Methods of conjugating an agent to a thiol moiety in a protein that contains at least one trisulfide bond

Abstract

The invention provides improved methods of conjugating an agent to a thiol moiety in a protein that contains at least one disulfide bond and at least one trisulfide bond. Exemplary embodiments include the production of antibody drug conjugates substantially free of impurities created in the presence of reactive sulfide moieties in the production processes.

1 .- 81 . (canceled) 
     
     
         82 . A method for converting trisulfide bonds to disulfide bonds in an isolated antibody comprising the steps of:
 (a) reducing at least one isolated antibody containing at least one trisulfide bond in a solution with TCEP at a pH between about pH 5.0 and about pH 8;   (b) contacting the solution with nitrogen gas; and,   (c) conjugating an auristatin, or a derivative thereof, to the reduced antibody to form an antibody-drug conjugate (ADC).   
     
     
         83 . The method of  claim 82 , wherein the reducing step comprises contacting the isolated protein with a chemical reducing agent in a concentration of about 0.5 to about 8 mM. 
     
     
         84 . The method of  claim 82 , wherein the molar ratio of TCEP to the isolated protein is about 3:1. 
     
     
         85 . The method of  claim 82 , wherein the contacting comprises bubbling nitrogen gas through the composition. 
     
     
         86 . (canceled) 
     
     
         87 . The method of  claim 82 , wherein the contacting comprises sparging the composition with nitrogen gas at a rate of about 50 cubic centimeters per minute. 
     
     
         88 . The method of  claim 82 , wherein the contacting comprises mixing the composition in the presence of nitrogen gas at a rate that is about 300% greater than the optimal mixing rate for the reducing step. 
     
     
         89 . The method of  claim 82 , wherein the contacting is conducted at a pH between about 5.5 and about 7.5. 
     
     
         90 . The method of  claim 82 , wherein the contacting is conducted at a temperature between about 20° C. and about 30° C. 
     
     
         91 . The method of  claim 82 , wherein a ratio of the surface area to the volume of the composition is about 2. 
     
     
         92 .- 96 . (canceled) 
     
     
         97 . The method of  claim 82 , wherein the contacting comprises sparging a vessel containing the composition with the nitrogen gas. 
     
     
         98 . The method of  claim 82 , wherein the contacting is conducted in the presence of at least one of a tween and an antifoaming agent. 
     
     
         99 . (canceled) 
     
     
         100 . The method of  claim 98 , wherein the antifoaming agent is at least one of Antifoam-A, Antifoam-C, and a poloxamer. 
     
     
         101 . The method of  claim 82 , wherein the antibody is an antibody fragment. 
     
     
         102 .- 112 . (canceled) 
     
     
         113 . The method of  claim 82 , wherein the antibody is an IgG1 monoclonal antibody which has 4 interchain sulfide bonds comprising two sulfide bonds in the hinge region connecting heavy chains, and one sulfide bond between each light chain and heavy chain. 
     
     
         114 . The method of  claim 82 , wherein the reducing step reduces only interchain disulfide bonds and not intrachain bonds. 
     
     
         115 . The method of  claim 82 , wherein the auristatin is MMAE or MMAF. 
     
     
         116 . The method of  claim 82 , wherein the therapeutic agent comprises a linker moiety adapted to link the at least one thiol group of the antibody to the therapeutic agent. 
     
     
         117 . The method of  claim 116 , wherein the linker moiety is a cleavable linker or a non-cleavable linker. 
     
     
         118 .- 124 . (canceled) 
     
     
         125 . The method of  claim 82 , wherein a dimer of the auristatin, or derivative thereof, is formed in the conjugating step and the method further comprises separating the ADC from the dimer to form a purified ADC. 
     
     
         126 . The method of  claim 82 , wherein the antibody-therapeutic agent conjugate is one of aCD22-val-cit-MMAE, aCD22-val-cit-MMAF, aLy6E-val-cit-MMAE, aLy6E-val-cit-MMAF, aCD79b-val-cit-MMAE, aCD79b-val-cit-MMAF, aNaPi2b-val-cit-MMAE, aNaPi2b-val-cit-MMAF, aMUC16-val-cit-MMAE, a MUC16-val-cit-MMAF, sSTEAP1, and aETBR.