Claims for Patent: 8,946,395

Summary for Patent: 8,946,395

Title:	Purification of proteins using hydrophobic interaction chromatography
Abstract:	The present invention is directed to methods for purifying a protein of interest, e.g., an antibody, from a sample comprising the protein of interest and at least one impurity, e.g., an aggregate, by employing a hydrophobic interaction chromatography (HIC) method that allows for binding of both the protein of interest and the at least one impurity under strong binding conditions. The present invention is based, at least in part, on the finding that both flow through and bind-elute techniques can be combined to achieve greater purification and recovery of a protein of interest, e.g., an antibody, under isocratic wash conditions and strong binding conditions.
Inventor(s):	Herigstad; Matthew Omon (Charlestown, MA), Rich; Linda E. (Worcester, MA), Lu; Stephen Ming-teh (Worcester, MA), Ramasubramanyan; Natarajan (Westborough, MA)
Assignee:	AbbVie Inc. (North Chicago, IL)
Application Number:	14/077,574
Patent Claims:	1. A method for producing a preparation comprising a protein of interest and having a reduced level of at least one impurity, said method comprising: (a) contacting a sample comprising the protein of interest and at least one impurity, to a hydrophobic interaction chromatography (HIC) media, in the presence of a load buffer such that (i) a portion of the protein of interest binds to the HIC media at a Kp of greater than 20 and (ii) a substantial portion of the at least one impurity binds to the HIC media; (b) collecting a flow through fraction comprising the protein of interest unbound to the HIC media; (c) washing the HIC media with a wash buffer such that a substantial portion of the protein of interest bound to the HIC media is released from the media, wherein the salt concentration and/or the pH of the wash buffer are within 20% of the salt concentration and/or pH of the load buffer; and (d) collecting a wash fraction comprising the protein of interest released from the HIC media, wherein each of the flow through and wash fractions comprise the protein of interest and have a reduced level of the at least one impurity. 2. The method of claim 1, wherein the protein of interest is an antibody or antigen-binding fragment thereof, a soluble protein, a membrane protein, a structural protein, a ribosomal protein, an enzyme, a zymogen, an antibody molecule, a humanized antibody or antigen-binding portion thereof, a human antibody or antigen-binding portion thereof, a chimeric antibody or antigen-binding portion thereof, a multivalent antibody, a cell surface receptor protein, a transcription regulatory protein, a translation regulatory protein, a chromatin protein, a hormone, a cell cycle regulatory protein, a 0 protein, a neuroactive peptide, an immunoregulatory protein, a blood component protein, an ion gate protein, a heat shock protein, an antibiotic resistance protein, a functional fragment of any of the preceding proteins, an epitope-containing fragment of any of the preceding proteins, and combinations thereof. 3. The method of claim 1, wherein the portion of the protein of interest binds to the HIC media at a Kp of greater than 50. 4. The method of claim 1, wherein the portion of the protein of interest binds to the HIC media at a Kp of greater than 100. 5. The method of claim 1, wherein the portion of the protein of interest binds to the HIC media at a Kp of greater than 200. 6. A method for producing a preparation comprising adalimumab and having a reduced level of at least one impurity, said method comprising: (a) contacting a sample comprising adalimumab and at least one impurity, to a HIC media, in the presence of a load buffer such that (i) a portion of the adalimumab in the sample binds to the HIC media at a Kp of greater than 20 and (ii) a substantial portion of the at least one impurity binds to the HIC media; (b) collecting a flow through fraction comprising the adalimumab unbound to the HIC media; (c) washing the HIC media with a wash buffer such that a substantial portion of the adalimumab bound to the HIC media is released from the media, wherein the salt concentration and/or the pH of the wash buffer are within 20% of the salt concentration and/or pH of the load buffer; and (d) collecting a wash fraction comprising the adalimumab released from the HIC media, wherein each of the flow through and wash fractions comprise adalimumab and have a reduced level of the at least one impurity. 7. The method of claim 6, wherein a substantial portion of the impurity bound to the HIC media remains bound upon washing with the wash buffer. 