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Process for the reduction of endotoxins in a plasmid preparation using a carbohydrate non-ionic detergent with silica chromatography


Title: Process for the reduction of endotoxins in a plasmid preparation using a carbohydrate non-ionic detergent with silica chromatography.
Abstract: The present invention provides methods for the reduction of endotoxins in a plasmid preparation using a carbohydrate non-ionic detergent with silica chromatography. ...



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USPTO Applicaton #: #20090240044 - Class: 536 254 (USPTO) - 09/24/09 - Class 536 
Inventors: Kevin Bernard Ray, Carol Ann Kreader, Fuqiang Chen, David Eric Cutter

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The Patent Description & Claims data below is from USPTO Patent Application 20090240044, Process for the reduction of endotoxins in a plasmid preparation using a carbohydrate non-ionic detergent with silica chromatography.

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.

BACKGROUND OF THE INVENTION

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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.

SUMMARY

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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.

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.

DEFINITIONS

The following definitions are provided in order to aid the reader in understanding the detailed description of the present invention.

The term “chaotropic substance,” as used in the present specifications, means every substance which is able to alter the secondary and/or tertiary and/or quaternary structure of a polymer without affecting the primary structure.

Examples of chaotropic substances are isothiocyanate salts, sodium iodide, sodium perchlorate, guanidinium salts, alkali salts and urea. Chaotropic substances are known to alter the secondary structure of polymers in general and/or nucleic acids in particular. This alteration can be measured in the decrease of the melting point of double stranded DNA. All kinds of nucleic acids, single stranded DNA, double stranded circular closed DNA, double stranded linear DNA and RNA can be immobilized on silica material under appropriate chaotropic conditions.

The optimal chaotropic conditions, e.g. kind and concentration of the chaotropic substance, for the immobilization of nucleic acids to silica material vary among the different species of nucleic acids. Typical binding conditions for plasmid DNA utilize 1 to 8 M solutions of guanidinium hydrochloride or guanidinium thiocyanate, displaying a pH of 4 to 7. The particular optimum depends mainly on the viscosity of the mixture, the content of proteins and other substances. In general, however, under the conditions, when circular double stranded DNA is bound, linear double stranded DNA with a similar size is also bound.

The term “substantially free of chaotropic substances” as used herein means that the concentration of chaotropic substances in the elution solution is sufficiently low that the binding matrix no longer binds the nucleic acids being purified. The concentration of chaotropic substance in the elution solution is preferably no higher than about 200 mM, more preferably no higher than about 50 mM, and most preferably is about zero. The elution solution is preferably water, more preferably deionized or distilled water, even more preferably nanopure endotoxin-free water.

The term “detergent” means an amphipathic molecule that contains both hydrophobic and hydrophilic groups. These molecules contain a polar, hydrophilic group (head) at the end of a long hydrophobic carbon chain (tail). The term “non-ionic detergent” means a detergent molecule that contains an uncharged, hydrophilic head group(s). The term “carbohydrate non-ionic detergent” as used herein means an uncharged detergent molecule in which the uncharged hydrophilic head group is or is derived from a carbohydrate molecule, which includes monosaccharides, oligosaccharides and polysaccharides.

The term “alkyl” embraces linear or branched radicals having one to about twenty carbon atoms or, preferably, six to about twelve carbon atoms. The term “cycloalkyl” embraces saturated carbocyclic radicals having three to twelve carbon atoms. More preferred cycloalkyl radicals are “lower cycloalkyl” radicals having three to about eight carbon atoms. Examples of such radicals include cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl. The term “alkylcycloalkyl” embraces radicals having one or more alkyl radicals attached to a cycloalkyl radical. The term “alkylcarbonyl” embraces an alkyl radical, as defined above, attached to a carbonyl radical. Examples of such radicals include substituted or unsubstituted n-decanoylcarbonyl and n-octanoylcarbonyl.

The term “BAC” (Bacterial Artificial Chromosome) describes a cloning vector based on bacterial mini-F plasmids.

The term “comprising” means “including the following elements but not excluding others.”

Methods

Among its several embodiments, the present invention provides a method for the reduction of endotoxin levels in nucleic acids originating from natural, genetic engineering or biotechnological biological sources, comprising the following steps: a) preparation of a solution which comprises a medium of the nucleic acids to be purified, a binding solution and a carbohydrate non-ionic detergent; b) application of the solution from step a) to a binding matrix; c) washing of the binding matrix from step b) with one or more wash solutions, wherein the wash solutions comprise, alone or in combination, a binding solution, an alcohol solution and optionally a carbohydrate non-ionic detergent; and d) elution of the nucleic acids from the binding matrix of step c).

