Simultaneous reversed-phase and anion-exchange method scouting with a dual system for mRNA impurity determination

Importance of the Topic

The rapid expansion of mRNA-based therapies and vaccines has heightened the need for robust analytical methods to detect impurities generated during in vitro transcription. High-resolution techniques are essential to ensure product quality, safety, and regulatory compliance in research, development, and quality control laboratories. The simultaneous application of reversed-phase and ion-exchange chromatographic modes offers comprehensive impurity profiling in a single workflow, increasing efficiency and data consistency.

Aims and Study Overview

This work aimed to identify optimal chromatographic conditions for simultaneous detection of post-transcriptional impurities in mRNA using ion pairing reversed-phase (IP-RP) and ion-exchange (IEX) chromatography on a dual-system LC platform. Key objectives included:

• Screening multiple mobile phase compositions and column temperatures for both separation modes.
• Minimizing analysis time through automated method scouting.
• Comparing purified and non-purified mRNA samples under selected conditions.

Methodology and Instrumentation Used

The study employed a Thermo Scientific Vanquish Duo dual-system LC fitted with a solvent extension kit for automated scouting. Separation modes were run concurrently using:

• DNAPac RP column (2.1×100 mm, 4 µm) for IP-RP analysis at 0.4 mL/min and 50 °C.
• DNAPac PA200 RS column (4.6×150 mm, 4 µm) for IEX analysis at 1.0 mL/min and temperatures of 30–80 °C.
• Ion-pairing reagents including heptafluorobutyric acid and diisopropylethylamine salts with acetonitrile gradients.
• IEX buffers composed of tris/0.8 M perchlorate with 20% organic content.
• Injection volumes of 1 µL and 0.3 µL to standardize mRNA load.
• UV detection at 260 nm and data collection at 10 Hz.

Main Results and Discussion

Automated scouting of nine IP-RP and eight IEX conditions yielded several promising methods for mRNA impurity separation. Key findings include:

• Optimal IP-RP condition using 25 mM heptafluoroacetic acid provided sharp mRNA peaks and effective impurity resolution.
• Best IEX conditions employed 40 mM tris-perchlorate with 20% acetonitrile at elevated temperatures, revealing detailed impurity profiles.
• Simultaneous dual-mode operation reduced analysis time and ensured direct comparability of results.
• Relative peak area measurements for purified mRNA were consistent between IP-RP (92.7%) and IEX (90.97%) modes, demonstrating reliable purity estimation.
• IEX chromatography identified additional impurity details beyond IP-RP capabilities.

Benefits and Practical Applications

The integrated dual-system approach significantly streamlines method development for mRNA analytics by:

• Reducing instrument time and user intervention through automated mobile phase and temperature scouting.
• Providing comprehensive impurity profiles in a single run to support faster decision-making in formulation and purification workflows.
• Enabling flexible adaptation of selected methods to diverse mRNA constructs and purification platforms.

Future Trends and Potential Applications

Continued advancements are expected to further enhance mRNA analysis, including:

• Integration with mass spectrometry for structural characterization of impurities.
• Artificial intelligence-driven method optimization and predictive modeling.
• Development of novel stationary phases and ion-pairing agents to improve resolution of complex mRNA variants.
• High-throughput platforms for large-scale vaccine and therapeutic development.

Conclusion

The Thermo Scientific Vanquish Duo platform with the Method Scouting Kit enables efficient, simultaneous IP-RP and IEX impurity profiling of mRNA in a single instrument. Selected conditions offer robust peak shapes, detailed impurity detection, and consistent purity estimation, underscoring the value of dual-mode LC in mRNA method development and quality control.

Used Instrumentation

• Thermo Scientific Vanquish Duo Dual LC system with Solvent Extension Kit
• DNAPac RP column (2.1×100 mm, 4 µm)
• DNAPac PA200 RS column (4.6×150 mm, 4 µm)
• Autosampler maintained at 4 °C and UV detector set at 260 nm

Reference

Application Note by Andrei Ispan D., Lovejoy K., Patzelt S., De Pra M. – Simultaneous reversed-phase and anion-exchange method scouting with a dual system for mRNA impurity determination, Thermo Fisher Scientific, 2022.