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, whereby a zwitterionic detergent selected from the group of amine oxides or mixtures thereof is added 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 type of zwitterionic detergent is added during anion exchange chromatographic purification of nucleic acids using membrane or monolith-based sorbents. [Background technology]

[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 purification 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 in high yield and high quality are receiving 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 cells at a number of about 3.5x106 copies per cell (Escherichia coli and Salmonella Typhimurium cells and Mol.Biology, JL Ingraham et al. Eds., 1987, ASM), thus exceeding the number of 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 a number of undesirable side reactions (Morrison and Ryan, 1987, Ann. Rev. Med. 38, 417-432;Boyle et al. 1998, DNA and Cell Biology, 17, 343-348). If the plasmid DNA is intended to be used for gene therapy or vaccines, 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. Known methods for reducing endotoxin levels are based on multiple purification steps, often using anion exchange chromatography.

[0005] 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 the 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 the 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 salt-free washing solutions and adsorbents having functional groups attached to tentacles.

[0010] WO2009 / 129524 discloses a suitable protocol for purifying plasmid DNA in which the plasmid DNA is contacted with a zwitterionic detergent in a small-scale parallel format.

[0011] No. 6,428,703 describes a method for purifying biological macromolecules by contacting them with a non-ionic detergent and performing a chromatographic purification.

[0012] US2005 / 245733 reports a method for reducing endotoxins in plasmid preparations using silica chromatography or organic polymeric resins and carbohydrate non-ionic detergents.

[0013] 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.

[0014] Downstream processes in the biopharmaceutical and biotechnology industries mostly rely on chromatography steps using bead-based resins in packed bed columns as stationary phases. The resins typically have diameters between 30 and 500 μm and generally provide an efficient chromatographic technique with high binding capacity. However, the method 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 intraparticle 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 molecules and 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.

[0015] 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.

[0016] It has been found that endotoxin clearance in nucleic acid purification using membrane or monolith-based chromatography matrices can be dramatically improved in combination with the use of zwitterionic detergents selected from the amine oxide group, without compromising the nucleic acid yield or the removal of host cell proteins. Selected types of detergents proved to be particularly effective in combination with certain high-productivity chromatography membrane or monolith materials. The examples provided for the proposed solution show its unique potential to enhance plasmid production at large scale over existing approaches based on bead-based materials with commonly used alternative detergents such as Triton™ X100. Summary of the Invention

[0017] Thus, the present invention is directed to a method for the depletion or removal of endotoxins from nucleic acids, comprising: a) providing a sample containing the nucleic acid and endotoxin; b) subjecting the sample of step a) to chromatographic separation on a membrane or monolith containing anion exchange groups. Thereby, the sample is contacted with a zwitterionic surfactant selected from the group of amine oxides. In a preferred embodiment, step b) comprises: i) loading the sample containing the nucleic acid and endotoxin onto a membrane or monolith containing an anion exchange group; 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.

[0018] In one embodiment, in step ii) the nucleic acid is contacted with the zwitterionic detergent by washing the membrane or monolith with a wash buffer comprising the zwitterionic detergent. In a preferred embodiment, the zwitterionic surfactant is an amine oxide. In a highly preferred embodiment, it is N,N-dimethyltetradecylamine N-oxide. In a preferred embodiment, the nucleic acid comprises or consists of plasmid DNA. In a preferred embodiment, the nucleic acid is contacted with a solution containing 0.01 to 10% (w / v) of a zwitterionic surfactant. In a preferred embodiment, the anion exchange capture material is a membrane. In a highly 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.

[0019] In one embodiment, the process of the invention provides nucleic acids that are as or more effectively endotoxin-depleted than the same process using only Triton® X100 as the only detergent.

[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 should 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, and so forth.

[0021] 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.

[0022] 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" it 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.

[0023] 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.

[0024] 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.

[0025] Plasmids are typically epigenomic circular DNA molecules with a length between 4 and 20 kB, which corresponds to a molecular weight between 2.6×10 6 and 13.2×10 6 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.

[0026] 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).

[0027] 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.

[0028] 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.

[0029] 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.

[0030] The term "batch" refers to a quantity of plasmid or nucleic acid material that is intended to have uniform characteristics and quality within defined limits and is produced according to a single manufacturing instruction during the same production cycle with a defined start and end point. In the case of continuous processes, a batch is typically defined based on a time and / or quantity strategy.

[0031] The terms "matrix" or "chromatographic matrix" are used interchangeably herein and refer to the solid phase through which a sample passes during 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, a bead-based resin, or a monolith, preferably the substrate is a membrane or monolith, most preferably a membrane.

[0032] 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.

