Endotoxin adsorbent

Abstract

This adsorbent for endotoxin in a culture medium contains at least one selected from the group consisting of anionic clay, a solid solution of anionic clay, and magnesium oxide, and can effectively adsorb ET in the culture medium. Representative examples of the anionic clay include hydrotalcite. The endotoxin adsorbent may further contain an adsorbent comprising a base material and a cationic group including a nitrogen atom. The adsorbent comprising a base material and a cationic group including a nitrogen atom is preferably a cellulose having an amino group.

Description

Endotoxin adsorbent

[0001] The present invention relates to an adsorbent for endotoxins in culture media, and to a method for producing culture media from which endotoxins have been removed using the same.

[0002] Endotoxin (ET) (hereinafter sometimes referred to as "ET") is a toxic substance, specifically lipopolysaccharide (LPS), a component of the outer membrane of Gram-negative bacteria. ET is composed of polysaccharide and lipid A, with lipid A being the primary contributor to its toxicity. When ET is taken into the body through contamination of injectable solutions, it can cause fever and shock reactions. Therefore, the Japanese Pharmacopoeia stipulates that the ET concentration in injectable solutions should be 10-100 pg / ml (0.1-1.0 endotoxin unit (EU) / ml) or less. In recent years, proteins and nucleic acids produced by culturing cells or microorganisms are often used as pharmaceuticals, but if ET is present in the culture medium, it is easy for ET to contaminate proteins and nucleic acids separated and purified from the culture. Therefore, it is desirable to remove ET from the culture medium used for cultivation, as well as from the culture medium during and after cultivation. However, removing ET from culture medium is extremely difficult. This is because culture media contain a high concentration of various substances such as proteins, amino acids, nucleic acids, sugars, inorganic salts, and vitamins. After culturing, the culture media also contains the target protein and nucleic acids. However, ET, a lipopolysaccharide, contains anionic heterosaccharides such as N-acetylgalactosamine and N-acetylglucosamine, as well as anionic hydrogen phosphate ions. Therefore, when attempting to adsorb ET, acidic proteins, anionic nucleic acids with phosphate groups, and anions derived from inorganic salts are also adsorbed, reducing the efficiency of ET adsorption. Consequently, ET adsorption from culture media is very inefficient.

[0003] As one of the methods for removing ET, a method using various ET adsorbents is known. For example, Patent Document 1 discloses an ET adsorbent comprising cellulose nanofibers having a cationic group having a nitrogen atom. As described above, since ET, which is a lipopolysaccharide, has anionic hetero sugars and hydrogen phosphate anions, generally, when an ET adsorbent having a cationic group is used for removing ET from a material containing a substance having a cationic group, competition for ET adsorption occurs between the ET adsorbent and the substance having a cationic group, and ET cannot be sufficiently removed. Nevertheless, the ET adsorbent of Patent Document 1 can sufficiently remove ET from a material containing a substance having a cationic group.

[0004] In addition, an adsorbent used for removing ET from medical materials is preferably made of an inorganic material that is difficult to denature even by high-temperature sterilization or radiation sterilization. As a document that discloses an ET adsorbent made of an inorganic material, for example, there is Patent Document 2. Patent Document 2 discloses that by bringing a liquid containing ET into contact with basic magnesium carbonate, ET can be effectively adsorbed and removed while suppressing the adsorption of useful substances such as proteins.

[0005] However, the ET adsorbents of Patent Documents 1 and 2 cannot adsorb ET from the medium with a practically sufficient efficiency.

[0006] International Publication No. 2017 / 018524 International Publication No. 2023 / 027044

[0007] An object of the present invention is to provide an ET adsorbent capable of effectively adsorbing ET in a medium.

[0008] To solve the above problems, the present inventors conducted extensive research and obtained the following findings. (1) Anionic clay, a solid solution of anionic clay, and magnesium oxide can efficiently adsorb ET from a medium for culturing cells or microorganisms and a medium after culturing cells or microorganisms. (2) Anionic clay, a solid solution of anionic clay, and magnesium oxide adsorb very little protein in the medium. (3) By combining anionic clay, a solid solution of anionic clay, and / or magnesium oxide with an ET adsorbent having a cationic group containing a nitrogen atom, the adsorption of protein in the medium is further reduced.

[0009] The present invention has been completed based on the above findings and provides the following [1] to [7]. [1] An adsorbent for endotoxin in a medium, comprising at least one selected from the group consisting of anionic clay, a solid solution of anionic clay, and magnesium oxide. [2] The anionic clay is represented by the following formula 1 [M ２＋ M ３＋ (OH) ２ｍ＋２ｎ (X Ｚ－ ) ｎ／ｚ ·bH ２ O Formula 1 (In Formula 1, m and n are independently of each other, m / n is an integer of 1 to 10, b is an integer of 0 to 10, and (X Ｚ－ ) ｎ／ｚ is an anion in which z is an integer of 1 to 10, M ２＋ is a divalent metal atom, M ３＋[1] The endotoxin adsorbent described in [1] is a substance represented by ) where is a trivalent metal atom. [3] The endotoxin adsorbent described in [1] or [2] further comprises an adsorbent comprising a cationic group containing a nitrogen atom and a substrate. [4] The endotoxin adsorbent described in [3] wherein the adsorbent comprising a cationic group containing a nitrogen atom and a substrate is cellulose having an amino group. [5] The endotoxin adsorbent described in any of [1] to [4] for adsorbing endotoxins while suppressing the adsorption of proteins in the culture medium. [6] A column for removing endotoxins from a culture medium, comprising a column packed with the endotoxin adsorbent described in any of [1] to [4]. [7] A method for producing a culture medium from which endotoxins have been removed, comprising the step of bringing the endotoxin adsorbent described in any of [1] to [4] into contact with the culture medium. [8] A method for producing a culture medium in which proteins remain and endotoxins are removed, comprising the step of contacting the culture medium with an endotoxin adsorbent described in any of [1] to [4]. [9] A method for removing endotoxins from a culture medium, comprising the step of contacting the culture medium with an endotoxin adsorbent described in any of [1] to [4].

