Claims for Patent: 7,825,223

Summary for Patent: 7,825,223

Title:	Methods of purifying anti A .beta. antibodies
Abstract:	The present application provides methods of purifying A.beta. binding proteins having a Fc region, for example, anti-A.beta. antibodies or antibody fusions, by adsorbing the A.beta. binding protein 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 A.beta. binding protein. The present application also features methods of eluting the purified A.beta. binding protein 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:	11/454,772
Patent Claims:	1. A method for purifying an A.beta. binding protein having an Fc region from a source liquid comprising the protein and one or more impurities, wherein the A.beta. binding protein is an anti-A.beta. antibody, comprising the steps of: adsorbing the A.beta. binding protein to an Fc binding agent; washing the Fc binding agent bound to the A.beta. binding 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 A.beta. binding 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 antibody is selected from the group consisting of: an antibody fusion, a murine antibody, a chimeric antibody, a humanized antibody and a human antibody. 5. The method of claim 1, wherein the A.beta. binding protein having an Fc region is recombinantly produced. 6. The method of claim 1, wherein the A.beta. binding protein having an Fc region is recombinantly produced in a Chinese Hamster Ovary (CHO) cell. 7. The method of claim 1, wherein the Fc binding agent comprises one or more of Protein A and Protein G. 8. The method of claim 1, wherein the Fc binding agent is immobilized on a solid phase. 9. The method of claim 8, wherein the solid phase comprises one or more of a bead, a gel, a resin, and a particle. 10. 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. 11. The method of claim 1, wherein the buffer solution containing the CaCl.sub.2 has a pH value in a range between about 7.3 to about 7.7. 12. 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. 13. The method of claim 12, wherein the buffer solution has a CaCl.sub.2 concentration in a range between about 1.5 M to about 2.0 M. 14. The method of claim 1, wherein the buffer solution containing CaCl.sub.2 comprises at 1 ast about 1.5 M to about 2.5 M CaCl.sub.2. 15. The method of claim 1, wherein the buffer solution containing CaCl.sub.2 comprises about 2 M CaCl.sub.2. 16. The method of claim 1, wherein the steps of adsorbing the A.beta. binding 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. 17. 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. 18. The method of claim 1, wherein the one or more impurities comprise an undesired species of the protein having an Fc region. 19. The method of claim 18, wherein the undesired species of the A.beta. binding protein 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. 20. The method of claim 1, wherein the step of recovering the A.beta. binding 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. 21. The method of claim 20, wherein the elution buffer has a pH in a range from about 2.0 to about 4.0. 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 an additional purification 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 moiety that binds selectively or preferentially to the Fc region of the antibody in the source liquid. 39. The method of claim 1, further comprising washing the Fc binding agent bound to the A.beta. binding protein with a buffer solution containing NaCl at a concentration of at least about 10 mM after washing with the CaCl.sub.2. 40. The method of claim 1, wherein the one or more impurities comprise one or more of an intron read-through variant species (IRT), an under disulfide-bonded species (UDB), and a low molecular weight species (LMW), and the one or more impurities is reduced to a level of at least about 2-fold lower than that present in the source liquid. 41. A method for purifying an A.beta. binding protein having an Fc region from a source liquid comprising the A.beta. binding protein and one or more impurities, wherein the A.beta. binding protein is a humanized 3D6 antibody and wherein the one or more impurities comprise one or more intron read through variant species (IRT), the method comprising the steps of: adsorbing the A.beta. binding protein 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 A.beta. binding protein from the affinity ligand in an elution solution, wherein the A.beta. binding protein having an Fc region is purified from the source liquid. 42. The method of claim 41, wherein the buffer solution containing the CaCl.sub.2 has a pH value in a range between about 4 to about 8. 43. The method of claim 41, wherein the buffer solution containing the CaCl.sub.2 has a pH value in a range between about 7.3 to about 7.7. 44. The method of claim 41, wherein the buffer solution has a CaCl.sub.2 concentration in a range between about 1 M to about 3 M. 45. The method of claim 44, wherein the buffer solution has a CaCl.sub.2 concentration in a range between about 1.5 M to about 2.5 M. 46. The method of claim 41, wherein the buffer solution containing the CaCl.sub.2 comprises about 2 M CaCl.sub.2. 47. The method of claim 41, wherein the A.beta. binding protein having an Fc region is recombinantly produced. 48. The method of claim 47, wherein the A.beta. binding protein having an Fc region is recombinantly produced in a Chinese Hamster Ovary (CHO) cell. 49. The method of claim 41, wherein the Fc binding agent comprises one or more of Protein A and Protein G. 50. The method claim 41, wherein the Fc binding agent is immobilized on a solid phase. 51. The method of claim 41, wherein the steps of adsorbing the A.beta. binding 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. 52. The method of claim 41, wherein the step of recovering the A.beta. binding 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. 53. The method of claim 52, wherein the elution buffer has a pH in a range from about 2.0 to about 4.0. 54. The method of claim 41, 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 41, wherein the method further comprises an additional purification 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 41, wherein the elution solution containing the A.beta. binding 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. 57. The method of claim 41, 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 41, wherein the affinity ligand is a moiety that binds selectively or preferentially to the Fc region of the antibody in the source liquid. 