Flash Characterization of Antibodies via Microdroplet Reactions in an Unmodified Jet Stream Source

Importance of the Topic

Rapid and reliable characterization of monoclonal antibodies is essential for biopharmaceutical research, development, and quality control. Conventional methods involving bulk digestion, reduction, and deglycosylation often require tens of minutes and consume significant amounts of enzymes and antibodies. The adoption of microdroplet reactions in electrospray ionization sources promises ultrafast kinetics, minimal reagent use, and streamlined workflows for high-throughput antibody analysis.

Study Objectives and Overview

This work presents “Flash Characterization,” an optimized approach for antibody analysis using microdroplet reactions in an unmodified Agilent Jet Stream ESI source. Key aims included (1) establishing rapid IdeS proteolysis and disulfide reduction protocols in microdroplets, (2) determining optimal reagent concentrations and flow rates, and (3) validating the approach across a diverse set of therapeutic antibodies.

Methodology and Instrumentation

Samples of NIST IgG1 and seven commercial monoclonal antibodies were prepared at 0.5 mg/mL in ammonium bicarbonate buffer. IdeS enzyme and reducing agents (TCEP, DTT, triethylphosphine) were diluted in the same buffer. A 1290 Infinity II LC system delivered sample and reagent microdroplets directly into a 6546 Q-TOF mass spectrometer operating in positive ESI mode. No chromatographic column was used; the autosampler exit line fed the Jet Stream source. Microdroplet reaction conditions were optimized by varying buffer concentration (5–100 mM), flow rate (15–200 µL/min), enzyme-to-antibody ratio, and nebulizer pressure.

Main Results and Discussion

Optimal conditions comprised 5 mM ammonium bicarbonate at pH 7, a 25 µL/min flow rate, and standard Jet Stream source settings. Under these parameters, IdeS digestion achieved ≥85 % conversion of intact IgG1 within microseconds, with a precision of 4.3 % over ten replicates. Panel testing revealed similarly high efficiencies (77–90 %) for six other antibodies, while a control lacking the IdeS cleavage motif showed no reaction. Disulfide bond reduction with TCEP, DTT, or triethylphosphine under microdroplet conditions yielded heavy and light chains with efficiencies around 87 %.

Benefits and Practical Applications

• Sub-minute reaction times versus traditional 30+ min incubations
• Significant reduction in enzyme and sample consumption
• Minimal method development and no additional hardware modifications
• High reproducibility and broad applicability across antibody subtypes

Future Trends and Potential Applications

Emerging directions include integrating microdroplet reactions with automated high-throughput screening, expanding reagent libraries for glycan and PTM mapping, and applying the approach to other biomolecule classes such as proteins and nucleic acids. Further work may focus on inline separation coupling and real-time reaction monitoring.

Conclusion

The Agilent Jet Stream microdroplet workflow delivers ultrafast, cost-effective antibody characterization with performance comparable to conventional methods. Its simplicity, speed, and robustness make it an attractive platform for routine biopharma analysis and accelerated method development.

References

Gunawardena H. P., et al. Rapid Characterization of Antibodies via Automated Flow Injection Coupled with Online Microdroplet Reactions and Native-pH Mass Spectrometry. Anal. Chem. 2023, 95(6), 3340–3348.