Nanoscopic Understanding of Protein Transport and Structure Dynamics in Charged Gels for Protein Chromatography

University Of Virginia Main Campus, Charlottesville VA

Investigators

Abstract

1032727 Carta Intellectual Merit The broad objectives of this research are a fundamental understanding of protein transport in charged gels for protein chromatography and the development of new matrices incorporating flexible polymers in a rigid support matrix. Although protein diffusion in neutral gels is generally well understood, our basic knowledge of how adsorbed biomolecules diffuse in oppositely charged matrices is currently extremely limited. New matrices obtained by incorporating charged dextran polymer grafts in porous supports and exhibiting unique protein transport properties have been developed in prior work. However, the underlying transport mechanisms are unknown, which prevents the development of optimized materials and their description with fundamentally sound models. Thus, the proposed research has three main goals: - To determine the structure and dynamic behavior of dextran-grafted matrices by dynamic light scattering (DLS) in order to understand fluctuations and structure dynamics of polymer chains in these systems. - To study protein transport in these materials at the nanoscopic scale using single molecule tracking techniques to determine the mobility of individual protein molecules in matrices functionalized with charged polymers. - To develop molecular models at multiple length scales to describe the structure and dynamic behavior of polymer functionalized matrices as well as their interactions with proteins, and use these results to elucidate the experiments in goal 2 and construct physically accurate conceptual and phenomenological models for practical rate predictions. Collectively, the components of this research will provide a yet unrealized level of understanding of transport phenomena in these complex media, the basis to develop new, more effective materials, and models to aid the design and optimization of chromatographic processes. Broader Impact: Advances in the biomedical sciences have raised expectations for new biologics-based drugs for the prevention, treatment, and cure of serious diseases and, indeed, new protein based biopharmaceuticals are having a huge impact on the lives of people. However, separating and purifying these biomolecules is complicated by their slow diffusion, which currently limits the performance of industrial biochromatography. The proposed project has the potential of transforming downstream processing of biopharmaceuticals by introducing new, more effective stationary phases as well as an unprecedented level of understanding of transport phenomena, thereby enhancing process understanding and control as well as reducing dramatically the time and effort needed for process development and design. Research and education will be integrated by incorporating the results of this project in educational modules for college level students, by strengthening the education of graduate students with synergistic activities in experiments and molecular modeling, and through a Short Course in Protein Chromatography for industry professionals held annually at UVa. Outreach will include developing laboratory demonstrations for use in sessions for the Upward Bound program for high school students, by facilitating participation of minority undergraduate students in our Short Course, and by recruiting graduate students from underrepresented groups.