Ion Mobility-Mass Spectrometry Reveals the Structures and Stabilities of Biotherapeutic Antibody Aggregates

Significance of the topic

Analysis of antibody aggregates is critical for ensuring safety, efficacy, and shelf-life of biotherapeutic monoclonal antibodies. Aggregation can alter higher-order structure, trigger immunogenicity, and reduce potency. Rapid, sensitive methods that reveal both quantitative and structural information on aggregate forms are essential in early screening and quality control.

Objectives and study overview

This study aims to apply ion mobility-mass spectrometry (IM-MS) combined with collision-induced unfolding (CIU) to characterize the size, structure, and stability of antibody monomers and dimers under native and stress conditions. Two model IgG1 antibodies (α and β) are evaluated following thermal and pH-induced stress to monitor aggregate formation and conformational changes over time.

Použitá instrumentace

• Waters Synapt G2 quadrupole-ion mobility-time-of-flight (q-IM-ToF) platform
• Agilent 6560 ion mobility quadrupole-time-of-flight (IM-q-ToF) instrument

Methodology and instrumentation

IM-MS separates ions by collision cross section (CCS) to distinguish monomers and oligomers. CIU experiments progressively increase collision voltage, inducing unfolding transitions that produce characteristic fingerprints of structural features. Samples were stressed at 50 °C (thermal) and pH 3 (chemical). CCS calibration used standard proteins and computational models of solution-phase IgGs, with trajectory methods applied via GROMACS and Collidoscope to predict gas-phase conformations.

Main results and discussion

• Distinct monomer and dimer populations were resolved, with CIU fingerprints showing multiple unfolding features sensitive to stress conditions.
• Thermal stress induced modest ground-state CCS shifts (<2%) and affected later CIU features, while pH 3 stress led to larger CCS increases (6–10%) and early feature destabilization.
• IgG1α exhibited greater sensitivity to heat, whereas IgG1β was more prone to pH-induced unfolding and aggregation.
• Inter-instrument comparison highlighted systematic CCS differences, but consistent trends across both platforms.
• Computational docking and CCS calculations supported experimental assignments of compact and extended dimer conformers, revealing potential CDR-driven assembly interfaces.

Benefits and practical applications

• IM-MS and CIU provide rapid, low-sample-consumption assays for detailed higher-order structure analysis and stability profiling.
• Methodology enables early screening of candidate mAbs for aggregation propensity and structural integrity.
• Techniques support formulation optimization, process development, and quality control in biopharmaceutical workflows.

Future trends and applications

• Integration with high-throughput screening to accelerate antibody lead selection.
• Advanced computational modeling to refine structure–function correlations for aggregates.
• Expansion to other biotherapeutic modalities, such as bispecifics and antibody–drug conjugates.
• Development of standardized CIU libraries for comparative stability assessment across platforms.

Conclusion

This work demonstrates that IM-MS coupled with CIU delivers both quantitative and structural insights into antibody aggregation under diverse stress conditions. The approaches distinguish subtle conformational changes in monomeric and dimeric species, highlighting sequence-dependent aggregation mechanisms. These capabilities offer valuable tools for biotherapeutic screening, formulation design, and quality assurance.

Reference

• Roberts CJ, et al. Trends Biotechnol. 2014;32(7):372–380.
• Moussa EM, et al. J Pharm Sci. 2016;105(2):417–430.
• Paul R, et al. Pharm Res. 2012;29(8):2047–2059.
• Polasky DA, et al. Anal Chem. 2019;91:3147–3155.
• Vallejo DD, et al. Anal Chem. 2019;91(13):8137–8145.