Selectivity evaluation and separation of human immunoglobulin G, Fab and Fc fragments with mixed-mode resins

•Adsorption selectivity of hIgG and fragments with mixed-mode resins was studied.•Four mixed-mode resins with different ligands were evaluated.•Adsorption selectivity could be improved by the control of pH.•Isothermal titration calorimetry was used to evaluate the molecular interactions.•Operation conditions were optimized for the separation of hIgG and Fc.

Adsorption selectivity is critical important for mixed-mode chromatography with specially-designed ligands. Human immunoglobulin G (hIgG), Fc and Fab fragments were used in the present work to evaluate adsorption behavior and binding selectivity of four mixed-mode resins with the ligands of 4-mercatoethyl-pyridine (MEP), 2-mercapto-1-methylimidazole (MMI), 5-aminobenzimidazole (ABI) and tryptophan-5-aminobenzimidazole (W-ABI), respectively. The resins showed an obvious pH-dependent adsorption behavior. High adsorption capacities were found at neutral pH for hIgG, Fc and Fab, and almost no adsorption happened under acidic conditions. An adsorption selectivity index was proposed to evaluate separation efficiency. High specificity of hIgG/Fc was found at pH 8.9 for MEP resin, and for W-ABI resin at pH 8.0 and 8.9. In addition, isothermal titration calorimetry was used to evaluate ligand-protein interactions. Finally, the separation of hIgG and Fc (1:1) was optimized with mixed-mode resins, and the best separation performance was obtained with W-ABI-based resin. Loading at pH 8.0 resulted in the flow through of Fc with purity of 90.4% and recovery of 98.8%, while elution at pH 3.6 provided hIgG with purity of 99.7% and recovery of 86.5%.