Muca Renata, Marek Wojciech, Żurawski Marek, Piątkowski Wojciech, Antos Dorota
Department of Chemical and Process Engineering, Rzeszów University of Technology, Powstańców Warszawy Ave. 6, 35-959, Rzeszów, Poland.
NanoTemper Technologies Sp.z.o.o. Bobrzyńskiego str. 14, 30-348, Kraków, Poland.
J Chromatogr A. 2017 Apr 7;1492:79-88. doi: 10.1016/j.chroma.2017.02.073. Epub 2017 Mar 1.
Adsorption behavior of unstable proteins, i.e., bovine serum albumin and α-lactalbumin, has been studied on a hydrophobic interaction chromatography medium under mass overloading conditions at different kosmotropic salt concentrations in the mobile phase. A mechanistic model has been formulated and used to describe kinetics and thermodynamics of protein interactions with the adsorbent surface. The model assumed two-site binding adsorption and reversible protein unfolding, which allowed predicting the inhibition of protein unfolding at high column loadings. A simplified procedure for the determination of model parameters has been developed, which was based on the inverse method. The model was successfully used to reproduce the pattern of chromatographic elution as well as the course of breakthrough curves. The model formulation was supported by Nano Differential Scanning Fluorimetry measurements, which were exploited to determine the protein stability in the liquid and adsorbed phases at different column loadings and salt concentrations.
已在流动相中不同促盐析盐浓度下，在质量过载条件下，研究了不稳定蛋白质（即牛血清白蛋白和α-乳白蛋白）在疏水相互作用色谱介质上的吸附行为。已建立并使用了一个机理模型来描述蛋白质与吸附剂表面相互作用的动力学和热力学。该模型假定为双位点结合吸附和可逆蛋白质解折叠，这使得能够预测在高柱负载下蛋白质解折叠的抑制情况。已开发出一种基于反演法的简化模型参数测定程序。该模型成功用于重现色谱洗脱模式以及穿透曲线的过程。纳米差示扫描荧光法测量支持了模型的建立，该测量用于确定在不同柱负载和盐浓度下液体相和吸附相中蛋白质的稳定性。
