Abstract
Therapeutic monoclonal antibodies (mAbs) are prone to degradation viaaggregation and fragmentation. In this study, forced degradation of trastuzumab (TmAb) was explored in saline and in-vitro models having H2O2 and exposed to UV light (case study 1), both bleomycin (BML) formulation and ferrous ions (Fe2+) (case study 2), and sodium hypochlorite (NaOCl) (case study 3). Size exclusion chromatography, dynamic light scattering, spectroscopic analysis, and fluorescence microscope image processing was carried out for characterizing TmAb degradation. Saline samples containing TmAb and 0.1% H2O2 incubated at 40ºC for 1h in the presence of UV light showed increased monomer loss by more than 40% compared to TmAb sample without H2O2 exposed to UV light. Saline containing TmAb having both 0.1-unit BML and 0.25mM Fe2+ showed increased monomer loss by more than 50% compared to TmAb in saline having only Fe2+ or BML. A higherTmAb degradation was also observed in saline containing 0.01% NaOCl compared to saline without NaOCl. Samples containing aggregates of mAb showed altered protein structure. Degradation of TmAb in saline increased with time, temperature, and concentrations of H2O2, Fe2+, and NaOCl. At different analysis time points, TmAb monomer loss was higher in saline compared to human serum filtrate, an in-vitro model. Aggregate particles (> 2µm size) of TmAb were also observed in serum containing both Fe2+ and BML. It can be concluded that rapid TmAb degradation significantly enhanced due to various stress factors, and the aggregates couldresult in enhanced immunogenic risk to the patients.
Published Version
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