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

Importance of the Topic

Messenger RNA based applications in vaccines and therapeutics require rigorous impurity profiling to ensure product safety and efficacy. High resolution chromatographic methods provide quantitative and qualitative analysis of residual nucleotides, dsRNA and other impurities arising during synthesis and purification.

Objectives and Overview of Study

This study aimed to develop and compare simultaneous scouting approaches for ion-pairing reversed-phase and anion-exchange liquid chromatography using a dual-LC platform. By evaluating multiple buffers, pH values and temperatures on a single instrument, the workflow seeks to accelerate method development and maximize impurity detection.

Methodology and Used Instrumentation

• Chromatographic strategies: Ion-pairing reversed-phase (IP-RP) and anion-exchange (IEX) separation modes were tested in parallel.
• Dual-LC system: Thermo Scientific Vanquish Duo UHPLC with two independent pumps, dual column compartments, dual detectors and automated solvent selection kits.
• Columns: DNAPac RP (2.1 × 100 mm, 4 μm) for IP-RP, DNAPac PA200RS (4.6 × 150 mm, 4 μm) for IEX.
• Mobile phases: Nine IP agents (TEAA, HAA, DIPEA) at varying pH and eight IEX eluents (Tris with NaCl or NaClO4, with or without acetonitrile).
• Sample prep: In vitro transcribed mRNA (2500 nts) before and after magnetic bead purification. Quantitation via Qubit RNA assay.
• Data acquisition: Thermo Scientific Chromeleon 7.3 CDS with custom variables for automated scouting.

Main Results and Discussion

• IP-RP mode at 50 °C identified 25 mM hexylamine acetate at pH 8.5 as optimal for sharp mRNA peaks and impurity separation. TEAA and DIPEA showed poor retention or high background at high pH.
• IEX mode at 80 °C using 40 mM Tris buffer with 0.8 M perchlorate and 20% acetonitrile achieved complete mRNA elution and good peak shape; NaCl-based gradients were insufficient.
• Purified and non-purified mRNA profiles compared, demonstrating the methods can assess purification efficiency by monitoring impurity peaks at low retention times.
• Estimated mRNA purity was approximately 91% in both IP-RP and IEX methods for the purified sample.

Benefits and Practical Applications

• Simultaneous method scouting of two separation chemistries reduces development time and instrumentation needs.
• High resolution impurity profiling supports quality control of mRNA therapeutics.
• Flexibility to evaluate denaturing conditions for secondary structure linearization.
• Use of corrosive phases enabled by biocompatible flowpaths expands eluent choices.

Future Trends and Potential Applications

• Integration with mass spectrometry for direct impurity identification.
• Automated AI-driven scouting workflows and real-time method optimization.
• Expansion to other nucleic acid modalities delivered in lipid nanoparticles.
• Novel stationary phases and mixed-mode columns for enhanced selectivity.

Conclusion

The dual-LC approach successfully enabled rapid, parallel scouting of IP-RP and IEX methods for mRNA impurity analysis. The selected conditions provide robust, high-resolution separation and support efficient method development for mRNA quality control.

References

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