Removal of leaked affinity purification ligand

Abstract

The invention provides for the removal of a large fraction of contaminants from protein preparations while maintaining a high level of recovery using tentacle anion exchange matrix chromatography medium. Using the methods of the invention, leached affinity chromatography contaminants can be removed from recombinant protein preparations.

What is claimed is: 
     
         1 . A method for purifying a recombinant protein from a sample containing the recombinant protein and a second protein that binds to the protein, comprising subjecting the sample to a tentacle anion exchange matrix chromatography medium under conditions whereby the recombinant protein binds to the tentacle anion exchange matrix chromatography medium, followed by eluting the recombinant protein bound to the chromatography medium in an eluant, whereby at least 85% of the recombinant protein is recovered in the eluant and at least 75% of the second protein is removed from the eluant. 
     
     
         2 . The method of  claim 1 , wherein the tentacle anion exchange matrix chromatography medium contains a strong anion functional group. 
     
     
         3 . The method of  claim 2 , wherein the strong anion functional group is trimethyl-ammoniumethyl (TMAE). 
     
     
         4 . The method of any one of  claims 1  to  3 , wherein the resin substrate of the tentacle anion exchange matrix chromatography medium is a methacrylate polymeric resin or a polyvinylstyrene polymeric resin. 
     
     
         5 . The method of any one of  claims 1  to  4 , wherein the chromatography medium is Fractogel® EMD TMAE HiCap. 
     
     
         6 . The method of any one of  claims 1  to  5 , wherein the recombinant protein contains a C H 2/C H 3 region of an antibody. 
     
     
         7 . The method of  claim 6 , wherein the second protein is Protein A or Protein G. 
     
     
         8 . The method of any one of  claims 1  to  7 , wherein the sample is obtained from affinity purification of the protein over a Protein A chromatography medium. 
     
     
         9 . The method of any one of  claims 6  to  8 , wherein the recombinant protein is an antibody or an Fc fusion protein. 
     
     
         10 . The method of  claim 9 , wherein the recombinant protein is a tumor necrosis factor receptor Fc fusion protein. 
     
     
         11 . The method of  claim 10 , where the protein is etanercept. 
     
     
         12 . The method of  claim 11 , wherein the sample is subjected to the tentacle anion exchange matrix chromatography medium at about pH 8. 
     
     
         13 . The method of any one of  claims 1  to  12 , wherein after the recombinant protein is bound to the tentacle anion exchange matrix chromatography medium and before the recombinant protein is eluted, the tentacle anion exchange matrix chromatography medium is subjected to a wash step. 
     
     
         14 . The method of  claim 13 , wherein the wash step comprises washing the tentacle anion exchange matrix chromatography medium with a buffered solution at about pH 8. 
     
     
         15 . The method of  claim 14 , wherein the buffered solution consists essentially of 25 mM Tris(hydroxymethyl)aminomethane (Tris) at about pH 8. 
     
     
         16 . The method of any one of  claims 1  to  15 , wherein the recombinant protein is eluted from the tentacle anion exchange matrix chromatography medium in an elution buffer at a pH of from about 7.2 to about 7.5. 
     
     
         17 . The method of  claim 15 , wherein the elution buffer is 25 mM Tris HCl, pH 7.2. 
     
     
         18 . The method of  claim 15 , wherein the elution buffer is 25 mM Tris HCl, pH 7.5 and NaCl from about 150 mM to about 200 mM. 
     
     
         19 . The method of any one of  claims 1  to  18 , wherein the recombinant protein is subjected to a further purification step before or after the tentacle anion exchange matrix chromatography. 
     
     
         20 . The method of any one of  claims 1  to  19 , wherein the recombinant protein is formulated in a pharmaceutical composition.