Selective reduction of proteins

Abstract

The present invention provides a method for making uncapped cysteine protein preparations, including uncapped engineered cysteine antibody preparations. The methods include, inter alia, contacting a reducing agent with engineered cysteine antibody molecules, each of the antibody molecules having at least one capped engineered cysteine residue and at least one interchain disulfide bond and reacting the reducing agent with the antibody molecules under conditions sufficient to uncap engineered cysteine residues and form cap byproducts. The method also includes removing the bap byproduct during the reduction reaction. Substantially all of the interchain disulfide bonds present in the antibody molecules prior to reduction are retained following reduction. Antibody conjugates and methods for preparing antibody conjugates using uncapped antibody preparations are also described.

1 .- 43 . (canceled) 
     
     
         44 . An uncapped engineered cysteine antibody preparation prepared according to a method for the selective reduction of one or more capped engineered cysteine residues in intact antibodies, the method comprising:
 a) contacting a reducing agent with antibody molecules, each of the antibody molecules having at least one capped engineered cysteine residue and at least three heavy-light and heavy-heavy inter-chain disulfide bonds;   b) reacting the reducing agent with the antibody molecules under conditions sufficient to uncap engineered cysteine residues and form cap byproducts between the reducing agent and one or more cap moieties of the antibody molecules; and   c) removing the cap byproducts during the reduction reaction;   whereby an uncapped engineered cysteine antibody preparation is formed and at least about 80% of the heavy-light and heavy-heavy inter-chain disulfide bonds present are retained in the uncapped engineered cysteine antibody preparation.   
     
     
         45 . The uncapped engineered cysteine antibody preparation of  claim 44 , wherein the method comprises supplementing the reduction reaction with additional reducing agent while removing the cap byproduct. 
     
     
         46 . The uncapped engineered cysteine antibody preparation  claim 44  comprising two or more members selected from the group consisting of:
 an antibody molecule having at least two uncapped engineered cysteine residues and no capped engineered cysteine residues; 
 an antibody molecule having at least two capped engineered cysteine residues and no uncapped engineered cysteine residues; and 
 an antibody molecule having at least one capped engineered cysteine residue and at least one uncapped engineered cysteine residue. 
 
     
     
         47 . The uncapped engineered cysteine antibody preparation of  claim 44 , wherein removing the cap byproduct during the reduction reaction comprises dialysis or diafiltration. 
     
     
         48 . The uncapped engineered cysteine antibody preparation of  claim 44 , wherein:
 each antibody molecule prior to the reduction reaction comprises four heavy-light and heavy-heavy inter-chain disulfide bonds;   each antibody molecule has at least two engineered cysteine residues, or   each antibody molecule has four engineered cysteine residues.   
     
     
         48 . The uncapped engineered cysteine antibody preparation of  claim 44 , wherein:
 the engineered cysteine residues are present in the heavy constant region of the antibody molecule.   the engineered cysteine residues are present in the heavy chain or light chain variable region of the antibody molecule.   
     
     
         49 . The uncapped engineered cysteine antibody preparation of  claim 44 , wherein:
 the reducing agent is selected from the group consisting of cysteine, cysteamine, (3-mercaptoethanol, 2-mercaptoethanesulfonic acid sodium salt, and mixtures thereof, or the reducing agent is cysteine.   
     
     
         50 . The uncapped engineered cysteine antibody preparation of  claim 49 , wherein:
 the method comprises maintaining the reducing agent at a concentration from about 5 times to about 15 times greater than the concentration of the antibody during the reduction reaction; or   the method comprises maintaining the reducing agent at a concentration from about 5 times to about 10 times greater than the concentration of the antibody during the reduction reaction.   
     
     
         51 . The uncapped engineered cysteine antibody preparation of  claim 49 , wherein the concentration of cysteine is maintained at a concentration of 0.5 mM to about 1.5 mM during the reduction reaction. 
     
     
         52 . The uncapped engineered cysteine antibody preparation of  claim 44 , wherein the concentration of the cap byproduct is maintained below the concentration at which re-capping prevents further activation of engineered cysteine residues. 
     
     
         53 . The uncapped engineered cysteine antibody preparation of  claim 44 , wherein the uncapped engineered cysteine antibody preparation is a monoclonal antibody preparation. 
     
     
         54 . The uncapped engineered cysteine antibody preparation of  claim 44 :
 further comprising the step of removing residual reducing agent from the uncapped engineered cysteine antibody preparation;   further comprising the step of purifying the uncapped engineered cysteine antibody preparation; or   further comprising combining uncapped antibody with a drug-linker compound under conditions sufficient to form antibody-drug conjugate.   
     
     
         55 . The uncapped engineered cysteine antibody preparation of  claim 54 , wherein:
 the average drug load of the antibody drug conjugate is about 3.6 to 4.2 drug moieties per antibody; or   the average drug load of the antibody drug conjugate is about is about 2 drug moieties per antibody.   
     
     
         56 . The uncapped engineered cysteine antibody preparation of  claim 54 , wherein the antibody drug conjugate is in solution. 
     
     
         57 . The uncapped engineered cysteine antibody preparation of  claim 44 , wherein:
 at least about 85% of the heavy-light and heavy-heavy inter-chain disulfide bonds present in the antibody molecules prior to the reduction reaction are retained in the uncapped cysteine antibody preparation; or   at least about 90% of the heavy-light and heavy-heavy inter-chain disulfide bonds present in the antibody molecules prior to the reduction reaction are retained in the uncapped cysteine antibody preparation.   
     
     
         58 . An activated engineered cysteine residue prepared according to a method for the selective activation of engineered cysteine residues of engineered cysteine antibodies, said method comprising:
 (a) diafiltering a mixture of engineered cysteine antibodies and buffer, and   (b) adding a reducing agent to said mixture in a concentration and at a rate to activate a portion of available engineered cysteine residues while retaining at least 80% of all inter-chain disulfide bonds present in the engineered cysteine antibodies prior to (b); and   (c) maintaining diafiltration of said mixture during step (b);   to selectively activate the engineered cysteine residue.   
     
     
         59 . The activated engineered cysteine residue of  claim 58 , wherein less than 20% of said inter-chain disulfide bonds are converted to a pair of free thiols. 
     
     
         60 . The activated engineered cysteine residue of  claim 58 , wherein said reducing agent is cysteine. 
     
     
         61 . The activated engineered cysteine residue of  claim 58 , wherein at least 80% of inter-chain disulfide bonds present in the engineered cysteine antibodies are retained prior to step (b). 
     
     
         62 . The activated engineered cysteine residue of  claim 59 , wherein:
 less than 15% of said inter-chain disulfide bonds are converted to a pair of free thiols; or
 less than 10% of said inter-chain disulfide bonds are converted to a pair of free thiols.