8. The method of claim 6, wherein the flow through and/or wash fractions are substantially free of the at least one impurity. 9. The method of claim 6, wherein the at least one impurity is an aggregate of the protein of interest. 10. The method of claim 9, wherein the aggregate of the adalimumab is selected from the group consisting of a multimer, a dimer, a trimer, a tetramer, an oligomer or other high molecular weight species. 11. The method of claim 10, wherein the aggregate of adalimumab is selected from the group consisting of multimer 1, multimer 2 and multimer 3. 12. The method of claim 6, wherein the impurity is a process-related impurity. 13. The method of claim 12, wherein the process-related impurity is selected from the group consisting of a host cell protein, a host cell nucleic acid, a media component, and a chromatographic material. 14. The method of claim 6, wherein the impurity is an acidic species (AR). 15. The method of claim 14, wherein the acidic species (AR) is selected from the group consisting of AR1, AR2, a charge variant, a structure variant, a fragmentation variant, a process-related impurity and a product-related impurity. 16. The method of claim 6, further comprising repeating steps (a)-(d) at least 1 time using the flow through fraction, wash fraction, or combination thereof having a reduced level of the at least one impurity. 17. The method of claim 6, wherein the flow through fraction and the wash fraction are combined. 18. The method of claim 6, wherein the portion of the adalimumab that binds to the HIC media is at least 20% of the adalimumab in the sample. 19. The method of claim 6, wherein the at least one impurity binds to the HIC media at a Kp of greater than 250. 20. The method of claim 6, wherein the HIC media either (i) comprises at least one hydrophobic ligand, optionally selected from the group consisting of alkyl-, aryl-ligands, and butyl, hexyl, phenyl, octyl, or polypropylene glycol ligands. 21. The method of claim 6, wherein the load buffer and/or wash buffer comprise a salt selected from the group consisting of a sulfate salt, a citrate salt, ammonium sulfate, sodium sulfate, sodium chloride, ammonium chloride, sodium bromide and a combination thereof. 22. The method of claim 6, wherein 100 g to 800 g of the sample are contacted per one liter of HIC media. 23. The method of claim 6, wherein a precursor sample comprising the adalimumab has been subjected to affinity chromatography to generate the sample. 24. The method of claim 6, wherein a precursor sample comprising the adalimumab has been subjected to ion exchange chromatography to generate the sample. 25. The method of claim 6, wherein a precursor sample comprising the adalimumab has been subjected to mixed mode chromatography to generate the sample. 26. The method of claim 6, wherein a precursor sample comprising the adalimumab has been subjected to a filtration step to generate the sample. 27. The method of claim 6, wherein the HIC media has a dynamic binding capacity of at least 2 g. 28. The method of claim 6, wherein adalimumab binds to the HIC media at a Kp of greater than 50. 29. The method of claim 6, wherein the salt concentration and/or the pH of the wash buffer are within about 15% of the salt concentration and/or pH of the load buffer. 30. The method of claim 6, wherein the salt concentration and/or the pH of the wash buffer are within about 10% of the salt concentration and/or pH of the load buffer. 31. The method of claim 6, wherein the salt concentration and/or the pH of the wash buffer are within about 5% of the salt concentration and/or pH of the load buffer. 32. The method of claim 6, wherein the salt concentration and/or the pH of the wash buffer and the salt concentration and/or pH of the load buffer are isocratic. 33. The method of claim 6, wherein the impurity is a product-related substance. 34. The method of claim 33, wherein the product-related substance is selected from the group consisting of a charge variant, an acidic variant, a basic variant, a lysine variant species, an aggregate of the protein of interest, a fragment of the protein of interest, an Fc fragment of the protein of interest, a Fab fragment of the protein of interest, a modified protein, a deamidated protein, and a glycosylated protein. 35. The method of claim 6, further comprising repeating steps (a)-(d) at least 5, 10, or 20 times using the flow through fraction, wash fraction, or combination thereof having a reduced level of the at least one impurity. 