Methods for reducing endotoxin levels in nucleic acid purification

Abstract

The present invention relates to a method for reducing endotoxin levels or removing endotoxins from nucleic acids, in which a non-ionic surfactant selected from the group of alkyl glycosides and secondary alcohol alkoxylates or mixtures thereof is added prior to or during anion exchange chromatographic purification of nucleic acids using membrane or monolith-based sorbents.

Description

[Technical field]

[0001] The present invention relates to a method for reducing endotoxin levels or removing endotoxins from nucleic acids, for which a non-ionic detergent is added during ion exchange chromatographic purification of nucleic acids using membrane or monolith-based sorbents.

[0002] The demand for rapid and efficient methods to obtain high purity nucleic acids such as plasmid DNA from biological sources is constantly increasing due to the growing importance of recombinant DNA for exogenous expression or therapeutic applications. In particular, the demand for production methods that can be carried out on a large scale is also increasing. The use of high purity plasmid DNA is crucial for various applications such as polymerase chain reaction (PCR) amplification, DNA sequencing, in vitro mRNA synthesis, and transgene subcloning. Therefore, protocols for generating plasmid DNA with high yield and high quality are attracting serious attention.

[0003] Many known methods for the purification of nucleic acids such as plasmid DNA, especially for relatively large scale purification, include a chromatographic purification step. The efficiency of this step also generally determines the efficiency and effectiveness of the manufacturing process.

[0004] Further problems, especially in the purification of plasmid DNA, are caused by the impurities from which the plasmid DNA is separated. These are, first of all, genomic DNA and RNA. Another impurity when purifying nucleic acids is endotoxin. Endotoxin is lipopolysaccharide (LPS), which is located in the outer membrane of gram-negative host cells, such as Escherichia coli. During lysis of the cells, LPS and other membrane constituents are released in addition to the plasmid DNA. Endotoxin can be present in the cell at a number of approximately 3.5x106 copies per cell (Escherichia coli and Salmonella Typhimurium cells and Mol. Biology, JL Ingraham et al. Eds., 1987, ASM) and thus outnumbers the plasmid DNA molecules by more than 104 times. For this reason, plasmid DNA obtained from gram-negative host cells often contains large amounts of endotoxin. However, these substances cause many undesirable side reactions (Morrison and Ryan, 1987, Ann. Rev. Med. 38, 417-432;Boyle et al. 1998, DNA and Cell Biology, 17, 343-348). When plasmid DNA is intended to be used, for example, for gene therapy, it is very important that no inflammatory or necrotic side reactions occur due to impurities. Therefore, there is a great demand for an effective method to reduce the concentration of endotoxin to the lowest possible level.

[0005] Known methods for reducing endotoxin levels are based on multiple purification steps, often using anion exchange chromatography. First, the host cells are digested by known methods, such as, for example, alkaline lysis. Other lysis methods are also suitable, such as, for example, the use of high pressure, boiling lysis, the use of detergents, or digestion with lysozyme.

[0006] The resulting plasmid DNA in the medium, "clarified lysate," is contaminated primarily by smaller cellular constituents, chemicals from previous processing steps, RNA, proteins, and endotoxins. Removal of these impurities often requires multiple subsequent purification steps, anion exchange chromatography being one possibility.

[0007] The disadvantage of anion exchange chromatography is that a significant amount of endotoxin is bound together with plasmid DNA and cannot be sufficiently separated by this method. Therefore, in order to reduce the endotoxin level, further purification steps are required, such as a chromatographic step (gel filtration) or precipitation with isopropanol, ammonium acetate or polyethylene glycol. For example, a purification method that combines a chromatographic method, such as anion exchange chromatography, with an additional endotoxin removal step makes it possible to obtain plasmid DNA with an endotoxin content of less than 50EU / mg of plasmid DNA. However, this type of method is usually complicated, time-consuming, and has limited suitability for the purification of relatively large amounts of DNA.

[0008] WO95 / 21179 describes a method for reducing endotoxin levels, in which clarified lysate is first pre-incubated with an aqueous salt solution and a detergent. This is followed by purification by ion exchange chromatography, in which the ion exchange material is washed with additional salt solution, and plasmid DNA is eluted and subsequently further purified, for example, by isopropanol precipitation. This method also has the above-mentioned disadvantages.

[0009] US6617443 describes a method for removing endotoxins from nucleic acid preparations using a salt-free washing solution and an adsorbent having functional groups attached to tentacles. WO2009 / 129524 discloses a method for purifying plasmid DNA comprising contacting the plasmid DNA with a zwitterionic detergent. No. 6,428,703 describes a method for purifying biological macromolecules by contacting them with a non-ionic detergent and performing a chromatographic purification.

[0010] All of these documents show methods for purifying plasmid DNA from endotoxins. Nevertheless, there is a need for a process that combines enhanced performance with high efficacy.

[0011] Downstream processes in the biopharmaceutical and biotechnology industries mostly rely on chromatography steps using bead-based resins in packed bed columns as stationary phase. The resins typically have a diameter between 30 and 500 μm and generally provide an efficient chromatographic technique with high binding capacity. However, the process is rather slow and represents a significant cost in the production of biomolecules, since the transport of solute molecules to the binding sites inside the resin pores is limited by intra-particle diffusion. The pressure drop across the column is high even at low flow rates and increases during processing due to bed compaction and column clogging. As a result, several other innovative stationary phases, including monoliths and membranes, have been developed in recent decades as viable alternatives to classical chromatographic supports. The main advantage of using membranes or monoliths is due to the short diffusion times, since the interactions between the molecules and the active sites in the membrane or monolith occur in the convective through-pores rather than in stagnant fluid inside the resin pores. Thus, membrane and monolith chromatography have the potential to operate at high flow rates and low pressure drop. However, as described above, membrane or monolith-based chromatography may exhibit different chromatographic behavior and therefore different separation characteristics due to, among other things, the absence of pore diffusion and higher flow rates.

[0012] It has been found that by carrying out the purification of plasmid DNA using an anion exchange membrane or monolith in combination with a type of non-ionic detergent, the high performance when using a membrane or monolith as a chromatographic matrix can be combined with high efficiency. It has further been found that using the process of the present invention, the subsequent determination of residual endotoxin can be carried out without the interference of detergents.

[0013] Thus, the present invention provides a) providing a sample comprising said nucleic acid and endotoxin; b) subjecting the sample of step a) to a chromatographic separation on a membrane or monolith comprising anion exchange groups; Thereby, the sample is contacted with a non-ionic surfactant selected from the group of alkyl glycosides and secondary alcohol alkoxylates or mixtures thereof; The present invention is directed to a method for the depletion or removal of endotoxins from nucleic acids, comprising:

[0014] In a preferred embodiment, step b) comprises: i) loading the sample containing the nucleic acids and endotoxins onto a membrane or monolith containing anion exchange groups; ii) washing the membrane or monolith with a wash buffer; iii) eluting the nucleic acid bound to the membrane or monolith with an elution buffer; Includes.

