Comparison of capillary electrophoresis-based methods for the analytical characterization of purity and stability of in vitro transcribed mRNA

Jul 19, 2024Journal of pharmaceutical and biomedical analysis

Comparing capillary electrophoresis methods for measuring purity and stability of lab-made mRNA

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Abstract

The Sciex RNA 9000 Purity and Integrity kit achieved the highest selectivity and resolving power among the four mRNA analytical methods evaluated.

Capillary electrophoresis (CE) methods were compared for assessing the purity and stability of mRNA therapeutics.
All four methods evaluated are suitable for characterizing in vitro transcribed (IVT) mRNA, depending on the specific application.
The Agilent RNA 6000 Nano Kit, Revvity RNA Reagent Kit, and Agilent HS RNA Kit offered faster analysis times but lower selectivity and resolution.
The Sciex RNA 9000 method is recommended for high-resolution, in-depth sample characterization due to its superior selectivity and resolving power.

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Messenger RNA (mRNA) is rapidly growing as a therapeutic modality for vaccination and the treatment of a wide range of diseases. As a result, there is an increased demand for mRNA-based analytical methods capable of assessing purity and stability, which are considered critical quality attributes (CQAs). In recent decades capillary electrophoresis (CE) has emerged alongside liquid chromatography (LC) as an important tool for the assessment of purity and stability of mRNA therapeutics. CE offers a variety of advantages over conventional LC or gel-based analytical methods, including reduced injection volume, increased resolution, and increased separation efficiency. In this study we compared CE-based analytical methods: the Agilent RNA 6000 Nano Kit, the Revvity RNA Reagent Kit, the Sciex RNA 9000 Purity and Integrity Kit, and the Agilent HS RNA Kit. These methods were evaluated on their vendor-recommended instruments: the Bioanalyzer, LabChip GXII, PA800 Plus, and Fragment Analyzer, respectively. We assessed the ability of these methods to measure mRNA integrity, purity, and stability. Furthermore, several parameters for each method were also assessed: selectivity, precision, resolution, analysis time, and ease of use. Based on our results, all four methods are suitable for use in the characterization of in vitro transcribed (IVT) mRNA, depending on the intended application. The Sciex RNA 9000 Purity and Integrity kit method achieved the highest selectivity and resolving power compared with the other methods, making it the most suitable for high-resolution, in-depth sample characterization. In comparison, the Agilent RNA 6000 Nano Kit, Revvity RNA Reagent Kit, and Agilent HS RNA Kit achieved lower selectivity and resolution, but their faster analysis times make them more suitable for high-throughput and screening applications.

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Comparison of capillary electrophoresis-based methods for the analytical characterization of purity and stability of in vitro transcribed mRNA

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