Protein partition coefficients can be estimated efficiently by hybrid shortcut calculations.
Protein partition coefficients can be estimated efficiently by hybrid shortcut calculations.
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DOI：
10.1016/j.jbiotec.2016.06.032
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发表时间：
2016-09
影响因子：
4.1
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
在双水相系统（ATPS）中提取治疗性蛋白质（如单克隆抗体）是下游工艺中常见的成本密集型色谱纯化步骤的合适替代方案。因此，可以使用置换剂（另外的盐）选择性地改变蛋白质分配，以允许成功纯化靶蛋白。在这项工作中，提出了一种新的快捷策略，用于计算蛋白质分配系数的聚合物-盐ATPS。所需的蛋白质-溶质（相形成组分，置换剂）相互作用被组成梯度多角度光散射（CG-MALS）测量的交叉维里系数B23所覆盖。使用该快捷计算可以有效测定PEG-柠檬酸盐和PEG-磷酸盐ATPS中靶蛋白免疫球蛋白G（IgG）和杂质人血清白蛋白（HSA）的分配系数，与蛋白质浓度无关。我们证明，选择一个合适的置换剂，允许选择性纯化IgG从HSA是由B23访问。基于通过第二渗透维里系数B22 a覆盖的CG-MALS确定蛋白质-蛋白质相互作用，能够进一步优化ATPS防止蛋白质沉淀。结果表明，我们的方法有助于有效的下游加工开发。
The extraction of therapeutic proteins like monoclonal antibodies in aqueous two-phase systems (ATPS) is a suitable alternative to common cost intensive chromatographic purification steps within the downstream processing. Thereby the protein partitioning can be selectively changed using a displacement agent (additional salt) in order to allow for a successful purification of the target protein. Within this work a new shortcut strategy for the calculation of protein partition coefficients in polymer-salt ATPS is presented. The required protein-solute (phase-forming component, displacement agent) interactions are covered by the cross virial coefficientB23measured by composition gradient multi-angle light scattering (CG-MALS). Using this shortcut calculation allows for an efficient determination of the partition coefficients of the target protein immunoglobulin G (IgG) and the impurity human serum albumin (HSA) within PEG-citrate and PEG-phosphate ATPS independently on the protein concentration. We demonstrate that the selection of a suitable displacement agent allowing for a selective purification ofIgGfromHSAis accessible byB23. Based on the determination of the protein–protein interactions via CG-MALS covered by the second osmotic virial coefficientB22a further optimization of ATPS preventing protein precipitation is enabled. The results show that our approach contributes to an efficient downstream processing development.