Suárez-Patiño Sandra Fernanda, Bernardino Thaissa Consoni, Núñez Eutimio Gustavo Fernández, Astray Renato Mancini, Pereira Carlos Augusto, Soares Hugo R, Coroadinha Ana S, Jorge Soraia Attie Calil
Laboratório de Imunologia Viral, Instituto Butantan, Av. Vital Brasil 1500, São Paulo, 05503-900, Brazil.
Grupo de Engenharia Bioquímica, Escola de Artes, Ciências e Humanidades (EACH), Universidade de São Paulo, São Paulo, Brazil.
Cytotechnology. 2019 Oct;71(5):949-962. doi: 10.1007/s10616-019-00337-y. Epub 2019 Aug 17.
The production of biopharmaceuticals as vaccines in serum-free media results in reduced risk of contamination and simpler downstream processing. The production of enveloped viruses and viral vectors such as Semliki Forest Virus (SFV) typically requires lipids that are provided by supplementation with animal serum, so production under serum-free conditions is challenging. In this work, the capacity to deliver genetic material of SFV-viral replicon particles (SFV-VRPs) produced in BHK-21 cells adapted to serum-free medium (BHK/SFM) was evaluated. Three transgenes were evaluated: GFP used as a model protein, while hepatitis C virus nonstructural protein 3 protease domain (HCV-NS3p) and rabies virus glycoprotein (RVGP) were selected based on their distinct nature (enzyme and glycoprotein, respectively). BHK/SFM cells produced a sevenfold higher number of SFV-VRPs, as determined by qRT-PCR. These particles showed similar capacities of infecting BHK/FBS or BHK/SFM cells. GFP expression was evaluated by flow cytometry, HCV-NS3p activity by enzymatic assay, and RVGP expression by ELISA and Western Blot. Expression analysis revealed higher levels of GFP and HCV-NS3p in BHK/SFM, while the levels of RVGP were similar for BHK/SFM and BHK/FBS. In conclusion, the BHK/SFM cells showed increased SFV-VRP production yields, without affecting vector infectivity or heterologous gene expression, hence validating the use of BHK/SFM for industrial applications.
在无血清培养基中生产作为疫苗的生物制药产品可降低污染风险并简化下游加工过程。生产包膜病毒和病毒载体，如Semliki森林病毒（SFV），通常需要通过添加动物血清来提供脂质，因此在无血清条件下生产具有挑战性。在这项工作中，评估了在适应无血清培养基的BHK-21细胞（BHK/SFM）中产生的SFV病毒复制子颗粒（SFV-VRPs）传递遗传物质的能力。评估了三个转基因：用作模型蛋白的绿色荧光蛋白（GFP），而丙型肝炎病毒非结构蛋白3蛋白酶结构域（HCV-NS3p）和狂犬病病毒糖蛋白（RVGP）则根据它们的不同性质（分别为酶和糖蛋白）进行选择。通过qRT-PCR测定，BHK/SFM细胞产生的SFV-VRPs数量高出七倍。这些颗粒显示出感染BHK/FBS或BHK/SFM细胞的相似能力。通过流式细胞术评估GFP表达，通过酶促测定评估HCV-NS3p活性，通过ELISA和蛋白质印迹评估RVGP表达。表达分析显示BHK/SFM中GFP和HCV-NS3p的水平较高，而BHK/SFM和BHK/FBS中RVGP的水平相似。总之，BHK/SFM细胞显示出SFV-VRP产量增加，而不影响载体感染性或异源基因表达，因此验证了BHK/SFM在工业应用中的用途。
