DAR Distribution Determination for Antibody-drug-conjugates (ADCs) by LC-HRMS in In Vitro and In Vivo Studies

The drug-to-antibody ratio (DAR) is an important characteristic of antibody-drug conjugates (ADCs). It is essential to determine and identify the change of the DAR distribution and DAR value in drug metabolism and pharmacokinetics (DMPK) investigations. In this study, commercial HER2-targeting ADC, trastuzumab deruxtecan (T-Dxd, DS-8201a) was incubated in plasma for in vitro stability study and dosed to SD rat for in vivo pharmacokinetics study to evaluate change of DAR value and DAR distribution, and the correlation between in vitro and in vivo biotransformation. Immunoaffinity capture method with streptavidin magnetic beads and biotinylated anti-human IgG was used to extract the ADCs in biological matrices such as plasma. Reverse Phase Liquid Chromatography (RPLC) coupled with tandem High Resolution Mass Spectrometry (HRMS) was utilized for the analysis.