Departament of Chemical Engineering, Federal University of São Carlos, São Carlos, SP, 13565-905, Brazil.
Laboratory of Viral Immunology, Butantan Institute, São Paulo, SP, 05503-900, Brazil.
Appl Microbiol Biotechnol. 2018 Jun;102(11):4773-4783. doi: 10.1007/s00253-018-8962-0. Epub 2018 Apr 19.
The transmembrane rabies virus glycoprotein (RVGP) is the main antigen of vaccine formulations used around the world to prevent rabies, the most lethal preventable infectious disease known. The objective of this work was to evaluate the potential of a bioreactor using wave-induced agitation in the initial steps of scaling up the rRVGP production process by a Drosophila melanogaster S2 cell line to produce rRVGP in sufficient quantities for immunization and characterization studies. Taking advantage of some remarkable features recognized in Drosophila S2 cells for scaling the culture process, a robust recombinant lineage (S2MtRVGPH-His) engineered by our group for the expression of rRVGP using a copper-inducible promoter was used in the bioreactor cultures. The WAVE Bioreactor was chosen because it represents an innovative approach to the cultivation of animal cells using single-use technology. For that purpose, we firstly established a procedure for culturing the S2MtRVGPH-His lineage in 100 mL Schott flasks. Using an inoculum of 5 × 10 cells/mL in culture medium (Sf900-III) induced with solution of CuSO (0.7 mM) and a convenient pH range (6.2-7.0), optimal parameter values such as time of induction (72 h) and temperature (28 °C) to increase rRVGP production could be defined. This procedure was reproduced in culture experiments conducted in a WAVE Bioreactor™ 2/10 using a 2 L Cellbag. The results in Schott flasks and in WAVE Bioreactor™ were very similar, yielding a maximum titer of rRVGP above of 1 mg.L. The immunization study showed that the rRVGP produced in the bioreactor was of high immunogenic quality.
跨膜狂犬病病毒糖蛋白（RVGP）是世界范围内用于预防狂犬病的疫苗制剂的主要抗原，狂犬病是已知最致命的可预防传染病。本工作的目的是评估使用波激搅拌的生物反应器在使用黑腹果蝇 S2 细胞系扩大 rRVGP 生产过程中的初始步骤中的潜力，以生产足够数量的 rRVGP 用于免疫和特征研究。利用在果蝇 S2 细胞中为放大培养过程而识别出的一些显著特征，我们小组利用铜诱导启动子设计了一个稳健的重组系（S2MtRVGPH-His）用于表达 rRVGP，该系用于生物反应器培养。选择 WAVE 生物反应器是因为它代表了一种使用一次性技术培养动物细胞的创新方法。为此，我们首先建立了在 100 mL Schott 瓶中培养 S2MtRVGPH-His 系的程序。在诱导培养基（Sf900-III）中使用 5×10 个细胞/mL 的接种物，并用 0.7 mM 的 CuSO 溶液诱导，并将 pH 值控制在 6.2-7.0 范围内，可以定义增加 rRVGP 生产的最佳参数值，如诱导时间（72 h）和温度（28°C）。该程序在 WAVE 生物反应器 2/10 中使用 2 L Cellbag 进行的培养实验中得到了复制。Schott 瓶和 WAVE 生物反应器中的结果非常相似，rRVGP 的最大滴度超过 1 mg.L。免疫研究表明，在生物反应器中生产的 rRVGP 具有很高的免疫原性。
