Composition for inhibiting denaturation of protein

Abstract

The present invention provides a composition for inhibiting the denaturation of a protein, with which the denaturation of a wide range of proteins can be inhibited. The present invention relates to a composition for inhibiting the denaturation of a protein, the composition comprising: at least one carbohydrate selected from the group consisting of trehalose, sucrose, maltitol, and isomaltitol; and at least one amine compound selected from the group consisting of glutamic acid, aspartic acid, arginine, proline, serine, choline, and salts thereof.

Description

Composition for inhibiting protein denaturation

[0001] The present invention relates to a composition for inhibiting protein denaturation.

[0002] Proteins are widely used in fields such as therapeutic drugs, diagnostic agents, vaccines, and foods. However, proteins are easily denatured when used alone, and their function is impaired when exposed to heat, long-term storage, or repeated freeze-thaw cycles. Attempts have been made to improve protein-containing compositions in order to prevent protein denaturation.

[0003] It has been known that carbohydrates and amino acids can suppress protein denaturation. For example, Patent Document 1 discloses a method for stabilizing an Fc variant protein using carbohydrates, cationic amino acids, anions, and polysorbates. Patent Document 2 discloses a method for stabilizing an anti-TNFα antibody using a polyol, a surfactant, and arginine. However, the types of proteins that can be stabilized by these methods are limited.

[0004] JP 2009-525986 A JP 2022-097600 A

[0005] An object of the present invention is to provide a composition for inhibiting the denaturation of proteins, which can inhibit the denaturation of a wide range of proteins.

[0006] The present inventors have discovered that protein denaturation can be suppressed by combining a specific carbohydrate with an amine compound, and have completed the present invention.

[0007] That is, the present invention relates to a composition for inhibiting protein denaturation, which comprises one or more carbohydrates selected from the group consisting of trehalose, sucrose, maltitol, and isomaltitol, and one or more amine compounds selected from the group consisting of glutamic acid, aspartic acid, arginine, proline, serine, choline, and salts thereof.

[0008] The composition for inhibiting protein denaturation preferably inhibits protein denaturation due to heating or freeze-thawing.

[0009] The composition for inhibiting protein denaturation preferably inhibits protein aggregation.

[0010] The protein is preferably one or more selected from the group consisting of an antibody, a cytokine, an allergen, and an enzyme.

[0011] The protein is preferably one or more selected from the group consisting of anti-IFNα antibody, anti-IFNγ antibody, anti-TNFα antibody, anti-IL-17A antibody, and anti-IL-18 antibody.

[0012] The protein is preferably one or more selected from the group consisting of IFNα, IFNγ, IL-18, and TNFα.

[0013] The protein is preferably a cedar pollen allergen.

[0014] The protein is preferably one or more selected from the group consisting of lactate dehydrogenase, glycosyltransferase, glycolytic enzyme, malate dehydrogenase, and protease.

[0015] The protein is preferably a virus-derived protein or a bacterial-derived protein.

[0016] The protein is preferably a blood coagulation factor or albumin.

[0017] The molar ratio of carbohydrate to amine compound in the composition for inhibiting protein denaturation is preferably 40:1 to 1:40.

[0018] The composition for inhibiting protein denaturation is preferably in a liquid form, and the total concentration of the carbohydrate and the amine compound in the composition is preferably 0.20 mol / L to 3.50 mol / L.

[0019] The composition for inhibiting protein denaturation preferably inhibits protein denaturation synergistically with the protein denaturation inhibiting effect of the carbohydrate and the protein denaturation inhibiting effect of the amine compound.

[0020] The present invention also relates to a method for inhibiting protein denaturation, which comprises bringing a protein into the presence of the composition for inhibiting protein denaturation.

[0021] The present invention also relates to a protein preparation comprising a protein and the composition for inhibiting protein denaturation.

[0022] The present invention also relates to a method for inhibiting the denaturation of a protein, which comprises causing the protein to coexist with one or more carbohydrates selected from the group consisting of trehalose, sucrose, maltitol, and isomaltitol, and one or more amine compounds selected from the group consisting of glutamic acid, aspartic acid, arginine, proline, serine, choline, and salts thereof.

[0023] The present invention also relates to a protein preparation comprising one or more carbohydrates selected from the group consisting of trehalose, sucrose, maltitol, and isomaltitol; one or more amine compounds selected from the group consisting of glutamic acid, aspartic acid, arginine, proline, serine, choline, and salts thereof; and a protein.

[0024] The composition for inhibiting protein denaturation of the present invention can inhibit the denaturation of a wide range of proteins.

[0025] <<Composition for Inhibiting Protein Denaturation>> The present invention relates to a composition for inhibiting protein denaturation, comprising one or more carbohydrates selected from the group consisting of trehalose, sucrose, maltitol, and isomaltitol, and one or more amine compounds selected from the group consisting of glutamic acid, aspartic acid, arginine, proline, serine, choline, and salts thereof. The combination of the carbohydrate and the amine compound can inhibit the denaturation of a wide range of proteins.

[0026] <Carbohydrates> The carbohydrates used in the present invention are selected from the group consisting of trehalose, sucrose, maltitol, and isomaltitol. These carbohydrates may be used alone or in combination of two or more. Among these, trehalose, sucrose, and maltitol are preferred, with trehalose being more preferred. These carbohydrates may also be derivatives in which substituents have been introduced into the hydrogen atoms of some of the hydroxyl groups. Examples of the substituents include carboxylic acid esters such as acetate esters and benzoic acid; sulfate esters; fatty acid esters such as laurate esters, myristate esters, palmitate esters, stearates, oleate esters, linoleate esters, and linolenate esters; and ethers such as methyl ether, benzyl ether, trityl ether, methylsilyl ether, and dodecyl ether.

[0027] The concentration of the carbohydrate in the composition for inhibiting protein denaturation is preferably 0.10 to 3.00 mol / L, more preferably 0.40 to 1.60 mol / L, and even more preferably 0.60 to 1.20 mol / L.

[0028] Furthermore, when the composition for inhibiting protein denaturation is mixed with a protein to obtain a protein preparation, the concentration of carbohydrates in the protein preparation is preferably 0.10 to 3.00 mol / L, more preferably 0.40 to 1.60 mol / L, and even more preferably 0.60 to 1.20 mol / L.

[0029] In the present invention, carbohydrates, as hydrogen bond donors, may form NADES (Natural Deep Eutectic Solvent) with amine compounds, as hydrogen bond acceptors, to suppress protein denaturation and stabilize the protein; however, the present invention is not limited to this mechanism.

[0030] <Amine Compound> The amine compound in the present invention is selected from the group consisting of glutamic acid, aspartic acid, arginine, proline, serine, choline, and salts thereof. Examples of the salts constituting the amine compound include sodium salts, potassium salts, hydrochlorides, chlorides, etc.

[0031] These amine compounds may be used alone or in combination of two or more. Among these, glutamic acid, aspartic acid, arginine, proline, serine, and salts thereof are preferred, and glutamic acid, aspartic acid, arginine, and salts thereof are more preferred. In the present invention, the amine compound may function as a hydrogen bond acceptor when forming NADES, but the present invention is not limited to this mechanism.

[0032] The combination of carbohydrate and amine compound is not particularly limited, but the combination of trehalose and an amine compound selected from the group consisting of arginine, aspartic acid, serine, and glutamic acid, or a salt thereof; the combination of sucrose and an amine compound selected from the group consisting of choline, arginine, and glutamic acid, or a salt thereof; the combination of maltitol and an amine compound selected from the group consisting of arginine, proline, and glutamic acid, or a salt thereof; the combination of isomaltitol and arginine, or a salt thereof, is more preferred, and the combination of maltitol and potassium glutamate or sodium glutamate, trehalose and potassium glutamate or sodium glutamate, trehalose and potassium aspartate or sodium aspartate, or maltitol and arginine hydrochloride is even more preferred, and the combination of maltitol and sodium glutamate, trehalose and sodium glutamate, or trehalose and sodium aspartate is even more preferred. These combinations are likely to achieve the effect of inhibiting protein denaturation.

