Monteiro Francisca, Bernal Vicente, Chaillet Maxime, Berger Imre, Alves Paula M
iBET, Instituto de Biologia Experimental e Tecnológica, Oeiras, Portugal; Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal.
Departamento de Bioquímica y Biología Molecular B y Imunología, Facultad de Química, Campus Internacional de Excelencia "Mare Nostrum", Universidad de Murcia, Murcia, Spain.
J Biotechnol. 2016 Sep 10;233:34-41. doi: 10.1016/j.jbiotec.2016.06.029. Epub 2016 Jul 1.
The recent approval of vaccines and gene therapy products for human use produced in the Insect Cell-Baculovirus Expression Vector System (IC-BEVS) underlines the high potential and versatility of this platform. The interest in developing robust production processes emerges to cope with manufacturing pressure, as well as stringent product quality guidelines. Previously, we addressed the impact of the baculovirus infection on the physiology of insect host cell lines, identifying key cellular pathways enrolled in heterologous gene/protein expression. In the present work, this knowledge was applied to design tailored media supplementation schemes to boost IC-BEVS production yields and quality of enveloped viral particles: influenza VLPs (Inf-VLP) and baculovirus vectors (BV). The addition of reduced glutathione, antioxidants and polyamines increased the cell specific yields of baculovirus particles up to 3 fold. Cholesterol was identified as the most critical system booster, capable of improving 2.5 and 6-fold cell specific yields of BV and Inf-VLPs, respectively. Surprisingly, the combination of polyamines and cholesterol supplementation improved baculovirus stock quality, by preventing the accumulation of non-infectious particles during viral replication while selectively increasing infectious particles production. In addition, the specific yields of both enveloped viral particles, BVs and Inf-VLPs, were also increased. The correlation between supplement addition and systems productivity was extensively analyzed, providing a critical assessment on final product quantity and quality as drivers of bioprocess optimization efforts.
近期，昆虫细胞-杆状病毒表达载体系统（IC-BEVS）生产的用于人类的疫苗和基因治疗产品获得批准，这凸显了该平台的巨大潜力和多功能性。为应对生产压力以及严格的产品质量准则，人们对开发稳健的生产工艺产生了浓厚兴趣。此前，我们研究了杆状病毒感染对昆虫宿主细胞系生理的影响，确定了参与异源基因/蛋白质表达的关键细胞途径。在本研究中，我们运用这些知识设计了定制的培养基补充方案，以提高IC-BEVS生产包膜病毒颗粒（流感病毒样颗粒（Inf-VLP）和杆状病毒载体（BV））的产量和质量。添加还原型谷胱甘肽、抗氧化剂和多胺可使杆状病毒颗粒的细胞特异性产量提高3倍。胆固醇被确定为最关键的系统促进剂，分别能够将BV和Inf-VLP的细胞特异性产量提高2.5倍和6倍。令人惊讶的是，多胺和胆固醇补充剂的组合提高了杆状病毒储备的质量，通过防止病毒复制过程中非感染性颗粒的积累，同时选择性地增加感染性颗粒的产生。此外，包膜病毒颗粒BV和Inf-VLP的特异性产量也有所提高。我们广泛分析了补充剂添加与系统生产力之间的相关性，为作为生物工艺优化努力驱动力的最终产品数量和质量提供了关键评估。
