Ammonium ion adsorption material and method for regenerating culture solution

Abstract

To provide an ammonium ion adsorbent that can remove ammonium ions in a cell culture medium to recycle the culture medium, while greatly reducing an influence on the metal ion composition in the culture medium and eliminating an adverse effect on the cells.SOLUTION: An ammonium ion adsorbent supports Na ions and K ions. A mol ratio of the Na ions and K ions supported on the adsorbent (K / Na value) is 0 or more and 50 or less.SELECTED DRAWING: Figure 3

Description

[Technical Field]

[0001] The present invention relates to an ammonium ion adsorbent material and a method for regenerating a culture solution, more specifically to an ammonium ion adsorbent material adjusted so that the composition of supported metal ions falls within a predetermined range, and a method for regenerating a culture solution containing ammonium ions using the ammonium ion adsorbent material. [Background technology]

[0002] In recent years, particularly in the fields of pharmaceuticals and regenerative medicine, there has been a demand for the establishment of efficient and inexpensive culture methods for various cells, with the aim of improving the efficiency of cell-based pharmaceutical production and ensuring a stable supply of totipotent stem cells and pluripotent stem cells, which are used as materials in regenerative medicine. During the cell culture process, it is known that waste products that are detrimental to cell growth efficiency increase in the culture medium due to cellular metabolism and chemical decomposition of unstable nutrients in the culture medium. Substances that are detrimental to cell growth efficiency include ammonia and ammonium ions (NH4 + ) is known to cause ammonium ions in the culture environment, and conventional measures to remove ammonium ions from the culture environment have been to replace part or all of the culture medium in a short period of time. However, the need to replace expensive culture medium in a short period of time is a major factor in determining the high costs involved in the production of cell-based pharmaceuticals and regenerative medicine. In principle, removing waste products such as ammonium ions and replenishing consumed nutrients allows for the reuse of the culture medium, thereby reducing production costs. In light of these challenges, a method of adsorbing and removing ammonium ions from the culture medium using a cation-adsorbing material has been used. However, because cell culture medium contains a large excess of metal ions such as sodium and potassium ions relative to the ammonium ion concentration, which can adversely affect cell growth, these cations may be preferentially adsorbed over ammonium ions. Furthermore, calcium and magnesium ions, which are added to the culture medium at relatively low concentrations, may also be preferentially adsorbed by the cation-adsorbing material over ammonium ions. Furthermore, zeolites used as cation-adsorbing materials adsorb cations in the culture medium through ion exchange, resulting in the release of cations carried by the zeolite into the culture medium.

[0003] Therefore, when treated with conventional cation adsorbents, metal ions essential for cell culture are preferentially adsorbed and exchanged along with ammonium ions. Furthermore, the metal ions eluted by cation exchange significantly alter the metal ion composition in the culture medium. The metal ion composition of a culture medium is optimized to maintain cell viability, and altering this composition, particularly the balance between sodium and potassium ions, through the use of cation adsorbents, is known to reduce cell growth efficiency. Thus, selective adsorption of ammonium ions in spent culture medium and regeneration of spent culture medium present numerous technical challenges. Despite the various proposed solutions, none have yet been successfully implemented in the industrial setting. Patent Document 1 discloses a method for removing ammonium ions from a used culture solution by treating the solution with potassium-loaded zeolite, hydrogen-loaded zeolite, or a strongly acidic ion exchange resin. However, as mentioned above, treating the culture solution with zeolite or ion exchange resin has the problem of affecting the ion composition in the culture solution. Thus, in order to regenerate the culture solution, it is necessary not only to efficiently remove ammonium ions but also to minimize the effect on the metal ion composition in the culture solution. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Japanese Patent Application Publication No. 2020-39258 Summary of the Invention [Problem to be solved by the invention]

[0005] One object of the present invention is to provide an ammonium ion adsorbent material capable of highly selectively adsorbing ammonium ions from a solution containing ammonium ions. Another object of the present invention is to provide an ammonium ion adsorbent material capable of removing and regenerating ammonium ions in a cell culture solution while significantly reducing the effect on the metal ion composition in the culture solution and eliminating adverse effects on cells. Another object of the present invention is to provide a method for highly efficient adsorption of ammonium ions from a solution.Another object of the present invention is to provide a method for highly efficient removal of ammonium ions from a culture medium and regenerating the cell culture medium. Another object of the present invention can be to provide a removal device capable of removing ammonium ions from a solution such as a culture medium with high efficiency. [Means for solving the problem]

[0006] In order to solve the above problems, the present inventors have conducted extensive research and discovered that an ammonium ion adsorbent material carrying Na ions and K ions at a predetermined molar ratio (K / Na value) can adsorb and remove ammonium ions from a culture medium while significantly reducing the effect on the metal ion composition in the culture medium and eliminating adverse effects on cells, leading to the present invention.

[0007] Specifically, the present invention can be embodied in the following aspects. [1] An ammonium ion adsorption material that supports Na ions and K ions, wherein the molar ratio of Na ions to K ions supported by the adsorption material (K / Na value) is greater than 0 and is 50 or less. [2] The ammonium ion adsorption material according to [1], which contains Si atoms and Al atoms in a molar ratio (Si / Al value) in the range of 1 or more and 100 or less. [3] The ammonium ion adsorption material according to [1] or [2], wherein the ammonium ion adsorption material is a zeolite. [4] The ammonium ion adsorption material according to [3], wherein the zeolite is an FER type zeolite or an LTL type zeolite. [5] preparing an adsorbent material precursor; a step of contacting the adsorbent material precursor with an aqueous inorganic ion solution containing K ions and / or Na ions to adjust the molar ratio of Na ions to K ions (K / Na value) carried by the adsorbent material precursor to a range exceeding 0 and not exceeding 50; A method for producing the ammonium ion adsorption material according to any one of [1] to [4] above, comprising: [6] A method for adsorbing ammonium ions, comprising contacting the ammonium ion adsorption material according to any one of [1] to [4] above with a solution containing ammonium ions, thereby adsorbing the ammonium ions. [7] A method for regenerating a culture solution, comprising treating the culture solution containing ammonium ions by contacting the culture solution with the ammonium ion adsorption material according to any one of [1] to [4] above. [8] An apparatus for removing ammonium ions, comprising the ammonium ion adsorption material according to any one of [1] to [4] above. [Effects of the Invention]