[10] A method for removing endotoxins from a culture medium while suppressing the adsorption of proteins in the culture medium to the endotoxin adsorbent, comprising the step of contacting the culture medium with an endotoxin adsorbent described in any of [1] to [4].

[11] Use of at least one selected from the group consisting of anionic clay, a solid solution of anionic clay, and magnesium oxide as an endotoxin adsorbent in a culture medium.

[12] The use according to

[11] , wherein the endotoxin adsorbent in a culture medium is for adsorbing endotoxins in the culture medium while suppressing the adsorption of proteins in the culture medium.

[0010] Generally, it is difficult to efficiently adsorb and remove ET from culture media. As shown in the Examples section, for example, magnesium carbonate, an ET adsorbent described in Patent Document 2, efficiently adsorbs ET in water, but its adsorption rate for ET in culture media is extremely low, approximately 1.5% of the adsorption rate in water. The ET adsorbent of the present invention can effectively adsorb ET from culture media containing high concentrations of various substances such as proteins, amino acids, nucleic acids, sugars, inorganic salts, and / or vitamins. Therefore, it can be suitably used for adsorption and removal of ET from culture media. Furthermore, since the ET adsorbent of the present invention adsorbs very little protein, the adsorption and removal of proteins, which are culture media components necessary for the growth of cells and microorganisms, is suppressed. In this respect as well, it is suitable for adsorption and removal of ET from culture media. Moreover, when the culture supernatant, which is the culture medium during or after culture, is used as the treatment solution, ET can be efficiently adsorbed while suppressing the adsorption of the generated target protein. Therefore, it can be used to adsorb and remove ET from the culture medium before separating the target protein after culturing, or to continue culturing while adsorbing and removing ET from the culture supernatant during culturing.

[0011] Furthermore, when using ET adsorbents in culture media for the manufacture of medical materials, the ET adsorbents must be sterilized at high temperatures, high temperatures and high pressure, or by radiation. In this regard, the ET adsorbent of the present invention is an inorganic material, and therefore is not easily degraded by high-temperature sterilization, high-temperature and high-pressure sterilization, or radiation sterilization, making it suitable for ET adsorption in culture media used in the manufacture of medical materials. In addition, since inorganic materials are generally highly hydrophilic, the ET adsorbent of the present invention can efficiently remove ET from aqueous culture media.

[0012] Furthermore, when anionic clay, a solid solution of anionic clay, and / or magnesium oxide are used in combination with an ET adsorbent having a cationic group containing a nitrogen atom, the adsorption of proteins in the culture medium can be suppressed even more effectively.

[0013] The present invention will be described in detail below. (1) Endotoxin Adsorbent The ET adsorbent of the present invention is an ET adsorbent in a culture medium, that is, an ET adsorbent for adsorbing ET in a culture medium, comprising at least one selected from the group consisting of anionic clay, a solid solution of anionic clay, and magnesium oxide. The anionic clay, a solid solution of anionic clay, and magnesium oxide may be used in granular form (including powder form). Alternatively, they may be used in the form of a film, a lump (for example, columnar), etc.

[0014] In the present invention, the anionic clay is a layered double hydroxide having an anion exchange function, in which anions and water exist between layers made of positively charged metal hydroxides. The metal hydroxide may be one or two or more types, preferably two types. The anionic clay may be a natural product, a modified natural product, or a synthetic product. The anionic clay may have a crystalline structure or be amorphous. Natural anionic clays often have a crystalline structure. Layered double hydroxides, also known as anionic clays, include menesite, pyrouroite, schogrenite, stichite, barbertonite, tachovite, reevesite, desorterusite, motokoliite, weemlandite, meixnerite, kallingite, chloromagalumite, carrboydite, honesite, woodwardite, iowaite, hydroonesite, and mount caysite.

[0015] In the present invention, a preferred form of anionic clay is represented by the following formula 1: [Mm ２＋ Mn ３＋ (OH) ２ｍ＋２ｎ ] (X Ｚ－ ) ｎ／ｚ ・bH ２ O Equation 1 In Equation 1, m and n are independent of each other, and m / n is an integer from 1 to 10, preferably an integer from 1 to 6, more preferably an integer from 2 to 4, and most preferably 3. b is an integer from 0 to 10, preferably an integer from 2 to 6, for example 4. (X Ｚ－ ) ｎ／ｚThis is an anion in which z is an integer from 1 to 10, preferably an integer from 1 to 6.

[0016] (X Ｚ－ ) ｎ／ｚ As for, Cl － , Br － , I － In addition to halide ions such as CO ３ ２－ NO ３－ , OH － SO ４２－ SiO ３２－ ,CrO ４２－ , BO ３２－ MnO ４－ , HGaO ３２－ HVO ４２－ , ClO ４－ , BO ３２－ , pillaring anion (V １０ O ２８６－ Mo ７ O ２４６－ Examples include monocarboxylates (such as acetate), dicarboxylates (such as oxalate), alkyl sulfonates (such as lauryl sulfonate), and halide ions, as well as CO2. ３ ２－ M is preferable. ２＋ It is a divalent metal atom, Be ２＋ Mg ２＋ ,Cd ２＋ Ni ２＋ Co ２＋ , Zn ２＋ Fe ２＋ Mn ２＋ , Cd ２＋ Ca ２＋ Among them, Mg ２＋ M is preferable. ３＋ Al is a trivalent metal atom. ３＋ Ga ３＋ Ni ３＋ Co ３＋ Fe ３＋ Mn ３＋ , Cr ３＋ , V ３＋ Ti ３＋ In ３＋ Among them, Al３＋ This is preferable. The anionic clay is preferably one in which anions and water are present between layers made of positively charged Mg and Al hydroxides.

