基于聚多巴胺功能化的膜色谱介质制备及其在蛋白分离中的应用

发布时间：2018-03-12 13:22

本文选题：膜色谱　切入点：膜色谱介质　出处：《中国科学院大学(中国科学院过程工程研究所)》2017年博士论文　论文类型：学位论文

【摘要】：膜色谱结合了膜过滤高通量和液相色谱高选择性的特点,能够高效地从复杂料液如发酵液、血浆、乳清中获得纯度较高的生物活性分子,因此在生物产品制备领域具有广阔的应用潜力。然而,现阶段膜色谱介质(基膜材料)存在稳定性较差、机械性能欠佳及其制备工艺复杂、不易控制等问题。本研究采用简单易操作的聚多巴胺(PDA)涂覆对机械强度较高的微滤膜进行活化,然后以PDA为中间功能层偶联膜色谱配基构建多种膜色谱介质,并应用于蛋白分离纯化和酶的固定化。(1)通过将PDA涂覆于亲水性聚醚砜膜(PES)形成中间功能层,然后偶联三种不同的配基(聚乙烯亚胺、十二硫醇和组氨酸)制备阴离子交换、疏水和亲和膜色谱介质。处理免疫球蛋白G/人血清白蛋白(IgG/HSA)混合液时,通过阴离子交换膜色谱分离可获得纯度为96.7%的HSA;经过疏水膜色谱分离可获得纯度为94.6%的IgG;通过亲和膜色谱分离,可获得的纯度接近100%的IgG。同时经PDA改性,可有效提高膜介质的亲水性并降低其非特异性吸附。(2)通过将PDA涂覆于疏水性聚偏二氟乙烯膜(PVDF),对其进行亲水改性(膜表面接触角由原来的116°下降至61.3°),然后偶联聚丙烯胺配基制备得到耐盐型阴离子(STAE)交换膜色谱介质,可用于高盐环境下单克隆抗体的高效精制。自制STAE膜色谱介质在含有150 mM NaCl的缓冲液中可以保留75%的蛋白吸附容量。另外,Langmuir等温吸附模型比Freundlich吸附模型更适合模拟蛋白在STAE膜表面的吸附。当流速为10-100膜体积(MV)/min时,膜色谱介质的蛋白吸附容量基本不受流速的影响。而且所制备STAE膜色谱介质的机械性能、稳定性、分离效率和重复使用性均优于商业化产品。(3)以涂覆于疏水膜的PDA为平台,可实现阴离子交换配基的快速匹配,构建适用于特定分离体系的膜色谱介质。同时通过优化流速、洗脱盐浓度、缓冲液pH、进样体积和配基密度等条件,实现了一步法从血浆沉淀Ⅳ中分离得到纯度为94.6%的α1-抗胰蛋白酶(AAT),其质量回收率和活性收率分别为94.2%和96.6%,该纯化效果优于采用商业化产品获得的结果以及文献报道结果。本研究建立了一种通过设计膜色谱介质实现复杂料液中快速纯化高纯度生物药物的新方法。(4)采用自制的阴离子交换膜色谱介质和金属螯合膜色谱介质,对发酵料液中的漆酶进行同步纯化和固定化以构建酶膜反应器。两种膜色谱介质上固定的漆酶均具有较高的纯度。膜表面漆酶的活力和比活力不受固定化过程中操作流速的影响,流穿式固定化模式比传统的浸泡模式更高效。所制备的酶膜反应器具有较好的稳定性、可重复性和良好的双酚A(BPA)去除能力。由于金属离子与漆酶结合更牢固,因此处理BPA料液时,用金属螯合膜色谱介质构建的酶膜反应器没有蛋白泄漏。研究发现当堆叠四层膜处理(BPA)时,用金属螯合膜色谱介质构建的酶膜反应器对BPA的去除率可提高至99.2%;当料液处理通量提高至50L/m~2h时,BPA的去除效率(473.0 rmg/m~2h)高于文献中报道的结果。
[Abstract]:Membrane chromatography combined with the characteristics of the high selectivity of the membrane filtration flux and liquid chromatography, which can efficiently plasma from the complex liquid such as liquid fermentation, and obtain bioactive molecules with high purity whey, so in the field of preparation of biological products with a wide range of potential applications. However, the present stage membrane chromatography medium (basal material) there is poor stability, poor mechanical properties and the preparation process is complex, not easy to control. This research adopts a simple and easy operation of the polydopamine (PDA) microfiltration membrane coating on the mechanical strength of high activation, then using PDA as the intermediate function layer coupling membrane chromatography ligand to construct various membrane chromatography media, immobilization and application in the separation and purification of protein and enzyme. (1) by PDA coated with hydrophilic polyether sulfone (PES) to form an intermediate function layer, and then coupling three different ligands (polyethyleneimine, twelve thiol and histidine) preparation Anion exchange and hydrophobic membrane affinity chromatography medium. Treatment of immunoglobulin G/ in human serum albumin (IgG/HSA) mixture, separation can be obtained with purity of 96.7% HSA by anion exchange membrane chromatography; through the hydrophobic membrane chromatography separation can be obtained with purity of 94.6% IgG; isolated by pro and membrane chromatography, the purity can get close to 100% IgG. and modified by PDA, which can effectively improve the hydrophilicity of the membrane medium and reduce the nonspecific adsorption. (2) the PDA is coated