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Biological macromolecules, especially therapeutic proteins, are delicate and highly sensitive to denaturation from stresses encountered during the manufacture of dosage forms. Thin-film freeze-drying (TFFD) and spray freeze-drying (SFD) are two processes used to convert liquid forms of protein into dry powders. In the production of inhalable dry powders that contain proteins, these potential stressors fall into three categories based on their occurrence during the primary steps of the process: (1) droplet formation (e.g., the mechanism of droplet formation, including spray atomization), (2) freezing, and (3) frozen water removal (e.g., sublimation). This study compares the droplet formation mechanism used in TFFD and SFD by investigating the effects of spraying on the stability of proteins, using lactoferrin as a model. This study considers various perspectives on the denaturation (e.g., conformation) of lactoferrin after subjecting the protein solution to the atomization process using a pneumatic two-fluid nozzle (employed in SFD) or a low-shear drop application through the nozzle. The surface activity of lactoferrin was examined to explore the interfacial adsorption tendency, diffusion, and denaturation process. Subsequently, this study also investigates the secondary and tertiary structure of lactoferrin and the quantification of monomers, oligomers, and, ultimately, aggregates. The spraying process affected the tertiary structure more negatively than the tightly woven secondary structure, resulting in the peak position corresponding to the tryptophan (Trp) residues red-shifting by 1.5 nm. This conformational change can either (a) be reversed at low concentrations via relaxation or (b) proceed to form irreversible aggregates at higher concentrations. Interestingly, when the sample was allowed to progress into micrometer-sized aggregates, such a dramatic change was not detected using methods such as size-exclusion chromatography, polyacrylamide gel electrophoresis, and dynamic light scattering at 173°. A more complete understanding of the heterogeneous protein sample was achieved only through a combination of 173 and 13° backward and forward scattering, a combination of derived count rate measurements, and microflow imaging (MFI). After studying the impact of droplet formation mechanisms on aggregation tendency of lactoferrin, we further investigated two additional model proteins with different surface activity: bovine IgG (serving as a non surface-active negative reference), and β-galactosidase (another surface-active protein). The results corroborated the lactoferrin findings that spray-atomization-related stress-induced protein aggregation was much more pronounced for proteins that are surface active (lactoferrin and β-galactosidase), but it was minimal for non-surface-active protein (bovine IgG). Finally, compared to the low-shear dripping used in the TFFD process, lactoferrin underwent a relatively fast conformational change upon exposure to the high air-water interface of the two-fluid atomization nozzle used in the SFD process as compared to the low shear dripping used in the TFFD process. The interfacial-induced denaturation that occurred during spraying was governed primarily by the size of the atomized droplets, regardless of the duration of exposure to air. The percentage of denatured protein population and associated activity loss, in the case of β-galactosidase, was determined to range from 2 to 10% depending on the air-flow rate of the spraying process.

This study was designed to test the feasibility of using thin-film freezing (TFF) to prepare aerosolizable dry powders of plasmid DNA (pDNA) for pulmonary delivery. Dry powders of pDNA formulated with mannitol/leucine (70/30, w/w) at various of drug loadings, solid contents, and solvents were prepared using TFF, their aerosol properties (i.e., mass median aerodynamic diameter (MMAD) and fine particle fraction (FPF)) determined, and selected powders were used for further characterization. Of the nine dry powders prepared, their MMAD values were about 1-2 mm, with FPF values (delivered) of 40-80%. The aerosol properties of the powders were inversely correlated with the pDNA loading and the solid content in the pDNA solution before thin-film freezing. Powders prepared with Tris-EDTA (TE) buffer or cosolvents (i.e., 1,4 dioxane or t-butanol in water), instead of water, showed slightly reduced aerosol properties. Ultimately, powders prepared with pDNA loading at 5% (w/w), 0.25% of solid content, with or without TE were selected for further characterization due to their overall good aerosol performance. The pDNA powders exhibited a porous matrix, crystalline structure, with a moisture content of

Thin-film freeze-drying is a dry powder engineering technology that involves in a rapid thin-film freezing (TFF) process followed by lyophilization to remove solvent. TFF process has been successfully used to produce pharmaceutical powders of the small and large molecule drugs as well as live organisms. This paper provides a comprehensive review of the pharmaceutical applications of TFF powders of small molecule drugs intended for pulmonary delivery or oral administration. The powders produced by the TFF process often exhibited unique physical properties such as amorphous morphology, high porosity, brittle matrix structure, and high specific surface area, and the powders also often contain loosely connected micron or submicron particles or aggregates. The TFF process parameters and formulation compositions directly affect the physical properties of the powders. These physical properties render TFF powders desirable aerodynamic properties for efficient pulmonary delivery by oral inhalation and help enhance the dissolution of poorly water-soluble small molecule drugs in the powders and thus improve their bioavailability after oral administration.