[0015] In one embodiment, the sample is contacted with a non-ionic detergent prior to step b). In another embodiment, the nucleic acid is contacted with the non-ionic detergent by washing the membrane or monolith in step ii) with a wash buffer comprising the non-ionic detergent.

[0016] Preferably, the detergent is added to the sample and / or wash buffer so as to be present therein at a concentration ranging from 0.01% to 10% (w / v). In a preferred embodiment, the non-ionic surfactant is an alkyl glycoside. In a highly preferred embodiment, it is a C8-16 alkyl glycoside. In a preferred embodiment, the nucleic acid comprises or consists of plasmid DNA.

[0017] In a preferred embodiment, the nucleic acid is contacted with a solution containing 0.01 to 10% (w / v) of a non-ionic surfactant. In a preferred embodiment, the membrane is a hydrogel membrane. In a preferred embodiment, step ii) comprises two or more washing steps, one of which is performed with a washing buffer comprising ethanol.

[0018] In one embodiment, the process of the invention provides nucleic acids that are more effectively depleted of endotoxins than the same process, except that Triton® X100 is used as the only detergent.

[0019] In one embodiment, the process further comprises the step c) of detecting residual endotoxin in the nucleic acid resulting from step b). In a preferred embodiment, the detection in step c) is performed by an LAL assay or a recombinant factor-based assay, in particular by an LAL assay. In a preferred embodiment, the detection in step c) is carried out directly on the eluate of the chromatographic separation, without further treatment of the eluate.

[0020] definition Before describing the invention in detail, it is to be understood that the invention is not limited to particular compositions or process steps, as such may vary. It must be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a ligand" includes a plurality of ligands, and reference to "an antibody" includes a plurality of antibodies, etc. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. For purposes of the present invention, the following terms are defined as described herein.

[0021] The nucleic acids to be purified according to the method of the present invention, also referred to as target nucleic acids, by depletion or removal of endotoxins, include DNA, RNA and chimeric DNA / RNA molecules, and may be from any biological source, including eukaryotic and prokaryotic cells, or may be synthetic. The nucleic acids to be purified include chromosomal DNA fragments, ribosomal RNA, mRNA, snRNAs, tRNA, plasmid DNA, viral RNA or DNA, synthetic oligonucleotides, ribozymes, etc. Of particular interest is plasmid DNA encoding a therapeutic gene. By "therapeutic gene" is intended to include functional genes or gene fragments that can be expressed in a suitable host cell and complement defective or under-expressed genes in the host cell, as well as genes or gene fragments that, when expressed, inhibit or suppress the function of a gene in the host cell, including, for example, antisense sequences, ribozymes, transdominant inhibitors, etc. Thus, by way of example, viral DNA or RNA may be purified from prokaryotic or eukaryotic viruses, where viral particles are first purified according to conventional techniques from cultures or cells permissive to viral infection, such as bacterial, insect, yeast, plant or mammalian cell cultures.

[0022] The term "plasmid DNA" refers to any separate cell-derived nucleic acid entity that is not a part or fragment of the primary genome of a host cell. As used herein, the term "plasmid" may refer to either a circular or linear molecule composed of DNA or DNA derivatives. The term "plasmid DNA" may refer to either a single-stranded or double-stranded molecule. Plasmid DNA includes naturally occurring plasmids as well as recombinant plasmids that encode genes of interest, including, for example, marker genes or therapeutic genes. Plasmids are typically epigenomic circular DNA molecules with a length between 4 and 20 kB, which corresponds to a molecular weight between 2.6x10 and 13.2x10 daltons that often allows autonomous replication in the producing cell. Even in their compact form (supercoiled), plasmid DNA molecules usually have a size of several hundred nm.

[0023] As used herein, and unless otherwise stated, the term "sample" refers to any composition or mixture containing nucleic acid. Samples may be derived from biological or other sources. Biological sources include eukaryotic and prokaryotic sources, such as plant and animal cells, tissues and organs. Samples may also include diluents, buffers, detergents, and contaminating species, debris, etc., that are found mixed with the target molecule. Samples may be "partially purified" (i.e., subjected to one or more purification steps, such as filtration steps) or may be obtained directly from a host cell or organism that produces nucleic acid (for example, a sample may include harvested cell culture fluid).

[0024] As used herein, the term "impurity" or "contaminant" refers to any foreign or undesirable molecule, including one or more host cell proteins, endotoxins, lipids, and one or more additives, that may be present in a sample containing nucleic acid to be separated from one or more of the foreign or undesirable molecules using the process of the present invention. One of the contaminants that is depleted or removed using the process of the present invention is endotoxin.

[0025] As used interchangeably herein, the terms "purifying," "separating," or "isolating" refer to increasing the purity of a target nucleic acid from a composition or sample that contains the target nucleic acid and one or more impurities. Typically, the purity of the target nucleic acid is increased by removing (completely or partially) at least endotoxins from the composition.

[0026] The term "chromatography" refers to any type of technique for separating an analyte of interest (e.g., a target nucleic acid) from other molecules present in a sample. Most often, the target nucleic acid is separated from other molecules as a result of differences in the rate at which individual molecules of a mixture bind to and / or migrate through a chromatographic matrix under the influence of a mobile phase.

[0027] The terms "matrix" or "chromatographic matrix" are used interchangeably herein and refer to a solid phase through which a sample passes during a chromatographic separation. A matrix typically comprises a substrate and a ligand covalently bound to the substrate. The matrix of the present invention comprises or consists of a membrane or monolith, preferably the substrate is a membrane or monolith, most preferably a membrane.

[0028] A "ligand" is a functional group that is typically part of a chromatography matrix that is attached to the matrix substrate and determines the binding and interaction properties of the matrix. Examples of "ligands" include, but are not limited to, ion exchange groups, hydrophobic interaction groups, hydrophilic interaction groups, thiophilic interaction groups, metal affinity groups, affinity groups, bioaffinity groups, and mixed mode groups (combinations of the foregoing). It is also possible for a ligand to have more than one binding / interaction property. The matrix of the present invention comprises at least anion exchange groups. These may be, for example, strong anion exchange groups such as trimethylammonium chloride, or weak anion exchange groups such as N,N diethylamino or DEAE. In addition, the matrix may further comprise other types of ligands so that the matrix is ​​a mixed mode matrix. Such ligands may have hydrophobic interaction groups such as, for example, phenyl, butyl, propyl, hexyl, etc.

