Suppression of Electrostatic Mediated Antibody Liquid-Liquid Phase Separation by Charged and Noncharged Preferentially Excluded Excipients.

Isotonic concentrations of inert cosolutes or excipients are routinely used in protein therapeutic formulations to minimize physical instabilities including aggregation, particulation, and precipitation that are often manifested during drug substance/product manufacture and long-term storage. Despite their prevalent use within the biopharmaceutical industry, a more detailed understanding for how excipients modulate the specific protein-protein interactions responsible for these instabilities is still needed so that informed formulation decisions can be made at the earliest stages of development when protein supply and time are limited. In the present report, subisotonic concentrations of the five common formulation excipients, sucrose, proline, sorbitol, glycerol, arginine hydrochloride, and the denaturant urea, were studied for their effect on the room temperature liquid-liquid phase separation of a model monoclonal antibody (mAb-B). Although each excipient lowered the onset temperatures of mAb-B liquid-liquid phase separation to different extents, all six were found to be preferentially excluded from the native state monomer by vapor pressure osmometry, and no apparent correlations to the excipient dependence of mAb-B melting temperatures were observed. These results and those of the effects of solution pH, addition of salt, and impact of a small number of charge mutations were most consistent with a mechanism of local excipient accumulation, to an extent dependent on their type, with the specific residues that mediate mAb-B electrostatic protein-protein interactions. These findings suggest that selection of excipients on the basis of their interaction with the solvent exposed residues of the native state may at times be a more effective strategy for limiting protein-protein interactions at pharmaceutically relevant storage conditions than choosing those that are excluded from the residues of the native state interior.