Protein purification using displacement chromatography

Abstract

Disclosed herein are compositions and methods for the isolation and purification of proteins from a sample. In particular, the present invention relates to compositions and methods for isolating and purifying proteins incorporating a displacement chromatographic step. The present invention is also directed toward pharmaceutical compositions comprising one or more antibodies purified by a method described herein.

We claim: 
     
         1 . A method for reducing process-related impurities and/or product-related substances in a preparative scale sample of a protein of interest comprising at least one process-related impurities and/or product-related substance, the method comprising:
 (a) contacting the sample to a chromatography media under conditions wherein the protein of interest binds to the chromatography media;   (b) displacing the protein of interest bound to the chromatography media with a displacer buffer comprising at least one displacer molecule; and   (c) collecting a chromatography sample,   
       wherein the chromatography sample comprises a reduced amount of process-related impurities and/or product-related substances. 
     
     
         2 . The method of  claim 1 , wherein the chromatography media is an ion exchange adsorbent material. 
     
     
         3 . The method of  claim 1 , wherein the chromatography media is a multimodal adsorbent material comprising cation exchange and hydrophobic interaction functional groups. 
     
     
         4 . The method of  claim 1 , wherein the pH of the displacing wash buffer is lower than the isoelectric point of the protein of interest. 
     
     
         5 . The method of  claim 1 , wherein the displacer in the wash buffer carries positive charge and wherein the concentration of the displacer in the wash buffer is greater than 0.1 mM. 
     
     
         6 . The method of  claim 2 , wherein the cation exchange (CEX) adsorbent material is selected from the group consisting of a CEX resin and a CEX membrane adsorber. 
     
     
         7 . The method of  claim 4 , wherein the pH of the displacing wash buffer is in the range of about 6.0 to about 8.0. 
     
     
         8 . The method of  claim 1 , wherein the conductivity of the wash buffer is in the range of about 2 to about 20 mS/cm. 
     
     
         9 . The method of  claim 1 , wherein the column length is in the range of about 10 cm to about 30 cm and the flow residence time is in the range of about 5 min to about 25 min. 
     
     
         10 . The method of  claim 5 , wherein the cationic displacer in the wash buffer is a quaternary ammonium salt and the concentration of the displacer in the wash buffer is in the range of 0.1 to 10 mM. 
     
     
         11 . The method of  claim 5 , wherein the cationic displacer in the wash buffer is protamine sulfate and the concentration of the protamine sulfate in the wash buffer is in the range of 0.1 to 5 mM. 
     
     
         12 . The method of  claim 1 , wherein two or more displacing buffers consisting of different displacer concentrations are used. 
     
     
         13 . The method of  claim 12 , wherein the first displacing buffer containing lower displacer concentration than the second or later displacing buffer. 
     
     
         14 . The method of  claim 1 , wherein the displacement operation is run in two-step displacement chromatography mode. 
     
     
         15 . The method of  claim 1 , wherein the displacement operation is run in multiple-step or linear displacement chromatography mode. 
     
     
         16 . The method of  claim 14 , wherein the first displacing buffer containing 0.5 mM Expell SP1™. 
     
     
         17 . The method of  claim 14 , wherein the first displacing buffer containing 0.25 mM protamine sulfate. 
     
     
         18 . A composition comprising a protein of interest and a reduced level of protein aggregates. 
     
     
         19 . A composition comprising a protein of interest and a reduced level of protein fragments. 
     
     
         20 . A composition comprising a chromatographic sample of  claim 1 , wherein the composition comprises a reduced level of host cell proteins. 
     
     
         21 . A composition comprising a chromatographic sample of  claim 1 , wherein the composition comprises levels of basic variants that differ from the starting load material. 
     
     
         22 . The method of  claim 1 , wherein the protein of interest is an anti-TNFα antibody.