Biocompatible polymer compositions and uses thereof

Abstract

Provided are: a biocompatible polymer composition having excellent handling properties, which can be used as a protein stabilizer having excellent protein stabilizing ability, a blocking agent having excellent sensitizing effect, and a cell culture medium additive capable of culturing antibody-producing cells having excellent antibody producibility and cell viability; provided is a biocompatible polymer composition which contains water and a vinyl polymer (A) having an amine oxide group and a mass average molecular weight of 5,000 or more, and which has a surface tension of 30-50 dyn / cm at 25 DEG C in a 1% aqueous solution.

Description

Technical Field

[0001] The present disclosure relates to a biocompatible polymer composition and uses thereof. Background Art

[0002] Proteins maintain their higher-order structures through weak non-covalent polar interactions such as Van der Waals interactions, hydrogen bonds, and electrostatic interactions within the polypeptide chain, and various functions are manifested through this higher-order structure. These combinations change according to various external factors (such as temperature, pH value, salt concentration, protein concentration, and the coexistence of surfactants, modifiers, divalent metals, proteases, etc.), and depending on the situation, the protein structure is destroyed and loses its original function. Therefore, the stability of a protein can be renamed as the ability to maintain a functional higher-order structure.

[0003] For this reason, it is necessary to stabilize proteins in aqueous solutions, and studies have been conducted on optimizing storage conditions such as storing the protein at a predetermined temperature in a buffer solution, or using stabilizers (e.g., other proteins such as albumin; polyols such as glycerol, polyethylene glycol, and sucrose; amino acids such as glutamic acid and lysine; reducing agents such as glutathione and dithiothreitol; metal chelators such as ethylene diamine tetraacetic acid (EDTA); organic acid salts such as citric acid; heavy metal salts, substrates, and coenzymes) alone or in combination (Non-Patent Document 1). In particular, as stabilizers for synthetic compounds, for example, Patent Document 1 discloses a method of using a polymer having a phosphorylcholine group, Patent Document 2 discloses a method of using a polymer having a polyethylene glycol moiety, Patent Document 3 discloses a method of using a low molecular weight amine oxide compound, and Patent Document 4 discloses a method of using a low molecular weight sulfobetaine compound. In addition, Patent Document 5 discloses a method using a polymer amine oxide compound.

[0004] However, these methods may not provide satisfactory retention rates or stable periods depending on the type of protein, and further improvements are required.

[0005] In bioassay, methods for detecting and visualizing deoxyribonucleic acid (DNA) or protein by specific adsorption based on antigen-antibody reaction are widely known in Southern blotting, Western blotting, enzyme-linked immunosorbent assay (ELISA), immunostaining, etc. Antibodies also undergo nonspecific adsorption reactions with proteins other than the target protein. In order to prevent such nonspecific adsorption, a pretreatment is performed by covering the surface of the object to be detected or measured with a blocking agent to prevent nonspecific adsorption, but without hindering specific adsorption.

[0006] As blocking agents, for example, Patent Document 6 and Non-Patent Document 2 disclose biologically derived proteins such as normal serum, bovine serum albumin, gelatin, and skimmed milk.

[0007] In addition, patent documents 7, 8, and 9 disclose surfactants called TWEEN (registered trademark) 20, hydroxyalkyl cellulose, polyvinyl alcohol, and copolymers of (meth) acryloyl morpholine and other monomers. In addition, patent document 10 and non-patent document 3 disclose a method of using a copolymer having a phosphorylcholine group in the side chain, and patent document 5 discloses a method of using a high molecular weight amine oxide compound. However, proteins derived from organisms such as those disclosed in patent document 6 or non-patent document 2 have the potential problem of easily producing performance deviations. Furthermore, compounds such as those disclosed in patent documents 5, 7, 8, 9, and 10 are not satisfactory from the perspective of the sensitization effect of antigen-antibody reactions, and further improvements are required. In addition, the copolymer disclosed in patent document 10 requires multiple reactions or the accompanying purification when synthesizing the monomers of the raw materials, and there are problems in productivity.

[0008] In the cell culture of animal cells, useful physiologically active substances (hormones, neurotransmitters, cytokines, vitamins or minerals, enzymes, nucleic acids, etc.) represented by monoclonal antibodies are mostly industrially mass-produced by mass culture. At this time, animal sera such as fetal bovine serum (FBS) are frequently used as proliferation factors. Specifically, a culture solution in which about 10% to 20% of animal sera such as fetal bovine serum (FBS) are added to synthetic basal culture media such as Roswell Park Memorial Institute (RPM1) 1640, Eagle's minimum essential medium (MEM), and Hams F12 (Ham's F12) is usually used. However, the supply of animal sera such as fetal bovine serum (FBS) is limited and usually expensive. As a result, the cost of the culture medium increases, and the manufacturing cost of the physiologically active substance increases. In addition, there is also the disadvantage that it is difficult to separate the target physiologically active substance from the culture solution containing serum-derived proteins. Furthermore, quality deviations between batches are found in serum. Therefore, before using serum, it is necessary to fully perform batch inspection and select the serum that can be used, but a lot of labor is required when selecting the serum. In addition, regarding serum derived from animals, in addition to problems related to diseases related to prions such as mad cow disease, bovine spongiform encephalopathy, infectious spongiform encephalopathy, and Creutzfeldt-Jakob disease, since serum derived from animals cannot be sterilized using autoclaves, it is possible to be contaminated by viruses or mycoplasmas, resulting in risks (or safety aspects) such as infectious diseases. In order to solve this problem, research on culture media (serum-free culture media) without using serum has been carried out. For example, in patent document 11, it is disclosed that animal cells can produce useful substances at high concentrations by adding glycylglycine to the culture media. In addition, in patent document 12, it is disclosed that when cell culture is carried out in a culture medium containing a polymer having a phospholipid-like structure, physiologically active substances can be efficiently produced. In addition, patent document 13 discloses that a culture medium containing hydrophobic D-amino acids promotes cell proliferation and increases the production of useful substances. However, many such culture media require further improvements in the proliferation, viability and antibody productivity of antibody-producing cells. Furthermore, serum substitutes are also mostly derived from natural products, have limited supply, and are usually expensive. Based on the above situation, it is desired to develop a serum substitute that replaces fetal bovine serum (FBS).Patent document 5 discloses that when cells are cultured in a culture medium containing a high molecular weight amine oxide compound, cell aggregates are well formed or the proliferation and viability of antibody-producing cells are improved, but there is room for improvement in the performance of the antibody-producing cells and there is a problem of deteriorated handling due to foaming.

[0009] Prior art literature

[0010] Patent Literature

[0011] Patent Document 1: Japanese Patent Application Laid-Open No. 10-45794

[0012] Patent Document 2: Japanese Patent Laid-Open No. 10-237094

[0013] Patent Document 3: Japanese Patent Publication No. 2007-509164

[0014] Patent Document 4: Japanese Patent Publication No. 2007-516281

[0015] Patent Document 5: Japanese Patent Application Publication No. 2020-38059

[0016] Patent Document 6: International Publication No. 2016 / 052690

[0017] Patent Document 7: Japanese Patent Application Publication No. 2004-219111

[0018] Patent Document 8: Japanese Patent Laid-Open No. 04-019561

[0019] Patent Document 9: Japanese Patent Application Publication No. 2008-209114

[0020] Patent Document 10: Japanese Patent Laid-Open No. 07-083923

[0021] Patent Document 11: Japanese Patent Laid-Open No. 7-23780

[0022] Patent Document 12: Japanese Patent Laid-Open No. 4-304882

[0023] Patent Document 13: Japanese Patent Application Publication No. 2015-027265

[0024] Non-patent literature

[0025] Non-patent literature 1: Freshman Chemistry Experimental Lecture 1 Protein I Isolation, Purification, and Properties Chapter 16

[0026] Non-patent literature 2: "Watanabe-Nakagenase Antibody Method" published by Gakuji Planning Co., Ltd. in 2002

[0027] Non-patent document 3: Polymer Science, Vol. 35, No. 7, 423 (1978) Summary of the invention

[0028] Problems to be solved by the invention

[0029] In view of the above circumstances, an object of the present disclosure is to provide a biocompatible polymer composition with excellent handling properties, which can be used as a protein stabilizer with excellent protein stabilization ability, a blocking agent with excellent sensitization effect, and a cell culture medium additive capable of culturing antibody-producing cells with excellent antibody productivity and cell activity.