[0033] The concentration of the amine compound in the composition for inhibiting protein denaturation is preferably 0.06 to 3.30 mol / L, more preferably 0.13 to 2.60 mol / L, and particularly preferably 0.21 to 1.90 mol / L.

[0034] Furthermore, when the composition for inhibiting protein denaturation is mixed with a protein to obtain a protein preparation, the concentration of the amine compound in the protein preparation is preferably 0.06 mol / L to 3.30 mol / L by weight, more preferably 0.13 to 2.60 mol / L, and particularly preferably 0.21 to 1.90 mol / L.

[0035] In the composition for inhibiting protein denaturation, the molar ratio of carbohydrate to amine compound is preferably 40:1 to 1:40, more preferably 20:1 to 1:20, and even more preferably 10:1 to 1:10. If the amount of carbohydrate is greater or less than the molar ratio of carbohydrate to amine compound of 40:1 to 1:40, the molar concentration of carbohydrate will be too high or too low, and the protein denaturation inhibitory effect will tend not to be observed.

[0036] When the composition for inhibiting protein denaturation is in a liquid form, the total concentration of the carbohydrate and the amine compound is preferably 0.20 to 3.50 mol / L, more preferably 0.80 to 3.00 mol / L, and even more preferably 1.00 to 2.50 mol / L. When the composition for inhibiting protein denaturation is in a solid form, the total concentration of the carbohydrate and the amine compound is preferably 0.05 to 3.50 mol / kg, more preferably 0.10 to 2.00 mol / kg, and even more preferably 0.20 to 1.50 mol / kg.

[0037] <Form of composition for inhibiting denaturation> The form of the composition for inhibiting protein denaturation is not particularly limited, and may be either liquid or solid. Examples of liquid forms include aqueous solutions, suspensions, and slurries. Examples of solid forms include powders, granules, and tablets. Among these, the liquid form is preferred from the viewpoints of ease of production and storage stability.

[0038] When the composition for inhibiting protein denaturation is in liquid form, its pH is preferably 4 to 9, more preferably 5 to 8. Under these pH conditions, there is little possibility of precipitation and the activity of the protein is also little affected. The pH of the composition for inhibiting protein denaturation can be adjusted with an acid such as hydrochloric acid or sulfuric acid, or a base such as sodium hydroxide or potassium hydroxide.

[0039] The composition for inhibiting protein denaturation can be produced by mixing the components in any order. After mixing the components, the mixture may be brought into contact with a porous material or passed through a filter to be filtered or sterilized.

[0040] <Optional Components> In addition to the carbohydrate and the amine compound, the composition for inhibiting protein denaturation may optionally contain components such as a pH adjuster, a surfactant, a thickener, an emulsifier, inorganic salts, a preservative, a solvent, an excipient, metals, a filter aid, etc. The content of these components is not particularly limited and can be selected at will by those skilled in the art, but can be, for example, 0.001 to 90 wt % of the composition for inhibiting protein denaturation, preferably 0.01 to 50 wt %, and more preferably 0.1 to 30 wt %.

[0041] Examples of pH adjusters include ascorbic acid, acetic acid, dehydroacetic acid, lactic acid, citric acid, gluconic acid, succinic acid, tartaric acid, fumaric acid, malic acid, and adipic acid, as well as sodium (Na), calcium (Ca), and potassium (K) salts of these organic acids, as well as carbonic acid, phosphoric acid, and pyrophosphoric acid, as well as Na and K salts of these inorganic acids.

[0042] Examples of surfactants include nonionic surfactants, anionic surfactants, and cationic surfactants. Specific examples of nonionic surfactants include polysorbate 20, polysorbate 80, polyoxyethylene sorbitan fatty acid esters, sorbitan monolaurate, sorbitan monooleate, polyoxyethylene polyoxypropylene glycol, and polyoxyethylene sorbitan monooleate. Specific examples of anionic surfactants include dioctanoyl sodium sulfate and sodium lauryl sulfate. Among these, nonionic surfactants are preferred, with polysorbate 20 and polysorbate 80 being more preferred. The concentration of the surfactant in the composition for inhibiting protein denaturation is preferably 0.005 to 5 (w / v)%, more preferably 0.01 to 3 (w / v)%, and even more preferably 0.05 to 1 (w / v)%.

[0043] Examples of thickeners and excipients include gums, alginic acid, alginic acid derivatives, pectin, carrageenan, curdlan, pullulan, gelatin, cellulose derivatives, agar, tamarind, psyllium, glucomannan, polyethylene glycol, mineral oil, and silicone oil.

[0044] Examples of emulsifiers include glycerin fatty acid esters, polyglycerin fatty acid esters, sucrose fatty acid esters, propylene glycol fatty acid esters, sorbitan fatty acid esters, lecithin, enzymatically hydrolyzed lecithin, and saponin.

[0045] Examples of inorganic salts include sodium chloride, ammonium sulfate, sodium sulfate, calcium chloride, and polymerized phosphates.

[0046] Preservatives include propionic acid, propionate salts, sulfite salts, benzoate salts, sorbic acid, sorbate salts, polylysine, glycine, acetate salts, etc. Salts include sodium (Na) salts, calcium (Ca) salts, and potassium (K) salts, etc.

[0047] Examples of the solvent include water, ethanol, methanol, isopropanol, etc. The solvent may contain a buffer component such as phosphoric acid, citric acid, glycine, or Tris.

[0048] Excipients include hyaluronic acid, methylcellulose, carboxymethylcellulose, hydroxypropylmethylcellulose, polyvinyl alcohol, polyethylene glycol, triglycerides, glycerides, propylene glycol, polyvinylpyrrolidone, and the like.

[0049] <Evaluation of Protein Denaturation Inhibition> Protein denaturation inhibition can be evaluated by the following method. That is, a protein preparation containing the protein denaturation inhibition composition of the present invention is denatured, and protein activity (A) is measured. In addition, a control is subjected to the same denaturation treatment, and protein activity (B) is measured. When A exceeds B, it can be determined that protein denaturation has been inhibited. An example of the control is a preparation containing the same protein as the test sample but not containing the denaturation inhibition composition.

[0050] When evaluating the inhibition of protein denaturation, examples of denaturing treatments for protein formulations include heating and freeze-thawing. Heating conditions are preferably 30 to 100°C, more preferably 35 to 75°C, and even more preferably 37°C, 40°C, 70°C, or 73°C. Heating time is preferably 1 hour to 3 days, more preferably 1 to 24 hours, even more preferably 1 to 6 hours, and even more preferably 2 hours or 5 hours. Freezing and thawing conditions include, for example, freezing at -80°C for 1.5 to 2 hours and thawing at room temperature (approximately 20°C) repeated 10 times.

[0051] Methods for measuring protein activity include methods that utilize antigen-antibody reactions such as EIA and ELISA, protein aggregation assays, measurement of protein enzymatic activity, etc. When competitive EIA or competitive ELISA is used to measure protein activity, protein activity is measured as a competitive inhibition rate (%).

[0052] When protein activity is measured by a method utilizing an antigen-antibody reaction, such as EIA or ELISA, the ratio A / B of protein activity (A) to protein activity (B) is greater than 1, preferably 1.05 or more, more preferably 1.1 or more, even more preferably 1.2 or more, still more preferably 1.5 or more, and particularly preferably 2 or more, 5 or more, 10 or more, or 100 or more.

[0053] When protein activity is measured by a protein aggregation assay, the ratio A / B of protein activity (A) to protein activity (B) is preferably less than 1, more preferably 0.5 or less, even more preferably 0.3 or less, and even more preferably 0.1 or less.

