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.

1 . (canceled) 
     
     
         2 . A method for making a pharmaceutical grade preparation comprising adalimumab, wherein the adalimumab is purified from a fermentation harvest of cultured Chinese hamster ovary (CHO) cells expressing adalimumab, and the method comprises:
 a) binding said expressed adalimumab to a Protein A resin at a loading amount of no less than 32 g/L, and   b) eluting the bound adalimumab at an elution pH of 3.2-3.6 and holding the resulting eluate under acid condition for at least 1 hour, wherein adalimumab monomer level after said holding and prior to any further purification steps is at least 96.1%.   
     
     
         3 . The method of  claim 2 , wherein said further purification steps comprise an ion exchange chromatography. 
     
     
         4 . The method of  claim 3 , wherein said ion exchange chromatography is cation exchange chromatography. 
     
     
         5 . The method of  claim 2 , wherein said further purification steps comprise a hydrophobic interaction chromatography. 
     
     
         6 . The method of  claim 2 , wherein said further purification steps comprise an ion exchange chromatography, a hydrophobic interactive chromatography or a combination thereof. 
     
     
         7 . The method of  claim 2 , further comprising determining the monomer level of adalimumab purified according to said method. 
     
     
         8 . The method of  claim 2 , further comprising determining the protein A level in the pharmaceutical grade preparation. 
     
     
         9 . The method of  claim 2 , wherein said elution pH is 3.2. 
     
     
         10 . A method for making a pharmaceutical grade preparation comprising adalimumab, wherein said adalimumab is purified from a fermentation harvest of cultured CHO cells expressing adalimumab, and the method comprises:
 a) binding said expressed adalimumab to a Protein A resin, wherein said expressed adalimumab is loaded onto said Protein A resin at a load amount that is 10 to 20% less than the loading amount that results in 10% breakthrough, and   b) eluting the bound adalimumab at an elution pH of 3.2-3.6 and holding the resulting eluate under acid condition for at least 1 hour, wherein adalimumab monomer level after said holding and prior to any further purification steps is at least 96.1%.   
     
     
         11 . The method of  claim 10 , wherein said further purification steps comprise an ion exchange chromatography. 
     
     
         12 . The method of  claim 11 , wherein said ion exchange chromatography is cation exchange chromatography. 
     
     
         13 . The method of  claim 10 , wherein said further purification steps comprise a hydrophobic interaction chromatography. 
     
     
         14 . The method of  claim 10 , wherein said further purification steps comprise an ion exchange chromatography, a hydrophobic interactive chromatography or a combination thereof. 
     
     
         15 . The method of  claim 10 , further comprising determining the monomer level of adalimumab purified according to said method. 
     
     
         16 . The method of  claim 10 , further comprising determining the protein A level in the pharmaceutical grade preparation. 
     
     
         17 . The method of  claim 10 , wherein said elution pH is 3.2. 
     
     
         18 . The method of  claim 2 , wherein the yield of adalimumab is at least 90%. 
     
     
         19 . The method of  claim 10 , wherein the yield of adalimumab is at least 90%.