Production and pharmaceutical formulation of plasmid DNA vaccines

Abstract

Research leading to the thesis ‘Production and pharmaceutical formulation of plasmid DNA vaccines‘ can be divided into two parts. The first part describes the development of a Good Manufacturing Practice (GMP) compliant plasmid DNA production process of pDNA vaccines for the treatment of Human papilloma viruses (HPV) 16 induced malignancies. Furthermore, this thesis focuses on the development of pDNA-polyplex formulations to further improve the transfection efficiency and immunogenicity of intradermally delivered DNA vaccines. The pharmaceutical development of the pDNA vaccine pVAX TTFC E7SH consisted of the improved upstream processing and downstream processing of pDNA. It was shown that the introduction of a transposon from a well-defined host cell in a pDNA vector can occur. In order to prevent costly rejection of clinical material, both the quality control of the source pDNA, the MCB and in-process controls during manufacture of pDNA bulk drug must be sensitive enough to detect a contamination as early as possible. We show that the freeze-dried pDNA formulation has a shelf life stability for more than 5 years when stored at -20°C. To determine if pDNA transfection can be further improved by formulating pDNA into polyplexes, we screened a broad panel of polymers with distinct differences in molecular structure and characteristics. We measured ex vivo human skin transfection efficiency and polymer characteristics (size, PDI, charge) for this panel of polyplex formulations. Based on the finding in this study, PAA-PEG based polyplexes are the most promising candidates for improving pDNA transfection efficiency. Because screening of a large library of polyplexes is a very time-consuming process, the High Content Screening (HCS) method was chosen as an application in our development study. We initially screened for the optimal polymer/pDNA ratio of Poly (amido amine)-polyplexes. In order to obtain a clinically feasible, stable pharmaceutical formulation we investigated the use of an improved buffer system. When formulated in l-histidine buffer, the transfection efficiency of PAA-polyplexes in vitro and in an ex vivo human skin model is enhanced compared to the same polyplexes dispersed in HEPES buffer. The better buffering capacity of l-histidine in the lower pH region of endosomal acidification, together with a higher concentration of cationic charge carriers (polymer and protonated histidine) is hypothesized to result in a more pronounced proton sponge effect and higher polymer-endosomal membrane interactions, giving rise to increased efficiency of endosomal escape and thus transfection. To investigate if lyophilization can preserve our PEGylated PAA-polyplexes, polyplexes formulated with potential lyoprotectants (trehalose, sucrose or HPßCD) and with or without l-histidine-buffer were assessed for physico-chemical characteristics (appearance, size, PDI and charge) as well as transfection efficiency before lyophilization and after reconstitution of the freeze-dried products. The PAA-PEG polyplex formulation with 10 % sucrose in WFI showed a comparable transfection efficiency before lyophilization and after reconstitution of the freeze-dried product. Apparently, the addition of an L-histidine buffer to the freeze-dried polyplexes instead of building it into the lyophilized formulation results in increased transfection efficiency.