Method of purifying cell-binding agent-cytotoxic agent conjugates with a PVDF membrane

Patent metadata

Field	Value
Publication number	US-10035817-B2
Application number	US-201314430701-A
Country	US
Kind code	B2
Filing date	Oct 4, 2013
Priority date	Oct 4, 2012
Publication date	Jul 31, 2018
Grant date	Jul 31, 2018

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.

First claim

The invention claimed is:

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° C. to about 8° 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° C. to about 24° C.

32. The process of claim 1 , wherein the contacting in step (a) occurs at a temperature of about 0° C. to about 15° 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 r

Assignees

• Immunogen Inc

Inventors

• Chen Xiaoxi Kevin
• Li Xinfang

Classifications

• A61P35/00

  Antineoplastic agents · CPC title

• B01D15/325

  Reversed phase · CPC title

• A61K47/6867

  the tumour determinant being from a cell of a blood cancer · CPC title

• B01D15/3804

  Affinity chromatography · CPC title

• B01D15/361

  Ion-exchange · CPC title