title: Large-scale purification of pharmaceutical-grade plasmid DNA using tangential flow filtration and multi-step chromatography.
abstract: The demand for pharmaceutical-grade plasmid DNA in vaccine applications and gene therapy has been increasing in recent years. In the present study, a process consisting of alkaline lysis, tangential flow filtration, purification by anion exchange chromatography, hydrophobic interaction chromatography and size exclusion chromatography was developed. The final product met the requirements for pharmaceutical-grade plasmid DNA. The chromosomal DNA content was <1 μg/mg plasmid DNA, and RNA was not detectable by agarose gel electrophoresis. Moreover, the protein content was <2 μg/mg plasmid DNA, and the endotoxin content was <10 EU/mg plasmid DNA. The process was scaled up to yield 800 mg of pharmaceutical-grade plasmid DNA from approximately 2 kg of bacterial cell paste. The overall yield of the final plasmid DNA reached 48%. Therefore, we have established a rapid and efficient production process for pharmaceutical-grade plasmid DNA.
authors: National Engineering Laboratory for AIDS Vaccine, Jilin University, Changchun 130012, China.
authors: Bo
authors: B
authors: Sun
authors: XiangHui
authors: X
authors: Yu
authors: Yuhe
authors: Y
authors: Yin
authors: Xintao
authors: X
authors: Liu
authors: Yongge
authors: Y
authors: Wu
authors: Yan
authors: Y
authors: Chen
authors: Xizhen
authors: X
authors: Zhang
authors: Chunlai
authors: C
authors: Jiang
authors: Wei
authors: W
authors: Kong
mesh_terms: D002845:Chromatography / Q000379:methods*; D002876:Chromosomes, Bacterial; D004247:DNA / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D004340:Drug Contamination; D005374:Filtration / Q000379:methods*; D015316:Genetic Therapy; D010957:Plasmids / Q000096:biosynthesis / Q000302:isolation & purification* / Q000592:standards; D012313:RNA / Q000032:analysis; D019444:Vaccines, DNA
chemical_list: D019444:Vaccines, DNA; D012313:RNA; D004247:DNA
keywords: Alkaline lysis; Chromatography; DNA vaccine; Gene therapy; Purification