[0017] Among them, the most preferred layered double hydroxide is Mg(OH) ２ Mg ２＋ A part of Al ３＋ The present invention has a layered structure in which layers of anions and water exist between layers of positively charged octahedron layers formed by substitution. ２ Mg ２＋ A part of Al ３＋ A layered double hydroxide having a layered structure in which layers of anions and water exist between layers of positively charged octahedrons formed by substitution is represented by the following formula 2. [Mg ｍ ２＋ Al ｎ ３＋ (OH) ２ｍ＋２ｎ ] (X Ｚ－ ) ｎ／ｚ ・bH ２ O Equation 2 In Equation 2, m, n, b, and (X Ｚ－ ) ｎ／ｚ This is the same as equation 1.

[0018] Solid solutions of anionic clays are obtained by heat-treating anionic clays at 150 to 700°C, resulting in a disordered anionic clay structure.

[0019] The ET adsorbent of the present invention may include an adsorbent comprising a cationic group containing a nitrogen atom and a substrate, in addition to anionic clay, a solid solution of anionic clay, and / or magnesium oxide (this adsorbent can adsorb ET). This makes it possible to suppress protein adsorption more effectively. The anionic clay, a solid solution of anionic clay, and / or magnesium oxide and the adsorbent comprising a cationic group containing a nitrogen atom and a substrate may be composite, mixed (i.e., uniformly contained without composite), or contained as separate parts (i.e., heterogeneously). A composite is formed by the anionic clay, a solid solution of anionic clay, and / or magnesium oxide and the adsorbent comprising a cationic group containing a nitrogen atom and a substrate being composited by some bonding or adsorption such as electrostatic adsorption. Embodiments in which the two exist as separate parts include stacked embodiments and embodiments in which they are used in combination, such as packing both into separate columns and sequentially passing the culture medium through both columns.

[0020] Examples of cationic groups containing a nitrogen atom include amino groups, quaternary ammonium groups, imino groups, amidine groups, guanidino groups, imidazole groups, quaternary imidazolium groups, pyridyl groups, and quaternary pyridinium groups. The amino group is a functional group (primary amino group) obtained by removing one hydrogen atom from ammonia (-NH₄). ２ The functional group may be a primary amine with one hydrogen atom removed (secondary amino group) (-NHR), or a secondary amine with one hydrogen atom removed (tertiary amino group) (-NRR'). Cationic groups containing a nitrogen atom include aliphatic groups, alicyclic groups, aromatic groups, and heterocyclic groups. Among these, aliphatic groups are preferred.

[0021] Examples of cationic compounds containing nitrogen atoms that serve as donors of cationic groups containing nitrogen atoms include ammonia, amidine, amines (primary, secondary, and tertiary amines), quaternary ammonium salts, quaternary imidazolium salts, and quaternary pyridinium salts.

[0022] A cationic group containing a nitrogen atom is a cationic group derived from a cationic group donor containing a nitrogen atom, i.e., a cationic group derived from a cationic compound containing a nitrogen atom. The donor of a cationic group containing a nitrogen atom may contain one cationic group containing a nitrogen atom, or two or more. For example, if the cationic compound containing a nitrogen atom is an amine, it may be a monovalent amine, a divalent amine, or a polyvalent amine with more than one nitrogen atom. The cationic compound containing a nitrogen atom may be a polymer or a nonpolymer.

[0023] Monovalent amines include aliphatic amines, particularly alkylamines (primary amines such as methylamine, ethylamine, propylamine, butylamine, pentylamine, hexylamine, heptylamine, coconutamine, octylamine, nonylamine, decylamine, laurylamine, stearylamine, and oleylamine; secondary amines such as dimethylamine, diethylamine, dipropylamine, dibutylamine, dipentylamine, dihexylamine, diheptylamine, dioctylamine, dinonylamine, and didecylamine) N,N-dimethylethylamine, tripropylamine, tributylamine, diisopropylethylamine, N,N-dimethylethylamine, N,N-dimethylpropylamine, N,N-dimethylbutylamine, N,N-dimethylpentylamine, N,N-dimethylhexylamine, N,N-dimethylheptylamine, N,N-dimethyloctylamine, N,N-dimethylnonylamine, N,N-dimethylcoconutamine, N,N-dimethyldecylamine, N,N-dimethyllaurylamine, N,N-dimethyl Tertiary amines such as risthylamine, N,N-dimethylpalmitylamine, N,N-dimethylstearylamine, dimethylbehenylamine, didecylmonomethylamine, and trioctylamine; aromatic amines (primary amines such as aniline and toluidine); heterocyclic amines (secondary amines such as pyrrolidine, piperidine, morpholine, and imidazole; tertiary amines such as pyridine, 2,4,6-trimethylpyridine (collidine), 2,6-lutidine, quinoline, N-methylmorpholine, and N-ethylmorpholine) (d); alkanolamines or amino alcohols (primary amines such as monomethanolamine, monoethanolamine, monoisopropanolamine, 2-amino-2-methyl-1-propanol, 2-amino-2-methyl-1,3-propanediol, 3-amino-1,2-propanediol, 3-dimethylamino-1,2-propanediol, tris(hydroxymethylamino)methane; secondary amines such as diethanolamine, diisopropanolamine, N-methylethanolamine, N-ethylethanolamine;Examples include tertiary amines such as triethanolamine, triisopropanolamine, N-dimethylaminoethanol, and N-diethylaminoethanol.

