Additivity of Transfer Free Energies Enables the Description of Complex Protein Formulations in Implicit Solvent Molecular Dynamics Simulations.

A complex 3D structure and the surrounding environment determine the function and stability of a protein. Various osmolytes can be added to a protein drug formulation to stabilize the native protein structure by preventing unfolding and aggregation. In this context, the concept of transfer free energy, which represents the change in chemical potential of a protein being transferred from water to an osmolyte solution, has emerged as a powerful tool to elucidate the energetics involved in the protein-osmolyte interaction. In the present work, we experimentally determine the transfer free energies for the excipients sodium chloride, arginine hydrochloride, and polysorbate 20, which are frequently used in pharmaceutical protein formulations. We show that these excipients display distinct patterns of exclusion or interaction toward different moieties on the protein surface. Furthermore, we report that the free energy cost for transferring a protein to a formulation composed of multiple components can be calculated by summing up the contributions of the individual components. This finding suggests that additivity applies to the transfer free energies. We demonstrate that this additive behavior can be leveraged to accurately and efficiently model complex protein formulations. Additionally, we discuss how transfer free energies can be incorporated within implicit solvent molecular dynamics calculations, providing a direct link between experiments and simulations. Our molecular dynamics results show good agreement with experimental data for lysozyme, interferon α-2a, and granulocyte colony-stimulating factor, for both single- and multicomponent matrices, demonstrating the validity of our approach.