Validation and international regulatory experience for a mycoplasma touchdown PCR assay.

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.