Real-time quantitative PCR for retrovirus-like particle quantification in CHO cell culture.
Chinese hamster ovary (CHO) cells have been widely used to manufacture recombinant proteins intended for human therapeutic uses. Retrovirus-like particles, which are apparently defective and non-infectious, have been detected in all CHO cells by electron microscopy (EM). To assure viral safety of CHO cell-derived biologicals, quantification of retrovirus-like particles in production cell culture and demonstration of sufficient elimination of such retrovirus-like particles by the down-stream purification process are required for product market registration worldwide. EM, with a detection limit of 1x10(6) particles/ml, is the standard retrovirus-like particle quantification method. The whole process, which requires a large amount of sample (3-6 litres), is labour intensive, time consuming, expensive, and subject to significant assay variability. In this paper, a novel real-time quantitative PCR assay (TaqMan assay) has been developed for the quantification of retrovirus-like particles. Each retrovirus particle contains two copies of the viral genomic particle RNA (pRNA) molecule. Therefore, quantification of retrovirus particles can be achieved by quantifying the pRNA copy number, i.e. every two copies of retroviral pRNA is equivalent to one retrovirus-like particle. The TaqMan assay takes advantage of the 5'-->3' exonuclease activity of Taq DNA polymerase and utilizes the PRISM 7700 Sequence Detection System of PE Applied Biosystems (Foster City, CA, U.S.A.) for automated pRNA quantification through a dual-labelled fluorogenic probe. The TaqMan quantification technique is highly comparable to the EM analysis. In addition, it offers significant advantages over the EM analysis, such as a higher sensitivity of less than 600 particles/ml, greater accuracy and reliability, higher sample throughput, more flexibility and lower cost. Therefore, the TaqMan assay should be used as a substitute for EM analysis for retrovirus-like particle quantification in CHO cell-based production system.
de Wit
C
C
Department of Cell Culture and Fermentation R&D, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, USA.
Fautz
C
C
Xu
Y
Y
D000818:Animals; D001685:Biological Factors / Q000096:biosynthesis / Q000235:genetics*; D016466:CHO Cells / Q000821:virology*; D018929:Cell Culture Techniques / Q000592:standards*; D006224:Cricetinae; D006801:Humans; D008854:Microscopy, Electron; D016133:Polymerase Chain Reaction / Q000379:methods*; D011786:Quality Control; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D012044:Regression Analysis; D015203:Reproducibility of Results; D012190:Retroviridae / Q000302:isolation & purification* / Q000648:ultrastructure; D014162:Transfection
D001685:Biological Factors; D011994:Recombinant Proteins