Puente-Massaguer Eduard, Strobl Florian, Grabherr Reingard, Striedner Gerald, Lecina Martí, Gòdia Francesc
Departament d'Enginyeria Química, Biològica i Ambiental, Universitat Autònoma de Barcelona, 08193 Barcelona, Spain.
Austrian Centre of Industrial Biotechnology (acib GmbH), 1010 Vienna, Austria.
Nanomaterials (Basel). 2020 Aug 12;10(8):1580. doi: 10.3390/nano10081580.
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 × 10 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.
在杆状病毒表达载体系统（BEVS）中，High Five细胞是生产病毒样颗粒（VLP）的优良宿主。然而，由于杆状病毒与这些纳米颗粒在物理化学性质上存在相似性，高滴度杆状病毒的同时产生会妨碍这些纳米颗粒的纯化。在本研究中，首先对基于转染试剂聚乙烯亚胺（PEI）的瞬时基因表达（TGE）方法进行了优化，以在摇瓶水平上生产HIV-1 VLP。此外，在生物反应器规模上成功证明了通过TGE在High Five细胞中生产VLP，与摇瓶相比，其最大活细胞浓度更高（5.1×10个细胞/毫升），转染效率相同，Gag-eGFP VLP产量提高了1.8倍。High Five细胞的代谢分析表明，与未转染的细胞培养物相比，主要代谢物的消耗减少，当天冬酰胺耗尽时，几种氨基酸的摄取率增加。通过纳米颗粒跟踪分析和流式病毒测定法对所生产的VLP进行质量评估，结果显示其平均大小为100-200纳米，与文献中报道的未成熟HIV-1病毒一致。总体而言，这项工作表明High Five/TGE系统是生产基于VLP的候选疫苗和其他重组蛋白的合适方法。
