Multi-Reflecting Time of Flight-MS: A Novel Technology for In-depth mAb Subunit Characterization

Importance of the Topic

The detailed subunit characterization of monoclonal antibodies (mAbs) is critical for ensuring the safety, efficacy, and consistency of biotherapeutic products.
By dividing a 150 kDa mAb into ~25 kDa subunits, analysts gain higher sensitivity to subtle variants and modifications than intact‐level analysis.
This approach supports quality control, comparability studies, and regulatory compliance in pharmaceutical development.

Goals and Study Overview

This study demonstrates the application of a multi‐reflecting time‐of‐flight mass spectrometer (Xevo MRT MS) coupled to UPLC for in‐depth analysis of mAb subunits derived from the NIST reference mAb.
Using an integrated informatics workflow (waters_connect INTACT Mass App), the work aims to assess sensitivity, mass resolution, accuracy, and reproducibility for routine subunit analysis.

Methodology and Instrumentation

Samples of NIST mAb subunit standard were digested with IdeS and reduced, yielding light chain, Fd′ and Fc/2 fragments.
Ten replicate 250 ng injections were analyzed by reversed‐phase UPLC (ACQUITY Premier UPLC C4 column, 0.4 mL/min, 60 °C) with UV 280 nm detection.
Mass spectrometry was performed on the Xevo MRT system in positive mode across m/z 400–4000 at 1 Hz scan rate, delivering up to 100 k resolving power independent of scan speed.
The dual‐gain ADC detector provided high dynamic range and sub-ppm mass accuracy.

Main Results and Discussion

Chromatographic separation of the three subunits was achieved with high retention time consistency across ten replicates.
Raw spectra displayed well‐resolved charge envelopes; high resolution permitted isotopic spacing recognition even when monoisotopic peaks were not visually apparent.
BayesSpray deconvolution in the INTACT Mass App yielded average mass errors below 2 ppm for all species, many at sub-ppm levels.
Statistical evaluation of the Fd′ subunit across replicates showed mean mass error ~220 ppb (RMS 320 ppb) and <1 % intensity RSD.
Lower‐abundance Fc glycoforms were consistently detected and quantified, demonstrating sensitivity to minor variants.

Benefits and Practical Applications

• The Xevo MRT MS delivers high sensitivity, dynamic range, and sub-ppm mass accuracy for detailed mAb subunit profiling.
• Automated data acquisition and processing via a compliance-ready app reduce manual effort and error risk.
• Reproducible chromatography and mass measurements enable robust comparability and statistical analysis across batches.
• Detection of low-abundance modifications supports product quality monitoring and regulatory submissions.

Future Trends and Opportunities

Integration of higher throughput UPLC methods and faster MS scanning may further accelerate subunit analyses.
Expansion of informatics workflows with AI-driven variant annotation could enhance interpretation of complex proteoforms.
Combining multi-reflection TOF with ion mobility separation offers the potential to resolve isomeric modifications.
Application to other biotherapeutics, such as antibody–drug conjugates, will broaden utility in biopharma development.

Conclusion

This work demonstrates that multi‐reflecting TOF MS coupled to UPLC and integrated informatics provides a robust, sensitive, and accurate platform for mAb subunit characterization.
The approach supports critical decision‐making in biotherapeutic development by delivering reproducible data on product variants with minimal manual intervention.

References

1. Waters Corporation. Xevo MRT Multi Reflecting Time of Flight Mass Spectrometer. Product overview and specifications.
2. Reid L, Daly ME, Gethings LA, Kirk J. Metabolomics Workflow using a Xevo™ MRT Mass Spectrometer. Waters Application Note, 720008552; October 2024.
3. Shion H, Boyce P, Berger SJ, Yu YQ. INTACT Mass™ Application for Rapid Mass Confirmation and Purity Assessment of Biotherapeutics. Waters Application Note, 720007547; February 2022.