Characterisation of the glycosylation of Enbrel: Etanercept by high resolution mass spectrometry

Therapeutic glycoproteins such as monoclonal antibodies, fusion proteins and cytokines exhibit complex and heterogeneous glycosylation patterns that directly impact their safety, bioactivity, stability and pharmacokinetics. Accurately characterising these glycosylation profiles is therefore critical, yet challenging, due to both micro- and macro-heterogeneity arising from their production systems.

This application note presents a comprehensive analytical workflow based on high resolution mass spectrometry (HRMS) to characterise both N- and O-glycosylation of Etanercept. By combining multiple orthogonal techniques across different structural levels, this approach enables a detailed and reliable understanding of glycosylation. The full application note is available for download below.

KEY HIGHLIGHT OF THE METHOD

• Comprehensive multi-level workflow combining released glycans, peptide mapping and subunit analysis
• N-glycan profiling achieved within one day using RapiFluor-MS labelling
• High identification accuracy with mass errors below 3 ppm
• Excellent reproducibility with ~1.5% mean RSD across independent preparations
• Quantification of sialylation from as little as 10 µg of protein with consistent results (~7.0–7.1%)

HOW THIS SUPPORTS YOUR ANALYTICAL STRATEGY?

This integrated HRMS-based workflow provides a robust framework for addressing glycosylation complexity in therapeutic proteins. It enables consistent characterisation across multiple analytical levels, improving confidence in structural and functional assessments.

• Enables thorough characterisation of both N- and O-glycosylation profiles
• Supports reliable identification and localisation of glycosylation sites at amino-acid level
• Facilitates consistent batch-to-batch comparison through reproducible quantification
• Improves interpretation of glycoprotein heterogeneity through orthogonal analytical approaches
• Allows analysis from low sample quantities within reduced timelines

TYPICAL APPLICATIONS

• Characterisation of therapeutic glycoproteins including monoclonal antibodies, fusion proteins and recombinant proteins
• Glycosylation profiling for biopharmaceutical development and quality assessment
• Site-specific glycosylation analysis using peptide mapping approaches
• Sialylation profiling and quantification for consistency evaluation
• Applicable to other glycoproteins such as mAbs, ADCs and recombinant DNA-derived proteins