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

Significance of the Topic

The purity of messenger RNA is critical for both research and therapeutic applications. Impurities such as truncated transcripts, double-stranded RNA, and residual DNA can impact biological efficacy and safety. High-performance liquid chromatography (HPLC) offers high resolution and quantitation capabilities essential for evaluating mRNA quality. Simultaneous screening of reversed-phase and anion-exchange separations accelerates method development and ensures comprehensive impurity profiling.

Objectives and Study Overview

This study aims to identify optimal chromatographic conditions for detecting post-transcriptional impurities in mRNA using ion pairing reversed-phase (IP-RP) and anion-exchange (IEX) chromatography on a single dual LC system. Automated scouting of multiple mobile phase and temperature parameters was conducted to streamline method selection.

Methodology and Instrumentation

The Thermo Scientific Vanquish Duo with a Solvent Extension Kit performed simultaneous scouting. Purified and non-purified mRNA (~2500 nt) were analyzed on DNAPac RP (2.1×100 mm, 4 µm) and DNAPac PA200 RS (4.6×150 mm, 4 µm) columns. IP-RP scouting included nine buffer conditions (e.g., TEAA, HAA, DIPEA) at 50 °C, while IEX scouting tested eight eluent systems (e.g., Tris/perchlorate, NaOH) at 30–80 °C. Flow rates were 0.4 mL/min (IP-RP) and 1.0 mL/min (IEX). UV detection at 240 nm (IP-RP) and 235 nm (IEX) monitored analytes.

Main Results and Discussion

Multiple conditions yielded sharp mRNA peaks and resolved impurity profiles. IP-RP condition with 25 mM hexylammonium acetate (HAA) at 50 °C provided a clear distinction between main transcript and byproducts. IEX method using 40 mM Tris/0.8 M perchlorate with 20% acetonitrile at 80 °C offered detailed impurity resolution. Both modes delivered similar mRNA purity estimates (≈91% relative area). The dual system reduced scouting time and simplified method development.

Benefits and Practical Applications

• Time-efficient simultaneous screening of two chromatographic modes
• Minimal sample and solvent consumption through automated scouting
• Flexible adaptation of selected methods for different mRNA samples
• Enhanced impurity profiling supports quality control in biopharmaceutical production

Future Trends and Potential Applications

Advancements in stationary phase chemistry and buffer design may further improve resolution of complex mRNA impurity profiles. Integration with mass spectrometry could enable structural identification of impurities. Automated platforms with expanded temperature and solvent ranges will accelerate therapeutic mRNA development and regulatory compliance.

Conclusion

The dual LC method scouting approach effectively identifies robust IP-RP and IEX conditions for mRNA impurity analysis. Selected methods demonstrated high resolution, reproducibility, and comparable purity estimations. This streamlined workflow supports rapid method development in research and biopharmaceutical quality control.

Reference

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