PDF(Pubmed)
Abstract：
Herpes simplex virus type 1 (HSV-1) plays an important role in the field of gene therapy and viral vaccines, especially as an oncolytic virus. However, the mass production of HSV-1 viral vectors remains a challenge in the industry. In this study, a microcarrier-mediated serum-reduced medium culture was used to improve the bioprocess of HSV-1 production and increase HSV-1 yields. The composition of the culture media, which included a basal medium, serum concentration, and glutamine additive, was optimized. The process was successfully conducted in a 1 L bioreactor, and virus production was threefold greater than that of conventional processes with a 10% serum medium. The bead-to-bead transfer process was also developed to further increase scalability. In spinner flasks, the detachment rate increased from 49.4 to 80.6% when combined agitation was performed during digestion; the overall recovery proportion increased from 37.9 to 71.1% after the operational steps were optimized. Specifically, microcarrier loss was reduced during aspiration and transfer, and microcarriers and detached cells were separated with filters. Comparable cell growth was achieved with the baseline process using 2D culture as the inoculum by exchanging the subculture medium. To increase virus production after bead-to-bead transfer, critical parameters, including shear stress during digestion, TrypLE and EDTA concentrations in the subculture, and the CCI, were identified from 47 parameters via correlation analysis and principal component analysis. The optimized bead-to-bead transfer process achieved an average of 90.4% overall recovery and comparable virus production compared to that of the baseline process. This study is the first to report the optimization of HSV-1 production in Vero cells cultured on microcarriers in serum-reduced medium after bead-to-bead transfer. KEY POINTS: • An HSV-1 production process was developed that involves culturing in serum-reduced medium, and this process achieved threefold greater virus production than that of traditional processes. • An indirect bead-to-bead transfer process was developed with over 90% recovery yield in bioreactors. • HSV-1 production after bead-to-bead transfer was optimized and was comparable to that achieved with 2D culture as inoculum.
摘要：
单纯疱疹病毒1型(HSV-1)在基因治疗和病毒疫苗,尤其是作为溶瘤病毒.然而,HSV-1病毒载体的大规模生产仍然是工业上的挑战。在这项研究中,使用微载体介导的血清减少培养基培养来改善HSV-1生产的生物过程并提高HSV-1产量。培养基的组成，其中包括基础培养基，血清浓度,和谷氨酰胺添加剂，已优化。该过程在1L生物反应器中成功进行，病毒产量比使用10％血清培养基的常规方法高三倍。还开发了珠子到珠子的转移过程以进一步提高可扩展性。在旋转烧瓶中，在消化过程中进行联合搅拌时，分离率从49.4％增加到80.6％；优化操作步骤后，总回收率从37.9％增加到71.1％。具体来说,微载体损失在抽吸和转移过程中减少，微载体和分离细胞用过滤器分离。使用2D培养作为接种物的基线过程通过交换传代培养培养基实现了相当的细胞生长。为了增加珠子到珠子转移后的病毒产量，关键参数,包括消化过程中的剪切应力，继代培养中的胰蛋白酶和EDTA浓度，通过相关分析和主成分分析，从47个参数中识别出CCI。与基线方法相比,优化的珠子到珠子转移方法实现了平均90.4%的总体回收率和相当的病毒产量。这项研究首次报道了在珠子到珠子转移后，在血清减少的培养基中在微载体上培养的Vero细胞中HSV-1生产的优化。开发了HSV-1生产工艺,包括在血清减少的培养基中培养,这个过程比传统工艺实现了三倍的病毒产量。•开发了间接的珠子到珠子转移过程,在生物反应器中具有超过90%的回收率。•优化珠子到珠子转移后的HSV-1生产,并且与使用2D培养作为接种物实现的生产相当。
