Spray drying of fenofibrate loaded nanostructured lipid carriers

The conversion of aqueous dispersion of nanostructured lipid carriers (NLCs) into dry powder by spray drying could be a useful approach to render NLCs with better physical chemical stability than the aqueous dispersion. In this study, aqueous NLC dispersion containing fenofibrate was converted into dry, easily reconstitutable powder using spray drying. A central composite face centered design (CCFD) was used to investigate the influence of the ratio of lipid to protectant (mannitol and trehalose) and crystallinity of spray-dried powder on the particle size, yield and residual moisture content of the dried powder. A linear relationship (R² = 0.9915) was established between the crystalline content of the spray-dried powders against the ratio of mannitol to trehalose from 3:7 to 10:0 (w/w). Spray drying of NLC aqueous dispersion using a mannitol and trehalose mixture resulted in an increase in particle size of the NLCs after reconstitution in water as compared to that in the initial aqueous dispersion. The decrease in crystallinity of the dry powder by reducing the ratio of mannitol to trehalose could improve the reconstitution of the NLCs in water. However the yield and residual moisture content of dry powder decreased with an increase in the ratio of mannitol to trehalose. Lipid nanoparticles were able to retain the drug incorporation and the prolonged drug release profile after spray drying. The experimental model was robust, and suggested that spray drying is a viable technique for the conversion of NLCs into dry powder.     

The Effect of Formulation Excipients on Protein Stability and Aerosol Performance of Spray-Dried Powders of a Recombinant Humanized Anti-IgE Monoclonal Antibody1

Purpose. To study the effect of trehalose, lactose, and mannitol on the biochemical stability and aerosol performance of spray-dried powders of an anti-IgE humanized monoclonal antibody. Methods. Protein aggregation of spray-dried powders stored at various temperature and relative humidity conditions was assayed by size exclusion chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis. Protein glycation was determined by isoelectric focusing and affinity chromatography. Crystallization was examined by X-ray powder diffraction. Aerosol performance was assessed as the fine particle fraction (FPF) of the powders blended with coarse carrier lactose, and was determined using a multiple stage liquid impinger. Results. Soluble protein aggregation consisting of non-covalent and disulfide-linked covalent dimers and trimers occurred during storage. Aggregate was minimized by formulation with trehalose at or above a molar ratio in the range of 300:1 to 500:1 (excipient:protein). However, the powders were excessively cohesive and unsuitable for aerosol administration. Lactose had a similar stabilizing effect, and the powders exhibited acceptable aerosol performance, but protein glycation was observed during storage. The addition of mannitol also reduced aggregation, while maintaining the FPF, but only up to a molar ratio of 200:1. Further increased mannitol resulted in crystallization, which had a detrimental effect on protein stability and aerosol performance. Conclusions. Protein stability was improved by formulation with carbohydrate. However, a balance must be achieved between the addition of enough stabilizer to improve protein biochemical stability without compromising blended powder aerosol performance.     

Dry powder inhalers of gentamicin and leucine: formulation parameters, aerosol performance and in vitro toxicity on CuFi1 cells

The high hygroscopicity of gentamicin (G) as raw material hampers the production of respirable particles during aerosol generation and prevents its direct use as powder for inhalation in patients suffering from cystic fibrosis (CF). Therefore, this research aimed to design a new dry powder formulation of G studying dispersibility properties of an aminoacid, L-leucine (leu), and appropriate process conditions. Spray-dried powders were characterized as to water uptake, particle size distribution, morphology and stability, in correlation with process parameters. Aerodynamic properties were analyzed both by Single Stage Glass Impinger and Andersen Cascade Impactor. Moreover, the potential cytotoxicity on bronchial epithelial cells bearing a CFTR F508/F508 mutant genotype (CuFi1) were tested. Results indicated that leu may improve the aerosol performance of G-dried powders. The maximum fine particle fraction (FPF) of about 58.3% was obtained when water/isopropyl alcohol 7:3 system and 15-20% (w/w) of leu were used, compared to a FPF value of 13.4% for neat G-dried powders. The enhancement of aerosol efficiency was credited both to the improvement of the powder flowability, caused by the dispersibility enhancer (aminoacid), and to the modification of the particle surface due to the influence of the organic co-solvent on drying process. No significant degradation of the dry powder was observed up to 6 months of storage. Moreover, particle engineering did not affect either the cell viability or cell proliferation of CuFi1 over a 24 h period.     

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.     

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.     

