Trastuzumab deruxtecan (T-DXd, Enhertu®) is a HER2-targeting antibody-drug conjugate (ADC) composed of trastuzumab, a cleavable tetrapeptide linker, and a topoisomerase I inhibitor payload (DXd). With a high drug-to-antibody ratio (DAR ~8) and tumor-selective payload release, Enhertu shows strong efficacy in HER2-positive cancers. However, its structural complexity requires robust analytical methods to evaluate product quality and linker stability during development.

We developed an integrated workflow combining HPLC-based physicochemical assessment with LC-MS/MS-based linker stability analysis. SEC- and HIC-HPLC evaluated monomer content, aggregation, and DAR distribution, while intact mass analysis confirmed batch homogeneity and conjugation profiles. A reference batch with acceptable quality attributes was selected for further testing.

Linker stability was evaluated both in vitro and in vivo. In vitro, T-DXd was incubated in mouse serum to monitor time-dependent DXd release. In vivo, released free DXd in serum was quantified via LC-MS/MS after intravenous dosing in mice. Results showed gradual, time-dependent linker cleavage under physiological conditions, suggesting partial instability during systemic circulation.

This integrated approach offers a reliable platform for comprehensive ADC quality and stability assessment. It supports pharmacokinetic interpretation and applies to formulation development, lot-release testing, and linker screening during ADC development.