title: Development of a Vero cell DNA reference standard for residual DNA measurement in China.
abstract: This collaborative study developed a Vero cell DNA reference for standardizing dot blot hybridization, an assay widely employed to measure residual DNA contents of viral vaccines prepared with Vero cells. High purity of Vero cell DNA was extracted and characterized by Hind III enzyme digestion and DNA sequencing. Then, with a cooperative calibration, the concentration of Vero cell DNA reference bulk solution was determined (64.0 ± 1.9 μg/mL, OD 260/OD 280 = 1.87) and diluted (40 ng/mL) with Tris-EDTA buffer containing bovine serum albumin as freeze-dried excipients. With industrial filling apparatus, the diluted bulk was loaded into ampoules (0.5 mL each) which were heat sealed after nitrogen filling. Finally, a collaborative study showed that the Vero cell DNA reference could reach a sensitivity of 1 to 5 pg/dot and maintained good stability after accelerated destruction test. The successful establishment of the Vero cell DNA quantitative reference will facilitate the standardization of dot blot hybridization for testing residual host cell DNA.
authors.affiliation: National Institutes for Food and Drug Control; Key Laboratory of the Ministry of Health for Research on Quality and Standardization of Biotech Products; Beijing, PR China.
authors.forename: Shouchun
authors.initials: S
authors.lastname: Cao
authors.forename: Guanmu
authors.initials: G
authors.lastname: Dong
authors.forename: Jianrong
authors.initials: J
authors.lastname: Tang
authors.forename: Jia
authors.initials: J
authors.lastname: Li
authors.forename: Jinghua
authors.initials: J
authors.lastname: Liu
authors.forename: Leitai
authors.initials: L
authors.lastname: Shi
authors.forename: Changgui
authors.initials: C
authors.lastname: Li
authors.forename: Junzhi
authors.initials: J
authors.lastname: Wang
mesh_terms: D000818:Animals; D002681:China; D002522:Chlorocebus aethiops; D004247:DNA / Q000032:analysis*; D009693:Nucleic Acid Hybridization / Q000379:methods*; D011786:Quality Control; D012015:Reference Standards; D013678:Technology, Pharmaceutical / Q000379:methods*; D014709:Vero Cells; D014765:Viral Vaccines / Q000737:chemistry*
chemical_list: D014765:Viral Vaccines; D004247:DNA
keywords: Vero cell genomic DNA; dot blot; quantitative reference standard; standardization