High Throughput Screening and Characterization of Bispecifics Using Native Ion Mobility Mass Spectrometry

Significance of the topic

Bispecific antibodies (bsAbs) combine two different antigen‐binding specificities in a single molecule, offering new therapeutic modalities. Their structural heterogeneity and assembly pathways pose analytical challenges during development, where rapid screening of intact mass, conformational states, and purity is essential.

Objectives and study overview

• Establish a high‐throughput native and denaturing ion mobility–MS (IM‐MS) workflow for intact bsAb screening.
• Profile individual IgG subclasses (IgG1–IgG4) to build reference collision cross‐sectional (CCS) databases.
• Monitor formation and heterogeneity of a model bsAb generated by Fab‐arm exchange under mild reduction.

Methodology and instrumentation

The workflow uses buffer‐exchanged antibody samples in 200 mM ammonium acetate (pH 7) delivered by a nanoLC interface at 0.4 µL/min. Native MS and IM separation are performed on an Agilent 6560 Ion Mobility Q‐TOF. Data acquisition alternates between low/high collision energies to capture both intact mass and fragmentation information. Drift times and CCS values are extracted using the IM‐MS Browser and BioConfirm software with maximum entropy deconvolution and IM molecular feature extraction (iMFE).

Used instrumentation

• Agilent 6560 Ion Mobility Q‐TOF Mass Spectrometer
• G1992A nano-LC interface with 200 mM ammonium acetate mobile phase
• Micro Bio‐Spin 30 columns for buffer exchange
• Agilent MassHunter Qualitative Analysis and BioConfirm software

Results and discussion

Native IM‐MS distinguished IgG1–IgG4 isotypes by unique drift time distributions and CCS values. The model bsAb, produced by incubating two IgG1 variants in reduced glutathione, exhibited distinct charge envelopes and two dominant IMS conformers, reflecting heterodimer and homodimer species. Alternating collision energy frames enabled simultaneous intact mass measurement and MS/MS data for sequence confirmation. Rapid acquisition (<2 min/sample) allowed clear differentiation of parental mAbs and resulting bsAb variants.

Benefits and practical applications

• High‐throughput, label‐free screening of intact bsAb mass and conformation.
• Clear resolution of subclass and heterodimer species without extensive sample preparation.
• Integration of IM separation enhances structural characterization in QA/QC and lead selection.

Future trends and opportunities

Advances may include use of alternative drift gases to improve separation, expanded automated scheduling and data analysis pipelines, and incorporation of machine‐learning algorithms for rapid pattern recognition. Coupling IM‐MS with higher‐order MS/MS workflows and online chromatography will support detailed mapping of complex biologics and multimeric assemblies.

Conclusion

Native and denaturing IM‐MS on the Agilent 6560 platform provides a powerful approach for high‐throughput screening and detailed characterization of bsAbs. The workflow offers rapid insight into mass heterogeneity, conformational landscapes, and assembly efficiency, supporting accelerated development of next‐generation biotherapeutics.

References

• Spiess C, Zhai Q, Carter PJ. Alternative molecular formats and therapeutic applications for bispecific antibodies. Mol Immunol. 2015;67(2):95–106.
• Kontermann RE, Brinkmann U. Bispecific antibodies. Drug Discov Today. 2015;20(7):838–847.
• Debaene F, et al. Time‐resolved native ion‐mobility mass spectrometry to monitor dynamics of IgG4 Fab arm exchange and “bispecific” monoclonal antibody formation. Anal Chem. 2013;85(20):9785–9792.