# Investigation on a Freeze-Drying Process for Long-Term Stability of mRNA-LNPs

## TL;DR

This study explores freeze-drying methods to improve the long-term stability of mRNA-LNPs vaccines at room and refrigerated temperatures.

## Contribution

The study identifies optimal excipient and buffer combinations for freeze-drying mRNA-LNPs to maintain stability and functionality.

## Key findings

- 20% sucrose and trehalose with Tris or histidine buffers preserved mRNA-LNPs properties during freeze-drying.
- Formulation F1 (20% sucrose with 5 mM Tris) showed the best stability and transfection efficiency at both 4°C and 20°C.
- Storage at 20°C caused progressive destabilization, with over 60% encapsulation loss after six months.

## Abstract

Background: Thermostability remains a key bottleneck for equitable access to mRNA-LNPs vaccines, mainly due to cold-chain requirements. Objectives and methods: Here, we optimized freeze-drying formulations by screening excipients (sugars, sugar alcohols, and proteins) and buffers to preserve mRNA-LNPs as solid formulations under ambient and refrigerated conditions. Physicochemical properties (size, polydispersity index [PDI], and encapsulation efficiency [EE]) and functional integrity, assessed by fluorescence-based in vitro transfection assays, were evaluated during long-term storage of up to six months. Results: Preliminary screening identified 20% sucrose and trehalose with Tris or histidine buffers as optimal for preserving physicochemical properties during freeze-drying, including high encapsulation efficiency (>90%), particle size (~200 nm), and low polydispersity (PDI < 0.2). Mannitol, gelatin, and PBS-based buffers showed adverse effects. At 4 °C, formulations F1–F3 maintained physicochemical stability and functional transfection activity for up to four months. In contrast, 20 °C storage caused progressive destabilization, with increased size, PDI, and encapsulation loss (>60% by six months). Among all formulations, 20% sucrose with 5 mM Tris (F1) showed the most robust preservation of physicochemical integrity and in vitro transfection efficiency under refrigerated and ambient conditions. Conclusions: Sugars outperformed sugar alcohols and gelatin as cryoprotectants. All formulations were stable, including functionally active at 4 °C for up to four months, while a sucrose/Tris formulation retained acceptable stability at 20 °C. Overall, the results demonstrate the feasibility of storing mRNA drug products as solid formulations at non-freezing temperatures.