Rapid Screening of Bispecific Antibodies and Antibody Impurities using In-source Collision Induced Unfolding coupled with IM-MS

Significance of the Topic

In pharmaceutical development, rapid and reliable detection of bispecific antibody variants and their impurities is critical for quality control and ensuring therapeutic efficacy. Traditional size-exclusion chromatography can be time-consuming and lack resolution, whereas ion mobility-mass spectrometry (IM-MS) combined with collision-induced unfolding (CIU) offers structural insights and faster screening.

Objectives and Study Overview

This study evaluated the capability of an Agilent 6560C IM-QTOF with in-source activation to differentiate bispecific antibody samples (bsAb1, bsAb2, bsAb3.2, bsAb4.2) and detect non-covalent aggregates or engineering variants. Standard monoclonal antibodies (Herceptin, NIST mAb) and enzymatically digested fragments (IdeS, PNGase) served as controls for method development and fingerprint validation.

Instrumentation Used

• Agilent 6560C IM-QTOF instrument equipped with in-source collision activation hardware
• AJS electrospray ionization source with micro-nebulizer and syringe pump sample delivery
• Sample preparation: buffer exchange into 200 mM ammonium acetate using Bio-Rad P-6 spin columns
• CIU parameters: in-source collision voltage ramp from 0 V to 430 V in 10 V increments, 12s per voltage step
• Data analysis performed with CIUSuite software for RMSD, CIU50 determination, and linear discriminant classification

Main Results and Discussion

• Abundant charge states +25 to +28 produced distinct CIU fingerprints corresponding to conformational transitions and collision cross sections (CCS).
• Average within-sample RMSD values (~2.7–4.5%) indicated high repeatability across triplicates, while cross-comparison RMSD (10.1–12.6%) confirmed structural differences among bsAb variants.
• Engineered disulfide bonds in Fv domains (bsAbx.2 series) increased gas-phase stability, evidenced by higher CIU50 voltages and altered unfolding pathways.
• IdeS digestion improved resolution of Fab and Fc regions, enhancing differentiation between Herceptin and NIST mAb (p-value < 0.01).
• Non-covalent dimers dissociated into monomers at elevated in-source CE voltages, enabling impurity detection and assessment of covalent linkage in engineered variants.
• Linear discriminant analysis of combined charge-state CIU curves achieved accurate classification of blind samples, demonstrating the method’s suitability for variant identification.

Advantages and Practical Applications

• Significantly reduced analysis time compared to chromatographic methods.
• High structural specificity through CIU fingerprints and CCS measurements.
• Ability to detect and characterize monomers, aggregates, and engineered modifications in a single experiment.
• Potential integration into QC workflows for rapid batch screening of bispecific antibodies and biosimilars.

Future Trends and Applications

• Automated CIU screening coupled with machine learning for enhanced pattern recognition and classification of complex biologics.
• Extension to other multi-domain proteins, antibody–drug conjugates, and nanobody formats.
• Integration with high-throughput sample introduction and real-time data processing in regulated environments.
• Development of standardized CIU databases for cross-laboratory comparisons and regulatory filings.

Conclusion

IM-MS with in-source CIU on the Agilent 6560C platform provides a rapid, reproducible, and detailed approach for screening bispecific antibodies and detecting impurities. The method offers robust classification capabilities and structural insights, making it a valuable tool for research and quality control in biopharmaceutical development.

References

• Boucher L. E. et al., MABS 2023, 15(1), 2195517
• Gadkari V. V. et al., Analyst 2023, 148(2), 391–401
• Kurulugama R. T. et al., ASMS 2022, TP 298