Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
1999-10-07
2002-06-25
Naff, David M. (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S174000, C536S022100
Reexamination Certificate
active
06410274
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a process for purification of plasmid DNA. The process is particularly concerned with an improved method of separation of plasmid DNA on an anion exchange matrix.
BACKGROUND OF THE INVENTION
The development of gene therapy has increased the demand for highly purified gene vectors such as plasmid DNA. The problem with the purification of plasmid DNA is to completely remove other cell components such as host proteins, endotoxins, chromosomal DNA and RNA. For molecular biology purposes plasmid DNA is purified in laboratory scale in several steps including centrifugation, extraction with toxic solvents, enzyme treatment and chromatographic separation.
A common purification protocol can be described as follows: After harvesting and lysis of the cells, the crude lysate is centrifuged to separate cell debris. The lysate is further treated with organic solvents, such as phenol/chloroform to extract proteins. The content of RNA is reduced by treatment with enzymes like RNase. Finally the plasmid DNA is purified by centrifuging the lysate to equilibrium in cesium chloride-ethidium bromide density gradients. Alternatively or in addition chromatography purification can be used. There are variations of these type of steps in other protocols. However, these type of purification methods are not suitable for large scale production of pharmaceutical grade plasmid.
The disadvantages with known purification methods are several. The density gradient step is cumbersome and not suited for large scale production. There is also an introduction of carcinogenic and expensive chemicals which are hazardous to work with and which have to be eliminated from the final product. When RNase is added to hydrolyze RNA or degrade RNA into ribonucleotide subunits there is always a risk of adding unwanted proteins including DNase from other animals. For the production of pharmaceutical grade DNA unwanted proteins should be avoided.
Different chromatographic methods have been used for plasmid DNA purification, such as size exclusion chromatography, or gel filtration, hydroxyapatite, ion exchange and reversed phase chromatography. Size exclusion chromatography or gel filtration is not practical as a first chromatography step as very large columns are needed. Hydroxyapatite chromatography has other disadvantages. The matrix is difficult to regenerate and does not have a high capacity for plasmids. Besides, diafiltration has to be used to change the buffer system. With reversed phase chromatography the use of organic solvents makes the method difficult to use in large scale.
Ion exchange chromatography is the most commonly used chromatography method. Plasmid DNA, chromosomal DNA and RNA all bind to anion exchangers as they have similar charge properties. Most of the commercial kits available use RNase to hydrolyse RNA in the clear lysate before applying the sample to the column. In most of the purification protocols used, the sample has either been treated with RNase or heated or isopropanol precipitated or phenol extracted. WO 96/36706 and WO 95/21178 are examples of these methods. For large scale production precipitation and extraction are difficult to carry out. The RNase used has to be of high grade purity produced from reliable sources.
If the lysate prepared by alkaline lysis method is applied directly to the ion exchanger without further treatment, plasmid, chromosomal DNA and most of the contaminating material including RNA and pigment bind to the column matrix. As the content of RNA of the lysate is about 80% and chromosomal DNA is about 2-20% (depending on the lysing method) and plasmid DNA is only 1-2%, the capacity of the column for binding plasmid DNA is greatly reduced due to the fact that the binding sites are occupied by RNA and chromosomal DNA. Thus, the amount of contaminating material has to be reduced in the lysate in some manner before the lysate is added to the chromatography matrix.
SUMMARY OF THE INVENTION
The object of the present invention is to provide an improved process for large scale purification of plasmid DNA to obtain plasmid DNA of pharmaceutical grade.
A further object of the invention is to present a purification method which is simple and effective without the draw-backs of known methods as mentioned above.
The objects of the invention are achieved by the process for purification as claimed in the claims. According to the invention a process for purification of plasmid DNA by separation on an insoluble matrix is obtained. The process comprising:
a)lysing cells containing said plasmid DNA to obtain a lysate;
b) adding divalent alkaline earth metal ions to the lysate from a);
c) removing precipitated compounds from the lysate from step b);
d) contacting the lysate from step c) with an anion exchange matrix to obtain purified plasmid DNA.
REFERENCES:
patent: 5561064 (1996-10-01), Marquet et al.
patent: 9636706 (1996-11-01), None
Raymond, et al., Analytical Biochemistry, vol. 173, pp. 125-133 (1988).
Amersham Pharmacia Biotech AB
Birch & Stewart Kolasch & Birch, LLP
Naff David M.
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