[0029] The ligands may be attached to the matrix substrate by any type of covalent attachment, e.g., OH, NH 2 This can be carried out by directly binding a functional group to a suitable residue on the substrate, such as carboxyl, phenol, anhydride, aldehyde, epoxide or thiol, etc. It is also possible to attach the ligand via a suitable linker. It is also possible to produce the matrix by polymerizing a monomer that contains the ligand and a polymerizable moiety. Examples of matrices produced by polymerization of suitable monomers are polystyrene, polymethacrylamide or polyacrylamide based matrices produced by polymerizing suitable styrol or acryloyl monomers.

[0030] In another embodiment, the stationary phase can be produced by grafting ligands onto or from a substrate. Grafting from a process using controlled free radical polymerization is suitable, for example, the method of atom transfer free radical polymerization (ATRP). For example, a highly preferred one-step grafting from the polymerization reaction of acrylamide, methacrylate, acrylate, methacrylate, etc. functionalized with ionic, hydrophilic or hydrophobic groups can be initiated by cerium (IV) on a hydroxyl-containing support without activating the support.

[0031] When a chromatography matrix is ​​used in a chromatographic separation, a means for holding the matrix is ​​typically used in a separation device, also called a housing. In one embodiment, the device comprises a housing having an inlet and an outlet and a fluid path between the inlet and the outlet. In a preferred embodiment, the device is a chromatography column. Chromatography columns are known to those skilled in the art. They typically comprise a cylindrical tube or cartridge filled with a stationary phase and a filter and / or a means for fixing the stationary phase in the tube or cartridge and optionally a connection for transporting solvent to and from the tube or cartridge. The size of the chromatography column varies depending on the application, for example, analysis or preparative. In one embodiment, the column or generally the separation device is a single-use device.

[0032] Thus, the term "anion exchange matrix" as used herein refers to a chromatography matrix that possesses at least anion exchange groups, i.e., typically has one or more types of ligands that are positively charged under the chromatographic conditions employed, such as quaternary amino groups.

[0033] A "buffer" is a solution that resists changes in pH due to the action of its acid-base conjugate components. For example, various buffers that can be employed depending on the desired pH of the buffer are described in Buffers. A Guide for the Preparation and Use of Buffers in Biological Systems, Gueffroy, D., ed. Calbiochem Corporation (1975). Non-limiting examples of buffers include MES, MOPS, MOPSO, Tris, HEPES, phosphate, acetate, citrate, succinate, and ammonium buffers, and combinations thereof. According to the present invention, the term "buffer" or "solvent" is used for any liquid composition used for loading, washing, elution, re-equilibration, stripping and / or disinfection of a chromatography matrix.

[0034] When "loading" a chromatography column in binding and elution mode, a sample or composition containing a target molecule and one or more impurities is loaded onto the chromatography column. In preparative chromatography, the sample is preferably loaded directly without the addition of a loading buffer. If a loading buffer is used, the buffer has a composition, conductivity and / or pH such that the target nucleic acid is bound to the stationary phase, but ideally all impurities such as endotoxin do not bind to the column and flow through. Typically, the loading buffer, if used, has the same or similar composition as the equilibration buffer used to prepare the column for loading. The final composition of the sample loaded onto the column is called the feed, which may include the sample and the loading buffer, but is preferably only the sample.

[0035] "Washing" or "washing" a chromatography matrix means passing a suitable liquid, e.g., a buffer, through or over the matrix. Typically, washing is used to remove weakly bound contaminants from the matrix in bind / elution mode before eluting the target molecule. In addition, washing steps can be used to reduce the level of residual detergent, enhance viral clearance, and / or modify the conductivity carryover during elution.

[0036] "Eluting" a molecule (e.g., a target nucleic acid) from a matrix means that the molecule is removed from it. Elution may be accomplished by changing solution conditions such that a buffer different from the loading buffer and / or wash buffer competes with the molecule of interest for ligand sites on the matrix, or by altering the equilibrium of the target molecule between the stationary and mobile phases such that the target molecule is preferentially present in the elution buffer. A non-limiting example is eluting molecules from an ion exchange resin by altering the ionic strength of the buffer surrounding the ion exchange material so that it competes with the molecule for charged sites on the ion exchange material.

[0037] Membranes as a chromatographic matrix can be distinguished from particle-based chromatography by the fact that the interaction between solutes, e.g., target nucleic acids or contaminants, and the matrix does not occur in the dead-end pores of the particles, but mainly in the throughpores of the membrane. Exemplary types of membranes are flat sheet systems, stacked membranes, microporous polymer sheets incorporating cellulose, polystyrene or silica-based membranes, as well as radial flow cartridges, hollow fiber modules and hydrogel membranes. Preferred are hydrogel membranes. Such membranes include a membrane carrier and a hydrogel formed within the pores of the carrier. The membrane carrier provides mechanical strength to the hydrogel. The hydrogel determines the properties of the final product, such as pore size and binding chemistry.

[0038] The membrane carrier can consist of any porous membrane, such as polymeric membranes, ceramic-based membranes, and woven or non-woven fibrous materials. Suitable polymeric materials for the membrane carrier are cellulose or cellulose derivatives, as well as other preferred inert polymers such as polyethylene, polypropylene, polybutylene terephthalate, or polyvinylidene difluoride.

[0039] Hydrogels may be formed through the in situ reaction of one or more polymerizable monomers with one or more crosslinkers and / or one or more crosslinkable polymers, preferably forming a crosslinked gel with macropores. Suitable polymerizable monomers include monomers containing vinyl or acrylic groups. Preferred are monomers containing additional functional groups that either directly form the ligands of the matrix or are suitable for attaching ligands. Suitable crosslinkers are compounds containing at least two vinyl or acrylic groups. Further details on suitable membrane supports, monomers, crosslinkers, etc., as well as suitable production conditions, can be found in WO04073843 and WO2010 / 027955. Particularly preferred are membranes made from inert, flexible fibrous web carriers, such as Natrix® Q chromatography membranes (Merck KGaA, Germany), which contain porous polyacrylamide hydrogels with quaternary ammonium groups (strong anion exchange groups) assembled within and around the fibrous web carrier.

[0040] Depending on the membrane device used, the respective processes are carried out with different operating principles such as dead-end operation, cross-flow operation and radial flow operating systems. Dead-end operation is preferred.

[0041] Examples of suitable membranes for use in the method of the present invention are: - Membranes with a polyethersulfone (PES) based support and a cross-linked polymer coating functionalized with quaternary ammonium groups (strong anion exchange groups), such as Mustang® Q (Pall). - Membranes made from stabilized reinforced cellulose functionalized with quaternary ammonium groups (strong anion exchange groups) or DEAE groups (diethylaminoethyl, a weak ion exchange group), such as Sartobind® membranes (Sartorius). - Membranes made from stabilized reinforced cellulose containing hydrogels with quaternary ammonium groups (strong ion exchange groups), such as the Sartobind® Jumbo membrane (Sartorius), which is made from stabilized reinforced cellulose functionalized with quaternary ammonium groups (strong anion exchange groups). -Membranes made from fine fiber nonwoven scaffolds containing hydrogels with quaternary ammonium groups (strong anion exchange groups), such as 3M™ Emphaze™ AEX Hybrid Purifier (3M). -Membranes made from an inert, flexible fibrous web carrier that contains a porous polyacrylamide hydrogel with quaternary ammonium groups (strong anion exchange groups) inside and around the fibrous web carrier, such as Natrix® Q chromatography membrane (Merck KGaA, Germany).

