聚合物接枝型疏水性电荷诱导层析介质设计及蛋白质吸附机理研究

发布时间：2018-06-25 20:32

本文选题：疏水性电荷诱导层析 + 聚合物接枝　；参考：《浙江大学》2017年博士论文

【摘要】：疏水性电荷诱导层析(Hydrophobic charge-induction chromatography,HCIC)是一种新型抗体分离技术,其配基兼有疏水、静电、氢键等多种相互作用,具有选择性好、耐盐性高、洗脱条件温和以及价格相对低廉等特点,已有一些成功应用实例。由于配基与抗体结合力较弱,使得HCIC介质在高流速下对抗体的动态结合载量较低,而聚合物接枝的离子交换层析介质可以显著提高蛋白的吸附容量、传质速率和动态结合载量。因此本文以高效抗体分离为目标,设计并制备了两种聚合物接枝型HCIC介质,比较蛋白吸附和传质性能,分析层析过程的固相条件(聚合物接枝、配基密度、接枝密度)和液相条件(pH和添加盐)的影响,并借助逆体积排阻层析(Inverse size exclusion chromatography,ISEC)、等温滴定量热仪(Isothermal titration calorimetry,ITC)和共聚焦激光扫描显微镜(Confocal laser scanning microscopy,CLSM)进行了微观分析,以指导新型介质设计。主要结果如下:首先以葡聚糖接枝的琼脂糖凝胶为基质,以2-巯甲基咪唑(MMI)为配基,优化了配基偶联条件,制备了不同配基密度的葡聚糖接枝MMI介质。相比非接枝MMI介质,配基密度提高了 50%左右,达到2000μmol/g。以hIgG为模型蛋白,考察了系列葡聚糖接枝MMI介质的静态吸附、吸附动力学和层析柱动态吸附性能。结果表明,在近中性pH下,葡聚糖接枝介质对蛋白的吸附容量、结合力、传质速率和动态结合载量随着配基密度增加而持续增加,葡聚糖接枝MMI介质相比非接枝MMI介质具有更高的蛋白吸附容量、传质速率和动态结合载量。在pH 7.0～8.9时,饱和吸附容量保持在107.5～110.0 mg/g,受pH影响较小;在100 cm/h线性流速下hIgG动态结合载量达到38.3mg/g;在弱酸性条件下,葡聚糖接枝MMI介质的蛋白吸附容量和传质速率下降更显著,有利于蛋白解吸。此外,葡聚糖接枝MMI介质吸附能力受盐浓度的影响较小,但传质速率受盐浓度的影响较大。其次,针对葡聚糖接枝介质中HCIC配基同时存在于接枝层和基质孔道表面上的局限,利用表面引发的电子转移再生活化剂的原子转移自由基聚合反应,制备了不同接枝密度和配基密度的聚甲基丙烯酸缩水甘油酯(GMA)接枝MMI介质。结果表明,高接枝密度和中等配基密度的聚GMA接枝MMI介质对hIgG具有更高的饱和吸附容量和传质速率。相比葡聚糖接枝MMI介质以及商业化HCIC介质MEP HyperCel,聚GMA接枝介质对hIgG具有更高动态结合载量。hIgG动态结合载量受流速影响较小,线性流速300 cm/h下可达34.6 mg/g,且具有明显的pH依赖吸附和耐盐吸附特性。此外,考察了从混合蛋白体系(hIgG/HSA=1:4)中分离hIgG和从CHO细胞培养液中分离单克隆抗体,聚GMA接枝HCIC介质均表现出良好的抗体分离和重复使用性能,50个周期循环使用的抗体纯度和收率分别稳定在98.5～99.8%和 90.2～96.9%。最后,为了从微观尺度揭示聚合物接枝MMI介质的吸附及传质机制,利用ISEC考察了介质孔结构的影响,利用ITC研究了不同蛋白(hIgG和BSA)的吸附热,利用CLSM实时观测了单个介质颗粒内蛋白的吸附、传质及洗脱过程。ISEC结果表明,不加盐时葡聚糖接枝链和聚GMA接枝链在基质表面形成三维空间分布,有助于提高蛋白吸附容量;高盐浓度下,接枝链出现塌缩,且葡聚糖链的塌缩程度更明显,削弱了链传递效应,降低了蛋白传质速率。ITC结果表明,对于hIgG,在pH 8时,配基密度和盐浓度的增加会显著增加葡聚糖接枝MMI介质吸附hIgG的焓变和熵变,说明疏水相互作用增强;弱酸条件下,焓变和熵变急剧下降为负值,说明静电排斥作用主导了蛋白的解吸;对于BSA,吸附容量随着盐浓度的增加而显著下降,且焓变和熵变值为负,表明主要通过静电吸引力吸附BSA。CLSM结果表明,hIgG和BSA在葡聚糖接枝、聚GMA接枝和非接枝MMI介质内,均体现出均质扩散控制的传质机理;hIgG在高配基密度葡聚糖接枝和中等配基密度聚GMA接枝MMI介质内体现了更高的荧光强度;相比hIgG,BSA达到MMI介质内核所需时间更短;pH4.0时,中等配基密度聚GMA接枝MMI介质内的荧光强度下降最为明显;以上结果与宏观吸附、传质及分离性能相匹配。本文从提高HCIC介质的蛋白载量出发,制备了两种新型聚合物接枝HCIC介质,探讨了聚合物接枝、配基密度、接枝密度、pH、添加盐和蛋白性质对聚合物接枝HCIC介质吸附和传质性能的影响,并利用ISEC、ITC、CLSM等手段从微观角度揭示了蛋白吸附和传质机理,结果显示孔内接枝可以明显提高HCIC介质对抗体的吸附容量、传质速率及动态载量,显示出良好的抗体分离应用前景。
[Abstract]:Hydrophobic charge induced chromatography (Hydrophobic charge-induction chromatography, HCIC) is a new type of antibody separation technology. Its ligand has a variety of interactions, such as hydrophobic, electrostatic and hydrogen bonds, with good selectivity, high salt tolerance, mild elution conditions and relatively low price. With weak antibody binding force, the dynamic binding capacity of HCIC medium to the antibody is low at high velocity, and the adsorption capacity, mass transfer rate and dynamic load of the protein can be significantly increased by the polymer graft ion exchange chromatography medium. Therefore, two kinds of polymer graft type HCIC were designed and prepared by high efficiency antibody separation. Medium, the adsorption and mass transfer performance of protein were compared, and the effects of solid state conditions (polymer