Freeze-drying, or lyophilization, is widely used to produce pharmaceutical solids from temperature-sensitive materials but the process is time and energy inefficient. Herein, using E. coli as a model live organism, whose viability in dry powders is highly sensitive to the water content in the powders, we demonstrated that the drying rate of thin-film freeze-drying (TFFD) is significantly higher than that of the conventional shelf freeze-drying, likely because the large total surface area from the loosely stacked frozen thin films and the low thickness of the thin-films enable faster and more efficient mass transfer during freeze-drying. The highly porous nature and high specific surface area of the thin-film freeze-dried powders may have contributed to the faster mass transfer as well. Moreover, we demonstrated that TFFD can be applied to produce dry powders of E. coli and L. acidophilus with minimum bacterial viability loss (i.e., within one log reduction), and the L. acidophilus dry powder is suitable for intranasal delivery. It is concluded that TFFD technology is promising in addressing the time-and cost-inefficient issue of conventional shelf freeze-drying.

AS01

AS01Bis a liposomal formulation of two immunostimulants namely 3-O-desacyl-4’-monophosphoryl lipid A (MPL) and QS-21. The liposomal formulation of AS01Breduces the endotoxicity of MPL and the lytic activity of QS-21; however, it renders the adjuvant sensitive to accidental slow freezing. The liposomal formulation also represents a major challenge towards the formulation of dry powders of vaccines containing AS01B. In the present study, we tested the feasibility of applying thin-film freeze-drying (TFFD) to engineer dry powders of the AS01Bliposomal adjuvant alone or vaccines containing AS01Bas an adjuvant. Initially, we showed that after the AS01Bliposomal adjuvant was subjected to TFFD using sucrose as a stabilizer at 4%w/v, the particle size distribution of AS01Bliposomes reconstituted from the dry powder was identical to the liquid adjuvant before drying. We then showed using ovalbumin (OVA) as a model antigen adjuvanted with AS01B(AS01B/OVA) that subjecting the AS01B/OVA vaccine to TFFD and subsequent reconstitution did not negatively affect the AS01Bliposome integrity, nor the immunogenicity of the vaccine. Importantly, the thin-film freeze-dried vaccine was not sensitive to repeated freezing-and-thawing. Finally, the feasibility of using TFFD to prepare dry powders of AS01B-adjuvanted vaccines was further confirmed using AS01B-adjuvanted Fluzone Quadrivalent and Shingrix, which contains AS01B. It is concluded that the TFFD technology can enable the formulation of AS01B-adjuvanted vaccines as freezing-insensitive dry powders in single-vial presentation.

Freeze-drying, or lyophilization, is widely used to produce pharmaceutical solids that contain temperature-sensitive materials. Herein, using Escherichia coli as a model live organism, whose viability in dry powders is highly sensitive to the water content in the powders, we demonstrated that the drying rate from the frozen thin films generated by thin-film freezing (TFF) is significantly faster than from the bulk frozen solids in conventional shelf freeze-drying. This is likely because the loosely stacked frozen thin films provided a larger solid-air interface and the low thickness of the thin films provided a low mass transfer resistance. The highly porous microstructure and high specific surface area of the thin-film freeze-dried powders may also be related to the faster drying observed. Moreover, we demonstrated that TFF can be applied to produce dry powders of E. coli, a Gram-negative bacterium, or Lactobacillus acidophilus, a Gram-positive bacterium, with minimum bacterial viability loss (i.e., within one log reduction). It is concluded that the TFF technology is promising in accelerating water removal from frozen samples.

Oral drug therapy requiring large quantities of active pharmaceutical ingredients (APIs) can cause a substantial pill burden, which can increase nonadherence and worsen healthcare outcomes. Maximizing the drug loading of APIs in oral dosage forms is essential to reduce pill burden. This can be challenging for poorly water-soluble APIs without compromising performance. We show a promising strategy for maximizing the drug loading of pH-dependent APIs in amorphous solid dispersions (ASDs) produced by hot-melt extrusion (HME) without compromising their dissolution performance. We examine potential increases in the drug loading (w/w) of telmisartan in ASDs by incorporating bases to modify pH during HME. Telmisartan is a weakly acidic, poorly water-soluble API with pH-dependent solubility. It is practically insoluble at physiological pH, but its solubility increases exponentially at pH values above 10. Telmisartan was extruded with the polymer Soluplus and various bases. With no base, the maximum drug loading achieved by extrusion was only 5% before crystalline telmisartan was detected. Including a strong, water-soluble base (NaOH or KOH) increased the maximum amorphous drug loading to 50%. These results indicate that telmisartan has pH-dependent solubility in a molten polymer, similar to that in an aqueous solution. We also examine the stability of Soluplus when extruded with a strong base, using solid-state nuclear magnetic resonance (ssNMR) to determine that NaOH (but not KOH) causes degradation by hydrolysis. Supersaturation was maintained for at least 20 h during dissolution testing of a 50% telmisartan ASD in biorelevant media.

