title: Selectivity evaluation and separation of human immunoglobulin G, Fab and Fc fragments with mixed-mode resins.
abstract: 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%.
authors.affiliation: Key Laboratory of Biomass Chemical Engineering of Ministry of Education, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China.
authors.forename: Ying-Di
authors.initials: YD
authors.lastname: Luo
authors.affiliation: Key Laboratory of Biomass Chemical Engineering of Ministry of Education, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China.
authors.forename: Qi-Lei
authors.initials: QL
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Biomass Chemical Engineering of Ministry of Education, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China.
authors.forename: Xiao-Ming
authors.initials: XM
authors.lastname: Yuan
authors.affiliation: Key Laboratory of Biomass Chemical Engineering of Ministry of Education, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Shi
authors.affiliation: Key Laboratory of Biomass Chemical Engineering of Ministry of Education, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China.
authors.forename: Shan-Jing
authors.initials: SJ
authors.lastname: Yao
authors.affiliation: Key Laboratory of Biomass Chemical Engineering of Ministry of Education, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China. Electronic address: lindq@zju.edu.cn.
authors.forename: Dong-Qiang
authors.initials: DQ
authors.lastname: Lin
mesh_terms: D000327:Adsorption; D001562:Benzimidazoles / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D006801:Humans; D007093:Imidazoles / Q000737:chemistry; D007140:Immunoglobulin Fab Fragments / Q000302:isolation & purification*; D007141:Immunoglobulin Fc Fragments / Q000302:isolation & purification*; D007074:Immunoglobulin G / Q000302:isolation & purification*; D008024:Ligands; D011725:Pyridines / Q000737:chemistry; D012117:Resins, Synthetic / Q000737:chemistry; D014364:Tryptophan / Q000031:analogs & derivatives
chemical_list: C107674:2-mercapto-1-methylimidazole; D001562:Benzimidazoles; D007093:Imidazoles; D007140:Immunoglobulin Fab Fragments; D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D008024:Ligands; D011725:Pyridines; D012117:Resins, Synthetic; D014364:Tryptophan; C059092:5-aminobenzimidazole; C027391:2-aminobenzimidazole; C023666:pyridine
keywords: Adsorption selectivity; Fab fragment; Fc fragment; Human immunoglobulin G; Mixed-mode chromatography; Separation