9a9499d57f41d1b2ce40b7cfcca366bd pmc_32806511.pdf 4bae6e469be6f71281fc16b2016115fc59c21ec8 pmc_32806511.pdf 9f44f8e38efd7a13f0e9333c02246302a6bc1a29b3fbe92095cd3c9279fefff6 pmc_32806511.pdf Title: PEI-Mediated Transient Transfection of High Five Cells at Bioreactor Scale for HIV-1 VLP Production Subject: High Five cells are an excellent host for the production of virus-like particles (VLPs) with the baculovirus expression vector system (BEVS). However, the concurrent production of high titers of baculovirus hinder the purification of these nanoparticles due to similarities in their physicochemical properties. In this study, first a transient gene expression (TGE) method based on the transfection reagent polyethylenimine (PEI) is optimized for the production of HIV-1 VLPs at shake flask level. Furthermore, VLP production by TGE in High Five cells is successfully demonstrated at bioreactor scale, resulting in a higher maximum viable cell concentration (5.1 106 cell/mL), the same transfection efficiency and a 1.8-fold increase in Gag-eGFP VLP production compared to shake flasks. Metabolism analysis of High Five cells indicates a reduction in the consumption of the main metabolites with respect to non-transfected cell cultures, and an increase in the uptake rate of several amino acids when asparagine is depleted. Quality assessment by nanoparticle tracking analysis and flow virometry of the VLPs produced shows an average size of 100–200 nm, in agreement with immature HIV-1 viruses reported in the literature. Overall, this work demonstrates that the High Five/TGE system is a suitable approach for the production of VLP-based vaccine candidates and other recombinant proteins. Keywords: High Five cells; transient gene expression; polyethylenimine; virus-like particle; bioreactor Author: Eduard Puente-Massaguer, Florian Strobl, Reingard Grabherr, Gerald Striedner, Martí Lecina and Francesc Gòdia Creator: LaTeX with hyperref package Producer: pdfTeX-1.40.18 CreationDate: Wed Aug 12 07:05:31 2020 CEST ModDate: Wed Aug 12 07:05:31 2020 CEST Custom Metadata: yes Metadata Stream: no Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 16 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 3833163 bytes Optimized: no PDF version: 1.5 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- MITBVM+URWPalladioL-Ital Type 1 Custom yes yes no 125 0 ZTFLRV+URWPalladioL-Bold Type 1 Custom yes yes no 126 0 IOVOOE+VnURWPalladioL-Bold Type 1 Custom yes yes no 127 0 GMUNRX+URWPalladioL-Roma Type 1 Custom yes yes no 128 0 TZLIJD+VnURWPalladioL Type 1 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