Standardized large-scale H-1PV production process with efficient quality and quantity monitoring

• Published in: Journal of virological methods 2016-03, Vol.229, p.48-59
• Main Authors: Leuchs, Barbara, Roscher, Mandy, Müller, Marcus, Kürschner, Kathrin, Rommelaere, Jean
• Format: Article
• Language: English
• Subjects: Capsid-ELISA; Cell Line; Centrifugation, Density Gradient - methods; Centrifugation, Density Gradient - standards; Characterization; Disinfection - methods; Epithelial Cells - virology; Filtration - methods; Filtration - standards; Humans; Large-scale production; Parvovirinae - growth & development; Parvovirinae - isolation & purification; Protoparvovirus H-1PV; Purification; Viral Load - methods; Virus Cultivation - methods; Virus Cultivation - standards
• ISSN: 0166-0934; 1879-0984
• DOI: 10.1016/j.jviromet.2015.11.022

•Development of a reproducible, effective, and scalable process for H-1PV production.•Standardization of infectious H-1PV particle purification through successive steps and continuous Iodixanol gradient ultracentrifugation.•Preparation of infectious virus-free batches of empty H-1PV by cesium chloride gradient ultracentrifugation followed by UV irradiation.•Generation of a monoclonal antibody recognizing assembled H-1PV capsids.•Development of a new ELISA for capsid quantification. 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 (1×103 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 (1×1010PFU/ml) or a continuous gradient (3×1011PFU/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 (1×1014physicalparticles/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.