ATE: Integrated characterization of a lysine-linked antibody-drug conjugate by native intact mass analysis and peptide mapping performed on a hybrid quadrupole-Orbitrap mass spectrometer with high mass range

Significance of the topic

Antibody-drug conjugates (ADCs) combine monoclonal antibodies with potent cytotoxic drugs to improve therapeutic selectivity.
Characterizing drug-to-antibody ratio (DAR) and conjugation sites is critical for efficacy, safety and quality control.
ADCs present significant analytical challenges due to heterogeneity in glycosylation, drug loading and linker chemistry.

Objectives and overview of the study

This work integrates native intact mass analysis and peptide mapping in a single analytical workflow using a hybrid quadrupole-Orbitrap mass spectrometer.
The primary goals are accurate measurement of the average DAR, profiling of glycoforms and linker-only species, and identification of lysine conjugation sites.

Methodology and instrumentation

A combined approach on a Thermo Scientific Q Exactive Plus with High Mass Range mode enabled native SEC-MS and high-resolution peptide mapping.

• Sample preparation: Trastuzumab Emtansine ADC in formulation buffer was analyzed directly without pretreatment for native MS and after SMART Digest and DTT reduction for peptide mapping.
• Native analysis: Online size-exclusion chromatography (SEC) coupled to MS up to m/z 8000, 10 microscans and resolution settings up to 70,000; data deconvoluted with ReSpect in BioPharma Finder.
• Peptide mapping: 60 min reverse-phase gradients, trypsin digestion, Q Exactive Plus at 70,000 resolution; data searched with MassAnalyzer allowing variable modifications: N-glycans, deamidation, oxidation, MCC-DM1 and dead linker.

Main results and discussion

Native intact MS revealed a broad distribution of 0–8 linker-drug attachments corresponding to major glycoforms (G0F/G0F, G0F/G1F, G1F/G1F, G1F/G2F).
The average DAR was determined as 3.65, consistent with prior observations.
A distinct linker-only species was detected with a mass shift of ~220.8 Da, indicating non-reactive dead linkers.
Peptide mapping achieved 100 % sequence coverage and identified 22 of 44 lysine conjugation sites for MCC-DM1, plus several lysines modified by linker-only attachments.
Conjugated peptides exhibited increased retention times and eluted as doublets, reflecting stereoisomerism of the DM1 payload.
HCD fragmentation generated unique signature ions enabling confident identification of linker and drug-modified peptides.

Advantages and practical applications of the method

• Single-platform workflow reduces sample handling and variability.
• Native MS improves separation of co-occurring species and reveals heterogeneity in glycosylation and drug loading.
• Peptide mapping delivers site-specific information and signature fragments for quality control.
• The combined strategy supports comprehensive ADC characterization in biopharmaceutical development and QC environments.

Future trends and applications

Advancements may include integration of ion mobility spectrometry for additional separation of isomers, automation of data analysis pipelines, and extension to other ADC constructs and biotherapeutics.
Improvements in mass spectrometer sensitivity and resolution will further enhance site-specific quantitation and trace-level detection of variant species.

Conclusion

The integrated use of native intact mass analysis and peptide mapping on a high-resolution quadrupole-Orbitrap platform enables detailed characterization of lysine-linked ADCs.
This approach delivers accurate DAR measurements, resolves glycoform and linker heterogeneity, and localizes drug conjugation sites in a streamlined workflow suitable for biopharmaceutical research and quality control.

References

• Marcoux J, Champion T, Colas O et al. Native mass spectrometry and ion mobility characterization of trastuzumab emtansine, a lysine-linked antibody drug conjugate. Protein Sci. 2015;24(8):1210–1223.
• Luo Q, Chung HH, Borths C et al. Structural characterization of a monoclonal antibody–maytansinoid immunoconjugate. Anal Chem. 2016;88(1):695–702.