[0008] The present invention provides an ammonium ion adsorption material capable of highly selectively adsorbing ammonium ions from a solution containing ammonium ions, and a method for producing the same. The present invention also provides an ammonium ion adsorption material capable of removing and regenerating ammonium ions from a cell culture solution while significantly reducing the impact on the metal ion composition in the culture solution and eliminating adverse effects on cells, and a method for producing the same. The present invention also provides a method for adsorbing ammonium ions in a solution and a method for regenerating a cell culture solution using an ammonium ion adsorption material having the above-described properties. [Brief explanation of the drawings]

[0009] [Figure 1] This is the chemical structural formula showing the skeleton of a typical LTA-type zeolite. [Figure 2] This is the framework structure of a typical LTA-type zeolite. [Figure 3] 1 is a diagram showing an example of an ammonium ion removal device of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0010] Hereinafter, embodiments of the present invention will be described in detail. Note that the preferred and more preferred embodiments exemplified below can be used in appropriate combination with each other regardless of expressions such as "preferable" or "more preferred." Furthermore, the descriptions of numerical ranges are merely examples, and ranges obtained by appropriately combining the upper and lower limits of each range and the numerical values ​​of the examples can also be preferably used. Furthermore, terms such as "contain" or "comprise" may be read as "essentially consisting of" or "consisting only of."

[0011] [Ammonium ion adsorption material] One aspect of the present invention is an ammonium ion adsorbent, in which the molar ratio of Na ions to K ions (K / Na value) supported by the adsorbent is greater than 0 and less than or equal to 50. In this specification, "adsorption" by an ammonium ion adsorbent refers to a state in which ions such as sodium (Na) ions, potassium (K) ions, and ammonium (NH4) ions are incorporated, held, contained, or supported on the surface or within the pores of the ammonium ion adsorbent through ionic bonds with charges held by the skeleton of the ammonium ion adsorbent, or through physical adsorptive forces such as van der Waals forces or chemical adsorptive forces such as covalent bonds with the ammonium ion adsorbent. Furthermore, "ion exchange" using an ammonium ion adsorbent refers to the release of adsorbed ions into a solution by the ammonium ion adsorbent, and the ammonium ion adsorbent instead adsorbing ions present in the solution.

[0012] The ammonium ion adsorbent material of the present invention is obtained by adjusting the K / Na value of the adsorbent precursor. Examples of the adsorbent precursor include porous inorganic materials obtained by calcining a crystalline compound containing an oxide of silicon (Si) and aluminum (Al). Examples of porous inorganic materials include, but are not limited to, zeolites (synthetic and natural), ferrite, mullite, bentonite, montmorillonite, and kaolin. Preferred porous inorganic materials are zeolites (synthetic and natural), more preferably LTA, BEA, FAU, LTL, MFI, HEU, MWW, FER, and MOR zeolites, particularly preferably LTA, MOR, HEU, FAU, FER, and LTL zeolites, even more preferably FER and LTL zeolites, and most preferably FER zeolites. As the adsorbent material precursor, for example, Na ion-supported FER type zeolite (Na-FER), K ion-supported FER zeolite (K-FER), K ion-supported LTL type zeolite (K-LTL), etc. can be used.

[0013] The molar ratio of Si atoms to Al atoms (Si / Al value) contained in the ammonium ion adsorption material of the present invention is, for example, 1 to 100, preferably 1 to 50, more preferably 2 to 20, and particularly preferably 3 to 15. Aluminosilicates such as zeolites can adsorb larger amounts of cations by increasing the Al atom content, but it is known that this also destabilizes the structure. In order to achieve both the adsorption capacity for cations such as ammonium ions (described below) and structural stability, the Si atom to Al atom content ratio is desirably within the above-mentioned numerical range. In addition to Si atoms and Al atoms, other metals or metal compounds may be contained in the structure of the ammonium ion adsorption material. The other metals or metal compounds are composed of metal elements selected from Groups 4A to 7A, 8, and 1B to 3B, more preferably nickel (Ni), titanium (Ti), silver (Ag), copper (Cu), zinc (Zn), and tin (Sn). Examples of metal compounds include oxides, nitrides, carbides, and borides. The mass proportion of the other metals or metal compounds contained in the structure of the ammonium ion adsorption material may be 0.1 to 30 mass%, preferably 1 to 20 mass%, based on 100 mass% of the ammonium ion adsorption material.

[0014] The ammonium ion adsorbent material of the present invention supports Na ions and K ions at a predetermined molar ratio (K / Na value) to reduce the influence on the metal ion composition in the culture solution. Specifically, the K / Na value is, for example, 0 to 50, preferably 0.001 to 20, more preferably 0.01 to 10, particularly preferably 0.15 to 8, even more preferably 0.2 to 5, and most preferably 0.5 to 2. For example, a K / Na value of 50 refers to a case where the amount of Na ions is 1 mole and the amount of K ions is 50 moles. Furthermore, in order for the K / Na value to have an appropriate effect on the ammonium ion selectivity of the ammonium ion adsorbent material, the sum of the amounts of Na ions and K ions supported in the ammonium ion adsorbent material is, as a percentage of the total amount of all cations supported by the ammonium ion adsorbent material, i.e., metal ions, hydrogen ions, and ammonium ions, for example, 50% or more, preferably 70% or more, more preferably 80% or more, especially preferably 90% or more, and especially preferably 95% or more.

