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.

Evaluation of Gallium Citrate Formulations against a Multidrug-Resistant Strain of Klebsiella pneumoniae in a Murine Wound Model of Infection.

Skin and soft tissue infections (SSTIs) are a common occurrence in health care facilities with a heightened risk for immunocompromised patients. Klebsiella pneumoniae has been increasingly implicated as the bacterial agent responsible for SSTIs, and treatment can be challenging as more strains become multidrug resistant (MDR). Therefore, new treatments are needed to counter this bacterial pathogen. Gallium complexes exhibit antimicrobial activity and are currently being evaluated as potential treatment for bacterial infections. In this study, we tested a topical formulation containing gallium citrate (GaCi) for the treatment of wounds infected with K. pneumoniae. First, the MIC against K. pneumoniae ranged from 0.125 to 2.0 µg/ml GaCi. After this in vitro efficacy was established, two topical formulations with GaCi (0.1% [wt/vol] and 0.3% [wt/vol]) were tested in a murine wound model of MDR K. pneumoniae infection. Gross pathology and histopathology revealed K. pneumoniae-infected wounds appeared to close faster with GaCi treatment and were accompanied by reduced inflammation compared to those of untreated controls. Similarly, quantitative indications of infection remediation, such as reduced weight loss and wound area, suggested that treatment improved outcomes compared to those of untreated controls. Bacterial burdens were measured 1 and 3 days following inoculation, and a 0.5 to 1.5 log reduction of CFU was observed. Lastly, upon scanning electron microscopy analysis, GaCi treatment appeared to prevent biofilm formation on dressings compared to those of untreated controls. These results suggest that with more preclinical testing, a topical application of GaCi may be a promising alternative treatment strategy for K. pneumoniae SSTI.

Using spectroscopic and microscopic methods to probe the structural stability of human adenovirus type 4.

Adenovirus serotype 4 (Ad4) is a major cause of Ad-associated human diseases. Ad4 is also considered to be a potential delivery vector for gene therapy. In this study, multiple spectroscopic techniques together with transmission electron microscopy (TEM) were employed to probe viral stability and to improve pharmaceutical formulations of Ad4-based vaccines and DNA carriers. Perturbations of secondary, tertiary and quaternary structure of Ad4 proteins induced by elevated temperatures over a wide pH range (3-8) were analyzed using circular dichroism, UV absorption and intrinsic and extrinsic fluorescence spectroscopy as well as static and dynamic light scattering. The spectroscopic results obtained indicate a decrease in Ad4 stability as pH increases from 4 to 8, similar to the behavior reported previously for Ad2 and Ad5, although the Ad4 virion appears to possess slightly more tolerance to thermal stress. An empirical phase diagram (EPD) approach was used to summarize the data in the form of a colored map. In addition, the different physical states of Ad4 identified by the EPD were confirmed by TEM images. The results obtained in this study reveal both structural similarities among three commonly employed Ad subtypes (2, 4 and 5) as well as unique properties of Ad4.

Biophysical characterization of rotavirus serotypes G1, G3 and G4.

The stability of attenuated virus vaccines has traditionally been assessed by a plaque assay to measure the virus's loss of replication competency in response to a variety of environmental perturbations. Although this method provides information regarding the impact of the vaccine formulation, it involves an empirical approach to evaluate stability. Biophysical studies on the other hand have the potential to provide insight into the mechanisms of inactivation of a viral vaccine in response to a variety of stressed conditions. Herein, we have employed a variety of spectroscopic techniques (i.e., circular dichroism, fluorescence spectroscopy and dynamic light scattering) for a comprehensive examination of the thermal stability of three live-attenuated human-bovine reassortant rotavirus strains (G1, G3 and G4) in the 5-8 pH range. The spectroscopic methods employed are not specific and response changes reflect an average change over the entire virus structure. The present work, however, suggests the utility of these methods in early formulation of rotaviral vaccines due to their ability to identify regions of marginal stability over which high throughput excipient screening assays can be designed. We have further shown that these methods are sufficiently sensitive to differentiate the stability of the three homologous G-subtypes differing only in the composition of their surface antigenic proteins. The data from these spectroscopic methods are also compared to biological activity using a tissue culture viral infectivity assay. Partial correlation between the structural alterations and losses in activity are observed, further suggesting the utility of biophysical studies in early formulation studies of rotavirus vaccines.

Drying-induced variations in physico-chemical properties of amorphous pharmaceuticals and their impact on stability (I): stability of a monoclonal antibody.

