(498b) Manufacturing Process Development of Gesicles

Gesicles are small cellular vesicles pseudotyped with the glycoprotein of the vesicular stomatitis virus (VSV-G). These particles are used to efficiently deliver nucleic acids and proteins in several human cell lines. Actually, gesicles are produced by transient transfection of HEK 293A adherent cells using plasmid containing VSV-G DNA sequence, in serum containing media and are purified using iodixanol gradients. Serum-free suspension cultures and chromatographic methods are however more suitable for gesicles manufacturing due to its readiness in upstream/downstream processing and scale-up. Here, we propose to develop a production process of gesicles in serum-free media, using suspension cells and on the other hand, to set up a purification process that can easily be scaled up for industrial purposes. Firstly, in order to produce gesicles, three parameters were investigated: cell density, DNA concentration and DNA/polyethylenimine (PEI) ratio. The highest level of gesicles production was obtained with a cell density of 3x10⁶ cell/mL, a plasmid concentration of 2 µg/mL and a DNA:PEI ratio of 1:4. The production kinetic of gesicles was also performed, showing that the highest quantity of gesicles can be harvested 3 days after transfection. The effect of temperature and the addition of cell culture supplements were evaluated in order to increase the quantity of gesicles produced. Compare to the standard production described above, the gesicles production increase when cells were transfected at 37°C and that 2 mM of sodium butyrate was added in media 18 h later. Secondly, a purification process was established. Gesicles are first cleared and concentrated by 20% sucrose cushion ultracentrifugation and then purified by chromatography. Two types of chromatography column were investigated, a strong anion exchange chromatography (AEX) and a heparin affinity column. Experiments have revealed that heparin purification is better for gesicles purification according to recovery observed with western blot and gesicles activity assay. The spherical shape of purified gesicles was observed using transmission electron microscopy (TEM). Dynamic light scattering analysis showed also that gesicles have a mean size of 180 nm in PBS solution. In conclusion, gesicles are spherical vesicles that can be produced with HEK 293 F suspension cells and be purified with a scalable purification method.