CROSS REFERENCE TO RELATED APPLICATIONS
This application is a continuation of application Ser. No. 11/108,317 filed on Apr. 18, 2005, which claims the benefit of Provisional Application Ser. No. 60/565,026 filed on Apr. 23, 2004, each of which is hereby incorporated by reference in its entirety.
FIELD OF THE INVENTION
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The present invention relates to methods for the reduction of endotoxins in a plasmid preparation using a carbohydrate non-ionic detergent with silica chromatography.
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OF THE INVENTION
The invention relates to a method for reducing endotoxin levels or removing endotoxins from biological material. The method according to the invention enables, for example, high-purity plasmid DNA to be obtained from natural sources, in particular bacterial sources.
The demand for rapid and efficient methods for obtaining high-purity plasmid DNA from biological sources is constantly increasing owing to the increasing importance of recombinant DNA for exogenous expression or therapeutic applications. In particular, the demand for purification methods that can also be carried out on a larger scale is also increasing.
Virtually all known methods for the purification of, in particular, relatively large amounts of plasmid DNA include a chromatographic purification step. The efficiency of this step generally also determines the efficiency and effectiveness of the purification.
Plasmids are epigenomic circular DNA molecules having a length of between 4 and 20 kB, which corresponds to a molecular weight of between 2.6×106 and 13.2×106 daltons. Even in their compact form (supercoiled), plasmid DNA molecules normally have a size of several hundred nanometers. Owing to these dimensions, they are larger than the pores of most chromatography materials. This in turn causes, inter alia, the poor binding capacities of the separating materials generally used for plasmid DNA.
A further problem in the purification of plasmid DNA is caused by the impurities from which the plasmid DNA is to be separated. These are firstly genomic DNA and RNA. Exactly like plasmid DNA, these molecules have a strongly anionic character and thus a very similar binding behavior to separating materials.
The removal of endotoxins is at least as complex. Endotoxins are lipopolysaccharides (LPSs) which are located on the outer membrane of Gram-negative host cells, such as, for example, Escherichia coli. During lysis of the cells, LPSs and other membrane constituents are dissolved out, in addition to the plasmid DNA. Endotoxins are present in cells in a number of approximately 3.5×106 copies per cell (Escherichia Coli and Salmonella typhimurium, Cell. and Mol. Biology, J. L. Ingraham et al. Eds., 1987, ASM) and thus exceed the number of plasmid DNA molecules by a factor of more than 104. For this reason and the fact that lipopolysaccharides are high molecular polyanions which tend to co-migrate with DNA on chromatographic matrices, plasmid DNA obtained from Gram-negative host cells often contains large amounts of endotoxins. These substances result in a number of undesired side reactions in further usage of the plasmid DNA (Morrison and Ryan, 1987, Ann. Rev. Med. 38, 417-432; Boyle et al. 1998, DNA and Cell Biology, 17, 343-348). If it is intended to employ the plasmid DNA for, for example, gene therapy, it is of extreme importance that, for example, inflammatory or necrotic side reactions due to the impurities do not occur. There is therefore a great demand for effective methods for reducing endotoxin concentrations to the lowest possible levels.
Known methods for reducing endotoxin levels are based on a plurality of purification steps, frequently using silica supports, glass powder or hydroxyapatite, and on reverse-phase, affinity, size-exclusion and/or anion-exchange chromatography, and are lengthy and tedious.
Firstly, the host cells are digested by known methods, such as, for example, alkaline lysis. However, other lysis methods, such as, for example, the use of high pressure, boiling lysis, the use of detergents or digestion by lysozyme, are also known. The resultant alkaline lysate is neutralized and then centrifuged or filtered to remove any precipitate.
The plasmid DNA in the medium obtained in this way, a “cleared lysate”, is principally contaminated by relatively small cell constituents, chemicals from the preceding treatment steps, RNA, proteins and endotoxins. The removal of these impurities usually requires a plurality of subsequent purification steps. Purification by means of anion-exchange chromatography has proven particularly advantageous.
However, the dynamic binding capacity of most anion exchangers for plasmid DNA is only about 0.4 mg/ml of separating material. The reason for this low value is that the functional groups are bonded to the support directly or via short spacers and consequently are only available to a limited extent for interactions with the large plasmid DNA molecules.
