Analysis of Cysteine-Conjugated Antibody Drug Conjugates (ADCs) Using a Native SEC LC-MS Workflow on the SYNAPT XS

Importance of the Topic

Antibody–drug conjugates (ADCs) represent a leading class of targeted biotherapeutics. Accurate measurement of the drug-to-antibody ratio (DAR) is vital because it directly influences ADC efficacy, pharmacokinetics, and safety. Native mass spectrometry coupled with size-exclusion chromatography (SEC) preserves noncovalent interactions and enables reliable DAR determination in intact cysteine-conjugated ADCs.

Objectives and Study Overview

This application brief evaluates an analytical-scale native SEC LC-MS workflow on the SYNAPT XS system for cysteine-conjugated ADCs with varying drug loads. The study benchmarks DAR and drug distribution measurements against historical data from hydrophobic interaction chromatography (HIC) and previous-generation Q-Tof platforms to demonstrate robustness and comparability.

Methodology and Instrumentation

• SEC Column: ACQUITY UPLC Protein BEH SEC, 200 Å, 1.7 µm, 2.1 × 150 mm
• Mobile Phase: 50 mM ammonium acetate, isocratic flow at 0.1 mL/min, 10-minute runtime
• LC System: ACQUITY UPLC H-Class PLUS
• MS Detection: SYNAPT XS high-resolution Q-Tof, native ESI conditions
• Software: MassLynx 1.4.2 for acquisition; UNIFI 1.9.4 (waters_connect) for automated data processing and DAR calculation

Main Results and Discussion

Native SEC LC-MS produced narrow, high m/z charge envelopes with clear glycoform and drug-load peak patterns. Deconvoluted spectra revealed discrete species corresponding to 0, 2, 4, 6, and 8 drug attachments per antibody. Calculated DAR values and drug-loading distributions aligned closely with HIC-UV data and previous Q-Tof analyses (Xevo G2-S, Vion IMS) across multiple batches and both glycosylated and deglycosylated samples, confirming method reproducibility.

Benefits and Practical Applications

• No pre-analysis sample preparation (e.g., buffer exchange or deglycosylation) required.
• Automated DAR and drug distribution measurement within the UNIFI informatics platform.
• Routine, robust native LC-MS workflow suitable for comparability studies and lot release testing.

Future Trends and Potential Applications

• Broad adoption of native SEC–MS for intact bioconjugate characterization in R&D and QC.
• Integration of advanced ion mobility separation for enhanced structural insights.
• Real-time process monitoring of ADC manufacturing using inline SEC-MS.
• Expansion to multi-drug and site-specific conjugate analysis with high-resolution platforms.

Conclusion

The analytical-scale native SEC LC-MS workflow on the SYNAPT XS provides consistent, high-throughput DAR measurements for cysteine-conjugated ADCs. It delivers comparable performance to orthogonal HIC methods and earlier Q-Tof systems while streamlining sample handling and data processing.

References

1. Shion H, Birdsall R, Cubbedge S, Chen W. Integrated Informatics Workflows for Automated Assessment of ADC Comparability Using LC-UV and LC-UV/MS. Waters Application Note 720005306EN, 2015.
2. Shion H, Yu Y, Chen W. Analytical-Scale Native SEC-MS for ADC Characterization. Waters Application Note 720006368EN, 2018.
3. Shion H, Yu Y, Chen W. Analysis of ADCs by Native Mass Spectrometry on the BioAccord System. Waters Application Note 720006570EN, 2019.