Analytical Challenges and Case Studies for Bioanalysis of Antibody-Drug Conjugates (ADCs)

Antibody-Drug Conjugates (ADCs) combine biologic specificity with small-molecule potency, creating unique challenges for pharmacokinetic characterization of a heterogeneous mixture that evolves in vivo. Conventional LC-MS/MS approaches often trade sensitivity (Bottom-Up) for structural fidelity (Top-Down), making a single workflow insufficient.
We evaluated a hybrid LC-MS toolkit to address these limitations and present three case studies that illustrate the challenges and constraints in method development. A streamlined Bottom-Up method enabled simultaneous quantitation of “Total Antibody” and “Conjugated Drug” from one digestion, but peptide-level data masked structural changes. Intact mass analysis was critical to reveal conjugation profiles and dynamic Drug-to-Antibody Ratio (DAR) shifts over time.
However, Top-Down approaches faced limitations: high drug loading and certain conjugation sites caused fragmentation or precipitation, requiring alternative Bottom-Up or Middle-Up strategies.
These cases highlight that ADC bioanalysis demands a fit-for-purpose integration of digestion-based quantitation and intact profiling to ensure accurate characterization throughout development.