Remdesivir dry powder for inhalation was previously developed using thin film freezing (TFF). A single-dose 24-h pharmacokinetic study in hamsters demonstrated that pulmonary delivery of TFF remdesivir can achieve plasma remdesivir and GS-441524 levels higher than the reported EC50s of both remdesivir and GS-441524 (in human epithelial cells) over 20 h. The half-life of GS-4412524 following dry powder insufflation was about 7 h, suggesting the dosing regimen would be twice-daily administration. Although the remdesivir-Captisol® (80/20 w/w) formulation showed faster and greater absorption of remdesivir and GS-4412524 in the lung, remdesivir-leucine (80/20 w/w) exhibited a greater Cmax with shorter Tmax and lower AUC of GS-441524, indicating lower total drug exposure is required to achieve a high effective concentration against SAR-CoV-2. In conclusion, remdesivir dry powder for inhalation would be a promising alternative dosage form for the treatment of COVID-19 disease., Graphical abstract Unlabelled Image

Thin-film freeze-drying (TFFD) is a rapid freezing and then drying technique used to prepare inhalable dry powders from the liquid form for drug delivery to the lungs. We report the preparation of aerosolizable dry powders of monoclonal antibodies (mAbs) by TFFD. We first formulated IgG with lactose/leucine (60:40 w/w) or trehalose/leucine (75:25). IgG 1% (w/w) formulated with lactose/leucine (60:40 w/w) in phosphate buffered saline (PBS) (IgG-1-LL-PBS) and processed by TFFD was found to produce the powder with the most desirable aerosol properties. We then replaced IgG with a specific antibody, anti-programmed cell death protein (anti-PD-1 mAb), to prepare a dry powder (anti-PD1-1-LL-PBS), which performed similarly to the IgG-1-LL-PBS powder. The aerosol properties of anti-PD1-1-LL-PBS were significantly better when TFFD was used to prepare the powder as compared to conventional shelf freeze-drying (shelf FD). The dry powder had a porous structure with nanoaggregates. The dry powder had a Tg value between 39-50 °C. When stored at room temperature, the anti-PD-1 mAb in the TFFD powder was more stable than that of the same formulation stored as a liquid. The addition of polyvinylpyrrolidone (PVP) K40 in the formulation was able to raise the Tg to 152 °C, which is expected to further increase the storage stability of the mAbs. The PD-1 binding activities of the anti-PD-1 mAbs before and after TFFD were not different. While protein loss, likely due to protein binding to glass or plastic vials and the TFF apparatus, was identified, we were able to minimize the loss by increasing mAb content in the powders. Lastly, we show that another mAb, anti-TNF-α, can also be converted to a dry powder with a similar composition by TFFD. We conclude that TFFD can be applied to produce stable aerosolizable dry powders of mAbs for pulmonary delivery.

Freezing techniques are an essential part of biologics manufacturing processes, yet the formation of ice/water interfaces can impart detrimental effects on proteins. However, the absence of chemical and structural differences between ice and liquid water poses the question as to why ice can destabilize proteins. We hypothesize that the destabilizing stress of the ice-liquid water interface does not originate from the ice-water system itself but rather from the air microbubbles present during the freezing process. As the temperature decreases, the dissolved air is expelled from the ice crystal lattices in the form of microbubbles and is subsequently trapped by the advancing ice front. This newly formed air-water interface represents an additional interfacial area for the proteins to be adsorbed onto and denatured. The result showed that freezing at ∼ 1 K/s led to the formation of small circular microbubbles with diameters ranging from 100 µm to 500 µm. In contrast, slower freezing resulted in the formation of larger, elongated millimeter-size bubbles. The reduction of the number of microbubbles was carried out by the deaeration process using agitation under reduced pressure at 20 kPa. The resulting deaerated (i.e., low dissolved air) protein samples were frozen and monitored for the formation of subvisible aggregates using micro-flow imaging (MFI). The results demonstrated that deaerating the samples prior to intermediate freezing (i.e., TFF) reduced the number of aggregates for both highly surface-active and low surface-active proteins (lactoferrin and bovine IgG, respectively). This reduction was more pronounced in spray freeze drying (SFD) than thin-film freezing (TFF), and less apparent in conventional lyophilization.

