Assessing Protein and Payload Stability of Antibody Drug Conjugate Brentuximab Vedotin in Monkey Plasma

Significance of the Topic

Antibody–drug conjugates (ADCs) represent a rapidly growing class of targeted cancer therapies combining the selectivity of monoclonal antibodies with the cytotoxic potency of small-molecule payloads. Precise quantification of both the antibody surrogate peptides and payload stability in biological matrices is critical for preclinical pharmacokinetics, safety assessment, and translational drug development.

Objectives and Study Overview

This work presents a fully automated hybrid ligand-binding assay/liquid chromatography–mass spectrometry (LBA/LC-MS) workflow for quantifying brentuximab vedotin and its payload in monkey plasma. Key aims include:

• Developing an automated immunoaffinity purification using the Agilent AssayMAP Bravo platform.
• Establishing LC separation on the Agilent 1290 Infinity II Bio LC.
• Performing sensitive MRM quantitation on the Agilent 6495 triple quadrupole MS.
• Evaluating peptide and payload stability over a seven-day incubation at 37 °C.

Methodology and Instrumentation

Monkey plasma spiked with brentuximab vedotin and stable isotope–labeled trastuzumab was processed on the AssayMAP Bravo using streptavidin and Protein A cartridges.

• Peptide analysis: On-cartridge trypsin digestion with reduction and alkylation followed by LC-MS injection.
• Payload analysis: On-cartridge papain release of MMAE payload, combined with acetonitrile wash, and LC-MS injection.

Instrumentation:

• Agilent AssayMAP Bravo Protein and Peptide Sample Prep System.
• Agilent 1290 Infinity II Bio LC (high-speed pump, multisampler, column compartment, EC-C18 column, 60 °C).
• Agilent 6495 triple quadrupole LC/MS with Jet Stream ESI source.

Results and Discussion

Optimized MRM transitions and source parameters yielded high sensitivity for surrogate peptide (VVSVLTVLHQDWLNGK) and payload (MMAE). Calibration curves were linear over 0.39–50 µg/mL for both targets (R2 > 0.995). Stability assessment showed:

• Surrogate peptide levels remained within 10 % of the nominal 50 µg/mL after 7 days.
• Payload concentration declined by ~40 % after 1 day and an additional ~25 % by day 3, then stabilized through day 7.

Benefits and Practical Applications

This hybrid workflow offers:

• High assay sensitivity and specificity without bespoke antibodies.
• Automated sample processing for throughput and reproducibility.
• Universal surrogate peptide applicability across human IgG-based ADCs.
• Minimal method development time to support early-stage ADC discovery.

Future Trends and Opportunities

Emerging directions include:

• Integration of high-resolution MS for deeper proteoform profiling.
• Multiplexed workflows to assess multiple ADC candidates in parallel.
• Application to patient-derived samples for translational biomarker studies.
• Advancements in cartridge chemistries for faster on-cartridge reactions.

Conclusion

The described Agilent LBA/LC-MS workflow provides a robust, sensitive, and automatable solution for quantitative ADC bioanalysis in preclinical plasma samples, streamlining ADC development and enabling informed design decisions.

References

1. Global Market Insights. Antibody Drug Conjugates Market Report, 2023–2032.
2. Jenkins R. et al. Recommendations for Validation of LC–MS/MS Bioanalytical Methods for Protein Biotherapeutics. AAPS J. 2015, 17(1):1–16.
3. Dailymed National Library of Medicine. Adcetris (brentuximab vedotin) Injection. 2023.
4. European Medicines Agency. Adcetris EPAR Summary. Accessed 2018.
5. Agilent Technologies. A Streamlined Drug-to-Antibody Ratio Determination Workflow for ADCs. Application Note, 2019.