Wash solution and method for affinity chromatography

Abstract

The invention provides a washing method for affinity chromatography in which a wash solution comprising arginine, or an arginine derivative, and a nonbuffering salt, preferably at high pH, greater than 8.0, is effective in removing impurities, such as high molecular weight species and host cell proteins, while also increasing product concentration in the eluate and maintaining a high percent yield of recovered product.

What is claimed is: 
     
         1 . A method of producing a purified protein of interest using an affinity chromatography (AC) matrix to which the protein of interest is bound, the method comprising:
 (a) loading a mixture comprising the protein of interest onto the AC matrix; and   (b) washing the AC matrix with one or more wash solutions, wherein at least one wash solution comprises (i) arginine, or an arginine derivative, and (ii) a nonbuffering salt, wherein the pH of the at least one wash solution is greater than 8.0; prior to elution of the protein of interest from the AC matrix.   
     
     
         2 . The method of  claim 1 , further comprising
 (c) eluting the protein of interest from the AC matrix.   
     
     
         3 . The method of  claim 1 , wherein the AC matrix is a Protein A column, a Protein G column, a Protein A/G column or a Protein L column. 
     
     
         4 . The method of  claim 1 , wherein the AC matrix is a Protein A column. 
     
     
         5 . The method of  claim 1 , wherein the at least one wash solution comprises arginine-HCl. 
     
     
         6 . The method of  claim 1 , wherein the nonbuffering salt is a halogen salt. 
     
     
         7 . The method of  claim 6 , wherein the nonbuffering salt is sodium chloride (NaCl). 
     
     
         8 . The method of  claim 1 , wherein the protein of interest is an antibody, antibody fragment, or Fc fusion protein. 
     
     
         9 . The method of  claim 1 , wherein the protein of interest is an antibody or antibody fragment that binds to the Protein A column. 
     
     
         10 . The method of  claim 1 , wherein the one or more wash solutions remove impurities from the AC matrix. 
     
     
         11 . A method of producing a purified antibody, or antibody fragment, using a Protein A column, the method comprising
 (a) loading a mixture comprising the antibody, or antibody fragment, onto the Protein A column;   (b) washing the Protein A column with a wash solution comprising (i) Arginine-HCl, at a concentration in a range of 0.05-0.85 M, and (ii) sodium chloride, at a concentration in a range of 0.1-2.0 M, wherein the pH of the wash solution is greater than 8.0, and wherein the wash solution removes impurities from the Protein A column; and   (c) eluting the antibody, or antibody fragment, from the Protein A column.   
     
     
         12 . The method of  claim 1 , wherein the arginine or arginine derivative is at a concentration in a range of 0.05-0.85 M. 
     
     
         13 . The method of  claim 12 , wherein the arginine or arginine derivative is at a concentration in a range of 0.1-0.5 M. 
     
     
         14 . The method of  claim 13 , wherein the arginine or arginine derivative is at a concentration of or about 0.25 M. 
     
     
         15 . The method of  claim 1 , wherein the nonbuffering salt is at a concentration in a range of 0.1-2.0 M. 
     
     
         16 . The method of  claim 15 , wherein the nonbuffering salt is at a concentration in a range of 0.5-1.5 M. 
     
     
         17 . The method of  claim 16 , wherein the nonbuffering salt is at a concentration of or about 1 M. 
     
     
         18 . The method of  claim 1 , wherein the pH of the at least one wash solution is at least 8.5. 
     
     
         19 . The method of  claim 1 , wherein the pH of the at least one wash solution is in a range of 8.1-9.5. 
     
     
         20 . The method of  claim 19 , wherein the pH of the at least one wash solution is in a range of about 8.5-9.5. 
     
     
         21 . The method of  claim 20 , wherein the pH of the at least one wash solution is 9.0. 
     
     
         22 . The method of  claim 10 , wherein the impurities comprise high molecular weight (HMW) species. 
     
     
         23 . The method of  claim 10 , wherein the impurities comprise host cell proteins (HCPs).