Method for reducing nucleic acid and nucleic acid adsorption filter

Abstract

The objective of the present invention is to provide a method capable of effectively and easily reducing the amount of a specified impurity in a liquid. The method for reducing the amount of nucleic acid in a liquid according to the present invention is characterized by including a step for bringing the liquid into contact with a water-insoluble magnesium compound, thereby causing at least a part of the nucleic acid to be adsorbed to the water-insoluble magnesium compound. Another objective of the present invention is to provide an adsorbing filter useful for purifying a useful substance such as an antibody or an antibody-like molecule as a material for purification that can easily maintain yield of a product of interest at a high level and is capable of efficiently removing an impurity due to low adsorption of an antibody or an antibody-like molecule but high adsorption ability for DNA or the like. The adsorption filter according to the present invention is characterized by including a layer that contains a water-insoluble magnesium compound.

Description

[Technical field]

[0001] The present invention relates to a method for effectively and simply reducing the amount of nucleic acids in a liquid, since such a filter has excellent adsorption ability for nucleic acids and the like, but low adsorption ability for antibodies and antibody-like molecules, and to an adsorption filter that is useful for purifying useful substances used as main components of biopharmaceuticals, such as antibodies and antibody-like molecules. [Background technology]

[0002] Since the transformation method using a plasmid containing a gene encoding a target protein was put to practical use, proteins have mainly been produced by the transformation method. For example, the transformation method using human genes can produce antibodies and antibody-like molecules consisting of only the parts of antibodies necessary for antigen recognition and cytotoxicity. In addition, in the production of viruses and virus-like particles that can be used as vaccines or gene therapy vectors, it is common to infect cultured cells or fertilized chicken eggs with the virus and increase the virus or virus-like particles in the culture medium or body fluid. Useful substances such as proteins and viruses are first separated from the culture medium or the like by filtration to remove solid matter such as cells and cell fragments, and then purified by chromatography or the like.

[0003] Impurities other than useful substances are contaminants such as proteins, nucleic acids, and lipids derived from hosts or living organisms, and among them, nucleic acids increase the viscosity of the liquid, making it difficult to filter, and can foul chromatography carriers and membranes, reducing purification efficiency. In addition, nucleic acids derived from some hosts, such as immortalized cells, pose a risk of causing cancer in patient cells, so they must be removed without fail.

[0004] Proteins and viruses are also used in testing and diagnosis. For example, to identify a virus, detection using antigen-antibody reactions is used, and diagnostic drugs are developed based on the virus's base sequence. In addition, to ensure the safety of pharmaceuticals derived from human serum, tests are conducted for viruses and bacteria that may be contaminating raw materials and final products. When detecting trace amounts of viruses, etc. using antigen-antibody reactions or nucleic acid amplification tests, it is possible to improve sensitivity and accuracy by removing nucleic acids derived from the host or living organism other than the target virus, etc.

[0005] For example, Non-Patent Document 1 shows an example of using nucleases to degrade nucleic acids and produce a Vero cell-derived vaccine; however, nucleases are expensive and require a process to remove the nucleases and fragmented nucleic acids.

[0006] Patent Document 1 shows that zeolite with barium sulfate formed on the surface can adsorb viruses from cell culture fluid. However, it does not show the adsorption and removal of impurities such as contaminants derived from host cells, such as proteins, nucleic acids, and lipids. Furthermore, there is a limit to the amount of poorly water-soluble inorganic compounds that can be supported on the carrier surface.

[0007] Patent Document 2 describes that a composite depth filter medium containing silica, alumina, or the like as a filter aid can reduce the total organic carbon (TOC) of a cell culture solution while recovering monoclonal antibodies (mAbs) with high efficiency. However, data also shows that the medium cannot reduce turbidity or DNA concentration.

[0008] Patent Document 3 discloses that adding a cationic solution and anionic solution to a cell culture medium to produce a water-insoluble solid and separating the produced solid by centrifugation reduces the turbidity of the supernatant while maintaining the antibody concentration. However, this method is subject to limitations in downstream purification process conditions, such as an increase in the amount of liquid and an increase in the ionic strength in the liquid.

[0009] Non-Patent Document 2 shows the usefulness of removing DNA and histones, but precise pH control is required to achieve both yield and impurity removal.

[0010] Patent Document 4 shows that a depth filter layer containing hydrotalcite binds to DNA but has a relatively low binding ability to IgG. However, according to the experimental findings of the present inventors, hydrotalcite also exhibits a certain degree of adsorptivity to antibodies and the like, and is therefore not suitable for large-scale purification requiring a yield of 80% or more. [Prior art documents] [Patent documents]

[0011] [Patent Document 1] JP 2009-42074 A [Patent Document 2] JP 2016-530993 A [Patent Document 3] JP 2009-508486 A [Patent Document 4] JP 2011-530405 A [Non-patent literature]

[0012] [Non-Patent Document 1] Si-Ming Li et al., Biologicals, 42(2014), 271-276 [Non-Patent Document 2] Pete Gagnon et al., Journal of Chromatography A, 1374(2014), 145-155 Summary of the Invention [Problem to be solved by the invention]

[0013] As described above, various techniques have been developed for roughly purifying a target product by adsorbing impurities onto a solid. However, for example, in the invention described in Patent Document 3, the formation of the water-insoluble solid takes time, so it is not suitable for mass purification of antibodies, etc., and there is a risk of problems such as clogging of pipes, etc., due to the fact that fine crystals of the water-insoluble solid grow in the later stage after separation from cells, etc., or new crystals of the water-insoluble solid are formed in the later stage. In addition, high ionic strength may hinder various analyses. If the concentration of each ion is reduced to prevent this, a sufficient amount of water-insoluble solid is not formed, and impurities cannot be effectively removed. Therefore, the present invention aims to provide a method for effectively and simply reducing the amount of specific impurities in a liquid, and an adsorption filter that has excellent adsorption ability for DNA, etc., but low adsorption ability for antibodies and antibody-like molecules, and is therefore useful for purifying useful substances that are used as the main components of biopharmaceuticals, such as antibodies and antibody-like molecules. [Means for solving the problem]

[0014] The present inventors have conducted extensive research to solve the above problems, and as a result have found that by contacting a purification target containing impurities such as nucleic acids with a water-insoluble magnesium compound, the amounts of cell-derived nucleic acids, nucleic acid-binding proteins, and lipopolysaccharides can be reduced, and the burden of subsequent purification of a target useful substance can be reduced, thereby completing the present invention. The present invention will now be described.

[0015] [1] A method for reducing the amount of nucleic acid in a liquid, comprising: contacting the liquid with a water-insoluble magnesium compound to adsorb at least a portion of the nucleic acid to the water-insoluble magnesium compound; and then separating said liquid from said water-insoluble magnesium compound. [2] The method according to [1], wherein the water-insoluble magnesium compound is one or more selected from magnesium carbonate, magnesium hydroxide, magnesium oxide, magnesium silicate, and magnesium phosphate. [3] The method according to [1] or [2], wherein the liquid further contains a useful substance. [4] The method according to any one of [1] to [3] above, wherein the liquid and the water-insoluble magnesium compound are separated by filtration. [5] The method according to [3] above, wherein the useful substance is one or more useful substances selected from useful proteins, viruses, and virus-like particles. [6] The method according to [5], wherein the useful protein is one or more useful proteins selected from an antibody, an antibody-like molecule, a hormone, an enzyme, a growth factor, a blood protein, and an antibody-binding protein. [7] The method according to any one of [1] to [6] above, wherein the liquid is a cell culture medium or a body fluid.

[0016] [8] An adsorption filter having a layer containing a water-insoluble magnesium compound. [9] The adsorption filter according to [8], having a layer made of water-insoluble magnesium compound particles.

[10] The adsorption filter according to [8], having a layer containing a water-insoluble magnesium compound and a water-insoluble medium.

[11] The adsorption filter according to

[10] , wherein the material of the water-insoluble medium is one or more selected from polysaccharides, synthetic polymers, and inorganic substances.

[12] The adsorption filter according to

[11] , wherein the polysaccharide is one or more selected from the group consisting of cellulose, cellulose acetate, nitrocellulose, agarose, and chitosan.

[13] The adsorption filter according to

[11] , wherein the synthetic polymer is one or more selected from polyacrylonitrile, polyester, polyethersulfone, polypropylene, and polytetrafluoroethylene.

[14] The adsorption filter according to

[11] , wherein the inorganic material is one or more selected from glass, silica, alumina, zirconia, and barium titanate.

