Thermo Scientific Glycan Analysis for Biotherapeutics

Importance of the Topic

Glycan characterization underpins quality, safety and efficacy of biotherapeutic proteins. Variations in glycosylation patterns can alter immunogenicity, half-life and biological activity. Robust analytical workflows are essential to support drug development, manufacturing control and regulatory compliance.

Objectives and Overview

This work presents four complementary workflows for comprehensive glycan analysis in biotherapeutics:

• Intact glycoprotein profiling to define overall glycoform distribution
• Glycopeptide mapping to locate glycosylation sites and quantify site-specific heterogeneity
• Free glycan analysis for structural identification and relative quantitation of released glycans
• Monosaccharide analysis to determine individual sugar composition and content

Methodology and Instrumentation Used

Each workflow integrates optimized sample preparation, chromatographic separation and mass spectrometry or specialized detection:

• Intact protein analysis employs hydrophobic interaction, ion exchange or reverse-phase UHPLC columns coupled to high-resolution accurate-mass Orbitrap or Exactive EMR instruments and Protein Deconvolution software for glycoform mass assignment
• Glycopeptide mapping uses SMART Digest trypsin kits for rapid digestion, Vanquish UHPLC with Accucore or Acclaim C18 columns, and Orbitrap Fusion Lumos Tribrid MS combining HCD and ETD modes with PepFinder software for site localization
• Free N-glycan workflows release glycans enzymatically, label fluorescently or with TMT tags, separate on GlycanPac AXH-1/AXR-1 columns and analyze by fluorescence or MS with SimGlycan for structure prediction
• Monosaccharide composition is determined after enzymatic release and acid hydrolysis by HPAE-PAD on CarboPac columns using Dionex ICS systems for high sensitivity and selectivity

Main Results and Discussion

Intact profiling revealed distinct glycoform clusters and variant fragments in monoclonal antibodies. Deconvoluted high-resolution spectra enabled quantitation of major glycan species such as G0F, G1F and sialylated forms. Glycopeptide mapping localized glycans to specific tryptic peptides, providing relative abundance of each glycoform at each site. Free glycan analysis delivered detailed isomer separation and quantitation of neutral, monosialylated and disialylated structures. Monosaccharide assays confirmed stoichiometric ratios of fucose, galactose and sialic acid derivatives across batches, demonstrating process consistency and critical quality attributes.

Benefits and Practical Applications

• Streamlined workflows reduce sample preparation time and increase throughput
• High-resolution mass spectrometry and optimized chromatography deliver confident glycan identification and quantitation
• Software automation improves data processing consistency and speeds decision making
• Techniques support comparability studies, biosimilar development and lot release testing

Future Trends and Opportunities

Advances in microfluidic UHPLC, ion mobility separation and artificial intelligence for spectral interpretation will further enhance glycan profiling depth and speed. Multiplexed labeling strategies and single-cell glycomics promise to expand understanding of glycosylation heterogeneity in complex biological systems. Integration with real-time process analytics could enable in-line glycosylation monitoring during biomanufacturing.

Conclusion

Comprehensive glycan analysis requires a combination of intact, peptide, released glycan and monosaccharide workflows. The described platform leverages optimized sample handling, high-performance chromatography and state-of-the-art mass spectrometry to deliver detailed structural and quantitative information critical for biotherapeutic development and quality control.

Reference

Thermo Fisher Scientific application note on glycan analysis for biopharmaceuticals provides detailed methodology and instrument specifications.