[0015] The ammonium ion adsorption material of the present invention may support, in addition to Na ions and K ions, cations typically incorporated into cell culture media. Specifically, the cations are ion-exchangeable cationic species and are typically selected from monovalent or divalent metal atoms, preferably metal atoms selected from Groups 1A-7A, 8, and 1B-3B, and more preferably calcium (Ca) ions, magnesium (Mg) ions, zinc (Zn) ions, iron (Fe) ions, copper (Cu) ions, and cobalt (Co) ions. The ammonium ion adsorption material of the present invention may also support hydrogen ions in solution. The ammonium ion adsorption material may contain these cations in an amount of, for example, 50% or less, preferably 20% or less, more preferably 10% or less, and particularly preferably 5% or less of the sum of the amounts (mol) of Na ions and K ions supported by the ammonium ion adsorption material, while maintaining the K / Na value of the Na ions and K ions supported by the ammonium ion adsorption material within the above range.

[0016] The ammonium ion adsorption material of the present invention may be formed into a compact by granulating a porous inorganic material such as zeolite with a binder. The binder is not particularly limited, and any material suitable for aiding the granulation of the porous inorganic material can be used. The amount of binder added may be appropriately adjusted to an amount that allows granulation. Binder materials include organic and inorganic materials. Examples of organic materials include dextrin, carboxymethyl cellulose, hydroxyethyl cellulose, xanthan gum, acrylic resin, polystyrene, polyvinyl alcohol, and carboxyvinyl polymer. Examples of inorganic materials include bentonite, sepiolite, smectite, silica, and diatomaceous earth. A more specific method for producing the ammonium ion adsorption material of the present invention will be described later.

[0017] When a porous inorganic material is used as the adsorbent for ammonium ions, its pore diameter (measured by gas adsorption) is, for example, 0.01 to 100 Å, preferably 0.1 to 50 Å, and more preferably 1 to 10 Å. Furthermore, the porous inorganic material preferably has a three-dimensional network structure and includes (a) an oxide ceramic layer formed on a part or all of the surface, and (b) a non-oxide ceramic portion formed in a portion other than the ceramic layer. It is particularly preferred that the pores in the porous inorganic material are through-holes (communicating pores). Furthermore, the average particle diameter of the porous inorganic material of the present invention, as measured by the d50% median diameter laser diffraction / scattering method, is, for example, 0.01 μm to 100 mm, preferably 0.1 to 50 mm, and more preferably 1 μm to 20 mm.

[0018] Other additives contained in the ammonium ion adsorption material of the present invention include end-capping agents such as trimethylchlorosilane, trimethylmethoxysilane, trimethylethoxysilane, and hexamethyldisilazane. The other additives may be bound to the surface, the interior of the pores, etc. of the porous inorganic material of the present invention.

[0019] Zeolite, which is preferably used as an adsorption material for ammonium ions, will be described in detail below. "Zeolite" is a crystalline aluminosilicate containing silicon dioxide (silica) and / or aluminum oxide (alumina), and is a porous crystal also called an oxide ceramic porous material. Generally, the basic unit of the crystal structure (also called framework structure) of zeolite is a tetrahedron consisting of four oxygen atoms surrounding a silicon atom or an aluminum atom (TO4 tetrahedron structure, where T is Si and / or Al), and these are connected in a three-dimensional direction to form the crystal structure. Generally, synthetic zeolites carry cations in their crystal structure, and these cations compensate for the negative charge in the crystal structure composed of aluminosilicate, thereby making up for the lack of positive charge. The general composition of zeolite is represented by the following formula (I): M z+ [(SiO2) x (Al2O3)y ] z- (I) (In formula (I), M is an ion-exchangeable cation species supported in the crystal structure, and typically represents a monovalent or divalent metal, z is the valence of M, and x and y are any integers.) Preferably, M is a metal atom selected from Groups 1A to 7A, 8, and 1B to 3B as described above, and more preferably an Na ion, a K ion, a Ca ion, an Mg ion, a Zn ion, an Fe ion, a Cu ion, or a Co ion. The zeolite represented by the above formula (I) may also be expressed as a general formula including a hydrate. Zeolite may also be one in which all or some of the silicon atoms or aluminum atoms in the zeolite framework have been substituted with other atoms such as phosphorus (P), boron (B), gallium (Ga), and titanium (Ti).

[0020] The crystal structure of the zeolite that can be used in the present invention is not particularly limited, and examples include various crystal structures represented by three-letter structure codes defined by the International Zeolite Association. A preferred embodiment of the crystal structure used in the present invention, as expressed by the structure code, is preferably at least one selected from the group consisting of LTA, BEA, FAU, LTL, MFI, HEU, MWW, FER, and MOR types, more preferably at least one selected from the group consisting of LTA, MOR, HEU, FAU, FER, and LTL types, even more preferably FER or LTL, and particularly preferably FER. The FAU zeolite used in the present invention may be either X or Y type. Figure 1 shows a chemical structural formula of a typical LTA zeolite skeleton, and Figure 2 shows its skeleton structure.

[0021] Zeolites have specific pore sizes, surface electric fields, ion exchange capacity, solid acidity, and adsorption capacity, and are used in applications such as desiccants, adsorbents, molecular sieve separation agents, ion exchange agents, and catalysts. The pore size (measured by gas adsorption) of the zeolites used here is, for example, 0.01 to 100 Å, preferably 0.1 to 50 Å, and more preferably 1 to 10 Å. When using zeolite powder, the average particle size of the zeolite, as measured by the d50% median diameter laser diffraction / scattering method, is, for example, 0.01 to 100 μm, preferably 0.1 to 50 μm, and more preferably 1 to 20 μm.

