Advancing ADC Characterization: SECBased Native DAR and Drug Distribution Analysis Using Multi-Reflecting TOF-MS and INTACT Mass Application

Significance of the Topic

Antibody-drug conjugates (ADCs) combine targeted antibody specificity with potent cytotoxic payloads, representing a major class of biotherapeutics. Accurate assessment of drug-to-antibody ratio (DAR) and glycoform distribution is critical for evaluating ADC efficacy, stability, and safety. Native mass spectrometry coupled with size-exclusion chromatography (SEC) preserves protein structure and enables detailed characterization in a label-free manner within a single run.

Aims and Study Overview

This study evaluates an integrated workflow using the ACQUITY Premier UPLC System and the Xevo MRT Mass Spectrometer along with the INTACT Mass Application within the waters_connect platform. The objectives are to demonstrate concurrent glycoform profiling and DAR analysis of monoclonal antibodies (mAbs) and cysteine-conjugated ADCs using native SEC-MS, and to highlight automated data processing for streamlined review and reporting.

Methodology and Instrumentation

Sample preparation involved dilution of Herceptin (trastuzumab) and ENHERTU™ (fam-trastuzumab deruxtecan-nxki) to 2 µg/µL in 10 mM ammonium acetate. Native SEC separation was performed on an ACQUITY UPLC Protein BEH SEC column (200 Å, 1.7 µm, 2.1 × 150 mm) at 0.1 mL/min and 30 °C. Mass analysis employed a Waters Xevo MRT TOF mass spectrometer in positive ion mode (500–8000 m/z, 1 Hz scan rate) with source and desolvation temperatures of 100 °C and 450 °C, respectively. Data acquisition, automated deconvolution and DAR calculations were conducted using INTACT Mass Application version 1.9.

Key Results and Discussion

• Herceptin Analysis: SEC-MS chromatogram showed a dominant monomer peak with a clear native charge envelope. Deconvoluted spectra revealed a distribution of biantennary N-glycoforms, confirming method suitability for glycoform profiling.
• ENHERTU Characterization: Native SEC-MS enabled preservation of intact ADC structure despite reduced interchain disulfides. Automated deconvolution identified major species carrying eight payloads (D8) and minor D6 species. Calculated DAR of 7.97 matched expected values and literature.

Benefits and Practical Applications

• Fully automated DAR and drug distribution calculations reduce manual intervention and interpretation errors.
• High-resolution native SEC-MS provides simultaneous glycoform profiling, aggregate detection, and DAR assessment in one analysis.
• Accessible workflow supports analysts with varying LC-MS expertise and accelerates both early research and QC release assays.

Used Instrumentation

• ACQUITY™ Premier UPLC System
• Xevo™ MRT Multi-Reflecting TOF Mass Spectrometer
• waters_connect™ Platform with INTACT Mass Application v1.9

Future Trends and Potential Applications

• Integration of AI-driven data analysis for more rapid interpretation of intact mass spectra.
• Extension of the workflow to other bioconjugates such as PEGylated proteins and oligonucleotide therapeutics.
• Development of compliance-ready solutions to streamline regulatory submissions and release testing.
• Continuous improvement of mass analyzer performance to increase sensitivity for low-abundance variants.

Conclusion

The combination of native SEC-MS on the ACQUITY Premier UPLC and Xevo MRT system with the automated INTACT Mass Application provides a robust, sensitive, and efficient platform for comprehensive characterization of mAbs and ADCs. This approach ensures high confidence in glycoform profiling and DAR determination, supporting critical quality assessments across discovery and development.

References

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