[0024] Examples of polyhydric amines include aliphatic diamines such as ethylenediamine, tetramethylethylenediamine, tetramethylenediamine, and hexamethylenediamine; 4,4'-diamino-3,3'dimethyldicyclohexylmethane, 3-(diethylamino)propylamine, and 3-(dimethylamino) Examples include alicyclic diamines such as -1-propylamine, N,N,N',N'-tetramethyl-1,6-diaminohexane, diaminecyclohexane, and isophoronediamine; aromatic diamines such as phenylenediamine, diaminonaphthalene, and xylylenediamine; heterocyclic diamines such as piperazine and 1,4-bis(3-aminopropyl)piperazine; trivalent or higher aliphatic amines such as diethylenetriamine, triethylenetetramine, tetraethylenepentamine, pentaethylenehexamine, hexaethylenepentamine, N,N'-dimethyldipropylenetriamine, tris(2-aminoethyl)amine, tris(3-aminopropyl)amine, and guanidine; and trivalent or higher aromatic amines such as melamine. Furthermore, examples of polyhydric amines include polyethyleneimine, polyvinylamine, polyallylamine, methoxypoly(oxyethylene / oxypropylene)-2-propylamine, polyoxypropylenediamine, polyetheramine, triethylene glycoldiamine, trimethylolpropanepoly(oxypropylene)triamine, glycerylpoly(oxypropylene)triamine, amino acids (especially basic amino acids such as lysine, arginine, histidine, ornithine, and tryptophan), polymers of amino acids (especially polymers of basic amino acids such as polylysine, polyarginine, polyhistidine, polyornithine, and polytryptophan), and polymers having amino groups such as polycreatinine. The polymers may be linear or branched.

[0025] Examples of quaternary ammonium salts include glycidyltrimethylammonium salt (hydrochloride, hydrobromide, etc.). Additionally, quaternary amines obtained by alkylating the tertiary amines exemplified above can also be used. Examples of quaternary imidazolium salts include 1-decyl-3-methylimidazolium salt, 1-methyl-3-octylimidazolium salt, and 1-methyl-benzoimidazolium salt (hydrochloride, hydrobromide, etc.). Examples of quaternary pyridinium salts include butylpyridinium salt and dodecylpyridinium salt (hydrochloride, hydrobromide, etc.).

[0026] Aliphatic amines are preferred as cationic compounds containing nitrogen atoms. One or more cationic compounds containing nitrogen atoms can be used. When using two or more cationic compounds, the substrates into which each cationic group is introduced may be mixed and used, or substrates into which two or more cationic groups are introduced may be used.

[0027] Furthermore, the cationic properties can be enhanced by further modification after introducing a cationic group containing a nitrogen atom into the substrate. For example, cationic properties can be enhanced by quaternizing an amino group. Examples of compounds used to quaternize an amino group include chloromethyloxirane (epichlorohydrin), glycidyl methacrylate, glycidyl acrylate, diglycidyl ether, epibromohydrin, epoxy group donors such as ethylene glycol diglycidyl ether, p-toluenesulfonic acid chloride, 2-fluoro-1-methylpyridinium, chloroacetyl chloride, hexamethylene diisocyanate, m-xylene diisocyanate, toluene-2,4-diisocyanate, iodomethane, and iodoethane. Cationic properties can also be enhanced by adding and introducing a cationic group containing a nitrogen atom. One method for introducing a cationic group containing a nitrogen atom involves activating the introduced cationic group with an activator (for example, a compound that quaternizes an amino group), and then reacting it with a cationic compound containing a nitrogen atom, which is the same as or different from the already introduced cationic group. The cationic compounds containing a nitrogen atom that can be used are those exemplified above. One or more compounds can be used to react in order to enhance the cationicity.

[0028] Examples of base materials include acrylate polymers (such as polymethyl methacrylate, poly(2-methoxyethyl acrylate), and polytetrahydrofurfuryl acrylate), vinyl polymers such as polymethyl vinyl ether, ethylene-vinyl alcohol copolymer, polyacrylonitrile, polyvinyl alcohol, ethylene-vinyl acetate copolymer, polystyrene, polyethylene, polypropylene, and polyvinylidene fluoride; sulfonyl group-containing polymers such as polysulfone, polyethersulfone, and polyarylethersulfone; polycarbonate; cellulose derivatives such as cellulose acetate; polylactide; polyglycolide; polycaprolactone; polyethylene oxide-poly(terephthalate butylene) copolymer; polyethylene glycol; and organic synthetic polymers such as epoxy resins. Examples of organic natural polymers include glucans (starch, cellulose (crystalline cellulose, fibrous cellulose, etc.), curdlan, pullulan, glycogen, etc.), chitosan, chitin, agarose or agar, gelatin, collagen, and lignin. Inorganic materials such as silica (silica gel, silica monolith, etc.), diatomaceous earth, perlite, zeolite, vermiculite, kaolinite, sepiolite, mineral shirasu, and shirasu porous glass can also be used. Among these, cellulose is preferred due to its good processability and handling properties, and fibrous cellulose and crystalline cellulose are more preferred. Examples of fibrous cellulose include cellulose nanofibers, cellulose microfibers, and cellulose fibers. Crystalline cellulose is obtained by extracting and purifying the crystalline region of a cellulosic substance, and commercially available products include Comprecel® from Mingtai Chemical Co., Ltd. and CEOLUS® from Asahi Kasei Corporation. One or more types of materials can be used for the base material. When using two or more types, they may be mixed to form the base material, or different materials may be used for each part of the base material, such as by laminating different materials.

[0029] Cationic groups containing nitrogen atoms can be introduced by known methods, for example, into the hydroxyl groups of a substrate. In this case, the hydroxyl groups of the substrate are activated with an activator and then reacted with a cationic compound containing nitrogen atoms. If the cationic compound containing nitrogen atoms itself has a reactive group, pretreatment with an activator is not necessarily required.

[0030] Examples of activators include chloromethyloxirane (epichlorohydrin), glycidyl methacrylate, glycidyl acrylate, diglycidyl ether, epibromohydrin, epoxy group donors such as ethylene glycol diglycidyl ether, p-toluenesulfonic acid chloride, 2-fluoro-1-methylpyridinium, chloroacetyl chloride, hexamethylene diisocyanate, m-xylene diisocyanate, and toluene-2,4-diisocyanate. One or more activators can be used.

