HIGHLY PURE PLASMID DNA PREPARATIONS
The present disclosure generally relates to highly pure plasmid compositions having low, or undetectable, levels of colanic acid and other contaminants made by a process that includes the steps of obtaining a crude lysate of a plasmid DNA from a bacteria that makes colanic acid; treating the partially purified or purified plasmid DNA with a polypeptide that digests colanic acid under conditions that digest the colanic acid; and purifying the plasmid DNA from the digested colanic acid and the colanic acid degrading enzyme by one or more chromatography steps.
1 . A process for the purification of plasmid DNA comprising the steps of:
obtaining a crude lysate of a plasmid DNA from a bacteria that makes colanic acid; treating the purified plasmid DNA with a polypeptide that digests colanic acid under conditions that digest the colanic acid; and
purifying the plasmid DNA from the digested colanic acid and the colanic acid degrading enzyme by one or more chromatography steps.
2 . The process of claim 1 , wherein the step of separating the plasmid DNA from the digested colanic acid comprises anion exchange chromatography.
3 . The process of claim 1 , wherein following the step of separating the plasmid DNA from the digested colanic acid, the method further comprises the step of washing the plasmid DNA by tangential flow filtration.
4 . The process of claim 1 , wherein the chromatography step purifies the plasmid DNA and is differential elution anion exchange chromatography.
5 . The process of claim 1 , wherein each of the one or more chromatographic steps can be performed more than once.
6 . The process of claim 1 , wherein the isolated plasmid DNA is obtained from crude bacterial lysate, a partially purified bacterial lysate, and an aqueous solution containing extracted bacterial nucleic acid by centrifugation and alkaline lysis.
7 . The process of claim 1 , wherein the plasmid DNA is pre-treated to at least partially remove endotoxin prior to digesting the colanic acid.
8 . The process of claim 1 , wherein the polypeptide is a recombinant polypeptide.
9 . The process of claim 1 , wherein the polypeptide has colanic acid digesting activity and comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% or 100% homology to SEQ ID NO: 1 or SEQ ID NO: 2, and conservative amino acid substitutions thereof.
10 . The process of claim 1 , further comprising the step of measuring at least one of the digested colanic acid or the colanic acid digesting enzyme in the purified plasmid DNA.
11 . The process of claim 10 , wherein at least one of the digested colanic acid or the colanic acid digesting enzyme are measured by an enzyme linked immunosorbent assay (ELISA), immunoprecipitation, an enzyme immunoassay (EIA), a radioimmunoassay (RIA), a fluorescent immunoassay, a chemiluminescent assay, an agglutination assay, a nephelometric assay, a turbidimetric assay, a Western Blot, a competitive immunoassay, a noncompetitive immunoassay, a homogeneous immunoassay, a heterogeneous immunoassay, a bioassay, or a reporter assay such as a luciferase or other equivalent chemiluminescent assay.
12 . The process of claim 1 , wherein the plasmid DNA is a gram negative bacterial plasmid DNA.
13 . The process of claim 1 , wherein the plasmid DNA expresses a bifunctional shRNA.
14 . The process of claim 1 , wherein the bacteria is a high copy-number plasmid containing bacteria.
15 . The process of claim 1 , wherein the bacteria is not E. coli X1776.
16 . A process for the purification of plasmid DNA comprising:
obtaining a crude lysate of a plasmid DNA from a bacteria that makes colanic acid;
treating the purified plasmid DNA with a colanic acid digesting enzyme in solution under conditions that digest the colanic acid; and
purifying plasmid DNA by differential elution anion exchange chromatography to separate in one step the plasmid DNA, the digested colanic acid and the colanic acid digesting enzyme; and
measuring the digested colanic acid and the colanic acid digesting enzyme in the purified plasmid DNA.
17 . The process of claim 16 , wherein the step of separating the plasmid DNA from the digested colanic acid by differential elution anion exchange chromatography is further defined as:
washing the column with a buffer has a conductivity of about 50 to 55 mS/cm, 51, 52, 53, 53.5, 54, 54.5 or 55 mS/cm;
an optional second wash with a buffer that has a conductivity of 65 to 70, 66, 67, 67.5, 68, 69 or 70 mS/cm; and
eluting the plasmid DNA using a buffer that has a conductivity of 75 to 80, 76, 77, 77.5, 78, 87.5, 78.8, 78.9, 79, or 80 mS/cm.
