(E-191) Thermal Stability of mRNA-Lipid Nanoparticles

Introduction::
A critical prerequisite to bypassing distribution constraints within the manufacturing to patient administration pipeline of mRNA-LNPs is extending stability duration. A better understanding of the intrinsic and extrinsic mechanisms of mRNA-LNPs (thermo)stability is conducive to prolonging storage in higher temperatures. Research has mainly focused on large-scale screening and correlating structure-activity relationships (SAR) to facilitate the design of potent lipids without considering storage conditions as an important parameter in drug product development. Here we investigate the effect of different structural features in ionizable lipids on the integrity of the mRNA payload and biophysical properties of nanoparticles stored under different conditions for improved lipid design.




Materials and Methods::
Six novel ionizable lipids were synthesized to incorporate structurally diverse headgroup moieties. MC3, SM102, and ALC-0315 were used as controls. Luciferase (Luc) mRNA-encapsulated LNPs were formulated at an N:P ratio of 4 and a total lipid concentration of 15 mM comprised of the ionizable lipid/DSPC/cholesterol/PEG2000 (50/10/38.5/1.5 mol %) solubilized in ethanol. The aqueous phase containing Luc mRNA was prepared in different buffer systems (pH 4) then mixed with the ethanolic solution at a 3:1 ratio using the Ignite NanoAssemblr^TM. LNPs were diluted in sucrose buffer and ultrafiltrated using Amicon Ultra-15 (MWCO 10 kDa) for removal of ethanol and neutralization of pH. LNPs were then subjected to different storage temperatures (4 ˚C, 25 ˚C, 37 ˚C) for 4 months. The pH, size, encapsulation efficiency, ζ-potential (pH 7.5), mRNA integrity, lipid integrity, lipid oxidation, and bioactivity were characterized at 0, 1, 3, 4, 8, and 16 weeks for each storage condition.




Results, Conclusions, and Discussions::
Results showed unchanging physicochemical characteristics for each of the LNPs throughout the study indicating colloidal stability as a function of time and temperature. Lipid- and temperature-dependent effects strongly correlated with loss of potency and mRNA integrity. mRNA integrity, and subsequent protein expression, significantly decreased irrespective of the ionizable lipid when LNPs were stored at 37 ˚C. In contrast, LNP potency was maintained at relatively high levels up to 4 weeks when stored at 4 and 25 ˚C. However, an observed decrease in potency occurred on a much faster timescale for some lipids, which negatively correlated with specific features and parameters such as head group and buffer composition. Lipid integrity measured by liquid chromatography/mass spectrometry showed minimal time- and temperature-induced degradation ultimately motivating our hypothesis that the loss of potency may be attributed to interactions between the mRNA and lipid headgroup that negatively affects mRNA integrity. In conclusion, this study confirmed a structure-dependent influence of ionizable lipids on the protein expression ability of LNPs that is mainly driven by the intrinsic instability of the mRNA and chemical interactions with the headgroups. Understanding the mechanisms of instability are crucial in employing mitigating strategies to develop thermoresistant mRNA-LNPs. Future studies will adopt a design of experiments-based optimization approach to explore extrinsic parameters affecting LNP thermal stability such as cryoprotectants, buffers, and concentrations.






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