The Complexity of ADCs

ADCs are intricate molecules comprising three main components:

1. Monoclonal Antibody: A targeting agent that binds specifically to an antigen such as that expressed on the surface of cancer cells.

2. Linker: A chemical bridge attaching the payload to the antibody, which is cleaved once the ADC arrives to its target.

3. Payload: A potent drug designed to kill target cells. Commonly, several copies of the payload are attached to a single antibody.

Analyzing Antibody Drug Conjugates (ADCs) is crucial due to their distinct structure, mechanism of action, and therapeutic applications. This complexity presents unique analytical challenges, which are important for the following reasons:

• Quantifying intact ADCs and free payloads: Ensures precise measurement of the therapeutic agent and any unbound cytotoxic drug. This is critical for evaluating efficacy and minimizing off-target toxicity. Accurate quantification helps maintain the balance between potency and safety.

• Characterizing the drug-to-antibody ratio (DAR): The DAR directly influences the pharmacokinetics, biodistribution, and therapeutic effectiveness of ADCs. Monitoring this ratio is essential for consistency, quality control, and compliance with regulatory standards.

• Detecting and quantifying impurities or degradation products: Identifying impurities and degradation products is vital for maintaining product stability and patient safety. It ensures the ADC retains its intended therapeutic properties throughout its shelf life and does not produce harmful byproducts.

Addressing these challenges is fundamental to developing safe, effective, and high-quality ADC therapies. HRMS provides a comprehensive solution, enabling precise and reliable analysis across the ADC development pipeline.

Why HRMS Outshines Traditional Methods

Traditional ligand-binding assays (LBAs) have been widely used in ADC analysis. However, they come with significant limitations:

• Limited Specificity: LBAs rely on antibody-antigen affinity, which can result in cross-reactivity.

• Single Analyte Focus: Separate assays are often required for each analyte.

• Higher Variability: Variability in antibody affinity impacts assay robustness.

HRMS, on the other hand, offers several advantages:

1. Unmatched Selectivity and Sensitivity HRMS directly measures analytes based on their unique mass-to-charge ratios, ensuring high specificity. It enables simultaneous quantification of intact ADCs, free payloads, and other analytes in a single run.

2. Comprehensive Data Acquisition HRMS captures detailed spectral data, allowing for in-depth characterization of ADCs, including DAR profiling and impurity analysis.

3. Reduced Variability Unlike LBAs, HRMS is less dependent on biological reagents, offering greater reproducibility and consistency across assays.

4. Matrix Compatibility HRMS, coupled with advanced LC methods, ensures accurate analysis even in complex biological matrices like plasma or serum.

5. Regulatory Compliance HRMS delivers traceable and reproducible data, making it indispensable for regulatory submissions and quality control in ADC manufacturing.

Applications of HRMS in ADC Method Development 

1. Quantification of ADCs and Payloads 
   HRMS enables precise measurement of both intact ADCs and their free payloads. Recent studies, such as Zhang et al. (2019), have demonstrated its accuracy in quantifying these components even in complex matrices. Accurate quantification helps assess the consistency of ADC production and ensures the correct dosing of active components during preclinical and clinical studies. 

2. Drug-to-Antibody Ratio (DAR) Analysis 
   DAR is a critical quality attribute of ADCs, influencing their efficacy and safety. HRMS provides accurate DAR profiling by resolving the heterogeneous nature of ADC populations, as highlighted by Beck et al. (2020). The DAR significantly impacts the pharmacokinetics, biodistribution, and potency of ADCs, and HRMS ensures a precise determination of DAR for product consistency and regulatory compliance. 

3. ADC Impurities 
   HRMS is a powerful tool for identifying and quantifying impurities, including unbound payloads, linker degradation products, and aggregation species. These impurities can compromise product safety, stability, and efficacy. HRMS's high sensitivity and specificity enable detailed impurity profiling, which is essential for maintaining ADC quality and adhering to regulatory standards. 

4. Overcoming Matrix Effects 
   Complex biological matrices, such as plasma, pose significant challenges in ADC analysis. HRMS, when combined with optimized liquid chromatography (LC) methods, effectively addresses matrix interferences. As demonstrated by Li et al. (2018), this ensures reliable and reproducible results. 

