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

Significance of the Topic

Microdroplet reactions enable rapid biochemical modifications of antibodies within microseconds, offering significant advantages over conventional bulk assays that require tens of minutes. This approach reduces sample and reagent consumption, accelerates analysis turnaround and supports high-throughput workflows essential for biopharmaceutical development and quality control.

Objectives and Study Overview

This study aimed to develop a “Flash Characterization” workflow for monoclonal antibodies by integrating enzyme digestion and disulfide reduction within microdroplets generated in an unmodified JetStream electrospray source. Conditions were optimized for IdeS cleavage and chemical reduction of disulfide bonds and then applied to a panel of commercial antibodies to evaluate robustness and general applicability.

Methodology

Antibodies such as NIST IgG1 were prepared at 0.5 mg/mL in 5 mM ammonium bicarbonate. IdeS and reducing agents (TCEP, TEP, DTT) were added to the sample stream. A flow injection analysis program on an Agilent 1290 Infinity II LC introduced alternating 1 µL segments of antibody and reagent into the JetStream ESI source. Reaction products were detected by an Agilent 6546 Q-TOF operating in positive AJS mode, and data were processed with MassHunter software.

Applied Instrumentation

• UHPLC: Agilent 1290 Infinity II with flow injection analysis
• Mass spectrometer: Agilent 6546 Q-TOF with JetStream ESI source
• Software: Agilent MassHunter Quantitative Analysis and BioConfirm v10

Main Results and Discussion

Optimization studies identified 5 mM ammonium bicarbonate at a flow rate of 25 µL/min as optimal for IdeS-mediated F(ab’)2 generation, achieving >85% yield with good peak shape. Using 1 unit of IdeS per reaction gave rapid but sufficient cleavage, while 2–3 units further improved yield. Ten replicates showed an average conversion of 85% with 4.3% RSD. Extending the workflow to seven commercial antibodies produced F(ab’)2 yields between 83% and 90% with RSD values below 7%. Microdroplet reduction using TCEP, TEP or DTT reached efficiencies of 85–87% within microseconds.

Benefits and Practical Applications

Flash Characterization drastically reduces analysis time from minutes to microseconds and minimizes enzyme and reagent consumption. The method enables high-throughput structural assessment of antibodies for research, development and quality control, facilitating faster decision-making in biopharmaceutical workflows.

Future Trends and Possibilities

Future developments may include integration with automated sample handling for fully online antibody profiling, expansion to other protein classes, coupling with front-end separations and real-time monitoring of bioprocess streams.

Conclusion

Microdroplet-based Flash Characterization in an unmodified JetStream source offers an ultrafast, high-yield and reproducible platform for antibody digestion and reduction, delivering significant time and cost savings in analytical workflows.

References

• Gunawardena H.P. et al. Anal. Chem. 2023, 95, 3340–3348.
• Lau J.; Knierman M.; Zhao H.; Gunawardena H. Flash Characterization of Antibodies via Microdroplet Reactions in an Unmodified JetStream Source. ASMS Poster MP 616, 2023.