(T1130-11-60) Investigating the Impact of Drying Processes on RNA Lipid Nanoparticles

Purpose: mRNA vaccines have demonstrated exceptional efficacy in mitigating illness and enhancing public health during the COVID-19 pandemic. However, due to stability concerns, both naked mRNA and mRNA lipid nanoparticle (LNP) vaccines need to be stored under freezing conditions, which can limit their accessibility. To overcome this limitation, solid-state LNPs have been investigated as a potential solution, as they are known to be more stable at room temperature compared to their liquid counterparts. The primary objective of this study is to investigate the influence of various drying techniques, including lyophilization, spray drying, and spray freeze drying (SFD), on RNA lipid nanoparticles (LNPs). The aim is to enhance the stability of these LNPs, which may facilitate the development of effective mRNA vaccines that can be easily accessible to people globally. By improving the stability of RNA LNPs, this research may contribute to the advancement of mRNA vaccine technology, and ultimately, help in mitigating the ongoing pandemic by making vaccines more widely available.

Methods: To formulate mRNA or yeast RNA LNPs, EGFP mRNA in citrate buffer was combined with ionizable cationic lipid, cholesterol, PEG, and phospholipids in ethanol. The resulting mixture was dialyzed, and Sucrose was added before the LNPs were subjected to lyophilization, spray drying, or spray freeze drying. The LNPs were then analyzed for particle size and polydispersity index using Dynamic Light Scattering (DLS) and for RNA encapsulation efficiency using the Quant-iT RiboGreen Assay. RNA integrity was measured using the Agilent 5200 Fragment Analyzer with the Agilent HS RNA Kit, and PROSize 3.0 software was used to determine the RNA integrity percentage.

Results: RNA LNPs had particle sizes ranging from 75-85 nm post fabrication, which increased to 90-110 nm after sucrose addition. There were no significant differences in particle size before drying (spray drying, lyophilization, or spray freeze drying), as shown in Figure 2A: i-a, ii-a, iv-a. However, varying the sucrose concentration in the formulation led to changes in particle sizes. RNA LNP formulation with 10% w/v sucrose did not show a substantial increase in particle size after lyophilization or spray freeze drying (Figure 2A: ii-a to v-a). Decreasing the sucrose concentration to 2.5% w/v and 5% w/v increased particle size to 120-160 nm in either case (lyophilization or SFD) as indicated in Figure 2A iii-a and v-a. Increasing the sucrose concentration to 20% also increased the particle size to 130-160 nm in either case (Figure 2A: iii-a and v-a). However, spray drying with 10% w/v sucrose increased the particle size of RNA LNPs to 400-425nm (Figure 2A: i-a). Lyophilization and SFD had similar trends of particle sizes, while spray drying led to an approximately four-fold increase in particle sizes (Figure 2A i-a to v-a). Polydispersity indices for all formulations were acceptable with values below 0.3 (Figure 2A: i-a to v-a). Most formulations exhibited a monomodal particle size distribution (except spray dried RNA LNP with 10% sucrose and SFD LNPs with 2.5% sucrose) as indicated in Figure 2A: i-b to v-b. No substantial difference in mRNA integrity was found post lyophilization, but reduction was observed in cases of spray drying and SFD. The percent encapsulation for RNA in LNPs before lyophilization, spray drying, or spray freeze drying were found to be between 90-93%, with no significant differences in encapsulation efficiencies before drying (Figure 2B: i, ii, iv). Different drying techniques led to differences in encapsulation efficiencies. Varying sucrose concentration also led to differences in percent encapsulation, with 2.5, 5, 10, and 20% w/v sucrose formulations demonstrating encapsulation efficiencies of 58.76%, 83.12%, 85.56%, and 50%, respectively, post lyophilization (Figure 2B: iii). Spray drying with 10% w/v sucrose decreased the encapsulation efficiency to about 42.37% (Figure 2B: i). Furthermore, spray freeze drying led to encapsulation efficiencies of 27.63%, 47.09%, 59.94% and 52.30% for 2.5, 5, 10, and 20% w/v sucrose, respectively (Figure 2B: vi). Smaller particle sizes in lyophilization and spray freeze drying were likely due to extended drying times, allowing for slow water sublimation in both methods (Figure 3B and 3C). Conversely, the rapid drying process and high temperatures in spray drying (as shown in Figure 3A) could cause lipid phase transitions and subsequent particle aggregation. Encapsulation efficiency reduction in SFD (as depicted in Figure 3C) could be due to the formation of relatively small particles following atomization and instantaneous freezing, resulting in a larger surface area exposed to stresses during drying resulting in reduced encapsulation

Conclusion: Optimal RNA LNPs with high encapsulation efficiency and desirable particle size were successfully formulated. LNPs formulated with 10% sucrose showed no significant change in particle size and encapsulation efficiency after lyophilization. However, spray drying had a negative effect on RNA LNPs, increasing their particle size and reducing encapsulation efficiency. Similarly, spray freeze drying had comparable particle sizes to lyophilization, but resulted in decreased encapsulation efficiencies.

References: Schoenmaker, L., et al. (2021). "mRNA-lipid nanoparticle COVID-19 vaccines: Structure and stability." International Journal of Pharmaceutics 601: 120586.

Acknowledgements: This work was performed under a Project Award Agreement from the National Institute for Innovation in Manufacturing Biopharmaceuticals (NIIMBL) and financial assistance award 70NANB21H085 from the U.S. Department of Commerce, National Institute of Standards and Technology.


Graphical Abstract for study design


Impact of drying techniques and varying sucrose ratios on A) Particle sizes and B) Encapsulation efficiencies of lipid nanoparticles encapsulating yeast RNA


Graphical illustration for the mechanism of particle formation using various drying techniques