[0022] [Characteristics of ammonium ion adsorption materials] The ammonium ion adsorbent material of the present invention has the property of adsorbing ammonium ions in a solution, and in particular, the ammonium ion adsorbent material of the present invention has the property of selectively adsorbing substances that are present in a cell culture solution and are detrimental to cell growth efficiency, particularly ammonium ions, and removing them from the culture solution. Here, the term "solution" refers to a liquid containing ammonium ions, and the type thereof is not particularly limited. The term "culture medium" refers to a solution in which cells can be cultured, and the type thereof is not particularly limited, and the culture medium contains ammonium ions. The cells cultured in the culture medium are not particularly limited. The ammonium ion-adsorbing material of the present invention can be used for the regeneration of culture medium for culturing cells used in regenerative medicine, such as totipotent or pluripotent stem cells (iPS cells, ES cells, etc.), or cells differentiated from these, or cells used for antibody production, such as CHO cells, or for the pretreatment or regeneration of any culture medium for culturing immortalized cells, immune cells, primary cultured cells, etc., to remove noise from test results due to ammonium ions and maintain quality.

[0023] The ammonium ion adsorbent of the present invention captures ammonium ions through adsorption or ion exchange. In the ion exchange process, the ammonium ion adsorbent supports cations such as sodium (Na) and potassium (K) ions on its surface or within its pores. Through ion exchange with cations such as ammonium ions present in the culture solution, the material releases Na and K ions into the culture solution and adsorbs ammonium ions instead. Without being bound by theory, for example, in the case of a conventional cation adsorbent that supports only K ions, when Na ions in the culture solution are captured, K ions are instead released into the culture solution, resulting in a change in the metal ion composition of the culture solution. On the other hand, the ammonium ion adsorbent of the present invention is adjusted to support K and Na ions at a predetermined molar ratio so that the ion exchange reaction between K and Na ions is saturated, i.e., even if K and Na ions are released due to the uptake of K and Na ions, the final metal ion composition ratio of the culture solution is equivalent to that before treatment. Therefore, the selective uptake of ammonium ions by the ammonium ion adsorbent of the present invention is improved compared to conventional adsorbents. The change rate of K ions and Na ions in the solution before and after the adsorption treatment of ammonium ions by the ammonium ion adsorbent (concentration ratio (%) = (metal ion concentration in the solution after treatment with the ammonium ion adsorbent) / (metal ion concentration in the solution before treatment with the ammonium ion adsorbent) × 100) is, for example, within the range of 50% to 150%, preferably 70% to 130%, more preferably 75% to 125%, and particularly preferably 90% to 110%.

[0024] When ammonium ions are taken up by ion exchange, Na ions, K ions, etc. carried by the ammonium ion adsorbent material are released into the solution. The amount of ammonium ions in the solution (e.g., 4 mM) is sufficiently smaller than the total amount of metal ions in the culture medium (e.g., in DMEM (product code: 045-30285, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), the total amount of metal ions is approximately 162.5 mM, of which approximately 154.5 mM is Na ions). Therefore, without being bound by theory, it is believed that the metal ions released by exchange with ammonium ions do not significantly affect the metal ion composition ratio in the solution.

[0025] Furthermore, determining the appropriate K / Na value of an ammonium ion adsorbent to saturate the ion exchange reaction depends on the ion preference of the adsorbent precursor. Therefore, the appropriate K / Na value of an ammonium ion adsorbent is not necessarily the same as the ion composition ratio in the culture solution. For example, FER-type zeolite has a higher preference for K ions than Na ions. Therefore, when FER-type zeolite is used as the adsorbent precursor, the optimal K ion ratio for culture solution regeneration is one that is sufficiently higher than the composition ratio in the culture solution.

[0026] Furthermore, in addition to Na ions and K ions, the culture solution also contains metal cations such as calcium (Ca) ions, magnesium (Mg) ions, zinc (Zn) ions, iron (Fe) ions, copper (Cu) ions, and cobalt (Co) ions. For example, in the culture solution, FER zeolite has an extremely high preference for K ions and Na ions compared to these metal ions. On the other hand, LTL zeolite and LTA zeolite have a higher preference for these metal ions compared to FER zeolite in the culture solution, and are relatively easy to adsorb. Therefore, when the adsorbent material precursor has such properties, in addition to adjusting the K / Na value, it may be used as an adsorbent material for ammonium ions, adjusted so as to support these metal ions and hydrogen ions in predetermined amounts as described above. The rate of change of these metal ions in the solution before and after the ammonium ion adsorption treatment with the ammonium ion adsorption material (concentration ratio (%) = (metal ion concentration in the solution after treatment with the ammonium ion adsorption material) / (metal ion concentration in the solution before treatment with the ammonium ion adsorption material) × 100) is, for example, within a range of 90% or more and 110% or less, preferably within a range of 95% or more and 105% or less, more preferably within a range of 97% or more and 103% or less, and particularly preferably within a range of 98% or more and 102% or less.

[0027] [Applications of ammonium ion adsorption materials] Due to the above-described properties, the ammonium ion adsorbent material of the present invention is useful for compositions for adsorbing and removing ammonium ions from solutions containing ammonium ions, and for methods for adsorbing ammonium ions using the same. In particular, the material is useful for compositions for adsorbing and removing excess ammonium generated in a cell culture solution due to cellular metabolism or chemical decomposition of unstable nutrients in the culture solution during cell culture, and for restoring or pretreating the solution to a state suitable for cell growth, from which unnecessary ammonium ions have been removed, and for methods for regenerating the culture solution using the same. Details of the solution, culture solution, and cells are as described above. The ammonium ion adsorption material of the present invention can also be used in the ammonium ion removal device described below.

