4. Conclusions
Considering that yellow fever virus is endemic in many countries in Africa and Latin America and that it could spread in the near future to the other continents where the mosquito vector is already present, a new vaccine is needed to overcome the limited production capacity of the current vaccine manufactured in embryonated eggs. Recombinant virus-like particles (VLPs) are an alternative in this context. In the present work we have shown that HEK293 cells can stably express YF VLPs, and that production process intensification can be achieved by combining two different strategies: (i) sequentially enriching cell pools by FACS to get more homogeneous populations of stable high producers; (ii) developing continuous perfusion processes that can provide higher volumetric productivities, increasing production capacity and decreasing production costs. To perform perfusion cultivations for VLP production, the membrane-based ATF system presented problems of fouling causing underfeeding of the culture and resulting in undesirable product retention. On the other hand, an inclined lamella settler showed promising results and should be deeper explored as a cell retention device for the production of VLPs. Purification by a very simple chromatography technique (steric exclusion chromatography) allowed achieving high purities in a single step. The purified particles were analyzed by different techniques, and their identity and 3D-structure was confirmed.