title: Evaluation of infectivity and reverse transcriptase real-time polymerase chain reaction assays for detection of xenotropic murine leukemia virus used in virus clearance validation.
abstract: Infectivity and reverse transcriptase quantitative real-time polymerase chain reaction (qRT-PCR) assays have been optimized and validated for xenotropic murine leukemia virus (X-MuLV) detection. We have evaluated the assays systematically with regard to specificity, linearity, lower limit of detection (LLOD), lower limit of quantification (LLOQ), and precision. Both assays are specific for X-MuLV detection, with a linear detection range of 0.6-5.6 log(10) TCID(50)/mL for the infectivity assay, and 1.4-6.5 log(10) particles/mL for the qRT-PCR assay. The LLOD and LLOQ of the infectivity and the qRT-PCR assays are determined as 0.5 and 1.0 log(10)/mL, and 1.4 and 2.2 log(10)/mL. The inter-assay repeatability of qRT-PCR assay (4.2% coefficient of variation [CV]) is higher than the infectivity assay (7.9% CV). We have shown that both assays are closely correlated (r = 0.85, P < 0.05, n = 22). The particle/infectivity ratio is determined as 66. Both assays were applied to evaluate virus removal using virus clearance samples of chromatographic and filtration processes. Here, we have demonstrated that the qRT-PCR assay is much faster in testing and is approximately 8-fold more sensitive than the infectivity assay. Therefore, the qRT-PCR assay can replace the infectivity assay in many cases, but both assays are complementary in elucidating the mechanism of virus inactivation and removal in virus clearance validation.
authors.affiliation: Pathogen Safety Department, Global Biologics Development, Bayer HealthCare Pharmaceuticals, Berkeley, CA, USA.
authors.forename: Mohammad
authors.initials: M
authors.lastname: Anwaruzzaman
authors.affiliation: Pathogen Safety Department, Global Biologics Development, Bayer HealthCare Pharmaceuticals, Berkeley, CA, USA.
authors.forename: Wensheng
authors.initials: W
authors.lastname: Wang
authors.affiliation: Pathogen Safety Department, Global Biologics Development, Bayer HealthCare Pharmaceuticals, Berkeley, CA, USA.
authors.forename: Eunice
authors.initials: E
authors.lastname: Wang
authors.affiliation: Pathogen Safety Department, Global Biologics Development, Bayer HealthCare Pharmaceuticals, Berkeley, CA, USA.
authors.forename: Lolita
authors.initials: L
authors.lastname: Erfe
authors.affiliation: Pathogen Safety Department, Global Biologics Development, Bayer HealthCare Pharmaceuticals, Berkeley, CA, USA.
authors.forename: Janice
authors.initials: J
authors.lastname: Lee
authors.affiliation: Pathogen Safety Department, Global Biologics Development, Bayer HealthCare Pharmaceuticals, Berkeley, CA, USA. Electronic address: shengjiang.liu@bayer.com.
authors.forename: Shengjiang
authors.initials: S
authors.lastname: Liu
mesh_terms: D000818:Animals; D002415:Cats; D002460:Cell Line; D009052:Leukemia Virus, Murine / Q000302:isolation & purification / Q000472:pathogenicity*; D057230:Limit of Detection; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D014774:Virulence
keywords: Chromatography; Infectivity assay; Reverse transcriptase real-time PCR assay; Virus clearance; Virus filtration; Xenotropic murine leukemia virus