Lactic acid adsorbent, lactic acid removal device, and lactic acid removal method

Abstract

To provide a novel technique for removing lactic acid.SOLUTION: A lactic acid adsorbent 1 comprises a support 2 represented by formula (1): TixZr1-xO2 [in formula (1), x represents a number of 0 or more and less than 1], and a boronic acid 4 bound to the support 2 directly or via a linker.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a lactic acid adsorbent, a lactic acid removal device, and a lactic acid removal method. [Background technology]

[0002] In recent years, there has been a demand for efficient artificial mass cultivation of cells and microorganisms in fields such as pharmaceutical manufacturing and regenerative medicine. Examples of cells that require mass cultivation include antibody-producing cells such as Chinese hamster ovary cells (CHO cells), stem cells such as embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells), and differentiated cells derived from stem cells. If these cells could be stably cultured in large quantities over a long period of time, it would be possible to efficiently produce biological substances such as monoclonal antibodies and differentiated tissues derived from pluripotent stem cells.

[0003] One method for industrially mass-cultivating cells or microorganisms is suspension agitation culture using a culture tank such as a spinner flask. However, suspension agitation culture tends to require large-scale facilities. Therefore, increasing the culture density of cells is an effective way to reduce costs. However, it is known that increasing the culture density suppresses cell proliferation. This is because the concentration of waste products (metabolites) in the culture solution (liquid medium) increases as the cell density increases, which reduces the proliferation activity of cells. Lactic acid is known to be a typical waste product that affects cells.

[0004] Therefore, in order to stably grow cells at high density, it is desirable to remove lactic acid that accumulates in the culture solution. In response to this, for example, Patent Document 1 discloses a cell culture device in which a cell culture vessel and a composition adjusting solution vessel are connected by a liquid feed line equipped with a culture solution composition adjusting membrane that allows components to permeate depending on the concentration difference. In this cell culture device, waste products that accumulate in the culture solution move to the composition adjusting solution side, thereby decreasing their concentration in the culture solution. At the same time, nutrients whose concentrations decrease during culture are replenished by moving from the composition adjusting solution to the culture solution. This maintains the environment in the culture solution in a state suitable for cell culture. Note that the culture solution itself was used as the composition adjusting solution. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. 2015 / 122528 Summary of the Invention [Problem to be solved by the invention]

[0006] The cell culture device disclosed in Patent Document 1 removes waste products from the culture solution using the principle of dialysis. Therefore, to achieve sufficient waste removal, the volume of the composition adjusting solution tank was set to be 10 times or more the volume of the cell culture tank. This posed a problem: the required volume of liquid was enormous, resulting in high costs. In particular, when using the culture solution itself as the composition adjusting solution, a large amount of expensive culture solution is consumed, further increasing costs. Another problem is that when dialysis technology is used to remove waste products, the structure of the culture device becomes complex. Furthermore, removal of lactic acid is often desired not only in culture solutions for cells, etc., but also in other solution systems. Therefore, a novel lactic acid removal technology using a method other than dialysis technology is strongly desired.

[0007] The present invention has been made in view of these circumstances, and one of its objects is to provide a novel technology for removing lactic acid. [Means for solving the problem]

[0008] In order to solve the above problems, one aspect of the present invention is a lactic acid adsorbent. This lactic acid adsorbent is represented by the formula (1): Ti x Zr 1-x It comprises a support represented by O2 [in formula (1), X is a number of 0 or more and less than 1], and a boronic acid bound to the support directly or via a linker.

[0009] Another aspect of the present invention is a lactic acid removal device, which includes a container for containing a solution containing lactic acid, and the lactic acid adsorbent of the above aspect that contacts the solution.

[0010] Another aspect of the present invention is a method for removing lactic acid, which comprises contacting the lactic acid adsorbent of the above aspect with a solution containing lactic acid.

[0011] Any combination of the above components, or mutual substitution of the components or expressions of the present invention between methods, devices, systems, etc., are also valid aspects of the present invention. [Effects of the Invention]

[0012] According to the present invention, a novel technique for removing lactic acid can be provided. [Brief explanation of the drawings]

[0013] [Figure 1] FIG. 2 is a schematic diagram showing an example of a synthesis process for a lactic acid adsorbent according to an embodiment. [Figure 2] FIG. 1 shows other examples of boronic acids. [Figure 3] FIG. 1 is a schematic diagram showing another example of a synthesis process for a lactic acid adsorbent. [Figure 4] FIG. 1 is a diagram showing how a lactic acid adsorbent adsorbs lactic acid. [Figure 5] 5(A) to 5(D) are schematic diagrams for explaining a method for removing lactic acid according to an embodiment. [Figure 6]FIG. 1 is a schematic diagram for explaining a lactic acid removal method according to an embodiment. [Figure 7] FIG. 1 is a graph showing the lactic acid adsorption rate and glucose adsorption rate of a lactic acid adsorbent in an aqueous solution of lactic acid and glucose, and the pH of the aqueous solution after lactic acid adsorption. [Figure 8] Fig. 8(A) is a graph showing the lactate adsorption rate of a lactate adsorbent in a cell culture solution, Fig. 8(B) is a graph showing the glucose adsorption rate of a lactate adsorbent in a cell culture solution, and Fig. 8(C) is a graph showing the pH of the cell culture solution after lactate adsorption in the cell culture solution. DETAILED DESCRIPTION OF THE INVENTION

[0014] The present invention will be described below based on preferred embodiments with reference to the drawings. The embodiments are illustrative and do not limit the invention, and all features and combinations thereof described in the embodiments are not necessarily essential to the invention. Identical or equivalent components, parts, and processes shown in each drawing are designated by the same reference numerals, and redundant description will be omitted where appropriate. The scale and shape of each part shown in each drawing are set for convenience to facilitate explanation and should not be interpreted as limiting unless otherwise specified. Furthermore, when terms such as "first" and "second" are used in this specification or claims, these terms do not indicate any order or importance, but are intended to distinguish one configuration from another. Furthermore, some components that are not important for explaining the embodiments are omitted from each drawing.

