Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/19—Cytokines; Lymphokines; Interferons
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/35—Allergens
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
  • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
  • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
  • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
  • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

• the present invention relates to a composition for inhibiting protein denaturation.
• Proteins are widely used in fields such as therapeutic drugs, diagnostic agents, vaccines, and food. However, proteins are easily denatured when used alone, and can be denatured and lose their function when subjected to heating, long-term storage, or multiple freeze-thaw cycles. Attempts have been made to improve compositions containing proteins in order to prevent protein denaturation.
• Patent Document 1 discloses a method for stabilizing an Fc mutant protein using carbohydrates, cationic amino acids, anions, and polysorbates.
• Patent Document 2 discloses a method for stabilizing an anti-TNF ⁇ antibody using polyols, surfactants, and arginine. The types of proteins that can be stabilized by these methods are limited.
• the objective of the present invention is to provide a composition for inhibiting protein denaturation that can inhibit the denaturation of a wide range of proteins.
• the inventors discovered that protein denaturation can be suppressed by combining a specific carbohydrate with an amine compound, and thus completed the present invention.
• 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.
• 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.
• composition for inhibiting protein denaturation preferably inhibits protein denaturation caused by heating or freezing and thawing.
• composition for inhibiting protein denaturation preferably inhibits protein aggregation.
• the protein is one or more selected from the group consisting of antibodies, cytokines, allergens, and enzymes.
• the protein is one or more selected from the group consisting of anti-IFN ⁇ antibody, anti-IFN ⁇ antibody, anti-TNF ⁇ antibody, anti-IL-17A antibody, and anti-IL-18 antibody.
• the protein is one or more selected from the group consisting of IFN ⁇ , IFN ⁇ , IL-18, and TNF ⁇ .
• the protein is preferably a cedar pollen allergen.
• the protein is one or more selected from the group consisting of lactate dehydrogenase, glycosyltransferase, glycolytic enzyme, malate dehydrogenase, and protease.
• the protein is preferably a viral or bacterial protein.
• the protein is preferably a blood clotting factor or albumin.
• the molar ratio of carbohydrate to amine compound in the protein denaturation inhibition composition is preferably 40:1 to 1:40.
• the total concentration of the carbohydrate and amine compound in the protein denaturation inhibition composition is preferably 0.20 mol/L to 3.50 mol/L.
• composition for inhibiting protein denaturation preferably inhibits protein denaturation synergistically with the protein denaturation inhibitory effect of the carbohydrate and the protein denaturation inhibitory effect of the amine compound.
• the present invention also relates to a method for inhibiting protein denaturation, characterized in that the protein is coexistent with the composition for inhibiting protein denaturation.
• the present invention also relates to a protein preparation comprising a protein and the above-mentioned composition for inhibiting protein denaturation.
• the present invention also relates to a method for producing a food product comprising: one or more carbohydrates selected from the group consisting of trehalose, sucrose, maltitol, and isomaltitol; and
• the present invention relates to a method for inhibiting the denaturation of a protein, which comprises allowing one or more amine compounds selected from the group consisting of glutamic acid, aspartic acid, arginine, proline, serine, choline, and salts thereof to coexist with the protein.
• the present invention also relates to a method for producing a saccharide composition comprising the steps of: one or more carbohydrates selected from the group consisting of trehalose, sucrose, maltitol, and isomaltitol;
• the present invention relates to a protein formulation comprising 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.
• composition for inhibiting protein denaturation of the present invention can inhibit the denaturation of a wide range of proteins.
• 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.
• carbohydrates selected from the group consisting of trehalose, sucrose, maltitol, and isomaltitol
• 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.
• the carbohydrate in the present invention is 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, and trehalose is more preferred. These carbohydrates may also be derivatives in which a substituent has been introduced into the hydrogen atom of a part of the hydroxyl group.
• substituents examples include carboxylate esters such as acetate and benzoic acid; sulfate esters; fatty acid esters such as laurate, myristic acid ester, palmitic acid ester, stearate, oleate, linoleic acid ester, and linolenic acid ester; and ethers such as methyl ether, benzyl ether, trityl ether, methylsilyl ether, and dodecyl ether.
