Glycosylation and Sialic Acid Analysis of Biotherapeutic Glycoproteins

Importance of the topic

Protein glycosylation, particularly N-linked glycans and their terminal sialic acids, critically influences the pharmacokinetics, pharmacodynamics and immunogenicity of biotherapeutic glycoproteins. Rigorous glycan profiling is therefore essential for defining and monitoring critical quality attributes (CQAs) during development, manufacturing and quality control of monoclonal antibodies, Fc-fusion proteins and other glycoprotein drugs.

Objectives and overview

This whitepaper outlines a streamlined workflow for rapid release, labeling, chromatographic separation and detection of N-glycans and sialic acid species in biotherapeutic proteins. It compares novel InstantPC fluorescent labeling with traditional 2-AB chemistry, demonstrates hydrophilic interaction liquid chromatography (HILIC) with fluorescence (FLD) and mass spectrometry (MS) detection, and presents both total sialic acid quantitation and linkage-specific profiling.

Methodology and instrumentation

• Sample preparation: Gly-X in-solution technology integrates protein denaturation, PNGase F digestion and rapid dye labeling (InstantPC or 2-AB Express) in 45 min to 2 h. Automation is feasible on liquid handlers such as Agilent AssayMAP Bravo.
• Labeling chemistries: InstantPC employs NHS-based dyes for enhanced FLD and MS response; 2-AB Express uses on-matrix reductive amination for continuity with legacy datasets.
• Separation and detection: Agilent 1290 Infinity II LC with AdvanceBio Glycan Mapping amide HILIC columns (1.8 µm particles) operates at 0.5 mL/min; FLD detects InstantPC at ex/em 285/345 nm or 2-AB at 260/430 nm; structural confirmation via Q-TOF MS (e.g., 6545XT).
• Sialic acid analysis: Total sialic acid is quantified using a plate-based fluorescence assay (Ex 530 nm/Em 590 nm), while detailed profiling of Neu5Ac and Neu5Gc is achieved by DMB derivatization followed by reverse-phase UHPLC-FLD/MS.

Main results and discussion

• Gly-X sample prep achieved >99% N-glycan release within 5 minutes for monoclonal antibodies such as rituximab and cetuximab.
• InstantPC labeling provided up to tenfold greater FLD and MS signal compared to 2-AB, improving the detection of low-abundance and sialylated species.
• HILIC separations resolved structural isomers and differentiated α(2,3) versus α(2,6) sialic acid linkages by characteristic retention shifts.
• Quantitative reproducibility was high, with intra-run %CVs below 5% for glycan relative abundances and operator-to-operator repeatability for total sialic acid within 7% CV.
• DMB profiling effectively distinguished Neu5Ac and Neu5Gc, enabling monitoring of potentially immunogenic non-human sialic acid residues.

Benefits and practical applications

• High-throughput, robust workflows support routine glycan analysis in biopharmaceutical R&D and QC laboratories.
• Enhanced FLD and MS sensitivity accelerates characterization of CQAs such as fucosylation, galactosylation and sialylation patterns.
• Automated sample handling reduces hands-on time and improves consistency for regulatory compliance.
• Comprehensive sialic acid assays facilitate assessment of immunogenic epitopes and lot-to-lot product consistency.

Future trends and potential applications

Emerging labeling chemistries, ultra-fast LC columns and high-resolution MS will further decrease analysis times and increase sensitivity. Integration with automated workflows and AI-driven data analysis promises real-time process monitoring in continuous biomanufacturing. Additionally, advances in site-specific glycopeptide analysis and glycoengineering are expected to complement released-glycan profiling for deeper insights into structure–function relationships.

Conclusion

The combination of rapid Gly-X sample preparation, InstantPC or 2-AB Express labeling, HILIC-FLD/MS analysis and sialic acid quantitation provides a versatile, high-performance platform for detailed glycan characterization of biotherapeutics. Adoption of these methods enhances analytical throughput, sensitivity and reproducibility, supporting robust development, manufacturing and quality control of glycoprotein drugs.

Reference

• Khoury M, et al. Sci Rep. 2011;1:90.
• Planinc A, et al. Anal Chim Acta. 2016;921:13–27.
• Walsh G. Nat Biotechnol. 2014;32(10):992–1000.
• Liu H. J Pharm Sci. 2015;104(6):1866–1884.
• Jones A. BioPharm Int. 2017;30:20–25.
• Higel F, et al. Eur J Pharm Biopharm. 2016;100:94–101.
• Abes R, Teillaud J-L. Pharmaceuticals. 2010;3:146–157.
• Ghaderi D, et al. Biotechnol Genet Eng Rev. 2012;28:147–175.