[0015] 1 is a schematic diagram showing an example of a synthesis process for a lactic acid adsorbent 1 according to an embodiment. The lactic acid adsorbent 1 according to this embodiment includes a support 2 and a boronic acid 4.

[0016] The support 2 is a composite of titania and zirconia or zirconia alone, represented by the following chemical formula (1): The support 2 may contain a substance other than the compound represented by formula (1). Formula (1): Ti x Zr 1-x O2 [In formula (1), X is a number equal to or greater than 0 and less than 1]

[0017] The boronic acid 4 before being bound to the support 2 is, for example, represented by the following chemical formula (2). Formula (2): Ar-B(OH)2 [In formula (2), Ar is an aryl group substituted with a first functional group 4a]

[0018] The support 2 has multiple hydroxy groups, i.e., multiple surface hydroxyl groups, on its surface. Before bonding to the support 2, the boronic acid 4 has a first functional group 4a capable of reacting with the surface hydroxyl groups. Figure 1 shows 4-carboxyphenylboronic acid (4-CPBA) as an example of the boronic acid 4. 4-CPBA has a carboxyl group as the first functional group 4a. When a mixture of the support 2 and 4-CPBA is subjected to a known process such as solvothermal synthesis, the multiple carboxyl groups of the 4-CPBA react with the surface hydroxyl groups of one support 2. As a result, multiple 4-CPBAs are directly bonded to the support 2, and the lactic acid adsorbent 1 is obtained.

[0019] FIG. 2 shows other examples of boronic acid 4. Other examples of boronic acid 4 include 3-aminophenylboronic acid (3-APBA), 4-vinylphenylboronic acid (4-VPBA), and 3-acrylamidophenylboronic acid (3-AAPBA). Therefore, the first functional group 4a includes not only a carboxyl group but also an amino group, a vinyl group, and an acryloyl group. For example, boronic acid 4 is at least one selected from the group consisting of 4-CPBA, 3-APBA, 4-VPBA, and 3-AAPBA. For example, first functional group 4a is at least one selected from the group consisting of a carboxyl group, an amino group, a vinyl group, and an acryloyl group.

[0020] When the first functional group 4a is a carboxyl group, the boronic acid 4 can be directly bound to the support 2 by reaction between the carboxyl group and a surface hydroxyl group of the support 2. On the other hand, when the first functional group 4a is an amino group, a vinyl group, or an acryloyl group, the boronic acid 4 is bound to the support 2 via a linker. Note that even when the first functional group 4a is a carboxyl group, the boronic acid 4 may be bound to the support 2 via a linker.

[0021] FIG. 3 is a schematic diagram showing another example of the synthesis process for a lactic acid adsorbent 1. The linker 6 has a second functional group 6a capable of reacting with the surface hydroxyl group of the support 2 and a third functional group 6b capable of reacting with the first functional group 4a of the boronic acid 4. FIG. 3 shows 3-aminopropyltriethoxysilane (APTES) as an example of the linker 6. Also, 4-CPBA is shown as an example of the boronic acid 4. Before bonding to the support 2 and the boronic acid 4, APTES has an ethoxy group as the second functional group 6a and an amino group as the third functional group 6b.

[0022] First, the support 2 and the linker 6 are mixed and subjected to a known treatment, whereby one ethoxy group on each of the multiple APTES reacts with a surface hydroxyl group on the support 2. As a result, multiple APTES molecules are bonded to one support 2. Furthermore, the ethoxy groups on two adjacent APTES molecules react with each other, bonding the adjacent APTES molecules together. This results in a complex between the support 2 and multiple APTES molecules. Next, a known treatment is applied to a mixture of this complex and 4-CPBA, whereby the carboxyl group of 4-CPBA reacts with the amino group of APTES, bonding 4-CPBA and APTES in a one-to-one ratio. As a result, multiple 4-CPBA molecules are bonded to the support 2 via APTES, and a lactic acid adsorbent 1 is obtained.

[0023] The third functional group 6b can be appropriately selected depending on the type of the first functional group 4a. Examples of combinations of the first functional group 4a and the third functional group 6b include a combination in which one is a hydroxyl group and the other is a carboxyl group, a combination in which one is an amino group and the other is a carboxyl group, a combination in which one is a vinyl group and the other is an amino group, and a combination in which one is an acryloyl group and the other is an amino group.

[0024] According to the lactic acid adsorbent 1 of this embodiment, by bringing the lactic acid adsorbent 1 into contact with a solution containing lactic acid, lactic acid in the solution can be removed from the solution. FIG. 4 is a diagram showing how the lactic acid adsorbent 1 adsorbs lactic acid. When the lactic acid adsorbent 1 comes into contact with a solution containing lactic acid, boronic acid 4 and lactic acid bond to each other, as shown in FIG. 4. This allows lactic acid to be removed. Furthermore, when the solution contains glucose as an active ingredient, the lactic acid adsorbent can highly selectively adsorb lactic acid, which is the target for removal, compared to glucose, which should remain in the solution. A culture solution of cells or microorganisms is an example of a solution containing glucose. The type of culture solution is not particularly limited.

[0025] Layered double hydroxides (LDHs) and layered double oxides (LDOs) are known as lactate adsorbents. LDHs and LDOs have multiple metal hydroxide layers when in contact with a solution containing lactic acid, allowing lactate to be adsorbed between the metal hydroxide layers. However, LDHs and LDOs have the property of randomly capturing not only lactate but also negatively charged ions in the solution. Therefore, when using LDHs and LDOs as lactate adsorbents, improving the adsorption selectivity for lactate is desirable.

