title: A replication-competent adenovirus assay for E1-deleted Ad35 vectors produced in PER.C6 cells.
abstract: The presence of replication-competent adenovirus (RCA) is a safety concern for biologics based on recombinant adenoviruses and RCA testing is therefore mandatory for release of clinical material. RCA, which arises from homologous recombination between Ad5 vectors and HEK-293 cells, can be eliminated by the use of PER.C6 cells in combination with a matched vector. However, little is known on RCA formation with vectors based on adenovirus serotypes other than Ad5 and reliable RCA assays to test them are generally lacking. Here we report on the development and qualification of a sensitive RCA assay for Ad35, a promising alternative to Ad5 vectors. The assay is able to detect 1 RCA in 3x10(10) vector particles with 95% confidence, thus meeting current FDA requirements, and can discriminate between RCA and other rare CPE-causing entities, including helper dependent E1 positive particles (HDEP). Using this assay, the first batches of Ad35 vectors produced in PER.C6 cells were analysed and found to be free of RCA and HDEP. Based on the statistical model used, we anticipate that our approach to RCA assay development can be broadly applicable to other adenoviral vectors.
authors.affiliation: Crucell Holland BV, P.O. Box 2048, 2301CA Leiden, The Netherlands. g.marzio@crucell.com
authors.forename: G
authors.initials: G
authors.lastname: Marzio
authors.forename: E
authors.initials: E
authors.lastname: Kerkvliet
authors.forename: J A
authors.initials: JA
authors.lastname: Bogaards
authors.forename: S
authors.initials: S
authors.lastname: Koelewijn
authors.forename: A
authors.initials: A
authors.lastname: De Groot
authors.forename: L
authors.initials: L
authors.lastname: Gijsbers
authors.forename: G J
authors.initials: GJ
authors.lastname: Weverling
authors.forename: R
authors.initials: R
authors.lastname: Vogels
authors.forename: M
authors.initials: M
authors.lastname: Havenga
authors.forename: J
authors.initials: J
authors.lastname: Custers
authors.forename: M G
authors.initials: MG
authors.lastname: Pau
authors.forename: J Y
authors.initials: JY
authors.lastname: Guichoux
authors.forename: J
authors.initials: J
authors.lastname: Lewis
authors.forename: J
authors.initials: J
authors.lastname: Goudsmit
mesh_terms: D000256:Adenoviridae / Q000235:genetics* / Q000254:growth & development; D017498:Adenovirus E1 Proteins / Q000235:genetics*; D001681:Biological Assay / Q000379:methods*; D002460:Cell Line; D045744:Cell Line, Tumor; D017353:Gene Deletion; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D016133:Polymerase Chain Reaction; D015203:Reproducibility of Results; D014779:Virus Replication
chemical_list: D017498:Adenovirus E1 Proteins