Specific detection of residual CHO host cell DNA by real-time PCR.
Chinese hamster ovary cells have been widely used to manufacture recombinant proteins for human therapeutic use. A sensitive quantitative real-time polymerase chain reaction assay for the detection of residual Chinese hamster (Cricetulus griseus) DNA is presented in this paper. The assay is reasonably affordable and can be adapted for high-throughput screening using 96-well format. Real-time PCR primers were designed to amplify a 150bp region of a genomic fragment from hamster DNA. The specificity of the probe was evaluated in real-time PCR reactions using genomic DNA from mouse fibroblast, human kidney and hamster ovary cell lines as template. Sensitivity of real-time PCR was compared on genomic DNA from hamster cell line CHO DG44. These primers can be used in real-time PCR reactions to detect presence of contaminating hamster DNA in purified protein samples down to sensitivity of 300fg genomic DNA.
Nissom
Peter Morin
PM
Bioprocessing Technology Institute, #06-01 Centros, 20 Biopolis Way, 138668 Singapore, Singapore. peter_morin@bti.a-star.edu.sg
D000818:Animals; D001483:Base Sequence; D016466:CHO Cells / Q000737:chemistry* / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D004247:DNA / Q000032:analysis* / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics; D004340:Drug Contamination; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods / Q000706:statistics & numerical data; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D012680:Sensitivity and Specificity
D017931:DNA Primers; D011994:Recombinant Proteins; D004247:DNA