[0026] In contrast, the lactic acid adsorbent 1 of this embodiment adsorbs lactic acid using boronic acid 4. Boronic acid 4 has the property of binding to hydroxy acids such as lactic acid. Therefore, it has higher adsorption selectivity for lactic acid than LDH or LDO. Therefore, by using the lactic acid adsorbent 1 of this embodiment, lactic acid can be efficiently removed from a solution. Furthermore, the boronic acid 4 is bound to the support 2. Therefore, the boronic acid 4 can be more easily recovered than when the boronic acid 4 is simply added to a solution. Therefore, by using the lactic acid adsorbent 1 of this embodiment, lactic acid can be easily removed from a solution.

[0027] Furthermore, when a solution contains glucose as an active ingredient, the lactic acid adsorbent 1 of this embodiment can highly selectively adsorb lactic acid, which is the target for removal, compared to glucose, which should be left in the solution. A culture solution is an example of an aqueous solution containing glucose. The type of culture solution is not particularly limited. The lactic acid adsorbent 1 can also be used in combination with other adsorbents for cellular waste products.

[0028] In formula (1), X is preferably 0.3 or more and 0.5 or less, which can improve the lactic acid adsorption capacity of the lactic acid adsorbent 1. In addition, the adsorption selectivity for lactic acid can be further increased.

[0029] The amount of lactic acid adsorbent 1 added to the solution, in other words, the concentration of the lactic acid adsorbent in the solution, can be set appropriately based on the designer's empirical knowledge or experiments or simulations, etc.

[0030] When the target for lactic acid removal is a culture medium, the cells and microorganisms cultured in this culture medium are not particularly limited. Examples of cells include pluripotent stem cells and differentiation-induced cells such as human iPS cells, human ES cells, and human Muse cells; somatic stem cells such as mesenchymal stem cells (MSCs) and nephron progenitor cells; tissue cells such as human proximal tubule epithelial cells, human distal tubule epithelial cells, and human collecting duct epithelial cells; antibody-producing cell lines such as human embryonic kidney cells (HEK293 cells); and antibody-producing cell lines derived from non-human animals such as Chinese hamster ovary cells (CHO cells) and insect cells (SF9 cells).

[0031] (Lactic acid removal method) The lactic acid removal method according to this embodiment involves bringing the above-described lactic acid adsorbent 1 into contact with a solution containing lactic acid, thereby allowing the lactic acid to be adsorbed by the lactic acid adsorbent 1. The method for bringing the lactic acid adsorbent into contact with the solution is not particularly limited, but the following embodiments are exemplified. Figures 5(A) to 5(D) and 6 are schematic diagrams illustrating the lactic acid removal method according to this embodiment. The following description will be given taking the removal of lactic acid from a culture solution 14 as an example, but the removal of lactic acid from other solutions can also be carried out in a similar manner.

[0032] As shown in FIG. 5(A), in a first embodiment, an adsorption module 5 is prepared in which a column 7 is filled with a lactate adsorbent 1. The column 7 has an inlet 7a and an outlet 7b that communicate between the inside and outside of the column 7. The adsorption module 5 is connected to a culture vessel 10, such as a spinner flask, via a circulation path 8. The circulation path 8 includes an outgoing path 8a that connects the culture vessel 10 with the inlet 7a of the column 7, and a returning path 8b that connects the outlet 7b of the column 7 with the culture vessel 10. A pump 12 is connected to the outgoing path 8a. A culture solution 14 and cells 16 are accommodated in the culture vessel 10. The pump 12 may be disposed in the returning path 8b.

[0033] When the pump 12 is driven, the culture solution 14 is sucked from the culture vessel 10 and sent into the column 7 of the adsorption module 5 via the outgoing path 8a. The culture solution 14 sent into the column 7 is returned into the culture vessel 10 via the returning path 8b. As the culture solution 14 circulates between the culture vessel 10 and the adsorption module 5, it comes into contact with the lactic acid adsorbent 1 packed in the column 7. At this time, the lactic acid in the culture solution 14 is adsorbed by the lactic acid adsorbent 1. As a result, the lactic acid in the culture solution 14 is removed.

[0034] A filter (not shown) is provided at the end of the outgoing path 8a that is connected to the culture vessel 10. This prevents the cells 16 from flowing toward the adsorption module 5. In the process of circulating the culture solution 14 between the culture vessel 10 and the adsorption module 5, components such as glucose and proteins necessary for culturing the cells 16 may be replenished to the culture solution 14.

[0035] That is, in the first embodiment, lactic acid in the culture solution 14 is removed by using a culture device comprising an adsorption module 5 having a lactic acid adsorbent 1, a culture vessel 10 that houses cells 16 or microorganisms and a culture solution 14, and a circulation path 8 that connects the adsorption module 5 to the culture vessel 10 and circulates the culture solution 14. This culture device can also be described as a lactic acid removal device that comprises the adsorption module 5, a vessel that houses the solution, and the circulation path 8 that connects the adsorption module 5 to the vessel and circulates the solution.

[0036] As shown in FIG. 5(B), in the second embodiment, a lactic acid adsorbent 1 is supported on the inner wall surface of a culture vessel 10. A culture solution 14 and cells 16 are contained in the culture vessel 10. Therefore, the culture solution 14 comes into contact with the lactic acid adsorbent 1 exposed on the inner wall surface of the culture vessel 10. This allows lactic acid in the culture solution 14 to be adsorbed onto the lactic acid adsorbent 1. Examples of the culture vessel 10 include a spinner flask, a petri dish, a well plate, a cell culture insert, and a microsphere.