Another disadvantage of anion-exchange purification is that high salt is required to elute DNA from anion-exchange matrices, which requires additional steps to remove the salt for utilization of the DNA in downstream applications.
A further disadvantage of conventional anion-exchange chromatography is that a considerable amount of endotoxins is bound together with the plasmid DNA and cannot be separated off in this way. Plasmid DNA with endotoxin proportions of greater than 5000 EU/mg of plasmid DNA is often obtained. In order to reduce the endotoxin levels, further purification steps, such as, for example, chromatographic steps (gel filtration) or precipitation with isopropanol, ammonium acetate or polyethylene glycol, are therefore necessary. Purification methods which combine chromatographic methods, such as, for example, anion-exchange chromatography, and additional endotoxin removal steps, enable plasmid DNA having an endotoxin content of less than 50 EU/mg of plasmid DNA to be obtained. However, methods of this type are usually complex, time-consuming and of only limited suitability for the purification of relatively large amounts of DNA.
A method to reduce the levels of bacterial lipopolysaccharides in plasmid DNA by treatment with the detergent n-octyl-β-D-thioglucopyranoside and polymyxin-B chromatography has been described (I. P. Wicks, et al., Human Gene Therapy, 6, 317-323 (1995)).
U.S. Pat. No. 6,617,443 describes a process using a salt-free detergent solution and subsequent anion exchange chromatography to remove endotoxins from a nucleic acid preparation.
U.S. Pat. No. 5,747,663 describes a process for the removal of endotoxins from nucleic acids by pre-incubation of the nucleic acid with an aqueous salt solution and detergents, followed by treatment with anion exchange materials.
U.S. Pat. No. 5,990,301 describes a process for the purification of nucleic acids for use in gene therapy that includes treating a lysate with a non-ionic detergent followed by anion exchange.
U.S. Pat. No. 6,297,371 describes a process for the purification of nucleic acids for use in gene therapy that includes treating a lysate with a non-ionic detergent followed by anion exchange.
U.S. Pat. No. 6,194,562 describe a process for the removal of endotoxins from nucleic acids using silica-based materials, such as silica gel particles, magnetic silica particles, or diatomaceous earth.
U.S. Pat. No. 6,428,703 describes a process for purifying biological macromolecules from starting materials and for the removal of endotoxins through the use of anion exchange chromatography utilizing a polyethylene glycol non-ionic surfactant.
U.S. Pat. No. 6,011,148 describes a process for producing highly purified compositions of nucleic acids with low endotoxin levels by using tangential flow ultrafiltration.
US 2003/0204077 describes a process for the isolation of RNA from eukaryotic cells involving the use of an extraction reagent which may contain one of several non-ionic detergents.
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OF THE INVENTION
A need however remains for an improved method for the purification of nucleic acids, in particular plasmid DNA, which provides plasmid DNA having an endotoxin content of less than about 100 EU/mg plasmid DNA. A special need exists for such a method to provide the purification of plasmid DNA with reduced endotoxin levels that is simpler and faster than existing methods. To address the continuing need for purified plasmid DNA, methods to achieve that end are herein reported. The present invention provides methods for the reduction of endotoxin levels in nucleic acid preparations using a carbohydrate non-ionic detergent in combination with silica chromatography to meet this need.
Among its several embodiments, the present invention provides a method for reducing endotoxin levels in a nucleic acid solution. The method comprises contacting the nucleic acid solution with a carbohydrate non-ionic detergent selected from the group consisting of an alkyl thiomaltoside and a sucrose monoalkyl ester. The method further comprises contacting the resultant solution with an inorganic binding matrix and washing the resultant binding matrix to obtain a nucleic acid composition having an endotoxin content of less that 100 EU/mg.
In another embodiment, the present invention further provides a kit for reducing endotoxin levels in a nucleic acid solution. The kit comprises a binding solution, a carbohydrate non-ionic detergent, and an inorganic binding matrix.
Further scope of the applicability of the present invention will become apparent from the detailed description provided below. However, it should be understood that the following detailed description and examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.
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OF THE INVENTION
The following detailed description is provided to aid those skilled in the art in practicing the present invention. Even so, this detailed description should not be construed to unduly limit the present invention as modifications and variations in the embodiments discussed herein can be made by those of ordinary skill in the art without departing from the spirit or scope of the present inventive discovery.
The contents of each of the references cited herein, including the contents of the references cited within these primary references, are herein incorporated by reference in their entirety.