Patent ID: 6197553
Filing Date: 2001-03-06
Classification: C12N

Abstract:
A process for isolation and purification of plasmid DNA from a large scale microbial cell fermentation with a volume of greater than about 5 liters, comprising:a) harvesting the cells;b) adding a lysis solution to the harvested cells, resulting in a cell slurry;c) heating the cell slurry of step b) in a flow-through heat exchange apparatus wherein the flow rate of the cell slurry through the heat exchange apparatus is adjusted such that the temperature of the cell slurry upon exiting the heat exchange apparatus is from about 70.degree. C. to about 80.degree. C. so as to denature cellular proteins, such that a crude lysate is formed;d) centrifuging the crude lysite to yield a pellet and a supernatant;e) filtering and diafiltering the supernatant of step d) to provide a filtrate containing the plasmid DNA;f) contacting the filtrate of step e) with an anion exchange matrix;g) eluting and collecting the plasmid DNA from the anion exchange matrix;h) contacting the plasmid DNA from step g) with a reversed phase high performance liquid chromatography matrix;i) eluting and collecting the plasmid DNA from the reversed phase high performance liquid chromatography matrix of step h);j) optionally concentrating and/or diafiltering the product of step i) into a pharmaceutically acceptable carrier;k) optionally sterilizing the DNA product;wherein:the lysis solution of step b) is modified STET buffer and lysozyme, the modified STET buffer consisting essentially of about 50 mM TRIS, about 50-100 mM EDTA, about 8% (w/v) sucrose and about 2% Triton X-100.