Protein-based therapeutics typically require high concentrations of the active protein, which can lead to protein aggregation and high solution viscosity. Such solution behaviors can limit the stability, bioavailability, and manufacturability of protein-based therapeutics and are directly influenced by the charge of a protein. Protein charge is a system property affected by its environment, including the buffer composition, pH, and temperature. Thus, the charge calculated by summing the charges of each residue in a protein, as is commonly done in computational methods, may significantly differ from the effective charge of the protein as these calculations do not account for contributions from bound ions. Here, we present an extension of the structure-based approach termed site identification by ligand competitive saturation-biologics (SILCS-Biologics) to predict the effective charge of proteins. The SILCS-Biologics approach was applied on a range of protein targets in different salt environments for which membrane-confined electrophoresis-determined charges were previously reported. SILCS-Biologics maps the 3D distribution and predicted occupancy of ions, buffer molecules, and excipient molecules bound to the protein surface in a given salt environment. Using this information, the effective charge of the protein is predicted such that the concentrations of the ions and the presence of excipients or buffers are accounted for. Additionally, SILCS-Biologics also produces 3D structures of the binding sites of ions on the proteins, which enable further analyses such as the characterization of protein surface charge distribution and dipole moments in different environments. Notable is the capability of the method to account for competition between salts, excipients, and buffers on the calculated electrostatic properties in different protein formulations. Our study demonstrates the ability of the SILCS-Biologics approach to predict the effective charge of proteins and its applicability in uncovering protein-ion interactions and their contributions to protein solubility and function.
Keywords: Debye−Hückel−Henry charge; buffer; excipient; formulation; ion; molecular dynamics; salt.