[0037] Examples of methods for supporting the lactic acid adsorbent 1 on the inner wall surface of the culture vessel 10 include a method of adhering the lactic acid adsorbent 1 to the inner wall surface of the culture vessel 10, and, if the culture vessel 10 is made of resin, a method of molding the culture vessel 10 from a resin pre-mixed with the lactic acid adsorbent 1. That is, in the second aspect, lactic acid in the culture solution 14 is removed by using a culture device comprising the culture vessel 10 and the lactic acid adsorbent 1 supported on the inner wall surface of the culture vessel 10. This culture device can also be referred to as a lactic acid removal device comprising a container for accommodating a solution and the lactic acid adsorbent 1 supported on the inner wall surface of the container.

[0038] As shown in FIG. 5(C), in a third embodiment, a culture vessel 10 has a structure in which the interior of the vessel is divided into an upper section 10a and a lower section 10b by a diaphragm 18 such as a porous membrane. An example of such a culture vessel 10 is a cell culture insert. A culture solution 14 and cells 16 are accommodated in the upper section 10a, and a culture solution 14 and a lactate adsorbent 1 are accommodated in the lower section 10b. The culture solution 14 can pass through the diaphragm 18 to move between the upper section 10a and the lower section 10b. On the other hand, the cells 16 and the lactate adsorbent 1 cannot pass through the diaphragm 18.

[0039] In this structure, the culture solution 14 comes into contact with the lactic acid adsorbent 1 accommodated in the lower stage 10b. This allows lactic acid in the culture solution 14 to be adsorbed by the lactic acid adsorbent 1. That is, in the third embodiment, lactic acid in the culture solution 14 is removed by using a culture device including a culture vessel 10, a lactic acid adsorbent 1, and a diaphragm 18 that divides the interior of the culture vessel 10 into a first space in which the lactic acid adsorbent 1 is accommodated and a second space in which cells 16 are accommodated. This culture device can also be referred to as a lactic acid removal device that includes a vessel in which a solution is accommodated, the lactic acid adsorbent 1, and the diaphragm 18 that divides the interior of the vessel into the first space in which the lactic acid adsorbent 1 is accommodated and a second space in which entry of the lactic acid adsorbent is restricted.

[0040] As shown in FIG. 5(D), in a fourth embodiment, a particulate lactic acid adsorbent 1 is dispersed, precipitated, or suspended in a culture solution 14. This allows lactic acid in the culture solution 14 to be adsorbed by the lactic acid adsorbent 1. Note that the lactic acid adsorbent 1 preferably has a predetermined size or larger, for example, 10 μm or larger, to prevent it from being phagocytosed by cells 16. That is, in the fourth embodiment, lactic acid in the culture solution 14 is removed by using a culture device including a culture vessel 10 and a lactic acid adsorbent 1 added to the culture solution 14 in the culture vessel 10. This culture device can also be referred to as a lactic acid removal device including a container for accommodating a solution and a lactic acid adsorbent 1 added to the solution in the container.

[0041] Fig. 6 shows a culture device 20 according to a fifth embodiment. Dashed lines with arrows in Fig. 6 indicate inputs and outputs of control-related signals. The culture device 20 mainly comprises a culture vessel 22, a path structure 24, a monitoring device 26, an adjusting device 28, a control device 30, and an agitating device 32.

[0042] The culture vessel 22 contains a culture solution and cells, i.e., a cell suspension. The path structure 24 has a supply pipe 40 and a circulating pipe 42. The supply pipe 40 and the circulating pipe 42 constitute a circulation path 38. One end of the supply pipe 40 is inserted into the culture vessel 22. The other end of the supply pipe 40 is connected to a dialysis module 46, which will be described later. One end of the circulating pipe 42 is inserted into the culture vessel 22. A filter (not shown) may be provided at one end of the circulating pipe 42. The other end of the circulating pipe 42 is connected to the dialysis module 46, which will be described later. A circulation pump 44 is connected to the middle of the circulating pipe 42. When the circulation pump 44 is driven, the culture solution that has been dialyzed in the dialysis module 46 is sent to the culture vessel 22 via the supply pipe 40. Furthermore, the culture solution contained in the culture vessel 22 is sent to the dialysis module 46 via the circulating pipe 42. The circulation pump 44 may be disposed in the supply pipe 40.

[0043] The monitoring device 26 detects the state of the culture solution and cells contained in the culture vessel 22 using various sensors and transmits a signal indicating the detection result to the control device 30. Examples of sensors included in the monitoring device 26 include a temperature sensor, a dissolved oxygen sensor, a pH sensor, a liquid level sensor, a glucose concentration sensor, and a lactate concentration sensor. The monitoring device 26 may also include a camera. Images captured by the camera are transmitted to the control device 30. The camera allows the state of the cells contained in the culture vessel 22 to be observed.

[0044] The adjustment device 28 has a dialysis module 46, a dialysate tank 48, a dialysis supply pump 50, a dialysis reflux pump 52, and an adsorption module 5. The dialysis module 46 removes waste products and the like contained in the culture solution supplied from the culture vessel 22 via the reflux pipe 42, and supplies components necessary for cell growth to the culture solution. The culture solution whose components have been adjusted in the dialysis module 46 is returned to the culture vessel 22 via the supply pipe 40.

[0045] The dialysate tank 48 contains dialysate. The dialysate contains glucose, amino acids, vitamins, inorganic salts, etc. The dialysate is, for example, DMEM. The dialysis module 46 and the dialysate tank 48 are connected to each other by a dialysis supply pipe 54 and a dialysis return pipe 56. The dialysis supply pump 50 is connected to the dialysis supply pipe 54 and supplies the dialysate stored in the dialysate tank 48 to the dialysis module 46. The dialysis return pump 52 is connected to the dialysis return pipe 56 and returns the dialysate containing waste products, etc. removed from the culture solution in the dialysis module 46 to the dialysate tank 48.