Polyethylene glycol-induced precipitation of interferon alpha-2a followed by vacuum drying: Development of a novel process for obtaining a dry, stable powder

Feasibility studies were performed on the development of a novel process based on polyethylene glycol (PEG)-induced precipitation of proteins followed by vacuum drying in the presence of sugars to obtain dry protein powders. Apparent solubility of interferon alpha-2a (IFNalpha2a) was determined in the presence of various PEGs and the effect of solution pH, ionic strength, and temperature was investigated. IFNalpha2a precipitate was dried at a shelf temperature of 25 degrees C at 100 mTorr either as it is or in the presence of mannitol and/or trehalose. The dried IFNalpha2a formulations were subjected to accelerated stability studies at 40 degrees C (3 months), and the stability was compared with that of a similar lyophilized formulation. The results indicated that more than 90% of the protein could be precipitated using 10% wt/vol PEG the protein could be precipitated using 10% wt/vol PEG 1450 at pH 6.5 at a solution ionic strength of 71 mM. Vacuum drying of the precipitate only resulted in the formation of insoluble aggregates of IFNalpha2a; however, this was prevented by the addition of either mannitol or trehalose. The addition of excess mannitol resulted in low residual moisture content and better handling of the final dried product. Accelerated storage stability did not show any aggregation and showed less than 5% formation of oxidized IFNalpha2a in the dried formulation containing IFNalpha2a: trehalose: mannitol in a 1:10:100 wt/wt ratio upon storage at 40 degrees C for 3 months. The stability of this vacuum dried formulation was comparable with that of a similar lyophilized formulation.     

Effect of Spray Drying and Subsequent Processing Conditions on Residual Moisture Content and Physical/Biochemical Stability of Protein Inhalation Powders

Purpose. To understand the effect of spray drying and powder processing environments on the residual moisture content and aerosol performance of inhalation protein powders. Also, the long-term effect of storage conditions on the powder's physical and biochemical stability was presented. Methods. Excipient-free as well as mannitol-formulated powders of a humanized monoclonal antibody (anti-IgE) and recombinant human deoxyribonuclease (rhDNase) were prepared using a Buchi 190 model spray dryer. Residual moisture content and moisture uptake behavior of the powder were measured using thermal gravimetric analysis and gravimetric moisture sorption isotherm, respectively. Protein aggregation, the primary degradation product observed upon storage, was determined by size-exclusion HPLC. Aerosol performance of the dry powders was evaluated after blending with lactose carriers using a multi-stage liquid impinger (MSLI). Results. Spray-dried powders with a moisture level (~ 3%) equivalent to the freeze-dried materials could only be achieved using high-temperature spray-drying conditions, which were not favorable to large-male manufacturing, or subsequent vacuum drying. These dry powders would equilibrate with the subsequent processing and storage environments regardless of the manufacturing condition. As long as the relative humidity of air during processing and storage was lower than 50%, powders maintained their aerosol performance (fine particle fraction). However, powders stored under drier conditions exhibited better long-term protein biochemical stability. Conclusions. Manufacturing, powder processing, and storage environments affected powder's residual moisture level in a reversible fashion. Therefore, the storage condition determined powder's overall stability, but residual moisture had a greater impact on protein chemical stability than on powder physical stability.     

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.     

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.     

Physical characteristics and aerosolization performance of insulin dry powders for inhalation prepared by a spray drying method

The objective of this study was to investigate the influence of formulation excipients on the physical characteristics and aerosolization performance of insulin dry powders for inhalation. Insulin dry powders were prepared by a spray drying technique using excipients such as sugars (trehalose, lactose and dextran), mannitol and amino acids (L-leucine, glycine and threonine). High performance liquid chromatography and the mouse blood glucose method were used for determination of the insulin content. The powder properties were determined and compared by scanning electron microscopy, thermo-gravimetric analysis and size distribution analysis by a time-of-flight technique. The in-vitro aerosolization behaviour of the powders was assessed with an Aerolizer inhaler using a twin-stage impinger. Powder yield and moisture absorption were also determined. Results showed that there was no noticeable change in insulin content in any of the formulations by both assay methods. All powders were highly wrinkled, with median aerodynamic diameters of 2-4 microm, and consequently suitable for pulmonary administration. The tapped density was reduced dramatically when glycine was added. The powders containing mannitol, with or without L-leucine, were less sensitive to moisture. The highest respirable fraction of 67.3 +/- 1.3% was obtained with the formulation containing L-leucine, in contrast to formulations containing glycine and threonine, which had a respirable fraction of 11.2 +/- 3.9% and 23.5 +/- 2.5%, respectively. In addition, powders with good physical properties were achieved by the combination of insulin and trehalose. This study suggests that L-leucine could be used to enhance the aerosolization behaviour of the insulin dry powders for inhalation, and trehalose could potentially be used as an excipient in the formulations.     

