Comparative Analysis of mRNA Degradation Kinetics Using Chromatographic and Electrophoretic Methods

The effects of temperature and lipid nanoparticle (LNP) encapsulation on mRNA chemical stability were assessed for two model mRNA (EGFP mRNA, 996 nt; Fluc mRNA, 1929 nt) using capillary electrophoresis (CE) and ion pair reversed phase high performance liquid chromatography (IP-RP-HPLC) to measure mRNA integrity. The apparent degradation rates for unencapsulated ("naked") mRNA were method-dependent for Fluc mRNA, with rates up to 50% greater when measured using CE compared to IP-RP-HPLC. This discrepancy is attributed to differences in mass resolution, separation mechanisms and/or linearity of the two methods and was not observed for EGFP mRNA. The apparent reaction order for naked mRNA degradation varied with storage temperature, being zero-order at 35 °C and first-order at 50 °C. Encapsulation of mRNA using LNPs slowed mRNA degradation by up to 9-fold relative to naked mRNA controls stored under the same conditions. Encapsulation also induced changes in thermal unfolding patterns. Overall, the results show the benefits and limitations of each analytical method for determining mRNA degradation kinetics and the effects of temperature and LNP encapsulation on mRNA stability.