Agilent Biocolumns - Glycan Analysis - Application Compendium

Significance of the Topic

Glycosylation is a critical post-translational modification affecting protein folding, stability, serum half-life and immune recognition, particularly in biotherapeutics such as monoclonal antibodies (mAbs). Precise characterization of N-linked glycans is essential for ensuring consistent efficacy, safety and regulatory compliance during biopharmaceutical development and manufacturing.

Objectives and Overview of Study

This compendium provides an integrated guide to high-throughput workflows and instrumentation for profiling, mapping and quantifying released N-linked glycans. It covers: sample preparation strategies for enzymatic glycan release and fluorescent labeling; HILIC-based chromatographic separation with fluorescence detection (FLD) or mass spectrometry (MS); application notes demonstrating streamlined, sensitive and reproducible analyses across various glycoproteins; and available standards and reference libraries.

Methodology and Instrumentation

• Glycan release: enzymatic deglycosylation with PNGase F in-solution or on AssayMAP Bravo cartridges.
• Fluorescent labeling: reductive amination using 2-aminobenzamide (2-AB) or instant glycosylamine tagging with InstantPC.
• Sample cleanup: HILIC solid-phase extraction cartridges or ProZyme cleanup modules for 2-AB/InstantPC removal.
• Chromatography: Agilent AdvanceBio Glycan Mapping columns in 1.8 µm (UHPLC) or 2.7 µm (HPLC) formats, optimised for HILIC separation of complex glycan structures.
• Detection: Agilent 1260/1290 Infinity LC with FLD (Ex/Em 260/430 nm for 2-AB; 285/345 nm for InstantPC), optionally coupled to Agilent 6545XT or 6550 Q-TOF MS for structural confirmation and MS-based quantitation.
• Automation: Agilent AssayMAP Bravo platform with ProZyme GlykoPrep kits for high-throughput, reproducible sample preparation.

Main Results and Discussion

Methods deliver fast, high-resolution separations of glycans from mAbs, fetuin, dextran ladders and other glycoproteins within 10–20 minutes. The 1.8 µm UHPLC columns achieved up to 40 % faster run times versus competitive sub-2 µm columns, without compromising peak capacity (≥200) or resolution of critical isomers (e.g., G0F/Man5, G1F isoforms). InstantPC labeling enhanced MS sensitivity by up to 10-fold versus 2-AB, enabling detection of low-abundance species and MS-based relative quantitation with <1 % variance. AssayMAP Bravo automation processed 96 samples in parallel with <6 % RSD for peak areas. Application notes demonstrate workflows for sialic acid profiling, glycan mapping of biosimilars, and high-throughput screening of cell culture clones.

Benefits and Practical Applications

These integrated solutions allow biopharma laboratories to:

• Increase sample throughput via automation and rapid LC methods.
• Ensure reproducible, high-resolution glycan profiling.
• Combine FLD and MS for both quantitative and structural data.
• Support biosimilar comparability, clone selection and QC testing.

Future Trends and Possibilities for Use

Advances in glycan labeling chemistry (e.g., brighter, more ionizable tags), microfluidic automation and integration with high-resolution MS/MS and ion mobility promise even greater sensitivity, structural elucidation and throughput. Emerging artificial intelligence tools for data analysis could further accelerate glycan annotation, quantitation and pattern recognition across large sample sets.

Conclusion

Agilent’s end-to-end glycan analysis platform—from AssayMAP Bravo sample preparation through AdvanceBio Glycan Mapping columns and FLD/MS detection—provides robust, high-throughput solutions for accurate, reproducible N-glycan profiling and quantitation across diverse glycoproteins.

Reference

• Harvey DJ, et al. Proteomics. 2009;9(15):3796–3801.
• Varki A, et al. Glycobiology. 2015;25(12):1323–1324.
• Ceroni A, et al. J Proteome Res. 2008;7(4):1650–1659.
• Lee EU, et al. J Biol Chem. 1989;264(23):13848–13855.
• Anthony RM, et al. Science. 2008;320(5874):373–376.