Stress testing of Humira (adalimumab) monitored by Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS)

Monoclonal antibodies are a rapidly expanding class of biotherapeutics, whose structural integrity is critical to their function and stability. However, their complex higher-order structure and sensitivity to degradation pathways make detailed characterisation challenging, particularly when assessing structural changes under stress conditions. Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS) offers a powerful solution-based approach to monitor structural dynamics at the amino acid level, providing insights beyond conventional analytical techniques.

In this application note, HDX-MS is used to evaluate the impact of heat stress on Humira® (adalimumab), demonstrating its relevance for stability and comparability studies.

KEY HIGHLIGHTS OF THE METHOD

• Detection of structural changes at the amino acid level using HDX-MS in solution
• Ability to analyse protein structure and dynamics across time scales ranging from seconds to hours
• High sequence coverage achieved (up to 100% for the light chain, 98.7% for the heavy chain) with strong peptide redundancy
• Comparison of stressed versus reference samples enables identification of localised structural alterations
• Method compatible with complex samples, tolerating impurities, buffers and near-physiological conditions

HOW THIS SUPPORTS YOUR BIOTHERAPEUTIC DEVELOPMENT PROGRAMME?

HDX-MS provides detailed structural insights that complement conventional physico-chemical techniques, enabling a deeper understanding of protein behaviour under stress conditions.

• Supports stability studies by detecting subtle structural modifications induced by stress
• Enhances comparability assessments between stressed and reference products
• Enables localisation of structural changes to specific amino acid regions
• Improves interpretation of higher-order structure changes impacting protein function
• Facilitates analysis under near-physiological conditions with low sample requirements

This approach bridges the gap between traditional analytical and structural biology techniques for biotherapeutics

APPLICATIONS

• Stress testing studies of monoclonal antibodies under accelerated degradation conditions
• Stability studies of therapeutic proteins
• Comparative analysis of biotherapeutics (e.g. biosimilarity assessment)
• Investigation of protein structure and interaction dynamics
• Identification of structurally impacted regions such as complementarity-determining regions (CDRs)