of a species of said protein in the elution solution. 58. The method of claim 42, wherein the impurities further comprise at least one of under disulfide bonded species (UDB) and low molecular weight species (LMW). 59. The method of claim 42, wherein the one or more intron read-through variant species (IRT) is reduced to a level of at least about 2-fold lower than that present in the source liquid. 60. The method of claim 42, further comprising washing the Fc binding agent bound to the protein having an Fc region with a buffer solution containing NaCl at a concentration selected from 5 mM or 10 mM after washing with the CaCl.sub.2, wherein the one or more impurities comprise one or more intron read through variant species (IRT) or host cell protein. 61. A method for purifying a protein having an Fc region from a source liquid comprising the protein and one or more impurities, wherein the protein having an Fc region is an antibody, the method comprising the steps of: adsorbing the protein having an Fc region to an affinity ligand which is an Fc binding agent; washing the affinity ligand with a buffer solution containing CaCl.sub.2 at a concentration from about 0.5 M to about 3 M to reduce the one or more impurities; and recovering the protein having an Fc region from the affinity ligand in an elution solution, wherein the protein having an Fc region is purified from the source liquid. 62. The method of claim 61, 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). 63. The method of claim 61, 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. 64. The method of claim 61, wherein the antibody is a humanized antibody. 65. The method of claim 61, wherein the antibody is a humanized IL-13 antibody. 66. The method of claim 61, wherein the protein having an Fc region is recombinantly produced. 67. The method of claim 66, wherein the protein having an Fc region is recombinantly produced in a Chinese Hamster Ovary (CHO) cell. 68. The method of claim 61, wherein the Fc binding agent is a protein selected from the group consisting of: Protein A, Protein G and Protein A/G. 69. The method of claim 61, wherein the Fc binding agent is immobilized on a solid phase. 70. The method of claim 61, wherein the buffer solution containing the CaCl.sub.2 has a pH value in a range between about 4 to about 8. 71. The method of claim 61, wherein the buffer solution containing the CaCl.sub.2 has a pH value in a range between about 4.5 to about 7.5. 72. The method of claim 61, wherein the buffer solution has a CaCl.sub.2 concentration in a range between about 1 M to about 3 M. 73. The method of claim 61, wherein the buffer solution has a CaCl.sub.2 concentration in a range between about 1.5 M to about 2.5 M. 74. The method of claim 61, wherein the buffer solution containing CaCl.sub.2 comprises about 2 M CaCl.sub.2. 75. The method of claim 61, wherein the steps of adsorbing the protein having an Fc region to an Fc binding agent and washing the Fc binding agent are performed at a temperature in the range between about 2.degree. C. to about 24.degree. C. 76. The method of claim 61, 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. 77. The method of claim 61, wherein the one or more impurities comprise an undesired species of the protein having an Fc region. 78. The method of claim 61, wherein the step of recovering the protein having an Fc region 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. 79. The method of claim 61, 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). 80. The method of claim 61, wherein the method further comprises a step selected from the group consisting of: hydroxyapatite chromatography, dialysis, affinity chromatography, ammonium sulphate precipitation, ethanol precipitation, reverse phase HPLC (RP-HPLC), and chromatofocusing. 81. The method of claim 61, 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. 82. The method of claim 61, 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. 83. The method of claim 61, 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. 84. The method of claim 61, 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. 85. The method of claim 61, further comprising washing the Fc binding agent bound to the protein having an Fc region with a buffer solution containing NaCl at a concentration of from 0.75 M to 2.0 M after washing with the CaCl.sub.2, wherein said one or more impurities is selected from the group consisting of: intron read through variant species (IRT), under disulfide bonded species (UDB), low molecular weight species (LMW), host cell protein and host cell DNA. 86. The method of claim 61, wherein said one or more impurities is selected from the group consisting of: intron read through variant species (IRT), under disulfide-bonded species (UDB), and low molecular weight species (LMW), and wherein the one or more impurities is reduced to a level of at least about 2-fold lower than that present in the source liquid.

Details for Patent 7,820,799

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132	06/04/1986		2025-06-17
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132			2025-06-17
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	Injection	103132			2025-06-17