• carboxylate esters such as acetate and benzoic acid
• sulfate esters examples include fatty acid esters such as laurate, myristic acid ester, palmitic acid ester, stearate, oleate, linoleic acid ester, and linolenic acid ester
• ethers such as methyl ether, benzyl ether, trityl ether, methylsilyl ether, and dodecyl ether
• the concentration of 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.
• the concentration of carbohydrate 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.
• carbohydrates acting as hydrogen bond donors, may form NADES (Natural Deep Eutectic Solvent) with amine compounds acting as hydrogen bond acceptors to inhibit protein denaturation and stabilize the protein, but the present invention is not limited to this mechanism.
• NADES Natural Deep Eutectic Solvent
• 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.
• the salts constituting the amine compound include sodium salts, potassium salts, hydrochlorides, chlorides, etc.
• 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.
• the amine compound may function as a hydrogen bond acceptor in forming NADES, but the present invention is not limited to this mechanism.
• the combination of the carbohydrate and the amine compound is not particularly limited, but a combination of trehalose and an amine compound selected from the group consisting of arginine, aspartic acid, serine, and glutamic acid, or a salt thereof; a combination of sucrose and an amine compound selected from the group consisting of choline, arginine, and glutamic acid, or a salt thereof; a combination of maltitol and an amine compound selected from the group consisting of arginine, proline, and glutamic acid, or a salt thereof; a combination of isomaltitol and arginine, or a salt thereof, is more preferred, and a 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 more preferred, and a combination of maltitol and sodium gluta
• 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.
• 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.
• 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 is too high or too low, and the effect of inhibiting protein denaturation tends not to be observed.
• 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.
• 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.
• the form of the composition for inhibiting protein denaturation is not particularly limited, and may be either liquid or solid.
• liquid forms include aqueous solutions, suspensions, and slurries.
• solid forms include powders, granules, tablets, and the like. Among these, the liquid form is preferred from the viewpoints of ease of production and storage stability.
• the composition for inhibiting protein denaturation 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 little effect on protein activity.
• 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.
• the composition for inhibiting protein denaturation can be produced by mixing the components in any order. After mixing the components, they may be filtered or sterilized by contacting them with a porous material or passing them through a filter.
• the composition for inhibiting protein denaturation may optionally contain components such as a pH adjuster, a surfactant, a thickener, an emulsifier, inorganic salts, preservatives, a solvent, an excipient, metals, a filter aid, etc.
• the content of these components is not particularly limited and can be selected by those skilled in the art in any amount, but can be, for example, 0.001 to 90% by weight, preferably 0.01 to 50% by weight, and more preferably 0.1 to 30% by weight in the composition for inhibiting protein denaturation.
• 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 the 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 the Na and K salts of these inorganic acids.
• 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, and polysorbate 20 and polysorbate 80 are 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)%.
• Thickening agents 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.
• 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.
• Inorganic salts include sodium chloride, ammonium sulfate, sodium sulfate, calcium chloride, and polymeric phosphates.
• 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.
• Solvents include water, ethanol, methanol, isopropanol, etc.
• the solvent may contain buffer components such as phosphoric acid, citric acid, glycine, Tris, etc.
• Excipients include hyaluronic acid, methylcellulose, carboxymethylcellulose, hydroxypropylmethylcellulose, polyvinyl alcohol, polyethylene glycol, triglycerides, glycerides, propylene glycol, polyvinylpyrrolidone, etc.
• Inhibition of protein denaturation can be evaluated by the following method. That is, a protein preparation containing the composition for inhibiting protein denaturation of the present invention is denatured, and protein activity (A) is measured. The same denaturation treatment is also performed on a control, and protein activity (B) is measured. When A exceeds B, it can be determined that protein denaturation is inhibited.
• An example of the control is a preparation containing the same protein as the test sample, but not containing the composition for inhibiting denaturation.