[0030] Technical means of solving problems

[0031] That is, the present disclosure relates to the following [1] to

[14] .

[0032] [1] A biocompatible polymer composition comprising a vinyl polymer (A) having an amine oxide group and a mass average molecular weight of 5,000 or more and water, wherein the surface tension of a 1% aqueous solution at 25° C. is 30 dyn / cm to 50 dyn / cm.

[0033] [2] The biocompatible polymer composition according to the present invention, wherein the osmotic pressure of the 1% aqueous solution is 1 mOsm / kg to 20 mOsm / kg.

[0034] [3] The biocompatible polymer composition further comprising a compound (B) having a zwitterionic structure and a molecular weight of 500 or less (excluding surfactants and monomers).

[0035] [4] The biocompatible polymer composition described above, wherein the ratio of the compound (B) is 0.5 to 10 parts per 100 parts of the vinyl polymer (A).

[0036] [5] The biocompatible polymer composition, wherein the vinyl polymer (A) having an amine oxide group and a mass average molecular weight of 5,000 or more has at least one structure represented by the following general formulas 1 to 3.

[0037] [Chemistry 1]

[0038] Formula 1

[0039]

[0040] [Chemistry 2]

[0041] Formula 2

[0042]

[0043] [Chemistry 3]

[0044] Formula 3

[0045]

[0046] [In general formulas 1 to 3,

[0047] X represents a divalent bonding group,

[0048] y represents 0 or 1,

[0049] R 1 represents an alkylene group having 1 to 6 carbon atoms,

[0050] R 2 and R 3 each independently represents an alkyl group having 1 to 4 carbon atoms,

[0051] R 4 represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms,

[0052] R 5 ~R 9 The four of them are independently a hydrogen atom or an alkyl group having 1 to 6 carbon atoms, and R 5 ~R 9 One of the following represents a bonding position to the main chain of the vinyl polymer (A),

[0053] * indicates the bonding position to the main chain of the vinyl polymer (A)]

[0054] [6] The biocompatible polymer composition, wherein the compound (B) has at least one structure represented by the following general formula 4 to general formula 9.

[0055] Formula 4

[0056] [Chemistry 4]

[0057]

[0058] Formula 5

[0059] [Chemistry 5]

[0060]

[0061] Formula 6

[0062] [Chemistry 6]

[0063]

[0064] Formula 7

[0065] [Chemistry 7]

[0066]

[0067] Formula 8

[0068] [Chemistry 8]

[0069]

[0070] Formula 9

[0071] [Chemistry 9]

[0072]

[0073] [In general formulas 4 to 9,

[0074] R 11 , R 12 , R 13 Each independently represents an alkyl group or a hydroxyalkyl group having 1 to 5 carbon atoms; R 11 , R 12 and R 13 Any two of them can be connected to form a ring;

[0075] R 14 represents an alkylene group having 1 to 4 carbon atoms,

[0076] Z stands for -COO - or SO3 - ,

[0077] R 15 represents a hydrogen atom or a methyl group,

[0078] R 16 represents an alkylene group having 1 to 4 carbon atoms,

[0079] R 17 ~R 21 represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms,

[0080] R 22 represents an alkylene group having 1 to 4 carbon atoms]

[0081] [7] A protein stabilizer comprising the biocompatible polymer composition.

[0082] [8] A method for stabilizing a protein, characterized in that the protein stabilizer is allowed to coexist with the protein.

[0083] [9] A sealing agent comprising the biocompatible polymer composition.

[0084]

[10] A method for detecting a target substance in a test object based on in vitro diagnosis, using the blocking agent.

[0085]

[11] A cell culture medium additive comprising the biocompatible polymer composition.

[0086]

[12] A cell culture medium comprising the cell culture medium additive.

[0087]

[13] The cell culture medium, wherein the concentration of the biocompatible polymer composition in the cell culture medium is 0.01 mass % or more and 1 mass % or less.

[0088]

[14] A method for producing a cell aggregate or a physiologically active substance, characterized in that cells are cultured using the cell culture medium.

[0089] Effects of the Invention

[0090] The present disclosure provides a biocompatible polymer composition with excellent handling properties, which can be used as a protein stabilizer with excellent protein stabilization ability, a blocking agent with excellent sensitization effect, and a cell culture medium additive capable of culturing antibody-producing cells with excellent antibody productivity and cell activity. DETAILED DESCRIPTION

[0091] <Biocompatible polymer composition>

[0092] The biocompatible polymer composition disclosed herein comprises a vinyl polymer (A) having an amine oxide group and a mass average molecular weight of 5,000 or more and water, and the surface tension of a 1% aqueous solution at 25° C. is 30 dyn / cm to 50 dyn / cm. By adopting such a structure, an excellent protein stabilization effect or an effect as a blocking agent and a cell culture medium additive, etc. is exhibited.

[0093] <Vinyl polymer (A)>

[0094] The biocompatible polymer composition disclosed herein includes a vinyl polymer (A) having an amine oxide group and having a mass average molecular weight of 5,000 or more.

[0095] Specifically, the vinyl polymer (A) preferably includes at least one structure represented by Formulae 1 to 3, and particularly preferably includes a structure represented by Formula 1. When the amine oxide group is a group represented by the above formula, the polymer can exhibit suitable water solubility.

[0096] The vinyl polymer (A) is preferably a copolymer, and may be any of a block copolymer, a random copolymer, and an alternating copolymer.

[0097] The vinyl polymer (A) having an amine oxide group can be obtained, for example, by the following method.

[0098] (1) A method of obtaining the polymer as a product obtained by polymerizing a tertiary amino group-containing monomer (a) and other monomers as necessary to obtain a vinyl polymer, and then reacting the tertiary amino group with an oxidizing agent.

[0099] (2) A method of obtaining the copolymer in the form of a monomer (a) which is a reaction product of a tertiary amino group-containing monomer and an oxidizing agent (an amine oxide group-containing monomer), and other monomers.

[0100] By reacting a tertiary amino group with an oxidizing agent (hereinafter also referred to as an oxidizing agent), an amine oxide group can be introduced into a polymer or a monomer. Hereinafter, such a reaction is also referred to as "oxidizing".

[0101] Among the above methods, the method (1) is preferred because side reactions are less likely to occur. In the method (1), it is preferred to polymerize a monomer other than the tertiary amino group-containing monomer (a).

[0102] <Tertiary amino group-containing monomer (a)>

[0103] Among the tertiary amino group-containing monomers (a) as the precursor before oxidation, the monomers for forming the structure of the general formula 1 include, for example:

[0104] N,N-dimethylaminoethyl (meth)acrylate, N,N-diethylaminoethyl (meth)acrylate, N,N-dimethylaminopropyl (meth)acrylate, N,N-diethylaminopropyl (meth)acrylate, N,N-dimethylaminoethyl (meth)acrylamide, N,N-diethylaminoethyl (meth)acrylamide, N,N-dimethylaminopropyl (meth)acrylamide, N,N-diethylaminopropyl (meth)acrylamide, N,N-dimethylaminopropionic acid vinyl ester, N,N-diethylaminopropionic acid vinyl ester, N,N-dimethyl acrylamide, N,N-dimethylallylamine, p-dimethylaminomethylstyrene, p-dimethylaminoethylstyrene, p-diethylaminomethylstyrene, p-diethylaminoethylstyrene, N,N-dimethylvinylamine, N,N-diethylvinylamine, N,N-diphenylvinylamine, or a reaction product of an acid anhydride containing an unsaturated group such as maleic anhydride, itaconic anhydride, citraconic anhydride and N,N-dimethyl-1,3-propylenediamine, etc., a reaction product of an unsaturated compound containing an epoxy group such as glycidyl (meth)acrylate and N,N-dimethyl-1,3-propylenediamine, etc.

