Cation exchange chromatography (methods)

Abstract

The present invention provides improved methods of protein purification using CEX chromatography. Such methods generally comprise the steps of: contacting a protein of interest (e.g., an antibody) with a cation exchange resin at a first pH, that is less than the pI of the most acidic isoform of the protein of interest, such that the protein of interest binds to the resin; washing the cation exchange resin at a second pH that is greater than the first pH, but less than the pI of the most acidic isoform of the protein of interest; and eluting the protein of interest from the resin at a third pH that is about equal to or less than the first pH. The methods of the invention are particularly useful for the commercial purification of recombinant therapeutic proteins (e.g., antibodies).

1 . A method of purifying a protein of interest from a mixture comprising the protein of interest and one or more contaminants, comprising:
 (a) determining the pI of the most acidic isoform of the protein of interest;   (b) contacting the protein of interest with a cation exchange resin at a first pH that is less than the pI of the most acidic isoform of the protein of interest, such that the protein of interest binds to the resin;   (c) washing the cation exchange resin at a second pH that is greater than the first pH, but less than the pI of the most acidic isoform of the protein of interest; and   (d) eluting the protein of interest from the resin at a third pH that is about equal to or less than the first pH, thereby purifying the protein of interest.   
     
     
         2 . The method of  claim 1 , wherein the first pH is about 6.2. 
     
     
         3 . The method of  claim 1 , wherein the second pH is about 0.1 to about 1.2 pH units less than the pI of the most acidic isoform of the protein of interest. 
     
     
         4 . The method of  claim 1 , wherein the third pH is about 4.5. 
     
     
         5 . The method of  claim 4 , wherein the mixture comprises clarified bulk. 
     
     
         6 . The method of  claim 4 , wherein the clarified bulk comprises a cell culture supernatant. 
     
     
         7 . The method of  claim 6 , wherein the supernatant is from a mammalian, bacterial or fungal cell culture. 
     
     
         8 . The method of  claim 6 , wherein the supernatant is from a Chinese Hamster Ovary (CHO) cell culture. 
     
     
         9 . The method of  claim 8 , wherein the contaminant comprises fragments or aggregates of the protein of interest. 
     
     
         10 . The method of  claim 9 , wherein the protein of interest is an antibody. 
     
     
         11 . The method of  claim 10 , wherein the antibody is a monoclonal antibody. 
     
     
         12 . The method of  claim 11 , wherein the monoclonal antibody is selected from the group consisting of a human, humanized and chimeric antibody.