Strategies for Antibody-Drug Conjugates Bioanalysis

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.

Key Considerations in ADC Bioanalysis

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.

2. Dynamic DAR Changes In Vivo

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.

3. Selection of Relevant Analytes

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:

• Total antibodies
• Conjugated antibodies
• Free payload
  
  

A broad analyte strategy supports a clearer understanding of exposure–response relationships.

Conventional Analytical Approaches

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.

Hybrid LC-MS/MS: An Evolving Standard

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:

• Adaptability to humanized or fully human IgG1 and IgG4 backbones
• Shorter development timelines
• Reduced reliance on specialized antibody reagents
• Broader analytical coverage, including total antibody, conjugated antibody, and released payload
  
  

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.

A General-Purpose Non-Clinical Method for ADC Bioanalysis

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:

• Antibody quantification through characteristic peptide segments of the IgG1 or IgG4 constant region in animal matrices
• Drug quantification through enzymatic cleavage of peptide-based linkers to release the payload, enabling DAR evaluation in vivo

This strategy allows simultaneous measurement of:

• Total antibodies
• Conjugated drug
• Free drug
  
  

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.

How a CRO Can Help ADC Development