Viral Inactivation During Purification of Antibodies

Abstract

Described herein are methods for isolating and purifying antibodies from a sample matrix. One aspect of the present disclosure is directed to viral reduction/inactivation of samples generated in the various steps of antibody purification. In a particular aspect, methods herein employ an acidification step followed by one or more chromatography steps. The chromatography steps can include one or more of the following chromatographic procedures: ion exchange chromatography, affinity chromatography, and hydrophobic interaction chromatography.

What is claimed is: 
     
         1 . A method for producing a host cell-protein (HCP) reduced antibody preparation from a sample mixture comprising an antibody, an HCP and a viral particle obtained from a fermentation harvest of Chinese Hamster Ovary (CHO) cells expressing the antibody, wherein said preparation comprises a decreased number of viral particles or decreased viral activity in comparison to the sample mixture, said method comprising:
 subjecting said sample mixture to protein A affinity chromatography and eluting the antibody bound to protein A at a pH of 3 to 4, wherein the pH of the sample mixture or the eluted antibody is reduced to pH 3 to 4 for 0.5 to 2 hours; and   further applying the processed sample mixture to an ion exchange chromatography step, a hydrophobic interaction chromatography step, or a combination thereof.   
     
     
         2 . The method of  claim 1 , wherein said reduction in pH is accomplished by admixing a suitable acid with said sample mixture, and wherein said suitable acid is selected from the group consisting of citric acid, phosphoric acid, acetic acid, and caprylic acid. 
     
     
         3 . The method of  claim 1 , wherein said ion exchange resin is either an anion exchange resin or a cation exchange resin. 
     
     
         4 . The method of  claim 3 , wherein said ion exchange resin is a cation exchange resin. 
     
     
         5 . The method of  claim 4 , wherein said cation exchange resin comprises a substituted matrix wherein the substituents are selected from the group consisting of carboxymethyl, sulfoethyl, sulfopropyl, SO 3   − , phosphate, and sulfonate. 
     
     
         6 . The method of  claim 5 , wherein said substituent is SO 3   − . 
     
     
         7 . The method of  claim 3 , wherein said ion exchange resin is an anion exchange resin. 
     
     
         8 . The method of  claim 7 , wherein said anion exchange resin comprises a substituted matrix wherein the substituents are selected from the group consisting of diethylaminoethyl, quaternary aminoethyl, and quaternary amine groups. 
     
     
         9 . The method of  claim 8 , wherein said substituent is a quaternary amine. 
     
     
         10 . The method of  claim 1 , wherein said ion exchange step comprises a first ion exchange step and a second ion exchange step. 
     
     
         11 . The method of  claim 10 , wherein said first ion exchange step is a cation exchange step followed by a second anion exchange step. 
     
     
         12 . The method of  claim 10 , further comprising an intermediate step, wherein said intermediate step is a filtration step occurring between said first and said second ion exchange step. 
     
     
         13 . The method of  claim 12 , wherein said filtration step is accomplished by capture ultrafiltration/diafiltration. 
     
     
         14 . The method of  claim 1 , wherein said HIC chromatography comprises a resin comprising a substituted matrix wherein the substituents consist of one or more hydrophobic groups. 
     
     
         15 . The method of  claim 14 , wherein said one or more hydrophobic groups are selected from the group consisting of alkyl-groups, aryl-groups, and a combination thereof. 
     
     
         16 . The method of  claim 14 , wherein said substituents are selected from the group consisting of phenyl, 3-octoxypropane-1,2-diol, ether, propyl, methyl, phenyl and butyl groups. 
     
     
         17 . The method of  claim 16 , wherein said resin comprises an agarose matrix comprising phenyl substituents. 
     
     
         18 . The method of  claim 1 , further comprising a filtration step to remove viral particles and to facilitate buffer exchange. 
     
     
         19 . The method of  claim 1 , wherein said preparation is substantially free of HCPs. 
     
     
         20 . The method of  claim 1 , further comprising determining the residual amount of protein A level produced according to said method. 
     
     
         21 . The method of  claim 1 , wherein said antibody is adalimumab. 
     
     
         22 . The method of  claim 21 , wherein said adalimumab is pharmaceutical grade.