title: Comparison of electron microscopic techniques for enumeration of endogenous retrovirus in mouse and Chinese hamster cell lines used for production of biologics.
abstract: Murine myeloma and Chinese hamster ovary cells are used widely in the manufacture of recombinant proteins for biopharmaceuticals. However, rodent cell lines express endogenous retrovirus, which necessitates appropriate design of purification processes to remove virus in excess of the calculated maximum retroviral load. Currently, electron microscopy is the method of choice for determination of retroviral titre in bulk harvest. In this study we compared three electron microscopy techniques to determine retroviral titre in bulk harvest. These were direct negative stain, negative stain after sucrose-density purification and thin section electron microscopy of pelleted supernatant. The study demonstrated that the level of C-type retrovirus associated with cells was predictive of the viral load in cell culture supernatants. The most accurate method for quantifying viral load was direct counting, followed by thin section of pelleted supernatant and negative stain after sucrose concentration. The most practical method was thin section of resuspended pelleted supernatant, which gave improved detection limits.
authors.affiliation: Q-One Biotech Ltd, Todd Campus, West of Scotland Science Park, G20 0XA, Glasgow, UK.
authors.forename: G G
authors.initials: GG
authors.lastname: Reid
authors.forename: E W
authors.initials: EW
authors.lastname: Milne
authors.forename: L W
authors.initials: LW
authors.lastname: Coggins
authors.forename: N J
authors.initials: NJ
authors.lastname: Wilson
authors.forename: K T
authors.initials: KT
authors.lastname: Smith
authors.forename: A J
authors.initials: AJ
authors.lastname: Shepherd
mesh_terms: D000818:Animals; D001688:Biological Products / Q000302:isolation & purification; D016466:CHO Cells; D002460:Cell Line; D002499:Centrifugation, Density Gradient; D006224:Cricetinae; D004340:Drug Contamination; D020077:Endogenous Retroviruses / Q000302:isolation & purification* / Q000648:ultrastructure*; D051379:Mice; D008854:Microscopy, Electron / Q000379:methods*; D014773:Virology / Q000379:methods*
chemical_list: D001688:Biological Products