[0031] The reaction between the substrate and the activator, and the reaction between the substrate or activated substrate and the cationic compound, can be carried out, for example, at approximately 10 to 100°C for approximately 0.1 to 48 hours. If necessary, a suitable catalyst and solvent may be used. Water is usually used as the solvent, but alcohols such as methanol, ethanol, 2-propanol, ethylene glycol monomethyl ether (2-methoxyethanol), aprotic polar solvents such as dimethylformamide and dimethyl sulfoxide can also be used.

[0032] The amino group content of the adsorbent, which comprises a cationic group containing a nitrogen atom and a substrate, can be set to an anion exchange capacity (AEC) of 0.01 meq / dry·g or more, 0.05 meq / dry·g or more, 0.1 meq / dry·g or more, 0.2 meq / dry·g or more, or 0.5 meq / dry·g or more. Within this range, ET can be sufficiently adsorbed and removed. Alternatively, it can be set to 20 meq / dry·g or less, 10 meq / dry·g or less, 5 meq / dry·g or less, or 1 meq / dry·g or less. Within this range, ET can be efficiently removed while suppressing protein adsorption. In this invention, the ion exchange capacity is a value measured by pH titration.

[0033] The ratio of anionic clay, a solid solution of anionic clay, and / or magnesium oxide to an adsorbent comprising a cationic group containing a nitrogen atom and a substrate can be 0.001 parts by mass or more, 0.01 parts by mass or more, 0.05 parts by mass or more, or 0.1 parts by mass or more of the adsorbent comprising a cationic group containing a nitrogen atom and a substrate, per 1 part by mass of the total amount of anionic clay, a solid solution of anionic clay, and / or magnesium oxide, and can also be 100 parts by mass or less, 50 parts by mass or less, 30 parts by mass or less, 20 parts by mass or less, or 10 parts by mass or less.

[0034] The ET adsorption rate of the ET adsorbent of the present invention may be 90% or more, particularly 94% or more, particularly 96% or more, particularly 98% or more, particularly 99% or more, particularly 99% or more, particularly 99.5% or more, whether or not the adsorbent comprises a cationic group containing a nitrogen atom and a substrate. For example, when 1 w / v% of the test ET adsorbent is used in a 2% D-MEM medium containing 32 to 43 EU / ml of ET or 10% RPMI-1640 medium containing 10% serum, and circulated through the medium at a flow rate of 1 mL / hour for 1 hour using a column method. The upper limit of the ET adsorption rate may be 100%.

[0035] The protein adsorption rate of the ET adsorbent of the present invention may be 50% or less, particularly 30% or less, particularly 20% or less, and particularly 10% or less, when 1 w / v% of the test ET adsorbent is used in a 2% D-MEM medium containing 32 to 43 EU / ml of serum, and circulated through it for 1 hour at a flow rate of 1 mL / hour using a column method, whether or not the adsorbent comprises a cationic group containing a nitrogen atom and a substrate. The lower limit of the protein adsorption rate may be 0%.

[0036] Applications The ET adsorbent of the present invention is an ET adsorbent for adsorbing ET in a culture medium or an ET adsorbent for removing ET from a culture medium. The culture medium can be any culture medium for culturing cells, microorganisms, etc. The culture medium targeted by the present invention is usually a liquid culture medium. The liquid culture medium only needs to be fluid, and may also be a viscous liquid culture medium. Examples of culture media include cell culture media and microbial culture media. Both inorganic and organic culture media are targeted, but organic culture media are preferred because they often contain many types of proteins in high concentrations as culture medium components. The culture medium may contain proteins, amino acids, nucleic acids, sugars, inorganic salts, and / or vitamins. The culture medium can be the culture medium before culturing, during culturing, or the culture supernatant after culturing. Furthermore, the culture medium, whether used before culturing, during culturing, or after culturing, preferably has a protein concentration of 0.01% by mass or more, 0.1% by mass or more, 1% by mass or more, or 2% by mass or more, and is preferably 50% by mass or less, 30% by mass or less, 20% by mass or less, or 10% by mass or less.

[0037] The ET adsorbent of the present invention can be used as an ET adsorbent for adsorbing ET in a culture medium while suppressing or without adsorbing proteins in the culture medium. Furthermore, the ET adsorbent of the present invention can be used by packing it into a column. A column packed with the ET adsorbent of the present invention can be used as a column for removing ET from a culture medium. Furthermore, this column can be used as a column for removing ET from a culture medium while suppressing or without adsorbing proteins in the culture medium.

[0038] (2) ET removal method By bringing the ET adsorbent of the present invention into contact with a culture medium, ET in the culture medium is adsorbed onto the ET adsorbent. This makes it possible to remove ET from the culture medium. After that, the culture medium from which ET has been removed and the ET adsorbent that has adsorbed ET can be separated. In other words, the ET removal method of the present invention is a method that includes the step of bringing the ET adsorbent of the present invention into contact with a culture medium. Furthermore, the method may also include the step of separating the culture medium from which ET has been removed from the ET adsorbent that has adsorbed ET, for example, the step of recovering the culture medium from which ET has been removed from a mixture of the ET adsorbent of the present invention and the culture medium. In other words, the ET removal method of the present invention is a method for producing a culture medium from which ET has been removed. The ET removal method of the present invention may also be a method for removing endotoxins from a culture medium while suppressing the adsorption of proteins in the culture medium, including the step of bringing the ET adsorbent of the present invention into contact with a culture medium. In other words, the ET removal method of the present invention may be a method for producing a culture medium containing protein from which ET has been removed, or a method for producing a culture medium in which protein remains and ET has been removed, comprising the step of bringing the ET adsorbent of the present invention into contact with the culture medium.

[0039] The culture medium to be processed may be the culture supernatant after the start of culture, in which case it may contain one or more target substances. At least one of the target components may be a protein. Both the protein in the culture medium and the protein for production may have a protein portion and include simple proteins, glycoproteins, lipoproteins, metalloproteins, heme proteins, phosphoproteins, etc. Among these, simple proteins and glycoproteins are preferred.

