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Current RNA vaccines against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) are limited by instability of both the RNA and the lipid nanoparticle delivery system, requiring storage at -20°C or -70°C and compromising universally accessible vaccine distribution. This study demonstrates the thermostability and adaptability of a nanostructured lipid carrier (NLC) delivery system for RNA vaccines that has the potential to address these concerns. Liquid NLC alone is stable at refrigerated temperatures for ≥1 year, enabling stockpiling and rapid deployment by point-of-care mixing with any vaccine RNA. Alternatively, NLC complexed with RNA may be readily lyophilized and stored at room temperature for ≥8 months or refrigerated temperature for ≥21 months while still retaining the ability to express protein

Spray drying is a technique that can be used to stabilize biopharmaceuticals, such as vaccines, within dry particles. Compared to liquid pharmaceutical products, dry powder has the potential to reduce costs associated with refrigerated storage and transportation. In this study, spray drying was investigated for processing an adjuvanted tuberculosis subunit vaccine, formulated as an oil-in-water nanoemulsion, into a dry powder composed of microparticles. Applying in-silico approaches to the development of formulation and processing conditions, successful encapsulation of the adjuvanted vaccine within amorphous microparticles was achieved in only one iteration, with high retention (>90%) of both the antigen and adjuvant system. Moisture-controlled stability studies on the powder were conducted over 26 months at temperatures up to 40 °C. Results showed that the powder was physically stable after 26 months of storage for all tested temperatures. Adjuvant system integrity was maintained at temperatures up to 25 °C after 26 months and after one month of storage at 40 °C. The spray-dried product demonstrated improved antigen thermostability when stored above refrigerated temperatures as compared to the liquid product. These results demonstrate the feasibility of spray drying as a method of encapsulating and stabilizing an adjuvanted vaccine.

Protein aggregation and particle formation have been observed when protein solutions contact hydrophobic interfaces, and it has been suggested that this undesirable phenomenon may be initiated by interfacial adsorption and subsequent gelation of the protein. The addition of surfactants, such as polysorbate 20, to protein formulations has been proposed as a way to reduce protein adsorption at silicone oil-water interfaces and mitigate the production of aggregates and particles. In an accelerated stability study, monoclonal antibody formulations containing varying concentrations of polysorbate 20 were incubated and agitated in pre-filled glass syringes (PFS), exposing the protein to silicone oil-water interfaces at the siliconized syringe walls, air-water interfaces, and agitation stress. Following agitation in siliconized syringes that contained an air bubble, lower particle concentrations were measured in the surfactant-containing antibody formulations than in surfactant-free formulations. Polysorbate 20 reduced particle formation when added at concentrations above or below the critical micelle concentration (CMC). The ability of polysorbate 20 to decrease particle generation in PFS corresponded with its ability to inhibit gelation of the adsorbed protein layer, which was assessed by measuring the interfacial diffusion of individual antibody molecules at the silicone oil-water interface using total internal reflectance fluorescence (TIRF) microscopy with single-molecule tracking.

Silicone oil is used as a lubricant in glass pre-filled syringes (PFS) but can contribute to the generation of particles within protein formulations in PFS. To mitigate the production of such particles, various silicone oil coating processes have been proposed. In this study, three siliconization methods (the "covalent" method, the "baked silicone oil" method, and the "liquid silicone oil" method) were used to coat glass syringes with silicone oil. Glide forces were determined for syringes coated by each method. Then, a monoclonal antibody formulation or a buffer solution were incubated in the coated syringes in either the presence or absence of an air bubble, and the syringes were rotated end-over-end to induce air bubble movement within the syringe. The particle concentrations were measured throughout the incubation period using flow microscopy. The coating method did not affect particle concentrations measured in buffer alone, nor did the coating method affect particle concentrations measured in antibody formulations in the absence of an air bubble. Particle concentrations were influenced by the syringe coating method in protein formulations agitated in the presence of an air bubble, with the most particles formed in syringes lubricated with liquid silicone oil. Fewer particles were produced in syringes lubricated with baked silicone oil, and the fewest particles were produced in syringes with covalently-attached silicone oil. However, the glide forces measured in syringes coated with silicone oil by each method are inversely correlated with the measured particle concentrations.

The stability of therapeutic proteins formulated in prefilled syringes (PFS) may be negatively impacted by the exposure of protein molecules to silicone oil-water interfaces and air-water interfaces. In addition, agitation, such as that experienced during transportation, may increase the detrimental effects (i.e., protein aggregation and particle formation) of protein interactions with interfaces. In this study, surfactant-free formulations containing either a monoclonal antibody or lysozyme were incubated in PFS, where they were exposed to silicone oil-water interfaces (siliconized syringe walls), air-water interfaces (air bubbles), and agitation stress (occurring during end-over-end rotation). Using flow microscopy, particles (≥2 μm diameter) were detected under all conditions. The highest particle concentrations were found in agitated, siliconized syringes containing an air bubble. The particles formed in this condition consisted of silicone oil droplets and aggregated protein, as well as agglomerates of protein aggregates and silicone oil. We propose an interfacial mechanism of particle generation in PFS in which capillary forces at the three-phase (silicone oil-water-air) contact line remove silicone oil and gelled protein aggregates from the interface and transport them into the bulk. This mechanism explains the synergistic effects of silicone oil-water interfaces, air-water interfaces, and agitation in the generation of particles in protein formulations.