Fundamentals of Retention and Transport in Protein Ion-Exchange Chromatography

点击翻译按钮获取中文摘要

This project investigates how protein ion-exchange behavior is related to the structure of the proteins and of the stationary phase. A quantitative mechanistic understanding is sought to facilitate the development of more systematic chromatographic design procedures than are currently possible. The goal is to aid in design of new sorbent materials, which may ultimately result in stationary phases custom-designed for particular separations.The research covers several aspects of preaparative protein ion-exchange chromatography. It extends prior measurements of both linear retention and full isotherms, including measurements on closely related protein mutants to probe effects of protein structure and of multicomponent isotherms to investigate the role of protein-protein interactions. Uptake experiments employ scanning confocal microscopy to monitor intraparticle concentration profiles in real time. These observations of uptake and retention are analyzed in terms of stationary-phase structure, which is characterized in terms of both pore-size distribution and internal topography, determined respectively by inverse size-exclusion chromatography and atomic force microscopy.These various measurements are used to construct mechanistic models of transport and retention within chromatographic particles, and hence of column performance. Thus, by measuring and modelling quantitatively the key mechanisms underlying protein chromatography in real commercial stationary phases, this work may be applied to industrial bioprocessing and can facilitate improving current procedures.