[0105] The tertiary amino group-containing monomer (a) is preferably a (meth)acrylate monomer, more preferably N,N-dialkylaminoethyl (meth)acrylate or N,N-dialkylaminopropyl (meth)acrylate. In the present disclosure, "(meth)acrylic acid" means both "acrylic acid" and "methacrylic acid", and "(meth)acrylate" means both "acrylate" and "methacrylate".

[0106] Examples of the tertiary amino group-containing monomer (a) for forming the structure of Formula 2 include:

[0107] 1-vinylimidazole, 2-methyl-1-vinylimidazole, 4-methyl-1-vinylimidazole, 5-methyl-1-vinylimidazole, 2-lauryl-1-vinylimidazole, 4-(tert-butyl)-1-vinylimidazole.

[0108] Examples of the tertiary amino group-containing monomer (a) for forming the structure of Formula 3 include:

[0109] 2-vinylpyridine, 3-vinylpyridine, 4-vinylpyridine, 2-methyl-3-vinylpyridine, 2-methyl-4-vinylpyridine, 3-methyl-4-vinylpyridine, 2-methyl-5-vinylpyridine, 3-methyl-5-vinylpyridine, 4-methyl-5-vinylpyridine, 2-lauryl-4-vinylpyridine, 2-lauryl-5-vinylpyridine, 2-(tert-butyl)-4-vinylpyridine, 2-(tert-butyl)-5-vinylpyridine.

[0110] <Other monomers>

[0111] When obtaining the vinyl polymer (A), in addition to the tertiary amino group-containing monomer (a), other monomers having one ethylenically unsaturated group in one molecule may be used. By introducing a structure based on other monomers, the polarity or glass transition temperature (Tg) can be appropriately controlled, and the solvent solubility can be controlled.

[0112] For example, we can cite:

[0113] A monomer having an alkyl group having 1 to 18 carbon atoms;

[0114] Acrylic (meth)acrylates such as methoxyethyl (meth)acrylate; Aromatic (meth)acrylates such as benzyl (meth)acrylate;

[0115] A monomer having a hydroxyl group such as 2-hydroxyethyl (meth)acrylate;

[0116] Monomers having a carboxylic acid group such as maleic acid, acrylic acid, methacrylic acid, or an anhydride thereof;

[0117] Monomers having primary to tertiary amide groups, such as N-vinyl-2-pyrrolidone; monomers having quaternary amino groups, such as (meth)acrylic acid dimethylaminoethyl methyl chloride salt, trimethyl-3-(1-(meth)acrylamide-1,1-dimethylpropyl)ammonium chloride, trimethyl-3-(1-(meth)acrylamidepropyl)ammonium chloride, and trimethyl-3-(1-(meth)acrylamide-1,1-dimethylethyl)ammonium chloride;

[0118] Monomers having a polyether chain such as polyethylene glycol (meth)acrylate; (meth)acrylate monomers having a high molecular weight structure in the side chain such as ethylenically unsaturated compounds having a polyester chain such as lactone-modified (meth)acrylate; aromatic vinyl monomers such as styrene;

[0119] (Meth) acrylonitrile and other ethylene-unsaturated monomers containing a nitrile group;

[0120] Fatty acid vinyl ester compounds such as vinyl acetate;

[0121] Vinyl ethers such as butyl vinyl ether are ethylenically unsaturated monomers.

[0122] Among them, from the viewpoint of introducing a hydrophobic group, it is preferred to use a monomer (b) having an alkyl group having 1 to 18 carbon atoms. When the vinyl polymer (A) contains a structural unit based on the monomer (b) having an alkyl group having 1 to 18 carbon atoms, the content thereof is preferably 5% to 70% by mass, more preferably 10% to 60% by mass, and further preferably 15% to 50% by mass, relative to 100% by mass of the total monomer units constituting the vinyl polymer (A).

[0123] <Oxidation>

[0124] The tertiary amino group derived from the tertiary amino group-containing monomer (a) can be oxidized by using various oxidizing agents. In this way, an oxidized amine group can be introduced into the vinyl polymer (A). The oxidization can be performed before or after the polymerization of the vinyl polymer (A). For the oxidization before polymerization, an oxidizing agent is added to a solution containing the tertiary amino group-containing monomer (a) and the reaction is carried out at a temperature in the range of 20°C to 100°C for 0.1 to 100 hours, preferably 1 to 50 hours, thereby oxidizing the tertiary amino group. For the oxidization after polymerization, an oxidizing agent is added to a solution containing a vinyl polymer obtained by polymerizing a monomer that necessarily contains the tertiary amino group-containing monomer (a), and the reaction is carried out at a temperature in the range of 20°C to 100°C for 0.1 to 100 hours, preferably 1 to 50 hours, thereby oxidizing the tertiary amino group.

[0125] As the oxidizing agent, an oxidizing agent such as peroxide or ozone can be used. Examples of the peroxide include hydrogen peroxide, ammonium persulfate, sodium persulfate, peracetic acid, m-chloroperbenzoic acid, benzoyl peroxide, tert-butyl hydroperoxide, etc., preferably hydrogen peroxide, which is usually used in the form of an aqueous solution.

[0126] <Mass average molecular weight (Mw)>

[0127] In the present disclosure, and as known in the art, mass average molecular weight and weight average molecular weight are used interchangeably, and both are expressed as Mw. The mass average molecular weight of the vinyl polymer (A) is 5,000 or more, preferably 5,000 to 500,000. By having a molecular weight in the above range, an appropriate surface tension can be adjusted when an aqueous solution is prepared, and further, when added to a protein or cell culture medium as an aqueous solution, the operation becomes easy from the viewpoint of defoaming properties.

[0128] Since the biocompatible polymer composition of the present disclosure is added to substances such as proteins, antibodies, and cells for use, the effect produced at the interface between substances is important. In the present disclosure, by making the surface tension of the biocompatible polymer composition into a 1% aqueous solution into an appropriate range, protein stability, blocking properties, and the production of monoclonal antibodies or physiologically active substances during cell culture can be improved. The details are not certain, but it is considered that appropriate surface tension or biocompatible polymers that provide appropriate surface tension contribute to the interaction between the vinyl polymer (A) and proteins or cells.

[0129] <Water>

[0130] The biocompatible polymer composition of the present disclosure includes water in addition to the vinyl polymer (A) having an amine oxide group. As water, for example, sterilized purified water, purified water, ion exchange water, distilled water, pure water, ultrapure water, etc., there is no particular limitation, preferably sterilized purified water. In order to prevent the effects of other components contained in the biocompatible polymer composition from being hindered, the water used is preferably improved by operations such as the removal of impurity ions using an ion exchange resin, the removal of foreign matter using a filter, and distillation to improve the purity of water. In the following description, as long as there is no special explanation, water refers to sterilized purified water.

[0131] <Surface tension (dyn / cm)>

[0132] The surface tension of the biocompatible polymer composition of the present disclosure at 25° C. when prepared as a 1% aqueous solution is in the range of 30 dyn / cm to 50 dyn / cm, and more preferably in the range of 30 dyn / cm to 48 dyn / cm. In the present disclosure, the concentration of the aqueous solution of the biocompatible polymer composition is expressed as a percentage, i.e., a percentage calculated by taking the mass of the dissolved vinyl polymer (A) or the total mass of (A) and (B) dissolved when the composition contains compound (B) as the numerator and the mass of water in which these components are dissolved as the denominator. Depending on the context, the water providing the denominator in the expression of the concentration sometimes includes the water contained in the biocompatible polymer composition product itself, and sometimes includes the additional water when the biocompatible polymer composition product is diluted with additional water. For example, in the above text, the surface tension of the biocompatible polymer composition when made into a 1% aqueous solution is 30 dyn / cm to 50 dyn / cm. This means that it is not limited to the biocompatible polymer composition itself being a 1% aqueous solution. When water is added or removed from the biocompatible polymer composition to adjust the (A) component and the (B) component to a 1% aqueous solution, the surface tension is also 30 dyn / cm to 50 dyn / cm.

[0133] Surface tension is a factor that affects the interface between substances and can be adjusted by adding ionic compounds such as sodium chloride and potassium chloride, nonionic compounds such as propylene glycol and glycerin, surfactants, etc., but in one embodiment of the present disclosure, a compound (B) having a zwitterionic structure and a molecular weight of 500 or less described later is used.