• examples of denaturation treatments for protein preparations include heating and freezing and 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 times are 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.
• Methods for measuring protein activity include methods that utilize antigen-antibody reactions, such as EIA and ELISA, protein agglutination assays, and measuring the enzyme activity of proteins.
• EIA and ELISA antigen-antibody reactions
• protein agglutination assays protein agglutination assays
• enzyme activity of proteins When competitive EIA or competitive ELISA is used to measure protein activity, protein activity is measured as the competitive inhibition rate (%).
• 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.
• the ratio A/B of protein activity (A) to protein activity (B) is preferably less than 1, more preferably less than 0.5, even more preferably less than 0.3, and even more preferably less than 0.1.
• 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.
• composition for inhibiting protein denaturation inhibits protein denaturation synergistically with the protein denaturation inhibiting effect of carbohydrates and the protein denaturation inhibiting effect of amine compounds.
• Whether the protein denaturation inhibition 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 denatured, and 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 denaturation treatment, and protein activity (B 1 ) is measured. As a comparative control 2, a preparation containing the same protein and amine compound as the test sample but not containing a carbohydrate is subjected to the same denaturation treatment, and protein activity (B 2 ) is measured.
• A>B 1 and A>B 2 it can be determined that the protein denaturation inhibition is synergistic. It is preferable that A>B 1 x 1.5 and A>B 2 x 1.5, more preferably that A>B 1 x 2 and A>B 2 x 2 , even more preferably that A>B 1 +B 2, even more preferably that A>(B 1 +B 2 ) x 2, and particularly preferably that A>(B 1 +B 2 ) x 10.
• the composition for inhibiting protein denaturation can inhibit protein denaturation even after the above-mentioned denaturation treatment, and therefore proteins can be stably stored for long periods of time under mild conditions that are unlikely 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%.
• the storage period of the protein preparation at 4°C is preferably one year or more, and more preferably two years or more.
• the protein preparation of the present invention is characterized by comprising a protein and the above-mentioned composition for inhibiting protein denaturation.
• the protein whose denaturation is inhibited is not particularly limited, and for example, the denaturation of proteins commonly used as antibodies, cytokines, allergens, enzymes, blood coagulation and fibrinolytic factors, hormones, etc. can be inhibited.
• 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.
• the animal from which the antibody is derived is not particularly limited, and examples thereof include non-human animals such as mice, rats, rabbits, sheep, goats, and chickens, in addition to humans.
• the antibody may be a chimeric antibody or a humanized antibody that combines a sequence derived from a non-human animal with a sequence derived from a human, or may be a fully human antibody.
• the antibody examples include minibodies, Fabs, diabodies, Fab', triabodies, scFv-Fc, scFv, F(ab') 2 , and Fv, in addition to antibodies that include a complete constant region and a complete variable region.
• the antibody may be a conjugate of 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 thereof include nucleic acids, proteins, and sugars. Specific examples of antigens include IFN ⁇ , IFN ⁇ , TNF ⁇ , IL-18, IL-17, and leukocyte surface antigen proteins.
• Cytokines include IFNs such as IFN ⁇ , IFN ⁇ , IFN ⁇ , IFN ⁇ , IFN ⁇ , IFN ⁇ , IFN ⁇ , and IFN ⁇ ; TNFs such as TNF ⁇ and TNF ⁇ ; 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, PDGF, etc.
• IFNs such as IFN ⁇ , IFN ⁇ , IFN ⁇ , IFN ⁇ , IFN ⁇ , IFN ⁇ , IFN ⁇ , and IFN ⁇
• TNFs such as TNF ⁇ and TNF ⁇
• interleukins (ILs) such as IL1, IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL11, IL
• Allergens are not limited as long as they are 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, silver grass, and red pine.
