Isoform separation of proteins by mixed-mode chromatography

•Two mixed-mode resins, Capto MMC and Capto adhere, were used to separate misfolded etanercept and BSA oligomers.•Simultaneous gradient of pH and salt concentration on Capto MMC resulted in separation of native etanercept.•Combination gradient of salt and arginine on Capto adhere resulted in separation of native etanercept.•Salt elution on Capto MMC and Capto adhere resulted in separation of BSA monomer.•Arginine wash on Capto MMC and Capto adhere resulted in removal of BSA oligomers.

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 misfolded 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.Another model protein, BSA, containing several oligomeric species, was also subjected to Capto adhere or Capto MMC chromatography using either NaCl or arginine gradient elution, with a greater recovery by arginine gradient. The oligomers were effectively separated on these mixed-mode columns using either gradient elution, eluting in later fractions similar to etanercept misfolded species.