An essential step in pharmaceutical product development is screening for contamination with adventitious agents, and there is desire to develop highly sensitive assays to detect adventitious viral nucleic acid. This study sought to examine the nucleic acid extraction efficiency of three viral candidates in relevant background matrices using four different extraction methods. Three model adventitious viruses, Minute virus of Mice, Porcine Circovirus, and Feline Leukemia Virus, were diluted within a variety of background matrices relevant to pharmaceutical production methods. Upon extraction, the nucleic acid was quantified using droplet digital PCR methods. Four nucleic acid extraction methods were assessed, including commercially available kits and manual extraction methods. Each method recovered nucleic acid post-extraction for each of the model viruses within the tested background matrices. The silica-column based method recovered a greater amount of viral nucleic acid, compared to the other methods tested. Similar trends were observed when model virus was diluted in bioreactor supernatant, which replicates industry testing conditions and provides details on which extraction methods might be used in Next Generation Sequencing and PCR methods for detecting contamination within pharmaceutical products.

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使用市售方法有效提取外来病毒核酸

药品开发的一个重要步骤是筛选外来因子的污染，并且需要开发高灵敏度的测定法来检测外来病毒核酸。本研究试图使用四种不同的提取方法检查相关背景基质中三种候选病毒的核酸提取效率。三种模型外来病毒，即小鼠微小病毒、猪圆环病毒和猫白血病病毒，在与药物生产方法相关的各种背景基质中进行稀释。提取后，使用液滴数字 PCR 方法对核酸进行定量。评估了四种核酸提取方法，包括市售试剂盒和手动提取方法。每种方法都回收了测试背景基质中每种模型病毒的提取后核酸。与其他测试方法相比，基于硅胶柱的方法回收了更大量的病毒核酸。当模型病毒在生物反应器上清液中稀释时，观察到了类似的趋势，这复制了行业测试条件，并提供了下一代测序和 PCR 方法中可使用哪些提取方法来检测药品内污染的详细信息。