Isoform separation of proteins by mixed-mode chromatography

摘要

Mixed-mode chromatography uses a multimodal functional resin, mainly composed of electrostatic and aromatic/hydrophobic groups. Here we have tested 2 mixed-mode resins, anion-exchange Capto adhere and cation-exchange Capto MMC, using 2 model proteins, i.e., an Fc-fusion etanercept, and bovine serum albumin (BSA). When etanercept was produced in Chinese hamster ovary cells, a large amount of mis-folded species was generated. A novel technology to achieve effective separation of the misfolded or aggregated species has been developed in this study using these mixed-mode columns and elution conditions that combine pH change and NaCl or arginine at different concentrations. Etanercept, which has been purified by Protein-A chromatography, was bound to the Capto MMC or Capto adhere columns under various conditions and eluted by modulating the pH and salt or arginine concentration. The misfolded species occurred in the fractions at higher salt or arginine concentrations, most likely reflecting stronger electrostatic and hydrophobic interactions of the misfolded species with these mixed-mode resins.