• 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
• the enzyme is not particularly limited as long as it is a protein that has the activity of catalyzing a chemical reaction, and examples thereof include sugar-degrading enzymes such as ⁇ -amylase, ⁇ -amylase, glucoamylase, isoamylase, maltogenic amylase, pullulanase, maltotriohydrolase, dextranase, glucose isomerase, cellulase, xylanase, hemicellulase, mannanase, pectinase, pectin methylesterase, invertase, lactase, inulinase, ⁇ -galactosidase, chitinase, chitosanase, alginate lyase, glucose oxidase, glucose dehydrogenase, cyclodextrin glucanotransferase, and transglucosidase.
• glycosyltransferases such as 6- ⁇ -glucanotransferase, glucosyltransferase, galactosyltransferase, sialyltransferase, N-acetylglucosaminyltransferase, and mannosyltransferase; proteolytic enzymes such as protease, peptidase, and collagenase; amino acid decomposition 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 decomposition enzyme, and urate oxidase.
• glycosyltransferases such as 6- ⁇ -glucanotransfera
• the protein concentration in the protein preparation 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.
• the pH of the protein preparation is preferably 4 to 9, more preferably 5 to 8. Under these pH conditions, the likelihood of precipitation is low and the effect on protein activity is also small.
• the pH of the protein preparation can be adjusted with an acid such as hydrochloric acid or sulfuric acid, or a base such as sodium hydroxide or potassium hydroxide.
• 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 are as described above.
• the content of these optional components is not particularly limited and can be selected by those skilled in the art in any amount, but can be, for example, 0.001 to 90% by weight in the protein preparation, preferably 0.01 to 50% by weight, and more preferably 0.1 to 30% by weight.
• the protein preparation can be produced by mixing the components in any order. After mixing the components, they may be filtered or sterilized by contacting them with a porous material or passing them through a filter. A powdered protein preparation can also be obtained by freeze-drying a liquid protein preparation.
• the method for inhibiting protein denaturation of the present invention is characterized in that the protein is coexisted with the composition for inhibiting protein denaturation.
• the denaturation of the protein 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.
• the method for causing the protein to coexist with the composition for inhibiting protein denaturation 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.
• the coexistence time of the protein and the composition for inhibiting protein denaturation may be determined according to the use of the protein and the storage period, but is preferably 30 minutes or more, and more preferably 1 hour or more. There is no particular upper limit to the coexistence time, and protein denaturation is inhibited for a long period of time, but it is generally 2 years or less. By keeping the protein in a state where it coexists with the composition for inhibiting protein denaturation, protein denaturation can be inhibited even during long-term storage.
• 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. In general, protein denaturation can be inhibited even at relatively high temperatures at which protein denaturation may occur. After the protein and the composition for inhibiting protein denaturation coexist, the protein can be optionally purified or diluted.
• the protein preparations of the present invention can be used in a wide range of applications depending on the type of protein contained therein.
• Applications include 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 bio-preparations.
• Anti-human IFN ⁇ monoclonal antibody denaturation inhibition test A carbohydrate and an amine compound were mixed to the concentrations shown 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 IFN ⁇ (MAb-IFN ⁇ -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 preparation.
• the antibody concentration in the protein preparation was 70 ⁇ g/mL.
• the protein preparation was heated at 73° C. for 5 hours.
• Comparative Example 1-1-1 the competitive inhibition rate was measured without heating the protein preparation without including carbohydrates or amine compounds, and the competitive inhibition rate was 87%.
• Comparative Example 1-1-2 the protein preparation was heated without including carbohydrates or amine compounds. As a result, the competitive inhibition rate was -5%.
• the competitive inhibition rates were higher than in the comparative examples in which the same concentrations of carbohydrates or amine compounds were used alone.
• 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 being combined with carbohydrates.
• Examples 1-3-1 to 1-3-11 the competitive inhibition rate was examined 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, a high competitive inhibition rate was observed when the molar ratio of maltitol to arginine hydrochloride was other than 1:1, similar to when the molar ratio was 1:1.
• Example 1-5-1 to 1-5-4 the total concentration of trehalose and potassium glutamate was lower than in Example 1-4-2, but like Example 1-4-2, the competitive inhibition rate was higher than in the comparative examples in which the same concentrations of carbohydrate or amine compound were used alone.
