Process for preparing cell-binding agent-cytotoxic agent conjugates

Abstract

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;
 (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. 
 
     
     
         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 1.5:1 to 12.5:1, 2:1 to 8:1, or 4:1 to 6:1. 
     
     
         6 - 7 . (canceled) 
     
     
         8 . The process of  claim 5 , wherein the molar ratio of the buffering agent to the bifunctional crosslinking reagent is 5:1. 
     
     
         9 . 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. 
     
     
         10 - 11 . (canceled) 
     
     
         12 . The process of  claim 9 , wherein the buffer solution has a pH of 8.2. 
     
     
         13 . 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. 
     
     
         14 - 15 . (canceled) 
     
     
         16 . The process of  claim 1 , wherein the buffer solution further comprises sodium chloride. 
     
     
         17 . The process of  claim 1 , wherein the buffer solution further comprises an organic solvent. 
     
     
         18 - 21 . (canceled) 
     
     
         22 . The process of  claim 1 , wherein molar excess amount of the cytototoxic agent relative to the bifunctional crosslinking reagent is used in the reaction of step (a). 
     
     
         23 - 34 . (canceled) 
     
     
         35 . 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. 
     
     
         36 - 38 . (canceled) 
     
     
         39 . 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. 
     
     
         40 - 41 . (canceled) 
     
     
         42 . The process of  claim 1 , wherein the solution in step (b) further comprises an organic solvent. 
     
     
         43 - 46 . (canceled) 
     
     
         47 . 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. 
     
     
         48 - 59 . (canceled) 
     
     
         60 . The process of  claim 1 , wherein the molar ratio of the bifunctional crosslinking reagent to the cell-binding agent is from 2 to 10. 
     
     
         61 - 63 . (canceled) 
     
     
         64 . 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. 
     
     
         65 - 67 . (canceled) 
     
     
         68 . The process of  claim 1 , wherein the cell-binding agent is an antibody. 
     
     
         69 - 70 . (canceled) 
     
     
         71 . The process of  claim 68 , 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-EGFRvIII 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.