Adventitious Virus Contamination Testing: Massively Parallel Sequencing in a Multimodal Solution for Biopharmaceutical Safety Testing

V irus contamination of commercially valued cell cultures can be a health risk to the general population and imposes financial burdens on manufacturing and biopharmaceutical companies. We investigated the use of massively parallel sequencing/next-generation sequencing (MPS/NGS) and bioinformatics for the detection and subsequent identification of adventitious contamination. Specifically, the Illumina® HiSeq 2000 instrument, in the 2 × 100 base pair (bp) paired end (PE) run configuration, was used to determine the identity and limit of detection of a DNA virus spiked into a virus vaccine, and an RNA virus spiked into a mammalian master cell bank (MCB). This configuration provided sufficient sequence read lengths and depth of coverage to detect and identify spiked-in SV40 and measles viruses in a background of vaccine, MCB, and host nucleic acid that make up the bulk of these samples. Furthermore, the detection of 30 SV40 reads within one tested sample suggested a < 1 plaque forming unit (pfu) sensitivity in a background of 2.8 × 107 infectious adenovirus serotype 5 (Ad5) particles. Results from subsequent virus vaccine testing suggested the presence of non-viable virus DNA contamination in the sample. Therefore, we propose that a multimodal approach, in which broad-range screening for known or unknown adventitious agents by MPS/NGS is complemented by targeted virus detection assays (e.g., PCR-based and infectivity assays), should provide the most useful safety monitoring information. We suggest that MPS/NGS and the accompanying bioinformatics is a sensitive, broad-range, and long-lasting tool with the ability to improve upon existing biosafety testing within a larger testing program. Introduction Biosafety testing of biologics produced in cell substrates is necessary to determine the presence of adventitious virus contamination. Cell-based infectivity assays, targeted nucleic acid amplification techniques, and broadrange hybridization-based microarray tests are the current industry standards utilized to detect these contaminants. Over time, these assays have been developed to detect contaminants of pre-established interest. In some instances, however, these tests may not be suff icient to detect very low levels of viral contamination, or to identify the species of the contaminant. Biosafety testing can be more effective by introducing an additional step that can provide a high degree of sensitivity, detect and identif y virus contaminants not directly targeted— and at low levels— and identify the species and variants of the contaminant. MPS/NGS offers the ability to detect all existing nucleotides, RNA, and DNA from adventitious virus contamination, thus fine-tuning the degree of sensitivity by increasing or decreasing the depth of coverage. With a high depth of coverage, the sensitivity is sufficient to detect very low levels of DNA contamination. In addition, keen sensitivity enables the potential for identification of contaminants Ad5 Cells