title: Real time quantitative PCR as a method to evaluate simian virus 40 removal during pharmaceutical protein purification.
abstract: Continuous cell lines used for pharmaceutical protein manufacturing have the potential to be contaminated by viruses. To ensure the safety of pharmaceutical proteins derived from continuous cell lines, validation of the ability of the manufacturing process to clear potential contaminating viruses is required for product registration. In this paper, a real time quantitative PCR method has been applied to the evaluation of simian virus 40 (SV40) removal during chromatography and filtration procedures. This method takes advantage of the 5'-3' exonuclease activity of Taq DNA polymerase and utilizes the PRISM 7700 sequence detection system of PE Applied Biosystems for automated SV40 DNA quantification through a dual-labeled fluorogenic probe. This method provides accurate and reproducible quantification of SV40 DNA. The SV40 clearance during chromatography and filtration procedures determined by this method is highly comparable with that determined by the cell-based infectivity assay. This method offers significant advantages over cell-based infectivity assays, such as higher sensitivity, greater reliability, higher sample throughput and lower cost. This method can be potentially used to evaluate the clearance of all model viruses during chromatography and filtration procedures. This method can be used to substitute cell-based infectivity assays for process validation of viral removal procedures and the availability of this method should greatly facilitate and reduce the cost of viral clearance evaluations required for new biologic product development.
authors.affiliation: Department of Cell Culture and Fermentation Research and Development, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, USA.
authors.forename: L
authors.initials: L
authors.lastname: Shi
authors.forename: L A
authors.initials: LA
authors.lastname: Norling
authors.forename: A S
authors.initials: AS
authors.lastname: Lau
authors.forename: S
authors.initials: S
authors.lastname: Krejci
authors.forename: A J
authors.initials: AJ
authors.lastname: Laney
authors.forename: Y
authors.initials: Y
authors.lastname: Xu
mesh_terms: D000911:Antibodies, Monoclonal / Q000302:isolation & purification; D002845:Chromatography / Q000379:methods; D004340:Drug Contamination; D004345:Drug Industry / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011994:Recombinant Proteins / Q000302:isolation & purification*; D013539:Simian virus 40 / Q000302:isolation & purification*
chemical_list: D000911:Antibodies, Monoclonal; D011994:Recombinant Proteins