title: Preliminary Evaluation of Next-Generation Sequencing Performance Relative to qPCR and In Vitro Cell Culture Tests for Human Cytomegalovirus.
abstract: To compare the performances of conventional in vitro indicator cell culture, quantitative polymerase chain reaction (qPCR), and next-generation sequencing (NGS) as detection methods for adventitious agents, a preliminary assessment was performed using human cytomegalovirus (HCMV) as a model virus. HCMV was spiked into a crude viral harvest at various concentrations and inoculated onto MRC-5 cell monolayers. The cultures were observed for cytopathic effects (CPEs) as per the compendial method requirements, and samples were taken at various time points for analysis by qPCR or NGS. When using NGS, the detection of HCMV is 10 fold more sensitive than observed using the conventional CPE endpoint method. It may be possible for qPCR to achieve the detection level demonstrated by NGS, but further optimization of the technique would be required. In addition, NGS was able to detect HCMV in the initial inoculum when it was spiked in at 10 CCID50/mL, suggesting the potential to eliminate cell culture amplification with an NGS-based assay. This study highlights the advantage of NGS as a surrogate broad-spectrum technology for the detection of adventitious agents in biologics.
  Human cytomeglovirus (HCMV) is highly prevalent in the general population and can lead to serious health issues in both immumocompromised individuals and/or newborns. The testing of HCMV in biological materials is stipulated by multiple regulatory agencies where HCMV is a potential risk. This test involves inoculating cell lines that are susceptible to HCMV infection, incubating the cultures for 28 days, and observing the cells for signs of viral infection. Next-generation sequencing and quantitative polymerase chain reaction (qPCR) are two technologies that can potentially shorten the extended 28 day cell culture incubation. In this study, we compared the sensitivity of the compendial cell culture assay with NGS and qPCR for the detection of HCMV. Our results show that NGS can potentially achieve a detection limit that is 10 times more sensitive than the cell culture assay. In addition, NGS was able to detect HCMV in the initial inoculum, potentially eliminating the need for cell culture amplification of the virus. Finally, sequence data generated by NGS directly demonstrate the presence of HCMV, and such information can serve as the foundation for any follow-up investigation.
authors.affiliation: Microbiology & Virology Platform, Department of Analytical Research & Development North America, Sanofi Pasteur, Toronto, Ontario, Canada; and siemon.ng@sanofipasteur.com.
authors.forename: Siemon H S
authors.initials: SH
authors.lastname: Ng
authors.affiliation: Microbiology & Virology Platform, Department of Analytical Research & Development North America, Sanofi Pasteur, Toronto, Ontario, Canada; and.
authors.forename: Ali
authors.initials: A
authors.lastname: Azizi
authors.affiliation: Microbiology & Virology Platform, Department of Analytical Research & Development North America, Sanofi Pasteur, Toronto, Ontario, Canada; and.
authors.forename: Kerrie
authors.initials: K
authors.lastname: Edamura
authors.affiliation: Microbiology & Virology Platform, Department of Analytical Research & Development North America, Sanofi Pasteur, Toronto, Ontario, Canada; and.
authors.forename: Rebecca J
authors.initials: RJ
authors.lastname: Malott
authors.affiliation: Microbiology & Virology Platform, Department of Analytical Research & Development North America, Sanofi Pasteur, Toronto, Ontario, Canada; and.
authors.forename: Robert L
authors.initials: RL
authors.lastname: Charlebois
authors.affiliation: Microbiology & Virology Platform, Department of Analytical Research & Development North America, Sanofi Pasteur, Toronto, Ontario, Canada; and.
authors.forename: Carine
authors.initials: C
authors.lastname: Logvinoff
authors.affiliation: Microbiology & Virology Platform, Department of Analytical Research & Development North America, Sanofi Pasteur, Toronto, Ontario, Canada; and.
authors.forename: Martha
authors.initials: M
authors.lastname: Schreiber
authors.affiliation: Product Conception and Development, Sanofi Pasteur, Marcy L'Etoile, France.
authors.forename: Laurent
authors.initials: L
authors.lastname: Mallet
authors.affiliation: Microbiology & Virology Platform, Department of Analytical Research & Development North America, Sanofi Pasteur, Toronto, Ontario, Canada; and.
authors.forename: Lucy
authors.initials: L
authors.lastname: Gisonni-Lex
mesh_terms: D001688:Biological Products / Q000032:analysis*; D001702:Biopharmaceutics / Q000379:methods*; D018929:Cell Culture Techniques; D002460:Cell Line; D003587:Cytomegalovirus / Q000235:genetics* / Q000254:growth & development / Q000302:isolation & purification; D003588:Cytopathogenic Effect, Viral; D004279:DNA, Viral / Q000235:genetics*; D004340:Drug Contamination / Q000517:prevention & control*; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D016133:Polymerase Chain Reaction; D015203:Reproducibility of Results; D013997:Time Factors; D014773:Virology / Q000379:methods*
chemical_list: D001688:Biological Products; D004279:DNA, Viral
keywords: Adventitious agent detection; HCMV; Herpesviridae; Human cytomegalovirus; Illumina sequencing; Next-generation sequencing; qPCR