Improving the Understanding and Application of Multi-Modal Chromatography

由美国国家科学基金会颁发的化学和生物分离项目奖支持伦斯勒理工学院的Steven Cramer教授和他的同事Shekhar Garde和Curt Breneman对多模态（MM）色谱系统中选择性本质的更深入理解。该项目将采用最先进的新型多模态配体和材料库与蛋白质库相结合，详细研究MM配体-蛋白质相互作用。自由溶液MM配体与一组标记的模型蛋白的结合将使用NMR来确定结合动力学和配体在蛋白质上的结合位点。将对天然蛋白质进行化学修饰，以产生具有各种表面性质组合的多种蛋白质阶梯。在一系列操作条件下，将对这些蛋白质以及一组市售蛋白质进行色谱实验，以研究蛋白质保留行为，并确定这些MM系统的合适等温模型。为了补充实验，将进行溶液中蛋白质-配体结合的大规模MD模拟，以及蛋白质-多模态树脂相互作用的粗粒度建模方法。这些模拟将提供配体结合位点、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和粗粒度模拟也将提供重要的数据，用于生成新的分子描述符。预测定量结构性质关系（QSPR）模型的蛋白质亲和力和等温线参数将确定使用色谱数据和新的分子描述符。该项目将对蛋白质纯化的MM色谱技术的发展和实施产生重大影响。这项工作将促进下一代MM色谱系统的设计，并将为MM色谱提供预测工具，这将促进这些系统纯化生物治疗药物的方法开发。改进的MM色谱系统的发展将有可能减少纯化治疗性蛋白质所需的下游处理步骤的数量，降低生产生物制药的成本。此外，具有独特选择性的多模态分离系统将为质谱分析蛋白质提供新的前端分离。最后，对蛋白质与各种类型的多模态配体和表面的相互作用模式的深入了解将对包括生物材料、生物传感器和药物发现在内的分离领域产生潜在的影响。本研究也将对化学工程专业研究生和本科生的教育产生重要影响。建议的研究将在生物技术与跨学科研究中心进行，该中心为研究生和本科生提供优秀的多学科培训机会。学生将接触到细胞培养，溶液核磁共振光谱，色谱，分子模拟和化学计量学。该项目的研究成果也将被纳入PI最近开发的化学工程高级实验室色谱实验以及色谱分离过程课程中。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，将激发分子馆项目的新方面，它使用动画电影来教授和激励各级学生关于分子迷人世界的知识。

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.