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.