[0028] [Method for manufacturing ammonium ion adsorption material] The adsorption material of ammonium ions of the present invention can be prepared by the following steps: (1) providing an adsorbent material precursor; (2) contacting the adsorbent material precursor with an aqueous inorganic ion solution containing K ions and / or Na ions to adjust the molar ratio of Na ions to K ions (K / Na value) carried by the adsorbent material precursor to a range exceeding 0 and not exceeding 50; It is produced by a production method including the steps of: Specifically, the adsorbent material is produced by passing an aqueous inorganic ion solution containing Na ions and / or K ions through a precursor of the adsorbent material, such as a porous inorganic material, or by immersing the precursor of the adsorbent material in an aqueous inorganic ion solution containing Na ions and / or K ions, thereby controlling the K / Na value within a suitable range. As described above, suitable ranges for the K / Na value include, for example, 0 to 50, preferably 0.001 to 20, more preferably 0.01 to 10, particularly preferably 0.1 to 8, even more preferably 0.2 to 5, and most preferably 0.5 to 2. In this process, shaking, stirring, heating, and the like may be combined appropriately to increase the efficiency of substitution of the metal carried by the adsorbent material for ammonium ions.

[0029] The inorganic ion aqueous solution may also contain metal ions that are typically added to cell culture media, such as Ca ions, Mg ions, Zn ions, Fe ions, Cu ions, and Co ions. In this case, as described above, while maintaining the K / Na value of the ammonium ion adsorbent at the above-mentioned value, it is desirable to control the content of these metal ions to 50% or less, preferably 20% or less, more preferably 10% or less, and particularly preferably 5% or less of the sum of the amounts (mol) of Na ions and K ions supported.

[0030] The ammonium ion adsorbent material having the supported metal ion composition adjusted as described above may be coated with a resin such as polyvinyl alcohol or alginic acid, or a bio-derived gel such as collagen or gelatin, and molded into, for example, a particle shape. The ammonium ion adsorbent material may also be kneaded with a ceramic binder, a resin binder, a bio-derived gel, or the like to form a molded body. Examples of ceramic binders include alumina binders and collidal silica, examples of resin binders include alginic acid, polyvinyl alcohol, and carboxymethyl cellulose, and examples of bio-derived gels include collagen and gelatin.

[0031] [Method for adsorbing ammonium ions] The ammonium ion adsorbent material of the present invention can adsorb ammonium ions by contacting the ammonium ion adsorbent material with a solution containing ammonium ions. The solution is not particularly limited, but a cell culture solution is preferred. When a cell culture solution is used as the solution, the ammonium ions in the culture solution can be adsorbed and removed, and the culture solution can be regenerated, as shown below.

[0032] [How to regenerate culture fluid] The culture medium can be regenerated by treating a cell culture solution with the ammonium ion adsorbent material of the present invention. Specifically, the ammonium ion adsorbent material is brought into contact with the culture medium by immersing, suspending, or dispersing the ammonium ion adsorbent material in the culture medium by stirring, or by passing the culture medium through the ammonium ion adsorbent material. Upon contact with the culture medium, the ammonium ion adsorbent adsorbs ammonium ions in the culture medium, or releases metal ions (such as Na ions and K ions) carried by the ammonium ion adsorbent material into the culture medium by ion exchange, and instead selectively adsorbs ammonium ions in the culture medium. Details of the culture medium and cells are as described above.

[0033] In regenerating the culture medium, the ammonium ion adsorbent material may be embedded in resin (PS, PP, PET, etc.) or glass, and the resin or glass embedded material may then be placed on the bottom or wall of the culture vessel or on the stirring paddle. Alternatively, a portion or all of the culture solution in the culture vessel may be removed and brought into contact with the ammonium ion adsorbent material or the embedded material. The culture solution regeneration process may also include resupplying consumed culture solution components, such as amino acids and metal ions. Furthermore, the ratio of the ammonium ion adsorbent material to be added to regenerate a culture solution containing waste products such as ammonium ions is, for example, in the range of 0.1 g to 1000 g, preferably 1 g to 500 g, more preferably 2 g to 200 g, and particularly preferably 5 g to 100 g per 1 L of culture solution.

[0034] [Ammonium ion removal device] The ammonium ion adsorption material of the present invention can be used in a removal device for removing ammonium ions from a solution containing the ammonium ions. The ammonium ion removal device includes an ammonium ion adsorbent. The ammonium ion removal device further includes a contactor that brings the ammonium ion adsorbent into contact with a solution containing ammonium ions, where the ammonium ion adsorbent adsorbs and removes ammonium ions from the solution. The configuration of the contactor is not particularly limited, and examples include containers such as beakers, flasks, petri dishes, well plates, and columns. The contactor functions as a reactor that constantly or temporarily brings the ammonium ion adsorbent into contact with the solution. The reactor may be either a batch or continuous reactor, such as a continuous tank reactor (CSTR) or a tubular reactor (PFR). The type of solution is not particularly limited, but when a cell culture solution is used as the solution, the culture solution can be regenerated using the removal device.

[0035] Figure 3 shows an example of an ammonium ion removal device. The ammonium ion removal device 1 shown in Figure 3 includes a container 2 composed of a column and a particulate ammonium ion adsorbent 3 housed inside the column, with the container 2 functioning as a contact point. A pressure-applying device, such as a pump, is located outside the removal device 1 and connected to the container 2. The pressure-applying device is connected to a solution placed outside the removal device 1 and drives the solution into the removal device 1. The solution sent into the removal device 1 passes through the interior of the container 2 in the direction indicated by the arrow in the figure. At this time, the solution comes into contact with the ammonium ion adsorbent 3 inside the container 2, allowing the ammonium ion adsorbent 3 to adsorb and remove the ammonium ions in the solution. The solution discharged from the container 2 can be collected, for example, in a container outside the removal device 1.

