Optimization of an Alum-Adsorbed Vaccine Powder Formulation for Epidermal Powder Immunization

Purpose. To develop stable and effective aluminum salt (alum)-adsorbed vaccine powder formulations for epidermal powder immunization (EPI) via a spray freeze-drying (SFD) process. Methods. Powder properties were determined using particle size analysis, tap density, and scanning electron microscopy. Alum coagulation was monitored via optical microscopy and particle sedimentation. Protein analysis was determined by the BCA protein assay, SDS-PAGE, and an enzyme immunoassay. In vivo immunogenicity and skin reactogenicity were performed on hairless guinea pigs and pigs, respectively. Results. SFD of hepatitis B surface antigen (HBsAg) adsorbed to aluminum hydroxide or aluminum phosphate using an excipient combination of trehalose/mannitol/dextran produced vaccine powders of dense particles and satisfactory powder flowability and hygroscopicity. This formulation also offered excellent long-term stability to the powder and the antigen. The two most important factors influencing alum particle coagulation are the freezing rate and the concentration of aluminum in the liquid formulation for SFD. The SFD vaccines, when delivered to hairless guinea pigs by EPI or injected intramuscularly after reconstitution, were as immunogenic as the original liquid vaccine. A further study showed that EPI with SFD alum-adsorbed diphtheria-tetanus toxoid vaccine was well tolerated, whereas needle injection of the liquid formulation caused persistent granuloma. Conclusions. Stabilization of alum-adsorbed vaccine by SFD has important implications in extending vaccination to areas lacking a cold chain for transportation and storage and may also accelerate the development of new immunization technologies such as EPI.     

Hepatitis-B surface antigen (HBsAg) powder formulation: process and stability assessment

The purpose of this study was to develop a hepatitis-B surface antigen (HBsAg) dry powder vaccine formulation suitable for epidermal powder immunization (EPI) via an efficient, scalable powder-formation process. Several HBsAg dry powder formulations were prepared using four different powder-formation methods: freeze-drying/compress/grind/sieve (FD/C/G/S), spray-drying (SD), agarose beads, and spray freeze-drying (SFD). Powder properties and physical stability were determined using particle size analysis, tap density measurement, scanning electron microscopy, optical microscopy, and moisture content analysis. Physical, chemical and biochemical stability of HBsAg was determined by dynamic light scattering, an enzyme immune assay, and immunogenicity in a mouse or hairless guinea pig model. Out of the four powder-formation methods evaluated SFD outperformed other methods in the following considerations: good process efficiency, flexible scalability, and desirable particle characteristics for skin penetration. The stress posed by SFD appeared to be mild as HBsAg in the dry form retained its potency and immunogenicity. Notably, the mechanism of fast freezing by SFD actually promoted the preservation of HBsAg nanoparticle size, in good correlation with long-term biochemical stability. Among several formulations screened, the formulation containing 10 microg HBsAg in 1-mg powder with a tertiary mixture of trehalose, mannitol, and dextran, exhibited excellent overall stability performance. In conclusion, HBsAg dry powder formulations suitable for EPI were successfully prepared using SFD. Further, a systematic formulation development strategy allowed the development and optimization of an HBsAg dry powder formulation, demonstrating excellent long-term physical, biochemical, and immunological stability.     

Stable high surface area lactate dehydrogenase particles produced by spray freezing into liquid nitrogen.

Enzyme activities were determined for lactate dehydrogenase (LDH) powder produced by lyophilization, and two fast freezing processes, spray freeze-drying (SFD) and spray freezing into liquid (SFL) nitrogen. The 0.25 mg/mL LDH aqueous feed solutions included either 30 or 100 mg/mL trehalose. The SFL process produced powders with very high enzyme activities upon reconstitution, similar to lyophilization. However, the specific surface area of 13 m(2)/g for SFL was an order of magnitude larger than for lyophilization. In SFD activities were reduced in the spraying step by the long exposure to the gas-liquid interface for 0.1-1s, versus only 2 ms in SFL. The ability to produce stable high surface area submicron particles of fragile proteins such as LDH by SFL is of practical interest in protein storage and in various applications in controlled release including encapsulation into bioerodible polymers. The SFL process has been scaled down for solution volumes <1 mL to facilitate studies of therapeutic proteins.     

