【作者】 贾国栋；

【导师】 孙彦；

【作者基本信息】 天津大学 ， 生物化工， 2009， 博士

【摘要】 本论文以色谱技术的两大要素——色谱柱和色谱介质——为研究对象,开展了蛋白质色谱过程强化的应用基础研究,包括制备型二维电场电色谱的构建和应用,超孔琼脂糖色谱介质的制备、接枝修饰和色谱性能表征两部分。在电色谱研究方面,首先构建了二维电色谱技术,即在填料室的横向和纵向同时外加电场,以在色谱柱内产生二维电动传递现象。然后分别以色素亲和色谱和金属螯合亲和色谱为研究对象,考查外加电场对介质动态吸附容量的影响。结果表明:在二维电色谱中可以获得比在一维电场中更高的蛋白质动态吸附容量。最后,利用填充Zn-Sepharose FF的二维电色谱柱,从酵母菌中纯化乙醇脱氢酶。与无电场作用时相比,生产能力提高一倍,进一步证明二维电色谱是一种有效的蛋白质色谱过程强化技术。在电色谱应用研究的基础上,开展电色谱传质机理的研究。首先利用甲基丙烯酸缩水甘油酯对TOYOPEARL HW-65介质进行接枝修饰,制得具有良好尺寸排阻色谱分离性能的“熵作用色谱”(EIC)介质。然后利用该介质研究了横向交变电场EIC中溶质的电色谱保留机理。结果表明,在接枝密度较小的固定相中,所有溶质分子均为电泳起主导作用;在接枝密度较高的固定相中,小分子溶质电泳起主导作用,而对于大分子溶质,则是浓差极化起主导作用。最后建立数学模型,求解溶质分子在不同体系中的浓差极化系数和固定相孔内电泳迁移率,说明浓差极化程度与溶质分子量和自由电泳迁移率成正相关性,而与分配系数和扩散系数成反相关性。在色谱介质研究方面,首先采用布尔马金式搅拌桨,在带有挡板的反应釜内实现超孔琼脂糖凝胶色谱介质从实验室小试规模到中试规模的放大制备。然后对超孔琼脂糖色谱介质进行了葡聚糖接枝修饰,考查其吸附和传质性能。研究结果表明:接枝式超孔色谱介质不仅具有较高的静态吸附容量,而且具有超高的孔扩散速率,在高流速色谱过程中仍保持很高的动态吸附容量和吸附效率,说明对超孔色谱介质进行接枝修饰,是一种非常有效的的改性方法。更多还原

【Abstract】 This thesis is focused on the process intensification of protein chromatography towards the objective of high performance preparation. The main approaches proposed here are the preparative electrochromatography and the superporous agarose gel. The development and application of preparative two-dimensional electrochromatography (2DEC) as well as the fabrication, grafting modification and adsorption performance of superporous agarose gel were all investigated in this work. The details are summarized as follows:A novel preparative 2DEC column with seven compartments was developed. The design gave promise to provide transverse and longitudinal electric field on the column in order to induce two-dimensional electrokinetic transports in the central gel compartment. The dye-ligand affinity chromatography and immobilized metal affinity chromatography (IMAC) were performed in the 2DEC respectively. It was confirmed that the dynamic binding capacity (DBC) of protein in 2DEC was promoted more efficiently than that in one-dimensional electrochromatography. Moreover, the results indicated that the electroosmotic flow on the charged adsorbent surface was the predominant electrokinetic transport in the affinity electrochromatography. The effect of ionic strength proved that low-salt concentration was more suitable for the electrochromatography. Then, alcohol dehydrogenase was purified by the 2DEC packed with Zn-Sepharose FF at loading volume which was 2-fold higher than that by the traditional IMAC. The results have proved that the 2DEC is an efficient technique for the process intensification of protein chromatography and promising for the high capacity protein separation in the downstream processing.The entropic interaction chromatographic (EIC) resins were fabricated by the grafting of glycidyl methacrylate to TOYOPEARL HW-65. The resins gave a high resolution of solutes during the separation range of 0.67 kD to 158 kD. The EIC resins of different grafting densities were used to investigate the transport mechanism in EIC with an oscillatory electric field perpendicular to mobile phase flow driven by pressure. The results demonstrated that the electrophoresis was the dominante electrokinetic transport in the EIC resin with a low grafting density for all the solutes employed in this research and in the resin with a high grafting density for the solutes of low molecular weights. However, the concentration polarization played a key role in the retention behaviors for the solutes of high molecular weights. The concentration polarization parameters and the electrophoretic mobilities in the station phase of solutes were calculated by a mathematical model developed. The concentration polarization parameters would increase with the increasing molecular weight and free electrophoretic mobilities but decrease with the increasing partition coefficient and free solution diffusivity.The scaling-up of the fabrication of superporous agarose gel (SA) was accomplished in a reactor of 10 L with Brumagin agitator and four baffles in it. Dextran chains of 40 kD were grafted into the pores of SA in order to improve the adsorption properties. Besides the increase of adsorption capacity led by the grafting of dextran chains, the protein uptake rate was also greatly increased. So, the grafted SA resin showed the high DBC and adsorption efficiency even at a high flow rate. Hence, it is considered that grafting modification was a prominent approach to improve the properties of superporous agarose gel.更多还原