Managing Dissolved Air Throughout Freeze-Thaw Process to Reduce Aggregation of Human Serum Albumin.

Freeze-thaw (FT) stress compromises protein stability through ice crystal formation and exposure to air-liquid and ice-liquid interfaces. This study investigated whether deaeration could mitigate Human Serum Albumin (HSA) aggregation by limiting gas-mediated interfacial stress. Tween 80 and trehalose were included to evaluate deaeration efficiency in the presence of common formulation components. The study demonstrated that deaeration using either PTFE or glass stirring consistently reduced dissolved oxygen (DO) from ~ 85% to ~ 20%, and that the extent of gas removal was independent of Tween 80 or trehalose concentration. SEC-HPLC analysis confirmed > 94% HSA monomer retention, with minimal loss attributable to deaeration. Most importantly, subvisible particles exhibited by microflow imaging (MFI) revealed the protective effect of dissolved gas reduction, with deaerated samples consistently showing lower aggregation (~ 1000 to ~ 2500 particles/mL) compared to untreated HSA samples with higher aggregation (~ 2000 to ~ 6500 particles/mL) following freezing at -80ºC. Crucially, thawing the protein samples under vacuum to prevent air redissolution preserved low dissolved air content through five FT cycles, resulting in lower aggregation than samples thawed at atmospheric pressure. The combination of deaeration process and thawing under vacuum serve as a holistic engineering approach to manage dissolved air content in protein solution and reduce the overall freeze-induced stress on HSA protein formulations.