Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1998-09-15
2004-05-04
Ketter, James (Department: 1636)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S023500, C435S259000
Reexamination Certificate
active
06730781
ABSTRACT:
The present invention relates to a novel process for purifying DNA. The process according to the invention enables pharmacologically utilizable double-stranded DNA to be purified rapidly. More specifically, the purification process according to the invention involves only diafiltration and chromatographic steps.
The techniques of gene and cell therapy are currently experiencing an extraordinary rate of growth. Nevertheless, these techniques involve the possibility of producing substantial quantities of DNA of pharmacological purity, in particular plasmid DNA. In fact, in these new therapies, the pharmaceutical often consists of DNA itself, and it is essential to be able to make this DNA in suitable quantities and to isolate and purify it in a manner which is appropriate for therapeutic use in man, in particular by way of the intravenous route.
These problems of quantity and purity have not been taken into account in the conventional methods for isolating DNA. As a result, it is not possible to adapt the methods which are employed in the laboratory for the purpose of purifying plasmid DNA within the pharmaceutical industry. Two of these laboratory methods are those which are most frequently employed and which give the best results. They consist of starting with a crude bacterial lysate and enriching it in plasmid DNA by removing the maximum possible quantity of contaminants. In particular, egg white lysozyme is used to break down the bacterial wall after which the lysate is centrifuged in order to remove the cell debris. The supernatant is then subjected to the action of a pancreatic RNase of animal origin, thereby removing the RNA, which at this time represents approximately 75% of the nucleic acids which are present.
The proteins are then precipitated with a phenol/chloroform/isoamyl alcohol mixture. The supernatant which is obtained following centrifugation is cleared of proteins and RNA but still contains large quantities of chromosomal DNA, which has to be removed during an additional step. This step consists of an ultracentrifugation in the presence of ethidium bromide and caesium chloride. The three types of nucleic acid, that is chromosomal DNA, plasmid DNA and RNA, vary in their ability to bind ethidium bromide. As a result, they separate into three distinct phases during ultracentrifugation on a caesium chloride gradient.
A variant of this protocol consists in following the action of the pancreatic RNase with a reduction in presence of an alkaline detergent, followed in turn by an extraction with phenol/chloroform. The DNA is then precipitated with ethanol, resuspended and reprecipitated with polyethylene glycol.
However, these two methods of obtaining a solution of plasmid DNA cannot be used for the industrial production of a product of pharmaceutical purity. Thus, the use of enzymes of animal origin poses a problem. Due to their origin, both the lysozyme and the pancreatic RNase entail the risk of introducing a viral contamination into the final product. Furthermore, the organic solvents are extremely toxic and have to be removed if it is desired to use the product as a pharmaceutical. These solvents also lead to a considerable increase in costs associated, in particular, with their storage, their use under conditions of maximum security and the removal of the toxic waste to which they give rise, and also because of the difficulty which is encountered in successfully verifying complete removal of such products from the final solution. As for the ethidium bromide, it is so toxic, mutagenic and teratogenic that its presence, even in traces, cannot be tolerated in a product that is meant for pharmaceutical purposes. The use of solvents, toxic reagents and also enzymes of animal origin is incompatible with an industrial process that conforms to good manufacturing practices.
The present invention describes a simple, and particularly efficient, novel process for purifying DNA. The process that is described in the present application enables a DNA of very high purity to be produced in large quantities. Particularly advantageously, the process that is described in the present application makes it possible to avoid using toxic organic solvents and enzymes of animal origin. It also makes it possible to dispense with large numbers of tedious centrifugations which are difficult to extrapolate and are of low yield because, in particular, of precipitation steps using PEG, ammonium acetate or CaCl
2
. The process according to the invention also enables large quantities of DNA (100 mg, 1 g, 10 g or more) to be obtained in a single batch, without any particular technical difficulty. Furthermore, the process according to the invention involves methods which are compatible with good manufacturing practices and makes it possible to obtain a DNA of pharmaceutical quality.
The invention relates, first of all, to a process for purifying double-stranded DNA, which process enables large quantities of plasmid DNA of pharmaceutical purity to be obtained very rapidly and involves a chromatographic step on a column of hydroxyapatite which is in ceramic form. While hydroxyapatite in crystalline form was already disclosed, the use of this hydroxyapatite was difficult and limited owing to its fragility. The ceramic form is much more resistant both physically and chemically.
Preferably, the process of the invention comprises two chromatographic steps, with at least one of them being a chromatographic step on hydroxyapatite.
Advantageously, the second chromatographic step is a step of affinity chromatography or ion exchange chromatography. The order in which the two chromatographic steps are carried out is immaterial.
According to one particularly preferred embodiment, the process according to the invention comprises a chromatographic step on a column of hydroxyapatite and a step of triple-helix affinity chromatography. Triple-helix affinity chromatography is based on using a support to which is covalently coupled an oligonucleotide which is able to form, by means of hybridization, a triple helix with a specific sequence which is present in the said DNA. The order in which the two chromatographic steps are carried out is immaterial.
According to another embodiment, the process of the invention comprises one step of chromatography on a column of hydroxyapatite and one step of ion exchange chromatography.
Advantageously, the process of the invention additionally comprises a diafiltration step. The latter is generally carried out prior to the chromatographic steps.
An important step in the process of the invention involves chromatography on a column of hydroxyapatite.
Hydroxyapatite is a complex calcium phosphate which includes ten calcium atoms. The ceramic form, which is more stable than the crystalline form, was developed by Bio-Rad Laboratories and Asahi Optical Co., Ltd. The ceramic compound has the same properties as the crystalline compound without having the physical limitations of the latter; while this material is particularly used in chromatography for purifying proteins, it offers advantages, and enables very good results to be obtained, in the purification of nucleic acids. It is macroporous, spherical and chemically and physically very stable and can be reused many times over without losing efficacy. This ceramic form can withstand high pressures, very high pH values, very rapid flows and organic solvents.
Chromatography on a column of ceramic hydroxyapatite is a special type of chromatography which is strictly neither affinity chromatography nor ion exchange chromatography. It imprints its properties to these two types of chromatography and it could be defined as to pseudo affinity chromatography and pseudo ion exchange chromatography.
The nucleic acids bind to the hydroxyapatite by virtue of interactions between the phosphate groups of the skeleton of the polynucleotide and the calcium residues of the support. The nucleic acids can be eluted differentially by varying the ionic strength of the phosphate buffers. The nucleic acids can thus be separated from the proteins and, among themse
Ollivier Monique
Wils Pierre
Finnegan Henderson Farabow Garrett & Dunner L.L.P.
Gencell S.A.
Ketter James
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