mAb Subunit Analysis Using the SYNAPT XS High Resolution Mass Spectrometer

Importance of the Topic

The structural and post-translational characterization of mAb subunits is critical in biopharmaceutical development and quality control. Detailed mass analysis at subunit level supports glycoform profiling, degradation monitoring, and ensures consistency of therapeutic performance.

Study Objectives and Overview

This study evaluates mAb subunit analysis using the SYNAPT XS mass spectrometer by comparing four mass resolution modes: sensitivity (Rs ~12 500), resolution (25 000), high resolution (56 000), and enhanced resolution (75 000). The aim was to assess mass accuracy, signal intensity, and relative variant quantification, and to explore advanced deconvolution approaches.

Methodology

Samples comprised the NISTmAb IdeS digestion standard and oxidatively stressed Trastuzumab subunits. Separation was performed by RP-LC on an ACQUITY UPLC I-Class PLUS with a BioResolve RP mAb Polyphenyl column at 80 °C using a formic acid/acetonitrile gradient. The SYNAPT XS was operated in positive ESI mode under four resolution settings. Data processing employed MaxEnt1 for average mass deconvolution and BayesSpray for monoisotopic mass extraction.

Instrumentation

• SYNAPT XS High Resolution Mass Spectrometer
• ACQUITY UPLC I-Class PLUS System
• Waters BioResolve RP mAb Polyphenyl Column (450 Å, 2.7 µm, 2.1 × 50 mm)
• MassLynx 4.1.2, waters_connect 1.9.7 and UNIFI Intact Mass software

Key Results and Discussion

All four resolution modes produced mass data with low ppm accuracy (mostly <10 ppm) and consistent relative abundance for scFc, LC, and Fd subunits after MaxEnt1 deconvolution. Sensitivity mode yielded the highest signal intensity, recommending it as default for routine subunit analysis. Enhanced resolution achieved monoisotopic peak resolution of ~25 kDa fragments. BayesSpray processing further improved mass accuracy to <2 ppm, though requiring higher sample loading due to reduced ion transmission at higher resolution.

Benefits and Practical Applications

• Flexible workflow accommodating routine attribute analysis and advanced structural studies (HDX, CIU).
• Consistent quantification of glycoforms and oxidative variants across resolution modes.
• Enhanced resolution combined with BayesSpray allows precise monoisotopic mass determination for detailed proteoform characterization.

Future Trends and Opportunities

Further development of high-resolution instrumentation and deconvolution algorithms will enable deeper insights into higher-order structures and low-abundance variants. Integration with ion mobility and emerging AI-driven data analysis promises more comprehensive biotherapeutic characterization with reduced sample requirements and faster throughput.

Conclusion

The SYNAPT XS platform offers versatile resolution settings for robust mAb subunit analysis. Sensitivity mode optimizes signal and accuracy for routine workflows, while enhanced resolution with BayesSpray deconvolution yields superior monoisotopic precision. This flexibility supports both standard QC assays and advanced structural investigations.

Reference

• Shion H, Quinn C, Berger S, Yu Y. Analysis of Cysteine-Conjugated ADCs Using a Native SEC LC-MS Workflow on the SYNAPT XS. Waters Application Note 720007026EN, 2020.
• Skilling J, Richardson K. Method and System of Identifying a Sample by Analyzing a Mass Spectrum by the Use of a Bayesian Inference Technique. EU Patent EP2558979A1, 2011.