Claims for Patent: 10,035,817

Summary for Patent: 10,035,817

Title:	Method of purifying cell-binding agent-cytotoxic agent conjugates with a PVDF membrane
Abstract:	The invention provides processes for preparing purified cell-binding agent cytotoxic agent conjugates comprising subjecting a mixture comprising a cell-binding agent cytotoxic agent conjugate and one or more impurities to a polyvinyl difluoride (PVDF) membrane to remove at least a portion of the impurities from the mixture, thereby providing a purified cell-binding agent cytotoxic agent conjugate.
Inventor(s):	Chen; Xiaoxi Kevin (Natick, MA), Li; Xinfang (Newton, MA)
Assignee:	IMMUNOGEN, INC. (Waltham, MA)
Application Number:	14/430,701
Patent Claims:	1. A process for preparing a purified cell-binding agent cytotoxic agent conjugate comprising: (a) contacting a cell-binding agent with a cytotoxic agent to form a first mixture comprising the cell-binding agent and the cytotoxic agent, then contacting the first mixture with a bifunctional crosslinking reagent comprising a linker, in a solution having a pH of about 4 to about 9, to provide a second mixture comprising the cell-binding agent cytotoxic agent conjugate comprising the cell-binding agent chemically coupled through the linker to the cytotoxic agent and one or more impurities; (b) adjusting the second mixture to have a pH of about 7 to about 9 to form a pH-adjusted second mixture; (c) subjecting the pH-adjusted second mixture to a polyvinyl difluoride (PVDF) membrane to remove at least a portion of the impurities, thereby providing a purified second mixture of the cell-binding agent cytotoxic agent conjugate; and (d) subjecting the purified second mixture after step (c) to tangential flow filtration, selective precipitation, non-adsorptive chromatography, adsorptive filtration, adsorptive chromatography, or a combination thereof, to further purify the cell-binding agent-cytotoxic agent conjugate from the impurities and thereby prepare a purified third mixture of the cell-binding agent-cytotoxic agent conjugate, wherein the purified third mixture comprises a reduced amount of the impurities as compared to the purified second mixture. 2. The process of claim 1, wherein step (c) is sequentially repeated two, three, or four times prior to step (d). 3. The process of claim 1, wherein adsorptive chromatography is utilized in step (d). 4. The process of claim 1, wherein the adsorptive chromatography is selected from hydroxyapatite chromatography, hydrophobic charge induction chromatography (HCIC), hydrophobic interaction chromatography (HIC), ion exchange chromatography, mixed mode ion exchange chromatography, immobilized metal affinity chromatography (IMAC), dye ligand chromatography, affinity chromatography, reversed phase chromatography, and combinations thereof. 5. The process of claim 4, wherein the adsorptive chromatography is ion exchange chromatography. 6. The process of claim 5, wherein the ion exchange chromatography is ceramic hydroxyapatite (CHT) chromatography. 7. The process of claim 1, wherein tangential flow filtration is utilized in step (d). 8. The process of claim 1, wherein the contacting in step (a) is effected by providing the cell-binding agent in a reaction vessel, adding the cytotoxic agent to the reaction vessel to form the first mixture comprising the cell-binding agent and the cytotoxic agent, and then adding the bifunctional crosslinking reagent to the first mixture. 9. The process of claim 1, further comprising holding the mixture between steps (a)-(b), steps (b)-(c), or steps (c)-(d) to release the unstably bound linkers from the cell-binding agent. 10. The process of claim 9, wherein the mixture is held for about 20 hours at a temperature of about 2.degree. C. to about 8.degree. C. 11. The process of claim 1, further comprising quenching the second mixture between steps (a)-(b) to quench any unreacted cytotoxic agent and/or unreacted bifunctional crosslinking reagent. 12. The process of claim 11, wherein the mixture is quenched by contacting the second mixture with a quenching reagent that reacts with the unreacted cytotoxic reagent. 13. The process of claim 12, wherein the quenching reagent is selected from 4-maleimidobutyric acid, 3-maleimidopropionic acid, N-ethylmaleimide, iodoacetamide, and iodoacetamidopropionic acid. 14. The process of claim 1, wherein the process further comprises subjecting the mixture to an ion exchange chromatography membrane between steps (a) and (b) or steps (b) and (c). 15. The process of claim 1, wherein the process further comprises subjecting the mixture to an ion exchange chromatography membrane between steps (c) and (d). 