[0054] When protein activity is evaluated by enzyme activity, the ratio A / B of protein activity (A) to protein activity (B) is greater than 1, preferably 1.05 or greater, more preferably 1.1 or greater, even more preferably 1.2 or greater, even more preferably 1.5 or greater, and particularly preferably 2 or greater, 5 or greater, 10 or greater, or 100 or greater.

[0055] It is preferable that the composition for inhibiting protein denaturation inhibits protein denaturation in a synergistic manner with the protein denaturation inhibiting effect of the carbohydrate and the protein denaturation inhibiting effect of the amine compound.

[0056] Whether or not the inhibition of protein denaturation is synergistic can be evaluated by the following method. That is, a protein preparation containing the composition for inhibiting protein denaturation of the present invention is subjected to a denaturing treatment, and the protein activity (A) is measured. In addition, as a comparative control 1, a preparation containing the same protein and carbohydrate as the test sample but not containing an amine compound is subjected to the same denaturing treatment, and the protein activity (B) is measured. １ As a comparative control 2, a preparation containing the same protein and amine compound as the test sample but not containing carbohydrates was subjected to the same denaturation treatment, and the protein activity (B ２ ) is measured. Then, A>B １ And A>B ２ When A≧B, it can be determined that the inhibition of protein denaturation is synergistic. １ ×1.5 and A≧B ２ × 1.5, and A≧B １ ×2 and A≧B ２ ×2, and A≧B １ +B ２ It is more preferable that A≧(B １ +B ２ ) × 2, and A ≥ (B １ +B ２ ) × 10 is particularly preferred.

[0057] The composition for inhibiting protein denaturation can inhibit protein denaturation even after the above-mentioned denaturation treatment, and therefore allows stable storage of proteins for long periods of time under mild conditions that are less likely to cause denaturation. The storage temperature of a protein preparation containing the composition for inhibiting protein denaturation of the present invention is preferably 4 to 40°C. The relative humidity during storage of the protein preparation is preferably 0 to 75%. Furthermore, the storage period of the protein preparation at 4°C is preferably one year or more, and more preferably two years or more.

[0058] <<Protein Preparation>> The protein preparation of the present invention is characterized by comprising a protein and the composition for inhibiting protein denaturation.

[0059] <Protein> In the present invention, the protein whose denaturation is inhibited is not particularly limited, and the denaturation of proteins commonly used as antibodies, cytokines, allergens, enzymes, blood coagulation and fibrinolytic factors, hormones, etc. can be inhibited.

[0060] The antibody is not particularly limited as long as it can specifically bind to an antigen, and may be any of IgG, IgA, IgM, IgD, and IgE. Furthermore, the animal from which the antibody is derived is not particularly limited, and examples thereof include humans and non-human animals such as mice, rats, rabbits, sheep, goats, and chickens. The antibody may be a chimeric antibody that combines a sequence derived from a non-human animal with a sequence derived from a human, a humanized antibody, or a fully human antibody. Furthermore, the antibody may include antibodies that contain a complete constant region and a complete variable region, as well as minibodies, Fabs, diabodies, Fab's, triabodies, scFv-Fc, scFvs, and F(ab's). ２ , Fv, etc. The antibody may be conjugated with a compound other than an antibody, such as a nucleic acid, a labeled compound, or a low-molecular-weight drug. The antigen to which the antibody specifically binds is not particularly limited, and examples include nucleic acids, proteins, and sugars. Specific examples of antigens include IFNα, IFNγ, TNFα, IL-18, IL-17, and leukocyte surface antigen proteins.

[0061] Examples of cytokines include IFNs such as IFNα, IFNβ, IFNγ, IFNδ, IFNω, IFNλ, IFNε, and IFNκ; TNFs such as TNFα and TNFβ; interleukins (ILs) such as IL1, IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL11, IL12, IL13, IL14, IL15, IL16, IL17, and IL18; CSF, EPO, EGF, FGF, and PDGF.

[0062] Allergens are not limited to substances that are allergens in humans and non-human animals, and examples include food allergens such as shrimp, crab, walnuts, wheat, buckwheat, eggs, milk, peanuts, almonds, abalone, squid, salmon roe, oranges, cashew nuts, kiwi fruit, beef, sesame, salmon, mackerel, soybeans, chicken, bananas, pork, matsutake mushrooms, peaches, yams, apples, and gelatin; and pollen allergens such as cedar, ragweed, Japanese silver grass, and red pine.

[0063] The enzyme is not particularly limited as long as it is a protein having the activity of catalyzing a chemical reaction, and examples thereof include glycolytic enzymes such as α-amylase, β-amylase, glucoamylase, isoamylase, maltogenic amylase, pullulanase, maltotriohydrolase, dextranase, glucose isomerase, cellulase, xylanase, hemicellulase, mannanase, pectinase, pectin methylesterase, invertase, lactase, inulinase, α-galactosidase, chitinase, chitosanase, alginate lyase, glucose oxidase, and glucose dehydrogenase, cyclodextrin glucanotransferase, and transglucosidase. Examples of enzymes that can be used include glycosyltransferases such as 6-α-glucanotransferase, glucosyltransferase, galactosyltransferase, sialyltransferase, N-acetylglucosaminyltransferase, and mannosyltransferase; proteolytic enzymes such as protease, peptidase, and collagenase; amino acid degrading enzymes such as glutaminase; lipid-related enzymes such as lipase, phospholipase, and esterase; and others such as catalase, urease, tannase, deaminase, alcohol dehydrogenase, lactate dehydrogenase (LDH), malate dehydrogenase, lysosome, alkaline phosphatase, DNA degrading enzyme, and urate oxidase.

[0064] The protein concentration in the protein formulation is preferably 0.0001 to 100 mg / mL, more preferably 0.001 to 50 mg / mL, and even more preferably 0.01 to 30 mg / mL. The protein concentration can also be relatively high, for example, 1 to 100 mg / mL or 10 to 100 mg / mL. It can also be relatively low, for example, 0.0001 to 1 mg / mL or 0.0001 to 0.1 mg / mL.

[0065] The pH of the protein formulation is preferably 4 to 9, more preferably 5 to 8. Under these pH conditions, there is little possibility of precipitation and the activity of the protein is also little affected. The pH of the protein formulation can be adjusted with an acid such as hydrochloric acid or sulfuric acid, or a base such as sodium hydroxide or potassium hydroxide.

[0066] In addition to the protein and the composition for inhibiting protein denaturation, the protein preparation may optionally contain components such as pH adjusters, thickeners, emulsifiers, inorganic salts, preservatives, solvents, excipients, metals, and filter aids. Specific examples of these components are as described above. The content of these optional components is not particularly limited and can be selected by those skilled in the art. For example, the content of these optional components in the protein preparation may be 0.001 to 90 wt %, preferably 0.01 to 50 wt %, and more preferably 0.1 to 30 wt %.

[0067] Protein formulations can be produced by mixing the components in any order. After mixing the components, they may be filtered or sterilized by contact with a porous material or by passing through a filter. Alternatively, powdered protein formulations can be obtained by lyophilizing liquid protein formulations.

[0068] <<Method for inhibiting protein denaturation>> The method for inhibiting protein denaturation of the present invention is characterized by causing a protein to coexist with the composition for inhibiting protein denaturation. Protein denaturation may be inhibited by the action of NADES formed from a carbohydrate and an amine compound in the composition for inhibiting protein denaturation, but the present invention is not limited to this mechanism.

[0069] The method for causing the protein and the composition for inhibiting protein denaturation to coexist is not particularly limited, as long as the protein, carbohydrate, and amine compound coexist. It is preferable to mix a liquid composition for inhibiting protein denaturation with a solution containing the protein to obtain a protein preparation, and to cause the protein and the composition for inhibiting protein denaturation to coexist in the preparation.

