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 tangential flow filtration, 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 6.5 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 selective precipitation, adsorptive filtration, or adsorptive chromatography 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,
with the proviso that if the first mixture is subjected to tangential flow filtration in step (b), the second mixture is not subjected to adsorptive chromatography in step (d).
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 steps (b) and (d).
4. 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, EGF, TGF-α, FGF, G-CSF, VEGF, MCSF, GM-CSF, and transferrin.
5. The process of claim 4 , wherein the cell-binding agent is an antibody.
6. The process of claim 5 , wherein the antibody is a monoclonal antibody.
7. The process of claim 6 , wherein the antibody is a humanized monoclonal antibody.
8. The process of claim 7 , wherein the antibody is selected from the group consisting of huN901, huMy9-6, huB4, huC242, trastuzumab, bivatuzumab, sibrotuzumab, CNTO95, huDS6, and rituximab.
9. The process of claim 1 , wherein the cytotoxic agent is selected from the group consisting of maytansinoids, taxanes, and CC1065.
10. The process of claim 9 , wherein the cytotoxic agent is a maytansinoid.
11. The process of claim 10 , wherein the maytansinoid comprises a thiol group.
12. The process of claim 11 , wherein the maytansinoid is N 2′ -deacetyl-N 2′ -(3-mercapto-1-oxopropyl)-maytansine (DM1).
13. The process of claim 11 , wherein the maytansinoid is N 2′ -deacetyl-N 2′ -(4-methyl-4-mercapto-1-oxopentyl)-maytansine (DM4).
14. 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.
15. The process of claim 1 , wherein the solution in step (c) comprises sucrose.
16. 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.
17. 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.
18. The process of claim 17 , wherein the mixture is held between steps (a)-(b).
19. The process of claim 17 , wherein the mixture is held between steps (b)-(c).
20. The process of claim 17 , wherein the mixture is held between steps (c)-(d).
21. 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 6.5 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, selective precipitation, adsorptive filtration, or adsorptive chromatography 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.
22. The process of claim 21 , 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.
23. The process of claim 21 , wherein adsorptive chromatography is utilized in step (b) and tangential flow filtration is utilized in step (d).
24. The process of claim 21 , 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, EGF, TGF-α, FGF, G-CSF, VEGF, MCSF, GM-CSF, and transferrin.
25. The process of claim 24 , wherein the cell-binding agent is an antibody.
26. The process of claim 25 , wherein the antibody is a monoclonal antibody.
27. The process of claim 26 , wherein the antibody is a humanized monoclonal antibody.
28. The process of claim 27 , wherein the antibody is selected from the group consisting of huN901, huMy9-6, huB4, huC242, trastuzumab, bivatuzumab, sibrotuzumab, CNTO95, huDS6, and rituximab.
29. The process of claim 21 , wherein the cytotoxic agent is selected from the group consisting of maytansinoids, taxanes, and CC1065.
30. The process of claim 29 , wherein the cytotoxic agent is a maytansinoid.
31. The process of claim 30 , wherein the maytansinoid comprises a thiol group.
32. The process of claim 31 , wherein the maytansinoid is N 2′ -deacetyl-N 2′ -(3-mercapto-1-oxopropyl)-maytansine (DM1).
33. The process of claim 31 , wherein the maytansinoid is N 2′ -deacetyl-N 2′ -(4-methyl-4-mercapto-1-oxopentyl)-maytansine (DM4).
34. The process of claim 22 , 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.
35. The process of claim 23 , wherein the solution in step (c) comprises sucrose.
36. The process of claim 24 , 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.
37. The process of claim 21 , 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.
38. The process of claim 37 , wherein the mixture is held between steps (a)-(b).
39. The process of claim 37 , wherein the mixture is held between steps (b)-(c).
40. The process of claim 37 , wherein the mixture is held between steps (c)-(d).