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. 2016 Apr 12;34(17):2035-43.
doi: 10.1016/j.vaccine.2015.12.020. Epub 2015 Dec 19.

Development of a candidate reference material for adventitious virus detection in vaccine and biologicals manufacturing by deep sequencing

Edward T Mee  1 , Mark D Preston  2 , Philip D Minor  3 , Silke Schepelmann  3 ; CS533 Study Participants
Collaborators, Affiliations

Development of a candidate reference material for adventitious virus detection in vaccine and biologicals manufacturing by deep sequencing

Edward T Mee et al. Vaccine. .

Abstract

Background: Unbiased deep sequencing offers the potential for improved adventitious virus screening in vaccines and biotherapeutics. Successful implementation of such assays will require appropriate control materials to confirm assay performance and sensitivity.

Methods: A common reference material containing 25 target viruses was produced and 16 laboratories were invited to process it using their preferred adventitious virus detection assay.

Results: Fifteen laboratories returned results, obtained using a wide range of wet-lab and informatics methods. Six of 25 target viruses were detected by all laboratories, with the remaining viruses detected by 4-14 laboratories. Six non-target viruses were detected by three or more laboratories.

Conclusion: The study demonstrated that a wide range of methods are currently used for adventitious virus detection screening in biological products by deep sequencing and that they can yield significantly different results. This underscores the need for common reference materials to ensure satisfactory assay performance and enable comparisons between laboratories.

Keywords: Adventitious virus; Collaborative study; Deep sequencing; Reference material; Vaccine.

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Figures

Fig. 1
Fig. 1
Number of target viruses detected by individual laboratories and methods. Horizontal hatched bars, target viruses detected in best replicate using 2 million reads. Solid black bar, target viruses detected in best replicate using all reads. Grey bar, target viruses detected in second replicate using all reads. White bar, target viruses detected in both replicates using all reads. Laboratories L01-B, L11, L13 and L15-B performed analysis only on the total read set.
Fig. 2
Fig. 2
Frequency of target virus detection in all methods. Left panel, Correlation between number of target viruses detected in 2,000,000 reads (median 20.5) and entire read set (median 22). Four laboratories performed analysis only on the total read set and these are shown left of the dotted line. Right panel, correlation between number of target viruses detected and read depth. Graph shows best fit and 95% confidence bands for regression line.
Fig. 3
Fig. 3
Consistency of virus detection across different methods and laboratories. Left panel, proportion of all methods detecting target viruses. Right panel, proportion of all laboratories detecting target viruses using best method. Grey shading indicates viruses not detected by real-time PCR.
Fig. 4
Fig. 4
Rank order of viruses using all methods. Top panel, ranking of target viruses. Bottom panel, ranking of all viruses, excluding results where no additional viruses were reported. Horizontal bars indicate median rank of viruses; open circles indicate individual data points. Solid circles and prefix NT indicate non-target viruses reported by three or more laboratories. Data points below dotted line indicate that virus was not detected – such viruses were assigned a rank order of 26 (for target viruses) or 31 (for all viruses) for plotting and calculation of median.
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References

    1. Victoria J.G., Wang C., Jones M.S., Jaing C., McLoughlin K., Gardner S. Viral nucleic acids in live-attenuated vaccines: detection of minority variants and an adventitious virus. J Virol. 2010;84(Jun (12)):6033–6040. pii:JVI.02690-09. - PMC - PubMed
    1. Cutrone R., Lednicky J., Dunn G., Rizzo P., Bocchetta M., Chumakov K. Some oral poliovirus vaccines were contaminated with infectious SV40 after 1961. Cancer Res. 2005;65(Nov (22)):10273–10279. pii:65/22/10273. - PubMed
    1. Gombold J., Karakasidis S., Niksa P., Podczasy J., Neumann K., Richardson J. 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(May (24)):2916–2926. pii:S0264-410X(14)00194-7. - PMC - PubMed
    1. Dubin G., Toussaint J.F., Cassart J.P., Howe B., Boyce D., Friedland L. Investigation of a regulatory agency enquiry into potential porcine circovirus type 1 contamination of the human rotavirus vaccine. Rotarix: approach and outcome. Hum Vaccin Immunother. 2013;9(Nov (11)):2398–2408. pii:25973. - PMC - PubMed
    1. Gilliland S.M., Forrest L., Carre H., Jenkins A., Berry N., Martin J. Investigation of porcine circovirus contamination in human vaccines. Biologicals. 2012;40(Jul (4)):270–277. pii:S1045-1056(12)00026-7. - PubMed

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