on the hydrophobic polyvinylidene fluoride membrane (two PVDF), the hydrophilic (membrane surface contact angle decreased from 116 degrees to 61.3 then the coupling degree), polypropylene amine ligands were prepared by salt type anion exchange membrane chromatography medium (STAE), can be used in high salt environment single clone antibody, purification. Homemade STAE membrane chromatography medium in buffer containing 150 mM NaCl can retain 75% of the protein The adsorption capacity of Langmuir. In addition, the adsorption isotherm model than the Freundlich adsorption model is more suitable for the simulation of proteins on the surface of the STAE film. When the film is 10-100 volume flow rate (MV) of /min, the effect of protein adsorption capacity of membrane chromatography medium is not affected by flow rate. The mechanical properties, and the preparation of STAE film color spectrum medium stability, the separation efficiency and repeatability are better than commercial products. (3) is coated with a hydrophobic film on the PDA platform, which can realize fast matching of anion exchange membrane chromatography medium ligand, applicable to the construction of specific separation system. At the same time by optimizing the elution velocity, salt concentration, buffer pH, sample volume and the ligand density and other conditions, the one-step separation is obtained with a purity of 94.6% alpha 1- antitrypsin IV in plasma from precipitation (AAT), the mass recovery and activity recovery were 94.2% and 96.6%, the purification effect is better than that of the commercial The product obtained by the results and literature reports. This study established a new method for rapid purification of high purity biological drugs through the design of complex liquid membrane chromatography medium. (4) using anion exchange membrane chromatography media and metal chelating membrane chromatography medium, simultaneous purification and immobilization of laccase fermentation material solution to the construction of the enzyme membrane reactor. Two kinds of membrane chromatography medium immobilized laccase were with high purity. The operation effect of membrane surface velocity of laccase activity and specific activity of the immobilized in the process flow, wear type fixed model than the traditional immersion mode more efficient. The enzyme membrane reaction the equipment has good stability, good repeatability and bisphenol A (BPA) removal capacity. Because the metal ions are firmly combined with laccase, so BPA liquid, enzyme membrane constructed by metal chelating membrane chromatography medium reaction Is no protein leakage. The study found that when stacked four layer membrane (BPA), constructed by metal chelating membrane chromatography medium enzyme membrane reactor on the removal rate of BPA can be increased to 99.2%; when the feed processing flux increased to 50L/m~2h, the removal efficiency of BPA (473 rmg/m~2h) is higher than the results reported in literature.

【学位授予单位】：中国科学院大学(中国科学院过程工程研究所)
【学位级别】：博士
【学位授予年份】：2017
【分类号】：TQ028.8;O652.63

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