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 method for producing a host cell-protein (HCP) reduced antibody preparation from a sample mixture comprising an antibody and at least one HCP, said method comprising:
 (a) subjecting said sample matrix to a reduction in pH thus forming a primary recovery sample, wherein said reduction in pH is from about 3 to about about 4;   (b) adjusting said primary recovery sample to a pH of about 6.0 to about 8 followed by contacting said primary recovery sample to an affinity chromatography resin and collecting an affinity chromatography sample;   (c) contacting said affinity chromatography sample to an ion exchange resin and collecting an ion exchange sample;   (d) contacting said ion exchange sample to a hydrophobic interactive chromatography (HIC) resin and collecting an HIC sample, wherein said HIC sample comprises said HCP-reduced antibody preparation.   
     
     
         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, acetic acid, caprylic acid, and the like. 
     
     
         3 . The method of  claim 1 , wherein said affinity chromatography resin is a Protein A resin. 
     
     
         4 . The method of  claim 3 , wherein said Protein A resin is MabSelect™ resin. 
     
     
         5 . The method of  claim 1 , wherein said ion exchange resin is either an anion exchange resin or a cation exchange resin. 
     
     
         6 . The method of  claim 5 , wherein said ion exchange resin is a cation exchange resin. 
     
     
         7 . The method of  claim 6 , wherein said cation exchange resin is selected from the group consisting of Fractogel, carboxymethyl (CM), sulfoethyl(SE), sulfopropyl(SP), phosphate(P) and sulfonate(S). 
     
     
         8 . The method of  claim 7 , wherein said cation exchange resin is Fractogel™ SO 3   − . 
     
     
         9 . The method of  claim 5 , wherein said ion exchange resin is an anion exchange resin. 
     
     
         10 . The method of  claim 9 , wherein said anion exchange resin is selected from the group consisting of Q sepharose, diethylaminoethyl (DEAE), quaternary aminoethyl(QAE), and quaternary amine(Q) groups. 
     
     
         11 . The method of  claim 11 , wherein said anion exchange resin is Q-sepharose. 
     
     
         12 . The method of  claim 1 , wherein said ion exchange step comprises a first ion exchange step and a second ion exchange step. 
     
     
         13 . The method of  claim 1 , wherein said HIC is accomplished using a column comprising one or more hydrophobic groups. 
     
     
         14 . The method of  claim 13 , wherein said one or more hydrophobic groups are selected from the group consisting of alkyl-, aryl-groups, and a combination thereof. 
     
     
         15 . The method of  claim 14 , wherein said column is selected from the group consisting of phenyl sepharose (such as Phenyl Sepharose™ 6 Fast Flow column, Phenyl Sepharose™ High Performance column), Octyl Sepharose™ High Performance column, Fractogel™ EMD Propyl, Fractogel™ EMD Phenyl columns, Macro-Prep™ Methyl, Macro-Prep™ t-Butyl Supports, WP HI-Propyl (C 3 )™ column, and Toyopearl™ ether, phenyl or butyl columns. 
     
     
         16 . The method of  claim 15 , wherein said column comprises phenyl sepharose. 
     
     
         17 . The method of  claim 1 , further comprising a filtration step, wherein said HIC sample is subjected to filtration to remove viral particles and to facilitate buffer exchange. 
     
     
         18 . The method of  claim 1 , wherein said HCP-reduced antibody preparation comprises either an anti-IL-12 antibody or an antigen-binding portion thereof, or an anti-TNFα antibody or an antigen-binding portion thereof. 
     
     
         20 . The method of  claim 18 , wherein said HCP-reduced antibody preparation is an anti-TNFα antibody or an antigen-binding portion thereof. 
     
     
         21 . The method of  claim 20 , wherein said anti-TNFα antibody or antigen-binding portion thereof is a humanized antibody, a chimeric antibody, or a multivalent antibody. 
     
     
         22 . The method of  claim 21 , wherein said anti-TNFα antibody or antigen-binding portion thereof is a humanized antibody. 
     
     
         23 . The method of  claim 1 , wherein said preparation is substantially free of HCPs. 
     
     
         24 . The method of  claim 1 , further comprising depth filtration. 
     
     
         25 . A pharmaceutical composition comprising an HCP-reduced antibody preparation produced by the method of  claim 1  and a pharmaceutically acceptable carrier. 
     
     
         26 . The pharmaceutical composition of  claim 27 , wherein said antibody is either an anti-IL-12 antibody or antigen-binding portion thereof, or an anti-TNFα antibody or antigen-binding portion thereof. 
     
     
         27 . The pharmaceutical composition of  claim 27 , wherein said composition is substantially free of HCPs.