Influenza vaccine powder formulation development: spray-freeze-drying and stability evaluation

The purpose of this study was to develop a spray-freeze-drying (SFD) process for preparing an influenza vaccine dry powder formulation suitable for epidermal powder immunization. After preformulation of two types of flu vaccines, their dry-powder formulations were prepared by SFD. Powder properties and physical stability were determined using particle size analysis, tap density measurement, scanning electron microscopy, optical microscopy, and moisture content analysis. Chemical and biochemical stability of vaccine antigens was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, single radial immunodiffusion assay, and in vivo immunogenicity in a mouse model. We demonstrated that SFD could produce high-density particles-a critical parameter for effective skin penetration. From the stability perspective, the stress posed by SFD was mild because the antigen in the dry powder retained its stability, potency, and immunogenicity. Among several formulations screened, we noted that formulation composition has a significant role in the powder's long-term physical and biochemical stability. One formulation, in particular, containing sub-unit vaccine (45 microg of antigen in 1 mg of powder) with a tertiary mixture of trehalose, mannitol, and dextran, exhibited excellent overall stability, including acceptable biochemical stability after being exposed to a highly humid environment. After all, we have not only demonstrated the suitability of SFD to prepare powders for epidermal powder immunization but also developed a systematic formulation development strategy that allowed the optimization of an influenza vaccine dry powder formulation. More important, this study led to the selection of a formulation system that had been successfully tested in a human clinical study.     

A comparison between spray drying and spray freeze drying for dry powder inhaler formulation of drug-loaded lipid-polymer hybrid nanoparticles

Lipid-polymer hybrid nanoparticles - polymeric nanoparticles enveloped by lipid layers - have emerged as a potent therapeutic nano-carrier alternative to liposomes and polymeric nanoparticles. Herein we perform comparative studies of employing spray drying (SD) and spray freeze drying (SFD) to produce inhalable dry-powder form of drug-loaded lipid-polymer hybrid nanoparticles. Poly(lactic-co-glycolic acid), lecithin, and levofloxacin are employed as the polymer, lipid, and drug models, respectively. The hybrid nanoparticles are transformed into micro-scale nanoparticle aggregates (or nano-aggregates) via SD and SFD, where the effects of (1) different excipients (i.e. mannitol, polyvinyl alcohol (PVA), and leucine), and (2) nanoparticle to excipient ratio on nano-aggregate characteristics (e.g. size, flowability, aqueous reconstitution, aerosolization efficiency) are examined. In both methods, PVA is found more effective than mannitol for aqueous reconstitution, whereas hydrophobic leucineis needed to achieve effective aerosolization as it reduces nano-aggregate agglomeration. Using PVA, both methods are equally capable of producing nano-aggregates having size, density, flowability, yield and reconstitutibility in the range ideal for inhaled delivery. Nevertheless, nano-aggregates produced by SFD are superior to SD in terms of their aerosolization efficiency manifested in the higher emitted dose and fine particle fraction with lower mass median aerodynamic diameter.     

Spray-Coating for Biopharmaceutical Powder Formulations: Beyond the Conventional Scale and Its Application

PURPOSE: Fluid-bed spray-coating process is widely used to prepare non-protein pharmaceutical solid dosage forms using macro-size seed particles (200-1000 microm) at kilogram batch sizes. In this study we developed a small-scale fluid-bed spray-coating process (20 g) to produce micro-sized vaccine powder formulations (40-60 microm) for epidermal powder immunization (EPI) METHODS: A bench-top spray coater was used to spray two vaccines, diphtheria toxoid (dT) and alum-adsorbed hepatitis-B surface antigen (Alum-HBsAg), onto crystalline lactose particles of 40-60 microm in diameter. Particle properties such as particle size, surface morphology, and degree of particle agglomeration were determined. Protein stability was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The immunogenicity of the vaccine was evaluated in vivo by needle injection and epidermal powder immunization (EPI) of mice or guinea pigs. RESULTS: Coating feasibility was demonstrated for both vaccine formulations containing different excipients. However, the nature of the vaccine antigen appeared to affect coating feasibility in terms of particle agglomeration considerably. Delivery of spray-coated dT and alum-HBsAg through EPI to mice and guinea pigs, respectively, generated significant antibody responses, at a level comparable to liquid formulation delivered subcutaneously through needle/syringe injection. CONCLUSIONS: The new spray-coating process represents an important technical advance and may provide a useful tool for developing high-valued biopharmaceutical powder formulations for novel applications. The strong in vivo performance of the coated dT and alum-HBsAg powders by EPI further demonstrated that spray-coating is a viable dry powder formulation process and the skin's epidermal layer presents an efficient vaccine delivery route.     