[0042] Monoliths or monolith adsorbents, like membranes, have through-holes, such as interconnected channels, that allow flow from one side of the monolith, through the monolith, to the other side of the monolith. As the mobile phase flows through these perforations, the molecules to be separated are transported by convection rather than by diffusion. Due to their structure, monolithic adsorbents exhibit flow rate independent of separation efficiency and dynamic capacity.

[0043] The monolith is typically formed in situ from the reactant solution and can have any shape or limited geometry, typically with a frit-free construction to ensure convenience of operation. Preferably, the material of the monolith has a bimodal pore structure of mesopores and macropores. The micron-sized macropores are through-holes and ensure fast dynamic transport and low back pressure in applications; the mesopores contribute to sufficient surface area and thus high loading capacity. The monoliths can be made from organic, inorganic or organic / inorganic hybrid materials, with organic polymer-based monoliths being preferred.

[0044] The synthesis of organic polymer monoliths is typically carried out by a one-step polymerization, which provides tunable porous structures with tailored functional groups. In general, a prepolymerization mixture consisting of monomers, crosslinkers, porogenic solvents, and initiators in appropriate ratios is polymerized in a suitable container, also called a mold, which determines the form of the monolith. Polymerization is typically initiated by the use of heat, UV radiation, microwaves, or gamma radiation in the presence of an initiator. After reacting at an appropriate temperature for a prescribed time, the resulting material is typically washed with a solvent to remove unreacted components and the porogenic solvent. Suitable organic polymers are polymethacrylates, polyacrylamides, polystyrenes, polyurethanes, etc., such as poly(methacrylic acid ethylene dimethacrylate), poly(glycidyl methacrylate-ethylene dimethacrylate) or poly(acrylamide-vinylpyridine-N,N'-methylenebisacrylamide).

[0045] Inorganic monoliths can be made from silica or other inorganic oxides. Preferably, they are made from silica. Silica monoliths are usually prepared via a sol-gel process with phase separation. This mainly involves hydrolysis, condensation, and polycondensation of silica precursors. Typically, tetraethoxysilane (TEOS) or tetramethyl orthosilicate (TMOS) is dispersed in a suitable solvent in the presence of a porogen (e.g., poly(ethylene glycol) (PEG)), followed by the sequential addition of a catalyst, acid or base, or a binary catalyst, acid and base. After reacting for a given time, the resulting gel-like product is washed with a solvent to remove unreacted precursors, porogens, and catalysts, followed by a suitable post-treatment, typically a thermal treatment.

[0046] The monolith may be modified with suitable functional groups, preferably at least ion exchange groups, to allow targeted interaction with samples containing target molecules and thus targeted separation. Typically, the monolith contains a housing, such as a column.

[0047] Alkyl glycosides, also called alkyl polyglycosides, comprise a sugar and an alkyl chain linked to the sugar, typically via the anomeric carbon. The sugars can be monosaccharides such as glucose or disaccharides or oligosaccharides such as maltose. Regardless of the type of sugar unit, the molecule is simply called a glycoside. Preferably, the sugar is glucose. The alkyl chain is preferably a linear, saturated alkyl chain having 8 to 16 C atoms. The alkyl glycosides used in the method of the invention can also be a mixture of two or more different alkyl glycosides having different sugar moieties and / or alkyl chains with different chain lengths. Preferred are alkyl glycosides with an alkyl chain length between 8 and 10 C atoms. Particularly preferred is Triton® CG-110 (Merck KGaA, German).

[0048] The secondary alcohol alkoxylates contain ethylene and / or propylene oxide chains to which secondary alcohols are attached. The secondary alcohols preferably have 8 to 18 carbons and the ethylene / propylene oxide chains preferably have 3 to 12 ethylene oxide and / or propylene oxide units. The secondary alcohol alkoxylates can also be mixtures of different secondary alcohol alkoxylates with different alcohol chains and / or different numbers of ethylene oxide and / or propylene oxide units. The preferred secondary alcohol alkoxylates used in the process of the present invention are 2-ethylhexanol ethylene oxide-propylene oxide copolymers according to Formula I. [ka] Formula I where m and n are numbers between 1 and 11, and m+n is 3 to 12. Such secondary alcohol alkoxylates are commercially available as Ecosurf® EH (Merck KGaA, Germany, or Dow Inc). Particularly preferred is Ecosurf® EH-9.

[0049] Other preferred secondary alcohol alkoxylates for use in the process of the present invention are secondary alcohol ethoxylates made from secondary alcohols having from 11 to 15 carbons and carrying from 3 to 12 ethylene oxide units. A particularly preferred group of such secondary alcohol ethoxylates is shown in formula II and contains 9 ethylene oxide units. [ka] Formula II Such a compound is commercially available as Tergitol® 15-S-9 (Merck KGaA, Germany).

[0050] Detailed Description The nucleic acids purified according to the method of the invention may be derived from either natural, genetically modified or biotechnological sources, such as, for example, prokaryotic cell cultures. If nucleic acids are purified from cell preparations, the cells are first digested by known methods, such as, for example, lysis. If the sample to be purified has already been pretreated in another way, digestion of lysis is unnecessary. For example, the sample may already have been previously purified from biological material by removal of cell debris and RNA precipitate, and may be obtained, for example, from a nucleic acid sample in the presence of a buffer, or alternatively from a nucleic acid solution formed after amplification and still containing endotoxin impurities. Filtration, precipitation or centrifugation steps may be necessary. The skilled person can select a suitable digestion method depending on the source of the nucleic acid to be purified. In any case, the sample to be purified should be in a medium that does not form precipitates or cause other undesirable side reactions to the addition of detergent solutions for the method according to the invention. The sample is preferably a lysate obtained from cells, such as, for example, a clarified lysate.

[0051] For purification of plasmid DNA from E. coli, the cells are firstly lysed by alkaline lysis, for example with a NaOH / SDS solution. The addition of an acidic potassium-containing neutralization buffer then causes the formation of a precipitate, which can be removed by centrifugation or filtration. The remaining clear supernatant, the clarified lysate, can be taken as starting material, i.e. as sample, for the method according to the invention. It is also possible to first concentrate or pre-purify the clarified lysate by known methods, such as dialysis or precipitation.

