Osmolyte-Based Formulations for Enhanced Thermal Stability of mRNA Drug Substance: A Systematic Screening and Optimization Study.

PURPOSE: Stability of messenger RNA (mRNA) is a critical factor in the development and storage of mRNA-based vaccines. In this study, we systematically examine the effects of addition of various osmolytes (and their combinations) on mRNA integrity during long-term storage and under thermal stress.

METHODS

Eleven osmolytes, belonging to subclasses of amino acids and derivatives (ectoine, methionine, L-cysteine, valine), polyols and sugars (sorbitol, trehalose and mannose), nucleosides (uridine), and vitamins and cofactors (nicotinamide and citric acid) have been examined by themselves as well in binary and ternary combinations. Their protective capacity has been evaluated. A concentration curve was developed where we assessed 0-30% weight/volume of the osmolytes in the mRNA vaccine formulations and analyzed the thermostability for 5-14 days in 40°C and 2.5 months at 4°C.

RESULTS

An optimized formulation of 80:20 taurine to uridine showed robust mRNA protection across 15-30% w/v concentrations (densitometric indices 0.833-0.875, p < 0.001) with exceptional stability at 1% w/v (0.966 ± 0.061) after 5 days at 40°C, while the ternary taurine-uridine-sorbitol system achieved peak performance at 5% w/v (0.917 ± 0.036). This established a concentration-dependent design principle for thermostable mRNA formulations with > tenfold improvement over untreated controls under accelerated thermal stress conditions.

CONCLUSIONS

Our results revealed that the effectiveness of each component depended not only on its identity but also on the composition of the formulation. Ternary mixtures did not always outperform binary systems. Our work demonstrates that osmolytes offer significant potential towards improving the shelf life and accessibility of mRNA vaccines.