Freezing-Induced Stress in mRNA-Lipid Nanoparticles During Lyophilization: Mechanistic Insights From Process and Formulation Studies

INTRODUCTION: Lyophilization is a promising strategy to enhance the long-term stability of messenger RNA lipid nanoparticles (mRNA-LNPs). However, lyophilization-induced stresses can impact product quality and the underlying mechanisms remain poorly understood. In this study, we systematically investigated stresses that arise during the freezing step, during the initial stage of the lyophilization process. METHODS: We examined the impact of different freezing protocols (freezing at 0.1, 0.5, and 1.5 K/min, plus controlled nucleation at -10°C) on mRNA-LNP stability. We also explored formulation strategies to mitigate freezing stress: (A) increasing mRNA-LNP concentration or adding empty LNPs to induce colloidal crowding, (B) adding Poloxamer 188 to reduce interfacial stress, (C) incorporating sucrose within LNPs to protect mRNA and reduce osmotic stress, and (D) adding NaCl or L-Methionine to modulate mRNA-lipid interactions. We evaluated particle size, polydispersity index, encapsulation efficiency (EE), mRNA integrity, and eGFP expression in HeLa cells. RESULTS: Faster freezing minimized LNPS particle size increase by trend but reduced EE. Controlled nucleation improved EE but increased LNP particle size. However, eGFP expression was more influenced by particle size than EE. CONCLUSION: These findings provide a mechanistic understanding of how freezing-induced stresses affect mRNA-LNP quality. We hypothesize that cryo-concentration caused by slow freezing leads to increasing size of LNP particles, while higher ice-liquid interfacial stress caused by fast freezing reduces EE. As these effects follow opposing trends, optimizing freezing conditions is crucial. Understanding these mechanisms will guide rational formulation and lyophilization process design for mRNA-LNPs.