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

基本信息

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

项目摘要

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.

该项目研究了蛋白质离子交换行为与蛋白质结构和固定相的结构如何相关。寻求一种定量的机械理解，以促进比目前更多的系统色谱设计程序的发展。目的是协助设计新的吸附剂材料，最终可能导致针对特定分离的固定相位的固定相。研究涵盖了预性蛋白质离子交换色谱的几个方面。它扩展了对线性保留率和全等温线的先前测量，包括对密切相关的蛋白质突变体的测量值，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。吸收实验采用扫描共聚焦显微镜实时监测颗粒内浓度谱。这些对摄取和保留率的观察是根据固定相结构的分析，这些结构的特征是孔径分布和内部地形，分别通过反向尺寸 - 分类色谱和原子力显微镜确定。这些测量用于各种测量值。构建色谱颗粒内运输和保留的机械模型，因此构建了色谱柱性能。因此，通过定量测量和建模实际商业固定阶段中蛋白质色谱的关键机制，这项工作可以应用于工业生物处理，并可以促进改善当前程序。