Process intensification for the production of yellow fever virus‐like particles as potential recombinant vaccine antigen

Yellow fever (YF) is a life‐threatening viral disease endemic in parts of Africa and Latin America. Although there is a very efficacious vaccine since the 1930s, YF still causes 29,000–60,000 annual deaths. During recent YF outbreaks there were issues of vaccine shortage of the current egg‐derived vaccine; rare but fatal vaccine adverse effects occurred; and cases were imported to Asia, where the circulating mosquito vector could potentially start local transmission. Here we investigated the production of YF virus‐like particles (VLPs) using stably transfected HEK293 cells. Process intensification was achieved by combining sequential FACS (fluorescence‐activated cell sorting) rounds to enrich the stable cell pool in terms of high producers and the use of perfusion processes. At shaken‐tube scale, FACS enrichment of cells allowed doubling VLP production, and pseudoperfusion cultivation (with daily medium exchange) further increased VLP production by 9.3‐fold as compared to batch operation mode. At perfusion bioreactor scale, the use of an inclined settler as cell retention device showed operational advantages over an ATF system. A one‐step steric exclusion chromatography purification allowed significant removal of impurities and is a promising technique for future integration of upstream and downstream operations. Characterization by different techniques confirmed the identity and 3D‐structure of the purified VLPs. Negative‐staining transmission electron microscopy (TEM) of SXC‐purified yellow fever virus‐like particles produced by HEK293 cells.