Department of Biochemical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300350, China; Key Laboratory of Systems Bioengineering and Frontiers Science Center for Synthetic Biology (Ministry of Education), Tianjin University, Tianjin 300350, China.
Department of Biochemical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300350, China.
J Chromatogr A. 2021 Feb 8;1638:461869. doi: 10.1016/j.chroma.2020.461869. Epub 2020 Dec 31.
Grafting functional polymer chains onto porous resins has been found to drastically increase both adsorption capacity and uptake rate in protein chromatography. In this work, 2-aminoethyl methacrylate (AEM) was used for grafting onto Sepharose FF gel, and six anion-exchangers of different polyAEM (pAEM) chain lengths (ionic capacities, ICs), FF-pAEM, were obtained for protein adsorption and chromatography. It was found that protein adsorption capacity (q) increased with increasing pAEM chain length, but the uptake rate, represented by the ratio of effective pore diffusivity to the free solution diffusivity (D/D), showed an up-down trend, reaching a peak value (D/D=0.55) at an IC of 313 mmol/L. Partial charge neutralization of the AEM-grafted resin of the highest IC (513 mmol/L) by reaction with sodium acetate produced three charge-reduced resins, FF-pAEM513-R. With reducing the charge density, the adsorption capacity kept unchanged and then decreased, but the uptake rate monotonically increased, reaching a maximum (about 2-fold increase) at a residual IC of 263 mmol/L. It is notable that, at the same IC, the charge-reduced resin (FF-pAEM513-R) presented similar or even higher values of q and D/D than its FF-pAEM counterpart. Particularly, at the same IC of 263 mmol/L, a ~50% enhancement of D/D was observed. Both adsorption capacity and uptake rate in the charge-reduced resin with a residual IC of 339 mmo/L (FF-pAEM513-R339) decreased more sharply with increasing NaCl concentration by comparison with FF-pAEM513, indicating its increased salt-sensitivity than FF-pAEM513. That is, charge reduction on the AEM-grafted resin could accelerate protein uptake at 0 mmol/L NaCl but decrease salt tolerance. Column breakthrough experiments showed that FF-pAEM513-R339 was favorable for high flow rate protein chromatography at low NaCl concentration (0 mmol/L), whereas FF-pAEM513 was a good choice in a wide range of salt concentrations at low flow rate. This research proved the excellent protein chromatography performance of the AEM-based anion-exchangers.
将功能聚合物链接枝到多孔树脂上已被发现可极大地提高蛋白质色谱中的吸附容量和上样速率。在这项工作中，使用 2-（甲基丙烯酰氨基）乙基甲基磺酸酯（AEM）接枝到 Sepharose FF 凝胶上，得到了六种不同聚 AEM（pAEM）链长（离子容量，IC）的阴离子交换剂 FF-pAEM，用于蛋白质吸附和色谱。结果发现，蛋白质吸附容量（q）随 pAEM 链长的增加而增加，但上样速率（以有效孔扩散率与自由溶液扩散率的比值 D/D 表示）呈上下波动趋势，在 IC 为 313 mmol/L 时达到峰值（D/D=0.55）。通过与乙酸钠反应使具有最高 IC（513 mmol/L）的 AEM 接枝树脂部分电荷中和，生成了三种电荷降低的树脂 FF-pAEM513-R。随着电荷密度的降低，吸附容量保持不变然后降低，但上样速率单调增加，在残余 IC 为 263 mmol/L 时达到最大值（约增加 2 倍）。值得注意的是，在相同的 IC 下，电荷降低的树脂（FF-pAEM513-R）的 q 和 D/D 值与相应的 FF-pAEM 相当或更高。特别是，在相同的 IC 为 263 mmol/L 时，观察到 D/D 约增加 50%。与 FF-pAEM513 相比，残余 IC 为 339 mmol/L（FF-pAEM513-R339）的电荷降低树脂的吸附容量和上样速率随 NaCl 浓度的增加而急剧下降，表明其对盐的敏感性比 FF-pAEM513 高。也就是说，AEM 接枝树脂上的电荷减少可在 0 mmol/L NaCl 下加速蛋白质上样，但降低耐盐性。柱突破实验表明，FF-pAEM513-R339 在低 NaCl 浓度（0 mmol/L）下有利于高通量蛋白质色谱，而 FF-pAEM513 在低流速下在较宽的盐浓度范围内是一个不错的选择。该研究证明了基于 AEM 的阴离子交换剂具有出色的蛋白质色谱性能。
