Determination of Drug-to-Antibody Ratio for Antibody-Drug Conjugates Purified from Serum

Significance of the Topic

Antibody-drug conjugates (ADCs) combine the specificity of monoclonal antibodies with the potency of small-molecule drugs. The average number of drugs attached per antibody, known as the drug-to-antibody ratio (DAR), critically influences ADC efficacy, safety, and pharmacokinetics. Reliable DAR determination in biological matrices such as serum is essential for development, quality control, and pharmacokinetic studies.

Objectives and Study Overview

This study presents an integrated workflow to automate ADC purification from serum and to determine DAR with minimal manual intervention. Key goals included reducing hands-on time, minimizing variability, and increasing sample throughput while maintaining high sensitivity and accuracy in DAR measurement across a range of ADC concentrations.

Methodology

• Affinity purification: HER2 extracellular domain was biotinylated and immobilized on streptavidin-coated AssayMAP cartridges. Rat serum spiked with a model ADC at defined concentrations (20–0.625 µg/mL) was processed on the Agilent AssayMAP Bravo using a controlled wash and elution protocol.
• Elution and sample preparation: ADC was eluted in acidic solution and neutralized with ammonium hydroxide to preserve intact mass.
• LC/MS analysis: Samples were analyzed on an Agilent 1290 Infinity UHPLC with a PLRP-S reversed-phase column operated at 80 °C using a water/acetonitrile gradient with 0.1 % formic acid. Eluate was directed into an Agilent 6550 Q-TOF mass spectrometer in positive ion mode with Dual Jet Stream ESI, acquiring high‐resolution mass spectra over 1,000–4,500 m/z.
• Data processing: Raw spectra were deconvoluted using Agilent MassHunter BioConfirm (maximum entropy algorithm), and DAR values and glycoform distributions were calculated with the MassHunter DAR Calculator.

Used Instrumentation

• Agilent AssayMAP Bravo automated liquid handler with streptavidin affinity cartridges
• Agilent 1290 Infinity UHPLC system with PLRP-S 150×2.1 mm column, 8 µm, 1000 Å
• Agilent 6550 Q-TOF accurate mass spectrometer with Dual Jet Stream electrospray source
• Agilent MassHunter BioConfirm and DAR Calculator software

Key Results and Discussion

• Purification performance: Automated affinity capture achieved high ADC recovery and purity from serum without detergents. Elution windows were free of interfering serum proteins.
• Spectral quality: Total ion chromatograms and extracted ion chromatograms showed consistent ADC peaks between 2.2 and 3.2 min. Deconvoluted spectra displayed seven DAR species (0–7), each with characteristic glycoform profiles.
• Reproducibility: At serum concentrations ≥1.25 µg/mL ADC, coefficient of variation (CV) for quantitation was <10 %. DAR values averaged 3.5±0.1 across all tested concentrations, closely matching the commercial reference.
• Lower limits: Acceptable deconvolution and DAR calculation were achieved down to 0.625 µg/mL serum concentration by injecting a larger fraction of the eluate, demonstrating the workflow's suitability for pharmacokinetic samples.

Benefits and Practical Applications

The automated workflow reduces manual labor, minimizes sample-to-sample variability, and lowers the risk of human error. It streamlines DAR determination for ADC development, quality control, and preclinical PK studies, and can be scaled to handle larger sample sets with minimal additional effort.

Future Trends and Opportunities

• Integration of deglycosylation steps to simplify spectra and improve sensitivity
• Further miniaturization and higher-throughput cartridge formats for larger PK studies
• Coupling with targeted quantitation methods for simultaneous measurement of free drug and ADC species
• Application to a broader range of ADC constructs and post‐translational modification profiles

Conclusion

This study demonstrates a robust, automated platform for serum-based ADC DAR measurement. By combining affinity purification, high-resolution LC/MS, and dedicated software tools, the workflow delivers high accuracy, reproducibility, and throughput, meeting the stringent requirements of biotherapeutic development and pharmacokinetic analysis.

References

1. Perez H. L., et al. Antibody-drug conjugates: current status and future directions. Drug Discovery Today. 2014;19(7):869–881.
2. Beck A., et al. Cutting-edge mass spectrometry methods for the multi-level structural characterization of antibody-drug conjugates. Expert Rev Proteomics. 2016;13(2):157–183.
3. Xu K., et al. Characterization of intact antibody-drug conjugates from plasma/serum in vivo by affinity capture capillary liquid chromatography-mass spectrometry. Anal Biochem. 2011;412:56–66.