Preparative Chromatography of Biological Macromolecules

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DESCRIPTION (provided by applicant): The proposed research seeks to improve the efficacy of preparative chromatographic separations of large biomacromolecules and assemblies, ranging from large proteins to viruses. Larger proteins such as monoclonal antibodies are increasingly found among licensed biotherapeutics, while viruses and virus-like particles are purified chromatographically as vaccines and as gene therapy vectors. The approaches that will be used to accomplish such improvements are based on developing a better quantitative and qualitative understanding of the mechanisms of adsorption, desorption and transport of large solutes in chromatographic media. The specific aims that will be addressed within this framework of seeking structure-performance relationships will employ chromatographic measurements and modification of stationary-phase surfaces, as well as several imaging methods, viz. electron tomography to determine the 3D structure of pore networks, scanning confocal microscopy to monitor uptake of solutes into stationary phases, and atomic force microscopy to observe the structure of adsorbate layers. The insights obtained from these methods will be coupled with appropriate modeling approaches, including simulations of solute diffusion in pore networks, modeling of uptake into chromatographic media, and colloidal modeling of adsorbate interactions. The experimental and modeling approaches will be used synergistically, and evaluated using several proteins and viruses on a variety of commercial stationary phases that are routinely used in preparative chromatography practice. Successful completion of the proposed research will facilitate efficient and rational approaches to stationary-phase selection for a particular application, and aid in methods for designing novel stationary phases.