[0036] The configuration of the ammonium ion removal device is not limited to the configuration shown in Fig. 3. The ammonium ion removal device is not particularly limited as long as it includes the ammonium ion adsorption material of the present invention, has a contact part that constantly or temporarily brings the ammonium ion adsorption material into contact with a solution, and is configured so that the ammonium ion adsorption material can adsorb and remove ammonium ions in the solution at the contact part. The ammonium ion removal device may be configured, for example, by adhering an ammonium ion adsorbent to the inner wall surface of a container such as a beaker, flask, petri dish, well plate, cell culture insert, or microsphere filled with a solution, in which case the interior of the container functions as the contact area. Alternatively, the ammonium ion removal device may be configured by immersing, dispersing, or suspending an ammonium ion adsorbent in a solution filled in a similar container as described above, in which case the interior of the container functions as the contact area. Furthermore, these ammonium ion removal devices may be configured such that a diaphragm such as a porous diaphragm is disposed to separate a compartment in the container where the solution is disposed from a compartment where the ammonium ion adsorbent is disposed, and the solution passes through the diaphragm to contact the ammonium ion adsorbent.

[0037] Specific examples of the present invention will be described below, but it should be clearly noted that these examples are not intended to limit the scope of the present invention. [Example]

[0038] (reagent) Details of the reagents used in the following examples and comparative examples are as follows: (Adsorbent material precursor) The following zeolites (1) to (3) were used as the adsorbent material precursors. (1) Na-FER The following ammonium ion-supported FER type zeolite (NH4 + The hydrogen ion-supported FER type zeolite (H-FER) was calcined at 500°C for 3 hours to obtain hydrogen ion-supported FER type zeolite (H-FER). A portion of this was ion-exchanged three times with a 10% NaCl aqueous solution to obtain Na-FER. NH4 + -FER: Tosoh Corporation Product code: HSZ-720 720NHA, Structure code: FER type, Crystal form: Ferrierite, Si / Almol ratio: 9.0, Pore diameter: 4.8 Å, Average particle size: 6 μm (2) K-FER The following products were used as K-FER: Tosoh Corporation Product Code: HSZ-720 720KOA, Structure Code: FER type, Crystal form: Ferrierite, Si / Almol ratio: 9.0, K / Na value: 67, Pore diameter: 4.8 Å, Average particle size: 20 μm (3) K-LTL The following products were used as K-LTL: Tosoh Corporation Product Code: HSZ-500 500KOA, Structure Code: LTL type, Crystal Form: L type, Si / Almol ratio: 3.05, K / Na value: >100, Pore size: 8 Å, Average particle size: 4 μm

[0039] (culture medium, cells, solvent, etc.) · Mock culture solution: An aqueous solution containing 155 mM Na ions, 1.8 mM Ca ions, 0.8 mM Mg ions, and 5.3 mM K ions was used as the mock culture solution. DMEM: Product code: 045-30285 Fujifilm Wako Pure Chemical Corporation ·Ca9-22 cells: JCRB0625 ·FBS:HyClone ATA31357 Cytiva 200mmol / L L-glutamine solution: Product code: 073-05391 Fujifilm Wako Pure Chemical Corporation

[0040] (Equipment used) Centrifuge: Model: H-19α Kokusan Co., Ltd. ·SEM-EDS: Model: JCM-6000 Plus JEOL Ltd. High-speed centrifuge: Model: Kubota model 6000 Kubota Shoji Co., Ltd. ICP Optical Emission Spectrometer: Model: SPS3100 SII Nano Technology Co., Ltd. Filter (0.2 μm pore size hydrophilic PTFE filter): Omnipore JGWP02500, Merck Ltd. Ammonia meter: Model: TiN-9001 Toko Chemical Research Institute Membrane filter (0.2 μm pore size): Model: 25AS020AS Toyo Roshi Co., Ltd. ·Living Cell Counting Kit:Cell Counting Kit-8 Dojindo Laboratories Microplate reader: Model: POWERSCAN HT DS Pharma Biomedical Co., Ltd.

[0041] [Test Example 1] Effect of ammonium ion adsorbent on metal ion concentration in culture solution (Method for preparing an adsorbent material for ammonium ions) (Comparative Examples 1 and 2) K-FER was used as Comparative Example 1, and K-LTL was used as Comparative Example 2 without adjusting the K / Na value. Hereinafter, the zeolites used in Comparative Examples 1 and 2 without ion adjustment may be referred to as unadjusted adsorption materials. Examples 1 and 3 2 g each of Na-FER (Example 1) and K-LTL (Example 3) was stirred for 24 hours in 200 mL of simulated culture medium as an aqueous solution of inorganic ions, and after centrifugation, 200 mL of simulated culture medium was added to the solid phase again and stirred. This process was repeated four times to produce ammonium ion adsorption materials with adjusted K / Na values. Examples 2 and 4 0.4 g each of K-FER (Example 2) and K-LTL (Example 4) was mixed with 40 mL of DMEM (an aqueous inorganic ion solution) and allowed to come into contact at 25°C for 24 hours. The resulting mixture was centrifuged at 3000 rpm for 5 minutes, and the supernatant was removed. The entire amount of the treated zeolite powder was again mixed with 40 mL of DMEM, allowed to come into contact at 25°C for 24 hours, and then centrifuged at 3000 rpm for 5 minutes to remove the supernatant. This process was repeated twice to produce an ammonium ion adsorption material with an adjusted K / Na value.

[0042] (Check K / Na values) The K / Na values ​​of the ammonium ion adsorption materials of Examples 1 to 4 were confirmed using SEM-EDS.

