Spray Freeze Drying for Solid Formulations of RNA Lipid Nanoparticles

Abstract

Spray-freeze drying (SFD) is a promising drying technique for stabilizing RNA lipid nanoparticles (RNA-LNPs) by converting them into solid-state formulations. This study examined the impact of the SFD process and the widely utilized stabilizing disaccharides, such as sucrose and trehalose, on the major properties (e.g. particle size, encapsulation efficiency) of RNA-LNPs post drying. Various process parameters, such as atomization, freezing, and drying temperature, were systematically evaluated, along with the impact of incorporating annealing post freezing step. Surface area measurements, solid-state particle size analysis, scanning electron microscopy, powder X-ray diffraction and solid-state NMR spectroscopy were employed to gain deeper insights into powder characteristics and matrix mobility. Our findings demonstrated that sucrose provided superior stabilization as compared to trehalose in our tested formulations and processes. Moreover, incorporation of the annealing process enhanced LNP stability. Results from the stability study revealed that the annealed 10% and 20% sucrose formulations maintained their stability, highlighting the potential of optimized SFD processing for producing solid formulations of RNA-LNPs.

Introduction

The global response to COVID-19 highlighted the critical role of mRNA vaccines in controlling the pandemic, with the rapid approval and success of Moderna’s SpikeVax® and Pfizer/BioNTech’s Comirnaty® by the regulatory agencies worldwide [1]. These vaccines demonstrated the potential of mRNA technology, in applications beyond COVID-19, as seen with the FDA’s approval of Moderna’s mRESVIA™, the first mRNA vaccine for respiratory syncytial virus [2]. However, the long-term stability of mRNA vaccines remains a challenge due to their sensitivity to factors such as temperature, pH, enzymes, oxygen, moisture, and lipid interactions [2]. Thus, current mRNA vaccines require sub-zero storage: Comirnaty® at − 90 °C to − 60 °C for 12 months, SpikeVax® at − 50 °C to − 15 °C for 9 months, and mRESVIA™ at − 40 °C to − 15 °C for 18 months, reflecting their susceptibility to degradation over temperature and time [3, 4].

Cold chain conditions are critical for preventing the hydrolysis of the phosphodiester bond in the RNA cargo, which plays a major role in its degradation. Furthermore, hydrolysis may also adversely affect the lipid system [2, 3]. To mitigate these challenges, drying technologies, including lyophilization, spray drying, and spray-freeze drying, have been explored to remove water and stabilize mRNA-LNP systems [2, 5,6,7,8,9,10,11,12].

Among these, lyophilization has been extensively investigated due to its ability to preserve the structural integrity of biological macromolecules [13]. Liangxia et al. demonstrated that lyophilized mRNA-LNPs designed for SARS-CoV-2 treatment exhibited long-term stability at 4 °C and 25 °C [14]. Similarly, Zhao et al. formulated mRNA-LNPs containing firefly luciferase mRNA and lyophilized them with 5% sucrose and trehalose, demonstrating their in vivo efficacy [15]. Further studies by Anindita et al. [16], Li et al. [17], and Shirane et al. [18] have also investigated the impact of lyophilization and excipients on the stability of mRNA-LNP formulations, underscoring the importance of drying technologies in this domain. However, lyophilization is often time-intensive and associated with high production costs [2]. In addition, the lyophilized product typically forms a poorly dispersible solid, limiting its suitability for alternative delivery routes such as pulmonary administration [19]. In contrast, spray drying is a continuous process but can present challenges for thermal-sensitive components due to the elevated temperatures involved [20]. Consequently, ongoing research is focusing on an alternative drying technique, spray-freeze drying, to address these limitations [9, 10, 21,22,23].

Spray-freeze drying (SFD) is a promising method for producing powders with improved flow properties compared to lyophilization, without the use of excessive heat [2, 22, 24, 25]. In this process, a liquid formulation is first atomized into fine droplets, which are then rapidly frozen with a cryogenic medium, such as liquid nitrogen [25]. SFD is particularly suitable for heat-sensitive formulations [20], such as RNA-LNPs. Stabilizing excipients such as sucrose or trehalose are commonly employed during this process to prevent freezing and drying related stresses and to further enhance the stability and performance of the final product [2].

