Osmotic Virial Coefficients as Access to the Protein Partitioning in Aqueous Two-Phase Systems.
Osmotic Virial Coefficients as Access to the Protein Partitioning in Aqueous Two-Phase Systems.
复制标题
DOI：
10.1002/jps.24602
复制
发表时间：
2015-11
影响因子：
3.8
通讯作者：
Christian Kress;Christoph Brandenbusch
中科院分区：
文献类型：
--
作者：
Christian Kress;Christoph Brandenbusch
使用双水相系统(ATPS)提取目标蛋白是治疗蛋白最先进的层析分离的一种有前途的替代方法。使用额外的盐作为置换剂可以影响ATP中的蛋白质分配行为，从而实现对目标蛋白质的选择性纯化。选择合适的ATPS用于蛋白质提取需要有关蛋白质-蛋白质相互作用(第二渗透维里系数B22)以及蛋白质与溶质(驱替剂和相形成组分)之间的相互作用(交叉维里系数B23)的信息。在这项工作中，考虑了免疫球蛋白G(IgG)在聚乙二醇磷酸盐ATPS中的分配行为和沉淀亲和力。考察了氯化钠和(NH4)2SO4的加入对免疫球蛋白分配的影响。根据静态光散射法测定了几种溶质[聚乙二醇、磷酸盐缓冲液、氯化钠和(NH4)2SO4]与免疫球蛋白的组合的B22和B23值，以了解抗体的亲和力和免疫球蛋白的分配行为。根据测得的B22和B23值，定性地估计了聚乙二醇磷酸盐ATPS中的免疫球蛋白沉淀亲和力和作为潜在驱替剂的溶质的适宜性。
A promising alternative to state of the art chromatographic separations of therapeutic proteins is the extraction of the target protein using an aqueous two-phase system (ATPS). The use of an additional salt working as a displacement agent can influence the protein partitioning behavior in ATPS and thus enable a selective purification of the target protein. The selection of a suitable ATPS for protein extraction requires information concerning the protein-protein interactions (second osmotic virial coefficient B22 ) as well as the interactions between protein and solute (displacement agent and phase-forming components) (cross virial coefficient B23 ). In this work, the partitioning behavior and the precipitation affinity of immunoglobulin G (IgG) is considered within a polyethylene glycol (PEG)-phosphate ATPS. The influence on IgG partitioning upon addition of NaCl and (NH4)2 SO4 was investigated. In order to access the IgG precipitation affinity and the IgG partitioning behavior, the B22 and B23 values were determined for several combinations of solute [PEG, phosphate buffer, NaCl, and (NH4)2 SO4 ] and IgG based on static light scattering measurements. A qualitative estimation of the IgG precipitation affinity and the suitability of a solute as potential displacement agent within the PEG-phosphate ATPS on the basis of the measured B22 and B23 values is presented.