Fundamentals of Retention and Transport in Protein Ion-Exchange Chromatography

本项目研究蛋白质离子交换行为如何与蛋白质和固定相的结构相关。 一个定量的机制的理解，寻求更系统的色谱设计程序比目前可能的发展。 目的是帮助设计新的吸附剂材料，这可能最终导致定制设计的固定相用于特定的separation.The研究涵盖了preparative蛋白质离子交换色谱的几个方面。 它扩展了线性保留和完整等温线的先前测量，包括对密切相关的蛋白质突变体的测量，以探测蛋白质结构和多组分等温线的影响，以研究蛋白质-蛋白质相互作用的作用。 摄取实验采用扫描共聚焦显微镜监测颗粒内浓度分布在真实的时间。 这些观测的吸收和保留的固定相结构，其特征在于在两个孔径分布和内部地形，分别由反相尺寸排阻色谱法和原子力microscopy.These不同的测量被用来构建色谱颗粒内的运输和保留的机理模型，因此列的性能。 因此，通过定量测量和建模的关键机制蛋白质色谱在真实的商业固定相，这项工作可以应用于工业生物处理，可以促进改善目前的程序。

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.