[0070] The coexistence time of the protein and the composition for inhibiting protein denaturation can be determined according to the use and storage period of the protein, but is preferably 30 minutes or more, more preferably 1 hour or more. There is no particular upper limit to the coexistence time, and protein denaturation can be inhibited for a long period of time, but it is generally 2 years or less. By keeping the protein and the composition for inhibiting protein denaturation in a coexistent state, protein denaturation can be inhibited even during long-term storage.

[0071] The temperature at which the protein and the composition for inhibiting protein denaturation coexist is not particularly limited, but is preferably 0 to 40°C, and more preferably 4 to 30°C. Protein denaturation can generally be inhibited even at relatively high temperatures at which protein denaturation can occur. After the protein and the composition for inhibiting protein denaturation coexist, the protein can be optionally purified, diluted, or the like.

[0072] The protein preparations of the present invention can be used in a wide range of applications depending on the type of protein contained therein, including medical and pharmaceutical products such as antibody drugs, vaccines, diagnostic agents, hormone preparations, enzyme preparations, and in vitro diagnostic agents; food and beverage products such as nutritional supplements and seasonings; industrial enzymes, detergents, cosmetics, quasi-drugs, animal feed, plant growth regulators, and plant biopreparations.

[0073] The present invention will be described below with reference to examples, but is not limited to the following examples. Hereinafter, "parts" and "%" mean "parts by weight" and "% by weight", respectively, unless otherwise specified.

[0074] (1) Anti-human IFNγ Monoclonal Antibody Denaturation Inhibition Test A carbohydrate and an amine compound were mixed to the concentrations listed in Tables 1 to 13 to prepare a composition for inhibiting protein denaturation. This composition for inhibiting protein denaturation was mixed with a phosphate buffer solution containing a mouse monoclonal antibody against IFNγ (MAb-IFNγ-15, an in-house preparation, Lot No. 010) in a volume ratio of 9:1 in a 0.5 mL Eppendorf tube (Eppendorf, product number 3810X) to prepare a protein formulation. The antibody concentration in the protein formulation was 70 μg / mL. The protein formulation was then heated at 73°C for 5 hours.

[0075] 15 μL of the heated protein preparation was dispensed into wells of a 96-well microplate (Corning, Costar 3513), followed by 135 μL of human IFNγ (400 pg / mL) as antigen. After mixing using a 12-channel multichannel pipette (Thermo Electron, Finnpipette), 100 μL was aliquoted and added to the wells of an immunomodule plate (Thermo Scientific, U16 maxisorp Loose Nunc-Immuno Module 469264) coated with MAb-IFNγ-15, and the antigen-antibody reaction was allowed to occur at room temperature for 1.5 hours. The plate was then washed, labeled with antibody, and measured for absorbance at 450 nm according to standard procedures.

[0076] Furthermore, as a reference test, 135 μL of human IFNγ (400 pg / mL) was added to a 96-well microplate containing 15 μL of phosphate buffer, and after mixing, 100 μL of the mixture was added to a well of an immunomodule plate on which MAb-IFNγ-15 had been immobilized, and the absorbance was measured.

[0077] The competitive inhibition rate was calculated using the following formula. The results are shown in Tables 1 to 13. Competitive inhibition rate (%) = (1 - (absorbance in each Example or Comparative Example / absorbance in the reference test)) x 100

[0078]

[0079] In Comparative Example 1-1-1, the competitive inhibition rate was measured without heating the protein preparation in the absence of carbohydrates and amine compounds, resulting in a competitive inhibition rate of 87%. In Comparative Example 1-1-2, the protein preparation was heated in the absence of carbohydrates and amine compounds. As a result, the competitive inhibition rate was -5%. In Examples 1-1-1 to 1-1-6, the competitive inhibition rates were higher than in the comparative examples in which the same concentrations of carbohydrates or amine compounds were used alone. In Comparative Examples 1-1-11 to 1-1-12, in which urea was used as the amine compound, the competitive inhibition rates were low despite the combination with carbohydrates.

[0080]

[0081] In Examples 1-2-1 to 1-2-3, the competitive inhibition rate was higher than that in the comparative examples in which the same concentration of saccharide or amine compound was used alone.

[0082]

[0083] In Examples 1-3-1 to 1-3-11, the competitive inhibition rate was investigated when the molar ratio of maltitol to arginine hydrochloride was changed, based on the results of Examples 1-1-1 to 1-1-6 in Table 1. As a result, as shown in Table 3, when the molar ratio of maltitol to arginine hydrochloride was other than 1:1, a high competitive inhibition rate was observed, similar to when the molar ratio was 1:1.

[0084]

[0085] In Examples 1-4-1 to 1-4-4, the competitive inhibition rate was higher than that in the Comparative Examples in which the same concentration of saccharide or amine compound was used alone.

[0086]

[0087] In Examples 1-5-1 to 1-5-4, the total concentration of trehalose and potassium glutamate was lower than that of Example 1-4-2, but as in Example 1-4-2, the competitive inhibition rate was higher than that of the comparative example in which the same concentration of carbohydrate or amine compound was used alone.

[0088]

[0089] In Examples 1-6-1 to 1-6-4, the total concentration of sucrose and potassium glutamate was lower than in Example 1-4-1, but similar to Example 1-4-1, the competitive inhibition rate was higher than in the Comparative Examples in which the same concentration of carbohydrate or amine compound was used alone. Furthermore, among the Examples, the competitive inhibition rate tended to be higher when the total concentration of carbohydrate and amine compound was higher.

[0090]

[0091] In Examples 1-7-1 to 1-7-10, the competitive inhibition rate was examined when the molar ratio of maltitol to potassium glutamate was changed, based on the results of Examples 1-4-1 to 1-4-3 in Table 4. As a result, as shown in Table 7, when the molar ratio of maltitol to potassium glutamate was 4:1 to 1:4, a higher competitive inhibition rate was observed than in Comparative Example 1-7-2, just as when the molar ratio was 1:1. Among Examples 1-7-1 to 1-7-5 and Examples 1-7-6 to 1-7-10, where the total concentration of carbohydrate and amine compound was constant, a tendency was observed for the competitive inhibition rate to be higher when the carbohydrate concentration was higher.

[0092]

[0093] In Examples 1-8-1 to 1-8-3, similarly to Examples 1-7-1 to 1-7-10, when the molar ratio of maltitol to potassium glutamate was 1:2 to 1:4, a higher competitive inhibition rate was observed than in Comparative Example 1-8-2, similarly to when the molar ratio was 1:1. Among the Examples, a tendency was observed that the higher the concentration of the amine compound, the higher the competitive inhibition rate.

[0094]

[0095] In Examples 1-9-1 to 1-9-5, the competitive inhibition rate was higher than that in the Comparative Examples in which the same concentration of carbohydrate or amine compound was used alone. Among the Examples, the higher the concentration of the amine compound, the higher the competitive inhibition rate tended to be.

[0096]

[0097] In Examples 1-10-1 to 1-10-5, the competitive inhibition rate was higher than that in the Comparative Examples in which the same concentration of carbohydrate or amine compound was used alone. Among the Examples, the higher the concentration of the amine compound, the higher the competitive inhibition rate tended to be.

[0098]

[0099] In Examples 1-11-1 and 1-11-2, the competitive inhibition rate was higher than that in the Comparative Examples in which the same concentrations of saccharide or amine compound were used alone.

[0100]

[0101] In Examples 1-12-1 to 1-12-8, the competitive inhibition rate was higher than in Comparative Examples in which the same concentration of carbohydrate was used alone. Furthermore, when Examples 1-12-5 to 1-12-8 were carried out under the same conditions as in Examples 1-12-1 to 1-12-4, except that potassium glutamate was replaced with monosodium glutamate, the results showed the same tendency as in Examples 1-12-1 to 1-12-4. Among the Examples, the higher the total concentration of carbohydrate and amine compound, the higher the competitive inhibition rate tended to be.

