Wang Xiuyan, Olszewska Malgorzata, Qu Jinrong, Wasielewska Teresa, Bartido Shirley, Hermetet Gregory, Sadelain Michel, Rivière Isabelle
*Cell Therapy and Cell Engineering Facility †Molecular Pharmacology and Chemistry Program §Center for Cell Engineering, Memorial Sloan Kettering Cancer Center, New York ‡Pall Life Sciences, Port Washington, NY.
J Immunother. 2015 Apr;38(3):127-35. doi: 10.1097/CJI.0000000000000072.
The successful genetic engineering of patient T cells with γ-retroviral vectors expressing chimeric antigen receptors or T-cell receptors for phase II clinical trials and beyond requires the large-scale manufacture of high-titer vector stocks. The production of retroviral vectors from stable packaging cell lines using roller bottles or 10- to 40-layer cell factories is limited by a narrow harvest window, labor intensity, open-system operations, and the requirement for significant incubator space. To circumvent these shortcomings, we optimized the production of vector stocks in a disposable fixed-bed bioreactor using good manufacturing practice-grade packaging cell lines. High-titer vector stocks were harvested over 10 days, representing a much broader harvest window than the 3-day harvest afforded by cell factories. For PG13 and 293Vec packaging cells, the average vector titer and the vector stocks' yield in the bioreactor were higher by 3.2- to 7.3-fold, and 5.6- to 13.1-fold, respectively, than those obtained in cell factories. The vector production was 10.4 and 18.6 times more efficient than in cell factories for PG13 and 293Vec cells, respectively. Furthermore, the vectors produced from the fixed-bed bioreactors passed the release test assays for clinical applications. Therefore, a single vector lot derived from 293Vec is suitable to transduce up to 500 patients cell doses in the context of large clinical trials using chimeric antigen receptors or T-cell receptors. These findings demonstrate for the first time that a robust fixed-bed bioreactor process can be used to produce γ-retroviral vector stocks scalable up to the commercialization phase.
使用表达嵌合抗原受体或T细胞受体的γ-逆转录病毒载体对患者T细胞进行成功的基因工程改造,用于II期及以后的临床试验,需要大规模生产高滴度的载体储备。使用滚瓶或10至40层细胞工厂从稳定的包装细胞系生产逆转录病毒载体,受到收获窗口狭窄、劳动强度大、开放式系统操作以及对大量培养箱空间的需求的限制。为了克服这些缺点,我们使用符合药品生产质量管理规范(GMP)级别的包装细胞系,在一次性固定床生物反应器中优化了载体储备的生产。高滴度载体储备在10天内收获,与细胞工厂提供的3天收获期相比,收获窗口要宽得多。对于PG13和293Vec包装细胞,生物反应器中的平均载体滴度和载体储备产量分别比细胞工厂中的高3.2至7.3倍和5.6至13.1倍。对于PG13和293Vec细胞,载体生产效率分别比细胞工厂高10.4倍和18.6倍。此外,从固定床生物反应器生产的载体通过了临床应用的放行测试分析。因此,在使用嵌合抗原受体或T细胞受体的大型临床试验中,源自293Vec的单个载体批次适合转导多达500个患者细胞剂量。这些发现首次证明,一种强大的固定床生物反应器工艺可用于生产可扩展至商业化阶段的γ-逆转录病毒载体储备。
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