[0040] Contact between the ET adsorbent of the present invention and the culture medium can be carried out, for example, by a batch method. The "batch method" is a method of contacting the ET adsorbent of the present invention and the culture medium by mixing them in a suitable container. The batch method may be carried out by standing, stirring, or shaking. The contact time can be, for example, 5 minutes to 120 hours, 30 minutes to 24 hours, 1 to 12 hours, or 2 to 4 hours. The temperature at the time of contact can be, for example, 5 to 80°C, 15 to 65°C, or 25 to 50°C. If the temperature is significantly higher than the optimal growth temperature for cells or microorganisms, it may be cooled before use for culture. After ET has been adsorbed onto the ET adsorbent of the present invention, the ET adsorbent of the present invention can be separated from the mixture by filtration or centrifugation.

[0041] Furthermore, contact between the ET adsorbent of the present invention and the culture medium can be achieved, for example, by a fluid separation method. A "fluid separation method" is a method of bringing the ET adsorbent of the present invention into contact with the culture medium by passing the culture medium through the ET adsorbent of the present invention. Specifically, for example, the ET adsorbent of the present invention can be packed into a column, and the culture medium can be passed through this column to bring the ET adsorbent of the present invention into contact with the culture medium. Also, for example, if the ET adsorbent of the present invention is formed in the shape of a filter, the ET adsorbent of the present invention can be brought into contact with the culture medium by passing the culture medium through this filter. Examples of membranes include membrane filters, hollow fiber membranes, and tubular membranes. Also, if the ET adsorbent of the present invention is formed in the shape of a column or the like, the ET adsorbent of the present invention can be brought into contact with the culture medium by passing the culture medium through this column or the like. Furthermore, the ET adsorbent of the present invention can be placed on filter paper, and the culture medium can be passed through it to bring the ET adsorbent of the present invention into contact with the culture medium.

[0042] The ET removal method of the present invention removes ET from the culture medium. The degree of ET removal is sufficient if the ET concentration or content of the culture medium after treatment (after contact with the ET adsorbent of the present invention) is lower than that before treatment (before contact with the ET adsorbent of the present invention). "ET is adsorbed or removed" means that the ET concentration or content of the culture medium obtained by treatment with the ET adsorbent is reduced to 50% or less, 30% or less, 20% or less, 10% or less, 8% or less, 6% or less, 2% or less, 1% or less, or 0.05% or less compared to before treatment. That is, the ET adsorption or removal rate may be 50% or more, 70% or more, 80% or more, 90% or more, 92% or more, 94% or more, 98% or more, 99% or more, or 99.5% or more. As a method for quantifying ET, the Limulus test using Limulus reagent can be mentioned. The Limulus test can be performed by conventional methods. Limulus testing can be performed using methods such as colorimetric, turbidimetric, or gelation methods.

[0043] "Proteins in the culture medium are not adsorbed or removed," "adsorption of proteins in the culture medium is suppressed," and "proteins remain in the culture medium" means that the protein content in the culture medium after treatment is maintained at 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, or 99% or more compared to before treatment. Proteins can be quantified by methods such as the ultraviolet absorption method, the Bradford method (Coomassie blue method), the Lowry method (phenol reagent method), or the bicinchoninic acid method (BCA method).

[0044] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these. (1) Production of cationized cellulose fiber (CF) Production Example 1 20 wet-g of wet cellulose fiber (Celish KY-100S; Lot. 64011; manufactured by Daicel Finechem Co., Ltd.) and a 10% (w / w) sodium hydroxide aqueous solution (10 g of sodium hydroxide (special grade; manufactured by Nacalai Tesque) dissolved in 90 mL of water) were placed in a 500 mL separable flask and stirred in a 30°C water bath for 1 hour. Next, 160 mL of chloromethyloxirane (special grade; manufactured by Wako Pure Chemical Industries, Ltd.) was added to the separable flask and stirred in a 30°C water bath for 2 hours. The stirring speed was kept constant. The reaction product was filtered by suction on a filter cloth (Toray Silk, mesh size 20 μm, manufactured by Toray Industries, Inc.) to obtain epoxy-activated cellulose fiber as solid content (filtration residue). The resulting epoxy-activated cellulose fiber was placed in a separable flask with a mixture of 0.55 g of butylamine (trade name "B0707"; Tokyo Chemical Industry Co., Ltd.), 35 mL of water, and 35 mL of denatured alcohol (trade name "AP-7"; Nippon Alcohol Sales Co., Ltd.), and stirred in a 45°C water bath for 4 hours. The reaction product was thoroughly washed on Toray Silk with ultrapure water until the pH of the washing solution became near neutral, and butylamine-immobilized cellulose fiber (hereinafter also referred to as BA-CF) was obtained as the solid component (filtration residue).

[0045] Manufacturing Example 2 In a 500 mL separable flask, 20 wet g of wet cellulose fiber (Celish KY-100S; Lot. 64011; manufactured by Daicel Finechem Co., Ltd.) and a 10% (w / w) sodium hydroxide aqueous solution (10 g of sodium hydroxide (special grade; manufactured by Nacalai Tesque) dissolved in 90 mL of water) were placed and stirred in a 30°C water bath for 1 hour. Next, 160 mL of chloromethyloxirane (special grade; manufactured by Wako Pure Chemical Industries, Ltd.) was added to the separable flask and stirred for a further 2 hours in a 30°C water bath. The stirring speed was kept constant. The reaction product was filtered by suction on a filter cloth (Toray Silk, mesh size 20 μm, manufactured by Toray Industries, Inc.) to obtain epoxy-activated cellulose fiber as solid matter (filtration residue). The obtained epoxy-activated cellulose fiber was placed in a separable flask and mixed with 0.55 g of N,N-dimethylbutylamine (trade name "D1506"; Tokyo Chemical Industry Co., Ltd.), 35 mL of water, and 35 mL of denatured alcohol (trade name "AP-7"; Nippon Alcohol Sales Co., Ltd.). The mixture was stirred in a 45°C water bath for 4 hours. The reaction product was thoroughly washed on Toray Silk with ultrapure water until the pH of the washing solution became near neutral, and N,N-dimethylbutylamine-immobilized cellulose fiber (hereinafter also referred to as DBA-CF) was obtained as the solid component (filtration residue).

