title: Determination of HSV-1 UL5 and UL29 gene copy numbers in an HSV complementing Vero cell line.
abstract: The genetic stability of transgenes is a critical characteristic used to assess constructed cell lines used for vaccine production. The evaluation of gene copy numbers by a qPCR method, is one of the most common approaches used to assess the consistency of transgenes in a constructed cell line. The cell line AV529-19 is a Vero-based cell line specifically engineered to express the HSV-1 UL5 and UL29 open reading frames. AV529-19 is used to support the replication of a defective HSV-2 viral candidate vaccine called HSV529. To assess the genetic stability of the UL5 and UL29 transgenes in AV529-19 cells, a digital PCR-based approach was developed. During characterization of the test method, the specificity, accuracy, and intermediate precision of the assay was investigated based on regulatory guidelines. The developed assay was used to monitor the stability of the transgenes in the manufactured AV529-19 cell lines by comparison of transgene copy numbers in the master cell bank (MCB) with their copy numbers in the extended cell bank (ECB). Results showed that the UL29 and UL5 transgenes are stable in that there are one and three copies of the UL29 and UL5 genes, respectively, per cell in both the AV529-19 MCB and ECB.
authors.affiliation: Microbiology & Virology Platform, Department of Analytical Research & Development North America, Sanofi Pasteur, 1755 Steeles Avenue West, Toronto, Ontario, Canada M2R 3T4. Electronic address: ali.azizi@sanofipasteur.com.
authors.forename: Ali
authors.initials: A
authors.lastname: Azizi
authors.forename: Francisca
authors.initials: F
authors.lastname: Aidoo
authors.forename: Lucy
authors.initials: L
authors.lastname: Gisonni-Lex
authors.forename: Bryan
authors.initials: B
authors.lastname: McNeil
mesh_terms: D000818:Animals; D002522:Chlorocebus aethiops; D004265:DNA Helicases / Q000235:genetics*; D019915:DNA Primase / Q000235:genetics*; D004261:DNA Replication / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics*; D018628:Gene Dosage; D018259:Herpesvirus 1, Human / Q000235:genetics*; D006801:Humans; D019076:Transgenes; D014709:Vero Cells; D014764:Viral Proteins / Q000235:genetics*
chemical_list: D004279:DNA, Viral; D004268:DNA-Binding Proteins; C045462:ICP8 protein, Simplexvirus; D014764:Viral Proteins; D019915:DNA Primase; C062021:helicase-primase, Human herpesvirus 1; D004265:DNA Helicases
keywords: Digital PCR; ECB; Gene copy number; HSV; MCB; Vaccine; Vero cells; dPCR; digital PCR; extended cell bank; master cell bank