Luo Ying-Di, Zhang Qi-Lei, Yuan Xiao-Ming, Shi Wei, Yao Shan-Jing, Lin Dong-Qiang
Key Laboratory of Biomass Chemical Engineering of Ministry of Education, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China.
Key Laboratory of Biomass Chemical Engineering of Ministry of Education, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China.
J Chromatogr B Analyt Technol Biomed Life Sci. 2017 Jan 1;1040:105-111. doi: 10.1016/j.jchromb.2016.11.036. Epub 2016 Nov 27.
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%.
对于使用特殊设计配体的混合模式色谱而言，吸附选择性至关重要。在本研究中，分别使用人免疫球蛋白G（hIgG）、Fc和Fab片段来评估四种混合模式树脂（其配体分别为4-巯基乙基吡啶（MEP）、2-巯基-1-甲基咪唑（MMI）、5-氨基苯并咪唑（ABI）和色氨酸-5-氨基苯并咪唑（W-ABI））的吸附行为和结合选择性。这些树脂表现出明显的pH依赖性吸附行为。在中性pH条件下，hIgG、Fc和Fab具有较高的吸附容量，而在酸性条件下几乎不发生吸附。提出了一个吸附选择性指数来评估分离效率。在pH 8.9时，MEP树脂对hIgG/Fc具有高特异性，在pH 8.0和8.9时，W-ABI树脂对hIgG/Fc具有高特异性。此外，使用等温滴定量热法评估配体与蛋白质的相互作用。最后，用混合模式树脂对hIgG和Fc（1:1）的分离进行了优化，基于W-ABI的树脂获得了最佳分离性能。在pH 8.0上样时，Fc流出，纯度为90.4%，回收率为98.8%，而在pH 3.6洗脱时，hIgG的纯度为99.7%，回收率为86.5%。
