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

Abstract

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 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).

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% H₂O₂ 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 H₂O₂ exposed to UV light. Saline containing TmAb having both 0.1-unit BML and 0.25 mM Fe²⁺ showed increased monomer loss by more than 50% compared to TmAb in saline having only Fe²⁺ 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 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 (> 2 µm size) of TmAb were also observed in serum containing both Fe²⁺ 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.