Tritosomes-Digestion for LC-MS Conjugated Payloads Quantitation: A Universal Approach for Dual-Payloads ADCs.

Bioanalytical methods to quantitate conjugated payloads are essential for assessing antibody-drug conjugate (ADC) stability and pharmacokinetics (PK). Dual-payload ADCs present analytical challenges; different linker chemistries can require complex digestion conditions to perform the cleavage. Developing separate methods for each linker combination can be time and resource demanding. Rat tritosomes-purified lysosomal fractions from Triton-treated rat liver-provide a comprehensive enzymatic mixture that mimics the lysosomal environment. The presented bioanalytical method combines immunoaffinity purification with tritosome-mediated digestion for simultaneous quantitation of dual-conjugated payloads. The method was applied to a model dual-payload ADC containing two different cytotoxic payloads, conjugated using different enzymatically cleavable linkers, with an unrelated DAR (drug-to-antibody ratio). Method validation in mouse plasma demonstrated excellent accuracy (bias ± 20%, LLOQ and ULOQ ± 25%) and precision (coefficient of variation CV% ≤ 20%, LLOQ and ULOQ ± 25%) across all concentration levels (lower to upper limit of quantitation, LLOQ to ULOQ) for both payloads, with 100% of quality control samples (QCs) meeting acceptance criteria for hybrid LC-MS/MS quantitation methods. This tritosome-based approach provides a unified, efficient platform for multi-payload ADC bioanalysis, eliminates linker-specific method optimization, and enables robust support for preclinical studies. The method has been tested for accuracy and precision on 4 different model ADCs and employed to quantify the conjugated payloads in in vivo samples from a homozygous hFcRn transgenic mouse model (Tg32) PK study, resulting in reliable data in accordance with total antibody measurements.