Asarnow David, Warford Ann, Fernandez Lewis, Hom Jonathan, Sandhu Gursaneep, Candichoy Zayda, Luna Geraldine, Goldman Mark, Rarich Randy
Department of Quality Control, Bayer HealthCare LLC, 800 Dwight Way, Berkeley, CA 94701, USA.
Biologicals. 2010 Mar;38(2):224-31. doi: 10.1016/j.biologicals.2009.11.006. Epub 2010 Feb 10.
A PCR assay was developed to monitor rFVIII production fermenters for mycoplasma contamination. The method uses a simple extraction procedure followed by a qualitative "touchdown" (TD) PCR protocol with primers specific to the 16S rRNA gene. The method has the capacity to detect a wide range of mycoplasma species. Validation was performed according to ICH guidelines and confirmed a limit of detection of between 579 and 1715 mycoplasma genome copies spiked per ml of sample, and a 1000-10,000-fold greater specificity compared to Gram-positive bacteria. In a comparability study, it was comparable in sensitivity to the current FDA-recommended broth and agar culture-based method down to one colony forming unit (cfu)/ml. The method was validated for its intended use as a replacement for culture-based mycoplasma detection during routine fermenter monitoring. Regulatory approvals for the method have been obtained in many of the major regions and activities are ongoing to address agency concerns regarding the comparative limit of detection of the method to culture-based assays.
开发了一种聚合酶链反应（PCR）检测方法，用于监测重组人凝血因子VIII（rFVIII）生产发酵罐中的支原体污染情况。该方法采用简单的提取程序，随后是针对16S rRNA基因的定性“降落式”（TD）PCR方案。该方法能够检测多种支原体物种。根据国际人用药品注册技术协调会（ICH）指南进行了验证，确认每毫升样品中加标的支原体基因组拷贝数的检测限在579至1715之间，与革兰氏阳性菌相比，特异性高1000至10000倍。在一项可比性研究中，其灵敏度与美国食品药品监督管理局（FDA）目前推荐的基于肉汤和琼脂培养的方法相当，低至每毫升一个菌落形成单位（cfu）。该方法经验证可用于在常规发酵罐监测期间替代基于培养的支原体检测方法。该方法已在许多主要地区获得监管批准，并且正在开展相关活动，以解决监管机构对该方法与基于培养的检测方法相比的检测限的担忧。
