MEETING DEMANDS FOR IMPROVED MS PERFORMANCE TO SUPPORT DEEPER PRODUCT LC-MS CHARACTERIZATION WITH MULTI-REFLECTING TOF MS TECHNOLOGY

Significance of the Topic

Liquid chromatography–mass spectrometry (LC-MS) remains the cornerstone technique for biotherapeutic characterization, enabling detection and monitoring of product variants that can impact development decisions, regulatory approval, and commercial success. The introduction of multi-reflecting time-of-flight (MRT-TOF) technology addresses the growing demand for higher mass resolution, improved mass accuracy, extended dynamic range, and faster acquisition rates, while reducing sample consumption.

Objectives and Study Overview

This work presents the application of a novel MRT detection-based Q-TOF mass spectrometer to three common biotherapeutic workflows:

• mAb subunit analysis
• Peptide mapping of a tryptic digest standard
• Synthetic antisense oligonucleotide characterization

Performance was assessed in terms of mass accuracy, resolution, dynamic range, sequence coverage, and automation through compliance-ready informatics.

Methodology

Three LC-MS workflows were optimized:

• Subunit Analysis: ACQUITY Premier LC with Protein BEH C4 (300 Å, 1.7 µm, 2.1 × 50 mm), gradient from 15 % to 80 % organic, column at 60 °C, 1 µL injections of 250 ng mAb subunit.
• Peptide Mapping: ACQUITY Premier LC with Peptide BEH C18 (1.7 µm, 2.1 × 100 mm), gradient from 3 % to 85 % organic, column at 40 °C, 200 ng tryptic digest, data processed by UNIFI™ Peptide Mapping App.
• Oligonucleotide Analysis: ACQUITY Premier LC with Oligonucleotide C18 (130 Å, 1.7 µm, 2.1 × 100 mm), TEA/HFIP mobile phases in negative ESI, gradient from 22 % to 60 % TEA/HFIP mix, 1 µL injections of a 25-mer antisense oligonucleotide, data processed by INTACT Mass App with BayesSpray deconvolution.

Instrumentation Used

• Waters Xevo™ MRT Q-TOF Mass Spectrometer featuring multi-reflecting TOF optics, offering ~100 000 FWHM resolution, sub-ppm mass accuracy, 4–5 orders of dynamic range, MS1/MS2 capability at up to 1 Hz acquisition.
• Waters ACQUITY™ Premier LC System with binary solvent management for high-throughput and robust chromatography.

Main Results and Discussion

• Subunit Analysis: Automated deconvolution yielded <2 ppm mass error for Fc glycoforms, average error ~0.22 ppm (RMS 0.32 ppm), intensity RSD <1.3 % across ten replicates.
• Peptide Mapping: Achieved 98 % sequence coverage for the mAb tryptic digest at 200 ng load, with high-confidence MS/MS confirmation using UNIFI workflows.
• Oligonucleotide Characterization: Confirmed 100 % sequence of a 25-mer antisense phosphorothioate oligonucleotide with <1 ppm mass error, quantification down to 0.02 ng on column and linear dynamic range spanning five orders with R² = 0.999 and RSD <10 %.

Benefits and Practical Applications

The MRT-TOF platform combines exceptional resolution and accuracy with high sensitivity and broad dynamic range, enabling confident detection of low-level variants and post-translational modifications. Coupled with streamlined, compliant-ready informatic pipelines, it supports accelerated workflows for product development, quality control, and regulatory submissions in both protein and nucleic acid therapeutic modalities.

Future Trends and Opportunities

• Further extension of ion flight path designs to achieve resolutions approaching one million FWHM.
• Integration of AI-driven data analysis for real-time variant identification and predictive quality assessments.
• Application to emerging modalities such as antibody-drug conjugates, gene therapies, and complex biologics requiring ultra-high confidence characterization.

Conclusion

The adoption of multi-reflecting TOF detection in a benchtop Q-TOF platform delivers next-generation performance for LC-MS characterization of biotherapeutics. Its combination of high mass resolution, sub-ppm accuracy, broad dynamic range, and automated informatics accelerates reliable product attribute analysis, ultimately enabling more informed decision-making across development and manufacturing.

References

• Waters Technologies Corporation. Xevo MRT Information brochure. 2024.
• Waters Technologies Corporation. Xevo MRT Brochure. 2025.
• Smith J, et al. Future of Multi-Reflecting TOF Technology: Approaching Million-Resolution. Int J Mass Spectrom. 2025.