Isolation and Purification of Antibodies Using Protein A Affinity Chromatography

Abstract

Disclosed herein are methods for the isolation and purification of antibodies wherein the use of an affinity chromatographic step results in an antibody composition sufficiently pure for pharmaceutical uses. The methods described herein comprise pH viral reduction/inactivation, ultrafiltration/diafiltration, affinity chromatography, preferably Protein A affinity, ion exchange chromatography, and hydrophobic chromatography. Further, the present invention is directed toward pharmaceutical compositions comprising one or more antibodies of the present invention.

What is claimed is: 
     
         1 . A pharmaceutical grade preparation comprising adalimumab, wherein the adalimumab is purified from a fermentation harvest of cultured cells expressing adalimumab by a method comprising:
 a) binding said expressed adalimumab to a Protein A resin, and   b) eluting the bound adalimumab at an elution pH of 3.2 and holding eluate under acid condition for 1 to 3 hours, wherein adalimumab monomer level after said holding is greater than 96.1%, and wherein the pharmaceutical grade preparation retains a residual amount of Protein A.   
     
     
         2 . The pharmaceutical grade preparation of  claim 1 , further comprising subjecting adalimumab to an ion exchange chromatography after said holding. 
     
     
         3 . The pharmaceutical grade preparation of  claim 2 , wherein said ion exchange chromatography is cation exchange chromatography. 
     
     
         4 . The pharmaceutical grade preparation of  claim 1 , further comprising subjecting adalimumab to a hydrophobic interaction chromatography after said holding. 
     
     
         5 . The pharmaceutical grade preparation of  claim 1 , further comprising after said holding, subjecting adalimumab to one or more chromatographic separations, wherein said one or more further chromatographic separations comprise an ion exchange chromatography, a hydrophobic interactive chromatography or a combination thereof. 
     
     
         6 . The pharmaceutical grade preparation of  claim 1 , further comprising determining the monomer level of adalimumab purified according to said method. 
     
     
         7 . The pharmaceutical grade preparation of  claim 1 , further comprising determining the protein A level in the pharmaceutical grade preparation. 
     
     
         8 . The pharmaceutical grade preparation of  claim 1 , wherein said cultured cells are Chinese Hamster Ovary (CHO) cells. 
     
     
         9 . A pharmaceutical grade preparation comprising adalimumab, wherein the adalimumab is purified from a fermentation harvest of cultured cells expressing adalimumab by a method comprising:
 a) binding said expressed adalimumab to a Protein A resin, and   b) eluting the bound adalimumab at an elution pH of 2.5-3.2 and holding eluate under acid condition for 1 to 3 hours, wherein adalimumab monomer level after said holding is greater than 92.3%, and wherein the pharmaceutical grade preparation retains a residual amount of Protein A.   
     
     
         10 . The pharmaceutical grade preparation of  claim 9 , further comprising subjecting adalimumab to an ion exchange chromatography after said holding. 
     
     
         11 . The pharmaceutical grade preparation of  claim 10 , wherein said ion exchange chromatography is cation exchange chromatography. 
     
     
         12 . The pharmaceutical grade preparation of  claim 9 , further comprising subjecting adalimumab to a hydrophobic interaction chromatography after said holding. 
     
     
         13 . The pharmaceutical grade preparation of  claim 9 , further comprising after said holding, subjecting adalimumab to one or more chromatographic separations, wherein said one or more further chromatographic separations comprise an ion exchange chromatography, a hydrophobic interactive chromatography or a combination thereof. 
     
     
         14 . The pharmaceutical grade preparation of  claim 9 , further comprising determining the monomer level of adalimumab purified according to said method. 
     
     
         15 . The pharmaceutical grade preparation of  claim 9 , further comprising determining the protein A level in the pharmaceutical grade preparation. 
     
     
         16 . The pharmaceutical grade preparation of  claim 9 , wherein said cultured cells are CHO cells.