Impact of Various Forced Oxidative Stress Factors in Rapid Degradation of mAb: Trastuzumab as a Case Study.

Purpose: Therapeutic monoclonal antibodies (mAbs) are prone to degradation via aggregation and fragmentation. In this study, forced degradation of trastuzumab (TmAb) was explored in saline and in-vitro models having HO and exposed to UV light (case study 1) both bleomycin (BML) formulation and ferrous ions (Fe) (case study 2) and sodium hypochlorite (NaOCl) (case study 3).

Methods: Size exclusion chromatography, dynamic light scattering, spectroscopic analysis, and fluorescence microscope image processing was carried out for characterizing TmAb degradation.

Results: Saline samples containing TmAb and 0.1% HO incubated at 40ºC for 1 h in the presence of UV light showed increased monomer loss by more than 40% compared to TmAb sample without HO exposed to UV light. Saline containing TmAb having both 0.1-unit BML and 0.25 mM Fe showed increased monomer loss by more than 50% compared to TmAb in saline having only Fe or BML. A higher TmAb 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 HO, Fe 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 (> 2 µm size) of TmAb were also observed in serum containing both Fe and BML.

Conclusion: It can be concluded that rapid TmAb degradation significantly enhanced due to various stress factors, and the aggregates could result in enhanced immunogenic risk to the patients.