title: Quantitative detection of residual porcine host cell DNA by real-time PCR.
abstract: All biological products are derived from complex living systems and are often mixed with large numbers of impurities. For reasons of safety, residual host-cell DNA must be eliminated during processing. To assay host-cell DNA content in biopharmaceutical products derived from porcine sources, this study applies the quantitative real-time polymerase chain reaction (Q-PCR) method. The optimized assay in this study is based on the pol region of the porcine endogenous retrovirus (PERV). Assay validation results demonstrate that the proposed assay has appropriate accuracy, preciseness, reproducibility, and sensitivity. Primer and probe specificity are evaluated in real-time Q-PCR reactions using genomic DNA from rabbit, mouse, cat, hamster, monkey, human cell, yeast, and Escherichia coli as templates. The sensitivity of real-time Q-PCR is determined using genomic DNA from the porcine kidney cell line. The reliable detection range is within 0.5-10(5) pg/reaction. The limit of quantitation is 500 fg. The sensitivity of the assay meets the authority criterion. Moreover, the assay is applied to determine the level of host-cell DNA in recombinant human coagulation factor IX (rhFIX) from transgenic pigs. The real-time Q-PCR assay is thus a promising new tool for quantitative detection and clearance validation of residual porcine DNA when manufacturing recombinant therapeutics.
authors.affiliation: Animal Technology Institute, Chunan, Miaoli, Taiwan.
authors.forename: Jen-Ting
authors.initials: JT
authors.lastname: Chang
authors.affiliation: Animal Technology Institute, Chunan, Miaoli, Taiwan.
authors.forename: Yu-Chen
authors.initials: YC
authors.lastname: Chen
authors.affiliation: Animal Technology Institute, Chunan, Miaoli, Taiwan.
authors.forename: Yu-Chi
authors.initials: YC
authors.lastname: Chou
authors.affiliation: Animal Technology Institute, Chunan, Miaoli, Taiwan. Electronic address: wsr@mail.atit.org.tw.
authors.forename: Shih-Rong
authors.initials: SR
authors.lastname: Wang
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D001483:Base Sequence; D004247:DNA / Q000032:analysis*; D017931:DNA Primers; D020077:Endogenous Retroviruses / Q000235:genetics; D005164:Factor IX / Q000737:chemistry / Q000235:genetics; D006801:Humans; D057230:Limit of Detection; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D013045:Species Specificity; D013552:Swine
chemical_list: D017931:DNA Primers; D011994:Recombinant Proteins; D005164:Factor IX; D004247:DNA
keywords: PERV; Q-PCR; Residual DNA; transgenic pig