[0134] The molecules on the surface of the liquid are strongly attracted by the molecules inside. As a result, the generated force acts in a way to generate the smallest possible liquid surface. The magnitude of the force is called surface tension, which is expressed by the unit of force per unit length. In the present disclosure, the surface tension is the surface tension when the liquid temperature is 25°C. At this temperature, the surface tension of pure water is 72 dyn / cm. That is, the biocompatible polymer composition has the effect of reducing surface tension.

[0135] (Measurement method of surface tension)

[0136] Examples of methods for measuring surface tension include the Wilhelmy method (plate method, vertical plate method), the duNouy method (ring method, circular ring method), the pendant drop method, the maximum bubble pressure method, and a method of measuring the contact angle and calculating it based on the Young's equation. Any method may be used, but in the present disclosure, the Wilhelmy method is used to measure the surface tension of the biocompatible polymer composition in a 1% aqueous solution state at 25°C.

[0137] <Osmotic pressure (mOsm / kg)>

[0138] The osmotic pressure of the biocompatible polymer composition disclosed herein when prepared as a 1% aqueous solution is preferably in the range of 1 mOsm / kg to 20 mOsm / kg, and more preferably in the range of 2 mOsm / kg to 17 mOsm / kg. If the osmotic pressure is in the above range, it can further improve protein stability, blocking properties, and the production of monoclonal antibodies or physiologically active substances during cell culture by synergistic action with the vinyl polymer (A) and the above surface tension.

[0139] The osmotic pressure is proportional to the number of dissolved particles mol ÷ the volume (L) of the solvent, and is expressed by the unit of osmotic concentration (osmolarity) Osm / L. In the case of an aqueous solution, the osmotic concentration can be expressed as Osm / kg. In particular, osmotic pressure is involved in the movement of water inside and outside cells, and osmotic pressure is generated by solutes such as electrolytes, sugars, and amino acids. In one embodiment of the present disclosure, the osmotic pressure of the biocompatible polymer composition is adjusted by containing a compound (B) described later. By making the osmotic pressure of 1% aqueous solution to be above 1mOsm / kg, damage to cells can be prevented. In addition, by making the osmotic pressure of 1% aqueous solution to be below 20mOsm / kg, the risk of intracellular dehydration is reduced.

[0140] <Compound (B)>

[0141] The biocompatible polymer composition disclosed in the present invention preferably also includes a compound (B) having a zwitterionic structure and a molecular weight of 500 or less. Wherein, surfactants and monomers (specifically, monomers used to make vinyl polymers (A)) are removed from compound (B). However, as long as it does not deviate from the main purpose of the present disclosure, the unintentional mixing of residual monomers, etc. is not prevented. In the present disclosure, the amount of compound (B) is preferably 0.5 to 10 parts, more preferably 1 to 5 parts relative to 100 parts of vinyl polymer (A). In one embodiment, the biocompatible polymer composition does not contain a surfactant. In one embodiment, the biocompatible polymer composition comprises a vinyl polymer (A), a compound (B), and water.

[0142] Specifically, compound (B) preferably comprises at least one structure represented by Formulae 4 to 9, and particularly preferably comprises a structure represented by Formulae 4 and / or 5. When compound (B) has a structure represented by the above formula, the biocompatible polymer composition can be easily made to have an appropriate surface tension.

[0143] The compound (B) can be synthesized, for example, using a compound having a tertiary amino group as a starting material.

[0144] For example, in the case of obtaining a compound having an amine oxide structure, an oxidizing agent such as the peroxide or ozone is used in a compound having a tertiary amino group.

[0145] In addition, for example, in the case of obtaining a compound having a carboxybetaine structure, a cyclic carboxylic acid ester such as β-propiolactone, γ-butyrolactone, δ-valerolactone, or a ω-halogenated alkylcarboxylic acid metal salt such as sodium 2-chloroacetate, sodium 2-bromoacetate, sodium 3-chloropropionate, sodium 3-bromopropionate, sodium 4-chlorobutyrate, sodium 4-bromobutyrate, sodium 5-chlorovalerate, sodium 5-bromovalerate is used as a compound having a tertiary amino group.

[0146] For example, in the case of obtaining a compound having a sulfobetaine structure, a cyclic sulfonate such as 1,2-ethane sultone, 1,3-propane sultone, or 1,4-butane sultone, or an ω-halogenated alkylsulfonic acid metal salt such as sodium 2-chloroethane sulfonate, sodium 2-bromoethane sulfonate, sodium 3-chloropropane sulfonate, sodium 3-bromopropane sulfonate, sodium 4-chlorobutane sulfonate, or sodium 4-bromobutane sulfonate is used as a compound having a tertiary amino group.

[0147] Examples of the compound for forming the structure of Formula 4 or Formula 5 include:

[0148] trimethylamine N-oxide, dimethyl-2-hydroxyethylamine N-oxide, N,N,N-trimethylglycine, dimethylethylammonium propanesulfonate, dimethyl-2-hydroxyethylammonium propanesulfonate, dimethylbenzylammonium propanesulfonate, and 3-(1-methylpiperidinium)-1-propanesulfonic acid.

[0149] Examples of the compound for forming the structure of Formula 6 or Formula 7 include:

[0150] Imidazole N-oxide, 2-methyl-imidazole N-oxide, imidazolinium betaine, and 3-(3-sulfopropyl)imidazolium internal salt.

[0151] Examples of the compound for forming the structure of Formula 8 or Formula 9 include:

[0152] Pyridine N-oxide, 1-(3-sulfopropyl)pyridinium internal salt, and 1-(4-sulfobutyl)pyridinium internal salt.

[0153] The various uses including protein stabilizers, blocking agents and cell culture medium additives are described in detail below.

[0154] <Protein stabilizer>

[0155] By making the biocompatible polymer composition of the present invention coexist with protein, the protein can be stably stored. The stabilizer and protein can coexist by being dissolved or dispersed in a common solvent. The solvent in which the stabilizer and protein coexist is preferably an aqueous solvent. As such a solvent, for example, water, tris buffered saline (TBS), and phosphate buffered saline (PBS) can be listed. Water may also contain a portion of organic solvents that can be mixed with water, such as methanol, ethanol, propanol, or tetrahydrofuran. Relative to the protein, the stabilizer of the present invention is preferably added 100 times by mass, more preferably 10,000 times by mass. In the case where the amount of stabilizer added is insufficient, the stabilization effect may not be manifested. For the same reason, the stabilizer, i.e., the biocompatible polymer composition, is preferably added at 0.01% or more, and more preferably at 0.1% or more, relative to the total mass of the protein solution.

[0156] <Sealing agent>

[0157] The biocompatible polymer composition disclosed in the present invention can be used as a blocking agent. A blocking agent is a component used to prevent nonspecific adsorption of antibody proteins to sites where specific antigens are not present and to enhance specific antigen-antibody reactions. Blocking agents can be used, for example, when detecting target substances in a test object in in vitro diagnosis. When a biocompatible polymer composition is used as a blocking agent, its use concentration is preferably 0.01% to 5% by mass, and more preferably 0.01% to 1% by mass. When the concentration of the biocompatible polymer composition is low, the effect of improving the efficiency of antigen-antibody reactions or reducing nonspecific reactions is small. When the concentration is high, it is possible to act in an inhibitory manner on antigen-antibody reactions. The blocking agent described herein is a component used when a test object containing an antigen is pretreated before an antibody reaction in order to prevent nonspecific adsorption of antibodies to sites where specific antigens are not present, or when added to a diluent for an antibody reaction in order to prevent nonspecific reactions of antibodies, and its form is not particularly limited.

[0158] The effects of these can be determined by adding the blocking agent and the antibody to an appropriate buffer and comparing the extent of nonspecific reaction when the composition is applied to an experimental method utilizing an immune reaction. More preferably, in an enzyme immunoassay (EIA), the blocking agent is diluted with a commonly used buffer, such as TBS or PBS, and the diluted substance is applied to a test object containing an antigen before an antibody reaction, for example, after incubation at 37°C for 1 hour to carry out an antibody reaction, thereby quantitatively measuring the extent of nonspecific adsorption of the antibody to the non-antigen.