[15] The adsorption filter according to any one of [8] to

[14] , wherein the water-insoluble magnesium compound is one or more selected from magnesium carbonate, magnesium hydroxide, magnesium oxide, and magnesium phosphate. Effect of the Invention

[0017] According to the method of the present invention, while the water-insoluble magnesium compound can adsorb at least nucleic acid, it does not adsorb or hardly adsorbs at least antibody or antibody-binding protein, so that the amount of cell-derived impurities such as nucleic acid can be reduced, and the target useful substance can be roughly purified.In addition, the water-insoluble magnesium compound is inexpensive, and the cell-derived impurities can be adsorbed by simply contacting the water-insoluble magnesium compound with the liquid containing the cell-derived impurities, so that the method of the present invention can be carried out easily and at low cost.In addition, the water-insoluble magnesium compound can improve the filtration efficiency as a filter aid, so that filtration can be performed in a short time. Furthermore, by simply passing a liquid containing impurities in addition to useful substances such as antibodies and antibody-like molecules through the adsorption filter of the present invention, impurities such as DNA can be adsorbed and removed, and a filtrate containing useful substances such as antibodies and antibody-like molecules while having a reduced impurity concentration can be obtained by simple operations. At the same time, host cells and their crushed products can be physically removed. In this way, water-soluble impurities such as DNA can be removed, and suspended matter such as cells can also be removed, thereby reducing the burden of subsequent purification by chromatography, etc. Therefore, the present invention is extremely useful industrially as it can contribute to the mass production of useful substances such as antibodies, the demand of which is expected to increase in the future. [Brief description of the drawings]

[0018] [Figure 1] FIG. 1 is a graph showing the filtrate yield when a water-insoluble magnesium compound was added to a CHO cell suspension and then filtered. [Diagram 2] FIG. 2 is a graph showing the filtrate yield when a water-insoluble magnesium compound was added to a suspension of HEK cells and then filtered. [Diagram 3] FIG. 3 is a schematic diagram of a layered basic magnesium carbonate loading device according to the present invention. [Figure 4]FIG. 4 is a schematic diagram of a dispersed basic magnesium carbonate loading device according to the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] The present invention relates to a method for reducing the amount of nucleic acid in a liquid by contacting a liquid containing nucleic acid with a water-insoluble magnesium compound, thereby adsorbing at least a part of the nucleic acid to the water-insoluble magnesium compound. The method of the present invention will be explained below in steps, but the present invention is not limited to the following specific examples.

[0020] 1. Preparation of impurity-containing liquid In this step, a liquid containing the nucleic acid to be reduced is prepared. This step is optional, and it is not necessary to perform this step when such a liquid has already been obtained. The liquid may be an aqueous solution or a suspension. An aqueous solution refers to a solution in which all components are dissolved in water as a solvent and does not substantially contain insoluble components, and a suspension refers to a liquid that may contain solutes but also contains insoluble components. Examples of insoluble components include cells, cell fragments, cell lysates, and aggregates of cell-derived components such as aggregated proteins. The insoluble components may be dispersed or precipitated in the solution.

[0021] Nucleic acids are biopolymers in which nucleotides consisting of bases, sugars, and phosphates are linked together by phosphodiester bonds, and exist in living organisms as RNA or DNA. Nucleic acids can be impurities, regardless of their composition or length, whether they are DNA or RNA. In addition, fragmented nucleic acids or those that form complexes with proteins, such as chromosomes, can be impurities. For example, if the liquid containing a useful substance is a culture medium, in addition to nucleic acids derived from cells, plasmids used for transient expression are also impurities. If the liquid containing a useful substance is a body fluid, in addition to nucleic acids derived from cells, cell-free DNA and RNA are also impurities. If the liquid containing a useful substance contains a virus, DNA and RNA leaked from the virus are also impurities.

[0022] Genomic nucleic acids have a long chain structure and often form complexes with proteins, which can cause problems such as high viscosity of liquids containing useful substances, accumulation or residue in purification materials, and reduced sensitivity and accuracy of analysis, so their removal is important. In cell culture fluids, it is important to remove long chain nucleic acids leaked by cell disruption or DNA present as fragments thereof.

[0023] The method of the present invention may reduce the amount of other cell-derived impurities in addition to nucleic acids. Examples of such impurities include nucleic acid-binding proteins and lipopolysaccharides. Nucleic acid-binding proteins are mainly DNA-binding proteins such as histones. Of the proteins called histones, four types of core histones are assembled in pairs to form histone octamers, which together with DNA form nucleosomes, and furthermore, linker histones are bound to the DNA between the nucleosomes. Therefore, the molecular weight of the DNA-binding proteins contained in the cell lysate or lysate is considered to be relatively large. Lipopolysaccharide is a complex of lipids and polysaccharides bound by covalent bonds, and is the main body of endotoxins that exist mainly as outer membrane components of gram-negative bacteria.

[0024] The liquid may contain a useful substance to be purified, such as useful proteins, such as antibodies, antibody-like molecules, antibody-binding proteins, enzymes, growth factors, hormones, cytokines, and blood proteins, as well as viruses and virus-like particles used in gene therapy, vaccine research, development, and production.

[0025] The "antibody or antibody-like molecule" that can be purified in the present invention may be any antibody or antibody-like molecule that is useful for industrial applications, and includes functional proteins having a polypeptide structure, including those containing a secondary structure such as an α-helix or β-sheet structure in the molecule, as well as those having a sugar chain, those modified with sugars, those modified by phosphorylation or tyrosination, and those coordinated with metals. In addition to naturally occurring proteins and peptides, they also include those produced by recombinant gene technology, those with improved functions, structures consisting of only functional sites, and those linked to different functional sites or those linked to the same functional sites. They also include those intramolecularly crosslinked by disulfide bonds between cysteine ​​residues in the molecule, those intermolecularly crosslinked by disulfide bonds between cysteine ​​residues in the molecule, those containing subunit structures noncovalently, proteins linked by chemical modification, and those functionalized by chemical modification of proteins or addition of functional molecules.

[0026] The antibody or antibody-like molecule is not particularly limited, and may be a polyclonal antibody, a monoclonal antibody, a human antibody, a humanized antibody, a chimeric antibody, a single-chain antibody, a heavy-chain antibody, a multivalent antibody, Fab, F(ab'), F(ab') 2 , Fc, Fc fusion protein, bispecific antibody, heavy chain (H chain), light chain (L chain), single chain Fv (scFv), sc(Fv) 2 , disulfide bond Fv (sdFv), diabody, antibody-like molecule target peptide (micro antibody), etc. In the present invention, these antibodies or antibody-like molecules may be Fc-containing proteins such as immunoglobulins and Fc fusion proteins having an Fc portion, and the above-mentioned Fab, F(ab'), F(ab') and 2 , Fc, heavy chain (H chain), light chain (L chain), single chain Fv (scFv), sc(Fv) 2 Any of these antibodies can be preferably targeted, including small molecular weight antibodies such as disulfide-linked Fv (sdFv), single-chain antibodies, heavy-chain antibodies, multivalent antibodies, bispecific antibodies, diabodies, and antibody-like molecular targeting peptides (microantibodies).

[0027] Examples of the enzyme include lipase, protease, steroid synthesis enzyme, kinase, phosphatase, xylanase, esterase, methylase, demethylase, oxidase, reductase, cellulase, aromatase, collagenase, transglutaminase, glycosidase, and chitinase.

[0028] Examples of growth factors include epidermal growth factor (EGF), insulin-like growth factor (IGF), transforming growth factor (TGF), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), vascular endothelial growth factor (VEGF), granulocyte-colony stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), platelet-derived growth factor (PDGF), erythropoietin (EPO), thrombopoietin (TPO), fibroblast growth factor (FGF), and hepatocyte growth factor (HGF).

[0029] Examples of hormones include insulin, glucagon, somatostatin, growth hormone, parathyroid hormone, prolactin, leptin, and calcitonin. Examples of cytokines include interleukins, interferons (IFNα, IFNβ, and IFNγ), and tumor necrosis factors (TNF).

[0030] Examples of blood proteins include thrombin, serum albumin, factor VII, factor VIII, factor IX, factor X, and tissue plasminogen activator.

[0031] The antibody-binding protein is not particularly limited as long as it is a protein that has specific binding ability to an antibody, and examples include protein A, protein G, protein L, Fcγ receptor, antibody-binding domains thereof, and mutants thereof that maintain or improve their binding ability to antibodies or antibody-like molecules.

[0032] The virus is not particularly limited as long as the virus itself or a part thereof is to be purified, but examples of non-enveloped viruses include adeno-associated virus, adenovirus, enterovirus, parvovirus, papovavirus, human papillomavirus, rotavirus, coxsackievirus, sapovirus, norovirus, poliovirus, echovirus, coronavirus, hepatitis A virus, hepatitis E virus, rhinovirus, astrovirus, etc. The adeno-associated virus has an AAV capsid serotype selected from the group consisting of AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, AAV-13, AAV-14, AAV-15, and AAV-16. Examples of enveloped viruses include retroviruses, lentiviruses, Sendai viruses, herpes simplex viruses, vaccinia viruses, measles viruses, baculoviruses, and influenza viruses.

[0033] Virus-like particles are made up of all or part of the viral coat proteins that mainly make up the capsid. Since they do not contain nucleic acids, there is no risk of infection, but because they can elicit an immune response, they can be used as an active ingredient in vaccines.

[0034] In the present invention, the liquid to be treated with the specific water-insoluble magnesium compound is not particularly limited as long as it contains the above-mentioned impurities, and may contain a useful substance. Examples of such liquids include culture liquid, culture supernatant, cultured cells, cell lysates, or cell lysates suspended in water or their extracts, biological extracts, liquids containing viruses or virus-like particles, and body fluids. In addition, the presence of a coexisting organic solvent is not prevented as an aqueous solution or suspension. The cells may be virus-infected cells. A liquid containing a virus or virus-like particles may be produced in a culture liquid or body fluid by infecting cultured cells or chicken fertilized eggs with the virus. In addition, the body fluid is a liquid that is present in the body of an animal and fills between tissues, in the body cavity, or in tubes or circulatory systems that are spread throughout the body, or a liquid that is secreted or excreted inside or outside the body, and may be, for example, chorioallantoic fluid separated after inoculating blood or a virus strain into the allantoic cavity of a chicken egg and culturing it.