Formulation of pH responsive peptides as inhalable dry powders for pulmonary delivery of nucleic acids

Nucleic acids have the potential to be used as therapies or vaccines for many different types of disease, but delivery remains the most significant challenge to their clinical adoption. pH responsive peptides containing either histidine or derivatives of 2,3-diaminopropionic acid (Dap) can mediate effective DNA transfection in lung epithelial cells with the latter remaining effective even in the presence of lung surfactant containing bronchoalveolar lavage fluid (BALF), making this class of peptides attractive candidates for delivering nucleic acids to lung tissues. To further assess the suitability of pH responsive peptides for pulmonary delivery by inhalation, dry powder formulations of pH responsive peptides and plasmid DNA, with mannitol as carrier, were produced by either spray drying (SD) or spray freeze drying (SFD). The properties of the two types of powders were characterised and compared using scanning electron microscopy (SEM), next generation impactor (NGI), gel retardation and in vitro transfection via a twin stage impinger (TSI) following aerosolisation by a dry powder inhaler (Osmohaler™). Although the aerodynamic performance and transfection efficacy of both powders were good, the overall performance revealed SD powders to have a number of advantages over SFD powders and are the more effective formulation with potential for efficient nucleic acid delivery through inhalation.     

Radio Frequency - Assisted Ultrasonic Spray Freeze Drying for Pharmaceutical Protein Solids

This study examined physical stability of spray freeze dried (SFD) bovine serum albumin (BSA) solids produced using the radio frequency (RF)-assisted drying technique. BSA formulations were prepared with varying concentrations of trehalose and mannitol, using an excipient-free formulation as control. These formulations were produced using either traditional ultrasonic spray freeze drying (SFD) or RF-assisted ultrasonic spray freeze drying (RFSFD). The dried formulations were then characterized using Karl Fischer moisture content measurement, powder X-ray diffraction (PXRD), size exclusion chromatography (SEC), and solid-state hydrogen/deuterium exchange with mass spectrometry (ssHDX-MS). Moisture content did not have a good correlation with the physical stability of the formulations measured by SEC. ssHDX-MS metrics such as deconvoluted peak areas of the deuterated samples showed a satisfactory correlation (R² = 0.914) with the SEC stability data. RFSFD improved the stability of formulations with 20 mg/ml of trehalose and no mannitol, and had similar stability with all other formulations as compared to SFD. This study demonstrated that RFSFD technique can significantly reduce the duration of primary drying cycle from 48.0 h to 27.5 h while maintaining or improving protein physical stability as compared to traditional lyophilization.     

Stabilization of Live Attenuated Influenza Vaccines by Freeze Drying,Spray Drying,and Foam Drying

Purpose

The goal of this research is to develop stable formulations for live attenuated influenza vaccines (LAIV) by employing the drying methods freeze drying, spray drying, and foam drying.

Methods

Formulated live attenuated Type-A H1N1 and B-strain influenza vaccines with a variety of excipient combinations were dried using one of the three drying methods. Process and storage stability at 4, 25 and 37°C of the LAIV in these formulations was monitored using a TCID₅₀ potency assay. Their immunogenicity was also evaluated in a ferret model.