[0159] <Cell culture medium additives>

[0160] The biocompatible polymer composition of the present disclosure can be used as a cell culture medium additive. As a cell culture medium, an existing known culture medium can be used. For example, various commercially available culture media (αMEM, MEM, Dulbecco's Modified Eagle Medium (Dulbecco's Modified Eagle Medium, DMEM), Iscove's Modified Dulbecco's Medium (Iscove's Modified Dulbecco's Medium, IMDEM), Roswell Park Memorial Institute (RoswellPark Memorial Institute, RPMI) 1640, DMEM / F12, etc.) or a combination of these can be cited. The concentration of the vinyl polymer (A) or the biocompatible polymer composition as a cell culture medium additive in the culture medium is preferably 0.001% by mass to 1% by mass, more preferably 0.01% by mass to 1% by mass. More preferably 0.1% by mass to 1% by mass. In the cell culture medium, it is preferred to add one or more of various growth factors (epithelial growth factor, insulin-like growth factor, nerve growth factor, hepatocyte growth factor, vascular endothelial growth factor, basic fibroblast growth factor, etc.), transferrin, steroid hormone, 2-mercaptoethanol, etc., various animal sera (fetal bovine serum (FBS) or bovine serum, etc.), serum substitutes, etc., as needed.

[0161] <Method for producing physiologically active substance or spheroid>

[0162] By culturing cells in a cell culture medium containing the biocompatible polymer composition of the present disclosure, physiologically active substances generated by cells and optionally secreted can be efficiently produced. In addition, by culturing cells in a cell culture medium containing the biocompatible polymer composition of the present disclosure, spheroids, i.e., cell aggregates, can be efficiently formed. Cell types with spheroid-forming ability are known to those skilled in the art. It is known that methods for forming spheroids by culturing such types of cells in, for example, round-bottom wells are known, but the maximum formation efficiency is not always obtained. The cell culture medium additive of the present disclosure can improve such suboptimal spheroid formation efficiency.

[0163] <Physiologically active substances>

[0164] The physiologically active substance of this embodiment is, for example, an antibody protein (hereinafter referred to as an antibody), albumin, a drug metabolizing enzyme, etc. The antibody produced here is not particularly limited, and is, for example, a mouse monoclonal antibody, a humanized monoclonal antibody, or a human monoclonal antibody. In addition, the class of immunoglobulin is not particularly limited, and is, for example, immunoglobulin G (IgG) (e.g., IgG1, IgG2, etc.). In addition, the drug metabolizing enzyme produced is not particularly limited, and examples thereof include alcohol dehydrogenase, aldehyde dehydrogenase, glutathione peroxidase, superoxide dismutase, monoamine oxidase, diamine oxidase, epoxide hydrolase, esterase, amidase, uridine diphosphate-glucuronosyl transferase (UDP-glucuronosyl transferase), glutathione S-transferase, γ-glutamyl transpeptidase, acetyltransferase, sulfotransferase, cytochrome P450, and the like.

[0165] In the culture method or production method of the present disclosure, a container or device commonly used for culture can be used during culture, for example, a multiwell plate, a culture dish, a culture flask, a spinner flask, a jar fermenter, a fermenter, a roller bottle, a hollow fiber, and a microcarrier.

[0166] <Cell>

[0167] The cells cultured using the culture method disclosed herein are not particularly limited as long as they are cells that can be used to produce physiologically active substances. Examples of cells that can produce antibodies include Chinese hamster ovary (CHO) cells, baby Syrian hamster kidney (BHK) cells, HepG2 cells, rodent myeloma cells (mouse myeloma cells such as SP2 / O cells and NSO cells), hybridoma cells, insect cells, and transformed cells into which foreign genes have been introduced. For example, hybridoma cells obtained by cell fusion of CHO cells, SP2 / O cells, or NSO cells can be used. Cells capable of producing drug-metabolizing enzymes include, for example, primary hepatocytes collected from the liver of an animal, hepatocytes induced by differentiation from stem cells such as embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), and mesenchymal stem cells, and cell lines derived from liver cancer such as HepG2 cells and HuH-7 cells.

[0168] The culture conditions in this embodiment may be the usual culture conditions for animal cells, for example, the conditions may be set to be 5 vol% CO 2 environment and 37°C.

[0169] When collecting cells from the culture medium, in the case of floating cells, for example, the culture medium is directly supplied to a centrifuge or a filter for collection. In the case of adherent cells, for example, 0.25% trypsin-0.02% EDTA solution is added to float the cells, and then the cells are collected by centrifugation or filtration.

[0170] Regarding physiologically active substances, particularly antibodies, produced by cell culture, when the substances are accumulated in the culture solution, the supernatant can be obtained by, for example, filtration or centrifugation, and then collected. In addition, in the case of substances accumulated in cells, for example, the cells obtained by filtration or centrifugation are subjected to homogenization, ultrasonic treatment, chemical treatment, etc. to obtain a supernatant for extracting the product.

[0171] The antibody can be separated and / or purified from the supernatant by appropriately combining known methods, for example, preferably ammonium sulfate precipitation, dialysis, ultrafiltration, electrophoresis, gel filtration, ion exchange chromatography, reverse phase chromatography, affinity chromatography, etc.

[0172] Example

[0173] The following examples are used to more specifically describe the embodiments of the present disclosure, but the following examples do not limit the scope of the present disclosure. "Parts" and "%" in the examples and comparative examples represent "parts by mass" and "% by mass", respectively, mol represents the amount of substance, and mol% represents the ratio of the amount of the target monomer in all monomers.

[0174] (Method for measuring mass average molecular weight (Mw))

[0175] The mass average molecular weight is a value measured by gel permeation chromatography (GPC) and converted to standard pullulan. As a measuring device and measuring conditions, the following condition 1 is used. For other matters, please refer to Japanese Industrial Standards (JIS) K7252-1~4:2008. For poorly soluble polymer compounds, the concentration that can be dissolved is measured under the following conditions. In addition, when it is difficult to measure the molecular weight of the vinyl polymer (A), the mass average molecular weight of the amine oxide precursor polymer can be set as the mass average molecular weight of the vinyl polymer (A). The mass average molecular weight of the amine oxide precursor polymer is a value measured by gel permeation chromatography (GPC) and converted to standard polystyrene, and the measuring device and measuring conditions are based on the following condition 2.

[0176] (Condition 1)

[0177] Column: A column formed by connecting OHpak SB-G, 3 OHpak SB-806M HQ, and OHpak SB-802.5HQ.

[0178] Carrier: 1 / 15 mol / L phosphate buffer, pH 7.0 (phosphate buffer powder 1 / 15 mol / L, pH 7.0 (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) dissolved in 1 L of ion exchange water)

[0179] Carrier flow rate: 1.0mL / min

[0180] Sample concentration: 0.5 mass%

[0181] Detector: RI (refractive index) detector

[0182] Injection volume: 0.1mL

[0183] (Condition 2)

[0184] Column: A column formed by connecting TSKgelSuperAW4000, TSKgelSuperAW3000 and TSKgelSuperAW2500.

[0185] Support: a mixture of N,N-dimethylformamide (1 L), triethylamine (3.04 g), and LiBr (0.87 g)

[0186] Measuring temperature: 40℃

[0187] Carrier flow rate: 0.6mL / min

[0188] (Measurement method of surface tension)

[0189] The biocompatible polymer composition was prepared as a 1% aqueous solution and measured at 25°C by the Wilhelmy method.

[0190] (Method for measuring osmotic pressure)

[0191] As a method for measuring osmotic pressure, the biocompatible polymer composition was made into a 1% aqueous solution to measure the osmotic concentration by measuring the freezing point depression of water according to the osmotic pressure measuring method described in the 16th revision of the Japanese Pharmacopoeia.

[0192] <Production of vinyl polymer (A)>

[0193] [Production Example 1]

[0194] (P-1)

[0195] Under nitrogen flow, 150 parts by mass of ethyl acetate as an organic solvent was charged into a reaction vessel including a thermometer, a stirrer, a nitrogen introduction tube, a reflux cooler, and a dropping tube, and heated at 80°C for 30 minutes under stirring. 70 parts by mass of N,N-dimethylaminoethyl methacrylate as a monomer, 30 parts by mass of butyl methacrylate, 0.3 parts by mass of azobisisobutyronitrile (AIBN) as a polymerization initiator, and 10 parts by mass of ethyl acetate as a solvent were charged into the dropping tube and added dropwise for 2 hours. After the addition was completed, heating was continued for 6 hours. Thereafter, the reaction was stopped by cooling to room temperature.

