Characterisation of the glycosylation of Humira (adalimumab) and Erbitux (Cetuximab) by high resolution mass spectrometry

Therapeutic monoclonal antibodies are glycosylated proteins whose glycosylation profiles strongly influence their safety, efficacy, stability and pharmacokinetics. Due to the complexity and heterogeneity of the glycan structures, their comprehensive characterisation requires robust and complementary analytical approaches.

This application note presents a multi-level strategy combining high-resolution mass spectrometry (HRMS) with orthogonal techniques to investigate glycosylation in adalimumab and cetuximab. By integrating analyses at released glycan, peptide and subunit levels, this workflow provides detailed insights into glycan composition and localisation, supporting advanced biopharmaceutical development

CORE ANALYTICAL PERFORMANCE

• Rapid N-glycan profiling achieved within one day using RapiFluor-MS labelling and HILIC-FLR-MS analysis
• High repeatability with minimal retention time drift (RSD_RT ≈ 0.08%) and robust quantification (RSD _%Area down to 0.89%)
• Detection of low abundance glycans (≈ 0.1%) with high sensitivity and accurate mass confirmation (error ~0.5 ppm)
• Quantification of sialic acids (Neu5Ac and Neu5Gc) from small sample amounts using DMB labelling and fluorescence detection

ANALYTICAL BENEFITS

This multi-level analytical approach enables a detailed understanding of glycosylation as a critical quality attribute, supporting informed decision-making throughout biopharmaceutical development. It:

• Provides detailed structural and site-specific glycosylation information across multiple analytical levels
• Ensures high comparability and consistency between analytical techniques and datasets
• Supports robust characterisation of biosimilars and innovator products
• Enables detection and quantification of critical glycan variants impacting efficacy and immunogenicity
• Facilitates rapid and sensitive analysis using small sample quantities
• Helps confirm glycosylation site localisation and molecular weight at subunit level

This integrated workflow strengthens confidence in glycosylation profiling across development stages.

WHERE THIS METHOD APPLIES?

• Characterisation of monoclonal antibodies glycosylation (e.g. adalimumab and cetuximab)
• Site-specific N-glycan profiling using peptide mapping approaches
• Subunit-level analysis to resolve glycosylation heterogeneity
• Quantification of sialic acid variants (Neu5Ac and Neu5Gc) in biotherapeutics
• Application to broader mAb and ADC development workflows