Sun Yang, Huang Lingling, Nie Jianqi, Feng Kai, Liu Yupeng, Bai Zhonghu
Institute of Microbial Engineering, School of Life Sciences, Henan University, Kaifeng, 475004, China.
Engineering Research Center for Applied Microbiology of Henan Province, Kaifeng, 475004, China.
AMB Express. 2022 May 14;12(1):58. doi: 10.1186/s13568-022-01398-7.
Herpes zoster is caused by reactivation of the varicella zoster virus (VZV). Researching and developing a herpes zoster vaccine will help to decrease the incidence of herpes zoster. To increase the bioreactor productivity, a serum-free HEK293 cell perfusion process with adenovirus vector herpes zoster (rAd-HZ) vaccine production was developed efficiently using the design of experiment (DoE) method. First, serum-free media for HEK293 cells were screened in both batch and semi-perfusion culture modes. Then, three optimal media were employed in a medium mixture design to improve cell culture performance, and the 1:1 mixture of HEK293 medium and MCD293 medium (named HM293 medium) was identified as the optimal formulation. On the basis of the HM293 medium, the relationship of critical process parameters (CPPs), including the time of infection (TOI), multiplicity of infection (MOI), pH, and critical quality attributes (CQAs) (adenovirus titer (Titer), cell-specific virus yield (CSVY), adenovirus fold expansion (Fold)) of rAd-HZ production was investigated using the DoE approach. Furthermore, the robust setpoint and design space of these CPPs were explored. Finally, the rAd-HZ production process with parameters at a robust setpoint (TOI = 7.2 × 10 cells/mL, MOI = 3.7, and pH = 7.17) was successfully scaled-up to a 3-L bioreactor with an alternating tangential flow system, yielding an adenovirus titer of 3.0 × 10 IFU/mL, a CSVY of 4167 IFU/cells, a Fold of 1117 at 2 days post infection (dpi). The DoE approach accelerated the development of a HEK293 serum-free medium and of a robust adenovirus production process.
带状疱疹由水痘-带状疱疹病毒（VZV）重新激活引起。研发带状疱疹疫苗有助于降低带状疱疹的发病率。为提高生物反应器的生产力，采用实验设计（DoE）方法高效开发了一种用于生产腺病毒载体带状疱疹（rAd-HZ）疫苗的无血清HEK293细胞灌注工艺。首先，在分批培养和半灌注培养模式下筛选用于HEK293细胞的无血清培养基。然后，在培养基混合设计中使用三种优化培养基来改善细胞培养性能，1:1的HEK293培养基和MCD293培养基混合物（命名为HM293培养基）被确定为最佳配方。在HM293培养基的基础上，使用DoE方法研究了关键工艺参数（CPPs），包括感染时间（TOI）、感染复数（MOI）、pH值，以及rAd-HZ生产的关键质量属性（CQAs）（腺病毒滴度（Titer）、细胞特异性病毒产量（CSVY）、腺病毒扩增倍数（Fold））。此外，还探索了这些CPPs的稳健设定点和设计空间。最后，将具有稳健设定点参数（TOI = 7.2×10个细胞/mL，MOI = 3.7，pH = 7.17）的rAd-HZ生产工艺成功放大到带有交替切向流系统的3-L生物反应器中，在感染后2天（dpi）产生的腺病毒滴度为3.0×10 IFU/mL，CSVY为4167 IFU/细胞，Fold为1117。DoE方法加速了HEK293无血清培养基和稳健腺病毒生产工艺的开发。
