Benzyl alcohol exacerbates freeze-thaw-induced aggregation of trastuzumab: elucidating mechanisms and formulation implications for clinical practice.

The increasing adoption of multi-dose formulations has improved equitable access to biologic therapies. Preservatives ensure the safety and efficacy of multi-dose formulations. While benzyl alcohol (BA)'s destabilizing effects on monoclonal antibodies (mAbs) are known, its stability under multifactorial stresses remains unclear. Given that preservatives are incorporated for long-term storage, understanding their impact on stability during extended use is critical. Using trastuzumab as a model mAb, this study systematically evaluated the stability of BA-containing formulations under various stress conditions, including long-term, accelerated, stress, light exposure, and repeated freeze-thaw cycles (FTCs). Analytical techniques (micro-flow imaging, chromatography, spectroscopy) and antimicrobial testing were employed. Key findings revealed severe destabilization of trastuzumab upon FTCs, with BA synergistically exacerbating damage through three mechanisms: (1) disruption of higher-order structure in solution; (2) cryoconcentration-enhanced BA-mAb interactions during freezing, coupled with BA-induced micro-ice crystal formation that increases destabilizing ice-water interfaces; and (3) thawing-induced stresses, including interfacial exposure, shear forces from concentration gradients, and methionine oxidation due to increased dissolved oxygen. These combined effects increased subvisible particles, soluble aggregates, and acidic variants. Antimicrobial testing identified 1.1 % BA as the optimal concentration, providing sufficient preservation while minimizing FTC-induced aggregation risks compared to 2 % BA. This study highlights the critical stability challenges of BA in multi-dose formulations and elucidates how BA synergizes with FTCs to compromise therapeutic antibody integrity. The findings emphasize avoiding frozen storage of BA-containing biologics and maintaining reconstituted products at 2-8 °C. These insights are vital for ensuring the stability, safety, and efficacy of multi-dose antibody formulations.