Characterization of Antibody Drug Conjugates (ADC) using 2-Dimension Liquid Chromatography (2D-LC) and Native Mass Spectrometric Technologies

Significance of the topic

Antibody-drug conjugates (ADCs) represent a key class of targeted cancer therapeutics combining monoclonal antibodies with potent cytotoxic agents. Reliable characterization of ADCs, especially their drug-to-antibody ratio (DAR) and structural integrity, is essential for ensuring safety, efficacy and reproducibility in research and quality control.

Objectives and Study Overview

This study describes the development of a novel two-dimensional liquid chromatography (2D-LC) workflow coupled with native mass spectrometry (MS) for comprehensive analysis of intact ADCs. The method employs hydrophobic interaction chromatography (HIC) for DAR-based fractionation, followed by size exclusion chromatography (SEC) under native conditions, enabling preservation of noncovalent complexes and accurate intact mass measurements.

Methodology and Instrumentation

• Sample: Lyophilized brentuximab vedotin (cysteine-linked ADC with DAR variants 0, 2, 4, 6, 8).
• 2D-LC System: Agilent 1290 Infinity II with multiple heart-cutting HIC (AdvanceBio HIC) and online SEC (AdvanceBio SEC) for native separations.
• Denaturing Analysis: PLRP-S reversed-phase column for detailed fragment profiling.
• MS Detection: Agilent 6545XT AdvanceBio LC/Q-TOF with extended mass range to m/z 30 000; MassHunter Data Acquisition 11.0, Qualitative Analysis 10.0 and BioConfirm 11.0 software.

Main Results and Discussion

• HIC Separation: Clear resolution of DAR variants (D0 to D8) under native buffer conditions allowed precise DAR determination by MS.
• Denaturing 2nd Dimension: Reversed-phase LC/MS revealed multiple degradation products (light and heavy chain fragments, partial drug loss) when organic solvents and acids were used.
• Native SEC-MS: Preserved intact ADC structures, minimized fragmentation, and delivered high-quality raw spectra for each DAR fraction. Deconvoluted spectra confirmed accurate masses corresponding to DAR=2 through DAR=6 species.

Benefits and Practical Applications

The integrated 2D-LC/native MS workflow provides robust, high-resolution separation, precise DAR quantification, intact mass confirmation, and minimal sample perturbation. This approach is well suited for biopharmaceutical development, quality control, and stability studies of ADCs and other complex protein therapeutics.

Future Trends and Potential Applications

• Adoption of ultrahigh-resolution MS for deeper structural characterization.
• Automation of multidimensional workflows for high-throughput screening.
• Extension of native 2D-LC/MS to study non-covalent protein complexes and other bioconjugates.

Conclusion

The presented 2D-LC/native MS strategy offers a powerful, sensitive and reproducible platform for detailed characterization of intact ADCs. By preserving native conformations and enabling accurate DAR and mass analysis, it streamlines ADC development and supports rigorous quality assessment.

Reference

1. Ross PL et al. Physical and Chemical Stability of Antibody Drug Conjugates: Current Status. Journal of Pharmaceutical Sciences. 2016;105:391–397.
2. Agilent Technologies. High Salt - High Reproducibility: Analysis of antibody drug conjugates using hydrophobic interaction chromatography with the Agilent 1290 Infinity II Bio LC System. Application note 5994-2691EN; 2020.
3. Agilent Technologies. Sensitive Native Mass Spectrometry of Macromolecules Using Standard Flow LC/MS. Application note 5994-1739EN; 2020.