The present study was conducted to investigate the impact of drying method and formulation on the storage stability of IgG1. Formulations of IgG1 with varying levels of sucrose with and without surfactant were dried by different methods, namely freeze drying, spray drying, and foam drying. Dried powders were characterized by thermal analysis, scanning electron microscopy, specific surface area (SSA) analysis, electron spectroscopy for chemical analysis (ESCA), solid state FTIR, and molecular mobility measurements by both isothermal calorimetry and incoherent elastic neutron scattering. Dried formulations were subjected to storage stability studies at 40 degrees C and 50 degrees C (aggregate levels were measured by size exclusion chromatography initially and at different time points). Both drying method and formulation had a significant impact on the properties of IgG1 powders, including storage stability. Among the drying methods, SSA was highest and perturbations in secondary structure were lowest with the spray-dried preparations. Sucrose-rich foams had the lowest SSA and the lowest protein surface accumulation. Also, sucrose-rich foams had the lowest molecular mobility (both fast dynamics and global motions). Stability studies showed a log-linear dependence of physical stability on composition. Preparations manufactured by "Foam Drying" were the most stable, regardless of the stabilizer level. In protein-rich formulations, freeze-dried powders showed the poorest storage stability and the stability differences were correlated to differences in secondary structure. In stabilizer-rich formulations, stability differences were best correlated to differences in molecular mobility (fast dynamics) and total protein surface accumulation.

Stabilization challenges and formulation strategies associated with oral biologic drug delivery systems.

Delivery of proteins to mucosal tissues of GI tract typically utilize formulations which protect against proteolysis and target the mucosal tissues. Using case studies from literature and the authors' own work, the in-process stability and solid state storage stability of biopharmaceuticals formulated in delivery systems designed for oral delivery to the GI tract will be reviewed. Among the range of delivery systems, biodegradable polymer systems for protection and controlled release of proteins have been the most studied; hence these systems will be covered in greater depth. These delivery systems include polymeric biodegradable microspheres or nanospheres that contain proteins or vaccines, which are designed to reduce the number of administrations/inoculations and the total protein dose required to achieve the desired biological effect. Specifically, this review will include a landscape survey of the systems that have been studied, the manufacturing processes involved, stability through the manufacturing process, key pharmaceutical formulation parameters that impact stability of the encased proteins, and storage stability of the encapsulated proteins in these delivery systems.

Gallium-based anti-infectives: targeting microbial iron-uptake mechanisms.

Microbes have evolved elaborate iron-acquisition systems to sequester iron from the host environment using siderophores and heme uptake systems. Gallium(III) is structurally similar to iron(III), except that it cannot be reduced under physiological conditions, therefore gallium has the potential to serve as an iron analog, and thus an anti-microbial. Because Ga(III) can bind to virtually any complex that binds Fe(III), simple gallium salts as well as more complex siderophores and hemes are potential carriers to deliver Ga(III) to the microbes. These gallium complexes represent a new class of anti-infectives that is different in mechanism of action from conventional antibiotics. Simple gallium salts such as gallium nitrate, maltolate, and simple gallium siderophore complexes such as gallium citrate have shown good antibacterial activities. The most studied complex has been gallium citrate, which exhibits broad activity against many Gram negative bacteria at ∼1-5µg/ml MICs, strong biofilm activity, low drug resistance, and efficacy in vivo. Using the structural features of specific siderophore and heme made by pathogenic bacteria and fungi, researchers have begun to evaluate new gallium complexes to target key pathogens. This review will summarize potential iron-acquisition system targets and recent research on gallium-based anti-infectives.

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.

Room temperature stabilization of oral, live attenuated Salmonella enterica serovar Typhi-vectored vaccines.

Foam drying, a modified freeze drying process, was utilized to produce a heat-stable, live attenuated Salmonella Typhi 'Ty21a' bacterial vaccine. Ty21a vaccine was formulated with pharmaceutically approved stabilizers, including sugars, plasticizers, amino acids, and proteins. Growth media and harvesting conditions of the bacteria were also studied to enhance resistance to desiccation stress encountered during processing as well as subsequent storage at elevated temperatures. The optimized Ty21a vaccine, formulated with trehalose, methionine, and gelatin, demonstrated stability for approximately 12 weeks at 37°C (i.e., time required for the vaccine to decrease in potency by 1log(10)CFU) and no loss in titer at 4 and 25°C following storage for the same duration. Furthermore, the foam dried Ty21a elicited a similar immunogenic response in mice as well as protection in challenge studies compared to Vivotif™, the commercial Ty21a vaccine. The enhanced heat stability of the Ty21a oral vaccine, or Ty21a derivatives expressing foreign antigens (e.g. anthrax), could mitigate risks of vaccine potency loss during long-term storage, shipping, delivery to geographical areas with warmer climates or during emergency distribution following a bioterrorist attack. Because the foam drying process is conducted using conventional freeze dryers and can be readily implemented at any freeze drying manufacturing facility, this technology appears ready and appropriate for large scale processing of foam dried vaccines.

