Solubilization of proteins in aqueous two‐phase extraction through combinations of phase‐formers and displacement agents
Solubilization of proteins in aqueous two‐phase extraction through combinations of phase‐formers and displacement agents
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DOI：
10.1016/j.ejpb.2016.11.016
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发表时间：
2017-03
影响因子：
4.9
通讯作者：
Christian Kress;G. Sadowski;Christoph Brandenbusch
中科院分区：
文献类型：
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作者：
Christian Kress;G. Sadowski;Christoph Brandenbusch
治疗性蛋白质的双水相萃取(ATPE)是一种很有前途的分离方法，可以替代成本较高的层析，目前仍是下游处理的主力。许多文献表明，在盐聚合物ATPE中使用氯化钠作为驱替剂，可以选择性地从人血清白蛋白(HSA，代表杂质)中提纯目标蛋白免疫球蛋白G(Ig G)。然而，只有在蛋白质在溶液中稳定而不是沉淀的情况下，才能获得高产率的目标蛋白质。在这项工作中，用组成梯度多角光散射(CG-MALS)测定了NaC l和聚乙二醇(Mw=2 0 0 0 g/m ol)对Ig G-Ig G相互作用的联合影响。此外，还表明驱替剂氯化钠对IgG在聚乙二醇2000-柠檬酸盐双水相体系(ATPS)中的溶解度有显著和有利的影响，这也可以通过这些先进的B22测量来获得。通过同时考虑免疫球蛋白溶解度数据和ATPS相行为的结果(特别是各相的体积分数)，可以选择过程定制的ATPS，包括确定最大蛋白饲料浓度。通过这种方法，ATPS的优化是可行的，提供了高产率和目标蛋白(IgG)的选择性。
The aqueous two-phase extraction (ATPE) of therapeutic proteins is a promising separation alternative to cost-intensive chromatography, still being the workhorse of nowadays downstream processing. As shown in many publications, using NaCl as displacement agent in salt-polymer ATPE allows for a selective purification of the target protein immunoglobulin G (IgG) from human serum albumin (HSA, represents the impurity). However a high yield of the target protein is only achievable as long as the protein is stabilized in solution and not precipitated. In this work the combined influence of NaCl and polyethylene glycol (Mw = 2000 g/mol) on theIgG-IgGinteractions was determined using composition gradient multi-angle light scattering (CG-MALS) demonstrating that NaCl induces a solubilization ofIgGin polyethylene glycol 2000 solution. Moreover it is shown that the displacement agent NaCl has a significant and beneficial influence on theIgGsolubility in polyethylene glycol 2000-citrate aqueous two-phase system (ATPS) which can also be accessed by these advancedB22measurements. By simultaneous consideration ofIgGsolubility data with results of the ATPS phase behavior (especially volume fraction of the respective phases) allows for the selection of process tailored ATPS including identification of the maximum protein feed concentration. Through this approach an ATPS optimization is accessible providing high yields and selectivity of the target protein (IgG).