Towards stable biologics: understanding co-excipient effects on hydrophobic interactions and solvent network integrity

The formulation of biologics for increased shelf life stability is a complex task that depends on the chemical composition of both the active ingredient and any excipients in solution. A large number of unique excipients are typically required to stabilize biologics. However, it is not well-known how these excipient combinations influence biologics stability. To examine these formulations at the molecular level, we performed molecular dynamics simulations of arginine - a widely used excipient with unique properties - in solution both alone and with equimolar concentrations of lysine or glutamate. We studied the effects of these mixtures on a hydrophobic polymer model to isolate excipient mechanisms on hydrophobic interactions relevant in both protein folding and aggregation, crucial phenomena in biologics stability. We observed that arginine is the most effective single excipient in stabilizing hydrophobic polymer folding, and its effectiveness is augmented by lysine or glutamate addition. We decomposed the free energy of polymer folding/unfolding to identify that the key source of arginine-lysine and arginine-glutamate synergy is a reduction in destabilizing polymer-excipient interactions. We additionally applied principles from network theory to characterize the local solvent network embedding the hydrophobic polymer. Through this approach, we found arginine supports a more highly connected and stable local solvent network than in water, lysine, or glutamate solutions. These network properties are preserved when lysine or glutamate are added to arginine solutions. Taken together, our results highlight important molecular features in excipient solutions that establish the foundation for rational formulation design.