• Example 1-6-1 to 1-6-4 the total concentration of sucrose and potassium glutamate was lower than in Example 1-4-1, but like 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. In addition, among the Examples, the competitive inhibition rate tended to be higher when the total concentration of carbohydrate and amine compound was higher.
• 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, as was the case when the molar ratio was 1:1.
• 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 concentration of carbohydrate was higher.
• Examples 1-8-1 to 1-8-3 similar 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, similar 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.
• Example 1-9-1 to 1-9-5 the competitive inhibition rate was higher than in the comparative examples in which the same concentration of carbohydrate or amine compound was used alone.
• the competitive inhibition rate tended to be higher when the concentration of the amine compound was higher.
• Example 1-10-1 to 1-10-5 the competitive inhibition rate was higher than in the comparative examples in which the same concentration of carbohydrate or amine compound was used alone.
• the competitive inhibition rate tended to be higher when the concentration of the amine compound was higher.
• Examples 1-12-1 to 1-12-8 the competitive inhibition rate was higher than in the comparative examples in which the same concentration of carbohydrate was used alone.
• Examples 1-12-5 to 1-12-8 were carried out under the same conditions except that potassium glutamate in Examples 1-12-1 to 1-12-4 was replaced with sodium glutamate, the same tendency as in Examples 1-12-1 to 1-12-4 was observed.
• Urea has a hydrogen atom bonded to a nitrogen atom with high electronegativity, and can function as a hydrogen bond donor.
• a deep eutectic solvent consisting of urea and choline chloride is also called Reline, and has been reported to have the effect of stabilizing proteins such as bovine serum albumin (Phys. Chem. Chem. Phys., 2022, 24, 5627-5637). Therefore, the antibody stabilizing effect of this Reline was compared with that of a composition for inhibiting protein denaturation consisting of trehalose and arginine hydrochloride. As a result, the competitive inhibition rate was low in Comparative Example 1-13-3 and Comparative Example 1-13-4, which used a composition combining urea and choline chloride. On the other hand, a high competitive inhibition rate was observed in Examples 1-13-1 to 1-13-3, which used the composition for inhibiting protein denaturation of the present invention.
• Anti-human TNF ⁇ monoclonal antibody denaturation inhibition test 1 A carbohydrate and an amine compound were mixed to give the concentrations shown 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 TNF ⁇ (MAb-TNF ⁇ -5, in-house production, 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 ⁇ g/mL.
• the protein preparation was heated at 73° C. for 5 hours.
• Examples 2-1 to 2-3 the competitive inhibition rate was higher than in the comparative examples in which the same concentration of carbohydrate or amine compound was used alone.
• the competitive inhibition rate tended to be higher when the total concentration of carbohydrate and amine compound was higher.
• Anti-human IFN ⁇ monoclonal antibody denaturation inhibition test 2 A carbohydrate and an amine compound were mixed to give the concentrations shown 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 IFN ⁇ (MAb-IFN ⁇ -43, in-house production, 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 ⁇ g/mL.
• the protein preparation was heated at 73° C. for 5 hours.
• the heated protein preparation was dispensed in 20 ⁇ L into the wells of a 96-well microplate, and 130 ⁇ L of human IFN ⁇ (2500 pg/mL) was further dispensed as an antigen.
• 100 ⁇ L was taken and added to the wells of an immunomodule plate on which MAb-IFN ⁇ -43 was immobilized, and an antigen-antibody reaction was caused at room temperature for 1.5 hours, followed by washing, addition of a labeled antibody, and measurement of absorbance at a wavelength of 450 nm according to a conventional method.
• the competitive inhibition rate was higher than in the comparative examples in which the same concentration of carbohydrate or amine compound was used alone.
• the competitive inhibition rate tended to be higher when the total concentration of carbohydrate and amine compound was higher.
• LDH Denaturation Inhibition Test A carbohydrate and an amine compound were mixed to the concentration shown in Table 16 to prepare a composition for inhibiting protein denaturation.