Spray-drying of proteins: effects of sorbitol and trehalose on aggregation and FT-IR amide I spectrum of an immunoglobulin G.

An immunoglobulin G (IgG) was spray-dried on a Buchi 190 laboratory spray-dryer at inlet and outlet air temperatures of 130 and 190 degrees C, respectively. The IgG solution contains initially 115 mg/ml IgG plus 50 mg/ml sorbitol. After dialysis, at least 80% of low molecular weight component was removed. After spray-drying the dialyzed IgG and immediate redissolution of the powder, an increase in aggregates from 1 to 17% occurred. A major shift towards increase beta-sheet structure was detected in the spray-dried solid, which, however, reverted to native structure on redissolution of the powder. A correlation between aggregation determined by size exclusion chromatography and alterations in secondary structure determined by Fourier transformation infra-red spectroscopy could not therefore be established. On spray-drying a non-dialyzed, sorbitol-containing IgG only some 0.7% aggregates were formed. The sorbitol is therefore evidently able to stabilize partially the IgG during the process of spray-drying. Addition of trehalose to the liquid feed produced quantitatively the same stabilizing action on the IgG during spray-drying as did the sorbitol. This finding again points towards a water replacement stabilization mechanism. The IgG spray-dried powder prepared from the dialyzed liquid feed showed continued substantial aggregation on dry storage at 25 degrees C. This was substantially less in the non-dialyzed, sorbitol-containing spray-dried powder. Addition of trehalose to both dialyzed and non-dialyzed system produced substantial improvement in storage stability and reduction in aggregate formation in storage. The quantitative stabilizing effect of the trehalose was only slightly higher than that of the sorbitol. Taken together, these results indicate that both the sorbitol and trehalose stabilize the IgG primarily by a water replacement mechanism rather than by glassy immobilization. The relevance of this work is its questioning of the importance of the usually considered dominance of glassy stabilization of protein in dried systems of high glass transition temperature, such as trehalose. The low glass transition temperature sorbitol produces almost equal process and storage stability in this case.     

Local and systemic delivery of high-molecular weight drugs by powder inhalation

The pulmonary route has recently attracted attention as a noninvasive administration route for peptide and protein drugs, and an insulin powder for inhalation was approved by authorities in Europe and the USA. The present study examined usefulness of insulin and gene powders for systemic and local inhalation therapy. We prepared several dry insulin powders by spray drying to examine the effect of additives on insulin absorption. Citric acid appears to be a safe and potent absorption enhancer for insulin in dry powder. However, in the powder with citric acid (MIC0.2 SD) insulin was unstable compared with the other powders examined. To improve insulin stability, a combination of insulin powder and citric acid powder was prepared (MIC Mix). MIC Mix showed hypoglycemic activity comparable to MIC0.2 SD while the insulin stability was much better than that of MIC SD. Next, dry insulin powders with mannitol were prepared with supercritical carbon dioxide (SCF); the powder thus prepared reduced blood glucose level rapidly and was more effective than that prepared by spray drying. Chitosan-pDNA complex powders as a pulmonary gene delivery system were also prepared with SCF and their in vivo activity was evaluated. The addition of chitosan suppressed the degradation of pCMV-Luc during preparation and increased the storage stability. The luciferase activity in mouse lung was evaluated after pulmonary administration of the powders. The chitosan-pDNA powder with an N/P ratio=5 increased the luciferase activity to 27 times that of the pCMV-Luc solution. These results suggest that gene powder with chitosan is a useful pulmonary gene delivery system.     