[0035] The cells to be cultured may be naturally occurring cells, but are preferably recombinant host cells. The host is a cell used to produce a useful substance, and is not particularly limited as long as it is an animal cell, plant cell, insect cell, or microbial cell that can be transformed with an expression vector or gene fragment containing a DNA encoding a useful protein, and can express the introduced DNA to produce a useful protein or can be infected with a virus. The genetic recombinant in the present invention is a host cell transformed by introducing an expression vector or gene fragment containing a base sequence encoding the amino acid sequence of a useful protein and a promoter that is operably linked to the base sequence and can function in the host.

[0036] The liquid may contain other impurities in addition to the impurities and useful substances. Examples of the other impurities include, but are not limited to, aggregated proteins, plasmids, medium components, plasmid DNA, etc. The solvent of the liquid may be a buffer solution.

[0037] 2. Treatment process with water-insoluble magnesium compounds In this step, the liquid containing the impurities is brought into contact with a water-insoluble magnesium compound, and at least a part of the impurities is adsorbed onto the water-insoluble magnesium compound, thereby reducing the amount of the impurities. As a result, when the liquid contains a useful substance, the amount of the impurities is reduced, and the useful substance is roughly purified. Note that "purification" refers to reducing the ratio of impurities to the useful substance in the liquid before contacting with the water-insoluble magnesium compound.

[0038] In the present disclosure, water-insoluble refers to the degree to which a magnesium compound dissolves within 30 minutes when powdered magnesium compound is placed in purified water and vigorously shaken for 30 seconds every 5 minutes at 20±5°C, specifically, the amount of purified water required to dissolve 1 g of the magnesium compound is 100 mL or more, and the amount of purified water is preferably 1000 mL or more.

[0039] The water-insoluble magnesium compound is not particularly limited as long as it is insoluble in water and has no or low adsorption capacity for a target useful substance such as an antibody, while adsorbing impurities such as nucleic acids derived from host cells. For example, basic magnesium carbonate (mMgCO3), which is precipitated by adding an alkali metal carbonate such as sodium carbonate or potassium carbonate to an aqueous solution of magnesium salt, can be used. 3 Mg(OH) 2 nH 2 O (m=3 or more and 5 or less, n=3 or more and 7 or less). As the water-soluble magnesium-containing compound, basic magnesium carbonate and / or magnesium oxide are preferable. On the other hand, according to the experimental findings of the present inventors, hydrotalcite:Mg 6 Al 2 CO 3 (OH) 16 4H 2Water-insoluble magnesium compounds containing metal ions other than magnesium, such as O, exhibit a certain degree of adsorptivity to antibodies, etc., and are therefore preferably not used as the water-insoluble magnesium compound in the present invention.

[0040] The size of the water-insoluble magnesium compound may be adjusted appropriately, and for example, the average particle size may be 0.1 μm or more and 1000 μm or less. If the average particle size is 1000 μm or less, the specific surface area of ​​the water-insoluble magnesium compound is sufficiently large, and impurities can be adsorbed more efficiently, and if the average particle size is 0.1 μm or more, excessive energy is not required for pulverization. In addition, from the viewpoint of handling when filling a column, the average particle size is preferably 1 μm or more, and more preferably 10 μm or more. In this disclosure, the average particle size is measured by a laser diffraction type particle size distribution measuring device, and the standard of the average particle size includes volume standard, weight standard, number standard, etc., and the volume standard is preferred.

[0041] The amount of the water-insoluble magnesium compound used may be adjusted depending on the amount of impurities in the liquid, and may be, for example, 0.01 g or more and 100 g or less of the water-insoluble magnesium compound may be used per 100 mL of the liquid. The ratio is preferably 15 g / 100 mL or less. In addition, the water-insoluble magnesium compound may be used in an amount of 0.01 w / v% or more and 100 w / v% or less of the liquid, and the ratio is preferably 1 w / v% or more and 15 w / v% or less.

[0042] The method of contacting the liquid with the water-insoluble magnesium compound may be appropriately selected. For example, the water-insoluble magnesium compound may be added to the liquid, followed by shaking or stirring. The temperature at this time may be room temperature, specifically, 0°C or higher and 40°C or lower. The temperature is preferably 1°C or higher, more preferably 10°C or higher or 15°C or higher, and is preferably 30°C or lower, and more preferably 25°C or lower. The contact time may be 1 second or longer and 10 hours or shorter.

[0043] 3.Separation process In this step, the water-insoluble magnesium compound to which at least a portion of the nucleic acid contained in the liquid has been adsorbed is separated from the liquid. The separation means is not particularly limited as long as it can separate the water-insoluble magnesium compound from the liquid, and examples of the separation means include centrifugation and filtration.

[0044] After the liquid and the water-insoluble magnesium compound are separated, the useful substance is mainly dispersed in the liquid, and the other impurities are all or partly adsorbed mainly to the water-insoluble magnesium compound. In addition, some of the useful substance may be adsorbed to the water-insoluble magnesium compound and some of the impurities may be dissolved in the liquid, but at least the total amount of the impurities in the liquid can be reduced, and the useful substance in the liquid is concentrated.

[0045] Alternatively, the water-insoluble magnesium compound may be packed in a column, and the liquid may be passed through the column to adsorb the impurities to the water-insoluble magnesium compound. In this case, adsorption of the impurities and separation of the liquid from the water-insoluble magnesium compound can be performed simultaneously. The amount of the water-insoluble magnesium compound packed in the column and the flow rate of the liquid are preferably adjusted within a range in which at least the impurities are sufficiently adsorbed by the water-insoluble magnesium compound.

[0046] In addition, the water-insoluble magnesium compound is also useful as a filter aid for improving filterability. In the present disclosure, filterability includes suppressing filter clogging and the like to perform filtration well. For example, when the water-insoluble magnesium compound is contacted with a liquid that is a suspension, and then the insoluble component containing the water-insoluble magnesium compound is separated by filtration, the clogging of the filter can be suppressed. Therefore, it is preferable that the water-insoluble magnesium compound is separated from the liquid by filtration.

[0047] By the above steps 2 and 3, at least a part of the impurities and relatively large insoluble components such as cells are separated and removed, and if useful substances are contained, it is preferable to further purify the resulting mixture by chromatography, but before that, the amount of the above impurities or other impurities may be further reduced by a general treatment step. The general treatment steps are described below.

[0048] 4. Activated carbon treatment process In this step, the liquid containing impurities is contacted with activated carbon. This step may be carried out before or after the above-mentioned step 2, or may be carried out simultaneously with the water-insoluble magnesium compound and activated carbon. However, the execution of this step is optional.

[0049] Activated carbon is made by burning charcoal or coconut shells to develop pores and make it porous, and has excellent adsorption properties. The typical specific surface area of ​​activated carbon is 800 m 2 / g or more, 2500m 2 / g or less.

[0050] The average pore diameter of the activated carbon is not particularly limited, but is usually 0.1 nm or more and 20 nm or less, preferably 0.5 nm or more and 5.0 nm or less, more preferably 2.0 nm or more and 5.0 nm or less, and even more preferably 3.0 nm or more and 5.0 nm or less. The average pore diameter of the activated carbon can be calculated from the nitrogen adsorption isotherm using the BJH method.

[0051] The purification method using the activated carbon of the present invention is not particularly limited, and examples thereof include a batch method, a membrane treatment method, and a column chromatography method, and an appropriate form of activated carbon is selected according to each method. If necessary, it can be used in the form of particles in which activated carbon is encapsulated in a porous polymer or gel, a membrane in which activated carbon is adsorbed, fixed or formed using a support material or fiber such as polypropylene or cellulose, or a cartridge.

[0052] The amount of activated carbon used may be adjusted depending on the concentration of impurities in the liquid to be treated, and for example, 0.5 g to 5 g of activated carbon may be used per 100 mL of liquid.

[0053] The contact method between the impurity-containing liquid and the activated carbon may be the same as that of the water-insoluble magnesium compound, in which the activated carbon is added to the impurity-containing liquid and then shaken or stirred, or the activated carbon may be packed in a column. When the present step 4 and the above step 2 are carried out simultaneously, the water-insoluble magnesium compound and the activated carbon may be mixed and used. After the impurity-containing liquid is brought into contact with the activated carbon, the liquid and the activated carbon are separated. The above step 3 may be carried out after the present step 4 and the above step 2 are carried out simultaneously.

[0054] 5. Treatment process with flocculant In this step, the liquid containing impurities is treated with a flocculant. This step may be carried out before or after the treatment step 2 with a water-insoluble magnesium compound and / or the treatment step 4 with activated carbon, or may be carried out simultaneously using a water-insoluble magnesium compound and / or activated carbon in combination with a flocculant. However, the implementation of this step is optional.

[0055] Examples of the flocculants include caprylic acid, polyamines, divalent cations, polyetherimines, chitosan, polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, poly(diallyldimethylammonium chloride) (pDADMAC), etc. Examples of the divalent cations include Ca 2+ , Mg 2+ , Cu 2+ , Co 2+ , Mn 2+ , Ni 2+ , B.E. 2+ , Sr 2+ , B.A. 2+ , Ra 2+ , Zn 2+ , Cd 2+ , Ag 2+ , Pd 2+ , Rh 2+These divalent cations can be used in the free state or as the hydrochloride, sulfate, citrate, etc.

