Analysis of Antibody Drug Conjugates (ADCs) by Native Mass Spectrometry on the BioAccord System

Importance of Analyzing Antibody Drug Conjugates (ADCs)

AntibodyDrugConjugates combine the targeting specificity of monoclonal antibodies with potent cytotoxic payloads and are emerging as a key class of cancer therapeutics. The drug to antibody ratio or DAR is a critical quality attribute that influences drug efficacy, safety, and pharmacokinetics. Accurate determination and monitoring of DAR across development and manufacturing stages is essential for ensuring consistent product performance.

Study Goals and Overview

This study demonstrates the capabilities of the BioAccord System for native mass spectrometry analysis of both cysteine conjugated and lysine conjugated ADCs. The primary objective was to assess DAR values and drug distribution profiles under non denaturing conditions and compare results with established orthogonal techniques and prior MS platforms.

Methodology and Instrumentation Used

The analysis employed analytical scale size exclusion chromatography under native conditions coupled directly to a compact oaTOF MS detector. Key elements included

• ACQUITY UPLC I Class PLUS System
• ACQUITY Protein BEH SEC Column 200Angstrom 1.7 micrometer 2.1 x 150 mm
• ACQUITY RDa Detector compact oaTOF MS
• UNIFI Scientific Information System for automated data acquisition, processing, and reporting
• Isocratic elution with 50 mM ammonium acetate over a ten minute run

Inline SEC facilitated buffer exchange and desalting, preserving native quaternary structures and yielding low charge state envelopes at high m/z ranges up to 7000.

Main Results and Discussion

For cysteine conjugated ADCs three levels of drug loading were analyzed without deglycosylation. DAR values obtained by native SEC MS matched those from hydrophobic interaction chromatography and prior QToF instruments. In the lysine conjugated ADC example of Kadcyla, the calculated average DAR of 3.46 closely agreed with the published value of 3.50. Deconvoluted spectra revealed distinct glycoforms and drug conjugation states. Automated peak integration and DAR calculation within UNIFI streamlined data processing and ensured reproducible results.

Benefits and Practical Implementation

The BioAccord System offers a small footprint, simplified user interface, and built in compliance features that lower the barrier for routine native MS analysis. Automated workflows for DAR determination reduce operator variability and enable consistent lot to lot comparisons. The platform supports regulated and non regulated environments and integrates seamlessly with existing QC processes.

Future Trends and Applications

Native SEC MS is poised to expand beyond DAR assessment to multiproduct attribute analysis, real time process monitoring, and high throughput screening of diverse biotherapeutic modalities. Integration with advanced informatics and AI driven spectral interpretation will further enhance speed, depth, and confidence in ADC characterization and comparability studies.

Conclusion

This work demonstrates that the BioAccord System provides a robust, accessible, and reproducible platform for native LC MS based analysis of antibody drug conjugates. Consistent DAR measurements, streamlined workflows, and flexible compliance options support its adoption across research and manufacturing settings.

References

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2. H Shion et al Analytical Scale Native SEC MS for Antibody Drug Conjugates ADCs Characterization Waters Application Note 720006368EN 2018
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