Effect of transcript length, temperature, and lyophilization on mRNA-lipid nanoparticle stability
Manasa Chillara1, Weibo Zhao1, Jonathan S Dordick1
1Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy, NY, 12180, USA; Center for Biotechnology & Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, 12180, USA.
Journal of Pharmaceutical Sciences
|May 3, 2026
Summary
Lyophilization significantly improves mRNA-lipid nanoparticle (LNP) stability and function over time. Freeze-drying protects mRNA-LNP formulations from degradation, enhancing therapeutic potential.
Area of Science:
- Biotechnology
- Pharmaceutical Sciences
- Drug Delivery
Background:
- mRNA-lipid nanoparticles (LNPs) are crucial for delivering genetic material.
- Lyophilization (freeze-drying) is a known method to enhance the long-term stability of pharmaceutical formulations.
- The specific impacts of lyophilization on mRNA-LNP physicochemical and functional properties require further investigation.
Purpose of the Study:
- To investigate the influence of mRNA length, formulation conditions, and lyophilization on mRNA-LNP stability.
- To assess the correlation between physicochemical properties and functional stability of mRNA-LNPs after lyophilization.
- To establish a basis for future modeling of mRNA-LNP stability.
Main Methods:
- Synthesized mRNA constructs of varying lengths (monomeric, dimeric, tetrameric EGFP).
- Optimized LNP composition and lyophilization conditions for mRNA-LNPs.
- Evaluated physicochemical properties and functional (EGFP expression) stability of both aqueous and lyophilized mRNA-LNPs under different storage conditions.
- Calculated inactivation rate constants using a first-order rate model.
Main Results:
- Physicochemical properties of mRNA-LNPs showed initial changes post-lyophilization but remained stable during storage.
- Functional stability was not reliably predicted by physicochemical properties.
- Aqueous mRNA-LNPs showed a rapid 50-fold decrease in EGFP expression within 28 days at room temperature.
- Lyophilized mRNA-LNPs exhibited significantly improved functional stability, with only a 6-fold loss in EGFP expression at 28 days and a 9-fold loss at 84 days.
- Lyophilization may induce LNP subpopulations, leading to functional heterogeneity.
Conclusions:
- Lyophilization substantially enhances the functional long-term stability of mRNA-LNPs compared to aqueous formulations.
- Physicochemical characterization alone is insufficient to predict the functional performance of lyophilized mRNA-LNPs.
- The study provides key data and a modeling framework for understanding and predicting mRNA-LNP stability, crucial for therapeutic development.
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