Processes for the Preparation of Highly Pure Plasmid Compositions

Abstract

The present disclosure generally relates to processes for preparing highly pure plasmid compositions. The processes generally involve treating a composition comprising plasmid DNA with a polypeptide to digest colanic acid. The treated plasmid DNA is then separated from the treated composition.

What is claimed is: 
     
         1 . A process for the purification of a plasmid DNA, the process comprising:
 treating an aqueous composition containing the plasmid DNA with an isolated polypeptide that digests at least one of colanic acid or endotoxin; and   separating the plasmid DNA from the aqueous composition.   
     
     
         2 . The process of  claim 1 , wherein the aqueous composition is selected from at least one of a crude bacterial lysate, a partially purified bacterial lysate, and an aqueous solution containing extracted bacterial nucleic acid. 
     
     
         3 . The process of  claim 1 , wherein the separation comprises combining the treated aqueous composition with a chromatographic material. 
     
     
         4 . The process of  claim 3 , wherein the chromatographic material is selected from at least one of an anion exchange chromatography resin, a cation exchange chromatography resin, a quaternary ammonium resin, a hydrophobic interaction chromatography resin, or an affinity chromatography resin. 
     
     
         5 . The process of  claim 4 , wherein the separation comprises combining the treated aqueous composition with an affinity chromatography resin, and thereafter combining the treated aqueous composition with a hydrophobic interaction chromatography resin. 
     
     
         6 . The process of  claim 1 , wherein the aqueous composition is pre-treated to partially remove endotoxin from the aqueous composition prior to treatment with the polypeptide, the pre-treatment comprising combining the aqueous composition with a chromatographic material. 
     
     
         7 . The process of  claim 5 , wherein the affinity chromatography resin comprises a boronic acid-based or boronate-containing compound. 
     
     
         8 . The process of  claim 1 , further comprising filtering the aqueous composition to further separate the endotoxin from the plasmid DNA. 
     
     
         9 . The process of  claim 8 , wherein the filtering is performed after eluting the treated aqueous composition from the chromatography material. 
     
     
         10 . The process of  claim 1 , wherein the polypeptide is a recombinant polypeptide. 
     
     
         11 . The process of  claim 1 , wherein the aqueous composition is pre-treated to partially remove endotoxin from the aqueous composition prior to treatment with the polypeptide, the pre-treatment comprising combining the aqueous composition with a chromatographic material; combined with an affinity chromatography resin after treatment with the polypeptide, and thereafter combined with a hydrophobic interaction chromatography resin; and filtered to further separate endotoxin from the plasmid DNA after eluting the plasmid DNA from the hydrophobic interaction chromatography resin. 
     
     
         12 . The process of  claim 1 , wherein the polypeptide comprises an amino acid sequence having at least 90% homology to SEQ ID NO: 1 or SEQ ID NO: 2, and conservative amino acid substitutions thereof. 
     
     
         13 . The process of  claim 1 , wherein the polypeptide comprises an amino acid sequence having at least 98% homology to SEQ ID NO: 1 or SEQ ID NO: 2, and conservative amino acid substitutions thereof. 
     
     
         14 . The process of  claim 1 , wherein the polypeptide is SEQ ID NO: 1 or SEQ ID NO: 2. 
     
     
         15 . The process of  claim 1 , wherein the plasmid DNA is a gram-negative bacterial plasmid DNA. 
     
     
         16 . A process for the purification of plasmid DNA, the process comprising:
 (a) pre-treating an aqueous composition containing plasmid DNA by combining the aqueous composition with an anion exchange resin;   (b) treating the pre-treated aqueous composition with an isolated polypeptide that digests at least one of colanic acid or endotoxin;   (c) separating the plasmid DNA from the aqueous composition, the separation comprising combining the treated aqueous composition with an affinity chromatography resin, and thereafter combining the treated aqueous composition with a hydrophobic interaction chromatography resin; and   (d) filtering the plasmid DNA.   
     
     
         17 . The process of  claim 16 , wherein the anion exchange resin comprises a quaternary ammonium resin. 
     
     
         18 . The process of  claim 16 , wherein the affinity chromatography resin comprises a boronic acid-based or boronate-containing compound. 
     
     
         19 . The process of  claim 16 , wherein the polypeptide comprises an amino acid sequence having at least 90% homology to SEQ ID NO: 1 or SEQ ID NO: 2, and conservative amino acid substitutions thereof. 
     
     
         20 . The process of  claim 16 , wherein the polypeptide comprises an amino acid sequence having at least 98% homology to SEQ ID NO: 1 or SEQ ID NO: 2, and conservative amino acid substitutions thereof. 
     
     
         21 . The process of  claim 16 , wherein the plasmid DNA is a gram negative bacterial plasmid DNA. 
     
     
         22 . The process of  claim 16 , wherein the polypeptide is a recombinant polypeptide.