[0033] The ligands can be attached to the substrate of the matrix by any type of covalent attachment. Covalent attachment can be performed by directly binding a functional group to a suitable residue on the substrate, such as, for example, OH, NH2, carboxyl, phenol, anhydride, aldehyde, epoxide, or thiol. 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.

[0034] In another embodiment, the stationary phase can be produced by grafting ligands onto or from the 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.

[0035] 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.

[0036] 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 packed 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 solvent delivery to and from the tube or cartridge. The size of the chromatography column varies depending on the application, for example analytical or preparative. In one embodiment, the column, or generally the separation device, is a single-use device.

[0037] Thus, the term "anion exchange matrix" as used herein refers to a chromatography matrix that possesses at least anion exchange groups, i.e., it typically has one or more types of ligands that are positively charged under the chromatographic conditions employed, such as quaternary amino groups.

[0038] 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 used 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.

[0039] 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.

[0040] 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.

[0041] 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.

[0042] "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 / elute mode before eluting the target molecule. In addition, washing steps can be used to reduce the level of residual detergent, enhance virus clearance, and / or modify the conductivity carryover during elution.

[0043] "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.

[0044] 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, causing the buffer to compete with the molecules for charged sites on the ion exchange material.

[0045] 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.

[0046] 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.

[0047] 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.

[0048] 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.

[0049] 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).

[0050] - 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).

[0051] - 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).

[0052] -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).

[0053] -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).

[0054] Monoliths or monolith adsorbents, like membranes, have through-holes, such as interconnected channels, that allow liquid to flow from one side of the monolith, through the monolith, to the other side of the monolith.

[0055] As the mobile phase flows through these through-holes, the molecules to be separated are transported by convection rather than by diffusion. Due to their structure, monolithic adsorbents exhibit separation efficiency and dynamic capacity that are independent of flow rate.

[0056] The monolith is typically formed in situ from the reactant solution and can have any shape or constrained geometry, typically a frit-free structure, which ensures convenience of operation. Preferably, the material of the monolith has a dual 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 are preferably located in the walls of the through-holes, contributing 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.

[0057] The synthesis of organic polymer monoliths is typically carried out by 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, microwave 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.

[0058] 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).

[0059] 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. The monolith can also be made by 3D printing.

[0060] 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 is contained in a housing, such as a column.

[0061] Particle-based resins for liquid chromatography are usually contained in particles that are packed together in a cylindrical cylinder called a column, forming a bed. Packed beds exhibit well-defined spaces between the particles, the so-called void volume, which primarily defines the liquid fluid permeability and hydrodynamic properties of the packed bed.

[0062] Particles typically consist of a cross-linked polymer matrix in the form of spheres, beads, or grains with a relatively uniform size to improve the chromatographic and hydrodynamic properties of the packed bed. They can have a dense structure with discontinuous or very small pores, but usually exhibit a porous multi-channel or network structure that creates a pore volume and additional surface area inside the particle. The particle surface area can be modified with a variety of functional groups suitable for chromatographic applications, either by using functional monomers in the backbone-polymer structure or by direct coupling of functional groups to the particle surface via ligands or short polymer structures (grafting).

[0063] Zwitterionic surfactants are amphoteric surfactants with a hydrophilic head bearing both anionic and cationic charged atomic groups, with a non-polar tail. The net charge and other physicochemical properties (viscosity, solubility, critical micelle formation) of zwitterionic surfactants change as the pH of the solution is adjusted. For a solution with a pH at the isoelectric point, the negative charge of a surfactant molecule exactly balances the positive charge of the same molecule, and the net charge of the surfactant molecule is zero. Amine oxides are classified as zwitterionic surfactants.

[0064] An amine oxide is a compound having the formula R1R2R3NO, where each R1, R2, and R3, independently of the others, is an optionally substituted C1-C30 hydrocarbon chain.

[0065] Particularly preferably used amine oxides are those in which R1 is C10-C18 alkyl and R2 and R3 are each independently C1-C4 alkyl, in particular C12-C16 alkyl dimethylamine oxides.

[0066] A particularly suitable amine oxide is N,N-dimethyltetradecylamine N-oxide (TDAO), also known as myristyldimethylamine-N-oxide (CAS number 3332-27-2).

[0067] 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. When 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.

[0068] 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.

[0069] The sample containing nucleic acids and endotoxins, and possibly other impurities, from which nucleic acids are to be purified and thus endotoxins are removed or depleted, is then subjected to a chromatographic separation on a membrane or monolith-based chromatography matrix containing anion exchange groups. For this purpose, the sample is loaded onto the chromatography matrix. The sample of the final composition that is loaded onto the matrix is ​​called the feed. The feed is preferably adjusted to an electrolytic conductivity between 40-90 mS / cm, and most preferably the feed is adjusted to a conductivity high enough to prevent binding of RNA to the anion exchange material, yet still allow for capture of target nucleic acid.