[0056] The amount of flocculant used may be adjusted depending on the concentration of impurities contained in the liquid to be treated, but for example, when the flocculant is polyamine or polyetherimine, a flocculant of 0.01 w / v% or more and 10 w / v% or less may be used, more preferably, a flocculant of 0.1 w / v% or more and 1 w / v% or less may be used. When the flocculant is caprylic acid, chitosan, polyethylene glycol, polyvinyl alcohol, or polyvinylpyrrolidone, a flocculant of 0.01 w / v% or more and 10 w / v% or less may be used, more preferably, a flocculant of 1 w / v% or more and 5 w / v% or less may be used. When the flocculant is pDADMAC, a flocculant of 0.01 w / v% or more and 0.1 w / v% or less may be used, more preferably, a flocculant of 0.1 w / v% or more and 0.5 w / v% or less may be used. When the flocculant is a divalent cation, it may be added in an amount such that the divalent cation concentration is 1 mM or more and 100 mM or less, more preferably 2 mM or more and 50 mM or less.

[0057] The method of contacting the impurity-containing liquid with the flocculant may be the same as that of the water-insoluble magnesium compound, in which the flocculant is added to the impurity-containing liquid and then shaken or stirred, or the flocculant may be packed in a column. When this step 5 is performed simultaneously with the treatment step 2 using a water-insoluble magnesium compound and / or the treatment step 4 using activated carbon, the flocculant may be mixed with the water-insoluble magnesium compound and / or the activated carbon and used. After contacting the impurity-containing liquid with the flocculant, the liquid and the flocculant are separated. The above step 3 may be performed after this step 5, the above step 2, and the above step 4 are performed simultaneously.

[0058] 6. Endonuclease treatment step In this step, the liquid containing impurities is treated with an endonuclease. This step may be performed before or after each of the above steps, or may be performed simultaneously with the endonuclease and one or more selected from a water-insoluble magnesium compound, activated carbon, and a flocculant. However, the execution of this step is optional.

[0059] Endonuclease is a type of DNA decomposition enzyme that has the ability to decompose even the center of a base sequence. Commercially available products include Benzonase (manufactured by Millipore) and Kaneka Endonuclease (manufactured by Kaneka).

[0060] The amount of endonuclease used may be adjusted depending on the concentration of DNA contained in the liquid, and is preferably 10 U / mL or more, more preferably 100 U / mL or more, relative to the liquid, while the upper limit is not particularly limited, and is preferably 10,000 U / mL or less.

[0061] 7.Other refining processes The above steps, especially the above treatment step 2, can be suitably used not only before or after general impurity reduction steps such as membrane or column treatment as shown below, but also when the above steps are performed before membrane or column treatment, it is expected to suppress the decrease in the adsorption capacity of the chromatography carrier, the decrease in separation ability, the decrease in processing speed due to back pressure increase, and the decrease in carrier life due to the decrease in cleaning and regeneration efficiency, and also in the membrane filtration process, it is expected to suppress the decrease in processing capacity per unit membrane area, the increase in back pressure, the decrease in processing speed, and the decrease in cleaning and regeneration efficiency. That is, it is also a preferred embodiment of the present invention to further subject the useful substance-containing liquid in which impurities have been reduced by the above steps to column treatment or membrane filtration treatment, in other words, the method of the present invention can be used as a pretreatment for column treatment or membrane filtration treatment.

[0062] The liquid containing the useful substance can be subjected to purification by column treatment such as chromatography. The chromatography used is not particularly limited as long as it is a method capable of recovering and purifying the target useful substance, but examples of the chromatography include anion exchange chromatography, cation exchange chromatography, hydrophobic chromatography, hydroxyapatite chromatography, mixed mode chromatography, and affinity chromatography, and these may be used alone or in combination. The above steps, especially the above treatment step 2, can be suitably used in the previous or subsequent steps of the chromatography step.

[0063] After the useful substance has been purified, the amount of solvent may be reduced to concentrate the useful substance, or the solvent may be exchanged.

[0064] Impurities can be quantified at any stage by absorbance analysis, electrophoresis, HPLC, or a commercially available assay kit. For example, nucleic acids can be quantified by absorbance analysis at the maximum absorption wavelength of nucleic acids, fluorescence analysis using a fluorescent reagent, emission analysis such as high performance liquid chromatography isotope dilution mass spectrometry (LC IDMS), inductively coupled plasma optical emission spectrometry (ICP OES) or inductively coupled plasma mass spectrometry (ICP MS), mass spectrometry, chromatography analysis, or a combination of these, or by gel electrophoresis, q-PCR, next-generation DNA sequencer, or other methods. In addition, the CHO HCP ELISA kit (manufactured by Cygnus) can be used to quantify host-derived proteins derived from CHO cells. If there is no assay kit for the desired impurity protein among the commercially available assay kits, the desired detection system can be created by immunizing animals such as chickens with the impurity protein. If some of the impurities other than the useful substances can be removed before the chromatography, the load on the chromatography can be reduced, and more efficient purification can be achieved.

[0065] The above-mentioned method for reducing nucleic acid can be carried out, for example, by using the adsorption filter according to the present invention. The adsorption filter according to the present invention has a layer containing a water-insoluble magnesium compound. In the present disclosure, the term "adsorption filter" refers to a filter that can adsorb at least a part of impurities such as DNA to reduce the amount thereof, and further refers to a filter that can physically block the movement of substances larger than the pores. Note that, with regard to the water-insoluble magnesium compound contained in the adsorption filter according to the present invention, the description of the water-insoluble magnesium compound in the above description of the method for reducing nucleic acid according to the present invention is to be used.

[0066] The layer containing a water-insoluble magnesium compound constituting the adsorption filter of the present invention (hereinafter referred to as the "water-insoluble magnesium compound-containing layer") is not particularly limited as long as it contains a water-insoluble magnesium compound, has a predetermined thickness, and has voids through which a liquid containing a useful substance such as an antibody can pass, and may or may not contain components other than the water-insoluble magnesium compound. The water-insoluble magnesium compound may be particles such as spherical particles processed to be porous or block-shaped crushed fragments. As described above, the filterability of the adsorption filter of the present invention is improved by including a water-insoluble magnesium compound-containing layer.

[0067] The water-insoluble magnesium compound-containing layer may be, for example, one made of a water-insoluble magnesium compound. A layer made of water-insoluble magnesium compound particles refers to a layer made substantially only of water-insoluble magnesium compound particles, and refers to a layer to which no components other than the water-insoluble magnesium compound particles are intentionally added, although unavoidable impurities and unavoidable mixtures are permitted.

[0068] In addition, as long as the layer contains a water-insoluble magnesium compound as a main component, it may be a layer containing a component other than the water-insoluble magnesium compound. Such other components are not particularly limited as long as they are water-insoluble media that are insoluble in water and have low adsorption capacity for the target useful substance, but for example, water-insoluble media consisting of one or more selected from activated carbon, polysaccharides, synthetic polymers, and inorganic substances are included, and water-insoluble media consisting of one or more selected from polysaccharides, synthetic polymers, and inorganic substances are preferred in terms of particularly low non-specific adsorption. The incorporation of a water-insoluble medium may improve the filtration characteristics, liquid permeability, and / or impurity removal ability of the water-insoluble magnesium compound-containing layer. It is preferable that the water-insoluble magnesium compound and the water-insoluble medium are uniformly dispersed in the water-insoluble magnesium compound-containing layer. The shape of the water-insoluble medium is not particularly limited, but it may be, for example, particles or fibers. The state of the blend of the water-insoluble magnesium compound and the water-insoluble medium is not particularly limited as long as at least a portion of the water-insoluble magnesium compound is exposed on the surface and can exhibit adsorption performance, and may be, for example, in a mixed state, an adhesive state, or a bonded state.

[0069] Examples of polysaccharides include celluloses such as cellulose, cellulose acetate, and nitrocellulose; agarose; and chitosan. Examples of synthetic polymers include polyacrylonitrile, polyester, polyethersulfone, polypropylene, and polytetrafluoroethylene. Examples of inorganic substances include diatomaceous earth, perlite, glass, silica, alumina, zirconia, and barium titanate.

[0070] When the water-insoluble magnesium compound-containing layer contains a water-insoluble medium, the amount of the water-insoluble medium used may be adjusted appropriately depending on the form and content of the insoluble matter contained in the liquid to be treated, the viscosity of the liquid to be treated, etc., but for example, the ratio of the water-insoluble medium to the total of the water-insoluble magnesium compound and the water-insoluble medium can be 1% by mass or more and 99% by mass or less. If the ratio is 1% by mass or more, the effects of improving filtration characteristics and liquid permeability can be more reliably obtained, and if the ratio is 99% by mass or less, the exposure of the water-insoluble magnesium compound to the surface can be ensured, and the effective amount of the water-insoluble magnesium compound, which is the main component, can be more reliably ensured. The above ratio is preferably 2% by mass or more, more preferably 5% by mass or more, and more preferably 50% by mass or less, and more preferably 30% by mass or less.

