The proposed renewal project builds upon our significant body of prior work in displacement chromatography in ion exchange (IEX) and hydrophobic interaction (HIC) systems as well as our new understanding of protein interactions in multimodal (MM) systems to create a hybrid separations technology that will produce highly selective and high capacity protein separations. This work will is based upon recent advances in our lab that have demonstrated that the use of selective displacers can result in dramatically enhanced selectivity in hydroxyapatite (HA) and other multimodal (MM) chromatographic systems. We will employ the tools of high-throughput screening (HTS) and molecular simulation to provide fundamental insight into the nature of this selective behavior and to address several critical issues related to the design of selective displacers for MM systems.
A robotic high-throughput displacer screening technique will be employed to screen for selective displacers in MM systems and to evaluate the effects of protein, displacer structure, mobile phase characteristics and displacer concentration. This work will employ protein and displacer libraries previously developed in our group to establish model protein pairs and potential displacers for further analysis. Multicomponent adsorption isotherms will also be generated to study competitive binding, synergistic interactions and hysteresis effects in these displacer-protein-MM systems. Column studies will be conducted to determine the level of enhanced selectivity and performance that can be achieved with this hybrid approach. A dynamic affinity plot analysis will also be carried out to examine if the enhanced selectivity and displacement-desorption transitions can be understood from a mass action perspective. A wide array of experimental and theoretical techniques will be employed to examine the interactions of selective displacers with immobilized MM ligands. Surface plasmon resonance spectroscopy will examine kinetic effects while isothermal titration calorimetry will provide insights into the underlying mechanisms involved in the selective displacement process in MM systems. A series of molecular simulations will be carried out to study the interactions of selective displacers with MM surfaces and to provide fundamental insight into the importance of various intermolecular interactions. Molecular simulations of multicomponent adsorption will also be carried out with small proteins in solutions of displacers and salt counter-ions to obtain further insights into competitive binding and the mechanism of the displacement process in MM systems. Finally, the use of selective displacers for addressing challenging biopharmaceutical separations in MM systems will be evaluated using complex biological mixtures provided by an industrial collaborator.
There is an urgent need to develop new technologies to meet the growing challenge for safe, low cost next generation biotherapeutics ranging from proteins (e.g., antibodies, enzymes) to polysaccharides (e.g., heparin, hyaluronic acid) to cellular based technologies. The proposed project will produce a powerful new hybrid separations technology to address this need. The insights obtained in the proposed research will aid in the development and implementation of improved selective displacement MM chromatographic systems for the purification of therapeutic proteins. This will potentially eliminate the need for expensive affinity chromatographic systems and will enable a reduction in the number of required downstream processing steps, with a significant impact on the costs of producing biopharmaceuticals. The fundamental studies described in this project will also provide important insights into modification of hydrophobicity/philicity and binding in complex multicomponent mixtures which will establish a new platform for deeper understanding of a range of systems where these effects are important (e.g. bioseparations, biosensors, biomaterials). Clearly, if successful the proposal will have a significant and broad impact on bioprocessing and a number of health related fields.
The proposed research will also have an important impact on the education of both graduate and undergraduate chemical engineering students as well as high school students. The Cramer laboratory has a long track record of producing chemical engineers with a first rate training in chromatographic bioprocessing. This training will continue with the students involved in this project. The proposed research will be carried out in the Center for Biotechnology and Interdisciplinary Studies which provides excellent multidisciplinary training opportunities for students and exposure to a wide range of experimental and simulation techniques. The research developed in this project will also be incorporated into a chemical engineering senior laboratory chromatography experiment recently developed 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.