Stressed stability and protective efficacy of lead lyophilized formulations of ID93+GLA-SE tuberculosis vaccine.

With the recent exception of coronavirus disease 2019 (COVID-19), tuberculosis (TB) causes more deaths globally than any other infectious disease, and approximately 1/3 of the world's population is infected with Mycobacterium tuberculosis (Mtb). However, encouraging progress in TB vaccine development has been reported, with approximately 50% efficacy achieved in Phase 2b clinical testing of an adjuvanted subunit TB vaccine candidate. Nevertheless, current lead vaccine candidates require cold-chain transportation and storage. In addition to temperature stress, vaccines may be subject to several other stresses during storage and transport, including mechanical, photochemical, and oxidative stresses. Optimal formulations should enable vaccine configurations with enhanced stability and decreased sensitivity to physical and chemical stresses, thus reducing reliance on the cold chain and facilitating easier worldwide distribution. In this report, we describe the physicochemical stability performance of three lead thermostable formulations of the ID93 + GLA-SE TB vaccine candidate under various stress conditions. Moreover, we evaluate the impact of thermal stress on the protective efficacy of the vaccine formulations. We find that formulation composition impacts stressed stability performance, and our comprehensive evaluation enables selection of a lead single-vial lyophilized candidate containing the excipient trehalose and Tris buffer for advanced development.

A flexible, thermostable nanostructured lipid carrier platform for RNA vaccine delivery.

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 in vivo. The thermostability of this NLC/RNA vaccine delivery platform could significantly improve distribution of current and future pandemic response vaccines, particularly in low-resource settings.

A self-amplifying RNA vaccine against COVID-19 with long-term room-temperature stability.

mRNA vaccines were the first to be authorized for use against SARS-CoV-2 and have since demonstrated high efficacy against serious illness and death. However, limitations in these vaccines have been recognized due to their requirement for cold storage, short durability of protection, and lack of access in low-resource regions. We have developed an easily-manufactured, potent self-amplifying RNA (saRNA) vaccine against SARS-CoV-2 that is stable at room temperature. This saRNA vaccine is formulated with a nanostructured lipid carrier (NLC), providing stability, ease of manufacturing, and protection against degradation. In preclinical studies, this saRNA/NLC vaccine induced strong humoral immunity, as demonstrated by high pseudovirus neutralization titers to the Alpha, Beta, and Delta variants of concern and induction of bone marrow-resident antibody-secreting cells. Robust Th1-biased T-cell responses were also observed after prime or homologous prime-boost in mice. Notably, the saRNA/NLC platform demonstrated thermostability when stored lyophilized at room temperature for at least 6 months and at refrigerated temperatures for at least 10 months. Taken together, this saRNA delivered by NLC represents a potential improvement in RNA technology that could allow wider access to RNA vaccines for the current COVID-19 and future pandemics.

Lyophilization Process Engineering and Thermostability of ID93 + GLA-SE, a Single-Vial Adjuvanted Subunit Tuberculosis Vaccine Candidate for Use in Clinical Studies.

Promising clinical efficacy results have generated considerable enthusiasm for the potential impact of adjuvant-containing subunit tuberculosis vaccines. The development of a thermostable tuberculosis vaccine formulation could have significant benefits on both the cost and feasibility of global vaccine distribution. The tuberculosis vaccine candidate ID93 + GLA-SE has reached Phase 2 clinical testing, demonstrating safety and immunogenicity as a two-vial point-of-care mixture. Earlier publications have detailed efforts to develop a lead candidate single-vial lyophilized thermostable ID93 + GLA-SE vaccine formulation. The present report describes the lyophilization process development and scale-up of the lead candidate thermostable ID93 + GLA-SE composition. The manufacture of three full-scale engineering batches was followed by one batch made and released under current Good Manufacturing Practices (cGMP). Up to 4.5 years of stability data were collected. The cGMP lyophilized ID93 + GLA-SE passed all manufacturing release test criteria and maintained stability for at least 3 months when stored at 37°C and up to 24 months when stored at 5°C. This work represents the first advancement of a thermostable adjuvant-containing subunit tuberculosis vaccine to clinical testing readiness.

Evaluation of the stability of a spray-dried tuberculosis vaccine candidate designed for dry powder respiratory delivery.

Particle engineering via spray drying was used to develop a dry powder presentation of an adjuvanted tuberculosis vaccine candidate. This presentation utilizing a trileucine-trehalose excipient system was designed to be both thermostable and suitable for respiratory delivery. The stability of the spray-dried vaccine powder was assessed over one year at various storage temperatures (-20, 5, 25, 40, 50 °C) in terms of powder stability, adjuvant stability, and antigen stability. A formulation without trileucine was included as a control. The results showed that the interior particle structure and exterior particle morphology of the powder was maintained for one year at 40 °C, while the control case exhibited a small extent of particle fusing under the same storage conditions. Moisture content was maintained, and powder solid state remained amorphous for all storage temperatures. Aerosol performance was assessed with a commercial dry powder inhaler in combination with a human mouth-throat model. The emitted dose and lung dose were maintained for all samples after one year at temperatures up to 40 °C. Nanoemulsion size and oil content of the adjuvant system were maintained after one year at temperatures up to 40 °C, and the agonist content was maintained after one year at temperatures up to 25 °C. The antigen was completely degraded in the control formulation at seven months of storage at 40 °C; by contrast, 45% of the antigen was still present in the trehalose-trileucine formulation after one year of storage at 50 °C. Comparatively, the antigen was completely degraded in a liquid sample of the vaccine candidate after only one month of storage at 37 °C. The spray-dried trehalose-trileucine vaccine powder clearly maintained its inhalable properties after one year's storage at high temperatures and improved overall thermostability of the vaccine.