[0052] The sample containing nucleic acids and endotoxins and potentially other impurities from which the nucleic acids are to be purified is then subjected to chromatographic separation on a membrane or monolith-based chromatography matrix containing anion exchange groups. For this, the sample is loaded onto the chromatography matrix. The sample of the final composition that is loaded onto the matrix is ​​also called the feed. In one embodiment of the invention, the feed does not contain any detergent. In another embodiment, the feed contains a non-ionic surfactant or a mixture thereof selected from the group of alkyl glycosides and secondary alcohol alkoxylates. Typically, the concentration of the non-ionic surfactant in the feed, if present, is between 0.01% and 10% (w / v), preferably between 0.1% and 1.5% (w / v). The non-ionic surfactant can be added directly to the sample before loading onto the column by in-line mixing or, preferably, added to the sample in a batch before loading. For this, the sample is preferably mixed with the detergent until it is dissolved. Mixing can be carried out for a time of, for example, between 5 and 60 minutes. Typically, feed preparation and chromatographic separations are also carried out at or around room temperature, however, it is also possible to work at temperatures between 5°C and 35°C.

[0053] The feed is preferably adjusted to an electrolytic conductivity between 40 and 90 mS / cm, most preferably between 75 and 85 mS / cm. The adjustment of the conductivity is performed by adding salt, salt concentrated solutions, or dilution with low conductivity buffer or neat water, respectively. For the adjustment of the conductivity of the feed by salt supplementation, preferably sodium chloride or potassium chloride is used, but any other salt commonly used in purification applications, such as, for example, salts from sulfates, acetates, carbonates / bicarbonates, phosphates or citrates, may be considered as well. The feed typically exhibits a pH value between 4.5 and 5.5, but the process may also be carried out on feeds exhibiting pH values ​​ranging from 4.0 up to 9.0.

[0054] Column equilibration and wash buffers are typically buffers that match the pH and conductivity of the feed loaded onto the chromatographic material. Typically, buffers with a pH below 6.0 and a conductivity between 40 and 90 mS / cm are selected, although buffers outside of that range are equally applicable. Low-conductivity wash buffers (<40 mS / cm) or detergent wash solutions made from pure water are particularly suitable. After loading, the matrix is ​​washed with at least one washing buffer. The washing buffer may be the same as the loading buffer or may be different from the loading buffer. The matrix may be washed with two, three or four different washing buffers. Optionally, one of the washing buffers contains a non-ionic surfactant selected from the group of alkyl glycosides and secondary alcohol alkoxylates or a mixture thereof. Typically, the concentration of the non-ionic surfactant in the washing buffer, if present, is between 0.01% and 10% (w / v), preferably between 0.1% and 1.5% (w / v).

[0055] If a non-ionic detergent is added to the wash buffer, it is preferably added to the first wash buffer, and in any case at least one further wash step is carried out after washing with a wash buffer containing the detergent. Preferably, the matrix is ​​washed with more than one wash buffer.

[0056] In another preferred embodiment, one wash buffer, preferably the last wash buffer, contains ethanol at a concentration between 10% and 25% (v / v). Preferably, the pH and ionic strength of the wash buffer are the same or similar to the pH and ionic strength of the equilibration / loading buffer.

[0057] Elution of the target nucleic acid is then performed using an elution buffer, which has a different pH and / or a different ionic strength than the equilibration / loading buffer. In one embodiment, it has a higher pH and / or a higher ionic strength than the equilibration / loading buffer. In one embodiment, the pH of the elution buffer is above pH 7, preferably between pH 8.5 and 9.5. In one embodiment, the elution buffer contains between 0.5M and 1.5M NaCl.

[0058] In either case, in the method of the invention, at least one non-ionic surfactant selected from the group of alkyl glycosides and secondary alcohol alkoxylates or a mixture thereof is added. As described above, detergents can be added to the feed and / or to the wash buffer. Preferably, the detergent is an alkyl glycoside, most preferably a C8-C16 alkyl glycoside, and a particularly preferred detergent is Triton® CG110.

[0059] By carrying out the methods of the present invention, target nucleic acids can be obtained with significantly lower endotoxin contamination compared to the contamination in the sample loaded onto the chromatography matrix. The final endotoxin level in the target nucleic acid depends on the initial endotoxin level. Using a target nucleic acid with an initial endotoxin level of approximately 1.3 106 EU / mg, the method of the present invention can achieve a final endotoxin level of the target nucleic acid of less than 30 EU / mg. Using a target nucleic acid with an initial endotoxin level of approximately 50.000 EU / mg, the method of the present invention can achieve a final endotoxin level of the target nucleic acid of less than 10 EU / mg.

[0060] It has been found that the method of the present invention typically exhibits better results than those achieved by carrying out the same method using other detergents typically recommended for bead-based applications such as Triton® X100 and Tween® 80 or Tween® 20.

[0061] In one embodiment, the method of the invention is carried out using only one or more non-ionic surfactants selected from the group of alkyl glycosides and secondary alcohol alkoxylates or mixtures thereof. No other detergents are added to the feed or wash buffers or at any other time during the chromatographic purification.

[0062] In one aspect of the present invention, the method further comprises an additional step for detecting residual endotoxin in the nucleic acid resulting from the purification of chromatography.Checking the quality of the nucleic acid product before its further use is typically highly relevant.Endotoxins can cause unwanted side effects, so controlling their removal or depletion is often crucial.Those skilled in the art are aware of methods for detecting endotoxins.

[0063] The Limulus-based detection assay, the LAL test, is generally regarded as the state-of-the-art in vitro detection method for endotoxins. Details of the LAL assay and other methods for detecting and measuring endotoxins are known to those skilled in the art. An example of an alternative method other than the LAL assay is a recombinant factor-based endotoxin detection kit, such as the recombinant factor C assay (Lonza). Further information can be found in EC Dullah, "Current trends in endotoxin detection and analysis of endotoxin-protein interactions", February 2016, Critical Reviews in Biotechnology 37(2):1-11.

[0064] Severe interference with the LAL assay as well as other endotoxin assays such as recombinant factor-based assays results from substances that interact with endotoxin forming "stealth" structures that shield the analyte from the LAL enzyme or recombinant enzyme, resulting in low endotoxin recovery (LER) and an underestimation of the actual endotoxin concentration. It is well known that detergents affect the detectability of endotoxins by forming micellar structures.

[0065] Being of the same amphipathic nature and similar structure, they are considered ideal partners for interaction. As a result, care must be taken when analyzing endotoxins in the final eluate samples, since the occurrence of LER effects caused by residual detergents must be excluded. It has been found that endotoxin assays such as LAL assays can be carried out on the products obtained using the method of the present invention without the occurrence of LER effect.Unexpectedly, the detergents used in the method of the present invention do not cause LER effect.In particular, alkyl glycosides and 2-ethylhexanol ethylene oxide-propylene oxide copolymers do not show any interference even when present at high concentrations. Consequently, in one aspect, the method of the invention comprises an additional step for detecting residual endotoxins in the nucleic acid resulting from the chromatographic purification directly in the eluate of the chromatography matrix, without any further treatment of the eluate.