[0196] Next, 150 parts of ethanol and 43 parts of 35% hydrogen peroxide as an oxidizing agent (equimolar to the N,N-dimethylaminoethyl methacrylate used) were added and reacted at 70° C. for 16 hours to oxidize the tertiary amino groups. Thereafter, the solvent was removed by a diaphragm pump to obtain a vinyl polymer having an amine oxide group.

[0197] [Manufacturing Examples 2 to 4 and Comparative Manufacturing Example 1]

[0198] (P-2~P-5)

[0199] Except having changed into the composition shown in Table 1, it carried out similarly to P-1, and obtained the vinyl polymer.

[0200] [Table 1]

[0201] Table 1

[0202]

[0203] The details of the abbreviations in Table 1 are as follows.

[0204] DEAEMA: N,N-diethylaminoethyl methacrylate

[0205] VP: Vinylpyridine

[0206] VI: Vinyl imidazole

[0207] ISTA: Isostearyl acrylate

[0208] BMA: Butyl Methacrylate

[0209] MMA: Methyl Methacrylate

[0210] St:Styrene

[0211] <Production of biocompatible polymer composition>

[0212] [Example 1-1]

[0213] (Additive 1)

[0214] The obtained vinyl polymer (P-1) was dissolved in water in an amount of 2 parts trimethylglycine (TMG) relative to 100 parts, thereby obtaining a biocompatible polymer composition having a concentration of 1% by mass. The surface tension was 43 dyn / cm, and the osmotic pressure was 2 mOsm / kg. In addition, the result of the defoaming property evaluation evaluated as an index of the treatment was ○ (good).

[0215] [Example 1-2 to Example 1-22, and Comparative Example 1-1 to Comparative Example 1-4]

[0216] (Additive 2 to Additive 26)

[0217] Except having changed into the composition shown in Table 2, it carried out similarly to Example 1-1.

[0218] [Defoaming property evaluation]

[0219] Typically, the biocompatible polymer composition is stirred before use, but if foam is generated during stirring, it will cause problems from the viewpoint of handling. Therefore, the defoaming property of the foam generated after stirring is used as an index of handling and evaluated by the following method.

[0220] 3 mL of a 1% aqueous solution of the biocompatible polymer composition was added to a 15 mL centrifuge tube, and the mixture was vigorously stirred by hand shaking and allowed to stand for 30 minutes. The height of the foam after standing was measured and evaluated according to the following criteria.

[0221] The height of the foam after 30 minutes of standing

[0222] ○: less than 3cm

[0223] ×: 3cm or more

[0224]

[0225] The details of the abbreviations in Table 2 are as follows.

[0226] TMG: Trimethylglycine

[0227] NDSB195: Dimethylethylammonium propanesulfonate

[0228] NDSB201: 3-(1-pyridine)propanesulfonic acid

[0229] NDSB211: Dimethyl-2-hydroxyethylammonium propanesulfonate

[0230] TMAO: Trimethylamine N-oxide

[0231] DMHEAO: Dimethyl-2-hydroxyethylamine N-oxide

[0232] PyO: Pyridine N-oxide

[0233] <Protein stability test of biocompatible polymer composition>

[0234] [Example 2-1 to Example 2-22] [Comparative Example 2-1 to Comparative Example 2-4]

[0235] Each compound was dissolved in PBS according to the composition ratio shown in Table 2 so as to have a solid content concentration of 0.1% by mass.

[0236] <Evaluation of protein stabilizers>

[0237] The obtained protein stabilizer was subjected to the following evaluations. The results are shown in Table 3.

[0238] [Preparation of enzyme aqueous solution for evaluation]

[0239] Horseradish peroxidase (HRP) labeled C-reactive protein (CRP) antibody (manufactured by Abcam) was diluted with 1 ml of the obtained protein stabilizer aqueous solution to 8.0 ng / ml and mixed to prepare an enzyme aqueous solution for evaluation. The enzyme aqueous solution for evaluation was fully sealed and stored at 40°C to prevent concentration changes.

[0240] [Evaluation of enzyme activity]

[0241] Using a peroxidase color development kit (manufactured by Sumitomo Bakelite), 10 ml of a color developing agent (3,3',5,5'-tetramethylbenzidine (TMBZ) solution) and 100 μl of a substrate solution (hydrogen peroxide solution) were mixed to obtain a color developing solution.

[0242] 100 μl of the color developing solution and 100 μl of the enzyme aqueous solution for evaluation are added to a 96-well plate and left to stand in the dark at 30°C for 20 minutes. 100 μl of stop solution (aqueous sulfuric acid solution) is added thereto to stop the reaction. The absorbance at 450 nm is measured using a plate reader multi-function microplate reader Mithras 2LB943 (manufactured by Berthold Technologies) to quantify the amount of TMBZ oxide produced by the reaction. The oxidation reaction of TMBZ is related to the activity of the HRP-labeled CRP antibody, so the absorbance obtained can be used as an indicator of enzyme activity.

[0243] [Evaluation of protein stabilization effect]

[0244] The enzyme activity evaluation was performed twice before storage of the evaluation enzyme aqueous solution and after storage at 40°C for 3 days, and the absorbance was measured. The ratio of the absorbance after storage to the absorbance before storage was calculated as 100% as the relative absorbance, and the protein modification was evaluated according to the following criteria.

[0245] Relative absorbance after storage at 40℃ for 3 days

[0246] ◎: 70% or more (extremely good)

[0247] ○: 50% or more and less than 70% (good)

[0248] △: 30% or more and less than 50% (usable)

[0249] ×: Less than 30% (cannot be used)

[0250] [Table 3]

[0251]

[0252] As shown in Table 3, a high protein stabilization effect was obtained by using the biocompatible polymer composition of the present disclosure as a protein stabilizer. On the other hand, Comparative Examples 2-1 to 2-4, which did not have a vinyl polymer (A) or had an aqueous solution whose surface tension was outside the range specified in the present disclosure, could not obtain a sufficient stabilization effect even when stored in the presence of protein.

[0253] <Evaluation of the Sensitizing Effect of Biocompatible Polymer Compositions as Blocking Agents>

[0254] [Example 3-1 to Example 3-22] [Comparative Example 3-1 to Comparative Example 3-4]

[0255] Each compound was dissolved in PBS at a composition ratio shown in Table 2 so that the solid content concentration was 1% by mass. The sensitization effect was evaluated as follows, and the difference in absorbance was used as a reference. The results are shown in Table 4.

[0256] [Antigen-antibody reaction test]

[0257] (Solid-phase antibody treatment of well plates)

[0258] The anti-human serum albumin (HSA) antibody as a solid phase antibody was adjusted to a concentration of 3 μg / ml using a PBS solution. The solid phase antibody solution was added to a polystyrene 96-well plate in units of 100 μl, and after standing at 4°C overnight, the solution was removed by suction, and then PBS-T solution was added in units of 200 μl and removed by suction, and the above operation was repeated 3 times.

[0259] (Closed Processing)

[0260] The adjusted blocking agent was added to the antibody-treated 96-well plate in units of 200 μl, and after standing at room temperature for 2 hours, the solution was removed by suction, and then PBS-T solution was added in units of 200 μl and removed by suction, and the operation was repeated 3 times.

[0261] (Antigen adsorption treatment)

[0262] The concentration of HSA as an antigen was adjusted using a PBS solution to 0 ng / ml, 25 ng / ml, 50 ng / ml, and 100 ng / ml. The HSA antigen solution was added to the 96-well plate treated with the solid phase antibody (and blocked) in units of 100 μl, and after standing at room temperature for 1 hour, the solution was removed by suction, and then PBS-T solution was added in units of 200 μl and removed by suction, and the operation was repeated 3 times.

