Top-down characterization of native monoclonal antibodies obtained with electron capture dissociation on Q-TOF instruments

Significance of the Topic

Monoclonal antibodies are a cornerstone of modern therapeutics, used in oncology, autoimmune diseases and beyond. Their structural complexity and susceptibility to post-translational modifications demand analytical methods that preserve intact proteoforms while delivering detailed sequence information. Top-down mass spectrometry can directly characterize whole antibodies, avoiding digestion and potential artifacts, but often suffers from low ion yields and limited sequence coverage. This study demonstrates that electron capture dissociation (ECD) combined with optimized collisional activation on an Agilent Q-TOF platform overcomes these limitations, enabling high-confidence mapping of complementarity-determining regions (CDRs) in native monoclonal antibodies.

Objectives and Study Overview

The primary aim was to apply top-down ECD to intact therapeutic antibodies to:

• Optimize supplemental vibrational activation to facilitate antibody unfolding and enhance ECD efficiency.
• Achieve extensive sequence coverage of light and heavy chains, with a focus on all CDRs, particularly CDR-3.
• Assess the competition between ECD and collision-induced dissociation (CID) and identify optimal tuning profiles.

Three reference antibodies (NIST mAb, SigmaMAb, Infliximab) were evaluated under identical conditions.

Methodology

Intact antibodies were buffer-exchanged into 100 mM ammonium acetate and infused by static nanospray at 1 mg/mL. ECD experiments scanned supplemental collision energies from 0 to 200 V to promote partial unfolding. Spectra were acquired in MS1 mode with a low-mass cutoff (~m/z 4000) to exclude low-mass contaminants. Real-time monitoring via ExDViewer guided selection of optimal activation voltages. Data analysis employed Agilent MassHunter Qualitative Analysis and ExDViewer for ion assignment, sequence coverage calculation and ECD efficiency evaluation.

Instrumentation

• Agilent 6545 and 6545XT AdvanceBio LC/Q-TOF mass spectrometers.
• Second-generation ExD cell (e-MSion, part of Agilent) featuring seven electrostatic lenses, dual ring magnets and an electron-emitting filament.
• Controlled by ExD Control software and calibrated with Agilent tune mix; tuning validated with Substance P and carbonic anhydrase standards.

Main Results and Discussion

Supplemental activation at ~100 V yielded the best balance between partial unfolding and ion signal intensity. Across the three antibodies:

• Light chains achieved 75–90 % sequence coverage, with confident identification of all three CDRs.
• Heavy chains reached 45–50 % coverage, with only CDR-3 mapped robustly.
• ECD outperformed CID at all tested energies when using an ECD-optimized tune; a second tune selectively cleaved interchain disulfide bonds, producing prominent intact light-chain fragments.
• SigmaMAb exhibited increased CID fragment intensity at higher collision energies under disulfide-targeting tunes, indicating competition between ECD and CID processes.

Benefits and Practical Applications

This top-down ECD approach offers:

• Rapid, digestion-free profiling of intact antibodies.
• High-confidence CDR mapping critical for biotherapeutic characterization and QA/QC.
• Capability to localize disulfide bonds and glycan attachments without extensive sample handling.

Future Trends and Opportunities

Advancements may include coupling ECD with higher-resolution platforms, applying real-time online monitoring in manufacturing environments, and extending the method to other large protein assemblies. Integration with automated data interpretation tools could further accelerate biopharmaceutical development and quality control workflows.

Conclusion

Electron capture dissociation on Q-TOF instruments, augmented with optimized collisional activation, provides a powerful top-down strategy for in-depth characterization of intact monoclonal antibodies. The method delivers substantial sequence coverage, particularly in light chains, and enables targeted disulfide bond cleavage, meeting the stringent demands of therapeutic antibody analysis.