[0102]

[0103] Urea has a hydrogen atom bonded to a highly electronegative nitrogen atom, and therefore can function as a hydrogen bond donor. A deep eutectic solvent consisting of urea and choline chloride, also known as Reline, has been reported to have the ability to stabilize proteins such as bovine serum albumin (Phys. Chem. Chem. Phys., 2022, 24, 5627-5637). Therefore, the antibody stabilization effect of Reline was compared with that of a protein denaturation inhibitor composition consisting of trehalose and arginine hydrochloride. As a result, Comparative Examples 1-13-3 and 1-13-4, which used compositions combining urea and choline chloride, showed low competitive inhibition rates. On the other hand, high competitive inhibition rates were observed in Examples 1-13-1 to 1-13-3, which used compositions for inhibiting protein denaturation of the present invention.

[0104] (2) Anti-human TNFα monoclonal antibody denaturation inhibition test 1 A carbohydrate and an amine compound were mixed to the concentrations listed in Table 14 to prepare a composition for inhibiting protein denaturation. This composition for inhibiting protein denaturation was mixed with a phosphate buffer solution containing a mouse monoclonal antibody against human TNFα (MAb-TNFα-5, manufactured in-house, LOT No. 006) in a volume ratio of 9:1 in a 0.5 mL Eppendorf tube to prepare a protein preparation. The antibody concentration in the protein preparation was 70 μg / mL. The protein preparation was then heated at 73°C for 5 hours.

[0105] After heating, 20 μL of the protein preparation was dispensed into wells of a 96-well plate, followed by 130 μL of human TNFα (25 pg / mL) as an antigen. After mixing using a 12-channel multichannel pipette, 100 μL was taken and added to the wells of an immunomodule plate onto which MAb-TNFα-5 had been immobilized. The antigen-antibody reaction was allowed to occur at room temperature for 1.5 hours, followed by washing, addition of a labeled antibody, and measurement of absorbance at 450 nm according to standard procedures.

[0106] As a reference test, 130 μL of human TNFα (25 pg / mL) alone was added as an antigen to a 96-well microplate containing 20 μL of phosphate buffer solution, and after mixing, 100 μL of the mixture was added to a well of an immunomodule plate on which MAb-TNFα-5 had been immobilized, and the absorbance was measured.

[0107] The competitive inhibition rate was calculated using the following formula. The results are shown in Table 14. Competitive inhibition rate (%) = (1 - (absorbance in each Example and Comparative Example / absorbance in the reference test)) x 100

[0108]

[0109] In Examples 2-1 to 2-3, the competitive inhibition rate was higher than that in the Comparative Examples in which the same concentration of carbohydrate or amine compound was used alone. Among the Examples, the competitive inhibition rate tended to be higher when the total concentration of carbohydrate and amine compound was higher.

[0110] (3) Anti-human IFNα monoclonal antibody denaturation inhibition test 2 A carbohydrate and an amine compound were mixed to the concentrations listed in Table 15 to prepare a composition for inhibiting protein denaturation. This composition for inhibiting protein denaturation was mixed with a phosphate buffer solution containing a mouse monoclonal antibody against human IFNα (MAb-IFNα-43, manufactured in-house, Lot No. 003) in a volume ratio of 9:1 in a 0.5 mL Eppendorf tube to prepare a protein preparation. The antibody concentration in the protein preparation was 70 μg / mL. The protein preparation was then heated at 73°C for 5 hours.

[0111] 20 μL of the heated protein preparation was dispensed into wells of a 96-well microplate, followed by 130 μL of human IFNα (2500 pg / mL) as an antigen. After mixing using a 12-channel multichannel pipette, 100 μL was removed and added to the wells of an immunomodule plate onto which MAb-IFNα-43 had been immobilized. The antigen-antibody reaction was allowed to occur at room temperature for 1.5 hours, followed by washing, addition of labeled antibody, and measurement of absorbance at 450 nm according to standard methods. Additionally, as a reference test, 130 μL of human IFNα (2500 pg / mL) alone was added as an antigen to a 96-well microplate containing 20 μL of phosphate buffer. After mixing, 100 μL of this mixture was added to a well of an immunomodule plate onto which an antibody against the same IFNα had been immobilized, and the absorbance was measured. The competitive inhibition rate was calculated using the following formula. The results are shown in Table 15. Competitive inhibition rate (%) = (1 - (absorbance in each Example and Comparative Example / absorbance in reference test)) x 100

[0112]

[0113] In Examples 3-1 to 3-4, the competitive inhibition rate was higher than that in the Comparative Examples in which the same concentration of carbohydrate or amine compound was used alone. Among the Examples, the competitive inhibition rate tended to be higher when the total concentration of carbohydrate and amine compound was higher.

[0114] (4) LDH Denaturation Inhibition Test A protein denaturation inhibition composition was prepared by mixing carbohydrates and amine compounds to the concentrations listed in Table 16. This protein denaturation inhibition composition was mixed with potassium phosphate buffer containing lactate dehydrogenase (LDH) (manufactured by Oriental Yeast Co., Ltd., product number 46776003) in a 0.5 mL Eppendorf tube at a volume ratio of 9:1 to prepare a protein preparation. The lactate dehydrogenase concentration in the protein preparation was 50 μg / mL. The protein preparation was then heated at 70°C for 2 hours. The enzyme activity of the heated protein preparation was measured using a kit (manufactured by Roche, product number 11644793001). The results are shown in Table 16.

[0115]

[0116] In Examples 4-1-1 to 4-1-6, the residual enzyme activity was higher than in the comparative examples in which the same concentration of saccharide or amine compound was used alone.

[0117] A composition for inhibiting protein denaturation was prepared by mixing carbohydrates and amine compounds to the concentrations listed in Table 17. This composition for inhibiting protein denaturation was mixed with potassium phosphate buffer containing lactate dehydrogenase (LDH) (manufactured by Oriental Yeast Co., Ltd., product number 46776003) in a 9:1 volume ratio in a 0.5 mL Eppendorf tube to prepare a protein preparation. The lactate dehydrogenase concentration in the protein preparation was 20 mg / mL. The protein preparation was then heated at 70°C for 2 hours. The enzyme activity of the heated protein preparation was measured using a kit (manufactured by Roche, product number 11644793001). The results are shown in Table 17.

[0118]

[0119] In Examples 4-2-1 and 4-2-2, the residual enzyme activity was higher than in the Comparative Examples in which the same concentrations of carbohydrate or amine compound were used alone. It was confirmed that the composition for inhibiting protein denaturation of the present invention exhibits the effect of inhibiting the activity decrease due to denaturation and aggregation caused by heat treatment, even for lactate dehydrogenase at a high concentration (20 mg / mL).

[0120] (5) Cedar pollen allergen denaturation inhibition test 1: A carbohydrate and an amine compound were mixed to the concentrations listed in Table 18 to prepare a composition for inhibiting protein denaturation. This protein denaturation inhibition composition was mixed with a phosphate buffer solution containing cedar pollen allergen protein (Cry j1) (manufactured in-house, Lot No. 180301) in a volume ratio of 9:1 in a 0.5 mL Eppendorf tube to prepare a protein preparation. The concentration of cedar pollen allergen protein in the protein preparation was 8 μg / mL. The protein preparation was then heated at 40°C for 23 hours. The amount of non-aggregated protein (ng / mL) in the heated protein preparation was measured using Cry j1 ELISA (manufactured in-house). The results are shown in Table 18.

[0121]

[0122] In Example 5-1, the amount of non-aggregated protein was greater than in the comparative example in which the same concentration of carbohydrate or amine compound was used alone.

