Top-down Monoclonal Antibody (mAb) Analysis Using CID and ETD Fragmentation and the UNIFI Scientific Information System

Importance of the Topic

Top-down analysis of monoclonal antibodies offers a direct route to verify amino- and carboxy-terminal sequences and locate post-translational modifications without peptide digestion. This orthogonal strategy complements traditional peptide mapping by providing intact mass context and streamlining the characterization of biotherapeutic proteins.

Objectives and Study Overview

This application study evaluates collision induced dissociation (CID) and electron transfer dissociation (ETD) fragmentation of trastuzumab subunits on a SYNAPT G2-Si HDMS mass spectrometer. Data are processed and reported automatically in the UNIFI Scientific Information System to confirm sequence coverage and identify glycoforms and other modifications.

Methodology and Instrumentation

• Trastuzumab digested with IdeS protease and reduced with DTT to yield LC, Fd, and Fc/2 fragments.
• Intact mass profiling and top-down MS-MS by targeted CID or ETD on selected charge states.
• Bayesian BayesSpray algorithm for deconvolution of raw and isotope-resolved spectra.

Used Instrumentation

• ACQUITY UPLC H-Class Bio System with Protein BEH C4 column, 80 °C, 0.4 mL/min gradient.
• SYNAPT G2-Si HDMS Mass Spectrometer in ESI+ mode with ion mobility separation capability.
• MassLynx v4.1 for acquisition and UNIFI v1.8 for data processing and reporting; ProSight PTM 2.0 for complementary annotation.

Main Results and Discussion

Intact mass deconvolution in UNIFI resolved LC, Fd, and Fc/2 glycoforms (G0, G0F, G1F, G2F). ETD fragmentation of the Fc/2 G0F precursor (765.8 m/z, 33+) yielded 42 c-ions and 41 z-ions (≈38% coverage). CID followed by ion mobility separation produced simplified spectra of singly charged b- and y-ions, assigning 19 b- and 31 y-ions (≈24% coverage) and facilitating terminal sequence confirmation. Exported .PUF files enabled cross-validation with ProSight PTM, showing equivalent fragment assignments.

Benefits and Practical Applications

• Automated intact mass and top-down workflows accelerate sequence verification and PTM localization.
• BayesSpray deconvolution and IMS separation reduce manual interpretation of complex spectra.
• Customizable UNIFI report templates enable standardized reporting across biopharmaceutical laboratories.

Future Trends and Opportunities

Integration of advanced fragmentation methods, deeper integration with AI-driven informatics, high-throughput screening of variant libraries, and enhanced PTM mapping algorithms will expand the utility of top-down mAb analysis for drug development and quality control.

Conclusion

The combination of SYNAPT G2-Si HDMS, BayesSpray deconvolution, and UNIFI automated workflows provides a robust platform for efficient top-down characterization of monoclonal antibodies, supporting comprehensive sequence confirmation and modification profiling.

References

• Waters Corporation. UNIFI Scientific Information System. Brochure. 2015. 720004686EN.
• Prosight PTM2.0. Proteomics Center of Excellence, Northeastern University.