Protein purification

Abstract

A method for purifying proteins by Protein A chromatography is described which comprises removing contaminants by washing the solid phase with various intermediate wash buffers.

1 . A method for purifying a protein, which comprises a C H   2 /C H   3  region, from a contaminated solution thereof by Protein A chromatography comprising: 
 (a) adsorbing the protein to Protein A immobilized on a solid phase;    (b) removing contaminants by washing the solid phase with a composition comprising detergent and salt; and    (c) recovering the protein from the solid phase.    
     
     
         2 . The method of  claim 1  wherein the solid phase comprises silica, glass, agarose, or polystyrene.  
     
     
         3 . The method of  claim 2  wherein the solid phase comprises silica or glass.  
     
     
         4 . The method of  claim 1  wherein the protein is an antibody or an immunoadhesin.  
     
     
         5 . The method of  claim 1  wherein the protein is an antibody.  
     
     
         6 . The method of  claim 5  wherein the antibody binds an antigen selected from the group consisting of HER2, vascular endothelial growth factor (VEGF), IgE; CD20, CD40, CD11a, tissue factor (TF), prostate stem cell antigen (PSCA), interleukin-8 (IL-8), epidermal growth factor receptor (EGFR), HER3, HER4, α4β7 or α5β3.  
     
     
         7 . The method of  claim 5  wherein the antibody is an anti-HER2 antibody.  
     
     
         8 . The method of  claim 5  wherein the antibody is an anti-IgE antibody.  
     
     
         9 . The method of  claim 1  wherein the detergent is polysorbate.  
     
     
         10 . The method of  claim 9  wherein the concentration of the polysorbate in the composition is from about 0.1 to about 1%.  
     
     
         11 . The method of  claim 1  wherein the salt is acetate or citrate.  
     
     
         12 . The method of  claim 1  wherein the composition is at about pH 4.5 to about 5.5.  
     
     
         13 . The method of  claim 1  wherein the concentration of the salt in the composition is from about 0.2 to about 0.6M.  
     
     
         14 . A method for purifying a protein, which comprises a C H   2 /C H   3  region, from a contaminated solution thereof by Protein A chromatography comprising: 
 (a) adsorbing the protein to Protein A immobilized on a solid phase;    (b) removing contaminants by washing the solid phase with a composition comprising a buffer at a concentration of greater than about 0.8M; and    (c) recovering the protein from the solid phase.    
     
     
         15 . The method of  claim 14  wherein the buffer is Tris acetate.  
     
     
         16 . The method of  claim 15  wherein the concentration of the Tris acetate buffer is from about 0.8 to about 1.5M.  
     
     
         17 . A method for purifying a protein, which comprises a C H   2 /C H   3  region, from a contaminated solution thereof by Protein A chromatography comprising: 
 (a) adsorbing the protein to Protein A immobilized on a solid phase;    (b) removing contaminants by washing the solid phase with a composition comprising salt and solvent; and    (c) recovering the protein from the solid phase.    
     
     
         18 . The method of  claim 17  wherein the solvent is hexylene glycol.  
     
     
         19 . A method for purifying a protein, which comprises a C H   2 /C H   3  region, from a contaminated solution thereof by Protein A chromatography comprising: 
 (a) adsorbing the protein to Protein A immobilized on a solid phase;    (b) removing contaminants by washing the solid phase with a composition comprising salt and a polymer; and    (c) recovering the protein from the solid phase.    
     
     
         20 . The method of  claim 19  wherein the polymer is polyethylene glycol.