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用于双水相萃取的混合模式配基制备及对IgG分配的影响

ISSN号：1003-9015
期刊名称：高校化学工程学报
时间：2013.2.1
页码：119-124
分类：TQ028.8[化学工程]
作者机构：[1]化学工程联合国家重点实验室浙江大学、浙江大学化学工程与生物工程学系,浙江杭州310027
相关基金：国家自然科学基金（21276228,21036005）;浙江省自然科学基金（LRl2806003）.
相关项目：抗体分离过程中的介质、方法和集成化研究

关键词：双水相系统, 混合模式配基, 2-巯基-1-甲基咪唑, 免疫球蛋白G, 分配系数, aqueous two-phase system, mixed-mode ligand, 2-mercapto-l-methyl-imidazole, immunoglobulin G, partition coefficient

中文摘要：

以2-巯基-1-甲基咪唑（MMI）为混合模式配基，人血免疫球蛋白G（IgG）为模型蛋白，探讨混合模式配基双水相系统分离抗体的新方法，主要包括MMI-聚乙二醇（PEG）的制备和IgG分配研究。首先优化了PEG4000的环氧氯丙烷活化反应条件，环氧氯丙烷和PEG比例为3．5：l，三氟化硼乙醚溶液加量为0．05mL．g-1PEG，开环反应时间为8h；40％氢氧化钠溶液加量为0．15mL·g-1PEG，闭环反应时间为4h，环氧基团接入量可达到460μmol-g-1PEG，偶联配基MMI，得到MMI-PEG混合模式配基。进一步考察了IgG在PEG4000／（NH4）2S04双水相系统中的分配，发现添加MMI-PEG配基有效提高了IgG分配系数，加入适量NaCl，分配系数达到了17．1。结果表明，混合模式配基双水相萃取可以实现IgG的上相富集，为抗体分离提供了一个新途径。

英文摘要：

With 2-mercapto-l-methyl-imidazole （MMI） as the mixed-mode ligand and human serum IgG as model protein, new aqueous two-phase system （ATPS） with mixed-mode ligand was developed and used for antibody separation. The preparation of MMI-polyethylene glycol （PEG） and the partitioning behaviors of IgG in ATPS were investigated. Firstly, the activation of PEG was studied, and the optimized conditions were obtained as follow： addition of BF3-CzHsOCzH5 of 0.05 mL.g-1 PEG, ring opening reaction time of 8 h, addition of NaOH （40%（wt）） of 0.15 mL.g-1 PEG and reaction time of 4 h at room temperature. The ratio of ECH and PEG is 3.5：1, and the epoxide content of activated PEG could reach 460 μmol.g-1 PEG. Then the PEG-expoxide was coupled with MMI to obtain the MMI-PEG as mixed-mode ligand. Finally, the partition behaviors of IgG in PEG4000/（NH4）2SO4 ATPS were studied. It was found that the MMI-PEG ligand could effectively increase the partition coefficient of IgG in the new ATPS. When some amount of NaC1 was added in the system, the partition coefficient could reach 17.1. The results demonstrate that the aqueous two-phase system with mixed-mode ligand could enrich the IgG into the top phase, which provides a new method for antibody separation.