[0263] (Labeled antibody adsorption treatment)

[0264] A80-129P (anti-human albumin antibody) as an HRP-labeled antibody was adjusted to 20 ng / ml using a PBS solution. The HRP-labeled HSA antibody solution was added to the 96-well plate after the blocking treatment (and antigen adsorption treatment) in units of 100 μl, and after standing at room temperature for 1 hour, the solution was removed by suction, and then PBS-T solution was added in units of 200 μl and removed by suction, and the above operation was repeated 3 times.

[0265] (Color reaction)

[0266] ABTS solution was added to the 96-well plate treated with the labeled antibody adsorption in units of 100 μl, and after standing at room temperature for 1 hour, 100 μl of stop solution was added to each well.

[0267] (Absorbance measurement)

[0268] The absorbance at 405 nm when the HSA antigen concentration was 100 ng / ml (absorbance A) and the absorbance at 405 nm when the HSA antigen concentration was 0 ng / ml (absorbance B) were measured. The enhancement effect when the biocompatible polymer composition was used as a blocking agent for ELISA was evaluated according to the following criteria.

[0269] [Evaluation criteria]

[0270] ◎: 1.5≦(Absorbance A-Absorbance B): Good

[0271] ○: 0.8<(Absorbance A-Absorbance B)<1.5: Usable

[0272] ×: (Absorbance A - Absorbance B) ≤ 0.8: Not usable

[0273] [Table 4]

[0274] Table 4 Sensitization effect Example 3-1 Sealing agent composition 1 ◎ Example 3-2 Sealing agent composition 2 ◎ Example 3-3 Sealing agent composition 3 ◎ Embodiment 3-4 Sealing agent composition 4 ◎ Embodiment 3-5 Sealing agent composition 5 ◎ Embodiment 3-6 Sealing agent composition 6 ◎ Embodiment 3-7 Sealing agent composition 7 ◎ Embodiment 3-8 Sealing agent composition 8 ◎ Embodiment 3-9 Sealing agent composition 9 ◎ Embodiment 3-10 Sealing agent composition 10 ◎ Example 3-11 Sealing agent composition 11 ◎ Example 3-12 Sealing agent composition 12 ◎ Example 3-13 Sealing agent composition 13 ◎ Embodiment 3-14 Sealing agent composition 14 ◎ Example 3-15 Sealing agent composition 15 ◎ Example 3-16 Sealing agent composition 16 ◎ Example 3-17 Sealing agent composition 17 ◎ Example 3-18 Sealing agent composition 18 ◎ Example 3-19 Sealing agent composition 19 ○ Example 3-20 Sealing agent composition 20 ○ Example 3-21 Sealing agent composition 21 ○ Example 3-22 Sealing agent composition 22 ◎ Comparative Example 3-1 Sealing agent composition 24 × Comparative Example 3-2 Sealing agent composition 25 × Comparative Example 3-3 Sealing agent composition 26 × Comparative Examples 3-4 Sealing agent composition 27 ×

[0275] According to the results in Table 4, the biocompatible polymer composition of the present disclosure was used as a blocking agent to obtain a sensitization effect in the ELISA test. On the other hand, Comparative Examples 3-1 and 3-4, which did not have a vinyl polymer (A) or had an aqueous solution with a surface tension outside the range specified in the present disclosure, could not obtain a sufficient sensitization effect as a blocking agent.

[0276] <Cell culture test of biocompatible polymer composition>

[0277] [Example 4-1 to Example 4-22] [Comparative Example 4-1 to Comparative Example 4-4]

[0278] Each compound was dissolved in a cell culture medium according to the composition ratio shown in Table 2, and the following evaluations were performed. The results are shown in Table 5. The following (1) evaluation of the production of physiologically active substances and (2) evaluation of cell activity were performed by adding a predetermined amount of a cell culture medium additive to each basal medium and pipetting to adjust the medium composition, and the evaluations were performed within the range of a final concentration of 0.01 mass % to 1 mass %.

[0279] (1) Evaluation of the production amount of physiologically active substances

[0280] <Evaluation of Antibody Production>

[0281] The culture medium uses Dynamis Medium (manufactured by Gibco), and L-Glutamin is added in a manner of 1.0 mass %. Hereafter, it is referred to as the basal medium. Two groups are prepared, and a cell culture medium additive containing the obtained biocompatible polymer composition is added in a manner of the concentration described in Table 5. In two culture media with different concentrations of cell culture medium additives containing biocompatible polymer compositions, CHO cell lines that introduce IgG genes and secrete IgG antibodies are added to prepare a cell suspension with a CHO cell line concentration of 1,000,000 cells / mL (cells / mL). 20 mL of the obtained cell suspension was sown into a 125 mL conical flask and cultured at 37 ° C. During the culture process, 15 mL of the supernatant was recovered once 3 to 4 days before the nutrient source was exhausted, replaced with 15 mL of new basal culture medium, and the operation was repeated 5 times. The cell culture fluid after repeated 5 medium changes was centrifuged to recover the culture medium supernatant. The amount of antibody in the culture medium was determined using a human IgG ELISA Quantitation Set manufactured by Bethyl Laboratories, Inc. In addition, the amount of DNA in the cell population after sedimentation was also quantified to calculate the antibody production per unit DNA amount. The score when the cell culture medium additive containing the biocompatible polymer composition is not added and cultured is set to 1, and the IgG antibody production is determined by the relative value at this time. The evaluation results are shown in Table 5.

[0282] ◎: 1.3 or above (very good)

[0283] ○: 1 or more to less than 1.3 (good)

[0284] ×: Less than 1 (bad)

[0285] <Evaluation of albumin production>

[0286] In 10% FBS-DMEM medium, a cell culture medium additive containing a biocompatible polymer composition is added in a manner to become the concentration described in Table 5, and stirred to prepare a medium composition. Human liver cancer cell line HepG2 cells are sown in a manner to become 250,000 cells / mL in a medium composition to which the cell culture medium additive containing a biocompatible polymer composition is added, and then dispensed into the wells of a 96-well U-bottom microplate in a manner to become 200 μL per well. Thereafter, this plate is cultured in a CO2 incubator (incubator) (37°C, 5% CO2) in a static state for 3 days. The culture fluid containing hepatocytes after 3 days of culture is recovered and centrifuged to recover the culture medium supernatant. The amount of albumin in the culture medium is determined using the Albumin ELISA Quantitation Set manufactured by Bethyl Laboratories, Inc. In addition, the amount of DNA in the cell population after sedimentation is also quantified to calculate the albumin production per unit amount of DNA. The score when the cells were cultured without adding a cell culture medium additive containing a biocompatible polymer composition was set as 1, and the albumin production was determined based on the relative value at this time.

[0287] ◎: 1.2 or above (very good)

[0288] ○: 1 or more to less than 1.2 (good)

[0289] ×: Less than 1 (bad)

[0290] <Evaluation of drug metabolizing enzyme activity>

[0291] A cell culture medium additive containing a biocompatible polymer composition is added to a 10% FBS-DMEM medium in a concentration as shown in Table 5, and the mixture is stirred to prepare a medium composition. Human liver cancer cell line HepG2 cells are seeded at 250,000 cells / mL in the medium composition to which the cell culture medium additive containing the biocompatible polymer composition is added, and then dispensed into the wells of a 96-well U-bottom microplate at 200 μL per well. Thereafter, the plate is cultured in a CO2 incubator (37°C, 5% CO2) in a static state for 3 days. After culturing for 3 days, the activity of drug metabolizing enzymes is evaluated. The activity of drug metabolizing enzymes is measured using P450-Glo TM CYP3A4 Assay Kit (P450-Glo TMCYP3A4 Assay Kits) (manufactured by Promega) was used for evaluation, and luciferin-IPA was used as the matrix. The culture medium was replaced with 10% FBS-DMEM medium containing the matrix and cultured for 4 hours. The cultured culture medium was transferred to a black plate for luminescence measurement, and the detection reagent (luciferin detection reagent) was added, and it was allowed to stand at room temperature for 20 minutes. The luminescence amount of the plate was measured using a plate reader (manufactured by Berthold). In addition, the amount of DNA in the cell population was also quantified, and the drug metabolizing enzyme activity value per unit amount of DNA was calculated. The score when the culture was carried out without adding a cell culture medium additive containing a biocompatible polymer composition was set to 1, and the drug metabolizing enzyme activity was determined by the relative value at this time. The evaluation results are shown in Table 5.