[0066] The invention is further illustrated by the following figures and examples, without, however, being limited thereto.

[0067] The entire disclosures of all applications, patents, and publications cited above and below, as well as corresponding application US63 / 229,666, filed May 8, 2021, are hereby incorporated by reference.

[0068] example The following examples represent practical applications of the present invention.

[0069] List of cleaning agents [Table 1]

[0070] Protocol for plasmid DNA capture NOTE 1: For each set of experiments testing an individual detergent, either as a wash or as a complement to the feed, use a new membrane device to avoid artifactual hardening from cross-contamination due to carryover of residual detergent between series of runs / experiments. Note 2: In small volume monolith or membrane screening devices where the hold-up of the system is disproportionately large, it is typical that very large volumes are used for wash 1, wash 2, elution, cleaning in place (CIP) and equilibration. At larger scales, these values ​​may be reduced and flow directions may be reversed to enhance the individual steps. This is standard practice and common knowledge to anyone skilled in the art.

[0071] Chromatography Materials [Table 2]

[0072] Protocol 1 of Natrix® Q - Capture from Feed A (20 kb Plasmid) after Detergent Treatment ·buffer [Table 3]

[0073] Chromatography methods [Table 4]

[0074] Plasmid feed The original 20kb plasmid lysate used as feed showed an initial endotoxin level of approximately 1,300,000EU / mg plasmid. The plasmid lysate filtered with 0.22μm PES medium was supplemented with 100mM NaCl, which is required for selective binding of pDNA. A defined amount of detergent was added to the lysate before plasmid capture with Natrix® Q. After gentle stirring at room temperature for 30min until the detergent was completely dissolved and a homogeneous mixture was reached, the sample was subsequently subjected to purification experiments.

[0075] Natrix® Q Protocol 2 - Capture from Feed B (8 kb Plasmid) after Detergent Treatment Chromatography buffers [Table 5]

[0076] Chromatography methods [Table 6]

[0077] Plasmid feed The original 8kb plasmid lysate used as feed showed an initial endotoxin level of approximately 50,000EU / mg plasmid. The plasmid lysate filtered with 0.22μm PES medium was supplemented with 175mM NaCl, which is required for selective binding of pDNA. A defined amount of detergent was added before plasmid capture with Natrix Q. After gentle stirring at room temperature for 30min until the detergent was completely dissolved and a homogenous mixture was reached, the sample was subsequently subjected to the purification experiment.

[0078] Natrix® Q Protocol 3 - Capture from Feed C (8 kb Plasmid) using Mild Detergent Wash Chromatography buffers [Table 7]

[0079] Chromatography methods [Table 8]

[0080] Plasmid feed The original 8 kb plasmid lysate used as feed showed an initial endotoxin level of approximately 275,000 EU / mg plasmid. The lysate was filtered using 0.22 μm PES media and supplemented with 175 mM NaCl, which is necessary for selective binding of pDNA.

[0081] Mustang® Q Protocol 1 - Capture from Feed C (8 kb Plasmid) using Wash with Active Detergent Chromatography buffers [Table 9]

[0082] Chromatography methods [Table 10]

[0083] Plasmid feed Purification tests were carried out with the original lysate filtered through 0.22 μm PES media and supplemented with 375 mM NaCl required for selective binding of pDNA.

[0084] Capture from Feed C (8 kb Plasmid) using CIMmultus® DEAE Protocol 1 - Mild Detergent Wash Chromatography buffers [Table 11]

[0085] Chromatography methods [Table 12]

[0086] Plasmid feed Purification tests were carried out with the original lysate filtered through 0.22 μm PES media and supplemented with 60 mM NaCl, necessary for selective binding of pDNA.

[0087] Endotoxin assay Endotoxin analysis was performed using the cartridge-based Limulus amebocyte lysate (LAL) Endosafe-PTS system from Charles River according to the manufacturer's instructions.

[0088] Analysis of plasmid DNA The purity and quantity of plasmid DNA in the original lysates and in the samples collected from the Natrix® Q capture studies were determined by our means of an analytical UV / HPLC method.

[0089] Analysis of cleaning agents Residual amounts of detergent in plasmid eluate fractions collected from Natrix® Q capture studies were measured using an analytical HPLC method as described below. The method allows direct analysis of plasmids without prior sample preparation to remove potentially interfering matrix components using, for example, solid phase extraction. The amount of detergent in the unknown eluate samples was calculated based on the analyte peak area using calibration curves obtained from standards of the individual detergents in the eluate buffer matrix. The suitability of the analytical method and the validity of the analytical results for real plasmid samples were demonstrated using spike recovery studies. To that end, the recovery of defined amounts of individual detergents (from capture studies without any detergent) spiked into plasmid eluate samples was verified.

[0090] [Table 13]

[0091] Test A - Lipopolysaccharide (LPS) spike detection in elution buffer using detergent The test was performed using a lyophilized E. Coli (O111:B4) endotoxin standard from Thermo Scientific (Cat# 1897398). The LPS standard was reconstituted with water to yield a nominal concentration of 50 EU / mL.

[0092] Part 1) 1.5M NaCl + 100mM Tris pH 8.0 Buffer Spike Recovery in Elution Buffer The detectability of LPS in the plasmid eluate buffer matrix (1.5 M NaCl + 100 mM Tris / HCl, pH 8.0) containing residual amounts of detergent was tested according to the following protocol: -Lyophilized LPS standards were dissolved in water resulting in an LPS stock solution containing nominal 50EU / mL endotoxin. - 20 μL of LPS stock solution was mixed with 100 μL of elution buffer supplemented with various amounts of different detergents. - The mixture was incubated at 30°C for 1 hour, then briefly centrifuged and mixed before final dilution by adding 880 μL of water. -Samples were analyzed directly for endotoxin using 5-0.05EU / mL cartridges and the recovered spike results were evaluated.

[0093] Part 2) 1M NaCl + 100mM Tris pH 9.0 Buffer Spike Recovery in Elution Buffer The detectability of LPS in the plasmid eluate buffer matrix (1 M NaCl + 100 mM Tris / HCl, pH 9.0) containing residual amounts of detergent was tested according to the following protocol: -Lyophilized LPS standards were dissolved in water resulting in an LPS stock solution containing nominal 50EU / mL endotoxin. - 20 μL of LPS stock solution was mixed with 100 μL of elution buffer supplemented with various amounts of different detergents. - The mixture was incubated at 30°C for 1 hour, then briefly centrifuged and mixed before final dilution by adding 880 μL of 25 mM Tris / HCl buffer, pH 7.0. -Samples were analyzed directly for endotoxin using 5-0.05EU / mL cartridges and the recovered spike results were evaluated.