[0046] An adsorption module 5, in which a column 7 is filled with a lactate adsorbent 1, is disposed in the dialysis return pipe 56. As the dialysate moves from the dialysis module 46 to the dialysate tank 48 via the dialysis return pipe 56, it comes into contact with the lactate adsorbent 1 filled in the column 7. At this time, lactic acid in the dialysate is adsorbed by the lactate adsorbent 1. As a result, lactic acid in the dialysate is removed. The adsorption module 5 may be disposed in the dialysis supply pipe 54 or in the dialysate tank 48. The dialysate tank 48 itself may function as the adsorption module 5. When the lactate adsorbent 1 is disposed in the dialysate tank 48, the lactate adsorbent 1 may be supported on the inner wall surface of the dialysate tank 48, for example, as in the second embodiment. Alternatively, as in the third embodiment, the interior of the dialysate tank 48 may be partitioned by a diaphragm 18 into a first space and a second space, and the lactate adsorbent 1 may be contained in the first space. Alternatively, the lactate adsorbent 1 may be added directly to the dialysate in the dialysate tank 48, as in the fourth embodiment.

[0047] The control device 30 performs various calculations and judgments based on various detection signals sent from the monitoring device 26, and controls the operation of each pump and the agitator 32. The control device 30 can be configured with a processor such as a CPU; ROM; RAM; I / F; auxiliary storage devices such as HDDs and SSDs; and input devices such as a keyboard, mouse, and touch panel. For example, the control device 30 can be a personal computer, tablet computer, or portable terminal device. The agitator 32 agitates the culture solution contained in the culture vessel 22. The agitator 32 can be configured with a known agitation mechanism such as a magnetic stirrer or spinner flask.

[0048] That is, in the fifth aspect, lactic acid in the culture solution is removed by using a culture device 20 including a culture container 22 that contains the culture solution, a dialysis module 46 connected to the culture container 22, a dialysis solution tank 48 connected to the dialysis module 46, and an adsorption module 5 that is arranged between the dialysis module 46 and the dialysis solution tank 48 and has a lactic acid adsorbent 1.

[0049] This culture apparatus 20 can also be described as a lactate removal device comprising a container for accommodating a solution containing lactic acid, a dialysis module connected to the container for transferring lactic acid from the solution to the dialysate, and an adsorption module 5 connected to the dialysis module. If the dialysate is considered to be a solution containing lactic acid, the fifth embodiment can be interpreted, like the first embodiment, as a lactate removal device comprising the adsorption module 5, a container for accommodating the solution (dialysis solution tank 48), and a circulation path (dialysis supply pipe 54, dialysis module 46, and dialysis return pipe 56) that connects the adsorption module 5 and the container and circulates the solution.

[0050] Preferably, the lactic acid adsorbent 1 is coated with a resin such as polyvinyl alcohol, or a bio-derived gel such as collagen, alginic acid, or gelatin. This prevents microparticles that may affect cells 16 from leaking from the lactic acid adsorbent 1 into the culture solution 14. Alternatively, the lactic acid adsorbent 1 is formed by kneading a ceramic binder, a resin binder, a bio-derived gel, or the like with a conjugate of the support 2 and boronic acid 4, or a conjugate of the support 2, a linker 6, and boronic acid 4. This also prevents microparticles from leaking out. Examples of ceramic binders include alumina binders and colloidal silica. Examples of resin binders include polyvinyl alcohol and carboxymethyl cellulose. Examples of bio-derived gels include collagen, alginic acid, and gelatin.

[0051] The method for detecting the lactic acid concentration in a solution is not particularly limited, but it is preferable to use a medium component analyzer. Alternatively, the lactic acid concentration can be detected by a colorimetric method using a predetermined measurement reagent, an enzyme electrode method utilizing the substrate specificity of an enzyme, or high performance liquid chromatography (HPLC).

[0052] As described above, the lactic acid adsorbent 1 according to this embodiment includes the support 2 represented by the above formula (1) and the boronic acid 4 bound to the support 2 directly or via a linker 6. This provides a novel lactic acid removal technology that can efficiently and easily remove lactic acid from a solution. Furthermore, when the solution contains glucose as an active ingredient, the lactic acid adsorbent 1 according to this embodiment can highly selectively adsorb the lactic acid to be removed compared to the glucose that should remain in the solution. Therefore, the lactic acid adsorbent 1 according to this embodiment is particularly useful for removing lactic acid from a culture solution.

[0053] Furthermore, according to this embodiment, unlike the case of removing lactic acid using conventional dialysis techniques, lactic acid can be removed without using a huge amount of solution. Therefore, lactic acid can be removed at low cost. In particular, when the solution is a culture solution for cells or the like, the amount of culture solution used can be reduced compared to conventional dialysis techniques. Since culture solutions are generally expensive, further cost reductions are possible. Furthermore, since lactic acid can be removed simply by contacting a lactic acid adsorbent with a lactic acid-containing solution, this embodiment allows for simplification of the structure of the culture device and the lactic acid removal device.

[0054] Furthermore, the removal of lactic acid allows for the mass cultivation of cells at high densities. Furthermore, the decrease in pH of the culture medium caused by lactic acid can be suppressed, which also makes it possible to mass cultivate cells at high densities. Furthermore, when the cells are pluripotent stem cells, the removal of lactic acid not only enables the mass cultivation of cells at high densities, but also maintains the cells in an undifferentiated state, i.e., their pluripotency (multipotency). Therefore, it is possible to obtain large quantities of cells suitable for the production of biological materials and the creation of differentiation-induced tissues. This reduces the costs required for pharmaceutical manufacturing and regenerative medicine.

