Glycan Analysis - Agilent BioHPLC Columns Application Compendium

Significance of the topic

The N-linked glycosylation of biotherapeutic proteins profoundly influences their safety, efficacy, pharmacokinetics, and immunogenicity.
Accurate characterization and quantification of glycan structures are mandated by regulatory authorities as critical quality attributes.
High-resolution separation, robust detection, and rapid workflows enable reliable monitoring throughout drug development and manufacturing.

Objectives and overview

This compendium reviews integrated methods for N-glycan analysis from sample preparation to data reporting.
It presents quick-start guides for HILIC separations on Agilent AdvanceBio Glycan Mapping columns, workflows for glycopeptide characterization by LC/Q-TOF, and comprehensive approaches for released glycan analysis with fluorescence and mass detection.
Comparisons of 2-AB and InstantPC labeling chemistries are included, along with strategies for MS-based identification, relative quantitation, and automation on the AssayMAP Bravo platform.

Methods and instrumentation

Sample preparation

• Denaturation (90 °C, 3 min) and rapid PNGase F digestion (50 °C, 5–15 min) with AdvanceBio Gly-X kits.
• Derivatization via reductive amination using 2-AB or InstantPC labels at 65 °C for 60 min on a HILIC solid support.
• On-matrix cleanup to remove excess dye without evaporation.

Chromatography and detection

• HILIC separations on Agilent AdvanceBio Glycan Mapping columns (1.8 µm and 2.7 µm) using UHPLC or HPLC systems.
• Fluorescence detection (Ex 260–285 nm/Em 430–345 nm) for robust quantitation.
• Mass detection via Agilent 6545XT LC/Q-TOF or compact LC/MSD XT single quadrupole for accurate mass profiling and MS/MS.
• Automated peptide mapping workflows for glycopeptide analysis on the 1290 Infinity II LC coupled to 6545 AdvanceBio Q-TOF.

Data analysis

• Agilent MassHunter BioConfirm software for peptide and released glycan workflows.
• Personal Compound Databases and Peptide Feature Extraction algorithms for compound identification.
• Mass Profiler and Report Builder for custom reporting and batch processing.

Main results and discussion

HILIC-based methods achieved high-resolution separations of labeled glycans and glycopeptides, resolving isomers and isoforms.
InstantPC labeling dramatically enhanced MS sensitivity, enabling detection of low-abundance components (<0.5 %) without sample concentration.
Comparisons between fluorescence- and MS-based relative quantitation demonstrated excellent concordance (RSDs <1 % for major glycans).
Automated sample preparation reduced total processing time from overnight to under two hours, with CVs below 1 % for key glycan species.
Glycopeptide mapping on HILIC columns outperformed conventional RP separation for hydrophilic peptides, delivering site-specific glycosylation profiles.

Benefits and practical applications

The integrated Agilent workflows offer:

• Rapid, high-throughput N-glycan and glycopeptide analysis for biopharmaceutical R&D and QC.
• Improved reproducibility and turnaround time via automation and on-matrix labeling.
• Robust fluorescence quantitation backed by MS confirmation to resolve ambiguous peaks.
• Versatility across instrumentation platforms and label chemistries (2-AB, InstantPC).

Future trends and applications

Emerging areas include further miniaturization and multiplexing of glycan assays, integration with high-throughput screening and process analytical technology (PAT), and advanced bioinformatics using machine learning for glycoform prediction.
Innovations in labeling reagents and column chemistries will expand sensitivity and resolution, while online MS/MS workflows may enable real-time CQA monitoring.

Conclusion

Agilent’s suite of sample preparation kits, HILIC columns, detection systems, and data software provides a comprehensive, reproducible, and scalable solution for N-glycan and glycopeptide characterization.
These workflows meet stringent regulatory requirements and streamline analytical operations from drug discovery to quality control.

Reference

1. Liu L. Antibody Glycosylation and its Impact on the 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. Mellquist JL et al. The amino acid following an Asn-X-Ser/Thr sequon is an important determinant of N-linked core glycosylation efficiency. Biochemistry. 1998;37(19):6833–6837.
4. Ghaderi D et al. N-Glycan profiles and antigenic potential of mammalian expression systems compared to human. Biotechnol Genet Eng Rev. 2012;28:147–175.