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Mixed-mode chromatography has been focused as a cost-effective new technique for antibody purification.Mixed-mode ligands possess a combination of multiple binding modes like hydrophobic,ionic and thiophilic interactions,which results in a variety of protein-ligand interactions,and the operation conditions often need to be optimized for the efficient separation of protein.Therefore,better understanding of the adsorption and desorption of IgG and impurities during the mixed-mode chromatography process is necessary,which would certainly enhance the separation efficiency and improve the process design of antibody purification.In this study,four typical mixed-mode resins with N-benzyl-N-methyl ethanol amine,2-benzamido-4-mercaptobutanoic acide,4-mercapto-ethyl-pyridine and phenylpropylamine as the ligands were investigated to characterize multi-functional interactions between ligand and protein.Immunoglobulin G (IgG),bovine serum albumin (BSA) and the binary mixture of BSA and IgG were used as the model feedstock to compare the separation behaviors by pH gradient elution.The comparison analysis showed mixed-mode resin with N-Benzyl-N-methyl ethanol amine as the ligand exhibited the best ability to separate IgG and BSA.The results indicated that for four resins tested ionic interaction might play the dominant role in the separation of IgG and BSA while the hydrophobic interactions and hydrogen bonding have some subsidiary effects.The pH stepwise elution and sample loading were optimized to improve the IgG purification from serum albumin containing feedstock.High purity (92.3 %) and high recovery (95.6 %) of IgG were obtained.The results indicated that mixed-mode chromatography would be a potential option for antibody purification with the control of loading and elution conditions.