[0046] Manufacturing Example 3 In a 500 mL separable flask, 20 wet g of wet cellulose fiber (Celish KY-100S; Lot. 64011; manufactured by Daicel Finechem Co., Ltd.) and a 10% (w / w) sodium hydroxide aqueous solution (10 g of sodium hydroxide (special grade; manufactured by Nacalai Tesque) dissolved in 90 mL of water) were placed and stirred in a 30°C water bath for 1 hour. Next, 160 mL of chloromethyloxirane (special grade; manufactured by Wako Pure Chemical Industries, Ltd.) was added to the separable flask and stirred for a further 2 hours in a 30°C water bath. The stirring speed was kept constant. The reaction product was filtered by suction on a filter cloth (Toray Silk, mesh size 20 μm, manufactured by Toray Industries, Inc.) to obtain epoxy-activated cellulose fiber as solid matter (filtration residue). The obtained epoxy-activated cellulose fiber was placed in a separable flask and mixed with 0.55 g of N,N-dimethyloctylamine (trade name "D1814"; Tokyo Chemical Industry Co., Ltd.), 35 mL of water, and 35 mL of denatured alcohol (trade name "AP-7"; Nippon Alcohol Sales Co., Ltd.). The mixture was stirred in a 45°C water bath for 4 hours. The reaction product was thoroughly washed on Toray Silk with ultrapure water until the pH of the washing solution became near neutral, and N,N-dimethyloctylamine-immobilized cellulose fiber (hereinafter also referred to as DOA-CF) was obtained as the solid component (filtration residue).

[0047] Production Example 4 In a 500 mL separable flask, 20 wet g of wet cellulose fiber (Celish KY-100S; Lot. 64011; manufactured by Daicel Finechem Co., Ltd.) and a 10% (w / w) sodium hydroxide aqueous solution (10 g of sodium hydroxide (special grade; manufactured by Nacalai Tesque) dissolved in 90 mL of water) were placed and stirred in a 30°C water bath for 1 hour. Next, 160 mL of chloromethyloxirane (special grade; manufactured by Wako Pure Chemical Industries, Ltd.) was added to the separable flask and stirred for a further 2 hours in a 30°C water bath. The stirring speed was kept constant. The reaction product was filtered by suction on a filter cloth (Toray Silk, mesh size 20 μm, manufactured by Toray Industries, Inc.) to obtain epoxy-activated cellulose fiber as solid content (filtration residue). The obtained epoxy-activated cellulose fiber was placed in a separable flask and mixed with 0.55 g of N,N-dimethyldodecylamine (trade name "D0002"; Tokyo Chemical Industry Co., Ltd.), 35 mL of water, and 35 mL of denatured alcohol (trade name "AP-7"; Nippon Alcohol Sales Co., Ltd.). The mixture was stirred in a 45°C water bath for 4 hours. The reaction product was thoroughly washed on Toray Silk with ultrapure water until the pH of the washing solution became near neutral, and N,N-dimethyldodecylamine-immobilized cellulose fiber (hereinafter also referred to as DDCA-CF) was obtained as the solid component (filtration residue).

[0048] (2) Preparation of ET adsorbent The substances shown in Table 1 were prepared as components of the ET adsorbent. KW-2000 is a solid solution of layered double hydroxide having a composition encompassed in Formula 2, Kyowa Mag 150 is magnesium oxide, and Kyoward 500SH and Catenaccio ABL are layered double hydroxides having a composition encompassed in Formula 2. The mixtures of Catenaccio ABL and cationized CF in Examples 5 to 8 were prepared by mixing the two components in a 1:1 mass ratio. The mixtures of Kyoward 500SH and cationized CF in Examples 9 to 12 were prepared by changing the mass ratio of the two components from 2:98 to 20:80.

[0049] (3) Preparation of Endotoxin-Containing Sterilized Water Endotoxin-Free Sterilized Water and Japanese Pharmacopoeia Endotoxin Standard (Fujifilm Wako Pure Chemical Industries, Ltd.) were added to a sterile 20 ml container and mixed with a vortex mixer to prepare endotoxin-containing sterile water. The endotoxin concentration was adjusted to approximately 120 EU / mL.

[0050] (4) Preparation of endotoxin-containing medium In a sterile 20 ml container, add 2% serum-prepared endotoxin-free D-MEM medium (low glucose) (containing L-glutamine and phenol red) (Fujifilm Wako Pure Chemical Industries, Ltd.) or 10% serum-prepared RPMI-1640 medium (containing L-glutamine and phenol red) (Fujifilm Wako Pure Chemical Industries, Ltd.) and Japanese Pharmacopoeia endotoxin standard (Fujifilm Wako Pure Chemical Industries, Ltd.), and mix with a vortex mixer to prepare endotoxin-containing 2% serum D-MEM medium (low glucose) (containing L-glutamine and phenol red) or endotoxin-containing 10% serum RPMI-1640 medium. The endotoxin concentration was adjusted to 32-43 EU / mL.

[0051] (5) Physical property evaluation (5-1) ET adsorption capacity The ET adsorption capacity of each adsorbent in water was measured. The ET adsorption capacity was evaluated by column method. Dry heat sterilizable equipment (conical beaker, volumetric pipette, pipette, glass filter, spatula, Limulus tube, tube cap) was thoroughly washed and then sterilized at 250°C for 4 hours. Syringes, membrane filters, and tips were used that had been sterilized by gamma irradiation beforehand. Each adsorbent was washed five times on a glass filter with 25 ml of 0.2 M NaOH / 95% EtOH. Then, the washing was repeated with sterile pure water until the filtrate became neutral.

