Flash Characterization of mAbs Using a Combination of Reagents via Automated On-Line Microdroplet Reaction

Importance of the Topic

Monoclonal antibodies (mAbs) are a cornerstone of biopharmaceutical development and require detailed structural characterization to ensure safety, efficacy, and batch-to-batch consistency.

Study Objectives and Overview

This work aimed to integrate enzymatic digestion, reduction, and deglycosylation of mAbs into a single automated, on-line microdroplet reaction. The goal was to assess reproducibility, reaction yield, and simplification of traditional multi-step workflows.

Methodology

• Antibody and reagent preparation: mAbs (including NIST IgG1, Herceptin, Sigmamab) diluted to 0.5 mg/mL in 5 mM ammonium bicarbonate; IdeS and GlycINATOR at 1 unit/µL; DTT at 10 mg/mL.
• Automated injection: Agilent 1290 Infinity II LC in flow injection mode executed multi-draw, mix, wait, and inject cycles using the Agilent Injection Program.
• Microdroplet reaction: sample and reagents condensed into microdroplets in the unmodified Agilent Jet Stream ESI source, enabling ultrafast enzymatic processes.
• Data acquisition: processed species detected by Agilent 6545XT AdvanceBio LC/Q-TOF MS; spectra analyzed with MassHunter and BioConfirm v12.

Used Instrumentation

• Agilent 1290 Infinity II UHPLC system operated in flow injection analysis mode.
• Unmodified Agilent Jet Stream electrospray ionization source in positive mode as microdroplet reactor.
• Agilent 6545XT AdvanceBio LC/Q-TOF mass spectrometer for high-resolution detection.
• Source parameters: drying gas 350 °C at 13 L/min, sheath gas 400 °C at 12 L/min, nebulizer 60 psi, capillary voltage 4500–5000 V, nozzle 2000 V.
• MS acquisition range m/z 1350–10000 at 2 spectra/s.

Results and Discussion

Microdroplet GlycINATOR deglycosylation selectively removed Fc N-glycans from NIST IgG1, Herceptin, and Sigmamab, retaining core GlcNAc residues. Combining IdeS cleavage with DTT reduction or GlycINATOR treatment in a single microdroplet reaction yielded clear Fc and Fab spectra, with fragment yields exceeding 90% and minimal residual intact mAb. Reaction times were reduced to milliseconds while maintaining high reproducibility.

Benefits and Practical Applications of the Method

• Ultrafast processing: enzymatic reactions complete within milliseconds.
• Resource efficiency: low reagent consumption and use of unmodified ESI source.
• High reproducibility: consistent fragment profiles across replicates.
• Workflow simplification: single automated protocol replaces multiple manual steps.

Future Trends and Opportunities

Expansion to other protein classes and post-translational modifications is anticipated. Integration with real-time data analysis and machine learning could further accelerate screening and quality control. Additional enzyme combinations or chemical modifiers may broaden analytical scope.

Conclusion

The automated microdroplet reaction platform demonstrated here offers a rapid, reproducible, and streamlined approach for mAb characterization. By integrating digestion, reduction, and deglycosylation into a single on-line workflow, this method reduces analysis time and resource use while providing high-quality structural information.

References

• Lau J., Zhao H., Knierman M. Flash Characterization of Antibodies via Microdroplet Reactions in an Unmodified Jet Stream Source. Agilent Application Note 5994-6752EN, 2023.
• Genovis. GlycINATOR product information. https://www.genovis.com/smartenzymes/antibody-deglycosylation/glycinator/