Antibody drug conjugates (ADCs) combine antibody specificity with potent small-molecule payloads, and their characterization requires methods that can capture the complexity of both components and the dynamic changes that occur in vivo. Robust bioanalysis is essential for understanding pharmacokinetics, ensuring safety, and supporting progression toward clinical development.
Non-clinical bioanalytical studies are key in defining ADC pharmacokinetics and pharmacodynamics. These analyses support a detailed evaluation of mechanisms of action, metabolic pathways, and PK or TK parameters, all of which contribute to the development of safe and effective therapies.
ADCs exhibit significant heterogeneity due to their complex structures and manufacturing processes. Variations in drug-to-antibody ratio (DAR), drug load distribution, linker chemistry, and antibody subclass may influence both efficacy and safety. Characterizing this heterogeneity is fundamental for reliable interpretation of preclinical results.
DAR values evolve dynamically following administration. Physiological factors such as pH, temperature, and enzyme activity affect linker stability and payload release. ADCs with different DAR values may show distinct half-lives or pharmacological activity, making DAR monitoring an important aspect of pharmacokinetic evaluation.
Unlike traditional small molecules or biologics, ADCs contain multiple components that change over time. Selecting analytes that accurately represent exposure and biological effect is therefore challenging.
The FDA’s 2024 guidance, Clinical Pharmacology Considerations for Antibody–Drug Conjugates, recommends that early-stage studies measure as many relevant analytes as possible, including:
A broad analyte strategy supports a clearer understanding of exposure–response relationships.
Bioanalysis of ADCs typically combines ligand-binding assays (LBAs) with LC-MS/MS to quantify both protein-based and small-molecule components.
Ligand-binding assays are widely used to measure total antibody, conjugated antibody, or conjugated drug. They require high-quality antibody reagents, which may be difficult to obtain early in development. LBAs may also be influenced by anti-drug antibodies in certain species.
LC-MS/MS remains the standard technology for quantifying free payload, offering high sensitivity and specificity through efficient chromatographic separation and mass spectrometric detection.
Affinity-capture or hybrid LC-MS/MS methods have gained prominence in ADC bioanalysis. By targeting characteristic peptide sequences within the antibody backbone, these methods reduce dependency on specific capture reagents and allow simultaneous quantification of multiple ADC-related analytes.
Key advantages during early research and development include:
As the number of ADC programs increases, LC-MS/MS-based approaches are becoming preferred due to their specificity, wider dynamic range, and regulatory acceptance.
Most ADCs in development incorporate humanized or fully human IgG1 or IgG4 antibodies, making them suitable for general-purpose assay strategies.
Crystal Bio Solutions has developed a general LC-MS/MS method that includes:
This strategy allows simultaneous measurement of:
These data support comprehensive pharmacokinetic characterization, DAR monitoring, and non-clinical pharmacological and toxicological evaluation.
To see the method performance, access to our case study A Generic LC-MS/MS Solution for ADC PK Bioanalysis.