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.
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