36. The method of claim 6, wherein the portion of the adalimumab that binds to the HIC media is at least 90% of the adalimumab in the sample. 37. The method of claim 6, wherein the substantial portion of the adalimumab released from the HIC media upon washing with the wash buffer is at least 20% of the amount of adalimumab bound to the HIC media. 38. The method of claim 6, wherein the substantial portion of the adalimumab released from the HIC media upon washing with the wash buffer is at least 50% of the amount of adalimumab bound to the HIC media. 39. The method of claim 6, wherein the substantial portion of the adalimumab released from the HIC media upon washing with the wash buffer is at least 100% of the amount of adalimumab bound to the HIC media. 40. The method of claim 6, wherein the accumulative yield of the adalimumab in the flow through fraction and/or wash fraction is at least 35%. 41. The method of claim 6, wherein the accumulative yield of the adalimumab in the flow through fraction and/or wash fraction is at least 50%. 42. The method of claim 6, wherein the accumulative yield of the adalimumab in the flow through fraction and/or wash fraction is at least 100%. 43. The method of claim 6, wherein the accumulative yield of the adalimumab in any one flow through fraction and/or wash fraction is at least 4%. 44. The method of claim 6, wherein the accumulative yield of the adalimumab in any one flow through fraction and/or wash fraction is at least 50%. 45. The method of claim 6, wherein the accumulative yield of the adalimumab in any one flow through fraction and/or wash fraction is at least 100%. 46. The method of claim 6, wherein the substantial portion of the at least one impurity that binds to the HIC media is at least 50% of the at least one impurity in the sample. 47. The method of claim 6, wherein the substantial portion of the at least one impurity that binds to the HIC media is at least 70% of the at least one impurity in the sample. 48. The method of claim 6, wherein the substantial portion of the at least one impurity that binds to the HIC media is at least 100% of the at least one impurity in the sample. 49. The method of claim 6, wherein the reduced level of the at least one impurity of the flow through fraction and/or wash fraction is at least 50% of the at least one impurity in the sample. 50. The method of claim 6, wherein the reduced level of the at least one impurity of the flow through fraction and/or wash fraction is at least 70% of the at least one impurity in the sample. 51. The method of claim 6, wherein the reduced level of the at least one impurity of the flow through fraction and/or wash fraction is at least 100% of the at least one impurity in the sample. 52. The method of claim 6, wherein the accumulative aggregate reduction of the at least one impurity in any one flow through fraction and/or wash fraction is at least 0.1%. 53. The method of claim 6, wherein the accumulative aggregate reduction of the at least one impurity in any one flow through fraction and/or wash fraction is at least 5.0%. 54. The method of claim 6, wherein the accumulative aggregate reduction of the at least one impurity in any one flow through fraction and/or wash fraction is at least 20%. 55. The method of claim 6, wherein the accumulative aggregate reduction of the at least one impurity in the flow through fraction and/or wash fraction is at least 0.1%. 56. The method of claim 6, wherein the accumulative aggregate reduction of the at least one impurity in the flow through fraction and/or wash fraction is at least 5.0%. 57. The method of claim 6, wherein the accumulative aggregate reduction of the at least one impurity in the flow through fraction and/or wash fraction is at least 20.0%. 58. The method of claim 6, wherein the level of the at least one impurity is reduced by at least 60%, 80% or 95% of the at least one impurity in the sample. 59. The method of claim 6, wherein the at least one impurity binds to the HIC media at a Kp of greater than 500. 60. The method of claim 6, wherein the at least one impurity binds to the HIC media at a Kp of greater than 1000. 61. The method of claim 6, wherein the adalimumab and the at least one impurity have a Kp ratio less than 1:10, 1:5 or 1:2. 62. The method of claim 6, wherein the K.sub.d for the binding of the adalimumab to the HIC media is at least 0.2, 0.4 or 0.6. 63. The method of claim 6, wherein the K.sub.d for the binding of the at least one impurity to the HIC media is less than or equal to 0.001. 64. The method of claim 6, wherein the K.sub.d for the binding of the at least one impurity to the HIC media is less than or equal to 0.01. 