Stability assessment of antibody-drug conjugate Trastuzumab emtansine in comparison to parent monoclonal antibody using orthogonal testing protocol

Abstract Antibody-drug conjugates (ADC) represent an emerging, novel class of biopharmaceuticals. The heterogeneity originating from the sophisticated structure requires orthogonal analytical techniques for quality and stability assessment of ADC to ensure safety and efficacy. In this study, the stability of Trastuzumab (recombinant humanized IgG1 mAb, targeting HER2 receptor) and its ADC with DM1 (anti-tubulin anticancer drug), Trastuzumab emtansine (T-DM1) were studied. SE-HPLC was used to monitor formation of aggregates and/or fragments of the monoclonal antibodies (mAb). Correlation with the results of reducing and non-reducing sodium dodecyl sulphate – polyacrylamide gel electrophoresis (SDS-PAGE) and dynamic light scattering (DLS) were performed to interpret the obtained results. RP-HPLC was used for assessment of the stability of DM1 in ADC while spectrophotometry was employed to determine drug antibody ratio (DAR) . The studied drugs were subjected to several stress conditions including pH, temperature, mechanical agitation and repeated freeze and thaw to generate possible degradation products and ensure suitability of the assay protocol. The degradation pattern and extent were demonstrated under the indicated stress conditions. The correlation between the results of SE-HPLC and those of SDS-PAGE and DLS ensured the validity of the orthogonal assay protocol and indicated aggregates that were not detected using SE-HPLC. Results showed clearly that T-DM1 is relatively less stable than its parent mAb. This was attributed to the presence of the drug-linker part that is attached to the mAb. RP-HPLC showed that the cytotoxic drug moiety is liable for degradation under the studied conditions resulting in alteration of DAR as well as formation of degradation products. This confirmed the need for more robust coupling chemistries for production of safe and effective ADC and highlighted the importance of orthogonal testing protocols for quality assessment. The assay protocol should be applicable for quality and stability assessment of various ADC.