[0070] 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.

[0071] Column equilibration and wash buffers are typically buffers that match the pH and conductivity of the feed loaded onto the chromatographic material. Typically, wash buffers with a pH of 7.5-9.0 and a conductivity of 5-90 mS / cm are selected, although buffers outside of that range are equally applicable.

[0072] After loading, the matrix is ​​washed with at least one wash buffer, which may be the same as the loading buffer or may be different from the loading buffer.

[0073] The matrix may be washed with two, three or four different washing buffers, at least one of which contains a zwitterionic detergent selected from the group of amine oxides or mixtures thereof. Typically, the concentration of the detergent in the washing solution is between 0.01% and 10% (w / v), preferably between 0.1% and 1.5% (w / v).

[0074] Detergent wash solutions made from low conductivity wash buffers (<40 mS / cm) with a pH range within + / - 1 unit of the isoelectric point of the zwitterionic detergent used are particularly suitable, although buffers outside that range are equally applicable. 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.

[0075] 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.

[0076] 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.

[0077] In any case, at least one wash step in the method of the invention uses a washing solution containing a zwitterionic surfactant, preferably the zwitterionic surfactant is selected from the group of amine oxides or mixtures thereof, the most preferred surfactant being N,N-dimethyltetradecylamine N-oxide.

[0078] The method of the present invention can be used for continuous, semi-continuous or batch chromatography using one or more chromatography columns. A variety of chromatography modes are known to those skilled in the art, and the teachings of the present invention can be easily adapted to each mode by those skilled in the art.

[0079] When nucleic acid purification according to the methods of the present invention is carried out using a zwitterionic detergent, target nucleic acids can be obtained with significantly lower endotoxin contamination compared to purification methods that omit the use of detergents. Depending on the actual type of anion exchange capture material used for nucleic acid purification, the method can result in anywhere from a 30- to 600-fold improvement in endotoxin reduction.

[0080] The types of AEX materials and particular classes of detergents are preferably selected for the methods disclosed herein and are particularly suitable for removing or depleting endotoxins from samples containing nucleic acids, such as plasmid DNA.

[0081] The starting material, i.e. sample, can be processed by the method of the present invention in a dosage volume ranging from 5 to 5000 liters with nucleic acid, e.g. plasmid DNA, in a concentration range of 0.02 to 1.0 mg / ml. The method is preferably applied on a scale of 50 to 500 L sample volume with a plasmid titer of 0.050 to 0.200 mg / mL. A total loading of 1 to 10 mg of nucleic acid, e.g. plasmid DNA, per mL volume of AEX membrane or monolith adsorber, and a flow rate of between 1 and 10 volumes of membrane or monolith device per minute are suitable for the method of the present invention.

[0082] Therefore, the method of the present invention is also suitable for large-scale purification of nucleic acids and therefore large-scale removal or depletion of endotoxins from said nucleic acid samples.The amount of nucleic acids present in the sample can also vary in a wide range and can be as much as 1 mg / mL.The method also allows for extremely high loading of up to 20 mg of nucleic acid per ml volume of membrane or monolith.

[0083] Since the method of the present invention allows the processing of various amounts of target nucleic acid, particularly plasmid DNA, batches of from 0.1 mg to 5 kg of nucleic acid can be subjected to chromatographic purification.

[0084] The final endotoxin level in the target nucleic acid pool depends on the initial endotoxin level: if the initial endotoxin level of the target nucleic acid is about 275,000 EU / mg, the method of the present invention allows the target nucleic acid to achieve a final endotoxin level as low as 10-40 EU / mg.

[0085] In one embodiment, the method of the invention is carried out by using only N,N-dimethyltetradecylamine N-oxide as the detergent, no other detergents are added to the feed or wash buffers, or at any time during the chromatographic purification. The invention is further illustrated by the following figures and examples, without, however, being limited thereto.

[0086] The entire disclosures of all applications, patents, and publications cited above and below, as well as corresponding patent application US 63 / 318,548, filed March 10, 2022, are hereby incorporated by reference.

[0087] example The following examples represent practical applications of the present invention. [Table 1]

[0088] Protocol for Plasmid DNA Capture Note: For small volume membrane screening devices where the hold-up of the system is disproportionately large, very large volumes are typically used for washing, elution, cleaning in place (CIP) and equilibration. At larger scales, these values ​​may be reduced and flow directions may be reversed to enhance individual steps. This is standard practice and common knowledge to anyone skilled in the art. [Table 2]

[0089] Natrix(R) Q Protocol [Table 3] [Table 4]

[0090] 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 through 0.22 μm PES media and supplemented with 175 mM NaCl, which is necessary for selective binding of pDNA.

