Method for large scale plasmid purification

Abstract

A process is disclosed for the large scale isolation and purification of plasmid DNA from large scale microbial fermentations. All three forms of plasmid DNA; supercoil (form I), nicked or relaxed circle (form II), and linearized (form III), are individually isolatable using the disclosed process. Highly purified DNA suitable for inclusion in a pharmaceutical composition is provided by the disclosed process.

What is claimed is:  
     
         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;    c) heating the microbial cells of step b) to a temperature between 70° C. and 100° C. in a flow-through heat exchanger to form a crude lysate;    d) centrifuging the crude lysate;    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;    i) eluting and collecting the plasmid 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; and    k) optionally sterilizing the DNA product.    
     
     
         2 . The process of  claim 1  wherein the lysis solution of step b) is modified STET buffer.  
     
     
         3 . The process of  claim 1  wherein the heating of step c) is to a temperature between 70° C. and 77° C.  
     
     
         4 . The process of  claim 1  wherein the lysis solution of step b) contains a sub-microgram concentration of lysozyme.  
     
     
         5 . The process of  claim 1  optionally including RNase treatment at any step following step a).  
     
     
         6 . An isolated and purified plasmid DNA obtained by the process of  claim 1 .  
     
     
         7 . The plasmid DNA of  claim 6  wherein said plasmid is suitable for administration to humans.  
     
     
         8 . The plasmid DNA of  claim 6  wherein said plasmid is suitable for administration to non-human animals.  
     
     
         9 . The plasmid DNA of  claim 6  wherein said plasmid is a polynucleotide vaccine.  
     
     
         10 . An isolated and purified plasmid DNA suitable for administration to humans.  
     
     
         11 . The plasmid DNA of  claim 10  wherein said plasmid DNA is a polynucleotide vaccine.