• This composition for inhibiting protein denaturation and a potassium phosphate buffer containing lactate dehydrogenase (LDH) (manufactured by Oriental Yeast Co., Ltd., product number 46776003) were mixed in a 0.5 mL Eppendorf tube at a volume ratio of 9:1 to prepare a protein preparation.
• the concentration of lactate dehydrogenase in the protein preparation was 50 ⁇ g/mL.
• the protein preparation was heated at 70° C. for 2 hours.
• the enzyme activity in the protein preparation after heating was measured using a kit (manufactured by Roche, product number 11644793001). The results are shown in Table 16.
• Example 4-1-1 to 4-1-6 the residual enzyme activity was higher than in the comparative examples in which the same concentration of carbohydrate or amine compound was used alone.
• a carbohydrate and an amine compound were mixed to the concentrations shown in Table 17 to prepare a composition for inhibiting protein denaturation.
• This composition for inhibiting protein denaturation was mixed with a potassium phosphate buffer solution containing lactate dehydrogenase (LDH) (manufactured by Oriental Yeast Co., Ltd., product number 46776003) in a volume ratio of 9:1 in a 0.5 mL Eppendorf tube to prepare a protein preparation.
• the concentration of lactate dehydrogenase in the protein preparation was 20 mg/mL.
• the protein preparation was then heated at 70°C for 2 hours.
• the enzyme activity in the protein preparation after heating was measured using a kit (manufactured by Roche, product number 11644793001). The results are shown in Table 17.
• Example 4-2-1 to 4-2-2 the residual enzyme activity was higher than in the comparative examples in which the same concentration of carbohydrate or amine compound was used alone. It was confirmed that the protein denaturation suppression composition of the present invention exerts an effect of suppressing the decrease in activity due to denaturation and aggregation caused by heat treatment, even for lactate dehydrogenase at a high concentration (20 mg/mL).
• Example 5-1 there was more non-aggregated protein than in the comparative example in which the same concentration of carbohydrate or amine compound was used alone.
• Example 6-1 there was more non-aggregated protein than in the comparative example in which the same concentration of carbohydrate or amine compound was used alone.
• 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 ⁇ g/mL) in a volume ratio of 9:1 in a 1.5 mL Eppendorf tube to prepare a protein preparation.
• cedar pollen allergen protein SBP manufactured by the company, Lot No. 180301, 900 ⁇ g/mL
• This protein preparation was added to a 96-well black plate at 98 ⁇ l/well, and 2 ⁇ l/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 protected from light with aluminum foil and incubated in an incubator at 37°C. The plate was removed over time and the fluorescence intensity at Ex550 nm/Em600 nm was measured. The fluorescence intensity after 1 hour of incubation was set to 100%, and the increase in fluorescence intensity over time was measured. The results are shown in Table 20 as the average value of three points ⁇ standard deviation (%).
• Comparative Example 7-1 by incubating cedar pollen allergen protein in D-PBS at 37°C, the fluorescence intensity increased over time, confirming the gradual occurrence of protein aggregation.
• Comparative Examples 7-2 to 7-5 in which carbohydrates or amine compounds were used alone, a certain degree of protein aggregation suppression effect was observed, but in Examples 7-1 to 7-2, the fluorescence intensity was suppressed to about 50% of that of Comparative Examples 7-2 to 7-5, confirming a strong effect of suppressing protein aggregation.
• Human IFN ⁇ Denaturation Inhibition Test 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 and a phosphate buffer solution containing human IFN- ⁇ (manufactured by the company, LOT No. 109025) were mixed in a 0.5 mL Eppendorf tube at a volume ratio of 99:1 to prepare a protein preparation.
• the concentration of IFN- ⁇ in the protein preparation was 80 ng/mL.
• the protein preparation was heated at 40° C. for 7 hours.
• the amount of active IFN- ⁇ (pg/mL) in the protein preparation after heating was measured by human IFN- ⁇ ELISA (manufactured by the company). The results are shown in Table 21.
