title: Production of clinical-grade plasmid DNA for human Phase I clinical trials and large animal clinical studies.
abstract: The use of plasmid DNA as vaccines for the treatment of cancer and infectious diseases is on the rise. In order to facilitate the manufacture of clinical-grade plasmid DNA for Phase I clinical trials, we developed a process whereby >200 mg plasmid could be produced in a single production run under Good Manufacturing Practices. A dedicated cleanroom (Class 10,000 with Class 100 biosafety cabinet) is utilized for production of the bacterial cell bank, fermentation, harvest/lysis of the biomass, and downstream purification. Fermentation requires three 16-18 h runs (approximately 12 L each) in shaker-flasks, yielding approximately 60 g bacterial paste following batch centrifugation. The biomass is alkaline-lysed, pooled, and the resulting flocculent precipitate is separated by a novel vacuum step, followed by depth-filtration. Downstream processing includes anion-exchange chromatography, utilizing Qiagen silica-based resin, and precipitation with isopropanol. Following precipitation, the DNA is harvested by centrifugation, dried, formulated, and sterile-filtered using a Sartorius Sartobran 150 filter prior to Final-Filling. All processing steps utilize sterilized, single-use components. This process results in a product manufactured according to regulatory guidelines. The plasmid DNA is sterile with >or=95% supercoiled DNA, an A260/A280 ratio>or=1.9, undetectable or extremely low residual endotoxin, RNA, genomic DNA, protein, and antibiotic. Residual solvent levels are negligible. The product yields the predicted profile upon restriction-enzyme digestion, is biologically active upon transfection and remains stable for several years at -20 degrees C. We have therefore developed a reproducible and cost effective process to manufacture clinical-grade plasmid DNA. This process can be adapted by other academic centers for human or large animal clinical trials.
authors.affiliation: Gene Transfer and Somatic Cell Engineering Facility, Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA.
authors.forename: Mark
authors.initials: M
authors.lastname: Przybylowski
authors.forename: Shirley
authors.initials: S
authors.lastname: Bartido
authors.forename: Oriana
authors.initials: O
authors.lastname: Borquez-Ojeda
authors.forename: Michel
authors.initials: M
authors.lastname: Sadelain
authors.forename: Isabelle
authors.initials: I
authors.lastname: Rivière
mesh_terms: D000818:Animals; D002482:Cellulose / Q000031:analogs & derivatives; D002852:Chromatography, Ion Exchange; D002874:Chromosome Mapping; D017321:Clinical Trials, Phase I as Topic / Q000592:standards*; D003470:Culture Media; D004247:DNA / Q000032:analysis / Q000096:biosynthesis*; D004345:Drug Industry; D004591:Electrophoresis, Polyacrylamide Gel; D005285:Fermentation; D006801:Humans; D007202:Indicators and Reagents; D010957:Plasmids / Q000235:genetics*; D019444:Vaccines, DNA / Q000032:analysis / Q000096:biosynthesis*
chemical_list: D003470:Culture Media; D007202:Indicators and Reagents; D019444:Vaccines, DNA; C005062:acetylcellulose; D002482:Cellulose; D004247:DNA