[0055] Furthermore, X in the above formula (1) is preferably 0.3 or more and 0.5 or less, which can enhance the lactic acid adsorption performance of the lactic acid adsorbent 1 and thereby enable more efficient removal of lactic acid.

[0056] The embodiments of the present invention have been described in detail above. The above-described embodiments merely illustrate specific examples of implementing the present invention. The content of the embodiments does not limit the technical scope of the present invention, and many design modifications, such as changing, adding, or deleting components, are possible within the scope of the inventive concept defined in the claims. A new embodiment with design modifications will combine the effects of the combined embodiments and modifications. In the above-described embodiments, content that allows such design modifications is emphasized by using notations such as "in this embodiment" or "in this embodiment," but design modifications are also permitted even in content without such notation. Any combination of the above-described components is also valid as an aspect of the present invention.

[0057] The embodiments may be specified by the following items. [1st item] Formula (1): Ti x Zr 1-x O2 [In formula (1), X is a number equal to or greater than 0 and less than 1] A support (2) represented by a boronic acid (4) attached directly or via a linker (6) to a support (2); Lactic acid absorbent (1). [Second item] In formula (1), X is 0.3 or more and 0.5 or less. The lactate adsorbent (1) described in item 1. [3rd item] contacting a solution containing lactic acid to adsorb the lactic acid in the solution; A lactate adsorbent (1) according to item 1 or 2. [4th item] The solution contains glucose, The lactate adsorbent (1) described in item 3. [Item 5] The solution is a cell or microbial culture (14); A lactate adsorbent (1) according to item 4. [Item 6] a container (10, 22) for containing a solution containing lactic acid; and a lactic acid adsorbent (1) according to any one of items 1 to 5 that contacts a solution. Lactic acid removal device. [Item 7] A method for producing a lactic acid adsorbent (1) according to any one of items 1 to 5, comprising contacting the lactic acid adsorbent (1) with a solution containing lactic acid. Lactic acid removal method. [Example]

[0058] Examples of the present invention will be described below, but these examples are merely illustrative examples for suitably explaining the present invention and do not limit the present invention in any way.

[0059] Lactic acid adsorbent 4-CPBA / Ti according to Test Example 1 (Ex. 1) 0.5 Zr 0.5 O2 synthesis] (Support Ti 0.5 Zr 0.5 (O2 synthesis) 7.95 g of 1,6-hexanediamine (HDA: Tokyo Chemical Industry Co., Ltd.) was dissolved in 790 mL of ethanol (Fujifilm Wako Pure Chemical Industries, Ltd.). 5.44 mL of purified water, 3.2 mL of 0.1 M KCl aqueous solution, 9.2 mL of Ti(O-iPr)4 (Sigma-Aldrich), 13.26 mL of Zr(O-Pr)4 (Sigma-Aldrich), and 10 mL of ethanol were added to this solution, in that order. The mixture was stirred at 1400 rpm at room temperature for 2 minutes and then allowed to stand for 24 hours. After standing for 24 hours, the resulting precipitate was washed with purified water. Next, 10 mL of purified water and 20 mL of ethanol were added to the precipitate. The mixture was stirred at 1400 rpm at room temperature for 30 minutes and then allowed to stand at 160 °C for 24 hours. After standing for 24 hours, the resulting product was washed with purified water and dried. The dried product was calcined at 500°C for 2 hours to obtain 0.28g of powder. The obtained powder was analyzed by a powder X-ray diffractometer (Rigaku MiniFlex600) to determine Ti 0.5 Zr 0.5 It was confirmed that the compound was O2, that is, X in formula (1) was 0.5.

[0060] (Binding of Boronic Acid 4-CPBA to Support) 0.28g Ti 0.5 Zr 0.5 8 mL of ethanol and 32 mL of pure water were added to the O2 particles. 2.2 g of 4-CPBA (Kanto Chemical Co., Ltd.) was added to the resulting solution. The mixture was stirred at 1500 rpm for 6 hours and then at 100 °C for 24 hours to separate the 4-CPBA and Ti. 0.5 Zr 0.5 After stirring for 24 hours, the resulting product was washed with pure water and dried under reduced pressure at 40°C. 0.5 Zr 0.5 The lactate adsorbent was subjected to CHN analysis and found to be composed of Ti. 0.5 Zr 0.5 The binding amount of 4-CPBA to O2 was confirmed to be 0.2 mmol / g.

[0061] Lactic acid adsorbent 4-CPBA / Ti according to Test Example 2 (Ex. 2) 0.7 Zr 0.3 O2 synthesis] A support powder was obtained in the same manner as in Test Example 1, except that the amount of Ti(O-iPr)4 added was 12.89 mL and the amount of Zr(O-Pr)4 added was 7.96 mL. 0.7 Zr 0.3 It was confirmed that the compound was O2, that is, X in formula (1) was 0.7. 0.7 Zr 0.3 Boronic acid was bound to the support in the same manner as in Test Example 1, except that O2 particles were used, and 4-CPBA / Ti 0.7 Zr 0.3 The lactate adsorbent was subjected to CHN analysis and found to be composed of Ti. 0.7 Zr 0.3 The binding amount of 4-CPBA to O2 was confirmed to be 0.05 mmol / g.

[0062] Lactic acid adsorbent 4-CPBA / Ti according to Test Example 3 (Ex. 3) 0.3 Zr 0.7O2 synthesis] A support powder was obtained in the same manner as in Test Example 1, except that the amount of Ti(O-iPr)4 added was 5.52 mL and the amount of Zr(O-Pr)4 added was 18.57 mL. 0.3 Zr 0.7 It was confirmed that the compound was O2, that is, X in formula (1) was 0.3. 0.3 Zr 0.7 Boronic acid was bound to the support in the same manner as in Test Example 1, except that O2 particles were used, and 4-CPBA / Ti 0.3 Zr 0.7 The lactate adsorbent was subjected to CHN analysis and found to be composed of Ti. 0.3 Zr 0.7 The binding amount of 4-CPBA to O2 was confirmed to be 0.14 mmol / g.

