Stable spray dried protein formulation and implementation in vaccine development

Proteins and peptides have escalated as an important class of therapeutic drugs for treating several chronic and acute illnesses. However, these macromolecules mostly have to be stored and transported under refrigeration as they are unstable at ambient temperatures. Limited storage stability also impedes their commercialisation. To increase their stability, biologicals are often formulated in dried state which generally requires stabilising excipients to protect proteins during drying. This thesis reports a successful development of biologically active and stable protein formulation using suitable excipients during spray drying. In this work, the ability of amino acids (ranging from large hydrophobic to small hydrophilic) and their combinations to stabilise proteins during spray drying was evaluated. On spray drying with the combination of arginine and glycine it could be shown that model proteins catalase and lysozyme maintain their activity on drying and remain stable after storage. It was shown that an influenza A (H1N1) vaccine (Pandemrix) in spray dried form is more stable compared to classical liquid vaccine which completely loses its activity especially when stored for 1 month at stress conditions whereas the dried formulation is found to be stable for more than 3 months under such harsh conditions. Surface activity of these amino acids protects the proteins from interfacial stress during spray droplet formation combined with water replacement mechanisms and creation of an amorphous matrix to ensure stability in the dried state. This combination is tailored to the needs of protein stabilisation during spray drying and may hence be utilised in dry powder formulation of biomolecules with superior stability characteristics. Stabilising and Protecting Solution (SPS; Leukocare, Germany) formulation technology which makes use of the stabilisation effect of amino acids was also evaluated as a potential spray drying protecting excipient. The effectiveness of SPS as a stabilising excipient for proteins during spray drying was established first using the model protein (ovalbumin) and then the results were transferred to influenza A (H1N1) vaccine. Several characterisation methods such as gel-electrophoresis, size exclusion chromatography, dynamic light scattering and a functional assay were used to optimise the dried formulation and to evaluate the long term storage stability. It could be shown by haemagglutination assay that the Pandemrix vaccine spray dried with SPS is stable and as functional as the original liquid vaccine. Terminal sterilisation was also possible for this formulation without harming the vaccine. Storage data showed high stability of protected vaccines at 2-8°C and 25°C/60% rH, for at least six months. The preclinical in vivo study (done in cooperation with Public Health England, Proton Down, UK) showed that effectiveness of the dried influenza vaccine is comparable to that of the original liquid vaccine. Animals receiving original Pandemrix exhibited expected levels of seroconversion after 21 days (prime) and 48 days (boost) as assessed by haemagglutination inhibition assay. Boost immunisation with SPS-protected vaccine resulted in a strong increase in seroconversion but had only minor effects in animals treated with non SPS-protected vaccine. The results indicate the potential utility of spray-dried and terminally sterilised vaccines stabilised by mixtures of amino acids which likely solve the problems associated with the long term instability and cold chain requirement of liquid vaccines.