摘要
在实验室有效获得100 mg临床试验等级的质粒DNA。摇床发酵3个批次,每批次4 L培养液,产生大约160 g的细菌沉淀物。碱裂解菌体,气浮法结合离心分离浮杂沉淀。裂解液经0.45/0.22μm囊式过滤器过滤。然后采用阴离子色谱介质富集质粒,异丙醇沉淀。重新溶解沉淀后采用100 kD切向流超滤处理,获得100 mg临床试验等级质粒DNA。体外、体内转染试验对质粒表达效果进行了鉴定。结果表明,纯化的质粒DNA几乎检测不到内毒素、RNA和蛋白质,A260/A280比值在1.82~1.86范围内。纯化的质粒DNA能够满足动物临床试验。
In order to facilitate the production of - 100 mg clinical-grade plasmid DNA in an academic laboratory environment, about 160 g bacterial paste was yielded through fermentation. The biomass was alkaline-lysed, and the flocculent precipitate was separated by air floatation, followed by centrifugation. After rey procedures,including depth-filtration, downstream processing, extraction tangential flow filtration (TFF) ,final product of 100 mg plasmid DNA was obtained. The product was biologically active upon in vitro and in vivo transfection. Besides,the plasmid DNA was proved as undetectable or extremely low residual endotoxin, RNA, protein, and the A260/A280 ratio between 1.82 to 1.86. Therefore,this process for plasmid purification can be adapted for animal clinical trials.
出处
《生物技术通报》
CAS
CSCD
北大核心
2009年第5期135-138,126,共5页
Biotechnology Bulletin
关键词
临床等级质粒DNA
色谱制备
质量控制
Clinical-grade plasmid DNA Chromatography preparation Quality control