Rodler Agnes, Ueberbacher Rene, Beyer Beate, Jungbauer Alois
a Department of Biotechnology , University of Natural Resources and Life Sciences , Vienna , Austria.
b Austrian Centre of Industrial Biotechnology , Vienna , Austria.
Prep Biochem Biotechnol. 2019;49(1):1-20. doi: 10.1080/10826068.2018.1487852. Epub 2019 Feb 8.
Hydrophobic interaction chromatography is a very popular chromatography method for purification of proteins and plasmids in all scales from analytical to industrial manufacturing. Despite this frequent use, the complex interaction mechanism and the thermodynamic aspects of adsorption in hydrophobic interaction chromatography are still not well understood. Calorimetric methods such as isothermal titration calorimetry and flow calorimetry can help to gain a deeper understanding of the adsorption strength, the influence of salt type and temperature. They can be used to study conformational changes of proteins, which are often associated with the adsorption in hydrophobic interaction chromatography. This review offers a detailed introduction into the thermodynamic fundamentals of adsorption in hydrophobic interaction chromatography with a special focus on the potential applications of isothermal titration calorimetry and flow calorimetry for studying specific problems and relationships of the adsorption behavior of proteins and its various influencing factors. Models for characterizing conformational changes upon adsorption are presented together with methods for assessing this problem for different proteins and stationary phases. All of this knowledge can contribute greatly to forming a sound basis for method development, process optimization and finding modelling strategies in hydrophobic interaction chromatography.
疏水作用色谱法是一种非常流行的色谱方法，可用于从分析规模到工业生产规模的蛋白质和质粒纯化。尽管这种方法被频繁使用，但疏水作用色谱法中复杂的相互作用机制以及吸附的热力学方面仍未得到很好的理解。等温滴定量热法和流动量热法等量热方法有助于更深入地了解吸附强度、盐类型和温度的影响。它们可用于研究蛋白质的构象变化，而这种变化通常与疏水作用色谱法中的吸附有关。本综述详细介绍了疏水作用色谱法中吸附的热力学基本原理，特别关注等温滴定量热法和流动量热法在研究蛋白质吸附行为的特定问题及其各种影响因素之间关系方面的潜在应用。文中介绍了用于表征吸附时构象变化的模型以及针对不同蛋白质和固定相评估该问题的方法。所有这些知识对于在疏水作用色谱法中形成方法开发、工艺优化和寻找建模策略的坚实基础有很大帮助。