Results

The thermal stability of H1N1 vaccine was significantly enhanced through application of unique formulation combinations and drying processes. Foam dried formulations were as much as an order of magnitude more stable than either spray dried or freeze dried formulations, while exhibiting low process loss and full retention of immunogenicity. Based on long-term stability data, foam dried formulations exhibited a shelf life at 4, 25 and 37°C of >2, 1.5 years and 4.5 months, respectively. Foam dried LAIV Type-B manufactured using the same formulation and process parameters as H1N1 were imparted with a similar level of stability.

Conclusion

Foam drying processing methods with appropriate selection of formulation components can produce an order of magnitude improvement in LAIV stability over other drying methods.

     

Drying-Induced Variations in Physico-Chemical Properties of Amorphous Pharmaceuticals and Their Impact on Stability II: Stability of a Vaccine

Objectives To investigate the impact of drying method on the storage stability of dried vaccine formulations. Materials and Methods A sucrose-based formulation of a live attenuated virus vaccine of a parainfluenza strain, with and without surfactant, was dried from by different methods; freeze drying, spray drying and foam drying. Dried powders were characterized by differential scanning calorimetry, specific surface area (SSA) analysis and by electron spectroscopy for chemical analysis (ESCA) to evaluate vaccine surface coverage in the dried formulations. Dried formulations were subjected to storage stability studies at 4, 25 and 37°C. The vaccine was assayed initially and at different time points to measure virus-cell infectivity, and the degradation rate constant of the vaccine in different dried preparations was determined. Results SSA was highest with the spray dried preparation without surfactant (∼ 2.8 m²/g) and lowest in the foam dried preparations (with or without surfactant) (∼ 0.1 m²/g). Vaccine surface coverage was estimated based on ESCA measurements of nitrogen content. It was predicted to be highest in the spray dried preparation without surfactant and lowest in the foam with surfactant. Stability studies conducted at 25°C and 37°C showed that the vaccine was most stable in the foam dried preparation with surfactant and least stable in spray dried preparations without surfactant and in all freeze dried preparations regardless of the presence of surfactant. Addition of surfactant did lower the SSA and vaccine surface coverage in freeze dried preparations but still did not improve storage stability. Conclusions In drying methods that did not involve a freezing step, good storage stability of Medi 534 vaccine in the dried form was found with low SSA and low vaccine surface accumulation, both of which integrate into low fraction of vaccine at the surface. Ice appears to be a major destabilizing influence.     

Bulk Dynamic Spray Freeze-Drying Part 1: Modeling of Droplet Cooling and Phase Change

In spray freeze-drying (SFD), the solution is typically dispersed into a gaseous cold environment producing frozen microparticles that are subsequently dried via sublimation. This technology can potentially manufacture bulk lyophilized drugs at higher rates compared with conventional freeze-drying in trays and vials because small frozen particles provide larger surface area available for sublimation. Although drying in SFD still has to meet the material collapse temperature requirements, the final characteristics of the respective products are mainly controlled by the spray-freezing dynamics. In this context, the main goal of this work is to present a single droplet spray-freezing model and validate it with previously published simulations and experimental data. For the investigated conditions, the droplet temperature evolutions predicted by the model agree with experiments within an error of ±10%. The proposed engineering-level modeling framework is intended to assist future development of efficient SFD processes and support scale up from laboratory to commercial scale equipment.     

Production of Inhalation Phage Powders Using Spray Freeze Drying and Spray Drying Techniques for Treatment of Respiratory Infections

Purpose

The potential of aerosol phage therapy for treating lung infections has been demonstrated in animal models and clinical studies. This work compared the performance of two dry powder formation techniques, spray freeze drying (SFD) and spray drying (SD), in producing inhalable phage powders.

Method

A Pseudomonas podoviridae phage, PEV2, was incorporated into multi-component formulation systems consisting of trehalose, mannitol and L-leucine (F1?=?60:20:20 and F2?=?40:40:20). The phage titer loss after the SFD and SD processes and in vitro aerosol performance of the produced powders were assessed.

