Effects of antimicrobial preservatives on protein folding stability and subvisible particle formation in monoclonal antibody trastuzumab

To prevent microbial contamination, antimicrobial preservatives need to be added in multi-dose biopharmaceuticals; however, it often introduces risks to protein stability, potentially compromising therapeutic efficacy. In this study, we investigated the effects of different preservatives (benzyl alcohol, m-cresol, phenoxyethanol, and benzalkonium chloride) on the biophysical stability of trastuzumab, a monoclonal antibody widely used for treatment of HER2 receptor-positive cancers. Among the preservatives tested, benzyl alcohol (1.0 % v/v) and m-cresol (0.3 % w/v) significantly reduced the monomeric content after 5 days of end-over-end agitation stress. Benzyl alcohol was associated with a surge in nano- to micro-sized particles (21-fold increase) and decreased thermal stability (ΔT_m: -5.39 °C). m-Cresol uniquely triggered visible particle formation (>100 µm) within 72 h, raising concerns for injectable biologics. Benzalkonium chloride (0.01 %-0.04 % w/v) exhibited inconsistent concentration-dependent behavior, initially showing increase in subvisible aggregates before stabilizing through micelle formation at higher concentrations, albeit with irreversible secondary structural shifts toward β-sheet motifs. Conversely, phenoxyethanol (0.5 % v/v) exhibited higher compatibility, preserved the monomeric content, and suppressed particle generation to baseline levels. These findings underscore the necessity of preservative-specific compatibility assessments in formulation design for therapeutic biologics, positioning phenoxyethanol as a promising candidate for trastuzumab preservation owing to the balance between its antimicrobial efficacy and minimal destabilization.