Chromatographic Purification With CIMmultus Oligo dT Increases mRNA Stability

Matevž Korenč, Nina Mencin, Jasmina Puc, Janja Skok, Kristina Šprinzar Nemec, Anže Martinčič Celjar, Pete Gagnon, Aleš Štrancar, Rok Sekirnik

Cell & Gene Therapy Insights 2021; 7(9), 1207–1216

Abstract:
One of the major challenges of mRNA based vaccines has been their requirement for distribution and storage at extremely low temperatures, indicating that exposure of mRNA to suboptimal physico-chemical conditions can result in degradation and loss of potency; it is unclear whether this is due to instability of mRNA drug substance, or LNP-encapsulated mRNA, or both. In this study we compare the stability of model mRNA drug substance (eGFP, 995 nt) prepared by affinity chromatography with the stability of mRNA purified by precipitation. We show that both purification methods lead to highly pure mRNA drug substance, however, mRNA purified by chromatography remains stable for 28 days at 37°C, whereas mRNA purified by precipitation is subject to significant degradation under the same storage conditions. We conclude that chromatography eliminates elements and/or conditions with adverse impact on the quality of mRNA to a greater extent than precipitation method and that choosing appropriate purification strategy is crucial not only to achieve target purity but also to obtain a stable product with retained integrity.

Thermal and freeze-thaw stability of a model RNA construct purified by various methods is evaluated by a range of analytical methods. Results indicate a stark contrast in stability, and discuss applicability of analytical methods for stability assessment.
  • How does chromatography affect RNA stability?
  • Can HPLC be used for RNA stability assessment?
  • Is precipitation a good approach for small-scale RNA purification?
  • How do HPLC, bioanalyser, AGE, and other methods compare?

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