From a process related standpoint, the freezing stage of spray freeze-drying (SFD) can include an annealing step, similar to that in conventional lyophilization. Annealing is a controlled thermal treatment in which the frozen material is maintained for a set period at a temperature above its initial freezing point but below its melting temperature. This treatment can encourage ice crystal growth, solute phase redistribution, and relaxation of the frozen matrix, which may in turn influence pore structure, mass transfer, and overall drying behavior. By contrast, in the absence of annealing, the frozen material proceeds directly without this intermediate hold, typically retaining the finer microstructure formed during initial freezing. Although the impact of annealing has been widely examined in traditional freeze-drying, its specific role and consequences in spray freeze-drying have received comparatively limited attention.

Although significant research has explored spray-freeze drying (SFD) for stabilizing and aerosolizing small molecules and protein therapeutics, its application to RNA-LNP systems remains limited [2]. Liang et al. demonstrated that the SFD using a dual-fluid nozzle preserved the integrity and biological activity of siRNA formulations, and Okuda et al. developed a lung-delivery siRNA powder using polyethyleneimine-based vectors [26,27,28]. Similarly, Miwata et al. reported promising gene-silencing and anti-tumor effects from a spray-freeze-dried intratracheal siRNA formulation containing chitosan, mannitol, and L-leucine [29]. Recently, Ogawa et al. produced an aerosolizable mRNA-LNPs using SFD without deliquescent excipients, achieving enhanced stability through improved interactions between mRNA and ionizable lipids [10]. Furthermore, a patent application highlighted the SFD’s ability to significantly reduce drying time compared to the traditional vial freeze drying (VFD), achieving comparable particle size, encapsulation efficiency, and mRNA integrity, underscoring its potential as an advanced drying method for the mRNA-LNP system [7].

While these studies highlight the potential of spray-freeze drying (SFD) as a valuable method for preserving sensitive macromolecules, there is a lack in mechanistic understanding of how processing parameters and excipients influence the properties of spray freeze dried RNA-LNPs. In this work, we investigated the effects of the SFD process and varying concentrations of sugar excipients, such as sucrose and trehalose, on particle size and encapsulation efficiency of RNA-LNPs, both pre- and post-drying. We used ribonucleic acid (RNA) from baker’s yeast (Saccharomyces cerevisiae) as a model RNA because it is widely accessible from major chemical suppliers with standardized quality controls. The outcomes of this study may facilitate the development of an optimized SFD process to produce stable RNA-LNPs formulations.

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Materials

(4-hydroxybutyl) azanediyl] bis (hexane-6,1-diyl)bis(2-hexyldecanoate), 2-[(polyethylene glycol)−2000]-N, N-ditetradecylacetamide, also known as ALC-0315 (ionizable lipid) and 1,2-Distearoyl-snglycero-3-phosphocholine (DSPC) and Methoxypoly(ethylene glycol) ditetradecylacetamide-2 K (ALC-0159 or PEG-DTA-2 K) were obtained from SINOPEG (Xiamen, Fujian, China). Cholesterol, sucrose, citric acid, sodium citrate and anhydrous ethanol, Triton X 100 and ribonucleic acid (RNA) from baker’s yeast (Saccharomyces cerevisiae) were purchased from Sigma Aldrich (St. Louis, MO). RiboGreen kit was purchased from Thermo Fischer Scientific (Eugene, Oregon, USA). Trehalose was purchased from Acros organics (Geel, Belgium). UltraPure™ Distilled Water DNAse-, RNAse-free (Grand Island, NY) was used for all experiments.

Arte, K.S., Sapkota, R., Patil, C.D. et al. Spray Freeze Drying for Solid Formulations of RNA Lipid Nanoparticles. AAPS PharmSciTech 27, 284 (2026). https://doi.org/10.1208/s12249-026-03516-1

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