Complete Characterization of a Cysteine- linked Antibody-Drug Conjugate Performed on a Hybrid Quadrupole-Orbitrap Mass Spectrometer with High Mass Range

Significance of the topic

Antibody–drug conjugates (ADCs) represent a cutting-edge class of biotherapeutics that combine the targeting specificity of monoclonal antibodies with potent cytotoxic drugs. Precise characterization of ADCs—particularly the distribution of drug-to-antibody ratio (DAR), glycoforms and conjugation sites—is essential for ensuring safety, efficacy and batch-to-batch consistency in pharmaceutical development and quality control.

Objectives and overview of the study

This work establishes a comprehensive analytical workflow to fully characterize a cysteine-linked ADC, Brentuximab vedotin. The study aims to demonstrate:

• Native MS intact analysis for direct DAR profiling of ADC assemblies under non-denaturing conditions.
• Denaturing LC-MS peptide mapping to localize conjugation sites and validate sequence coverage.
• Integration of both approaches on a single hybrid quadrupole-Orbitrap platform modified for high mass range (HMR) detection.

Methodology and instrumentation used

A Thermo Fisher Vanquish UHPLC was coupled to modified Q Exactive Plus and Q Exactive HF Orbitrap mass spectrometers upgraded with HMR mode to extend m/z transmission up to 8 000. Two complementary workflows were executed:

• Native intact LC-MS: Online SEC desalting (Waters BEH SEC, 50 mM NH₄OAc) and direct electrospray native conditions at R = 15 000 or 17 500 followed by ReSpect deconvolution and Sliding Window integration in BioPharma Finder.
• Denaturing peptide mapping: Reversed-phase LC (MAb-Pac RP, Acclaim RSLC C18) with 2–90 % ACN gradient, HCD MS/MS (R = 60 000 full MS, R = 15 000–17 500 for MS/MS) and data analysis with MassAnalyzer algorithm in BioPharma Finder (5 ppm tolerance).

Main results and discussion

Native analysis resolved eight distinct DAR species (DAR0–DAR8) of Brentuximab vedotin, yielding an average DAR of 4.07, consistent with literature. The spectra preserved non-covalent interchain bonds and revealed minor 762 Da losses corresponding to linker cleavage.

Peptide mapping achieved 99 % sequence coverage for light and heavy chains in a single 90 min run. HCD fragmentation identified all four cysteine-conjugation sites in the hinge region. The addition of the vcMMAE linker dramatically increased peptide hydrophobicity and retention time, yet HCD signature ions (e.g., m/z 718, 506, 321, 152 ) enabled confident detection of modified peptides.

Benefits and practical applications of the method

• Unified platform for both native and denaturing analyses reduces instrument footprint and sample handling.
• Native MS directly reports DAR distributions and maintains structural integrity of ADCs.
• Peptide mapping with HCD fragmentation reliably localizes drug conjugation sites and post-translational modifications.
• High specificity and sensitivity support QC release testing and biosimilar comparability studies.

Future trends and potential uses

Advances in Orbitrap speed and mass range will further improve resolution of high-DAR species and labile linkers. Integration with ion mobility MS may add conformational insights. Automated data processing pipelines and expanded signature-ion libraries will streamline ADC characterization in regulated environments.

Conclusion

The study demonstrates a robust single-platform workflow combining native high-mass-range LC-MS and denaturing peptide mapping to achieve complete characterization of a cysteine-linked ADC. Instrument modifications and optimized HCD methods enable accurate DAR profiling and site-specific conjugation mapping, providing critical tools for biopharmaceutical development and quality control.

Reference

1. Janin-Bussat MC, Dillenbourg M, Corvaia N, Beck A, Klinguer-Hamour C. Characterization of antibody drug conjugate positional isomers at cysteine residues by peptide mapping LC-MS analysis. J Chromatogr B Analyt Technol Biomed Life Sci. 2015;981-982:9-13.
2. Debaene F, Boeuf A, Wagner-Rousset E, Colas O, Ayoub D, Corvaïa N, Van Dorsselaer A, Beck A, Cianférani S. Innovative native MS methodologies for antibody drug conjugate characterization: high-resolution native MS and IM-MS for average DAR and DAR distribution assessment. Anal Chem. 2014;86(21):10674-10683.