Demonstrating Functional Equivalence of Pilot and Production Scale Freeze-Drying of BCG.

Process analytical technology (PAT)-tools were used to monitor freeze-drying of Bacille Calmette-Guérin (BCG) at pilot and production scale. Among the evaluated PAT-tools, there is the novel use of the vacuum valve open/close frequency for determining the endpoint of primary drying at production scale. The duration of primary drying, the BCG survival rate, and the residual moisture content (RMC) were evaluated using two different freeze-drying protocols and were found to be independent of the freeze-dryer scale evidencing functional equivalence. The absence of an effect of the freeze-dryer scale on the process underlines the feasibility of the pilot scale freeze-dryer for further BCG freeze-drying process optimization which may be carried out using a medium without BCG.

Improved storage stability and immunogenicity of hepatitis B vaccine after spray-freeze drying in presence of sugars.

The current hepatitis B vaccines need to be stored and transported under refrigerated conditions (2-8°C). This dependence on a cold-chain is highly challenging in areas where hepatitis B virus infections are endemic. To decrease the cold-chain dependency, powder formulations of the hepatitis B surface antigen (HBsAg) without aluminum were prepared by spray-freeze drying in the presence of either inulin or a combination of dextran and trehalose. The stability of HBsAg in the amorphous powder formulations was strongly improved during storage both at room temperature and at an elevated temperature (60°C), compared to a liquid plain and an aluminum hydroxide adjuvanted HBsAg formulation. Immunogenicity studies in mice showed that reconstituted powder formulations induced higher IgG immune responses after intramuscular administration than those induced after administration of unprocessed plain antigen. Although the immune response was not as high as after administration of aluminum adjuvanted HBsAg, the immune response to the reconstituted vaccines shifted towards a more balanced Th1/Th2 response compared to the aluminum containing HBsAg formulation.

A comparison between spray drying and spray freeze drying to produce an influenza subunit vaccine powder for inhalation.

The aim of this study was to investigate two different processes to produce a stable influenza subunit vaccine powder for pulmonary immunization i.e. spray drying (SD) and spray freeze drying (SFD). The formulations were analyzed by proteolytic assay, single radial immunodiffusion assay (SRID), cascade impactor analysis, and immunization studies in Balb/c mice. Proteolytic assay and SRID analysis showed that antigen integrity after SFD was best conserved when the formulation was buffered by Hepes buffer saline (HBS). Surprisingly, antigen integrity after SD was better conserved when the formulation was buffered by phosphate buffer saline (PBS) rather than by HBS. The dispersion from the dry powder inhaler, the Twincer, resulted in a fine particle fraction (aerodynamic particle size <5microm) of 37% and 23% for spray dried and spray freeze dried powders, respectively. Immunogenicity of both vaccine formulations (SFD/HBS and SD/PBS) was similar to conventional liquid formulation after i.m. immunization. In addition, compared to i.m. immunizations, the pulmonary immunization with the dry powders resulted in significantly higher IgG titers. Furthermore, both the formulations remained biochemically and physically stable for at least 3years of storage at 20 degrees C. Our results demonstrate that both optimized formulations are stable and have good inhalation characteristics.

Development of stable influenza vaccine powder formulations: challenges and possibilities.

Influenza vaccination represents the cornerstone of influenza prevention. However, today all influenza vaccines are formulated as liquids that are unstable at ambient temperatures and have to be stored and distributed under refrigeration. In order to stabilize influenza vaccines, they can be brought into the dry state using suitable excipients, stabilizers and drying processes. The resulting stable influenza vaccine powder is independent of cold-chain facilities. This can be attractive for the integration of the vaccine logistics with general drug distribution in Western as well as developing countries. In addition, a stockpile of stable vaccine formulations of potential vaccines against pandemic viruses can provide an immediate availability and simple distribution of vaccine in a pandemic outbreak. Finally, in the development of new needle-free dosage forms, dry and stable influenza vaccine powder formulations can facilitate new or improved targeting strategies for the vaccine compound. This review represents the current status of dry stable inactivated influenza vaccine development. Attention is given to the different influenza vaccine types (i.e. whole inactivated virus, split, subunit or virosomal vaccine), the rationale and need for stabilized influenza vaccines, drying methods by which influenza vaccines can be stabilized (i.e. lyophilization, spray drying, spray-freeze drying, vacuum drying or supercritical fluid drying), the current status of dry influenza vaccine development and the challenges for ultimate market introduction of a stable and effective dry-powder influenza vaccine.

Towards an oral influenza vaccine: comparison between intragastric and intracolonic delivery of influenza subunit vaccine in a murine model.

In this paper we investigated to which part of the gastro-intestinal (GI) tract, the upper or lower part, an oral influenza vaccine should be targeted to result in an effective immune response in mice. Our study demonstrates that without adjuvant substantial systemic but low respiratory mucosal immune responses were induced in mice after delivery of influenza subunit vaccine to the upper GI-tract (intragastric) as well as the lower GI-tract (intracolonically). When the vaccine was adjuvanted with Escherichia coli heat-labile enterotoxin (LT) these responses were significantly enhanced. Interestingly, intracolonic administration of vaccine with adjuvant also resulted in enhanced cellular immune responses and the desired Th1-skewing of these responses. Intragastric administration of the adjuvanted vaccine also increased T-helper responses. However, Th1-skewing was absent. In conclusion, the right combination of strong mucosal adjuvant (e.g. LT) and antigen delivery site (e.g. the lower part of the gastro-intestinal tract) might result in effective vaccination via the oral route.

Pulmonary delivery of an inulin-stabilized influenza subunit vaccine prepared by spray-freeze drying induces systemic, mucosal humoral as well as cell-mediated immune responses in BALB/c mice.

In this study pulmonary vaccination with a new influenza subunit vaccine powder was evaluated. Vaccine powder was produced by spray-freeze drying (SFD) using the oligosaccharide inulin as stabilizer. Immune responses after pulmonary vaccination of BALB/c mice with vaccine powder were determined and compared to those induced by intramuscular and pulmonary vaccination with a conventional liquid subunit vaccine. All vaccinations were performed without adjuvant. Pulmonary vaccination with liquid subunit vaccine resulted in systemic humoral (IgG) immune responses similar to intramuscular immunization. In contrast, the vaccine powder delivered by the pulmonary route, induced not only systemic humoral (IgG) responses, but also cell-mediated (Il-4, IFN-gamma) and mucosal immune responses (IgA, IgG). This study demonstrates that the combination of pulmonary antigen delivery and antigen powder production by SFD improves the immunogenic potential of (influenza subunit) antigen. In conclusion, vaccination with a non-adjuvanted SFD subunit vaccine powder by inhalation might be feasible and could be an alternative to conventional parenteral vaccine administration.

Rational design of an influenza subunit vaccine powder with sugar glass technology: preventing conformational changes of haemagglutinin during freezing and freeze-drying.

The development of a stable influenza subunit vaccine in the dry state was investigated. The influence of various carbohydrates, buffer types and freezing rates on the integrity of haemagglutinin after freeze-thawing or freeze-drying was investigated with a range of analytical and immunological methods. The use of fast freezing, Hepes buffer and carbohydrates (trehalose, inulin or dextran) as cryo- and lyoprotectants resulted in a significant reduction or even absence of conformational changes of HA as revealed by the used methods. The subunit vaccine in the powder was shown to remain immunogenic in an in vivo study in mice, using reconstituted powder. Moreover, the HA potency of the influenza subunit vaccine powder was stable for at least 26 weeks at room temperature.