Microparticle encapsulation of a tuberculosis subunit vaccine candidate containing a nanoemulsion adjuvant via spray drying.

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.

Development of a formulation platform for a spray-dried, inhalable tuberculosis vaccine candidate.

Protection against primarily respiratory infectious diseases, such as tuberculosis (TB), can likely be enhanced through mucosal immunization induced by direct delivery of vaccines to the nose or lungs. A thermostable inhalable dry powder vaccine offers further advantages, such as independence from the cold chain. In this study, we investigate the formulation for a stable, inhalable dry powder version of ID93 + GLA-SE, an adjuvanted subunit TB vaccine candidate, containing recombinant fusion protein ID93 and glucopyranosyl lipid A (GLA) in a squalene emulsion (SE) as an adjuvant system, via spray drying. The addition of leucine (20% w/w), pullulan (10%, 20% w/w), and trileucine (3%, 6% w/w) as dispersibility enhancers was investigated with trehalose as a stabilizing agent. Particle morphology and solid state, nanoemulsion droplet size, squalene and GLA content, ID93 presence, and aerosol performance were assessed for each formulation. The results showed that the addition of leucine improved aerosol performance, but increased aggregation of the emulsion droplets was demonstrated on reconstitution. Addition of pullulan preserved emulsion droplet size; however, the antigen could not be detected after reconstitution. The trehalose-trileucine excipient formulations successfully stabilized the adjuvant system, with evidence indicating retention of the antigen, in an inhalable dry powder format suitable for lung delivery.

Development and Testing of a Spray-Dried Tuberculosis Vaccine Candidate in a Mouse Model.

Converting a vaccine into a thermostable dry powder is advantageous as it reduces the resource burden linked with the cold chain and provides flexibility in dosage and administration through different routes. Such a dry powder presentation may be especially useful in the development of a vaccine towards the respiratory infectious disease tuberculosis (TB). This study assesses the immunogenicity and protective efficacy of spray-dried ID93+GLA-SE, a promising TB vaccine candidate, against Mycobacterium tuberculosis (Mtb) in a murine model when administered via different routes. Four administration routes for the spray-dried ID93+GLA-SE were evaluated along with relevant controls-1) reconstitution and intramuscular injection, 2) reconstitution and intranasal delivery, 3) nasal dry powder delivery via inhalation, and 4) pulmonary dry powder delivery via inhalation. Dry powder intranasal and pulmonary delivery was achieved using a custom nose-only inhalation device, and optimization using representative vaccine-free powder demonstrated that approximately 10 and 44% of the maximum possible delivered dose would be delivered for intranasal delivery and pulmonary delivery, respectively. Spray-dried powder was engineered according to the different administration routes including maintaining approximately equivalent delivered doses of ID93 and GLA. Vaccine properties of the different spray-dried lots were assessed for quality control in terms of nanoemulsion droplet diameter, polydispersity index, adjuvant content, and antigen content. Our results using the Mtb mouse challenge model show that both intranasal reconstituted vaccine delivery as well as pulmonary dry powder vaccine delivery resulted in Mtb control in infected mice comparable to traditional intramuscular delivery. Improved protection in these two vaccinated groups over their respective control groups coincided with the presence of cytokine-producing T cell responses. In summary, our results provide novel vaccine formulations and delivery routes that can be harnessed to provide protection against Mtb infection.

Effect of the siliconization method on particle generation in a monoclonal antibody formulation in pre-filled syringes.

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.

Surfactant Effects on Particle Generation in Antibody Formulations in Pre-filled Syringes.

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.

Protein aggregation and particle formation in prefilled glass syringes.

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.

Ionic strength affects tertiary structure and aggregation propensity of a monoclonal antibody adsorbed to silicone oil-water interfaces.

Therapeutic proteins formulated in prefilled syringes lubricated with silicone oil come in contact with silicone oil-water interfaces for their entire shelf lives. Thus, the interactions between protein and silicone oil were studied to determine the effect of silicone oil on a monoclonal antibody's stability, both at the interface and in the bulk solution. The influence of ionic strength on these interactions was also investigated through the addition of various monovalent and divalent salts to sample formulations. The tertiary structure of the antibody was perturbed when it adsorbed to the silicone oil-water interface in solutions at low ionic strength. However, the tertiary structure of the antibody at the interface was not perturbed when the ionic strength of the formulation was increased. Even at low ionic strength, the secondary structure of the antibody adsorbed to the silicone oil-water interface was retained, suggesting that at low ionic strength, the adsorbed antibody assumes a molten globule-like conformation. This partially unfolded species was aggregation-prone, especially during agitation. Silicone oil-induced aggregation of the antibody was inhibited at higher ionic strength.