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.