Results

A significant titer loss (~2 log) was noted for droplet generation using an ultrasonic nozzle employed in the SFD method, but the conventional two-fluid nozzle used in the SD method was less destructive for the phage (~0.75 log loss). The phage were more vulnerable during the evaporative drying process (~0.75 log further loss) compared with the freeze drying step, which caused negligible phage loss. In vitro aerosol performance showed that the SFD powders (~80% phage recovery) provided better phage protection than the SD powders (~20% phage recovery) during the aerosolization process. Despite this, higher total lung doses were obtained for the SD formulations (SD-F1?=?13.1?±?1.7?×?10⁴ pfu and SD-F2?=?11.0?±?1.4?×?10⁴ pfu) than from their counterpart SFD formulations (SFD-F1?=?8.3?±?1.8?×?10⁴ pfu and SFD-F2?=?2.1?±?0.3?×?10⁴ pfu).

Conclusion

Overall, the SD method caused less phage reduction during the powder formation process and the resulted powders achieved better aerosol performance for PEV2.

     

A Kinetic Model for Spray-Freezing of Pharmaceuticals

Spray freeze-drying (SFD), which includes spray-freezing into droplets and dynamic vacuum drying, presents a promising alternative approach to manufacture dried pharmaceuticals more efficiently than conventional vial freeze-drying. Without reliable predictive models for the SFD conditions of interest, any respective process development still relies on empirical approaches. In this work, we propose an improved modeling framework to describe the fast freezing (<1 s) that sub-millimeter droplets undergo in the present SFD process. The modeled freezing rate accounts for both the kinetics of ice growth and droplet heat transfer mechanisms. Computational fluid dynamics (CFD) simulations and experiments on bulk spray-freezing are combined to refine and validate the proposed reduced-order model. While this study is limited to water-sucrose solutions, the present modeling approach can be extended to other pharmaceutical excipients. For the cooling rates of interest, model results indicate that droplets with initial sucrose concentration higher than 20% w/w will transit to a glassy state before completion of crystallization and, consequently, devitrification is expected during post spray-freezing manipulation of the bulk material. In practice, such compact model does not only allow quantification of process parameters that cannot be measured in real time but also enable the choice of optimal spraying conditions for production of free-flowing, high-quality frozen droplets that meet the target product profile.     

Spray-freeze-drying for protein powder preparation: particle characterization and a case study with trypsinogen stability

This work investigates the use of spray freeze-drying (SFD) to produce protein loaded particles suitable for epidermal delivery. In the first part of the study, the effects of formulation and process conditions on particle properties are examined. Aqueous solutions of trehalose produce SFD particles in the size range 20-80 microm, with a smooth, textured surface, but having high internal porosity. The latter was visualized using SEM and a novel particle embedding and sectioning technique. Use of an annealing step during the freeze-drying cycle caused the particles to shrink, reducing hereby porosity and also the measured rate of moisture uptake into these amorphous particles. SFD pure mannitol was approximately 40% amorphous, but not hygroscopic. Incorporation of dextran 37,500 into a combined amorphous trehalose/mannitol formulation led to increased particle shrinkage and lower particle porosity on annealing. The model protein trypsinogen lost approximately 15% activity during SFD of solutions containing 50 mg/mL protein, but was only marginally aggregated (1.4%). It is suggested that trypsinogen forms an irreversible partially unfolded state or molten globule on SFD/rehydration. The pure protein was also partially inactivated without aggregation during atomization into air. Surprisingly, neither activity loss nor aggregation were detected on atomization of the protein solution into liquid nitrogen. Quench-freezing of small droplets may reverse the partial unfolding of trypsinogen occurring on atomization into air. The origin of the trypsinogen inactivation during SFD must therefore be the subsequent freeze-drying step of this multistep process. Isolated freeze drying of trypsinogen produces strong aggregation and equivalent inactivation. This result suggests that trypsinogen behaves differently during freeze drying from frozen droplets and from bulk solution in a vial. In the former case the protein forms an irreversible partially unfolded state, whereas in the latter case aggregates are formed. Trypsinogen inactivation during SFD could be completely prevented by the presence of trehalose in the formulation. Electron Spectroscopy for Chemical Analysis (ESCA) showed a high surface excess of the protein in the SFD particles, which was reduced on inclusion of Polysorbate 80, but not trehalose. Taken together, these results help to elucidate the complex destabilization behavior of trypsinogen during SFD.     