Formulation and stabilization of Francisella tularensis live vaccine strain

Francisella tularensis live vaccine strain (F. tularensis LVS), a promising vaccine candidate for protection against F. tularensis exposure, is a particularly thermolabile vaccine and difficult to stabilize sufficiently for storage under refrigerated conditions. Our preliminary data show that F. tularensis LVS can be stabilized in the dried state using foam drying, a modified freeze drying method, with sugar-based formulations. The process was conducted under mild drying conditions, which resulted in a good titer retention following processing. The inclusion of osmolytes in the growth media resulted in an acceleration of growth kinetics, although no change in osmotolerance was observed. The optimized F. tularensis formulation, which contained trehalose, gelatin, and Pluronic F68 demonstrated stability for approximately 1.5 weeks at 37°C (i.e., time required for the vaccine to decrease in potency by 1 log(10) colony forming unit) and for 12 weeks at 25°C. At refrigerator storage condition (4°C), stabilized F. tularensis LVS vaccine exhibited no activity loss for at least 12 weeks. This stabilization method utilizes conventional freeze dryers and pharmaceutically approved stabilizers, and thus can be readily implemented at many manufacturing sites for large-scale production of stabilized vaccines. The improved heat stability of the F. tularensis LVS could mitigate risks of vaccine potency loss during long-term storage, shipping, and distribution.     

A specific molar ratio of stabilizer to protein is required for storage stability of a lyophilized monoclonal antibody

The selection of the appropriate excipient and the amount of excipient required to achieve a 2-year shelf-life is often done by using iso-osmotic concentrations of excipients such as sugars (e.g., 275 mM sucrose or trehalose) and salts. Excipients used for freeze-dried protein formulations are selected for their ability to prevent protein denaturation during the freeze-drying process as well as during storage. Using a model recombinant humanized monoclonal antibody (rhuMAb HER2), we assessed the impact of lyoprotectants, sucrose, and trehalose, alone or in combination with mannitol, on the storage stability at 40 degrees C. Molar ratios of sugar to protein were used, and the stability of the resulting lyophilized formulations was determined by measuring aggregation, deamidation, and oxidation of the reconstituted protein and by infrared (IR) spectroscopy (secondary structure) of the dried protein. A 360:1 molar ratio of lyoprotectant to protein was required for storage stability of the protein, and the sugar concentration was 3-4-fold below the iso-osmotic concentration typically used in formulations. Formulations with combinations of sucrose (20 mM) or trehalose (20 mM) and mannitol (40 mM) had comparable stability to those with sucrose or trehalose alone at 60 mM concentration. A formulation with 60 mM mannitol alone provided slightly less protection during storage than 60 mM sucrose or trehalose. The disaccharide/mannitol formulations also inhibited deamidation during storage to a greater extent than the lyoprotectant formulations alone. The reduction in aggregation and deamidation during storage correlated directly with inhibition of unfolding during lyophilization, as assessed by IR spectroscopy. Thus, it appears that the protein must be retained in its native-like state during freeze-drying to assure storage stability in the dried solid. Long-term studies (23-54 months) performed at 40 degrees C revealed that the appropriate molar ratio of sugar to protein stabilized against aggregation and deamidation for up to 33 months. Therefore, long-term storage at room temperature or above may be achieved by proper selection of the molar ratio and sugar mixture. Overall, a specific sugar/protein molar ratio was sufficient to provide storage stability of rhuMAb HER2.     

Role of trehalose in moisture-induced aggregation of bovine serum albumin.

Moisture-induced aggregation has been identified as a key problem in the long term storage stability of therapeutic proteins. In the present work, we have investigated the impact of the disaccharide trehalose on the aggregation behavior of a model protein, bovine serum albumin (BSA) under moist conditions. About 50% aggregation of BSA was observed at a moisture level of 8mul/10mg protein. Including trehalose in the protein sample caused a significant reduction in aggregation. We address the probable mechanisms for the protective effect of trehalose by considering the various hypotheses that have been proposed in the literature. The techniques that have been used include denaturing and non-denaturing gel electrophoresis and tryptophan intrinsic fluorescence. The nature of the aggregates was studied by carrying out electrophoresis of the aggregated protein in the presence of reducing and chaotropic reagents. The interaction studies of aggregated BSA with Thioflavin T and CongoRed indicate the possibility of amyloid type of character in the former. These studies may explain the protective role of trehalose under conditions where the storage stability of therapeutic proteins is compromised.     

Development of directly compressible powders via co-spray drying.

