Improving the Understanding and Application of Multi-Modal Chromatography

这个由化学和生物分离计划授予的NSF奖项支持伦斯勒理工学院的Steven Cramer教授及其同事Shekhar Garde和Curt Breneman为建立对多模式(MM)色谱系统选择性本质的更深层次的理解所做的工作。这个项目将使用最先进的新型多模式配体和材料的库与蛋白质库相结合，详细研究MM配体与蛋白质的相互作用。自由溶液MM配体与一组标记的模型蛋白质的结合将使用核磁共振来研究，以确定结合动力学和蛋白质上的配体结合位置。将对天然蛋白质进行化学修饰，以产生一套具有各种表面性质组合的蛋白质梯形图。我们将在各种操作条件下对这些蛋白质和一组商业可获得的蛋白质进行层析实验，以研究蛋白质保留行为，并为这些MM系统确定合适的等温线模型。作为实验的补充，将进行大规模的分子动力学模拟蛋白质-配体在溶液中的结合，以及蛋白质-多峰树脂相互作用的粗粒度建模方法。这些模拟将提供关于配体结合位置、结合模式以及溶液中相互作用的动力学和热力学的数据。MD和粗粒度模拟还将提供重要的数据，这些数据将用于生成新的分子描述符。蛋白质亲和力和等温线参数的预测定量结构性质关系(QSPR)模型将使用层析数据和新的分子描述符来确定。该项目将对MM层析技术在蛋白质纯化方面的发展和实施产生重大影响。这项工作将促进下一代MM层析系统的设计，并将为MM层析提供预测工具，从而促进利用这些系统进行生物治疗药物纯化的方法开发。改进的MM层析系统的开发将有可能减少提纯治疗性蛋白质所需的下游加工步骤，从而降低生产生物制药的成本。此外，具有独特选择性的多模式分离系统将为蛋白质的质谱分析提供新的前端分离。最后，深入了解蛋白质与各种类型的多模式配体和表面的相互作用模式，将不仅对生物材料、生物传感器和药物发现等领域产生潜在的影响。这项拟议的研究也将对化学工程研究生和本科生的教育产生重要影响。拟议的研究将在生物技术和跨学科研究中心进行，该中心为研究生和本科生提供绝佳的多学科培训机会。学生将接触到细胞培养、溶液核磁共振光谱、层析、分子模拟和化学计量学。在这个项目中开展的研究还将被纳入由该研究所最近开发的一个化学工程高级实验室层析实验以及一个关于色谱分离过程的课程。最后，在这个项目中进行的模拟，以及分子相互作用的概念部分，将激发分子库项目的新方面，该项目使用动画电影来教授和启发各级学生关于迷人的分子世界。

0933169CramerThis NSF award by the Chemical and Biological Separations program supports work by Professor Steven Cramer and his colleagues Shekhar Garde and Curt Breneman at Rensselaer Polytechnic Institute to establish a deeper understanding of the nature of selectivity in multi-modal (MM) chromatographic systems. This project will employ a state of the art library of novel multi-modal ligands and materials in concert with protein libraries in a detailed study of MM ligand - protein interactions. The binding of free solution MM ligands to a set of labeled model proteins will be studied using NMR to determine binding kinetics and ligand binding sites on the proteins. Chemical modifications of native proteins will be carried out to generate a diverse set of protein ladders possessing various combinations of surface properties. Chromatographic experiments will be carried out with these proteins along with a set of commercially available proteins under a range of operating conditions to study protein retention behavior and to determine appropriate isotherm models for these MM systems. To complement the experiments, large scale MD simulations of protein-ligand binding in solution, and coarse-grained modeling approaches for protein - multi-modal resin interactions will be carried out. These simulations will provide data on ligand binding sites, modes of binding, and dynamics and thermodynamics of interactions in solution. MD and coarse grained simulations will also provide important data that will be used to generate novel molecular descriptors. Predictive quantitative structure property relationship (QSPR) models of protein affinity and isotherm parameters will be determined using the chromatographic data and novel molecular descriptors. The proposed project will have a significant impact on the development and implementation of MM chromatographic technology for protein purification. This work will facilitate the design of next generation MM chromatographic systems and will provide predictive tools for MM chromatography that will facilitate methods development with these systems for the purification of biotherapeutics. The development of improved MM chromatographic systems will potentially enable a reduction in the number of downstream processing steps required for the purification of therapeutic proteins, reducing the costs of producing biopharmaceuticals. Further, multi-modal separation systems with unique selectivities will provide new novel front end separations for the analysis of proteins by mass spectrometry. Finally, a deeper understanding of the modes of interactions of proteins with various classes of multi-modal ligands and surfaces will have potential implications beyond separations to fields including biomaterials, biosensors, and drug discovery. The proposed research will also have an important impact on the education of both graduate and undergraduate chemical engineering students. The proposed research will be carried out in the Center for Biotechnology and Interdisciplinary Studies which provides excellent multidisciplinary training opportunities for graduate and undergraduate students. Students will gain exposure to cell culture, solution NMR spectroscopy, chromatography, molecular simulations and chemometrics. The research developed in this project will also be incorporated into a chemical engineering senior laboratory chromatography experiment recently developed by the PI as well as a course on Chromatographic Separation Processes. Finally, simulations performed in this project, as well as conceptual parts of molecular interactions will motivate new aspects of the Molecularium project, which uses animation movies to teach and inspire students at all levels about the fascinating world of molecules.