[0123] (6) Cedar pollen allergen denaturation inhibition test 2: A carbohydrate and an amine compound were mixed to the concentrations listed in Table 19 to prepare a composition for inhibiting protein denaturation. This composition for inhibiting protein denaturation was mixed with a phosphate buffer solution containing cedar pollen allergen protein (Cry j2) (manufactured in-house, Lot No. 180301) in a volume ratio of 9:1 in a 0.5 mL Eppendorf tube to prepare a protein preparation. The concentration of cedar pollen allergen protein in the protein preparation was 2 μg / mL. The protein preparation was then heated at 40°C for 4 hours. The amount of non-aggregated protein (ng / mL) in the heated protein preparation was measured using Cry j2 ELISA (manufactured in-house). The results are shown in Table 19.

[0124]

[0125] In Example 6-1, the amount of non-aggregated protein was greater than in the comparative example in which the same concentration of carbohydrate or amine compound was used alone.

[0126] (7) Cedar pollen allergen denaturation inhibition test 3 A composition for inhibiting protein denaturation was prepared by mixing a carbohydrate and an amine compound to the concentrations shown in Table 20. This composition for inhibiting protein denaturation was mixed with a phosphate buffer solution containing cedar pollen allergen protein SBP (manufactured by the company, Lot No. 180301, 900 μg / mL) in a volume ratio of 9:1 in a 1.5 mL Eppendorf tube to prepare a protein preparation.

[0127] This protein preparation was added to a 96-well black plate at 98 μl / well, and 2 μl / well of PROTEOSTAT® Protein Aggregation Assay Reagent (Enzo), a reagent that binds to aggregated proteins and emits fluorescence, was added and gently mixed. The plate was then shielded from light with aluminum foil and incubated in an incubator at 37°C. The plate was removed over time, and the fluorescence intensity at Ex 550 nm / Em 600 nm was measured. The fluorescence intensity after 1 hour of incubation was defined as 100%, and the increase in fluorescence intensity over time was measured. The results are shown in Table 20 as the mean ± standard deviation (%) of three points.

[0128]

[0129] In Comparative Example 7-1, the cedar pollen allergen protein was incubated in D-PBS at 37°C, and the fluorescence intensity increased over time, confirming the gradual occurrence of protein aggregation. In Comparative Examples 7-2 to 7-5, in which a carbohydrate or an amine compound was used alone, a certain degree of inhibitory effect on protein aggregation was observed, but in Examples 7-1 and 7-2, the fluorescence intensity was suppressed to about 50% of that of Comparative Examples 7-2 to 7-5, confirming a strong inhibitory effect on protein aggregation.

[0130] (8) Test for inhibiting denaturation of human IFN-γ A carbohydrate and an amine compound were mixed to the concentrations shown in Table 21 to prepare a composition for inhibiting protein denaturation. This composition for inhibiting protein denaturation was mixed with phosphate buffer containing human IFN-γ (manufactured in-house, Lot No. 109025) in a 0.5 mL Eppendorf tube at a volume ratio of 99:1 to prepare a protein preparation. The concentration of IFN-γ in the protein preparation was 80 ng / mL. Next, the protein preparation was heated at 40°C for 7 hours. The amount of active IFN-γ (pg / mL) in the heated protein preparation was measured using human IFN-γ ELISA (manufactured in-house). The results are shown in Table 21.

[0131]

[0132] In Example 8-1, more active IFN-γ was obtained than in the Comparative Example in which the same concentration of carbohydrate or amine compound was used alone.

[0133] (9) Human TNF-α Denaturation Inhibition Test 2 A carbohydrate and an amine compound were mixed to the concentrations listed in Table 22 to prepare a composition for inhibiting protein denaturation. This composition for inhibiting protein denaturation was mixed with phosphate buffer containing human TNF-α (manufactured by our company, Lot No. 960506) in a 0.5 mL Eppendorf tube at a volume ratio of 99:1 to prepare a protein preparation. The concentration of human TNF-α in the protein preparation was 10 μg / mL. Next, the protein preparation was frozen at -80°C and then thawed at room temperature, a freeze-thaw cycle repeated 10 times. The amount of active TNF-α (ng / mL) in the heated protein preparation was measured using a human TNF-α ELISA (manufactured by our company). The results are shown in Table 22.

[0134]

[0135] In Example 9-1, the amount of active TNF-α was greater than in the comparative examples in which the same concentration of carbohydrate or amine compound was used alone.

[0136] (10) Anti-Human IFNγ Monoclonal Antibody Denaturation Inhibition Test: A carbohydrate and an amine compound were mixed to the concentrations listed in Tables 23 to 26 to prepare a protein denaturation inhibitory composition. This protein denaturation inhibitory composition was mixed with a phosphate buffer solution containing a mouse monoclonal antibody against IFNγ (MAb-IFNγ-15, in-house preparation, Lot No. 010) in a volume ratio of 9:1 in a 0.5 mL Eppendorf tube (Eppendorf, product number 3810X) to prepare a protein formulation. The antibody concentration in the protein formulation was 70 μg / mL. In the tests listed in Tables 23 and 24, 0.01% to 0.5% (w / v) polysorbate 80 or polysorbate 20 (both manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added. The protein formulation was then heated at 73°C for 5 hours.

[0137] 15 μL of the heated protein preparation was dispensed into wells of a 96-well microplate (Corning, Costar 3513), followed by 135 μL of human IFNγ (400 pg / mL) as antigen. After mixing using a 12-channel multichannel pipette (Thermo Electron, Finnpipette), 100 μL was aliquoted and added to the wells of an immunomodule plate (Thermo Scientific, U16 maxisorp Loose Nunc-Immuno Module 469264) coated with MAb-IFNγ-15, and the antigen-antibody reaction was allowed to occur at room temperature for 1.5 hours. The plate was then washed, labeled with antibody, and measured for absorbance at 450 nm according to standard procedures.

[0138] As a reference test, 135 μL of human IFNγ (400 pg / mL) was added to a 96-well microplate containing 15 μL of phosphate buffer, and after mixing, 100 μL of the mixture was added to a well of an immunomodule plate on which MAb-IFNγ-15 had been immobilized, and the absorbance was measured. The competitive inhibition rate was calculated using the following formula. The results are shown in Tables 23 to 26. Competitive inhibition rate (%) = (1 - (absorbance in each Example and Comparative Example / absorbance in reference test)) × 100

[0139]

[0140] As shown in Table 23, while 0.5 M trehalose alone (Comparative Example 10-3) and 0.5 M sodium glutamate alone (Comparative Example 10-4) yielded competitive inhibition rates of 1% and 3%, respectively, the 0.5 M protein denaturation-inhibiting composition comprising both (Example 10-1) yielded a high competitive inhibition rate of 39%, demonstrating a synergistic effect. Furthermore, while the competitive inhibition rates for polysorbate 80 alone were 1% to 12% (Comparative Examples 10-5 to 10-8), the combined use of polysorbate 80 and the 0.5 M protein denaturation-inhibiting composition synergistically improved the competitive inhibition rate (i.e., antibody stabilization effect) (Examples 10-2 to 10-5).

[0141] The synergistic effect of inhibiting protein denaturation was tested by two-way repeated-design analysis of variance using the software "Statcel 4" included in 4 Steps Excel Statistics (4th Edition) (Yanai Hisae, OMS Publishing).

[0142]

[0143] As shown in Table 24, the compositions for inhibiting protein denaturation containing polysorbate 20 (Examples 11-2 to 11-6) had improved competitive inhibition rates (i.e., antibody stabilizing effect) compared to the composition for inhibiting protein denaturation not containing polysorbate 20 (Example 11-1).

