Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
1997-12-08
2001-04-10
Lankford, Jr., Leon B. (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S252800, C435S259000, C536S025400, C536S025410
Reexamination Certificate
active
06214586
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to methods for purification of plasmid DNA, particularly large quantities of plasmid DNA, and more particularly to methods for separating plasmid DNA from genomic DNA that are capable of providing kilogram quantities of plasmid DNA substantially free of genomic DNA.
BACKGROUND OF INVENTION
Many genetic diseases are difficult or impossible to treat with small molecule drugs or with enzyme replacement therapy as with Gaucher's disease. Recent advances in biology have raised the possibility that gene therapy, replacement of a defective gene with a normal gene, may be possible. Thus researchers have been studying several promising methods to deliver normal copies of genes to cells containing defective ones.
One promising method of gene delivery to humans is to use a plasmid DNA/lipid complex. The plasmid, a closed circular form of bacterial DNA, contains the genetic material needed to correct a genetic defect.
In some cases, large quantities of plasmid DNA (“pDNA”) will be required to provide effective treatment to a population of patients afflicted with certain types of genetic diseases. Thus, recovery from the process must be high and the process must be scaleable to allow for efficient, cost effective production. Furthermore, the plasmid DNA will have to be very highly purified to allow for repeat dose administration of the complex to these patients. Because the plasmids used in clinical applications are produced typically by bacteria, e.g.,
E. Coli
, the purification process must efficiently separate bacterial endotoxin, protein, and genomic (chromosomal) DNA from plasmid DNA, at very large scale. Because bacterial chromosomal DNA and plasmid DNA are so similar the process needs to be particularly effective at removing this contaminant.
Current methods described in the literature, such as cesium chloride centrifugation, chromatography on hydroxyapatite, or chromatographic methods based on reverse phase or anion exchange HPLC have limitations that would make purification of kilogram quantities difficult, if not impossible.
Purification of pDNA by centrifugation using cesium chloride involves use of toxic and carciongenic compounds, making this method unsuitable for purification of products for use in humans. Further, this technique could not be scaled to kilogram quantities per run.
Hydroxyapatite chromatography suffers from drawbacks as well. Lot variability of hydroxyapatite media would make consistent purification of plasmid DNA difficult. Further, because hydroxyapatite has a double positive charge and cannot be deprotonated, the resin cannot be cleaned, making it unusuable as a technique for purifying clinical grade material.
HPLC (high pressure liquid chromatography) techniques also suffer from scaleability problems. HPLC columns capable of purifying kilogram quantities of pDNA are not currently manufactured.
Thus, new and better methods continue to be sought for purifying pDNA and which are scaleable to produce kilogram quantities of plasmid DNA of sufficient purity to be used as a therapeutic in humans.
SUMMARY OF THE INVENTION
The present invention provides a method for producing, in up to kilogram quantities, plasmid DNA, sufficiently pure for use as a human therapeutic. The method comprises separating plasmid DNA from genomic DNA by treating a solution containing both plasmid DNA from genomic DNA with at least 80% by weight saturation with ammonium sulfate, thereby precipitating the genomic DNA and leaving the plasmid DNA in solution.
In preferred embodiments of the invention, purified plasmid DNA is obtained that has less than 1% by weight genomic DNA by Southern Blot testing, more preferably no detectable genomic DNA, more preferably less than 0.2% by weight genomic DNA.
In one embodiment of the invention, a method for purifying plasmid DNA from a culture of cells comprises: lysing the cells; precipitating the bulk of contaminating cellular components to obtain a clarified lysate containing the plasmid DNA; concentrating the clarified lysate using a 100,000 molecular weight cutoff membrane; diafiltering the concentrated lysate into a suitable buffer; and precipitating from the diafiltered lysate both bacterial genomic DNA and RNA by the addition of ammonium sulfate, thereby providing a supernatant containing purified plasmid DNA. The lysing step can be performed on the cells directly from a fermenter or on the cells after the cells have been harvested by centrifugation then resuspended in a suitable buffer. Preferably, the step of lysing the cells and the step of precipitating the bulk of contaminating cellular components are both performed using static mixers.
In another embodiment of the invention, bacterial cells are lysed by simultaneously flowing a cell suspension and a lysis solution through a static mixer. Thousands of liters of a cell resuspension, containing hundreds of kilograms of cells can be lysed in a rapid and efficient manner using this technique. The bulk of contaminating cellular components including proteins, endotoxins and a substantial portion of the genomic DNA, are precipitated by simultaneously flowing the cell lysate and a precipitating solution through a static mixer. After removal of the precipitate by either filtration or centrifugation, the supernatant is ultrafiltered by tangential flow ultrafiltration using a 100,000 MW cutoff membrane. After the supernatant is concentrated, the material is diafiltered using a diafiltration buffer. When diafiltration is complete, ammonium sulfate is added to the retentate to a saturation of at least about 80%. After stirring, the precipitate, which contains RNA and bacterial genomic DNA, is removed by centrifugation. The resulting ammonium sulfate supernatant is loaded onto a column containing a reverse phase resin equilibrated with a buffer. After washing the column to reduce the amount of nicked plasmid, the plasmid DNA is eluted from the column. Finally, the pDNA containing reverse phase pool is loaded onto a column containing an anion exchange resin equilibrated with buffer. After washing the column to remove contaminants, the purified plasmid DNA is eluted from the column. At this stage, the plasmid DNA is highly purified and suitable for use in humans.
The methods of the present invention are capable of providing large (i.e., kilogram) quantities of clinical quality pDNA in a relatively efficient and cost-effective manner. Further, preferred embodiments of the present invention permits one to eliminate harvesting of the cells from the fermentor and to eliminate the separate precipitation of RNA with ammonium acetate.
DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS
The purification process of the present invention is capable of producing up to kilogram quantities of pDNA having sufficient purity for use as a human therapeutic. First, plasmid containing bacterial cells are lysed. Large volumes of cell suspensions can be lysed gently by use of static mixers as described in the U.S. patent application Ser. No. 08/632,203, filed on Apr. 15, 1996 now U.S. Pat. No. 5,837,529 and titled “Method of Lysing Cells,” the disclosure of which is hereby incorporated by reference. After collection, the precipitated lysate is clarified, e.g., by centrifugation.
After clarification, it is generally desirable to lower the salt concentration to an appropriate level. This procedure is preferably performed using a tangential flow ultrafiltration device. It has been found that much of the contaminating material can be removed in the filtrate during desalting. Commercially available tangential flow ultrafiltration devices are suitable for this operation. A membrane having a 100,000 molecular weight cutoff is preferable, because it will be able to process a wide range of different sized plasmids, but devices with other porosites could be used depending on the size of the plasmid. After concentration to a suitable volume, preferably the material is diafiltered into a buffer appropriate for further processing. Preferably, the clarified lysate is concentra
Dike Bronstein, Roberts & Cushman LLP
Genzyme Corporation
Lankford , Jr. Leon B.
Lazar Steven R.
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