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 and 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 not related 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.

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.