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首页> 外文会议>2002 International Symposium on Safety Science and Technology (2002 ISSST) Vol.3 Pt.A Oct 10-13, 2002 Tai'an, China >A Real-time Quantitative PCR-based Method for the Detection and Quantification of Xenotropic Murine Leukemia Virus (X-MuLV) and its Application in Evaluation of X-MuLV Removal during Pharmaceutical Protein Purification
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A Real-time Quantitative PCR-based Method for the Detection and Quantification of Xenotropic Murine Leukemia Virus (X-MuLV) and its Application in Evaluation of X-MuLV Removal during Pharmaceutical Protein Purification

机译：基于实时定量PCR的异嗜性鼠白血病病毒（X-MuLV）的检测和定量方法及其在药物蛋白质纯化过程中对X-MuLV去除率的评估中的应用

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Continuous mammalian cell lines have been widely used to manufacture biotechnology products withrnpharmaceutical applications. One of the common features to all protein pharmaceuticals derived from continuousrncell lines is the risk of viral contamination. To assure the safety of these cell line-derived protein pharmaceuticsrnwith regard to viral contamination, implementation of appropriate viral prevention procedures during production,rnapplication of a virus testing program for lot release and validation of the manufacturing process to clear virus arernrequired for marketing application/registration world-wide.rnXenotropic murine leukemia virus (X-MuLV) has been widely used as a model virus for viral clearancernevaluations for pharmaceutical protein manufacturing. Here, the development of a novel 搑eal time” quantitativernPCR-based X-MuLV detection and quantification method has been described. The method measures PCR productrnaccumulation through a dual-labeled fluorogenic probe (TaqMan probe). X-MuLV particle RNA (pRNA) isolatedrnfrom test samples was first reverse transcribed and then PCR amplified. The initial quantity of X-MuLV pRNA inrntest samples was determined using a known amount of X-MuLV standard control RNA fragment (sRNA) whichrnwas reverse transcribed from a cloned X-MuLV envelope gene fragment. This method provides accurate andrnreproducible quantification of X-MuLV pRNA over a linear dynamic range of at least 100,000-fold with arnquantification limit of approximately 1.5 pRNA copy ml-1 in test samples. This TaqMan assay is about 100-foldrnmore sensitive than the cell-based infectivity assay. The sample preparation procedure employed allows forrnefficient and consistent recovery of X-MuLV pRNA from test samples. High concentrations of protein andrncellular DNA present in test samples have been demonstrated to have no impact on X-MuLV quantification.rnReal Time Quantitative PCR has been applied to the evaluation of X-MuLV removal during chromatographyrnand filtration procedures used for therapeutic protein purification. The X-MuLV clearance during chromatographyrnand filtration procedures determined by this method is highly comparable with that determined by the cell-basedrninfectivity assay. This method offers significant advantages over cell-based infectivity assays, such as higherrnsensitivity, greater reliability, higher sample throughput and lower cost. This method can be used to substituterncell-based infectivity assays for process validation of viral removal procedures. The availability of this methodrnshould greatly facilitate and reduce the cost of viral clearance evaluations required for new biologic productrndevelopment.
机译：连续的哺乳动物细胞系已被广泛用于制造具有医药用途的生物技术产品。源自连续细胞系的所有蛋白质药物的共同特征之一是病毒污染的风险。为了确保这些细胞系蛋白药物在病毒污染方面的安全性，在生产过程中实施适当的病毒预防程序，应用病毒测试程序以进行批量释放以及验证生产过程以清除病毒，需要进行市场营销应用/注册异嗜性鼠白血病病毒（X-MuLV）已被广泛用作模型病毒，用于病毒清除评估药物蛋白的生产。在此，已经描述了基于“实时时间”定量rnPCR的新型X-MuLV检测和定量方法的开发。该方法通过双标记荧光探针（TaqMan探针）测量PCR产物的积累。从测试样品中分离出的X-MuLV颗粒RNA（pRNA）首先被逆转录，然后进行PCR扩增。使用已知量的X-MuLV标准对照RNA片段（sRNA）确定了X-MuLV pRNA检测样品的初始量，该片段是从克隆的X-MuLV包膜基因片段中反转录而来。该方法可在线性动态范围至少100,000倍内对X-MuLV pRNA进行准确且可再现的定量，测试样品中的定量限约为1.5 pRNA复制ml-1。 TaqMan分析的敏感性比基于细胞的传染性分析高100倍。所采用的样品制备程序可从测试样品中高效，稳定地回收X-MuLV pRNA。已证明测试样品中存在高浓度的蛋白质和细胞DNA对X-MuLV定量没有影响。实时定量PCR已用于评估层析和用于治疗性蛋白质纯化的过滤程序中X-MuLV的去除。用这种方法确定的色谱和过滤过程中的X-MuLV清除率与基于细胞的感染性测定所确定的清除率高度可比。与基于细胞的传染性测定相比，该方法具有明显的优势，例如灵敏度更高，可靠性更高，样品通量更高且成本更低。此方法可用于替代基于细胞的感染性测定法，用于病毒去除程序的过程验证。这种方法的可用性应该极大地促进和减少开发新的生物制品所需的病毒清除率评估的成本。

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