Continuous production of directly compressible powders was achieved by coprocessing acetaminophen and carbohydrates via spray drying. Binary and ternary powder mixtures containing drug substance and carbohydrates were prepared by co-spray drying and evaluated on spray drying processibility, powder hygroscopicity, flowability, and compactability. The influence of process parameters during spray drying on the compaction behaviour of drug/excipient mixtures was investigated via Heckel analysis. Erythritol, lactose, maltodextrin, and mannitol were efficient in co-spray drying with acetaminophen. However, lactose mixtures showed poor flowability. Spray dried mixtures containing mannitol and erythritol were characterised as non-hygroscopic, highly dense, and good flowing powders. Mannitol increased tablet tensile strength in contrast with the poor compactability of erythritol. Maltodextrin was selected for further experiments because it provided excellent tablet tensile strength. The use of erythritol, maltodextrin and mannitol in binary drug/excipient mixtures resulted in high process yields. Compacts of erythritol, mannitol, and maltodextrin were characterised by higher tablet tensile strength at higher spray drying temperatures due to the increased particle fragmentation of erythritol and mannitol mixtures and to the increased plastic deformation of maltodextrin formulations. A combination of erythritol, maltodextrin, and mannitol was selected for further formulation and process optimisation of co-spray dried powders for direct compression.     

Effects of additives on the stability of Humicola lanuginosa lipase during freeze-drying and storage in the dried solid

The effects of various classes of additives on the stability of a protein with a relatively hydrophobic surface, Humicola lanuginosa lipase (HLL), during lyophilization and storage in the dried solid, were investigated. Prior to lyophilization, it was found that 1 M trehalose or 1% (wt/vol) Tween 20 caused the protein to precipitate. Infrared spectroscopy indicated that trehalose "salted-out" native HLL, whereas Tween 20 induced non-native aggregates. Optimal recovery of native protein in the initial dried solid was obtained in the presence of additives which formed an amorphous phase and which had the capacity to hydrogen bond to the dried protein (e.g., trehalose and sucrose). Additives which crystallized during lyophilization (e.g., mannitol) or which remained amorphous, but were unable to hydrogen bond to the dried protein (e.g., dextran), afforded less stabilization relative to that seen in the absence of additives. Optimal storage stability in the dried solid required that both protein unfolding during lyophilization was minimized and that the formulation was stored at a temperature below its Tg value. Crystallization of sucrose during storage greatly reduced the storage stability of HLL. This was attributed to the increased moisture content and the reduced Tg value in the remaining amorphous phase containing the protein. Sucrose crystallization and the resulting damage to the protein were inhibited by decreasing the mass ratio of sucrose:protein.     

Stability of chitosan-pDNA complex powder prepared by supercritical carbon dioxide process

The present study examined the stability of a gene in powders prepared with supercritical carbon dioxide (CO₂) from the viewpoints of the ternary structure of DNA and in vivo transfection potential. An aqueous chitosan–pCMV-Luc complex solution containing mannitol was injected into the stream of a supercritical CO₂/ethanol admixture to precipitate a gene powder. The obtained gene powders and gene solutions were placed in stability chambers at 25 or 40 °C for 4 weeks. The integrity and transfection potency of the gene were examined by electrophoresis and in vivo pulmonary transfection study in mice. The supercritical CO₂ process decreased the supercoiled DNA during the manufacturing process; however, the decrease in the remaining supercoiled and open circular DNA in the powders during storage was much slower than that in solutions. In addition, the powders had higher transfection potency than the solutions containing the same amount of DNA. The effect of chitosan on the stability of DNA in solutions was not obvious in the solutions but it improved the stability of DNA in powders during manufacturing and storage. Thus, a gene powder with a cationic vector is a promising ready-to-use formulation for inhalation therapy of pulmonary diseases.     

Dextran or hydroxyethyl starch in spray-freeze-dried trehalose/mannitol microparticles intended as ballistic particulate carriers for proteins

The goal of this study was to clarify the effects of dextran 10 kDa on the properties of spray-freeze-dried microparticles for use with ballistic injectors. A novel carrier of trehalose, mannitol, and the polymer is known to maximize particle density. Measurements of T'(g) showed that the dextran anti-plasticizes the trehalose/mannitol, but also undergoes phase separation. The product temperature exceeded T'(g) during primary drying. The collapsed particles can therefore be explained by plastic flow of the freeze concentrate. DSC of the powder showed T(g) at 45 degrees C and, in the first scan, a wide endothermic melting peak caused by mannitol recrystallization. Catalase showed 35% activity loss on rehydration of its spray freeze-drying (SFD) powder, which was improved in the TM/D (3:3:4) formulation, but not up to that level seen with either trehalose or mannitol alone. The dextran 10 kDa, which is vital to maximize particle density, was therefore detrimental to protein integrity during SFD, as also found with a 65-72 kDa dextran. Hydroxyethyl starch (HES) 200 kDa gave similar, limited stabilizing effects on the protein. The proportion of polymer in the formulation should be low to minimize protein damage, whilst high enough to give required particle morphology and density.