Summer activity #3
Avoiding False Positives
Next-Generation Sequencing: Do Not Allow False Positives Hold You Back Anymore
From vaccines to cell & gene therapies, and monoclonal antibodies, ensuring the safety of the products is the major concern when it comes to the development of biologics. The use of animal-derived materials in manufacturing is a potential source of contamination. Ensuring that these materials are free of adventitious agents is crucial.
For many years, only in vivo and in vitro assays were used to screen the cell banks, virus stocks, serum, and intermediate of production, etc. in the production process. Recent studies [1, 2] and contamination events  have demonstrated the limitations of these assays and new approaches based on the detection of viral nucleic acids have been developed such as PCR/qPCR or Next Generation Sequencing (NGS). PCR is a useful alternative when dealing with a small number of targets, yet it remains challenging to implement for the detection of many targets.
Moreover, PCR cannot detect new or emerging viruses as it relies on previous knowledge of the target. Next-Generation Sequencing detects all nucleic acids present in a sample without prior knowledge of the sequence. The capability of NGS to detect new viruses has rapidly put this technology as a complemental test first, and nowadays as an alternative test for viral safety. However, a major concern about NGS is its potential to detect viral sequences that are not related to infectious viruses and thus to get a false positive result. These cautions from the manufacturers are understandable as a false positive result significantly impacts the production process with lengthy, unnecessary, and costly investigations.
At PathoQuest, we understand that a new method for quality control should bring advantages over the old ones by positively impacting the time to release and ease the processes of the producers. We have thus developed a specific NGS solution dedicated to the viral safety assessment of cell banks (Master and Working Cell Bank of production, Therapeutic Cells, etc.). This unique approach deals with the problem of false positives through increased specificity and biological relevance.
PathoQuest transcriptome service differentiates inert carryover (irradiated BVDV-2) from a truly replicating virus (infectious BVDV-2) by ruling out the sequences coming from the genome of the RNA virus and those coming from the messenger RNA. When standard NGS only assesses the presence of RNA and DNA nucleic acids from cell lysate and/or supernatant, with a high risk of false positive hits, our Transcriptome NGS solution relies on the detection of the viral messenger RNA. The viral messenger RNAs that are an intermediate of replication synthesized only during viral infection by all virus types .
This capability of distinguishing the inert genome from the active messenger RNA is a significant aspect of our solution to eliminate the risk of false positive. The second aspect resides in our proprietary data analysis pipeline that includes a smart step of hit invalidation. The goal of this step is to determine according to a list of defined and validated criteria the relevance of a hit and to classify this hit as a “positive hit” or a “background unspecific hit”.
Finally, as confidence in the solution used for quality control is key for all manufacturers of biologics, we have fully validated our method according to the different guidelines including ICHQ2R1 and 21 CFR Part 11. Should you be interested to know more about our validation strategy, do not miss next week’s regulatory summer camp activity (activity #4).
To summarize, with PathoQuest’s validated Transcriptome NGS solution dedicated to cell bank viral safety testing, you beneficiate from the power of NGS without the drawbacks of false positive detection. Our method allows a broad-spectrum detection of all replicating infectious viruses (RNA and DNA viruses) with better specificity and validated sensitivity as compared to standard NGS. This method is the solution to enter the program of in vivo assays replacement for your master cell banks or to accelerate the release of your therapeutic cells.
 Gombold J, Karakasidis S, Niksa P, Podczasy J, Neumann K, Richardson J, et al. Systematic evaluation of in vitro and in vivo adventitious virus assays for the detection of viral contamination of cell banks and biological products. Vaccine 2014;32:2916–26. https://doi.org/10.1016/j.vaccine.2014.02.021.
 Marcus-Sekura C, Richardson JC, Harston RK, Sane N, Sheets RL. Evaluation of the human host range of bovine and porcine viruses that may contaminate bovine serum and porcine trypsin used in the manufacture of biological products. Biologicals 2011;39:359–69. https://doi.org/10.1016/j.biologicals.2011.08.003.
 Victoria JG, Wang C, Jones MS, Jaing C, McLoughlin K, Gardner S, et al. Viral nucleic acids in live-attenuated vaccines: detection of minority variants and an adventitious virus. J Virol 2010;84:6033–40. https://doi.org/10.1128/JVI.02690-09.
 Brussel A, Brack B, Muth E, Zirwes R, Cheval J, Hebert C, Charpin JM, Marinaci A, Flan B, Ruppach H, Beurdeley P, Eloit M. Use of a new RNA next-generation sequencing approach for the specific detection of virus infection in cells. Biologicals. 2019 May;59:29-36. doi: 10.1016/j.biologicals.2019.03.008. Epub 2019 Apr 13. https://doi.org/10.1016/j.biologicals.2019.03.008