• Example 8-1 there was more active IFN- ⁇ than in the comparative example in which the same concentration of carbohydrate or amine compound was used alone.
• Human TNF ⁇ degeneration inhibition test 2 A carbohydrate and an amine compound were mixed to the concentrations shown in Table 22 to prepare a composition for inhibiting protein denaturation.
• This composition for inhibiting protein denaturation and a phosphate buffer solution containing human TNF- ⁇ (manufactured by the company, LOT No. 960506) were mixed in a 0.5 mL Eppendorf tube at a volume of 99:1 to prepare a protein preparation.
• the concentration of human TNF- ⁇ in the protein preparation was 10 ⁇ g/mL.
• the protein preparation was frozen at ⁇ 80° C. and then thawed at room temperature, a freeze-thaw process being repeated 10 times.
• the amount of active TNF- ⁇ (ng/ml) in the protein preparation after heating was measured by human TNF- ⁇ ELISA (manufactured by the company). The results are shown in Table 22.
• Example 9-1 there was more active TNF- ⁇ than in the comparative example in which the same concentration of carbohydrate or amine compound was used alone.
• Anti-human IFN ⁇ monoclonal antibody denaturation inhibition test A carbohydrate and an amine compound were mixed to the concentrations shown in Tables 23 to 26 to prepare a protein denaturation inhibition composition.
• This protein denaturation inhibition composition and a phosphate buffer containing a mouse monoclonal antibody against IFN ⁇ (MAb-IFN ⁇ -15, in-house preparation, LOT No. 010) were mixed in a volume ratio of 9:1 in a 0.5 mL Eppendorf tube (Eppendorf, product number 3810X) to prepare a protein preparation.
• the antibody concentration in the protein preparation was 70 ⁇ g/mL.
• the competitive inhibition rates for 0.5 M trehalose alone (Comparative Example 10-3) and 0.5 M sodium glutamate alone (Comparative Example 10-4) were 1% and 3%, respectively, but the 0.5 M protein denaturation suppression composition consisting of both (Example 10-1) had a high competitive inhibition rate of 39%, demonstrating a synergistic effect.
• the competitive inhibition rates for polysorbate 80 alone were 1% to 12% (Comparative Examples 10-5 to 10-8), the competitive inhibition rate (i.e., antibody stabilization effect) was synergistically improved when used in combination with the 0.5 M protein denaturation suppression composition (Examples 10-2 to 10-5).
• the protein denaturation suppression compositions containing polysorbate 20 had improved competitive inhibition rates (i.e., antibody stabilization effects) compared to the protein denaturation suppression composition not containing polysorbate 20 (Example 11-1).
• the competitive inhibition rate was higher when a composition for inhibiting protein denaturation 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) was used, demonstrating a synergistic effect.
• sodium aspartate showed the same effect as sodium glutamate.
• Examples 13-1 to 13-3 which used a composition for inhibiting protein denaturation consisting of trehalose and sodium glutamate, the change in absorbance over time was the same as that of the non-heated Comparative Example 13-1, and a synergistic stabilizing effect was observed.
• Anti-human IL-17A antibody 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 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.
• the competitive inhibition rate was reduced to 18% by heat treatment of the antibody (Comparative Example 14-2).
• the antigen binding was reduced by about 50% (Comparative Example 14-3), and the addition of sodium glutamate alone further reduced the antigen binding (Comparative Examples 14-4 to 14-6), which correlated with the increase in turbidity.
• a protein denaturation suppression composition consisting of trehalose and sodium glutamate (Examples 14-1 to 14-3)
• the antigen binding was the same as that of a non-heated antibody, and it was revealed that the protein denaturation suppression composition has a synergistic antibody protective effect compared to the case where a carbohydrate or an amine compound is used alone.
• the antibody used in this test is an example of an antibody preparation used in clinical practice, and this result shows that the protein denaturation suppression composition also has a protective effect on antibody preparations in clinical use.
