Process for preparing purified drug conjugates
The invention provides a process for preparing a cell-binding agent chemically coupled to a drug. The process comprises covalently attaching a linker to a cell-binding agent, a purification step, conjugating a drug to the cell-binding agent and a subsequent purification step.
1. A process for preparing a cell-binding agent-cytotoxic agent conjugate comprising the steps of:
(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 selective precipitation, adsorptive filtration, or adsorptive chromatography 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 (i) cell-binding agent chemically coupled through the linker to the cytotoxic agent, (ii) free cytotoxic agent, and (iii) reaction by-products, and
(d) subjecting the second mixture to tangential flow filtration to purify the cell-binding agents chemically coupled through the linkers to the cytotoxic agent from the other components of the second mixture and thereby prepare a purified second mixture of cell-binding agents chemically coupled through the linkers to the cytotoxic agent.
2. The process of claim 1 , wherein the adsorptive chromatography is selected from the group consisting of 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.
3. The process of claim 1 , wherein adsorptive chromatography is utilized in step (b).
4. The process of claim 3 , wherein the adsorptive chromatography is hydroxyapatite chromatography.
5. The process of claim 1 , wherein the solution in step (c) has a pH of from about 4 to about 6.
6. The process of claim 1 , wherein the solution in step (c) has a pH of from about 6.5 to about 9.
7. The process of claim 1 , wherein the solution in step (c) has a pH of less than 6.0 or a pH of greater than 6.5.
8. The process of claim 1 , wherein the cell-binding agent is selected from the group consisting of 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 α (TGF-α), fibroblast growth factor (FGF), granulocyte colony stimulating factor (G-CSF), vascular endothelial growth factor (VEGF), macrophage colony stimulating factor (M-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), and transferrin.
9. The process of claim 8 , wherein the cell-binding agent is an antibody.
10. The process of claim 9 , wherein the antibody is a chimeric antibody.
11. The process of claim 9 , wherein the antibody is a humanized antibody.
12. The process of claim 9 , wherein the antibody is a human monoclonal antibody.
13. The process of claim 9 , wherein the antibody is a mouse monoclonal antibody.
14. The process of claim 9 , wherein the antibody is a monoclonal antibody.
15. The process of claim 14 , wherein the antibody is a humanized monoclonal antibody.
16. The process of claim 15 , wherein the antibody is selected from the group consisting of huN901, huMy9-6, huB4, huC242, trastuzumab, bivatuzumab, sibrotuzumab, CNTO95, huDS6, and rituximab.
17. The process of claim 1 , wherein the cytotoxic agent is selected from the group consisting of maytansinoids, taxanes, and CC1065.
18. The process of claim 17 , wherein the cytotoxic agent is a maytansinoid.
19. The process of claim 18 , wherein the maytansinoid comprises a thiol group.
20. The process of claim 19 , wherein the maytansinoid is N 2′ -deacetyl-N 2′ -(3-mercapto-1-oxopropyl)-maytansine (DM1).
21. The process of claim 19 , wherein the maytansinoid is N 2′ -deacetyl-N 2′ -(4-methyl-4-mercapto-1-oxopentyl)-maytansine (DM4).
22. The process of claim 1 , wherein the cell-binding agent is chemically coupled to the cytotoxic agent via chemical bonds selected from the group consisting of disulfide bonds, acid labile bonds, photolabile bonds, peptidase labile bonds, thioether bonds, and esterase labile bonds.
23. The process of claim 1 , wherein the solution in step (c) comprises sucrose.
24. The process of claim 1 , wherein the solution in step (c) comprises a buffering agent selected from the group consisting of a citrate buffer, an acetate buffer, a succinate buffer, and a phosphate buffer.
25. The process of claim 1 , further comprising
(e) holding the mixture between at least one of steps a-b, steps b-c, and steps c-d to release the unstably bound linkers from the cell-binding agent.
