The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China; National Engineering Research Center of Cereal Fermentation and Food Biomanufacturing, Jiangnan University, Wuxi 214122, China.
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.
Vaccine. 2022 Mar 18;40(13):2036-2043. doi: 10.1016/j.vaccine.2022.02.024. Epub 2022 Feb 23.
The adenovirus vector vaccines induce humoral and cellular immune responses and have been used to develop vaccines for effective prevention of life-threating viruses, such as Ebola and Coronaviruses. High demand of vaccines worldwide requires optimization of the production process. Perfusion process increases cell concentration and volumetric productivity, so that it becomes the commonly used strategy in vaccine production In this study, we optimized and developed a perfusion process for the adenovirus-based zoster vaccine production efficiently. We first tested different perfusion strategies in shake flasks, showing semi-continuous strategies for optimal HEK 293 cell growth. We then evaluated three empirical key process parameters (cell concentration at the time of infection (VCC), multiplicity of infection (MOI), virus production pH) by the design of experiment (DoE) method, from which the robust setpoint (VCC 1.04 × 10 cells/mL, MOI 9, and virus production pH 7.17) was confirmed in both shake flask and 2 L benchtop bioreactor. In the bioreactor, we compared the performances of two perfusion systems, the commercially-available XCell ATF® system and a novel peristaltic pump-driven alternating tangential flow perfusion system (PATFP system) that we developed. During cell cultivation stage, both perfusion systems have comparable performances regarding viable cell concentration and cell viability. At 2 dpi, the PATFP system resulted in an adenovirus titer of 2.1 × 10 IFU/mL and cell-specific virus yield of 2,062 IFU/cell, reaching 75% and 77% of values for XCell ATF® system. This study demonstrates the perfusion process to be superior strategy for adenovirus-based vaccine production compared to the batch-mode strategy (1,467 IFU/cell). Furthermore, our PATFP system shows potential to be comparable to the XCell ATF® system, and it would become an alternative perfusion strategy for the vaccine production.
腺病毒载体疫苗可诱导体液和细胞免疫应答，已被用于开发针对埃博拉病毒和冠状病毒等致命病毒的有效疫苗。全球对疫苗的高需求要求优化生产工艺。灌注工艺可提高细胞浓度和体积生产率，因此成为疫苗生产中常用的策略。在本研究中，我们优化并开发了一种高效的基于腺病毒的带状疱疹疫苗生产灌注工艺。我们首先在摇瓶中测试了不同的灌注策略，结果表明半连续策略最适合 HEK 293 细胞的生长。然后，我们通过实验设计 (DoE) 方法评估了三个经验关键工艺参数（感染时细胞浓度 (VCC)、感染复数 (MOI)、病毒生产 pH 值），从中确定了在摇瓶和 2 L 台式生物反应器中稳健的设定点（VCC 为 1.04×10 个细胞/mL、MOI 为 9，病毒生产 pH 值为 7.17）。在生物反应器中，我们比较了两种灌注系统的性能，即市售的 XCell ATF®系统和我们开发的新型蠕动泵驱动的切向流交替灌注系统 (PATFP 系统)。在细胞培养阶段，两种灌注系统在活细胞浓度和细胞活力方面表现相当。在 2 dpi 时，PATFP 系统的腺病毒滴度为 2.1×10 IFU/mL，细胞特异性病毒产量为 2062 IFU/细胞，达到 XCell ATF®系统的 75%和 77%。这项研究表明，与批次式策略（1467 IFU/细胞）相比，灌注工艺是基于腺病毒的疫苗生产的优越策略。此外，我们的 PATFP 系统显示出与 XCell ATF®系统相媲美的潜力，它将成为疫苗生产的替代灌注策略。
