Subunit Level Top-down ECD Characterization of Biotherapeutic Proteins: Improving Mass Spectral Clarity with Fragment Ion Level Ion Mobility

Significance of the Topic

Electron Capture Dissociation is a top-down fragmentation technique that cleaves N–Cα bonds while preserving side-chain modifications, offering complementary information to CID.
Integrating ECD with cyclic ion mobility separation enhances fragment ion clarity and sequence coverage, critical for detailed structural characterization of biotherapeutic proteins and their post-translational modifications.

Objectives and Study Overview

This work aimed to optimize top-down ECD conditions on a SELECT SERIES Cyclic IMS system using bovine carbonic anhydrase II as a model. Subsequently, the performance was demonstrated on subunits of NISTmAb, namely scFc, light chain and Fd, to assess sequence coverage and analytical robustness.

Used Instrumentation

• Premier ACQUITY UPLC H-Class system with Quaternary Solvent Management.
• BioResolve RP mAb Polyphenyl column for subunit separation.
• SELECT SERIES Cyclic IMS mass spectrometer equipped with an e-MSion ECD cell in a pre-ion mobility configuration.
• MassLynx 4.2 and waters_connect for BayesSpray deconvolution; ProSite Lite2 for fragment annotation.

Methodology

Bovine carbonic anhydrase II was infused at 10 μM in 50:50 methanol water at 10 μL per minute to tune ECD cell voltages.
Subunits of the NIST mAb obtained via IdeS digestion and reduction were separated by reversed phase LC with a total run time of 10 minutes.
Targeted ECD and ECD-IMS experiments were performed to compare fragmentation efficiency, and data were deconvolved and annotated to determine sequence coverage.

Main Results and Discussion

For BCA, sequence coverage increased from 11% with CID to 76% with ECD and further to 81% when coupled to IMS, identifying 154 additional fragment ions.
Chromatographic analysis of NISTmAb subunits yielded retention times of 3.98 minutes for scFc, 5.31 minutes for light chain and 7.41 minutes for Fd.
Targeted ECD-IMS of the scFc subunit produced 52% sequence coverage at analytical LC flow rates, demonstrating effective performance beyond nanoscale infusion.

Benefits and Practical Applications

The combined ECD-IMS approach delivers higher sequence coverage and improved fragment clarity, facilitating confident mapping of labile modifications and structural heterogeneity in therapeutic proteins.
This method is adaptable for quality control, comparability studies and advanced structural analysis in biopharmaceutical development.

Future Trends and Potential Applications

Ongoing developments may include integration with high throughput platforms, automated data annotation and hybrid fragmentation workflows to further enhance depth of analysis.
Broader application to other classes of biotherapeutics, extensive PTM profiling and clinical biomarker characterization are anticipated as IMS technology matures.

Conclusion

Top-down ECD combined with cyclic ion mobility offers a robust analytical strategy for detailed subunit-level characterization of biotherapeutic proteins, achieving high sequence coverage and spectral clarity at both nanoscale and analytical flow regimes.

References

• Henry Shion et al. Subunit Level Top-down ECD Characterization of Biotherapeutic Proteins: Improving Mass Spectral Clarity with Fragment Ion Level Ion Mobility. Waters Corporation, 2021.