Top-down Analysis of Intact Antibodies under Denatured and Native Conditions on the Omnitrap Platform Coupled to an Orbitrap Mass Spectrometer

Significance of the topic

The detailed characterization of monoclonal antibodies (mAbs) is critical for therapeutic development, quality control and biosimilarity assessment. Top-down mass spectrometry preserves intact proteoforms and labile modifications, offering a direct route to full-sequence and post-translational modification analysis. The Omnitrap platform coupled to an Orbitrap mass spectrometer represents an innovative approach to enhance fragmentation efficiency and sequence coverage for intact mAbs under both native and denatured conditions.

Objectives and study overview

• Assess the performance of the Omnitrap-Q Exactive Plus setup for top-down analysis of intact mAbs.
• Compare fragmentation techniques including slow-heating CID with broadband excitation, electron capture dissociation (ECD), and collisionally activated ECD (CA-ECD).
• Quantify sequence coverage improvements and detection of Fc glycoforms (G0F, G1F) in heavy and light chains.

Methodology and instrumentation

• Sample preparation: Herceptin® mAb buffer-exchanged into 100 mM ammonium acetate for native analysis or 50:50 water/acetonitrile with 0.1% formic acid for denatured conditions; concentration at 5 µM.
• Ionization: Static nanoelectrospray using coated borosilicate emitters.
• Instrument configuration: Omnitrap platform integrated with Q Exactive Plus (Biopharma option), featuring an electron gun, H-atom source, HCD cell, quadrupole mass filter and Orbitrap analyzer.
• Fragmentation workflows: (a) slow-heating CID with broadband excitation of high-m/z first-generation fragments; (b) ECD of charge-reduced radical species; (c) collisionally-activated ECD on 49+ precursors; (d) pseudo MS³ combining ECD followed by CID of primary fragments.

Main results and discussion

• CID with broadband excitation improved signal-to-noise ratios for first-generation fragments, facilitating confident assignment of b and y ions.
• Collisionally activated ECD generated novel N-terminal b and c fragments, boosting ECD sequence coverage beyond conventional ECD alone.
• Overall sequence coverage in MS² CID experiments increased from 48% to 73% when internal fragments were included; CA-ECD primary fragment assignments reached 63%.
• Glycoform-containing C-terminal fragments (G0F, G1F) were identified consistently in both MS² CID and pseudo MS³ CA-ECD spectra.
• Data processing aided by the new PeakFinder software improved assignment confidence and streamlined spectral interpretation.

Benefits and practical applications

• Provides comprehensive top-down profiling of intact mAbs, preserving native structure and labile modifications.
• Enables robust quality control and comparability studies in biopharmaceutical development, including glycoform analysis.
• Combination of multiple fragmentation strategies yields high sequence coverage, improving detection of sequence variants and degradation products.

Future trends and opportunities

• Integration of additional fragmentation methods (UVPD, EThcD) on the Omnitrap for deeper proteoform coverage.
• Development of AI-driven data analysis pipelines for automated fragment assignment and proteoform characterization.
• Extension of the workflow to other complex biotherapeutics such as antibody-drug conjugates, bispecific antibodies and fusion proteins.
• Adoption of native MS approaches for studying higher-order structure and non-covalent complexes in real time.

Conclusion

The Omnitrap-Orbitrap platform demonstrates efficient top-down analysis of intact monoclonal antibodies under denatured and native conditions. Combining slow-heating CID, ECD and CA-ECD fragmentation enhances sequence coverage and glycoform detection. New data-processing tools further improve confidence in fragment assignments, presenting a powerful workflow for detailed biopharmaceutical characterization.