Native Top-Down Analysis of Intact Antibodies using Multiple Dissociation Techniques on a Tribrid Quadrupole Orbitrap Linear Ion Trap Mass Spectrometer

Importance of the Topic

Therapeutic monoclonal antibodies are key biopharmaceuticals for oncology and autoimmune diseases. Top-down mass spectrometry offers direct analysis of intact proteins, preserving labile modifications and complex architectures, which is critical for comprehensive structural characterization.

Objectives and Study Overview

The study aims to assess the performance of the Thermo Scientific Orbitrap Eclipse Tribrid mass spectrometer under native conditions for top-down analysis of intact antibodies. It compares multiple dissociation techniques and contrasts native versus denaturing workflows to maximize sequence coverage and structural insight.

Methodology and Instrumentation

Analysis was performed using direct infusion electrospray in High Mass Range MSn (HMRn) mode with the Orbitrap Eclipse Tribrid quadrupole–Orbitrap–linear ion trap platform. Multiple fragmentation methods were applied:

• High-energy collision dissociation (HCD)
• Electron transfer dissociation (ETD)
• Electron-transfer/higher-energy collision dissociation (EThcD)
• Ultraviolet photodissociation (UVPD)

Sample preparation involved buffer exchange of trastuzumab (Herceptin) into 200 mM ammonium acetate for native analysis and a 1:1 acetonitrile:water solution with 0.1 % formic acid for denaturing experiments. Data processing used BioPharma Finder, TD Validator, Xtract, and ProSight Lite.

Main Results and Discussion

Native top-down analysis under non-denaturing conditions achieved 43 % combined sequence coverage, while denaturing conditions provided 39.7 %. By integrating native and denaturing data, total coverage rose to 53.3 %, with light chain coverage reaching 67.9 % and heavy chain 46.4 %. Fragmentation maps revealed complementary cleavage patterns across techniques, and Venn diagram analysis highlighted overlapping and unique residue cleavages between native and denatured states.

Benefits and Practical Applications

This approach delivers direct molecular weight determination, comprehensive sequence confirmation, localization of post-translational modifications such as glycosylation, and disulfide mapping without extensive sample preparation. It supports quality control and characterization workflows in biopharmaceutical development and regulatory compliance.

Future Trends and Opportunities

Advances in top-down instrumentation will enable higher resolution and faster scan rates, facilitating routine analysis of larger and more complex biotherapeutics. Integration with improved software tools and workflows promises automated data interpretation, expanded PTM mapping, and broader adoption in industrial and research laboratories.

Conclusion

The Thermo Scientific Orbitrap Eclipse Tribrid mass spectrometer demonstrates outstanding capability for native top-down analysis of intact antibodies, leveraging multiple dissociation methods to achieve high sequence coverage and detailed structural insights in a streamlined workflow.

References

No external references provided in the original document.