In one embodiment, the biological source to be purified contains plasmid DNA.

In a further embodiment, the binding solution comprises a chaotropic substance.

In another embodiment, the chaotropic salt is preferably a guanidinium salt and even more preferably guanidinium hydrochloride.

In yet another embodiment the binding matrix is silica.

In still another embodiment, the carbohydrate non-ionic detergent is n-octyl-β-D-thioglucopyranoside.

In another embodiment, the present invention further provides a kit that is suitable for use in the reduction of endotoxin levels in nucleic acids according to a method comprising the following steps: a) preparation of a solution which comprises a medium of the nucleic acids to be purified, a binding solution and a carbohydrate non-ionic detergent; b) application of the solution from step a) to a binding matrix; c) washing of the binding matrix from step b) with one or more wash solutions, wherein the wash solutions comprise, alone or in combination, a binding solution, an alcohol solution and optionally a carbohydrate non-ionic detergent; and d) elution of the nucleic acids from the binding matrix of step c); wherein the kit comprises reagents, chromatographic binding matrices for separation of nucleic acids, aqueous buffer solutions, and substances for the removal of endotoxins.

The methods of the present invention provide one or more benefits. Utilization of a carbohydrate non-ionic detergent in combination with silica chromatography for the purification of plasmid DNA is useful as a simple and rapid method for the reduction of endotoxins. High levels of endotoxins have been shown to cause deleterious effects in downstream applications. Current methods for the preparation of plasmid DNA that contains low levels of endotoxins involve lengthy and tedious protocols that may involve difficult phase separations. Therefore the methods of the present invention provide processes that are quick and easy to perform, while retaining their efficiency to reduce endotoxins to desired levels. The non-ionic carbohydrate detergent utilized in the present invention is compatible with chaotropic substances, therefore allowing a one step process of binding plasmid DNA to a binding matrix while minimizing the absorption of endotoxins. In the final step of the methods described herein, a preferred method of elution is with a salt-free solution. This process avoids the necessity of precipitating the eluted plasmid DNA to remove salt, as other methods require. The ease of performing the present invention also decreases the risk of recontaminating the plasmid DNA with endotoxins or other impurities from the reagents added, the measuring device, or from the tube or bottle used to pellet the precipitate.

The methods of the present invention will have a number of uses. For example, the DNA purified by the methods described herein is ready for immediate use in downstream applications such as transfection, transformation, restriction digestion, ligation, sequencing and PCR. The present processes reduce the levels of endotoxins in the DNA, which can reduce transfection efficiencies in sensitive eukaryotic cell lines. The requirement for reduced levels of endotoxins is even more stringent in whole cell experiments, animal studies and human gene therapy. The presence of endotoxins in these applications can be responsible for inflammatory reactions and endotoxin shock, among other deleterious effects. The present invention provides a rapid and easy method to reduce endotoxins to a level at which they do not interfere with such sensitive applications.

The method according to the invention is particularly suitable for the purification of nucleic acids. These are single-stranded or double-stranded RNA or DNA, RNA/DNA hybrids, DNA fragments, oligonucleotides, amplified DNA or RNA, BACs, or in particular plasmid DNA. The size of the nucleic acids can be between 6 b/bp and 1000 kb/kbp.

The nucleic acids to be purified may originate from any natural, genetic engineering or biotechnological source, such as, for example, prokaryotic cell cultures. If nucleic acids from cell preparations are to be purified, the cells are firstly digested by known methods, such as, for example, lysis. If the material to be purified has already been pre-treated in another way, lytic digestion is unnecessary. For example, the medium can be obtained from biological material by removal of the cell debris and a precipitate of RNA from nucleic acid samples which have already been pre-purified and, for example, are present in buffer, or alternatively from nucleic acid solutions which have been formed after amplification and still contain endotoxin impurities. Filtration, precipitation or centrifugation steps may be necessary. The person skilled in the art is able to select a suitable digestion method depending on the source of the biological material. In any case, the sample to be purified should, for the method according to the invention, be present in a medium which does not form precipitates or cause other undesired side reactions on addition of the detergent solution. The medium is preferably a lysate obtained from cells, such as, for example, a cleared lysate.

For the purification of plasmid DNA from E. Coli, the cells are, for example, firstly lysed by alkaline lysis with NaOH/SDS solution. Addition of an acidic potassium-containing neutralization buffer then causes the formation of a precipitate, which can be removed by centrifugation or filtration. The clear supernatant remaining, the cleared lysate, can be employed as starting material, i.e. as medium, for the method according to the invention. It is also possible firstly to concentrate or pre-purify the cleared lysate by known methods, such as dialysis or precipitation.

