Comprehensive Characterization of Monoclonal Antibody and Antibody Drug Conjugate on a Hybrid Quadrupole- Orbitrap Mass Spectrometer

Significance of the Topic

The structural complexity and heterogeneity of monoclonal antibodies (mAbs) and antibody–drug conjugates (ADCs) require comprehensive analytical methods to ensure safety, efficacy and consistency during development and production. High-resolution mass spectrometry (MS) provides critical information on intact masses, subunit composition, post-translational modifications and drug-to-antibody ratios (DAR), supporting deep characterization of biotherapeutics.

Objectives and Overview

This study evaluates the performance of the Thermo Scientific™ Orbitrap Exploris™ 480 mass spectrometer equipped with the BioPharma Option for end-to-end characterization of mAbs and ADCs. Key aims include:

• Intact mass analysis under native and denaturing conditions.
• Top-down and middle-down subunit fragmentation.
• Peptide mapping for full sequence coverage and modification profiling.

Methodology and Instrumentation

Three complementary MS workflows were applied:

• Intact mass analysis: Native size-exclusion chromatography (SEC)-MS and reversed-phase (RP) LC-MS under denaturing conditions to profile glycoforms and charge states.
• Top-down/middle-down: RP LC-MS/MS analysis of reduced or IdeS-digested subunits for direct fragmentation of light chain, Fdʹ and Fc domains.
• Peptide mapping: Proteolytic digestion using SMART Digest™ kit followed by RP LC-MS/MS for sequence coverage and modification detection.

Data processing employed Thermo Scientific™ BioPharma Finder™ software with ReSpect deconvolution for intact mass and fragmentation assignment, using a mass tolerance of 10 ppm and a signal-to-noise cutoff of 7.

Used Instrumentation

• Thermo Scientific Orbitrap Exploris 480 with BioPharma Option.
• SEC LC system for native analysis and RP LC system for denatured workflows.
• SMART Digest™ kit for peptide digestion and FabRICATOR® (IdeS) enzyme for subunit generation.
• BioPharma Finder™ 3.2 software for data analysis and result visualization.

Main Results and Discussion

• Peptide Mapping: Achieved 100% sequence coverage of trastuzumab, enabling identification of deamidation sites and other modifications.
• Intact Mass Native Analysis: Obtained single-scan spectra at 30 000 resolution (m/z 200) with <7 ppm mass error, resolving individual glycoforms and charge states.
• Top-Down Subunit Analysis: Reduced mAb analysis at 240 000 resolution provided baseline-resolved spectra of light and heavy chains, with accurate mass measurements within 2 ppm.
• Middle-Down IdeS Digestion: Subunit fragmentation of Fc and Fdʹ domains yielded >50% residue cleavages, confirming domain-specific modifications.
• ADC Mimic Analysis: Native SEC-MS deconvolution revealed a distribution of 0–8 drug attachments; DAR calculation based on G0F/G1F glycoform gave accurate DAR values aligning with theoretical expectations.

Benefits and Practical Applications

This unified platform delivers:

• High mass accuracy and resolution across intact, subunit and peptide levels.
• Streamlined workflows for R&D and quality control in biopharmaceutical development.
• Comprehensive glycoform, modification and DAR profiling without multiple instruments.
• Enhanced structural insight to support comparability studies and regulatory submissions.

Future Trends and Potential Applications

Emerging directions include:

• Integration with ion mobility separation for conformational analysis of mAbs and ADCs.
• Automation and high-throughput sample handling for large-scale screening.
• Machine learning tools for rapid interpretation of complex MS data.
• Real-time monitoring of bioprocesses using native MS to track product quality attributes.

Conclusion

The Orbitrap Exploris 480 with BioPharma Option demonstrates outstanding performance for complete characterization of therapeutic antibodies and conjugates on a single platform. Its high resolution, mass accuracy and versatile workflows enable in-depth structural and compositional analysis, supporting robust development and quality assurance of biopharmaceuticals.