Publication date: Jan 26, 2025

Lipid nanoparticles (LNPs) have demonstrated their therapeutic potential as safe and effective drug delivery systems for nucleic acids during the COVID-19 pandemic. However, one of the main challenges during technical CMC (Chemistry, Manufacturing, and Controls) development is their long-term stability at temperatures of 2-8 ^0C or higher, which may be improved by the removal of water by lyophilization. In this study, we identified lyo-/cryo-protectants for freeze-dried mRNA-LNP formulations beyond conventional excipients such as sucrose and trehalose as T-modifiers using polyA as a surrogate. Hydroxypropyl-beta-cyclodextrin, Kollidon(R) 12 PF (PVP), and dextran 40 kDa were tested in combinations to best stabilize the mRNA-LNPs during the lyophilization process as well as during storage for up to 6 months at 2-8 ^0C, 25 ^0C/60 % r. h., and 40 ^0C/75 % r. h.. We also tested the formulation principle including protectants in- and outside of the LNPs. Formulations were assessed for size, PDI, encapsulation efficiency, and properties related to the lyophilized dosage form. While 10 % (w/V) sucrose formulations successfully stabilized LNPs during the lyophilization process, they were not suitable for storage at temperatures beyond 2-8 ^0C. The most promising formulations for storage at higher temperatures were identified as 9 % (w/V) trehalose + 1 % (w/V) PVP with only a small increase in size over 6 months at 25 ^0C maintaining PDI and encapsulation efficiency. Results were verified with eGFP-mRNA-LNPs and tested in cell culture experiments. This study may serve as guidance for formulation scientists to further optimize freeze-dried mRNA-LNP formulations and eventually eliminate the cold chain for mRNA-LNP products.

Semantics

Original Article