(M1430-02-09) Application of Sequential Window Acquisition of all Theoretical Fragment Ion Spectra Mass Spectrometry (SWATH-MS) for Identification of Tetrapeptide Linker Cleavage Profile of Antibody Drug Conjugate

Purpose: Advances in Antibody-Drug Conjugates (ADCs) underscore the need for robust bioanalytical methods. In particular, measuring the Drug-to-Antibody Ratio (DAR) change of bioanalytical sample is crucial for assessing ADC safety and efficacy, requiring precise quantification of the antibody-conjugated drug (acDrug) following linker cleavage. The purpose of this study is to explore the tetrapeptide linker cleavage profiles of Trastuzumab Deruxtecan (Enhertu) to understand efficienct cleavage of acDrug in standard/QC samples as well as in study samples by examining all possible linker cleavage forms of Trastuzumab Deruxtecan utilizing Sequential Window Acquisition of all Theoretical Fragment Ion Spectra Mass Spectrometry (SWATH-MS) with QTOF Mass Spectrometry method.

Methods: 4 µL of 1mg/mL Trastuzumab Deruxtecan was treated with 50 µL of 0.2 mg/mL papain in 50 mM Tris(hydroxymethyl)aminomethane (TRIS), incubated at 37°C for 2 hours, then neutralized with 10 µL of 2N HCl. The supernatant was analyzed via SWATH-MS with an accumulation time of 39 ms across 100 SWATH windows, using a declustering potential (DP) of 100 and collision energy (CE) of 15. Chromatographic separation occurred on a 2.1x100 mm Phenomenex Kinetex C18 column at 55°C and 0.3 mL/min. SCIEX QTOF 5600+ was used for MS analysis, with precursor ion mode scans on a SCIEX QTRAP 6500 set to DP at 140 and CE at 30.

Results: The method not only identified DXd, the payload of Trastuzumab Deruxtecan but also detected various chemical moieties such as Phenylalanine-Glycine-DXd (FG-DXd) and Glycine-Phenylalanine-Glycine-DXd (GFG-Dxd), which were also cleaved from Trastuzumab Deruxtecan by papain. The estimated amount of these new moieties were about 10% of the Dxd based on peak intensities.

Conclusion: SWATH-MS, combined with TOF-MS, provided high-resolution mass spectra and enabled more accurate identification of catabolite moieties from ADC. This method would be useful in developing and optimizing the bioanalytical method for the acDrug quantification of ADC.