(522b) Evaluation of Preferred Binding Orientations of Protein Therapeutics in Multimodal Chromatographic Systems Using Protein Fingerprinting and Dewetting Simulations

The identification of preferred binding regions and specific residues on proteins that interact with ligands and ligand coated surfaces is important for understanding the molecular basis for the purification of biological products from product related impurities. In this presentation we will discuss a variety of biophysical and simulation tools that can provide insights into these interactions in multimodal chromatographic systems. NMR with labelled proteins and ligand coated nanoparticles is employed to identify preferred binding domains for the Fc domain of antibodies and to examine the impact of multimodal ligand chemistry and ligand densities on this interaction. Umbrella sampling molecular simulations are then used to provide information on the contributions of ligand clustering to these interactions. High throughput protein surface footprinting using covalent labeling followed by LC/MS analysis is then employed to identify preferred binding domains for a relatively large number of model proteins as well as several new classes of protein biotherapeutics in multimodal systems as a function of pH. Finally, this data set is examined using a variety of simulation tools including dewetting calculations of protein and chromatographic surfaces to shed light on the nature of selectivity in these complex bioseparation systems.