Microbial and Viral Platforms (MVP), WuXi Biologics, 291 Fucheng Road, Hangzhou, 311106, China.
Appl Microbiol Biotechnol. 2024 Jun 19;108(1):383. doi: 10.1007/s00253-024-13193-4.
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.
单纯疱疹病毒 1 型（HSV-1）在基因治疗和病毒疫苗领域发挥着重要作用，特别是作为溶瘤病毒。然而，大规模生产 HSV-1 病毒载体仍然是该行业面临的挑战。在这项研究中，采用微载体介导的血清减少培养基培养来改进 HSV-1 生产的生物工艺并提高 HSV-1 的产量。优化了培养基的组成，包括基础培养基、血清浓度和谷氨酰胺添加剂。该过程在 1 L 生物反应器中成功进行，病毒产量比传统的 10%血清培养基工艺提高了三倍。还开发了珠对珠转移工艺以进一步提高可扩展性。在摇瓶中，当在消化过程中进行组合搅拌时，珠间的脱离率从 49.4%增加到 80.6%；通过优化操作步骤，总回收率从 37.9%增加到 71.1%。具体来说，在抽吸和转移过程中减少了微载体的损失，并使用过滤器将微载体和脱离的细胞分离。通过使用 2D 培养作为接种物来交换传代培养基，可以实现与基线工艺相当的细胞生长。为了在珠对珠转移后提高病毒产量，通过相关分析和主成分分析从 47 个参数中确定了消化过程中的剪切应力、传代中的 TrypLE 和 EDTA 浓度以及 CCI 等关键参数。优化的珠对珠转移工艺平均总回收率达到 90.4%，与基线工艺相比病毒产量相当。本研究首次报道了在血清减少培养基中培养的微载体上进行 HSV-1 生产的优化。 关键点： • 开发了一种涉及在血清减少培养基中培养的 HSV-1 生产工艺，该工艺的病毒产量比传统工艺提高了三倍。 • 在生物反应器中开发了间接的珠对珠转移工艺，回收率超过 90%。 • 优化了珠对珠转移后的 HSV-1 生产，与使用 2D 培养作为接种物的效果相当。
