The INTACT Mass Application in waters_connect™ Platform Streamlines ADC DAR and Drug Distribution Analysis

Importance of the topic

The precise measurement of drug-to-antibody ratio (DAR) and drug distribution is critical for the development and quality control of antibody-drug conjugates (ADCs). Automating these analyses reduces manual errors, accelerates decision making, and ensures compliance in biopharmaceutical workflows.

Study objectives and overview

This application brief evaluates the INTACT Mass App version 1.9, integrated within the waters_connect™ Platform, for automated LC-MS deconvolution, DAR calculation, and drug distribution profiling. Two FDA-approved ADCs—ENHERTU™ (cysteine-linked) and KADCYLA™ (lysine-linked)—serve as test cases to demonstrate accuracy, reproducibility, and workflow efficiency.

Methodology and instrumentation

Ultrahigh-resolution LC-MS data were acquired on the BioAccord™ System using:

• Native size exclusion chromatography-mass spectrometry (SEC-MS) for cysteine-conjugated ADCs
• Denaturing reversed-phase LC-MS (RPLC-MS) with PNGase F deglycosylation for lysine-conjugated ADCs

The INTACT Mass App 1.9 processed raw spectra, performed automated mass deconvolution, matched observed to theoretical masses with a “Drug” tag, and calculated DAR and drug distribution within the compliance-ready waters_connect Platform.

Main results and discussion

For ENHERTU™, native SEC-MS data deconvolution revealed a dominant D8 species (>98% relative abundance) and a calculated DAR of 7.97, consistent with literature. For KADCYLA™, RPLC-MS of deglycosylated samples yielded a DAR of 3.56, while native SEC-MS of untreated samples produced a DAR of 3.50. In both workflows, the automated calculations closely matched previously reported values obtained with the UNIFI™ App.

Benefits and practical applications

• Streamlines ADC characterization from early research through release testing
• Minimizes manual intervention and calculation errors
• Supports both denaturing and native separation methods
• Ensures data integrity and compliance in regulated environments

Future trends and applications

Ongoing developments may include support for more complex conjugation chemistries, expanded payload libraries, and integration with advanced data-mining tools. The automated platform is poised to accelerate ADC design, biosimilar comparison, and high-throughput screening in drug discovery.

Conclusion

INTACT Mass App 1.9 within the waters_connect Platform offers a robust, automated solution for ADC DAR and drug distribution analysis. It delivers reliable results across diverse conjugation types and LC-MS methods, enhancing throughput and compliance for biopharmaceutical laboratories.

Reference

1. Shion H et al. Waters Application Note 720007547 (2022)
2. Doneanu CE et al. Waters Application Note 720007546 (2022)
3. D’Esposito RJ et al. Waters Application Note 720008130 (2023)
4. Ranbaduge N et al. Waters Application Note 720007752 (2022)
5. Riccardi F et al. Front. Pharmacol. 14 (2023)
6. Shion H et al. Waters Application Note 720006570 (2019)
7. Martín M et al. Crit. Rev. Oncol. Hematol. 198 (2024)
8. Chen L et al. MAbs 8(7):1210–1223 (2016)