JP2011067202A - Stabilizer for aqueous protein solution, aqueous protein solution containing the same, and method for stabilization of protein - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a stabilizer for an aqueous protein solution capable of suppressing lowering of physiological activity of protein caused by aggregation, etc., of the protein in an aqueous solution and stabilizing the aqueous protein solution for a long time, and to provide a method for stabilizing the proteins by using the stabilizer. <P>SOLUTION: The stabilizer for the aqueous solution of the protein includes an organic compound (A) which fulfills following requirements (1) to (3): (1) when a proton is added to the molecule of the organic compound (A), a π-bond formed by a π electron of an atom (Z) and an atom (Y) bound to the atom (Z) has a resonance structure in at least one atom (Z) in the molecule, and there are at least four π electrons which are shared between the atom (Z) and the atom (Y) and are involved in the π-bond having the resonance structure; (2) the 0.1 mol/dm<SP>3</SP>solution of the organic compound (A) has an ionic strength of ≥0.1; and (3) the organic compound (A) has a molecular weight of <300. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a protein aqueous solution stabilizer, a protein aqueous solution containing the stabilizer, and a protein stabilization method using the stabilizer.

Proteins such as enzymes, antibodies, and peptides are widely used as detergents, diagnostic / testing agents, and pharmaceuticals. In these products, it is important that the physiological activity (titer) is not impaired during the manufacturing process and storage period. When protein is used as an aqueous solution, there is a problem that physiological activity cannot be maintained for a long time. Therefore, a method of supplying a purified protein preparation has been adopted as a method of supplying a protein without reducing its physiological activity.
As one method for stably extracting and purifying proteins, lyophilization is generally performed. Many proteins have the property of being easily inactivated by heat, but the freeze-drying method can stabilize the protein without applying heat.
However, the lyophilization method cannot be used for proteins that are denatured by dehydration, and has a drawback that it is likely to undergo alteration due to moisture absorption or oxidation during the lyophilization process. In addition, when a freeze-dried preparation is used by dissolving as an aqueous solution at the time of use, there is a problem that the aqueous solution in which the protein preparation is adjusted to a required concentration must be prepared each time.
For these reasons, techniques for stabilizing proteins in aqueous solutions have been disclosed. As a stabilizer for an aqueous solution of urease peroxidase, a technique for adding a polyhydric alcohol such as glycerin (Patent Document 1), an amino acid such as lysine such as bovine serum albumin or glucose, or an amino acid such as lysine is added to an aqueous solution containing cholesterol oxidase. Technology (patent document 2) etc. are mentioned.
However, it is difficult to say that these are methods for stabilizing a specific protein and are versatile, and are general-purpose stabilizers and stabilization methods that can be applied to all proteins and maintain their activity for a long period of time. There is no.

JP-A-6-70798 JP-A-8-187095

  Therefore, a protein aqueous solution stabilizer that can suppress a decrease in the physiological activity of the protein due to aggregation of the protein in the aqueous solution and stabilize the aqueous solution of the protein for a long period of time, and stabilization of the protein using this stabilizer The challenge is to provide a method.

The inventor of the present invention has arrived at the present invention as a result of studies to achieve the above object.
That is, this invention is a protein aqueous solution stabilizer, Comprising: The protein aqueous solution stabilizer containing the organic compound (A) which satisfy | fills the conditions of the following (1)-(3);
(1) When a proton is added to the molecule of the organic compound (A), at least one atom (Z) in the molecule has an atom (Z) and an atom (Y) bonded to the atom (Z). The π bond formed by π electrons has a resonance structure,
There are four or more π electrons that are atoms (Z) and atoms (Y) involved in the π bond of this resonance structure. (2) The ionic strength of the 0.1 mol / dm ³ solution is It is 0.1 or more. (3) The molecular weight is less than 300.
Moreover, the protein aqueous solution of this invention makes it a summary to contain the stabilizer of this protein aqueous solution, protein, and water.
Moreover, the protein stabilization method of this invention makes it a summary to mix the stabilizer of protein aqueous solution, protein, and water, and to stir for 1 minute-2 hours.

  The protein aqueous solution stabilizer of the present invention stabilizes the protein in the aqueous solution and does not decrease the physiological activity. Therefore, the protein aqueous solution containing the protein aqueous solution stabilizer of the present invention and the protein stabilization method of the present invention can maintain physiological activity for a long period of time.