16. The process of claim 1, wherein the one or more impurities are selected from the group of cytotoxic agent dimers, aggregates of the cell-binding agent cytotoxic agent conjugate, free cytotoxic agent, unconjugated linker, and mixtures thereof. 17. The process of claim 16, wherein the mixture comprises aggregates of the cell-binding agent cytotoxic agent conjugate as an impurity, and some portion of the aggregates of the cell-binding agent cytotoxic agent conjugate is removed from the mixture to provide the purified cell-binding agent cytotoxic agent conjugate. 18. The process of claim 16, wherein the mixture comprises free cytotoxic agent as an impurity, and some portion of the free cytotoxic agent is removed from the mixture to provide the purified cell-binding agent cytotoxic agent conjugate. 19. The process of claim 16, wherein the mixture comprises unconjugated linker as an impurity, and some portion of the unconjugated linker is removed from the mixture to provide the purified cell-binding agent cytotoxic agent conjugate. 20. The process of claim 1, wherein the pH of the pH-adjusted second mixture is about 7 to about 8. 21. The process of claim 1, wherein the pH of the pH-adjusted second mixture is about 8 to about 9. 22. The process of claim 21, wherein the pH of the pH-adjusted second mixture is about 8.5. 23. The process of claim 1, wherein at least 50% of the one or more impurities are removed from the pH-adjusted second mixture. 24. The process of claim 1, wherein at least 75% of the one or more impurities are removed from the pH-adjusted second mixture. 25. The process of claim 1, wherein at least 90% of the one or more impurities are removed from the pH-adjusted second mixture. 26. The process of claim 1, wherein the PVDF membrane is selected from a 0.22 micron pore size membrane, a 0.45 micron pore size membrane, and a dual layer 0.45/0.22 micron pore size membrane. 27. The process of claim 1, wherein the PVDF membrane is gamma irradiated. 28. The process of claim 1, wherein the contacting in step (a) occurs in a solution having a pH of about 7 to about 9. 29. The process of claim 1, wherein the solution in step (a) comprises a buffering agent selected from a citrate buffer, an acetate buffer, a succinate buffer, and a phosphate buffer. 30. The process of claim 28, wherein the solution in step (a) comprises a buffering agent selected from HEPPSO (N-(2-Hydroxyethyl)piperazine-N'-(2-hydroxypropanesulfonic acid)), POPSO (Piperazine-1,4-bis-(2-hydroxy-propane-sulfonic acid) dihydrate), HEPES (4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid), HEPPS (EPPS) (4-(2-hydroxyethyl)piperazine-1-propanesulfonic acid), TES (N-[tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid), and a combination thereof. 31. The process of claim 1, wherein the contacting in step (a) occurs at a temperature of about 16.degree. C. to about 24.degree. C. 32. The process of claim 1, wherein the contacting in step (a) occurs at a temperature of about 0.degree. C. to about 15.degree. C. 33. The process of claim 1, wherein the bifunctional crosslinking reagent is an acid labile linker, a disulfide containing linker, a photolabile linker, a peptidase labile linker, or an esterase labile linker. 34. The process of claim 1, wherein the bifunctional crosslinking reagent is a disulfide-containing cleavable linker. 35. The process of claim 1, wherein the bifunctional crosslinking reagent is a non-cleavable linker. 36. The process of claim 1, wherein the bifunctional crosslinking reagent comprises an N-succinimidyl ester moiety, an N-sulfosuccinimidyl ester moiety, a maleimido-based moiety, or a haloacetyl-based moiety. 37. The process of claim 34, wherein the bifunctional crosslinking reagent is selected from N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP), N-succinimidyl 4-(2-pyridyldithio)butanoate (SPDB), N-succinimidyl 4-(2-pyridyldithio)pentanoate (SPP), and N-succinimidyl-4-(2-pyridyldithio)2-sulfo butanoate (sulfo-SPDB). 38. The process of claim 35, wherein the bifunctional crosslinking reagent is selected from N-succinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate (SMCC), N-succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxy-(6-amidocaproa- te) (LC-SMCC), .kappa.-maleimidoundecanoic acid N-succinimidyl ester (KMUA), .gamma.-maleimidobutyric acid N-succinimidyl ester (GMBS), .beta.-maleimidopropyloxy-succinimidyl ester (BMPS), .epsilon.-maleimidocaproic acid N-hydroxysuccinimide ester (EMCS), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), N-(.alpha.-maleimidoacetoxy)-succinimide ester (AMAS), succinimidyl-6-(.beta.-maleimidopropionamido)hexanoate (SMPH), N-succinimidyl 4-(p-maleimidophenyl)-butyrate (SMPB), and N-(p-maleimidophenyl)isocyanate (PMPI), sulfo-Mal, PEG.sub.4-Mal and CX1-1. 