[0071] Since the layer only needs to pass liquid in one direction, for example, from the top to the bottom, the water-insoluble magnesium-containing compound constituting the layer, or the mixture of the water-insoluble magnesium-containing compound and the water-insoluble medium, does not need to be fixed on the surface of, for example, a support substrate, and may simply be deposited on the support substrate.In addition, a support substrate may also be provided for the water-insoluble magnesium compound-containing layer.In particular, when the water-insoluble magnesium compound is mainly composed of fine water-insoluble magnesium compounds, it is preferable to sandwich the water-insoluble magnesium compound-containing layer from above and below with support substrates such as membranes in order to prevent the leakage of fine particles from the adsorption filter.

[0072] The size and thickness of the water-insoluble magnesium compound-containing layer may be appropriately adjusted depending on the presence or absence of a water-insoluble medium, the amount of the water-insoluble medium, the amount of the liquid to be treated, etc., and are not particularly limited as long as the concentration of the target impurity can be reduced to a predetermined value or less. For example, the amount of the water-insoluble magnesium compound-containing compound constituting the water-insoluble magnesium compound-containing layer, or the total amount of the water-insoluble magnesium compound and the water-insoluble medium, can be 0.0001 times or more by mass and 1 times or less by mass with respect to the amount of the liquid to be treated. If the ratio is 0.0001 times or more by mass, there is less possibility of clogging or saturation of adsorbed impurities, and it can be said that good treatment is more reliably performed, and if the ratio is 1 times or less by mass, the treatment system does not become excessively large compared to the amount of the liquid to be treated. The above ratio is preferably 0.0005 times or more by mass, more preferably 0.001 times or more by mass, and more preferably 0.5 times or less by mass, and more preferably 0.1 times or less by mass. When the water-insoluble magnesium compound-containing layer has two or more layers, the above amount refers to the total amount of the water-insoluble magnesium compound and the like.

[0073] The adsorption filter according to the present invention may have two or more water-insoluble magnesium compound-containing layers. For example, the number of water-insoluble magnesium compound-containing layers may be 1 or more and 3 or less, preferably 2 or less, and may be 1. The thickness of the water-insoluble magnesium compound-containing layer may be, for example, 1 μm or more and 1000 cm or less.

[0074] Also, depending on the liquid-transport speed of the liquid to be treated to the water-insoluble magnesium compound-containing layer, the amount of the water-insoluble magnesium compound constituting the water-insoluble magnesium compound-containing layer, or the total amount of the water-insoluble magnesium compound and the water-insoluble medium, is preferably adjusted so that the contact time between the liquid to be treated and the water-insoluble magnesium compound is 10 seconds or more and 60 minutes or less. If the contact time is within this range, efficient impurity removal is achieved. The time is preferably 20 seconds or more, more preferably 60 seconds or more, and preferably 30 minutes or less, more preferably 20 minutes or less. When the water-insoluble magnesium compound-containing layer has two or more layers, the contact time refers to the total contact time with each layer.

[0075] The adsorption filter according to the present invention may have a layer other than the water-insoluble magnesium compound-containing layer. For example, a support layer having a pore size large enough to hold water-insoluble magnesium compound particles may be provided directly below the water-insoluble magnesium compound-containing layer. In addition, a general support substrate layer may be provided on the water-insoluble magnesium compound-containing layer or below the support layer. Examples of the material of such a support substrate layer include one or more water-insoluble media selected from activated carbon, polysaccharides, synthetic polymers, and inorganic substances, and one or more water-insoluble media selected from polysaccharides, synthetic polymers, and inorganic substances are preferred in terms of particularly low non-specific adsorption. Examples of polysaccharides include celluloses such as cellulose, cellulose acetate, and nitrocellulose; agarose; chitosan, etc. Examples of synthetic polymers include polyacrylonitrile, polyester, polyethersulfone, polypropylene, and polytetrafluoroethylene, etc. Examples of inorganic substances include diatomaceous earth, perlite, glass, silica, alumina, zirconia, and barium titanate, etc.

[0076] The pore size of the support substrate layer may be adjusted as appropriate, and may be, for example, 0.1 μm or more and 100 μm or less so that cell debris and the like are captured inside the support substrate layer and leakage of the water-insoluble magnesium compound is suppressed. Note that, for the pore size of the support substrate layer, if a catalog value for the product is available, the catalog value may be referred to, and if no catalog value is available, the average pore size may be directly determined from an enlarged photograph, or may be determined as an estimated value using a Gurley air permeability tester or the like.

[0077] For example, Fig. 1 shows a schematic diagram of a laminated basic magnesium carbonate-filled device including an adsorption filter according to the present invention. The device according to Fig. 1 is a depth filter device, and in a filter holder, a polytetrafluoroethylene (PTFE) filter 1, a depth filter 3, a water-insoluble magnesium compound-containing layer 2 made of a water-insoluble magnesium compound, and a PTFE filter 1 are arranged in this order from the bottom. Also, in the schematic diagram of a dispersion-type basic magnesium carbonate-filled device including an adsorption filter according to the present invention shown in Fig. 2, in a filter holder, a PTFE filter 1, a water-insoluble magnesium compound-containing layer 4 made of a mixture of a water-insoluble magnesium compound and a water-insoluble medium, and a PTFE filter 1 are arranged in this order from the bottom.

[0078] The adsorption filter of the present invention may be distributed as a finished product in which a water-insoluble magnesium compound-containing layer is formed in a filter holder, or when in use, a water-insoluble magnesium compound may be filled into the filter holder to form a water-insoluble magnesium compound-containing layer.

[0079] The form of the adsorption filter according to the present invention is not particularly limited, and for example, a layer containing a water-insoluble magnesium compound may be laminated on another filter. Alternatively, the water-insoluble magnesium compound may be made into a sheet-like water-insoluble magnesium compound-containing layer, and then molded into a pleated or hollow fiber shape. These water-insoluble magnesium compound-containing layers may be inserted into a package to form a depth filter, a syringe filter, or a liquid treatment cartridge.

[0080] The adsorption filter according to the present invention can adsorb cell-derived water-soluble impurities such as cell-derived DNA and histones, and can be used not only for the purification of target useful substances such as antibodies and antibody-like molecules, but also for physically filtering out cells, cell fragments, cell lysates caused by surfactants, etc. Specifically, by passing a liquid containing a useful substance through the adsorption filter according to the present invention, the above-mentioned impurities can be removed while purifying the useful substance. In addition, the filter can be suitably used not only in the front or back stages of general protein purification processes such as membrane and column treatment, but also in the front stage of membrane or column treatment, the adsorption capacity of the chromatography carrier can be reduced, the separation ability can be reduced, the processing speed can be reduced due to an increase in back pressure, and the carrier life can be reduced due to a decrease in cleaning and regeneration efficiency. In addition, in the membrane filtration process, the processing capacity per unit membrane area can be reduced, the back pressure can be increased, and the carrier life can be reduced due to a decrease in cleaning and regeneration efficiency. That is, it is also a preferred embodiment of the present invention to further subject useful substances such as antibodies or antibody-like molecules purified by the purification method of the present invention to column treatment or membrane filtration treatment, in other words, the adsorption filter of the present invention can be used in pretreatment of column treatment or membrane filtration treatment. In addition, the adsorption filter according to the present invention can also be used for pretreatment of sample liquids in tests and diagnoses in which the same substances as the above-mentioned useful substances are used as indicators for tests and diagnoses.

[0081] The conditions for passing a solution or suspension containing a useful substance through the adsorption filter of the present invention may be adjusted as appropriate, for example, the passing speed of the solution or suspension through the adsorption filter may be adjusted to 1 cm / hr or more and 10 m / hr or less. If the speed is 1 cm / hr or more, it can be said that the impurities are sufficiently adsorbed by the adsorption filter, and if the speed is 10 m / hr or less, the treatment efficiency can be sufficiently ensured. The speed is preferably 5 cm / hr or more, more preferably 10 cm / hr or more, and preferably 5 m / hr or less, more preferably 1 m / hr or less.

[0082] The temperature when the solution or suspension containing the target useful substance is passed through the adsorption filter of the present invention may be room temperature, specifically, it can be from 0° C. to 40° C. The temperature is preferably 1° C. or higher, more preferably 10° C. or higher or 15° C. or higher, and is preferably 30° C. or lower, more preferably 25° C. or lower.

[0083] In addition to removing cells from the liquid after passing through the adsorption filter according to the present invention, the concentration of impurities such as DNA and DNA-binding proteins is reduced. In order to further reduce the concentration of impurities from the liquid, the liquid may be treated with a conventional adsorbent such as activated carbon. In addition, the liquid may be further subjected to affinity chromatography, ion exchange chromatography, gel filtration chromatography, etc. to purify the target useful substance. If a part of the impurities can be removed before the chromatography, the load on the chromatography can be reduced, and more efficient purification is possible. In addition, if it is possible to reduce impurities by inserting a filter process between steps that does not impair the process yield, there is no need to prioritize the reduction of impurities at the expense of the yield of the chromatography step, and the purification yield can be improved throughout the entire manufacturing process. Furthermore, since impurities cause a decrease in the life and performance of purification materials such as column chromatography as residual contaminants, the introduction of a simple impurity removal process is expected to contribute to the reduction of manufacturing material costs through the extension of the life and maintenance of the performance of purification materials.

