Top-down sequencing of bispecific antibodies by electron capture dissociation on a timsOmni platform

Importance of the topic

Bispecific antibodies offer dual-target engagement, enhancing therapeutic precision. Detailed characterization of their sequence, disulfide connectivity and posttranslational modifications is critical for ensuring efficacy and safety. Top-down mass spectrometry with electron capture dissociation (ECD) preserves native proteoform integrity and reveals key structural features, supporting advanced biologics development.

Objectives and study overview

This study applies the Bruker timsOmni platform to perform top-down ECD sequencing on bispecific antibody fragments. By generating F(ab’)2 and Fab subunits, the work aims to map complementarity-determining regions (CDRs), locate disulfide bonds, and identify O-glycosylation sites, comparing intact subunit spectra with those of the assembled proteoform to streamline interpretation.

Methodology and instrumentation

Enzymatic digestion: IdeS and IgdE cleavage produced ~100 kDa F(ab’)2 and ~50 kDa Fab fragments. Controlled reduction/reoxidation with 2-mercaptoethylamine adjusted disulfide pairing. Separation by cation exchange, hydrophobic interaction chromatography and ESI-MS assessed fragment quality.

ECD fragmentation: Charge states (20+ to 50+) were isolated and trapped in the Omnitrap cell of the timsOmni IMS Q-Omnitrap ToF instrument. ECD at ~1 eV with 50 ms activation generated radical-driven backbone cleavages. DataAnalysis, OmniScape and custom software facilitated spectral processing.

Instrumentation:

• Bruker timsOmni IMS Q-Omnitrap ToF mass spectrometer
• Omnitrap cell with integrated electron gun
• Nanospray ionization, 2 s accumulation

Key results and discussion

• Clear ECD ion sequence ladders (E2RISL) were obtained for bsAb F(ab’)2 and Fab fragments, allowing direct spectral comparison.
• Light and heavy chain CDRs were unambiguously assigned with <5 ppm mass accuracy.
• O-glycosylation sites were localized on AR3C and AR4A light chains (Hex(1)HexNAc(1) on specific serine residues).
• N-terminal pyroglutamate formation on heavy chains was detected.
• Fragmentation of >25 kDa proteins achieved high sequence coverage and enabled disulfide bridge mapping, overcoming common top-down limitations.

Benefits and practical applications

This approach enables comprehensive proteoform profiling of bispecific antibodies, supporting quality control and regulatory compliance in biopharmaceutical production. It facilitates detection of mispaired chains, modifications and sequence variants, accelerating therapeutic development and troubleshooting.

Future trends and potential applications

• Incorporation of collision-induced unfolding (CIU) to generate conformers for MS3 fragmentation workflows.
• Pre-fragmentation ion mobility separation to resolve proteoform heterogeneity.
• Extension of top-down ECD strategies to larger antibody constructs and multispecific formats for enhanced structural insights.

Conclusion

The Bruker timsOmni platform delivers high-quality top-down ECD fragmentation for bispecific antibody fragments. Its combination of ion mobility, electron capture dissociation and advanced data analysis addresses previous challenges in proteoform characterization, enabling detailed mapping of therapeutic antibody structure and modifications.

References

• Radić L et al. Bispecific antibodies against the hepatitis C virus E1E2 envelope glycoprotein. Proc Natl Acad Sci U S A. 2025; DOI:10.1073/pnas.2420402122
• den Boer MA, Greisch JF, Tamara S, Bondt A, Heck AJR. Selectivity over coverage in de novo sequencing of IgGs. Chem Sci. 2020; DOI:10.1039/d0sc03438j