ADC Case Studies: Overcoming Challenges in Bioanalytical and CMC Characterization 

The multi-component structure of ADCs presents several technical challenges to developing assays for characterization, from optimizing component quantitation and assessing clinically significant immunogenicity to analyzing structure, function, and process-related impurities. Thus, the process of bringing an ADC to market demands multidisciplinary tools, a nuanced understanding of bioanalytical and chemistry, manufacturing, and controls (CMC) analytical requirements, and a robust strategy for assay development and validation.

Watch the webinar on ADC case studies below or continue on to read the full summary.

Unlocking the potential of ADCs 

The complexity of ADCs is reflected in the lengthy list of assays that should be included in the bioanalytical strategy for preclinical and clinical testing of these therapeutics. Common assays such as pharmacokinetics (PK), immunogenicity assessments, and biomarker testing are included but many ADC-specific assays are also required such as drug-to-antibody ratio (DAR), metabolite profiling, and drug-drug interactions (DDI). In addition, there are three major characteristics of ADCs that need to be assessed as part of the CMC analytical testing strategy – the targeting capability of its antibody, the potency of its payload, and the stability of its linker.  

Navigating bioanalytical challenges

Pharmacokinetics 

Pharmacokinetic (PK) assays are critical for understanding how intact ADC molecules behave in biological systems. These assays must quantify not just the intact ADC but also its components, including unconjugated antibodies, numerous iterations of the payload, and payload metabolites. Customization is key, as each ADC presents unique challenges dictated by its biology and the available reagents. In most cases, a free PK, where the antibody portion of the ADC contains at least one unoccupied target binding site, is preferred. However, knowing the total amount of ADC and the amount of ADC bound to target can be instrumental in understanding the full profile of the drug in the body in some cases. 

When developing a ligand binding assay for ADC or total antibody quantitation, it is also important to: 

• Understand what conditions may be required for optimal binding of the ADC or antibody to the capture antibody  

• Consider and test multiple reagent combinations to determine the optimal pairing that creates an assay that is independent and unencumbered by the number of conjugated payload molecules 

Payload quantitation

Developing assays to measure ADC payloads requires a risk-based approach that is dependent on the unique characteristics of each ADC. Quantitation methods such as liquid chromatography tandem mass spectrometry (LC-MS/MS) must ensure accuracy and sensitivity while accounting for factors such as linker stability and sample handling. For example, with some linkers, exposure to LED lighting can cause artificial release of the payload, thus overestimating the amount of circulating payload detected. In vivo metabolism and non-specific binding can also confound accurate payload quantitation. Thus, it is critical to determine the free payload present within the reference standard of the ADC. In addition, during method development, every effort should be made to evaluate the payload and linker to ensure that no release is caused by dilution, handling, or extraction methods used to prepare the biological sample for analysis.  

Immunogenicity assessment 

Immunogenicity assessments, including anti-drug antibody (ADA) and neutralizing antibody (NAb) assays, evaluate potential immune responses against the ADC after dosing. Given the complexity of ADCs, a bridging format where the fully conjugated ADC molecule is used for both capture and detection is the preferred starting point for an ADA assay as it allows for the broadest coverage and the best opportunity to pull down ADAs against all portions of the molecule. If the ADC has a naturally occurring payload, there is the potential for pre-existing antibodies in treatment naive patients. If this is the case, applying a robust statistical model is necessary for generating an accurate cut point. For detection of NAb, cell-based assays are the recommended format. A key step in NAb assay development is identifying a cell line that expresses the target. For both ADA and NAb assays, confirming availability and quality of critical reagents is one of the most important steps of assay implementation. 

Addressing CMC analytical considerations 

Structural analysis 

The structural complexity and inherent heterogeneity of ADCs demands advanced characterization techniques. High-resolution mass spectrometry (HRMS) has emerged as a powerful tool for analyzing both intact molecules and their subunits. Breaking ADCs into manageable components using enzymatic digestion enables detailed assessments of specific structural features. The data generated by HRMS is quite complex but can be deconvoluted by software such as BioPharma Finder to evaluate attributes such as drug-to-antibody ratio (DAR), conjugation position of drug molecules, presence of unconjugated antibody, and glycosylation patterns. These insights are critical as part of a control strategy for maintaining consistency and quality of the ADC product throughout development and manufacturing. 

Functional characterization

Functional assays, such as cytotoxicity and antibody-dependent cellular cytotoxicity (ADCC) tests, mimic the mechanism of action (MOA) of the ADC. These assays measure biological activity and potency, ensuring that the ADC maintains the intended specificity, binding to and destroying the target expressing cancer cells. Rigorous validation processes are needed to confirm the reliability of these tests.

Impurity analysis

Host cell proteins (HCPs) are process-related impurities that can compromise the safety, efficacy, and stability of ADCs, making HCP analysis a cornerstone of any ADC control strategy. Traditional enzyme-linked immunosorbent assays (ELISAs) are effective, but have limitations related to identification of individual HCPs, gaps in detection, and cost of reagents. Emerging orthogonal methods, such as liquid chromatography-mass spectrometry (LC-MS), offer enhanced sensitivity and the ability to identify and quantify specific impurities.

Conclusion

There has been significant growth in the prevalence of ADCs in the drug development space, with 12 FDA-approved therapeutics, over 370 programs in preclinical development, and more than 170 in clinical trials as of the beginning of 2025. At BioAgilytix, we have supported more than 20 sponsors with over 80 assays for ADCs. For small molecules, we have developed more than 500 assays and supported 10 approved programs. For monoclonal antibodies, we have developed more than 800 assays and supported 23 approved programs. This combined experience informs our approach to assay development, where each compound is unique and requires customized methodologies.  

To learn more about developing and implementing a robust bioanalytical and CMC analytical characterization strategy for ADCs, speak to a scientist.