[0292] ◎: 1.2 or above (very good)

[0293] ○: 1.0 or more and less than 1.2 (good)

[0294] ×: Less than 1 (bad)

[0295] <Gene expression analysis based on quantitative reverse transcription-polymerase chain reaction (qRT-PCR)>

[0296] In 10% FBS-DMEM medium, a cell culture medium additive containing a biocompatible polymer composition is added in a manner to become the concentration described in Table 5, and stirred to prepare a medium composition. Human liver cancer cell line HepG2 cells are sown in a manner to become 250,000 cells / mL in a medium composition to which the cell culture medium additive containing the biocompatible polymer composition is added, and then dispensed into the wells of a 96-well U-bottom microplate in a manner to become 200 μL per well. Thereafter, the plate is cultured in a CO2 incubator (37°C, 5% CO2) in a static state for 3 days. The culture fluid containing hepatocytes after 3 days of culture is recovered and centrifuged to recover the cells. All ribonucleic acids (RNA) are extracted from the recovered HepG2 cells using the RNeasy Micro Kit (manufactured by QIAGEN). Complementary DNA (cDNA) was synthesized from all extracted RNA by reverse transcription reaction using ReeverTra Ace qPCR RT Master Mix (manufactured by TOYOBO). qRT-PCR was performed using TaqMan probes (manufactured by Life Technologies, CYP3A4: Hs00604506_m1, Albumin: Hs00609411_m1) of cDNA and target genes (CYP3A4 and Albumin). GAPDH (manufactured by Life Technologies, Hs02786624_g1) was used as a reference gene. The relative expression level of each gene was calculated using the cycle threshold (Ct) method with GAPDH as the reference. The result when culturing without adding a cell culture medium additive containing a biocompatible polymer composition was set to 1, and the gene expression was determined by the relative value at this time. Table 5 shows the evaluation results.

[0297] ◎: 2 or more (very good)

[0298] ○: 1 or more and less than 2 (good)

[0299] ×: Less than 1 (bad)

[0300] (2) Evaluation of cell activity

[0301] <Adenosine triphosphate (ATP) activity>

[0302] In 10% FBS-DMEM medium, a cell culture medium additive containing a biocompatible polymer composition was added to the concentration described in Table 5, and stirred to prepare a medium composition. Human liver cancer cell line HepG2 cells were sown in a medium composition to which the cell culture medium additive containing a biocompatible polymer composition was added in a manner of 100,000 cells / mL, and then dispensed into the wells of a 96-well U-bottom microplate in a manner of 100 μL per well. Thereafter, the plate was cultured in a CO2 incubator (37°C, 5% CO2) in a static state for 5 days.

[0303] ATP activity was evaluated by performing ATP analysis 1 day and 5 days after the start of culture. Specifically, 100 μL of ATP reagent ("block" ATP assay reagent: manufactured by TOYO-B-Net) was added to the wells after culture, and 5 pipettings were performed. After standing at room temperature for 5 minutes, 100 μL of the obtained reagent-cell lysate mixture was aliquoted onto another plate and stirred for 1 minute. On the plate, the luminescence was measured using Mithras LB940 (manufactured by Berthold). The evaluation results are shown in Table 5.

[0304] ATP activity = (luminescence after 5 days of culture) / (luminescence after 1 day of culture) × 100

[0305] ◎: 150% ≤ ATP activity, ATP activity is quite high. (Very good)

[0306] ○: 100% ≤ ATP activity < 150%, ATP activity is high. (Good)

[0307] △: 50% ≤ ATP activity < 100%, ATP activity is slightly higher. (OK)

[0308] ×: ATP activity <50%, ATP activity is low. (Poor)

[0309] [Table 5]

[0310]

[0311] According to the results in Table 5, the cell culture medium additives of Examples 4-1 to 4-22 showed excellent antibody production or albumin production, and the activity of drug metabolizing enzymes, etc. On the other hand, the additives of Comparative Examples 4-1 to 4-4 showed that the antibody production, albumin production, and ATP activity were all inferior to those of the cell culture medium additives of Examples 4-1 to 4-22.

[0312] The biocompatible polymer composition of the present disclosure can be used as a protein stabilizer that can stably store the protein without impairing the function of the protein by coexisting with the protein.

[0313] In addition, by using it as a blocking agent, analysis utilizing antigen-antibody reaction such as Western blotting, immunohistostaining, ELISA, and immunochromatography can be performed with high sensitivity.

[0314] Furthermore, by adding it to a cell culture medium, the production of physiologically active substances can be increased, which is also useful in terms of reducing production costs and labor. For example, it can make a great contribution to the mass supply of biopharmaceuticals such as antibody pharmaceuticals.

Claims

1. A biocompatible polymer composition comprising a vinyl polymer (A) having an amine oxide group and a mass average molecular weight of 5,000 or more and water, wherein the surface tension of a 1% aqueous solution at 25° C. is 30 dyn / cm to 50 dyn / cm.

2. The biocompatible polymer composition according to claim 1, wherein the osmotic pressure of the 1% aqueous solution is 1 mOsm / kg to 20 mOsm / kg.

3. The biocompatible polymer composition according to claim 1 or 2, further comprising a compound (B) having a zwitterionic structure and a molecular weight of 500 or less (wherein, Surfactants and monomers are excluded). 4 . The biocompatible polymer composition according to claim 3 , wherein the ratio of the compound (B) is 0.5 to 10 parts per 100 parts of the vinyl polymer (A).

5. The biocompatible polymer composition according to any one of claims 1 to 4, wherein the vinyl polymer (A) has at least one structure represented by the following general formula 1 to general formula 3: [Chemistry 1] Formula 1 [Chemistry 2] Formula 2 [Chemistry 3] Formula 3 [In general formulas 1 to 3, X represents a divalent bonding group, y represents 0 or 1, R 1 represents an alkylene group having 1 to 6 carbon atoms, R 2 and R 3 each independently represents an alkyl group having 1 to 4 carbon atoms, R 4 represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms, R 5 ~R 9 The four of them are independently a hydrogen atom or an alkyl group having 1 to 6 carbon atoms, and R 5 ~R 9 One of the following represents a bonding position to the main chain of the vinyl polymer (A), * represents the bonding position to the main chain of the vinyl polymer (A).

6. The biocompatible polymer composition according to claim 3 or 4, wherein compound (B) has at least one structure represented by the following general formula 4 to general formula 9, Formula 4 [Chemistry 4] Formula 5 [Chemistry 5] Formula 6 [Chemistry 6] Formula 7 [Chemistry 7] Formula 8 [Chemistry 8] Formula 9 [Chemistry 9] [In general formulas 4 to 9, R 11 , R 12 , R 13 Each independently represents an alkyl group or a hydroxyalkyl group having 1 to 5 carbon atoms; R 11 , R 12 and R 13 Any two of them can be connected to form a ring; R 14 represents an alkylene group having 1 to 4 carbon atoms, Z stands for -COO - or SO3 - , R 15 represents a hydrogen atom or a methyl group, R 16 represents an alkylene group having 1 to 4 carbon atoms, R 17 ~R 21 represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms, R 22 represents an alkylene group having 1 to 4 carbon atoms].

7. A protein stabilizer comprising the biocompatible polymer composition according to any one of claims 1 to 6.

8. A method for stabilizing a protein, characterized in that: The protein stabilizer according to claim 7 is allowed to coexist with the protein.

9. A sealing agent comprising the biocompatible polymer composition according to any one of claims 1 to 6.

10. A method for detecting a target substance in a test object based on in vitro diagnosis, using the blocking agent according to claim 9.

11. A cell culture medium additive comprising the biocompatible polymer composition according to any one of claims 1 to 6. 12 . A cell culture medium comprising the cell culture medium additive according to claim 11 . 13 . The cell culture medium according to claim 12 , wherein the concentration of the biocompatible polymer composition in the cell culture medium is 0.01% by mass or more and 1% by mass or less.

14. A method for producing a cell aggregate or a physiologically active substance, characterized in that: Cells are cultured using the cell culture medium according to claim 12 or 13.

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