[0084] This application claims the benefit of priority based on Japanese Patent Application No. 2020-33132 and Japanese Patent Application No. 2020-33216, filed on February 28, 2020. The entire contents of the specifications of Japanese Patent Application No. 2020-33132 and Japanese Patent Application No. 2020-33216, filed on February 28, 2020, are incorporated by reference into this application. EXAMPLES

[0085] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to the following examples, and it is possible to carry out the present invention by making appropriate modifications within the scope of the present invention, and all such modifications are included in the technical scope of the present invention. In addition, the reagents used in the examples were commercially available products unless otherwise specified.

[0086] Example 1: Adsorption and removal of DNA using basic magnesium carbonate Basic magnesium carbonate was added at a ratio of 1 w / v% or 10 w / v% to salmon sperm DNA solution adjusted to 1 g / L, and the solution was stirred for 2 hours in a mix rotor, then centrifuged at 15,000 rpm for 5 minutes to collect the supernatant, obtaining a treated solution. The DNA concentration in the DNA solution before treatment and in the treated solution was determined from the UV260 nm absorbance. For comparison, an experiment was conducted in the same manner except that basic magnesium carbonate was not added. The results are shown in Table 1.

[0087] [Table 1]

[0088] As shown in the results in Table 1, it was demonstrated that basic magnesium carbonate can adsorb and remove DNA.

[0089] Example 2: Adsorption and removal of endotoxin using basic magnesium carbonate Basic magnesium carbonate was added at a ratio of 1 w / v% to the endotoxin solution adjusted to a concentration of 0.4 EU / mL, and the solution was stirred in a mix rotor for 2 hours, then centrifuged at 15,000 rpm for 5 minutes to collect the supernatant, yielding a treated solution. The endotoxin concentrations in the endotoxin solution before and after treatment were measured using a simplified endotoxin test system ("Endosafe (R) Measurements were made using a "Nexgen PTS" (manufactured by Charles River). For comparison, a similar experiment was conducted except that basic magnesium carbonate was not added. The results are shown in Table 2.

[0090] [Table 2]

[0091] As shown in the results in Table 2, it was found that basic magnesium carbonate can also adsorb and remove endotoxin.

[0092] Example 3: Adsorption and removal of DNA, HCP, and histones from culture supernatants derived from animal cells using basic magnesium carbonate 1 w / v% basic magnesium carbonate was added to the culture supernatant of animal cells containing monoclonal antibody (IgG), and the mixture was stirred at room temperature for 18 hours using a mix rotor. The treated solution was then centrifuged at 15,000 rpm for 5 minutes, and then filtered to recover the supernatant, obtaining a treated solution. The concentrations of antibodies, which are useful proteins, and DNA, which is an impurity, in the culture supernatant before and after the treatment were quantified. The concentrations of host cell-derived proteins (HCPs) and histones were also quantified. Specifically, the antibody concentration was measured by Protein A chromatography, the DNA concentration was measured using a host cell-derived DNA detection kit ("Host Cell DNA Kit D555T" manufactured by Cygnus) according to the attached protocol, the HCP concentration was measured using "CHO ​​Host Cell Protein ELISA Kit, 3rd Generation" manufactured by Cygnus according to the attached protocol, and histone H2A was treated with trypsin and then analyzed by LC-TOFMS (a combination of "UFLC Nexera X2" manufactured by Shimadzu Corporation and "Triple TOF6600" manufactured by ABSCIEX), and each was measured using Protein pilot and Markerview software. The results are shown in Table 3.

[0093] [Table 3]

[0094] As shown in the results in Table 3, it was found that basic magnesium carbonate was able to adsorb and remove HCPs and histones in addition to DNA from culture supernatants derived from IgG-containing animal cells while maintaining the IgG concentration.

[0095] Example 4: Adsorption and removal of DNA from animal cell culture suspensions using basic magnesium carbonate Basic magnesium carbonate (1 w / v% or 3 w / v%) was added to an animal cell-derived suspension containing a monoclonal antibody (IgG), and the mixture was stirred at room temperature for 1 hour using a mix rotor. Then, 15 g of the treated solution was filtered through a 0.2 μm membrane filter to obtain a treated solution. The IgG, DNA, and HCP contents in the resulting treated solution were measured in the same manner as in Example 3. For comparison, an experiment was also conducted in the same manner except that basic magnesium carbonate was not used. The results are shown in Table 4.

[0096] [Table 4]

[0097] As shown in the results in Table 4, it was found that basic magnesium carbonate can remove DNA even when added to a cell suspension. In addition, the more basic magnesium carbonate added, the more the filter clogging during filtration can be suppressed, indicating that basic magnesium carbonate functions as a filter aid.

[0098] Example 5: Removal of proteins from culture supernatants derived from Protein G-containing E. coli using basic magnesium carbonate To the protein G-containing E. coli culture supernatant, 1 w / v% or 10 w / v% basic magnesium carbonate and / or 0.67 w / v% activated charcoal were added, and the mixture was stirred for 2 hours in a mix rotor, then centrifuged at 15,000 rpm for 5 minutes to collect the supernatant, yielding a treated solution. The protein G concentration before and after treatment was measured using reversed-phase HPLC, and the total protein concentration was measured using a Pierce HPLC column (Thermo Scientific). TMAnalysis was performed using a 660 nm Protein Assay. The impurity protein concentration was calculated by subtracting the protein G concentration from the total protein concentration. The results are shown in Table 5.

[0099] [Table 5]

[0100] As shown in the results in Table 5, it was found that basic magnesium carbonate can remove impurity proteins even from E. coli culture supernatant containing protein G. It was also found that by combining it with activated carbon, the concentration of protein G was somewhat reduced, but impurity proteins could be further removed.

[0101] Example 6: Protein removal from protein G-containing Escherichia coli culture medium using basic magnesium carbonate Before the bacterial cell separation, 10 w / v% basic magnesium carbonate was added to the protein G-containing E. coli culture supernatant, which was then stirred for 2 hours in a mix rotor, and centrifuged at 15,000 rpm for 5 minutes to collect the supernatant, yielding a treated solution. The protein G concentration of the culture solution before and after the treatment was measured using reversed-phase HPLC, and the total protein concentration was measured using a Pierce HPLC column (Thermo Scientific). TM Analysis was performed using a 660 nm Protein Assay. The impurity protein concentration was calculated by subtracting the protein G concentration from the total protein concentration. The results are shown in Table 6.

[0102] [Table 6]

[0103] As shown in the results in Table 6, it was found that impurity proteins could be removed from the protein G-containing E. coli culture supernatant even if basic magnesium carbonate was added before cell disruption.

[0104] Example 7: Protein removal from culture supernatant of Brevibacillus choshinensis containing protein A by basic magnesium carbonate The pH of the culture supernatant of Brevibacillus choshinensis containing protein A was adjusted to 5, 10 w / v% basic magnesium carbonate and / or 0.67 w / v% activated carbon was added, and the mixture was stirred for 2 hours in a mix rotor, and then centrifuged at 15,000 rpm for 5 minutes to recover the supernatant, obtaining a treated solution. The protein A concentration of the culture solution before and after the treatment was measured by reverse phase HPLC, and the total protein concentration was measured by a Pierce HPLC column (Thermo Scientific). TM Analysis was performed using a 660 nm Protein Assay. The impurity protein concentration was calculated by subtracting the Protein A concentration from the total protein concentration. The results are shown in Table 7.

[0105] [Table 7]

[0106] As the results shown in Table 7 show, basic magnesium carbonate has an effect of removing impurity proteins even from protein A-containing E. coli culture supernatant, and although the concentration of protein A is somewhat reduced by combining it with activated carbon, a high impurity protein removal effect can be obtained.

[0107] Example 8: Removal of impurities from Protein A-containing Blevi-derived culture supernatant after acid dissociation treatment with basic magnesium carbonate The pH of the culture supernatant of Brevibacillus choshinensis containing protein A was adjusted to 5.4 with acetic acid and heat-treated at 60°C for 60 minutes. After each sample was centrifuged at 12,000 rpm for 5 minutes, the supernatant was collected. To the culture supernatant after the above treatment, 10 w / v% basic magnesium carbonate was added, stirred in a mix rotor for 2 hours, and then centrifuged at 15,000 rpm for 5 minutes to collect the supernatant, obtaining a treated solution. The protein A concentration of the culture solution before and after treatment was analyzed by reversed-phase HPLC, and the total protein concentration was analyzed by the Lowry method. The impurity protein concentration was calculated by subtracting the protein A concentration from the total protein concentration. The results are shown in Table 8.

[0108] [Table 8]

[0109] As the results shown in Table 8 show, impurity proteins can be removed even from acid-treated E. coli culture supernatant containing Protein A by adding basic magnesium carbonate, and it was found that a high impurity protein removal effect could be obtained by combining it with activated carbon, although the Protein A concentration was somewhat reduced.

[0110] Example 9: Adsorption and removal of DNA by water-insoluble magnesium compounds The salmon sperm DNA solution was treated and the DNA concentration was measured in the same manner as in Example 1, except that magnesium oxide, magnesium hydroxide, magnesium silicate or magnesium phosphate was used as the water-insoluble magnesium salt. The results are shown in Table 9.

[0111] [Table 9]

[0112] As shown in the results in Table 9, it was found that magnesium oxide, magnesium hydroxide, magnesium silicate and magnesium phosphate, as water-insoluble magnesium salts, also exhibited high DNA removal effects.

