(T1130-07-44) Encapsulating Vaccines via Spray Drying: Achieving Thermostability and Controlled Delivery for a Single-Shot Vaccination Strategy

Purpose: This work aims to develop thermostable, controlled-release, single-dose vaccines using spray-drying encapsulation technology. Current vaccines often require continuous refrigeration from the site of manufacture until administration. Further, most vaccines require multiple doses to be protective; however, this leads to poor compliance, thus limiting their effectiveness. To address these challenges, we are developing thermostable encapsulated vaccines that can withstand extreme storage conditions. This strategy will eliminate cold chain requirements and provide long-lasting protection with a single dose. We have selected a nanolipoprotein particle (NLPs) vaccine to be encapsulated using our spray drying technology. NLPs will be spray-dried with Generally Regarded as Safe (GRAS) excipients to produce a thermostable dry powder vaccine capable of withstanding extreme temperature and humidity conditions. These NLPs protect against Francisella tularensis (Ft), the bacteria that causes tularemia. NLPs allow co-delivering recombinant IglC protein, Ft LVS (live attenuated strain) LPS, and a cholesterol-anchored CpG adjuvant. NLPs are 10-25 nm discoidal membrane mimetics that self-assemble from purified lipoproteins and lipids, offering a natural, biocompatible platform for vaccine delivery. However, like most biologics, thermal stability remains a concern during vaccine storage and transportation. Our approach strengthens the potential of NLPs as a robust and flexible vaccine platform for F. tularensis and other biothreat pathogens. The current focus of this study is to stabilize the NLP vaccine by microencapsulating it with a combination of GRAS excipients (sugars and amino acids) and, ultimately, improve vaccine availability in remote regions of the world.

Methods: For encapsulating vaccines using spray drying, a combination of oligosaccharides and amino acids was selected. Based on our previous studies, a standard mixture of trehalose and L-leucine at different ratios was selected as the excipients for spray drying. Additionally, L-arginine monohydrochloride was investigated as a potential alternative to leucine due to its enhanced aqueous solubility. The details of the different spray-dried batches are summarized in Table 1. The optimized spray drying parameters were an inlet temperature of 110 °C, feed rate of 2.5 mL/min, N₂ flow rate of 1200 L/h, aspirator setting of 31 m³/h, and outlet temperature of 55–57 °C. The spray-dried vaccines were stored in sealed glass vials at 2–8 °C for further analysis. Particle size was measured by laser diffraction, and vaccine stability post-spray drying was assessed using size exclusion chromatography (SEC).

Results: All spray-dried batches achieved yields exceeding 60 %, demonstrating a consistently efficient drying process across different excipient combinations. A higher ratio of leucine and increased total excipient concentration resulted in improved powder yield, attributable to enhanced spray drying efficiency and the superior hydrophobic properties of leucine relative to trehalose. The spray-dried NLPs exhibited particle sizes ranging from 1.2 to 2.34 µm (Table 1); small and uniformly-sized particles will facilitate rapid and uniform resuspension, improve solubilization of excipient matrix due to increased surface area, and ensure safe and efficient injectability without risk of needle clogging or vascular occlusion. From the SEC analysis, pure NLPs exhibited a single unimodal peak (Figure 1A) after synthesis and lyophilization (dried for storage stability); however, spray-dried NLPs showed degradation despite variations in excipient ratio and total excipient concentration (Figure 1B). These findings indicate that the selected excipients failed to form a protective glassy matrix around NLPs during spray drying. Furthermore, a comprehensive compatibility study revealed that leucine was incompatible with NLPs (Figure 1C). Owing to its hydrophobic nature, leucine may intercalate into the NLP bilayer, potentially compromising bilayer integrity. Moreover, the inclusion of leucine also introduced challenges in resuspension in PBS due to its limited aqueous solubility. Consequently, L-arginine was explored as a replacement amino acid excipient owing to its superior solubility; however, spray drying with arginine also resulted in NLP dissociation and degradation. We hypothesize that shear stress during atomization and elevated temperatures (inlet and outlet) during spray drying contribute to the instability of NLPs.

Conclusion: SEC analysis demonstrates that trehalose, leucine, and arginine in different ratios and concentrations were insufficient to stabilize NLPs during the spray drying process. Given the different constituents of NLPs (lipids and proteins), multiple critical factors may be involved, including temperature, shear forces, and excipient physicochemical properties; achieving stable NLP formulations via spray drying remains challenging. Current efforts are focused on systematically evaluating the effects of shear and thermal stress during the spray drying process to identify optimal formulation parameters for producing thermostable dry powder NLP vaccines.

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Acknowledgements: This research work is supported through the project entitled “Encapsulation of Vaccine Candidates for Single-Shot Immunization and Vaccine Stability” (MCDC 22-06-008) by the Defense Threat Reduction Agency (DTRA), Joint Science and Technology Office (JSTO), Vaccines/Therapeutics Division (CBM).


Table 1. Different batches of spray drying of NLPs


Figure 1. SEC chromatogram of pure NLPs after synthesis and lyophilization (A), after spray drying with comparison to pure NLPs (B) and impact of leucine on NLPs (C)