39. The process of claim 1, wherein the cell-binding agent is selected from antibodies, interferons, interleukin 2 (IL-2), interleukin 3 (IL-3), interleukin 4 (IL-4), interleukin 6 (IL-6), insulin, epidermal growth factor (EGF), transforming growth factor alpha (TGF-.alpha.), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), a colony stimulating factor (G-CSF, MCSF, GM-CSF), and transferrin. 40. The process of claim 39, wherein the cell-binding agent is an antibody. 41. The process of claim 40, wherein the antibody is a monoclonal antibody. 42. The process of claim 41, wherein the antibody is a humanized monoclonal antibody. 43. The process of claim 39, wherein the cell-binding agent is an antibody selected from huB4, huC242, trastuzumab, bivatuzumab, sibrotuzumab, huDS6, rituximab, anti-CD33 antibody, anti-CD27L antibody, anti-Her2 antibody, anti-epidermal growth factor receptor (EGFR) antibody, anti-EGFRvIII antibody, Cripto, anti-CD138 antibody, anti-CD38 antibody, anti-ephrin type A receptor 2 (EphA2) antibody, integrin targeting antibody, anti-CD37 antibody, anti-folate receptor antibody, anti-Her3 antibody, B-B4 antibody and anti-insulin like growth factor I receptor (IGFIR) antibody. 44. The process of claim 1, wherein the cytotoxic agent is selected from maytansinoids, taxanes, and CC1065. 45. The process of claim 44, wherein the cytotoxic agent is a maytansinoid. 46. The process of claim 45, wherein the maytansinoid comprises a thiol group. 47. The process of claim 46, wherein the maytansinoid is N.sup.2'-deacetyl-N.sup.2'-(3-mercapto-1-oxopropyl)-maytansine (DM1) or N.sup.2'-deacetyl-N.sup.2'-(4-methyl-4-mercapto-1-oxopentyl)-maytansine (DM4). 48. The process of claim 1, wherein the cytotoxic agent is DM1, the bifunctional crosslinking agent is SMCC, and the cell-binding agent is huCD37-3 antibody. 49. The process of claim 1, wherein the cytotoxic agent is DM1, the bifunctional crosslinking agent is SMCC, and the cell-binding agent is EGFR-7R antibody. 50. The process of claim 1, wherein the cytotoxic agent is DM1, the bifunctional crosslinking agent is SMCC, and the cell-binding agent is an anti-EFGRvIII antibody. 51. The process of claim 1, wherein the cytotoxic agent is DM1, the bifunctional crosslinking agent is SMCC, and the cell-binding agent is an anti-CD27L antibody. 52. The process of claim 1, wherein the cytotoxic agent is DM1, the bifunctional crosslinking agent is SMCC, and the cell-binding agent is trastuzumab. 53. The process of claim 1, wherein the process further comprises (a) contacting a cell-binding agent with a cytotoxic agent to form a first mixture comprising the cell-binding agent and the cytotoxic agent, then contacting the first mixture with a bifunctional crosslinking reagent comprising a linker, in a solution having a pH of about 4 to about 9, to provide a second mixture comprising the cell-binding agent cytotoxic agent conjugate comprising the cell-binding agent chemically coupled through the linker to the cytotoxic agent and one or more impurities; (b) adjusting the second mixture to have a pH of about 7 to about 9 to form a pH-adjusted second mixture; (c) subjecting the pH-adjusted second mixture to a PVDF membrane to remove at least a portion of the impurities from the mixture, thereby providing a purified second mixture of the cell-binding agent cytotoxic agent conjugate; (d) quenching the purified second mixture after step (c) to quench any unreacted cytotoxic agent and/or unreacted bifunctional crosslinking reagent; (e) subjecting the quenched mixture after step (d) to a PVDF membrane to remove at least a portion of the impurities from the mixture, thereby providing a purified third mixture of the cell-binding agent cytotoxic agent conjugate; (f) holding the purified third mixture to release the unstably bound linkers from the cell-binding agent; (g) optionally subjecting the purified third mixture after step (f) to a PVDF membrane to remove at least a portion of the impurities from the mixture, thereby providing a purified fourth mixture of the cell-binding agent cytotoxic agent conjugate; and (h) subjecting the purified fourth mixture after step (g) to tangential flow filtration, selective precipitation, non-adsorptive chromatography, adsorptive filtration, adsorptive chromatography, or a combination thereof, to further purify the cell-binding agent-cytotoxic agent conjugate from the impurities and thereby prepare a purified third mixture of the cell-binding agent-cytotoxic agent conjugate, wherein the purified third mixture comprises a reduced amount of the impurities as compared to the purified second mixture. 54. The process of claim 53, wherein the pH-adjusted second mixture has a pH of about 8.5. 55. The process of claim 54, wherein the quenched mixture of step (e) has a pH of about 4.8. 