[0113] Example 10: Adsorption and removal of DNA from insulin solution by basic magnesium carbonate In the same manner as in Example 1, 1 w / v% basic magnesium carbonate was added to an insulin solution adjusted to 0.1 mg / mL, and the solution was stirred in a mix rotor for 1 hour, then centrifuged at 15,000 rpm for 5 minutes to collect the supernatant, yielding a treated solution. The insulin recovery rate was calculated by dividing the UV280nm absorbance of the solution before treatment by the UV280nm absorbance of the treated solution. As a result, the recovery rate of insulin was 90%, and it was found that insulin can be recovered in high yield even when treated with basic magnesium carbonate. As shown in Example 1, it is known that DNA can be efficiently removed by treating under the same conditions.

[0114] Example 11: Adsorption and removal of DNA from protease solution using basic magnesium carbonate In the same manner as in Example 1, basic magnesium carbonate was added to a 1 mg / mL protease aqueous solution at a concentration of 1 w / v%, and the solution was stirred in a mix rotor for 1 hour, and then centrifuged at 15,000 rpm for 5 minutes to collect the supernatant, thereby obtaining a treated solution. The protease recovery rate was calculated by dividing the UV280nm absorbance of the solution before treatment by the UV280nm absorbance of the treated solution. As a result, the recovery rate of the protease was 94%, indicating that the protease can be recovered in high yield even when treated with basic magnesium carbonate. As shown in Example 1, it has been found that DNA can be removed efficiently when treated under the same conditions.

[0115] Example 12: Adsorption and removal of DNA from epidermal growth factor (EGF) solution by basic magnesium carbonate In the same manner as in Example 1, basic magnesium carbonate was added to a 10 ng / mL aqueous solution of epidermal growth factor at a ratio of 1 w / v%, and the mixture was stirred in a mix rotor for 1 hour, then centrifuged at 15,000 rpm for 5 minutes to collect the supernatant, thereby obtaining a treatment solution. The EGF concentrations of the treatment solution and the solution before treatment were measured using a Human EGF Quantikine ELISA kit manufactured by R&D Systems, and the EGF recovery rate was calculated by dividing the EGF concentration after treatment by the EGF concentration before treatment. As a result, the recovery rate of EGF was 94%, indicating that EGF can be recovered in high yield even when treated with basic magnesium carbonate. As shown in Example 1, it has been found that DNA can be removed efficiently when treated under the same conditions.

[0116] Example 13: Adsorption and removal of DNA from human serum albumin solution by basic magnesium carbonate In the same manner as in Example 1, basic magnesium carbonate was added to a 1 mg / mL human serum albumin aqueous solution at a ratio of 1 w / v%, and the mixture was stirred in a mix rotor for 1 hour, and then centrifuged at 15,000 rpm for 5 minutes to collect the supernatant, thereby obtaining a treated solution. The human serum albumin recovery rate was calculated by dividing the UV280 nm absorbance of the treated solution by the absorbance of the solution before treatment. As a result, the recovery rate of human serum albumin was 90%, indicating that human serum albumin can be recovered in high yield even when treated with basic magnesium carbonate.

[0117] Example 14: Adsorption and removal of nucleic acids using basic magnesium carbonate The removal rates of plasmids and ribosomes were obtained in the same manner as in Example 1, except that a 1 μg / mL aqueous solution of a plasmid (which is a circular DNA), a 1 μg / mL aqueous solution of a DNA fragment, or a 1 μg / mL aqueous solution of a ribosome (which is a complex of nucleic acid and protein) was used. The results are shown in Table 10.

[0118] [Table 10]

[0119] As shown in the results in Table 10, it was found that plasmids, which are circular DNA, DNA fragments, and ribosomes, which are complexes of nucleic acids and proteins, could also be removed at a high rate by basic magnesium carbonate.

[0120] Example 15: Improving filterability of CHO cell suspensions with water-insoluble magnesium compounds Basic magnesium carbonate, magnesium hydroxide, or magnesium oxide was added to 400 μL of CHO cell suspension at a concentration of 10 wt%, stirred, and then centrifuged at 9000 G in a spin column for 1 minute. The filtrate yield was calculated by dividing the amount of cell suspension added by the amount of the filtrate obtained. As a result, insoluble components such as cells and water-insoluble magnesium compounds were filtered out while suppressing clogging. The filtration yield is also shown in Figure 1. As shown in the results in Figure 1, it was found that the addition of a water-insoluble magnesium compound could improve the filtrate yield of the CHO cell suspension by about six times.

[0121] Example 16: Improving filterability of HEK cell suspensions with water-insoluble magnesium compounds 10 wt% basic magnesium carbonate, magnesium hydroxide, or magnesium oxide was added to 400 μL of HEK cell suspension, stirred, and then centrifuged at 9000 G in a spin column for 1 minute. The filtrate yield was calculated by dividing the amount of cell suspension added by the amount of filtrate obtained. As a result, insoluble components such as cells and water-insoluble magnesium compounds were filtered out while suppressing clogging. The filtration yield is shown in Figure 2. As shown in the results in Figure 2, it was found that the addition of a water-insoluble magnesium compound could increase the filtrate yield of the HEK cell suspension by about 14-fold.

[0122] Example 17: Preparation of a layered basic magnesium carbonate loaded device A PTFE filter (Diba) with a thickness of about 1 mm was placed on the bottom of a filter holder with an inner diameter of 15 mm and a bed height of 4.5 mm, and a glass fiber depth filter (Millipore) with a thickness of about 0.5 mm was placed on top of it. 200 mg of basic magnesium carbonate (Kishida) was added in a state of being suspended in ultrapure water, and the ultrapure water was removed from the bottom of the filter holder using a syringe to fill the basic magnesium carbonate. The same PTFE filter (Diba) was placed on the basic magnesium carbonate layer formed to prepare a laminated basic magnesium carbonate-filled device. A schematic diagram of the laminated basic magnesium carbonate-filled device prepared in Example 17 is shown in FIG. 3. The filling volume of basic magnesium carbonate was 0.35 mL.

[0123] Example 18: Preparation of a Dispersed Basic Magnesium Carbonate-Filled Device A PTFE filter (Diba) with a thickness of about 1 mm was placed on the bottom of a filter holder with an inner diameter of 15 mm and a bed height of 4.5 mm. A dispersion liquid in which 20 mg of loosened glass fibers of a glass fiber depth filter (Millipore) with a thickness of about 0.5 mm and 200 mg of basic magnesium carbonate (Kishida) were suspended in ultrapure water was filled on top of the filter holder. After the ultrapure water was removed from the bottom of the filter holder using a syringe, the PTFE filter (Diba) was placed on the top of the formed glass fiber-basic magnesium carbonate mixed layer to prepare a dispersion-type basic magnesium carbonate-filled device. A schematic diagram of the dispersion-type basic magnesium carbonate-filled device prepared in Example 18 is shown in FIG. 4. The filling volume of basic magnesium carbonate in the basic magnesium carbonate-containing layer was 0.35 mL.

[0124] Comparative Example 1: Preparation of a dispersed hydrotalcite-filled device A PTFE filter (Diba) with a thickness of about 1 mm was placed at the bottom of a filter holder with an inner diameter of 15 mm and a bed height of 4.5 mm. A glass fiber depth filter (Millipore) with a thickness of about 0.5 mm was filled with a dispersion of 20 mg of loosened glass fibers and 200 mg of hydrotalcite (Wako) suspended in ultrapure water. After the ultrapure water was removed from the bottom of the filter holder using a syringe, the PTFE filter (Diba) was placed on top of the formed glass fiber-hydrotalcite mixed layer to prepare a dispersed hydrotalcite-filled device. The filling volume of the hydrotalcite in the hydrotalcite-containing layer was 0.35 mL.

[0125] Comparative Example 2: Preparation of a control device without filler A PTFE filter (Diba) with a thickness of about 1 mm was placed on the bottom of a filter holder with an inner diameter of 15 mm and a bed height of 4.5 mm. A glass fiber depth filter (Millipore) with a thickness of about 0.5 mm was placed on top of the PTFE filter (Diba), and another PTFE filter (Diba) was placed on top of the filter to prepare a control device.

[0126] Test example 1: Evaluation of impurity removal ability of each device Each device prepared in Examples 17-18 and Comparative Examples 1-2 was connected to a chromatography system ("AKTA Avant25" manufactured by GE Healthcare) to evaluate the impurity removal ability. Specifically, a CHO culture solution (20 mL) containing a monoclonal antibody (IgG) was fed. The weight of the fed culture solution was approximately 100 times the weight of the basic magnesium carbonate filled in the device of Examples 17-18. The feed rate was set to 0.35 mL / min so that the residence time for the basic magnesium carbonate filled in the device of Examples 17-18 and the hydrotalcite filled in the device of Comparative Example 1 was 1 minute. The culture solution that passed through the device was collected in 1.7 mL portions using the fraction collector of AKTA Avant25. The amount of IgG and impurities in the final fraction was measured by the method shown below. The IgG concentration was evaluated by a general chromatography system using a Protein A affinity column (TSKgel Protein A-5PW, manufactured by Tosoh Corporation). The content of host cell-derived protein (HCP) was measured using a host cell-derived protein detection kit ("CHO Host Cell Protein ELISA Kit, 3rd Generation" manufactured by Cygnus) according to the attached protocol. The DNA content was measured using a host cell-derived DNA detection kit ("CHO DNA Amplification Kit in Tubes" manufactured by Cygnus) according to the attached protocol. The results are shown in Table 11.

