Improving the Understanding and Application of Multi-Modal Chromatography

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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.