Fundamentals of Retention and Transport in Protein Ion-Exchange Chromatography

蛋白质离子交换色谱中保留和传输的基础知识

• 批准号: 9977120
• 负责人: Abraham Lenhoff
• 金额: $ 25万
• 依托单位: University of Delaware
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-01-01 至 2003-12-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9977120&HistoricalAwards=false
• 关键词: Fundamentals; Retention; Transport; Protein; Ion

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.

该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。 寻求定量机制的理解，以促进比目前可能的更系统的色谱设计程序的开发。 目标是帮助设计新的吸附剂材料，最终可能产生为特定分离定制设计的固定相。该研究涵盖了制备型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，以探测蛋白质结构的影响，以及多组分等温线的测量，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焦显微镜实时监测颗粒内浓度分布。 这些吸收和保留的观察结果是根据固定相结构进行分析的，固定相结构以孔径分布和内部形貌为特征，分别通过反尺寸排阻色谱和原子力显微镜确定。这些不同的测量用于构建色谱颗粒内传输和保留的机械模型，从而构建色谱柱性能的机械模型。 因此，通过定量测量和模拟实际商业固定相中蛋白质色谱的关键机制，这项工作可以应用于工业生物加工，并可以促进改进当前的程序。