title: Quantitative competitive reverse transcription-PCR as a method to evaluate retrovirus removal during chromatography procedures.
abstract: Chinese hamster ovary cells used for pharmaceutical protein production express non-infectious retrovirus-like particles. To assure the safety of pharmaceutical proteins, validation of the ability of manufacturing process to clear retrovirus-like particles is required for product registration. Xenotropic murine leukemia virus (X-MuLV) is often used as a model virus for validation studies. Some chromatography procedures used for pharmaceutical protein purification utilize low pH (< pH 4.0) elution buffers which readily inactivate X-MuLV. Therefore, cell-based infectivity assays are unable to evaluate the physical removal of X-MuLV by these chromatography procedures. To distinguish viral inactivation by low pH treatment from viral removal by chromatography, a quantitative competitive reverse transcription PCR method capable of quantifying both infectious and non-infectious X-MuLV has been developed. This method quantifies X-MuLV particles in chromatography pools by quantifying the X-MuLV particle RNA (pRNA). The difference between the amount of X-MuLV pRNA in the load pool and the product-containing elution pool represents the extent of X-MuLV removal. This method is an extremely powerful complement to cell based-infectivity assays as it allows physical removal of X-MuLV by chromatography and filtration procedures to be distinguished from X-MuLV inactivation when buffers with the ability to inactivate retrovirus are used.
authors.affiliation: Genentech, Inc., South San Francisco, CA 94080, USA.
authors.forename: A S
authors.initials: AS
authors.lastname: Lau
authors.forename: Y S
authors.initials: YS
authors.lastname: Lie
authors.forename: L A
authors.initials: LA
authors.lastname: Norling
authors.forename: J
authors.initials: J
authors.lastname: Sernatinger
authors.forename: M
authors.initials: M
authors.lastname: Dinowitz
authors.forename: C J
authors.initials: CJ
authors.lastname: Petropoulos
authors.forename: Y
authors.initials: Y
authors.lastname: Xu
mesh_terms: D000818:Animals; D001483:Base Sequence; D016466:CHO Cells / Q000821:virology*; D002845:Chromatography / Q000379:methods; D006224:Cricetinae; D003412:Cricetulus; D009052:Leukemia Virus, Murine / Q000235:genetics / Q000302:isolation & purification; D008969:Molecular Sequence Data; D012367:RNA, Viral / Q000032:analysis*; D012190:Retroviridae / Q000235:genetics / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*
chemical_list: D012367:RNA, Viral