[0052] 0.3 g of each washed adsorbent was packed into a 30 mL empty column. 30 mL of the endotoxin-containing sterile water prepared in section (3) Preparation of Endotoxin-Containing Sterile Water was circulated through the adsorbent-packed column at a flow rate of 1 mL / hour for 1 hour using a pump. The circulating aqueous solution was then collected and filtered through a 0.8 μm membrane filter. The filtrate was diluted 10 to 1000 times with water for injection (Otsuka distilled water). 0.2 mL of the diluted solution was added to test tubes containing Endospecy ES-24M (Seikagaku Corporation), a Limulus reagent, and thoroughly mixed using a vortex mixer. The test tubes were placed in an EG reader (SV-12; Seikagaku Corporation), and the residual ET concentration was determined by the colorimetric time method (Ee). Furthermore, the ET concentration (Es) in the endotoxin-containing sterile water before contact with the adsorbent was determined in the same manner as above: the endotoxin-containing sterile water was filtered through a 0.8 μm membrane filter, diluted 10 to 1000 times with sterile water for injection (Otsuka distilled water), and then determined by the colorimetric time method using the Limulus reagent described above. The ET adsorption rate was calculated according to the following formula: ET adsorption rate (%) = [(Es - Ee) / Es] × 100

[0053] ET adsorption capacity in culture medium The ET adsorption capacity of the endotoxin-containing culture medium prepared in section (4) Preparation of endotoxin-containing culture medium was measured. The evaluation of ET adsorption capacity was performed using the same procedure as described in section "ET adsorption capacity in water," except that endotoxin-containing culture medium was used instead of sterile water containing endotoxin.

[0054] The evaluation results for endotoxin adsorption capacity are shown in Tables 2 and 3. Magnesium carbonate effectively adsorbed endotoxins in water, but hardly adsorbed any endotoxins in the culture medium. In contrast, anionic clay, its solid solution, and magnesium oxide adsorbed endotoxins in the culture medium as efficiently as endotoxins in water (Examples 1-4). Furthermore, a mixture of anionic clay and cellulose fiber having anionic groups containing nitrogen atoms showed slightly lower adsorption capacity for endotoxins in the culture medium than in water, but the adsorption rate was still sufficiently high (Examples 5-12).

[0055] (5-2) Protein Adsorption Rate Test The protein adsorption rate of the adsorbent in each case was measured. The protein adsorption rate was evaluated by the column method. Dry heat sterilizable equipment (conical beaker, volumetric pipette, pipette, glass filter, spatula, Limulus tube, tube cap) was thoroughly washed and then sterilized at 250°C for 4 hours. Syringes, membrane filters, and tips were used that had been sterilized by gamma irradiation beforehand. Each adsorbent was washed five times on a glass filter with 25 mL of 0.2 M NaOH / 95% EtOH. Then, washing was repeated with sterile pure water until the filtrate was neutral. 0.3 g of the washed adsorbent was packed into a 30 mL empty column. 30 mL of the endotoxin-containing medium prepared in section (4) Preparation of Endotoxin-Containing Medium was circulated through the column packed with the adsorbent at a flow rate of 1 mL / hour for 1 hour using a pump. Next, the culture medium through which the solution had passed was collected, and the protein concentration was measured by the BCA method using a protein assay BCA kit (Fujifilm Wako Pure Chemical Industries, Ltd.) (Te). The protein concentration of the culture medium before contact with the adsorbent was also measured in the same manner (Ts). The protein adsorption rate was calculated according to the following formula: Protein adsorption rate (%) = [(Ts - Te) / Ts] × 100

[0056] Table 4 shows the evaluation results for protein adsorption rates. The protein adsorption rates of the adsorbents in Examples 1-12 and Comparative Example 1 were all 10% or less, indicating very little protein adsorption. By mixing the anionic clay with cellulose fiber having anionic groups containing nitrogen atoms, protein adsorption was further reduced (comparison between Example 4 and Examples 5-8, and comparison between Example 3 and Examples 9-12).

[0057] The ET adsorbent of the present invention can very efficiently adsorb ET from culture media, which conventional ET adsorbents could not adequately adsorb. Furthermore, because it adsorbs very little protein, it can adsorb and remove ET while suppressing the adsorption of proteins, which are culture media components necessary for the growth of cells and microorganisms, as well as proteins, which are the target products generated by cells and microorganisms. Therefore, it has very high commercial value.

Claims

1. An adsorbent for endotoxins in a culture medium, comprising at least one selected from the group consisting of anionic clay, a solid solution of anionic clay, and magnesium oxide.

2. The anionic clay is represented by the following formula 1: [M ２＋ M ３＋ (OH) ２ｍ＋２ｎ (X Ｚ－ ) ｎ／ｚ ·bH ２ O Formula 1 (In Formula 1, m and n are independent of each other, m / n is an integer of 1 to 10, b is an integer of 0 to 10, and (X Ｚ－ ) ｎ／ｚ is an anion in which z is an integer of 1 to 10, M ２＋ is a divalent metal atom, and M ３＋ is a trivalent metal atom.). The endotoxin adsorbent according to claim 1, which is a substance represented by the formula.

3. The endotoxin adsorbent according to claim 1 or 2, further comprising an adsorbent having a cationic group containing a nitrogen atom and a substrate.

4. The endotoxin adsorbent according to claim 3, wherein the adsorbent comprising a cationic group containing a nitrogen atom and a substrate is cellulose having an amino group.

5. An endotoxin adsorbent according to claim 1 or 2, for adsorbing endotoxins while suppressing the adsorption of proteins in the culture medium.

6. A column for removing endotoxins from a culture medium, comprising a column packed with the endotoxin adsorbent described in claim 1 or 2.

7. A method for producing a culture medium from which endotoxins have been removed, comprising the step of bringing the endotoxin adsorbent described in claim 1 or 2 into contact with the culture medium.

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