title: Standardized large-scale H-1PV production process with efficient quality and quantity monitoring.
abstract: The promising anticancer properties of rodent protoparvoviruses, notably H-1PV, have led to their clinical testing. This makes it necessary to produce highly pure, well-characterized virus batches in sufficient quantity. The present work focused on developing standardized production, purification, and characterization procedures as a basis for exploiting H-1PV both preclinically and in clinical trials for anticancer virotherapy. Two infection and two virus purification strategies were tested and the resulting virus preparations compared for their purity and full-, infectious-, and empty-particle contents. The adopted production process, which involves culturing and infecting NB-324K cells in 10-layer CellSTACK(®) chambers (1×10(3) infectious units per infected cell), is simple, scalable, and reproducible. Downstream processing to eliminate contaminating DNA and protein includes DNAse treatment, filtration, and two Iodixanol density-gradient centrifugations, the first gradient being a step gradient and the second, either a step (1×10(10) PFU/ml) or a continuous gradient (3×10(11) PFU/ml). A procedure was also developed for obtaining infectious particle-free preparations of empty virions for research purposes: cesium chloride density gradient centrifugation followed by UV irradiation (1×10(14) physical particles/ml). For quick, sensitive determination of physical particles (and hence, particle-to-infectivity ratios), a "Capsid-ELISA" was developed, based on a novel monoclonal antibody that specifically targets assembled capsids.
authors.affiliation: German Cancer Research Center (DKFZ), Tumor Virology F010, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany. Electronic address: B.Leuchs@dkfz.de.
authors.forename: Barbara
authors.initials: B
authors.lastname: Leuchs
authors.affiliation: German Cancer Research Center (DKFZ), Tumor Virology F010, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany.
authors.forename: Mandy
authors.initials: M
authors.lastname: Roscher
authors.affiliation: German Cancer Research Center (DKFZ), Tumor Virology F010, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany.
authors.forename: Marcus
authors.initials: M
authors.lastname: Müller
authors.affiliation: German Cancer Research Center (DKFZ), Tumor Virology F010, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany.
authors.forename: Kathrin
authors.initials: K
authors.lastname: Kürschner
authors.affiliation: German Cancer Research Center (DKFZ), Tumor Virology F010, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany.
authors.forename: Jean
authors.initials: J
authors.lastname: Rommelaere
mesh_terms: D002460:Cell Line; D002499:Centrifugation, Density Gradient / Q000379:methods* / Q000592:standards; D004203:Disinfection / Q000379:methods; D004847:Epithelial Cells / Q000821:virology; D005374:Filtration / Q000379:methods* / Q000592:standards; D006801:Humans; D019219:Parvovirinae / Q000254:growth & development* / Q000302:isolation & purification*; D019562:Viral Load / Q000379:methods; D014776:Virus Cultivation / Q000379:methods* / Q000592:standards
keywords: Capsid-ELISA; Characterization; Large-scale production; Protoparvovirus H-1PV; Purification