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.

Isotonic concentrations of excipients control the dimerization rate of a therapeutic immunoglobulin G1 antibody during refrigerated storage based on their rank order of native-state interaction.

Inert co-solutes, or excipients, are often included in protein biologic formulations to adjust the tonicity of liquid dosage forms intended for subcutaneous delivery. Despite the low concentration of their use, many of these excipients alter protein-protein interactions such as dimerization and aggregation rates of high concentration monoclonal antibody (mAb) therapeutics to varying extents during long-term refrigerated clinical storage, challenging the formulation scientist to make informed excipient selections at the earliest stages of development when protein supply and time are often limited. The objectives of this study were to better understand how isotonic concentrations of excipients influence the dimerization rates of a model mAb stored at refrigerated and room temperatures and explore protein sparing biophysical methods capable of predicting this dependence. Despite their prevalence of use in the biopharmaceutical industry, methods for assessing conformational stability such differential scanning calorimetry and isothermal equilibrium unfolding showed little predictive power and we highlight some of the assumptions and technical challenges of their use with mAbs. Conversely, measures of colloidal stability of the native-state such as preferential interaction coefficients measured by vapor pressure osmometry and solubility assessed by polyethylene-glycol induced precipitation correlated reasonably well with the mAb dimerization data and are most consistent with the excipients tested minimizing dimerization by interacting favorably with the residues comprising the protein-protein association interface.