Quality Evaluation of mRNA by Electrophoresis

Importance of the Topic

Reliable and rapid assessment of mRNA integrity and purity is critical in research and manufacturing of mRNA vaccines and therapeutics. Monitoring degradation products and off-target RNA helps ensure product efficacy and safety during transcription, capping, and purification steps.

Objectives and Study Overview

This study evaluated the performance of the Shimadzu MultiNA II microchip electrophoresis system for quality control of mRNA. Two main goals were addressed:

• Assess the system’s ability to detect mRNA degradation over controlled thermal treatments.
• Determine sensitivity for identifying low-level impurities in mixed mRNA samples.

Methodology and Used Instrumentation

RNA samples (CleanCap FLuc, Cas9, and Cre mRNAs) were prepared at defined concentrations and subjected to thermal denaturation at 90 °C for various durations to generate degradation profiles. Purity mixtures of Cas9 and Cre mRNAs were created at ratios ranging from 100:1 to 4:1. Samples were mixed 1:1 with an internal mobility marker, heated at 72 °C for 3 minutes, then analyzed by the MultiNA II MCE-301 system using the proprietary RNA kit and RNA 6000 Ladder for size calibration.

Used Instrumentation:

• Shimadzu MultiNA II MCE-301 microchip electrophoresis system
• MultiNA RNA Kit (marker solution and separation buffer)
• Thermo Fisher RNA 6000 Ladder

Main Results and Discussion

• Degradation Evaluation: A progressive loss of the 1,922 nt FLuc mRNA peak and concomitant increase in low-molecular-weight fragments were observed with longer heat treatment times, demonstrating clear separation of degradation products.
• Purity Assessment: Cre mRNA was detected at concentrations as low as 1 ng/µL when spiked into 100 ng/µL Cas9 mRNA at a 100:1 ratio. Electropherograms and gel images confirmed distinct peaks for both transcripts even at low impurity levels.

Benefits and Practical Applications

• Rapid Analysis: Each run completes in ~100 seconds, enabling high throughput.
• Automation: Minimal manual intervention reduces operator variability.
• Process Control: Suitable for in-line monitoring of mRNA synthesis and purification workflows.

Future Trends and Applications

Advances may include integration with automated sample handling for real-time quality feedback, expansion of analyte range (e.g., small RNAs and modified nucleotides), and coupling with bioinformatics tools for detailed impurity profiling. The trend toward on-demand mRNA manufacturing could drive further miniaturization and multiplexing of microchip electrophoresis platforms.

Conclusion

The Shimadzu MultiNA II microchip electrophoresis system offers a fast, automated, and sensitive method for assessing mRNA degradation and purity. Despite slightly lower resolution compared to capillary electrophoresis, its throughput and ease of use make it a valuable tool for quality assurance in mRNA production.

References

No external literature references were provided in the original application note.