(M0930-11-60) Improving the Stability of RNA Lipid Nanoparticles Using the Innovative Spray-Freeze Drying Technique

Purpose: The global response to the COVID-19 pandemic underscored the pivotal role of mRNA vaccines, such as Moderna's SpikeVax® and Pfizer/BioNTech's Comirnaty®, in mitigating viral transmission and restoring public health. Nevertheless, the requirement of sub-zero storage conditions to preserve their stability presents considerable logistical challenges. Our research is based on the hypothesis that spray-freeze drying (SFD) can improve the stability of mRNA lipid nanoparticle (LNP) systems by reducing water content, which is a primary initiator of degradation reactions. While lyophilization, a commonly explored method for drying mRNA LNP systems, results in products with poor flow properties unsuitable for alternative delivery methods such as pulmonary routes, SFD offers powders with superior flow characteristics, facilitating such novel delivery approaches. This study aims to optimize SFD parameters and aims at mechanistically understand the utilization of sugar excipients like sucrose or trehalose to enhance mRNA LNP encapsulation efficiency and structural integrity. By modulating particle size through controlled ice crystal formation during annealing and adjusting the concentration of protective sugars, we seek to minimize mRNA LNP exposure to degradation-inducing stresses. This advancement could lead to more stable, easily distributable, and administrable formulations, thereby significantly enhancing global vaccination efforts.

Methods: RNA loaded lipid nanoparticles were prepared using an Ignite NanoAssemblr with microfluidic cartridges. Lipids (ionizable lipid, DSPC, Cholesterol and PEG2000) were dissolved in ethanol at a molar ratio of 50:10:38.5:1.5. mRNA was dissolved in 50mM citrate buffer (pH 4) at 0.1 mg/mL. The lipid solution and the RNA solution were mixed at a flow rate of 12 mL/min and a flow rate ratio of 3:1 (aqueous: organic). Atomization step of spray-freeze drying was conducted using an ultrasonic nozzle at a 3-5 watt power input, over liquid nitrogen. Spray-frozen particles were dried using a benchtop lyophilizer with or without annealing. Critical quality attributes (CQAs) of mRNA LNPs such as particle size, zeta potential, polydispersity index (PDI), mRNA encapsulation, RNA integrity were assessed before and after spray freeze drying. Scanning electron microscopy was utilized to understand the surface morphology of spray-freeze dried particles.

Results: Immediately after formulation, no significant changes were observed in the particle size or polydispersity index (PDI) of the lipid nanoparticles (LNPs), with all RNA LNP systems measuring between 80-120 nm. Post spray-freeze drying, however, particle sizes increased across all formulations, though the extent of this increase varied with the sugar content. Formulations containing 2.5% sucrose or trehalose experienced a rise in particle size to 200-250 nm, while those with higher sugar contents of 5%, 10%, and 20% displayed more moderate increases, maintaining sizes within the range of 120-180 nm. The encapsulation efficiency of LNPs showed no initial significant difference post-formulation, but the atomization process during spray freezing led to a reduction by 5-15%, with further decreases during the drying process, particularly evident in formulations with lower sugar concentrations. For instance, those with 2.5% sugar experienced the most substantial declines in encapsulation efficiency, while higher sugar concentrations (10% and 20%) resulted in relatively smaller reductions, with efficiencies falling to 52% and 60% from initial values of 95% and 97%, respectively (Fig 1a and 1b). The introduction of an annealing step post-atomization led to a significant increase in encapsulation efficiencies by 50-100% across sucrose and trehalose formulations, as illustrated in Fig. 2a and 2b. This improvement in percent encapsulation due to annealing may stem from the enlargement of ice crystal size during the process, resulting in reduced surface area of LNPs exposed to the ice-air interface and thereby mitigating stress experienced by the LNPs. Notably, sucrose, acting as a stabilizer, demonstrated a more effective enhancement of product quality attributes compared to trehalose. Scanning Electron Microscopy (SEM) images revealed the presence of agglomerated particles at lower sugar concentrations. The morphology of particles produced via spray-freeze drying (SFD) suggests the potential for administration via a dry powder inhaler, highlighting their suitability for pulmonary delivery.

Conclusion: Spray-freeze drying (SFD) is a promising technique for drying RNA lipid nanoparticle (LNP) systems. The critical quality attributes (CQAs) of the spray-freeze dried formulations largely depend on the sugar content. Our study suggested that modifying the SFD process can enhance the CQAs of the RNA LNP systems. This study demonstrated that introducing annealing improves the encapsulation efficiency of RNA LNPs. The spray-freeze drying process produces particles with characteristics suitable for inhalation, suggesting the potential of this technology for formulating products that could be administered via alternative delivery routes. The current study serves as a proof-of-concept, demonstrating the viability of using SFD to formulate solid-state mRNA LNP systems.

References: Schoenmaker, Linde, et al. "mRNA-lipid nanoparticle COVID-19 vaccines: Structure and stability." International journal of pharmaceutics 601 (2021): 120586.
Kon, Edo, Uri Elia, and Dan Peer. "Principles for designing an optimal mRNA lipid nanoparticle vaccine." Current opinion in Biotechnology 73 (2022): 329-336.

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.


Figure 1. Impact of atomization and post-atomization drying on the encapsulation efficiency of yeast RNA LNPs containing a) sucrose and b) trehalose dried without annealing.


Figure 2. Impact of atomization and post-atomization drying on the encapsulation efficiency of yeast RNA LNPs containing a) sucrose and b) trehalose dried with annealing