[0144]

[0145] As shown in Table 25, the competitive inhibition rate was higher when a composition for inhibiting protein denaturation comprising trehalose and sodium glutamate was used (Examples 12-1 to 12-5) than when trehalose alone (Comparative Example 12-3) or sodium glutamate alone (Comparative Examples 12-4 to 12-8), and a synergistic effect was observed.

[0146]

[0147] As shown in Table 26, the competitive inhibition rate was higher when the protein denaturation-inhibiting composition comprising trehalose and sodium aspartate was used (Examples 13-1 to 13-3) than when trehalose alone (Comparative Examples 13-3 to 13-5) or sodium aspartate alone (Comparative Examples 13-6 to 13-8), demonstrating a synergistic effect. As an amine compound, sodium aspartate exhibited an effect similar to that of sodium glutamate.

[0148] (11) Malate dehydrogenase denaturation inhibition test A protein denaturation inhibition composition was prepared by mixing carbohydrates and an amine compound to the concentrations listed in Table 27. This protein denaturation inhibition composition and water containing recombinant microbial malate dehydrogenase (rMDH, manufactured by Oriental Yeast Co., Ltd.) at a concentration of 50 μg / mL were heat-treated at 90°C for 2.5 hours. The activity of malate dehydrogenase contained in the heat-treated solution was measured using an MDH enzyme activity measurement kit (manufactured by Bioassay Systems Co., Ltd.). The measured absorbance of the enzyme reaction product over time is shown in Table 27.

[0149]

[0150] The heated enzyme (Comparative Example 13-2) had a clearly reduced absorbance compared to the unheated enzyme (Comparative Example 13-1). When sodium glutamate alone was added to the enzyme and the mixture was heated (Comparative Examples 13-6 to 13-8), a further decrease in absorbance was observed. Furthermore, when trehalose alone was added (Comparative Examples 13-3 to 13-5), almost no difference in absorbance was observed compared to Comparative Example 13-2, indicating that trehalose alone did not provide a sufficient protective effect. On the other hand, in Examples 13-1 to 13-3, which used a composition for inhibiting protein denaturation comprising trehalose and sodium glutamate, the change in absorbance over time was the same as that of the unheated Comparative Example 13-1, demonstrating a synergistic stabilizing effect.

[0151] (12) Test 1 for inhibiting denaturation of anti-human IL-17A antibody A composition for inhibiting protein denaturation was prepared by mixing a carbohydrate and an amine compound to the concentrations shown in Table 28. An aqueous solution containing this composition for inhibiting protein denaturation and a humanized anti-human IL-17A antibody at a concentration of 1 mg / mL was heat-treated at 73°C for 5 hours. The degree of turbidity due to protein denaturation and aggregation after heat treatment was evaluated based on absorbance at 660 nm. The results are shown in Table 28.

[0152] Furthermore, the reactivity of the heat-treated antibodies to the antigen (human IL-17A) was measured by competitive ELISA. The measured competitive inhibition rates (%) are shown in Table 28.

[0153]

[0154] As shown in Table 28, the turbidity in Comparative Example 14-2 increased by approximately 3.3 times compared to Comparative Example 14-1. This indicates that the heat treatment caused antibody aggregation and turbidity. The addition of trehalose alone (Comparative Example 14-3) or sodium glutamate alone (Comparative Examples 14-4 to 14-6) further increased the turbidity and increased the absorbance. However, in the presence of a composition for inhibiting protein denaturation comprising 1 M trehalose and various concentrations of sodium glutamate, the turbidity was reduced from the same level as in the unheated Comparative Example 14-1 to approximately 1.8 times, depending on the concentration, and antibody protein aggregation was inhibited (Examples 14-1 to 14-3).

[0155] As shown in Table 28, heat treatment of the antibody reduced the competitive inhibition rate to 18% (Comparative Example 14-2). In the presence of 1 M trehalose, antigen binding was reduced by approximately 50% (Comparative Example 14-3). Addition of monosodium glutamate alone further reduced antigen binding (Comparative Examples 14-4 to 14-6), correlating with increased turbidity. However, in the presence of a composition for inhibiting protein denaturation comprising trehalose and monosodium glutamate (Examples 14-1 to 14-3), antigen binding was comparable to that of the unheated antibody, demonstrating that the composition for inhibiting protein denaturation exerts a synergistic protective effect on the antibody compared to the use of a carbohydrate or an amine compound alone. The antibody used in this test is an example of an antibody preparation used clinically, and these results demonstrate that the composition for inhibiting protein denaturation also exerts a protective effect on antibody preparations in clinical use.

[0156] (13) Test 2 for inhibiting denaturation of anti-human IL-17A antibody The same procedures as in Comparative Examples 14-1 to 14-6 and Examples 14-1 to 14-3 were performed, except that sodium aspartate was used instead of sodium glutamate as the amine compound, and the degree of turbidity due to protein denaturation and aggregation after heat treatment was evaluated based on absorbance at 660 nm. The results are shown in Table 29. Furthermore, the reactivity of the heat-treated antibody to the antigen (human IL-17A) was measured by competitive ELISA, and the results are shown in Table 29.

[0157]

[0158] As shown in Table 29, the turbidity in Comparative Example 15-3 increased by approximately 4.3 times compared to Comparative Example 15-1. This indicates that the heat treatment caused antibody aggregation and turbidity. When trehalose alone (Comparative Example 15-3) or sodium aspartate alone (Comparative Examples 15-4 to 15-6) was added, the turbidity increased more than in Comparative Example 15-2. However, in the presence of a composition for inhibiting protein denaturation comprising trehalose and various concentrations of sodium aspartate, the turbidity was suppressed from the same level as in unheated Comparative Example 15-1 to approximately 1.5 times, depending on the concentration, and antibody protein aggregation was suppressed (Examples 15-1 to 15-3).

[0159] As shown in Table 29, heat treatment of the antibody reduced the antigen binding activity to 9% (Comparative Example 15-2). In the presence of 1 M trehalose, the antigen binding activity was reduced to 47% (Comparative Example 15-3). The addition of monosodium glutamate alone further reduced the antigen binding activity (Comparative Examples 15-4 to 15-6), which correlated with the increase in turbidity. However, in the presence of a composition for inhibiting protein denaturation comprising 1 M trehalose alone and monosodium aspartate (Examples 15-1 to 15-3), the antigen binding activity was the same as that of the unheated antibody, demonstrating that the composition for inhibiting protein denaturation has a synergistic protective effect on the antibody compared to the use of a carbohydrate or an amine compound alone.

[0160] (14) Lactase Denaturation Inhibition Test A composition for inhibiting protein denaturation was prepared by mixing carbohydrates and an amine compound to the concentrations shown in Table 30. An aqueous solution containing this composition for inhibiting protein denaturation and lactase derived from the genus Kluyveromyces at a concentration of approximately 6 mg / mL was heat-treated at 50°C or 60°C for 2 hours. The degree of turbidity due to protein denaturation and aggregation after heat treatment was evaluated by absorbance at 660 nm. The results are shown in Table 30.

[0161] Furthermore, the lactase activity (residual activity (%)) after heat treatment relative to the lactase activity before and after heating was measured. The results are shown in Table 30.

[0162]

[0163] As shown in Table 30, in Comparative Example 16-2, cloudiness was observed, and the residual activity after heating was significantly reduced compared to Comparative Example 16-1. In contrast, in Example 16, cloudiness was suppressed, and the residual activity was improved.

[0164] (15) 6-α-glucanotransferase denaturation inhibition test 1 A composition for inhibiting protein denaturation was prepared by mixing a carbohydrate and an amine compound to the concentrations shown in Table 31. An aqueous solution containing this composition for inhibiting protein denaturation and 6-α-glucanotransferase derived from the genus Geobacillus at a concentration of approximately 25 mg / mL was heat-treated at 70°C for 2 hours. The degree of turbidity due to protein denaturation and aggregation after heat treatment was evaluated by absorbance at 660 nm. The results are shown in Table 31.

