Validation of a next generation sequencing method for adventitious virus detection: Demonstration of sensitivity in multiple cell lines.

ABSTRACT

In the past few years NGS has become the technology of choice to replace animal-based virus safety methods and this has been strengthened by the recent revision to the ICHQ5A virus safety chapter. Here we describe the validation of an NGS method using an agnostic analysis to detect and identify RNA virus and actively replicating DNA virus contaminants in cell banks. We report the results of the validation of each step in the sequencing process that established quality criteria to ensure consistent sequencing data. Furthermore, the validation of the analysis algorithm designed to identify virus specific sequences is described along with steps undertaken to ensure the integrity of the sequencing data from generation to analysis. Lastly, the validated sequencing and analysis systems were used to establish a limit of detection (LOD) for model viruses in cells that are commonly used in biomanufacturing. The LOD from these studies ranged from 1E+03 to 1E+04 genome copies and were dependent on the virus type with little variability between the different cell types. Thus, the validation of the NGS method for adventitious agent testing and the establishment of a general LOD for cell-based samples provides a suitable alternative to traditional virus detection methods.