کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
2406575 1103086 2007 12 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Production of clinical-grade plasmid DNA for human Phase I clinical trials and large animal clinical studies
کلمات کلیدی
موضوعات مرتبط
علوم زیستی و بیوفناوری ایمنی شناسی و میکروب شناسی ایمونولوژی
پیش نمایش صفحه اول مقاله
Production of clinical-grade plasmid DNA for human Phase I clinical trials and large animal clinical studies
چکیده انگلیسی

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 (∼12 L each) in shaker-flasks, yielding ∼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 ≥95% supercoiled DNA, an A260/A280 ratio ≥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 °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.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Vaccine - Volume 25, Issue 27, 28 June 2007, Pages 5013–5024
نویسندگان
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