Dry powder inhalers (DPIs) are one of the most widely used devices for treating respiratory diseases. Thin--film--freezing (TFF) is a particle engineering technology that has been demonstrated to prepare dry powder for inhalation with enhanced physicochemical properties. Aerosol performance, which is indicated by fine particle fraction (FPF) and mass median aerodynamic diameter (MMAD), is an important consideration during the product development process. However, the conventional approach for formulation development requires many trial-and-error experiments, which is both laborious and time consuming. As a state-of-the art technique, machine learning has gained more attention in pharmaceutical science and has been widely applied in different settings. In this study, we have successfully built a prediction model for aerosol performance by using both tabular data and scanning electron microscopy (SEM) images. TFF technology was used to prepare 134 dry powder formulations which were collected as a tabular dataset. After testing many machine learning models, we determined that the Random Forest (RF) model was best for FPF prediction with a mean absolute error of ± 7.251%, and artificial neural networks (ANNs) performed the best in estimating MMAD with a mean absolute error of ± 0.393 μm. In addition, a convolutional neural network was employed for SEM image classification and has demonstrated high accuracy (83.86%) and adaptability in predicting 316 SEM images of three different drug formulations. In conclusion, the machine learning models using both tabular data and image classification were successfully established to evaluate the aerosol performance of dry powder for inhalation. These machine learning models facilitate the product development process of dry powder for inhalation manufactured by TFF technology and have the potential to significantly reduce the product development workload. The machine learning methodology can also be applied to other formulation design and development processes in the future.

MF59® is an oil-in-water (O/W) nanoemulsion-based vaccine adjuvant that is often used in seasonal and pandemic influenza vaccines. We explored the feasibility of developing dry powders of vaccines adjuvanted with MF59 or AddaVax™, a preclinical grade equivalent of MF59 with the same composition and droplet size as MF59, by thin-film freeze-drying (TFFD). Liquid AddaVax alone was successfully converted to a dry powder by TFFD using trehalose as a stabilizing agent while maintaining the droplet size distribution of AddaVax after it was reconstituted. TFFD was then applied to convert liquid AddaVax-adjuvanted vaccines containing either a model antigen (e.g., ovalbumin) or mono-, bi-, and tri-valent recombinant hemagglutinin (rHA) protein-based H1 and/or H3 (universal) influenza vaccine candidates, as well as the MF59-containing Fluad® Quadrivalent influenza vaccine to dry powders. Both antigens and stabilizing agents affected the physical properties of the vaccines (e.g., mean particle size and particle size distribution) after the vaccines were subjected to TFFD. Importantly, the integrity and hemagglutination activity of the rHA antigens did not significantly change and the immunogenicity of reconstituted influenza vaccine candidates was maintained when evaluated in a mouse model. The vaccine dry powder was not sensitive to repeated freezing-and-thawing, in contrast to its liquid counterpart. It is concluded that TFFD can be applied to convert liquid vaccines containing MF59 or AddaVax to dry powders while maintaining the immunogenicity of the vaccines. Ultimately, TFFD technology may be used to prepare dry powders of multivalent universal influenza vaccines.

A bivalent Norovirus vaccine candidate has been developed that contains Norovirus strain GI.1 Norwalk-virus like particles (VLP) and strain GII.4 Consensus VLP adsorbed on aluminum (oxy)hydroxide. The Norwalk and Consensus antigens have different stability profiles, making it challenging to prepare a dry powder form of the Norovirus vaccine while maintaining the potency of both antigens. In the present study, we tested the feasibility of converting the vaccine from a liquid suspension to dry powders by thin-film freeze-drying (TFFD). With the proper amount of trehalose and/or sucrose as cryoprotectant (i.e. sucrose alone at 4.55% or 5.55%, w/v, or trehalose at 3–4% with 0.55% of sucrose), TFFD can be applied to successfully convert the Norovirus vaccine candidate into dry powders without causing antigen loss or particle aggregation, while maintaining the relative potency of both antigens within a specified acceptable range. In an accelerated stability study, the potency of the antigens was also maintained in the specified acceptable range after the dry powders prepared by TFFD in the presence of 5.55% (w/v) of sucrose were stored for eight weeks at 40 °C, 75% relative humidity. It is concluded that it is feasible to apply TFFD to convert the Norovirus vaccine from a liquid suspension to stable dry powders.