Non-ideal osmolality behavior in high-concentration protein formulations: mechanistic insights from freezing-point...

Subcutaneous delivery of high-concentration protein formulations is increasingly adopted to overcome injection volume limitations. However, osmolality, which is closely associated with injection pain and patient safety, remains poorly understood compared with viscosity. This study systematically investigates the non-ideal osmolality behavior of high-concentration protein formulations (i.e., 150 mg/mL monoclonal antibody A, bovine serum albumin, and lysozyme) containing six polyol or carbohydrate excipients including glycerol, sorbitol, mannitol, sucrose, trehalose, and raffinose, and elucidates the underlying mechanisms through both experimental and computational approaches. Osmolality determined by freezing-point depression (FPD) and vapor-pressure deficit (VPD) osmometry consistently exceeded theoretical predictions of 291 mOsm/L. Excipient molecular structure showed a strong effect: simple molecules such as glycerol produced the lowest osmolality, while complex saccharides such as raffinose yielded the highest. A quadratic correlation (R² ≥ 0.98) was observed between osmolality and excipient molecular weight. Molecular dynamics simulations further revealed that excipients with greater numbers of hydroxyl and ether groups enhanced solute-water hydrogen bonding, disrupted the water network, and reduced water crystallization and vaporization, thereby explaining the experimentally observed osmolality deviations. The detection constants of both FPD and VPD methods increased quadratically with the number of hydroxyl or ether groups, and the coexistence of proteins and excipients caused a synergistic, non-additive increase in osmolality. These findings reveal a mechanistic link between excipient molecular structure, hydrogen-bonding capacity, and non-ideal osmolality in high-concentration protein formulations, providing molecular-level insights to guide rational formulation design and improve the safety and tolerability of subcutaneous biotherapeutic delivery.