Drug to Antibody Ratio Analysis of Brentuximab Vedotin in Monkey Plasma

Importance of the Topic

ADCs combine a monoclonal antibody with a cytotoxic payload via a chemical linker. Monitoring the average drug to antibody ratio (DAR) is vital for understanding the pharmacokinetics, efficacy, and safety of ADC therapeutics. Reliable DAR analysis supports development, quality control, and clinical performance of these complex biologics.

Aims and Study Overview

This study presents a hybrid ligand binding assay (LBA) and liquid chromatography mass spectrometry (LC MS) workflow for intact DAR measurement of brentuximab vedotin in monkey plasma under native conditions. The authors aim to demonstrate an automated, sensitive, and reproducible process that tracks DAR stability over a seven day incubation period.

Methodology and Instrumentation

Sample preparation uses an Agilent AssayMAP Bravo platform for immunoaffinity purification of ADC from plasma. Eluted intact conjugate is analyzed using an Agilent 1290 Infinity II Bio LC system coupled to an AdvanceBio 6545XT LC Q TOF with jet stream electrospray. Key LC conditions include a Bio SEC 3 column operated isocratically with ammonium acetate buffer. MS acquisition employs native electrospray conditions for intact mass measurement and DAR deconvolution via BioConfirm software.

Key Results and Discussion

Under optimized native conditions, the intact brentuximab vedotin charge envelope centers around m/z 5804. Deconvolution reveals dominant DAR species at 2, followed by DAR 4 and DAR 0, resulting in an average DAR of 2.53 at day zero. Stability samples incubated at 37 C exhibit a rapid decrease in average DAR, falling by about 30 percent in the first 24 hours and another 25 percent by day three. The proportion of lower DAR species increases, indicating progressive payload release. DAR values stabilize from day three to day seven.

Benefits and Practical Applications

• The hybrid LBA LC MS workflow provides high specificity and sensitivity for intact DAR measurement without the need for custom reagents.
• Automation on the AssayMAP Bravo platform ensures reproducible sample preparation and reduces method development time.
• Native MS conditions preserve protein structure and offer accurate mass profiles for DAR distribution analysis.

Future Trends and Potential Applications

Emerging directions include extending this workflow to other ADC platforms and large protein conjugates, integrating higher resolution MS for minor species detection, and coupling with in vivo pharmacokinetic and payload quantification assays to build comprehensive ADC characterization pipelines.

Conclusion

The Agilent automated LBA LC MS platform delivers a robust solution for intact ADC DAR analysis under native conditions. The demonstrated workflow enables rapid, reproducible assessment of conjugate stability in biological matrices, supporting ADC research and development efforts.

References

• Global Market Insights. Antibody Drug Conjugates Market, 2023.
• Jenkins R et al. Recommendations for Validation of LC MS MS Bioanalytical Methods for Protein Biotherapeutics. AAPS Journal 2015;17(1):1-16.
• DailyMed. Adcetris Brentuximab Vedotin Injection Label, 2018.
• European Medicines Agency. Adcetris EPAR, 2018.