56. A process for preparing a purified cell-binding agent cytotoxic agent conjugate comprising: (a) contacting a cell-binding agent with a bifunctional crosslinking reagent to covalently attach a linker to the cell-binding agent and thereby prepare a first mixture comprising cell-binding agents having linkers bound thereto, (b) subjecting the first mixture to tangential flow filtration, selective precipitation, non-adsorptive chromatography, adsorptive filtration, adsorptive chromatography, or a combination thereof and thereby prepare a purified first mixture of cell-binding agents having linkers bound thereto, (c) conjugating a cytotoxic agent to the cell-binding agents having linkers bound thereto in the purified first mixture by reacting the cell-binding agents having linkers bound thereto with a cytotoxic agent in a solution having a pH of about 4 to about 9 to prepare a second mixture comprising the cell-binding agent-cytotoxic agent conjugate comprising the cell-binding agent chemically coupled to the cytotoxic agent through the linker, and further comprising one or more impurities, (d) adjusting the second mixture to have a pH of about 7 to about 9 to form a pH-adjusted second mixture, (e) subjecting the pH-adjusted second mixture to a polyvinyl difluoride (PVDF) membrane to remove at least a portion of the impurities, thereby providing a purified second mixture of the cell-binding agent cytotoxic agent conjugate; and subjecting the purified second mixture after step (e) to tangential flow filtration, selective precipitation, non-adsorptive chromatography, adsorptive filtration, adsorptive chromatography, or a combination thereof, to further purify the cell-binding agent-cytotoxic agent conjugate from the impurities and thereby prepare a purified third mixture of the cell-binding agent-cytotoxic agent conjugate, wherein the purified third mixture comprises a reduced amount of the impurities as compared to the purified second mixture. 57. The process of claim 56, wherein step (e) is sequentially repeated two, three, or four times prior to step (f). 58. The process of claim 56, wherein the process further comprises subjecting the mixture to an ion exchange chromatography membrane between steps (c) and (d) or steps (d) and (e). 59. The process of claim 56, wherein the process further comprises subjecting the mixture to an ion exchange chromatography membrane between steps (e) and (f). 60. The process of claim 56, wherein adsorptive chromatography is utilized in steps (b) and (f). 61. The process of claim 56, wherein tangential flow filtration is utilized in step (b) and adsorptive chromatography is utilized in step (f). 62. The process of claim 56, wherein adsorptive chromatography is utilized in step (b) and tangential flow filtration is utilized in step (f). 63. The process of claim 56, wherein the adsorptive chromatography is selected from hydroxyapatite chromatography, hydrophobic charge induction chromatography (HCIC), hydrophobic interaction chromatography (HIC), ion exchange chromatography, mixed mode ion exchange chromatography, immobilized metal affinity chromatography (IMAC), dye ligand chromatography, affinity chromatography, reversed phase chromatography, and combinations thereof. 64. The process of claim 63, wherein the adsorptive chromatography is ion-exchange chromatography. 65. The process of claim 64, wherein the ion-exchange chromatography is ceramic hydroxyapatite (CHT) chromatography. 66. The process of claim 56, wherein tangential flow filtration is utilized in steps (b) and (f). 67. The process of claim 56, wherein non-adsorptive chromatography is utilized in steps (b) and (f). 68. The process of claim 56, wherein the solution in step (c) comprises sucrose. 69. The process of claim 56, wherein the solution in step (c) comprises a buffering agent selected from a citrate buffer, an acetate buffer, a succinate buffer, and a phosphate buffer. 70. The process of claim 56, wherein the solution in step (c) comprises a buffering agent selected from HEPPSO (N-(2-Hydroxyethyl)piperazine-N'-(2-hydroxypropanesulfonic acid)), POPSO (Piperazine-1,4-bis-(2-hydroxy-propane-sulfonic acid) dihydrate), HEPES (4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid), HEPPS (EPPS) (4-(2-hydroxyethyl)piperazine-1-propanesulfonic acid), TES (N-[tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid), and a combination thereof. 71. The process of claim 56, further comprising (g) holding the mixture between at least one of steps (a)-(b), steps (b)-(c), steps (c)-(d), steps (d)-(e), and steps (e)-(f) to release the unstably bound linkers from the cell-binding agent. 