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J Chromatogr B Analyt Technol Biomed Life Sci. 2013 Oct 1;936:33-41. doi: 10.1016/j.jchromb.2013.07.029. Epub 2013 Aug 7.

Evaluation of mixed-mode chromatographic resins for separating IgG from serum albumin containing feedstock.

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Key Laboratory of Biomass Chemical Engineering of Ministry of Education, Department of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China.


Mixed-mode chromatography has been focused as a cost-effective new technique for antibody purification. In this study, four 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 tested and the multi-functional interactions between ligand and protein were discussed. 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 had 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.


BSA; CIP; CV; Gradient elution; HCIC; IgG; Immunoglobulin G; Mixed-mode chromatography; Purification; SEC-HPLC; Serum albumin; Step elution; bovine serum albumin; clean-in-place; column volume; hydrophobic charge induction chromatography; immunoglobulin G; mAbs; monoclonal antibodies; size exclusion chromatography–high performance liquid chromatography

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