• Anti-human IL-17A antibody denaturation inhibition test 2 The same procedures as in Comparative Example 14-1 to Comparative Example 14-6 and Example 14-1 to Example 14-3 were carried out, 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 antibody after heat treatment against the antigen (human IL-17A) was measured by competitive ELISA, and the results are shown in Table 29.
• the antigen binding activity of the antibody was reduced to 9% by heat treatment (Comparative Example 15-2).
• the antigen binding activity was reduced to 47% (Comparative Example 15-3), and the addition of sodium glutamate alone reduced the antigen binding activity further (Comparative Examples 15-4 to 15-6), which correlated with the increase in turbidity.
• compositions for inhibiting protein denaturation consisting of 1 M trehalose alone and sodium aspartate (Examples 15-1 to 15-3)
• the antigen binding activity was the same as that of a non-heated 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.
• Lactase Denaturation Inhibition Test A carbohydrate and an amine compound were mixed to the concentrations shown in Table 30 to prepare a composition for inhibiting protein denaturation.
• This composition for inhibiting protein denaturation and an aqueous solution containing lactase derived from the genus Kluyveromyces at a concentration of about 6 mg/mL were 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.
• 6- ⁇ -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. This composition for inhibiting protein denaturation and an aqueous solution containing 6- ⁇ -glucanotransferase derived from the genus Geobacillus at a concentration of about 25 mg/mL were heat-treated at 70° C. for 2 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 31.
• 6- ⁇ -glucanotransferase activity (residual activity (%)) after heat treatment was measured relative to the non-heated 6- ⁇ -glucanotransferase activity. The results are shown in Table 31.
• Example 17-2 As shown in Table 31, in Comparative Example 17-2, cloudiness was observed, and the residual activity after heating was significantly lower than in Comparative Example 17-1. In contrast, in Example 17, cloudiness was suppressed, and the residual activity was improved.
• 6- ⁇ -glucanotransferase denaturation inhibition test 2 A carbohydrate and an amine compound were mixed to prepare a composition for inhibiting protein denaturation.
• This composition for inhibiting protein denaturation and an aqueous solution containing 6- ⁇ -glucanotransferase derived from the genus Geobacillus at a concentration of about 25 mg/mL were freeze-dried to prepare a powder with the content shown in Table 32.
• the freeze-dried powder was kept at 70°C for 2 days in an incubator. The powder state was maintained even after storage, and there was no change in appearance due to moisture absorption. Thereafter, the 6- ⁇ -glucanotransferase activity after incubation (residual activity (%)) relative to the non-heated 6- ⁇ -glucanotransferase activity was measured. The results are shown in Table 32.
• 6- ⁇ -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 6- ⁇ -glucanotransferase derived from the genus Geobacillus at a concentration of about 45 mg/mL was freeze-dried to prepare a powder with the content shown in Table 33. The freeze-dried powder was stored in an incubator at 40° C. and a relative humidity of 75% for three months. The powder state was maintained even after storage, and no change in appearance due to moisture absorption occurred. Thereafter, the 6- ⁇ -glucanotransferase activity after incubation (residual activity (%)) was measured relative to the 6- ⁇ -glucanotransferase activity before incubation. The results are shown in Table 33.
• Serine protease denaturation inhibition test A carbohydrate and an amine compound were mixed to give the concentrations shown in Tables 34 to 35 to prepare a composition for inhibiting protein denaturation.
• 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 about 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 to 35.
• Enzyme activity was measured using fluorescently labeled casein included in the AAT Bioquest Amplite protease activity measurement kit. Specifically, the heat-treated sample was diluted 60-fold with purified water and added to a Corning 96-well black plate (3603) at 50 ⁇ L/well. Next, fluorescently labeled casein was diluted 100-fold with 80 mM phosphate buffer (pH 7.5) and added to the black plate at 50 ⁇ L/well. After incubation at 37°C for 15 minutes, the fluorescence intensity (Ex/Em: 490 nm/525 nm) was measured. In addition, non-heated proteolytic enzyme powder was diluted 2-fold with purified water from 50 ⁇ g/mL and added as a standard.

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