PT - JOURNAL ARTICLE AU - Tingting Cai AU - Liqi Shi AU - Huihui Guo AU - Ruixing Li AU - Weiqun Cao AU - Liang Shen AU - Mingshe Zhu AU - Yi Tao TI - <strong>Detection and characterization of in vitro payload-containing catabolites of non-cleavable ADCs by high-resolution mass spectrometry and multiple data-mining tools</strong> AID - 10.1124/dmd.122.001135 DP - 2023 Jan 01 TA - Drug Metabolism and Disposition PG - DMD-AR-2022-001135 4099 - http://dmd.aspetjournals.org/content/early/2023/03/06/dmd.122.001135.short 4100 - http://dmd.aspetjournals.org/content/early/2023/03/06/dmd.122.001135.full AB - The formation and accumulation of payload-containing catabolites (PCCs) from a non-cleavable ADC in targeted and normal tissues are directly associated with the therapeutic effect and toxicity of the ADC, respectively. Understanding the PPC formation is important for supporting the payload design and facilitating preclinical evaluation of ADCs. However, detection and identification of PCCs of a non-cleavable ADC are challenging due to their low concentrations and unknown structures. The main objective of this study was to develop and apply a generic LC-HRMS method for profiling PCCs in vitro. Non-cleavable ADCs, T-DM1 and ADC-1, were incubated in liver lysosomes, liver S9 and/or cancer cells followed by data acquisition using liquid chromatography-high resolution mass spectrometry (LC-HRMS). Profiling PCCs mainly relied on processing LC-HRMS datasets using untargeted background subtraction processing (PATBS) and targeted product ion filtering (PIF). As a result, 12 PCCs of T-DM1 were detected and structurally characterized in human liver lysosomal incubation, a majority of which consisted of MCC-DM1 and a few amino acids. Additionally, the incubation of ADC-1 in human, rat and money liver S9 and cancer cells generated one major and three very minor PCCs, verifying the payload design. The results demonstrate that PATBS enabled the comprehensive profiling of PCCs regardless of their molecular weighs, charge states, and fragmentations. As a complementary tool, PIF detected specific PCCs with superior sensitivity. The combination of the in vitro metabolism systems and the LC-HRMS method is a useful approach to profiling in vitro PCCs of non-cleavable ADCs in support of drug discovery programs. Significance Statement Profiling in vitro payload-containing catabolites (PCCs) of a non-cleavable ADC is important in support of the payload design and preclinical evaluation of ADC. However, currently used analytical approaches often fail to quickly provide reliable PCC profiling results. The work introduces a new LC-HRMS method for comprehensive and rapid detection and characterization of PCCs released from a non-cleavable or cleavable ADC in liver lysosomes and S9 incubations.