18 . The process of claim 16 , wherein following the step of separating the plasmid DNA from the digested colanic acid, the method further comprises the step of washing the plasmid DNA by tangential flow filtration.
19 . The process of claim 16 , wherein the bacteria is not E. coli strain X-1776.
20 . The process of claim 16 , wherein each of the one or more chromatographic steps can be performed more than once.
21 . The process of claim 16 , wherein the isolated plasmid DNA is obtained from crude bacterial lysate, a partially purified bacterial lysate, and an aqueous solution containing extracted bacterial nucleic acid by centrifugation and alkaline lysis.
22 . The process of claim 16 , wherein the plasmid DNA is pre-treated to at least partially remove endotoxin prior to digesting the colanic acid.
23 . The process of claim 16 , wherein the colanic acid digesting enzyme is a recombinant polypeptide.
24 . The process of claim 16 , wherein the colanic acid digesting enzyme comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% or 100% homology to SEQ ID NO: 1 or SEQ ID NO: 2, and conservative amino acid substitutions thereof.
25 . The process of claim 16 , wherein at least one of the digested colanic acid or the colanic acid digesting enzyme are measured by an enzyme linked immunosorbent assay (ELISA), immunoprecipitation, an enzyme immunoassay (EIA), a radioimmunoassay (RIA), a fluorescent immunoassay, a chemiluminescent assay, an agglutination assay, a nephelometric assay, a turbidimetric assay, a Western Blot, a competitive immunoassay, a noncompetitive immunoassay, a homogeneous immunoassay, a heterogeneous immunoassay, a bioassay, or a reporter assay such as a luciferase or other equivalent chemiluminescent assay.
26 . The process of claim 16 , wherein the plasmid DNA is a gram negative bacterial plasmid DNA.
27 . The process of claim 16 , wherein the plasmid DNA expresses a bifunctional shRNA.
28 . A method of detecting the presence of a colanic acid digesting enzyme comprising:
contacting the colanic acid digesting enzyme with an antibody that specifically binds the colanic acid digesting enzyme; and
detecting the binding of the antibody to the colanic acid digesting enzyme.
29 . The process of claim 28 , wherein the binding of the antibody to the colanic acid digesting enzyme is detected by an enzyme linked immunosorbent assay (ELISA), immunoprecipitation, an enzyme immunoassay (EIA), a radioimmunoassay (RIA), a fluorescent immunoassay, a chemiluminescent assay, an agglutination assay, a nephelometric assay, a turbidimetric assay, a Western Blot, a competitive immunoassay, a noncompetitive immunoassay, a homogeneous immunoassay, a heterogeneous immunoassay, a bioassay, or a reporter assay such as a luciferase or other equivalent chemiluminescent assay.
30 . A process for the purification of plasmid DNA comprising:
obtaining a partially purified plasmid DNA from a bacteria that makes colanic acid;
treating the purified plasmid DNA with a polypeptide that digests colanic acid under conditions that digest the colanic acid;
purifying the plasmid DNA from the digested colanic acid and the colanic acid degrading enzyme by one or more chromatography steps; and
further purifying the plasmid DNA under cGMP conditions.
31 . A process for the purification of plasmid DNA comprising:
obtaining a crude lysate of a plasmid DNA from a bacteria that makes colanic acid;
treating the purified plasmid DNA with a polypeptide that digests colanic acid under conditions that digest the colanic acid;
separating the plasmid DNA from the digested colanic acid by differential elution anion exchange chromatography; and
purifying the washed plasmid DNA by filtration.
32 . A plasmid DNA obtained by a process comprising the steps of:
obtaining a crude lysate of a plasmid DNA from a bacteria that makes colanic acid;
treating the purified plasmid DNA with a polypeptide that digests colanic acid under conditions that digest the colanic acid; and
purifying the plasmid DNA from the digested colanic acid and the colanic acid degrading enzyme by one or more chromatography steps.