The medium of the nucleic acids to be purified are combined with a binding solution and a carbohydrate non-ionic detergent to form the solution described in step a) of the present invention.

The binding substance used in this step of this embodiment of this method is selected for its capacity to promote the formation of a complex between the target nucleic acid and a binding matrix. In one embodiment of this aspect of the method, the binding substance is selected for its capacity to promote a link between the silica of a silica matrix and the target nucleic acid. In such a case, the binding agent is preferably selected from the group consisting of a chaotropic substance, a salt which is not a chaotropic agent, or a combination of the above. The proportions of each binding substance used depend upon how much of each other agent is present in the resulting binding solution. When only a non-chaotropic salt, such as sodium chloride, potassium chloride, or potassium acetate is used, the concentration of salt in the binding solution is preferably at least 500 mM. Smaller concentrations of non-chaotropic salts and other binding agents can be used where more than one binding substance is present in the binding solution. When only a chaotropic substance is used, the final concentration of chaotropic substance in the binding solution is preferably at least 100 mM, more preferably at least 200 mM, and most preferably at least 500 mM. The concentration of chaotropic substance in the binding solution formed in the practice of the present method is preferably between about 0.1 M and 12 M, but more preferably between about 1 M and 10 M and even more preferably between about 4 M and 8 M. When a chaotropic substance is the only binding substance in a binding solution, the concentration of chaotropic substance therein must be sufficiently high to cause the nucleic acid to form a complex with the binding matrix, but not so high as to substantially denature, degrade, or cause the target nucleic acid to precipitate out of the binding solution. Large molecules of double-stranded DNA, such as chromosomal DNA, are stable at chaotropic agent concentrations between 0.5 and 2 M, but are known to precipitate out of solution at chaotropic substance concentrations above 2 M (see, e.g. U.S. Pat. No. 5,346,994). Contrastingly, RNA and smaller molecules of DNA such as plasmid DNA, restriction or PCR fragments of chromosomal DNA, or single-stranded DNA remain undegraded and in solution at chaotropic substance concentrations between about 2 and 8 M.

Examples of suitable chaotropic substances are chaotropic salts selected from the group consisting of a guanidinium salt, urea, an alkali thiocyanate, an alkali halide and an alkali perchlorate. Further examples of chaotropic substances for use in the present invention are sodium perchlorate, guanidine hydrochloride, guanidine isothiocyanate (also referred to as guanidine thiocyanate), sodium trichloroacetate and potassium iodide in concentrations of, for example, from about 1 to 8 M. Also useful are concentrated solutions of salts, such as, for example, greater than about 1 M NaCl, KCl, LiCl, etc., reagents such as urea (utilized at, for example, greater than about 1 M), and combinations of such components. Preferred chaotropic agents for use in promoting the formation of a complex between the target nucleic acid and the binding matrix in a preferred embodiment of the method are guanidinium salts, and more preferably guanidine hydrochloride.

The carbohydrate non-ionic detergent useful in the methods and kits of the present invention is preferably an alkyl carbohydrate non-ionic detergent. Alkyl carbohydrate non-ionic detergents useful in the present invention are carbohydrate non-ionic detergents in which one or more of the carbohydrate hydroxyl or thiol groups is attached to an alkyl, cycloalkyl, alkylcycloalkyl or alkylcarbonyl group containing at least six carbon atoms.

More preferably, the alkyl carbohydrate non-ionic detergent is selected from the group consisting of alkyl thioglucosides, alkyl glucosides, alkyl thiomaltosides, alkyl maltosides and sucrose monoalkyl esters. A preferred class of alkyl carbohydrate non-ionic detergents useful in the present invention comprises alkyl glucoside (or glucopyranoside) compounds of formula (1)

wherein R is an alkyl, cycloalkyl or alkylcycloalkyl group containing at least six carbon atoms.

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stats Patent Info
Application #
US 20090240044 A1
Publish Date
09/24/2009
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File Date
12/31/1969
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Organic Compounds -- Part Of The Class 532-570 Series   Azo Compounds Containing Formaldehyde Reaction Product As The Coupling Component   Carbohydrates Or Derivatives   Nitrogen Containing   Dna Or Rna Fragments Or Modified Forms Thereof (e.g., Genes, Etc.)   Separation Or Purification Of Polynucleotides Or Oligonucleotides