OBJECTIVE To investigate the impact of benzyl alcohol(BA) concentration on etanercept formulation stability under multiple stress conditions, thereby supporting the long-term storage of etanercept.

METHODS Using etanercept as a model drug, this study systematically evaluated the stability of BA-containing multi-dose formulations under various stress conditions, including low temperature, room temperature, high temperature, light exposure, freeze-thaw cycles, and shaking. Techniques such as dynamic light scattering(DLS), differential scanning calorimetry(DSC), nano differential scanning fluorimetry(nanoDSF), intrinsic fluorescence(IF), size exclusion chromatography(SEC), and 8-anilino-1-naphthalenesulfonic acid were employed to assess formulation stability, and antibacterial activity was evaluated through bacteriostatic experiments.

RESULTS DLS and IF analysis showed that BA induced initial conformational unfolding immediately after addition to the formulation. DSC and nanoDSF results indicated that the stability of etanercept decreased with increasing BA concentration, making it more prone to aggregation. Further SEC-based stability assessments under multiple stress conditions revealed that BA may trigger degradation of etanercept, and such degradation likely exhibits BA concentration dependence.

CONCLUSION Within the framework of this study, 0.9% BA is the optimal concentration that balances antibacterial efficacy and minimization of aggregation risk. In addition, BA-containing etanercept formulations should be stored at 2–8 ℃.