title: Universal real-time PCR assay for quantitation and size evaluation of residual cell DNA in human viral vaccines.
abstract: Residual host cellular DNA (rcDNA) is one of the principal risk associated with continuous cell lines derived medicines such as viral vaccines. To assess rcDNA degradation, we suggest two quantitative real-time PCR assays designed to separately quantify target sequences shorter and longer than the 200 bp risk limit, the relative abundance of both targets reflecting the extent of rcDNA fragmentation. The conserved multicopy ribosomal 18S RNA gene was targeted to detect host cell templates from most mammalian cell substrates commonly used in the manufacture of human viral vaccines. The detection range of the method was assessed on purified DNA templates from different animal origins. The standard calibrator origin and structural conformation were shown crucial to achieve accurate quantification. Artificial mixtures of PCR products shorter and longer than 200 bp were used as a model to check the ability of the assay to estimate the fragment size distribution. The method was successfully applied to a panel of Vero cell derived vaccines and could be used as a universal method for determination of both content and size distribution of rcDNA in vaccines.
authors.affiliation: Agence Nationale de Sécurité du Médicament et des Produits de Santé (ANSM), Control Directorate, Biological Controls for Immunological Medicines and Biological Safety Department, 321 Avenue Jean Jaurès, F-69007, Lyon, France. Electronic address: murielle.andre@ansm.sante.fr.
authors.forename: Murielle
authors.initials: M
authors.lastname: André
authors.affiliation: Agence Nationale de Sécurité du Médicament et des Produits de Santé (ANSM), Control Directorate, Biological Controls for Immunological Medicines and Biological Safety Department, 321 Avenue Jean Jaurès, F-69007, Lyon, France.
authors.forename: Sylviane
authors.initials: S
authors.lastname: Reghin
authors.affiliation: Agence Nationale de Sécurité du Médicament et des Produits de Santé (ANSM), Control Directorate, Biological Controls for Immunological Medicines and Biological Safety Department, 321 Avenue Jean Jaurès, F-69007, Lyon, France.
authors.forename: Estelle
authors.initials: E
authors.lastname: Boussard
authors.affiliation: Agence Nationale de Sécurité du Médicament et des Produits de Santé (ANSM), Control Directorate, Biological Controls for Immunological Medicines and Biological Safety Department, 321 Avenue Jean Jaurès, F-69007, Lyon, France.
authors.forename: Laurent
authors.initials: L
authors.lastname: Lempereur
authors.affiliation: Agence Nationale de Sécurité du Médicament et des Produits de Santé (ANSM), Control Directorate, Biological Controls for Immunological Medicines and Biological Safety Department, 321 Avenue Jean Jaurès, F-69007, Lyon, France.
authors.forename: Stéphane
authors.initials: S
authors.lastname: Maisonneuve
mesh_terms: D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D006224:Cricetinae; D003412:Cricetulus; D004247:DNA / Q000235:genetics* / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D004285:Dogs; D004340:Drug Contamination / Q000517:prevention & control; D005240:Feasibility Studies; D006801:Humans; D061985:Madin Darby Canine Kidney Cells; D012337:RNA, Ribosomal, 18S / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D014709:Vero Cells; D014765:Viral Vaccines / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D014780:Viruses / Q000235:genetics* / Q000276:immunology / Q000378:metabolism
chemical_list: D012337:RNA, Ribosomal, 18S; D014765:Viral Vaccines; D004247:DNA; D015252:Deoxyribonucleases, Type II Site-Specific; C024412:GGTGA-specific type II deoxyribonucleases
keywords: Human vaccine purity; Residual DNA; Size distribution; qPCR