Annual Research & Review in Biology

Antibody–drug conjugates (ADCs) couple the target selectivity of a monoclonal antibody to the cytotoxic potency of a small-molecule payload, and the modality has matured from a single early approval to more than a dozen marketed products across haematological and solid tumours. Their defining analytical and pharmacokinetic feature is that an ADC is not a single chemical entity but a heterogeneous and dynamic mixture of species that differ in drug-to-antibody ratio (DAR) and that change continuously in the circulation through payload deconjugation, linker cleavage and antibody catabolism. This review critically examines how bioanalytical measurement and pharmacokinetic (PK) interpretation have co-evolved to address that complexity, and argues that the two disciplines are most usefully treated as one integrated problem rather than as sequential technical steps. The literature is synthesised around the definition of the measurable analytes, the relative strengths of ligand-binding assays, hybrid immunocapture liquid chromatography–tandem mass spectrometry and intact or native mass spectrometry, the in vivo characterisation of DAR and biotransformation, and the progression of PK modelling from empirical multi-analyte descriptions through target-mediated disposition models to multiscale and physiologically based frameworks. Convergent evidence supports several conclusions, including the dominant influence of linker and conjugation-site chemistry on systemic stability and the value of simultaneously quantifying total antibody, conjugated species and unconjugated payload. Substantial disagreement remains over which analyte best predicts efficacy and toxicity, how deconjugation should be parameterised, and how intratumoral distribution should inform dose. Reported relationships are frequently molecule-specific, and generalisation across payload classes is weakly supported. The analysis identifies priorities for standardising analyte nomenclature, strengthening the measurement-to-model link, and generating the mechanistic data required for dose optimisation under contemporary regulatory expectations. The modality now demands bioanalytical and PK strategies that are explicitly designed together from early discovery.