Characterisation of Adalimumab by high resolution mass spectrometry

Monoclonal antibodies are inherently complex and heterogeneous biomolecules, requiring multiple orthogonal analytical techniques to fully characterise their structural variants. Humira® (adalimumab), a widely used therapeutic antibody, exemplifies this complexity due to the presence of multiple molecular forms arising from recombinant production. Comprehensive characterisation is essential, as product heterogeneity can directly affect safety and efficacy.

In this application note, high resolution mass spectrometry is presented as a powerful and versatile approach to analyse adalimumab at different structural levels, from intact protein to peptide mapping and glycan profiling. The full technical details and results are available in the complete application note for download.

KEY HIGHLIGHTS OF THE METHOD

• Intact mass analysis enables determination of glycosylation profiles with mass accuracy typically below 20 ppm
• Rapid deglycosylation achieved in 10–15 minutes using Rapid PNGase F, simplifying spectra and enabling detection of additional modifications
• IdeS “middle-up” strategy generates ~25 kDa fragments, allowing improved chromatographic resolution and mass accuracy below 1 Da
• Peptide mapping delivers up to 100% sequence coverage with mass accuracy below 5 ppm
• N-glycan profiling workflow enables full analysis, including processing and reporting, within 24 hours

BENEFITS FOR YOUR ANALYTICAL STRATEGY

This multi-level mass spectrometry approach provides a comprehensive framework to characterise monoclonal antibodies with high precision and reproducibility. By combining complementary techniques, it enables both global and targeted insights into critical quality attributes.

• Enables thorough characterisation of heterogeneity impacting safety and efficacy
• Improves identification and quantification of variants such as oxidation, deamidation and glycosylation
• Provides high-resolution data for both intact molecules and subunits
• Facilitates localisation of modifications through subunit and peptide-level analysis
• Supports routine use through automated workflows and validated software solutions
• Enables monitoring during stability and stress studies, including oxidation profiling

APPLICATIONS

• Identity testing and molecular mass determination of monoclonal antibodies
• Glycosylation profiling and batch-to-batch consistency assessment
• Monitoring oxidation and stability through stress studies
• Targeted purity profiling via peptide mapping
• Characterisation of post-translational modifications (e.g. deamidation, pyroglutamate formation, glycosylation)