Preparation of Released N-Glycan Samples from Monoclonal Antibodies Using Agilent AdvanceBio Gly-X 2-AB Express for LC-Fluorescence Analysis

Significance of the Topic

Glycosylation is a critical quality attribute of biotherapeutic proteins that influences pharmacokinetics, pharmacodynamics and immunogenicity. Accurate profiling of N-linked glycans is therefore essential in the development, manufacturing and quality control of monoclonal antibodies and other glycoprotein therapeutics.

Objectives and Overview of the Article

This application note describes a streamlined workflow for released N-glycan sample preparation using the Agilent AdvanceBio Gly-X 2-AB Express kit. The protocol aims to reduce total processing time from over 24 hours to approximately two hours, while preserving labeling consistency and compatibility with historical 2-aminobenzamide (2-AB) data.

Methodology

The sample preparation comprises four main steps:

• Denaturation: Protein unfolding at 90 °C for 3 minutes in the presence of a proprietary reagent.
• Deglycosylation: Rapid release of N-linked glycans using PNGase F at 50 °C for 5 minutes.
• Labeling: On-matrix reductive amination with 2-AB at 65 °C for 60 minutes directly on a HILIC solid phase, eliminating any drying step.
• Cleanup: Sequential acetonitrile washes to remove excess dye, followed by elution of labeled glycan fractions with water.

Instrumentation Used

Analysis of labeled glycans was performed on an Agilent 1290 Infinity II system equipped with:

• High-speed binary pump (G7120A)
• Multisampler (G7167B)
• Multicolumn thermostat (G7116B)
• 1260 Infinity fluorescence detector (G1321B)

A HILIC AdvanceBio Glycan Mapping column (2.1 × 150 mm, 1.8 µm) was operated at 40 °C. Mobile phases consisted of 50 mM ammonium formate (pH 4.5) and acetonitrile under a gradient elution at 0.5 mL/min. Data acquisition and processing employed Agilent MassHunter software.

Main Results and Discussion

Chromatograms of 2-AB labeled N-glycans from MabThera and Enbrel displayed well-resolved peaks for major glycoforms (G0, G1F, G2F, sialylated species). Relative area measurements across three independent preparations of MabThera showed coefficient of variation below 1% for dominant peaks and below 10% near the detection limit. Comparative profiling of Enbrel matched results obtained with a legacy 2-AB protocol (ProZyme GlykoPrep), confirming equivalence to historic data while cutting sample prep time by over 90%.

Practical Benefits and Applications

The rapid Gly-X 2-AB Express workflow enables same-day glycan profiling, supporting accelerated decision-making in process development and lot release testing. On-matrix labeling is compatible with high-throughput and automated platforms, and retention of 2-AB chemistry ensures consistency with extensive legacy datasets.

Future Trends and Applications

Emerging directions include integration of rapid deglycosylation with online HILIC-MS, application of novel fluorescent and mass tags for enhanced sensitivity, fully automated sample-to-data pipelines and AI-driven glycan pattern recognition for real-time bioprocess monitoring.

Conclusion

The Agilent AdvanceBio Gly-X 2-AB Express kit delivers a fast, robust and reproducible method for released N-glycan analysis in under two hours. It maintains compatibility with established 2-AB labeling data and supports high-throughput workflows essential for modern therapeutic protein development.

References

1. Liu L. Antibody Glycosylation and Its Impact on Pharmacokinetics and Pharmacodynamics of Monoclonal Antibodies and Fc-Fusion Proteins. J Pharm Sci. 2015;104(6):1866–1884.
2. Reusch D, Tejada ML. Fc Glycans of Therapeutic Antibodies as Critical Quality Attributes. Glycobiology. 2015;25(12):1325–1334.
3. Ruhaak LR, et al. Glycan Labeling Strategies and Their Use in Identification and Quantification. Anal Bioanal Chem. 2010;397(8):3457–3481.
4. Kimzey M, et al. Development of a 5-Minute Deglycosylation Method for High Throughput N-Glycan Analysis by Mass Spectrometry. ProZyme Tech Note. 2019.