(342c) Theoretical Investigations of Multiple Weak Interactions in Multimodal Chromatography Using Molecular Dynamics Simulations and Protein Surface Characterization

Multimodal (MM) chromatography has emerged as an extremely promising form of chromatography. Recent studies published by our group and others have shown that an appropriate choice of ligand chemistry and fluid phase modifiers can modulate the different modes of interactions present in MM systems to achieve enhanced selectivities as compared to traditional single-mode chromatography. However, exciting as the experimental results might be, there is still a lack of mechanistic details in these MM systems which is essential to optimize desired separations. The goal of the work presented here is to provide fundamental understanding of the nature of water-mediated protein-ligand interactions at a molecular level which can provide significant insight into the design of MM ligands, the roles of synergy and the modulation of selectivity using fluid phase modifiers (FPMs).

A combination of theoretical techniques has been used to understand MM interactions. Molecular Dynamics (MD) simulations have been employed to gain molecular insights into how multiple weak interactions in MM systems work together to create binding selectivity. All-atom explicit MD simulations have been performed with two different MM ligands interacting with proteins and the results are analyzed to understand binding preferences of these MM ligands in terms of the interaction chemistry and their conformational flexibility. Simulations have also been performed with different fluid phase modifiers to understand how co-solutes affect protein-ligand binding and to shed light on the different mechanisms which govern their behavior.

Protein surface characterization techniques have also been employed as a fast, coarse-grained approach to map proteins based on their potential to interact with different modes. Protein-ligand binding in MM systems involves a combination of electrostatic and hydrophobic interactions. To accurately measure the individual contributions of both these modes, and their possible synergistic effects to the overall binding behavior requires the characterization of the molecules involved in binding in terms of their electrostatic and hydrophobic potential. While electrostatic interactions between two molecules can be easily calculated, hydrophobicity has been difficult to understand. Hence, hydrophobic characterization of proteins has been carried out here using recently developed methods which incorporate the “context-dependence” of hydrophobicity. The results from these calculations, together with the molecular understanding gained from MD simulations, have been used to explain chromatographic retention behavior of proteins in MM systems on different resins and under different fluid phase conditions.

The work presented here demonstrates the use of theoretical techniques, especially MD simulations, to probe atomistic details of protein-ligand binding which can shed light on and indeed predict protein retention behavior in chromatographic systems. Finally, the knowledge base created using these simulations can be used to select appropriate combinations of MM ligands and modifiers to achieve unique selectivities for challenging protein separations.