Process for preparing cell-binding agent-cytotoxic agent conjugates
The present invention provides a novel process for preparing a cell-binding agent cytotoxic agent conjugate. The process comprises the steps of: (a) reacting a cytotoxic agent with a bifunctional crosslinking reagent represented by the structural formula (I) or a salt thereof, in a buffer solution comprising a buffering agent to provide a first mixture comprising a cytotoxic agent-linker compound, wherein the buffer solution has high buffer capacity; and (b) reacting the first mixture comprising the cytotoxic agent-linker compound from step (a) with a cell-binding agent in a solution having a pH of 4 to 9 to provide a second mixture comprising the cell-binding agent cytotoxic agent conjugate. The cell-binding agent cytotoxic agent conjugates prepared according to the processes described herein are also included in the present invention.
1. A process for preparing a cell-binding agent cytotoxic agent conjugate comprising the steps of:
(a) reacting a cytotoxic agent with a bifunctional crosslinking reagent represented by the following structural formula:
or a salt thereof, in a buffer solution comprising a buffering agent to provide a first mixture comprising a cytotoxic agent-linker compound, wherein the buffer solution has high buffer capacity and the molar ratio of the buffering agent to the bifunctional crosslinking reagent is 2:1 to 8:1;
(b) reacting the cytotoxic agent-linker compound in the first mixture obtained from step (a) with a cell-binding agent in a solution having a pH of 4 to 9 to provide a second mixture comprising the cell-binding agent cytotoxic agent conjugate,
wherein the cell-binding agent is an antibody.
2. The process of claim 1 , wherein the cell-binding agent is prone to fragmentation.
3. The process of claim 1 , wherein the cytotoxic agent is a maytansinoid.
4. The process of claim 3 , wherein the cytotoxic agent is DM4.
5. The process of claim 1 , wherein molar ratio of the buffering agent to the bifunctional crosslinking reagent is 4:1 to 6:1.
6. The process of claim 5 , wherein the molar ratio of the buffering agent to the bifunctional crosslinking reagent is 5:1.
7. The process of claim 1 , wherein the buffer solution has a pH of 4 to 9, 7.5 to 8.5, 7.9 to 8.5, 8.0 to 8.4 or 8.1 to 8.3.
8. The process of claim 7 , wherein the buffer solution has a pH of 8.2.
9. The process of claim 1 , wherein the buffering agent is selected from the group consisting of a citrate buffer, an acetate buffer, a succinate buffer and a phosphate buffer or the buffering agent is selected from the group consisting of HEPPSO (N-(2-hydroxyethyl)piperazine-N′-(2-hydroxypropanesulfonic acid)), POPSO (piperazine-1,4-bis-(2-hydroxy-propane-sulfonic acid) dehydrate), HEPES (4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid), EPPS (4-(2-hydroxyethyl)piperazine-1-propanesulfonic acid), TES (N-[tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid), MES (2-(N-morpholino)ethanesulfonic acid) and a combination thereof.
10. The process of claim 1 , wherein the buffer solution further comprises sodium chloride.
11. The process of claim 1 , wherein the buffer solution further comprises an organic solvent.
12. The process of claim 1 , wherein molar excess amount of the cytotoxic agent relative to the bifunctional crosslinking reagent is used in the reaction of step (a).
13. The process of claim 1 , wherein the solution in step (b) has a pH of 5.0-9.0, 5.5-9.0, 6.0-9.0 or 6.5-9.0.
14. The process of claim 1 , wherein the solution in step (b) comprises a buffering agent selected from the group consisting of a citrate buffer, an acetate buffer, a succinate buffer and a phosphate buffer, or a buffering agent selected from the group consisting of HEPPSO (N-(2-hydroxyethyl)piperazine-N′-(2-hydroxypropanesulfonic acid)), POPSO (piperazine-1,4-bis(2-hydroxy-propane-sulfonic acid) dehydrate), HEPES (4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid), EPPS (4-(2-hydroxyethyl)piperazine-1-propanesulfonic acid), TES (N-[tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid), MES (2-(N-morpholino)ethanesulfonic acid) and a combination thereof.
15. The process of claim 1 , wherein the solution in step (b) further comprises an organic solvent.
16. The process of claim 1 , wherein concentration of the cell-binding agent in the solution of step (b) is 5 g/L to 100 g/L.
17. The process of claim 1 , wherein the molar ratio of the bifunctional crosslinking reagent to the cell-binding agent is from 2 to 10.
18. The process of claim 1 , wherein the process further comprises the step of quenching the reaction in step (b) by adjusting the pH of the solution to 5 or below.
19. The process of claim 1 , wherein the antibody is selected from huN901, huMy9-6, huB4, huC242, trastuzumab, bivatuzumab, sibrotuzumab, CNTO95, huDS6, rituximab, anti-Her2 antibody, anti-EGFR antibody, anti-CD27L antibody, anti-EGFRvIll antibody, anti-Cripto antibody, anti-CD138 antibody, anti-CD38 antibody, anti-EphA2 antibody, integrin targeting antibody, anti-CD37 antibody, anti-folate receptor antibody, anti-Her3 antibody, and anti-IGFIR antibody.