Multimodal (MM) Chromatography offers highly selective protein separation systems by taking advantage of hydrophobic, electrostatic and Van der Waals interactions. Due to the complexity of such protein-ligand interactions, there is a need for the molecular level understanding of the nature of proteins binding to different MM chromatographic systems. Recent work in our lab has shown the importance of specific domains towards binding for multi-domain molecules such as antibodies. In this work, we employ Nuclear Magnetic Resonance spectroscopy (NMR) to determine the amino acids involved in binding with chromatographic ligands. NMR titration experiments with MM ligands and isotopically enriched Fc domain of a monoclonal antibody were used to determine the relative binding affinities of interaction sites on the protein surface. Further, in order to mimic chromatographic surface, a pseudo solid-state resin system i.e. gold nanoparticles functionalized with Self Assembled Monolayers (SAM) presenting a MM ligand headgroup was used to study the binding. In order to study the co-operativity and avidity effects in these MM chromatographic systems binding was studied with gold nanoparticles functionalized with SAMs presenting different ligand densities of MM ligand headgroup. Further, molecular dynamics simulations with ligands in free solution around the protein were performed to gain additional molecular details on multimodal-ligand protein binding. The results presented in this poster demonstrate that NMR spectroscopy can be employed in concert with chromatography and molecular dynamics simulations to provide important fundamental insights into protein multimodal surface interactions.   <br/>

Investigation into molecular basis of protein interactions in multimodal chromatography using nuclear magnetic resonance spectroscopy and molecular dynamics simulations

255th National Meeting (2018)

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