A COMBINED WORKFLOW FOR IN-DEPTH CHARACTERIZATION OF CYSTEINE-CONJUGATED ANTIBODY DRUG CONJUGATES

Importance of the topic

Antibody drug conjugates (ADCs) represent a key class of targeted therapies that combine the specificity of monoclonal antibodies with the potency of cytotoxic drugs. Cysteine conjugation is widely used to attach payloads while preserving antibody structure. Detailed characterization of drug-to-antibody ratio (DAR), conjugation sites and positional isomers is critical to ensure consistent efficacy and safety in biopharmaceutical development.

Objectives and overview of the study

This work describes a unified analytical strategy to thoroughly characterize cysteine-conjugated ADCs. The goals were to measure DAR, resolve positional isoforms, analyze subunit composition and pinpoint conjugation sites using complementary chromatography-mass spectrometry methods.

Methodology and instrumentation

A multi-tiered workflow was applied:

• Intact ADC analysis by hydrophobic interaction chromatography with UV detection (HIC/UV) and native size exclusion chromatography–LC/MS (SEC-LC/MS)
• Subunit separation by reversed-phase LC/MS following reduction
• Positional isomer resolution via two-dimensional HIC/RPLC LC/MS
• Peptide mapping using LC/MSE for site identification

Used instrumentation

• Waters ACQUITY H-Class Bio UHPLC systems with 2D technology
• Waters Xevo G2-S and G2 QTof mass spectrometers
• Columns: ProteinPak HiRes HIC, UPLC Protein BEH C4, BEH SEC, and CSH C18

Main results and discussion

Strong agreement was observed between DAR values determined by HIC/UV and native SEC-LC/MS across three loading levels. Two-dimensional LC/MS enabled unambiguous separation and identification of positional isomers through deconvoluted subunit mass spectra. Peptide mapping identified thirteen cysteine conjugation sites and provided occupancy ratios, revealing heterogeneous modification patterns.

Benefits and practical applications

• Accurate DAR profiling supports robust quality control and lot consistency
• Positional isomer analysis informs structure–function relationships
• Site-specific peptide mapping ensures precise characterization for safety assessment

Future trends and opportunities

Future advancements may include higher-resolution mass analyzers, fully automated fraction collection and improved informatics for data interpretation. Miniaturized, high-throughput LC-MS platforms will further streamline ADC characterization. Extending this workflow to other conjugation chemistries and payloads can broaden its impact.

Conclusion

The integrated LC/UV/MS and peptide mapping workflow provides comprehensive insight into cysteine-conjugated ADCs by combining DAR measurement, isomer resolution and site localization. Such detailed analytics are essential for rigorous development and quality control of next-generation biotherapeutics.

References

1. Poster TP236 at the 61st ASMS Conference detailing HIC-UV, SEC-LC/MS and RP-LC/MS analysis
2. Poster T2265 at the 61st ASMS Conference detailing 2D LC/MS analysis