[0094] Test B - Detection of residual endotoxin in plasmid eluates in the presence of Tergitol® In this experiment, endotoxin recovery in actual plasmid eluate samples in the presence of defined amounts of Tergitol® 15-S-9 was investigated. Samples of plasmid eluate material obtained from a Natrix® Q capture run performed without any detergent were subsequently spiked with defined amounts of detergent and finally analyzed for endotoxin. - The starting material was an 8kb plasmid eluate pool w / o detergent, which exhibited an endotoxin level of approximately 200EU / mL. The eluate buffer matrix was equivalent to 1M NaCl + 100mM Tris, pH 9.0. - Detergent stock solution was prepared in elution buffer 1M NaCl + 100 mM Tris, pH 9.0.

[0095] -The pipetting scheme for preparation of spiked samples is as follows: [Table 14] - The mixture was incubated at 30°C for 5 min, then briefly centrifuged and mixed. -Before analysis, samples were diluted 204-fold with 25 mM Tris / HCL buffer, pH 7.0, and subjected to endotoxin measurement using a 5-0.05 EU / mL test cartridge.

[0096] Test C - Detection of endotoxins in plasmid eluates spiked with 200 ppm of neutral detergent In this experiment, the recovery of endotoxins in actual plasmid eluate samples in the presence of defined amounts of mild detergent was investigated. Samples of plasmid eluate material obtained from a Natrix® Q capture run performed without any detergent were subsequently spiked with defined amounts of detergent and finally analyzed for endotoxins. - The starting material was a 20kb plasmid eluate pool w / o detergent, which exhibited an endotoxin level of approximately 500EU / mL. The eluate buffer matrix was equivalent to 1.5M NaCl + 100mM Tris, pH 8.0. A detergent stock solution at -10,000 ppm was prepared in water.

[0097] -The pipetting scheme for preparation of spiked samples was as follows: [Table 15] - 490 μL of the plasmid eluate was mixed with 10 μL of the corresponding detergent stock solution. - The mixture was incubated overnight at 8°C for 16h and then at 30°C for 5min, followed by brief centrifugation and mixing. -Before analysis, the samples were diluted 1,000 times with water and finally subjected to endotoxin measurement using a 5-0.05EU / mL test cartridge.

[0098] result 1) Plasmid capture using Natrix® Q from detergent-treated Feed A (20 kb pDNA) Tables R1 (parts A and B) and R2 compare the results obtained from a plasmid DNA capture study with Natrix Q, where different detergents were tested on lysates pretreated according to Natrix® Q protocol 1. Membrane loading was 0.5 mg plasmid / mL membrane volume. The original 20 kb plasmid lysate used as feed showed an initial endotoxin level of approximately 1,300,000 EU / mg plasmid.

[0099] Table R1 - Part A: Analytical data for purification of a 20 kb plasmid testing various detergents as a complement to the feed. [Table 16]

[0100] Table R1 - Part B: Analytical data for purification of a 20 kb plasmid testing various detergents as a complement to the feed. [Table 17]

[0101] The endotoxin removal effectiveness observed with various detergents tested as a complement to feed pre-treatment is given in Table R2. Table R2. Factor of endotoxin reduction in plasmid eluate pool compared to baseline experiment performed without detergent. Values ​​listed are averages calculated from duplicate runs and replicates. [Table 18]

[0102] 2) Capture of plasmid from detergent-treated Feed B (8 kb pDNA) using Natrix® Q Table R3 (parts A and B) and Table R4 compare the results obtained from plasmid DNA capture studies with Natrix Q, in which various detergents were tested on lysates that had been pretreated according to Protocol 2 of Natrix Q. Membrane loading was 1.6 mg plasmid / mL membrane volume. The original 8 kb plasmid lysate used as feed showed initial endotoxin levels of approximately 50,000 EU / mg plasmid.

[0103] Table R3 - Part A. Analytical data for the purification of an 8 kb plasmid testing various detergents as complements to the wash buffer. [Table 19]

[0104] Table R3 - Part B. Analytical data for purification of an 8 kb plasmid testing various detergents as complements to the wash buffer. [Table 20]

[0105] The endotoxin removal effectiveness observed with various detergents tested as buffer complements is given in Table R4. Table R4. Factor of endotoxin reduction in the plasmid eluate pool compared to the baseline experiment performed without detergent. Values ​​listed are averages calculated from duplicate runs and replicates. [Table 21]

[0106] 3) Plasmid capture with Natrix Q from Feed C (8 kb pDNA) with detergent wash buffer tested Tables R5 (parts A and B) and R6 compare the results obtained from plasmid DNA capture studies with Natrix Q, where various detergents were tested as buffer complements following Natrix Q protocol 3. Membrane loading was 1.6 mg plasmid / mL membrane volume. The original 8 kb plasmid lysate used as feed showed initial endotoxin levels of approximately 275,000 EU / mg plasmid.

[0107] Table R5 - Part A. Analytical data for 8 kb plasmid eluate pools obtained using different wash buffers [Table 22]

[0108] Table R5 - Part B. Analytical data for 8 kb plasmid eluate pools obtained using various wash buffers [Table 23]

[0109] The effectiveness of endotoxin removal observed with various detergents tested as complements to the wash buffer is given in Table R6. Table R6. Factor of endotoxin reduction in the plasmid eluate pool compared to a baseline experiment performed without detergent. Values ​​shown are average values ​​calculated from duplicate runs and each replicate. [Table 24]

[0110] Residual host cell protein concentrations in the plasmid eluate pools obtained from Natrix Q runs using various detergent wash buffers are listed in Table 7. The results suggest that the lowest HCP impurity levels were in the plasmid eluate pools from the purification protocol based on the use of Triton CG110. Table R7. Removal of E. coli host cell proteins (HCPs) from plasmid DNA during the Natrix Q capture step. The table below compares the residual HCP concentrations measured in plasmid eluate pools from Natrix Q capture following wash protocols with different detergents. For each wash protocol, plasmid eluate pools collected from two consecutive runs were analyzed (referred to as run 1 and run 2). The HCP concentration in the original plasmid lysate amounted to 3,235 μg HCP / mg pDNA. [Table 25]

[0111] 4) Plasmid capture using Mustang® Q from Feed C (8 kb pDNA) with detergent wash buffer tested Tables R8 (parts A and B) and R7 compare the results obtained from plasmid DNA capture studies with Mustang® Q, where various detergents were tested as a complement to the wash buffer according to Protocol 1 of the Mustang® Q. Membrane loading was 1.6 mg plasmid / mL membrane volume. The original 8 kb plasmid lysate used as feed showed an initial endotoxin level of approximately 275,000 EU / mg plasmid. Table R8 - Part A. Analytical data for 8 kb plasmid eluate pools obtained using different wash buffers [Table 26]

[0112] Table R8 - Part B. Analytical data for 8 kb plasmid eluate pools obtained using various wash buffers [Table 27]