Distinct effects of sucrose and trehalose on protein stability during supercritical fluid drying and freeze-drying.

Supercritical fluid (SCF) drying has been proposed as an alternative for freeze-drying to stabilize proteins. Here we studied the influence of sucrose and trehalose during SCF drying on the protein stability and the physical powder characteristics of lysozyme and myoglobin formulations. The results obtained with SCF drying were compared with the results after freeze-drying of the same solutions. Aqueous protein solutions, with or without sugar, were sprayed into a SCF mixture of carbon dioxide and ethanol. The dried products were analyzed by residual water measurements, scanning electron microscopy, X-ray powder diffraction and differential scanning calorimetry. After reconstitution the protein structure was studied by UV/VIS, circular dichroism and fluorescence spectroscopy, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and bioactivity assay (lysozyme). The SCF dried and freeze-dried formulations showed comparable water contents, but their physical properties were substantially different. All freeze-dried cakes were amorphous with fully preserved protein structure. SCF dried sucrose-containing formulations showed agglomerated crystalline particles, whereas SCF dried trehalose-containing formulations appeared to consist of amorphous spherical particles. Particle morphology of excipients-free proteins was protein specific. Nearly all SCF dried lysozyme could be readily reconstituted, but for myoglobin significant fractions of SCF protein did not dissolve, especially in the absence of sugars. Covalent aggregation was not observed for the two proteins. For the recovered soluble fractions, the secondary protein structure was preserved. The tertiary structure was preserved for lysozyme, but not entirely for myoglobin. Surprisingly, during SCF drying trehalose was less protective than sucrose for myoglobin.     

Formation of Stable Submicron Protein Particles by Thin Film Freezing

Purpose Highly stable, submicron lactate dehydrogenase (LDH) and lysozyme particles may be produced by thin film freezing (TFF) of aqueous solutions followed by lyophilization. Methods The LDH activity was determined by measuring the decrease in absorbance of NADH over time for the reaction of pyruvate to lactate. For lysozyme the particle morphology was determined by scanning electron microscopy (SEM) and compared with the specific surface area (BET) and the particle size, as measured by laser light scattering, Results Protein particles with an average diameter of 300 nm and 100% enzyme activity upon reconstitution (for LDH) were formed by TFF. Droplets of protein solutions, 3.6 mm in diameter, spread upon impact with 223 and 133 K metal surfaces to form cylindrical disks with thicknesses of 200–300 μm. Calculated cooling rates of the disks of 10² K/s were confirmed experimentally with infrared measurements. Conclusions The cooling rates of 10² K/s, intermediate to those in lyophilization (1 K/min) and spray freeze-drying (SFD) (10⁶ K/s), were sufficiently fast to produce sub-micron protein particles with surface areas of 31–73 m²/g, an order of magnitude higher than in lyophilization. In addition, the low surface area/volume ratio (32–45 cm⁻¹) of the gas–liquid interface led to minimal protein adsorption and denaturation relative to SFD.     

Percutaneous absorption of indomethacin from transparent oil/water gels in rabbits.

The percutaneous absorption of indomethacin from transparent oil in water gels (TOW gels) has been studied in rabbits and compared with absorption from a hydrophilic gel and from a spray formulation. The area under the curve and the Cmax values in plasma were significantly higher for the TOW gels in comparison with the other formulations after single application. The pH of the aqueous phase of the TOW gels did not significantly influence the bioavailability. After multiple application the TOW gels induced a larger increase in AUC (vs first application) in comparison with the other formulations. None of the formulations without drug damaged the skin after multiple application. For indomethacin formulations skin damage was more pronounced with the hydrophilic gel than for the TOW gels and spray formulation.     

Spray freezing into liquid nitrogen for highly stable protein nanostructured microparticles.