grafting, ligand density, grafting density) and liquid phase conditions (pH and salt) were analyzed, and the inverse volume exclusion chromatography (Inverse size exclusion chromatography, ISEC), isothermal titration calorimeter (Isothermal titration calorimetry, ITC) and Co Confocal laser scanning microscopy (CLSM) was microanalyzed to guide the design of new medium. The main results were as follows: firstly, the agarose gel grafted by glucan was used as the matrix and 2- mercapto imidazole (MMI) as the ligand. The coupling conditions of the ligand were optimized, and the graft MM with different ligand density was prepared. I medium. Compared with non graft MMI medium, the density of the ligand was increased by 50% and 2000 mol/g. to hIgG as the model protein. The static adsorption, adsorption kinetics and dynamic adsorption properties of a series of glucan graft MMI media were investigated. The results showed that the adsorption capacity, binding force and transmission capacity of the gluconate graft medium on the near neutral pH showed that the adsorption capacity and binding force of the gluconate graft medium on the protein were transmitted. The mass rate and dynamic load increase continuously with the increase of the density of the coordination group. The MMI medium with glucan graft has higher protein adsorption capacity, mass transfer rate and dynamic binding load than non grafted MMI medium. At pH 7 ~ 8.9, the saturated adsorption capacity remains at 107.5 ~ 110 mg/ g, which is less affected by pH; hIgG at 100 cm/h linear flow velocity. Under the condition of weak acid, the adsorption capacity and mass transfer rate of glucan grafted MMI medium decreased more significantly, which was beneficial to protein desorption. In addition, the adsorption capacity of glucan graft MMI medium was less affected by salt concentration, but mass transfer rate was greatly influenced by salt concentration. Secondly, the graft mediate of glucan was applied to the glucan grafting medium. The HCIC ligand in the mass exists at the same time on the surface of the graft layer and the matrix pore. Using the surface induced electron transfer and the atom transfer radical polymerization of the living agent, the MMI medium with different grafting density and the density of polyglycidyl methacrylate (GMA) is prepared. The results show that the high grafting density and the medium distribution are high. The base density poly GMA grafted MMI medium has higher saturated adsorption capacity and mass transfer rate for hIgG. Compared with glucan graft MMI medium and commercial HCIC medium MEP HyperCel, GMA grafting medium has higher dynamic binding load on hIgG.HIgG dynamic binding load is less affected by flow velocity, and the linear velocity is 300 cm/h up to 34.6 mg/g. In addition, the separation of hIgG from the mixed protein system (hIgG/HSA=1:4) and the separation of monoclonal antibodies from the culture medium of CHO cells were investigated. The GMA graft HCIC media showed good antibody separation and reuse