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Its manufacture is based on embryonated chicken eggs or adherent Vero cells. Both processes are unsuitable for rapid and scalable supply. Here, we introduce a high-throughput workflow to identify suspension cells that are fit for the high-yield production of live YF17D-based vaccines in an intensified upstream process. The use of an automated parallel ambr15 microbioreactor system for screening and process optimization has led to the identification of two promising cell lines (AGE1.CR.pIX and HEKDyn) and the establishment of optimized production conditions, which have resulted in a &gt;100-fold increase in virus titers compared to the current state of the art using adherent Vero cells. The process can readily be scaled up from the microbioreactor scale (15 mL) to 1 L stirred tank bioreactors. The viruses produced are genetically stable and maintain their favorable safety and immunogenicity profile, as demonstrated by the absence of neurovirulence in suckling BALB\/c mice and consistent seroprotection in AG129 mice. In conclusion, the presented workflow allows for the rapid establishment of a robust, scalable, and high-yield process for the production of live-attenuated orthoflavivirus vaccines, which outperforms current standards. The approach described here can serve as a model for the development of scalable processes and the optimization of yields for other virus-based vaccines that face challenges in meeting growing demands.<\/jats:p>","DOI":"10.3390\/vaccines12070755","type":"journal-article","created":{"date-parts":[[2024,7,9]],"date-time":"2024-07-09T11:17:09Z","timestamp":1720523829000},"page":"755","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":5,"title":["Parallel Multifactorial Process Optimization and Intensification for High-Yield Production of Live YF17D-Vectored Zika Vaccine"],"prefix":"10.3390","volume":"12","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-2264-5569","authenticated-orcid":false,"given":"Sven","family":"G\u00f6bel","sequence":"first","affiliation":[{"name":"Bioprocess Engineering, Max Planck Institute for Dynamics of Complex Technical Systems, Sandtorstr. 1, 39106 Magdeburg, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0005-1448-4805","authenticated-orcid":false,"given":"Ozeir","family":"Kazemi","sequence":"additional","affiliation":[{"name":"KU Leuven Department of Microbiology, Immunology & Transplantation, Rega Institute, Molecular Vaccinology and Vaccine Discovery (MVVD), 3000 Leuven, Belgium"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0912-2632","authenticated-orcid":false,"given":"Ji","family":"Ma","sequence":"additional","affiliation":[{"name":"KU Leuven Department of Microbiology, Immunology & Transplantation, Rega Institute, Molecular Vaccinology and Vaccine Discovery (MVVD), 3000 Leuven, Belgium"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2711-7252","authenticated-orcid":false,"given":"Ingo","family":"Jordan","sequence":"additional","affiliation":[{"name":"ProBioGen AG, 13086 Berlin, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3889-2078","authenticated-orcid":false,"given":"Volker","family":"Sandig","sequence":"additional","affiliation":[{"name":"ProBioGen AG, 13086 Berlin, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jasmine","family":"Paulissen","sequence":"additional","affiliation":[{"name":"KU Leuven Department of Microbiology, Immunology & Transplantation, Rega Institute, Translational Platform Virology and Chemotherapy (TPVC), 3000 Leuven, Belgium"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4933-6448","authenticated-orcid":false,"given":"Winnie","family":"Kerstens","sequence":"additional","affiliation":[{"name":"KU Leuven Department of Microbiology, Immunology & Transplantation, Rega Institute, Translational Platform Virology and Chemotherapy (TPVC), 3000 Leuven, Belgium"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5785-8276","authenticated-orcid":false,"given":"Hendrik","family":"Thibaut","sequence":"additional","affiliation":[{"name":"KU Leuven Department of Microbiology, Immunology & Transplantation, Rega Institute, Translational Platform Virology and Chemotherapy (TPVC), 3000 Leuven, Belgium"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6538-1332","authenticated-orcid":false,"given":"Udo","family":"Reichl","sequence":"additional","affiliation":[{"name":"Bioprocess Engineering, Max Planck Institute for Dynamics of Complex Technical Systems, Sandtorstr. 1, 39106 Magdeburg, Germany"},{"name":"Bioprocess Engineering, Otto-von-Guericke University, Universit\u00e4tsplatz 2, 39106 Magdeburg, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8117-9166","authenticated-orcid":false,"given":"Kai","family":"Dallmeier","sequence":"additional","affiliation":[{"name":"KU Leuven Department of Microbiology, Immunology & Transplantation, Rega Institute, Molecular Vaccinology and Vaccine Discovery (MVVD), 3000 Leuven, Belgium"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2652-5943","authenticated-orcid":false,"given":"Yvonne","family":"Genzel","sequence":"additional","affiliation":[{"name":"Bioprocess Engineering, Max Planck Institute for Dynamics of Complex Technical Systems, Sandtorstr. 1, 39106 Magdeburg, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2024,7,9]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"601","DOI":"10.1056\/NEJMp1600297","article-title":"Zika Virus in the Americas\u2014Yet Another Arbovirus Threat","volume":"374","author":"Fauci","year":"2016","journal-title":"N. 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