Protein purification

Abstract

The present invention relates to a process for the purification of an antibody fragment from a periplasmic cell extract comprising a first cation exchange chromatography step and a second anion exchange chromatography step.

We claim: 
     
         1 . A process for the purification of an antibody fragment from periplasmic cell extract comprising:
 a) a first chromatography step to capture the antibody fragment wherein a mixture containing an antibody fragment at a concentration of at least 1.5 g/L is subjected to cation exchange chromatography and subsequently eluted to produce a first eluate containing the antibody fragment; and   b) a second chromatography step wherein the first eluate is subjected to anion exchange chromatography to capture impurities and produce a flow through containing the antibody fragment.   
     
     
         2 . The process according to  claim 1 , wherein the process comprises not more than two chromatography steps. 
     
     
         3 . The process according to  claim 1 , wherein all chromatography steps are performed on a chromatography column. 
     
     
         4 . The process according to  claim 1 , wherein the cation exchange chromatography is performed in elution mode. 
     
     
         5 . The process according to  claim 1 , wherein the first cation chromatography step comprises the following steps in sequential order:
 a) loading a mixture containing an antibody fragment onto the cation exchange column,   b) washing the cation exchange column with a wash buffer wherein during the washing the conductivity, pH and salt concentration of the buffer remains essentially unchanged, and   c) eluting the antibody fragment with an elution buffer.   
     
     
         6 . The process according to  claim 5 , wherein the pH of the washing buffer is identical to the pH of the mixture containing an antibody fragment prior to first chromatography step. 
     
     
         7 . The process according to  claim 5 , wherein the mixture containing an antibody fragment, prior to the first chromatography step has a pH of between 4.0 to 5.0. 
     
     
         8 . The process according to  claim 1 , wherein the cation exchange chromatography in the first chromatography step is performed at a flow rate of at least 300 cm/h. 
     
     
         9 . The process according to  claim 1 , wherein the cation exchange chromatography in the first chromatography step is performed at a conductivity of not more 6 mS/cm. 
     
     
         10 . The process according to  claim 1 , wherein the cation exchange chromatography in the first chromatography step is performed in a chromatography column comprising sulphonyl, sulphopropyl or carboxymethyl coupled to a resin. 
     
     
         11 . The process according to  claim 1 , wherein the cation exchange chromatography column in the first chromatography step is has a dynamic binding capacity for the antibody fragment of between 50 and 75 g/L resin. 
     
     
         12 . The process according to  claim 11 , wherein the cation exchange chromatography column resin in the first chromatography step has a mean particular size of at least 50 μm. 
     
     
         13 . The process according to  claim 1 , wherein in the cation exchange chromatography of the primary capture step between 5 and 100 g antibody fragment per liter resin are loaded. 
     
     
         14 . The process according to  claim 1 , wherein the anion exchange chromatography in the second chromatography step is performed on a resin comprising quaternary ammonium (Q), diethylaminoethyl (DEAE) or trimethylaminoethyl (TMAE). 
     
     
         15 . The process according to  claim 1 , wherein the antibody fragment is a Fab, Fab′ or scFv. 
     
     
         16 . The process according to  claim 15 , wherein the Fab or Fab′ binds specifically to VEGF-A, FcRn, OX40, glycoprotein IIb/IIIa receptor, C5, HER2/neu, TNFα, IL1β or CD40-L. 
     
     
         17 . The process according to  claim 16 , wherein the Fab or Fab′ is abciximab, ranibizumab, pexelizumab, CDP870, CDP484 or CDP7657. 
     
     
         18 . The process according to  claim 1 , wherein the antibody fragment recovered from the process contains host cell protein in an amount of not more than 150 parts per million. 
     
     
         19 . A process for the purification of an antibody fragment from periplasmic cell extract consisting essentially of:
 a) a first chromatography step to capture the antibody fragment wherein a mixture containing an antibody fragment at a concentration of at least 1.5 g/L is subjected to cation exchange chromatography and subsequently eluted to produce a first eluate containing the antibody fragment;   b) a first ultrafiltration applied to the first eluate;   c) a second chromatography step wherein the purified first eluate is subjected to anion exchange chromatography to capture impurities and produce a flow through containing the antibody fragment; and   d) a second ultrafiltration applied to the flow through.