[0165] Furthermore, the 6-α-glucanotransferase activity (residual activity (%)) after heat treatment relative to the non-heated 6-α-glucanotransferase activity was measured. The results are shown in Table 31.

[0166]

[0167] As shown in Table 31, in Comparative Example 17-2, cloudiness was observed, and the residual activity after heating was significantly reduced compared to Comparative Example 17-1. In contrast, in Example 17, cloudiness was suppressed, and the residual activity was improved.

[0168] (16) 6-α-glucanotransferase Denaturation Inhibition Test 2 A carbohydrate and an amine compound were mixed to prepare a composition for inhibiting protein denaturation. An aqueous solution containing this composition for inhibiting protein denaturation and Geobacillus-derived 6-α-glucanotransferase at a concentration of approximately 25 mg / mL was lyophilized to prepare a powder with the content shown in Table 32. The lyophilized powder was kept at 70°C in an incubator for 2 days. The powder state was maintained even after storage, and no change in appearance due to moisture absorption occurred. The 6-α-glucanotransferase activity (residual activity (%)) after incubation relative to the unheated 6-α-glucanotransferase activity was then measured. The results are shown in Table 32.

[0169]

[0170] As shown in Table 32, the residual activity after heating in Comparative Example 18-2 was significantly lower than that in Comparative Example 18-1. In contrast, the residual activity in Examples 18-1 to 18-3 improved and recovered to the same level as that in Comparative Example 18-1.

[0171] (17) 6-α-glucanotransferase Denaturation Inhibition Test 3 A carbohydrate and an amine compound were mixed to prepare a composition for inhibiting protein denaturation. An aqueous solution containing this composition for inhibiting protein denaturation and Geobacillus-derived 6-α-glucanotransferase at a concentration of approximately 45 mg / mL was lyophilized to prepare a powder with the content shown in Table 33. The lyophilized powder was stored in an incubator at 40°C and 75% relative humidity for 3 months. The powder state was maintained even after storage, and no change in appearance due to moisture absorption occurred. The 6-α-glucanotransferase activity (residual activity (%)) after incubation relative to the 6-α-glucanotransferase activity before incubation was then measured. The results are shown in Table 33.

[0172]

[0173] As shown in Table 33, the residual activity after 3 months of storage in Comparative Example 19 was significantly reduced. In contrast, the residual activity was improved in Examples 19-1 to 19-3.

[0174] (18) Serine protease denaturation inhibition test A composition for inhibiting protein denaturation was prepared by mixing a carbohydrate and an amine compound to the concentrations shown in Tables 34 and 35. An aqueous solution containing this composition for inhibiting protein denaturation and a serine protease derived from the genus Bacillus (Biophrase®, manufactured by Nagasevita Co., Ltd.) at a concentration of approximately 1 mg / mL was heat-treated at 70°C for 2 hours. The enzyme activity after heat treatment (residual activity (%)) was measured relative to the enzyme activity after heat treatment. The results are shown in Tables 34 and 35.

[0175] Enzyme activity was measured using fluorescently labeled casein contained in the AAT Bioquest Amplite protease activity measurement kit. Specifically, the heat-treated sample was diluted 60 times with purified water and added to a Corning 96-well black plate (3603) at 50 μL / well. Next, fluorescently labeled casein was diluted 100 times with 80 mM phosphate buffer (pH 7.5) and added to the black plate at 50 μL / well. After incubation at 37 ° C for 15 minutes, the fluorescence intensity (Ex / Em: 490 nm / 525 nm) was measured. In addition, unheated proteolytic enzyme bulk powder was prepared by 2-fold serial dilution with purified water from 50 μg / mL and added as a standard.

[0176]

[0177] As shown in Table 34, the residual activity of Comparative Example 20-2 was significantly reduced. In contrast, the residual activity was improved in Examples 20-1 to 20-3. A statistically significant synergistic effect was observed in Examples 20-1 and 20-2.

[0178]

[0179] As shown in Table 35, the residual activity of Comparative Example 21-2 was significantly reduced. In contrast, the residual activity of Examples 21-1 to 21-3 was improved. A statistically significant synergistic effect was observed in Examples 21-1, 21-2, and 21-3. Serine protease ("Biophrase®" manufactured by Nagase Vita Co., Ltd.) is used in the cosmetics industry as a pack agent to decompose and exfoliate aged keratin, and is also used industrially in detergent manufacturing and various cleaning agents. The results in Tables 34 to 35 suggest that compositions for inhibiting protein denaturation can be applied to cosmetic pack agents and cleaning agents.

Claims

1. A composition for inhibiting protein denaturation, comprising one or more carbohydrates selected from the group consisting of trehalose, sucrose, maltitol, and isomaltitol, and one or more amine compounds selected from the group consisting of glutamic acid, aspartic acid, arginine, proline, serine, choline, and salts thereof.

2. A composition for inhibiting protein denaturation as described in claim 1, which inhibits protein denaturation caused by heating or freezing and thawing.

3. A composition for inhibiting protein denaturation as described in claim 1 or 2, which inhibits protein aggregation.

4. A composition for inhibiting protein denaturation according to claim 1 or 2, wherein the protein is one or more selected from the group consisting of antibodies, cytokines, allergens, and enzymes.

5. A composition for inhibiting protein denaturation according to claim 1 or 2, wherein the protein is one or more selected from the group consisting of anti-IFNα antibody, anti-IFNγ antibody, anti-TNFα antibody, anti-IL-17A antibody, and anti-IL-18 antibody.

6. A composition for inhibiting protein denaturation according to claim 1 or 2, wherein the protein is one or more selected from the group consisting of IFNα, IFNγ, IL-18, and TNFα.

7. A composition for inhibiting protein denaturation as described in claim 1 or 2, wherein the protein is a cedar pollen allergen.

8. A composition for inhibiting protein denaturation as described in claim 1 or 2, wherein the protein is one or more selected from the group consisting of lactate dehydrogenase, glycosyltransferase, glycolytic enzyme, malate dehydrogenase, and protease.

9. A composition for inhibiting protein denaturation according to claim 1 or 2, wherein the protein is a virus-derived protein or a bacteria-derived protein.

10. A composition for inhibiting protein denaturation according to claim 1 or 2, wherein the protein is a blood coagulation factor or albumin.

11. A composition for inhibiting protein denaturation according to claim 1 or 2, wherein the molar ratio of carbohydrate:amine compound is 40:1 to 1:

40.

12. A composition for inhibiting protein denaturation according to claim 1 or 2, which is in a liquid form and has a total concentration of the carbohydrate and the amine compound of 0.20 mol / L to 3.50 mol / L.

13. A composition for inhibiting protein denaturation according to claim 1 or 2, which synergistically inhibits protein denaturation with the protein denaturation inhibitory effect of the carbohydrate and the protein denaturation inhibitory effect of the amine compound.

14. A method for inhibiting protein denaturation, comprising causing a protein to coexist with the composition for inhibiting protein denaturation described in claim 1 or 2.

15. A protein preparation comprising a protein and a composition for inhibiting protein denaturation described in claim 1 or 2.

16. A method for inhibiting the denaturation of a protein, comprising causing a protein to coexist with one or more carbohydrates selected from the group consisting of trehalose, sucrose, maltitol, and isomaltitol, and one or more amine compounds selected from the group consisting of glutamic acid, aspartic acid, arginine, proline, serine, choline, and salts thereof.

17. A protein preparation comprising: one or more carbohydrates selected from the group consisting of trehalose, sucrose, maltitol, and isomaltitol; one or more amine compounds selected from the group consisting of glutamic acid, aspartic acid, arginine, proline, serine, choline, and salts thereof; and a protein.

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