title: Validation of retroviral detection for rodent cell-derived products and gene therapy applications.
abstract: The availability of sensitive assays for detecting infectious murine retroviruses has become critical for the development and acceptance of a number of biopharmaceuticals, including monoclonal antibody-derived products and gene therapy vectors. Comparative studies demonstrated that the PG4 S+L- retrovirus infectivity test routinely yields higher titres than the mink cell test for xenotropic, amphotrophic and MCF murine retroviruses. A validation study for the PG4 S+L- assay demonstrated very good linearity (r2 of 0.95 to 0.99), reproducibility within a study (+/-0.35 log10 units), and precision between tests (+/-0.45 log10 units). Interference (or selectivity) in the presence of a non-specific antibody was insignificant (less than 0.2 log10 units). Sensitivity levels established from measurements as virus titres approach zero demonstrated a threshold value of 2-3 focus forming units (FFU)/ml. Two methods for increasing assay sensitivity were used including: (i) increased product samplings combined with a Poisson distribution analysis, and (ii) a 14-day co-cultivation with Mus dunni cells. Each of these methods was shown to increase sensitivity by at least one log10 unit. Murine retroviruses may also be detected by a less sensitive immunofluorescence assay (IFA) using specific monoclonal antibodies; this assay is essential for detecting certain recombinant ecotropic MuLVs. In summary, murine retroviral detection ranked by sensitivity is mink S+L- < IFA with monoclonal antibodies < PG4 S+L- < Mus dunni co-cultivation followed by PG4 S+L-.
authors.affiliation: Quality Biotech Inc., Camden, NJ 08104, USA.
authors.forename: J V
authors.initials: JV
authors.lastname: Hughes
authors.forename: K
authors.initials: K
authors.lastname: Messner
authors.forename: M
authors.initials: M
authors.lastname: Burnham
authors.forename: D
authors.initials: D
authors.lastname: Patel
authors.forename: E M
authors.initials: EM
authors.lastname: White
mesh_terms: D000818:Animals; D016466:CHO Cells / Q000821:virology; D002460:Cell Line / Q000821:virology; D006224:Cricetinae; D015316:Genetic Therapy; D009052:Leukemia Virus, Murine / Q000302:isolation & purification*; D051379:Mice; D008907:Mink; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014015:Tissue Banks / Q000592:standards*; D014779:Virus Replication