title: Transposon leads to contamination of clinical pDNA vaccine.
abstract: We report an unexpected contamination during clinical manufacture of a Human Papilomavirus (HPV) 16 E6 encoding plasmid DNA (pDNA) vaccine, with a transposon originating from the Escherichia coli DH5 host cell genome. During processing, presence of this transposable element, insertion sequence 2 (IS2) in the plasmid vector was not noticed until quality control of the bulk pDNA vaccine when results of restriction digestion, sequencing, and CGE analysis were clearly indicative for the presence of a contaminant. Due to the very low level of contamination, only an insert-specific PCR method was capable of tracing back the presence of the transposon in the source pDNA and master cell bank (MCB). Based on the presence of an uncontrolled contamination with unknown clinical relevance, the product was rejected for clinical use. In order to prevent costly rejection of clinical material, both in-process controls and quality control methods must be sensitive enough to detect such a contamination as early as possible, i.e. preferably during plasmid DNA source generation, MCB production and ultimately during upstream processing. However, as we have shown that contamination early in the process development pipeline (source pDNA, MCB) can be present below limits of detection of generally applied analytical methods, the introduction of "engineered" or transposon-free host cells seems the only 100% effective solution to avoid contamination with movable elements and should be considered when searching for a suitable host cell-vector combination.
authors.affiliation: Department of Pharmacy & Pharmacology, Slotervaart Hospital/The Netherlands Cancer Institute, Amsterdam, The Netherlands. Iris.vanderHeijden@slz.nl
authors.forename: I
authors.initials: I
authors.lastname: van der Heijden
authors.forename: R
authors.initials: R
authors.lastname: Gomez-Eerland
authors.forename: J H
authors.initials: JH
authors.lastname: van den Berg
authors.forename: K
authors.initials: K
authors.lastname: Oosterhuis
authors.forename: T N
authors.initials: TN
authors.lastname: Schumacher
authors.forename: J B A G
authors.initials: JB
authors.lastname: Haanen
authors.forename: J H
authors.initials: JH
authors.lastname: Beijnen
authors.forename: B
authors.initials: B
authors.lastname: Nuijen
mesh_terms: D004251:DNA Transposable Elements; D004269:DNA, Bacterial / Q000737:chemistry; D004340:Drug Contamination; D004926:Escherichia coli / Q000235:genetics*; D005285:Fermentation; D005822:Genetic Vectors; D057230:Limit of Detection; D009856:Oncogene Proteins, Viral / Q000235:genetics; D053918:Papillomavirus Vaccines / Q000096:biosynthesis* / Q000235:genetics; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011786:Quality Control; D012097:Repressor Proteins / Q000235:genetics; D015183:Restriction Mapping; D017422:Sequence Analysis, DNA; D019444:Vaccines, DNA / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D004251:DNA Transposable Elements; D004269:DNA, Bacterial; C068651:E6 protein, Human papillomavirus type 16; D009856:Oncogene Proteins, Viral; D053918:Papillomavirus Vaccines; D012097:Repressor Proteins; D019444:Vaccines, DNA