[0127] [Table 11]

[0128] As shown in the results in Table 11, it was demonstrated that it was difficult to remove impurity proteins and DNA derived from host cells using only a general depth filter (Comparative Example 2). When hydrotalcite, which is also used as an adsorbent, was used in addition to the depth filter (Comparative Example 1), impurity proteins and DNA derived from the host cells were relatively well removed, but the recovery rate of IgG, the target substance, was reduced, probably because it was also adsorbed. In contrast, when basic magnesium carbonate was used in addition to a depth filter, whether a layer made of basic magnesium carbonate was used (Example 17) or basic magnesium carbonate was dispersed in glass fibers derived from a depth filter (Example 18), it was shown that while IgG was adsorbed to a low extent, impurity proteins and DNA derived from host cells could be adsorbed and removed more efficiently than with hydrotalcite.

[0129] Test Example 2: Comparison with commercially available depth filters The filter device prepared in Example 17, Example 18, or Comparative Example 2, or a commercially available depth filter ("Millistack A1HC" manufactured by Merck Millipore) (Comparative Example 3) was connected to a rotary pump, and a 100 mg / L salmon sperm-derived DNA solution was passed through the device at a flow rate of 1 mL / min, and the fraction that passed through the device was collected. The DNA removal rate was calculated by measuring the UV260 nm absorbance before and after the flow. The results are shown in Table 12.

[0130] [Table 12]

[0131] As shown in the results in Table 12, it was demonstrated that it was difficult to remove DNA using a general depth filter that contained only a water-insoluble medium and no filler (Comparative Example 2) or a commercially available depth filter alone (Comparative Example 3). In contrast, it was shown that the filter device of the present invention having a layer containing basic magnesium carbonate can efficiently adsorb and remove DNA, which is an impurity.

[0132] Test Example 3: Evaluation of antibody recovery rate of filter devices The fraction that passed through the device was collected in the same manner as in Test Example 1, except that 1 g / L of human blood-derived polyclonal antibody (IgG) and 25 mM Tris-HCl buffer (pH 7.5) containing 500 mM sodium chloride were used instead of the culture supernatant. The IgG recovery rate was calculated by measuring the UV280 nm absorbance before and after passing the liquid through the device. The results are shown in Table 13.

[0133] [Table 13]

[0134] As shown in the results in Table 13, when hydrotalcite, which is also used as an adsorbent, was used (Comparative Example 1), IgG was probably adsorbed, and the IgG recovery rate was significantly impaired. In contrast, when the filter device of the present invention having a layer containing basic magnesium carbonate was used, IgG could be recovered satisfactorily.

[0135] Test Example 4: Evaluation of antibody recovery rate of filter devices Polyclonal antibody (IgG) derived from human blood was added to 25 mM Tris-HCl buffer (pH 7.5) to give a concentration of 1 g / L, and the solution was thoroughly stirred after adding sodium chloride to give a salt concentration of 0 mM, 100 mM, 500 mM, or 1000 mM. Basic magnesium carbonate or hydrotalcite was added to the resulting solution at a concentration of 1 wt%, and the solution was stirred for 1 hour and then centrifuged. The IgG recovery rate was calculated from the UV280 nm absorbance of the resulting solution. The results are shown in Table 14.

[0136] [Table 14]

[0137] As shown in the results in Table 14, the antibody recovery rate decreased as the salt concentration decreased, but the antibody recovery rate of basic magnesium carbonate was shown to be significantly higher than that of hydrotalcite.

[0138] Example 19: Liquid passing rate through a layered basic magnesium carbonate-packed device A PTFE filter (Diba) with a thickness of about 1 mm was placed at the bottom of a column with an inner diameter of 7 mm and a bed height of 25 mm. A glass fiber depth filter (Millipore) with a thickness of about 0.5 mm was filled with a dispersion of 20 mg of loosened glass fibers and 1000 mg of basic magnesium carbonate (Kishida) suspended in ultrapure water. After the ultrapure water was removed from the bottom of the filter holder using a syringe, the PTFE filter (Diba) was placed on top of the formed glass fiber-basic magnesium carbonate mixed layer to prepare a dispersion-type basic magnesium carbonate-filled device. The filling volume of basic magnesium carbonate in the basic magnesium carbonate-containing layer was 1 mL. As Comparative Example 4, a hydrotalcite-packed column was prepared in the same manner except that hydrotalcite was used instead of basic magnesium carbonate. The column prepared above was connected to a chromatography system ("AKTA Avant25" manufactured by GE Healthcare), and a 100 mg / L salmon sperm DNA solution was passed through the device at a flow rate of 0.1 mL / min (15.6 cm / hr), 1 mL / min (156 cm / hr), or 10 mL / min (1560 cm / hr), and the fraction that passed through the device was collected. The DNA removal rate was calculated by measuring the UV260 nm absorbance before and after the flow. The results are shown in Table 15.

[0139] [Table 15]

[0140] As the results shown in Table 15 show, even if the flow rate was changed, it was demonstrated that the filter device of the present invention having a layer containing basic magnesium carbonate could effectively remove DNA as an impurity.

[0141] Example 20: Layered magnesium hydroxide loaded device The DNA removal rate for each device was calculated in the same manner as in Example 19, except that magnesium phosphate (1000 mg) or magnesium hydroxide (1000 mg) was used instead of basic magnesium carbonate (1000 mg) and the flow rate was 1 mL / min (156 cm / hr). The results are shown in Table 16.

[0142] [Table 16]

[0143] As shown in the results in Table 16, it was demonstrated that the use of magnesium phosphate and magnesium hydroxide also effectively reduced the amount of DNA impurities.

[0144] Example 21: Water-insoluble media for use in devices The DNA removal rate of each device was calculated in the same manner as in Example 19, except that agarose, cellulose, cellulose acetate, activated carbon, diatomaceous earth, perlite, hydrotalcite, milled fiber, glass fiber, alumina, silica gel, zirconia, barium titanate, polyacrylonitrile, polyester, polyethersulfone, polypropylene, or PTFE was used as the water-insoluble medium. The results are shown in Table 17.

[0145] [Table 17]

[0146] As the results shown in Table 17 show, even when agarose, cellulose, cellulose acetate, activated carbon, diatomaceous earth, perlite, hydrotalcite, milled fiber, glass fiber, alumina, silica gel, zirconia, barium titanate, polyacrylonitrile, polyester, polyethersulfone, polypropylene, and PTFE are used as water-insoluble media, it was shown that the impurity DNA can be effectively removed by using the filter device of the present invention having a layer containing basic magnesium carbonate. [Explanation of symbols]

[0147] 1: PTFE filter 2: Water-insoluble magnesium compound-containing layer made of a water-insoluble magnesium compound 3: Depth filter 4: Water-insoluble magnesium compound-containing layer containing water-insoluble magnesium compound particles and a water-insoluble medium

Claims

1. 1. A method for reducing the amount of nucleic acid in a liquid, comprising: a step of adding a water-insoluble magnesium compound to the liquid, or filling a column with the water-insoluble magnesium compound and passing the liquid through the column, thereby adsorbing at least a portion of the nucleic acid to the water-insoluble magnesium compound; and then separating said liquid from said water-insoluble magnesium compound.

2. 2. The method of claim 1, wherein the water-insoluble magnesium compound is one or more selected from magnesium carbonate, magnesium hydroxide, magnesium oxide, magnesium silicate, and magnesium phosphate.

3. The method of claim 1 or 2, wherein the liquid further comprises a useful substance.

4. The method according to any one of claims 1 to 3, wherein the liquid and the water-insoluble magnesium compound are separated by filtration.

5. The method according to claim 3 , wherein the useful substance is one or more useful substances selected from useful proteins, viruses, and virus-like particles.

6. The method according to claim 5, wherein the useful protein is one or more useful proteins selected from the group consisting of antibodies, antibody-like molecules, hormones, enzymes, growth factors, blood proteins, and antibody-binding proteins.

7. The method according to any one of claims 1 to 6, wherein the liquid is a cell culture medium or a body fluid.

8. A nucleic acid adsorption filter comprising a layer containing a water-insoluble magnesium compound.

9. 9. The nucleic acid adsorption filter according to claim 8, which has a layer made of water-insoluble magnesium compound particles.

10. 9. The nucleic acid adsorption filter according to claim 8, which has a layer containing a water-insoluble magnesium compound and a water-insoluble medium.

11. 11. The nucleic acid adsorption filter according to claim 10, wherein the material of the water-insoluble medium is one or more selected from the group consisting of polysaccharides, synthetic polymers, and inorganic substances.

12. 12. The nucleic acid adsorption filter according to claim 11, wherein the polysaccharide is one or more selected from the group consisting of cellulose, cellulose acetate, nitrocellulose, agarose, and chitosan.

13. 12. The nucleic acid adsorption filter according to claim 11, wherein the synthetic polymer is one or more selected from the group consisting of polyacrylonitrile, polyester, polyethersulfone, polypropylene, and polytetrafluoroethylene.

14. 12. The nucleic acid adsorption filter according to claim 11, wherein the inorganic material is one or more selected from the group consisting of glass, silica, alumina, zirconia, and barium titanate.

15. 15. The nucleic acid adsorption filter according to any one of claims 8 to 14, wherein the water-insoluble magnesium compound is one or more selected from magnesium carbonate, magnesium hydroxide, magnesium oxide, and magnesium phosphate.

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