LOCALIZED CONFORMATION ANALYSES OF MUTATED HUMAN IgG1 BY HYDROGEN DEUTERIUM EXCHANGE MASS SPECTROMETRY AND DIFFERENTIAL SCANNING CALORIMETRY

Significance of the topic

Hydrogen deuterium exchange mass spectrometry (HDX MS) and differential scanning calorimetry (DSC) are essential techniques for investigating protein conformational dynamics and stability. They provide complementary information on local structural changes and global folding/unfolding processes, critical in biotherapeutics development and quality control.

Objectives and study overview

This study aimed to detect and localize conformational differences between non-mutated and site-specific mutated human IgG1 antibodies using HDX MS and DSC approaches. Two mutated IgG1 batches were compared to a control to identify mutation-induced alterations at the peptide level.

Methodology

• Online pepsin digestion at pH 2.5 and 0°C to minimize back-exchange
• Rapid peptide separation by nanoACQUITY UPLC within 10 minutes
• Mass analysis on a XEVO G2 Q-TOF mass spectrometer
• Automated deuterium uptake processing with DynamX software
• Nano DSC used to probe global stability and detect domain-specific transitions

Instrumentation used

• Waters nanoACQUITY UPLC with HDX manager
• Waters XEVO G2 Q-TOF mass spectrometer
• TA Instruments Nano DSC autosampler
• Leap robotic system for automated sample handling
• PLGS software for peptide identification
• DynamX software for HDX data processing

Major results and discussion

• Peptides adjacent to mutation sites exhibited increased deuterium uptake compared to control, indicating localized destabilization
• Differential and butterfly charts highlighted mutation-specific conformational changes with uptake differences exceeding 1 Da
• Peptides distal from mutation sites showed no significant uptake variation, confirming site-specific effects
• DSC analysis revealed a shift to lower transition temperatures in mutated samples, consistent with decreased stability in the affected domain

Benefits and practical applications

Combining HDX MS and DSC allows sensitive detection of local and global structural changes, supporting comparability studies of therapeutic antibodies, stability assessments during formulation optimization, and quality control in biopharmaceutical production.

Future trends and opportunities

• Integration of HDX MS with orthogonal structural techniques such as cryo-EM and NMR
• Advances in automation and high-throughput workflows for rapid comparability testing
• Application to more complex biologics, including bispecific antibodies and antibody-drug conjugates
• Enhanced data analytics and machine learning for predictive stability modeling

Conclusion

The study demonstrates that automated HDX MS workflows combined with DSC provide a robust platform to localize and quantify mutation-induced conformational changes in IgG1. This integrated approach enhances understanding of protein stability and supports the development and quality control of biotherapeutics.

Reference

1. Houde D et al. J. Pharm. Sci. 2011;100(6):2071-2086.