title: Multiplex RT Q-PCR assay for simultaneous quantification of three viruses used for validation of virus clearance by biopharmaceutical production.
abstract: Virus removal studies are used to insure the safety of biopharmaceutical products by quantitatively estimating the viral clearance capacity by the manufacturing process. Virus quantification assays are used to measure the log(10) clearance factor of individual purification unit operations in spike recovery studies. We have developed a multiplex RT Q-PCR assay that detects and quantifies three commonly used model viruses X-MuLV, SV40, and MMV simultaneously. This RT Q-PCR multiplex assay has a 6log(10) dynamic range with a limit of detection (LOD) of approximately 1 genome copy/microL. Amplification profiles are similar to existing singleplex assays. Overall, this RT Q-PCR multiplex assay is highly quantitative, accurately identifies multiple viruses simultaneously, and may prove useful to validate viral clearance of biological products in small scale studies.
authors.affiliation: Center for Drug Evaluation and Research, Food and Drug Administration, 10903 New Hampshire Ave., Silver Spring, MD 20993, USA. scott.lute@fda.hhs.gov
authors.forename: Scott
authors.initials: S
authors.lastname: Lute
authors.forename: Hua
authors.initials: H
authors.lastname: Wang
authors.forename: Davonie
authors.initials: D
authors.lastname: Sanchez
authors.forename: Janet
authors.initials: J
authors.lastname: Barletta
authors.forename: Qi
authors.initials: Q
authors.lastname: Chen
authors.forename: Kurt
authors.initials: K
authors.lastname: Brorson
mesh_terms: D000818:Animals; D001702:Biopharmaceutics / Q000379:methods / Q000592:standards*; D004279:DNA, Viral / Q000032:analysis*; D004340:Drug Contamination / Q000517:prevention & control; D018628:Gene Dosage; D016679:Genome, Viral; D009052:Leukemia Virus, Murine / Q000235:genetics / Q000302:isolation & purification; D008915:Minute Virus of Mice / Q000235:genetics / Q000302:isolation & purification; D004364:Pharmaceutical Preparations / Q000737:chemistry*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012680:Sensitivity and Specificity; D013539:Simian virus 40 / Q000235:genetics / Q000302:isolation & purification; D038301:Virus Inactivation / Q000187:drug effects; D014780:Viruses / Q000235:genetics / Q000302:isolation & purification
chemical_list: D004279:DNA, Viral; D004364:Pharmaceutical Preparations