[0091] Mustang® Q Protocol [Table 5] [Table 6]

[0092] Plasmid feed Purification tests were carried out with the original lysate filtered through 0.22 μm PES medium and supplemented with 375 mM NaCl, necessary for selective binding of pDNA. CIMmultus® DEAE Protocol [Table 7] [Table 8]

[0093] 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.

[0094] 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.

[0095] Residual amounts of detergent in the collected plasmid eluate fractions from the AEX capture studies were measured using an analytical HPLC method as described below. This method allows for direct analysis of the plasmid eluate without prior sample preparation to remove potentially interfering matrix components using, for example, solid phase extraction.

[0096] The amount of surfactant in unknown eluate samples was calculated based on the analyte peak area using calibration curves obtained from individual surfactant standards in the eluate buffer matrix.

[0097] 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. [Table 9]

[0098] result 1) Plasmid capture with Natrix® Q Tables R1 (parts A and B) and R2 compare the results obtained from the plasmid DNA capture test following the Natrix® Q protocol. The 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 10] [Table 11]

[0099] The effectiveness of endotoxin removal observed with pDNA capture using the Deviron™ wash protocol is shown in Table R2. [Table 12] Residual host cell protein concentrations in the plasmid eluate pools obtained from Natrix Q capture are shown in Table 3.

[0100] [Table 13]

[0101] 2) Plasmid capture with Mustang® Q Tables R4 (parts A and B) and R5 compare the results obtained from plasmid DNA capture studies using Mustang® Q. The 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 14] [Table 15]

[0102] The effectiveness of endotoxin removal observed with pDNA capture using the Deviron™ wash protocol is shown in Table R5. [Table 16]

[0103] The residual host cell protein concentrations in the plasmid eluate pools obtained from the Mustang® Q capture runs are listed in Table 6. [Table 17]

[0104] 3) Plasmid capture with CIMmultus® DEAE Tables R7 (parts A and B) and R8 compare the results obtained from plasmid DNA capture studies using CIMmultus® DEAE. 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 18] [Table 19]

[0105] The efficacy of endotoxin removal observed with pDNA capture using the Deviron™ wash protocol is given in Table R8. [Table 20]

[0106] Residual host cell protein concentrations in the plasmid eluate pools resulting from the CIMmultus® DEAE capture studies are listed in Table 9. [Table 21]

[0107] 4) Endotoxin clearance with Deviron™ compared to a common alternative approach using the mild detergent Triton™ X100 [Table 22]

Claims

1. A method for depleting or removing endotoxins from nucleic acids, a) To provide a sample containing the nucleic acid and endotoxin. b) The sample from step a) is subjected to chromatographic separation on a membrane or monolith containing anion exchange groups. Includes, This means that the sample is contacted with an amphoteric surfactant selected from a group of amine oxides or mixtures thereof, before or during chromatographic separation. The aforementioned method.

2. Step b) is, i) Loading the sample containing the nucleic acid and endotoxin onto a membrane or monolith containing anion exchange groups. ii) Wash the membrane or monolith with a washing buffer. iii) Elute nucleic acids bound to the membrane or monolith with an elution buffer. The method according to claim 1, characterized by including the following:

3. The method according to claim 1 or 2, characterized in that nucleic acids are brought into contact with the amphoteric surfactant by washing the membrane or monolith with a washing buffer containing an amphoteric surfactant.

4. The method according to claim 3, characterized in that the washing buffer containing the amphoteric surfactant contains an amphoteric surfactant in an amount between 0.01% and 10% (w / v).

5. The method according to claim 1, characterized in that the amphoteric surfactant used in the method of the present invention is a C12-C16 alkyldimethylamine oxide.

6. The method according to claim 1, characterized in that the amine oxide used in the method of the present invention is N,N-dimethyltetradecylamine N-oxide.

7. The method according to claim 1, characterized in that the nucleic acid includes or consists of plasmid DNA.

8. The method according to claim 1, characterized in that nucleic acids are brought into contact with a solution containing 0.01 to 10% (w / v) of an amphoteric surfactant.

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

10. The method according to claim 2, characterized in that step ii) includes two or more washing steps, one of which is performed using a washing buffer containing ethanol.

11. The method according to claim 1, characterized in that, in step b), the volume of the sample subjected to chromatographic separation is between 5 and 5000 liters and has a plasmid DNA concentration in the range of 0.02 to 1.0 mg / ml.

12. The method according to claim 1, characterized in that, in step b), the mass of nucleic acids subjected to chromatographic separation is in the range of 0.1 gm to 5 kg per batch.

13. The method according to claim 1, characterized in that in step b), 1 to 20 mg of nucleic acid is loaded per mL volume of a membrane or monolith containing anion exchange groups.

14. The method according to claim 1, characterized in that in step b), the chromatographic separation is performed at a flow rate between 1 and 10 volumes of the membrane or monolith per minute.