Liu Tao, Angelo James M, Lin Dong-Qiang, Lenhoff Abraham M, Yao Shan-Jing
Key Laboratory of Biomass Chemical Engineering of Ministry of Education, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China; Department of Chemical and Biomolecular Engineering, University of Delaware, Newark, DE 19716, USA.
Department of Chemical and Biomolecular Engineering, University of Delaware, Newark, DE 19716, USA.
J Chromatogr A. 2017 Sep 29;1517:44-53. doi: 10.1016/j.chroma.2017.07.090. Epub 2017 Aug 5.
The structural and functional properties of a series of dextran-grafted and non-grafted hydrophobic charge-induction chromatographic (HCIC) agarose resins were characterized by macroscopic and microscopic techniques. The effects of dextran grafting and mobile phase conditions on the pore dimensions of the resins were investigated with inverse size exclusion chromatography (ISEC). A significantly lower pore radius (17.6nm) was found for dextran-grafted than non-grafted resins (29.5nm), but increased salt concentration would narrow the gap between the respective pore radii. Two proteins, human immunoglobulin G (hIgG) and bovine serum albumin (BSA), were used to examine the effect of protein characteristics. The results of adsorption isotherms showed that the dextran-grafted resin with high ligand density had substantially higher adsorption capacity and enhanced the salt-tolerance property for hIgG, but displayed a significantly smaller benefit for BSA adsorption. Confocal laser scanning microscopy (CLSM) showed that hIgG presented more diffuse and slower moving adsorption front compared to BSA during uptake into the resins because of the selective binding of multiple species from polyclonal IgG; polymer-grafting with high ligand density could enhance the rate of hIgG transport in the dextran-grafted resins without salt addition, but not for the case with high salt and BSA. The results indicate that microscopic analysis using ISEC and CLSM is useful to improve the mechanistic understanding of resin structure and of critical functional parameters involving protein adsorption and transport, which would guide the rational design of new resins and processes.
采用宏观和微观技术对一系列葡聚糖接枝和未接枝的疏水电荷诱导色谱（HCIC）琼脂糖树脂的结构和功能特性进行了表征。通过反相尺寸排阻色谱（ISEC）研究了葡聚糖接枝和流动相条件对树脂孔径的影响。发现葡聚糖接枝树脂的孔径半径（17.6nm）明显低于未接枝树脂（29.5nm），但增加盐浓度会缩小各自孔径半径之间的差距。使用两种蛋白质，即人免疫球蛋白G（hIgG）和牛血清白蛋白（BSA），来研究蛋白质特性的影响。吸附等温线结果表明，具有高配体密度的葡聚糖接枝树脂对hIgG具有显著更高的吸附容量并增强了其耐盐性，但对BSA吸附的益处明显较小。共聚焦激光扫描显微镜（CLSM）显示，在树脂摄取过程中，由于多克隆IgG中多种物质的选择性结合，hIgG与BSA相比呈现出更分散、移动更慢的吸附前沿；在不添加盐的情况下，高配体密度的聚合物接枝可以提高hIgG在葡聚糖接枝树脂中的传输速率，但在高盐和BSA的情况下则不然。结果表明，使用ISEC和CLSM进行微观分析有助于提高对树脂结构以及涉及蛋白质吸附和传输的关键功能参数的机理理解，这将指导新型树脂和工艺的合理设计。
