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Freezing-Induced Stress in mRNA-Lipid Nanoparticles During Lyophilization: Mechanistic Insights From Process and
Anna Ruppl1, Andrei Hutanu2, Monika Köll-Weber3
1Department of Pharmaceutics, Institute of Pharmaceutical Sciences, University of Freiburg, Sonnenstr. 5, 79104, Freiburg I. Br., Germany. anna.ruppl@freenet.de.
Pharmaceutical Research
|February 19, 2026
Summary
Optimizing the freezing step in lyophilization is key for messenger RNA lipid nanoparticle (mRNA-LNP) stability. Understanding freezing-induced stresses helps improve mRNA-LNP formulation and process design for better product quality.
Area of Science:
- Biotechnology
- Pharmaceutical Sciences
- Materials Science
Background:
- Lyophilization (freeze-drying) is a critical technique for enhancing the long-term stability of messenger RNA lipid nanoparticles (mRNA-LNPs).
- However, stresses induced during lyophilization, particularly during freezing, can negatively impact mRNA-LNP product quality.
- The precise mechanisms underlying these freezing-induced stresses and their effects on mRNA-LNPs are not fully understood.
Purpose of the Study:
- To systematically investigate the stresses encountered during the freezing stage of lyophilization for mRNA-LNPs.
- To elucidate the impact of different freezing protocols and formulation strategies on mRNA-LNP stability.
- To provide a mechanistic understanding to guide rational design of mRNA-LNP formulations and lyophilization processes.
Main Methods:
- Examined the effects of various freezing rates (0.1, 0.5, 1.5 K/min) and controlled nucleation (-10°C) on mRNA-LNP stability.
- Investigated formulation strategies including colloidal crowding, addition of Poloxamer 188, sucrose incorporation, and salt/amino acid additives.
- Assessed particle size, polydispersity index, encapsulation efficiency (EE), mRNA integrity, and in vitro mRNA expression (eGFP).
Main Results:
- Faster freezing rates tended to minimize particle size increase but reduced encapsulation efficiency (EE).
- Controlled nucleation improved EE but led to increased particle size.
- In vitro mRNA (eGFP) expression was found to be more sensitive to particle size than to EE.
Conclusions:
- Hypothesize that slow freezing causes cryo-concentration, increasing particle size, while fast freezing induces higher interfacial stress, reducing EE.
- The opposing trends of particle size and EE with varying freezing rates highlight the need for optimized freezing conditions.
- Understanding these freezing-induced stress mechanisms is crucial for developing robust mRNA-LNP formulations and lyophilization processes.