59. The method of claim 41, wherein the impurities further comprise at least one of under disulfide bonded species (UDB) and low molecular weight species (LMW). 60. The method of claim 41, wherein the one or more impurities comprise one or more of an intron read-through variant species (IRT), an under disulfide-bonded species (UDB), and a low molecular weight species (LMW), and the one or more impurities is reduced to a level of at least about 2-fold lower than that present in the source liquid. 61. The method of claim 41, further comprising washing the Fc binding agent bound to the A.beta. binding protein with a buffer solution containing NaCl at a concentration of at least about 10 mM after washing with CaCl.sub.2. 62. The method of claim 41, wherein the humanized 3D6 antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO:1 and a heavy chain comprising the amino acid sequence of SEQ ID NO:2. 63. The method of claim 41, wherein the humanized 3D6 antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO:1 and a heavy chain comprising the amino acid sequence of residues 1-448 of SEQ ID NO:2. 64. The method of claim 41, wherein the humanized 3D6 antibody is of isotype IgM, IgG1, IgG2, IgG3, or IgG4. 65. The method of claim 64, wherein the humanized 3D6 antibody is of isotype human IgG1. 66. A method for purifying an A.beta. binding protein having an Fc region from a source liquid comprising the protein and one or more impurities, wherein the A.beta. binding protein is a humanized anti-A.beta. antibody, the method comprising the steps of: adsorbing the A.beta. binding protein 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 A.beta. binding protein from the affinity ligand in an elution solution. 67. The method of claim 66, 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). 68. The method of claim 66, wherein the antibody is selected from the group consisting of: 3D6, 10D5, 12A11, 266, 15C11, and 12B4. 69. The method of claim 66, wherein the A.beta. binding protein having an Fc region is recombinantly produced. 70. The method of claim 69, wherein the A.beta. binding protein having an Fc region is recombinantly produced in a Chinese Hamster Ovary (CHO) cell. 71. The method of claim 66, wherein the Fc binding agent comprises one or more of Protein A and Protein G. 72. The method of claim 66, wherein the Fc binding agent is immobilized on a solid phase. 73. The method of claim 66, wherein the buffer solution containing the CaCl.sub.2 has a pH value in a range between about 4 to about 8. 74. The method of claim 66, wherein the buffer solution containing the CaCl.sub.2 has a pH value in a range between about 7.3 to about 7.7. 75. The method of claim 66, wherein the buffer solution has a CaCl.sub.2 concentration in a range between about 1 M to about 3 M. 76. The method of claim 66, wherein the buffer solution containing CaCl.sub.2 comprises at least about 1.5 M to about 2.5 M CaCl.sub.2. 77. The method of claim 66, wherein the buffer solution containing CaCl.sub.2 comprises about 2 M CaCl.sub.2. 78. The method of claim 66, wherein the steps of adsorbing the A.beta. binding 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. 79. The method of claim 66, 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. 80. The method of claim 66, wherein the one or more impurities comprise an undesired species of the protein having an Fc region. 81. The method of claim 80, wherein the undesired species of the A.beta. binding protein 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 thereof. 82. The method of claim 66, wherein the step of recovering the A.beta. binding 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. 83. The method of claim 81, wherein the elution buffer has a pH in a range from about 2.0 to about 4.0. 84. The method of claim 66, 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). 85. The method of claim 66, wherein the method further comprises an additional purification step selected from the group consisting of: hydroxyapatite chromatography, dialysis, affinity chromatography, ammonium sulphate precipitation, ethanol precipitation, reverse phase HPLC (RP-HPLC), and chromatofocusing. 86. The method of claim 66, 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. 87. The method of claim 66, 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. 88. The method of claim 66, 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. 89. The method of claim 66, 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. 90. The method of claim 66, wherein the affinity ligand is a moiety that binds selectively or preferentially to the Fc region of the antibody in the source liquid. 91. The method of claim 66, further comprising washing the Fc binding agent bound to the A.beta. binding protein with a buffer solution containing NaCl at a concentration of at least about 10 mM after washing with the CaCl.sub.2. 92. The method of claim 66, wherein the one or more impurities comprise one or more of an intron read-through variant species (IRT), an under disulfide-bonded species (UDB), and a low molecular weight species (LMW), and the one or more impurities is reduced to a level of at least about 2-fold lower than that present in the source liquid. 93. The method of claim 66, further comprising washing the affinity ligand with a buffer solution containing NaCl at a concentration of from 0.75 mM to 2.0 mM prior to recovering the A.beta. binding protein. 94. The method of claim 66, wherein the humanized antibody is of isotype IgM, IgG1, IgG2, IgG3, or IgG4. 95. The method of claim 94, wherein the humanized antibody is of isotype human IgG1. 96. A method for purifying an A.beta. binding protein having an Fc region from a source liquid comprising the A.beta. binding protein and one or more impurities, the method comprising the steps of: adsorbing the A.beta. binding protein to an Fc binding agent comprising Protein A, wherein the A.beta. binding protein is a humanized 3D6 antibody comprising a light chain comprising the amino acid sequence of SEQ ID NO:1 and a heavy chain comprising the amino acid sequence of residues 1-448 of SEQ ID NO:2; washing the Fc binding agent bound to the A.beta. binding protein with a buffer solution containing CaCl.sub.2 at a concentration of from about 1.5 M to about 2 M to reduce the one or more impurities; and recovering the A.beta. binding protein from the Fc binding agent using an elution buffer having a pH in a range from about 2.5 to about 3.5, wherein the A.beta. binding protein having an Fc region is purified from the source liquid.

Details for Patent 7,825,223

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