72. A process for preparing a purified cell-binding agent cytotoxic agent conjugate comprising: (a) contacting a cell-binding agent with a bifunctional crosslinking reagent to covalently attach a linker to the cell-binding agent and thereby prepare a first mixture comprising cell-binding agents having linkers bound thereto; (b) conjugating a cytotoxic agent to the cell-binding agents having linkers bound thereto in the first mixture by reacting the cell-binding agents having linkers bound thereto with a cytotoxic agent to prepare a second mixture comprising the cell-binding agent-cytotoxic agent conjugate comprising the cell-binding agent chemically coupled through the linker to the cytotoxic agent and one or more impurities; (c) adjusting the second mixture to have a pH of about 7 to about 9 to form a pH-adjusted second mixture; (d) subjecting the pH-adjusted second mixture to a polyvinyl difluoride (PVDF) membrane to remove at least a portion of the impurities, thereby providing a purified second mixture of the cell-binding agent cytotoxic agent conjugate; and (e) subjecting the purified second mixture after step (d) to tangential flow filtration, selective precipitation, non-adsorptive chromatography, adsorptive filtration, adsorptive chromatography, or a combination thereof, to further purify the cell-binding agent-cytotoxic agent conjugate from the impurities and thereby prepare a purified third mixture of the cell-binding agent-cytotoxic agent conjugate, wherein the purified third mixture comprises a reduced amount of the impurities as compared to the purified second mixture. 73. The process of claim 72, wherein the first mixture is not subjected to purification between steps (a) and (b). 74. The process of claim 72, wherein step (d) is sequentially repeated two, three, or four times prior to step (e). 75. The process of claim 72, wherein the process further comprises subjecting the mixture to an ion exchange chromatography membrane between steps (b) and (c) or steps (c) and (d). 76. The process of claim 72, wherein the process further comprises subjecting the mixture to an ion exchange chromatography membrane between steps (d) and (e). 77. The process of claim 72, wherein adsorptive chromatography is utilized in step (e). 78. The process of claim 77, wherein the adsorptive chromatography is selected from hydroxyapatite chromatography, hydrophobic charge induction chromatography (HCIC), hydrophobic interaction chromatography (HIC), ion exchange chromatography, mixed mode ion exchange chromatography, immobilized metal affinity chromatography (IMAC), dye ligand chromatography, affinity chromatography, reversed phase chromatography, and combinations thereof. 79. The process of claim 78, wherein the adsorptive chromatography is ion-exchange chromatography. 80. The process of claim 79, wherein the ion-exchange chromatography is ceramic hydroxyapatite (CHT) chromatography. 81. The process of claim 72, wherein tangential flow filtration is utilized in step (e). 82. The process of claim 72, wherein non-adsorptive chromatography is utilized in step (e). 83. The process of claim 72, wherein the solution in step (b) comprises sucrose. 84. The process of claim 72, wherein the solution in step (b) comprises a buffering agent selected from a citrate buffer, an acetate buffer, a succinate buffer, and a phosphate buffer. 85. The process of claim 72, wherein the solution in step (b) comprises a buffering agent selected from HEPPSO (N-(2-Hydroxyethyl)piperazine-N'-(2-hydroxypropanesulfonic acid)), POPSO (Piperazine-1,4-bis-(2-hydroxy-propane-sulfonic acid) dihydrate), HEPES (4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid), HEPPS (EPPS) (4-(2-hydroxyethyl)piperazine-1-propanesulfonic acid), TES (N-[tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid), and a combination thereof. 86. The process of claim 72, further comprising (f) holding the mixture between at least one of steps (a)-(b), steps (b)-(c), steps (c)-(d), and steps (d)-(e) to release the unstably bound linkers from the cell-binding agent. 87. The process of claim 86, wherein the mixture comprises cytotoxic agent dimers as an impurity, and some portion of the cytotoxic agent dimers is removed from the mixture to provide the purified cell-binding agent cytotoxic agent conjugate. 88. The process of claim 87, wherein the cytotoxic agent dimer comprises ##STR00016## 89. The process of claim 87, wherein the cytotoxic agent dimer comprises ##STR00017## 90. The process of claim 87, wherein the cytotoxic agent dimer comprises ##STR00018##

Details for Patent 10,035,817

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Genentech, Inc.	RITUXAN	rituximab	Injection	103705	11/26/1997		2032-10-04
Genentech, Inc.	HERCEPTIN	trastuzumab	For Injection	103792	09/25/1998		2032-10-04
Genentech, Inc.	HERCEPTIN	trastuzumab	For Injection	103792	02/10/2017		2032-10-04