Applying cDDA-eXd on a timsOmni to the analysis of the Fab regions of antibodies originating from human serum

Importance of Topic

The detailed characterization of antibody fragments, especially the antigen-binding Fab regions, is crucial for biopharmaceutical development and quality control.
Understanding proteoform heterogeneity and post-translational modifications in Fabs directly impacts drug safety, efficacy, and stability assessment.

Objectives and Study Overview

This work evaluates a novel top-down approach combining charge-state‐resolved data‐dependent acquisition (cDDA) with electron capture dissociation (ECD) on a Bruker timsOmni platform.
The aim was to analyze a complex mixture of six human IgG1 Fabs derived from serum, achieving high sequence coverage and unambiguous proteoform identification on an LC-MS timescale.

Methodology and Instrumentation

• Sample Preparation: Six monoclonal IgG1 antibodies were enzymatically cleaved using hinge‐specific protease IgdE, yielding Fab fragments. Aliquots were resolved in 0.1% formic acid.
• Liquid Chromatography: Fabs were separated on a MAbPac Capillary Reversed-Phase column with a fast gradient (23–33% to 95% acetonitrile) to distribute charge states between 33+ and 37+.
• Mass Spectrometry: The timsOmni IMS Q-Omnitrap ToF instrument applied cDDA for on-the-fly charge deconvolution and dynamic precursor selection.
  Selected ions were accumulated in the Omnitrap cell and fragmented by ECD at low electron energy (~1 eV).
• Data Processing: Raw spectra were interpreted with DataAnalysis and OmniScape software, enabling high-fidelity isotopic profiling and sequence assignment.

Main Results and Discussion

The charge-resolved ECD strategy yielded rich fragmentation patterns for each Fab proteoform:

• High signal-to-noise TOF spectra were obtained by averaging kHz-rate scans, ensuring clear isotope clusters.
• Sequential MS2 acquisitions captured complementary c- and a-type ions, improving sequence coverage across light and heavy chains.
• Dynamic precursor scheduling minimized chimeric spectra by isolating non-overlapping charge windows.

The approach disentangled overlapping coeluting species, revealing subtle proteoform variants and modifications without digestion steps.

Benefits and Practical Applications

• Direct top-down analysis preserves intact mass and heterogeneity, bypassing peptide assembly.
• cDDA-ECD on the timsOmni allows untargeted profiling of complex mixtures, suitable for serum-derived or biopharmaceutical samples.
• High analytical confidence supports batch-to-batch comparability, detection of deamidation or glyco-variants in real time.
• Fast LC-MS timescale operation aligns with routine quality control workflows.

Future Trends and Potential Applications

Further enhancements may include:

• Integrating electron impact dissociation (EID) to access fragmentation channels inaccessible to ECD, boosting sequence coverage in challenging regions.
• Combining optimized LC gradients and ion mobility separation for deeper resolution of highly similar Fab proteoforms.
• Extending cDDA-ECD workflows to full antibodies, antibody-drug conjugates, and other therapeutic proteins for comprehensive characterization.

Conclusion

This study demonstrates that cDDA-ECD on a timsOmni platform enables robust, high-resolution top-down analysis of antibody Fab fragments.
By dynamically selecting charge states and applying ECD fragmentation, the method delivers unambiguous proteoform assignments on an LC-MS timescale.
Such capabilities are invaluable for biopharmaceutical R&D, QC, and advanced proteomics research.

Used Instrumentation

• Bruker timsOmni IMS Q-Omnitrap ToF mass spectrometer with integrated Omnitrap cell and electron gun
• MAbPac Capillary Reversed-Phase HPLC Column
• CaptiveSpray ion source
• In-source collision energy (isCID) set to 40 eV
• Dynamic charge-state DDA and low-energy ECD (~1 eV)

References

1. den Boer MA, Greisch JF, Tamara S, Bondt A, Heck AJR. Selectivity over coverage in de novo sequencing of IgGs. Chemical Science. 2020; DOI:10.1039/d0sc03438j.