[0063] [Synthesis of lactic acid adsorbent 4-CPBA / ZrO according to Test Example 4 (Ex. 4)] A support powder was obtained using the same procedure as in Test Example 1, except that Ti(O-iPr)4 was not added and the amount of Zr(O-Pr)4 added was 38.86 mL. PXRD confirmed that the obtained powder was ZrO2, i.e., X in formula (1) was 0. Next, boronic acid was bound to the support using the same procedure as in Test Example 1, except that ZrO2 particles were used, to obtain a lactic acid adsorbent consisting of 4-CPBA / ZrO2. CHN analysis of the obtained lactic acid adsorbent confirmed that the amount of 4-CPBA bound to ZrO2 was 0.08 mmol / g.

[0064] [Analysis of adsorption performance in aqueous solutions of lactic acid and glucose] Lithium lactate (Kanto Chemical Co., Ltd.) and glucose (Kanto Chemical Co., Ltd.) were added to pure water to prepare an aqueous solution with a glucose concentration of 1000 mg / L and a lactic acid concentration of 10 mM. A 1 M aqueous solution of sodium hydroxide was added to this aqueous solution to adjust the pH of the aqueous solution to 7.2. Then, 10 mL of this aqueous solution was dispensed into multiple 50 mL Erlenmeyer flasks. Additionally, 0.125 g of the lactic acid adsorbent for each test example was added to the aqueous solution in each flask. Therefore, the concentration of the lactic acid adsorbent was 0.0125 g / mL.

[0065] Each aqueous solution was shaken at 37°C and 150 rpm for 24 hours. After 24 hours of shaking, the lactic acid adsorbent and the aqueous solution were separated using a 0.1 μm filter. The lactic acid and glucose concentrations in the aqueous solution were measured using HPLC (JASCO Corporation). The pH of the aqueous solution was also measured using a multi-water quality meter (DKK-TOA Corporation). The adsorption rates of lactic acid and glucose for each lactic acid adsorbent were calculated using the following formula: Adsorption rate (%) = {(concentration before adsorption - concentration after adsorption) / concentration before adsorption} × 100

[0066] The results are shown in Figure 7. Figure 7 shows the lactic acid adsorption rate and glucose adsorption rate of the lactic acid adsorbent in an aqueous solution of lactic acid and glucose, and the pH of the aqueous solution after lactic acid adsorption. As shown in Figure 7, lactic acid was adsorbed in Test Examples 1-4. This confirmed that the boronic acid bound to the support functions as a lactic acid adsorbent. Furthermore, the greater the amount of boronic acid bound to the support, the higher the lactic acid adsorption rate. In particular, Test Examples 1 and 3 showed significantly higher lactic acid adsorption rates. This confirmed that lactic acid can be more efficiently adsorbed when X in formula (1) is 0.3 or more and 0.5 or less. Furthermore, in Test Examples 1-4, the glucose adsorption rate was extremely low. This confirmed that the boronic acid bound to the support can highly selectively adsorb lactic acid.

[0067] [Analysis of adsorption performance in cell culture medium] Lithium lactate (Fujifilm Wako Pure Chemical Industries, Ltd.) was added to pluripotent stem cell medium (StemFit® AK02N: Ajinomoto Co., Inc.) to prepare a culture medium with a glucose concentration of 250 mg / dL and a lactate concentration of 10 mM. 2.0 mL of this culture medium was dispensed into multiple 15 mL tubes (ThermoFisher Scientific®). Various amounts of the lactate adsorbent from Test Examples 1-3 were added to the culture medium in each tube. The amounts added were 0.025 g, 0.05 g, 0.1 g, and 0.2 g. The lactate adsorbent concentrations were 0.0125 g / mL, 0.025 g / mL, 0.05 g / mL, and 0.1 g / mL. A control culture medium containing no lactate adsorbent, i.e., a lactate adsorbent concentration of 0 g / mL, was also prepared.

[0068] The culture medium containing the lactate adsorbent was shaken at 37°C at 60 rpm for 24 hours. After 24 hours, the culture medium and the lactate adsorbent were separated using a 0.22 μm filter. The lactate concentration, glucose concentration, and pH of the culture medium were measured using a blood gas analyzer (ABL800 FLEX: Radiometer). The pH of the culture medium without the lactate adsorbent was also measured. The lactate adsorption rate and glucose adsorption rate for each lactate adsorbent were calculated based on the above formula.

[0069] The results are shown in Figures 8(A) to 8(C). Figure 8(A) is a graph showing the lactate adsorption rate of the lactate adsorbent in a cell culture solution. Figure 8(B) is a graph showing the glucose adsorption rate of the lactate adsorbent in a cell culture solution. Figure 8(C) is a graph showing the pH of the cell culture solution after lactate adsorption in the cell culture solution.

[0070] As shown in Figures 8(A) and 8(B), Test Examples 1-3 were able to adsorb lactic acid in the culture medium. This confirmed that the boronic acid bound to the support also functions as a lactic acid adsorbent for the culture medium. Furthermore, in Test Examples 1-3, the lactic acid adsorption rate tended to exceed the glucose adsorption rate. This confirmed that the boronic acid bound to the support can highly selectively adsorb lactic acid in the culture medium. In particular, Test Examples 1 and 3 showed significantly higher lactic acid adsorption rates. Furthermore, the higher the adsorbent concentration, the higher the lactic acid adsorption rate. This confirmed that when X in formula (1) is 0.3 or more and 0.5 or less, lactic acid can be more efficiently and highly selectively adsorbed in the culture medium. Furthermore, as shown in Figure 8(C), no significant change in pH was observed in Test Examples 1-3. The inventors also confirmed that Test Example 4 was also able to adsorb lactic acid in the culture medium.

