GLYCAN ANALYSIS SOLUTIONS - The Next Generationof Glycan Sample Preparation and Analysis

Significance of Glycan Analysis

Glycan structures on glycoproteins modulate critical biological functions such as folding, stability and cell signaling. A robust analytical workflow enables detailed characterization of these modifications, supporting biopharmaceutical development, quality control and fundamental research.

Objectives and Overview

This whitepaper presents a comprehensive workflow for glycan analysis at multiple structural levels. Techniques covered include intact protein profiling, subunit analysis, glycopeptide mapping and released glycan characterization. The study demonstrates integration of fluorescence and mass detection to streamline glycan identification, quantification and monitoring.

Methods and Instrumentation

Multiple complementary liquid chromatography and mass spectrometry approaches are combined in a modular workflow:

• Intact protein and subunit analysis using ACQUITY UPLC Glycoprotein BEH Amide columns (300 Å) with TUV or FLR detectors and QTof MS (Xevo G2-XS, SYNAPT G2-Si HDMS)
• Glycopeptide mapping after enzymatic digestion (IdeS or Lys-C) with reduction, alkylation and UPLC-MS (UNIFI, MassLynx)
• Released glycan profiling via RapiFluor-MS labeling, HILIC separation, FLR quantification and MS confirmation (ACQUITY QDa Detector)
• Monosaccharide and sialic acid composition by acid hydrolysis, fluorophore labeling and UPLC-FLR analysis (BEH C18 columns, Empower 3 software)

Main Results and Discussion

Installation of the new Glycoprotein BEH Amide column facilitated high-resolution separations of intact proteins, subunits and glycopeptides. The RapiFluor-MS reagent delivered rapid, 5-minute labeling and improved MS ionization, yielding up to 14-fold greater fluorescence response and hundreds to thousands-fold enhanced mass spectral sensitivity compared to traditional labels. Glycan profiling on the ACQUITY QDa enabled routine mass detection for process monitoring, while HILIC-µElution cleanup provided a streamlined sample prep of three steps in 30 minutes.

Benefits and Practical Applications

Adopting this integrated workflow supports accelerated biopharmaceutical development by reducing sample preparation time, improving detection of low-abundance glycans and enabling site-occupancy analysis. It aligns with GMP compliance and can be deployed for quality control, comparability studies and process optimization.

Future Trends and Potential Uses

Next-generation advances may include further automation of glycan labeling and cleanup, expansion of site-specific glycosylation mapping by advanced MS/MS methods, and integration of glycomic data into predictive models for biotherapeutic design. Enhanced software solutions will drive deeper structural insights and broader routine application.

Conclusion

This workflow demonstrates a holistic approach to glycan analysis combining UPLC separations with both fluorescence and mass spectrometry. The innovations in column technology and RapiFluor-MS labeling deliver higher sensitivity, faster turnaround and more confident glycan characterization suitable for research and industrial laboratories.

Used Instrumentation

Key instruments include: ACQUITY UPLC Systems (intact protein, subunit, glycopeptide, monosaccharide analysis), Xevo G2-XS QTof MS, SYNAPT G2-Si HDMS, ACQUITY QDa Detector, Empower 3, UNIFI and MassLynx software.