Messenger RNA represents a very attractive therapeutic option for a wide range of clinical applications. Current clinical efforts are focused on vaccination, peptide replacement therapy and treatment of genetic diseases. The clinical translation of mRNA therapeutics has been made possible through improvement of the production and delivery process of mRNA. We believe an investigational study is necessary to directly compare possible different mRNA formats. Therefore we examined the effect from several degrees of mRNA optimization including plasmid engineering, mRNA purification and base modifications on the biological activity, translatability and immunogenicity of the mRNA. Our data showed that for mRNA therapeutics a diverse degree of optimization is needed, which depends on the application. We observed that optimized packaged mRNA leads to enhanced in vitro and in vivo protein expression and a lower immunogenicity. For electroporation purposes, an effect on translation capacity of the mRNA could be seen by plasmid engineering. Although highly pure and non-immunogenic mRNA is favored for clinical application, it is not allowing high induction of T cell responses when delivered intravenously, warranting research into adjuvants for vaccine formulation.