[0113] The effectiveness of endotoxin removal observed with various detergents tested as complements to the wash buffer is given in Table R9. Table R9. Factor of endotoxin reduction in the plasmid eluate pool compared to the baseline experiment performed without detergent. Values ​​shown are average values ​​calculated from duplicate runs and each replicate. [Table 28]

[0114] Residual host cell protein concentrations in plasmid eluate pools resulting from Mustang® Q capture runs performed with various detergent wash buffers are listed in Table 10. The results confirm improved HCP clearance using the Triton® CG110 wash protocol. Table R10. Removal of E. coli host cell proteins (HCPs) from plasmid DNA during the Mustang® Q capture step. The table below compares the residual HCP concentrations measured in plasmid eluate pools from Mustang® Q capture following different detergent wash protocols. For each wash protocol, plasmid eluate pools collected from two consecutive runs were analyzed (referred to as Run 1 and Run 2). The HCP concentration in the original plasmid lysate amounted to 3,235 μg HCP / mg pDNA. [Table 29]

[0115] 5) Plasmid capture using CIMmultus® DEAE from Feed C (8 kbp DNA) with detergent wash buffer tested Tables R11 (parts A and B) and R12 compare the results obtained from a plasmid DNA capture study with CIMmultus® DEAE, where various detergents were tested as a complement to the wash buffer according to CIMmultus® DEAE protocol 1. The column loading was approximately 1 mg plasmid / mL column volume. The original 8 kb plasmid lysate used as feed showed an initial endotoxin level of approximately 275,000 EU / mg plasmid. Table R11 - Part A. Analytical data for 8 kbp plasmid eluate pools obtained using various wash buffers [Table 30]

[0116] Table R10 - Part B. Analytical data for 8 kb plasmid eluate pools obtained using various wash buffers [Table 31]

[0117] The effectiveness of endotoxin removal observed with various detergents tested as complements to the wash buffer is given in Table R12. Table R12. Factor of endotoxin reduction in the plasmid eluate pool compared to a baseline experiment performed without detergent. Values ​​shown are averages calculated from duplicate runs and each replicate. [Table 32]

[0118] Residual host cell protein concentrations in plasmid eluate pools resulting from CIMmultus® DEAE capture runs performed with various detergent wash buffers are listed in Table 13. The results confirm improved HCP clearance using the Triton® CG110 wash protocol. Table R13. Removal of E. coli host cell proteins (HCPs) from plasmid DNA during the CIMmultus® DEAE capture step. The table below compares the residual HCP concentrations measured in plasmid eluate pools from CIMmultus® DEAE capture following different detergent wash protocols. For each wash protocol, plasmid eluate pools collected from two consecutive runs were analyzed (referred to as Run 1 and Run 2). The HCP concentration in the original lysate amounted to 3,235 μg HCP / mg pDNA. [Table 33]

[0119] Endotoxin assay interference (endotoxin masking effect) Test A - Lipopolysaccharide (LPS) spike detection in elution buffer containing detergent Table R14 summarizes the recovery data observed for LPS in elution buffers containing various detergents. The data indicates that the occurrence of interference of the LAL assay with the detection of LPS is dependent on the type and concentration of residual detergent. Table R14. LPS spike recovery of different elution buffers containing different levels of different detergents according to the protocol for detection of LPS as detailed above. 20 μL of LPS stock solution was spiked into 100 μL of A) 1.5 M NaCl + 100 mM Tris, pH 8.0 or B) 1.0 M NaCl + 100 mM Tris, pH 9.0 elution buffer supplemented with different detergents at different concentrations. [Table 34]

[0120] Test B - Detection of residual endotoxin in plasmid eluates in the presence of Tergitol® Endotoxin recovery results from Test B using actual plasmid samples containing defined amounts of Tergitol® are given in Table R15. The data show that endotoxin detection is not interfered with in the presence of low concentrations of Tergitol® up to 45 ppm. Table R15. Endotoxin recovery in actual plasmid eluates containing defined residual amounts of Tergitol® 15-S-9 [Table 35]

[0121] Test C - Detection of endotoxin in plasmid eluate spikes containing 200 ppm neutral detergent Endotoxin recovery results from Test C using actual plasmid samples containing mild detergent are given in Table R16. Severely impaired endotoxin recovery was found for Triton® X100. Table R16. Endotoxin recovery in actual plasmid eluates containing defined residual amounts of mild detergent. [Table 36]

Claims

1. 1. A method for the depletion or removal of endotoxin from nucleic acids, comprising: a) providing a sample containing the nucleic acid and endotoxin; b) subjecting the sample of step a) to a chromatographic separation on a membrane or monolith containing anion exchange groups; whereby the sample is contacted with a non-ionic surfactant selected from the group of alkyl glycosides and secondary alcohol alkoxylates or mixtures thereof prior to or during chromatographic separation; The method comprising:

2. Step b) i) loading the sample containing the nucleic acids and endotoxins onto a membrane or monolith containing anion exchange groups; ii) washing the membrane or monolith with a wash buffer; iii) eluting the nucleic acids bound to the membrane or monolith with an elution buffer; 2. The method of claim 1, comprising:

3. 3. The method according to claim 1, wherein the sample is contacted with a non-ionic detergent before step b).

4. 4. The method of claim 3, wherein the sample subjected to chromatographic separation contains between 0.01% and 10% (w / v) of a non-ionic surfactant.

5. 3. The method of claim 1 or claim 2, wherein the nucleic acid is contacted with the non-ionic detergent by washing the membrane or monolith with a wash buffer comprising the non-ionic detergent.

6. 6. The method of claim 5, wherein the washing buffer containing a non-ionic detergent contains between 0.01% and 10% (w / v) of a non-ionic detergent.

7. 3. The method according to claim 1, wherein the nonionic surfactant is an alkyl glycoside.

8. 3. The method of claim 1 or 2, wherein the nucleic acid comprises or consists of plasmid DNA.

9. 3. The method according to claim 1, wherein the nucleic acid is contacted with a solution containing 0.01 to 10% (w / v) of a non-ionic surfactant.

10. 3. The method according to claim 1 or 2, characterized in that in step b) a membrane is used, preferably a hydrogel membrane.

11. 3. The method according to claim 1 or 2, characterized in that step ii) comprises two or more washing steps, one of which is carried out with a washing buffer comprising ethanol.

12. 3. The method of claim 1 or 2, wherein the method of the invention provides nucleic acids that are more effectively endotoxin-depleted than the otherwise identical process using Triton® X100 as the only detergent.

13. 3. The method according to claim 1 or 2, characterized in that the method further comprises a step c) of detecting residual endotoxin in the nucleic acid resulting from step b).

14. 3. The method according to claim 1 or 2, characterized in that the detection in step c) is carried out by LAL assay.

15. 3. The method according to claim 1, wherein the detection in step c) is carried out directly in the eluate of the chromatographic separation according to step b), without any further treatment of the eluate.