[0071] Lactic Acid Adsorbent 4-CPBA / APTES / Ti according to Test Example 5 0.5 Zr 0.5 O2 synthesis] (Binding of linker APTES to support) 1g of Ti 0.5 Zr 0.5 O2 was dissolved in 10 mL of dimethyl carbonate (DMC: Tokyo Chemical Industry Co., Ltd.) and stirred at room temperature at 1500 rpm for 30 minutes. To this solution, a DMC solution containing APTES (Tokyo Chemical Industry Co., Ltd.) adjusted to a concentration of 5.36 v / v% was added, and the mixture was stirred at 60 °C and 1500 rpm for 24 hours. After 24 hours of stirring, the reaction mixture was filtered through a 0.1 μm filter, and the residue was washed three times with 10 mL of DMC, followed by three times with 10 mL of ethanol. The washed residue was dried in vacuum at 40 °C for 12 hours to obtain the APTES / Ti complex. 0.5 Zr 0.5 Got O2.

[0072] (Binding of boronic acid 4-CPBA to the complex) 0.5 g of the complex was mixed with 5 mL of DMSO and stirred at room temperature at 1500 rpm for 30 minutes. To this solution, 5 mL of a DMSO mixture containing 0.25 g of 4-CPBA and 0.35 mL of 2,6-lutidine (Tokyo Chemical Industry Co., Ltd.) was added. Next, 5 mL of a DMSO mixture containing 0.402 g of 2-chloro-1-methylpyridinium iodide (Tokyo Chemical Industry Co., Ltd.) was added. The solution was then stirred at 40 °C and 1500 rpm for 24 hours. After 24 hours of stirring, the reaction mixture was filtered through a 0.1 μm filter, and the filter cake was washed three times with 10 mL of ethanol, followed by 10 mL of purified water. The washed filter cake was vacuum dried at 40 °C for 12 hours to obtain 4-CPBA / APTES / Ti. 0.5 Zr 0.5 A lactate adsorbent consisting of O2 was obtained.

[0073] Lactic Acid Adsorbent 4-CPBA / APTES / Ti according to Test Example 6 0.7 Zr 0.3 O2 synthesis] Ti as support 0.7 Zr 0.3 The same procedure as in Test Example 5 was used to prepare 4-CPBA / APTES / Ti 0.7 Zr 0.3 A lactate adsorbent consisting of O2 was synthesized.

[0074] Lactic acid adsorbent 4-CPBA / APTES / Ti according to Test Example 7 0.3 Zr 0.7 O2 synthesis] Ti as support 0.3 Zr 0.7 The same procedure as in Test Example 5 was used to prepare 4-CPBA / APTES / Ti 0.3 Zr 0.7 A lactate adsorbent consisting of O2 was synthesized.

[0075] [Analysis of adsorption performance in aqueous solution of lactic acid] Lithium lactate (Kanto Chemical Co., Ltd.) was added to pure water to prepare an aqueous solution with a lactic acid concentration of 10 mM. A 1M aqueous solution of sodium hydroxide was added to this solution to adjust the pH of the solution to 7.2. Then, 10 mL of this solution was dispensed into multiple 50 mL Erlenmeyer flasks. Furthermore, 0.25 g of the lactic acid adsorbent of Test Examples 5-7 was added to the solution in each flask. Therefore, the concentration of the lactic acid adsorbent was 0.025 g / mL.

[0076] Each aqueous solution was shaken at 37°C and 150 rpm for 24 hours. After 24 hours of shaking, the lactic acid adsorbent and the aqueous solution were separated using a 0.1 μm filter. The lactic acid concentration in the aqueous solution was measured using HPLC (JASCO Corporation). The adsorption rate of lactic acid for each lactic acid adsorbent was calculated based on the above formula. As a result, in Test Example 5, the lactic acid adsorption rate was 38%. In Test Example 6, the lactic acid adsorption rate was 9%. In Test Example 7, the lactic acid adsorption rate was 41%. This confirmed that boronic acid bound to a support via a linker functions as a lactic acid adsorbent. Furthermore, even when a linker is interposed between the support and the boronic acid, it was confirmed that lactic acid can be adsorbed more efficiently when X in formula (1) is 0.3 to 0.5. The inventors have also confirmed that the lactic acid adsorbent 4-CPBA / APTES / ZrO2 can also adsorb lactic acid. Furthermore, from the results for the lactic acid aqueous solution, it can be inferred that the boronic acid bound to the support via a linker also functions as a lactic acid adsorbent in the culture medium. [Explanation of symbols]

[0077] 1 Lactate adsorbent, 2 Support, 4 Boronic acid, 6 Linker, 14 Culture medium, 16 Cells.

Claims

1. Expression (1): Three ｘ Zr １－ｘ O ２ [In formula (1), X is a number equal to or greater than 0 and less than 1] A support represented by a boronic acid attached directly or via a linker to the support; Lactic acid absorbent.

2. In the formula (1), X is 0.3 or more and 0.5 or less. The lactic acid adsorbent according to claim 1.

3. contacting a solution containing lactic acid to adsorb lactic acid in the solution; The lactic acid adsorbent according to claim 1 or 2.

4. The solution contains glucose. The lactic acid adsorbent according to claim 3.

5. The solution is a cell or microbial culture medium. The lactic acid adsorbent according to claim 4.

6. a container for containing a solution containing lactic acid; and the lactic acid adsorbent according to claim 1 or 2, which is in contact with the solution. Lactic acid removal device.

7. A method for producing a lactic acid adsorbent according to claim 1 or 2, comprising contacting the lactic acid adsorbent with a solution containing lactic acid. Lactic acid removal method.

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