DETECTION OF VIRUSES IN CELL LINES USED TO MANUFACTURE BIOLOGICAL PRODUCTS

用于制造生物产品的细胞系中病毒的检测

• 批准号: 3770331
• 负责人: C J MARCUS-SEKURA
• 金额: --
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3770331
• 关键词: Baculoviridae; Bunyaviridae; CHO cells; Reoviridae; Retroviridae; Togaviridae; biological products; cytokine; electron microscopy; immunofluorescence technique; nucleic acid sequence; phorbols; recombinant proteins; tissue /cell culture; virus DNA; virus RNA; virus classification; virus cytopathogenic effect; virus genetics; virus replication; viruslike particle

It is of interest to CBER whether cell lines used for product production contain virus or virus-like particles or viral nucleic acid sequences or support the growth of viruses infectious for humans. In previous studies, we used electron microscopy to examine endogenous retroviral-like particles found in the Chinese Hamster Ovary (CHO) cell line which is used as the cel substrate for several recombinant products made in CHO cells. CHO cells contain endogenous retroviral-like particles visible by electron microscopy We attempted to determine whether growth factors or cytokines (which might be manufactured in CHO cells) might affect these endogenous particles. The results indicated increases in particles from 30-100% after treatment of CH cells with several cytokines (GM-CSF, IFN, TNF) or with the phorbol ester TPA. A manuscript describing these findings has been published in In Vitro Toxicology (1991). We have recently approached another cell substrate issue. Since many new recombinant biological products are produced using the Baculovirus expression system and use an insect cell line called Sf9; and relatively little is known about other viruses which can infect Sf9 cells, particularly those with human host range, we recently initiated a study to determine whether any viruses pathogenic for humans can infect Sf9 cells. We have examined a series of viruses for their ability to infect Sf9 cells, and protocols for evaluating whether Sf9 cells are free of viral contamination and suitable for use for production of biological products have been developed. We evaluated the ability of Sf9 cells to be infected by fifteen viruses chosen to represent several classes of arboviruses as well as several additional viruses with wide host range. St Louis Encephalitis virus (SLE) a flavivirus, can productively infect the SF9 cells. SLE causes CPE in the Sf9 cells (10-20% giant cells) and can also b detected in SF9 cells by immunofluorescence and electron microscopy. A manuscript is in press in J. Gen. virol. Many of the other viruses we teste apparently can survive for long periods in cell culture without causing apparent CPE, several can survive up to 8 weeks in media alone. This may present risks of contamination for products produced in Sf9 cells if appropriate testing is not performed.

CBER 感兴趣的是细胞系是否用于产品生产 含有病毒或病毒样颗粒或病毒核酸序列或 支持对人类具有传染性的病毒的生长。 在之前的研究中， 我们使用电子显微镜检查内源性逆转录病毒样颗粒 在用作细胞的中国仓鼠卵巢（CHO）细胞系中发现 CHO 细胞中制备的多种重组产品的底物。 CHO细胞 含有电子显微镜可见的内源性逆转录病毒样颗粒 我们试图确定生长因子或细胞因子（这可能 在 CHO 细胞中制造）可能会影响这些内源性颗粒。 这 结果表明，CH 处理后颗粒增加了 30-100% 具有多种细胞因子（GM-CSF、IFN、TNF）或佛波酯的细胞 TPA。描述这些发现的手稿已发表在《体外》杂志上 毒理学（1991）。 我们最近接触了另一种细胞基质 问题。 由于许多新的重组生物制品是使用 杆状病毒表达系统并使用称为 Sf9 的昆虫细胞系； 对于可感染 Sf9 的其他病毒知之甚少 细胞，特别是那些具有人类宿主范围的细胞，我们最近发起了一项 研究确定是否有任何对人类致病的病毒可以感染 Sf9 细胞。我们检查了一系列病毒感染 Sf9 的能力 细胞和评估 Sf9 细胞是否不含病毒的方案 污染少，适合用于生物制品生产 已被开发出来。 我们评估了Sf9细胞的感染能力 由十五种病毒选择代表几类虫媒病毒： 以及其他几种具有广泛宿主范围的病毒。圣路易斯 脑炎病毒 (SLE) 是一种黄病毒，可以高效感染 SF9 细胞。 SLE 会导致 Sf9 细胞（10-20% 巨细胞）中的 CPE，并且还可能导致 通过免疫荧光和电子显微镜在 SF9 细胞中检测到。一个 手稿正在 J. Gen. Virol 杂志上印刷。我们测试的许多其他病毒 显然可以在细胞培养物中长期存活而不会引起 明显的 CPE，其中一些仅在培养基中即可存活长达 8 周。 这可能 如果存在以下情况，Sf9 细胞生产的产品存在污染风险： 没有进行适当的测试。