Refolding Proteins Using a Chemically Controlled Redox State

Abstract

A method of refolding proteins expressed in non-mammalian cells present in concentrations of 2.0 g/L, or higher is disclosed. The method comprises identifying the thiol pair ratio and the redox butler strength to achieve conditions under which efficient folding at concentrations of 2.0 g/L or higher is achieved and can be employed over a range of volumes, including commercial scale.

1 - 24 . (canceled) 
     
     
         25 . A method of refolding a protein expressed in a non-mammalian expression system, said method comprising:
 (a) contacting said protein with a refold buffer to form a refold mixture, wherein said protein is present at a concentration of 2.0 g/L or greater in said refold mixture, and wherein said refold buffer comprises:
 (i) a redox component comprising a final thiol-pair ratio, wherein said final thiol-pair ratio has a range of 0.001 to 50, and wherein said thiol-pair ratio is calculated according to Equation 1: 
   
       
         
           
             
               
                 
                   
                     [ 
                     reductant 
                     ] 
                   
                   2 
                 
                 
                   [ 
                   oxidant 
                   ] 
                 
               
               ; 
             
           
         
         
           (ii) a thiol pair buffer strength of 2 mM or greater, wherein said thiol-pair buffer strength is calculated according to Equation 2:
   2[oxidant]+[reductant]; and 
 
           (iii) at least one of:
 a denaturant; 
 an aggregation suppressor; and 
 a protein stabilizer; and 
 
         
         (b) incubating said refold mixture. 
       
     
     
         26 . The method of  claim 25 , wherein said method further comprises:
 (c) isolating the refolded protein from step b).   
     
     
         27 . The method of  claim 25 , wherein said protein is present at a concentration of 2 to 40 g/L in said refold mixture. 
     
     
         28 . The method of  claim 27 , wherein said protein is present at a concentration of 10 to 20 g/L in said refold mixture. 
     
     
         29 . The method of  claim 25 , wherein said refold buffer comprises a denaturant, an aggregation suppressor, and a protein stabilizer. 
     
     
         30 . The method of  claim 25 , wherein said protein is an antibody. 
     
     
         31 . The method of  claim 25 , wherein said protein comprises an Fc domain. 
     
     
         32 . The method of  claim 25 , wherein said final thiol-pair ratio has a range of 1 to 25. 
     
     
         33 . The method of  claim 25 , wherein said thiol-pair buffer strength is greater than or equal to 5 mM. 
     
     
         34 . The method of  claim 25 , wherein said protein in step a) is present in a non-native limited solubility form at the time said protein is contacted with said refold buffer. 
     
     
         35 . The method of  claim 34 , wherein said non-native limited solubility form is an inclusion body. 
     
     
         36 . The method of  claim 25 , wherein said incubation is performed under non-aerobic conditions. 
     
     
         37 . The method of  claim 25 , wherein said non-mammalian expression system is a bacterial expression system or a yeast expression system. 
     
     
         38 . The method of  claim 37 , wherein said non-mammalian expression system is a bacterial expression system. 
     
     
         39 . The method of  claim 38 , wherein said bacterial expression system is an  E. coli  expression system. 
     
     
         40 . A method of refolding a protein expressed in an  E. coli  expression system, said method comprising:
 (a) contacting said protein with a refold buffer to form a refold mixture, wherein said protein is present at a concentration of 2 to 40 g/L in said refold mixture, and wherein said refold buffer comprises:
 (i) a redox component comprising a final thiol-pair ratio, wherein said final thiol-pair ratio has a range of 1 to 50, and wherein said thiol-pair ratio is calculated according to Equation 1: 
   
       
         
           
             
               
                 
                   
                     [ 
                     reductant 
                     ] 
                   
                   2 
                 
                 
                   [ 
                   oxidant 
                   ] 
                 
               
               ; 
             
           
         
         
           (ii) a thiol pair buffer strength between 2 mM and 20 mM, wherein said thiol-pair buffer strength is calculated according to Equation 2:
   2[oxidant]+[reductant]; and 
 
           (iii) at least one of:
 a denaturant; 
 an aggregation suppressor; and 
 a protein stabilizer; and 
 
         
         (b) incubating said refold mixture incubation under non-aerobic conditions. 
       
     
     
         41 . The method of  claim 40 , wherein said method further comprises:
 (c) isolating the refolded protein from step b).   
     
     
         42 . The method of  claim 40 , wherein said refold buffer comprises a denaturant, an aggregation suppressor, and a protein stabilizer. 
     
     
         43 . The method of  claim 40 , wherein said protein is present at a concentration of 10 to 20 g/L in said refold mixture. 
     
     
         44 . The method of  claim 40 , wherein said protein is an antibody. 
     
     
         45 . The method of  claim 40 , wherein said protein comprises an Fc domain. 
     
     
         46 . The method of  claim 40 , wherein said final thiol-pair ratio has a range of 1 to 25. 
     
     
         47 . The method of  claim 40 , wherein said thiol-pair buffer strength is between 5 mM and 20 mM. 
     
     
         48 . The method of  claim 47 , wherein said thiol-pair buffer strength is between 10 mM and 20 mM. 
     
     
         49 . The method of  claim 40 , wherein said protein in step a) is present in a non-native limited solubility form at the time said protein is contacted with said refold buffer. 
     
     
         50 . The method of  claim 49 , wherein said non-native limited solubility form is an inclusion body. 
     
     
         51 . The method of  claim 40 , wherein said denaturant is selected from the group consisting of urea, guanidinium salts, dimethyl urea, methylurea and ethylurea. 
     
     
         52 . The method of  claim 40 , wherein said aggregation suppressor is selected from the group consisting of arginine, proline, polyethylene glycols, non-ionic surfactants, ionic surfactants, polyhydric alcohols, glycerol, sucrose, sorbitol, glucose, Tris, sodium sulfate, potassium sulfate and osmolytes. 
     
     
         53 . The method of  claim 40 , wherein said protein stabilizer is selected from the group consisting of arginine, proline, polyethylene glycols, non-ionic surfactants, ionic surfactants, polyhydric alcohols, glycerol, sucrose, sorbitol, glucose, Tris, sodium sulfate, potassium sulfate and osmolytes. 
     
     
         54 . The method of  claim 40 , wherein said thiol-pairs comprise at least one thiol-pair selected from the group consisting of reduced glutathione, oxidized glutathione, cysteine, cystine, cysteamine, cystamine and beta-mercaptoethanol.