Dissolved oxygen effects on human growth hormone stability during freeze-thaw and metal-catalyzed oxidation

Dissolved oxygen (DO) has been proposed to influence protein stability during freeze-thaw (F/T) processing, yet its direct impact remains unclear. This study evaluated whether DO levels or vacuum degassing affect the stability of human growth hormone (hGH) during F/T. Buffers were degassed with or without vacuum-induced surface freezing (VISF). Degassing itself did not induce particle formation. Upon freezing substantial aggregation occurred, independent of the presence or absence of dissolved gas. To isolate oxygen effects, hGH solutions equilibrated with nitrogen, air, or oxygen were subjected to F/T cycles or frozen storage at -40°C. DO levels did not significantly influence turbidity, particle counts, intact LC-MS profiles, while only minor differences were observed by SEC. Under metal-catalyzed oxidative conditions, however, oxygen modified aggregation pathways depending on the catalyst. These findings demonstrate that DO alone does not substantially affect hGH stability during F/T but becomes relevant in the presence of catalytic oxidative stress. Protein destabilization during freezing is instead dominated by interfacial stresses, particularly those associated with VISF. Together, these results help to distinguish chemical and physical contributions of dissolved gases in protein formulations.