Rapid and Sensitive Detection of dsRNA Contaminants in mRNA Using the GTxResolve™ 250Å Slalom Chromatography Column

Significance of the Topic

Messenger RNA–based therapeutics have transformed vaccine development, cancer immunotherapy, and protein replacement strategies. A critical challenge in the in vitro transcription (IVT) synthesis of mRNA is the unintended formation of double-stranded RNA (dsRNA) contaminants. Even trace levels of dsRNA can activate innate immune receptors and compromise safety and efficacy of mRNA drugs. Rapid, highly sensitive detection of dsRNA is therefore essential for quality control and regulatory compliance in mRNA manufacturing.

Objectives and Study Overview

This study introduces a two-step analytical workflow combining selective enzymatic digestion and slalom chromatography to detect and quantify dsRNA impurities in mRNA samples. The goals are to (1) eliminate interference from single-stranded RNA (ssRNA) using RapiZyme™ Cusativin, (2) resolve intact dsRNA from ssRNA degradation products via the GTxResolve™ 250 Å Slalom Chromatography Column, and (3) demonstrate superior sensitivity and speed compared to traditional electrophoretic methods.

Methodology

• Preparation of dsRNA model: Sense and antisense RNA strands (3 kb cas9 gene segment) were generated by PCR-driven IVT, purified, and annealed to form dsRNA.
• Enzymatic digestion: Samples containing ssRNA or dsRNA were incubated with RapiZyme Cusativin (200 U, pH 9.0 buffer) at 30 °C for 1 h; enzyme inactivation at 75 °C for 15 min.
• Agarose gel electrophoresis: Comparative analysis using 1X TAE buffer, SYBR Gold staining, 50 V for 2 h 10 min.
• Slalom chromatography: Isocratic separation on a Waters ACQUITY™ Premier UPLC system with GTxResolve 250 Å column; mobile phase 1X TAE (pH 8.3); flow rate 1.3 mL/min; column temperature 40 °C; UV detection at 260 nm.

Used Instrumentation

• Waters ACQUITY Premier UPLC system (Quaternary Solvent Manager, TUV detector, FTN sample manager).
• GTxResolve 250 Å Slalom Chromatography Column, 2.5 µm, 4.6 × 300 mm with MaxPeak Premier packing.
• Bio-Rad PowerPac™ HV and Gel Doc™ EZ for agarose gel electrophoresis.
• Monarch® RNA Cleanup Kit, TURBO DNase™ I, and RapiZyme™ Cusativin for sample preparation.

Main Results and Discussion

• Slalom chromatography distinguished dsRNA from sense and antisense ssRNA with distinct peak profiles and minimal retention time shifts.
• RapiZyme Cusativin efficiently degraded ssRNA, while dsRNA peaks remained intact, confirming enzymatic selectivity.
• Detection of as little as 10 ng dsRNA was achieved (<6 min run time), representing >40-fold sensitivity improvement over agarose gels (≥400 ng).
• Column-to-column and batch-to-batch reproducibility was excellent, with low %RSD in retention times and peak areas.

Benefits and Practical Applications

• Rapid analysis (<6 min) accelerates process development and release testing of mRNA therapeutics.
• High sensitivity supports trace-level dsRNA quantification critical for safety assessments.
• Quantitative, scalable workflow integrates easily into quality control laboratories.
• Reduces sample consumption and assay time compared to gel electrophoresis and ELISA-based methods.

Future Trends and Opportunities

Further work may explore the impact of mRNA secondary and tertiary structures on slalom separation, integration with automated high-throughput platforms, and expansion to other nucleic acid contaminants. Optimization of flow rate, solvent viscosity, and column temperature could enhance resolution for longer RNA constructs and complex formulations.

Conclusion

The combined use of RapiZyme Cusativin and GTxResolve 250 Å Slalom Chromatography Column delivers a robust, sensitive, and reproducible platform for detecting dsRNA impurities in mRNA preparations. This approach ensures rapid quality control, supports regulatory requirements, and enhances the safety profile of mRNA-based therapeutics.

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