[0043] (Confirmation of changes in metal ion concentration) 1 g of each of the ammonium ion adsorption materials of Examples 1 to 4 and the unadjusted adsorption materials of Comparative Examples 1 and 2 was mixed with 100 ml of DMEM and allowed to come into contact for 24 hours at 25° C. The control in Table 1 below is a test group in which no ammonium ion adsorption material was used and 100 ml of DMEM alone was allowed to stand at 25° C. for 24 hours. Next, the culture medium and the ammonium ion adsorbent or unadjusted adsorbent were separated using a high-speed centrifuge at 11,000 rpm for 15 minutes, and the liquid phase was then filtered using a filter to prepare a sample for ICP atomic emission spectrometry analysis. Using an ICP atomic emission spectrometer, the concentrations of K ions, Ca ions, and Mg ions in DMEM were measured before and after treatment with the ammonium ion adsorbent or the unadjusted adsorbent, and the ratio (%) of the post-treatment concentration to the pre-treatment concentration was calculated. The concentration ratio (%) is expressed by the following formula (A). Concentration ratio (%) = (metal ion concentration after treatment with the ammonium ion adsorbent) / (metal ion concentration before treatment with the ammonium ion adsorbent) × 100 (A)

[0044] Based on the calculated concentration ratio, the influence of the adsorbent material used in each Example and Comparative Example on the metal ion concentration in the culture solution was evaluated based on the following evaluation criteria. <Effect on metal ion concentration> Excellent: The concentration ratio of K ions is within the range of 90 to 110, and the concentration ratios of Ca ions and Mg ions are each within the range of 95 to 105. ◯ (Good): The concentration ratio of K ions is within the range of 70 or more but less than 90 or more than 110 but not more than 128, and in addition, the concentration ratios of Ca ions and Mg ions are each within the range of 95 or more but not more than 105. △ (Acceptable): The concentration ratio of K ions is within the range of 50 or more but less than 70, or more than 128 but not more than 150, and in addition, the concentration ratios of Ca ions and Mg ions are each within the range of 95 or more but not more than 105, or the concentration ratio of K ions is within the range of 70 or more but not more than 128, and in addition, the concentration ratios of Ca ions and Mg ions are each within the range of 90 or more but not more than 110, and the above conditions of ◎ and ○ are not met. × (Not acceptable): Does not meet the above ◎, 〇, and △ conditions.

[0045] Table 1 TIFF0007727278000001.tif67145

[0046] As shown in Table 1, when zeolites without adjustment of the K / Na value of the supported metal ions (K-FER in Comparative Example 1 and K-LTL in Comparative Example 2) were used, the concentrations of K ions, Ca ions, and Mg ions in DMEM changed significantly. On the other hand, when the ammonium ion adsorbent materials of Examples 1 to 4 were used, the changes in the concentrations of these ions in DMEM were significantly reduced.

[0047] [Test Example 2] Ammonium ion removal activity of ammonium ion adsorbent material (Method for preparing an adsorbent material for ammonium ions) (Comparative Example 1) In Comparative Example 1, K-FER was used without adjusting the K / Na value. Example 1 2 g of Na-FER was stirred in 200 mL of simulated culture medium for 24 hours, and after centrifugation, 200 mL of simulated culture medium was added to the solid phase again and stirred. This process was repeated four times to produce an ammonium ion adsorption material with an adjusted K / Na value.

[0048] (Check K / Na values) The K / Na value of the ammonium ion adsorption material of Example 1 was confirmed using SEM-EDS.

[0049] (Confirmation of changes in ammonium ion concentration) 1 g each of the ammonium ion adsorption material of Example 1 and the unadjusted adsorption material of Comparative Example 1 was mixed with 100 mL of DMEM (NH3-DMEM) containing ammonium chloride at a final concentration of 4 mM, and the mixture was allowed to come into contact at 25°C for 24 hours. Next, the culture medium was separated from the ammonium ion adsorbent or unadjusted adsorbent using a high-speed centrifuge at 11,000 rpm for 15 minutes, and the liquid phase was then filtered. Using an ion electrode-type ammonia meter, the ammonium ion concentration in NH3-DMEM was measured before and after treatment with the ammonium ion adsorbent or unadjusted adsorbent. Using the measurement results, the amount of ammonium ion adsorbed (mmol / g) for each adsorbent used in the Examples and Comparative Examples was calculated. The amount of ammonium ion adsorbed (mmol / g) refers to the amount of ammonium ion (mol) adsorbed per 1 g of the adsorbent used in the Examples or Comparative Examples.

[0050] Based on the calculated adsorption amount of ammonium ions, the ammonium ion removal activity of each adsorbent material used in the Examples and Comparative Examples was evaluated based on the following evaluation criteria. <Ammonium ion removal activity> Good (Acceptable): The amount of ammonium ions adsorbed by the adsorbent material is 0.20 mmol / g or more. × (unacceptable): The amount of ammonium ions adsorbed by the adsorbent material is less than 0.20 mmol / g.

[0051] Table 2 TIFF0007727278000002.tif52143

[0052] As shown in Table 2, it was confirmed that the ammonium ion adsorption material with adjusted K / Na value used in Example 1 has ammonium ion removal activity similar to the adsorption material with unadjusted supported metal ions used in Comparative Example 1.

[0053] [Test Example 3] Improvement of cell proliferation efficiency by regenerating culture medium using ammonium ion adsorbent (Method for preparing an adsorbent material for ammonium ions) (Comparative Examples 1 and 2) K-FER was used as Comparative Example 1, and K-LTL was used as Comparative Example 2 without adjusting the K / Na value. Examples 1 and 3 2 g each of Na-FER (Example 1) and K-LTL (Example 3) was stirred for 24 hours in 200 mL of simulated culture medium as an aqueous solution of inorganic ions, and after centrifugation, 200 mL of simulated culture medium was added to the solid phase again and stirred. This process was repeated 3 to 4 times to produce an ammonium ion adsorption material with an adjusted K / Na value. Examples 2 and 4 0.4 g each of K-FER (Example 2) and K-LTL (Example 4) was mixed with 40 mL of DMEM (an aqueous inorganic ion solution) and allowed to come into contact at 25°C for 24 hours. The mixture after contact was centrifuged at 3000 rpm for 5 minutes using a centrifuge, and the supernatant was removed. The entire amount of the treated zeolite powder was again mixed with 40 mL of DMEM, allowed to come into contact at 25°C for 24 hours, centrifuged at 3000 rpm for 5 minutes using a centrifuge, and the supernatant was removed. This process was repeated twice to produce an ammonium ion adsorption material with an adjusted K / Na value.

