Application of aqueous two-phase systems to antibody purification: A multi-stage approach
Application of aqueous two-phase systems to antibody purification: A multi-stage approach
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DOI：
10.1016/j.jbiotec.2009.01.001
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发表时间：
2009-02-23
影响因子：
4.1
通讯作者：
Baecker, W.
中科院分区：
文献类型：
--
作者：
Rosa, P. A. J.;Azevedo, A. M.;Baecker, W.
研究了从中国仓鼠卵巢 (CHO) 细胞上清液中单阶段平衡水相两相萃取 (ATPE) 的人抗体。通过研究原料中的pH、离子强度、体积比和初始抗体浓度等实验条件，评估了聚乙二醇3350（PEG 3350）/磷酸盐水性两相体系（ATPS）的性能。有利于人免疫球蛋白G（IgG）提取的条件是低pH值、高NaCl浓度和高体积比。污染物去除百分比并不取决于 pH 值和 NaCl 浓度，但随着体积比的减小而增加。当使用0.4的体积比时，约66%的总污染物被去除。还通过模拟试管中的四级错流操作来研究多级平衡ATPE。根据IgG平衡曲线和各自的McCabe Thiele图，描述了逆流多级ATPE的预测优化方案。使用含有 10% (w/w) NaCl 的 PEG/磷酸盐 ATPS，分五个阶段且体积比为 0.4，可实现 89% 的 IgG 回收率和 75% 的蛋白质纯度。与在相同实验条件下进行的单阶段试验相比，这表明回收率和纯度显着提高，其中回收率达到 61%，蛋白质纯度达到 55%。根据CHO细胞上清液组分平衡曲线，观察到IgG可以从较高分子量(MW)组分中完全纯化，并从较低MW组分中部分纯化。 (c) 2009 Elsevier B.V. 保留所有权利。
The single-stage equilibrium aqueous two-phase extraction (ATPE) of human antibodies from a Chinese hamster ovary (CHO) cells supernatant was investigated. The performance of polyethylene glycol 3350 (PEG 3350)/phosphate aqueous two-phase systems (ATPS)was evaluated by studying several experimental conditions,such as pH, ionic strength, volume ratio and initial antibody concentration in the feed stock. The conditions that favoured the extraction of human immunoglobulin G (IgG) were low pH values, high NaCl concentration and high volume ratios. The percentage of contaminants removal did not depend on the pH value and NaCl concentration, increasing, however, as the volume ratio decreased. About 66% of total contaminants were removed when a volume ratio of 0.4 was used.The multi-stage equilibrium ATPE was also investigated by simulating a four stages cross-current operation in test tubes. According to the IgG equilibrium curves and respective McCabe Thiele diagrams, a predicted optimised scheme of a counter-current multi-stage ATPE was described. An IgG recovery yield of 89% and a protein purity of 75% can be achieved using a PEG/phosphate ATPS containing 10% (w/w) NaCl, five stages and a volume ratio of 0.4. This represents significant improvements in the recovery yield and purity when compared to a single-stage trial performed at the same experimental conditions, where a 61% recovery yield and 55% protein purity were attained. Based on the CHO cells supernatant components equilibrium curves, it was observed that IgG can be completely purified from the higher molecular weight (MW) components and partially purified from the lower MW components. (c) 2009 Elsevier B.V. All rights reserved.