Multimodal chromatography presents unique selectivity in bio-separations, but a fundamental understanding of differences in multimodal (MM) ligands’ behavior still eludes us. Recent simulations work in our lab on MM-ligand immobilized self assembled monolayer (SAM) surfaces has demonstrated that ligand flexibility and density differences can result in surfaces with relatively large patches of hydrophobicity and charge. These self-associated ligand patterns have also been found to be persistent in chromatographically relevant higher salt concentrations and could govern protein-surface interactions. Here we develop that premise using enhanced sampling in molecular dynamics simulations of these systems. First, INDirect Umbrella Sampling (INDUS) was used to study the hydrophobic nature of surfaces where immobilized MM-ligands were found to aggregate and create hydrophobic and charged patches, and then compared with low ligand density surfaces without ligand self-association. To further understand the importance of ligand clustering in protein adsorption, free energies of binding of model proteins to a series of SAM surfaces with varying MM-ligand densities were calculated from potential of mean force (PMF) measurements performed using umbrella sampling. The possibility that water-mediated interactions during binding and unbinding at the protein-SAM interaction interface show hysteresis-like behavior was also investigated. Correlations between the calculated free energies and corresponding patch distributions for those ligand surfaces, inform us about the role of ligand density driven self-association in adsorption. Our study presents an understanding of hydrophobic behavior in multimodal systems at different ligand densities and connects multimodal resin surface properties with density dependent behavior during protein adsorption, which in turn has implications for surface property driven multimodal resin design and selection.

Impact of multimodal ligand density on free energy of adsorption of proteins to ligand-immobilized SAM surfaces: A molecular dynamics study

ACS Fall 2021

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