Biophysical characterization and conformational stability of Ebola and Marburg virus-like particles.

The filoviruses, Ebola virus and Marburg virus, cause severe hemorrhagic fever with up to 90% human mortality. Virus-like particles of EBOV (eVLPs) and MARV (mVLPs) are attractive vaccine candidates. For the development of stable vaccines, the conformational stability of these two enveloped VLPs produced in insect cells was characterized by various spectroscopic techniques over a wide pH and temperature range. Temperature-induced aggregation of the VLPs at various pH values was monitored by light scattering. Temperature/pH empirical phase diagrams (EPDs) of the two VLPs were constructed to summarize the large volume of data generated. The EPDs show that both VLPs lose their conformational integrity above about 50°C-60°C, depending on solution pH. The VLPs were maximally thermal stable in solution at pH 7-8, with a significant reduction in stability at pH 5 and 6. They were much less stable in solution at pH 3-4 due to increased susceptibility of the VLPs to aggregation. The characterization data and conformational stability profiles from these studies provide a basis for selection of optimized solution conditions for further vaccine formulation and long-term stability studies of eVLPs and mVLPs.

Heat-stable measles vaccine produced by spray drying.

A combination of unique stabilizers and mild spray drying process conditions was employed to produce heat-stable measles vaccine powder. Live attenuated measles vaccine from Serum Institute of India was formulated with pharmaceutically approved stabilizers, including sugars, proteins, amino acids, polymers, surfactants, and plasticizers, as well as charged ions. In addition, the effects of buffer salt and pH on the storage stability of measles virus were examined. The potency of the dried vaccine stored at several temperatures was quantified by TCID(50) assay on Vero cells. As a comparison to other process methods, lead formulations were also subjected to freeze drying and foam drying. The optimized measles vaccine formulation tested at 37 degrees C was stable for approximately 8 weeks (i.e. time for 1 log TCID(50) loss). The measles titer decreased in a bi-phasic manner, with initial rapid loss within the first week but relative stability thereafter. Key stabilizers identified during the formulation screening processes were L-arginine, human serum albumin, and a combination of divalent cations. Spray drying was identified as the optimal processing method for the preparation of dried vaccine, as it generally resulted in negligible process loss and comparable, if not better storage stability, with respect to the other processes. Processing methods and formulation components were developed that produced a measles vaccine stable for up to 8 weeks at 37 degrees C, which surpassed the WHO requirement for heat stability of 1 week at that temperature.

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 degrees 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 (approximately 2.8 m(2)/g) and lowest in the foam dried preparations (with or without surfactant) (approximately 0.1 m(2)/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 degrees C and 37 degrees 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.

Expression and characterization of a low molecular weight recombinant human gelatin: development of a substitute for animal-derived gelatin with superior features.

Gelatin is used as a stabilizer in several vaccines. Allergic reactions to gelatins have been reported, including anaphylaxis. These gelatins are derived from animal tissues and thus represent a potential source of contaminants that cause transmissible spongiform encephalopathies. We have developed a low molecular weight human sequence gelatin that can substitute for the animal sourced materials. A cDNA fragment encoding 101 amino acids of the human proalpha1 (I) chain was amplified, cloned into plasmid pPICZalpha, integrated into Pichia pastoris strain X-33, and isolates expressing high levels of recombinant gelatin FG-5001 were identified. Purified FG-5001 was able to stabilize a live attenuated viral vaccine as effectively as porcine gelatin. This prototype recombinant gelatin was homogeneous with respect to molecular weight but consisted of several charge isoforms. These isoforms were separated by cation exchange chromatography and found to result from a combination of truncation of the C-terminal arginine and post-translational phosphorylation. Site-directed mutagenesis was used to identify the primary site of phosphorylation as serine residue 546; serine 543 was phosphorylated at a low level. A new construct was designed encoding an engineered gelatin, FG-5009, with point mutations that eliminated the charge heterogeneity. FG-5009 was not recognized by antigelatin IgE antibodies from children with confirmed gelatin allergies, establishing the low allergenic potential of this gelatin. The homogeneity of FG-5009, the ability to produce large quantities in a reproducible manner, and its low allergenic potential make this a superior substitute for the animal gelatin hydrolysates currently used to stabilize many pharmaceuticals.