(T1030-03-17) Lyoprotectants Affecting Product Stability of Lyophilized mRNA-Lipid Nanoparticles

Purpose: The mRNA lipid nanoparticle (LNP) vaccines for COVID-19 have emerged as a groundbreaking advancement to combat the global pandemic. However, these vaccines are required to be stored at an ultra-low temperature to maintain their efficacy and stability. The low-temperature storage requirement arises from the need to prevent LNP and mRNA instability which creates a burden for storage, transportation, distribution, and clinic usage. mRNA hydrolysis is the primary stability challenge for mRNA-LNP solution. Lyophilization of mRNA-LNP formulations presents a potential avenue for enhancing their stability. Investigations into the impact of lyoprotectants on post-lyophilization stability of freeze-dried mRNA-LNPs are limited. Thus, this study aims to comprehensively understand the influence of lyoprotectants on critical quality attributes—including particle size, polydispersity index (PDI), zeta potential, percentage of mRNA encapsulation, and transfection efficiency—of freeze-dried mRNA-LNPs.

Methods: mRNAs encoding firefly luciferase (FLuc mRNA) were loaded into LNPs composed of SM-102, DSPC, cholesterol, and PEG2000-DMG at a molar ratio of 50:10:38.5:1.5. This formulation was prepared through microfluidic mixing at a total flow rate (TFR) of 3 ml/min, with a flow rate ratio (FRR) of 1:3 for the organic to aqueous phase. Subsequently, the formulation underwent dialysis against 20mM Tris-HCl buffer at pH 7.4. Sucrose, trehalose, sucrose with trehalose at 1:1 (w/w), and sucrose with trehalose with PVP at 1:1:1(w/w) were added to mRNA-LNPs with 1:5, 1:10, and 1:20 weight ratio of total lipid to lyoprotectant. Glass transition temperature of the maximumly freeze-concentrate (Tg’) of each lyoprotectant were determined using differential scanning calorimetry (DSC). Lyophilization was conducted using LyoStar^TM 3 lyophilizer under controlled ice nucleation. Mean particle size, polydispersity index, and zeta potential were measured before and after lyophilization. Residual moisture of lyophilized cake was assessed using Karl Fischer titration. The morphology of mRNA-LNPs was visualized using transmission electron microscopy (TEM). The percent of mRNA encapsulation was determined by RiboGreen assay. In-vitro transfection of mRNA-LNPs before and after lyophilization was evaluated in Jurkat cells.

Results: mRNA -LNPs prepared via microfluidic mixing exhibited a mean size of 93nm and a PDI of 0.221. Different lyoprotectants showed variations in Tg’, with formulations containing trehalose and PVP exhibiting higher Tg’ compared to those with sucrose alone. Before lyophilization, the addition of various lyoprotectants had no significant impact on particle size distribution, zeta potential, or the percentage of mRNA encapsulated within mRNA-LNPs. However, post-lyophilization, mRNA-LNPs containing lyoprotectants showed lower residual moisture, with formulations at the lower lyoprotectant ratios exhibiting a higher residual moisture. Additionally, the particle size distribution and encapsulation efficiency of mRNA-LNPs varied depending on the lyoprotectant-to-lipid ratio and the specific lyoprotectant used during the freeze-drying process. Notably, formulations with sucrose 1:10, 1:20, sucrose 1:10, sucrose: trehalose=1:1 1:20, and sucrose: trehalose: PVP=1:1:1 1:20 exhibited smaller particle sizes, lower PDIs, and higher percentages of mRNA encapsulation compared with other formulations. Moreover, freeze-dried mRNA-LNPs with sucrose when using sucrose to LNP ratios higher than 10:1 demonstrated higher transfection efficiency compared to the liquid suspension after storage at 4C for 2 weeks. The variance in transfection efficiency was not attributable to mRNA degradation or variations in lyoprotectants.

Conclusion: Sucrose is effective in stabilizing mRNA-LNPs during freeze-drying when using sucrose to LNP ratios higher than 10:1, compared with Trehalose and the mixture with Sucrose and PVP with higher Tg. Transfection efficiency can differentiate the lyoprotectants for their protection effectiveness, although particle size, PDI, and percentage of mRNA encapsulation remain the same after lyophilization.

Acknowledgements: The authors would like to acknowledge the Center of Pharmaceutical Processing Research (CPPR) for financial support of this research.