High-Throughput ADC Analysis Using HRMS 

With the rapid advancement of ADC development, high-throughput workflows are essential to meet growing demand while maintaining data quality. According to a report by Dimension Market Research, the global Antibody Drug Conjugates (ADC) market is expected to expand from USD 13.7 billion in 2024 to USD 51.2 billion by 2033, representing a compound annual growth rate (CAGR) of 15.8% (GlobeNewswire). 

Similarly, BCC Research projects that the global ADC market will grow from $10.8 billion in 2023 to $47.0 billion by 2029, with a CAGR of 28.4% (BCC Research). 

These forecasts underscore the anticipated substantial growth in the ADC market in the coming years. HRMS is uniquely positioned to address this need: 

1. Automated Workflows 
   Integrating HRMS with automated liquid handling systems streamlines sample preparation, reducing variability and increasing throughput. 

2. Data Integration and Insights 
   Modern HRMS platforms incorporate AI-driven algorithms for spectral deconvolution and DAR profiling, accelerating data interpretation and improving decision-making.

3. Fast and Accurate Quantitation 
   Ultra-high-performance liquid chromatography (UHPLC) combined with HRMS enables rapid separation and quantitation of ADC components, including: 

• Intact ADCs. 

• Free payloads. 

• Catabolites in biological matrices.  

Applications & Benefits for Drug Development Pipelines 

HRMS is transforming workflows across ADC development stages: 

• Discovery and Screening: Rapidly analyze large candidate libraries. 

• Pharmacokinetics: Quantify ADCs and metabolites in complex biological samples. 

• Stability Testing: Detect degradation pathways to optimize formulation. 

• Batch Consistency: Ensure rigorous quality control and regulatory compliance. 

Incorporating HRMS into ADC workflows accelerates drug discovery and development by: 

• Reducing assay development timelines. 

• Enhancing confidence in data integrity. 

• Supporting regulatory submissions with comprehensive analytical data. 

​​​​​Conclusion 

High-Resolution Mass Spectrometry (HRMS) is revolutionizing ADC analytics, addressing the unique challenges posed by their complexity. Its precision, versatility, and scalability make HRMS an invaluable tool for advancing ADC therapeutics. By integrating HRMS into workflows, organizations can unlock new possibilities in ADC development, driving innovation and improving patient outcomes. 

Partner with Qmera for your analytical ADC needs! 

References 

1. Zhang, Y., et al. (2019). Quantitation of Antibody-Drug Conjugates Using HRMS. Analytical Chemistry, 91(12), 7489-7496. DOI:10.1021/acs.analchem.9b00938. 

2. Beck, A., et al. (2020). Strategies for the Analytical Characterization of ADCs. mAbs, 12(1), 178-192. DOI:10.1080/19420862.2020.1737892. 

3. Li, H., et al. (2018). Optimized LC-HRMS Methods for ADC Quantitation in Complex Matrices. Journal of Chromatography B, 1085, 45-53. DOI:10.1016/j.jchromb.2018.02.024. 

4. Cooley, M. (2025). The Need for HRMS for Quantitation of ADCs vs. QqQ. Bioanalysis Zone. 

5. Foreman, R. (2025). Optimized LC-MS/MS Methods for Quantifying ADC Payloads. Bioanalysis Zone 

6. Dimension Market Research: "Antibody Drug Conjugates Market Is Expected To Reach Revenue Of USD 51.2 Billion By 2033 at 15.8% CAGR," GlobeNewswire, September 5, 2024. Available at: https://www.globenewswire.com/news-release/2024/09/05/2941601/0/en/Antibody-Drug-Conjugates-Market-Is-Expected-To-Reach-Revenue-Of-USD-51-2-Bn-By-2033-at-15-8-CAGR-Dimension-Market-Research.html 

7. BCC Research: "Antibody-Drug Conjugates Markets Report," BCC Research, 2023. Available at: https://www.bccresearch.com/market-research/pharmaceuticals/antibody-drug-conjugates-markets-report.html