65. The method of claim 6, wherein the K.sub.d for the binding of the at least one impurity to the HIC media is less than or equal to 0.1. 66. The method of claim 6, wherein the K.sub.d for the binding of the at least one impurity to the HIC media is less than or equal to 0.2. 67. The method of claim 6, wherein the K.sub.d for the binding of the adalimumab protein of interest to the HIC media is less than 50, 25 or 5 times the K.sub.d for the binding of the at least one impurity to the HIC media. 68. The method of claim 6, wherein the adalimumab has a Qmax of at least 20, 50 or 100. 69. The method of claim 6, wherein the at least one impurity has a Qmax of at least 2, 20 or 40. 70. The method of claim 6, wherein the load buffer and/or wash buffer comprise a cation selected from the group consisting of Ba.sup.2+, Ca.sup.2+, Mg.sup.2+, Li.sup.+, Cs.sup.+, Na.sup.+, K.sup.+, Rb.sup.+, NH.sub.4.sup.+and a combination thereof. 71. The method of claim 6, wherein the load buffer and/or wash buffer comprise an anion selected from the group consisting of PO.sub.4.sup.3-, SO.sub.4.sup.2-, CH.sub.3CO.sub.3.sup.-, Cl.sup.-, Br.sup.-, NO.sub.3.sup.-, C1O.sub.4.sup.-, I.sup.-, SCN.sup.- and a combination thereof. 72. The method of claim 6, wherein the load buffer and/or wash buffer comprise a salt having a concentration of between 50 mM and 2000 mM. 73. The method of claim 6, wherein the load buffer and/or wash buffer have a pH between about 4.0 and 8.5. 74. The method of claim 6, wherein the load buffer and/or wash buffer have a pH between 5.0 and 7.0. 75. The method of claim 6, wherein the load buffer and/or wash buf 76. The method of claim 6, wherein the load buffer and/or wash buffer have a pH of 6.5. 77. The method of claim 6, wherein the load buffer and/or wash buffer have a pH of 8.5. 78. The method of claim 6, wherein 0.2 g to 120 g of the at least one impurity is contacted per one liter of HIC media. 79. The method of claim 6, wherein the sample has a protein concentration of 2 mg/ml to 50 mg/ml. 80. The method of claim 6, wherein the sample has an adalimumab a protein of interest concentration of 2 mg/ml to 50 mg/ml. 81. The method of claim 6, wherein the concentration of the at least one impurity in the sample is 0.01 to 5.0 mg/ml. 82. The method of claim 6, further comprising subjecting the preparation comprising adalimumab a protein of interest and having a reduced level of one impurity to affinity chromatography. 83. The method of claim 23 or 82, wherein the affinity chromatography is performed using affinity chromatographic media selected from the group consisting of Protein A, G, A/G and L media. 84. The method of claim 6, further comprising subjecting the preparation comprising adalimumab a protein of interest and having a reduced level of the at least one impurity to a filtration step. 85. The method of claim 26 or 84, wherein the filtration step is selected from the group consisting of a depth filtration step, a nanofiltration step, an ultrafiltration step, and an absolute filtration step, or a combination thereof. 86. The method of claim 6, wherein the HIC media has a dynamic binding capacity of at least 50 g. 87. The method of claim 6, wherein the HIC media has a dynamic binding capacity of at least 100 g. 88. The method of claim 6, wherein adalimumab binds to the HIC media at a Kp of greater than 100. 89. The method of claim 6, wherein adalimumab binds to the HIC media at a Kp of greater than 200. 90. The method of claim 6, further comprising subjecting the preparation comprising adalimumab and having a reduced level of the at least one impurity to ion exchange chromatography. 91. The method of claim 24 or 90, wherein ion exchange chromatography is performed using ion exchange chromatography media selected from the group consisting of a cation exchange media and an anion exchange media. 92. The method of claim 6, further comprising subjecting the preparation comprising adalimumab and having a reduced level of the at least one impurity to mixed mode chromatography.

Details for Patent 8,946,395

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Abbvie Inc.	HUMIRA	adalimumab	Injection	125057	12/31/2002		2033-10-18
Abbvie Inc.	HUMIRA	adalimumab	Injection	125057	02/21/2008		2033-10-18
Abbvie Inc.	HUMIRA	adalimumab	Injection	125057	04/24/2013		2033-10-18
Abbvie Inc.	HUMIRA	adalimumab	Injection	125057	09/23/2014		2033-10-18
Abbvie Inc.	HUMIRA	adalimumab	Injection	125057	11/23/2015		2033-10-18
Abbvie Inc.	HUMIRA	adalimumab	Injection	125057	03/09/2016		2033-10-18