Antibody-drug conjugates (ADCs) have emerged as one of the fastest-growing drug modalities in oncology, offering both precision and potency. Their unique structures allow them to bind to their targets while carrying and releasing potent toxins in controlled ways, boosting efficacy while reducing off-target effects.
Developers are moving quickly to capitalize on this potential. The global ADC drug market was estimated at $12.26 billion in 2024 and is projected to grow to $32.11 billion by 2033, according to a GVR Pharmaceuticals report, driven by demand for greater efficacy and reduced toxicity in cancer-targeting drugs.
But the complex molecular make-up of ADCs introduces challenges during early-stage drug development. Preclinical bioanalysis is crucial to safe and efficient drug development, and choosing the right platform is critical if sponsors are to bring new ADCs to market and into the hands of patients in need of new treatment options.
How do ADCs Work?
ADCs combine a monoclonal antibody, a cytotoxic payload, and a chemical linker. Among the many types of antibodies, humanized and full human IgGs, especially IgG1, are the most common in use.
Antibody targets are selected to deliver highly potent drugs to tumor cells while limiting toxicity to healthy tissue. Common targets include HER2 and TROP2, and payloads often involve tubulin or topoisomerase inhibitors.
The chemical linkers play a pivotal role, enabling attachment of cytotoxic payloads to the antibody until the target cell is reached. They can be both cleavable and non-cleavable—the former contains a trigger that breaks once it’s inside or near tumor cells, while the latter degrades slowly once it’s inside the cell. Put simply, cleavable linkers prioritize efficient release; non-cleavable ones prioritize stability and control. Most approved ADC drugs contain cleavable linkers, and, historically, many approved ADCs have used tubulin-binding payloads.
What ADC Bioanalysis Measures
Because of ADCs’ structural complexity, bioanalytical testing must track multiple molecular forms in circulation rather than a single drug entity. ADC bioanalysis typically focuses on three key analytes:
- Conjugated antibody: Quantifies the intact ADC that remains pharmacologically active. It is often considered the most direct indicator of therapeutic exposure, payload delivery to tumor tissue, and linker stability in circulation. A rapid decline can indicate premature linker cleavage and instability in plasma, resulting in payload release before the ADC reaches tumor cells.
- Total antibodies: Measure all antibody molecules, regardless of whether the payload remains attached. This includes fully conjugated ADCs, partially deconjugated ADCs, and completely deconjugated antibodies. The measurement reflects the overall antibody exposure, antibody pharmacokinetics, clearance rates, and target-mediated drug disposition (TMDD).
- Free payload: Quantifies the amount of cytotoxic drug released from the ADC. It is critically important for safety; high free payload levels can indicate premature linker cleavage, off-target toxicity, and metabolic instability.
Unlike conventional biologics, ADCs can lose payload over time, undergo linker cleavage, and generate multiple circulating molecular species. This makes bioanalysis substantially more complex than measuring a traditional monoclonal antibody.
ELISA vs. LC/MS-MS
Ligand-binding assays (ELISA) and LC-MS/MS are the two main platforms used for ADC bioanalysis, but each has benefits and drawbacks. ELISA is preferred for measuring total and conjugated antibodies because it offers high sensitivity, precision, and high throughput. Common assay formats include generic assays, target-binding assays, and anti-ID antibody assays.
LC-MS/MS is more commonly used for free payload analysis after protein precipitation. It can also quantify the total and conjugated antibodies through immunocapture, enzymatic digestion, and signature peptide analysis. A recent WuXi AppTec case study illustrates the challenge of ADC bioanalysis.
A case study
WuXi AppTec researchers highlighted potential issues with ELISA assays for an ADC with an MMAE payload attached via a cleavable linker. The goal was to measure both total antibody and conjugated antibody levels.
Several problems arose during sample analysis on the ELISA platform. There were abnormal ADC-to-total antibody ratios, weak assay signals, high background interference, and poor incurred sample reproducibility (ISR). The team repeatedly modified the assay design to address these issues, but although the final ELISA methods met formal validation criteria, the ISR performance remained low.
Researchers switched to an LC-MS-based workflow and optimized trypsin digestion conditions and linker cleavage conditions. The performance of this method was improved significantly, achieving nearly 100% ISR pass rates.
The case study demonstrated that ligand-binding assays remain the preferred analytical platform for analyzing total and conjugated antibodies in ADCs, but the format may not perform well in all cases. In this example, LC-MS served as an alternative analytical platform with unique strengths.
A Final Word
ADCs are an exciting and promising new tool in the fight against cancer. Their molecular design is both their strength—allowing precision and potency with limited off-target effects—and their greatest challenge during the development phase.
In practice, many ADC programs rely on complementary use of both ELISA and LC-MS/MS platforms, but knowing when to use which is crucial. By working with a trusted lab partner, developers and sponsors can ensure they are using the correct platform at the right stage for a specific candidate and avoid potential delays in the process.
