A METHOD FOR LARGE SCALE ISOLATION AND PURIFICATION OF PLASMID DNA

摘要

1. A process for large scale isolation and purification of plasmid DNA from large scale microbial cell fermentations, comprising: a) harvesting microbial cells from a large scale fermentation; b) adding to the harvested microbial cells a sufficient amount of a lysis solution; optionally not comprising lysozyme; c) heating the microbial cells of step b) to a temperature between 70 degree C and 100 degree C in a flow-through heat exchanger to form a crude lysate; d) centrifuging the crude lysate and collecting supernatant e) filtering and diafiltering the supernatant of step d) providing a filtrate; f) contacting the filtrate of step e) with an anion exchange matrix; g) eluting and collecting plasmid DNA from the anion exchange matrix; h) contacting the plasmid DNA from step g) with a reversed phase high performance liquid chromatography matrix; and i) eluting and collecting the plasmid DNA of step g) from the matrix. 2. The process of claim 1 wherein optionally concentrating and/or diafiltering the product of step i) into a pharmaceutically acceptable carrier. 3. The process of claim 2 wherein optionally sterilizing the DNA product. 4. The process of claims 1-3 wherein the heating of step c) is to a temperature between 70 degree C and 77 degree C. 5. The process of claims 1-3 wherein the lysis solution of step b) contains a sub-microgram concentration of lysozyme. 6. The process of claims 1-3 optionally including RNAsa treatment at any step following step a). 7. The process of claims 1-3 wherein the lysis solution of step b) is modified STET buffer. 8. The process of claims 1-3 wherein the lysis solution of step b) is modified STET buffer containing a sub-microgram concentration of lysozyme.