Improved Middle-Down Characterization of Antibodies Using Multiple Ion Activation Techniques and Ion-Ion Proton Transfer Reactions on a Modified Orbitrap Mass Spectrometer

Importance of the topic

Monoclonal antibodies play a crucial role in biotherapeutics and diagnostics. Detailed sequence characterization of antibody subunits supports quality control, comparability studies, and detection of sequence variants and post-translational modifications. Middle-down mass spectrometry bridges the gap between top-down and bottom-up approaches by preserving large fragments for structural analysis.

Objectives and overview of the study

This work evaluates the benefit of applying ion-ion proton transfer (IIPT) reactions to selected product-ion m/z windows following various fragmentation methods in a middle-down LC-MS3 workflow. The goal is to improve detection of high-mass sequence ions in the light chain (LC), Fd, and Fc/2 subunits of a NIST mAb standard.

Methodology and instrumentation

Sample preparation included IdeS proteolysis and reduction to yield ~25 kDa subunits. Online reversed-phase LC separated subunits prior to targeted MS2 and MS3 acquisition. A modified Thermo Scientific Orbitrap Fusion Lumos Tribrid mass spectrometer featured dual reagent inlets to introduce ETD and IIPT reagents simultaneously. Fragmentation modes covered ETD, CID, HCD, and UVPD, with subsequent IIPT applied to narrow m/z windows of product ions. Data processing was performed using QualBrowser, BioPharma Finder, and ProSight Lite.

Main results and discussion

Combining ETD with IIPT (Experiments 1–5) provided the most significant increase in sequence coverage, revealing long, high-mass fragment ions across all subunits. Aggregate coverage for these experiments reached up to ~92%, with extensive mapping of complementary N- and C-terminal fragments. In contrast, IIPT following CID or HCD yielded lower coverage (13–31%) due to the use of broader m/z windows that led to spectral congestion.

Benefits and practical applications of the method

- Enhanced detection of large sequence ions improves confidence in sequence assignment and PTM localization.
- Accelerated middle-down workflows support rapid comparability and QA/QC of biotherapeutic antibodies.
- The approach can be integrated into regulated laboratories for routine antibody characterization.

Future trends and opportunities

Applying IIPT to UVPD-generated product ions and refining m/z window widths are promising directions to further boost sequence coverage. Automation of targeted IIPT strategies and integration with advanced bioinformatics will enable higher throughput and deeper structural insights in antibody analysis.

Conclusion

This preliminary study demonstrates that targeted IIPT following ETD fragmentation substantially enhances middle-down sequence coverage of mAb subunits, enabling observation of large fragment ions that are otherwise obscured. Optimization of window selection and application to other activation methods will expand its utility in antibody characterization.

Used instrumentation

• Thermo Scientific UltiMate 3000 HPLC system with RP-4H column, flow rate 1.5 µL/min
• Modified Thermo Scientific Orbitrap Fusion Lumos Tribrid mass spectrometer with dual reagent inlet for ETD and IIPT
• Data analysis software: QualBrowser, BioPharma Finder 3.0, ProSight Lite

References

1. Houel S, Fornelli L, Srzentic K, et al. Middle-down analyses of monoclonal antibodies on Orbitrap Fusion Lumos Tribrid. Proceedings of the 16th Annual HUPO World Congress; 2017.
2. Anderson LC, English MA, Wang WH, et al. Protein derivatization and sequential ion/ion reactions to enhance sequence coverage by electron transfer dissociation mass spectrometry. Int J Mass Spectrom. 2015;377:717–724.
3. Mullen C, Schwartz JC, Syka JEP, et al. Improved top-down sequence coverage on a Tribrid mass spectrometer by ion-ion proton transfer reactions subsequent to ETD and UVPD. ASMS 66th Conf.; San Diego, 2018.
4. Sanders J, Holden D, et al. Reducing congestion in ultraviolet photodissociation spectra of large protein ions using proton transfer reactions. ASMS 66th Conf.; San Diego, 2018.