Department of Chemical and Process Engineering, Rzeszów University of Technology, Powstańców Warszawy Ave. 6, 35-959 Rzeszów, Poland.
School of Engineering & Applied Science, University of Virginia, Thornton Hall, P.O. Box 400259, Charlottesville, VA 22904-4259, United States.
J Chromatogr A. 2020 Aug 16;1625:461309. doi: 10.1016/j.chroma.2020.461309. Epub 2020 Jun 6.
The adsorption behavior of the model proteins: alpha-Lactalbumin, Bovine Serum Albumin, Lysozyme, and a monoclonal antibody, in single component and in binary mixtures, was investigated on two different hydrophobic interaction chromatography resins using both static and dynamic methods. A kinetic model of the adsorption process was developed, which accounted for protein unfolding and intermolecular interactions in the adsorbed phase. The latter incorporated positive cooperative interactions, resulting from preferred and multilayer adsorption on the adsorbent surface, as well as negative cooperative interactions attributed to exclusion effects due to size exclusion and repulsion. Cooperative adsorption resulted in negative or positive deviations from the Langmuir model for both single and multicomponent isotherms. The model was used to assess possible contributions of different adsorption mechanisms of proteins and their structurally different forms to the overall adsorption pattern, as well as to simulate chromatographic band profiles under different loading conditions. For proteins with unstable structure, the overall adsorption isotherm was dominated by binding of unfolded species at low surface coverage and by positive cooperative adsorption at high surface coverage. Furthermore, regardless of structural stability, exclusion effects influenced strongly adsorption equilibrium, particularly at low surface coverages. In case of chromatographic elution, i.e. under dynamic conditions, unfolding, negative cooperative adsorption, and kinetic effects governed the retention behavior and determined peak shapes, whereas the effect of positive cooperative adsorption was negligible.
采用静态和动态方法，在两种不同的疏水作用层析树脂上研究了模型蛋白（α-乳白蛋白、牛血清白蛋白、溶菌酶和单克隆抗体）在单一组分和二元混合物中的吸附行为。建立了吸附过程的动力学模型，该模型考虑了蛋白质在吸附相中的展开和分子间相互作用。后者包括由于在吸附剂表面上的优先和多层吸附而产生的正协同相互作用，以及由于尺寸排阻和排斥引起的排除效应而产生的负协同相互作用。协同吸附导致单组分和多组分等温线的朗缪尔模型出现负或正偏差。该模型用于评估不同的蛋白质吸附机制及其结构不同形式对整体吸附模式的可能贡献，以及模拟不同装载条件下的色谱带轮廓。对于结构不稳定的蛋白质，整体吸附等温线主要由低表面覆盖率下未展开物种的结合和高表面覆盖率下的正协同吸附所支配。此外，无论结构稳定性如何，排除效应都强烈影响吸附平衡，特别是在低表面覆盖率下。在色谱洗脱的情况下，即动态条件下，展开、负协同吸附和动力学效应控制着保留行为并决定了峰形，而正协同吸附的影响可以忽略不计。