[0054] (Check K / Na values) The K / Na values ​​of the ammonium ion adsorption materials of Examples 1 to 4 were confirmed using SEM-EDS.

[0055] (Confirmation of the effect of regenerating culture medium on improving cell proliferation efficiency) 1 g of each of the adsorption materials of Examples 1 to 4 and Comparative Examples 1 and 2 was mixed with 100 mL of DMEM (NH3-DMEM) containing ammonium chloride at a final concentration of 4 mM, and the mixture was allowed to come into contact at 25°C for 24 hours. After contact, each adsorption material was filtered from the mixture using a membrane filter, and the above-mentioned FBS was added to the filtrate to a final concentration of 10% and the above-mentioned 200 mmol / L L-glutamine to a final concentration of 4 mM to prepare a cytotoxicity test solution. Ca9-22 cells were cultured for 3 days using the cytotoxicity test solution. The control group in Table 3 below is a test group in which cells were cultured using the cytotoxicity test solution prepared by adding the above-mentioned FBS to a final concentration of 10% and the above-mentioned 200 mmol / L L-glutamine to a final concentration of 4 mM to 100 mL of NH3-DMEM without using any ammonium ion adsorbent. After 3 days of culture, the cell proliferation rate from before culture was calculated using a viable cell counting kit and a microplate reader, and the relative value (relative cell proliferation rate) was calculated when the cell proliferation rate in the control group was set at 100. The relative cell proliferation rate is expressed by the following formula (B). Relative cell proliferation rate = (cell proliferation rate of each test group) / (cell proliferation rate of the control group) × 100 (B)

[0056] Based on the calculated relative cell proliferation rate, the effect of the ammonia removal treatment using the adsorbent material used in each Example and Comparative Example on cell proliferation was evaluated based on the following criteria. <Effects on cell proliferation> 〇 (Good): Relative cell proliferation rate is 118 or higher △ (Acceptable): Relative cell proliferation rate is 105 or more and less than 118 × (unacceptable): Relative cell proliferation rate is less than 105

[0057] Table 3 TIFF0007727278000003.tif62155

[0058] As shown in Table 3, the cell proliferation rate was higher when the culture medium was treated with the ammonium ion adsorption materials of Examples 1 to 4 compared to when the culture medium was treated with zeolites (K-FER in Comparative Example 1 and K-LTL in Comparative Example 2) that had not been adjusted for the supported metal ions.

[0059] (comprehensive evaluation) A comprehensive evaluation was made of the suitability of each adsorbent material when used for regenerating a culture medium based on the above Test Examples 1 to 3. In Table 4 below, the "change in metal ion concentration" is based on the evaluation of Test Example 1, the "ammonium ion removal activity" is based on the evaluation of Test Example 2, and the "cell proliferation effect" is based on the evaluation of Test Example 3. The comprehensive evaluation was based on the following evaluation criteria. <Overall rating> ⊚ (Excellent): The evaluation results of Test Examples 1 to 3 were ⊚ (Excellent) only, or consisted of ⊚ (Excellent) and ○ (Good), and the material was particularly suitable for regenerating culture fluid. ◯ (Good): The evaluation results of Test Examples 1 to 3 consisted only of ◯ (Good), and the material was suitable for regenerating the culture medium. △ (Acceptable): The evaluation results of Test Examples 1 to 3 do not include × (unacceptable) and include △ (acceptable), and the material is usable for regenerating the culture medium. × (unacceptable): The evaluation results of Test Examples 1 to 3 include × (unacceptable), and the material is not suitable for regenerating the culture medium.

[0060] Table 4 TIFF0007727278000004.tif73155

[0061] As shown in Table 4, by adjusting the composition of the supported metal ions in the FER and LTL zeolites, particularly the K / Na value, it was possible to remove ammonium ions from the culture medium while reducing their effect on the metal ion composition in the culture medium. As a result, when cells were cultured using the culture medium after the regeneration treatment, the cell proliferation effect could be further improved.

Claims

1. An adsorption material for ammonium ions that supports Na ions and K ions, wherein the molar ratio of Na ions to K ions supported by the adsorption material (K / Na value) is greater than 0 and not more than 50, and the adsorption material for ammonium ions is FER-type zeolite or LTL-type zeolite.

2. The ammonium ion adsorption material according to claim 1 , which contains Si atoms and Al atoms in a molar ratio (Si / Al value) in the range of 1 or more and 100 or less.

3. providing an adsorbent material precursor; a step of contacting the adsorbent material precursor with an aqueous inorganic ion solution containing K ions and / or Na ions to adjust the molar ratio of Na ions to K ions (K / Na value) carried by the adsorbent material precursor to a range greater than 0 and not greater than 50; 3. A method for producing the ammonium ion adsorption material according to claim 1 or 2, comprising:

4. A method for adsorbing ammonium ions, comprising contacting the ammonium ion adsorption material according to claim 1 or 2 with a solution containing ammonium ions, thereby adsorbing the ammonium ions.

5. A method for regenerating a culture solution, comprising treating the culture solution containing ammonium ions by bringing the culture solution into contact with the ammonium ion adsorption material according to claim 1 or 2.

6. An apparatus for removing ammonium ions, comprising the ammonium ion adsorption material according to claim 1 or 2.

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