26. The process of claim 25 , wherein the mixture is held between steps a-b.
27. The process of claim 25 , wherein the mixture is held between steps b-c.
28. The process of claim 25 , wherein the mixture is held between steps c-d.
29. The process of claim 1 , wherein selective precipitation is utilized in step (b).
30. The process of claim 1 , wherein adsorptive filtration is utilized in step (b).
31. The process of claim 1 , wherein the bifunctional crosslinking reagent comprises an N-succinimidyl ester moiety.
32. The process of claim 1 , wherein the bifunctional crosslinking reagent comprises an N-sulfosuccinimidyl ester moiety.
33. The process of claim 1 , wherein the bifunctional crosslinking reagent comprises a maleimido-based moiety.
34. The process of claim 1 , wherein the bifunctional crosslinking reagent comprises a haloacetyl-based moiety.
35. The process of claim 1 , wherein the bifunctional crosslinking reagent is selected from the group consisting of N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP), N-succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxy-(6-amidocaproate), (LC-SMCC), κ-maleimidoundecanoic acid N-succinimidyl ester (KMUA), γ-maleimidobutyric acid N-succinimidyl ester (GMBS), ε-maleimidocaproic acid N-hydroxysuccinimide ester (EMCS), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), N-(α-maleimidoacetoxy)-succinimide ester (AMAS), succinimidyl-6-β-maleimidopropionamido)hexanoate (SMPH), N-succinimidyl 4-(p-maleimidophenyl)-butyrate (SMPB), N-(p-maleimidophenyl)isocyanate (PMPI), N-succinimidyl-4-(iodoacetyl)-aminobenzoate (SIAB), N-succinimidyl iodoacetate (SIA), N-succinimidyl bromoacetate (SBA), and N-succinimidyl 3-(bromoacetamido)propionate (SBAP).
36. The process of claim 1 , wherein the bifunctional crosslinking reagent is N-succinimidyl 4-(2-pyridyldithio)butanoate (SPDB).
37. The process of claim 1 , wherein the bifunctional crosslinking reagent is N-succinimidyl 4-(2-pyridyldithio)pentanoate (SPP).
38. The process of claim 1 , wherein the bifunctional crosslinking reagent is N-succinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate (SMCC).
39. The process of claim 1 , wherein the cell-binding agent is huN901 and the cytotoxic agent is N 2′ -deacetyl-N 2′ -(3-mercapto-1-oxopropyl)-maytansine (DM1).
40. The process of claim 39 , wherein the bifunctional crosslinking reagent is N-succinimidyl 4-(2-pyridyldithio)pentanoate (SPP).
41. The process of claim 1 , wherein the cell-binding agent is trastuzumab and the cytotoxic agent is N 2′ -deacetyl-N 2′ -(4-methyl-4-mercapto-1-oxopentyl)-maytansine (DM4).
42. The process of claim 41 , wherein the bifunctional crosslinking reagent is N-succinimidyl 4-(2-pyridyldithio)butanoate (SPDB).
43. The process of claim 1 , wherein the cell-binding agent is huB4 and the cytotoxic agent is N 2′ -deacetyl-N 2′ -(4-methyl-4-mercapto-1-oxopentyl)-maytansine (DM4).
44. The process of claim 43 , wherein the bifunctional crosslinking reagent is N-succinimidyl 4-(2-pyridyldithio)butanoate (SPDB).
45. The process of claim 1 , wherein the cell-binding agent is huDS6 and the cytotoxic agent is N 2′ -deacetyl-N 2′ -(4-methyl-4-mercapto-1-oxopentyl)-maytansine (DM4).
46. The process of claim 45 , wherein the bifunctional crosslinking reagent is N-succinimidyl 4-(2-pyridyldithio)butanoate (SPDB).
47. The process of claim 1 , wherein the antibody is trastuzumab and the maytansinoid is N 2′ -deacetyl-N 2′ -(3-mercapto-1-oxopropyl)-maytansine (DM1).
48. The process of claim 47 , wherein the bifunctional crosslinking reagent is N-succinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate (SMCC).