Monitoring Antibody Glycosylation at Intact and Subunit Levels Using a Single Quadrupole LC/MS

Importance of the Topic

Glycosylation is a critical quality attribute in biopharmaceuticals governing antibody stability, efficacy and safety. Robust monitoring of glycan variants ensures consistent product performance and supports regulatory compliance. LC/MS-based intact and subunit mass analysis offers a sensitive approach for glycoform profiling within quality control workflows.

Objectives and Study Overview

This study demonstrates the use of a single quadrupole LC/MS platform to monitor relative glycosylation levels in trastuzumab at both the intact molecule and reduced subunit (heavy and light chain) levels. The goals were to evaluate detection sensitivity, spectral quality and consistency of relative abundance measurements compared to high-resolution systems.

Methodology and Instrumentation

Trastuzumab was prepared at 250 ng/µL in 0.1% formic acid for intact analysis. For subunit profiling, the antibody was denatured with guanidine-HCl, reduced with DTT and quenched with formic acid. Separation employed a PLRP-S column (2.1×50 mm, 5 µm) on an Agilent 1290 Infinity II Bio LC system with a gradient from 10% to 60% acetonitrile in 8 minutes. Mass detection used an Agilent InfinityLab Pro iQ Plus single quadrupole MS in positive ESI mode, scanning m/z 1000–3000 for intact and 600–2400 for reduced forms. Data were processed using Agilent OpenLab CDS with deconvolution parameters optimized for minimum peak sets and charge state count thresholds.

Key Results and Discussion

Intact trastuzumab analysis resolved five major glycoforms with relative abundance profiles matching previously reported high-resolution data. Subunit analysis showed a single light chain peak and four heavy chain glycoforms (G0, G0F, G1F, G2F) with high spectral clarity. The observed mass errors were within ±250 ppm and relative quantitation reproducibility was demonstrated. Effective peak shape and reliable glycoform abundance detection highlight the single quadrupole system capability for routine monitoring.

Benefits and Practical Applications

• Cost-effective and compact LC/MS configuration suitable for QC environments
• Rapid assessment of glycosylation patterns without high-resolution instruments
• Streamlined sample preparation for intact and reduced antibody levels
• Traceable processing and reporting via integrated CDS software

Future Trends and Applications

Advancements in single quadrupole technology, combined with improved software deconvolution and AI-driven data analysis, will expand throughput and automation of glycoform profiling. Integration with bioprocess analytics and real-time monitoring platforms promises enhanced control over therapeutic glycosylation during manufacturing.

Conclusion

The Agilent 1290 Infinity II Bio LC coupled with the InfinityLab Pro iQ Plus single quadrupole MS provides a robust, accessible approach to antibody glycosylation analysis. It delivers reliable relative abundance data at both intact and subunit levels, supporting quality control in biopharmaceutical development and production.

References

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2. Wang D, Baudys J, Bundy J, Solano M, Keppel T, Barr J. Comprehensive analysis of the glycan component of SARS-CoV-2 spike proteins using signature ions-triggered EThcD mass spectrometry. Anal Chem. 2020;92(21):14730–14739.