performance, and the purity and yield of the 50 cycles used for the 50 cycles were stable, respectively. At the end of 98.5 ~ 99.8% and 90.2 ~ 96.9%., in order to reveal the adsorption and mass transfer mechanism of the polymer graft MMI medium from the microscale, the effect of the pore structure of the medium was investigated by ISEC. The adsorption heat of different proteins (hIgG and BSA) was studied by ITC. The adsorption, mass transfer and elution of the protein in a single medium particle were observed by CLSM in real time. .ISEC results show that the three-dimensional spatial distribution of glucan graft chain and poly GMA graft chain is formed on the surface of the matrix without salt, which helps to improve the protein adsorption capacity. Under high salt concentration, the graft chain appears collapsing, and the collapse degree of the glucan chain is more obvious, which weakens the chain transfer effect and reduces the mass transfer rate.ITC results to the hIg G, at pH 8, the increase in the density and salt concentration of the ligand significantly increases the enthalpy and entropy change of the adsorbed hIgG by the dextran graft MMI medium, indicating the enhancement of the hydrophobic interaction. Under the weak acid conditions, the enthalpy change and entropy change sharply decrease to negative values, indicating that the electrostatic repellent dominates the desorption of the protein; for BSA, the adsorption capacity increases with the increase of salt concentration. With the decrease, the enthalpy change and the entropy change value are negative, it is shown that the adsorption of BSA.CLSM mainly through electrostatic attraction shows that hIgG and BSA show the mass transfer mechanism of homogeneous diffusion control in the grafting of dextran, GMA graft and non grafted MMI medium; hIgG is embodied in the grafting of high ligand density glucan and the medium density poly GMA graft MMI medium. Compared with hIgG, the time required for BSA to reach the MMI medium is shorter than that of the MMI medium; when pH4.0, the fluorescence intensity in the medium density GMA graft MMI medium is the most obvious; the above results are matched with the macro adsorption, mass transfer and separation performance. In this paper, two new types of polymerization are prepared from the increase of the protein load of the HCIC medium. Grafted HCIC medium, the effects of polymer grafting, density, grafting density, pH, salt and protein properties on the adsorption and mass transfer properties of the polymer grafted HCIC medium were investigated. The mechanism of protein adsorption and mass transfer was revealed by means of ISEC, ITC, CLSM and other means. The results showed that the grafting in the hole could improve the antagonism of the HCIC medium. The adsorption capacity, mass transfer rate and dynamic load of the body showed good prospects for antibody separation.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：O631.3;TQ460.1

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