Processes for the Preparation of Highly Pure Plasmid Compositions
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.
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.