N-Glycan Analysis of Biotherapeutic Glycoproteins using AdvanceBio Gly-X InstantPC Sample Preparation and LC/FLD/MS

Significance of the topic

Glycosylation, specifically N-linked glycan profiling, critically influences the stability, efficacy and safety of biotherapeutic proteins.
Variations in glycan structures can alter immunogenicity, pharmacokinetics and pharmacodynamics of monoclonal antibodies and fusion proteins.
Rapid, sensitive and reproducible N-glycan analysis is therefore essential in biopharmaceutical development, quality control and regulatory compliance.

Objectives and study overview

This guide presents a comprehensive consumables workflow for N-glycan analysis using the Agilent AdvanceBio Gly-X InstantPC sample preparation kit and LC/FLD/MS detection.
The primary goal is to standardize and streamline glycan release, labeling, cleanup and analysis for high productivity and reliable quantitation.
The performance of the InstantPC-based workflow is demonstrated with rituximab (Rituxan) and etanercept (Enbrel) model glycoproteins.

Methodology and instrumentation used

The workflow comprises four key steps:

• Rapid denaturation (3 min at 90 °C) and PNGase F–mediated deglycosylation (5 min at 50 °C).
• InstantPC fluorescent labeling (60 min at 65 °C) without a drying step.
• Vacuum‐driven cleanup in a 96‐well plate (10–15 min) to remove excess reagents.
• UHPLC separation on an AdvanceBio Amide HILIC column coupled to FLD (Ex 285 nm/Em 345 nm) and Q-TOF MS detection.

Key instrumentation:

• Agilent 1260/1290 Infinity II LC systems with bio‐inert flow paths.
• AdvanceBio Amide HILIC columns (2.1×150 mm, 1.8 µm).
• Agilent 6545XT Q-TOF with Dual AJS ESI source and MassHunter BioConfirm software.

Main results and discussion

InstantPC labeling delivered markedly higher fluorescence and MS signal compared with traditional 2-AB labeling, enabling detection of low-abundance glycan species.
HILIC-FLD profiles of Rituxan and Enbrel showed well-resolved bi- and triantennary glycan peaks, including sialylated and fucosylated structures.
Relative percent area and reproducibility (n=4) exhibited low CVs (<1%) across major glycan species.
MS data supported confident glycan identification and structural assignment.

Benefits and practical applications

The Gly-X InstantPC platform enables complete sample processing and data acquisition in under 60 minutes.
The ambient-stable reagents and modular 96-well format support high throughput, minimal waste and flexible batch sizes.
The high sensitivity and dynamic range facilitate routine monitoring of glycan profiles during process development, release testing and comparability studies.

Future trends and potential applications

Automation of sample preparation in microplate and robotic platforms will further enhance throughput and reproducibility.
Development of novel fluorescent or isobaric tags may expand multiplexing and absolute quantitation capabilities.
Integration with AI-driven data analysis and glycan databases will accelerate glycoform characterization and biomarker discovery.
Expanding applications to cell-surface glycomics and glycoproteomics can deepen understanding of glyco-biology.

Conclusion

The Agilent AdvanceBio Gly-X InstantPC workflow represents a robust, fast and sensitive solution for N-glycan analysis of biotherapeutics.
By combining streamlined sample preparation with high-resolution HILIC-FLD/MS, the method addresses critical needs in biopharma R&D and QC, supporting efficient glycan profiling across diverse glycoprotein products.

References

• Agilent (2020) N-Glycan Analysis: Better Together. Brochure 5994-1647EN.
• Agilent (2023) Improved Hydrophilic Interaction Liquid Chromatography for LC/FLD/MS Analysis of Released N-Glycans. Application Note 5994-6916EN.
• Agilent (2019) AdvanceBio Gly-X N-Glycan Prep with InstantPC Kit. Flyer 5994-0918EN.
• Agilent (2020) Glycan Standards Technical Flyer 5994-2202EN.
• Agilent (2019) AdvanceBio Gly-X N-Glycan Prep with InstantPC User Manual 5994-1231EN.
• Xie Y. et al. (2021) High-throughput and high-sensitivity N-Glycan profiling: A platform for biopharmaceutical development and disease biomarker discovery. Analytical Biochemistry, 623:114205.
• Varki A. et al. (2015) Symbol Nomenclature for Graphical Representations of Glycans. Glycobiology 25(12):1323–1324.