Novel purification of antibodies using hydrophobic interaction chromatography

Abstract

Disclosed herein are compositions and methods for purifying antibody products from a sample matrix. In particular, the present invention relates to compositions and methods for purifying antibody products employing hydrophobic interaction chromatography media. In certain embodiments, the invention provides a method for reducing process-related impurities (e.g., host cell proteins), as well as product-related substances, including molecular weight variants (e.g., aggregates and fragments of the antibody product).

What is claimed is: 
     
         1 . A method for producing a preparation comprising a process-related impurity-reduced and/or product-related substance-reduced composition comprising a protein of interest from a sample mixture comprising a protein of interest and at least one process-related impurity and/or product-related substance, said method comprising:
 (a) subjecting said sample mixture to a hydrophobic interaction media and collecting the unbound fraction;   (b) contacting the said hydrophobic interaction media after loading with a solution that is substantially similar in hydrophobic interaction functionality to the load sample and collecting the wash;   
       wherein the flow through and/or wash fractions constitute the process-related impurity-reduced and/or product-related substance-reduced composition comprising a protein of interest preparation 
     
     
         2 . The method of  claim 1 , wherein prior to subjecting said sample mixture to a hydrophobic interaction chromatography media the sample mixture is subjected to an affinity chromatographic media. 
     
     
         3 . The method of  claim 2 , wherein the affinity chromatographic media is a Protein A, G, A/G, or L media. 
     
     
         4 . The method of  claim 2 , wherein the affinity chromatographic media is MabSuRe Protein A media. 
     
     
         5 . The method of  claim 1 , wherein prior to subjecting said sample mixture to a hydrophobic interaction chromatography media the sample mixture is subjected to an ion exchange chromatography media. 
     
     
         6 . The method of any one of  claim 5 , wherein the ion exchange media is selected from a cation exchange media and an anion exchange media. 
     
     
         7 . The method of  claim 6 , wherein the ion exchange media is an anion exchange media selected from media comprising diethylaminoethyl (DEAE), quaternary aminoethyl (QAE) and quaternary amine (Q) group ligands. 
     
     
         8 . The method of  claim 6 , wherein the ion exchange media is a cation exchange media selected from media comprising carboxymethyl (CM), sulfoethyl(SE), sulfopropyl(SP), phosphate(P) and sulfonate(S) ligands. 
     
     
         9 . The method of  claim 1 , wherein prior to subjecting said sample mixture to a hydrophobic interaction chromatography media the sample mixture is subjected to a mixed mode chromatography media. 
     
     
         10 . The method of  claim 9 , wherein the mixed mode media is CaptoAdhere resin. 
     
     
         11 . The method of  claim 1  wherein said antibody is a human antibody, humanized antibody, a chimeric antibody, or a multivalent antibody, or an antigen-binding portion thereof. 
     
     
         12 . The method of  claim 1 , wherein said HIC media comprises at least one hydrophobic ligand. 
     
     
         13 . The method of  claim 12 , wherein said at least one hydrophobic ligand is selected from the group consisting of alkyl-, aryl-ligands, and combinations thereof. 
     
     
         14 . The method of  claim 12 , wherein at least one hydrophobic group is selected from the group consisting of butyl, hexyl, phenyl, octyl, or polypropylene glycol ligands. 
     
     
         15 . The method of  claim 12 , wherein said HIC comprises a column. 
     
     
         16 . The method of  claim 16 , wherein said column is selected from the group consisting of: Capto Phenyl; Phenyl Sepharose™ 6 Fast Flow with low or high substitution; Phenyl Sepharose™ High Performance; Octyl Sepharose™ High Performance; Fractogel™ EMD Propyl; Fractogel™ EMD Phenyl; Macro-Prep™ Methyl; Macro-Prep™ t-Butyl columns; WP HI-Propyl (C3)™; Toyopearl™ ether, phenyl or butyl; ToyoScreen PPG; ToyoScreen Phenyl; ToyoScreen Butyl; ToyoScreen Hexyl; GE HiScreen Butyl FF; and HiScreen Octyl FF. 
     
     
         17 . The method of  claim 1 , wherein said hydrophobic interaction chromatography sample is subjected to a filtration step. 
     
     
         18 . The method of  claim 17 , further comprising a depth filtration step, nanofiltration step, ultrafiltration step or absolute filtration step or combination thereof. 
     
     
         19 . A pharmaceutical composition comprising a process-related impurity-reduced and/or product-related substance-reduced composition comprising a protein of interest and a pharmaceutically acceptable carrier 
     
     
         20 . The pharmaceutical composition of  claim 19 , wherein said composition is substantially free of product-related aggregates, product-related fragments, and HCPs.