Automated online deconjugation of antibody-drug conjugate for small molecule drug profiling.

Antibody-drug conjugates (ADCs) are designed by chemically linking highly potent cytotoxic small molecule drugs to monoclonal antibodies of unique specificity for targeted destruction of cancer cells. This innovative class of molecules incurs unique developmental challenges due to its structural complexity of having both small molecule and protein components. The stability of the small molecule payload on the ADC is a critical attribute as it directly relates to product efficacy and patient safety. This study describes the use of an end-to-end automated workflow for effective and robust characterization of the small molecule drug while it is conjugated to the antibody. In this approach, online deconjugation was accomplished by an autosampler user defined program and D size exclusion chromatography was utilized to provide separation between small molecule and protein species. The small molecule portion was then trapped and sent to the D for separation and quantification by reversed-phase liquid chromatography with identification of impurities and degradants by mass spectrometry. The feasibility of this system was demonstrated on an ADC with a disulfide-based linker. This fully automated approach avoids tedious sample preparation that may lead to sample loss and large assay variability. Under optimized conditions, the method was shown to have excellent specificity, sensitivity (LOD of 0.036 µg/mL and LOQ of 0.144 µg/mL), linearity (0.04-72.1 µg/mL), precision (system precision %RSD of 1.7 and method precision %RSD of 3.4), accuracy (97.4 % recovery), stability-indicating nature, and was successfully exploited to analyze the small molecule drug on a panel of stressed ADC samples. Overall, the workflow established here offers a powerful analytical tool for profiling the in-situ properties of small molecule drugs conjugated to antibodies and the obtained information could be of great significance for guiding process/formulation development and understanding pharmacokinetic/pharmacodynamic behavior of ADCs.