The objectives of this study were to produce nanostructured protein microparticles with the spray freezing into liquid (SFL) cryogenic process and to demonstrate a smaller degree of protein denaturation and aggregation than observed in spray freeze drying (SFD). Nanostructured microparticles were formed by atomization of an aqueous buffer solution containing bovine serum albumin (BSA) with and without excipients beneath the surface of a cryogenic liquid. Lyophilization was used to sublime the water in the frozen particles. The resulting BSA dry powder was characterized by size exclusion chromatography, Fourier-transform infrared spectroscopy, scanning electron microscopy (SEM), light scattering, and specific surface area analysis. SEM revealed highly porous microparticle with features smaller than 500 nm. The specific surface area of the BSA microparticles ranged from 19.2 to 97.7 m(2)/g as a function of the total protein and excipient content in the aqueous feed solution. SFL produced less denaturation and aggregation of protein monomer than SFD, despite the extremely high surface areas in both processes. The intense atomization and ultra-rapid freezing in the SFL process lead to nanostructured BSA microparticles with high surface areas. Protein denaturation and aggregation are reduced in SFL relative to SFD. The more rapid freezing in SFL lowers the time for proteins to aggregate or diffuse to water-air and water-ice interfaces where they may be denatured.     

Spray freeze drying for dry powder inhalation of nanoparticles

Formulating nanoparticles for delivery to the deep lung is complex and many techniques fail in terms of nanoparticle stability. Spray freeze drying (SFD) is suggested here for the production of inhalable nanocomposite microcarriers (NCM). Different nanostructures were prepared and characterized including polymeric and lipid nanoparticles. Nanoparticle suspensions were co-sprayed with a suitable cryoprotectant into a cooled, stainless steel spray tower, followed by freeze drying to form a dry powder while equivalent compositions were spray dried (SD) as controls. SFD-NCM possess larger specific surface areas (67–77 m²/g) and lower densities (0.02 g/cm³) than their corresponding SD-NCM. With the exception of NCM of lipid based nanocarriers, SFD produced NCM with a mass median aerodynamic diameter (MMAD) of 3.0 ± 0.5 μm and fine particle fraction (FPF ⩽ 5.2 μm) of 45 ± 1.6% with aerodynamic performances similar to SD-NCM. However, SFD was superior to SD in terms of maintaining the particle size of all the investigated polymeric and lipid nanocarriers following reconstitution (S_f/S_i ratio for SFD ≈ 1 versus >1.5 for SD). The SFD into cooled air proved to be an efficient technique to prepare NCM for pulmonary delivery while maintaining the stability of the nanoparticles.     

Atomic-Layer Deposition Processes Applied to Phage λ and a Phage-like Particle Platform Yield Thermostable,Single-Shot Vaccines

Especially in developing countries, the impact of vaccines can be limited by logistical obstacles associated with multiple dose regimens, pathogen variants, and challenges imposed by requirements for maintaining vaccines at low temperatures during shipping and storage. Thus, there is a need for vaccines that can be flexibly modified to address evolving pathogen landscapes, are stable outside of narrow “cold-chain” temperatures and require administration of only single doses. Here we demonstrate in proof-of-concept studies a vaccine platform that addresses these impediments to more widespread use of vaccines. The platform relies on bacteriophage-derived phage-like-particles (PLPs) that utilize a “plug-and-play” antigen delivery system that allows for fast, easy alteration of antigens on the surface of the PLPs. Thermostability of PLP-based vaccines can be achieved by embedding the PLPs within glassy particles produced by spray drying, and nanoscopic aluminum oxide layers applied using atomic layer deposition (ALD) can serve to control release of antigen in vivo, yielding vaccine formulations that elicit strong immune responses after administration of single doses. Bacteriophage λ was stabilized by spray drying to form powders that were incubated at 37 °C for up to a year without loss of infectious activity. PLPs derived from bacteriophage λ were expressed and purified from E. coli cultures, and an in vitro conjugation strategy was used to decorate specific PLP surface sites with T4-lysozyme, a model vaccine antigen. The resulting T4-lysozyme:PLP complexes (Lys-PLPs) were embedded in glassy dry powders formed by spray drying and coated with nanometer-thick layers of alumina deposited by ALD in a fluidized bed reactor. Alumina-coated Lys-PLP vaccines were stable for a least a month at 50 °C, and single doses of the alumina-coated vaccines elicited immune responses that were indistinguishable from responses generated by conventional two-dose, prime-and-boost dosing regimens of alum-adjuvanted Lys-PLP vaccines.