Varley D L, Hitchcock A G, Weiss A M, Horler W A, Cowell R, Peddie L, Sharpe G S, Thatcher D R, Hanak J A
Cobra Therapeutics Ltd, Keele, Staffs., U.K.
Bioseparation. 1999;8(1-5):209-17.
We describe a process for the commercial manufacture of therapeutic grade plasmid DNA. The industrially scaleable unit operations employed in this process are: (i) optimized alkaline lysis; (ii) bag filtration; (iii) expanded bed anion exchange chromatography; (iv) ultrafiltration, and (v) size exclusion chromatography. These steps are scaleable alternatives to current approaches to plasmid DNA isolation such as high speed centrifugation for feed-stock clarification and solvent precipitation for plasmid concentration, and an efficient alternative to conventional low through-put packed bed chromatography. The process produces plasmid DNA characterized by low level chromosomal DNA, RNA and endotoxin contamination without the use of flammable solvents or toxic reagents and is suitable for therapeutic administration.
我们描述了一种治疗级质粒DNA的商业化生产工艺。该工艺所采用的可进行工业规模放大的单元操作包括：（i）优化的碱裂解；（ii）袋式过滤；（iii）扩张床阴离子交换色谱；（iv）超滤，以及（v）尺寸排阻色谱。这些步骤是质粒DNA分离现有方法的可扩展替代方案，如用于原料澄清的高速离心和用于质粒浓缩的溶剂沉淀，并且是传统低通量填充床色谱的有效替代方法。该工艺生产的质粒DNA具有低水平的染色体DNA、RNA和内毒素污染，无需使用易燃溶剂或有毒试剂，适用于治疗给药。
