High-Throughput N-Glycans Profiling of Monoclonal Antibodies EG2-hFc and Rituximab Using the Agilent AdvanceBio Gly-X N-Glycan Prep with InstantPC Kit

Significance of the Topic

Glycosylation is a critical quality attribute of therapeutic glycoproteins that directly influences their safety, efficacy, stability, and immunogenicity. Routine, detailed profiling of N-glycans is essential for biopharmaceutical development, quality control, and regulatory compliance. Traditional glycan analysis workflows often involve lengthy sample preparation, limited throughput, and variable sensitivity, hindering rapid screening and process monitoring.

Objectives and Study Overview

This application note evaluates the Agilent AdvanceBio Gly-X N-glycan prep with InstantPC kit (GX96-IPC) for high-throughput, high-sensitivity profiling of N-glycans released from two monoclonal antibodies: EG2-hFc and rituximab. The goals were to streamline sample preparation to under one hour, achieve reproducible fluorescence detection, and demonstrate the method’s adaptability for routine glycan characterization in biopharmaceutical workflows.

Methods and Instrumentation

A rapid, 96-well plate-based workflow was developed:

• Denaturation: 40 µg antibody in HEPES buffer with Gly-X denaturant, 90 °C for 3 min.
• Deglycosylation: Addition of N-glycanase, incubation at 50 °C for 5 min.
• Derivatization: InstantPC dye labeling at 50 °C for 1 min.
• Purification: On-plate clean-up with formic acid/acetonitrile wash steps.
• Analysis: HILIC/FLD using Agilent 1260 Infinity II LC with AdvanceBio Glycan Mapping column (2.1×150 mm, 2.7 µm), excitation 285 nm, emission 345 nm.

Used Instrumentation

• AdvanceBio Gly-X N-glycan prep with InstantPC kit (GX96-IPC)
• Agilent 1260 Infinity II LC system with quaternary pump, autosampler, FLD detector
• AdvanceBio Glycan Mapping HILIC column
• AdvanceBio InstantPC maltodextrin ladder standard

Results and Discussion

InstantPC-labeled N-glycans from EG2-hFc and rituximab produced well-resolved peaks under a 30-min linear gradient. EG2-hFc displayed typical CHO-derived biantennary complex glycans, dominated by FA2G2 (33.1 %), FA2G1 (27.2 %), and sialylated FA2G2S1 (14.3 %). Rituximab exhibited higher neutral glycan content with FA2 (40.2 %), FA2G1 (35.7 %), FA2G2 (10.7 %), and low-abundance high-mannose species (M5–M7). The workflow achieved consistent reproducibility and sensitivity for sub-microgram glycoprotein loads.

Benefits and Practical Applications

• Complete sample prep to profiling in under one hour.
• High-throughput 96-well format with minimal manual handling.
• Sensitive FLD detection of InstantPC-labeled glycans.
• Robust, reproducible glycan quantitation for QC and process development.
• Easy adaptation to diverse glycoprotein biotherapeutics.

Future Trends and Opportunities

• Integration with mass spectrometry for structural elucidation.
• Automation and robotics for higher throughput in bioprocessing labs.
• Expansion to O-glycan and glycopeptide analysis platforms.
• Application of machine learning for glycan pattern recognition.
• Use in disease biomarker discovery and comparability studies.

Conclusion

The Agilent AdvanceBio Gly-X N-glycan prep with InstantPC kit offers a streamlined, sensitive, and reproducible approach for high-throughput N-glycan profiling of monoclonal antibodies. By reducing sample preparation time to under one hour and enabling clear HILIC/FLD separation of major glycan species, this workflow supports routine quality control, biopharmaceutical development, and research applications.

References

1. Bairoch A.; Apweiler R. The SWISS-PROT Protein Sequence Database and Its Supplement TrEMBL. Nucleic Acids Res. 2000, 28, 45–48.
2. Apweiler R.; Hermjakob H.; Sharon N. On the Frequency of Protein Glycosylation as Deduced from SWISS-PROT. Biochim. Biophys. Acta 1999, 1473, 4–8.
3. Khoury G. A.; Baliban R. C.; Floudas C. A. Proteome-Wide Post-Translational Modification Statistics: Swiss-Prot Curation. Sci. Rep. 2011, 1, 90.
4. Zhou Q.; Qiu H. Mechanistic Impact of N-Glycosylation on Therapeutic Proteins. J. Pharm. Sci. 2019, 108, 1366–1377.
5. Wada R.; Matsui M.; Kawasaki N. Influence of N-Glycosylation on Effector Functions and Thermal Stability of Glycoengineered IgG1. MAbs 2019, 11, 350–372.
6. Higel F.; Seidl A.; Sörgel F.; Friess W. N-Glycosylation Heterogeneity and Influence on Monoclonal Antibodies. Eur. J. Pharm. Biopharm. 2016, 100, 94–100.
7. Delobel A. Glycosylation of Therapeutic Proteins: A Critical Quality Attribute. Methods Mol. Biol. 2021, 2271, 1–21.
8. Goh J. B.; Ng S. K. Impact of Host Cell Line on Glycan Profile. Crit. Rev. Biotechnol. 2018, 38, 851–867.
9. Hossler P.; Khattak S. F.; Li Z. J. Optimal Protein Glycosylation in Mammalian Culture. Glycobiology 2009, 19, 936–949.
10. Zhang P. et al. Challenges of Glycosylation Analysis and Control in Therapeutic Drugs. Drug Discov. Today 2016, 21, 740–765.
11. Seeberger P. H. Monosaccharide Diversity. In Essentials of Glycobiology; Cold Spring Harbor, 2015; pp. 19–30.
12. Everest-Dass A. V. et al. Human Disease Glycomics: Glycosylation Analysis Part 1. Expert Rev. Proteomics 2018, 15, 165–182.
13. Melmer M. et al. HILIC Analysis of Fluorescence-Labeled N-Glycans. Anal. Bioanal. Chem. 2010, 398, 905–914.
14. Xie Y. et al. High-Throughput and High-Sensitivity N-Glycan Profiling. Anal. Biochem. 2021, 623, 114205.
15. Castelli M. S.; McGonigle P.; Hornby P. J. Pharmacology and Therapeutic Applications of Monoclonal Antibodies. Pharmacol. Res. Perspect. 2019, 7, e00535.
16. Bell A. et al. Differential Tumor-Targeting Abilities of Single-Domain Antibody Formats. Cancer Lett. 2010, 289, 81–90.
17. Pierpont T. M.; Limper C. B.; Richards K. L. Past, Present, and Future of Rituximab. Front. Oncol. 2018, 8, 163.
18. Smith M. R. Rituximab: Mechanisms of Action and Resistance. Oncogene 2003, 22, 7359–7368.
19. Yan J. et al. Streamlined Workflows for N-Glycan Analysis of Biotherapeutics. Agilent Technologies application note 5994-1348EN, 2019.
20. Liau B. Comparative Study of Rituximab Biosimilars Using High-Resolution LC/MS. Agilent Technologies application note 5994-1653EN, 2020.