Wei Zhaohui, Xia Yang, Su Yuning, Quan Yufen, Sun Liuliu, Wang Shanshan, Zhu Fangjian, Chen Zhenzhen, Tian Jun, Wang Wei-Chun, Zhou Weichang, Yu Haiyang
Process Development, WuXi Biologics, 108 Meiliang Road, Wuxi, 214092, China.
WuXi Biologics, 299 Fute Zhong Road, Waigaoqiao Free Trade Zone, Shanghai, 200131, China.
Biotechnol Prog. 2023 Jul-Aug;39(4):e3340. doi: 10.1002/btpr.3340. Epub 2023 Mar 27.
Perfusion culture is often performed with micro-sparger to fulfill the high oxygen demand from the densified cells. Protective additive Pluronic F-68 (PF-68) is widely used to mitigate the adverse effect in cell viability from micro-sparging. In this study, different PF-68 retention ratio in alternating tangential filtration (ATF) columns was found to be crucial for cell performance of different perfusion culture modes. The PF-68 in the perfusion medium was found retained inside the bioreactor when exchanged through ATF hollow fibers with a small pore size (50 kD). The accumulated PF-68 could provide sufficient protection for cells under micro-sparging. On the other hand, with large-pore-size (0.2 μm) hollow fibers, PF-68 could pass through the ATF filtration membranes with little retention, and consequently led to compromised cell growth. To overcome the defect, a PF-68 feeding strategy was designed and successfully verified on promoting cell growth with different Chinese hamster ovary (CHO) cell lines. With PF-68 feeding, enhancements were observed in both viable cell densities (20%-30%) and productivity (~30%). A threshold PF-68 concentration of 5 g/L for high-density cell culture (up to 100 × 10 cells/mL) was also proposed and verified. The additional PF-68 feeding was not observed to affect product qualities. By designing the PF-68 concentration of perfusion medium to or higher than the threshold level, a similar cell growth enhancement was also achieved. This study systematically investigated the protecting role of PF-68 in intensified CHO cell cultures, shedding a light on the optimization of perfusion cultures through the control of protective additives.
灌注培养通常使用微型气体分布器来满足高密度细胞对高氧气的需求。保护性添加剂普朗尼克F - 68（PF - 68）被广泛用于减轻微型气体分布对细胞活力的不利影响。在本研究中，发现交替切向流过滤（ATF）柱中不同的PF - 68保留率对于不同灌注培养模式下的细胞性能至关重要。当通过小孔径（50 kD）的ATF中空纤维进行交换时，发现灌注培养基中的PF - 68保留在生物反应器内部。积累的PF - 68可以在微型气体分布条件下为细胞提供足够的保护。另一方面，使用大孔径（0.2μm）的中空纤维时，PF - 68几乎没有保留地穿过ATF过滤膜，从而导致细胞生长受损。为了克服这一缺陷，设计了一种PF - 68补料策略，并成功验证了其对不同中国仓鼠卵巢（CHO）细胞系促进细胞生长的作用。通过PF - 68补料，活细胞密度（提高20% - 30%）和生产率（提高约30%）均有提高。还提出并验证了高密度细胞培养（高达100×10⁶细胞/mL）时PF - 68的阈值浓度为5 g/L。未观察到额外的PF - 68补料会影响产品质量。通过将灌注培养基中PF - 68的浓度设计为等于或高于阈值水平，也实现了类似的细胞生长增强。本研究系统地研究了PF - 68在强化CHO细胞培养中的保护作用，为通过控制保护性添加剂优化灌注培养提供了思路。