Hereinafter, the protein aqueous solution stabilizer is also simply referred to as a stabilizer. Further, the organic compound (A) is also simply referred to as the compound (A).
The present invention is an aqueous protein protein stabilizer, which comprises an organic compound (A) that satisfies the following conditions (1) to (3):
(1) When a proton is added to the molecule of the organic compound (A), at least one atom (Z) in the molecule has an atom (Z) and an atom (Y) bonded to the atom (Z). The π bond formed by π electrons has a resonance structure,
There are four or more π electrons that are atoms (Z) and atoms (Y) involved in the π bond of this resonance structure. (2) The ionic strength of the 0.1 mol / dm ³ solution is It is 0.1 or more. (3) The molecular weight is less than 300.

  If the proteins are stored as they are as an aqueous solution, they will cause aggregation, hydrolysis and the like, and the titer will be significantly reduced. However, in the present invention, the above compound (A) having a specific chemical structure is present. Can be used as a stabilizer for protein aqueous solutions.

The condition (1) will be described.
For example, when the organic compound (A) is a guanidine molecule represented by the following general formula (1), the carbon atom (I) is bonded to the nitrogen atoms (II), (III) and (IV). . When a proton is added to the guanidine molecule, the carbon atom (I) becomes a cation and has a vacant orbit, and the nitrogen atoms (II) to (IV) each have a lone pair of electrons in the sp ³ hybrid orbital. These three lone electron pairs can form a π bond with the carbon atom (I) and have a resonance structure.
That is, when the carbon atom (I) in the guanidine molecule is the atom (Z), the atom (Y) bonded to the atom (Z) is a nitrogen atom (II) to (IV), This atom (Z) {carbon atom (I)} and atom (Y) {nitrogen atoms (II) to (IV)} have 6 electrons that are involved in the π bond of this resonance structure. One.
When protons are added to the guanidine molecule, nitrogen atoms (II) to (IV) are bonded to carbon atom (I) and two hydrogen atoms, respectively, and each lone pair forms a π bond. Therefore, when the nitrogen atoms (II) to (IV) are the atoms (Z), they each have two π electrons involved in the π bond.
Therefore, guanidine satisfies the condition (1) because the carbon atom (I) satisfies the condition of the atom (Z).

  Examples of the compound satisfying the condition (1) include guanidinium salts, guanazine, guanamine, formamidine, heterocyclic compounds (pyrrole, imidazole, pyridine, pyrimidine, indole, quinoline, isoquinoline, purine, etc.), organic phosphoric acid ( For example, phosphoric acid esters of alcohols having 1 to 30 carbon atoms).

In the condition (2) above, the ionic strength I is obtained by adding the product of the squares of the molar concentration m and the charge z of each ion for the ionic species i in the solution, and halving it. And is represented by the following equation.
I = 1 / 2Σm _i z _i ²
That is, the ionic strength of the 0.1 mol / dm ³ solution being 0.1 or more indicates that the value of I calculated by the above equation is 0.1 or more.

Examples of the organic compound (A) satisfying the above conditions (1) to (3) of the present invention include a heterocyclic compound (A-1), an organic phosphoric acid (A-2), nitrogen having 1 to 20 carbon atoms, oxygen And a compound (A-3) having two or more sulfur atoms and a salt thereof (A-4).
Examples of the heterocyclic compound (A-1) include nitrogen-containing heterocyclic compounds, oxygen-containing heterocyclic compounds, sulfur-containing heterocyclic compounds, and phosphorus-containing heterocyclic compounds, such as pyrrole, imidazole, pyridine, pyrimidine, indole, Examples include quinoline, isoquinoline, purine, indole, quinoline, isoquinoline, furan, thiophene, oxazole, thiazole, isoxazole, and isothiazole.
Examples of the organic phosphoric acid (A-2) include organic phosphoric acids having 1 to 20 carbon atoms, such as methyl phosphoric acid, ethyl phosphoric acid, isopropyl phosphoric acid, butyl phosphoric acid, dimethyl phosphoric acid, diethyl phosphoric acid, dipropyl phosphoric acid, dibutyl phosphorous. Examples of the compound (A-3) having 2 or more of nitrogen, oxygen and sulfur atoms having 1 to 20 carbon atoms include compounds represented by the following general formula (2): acid, methyl pyrophosphate, ethyl pyrophosphate, etc. (A), an amidino group-containing compound, an amide group-containing compound and the like can be mentioned, and examples thereof include guanidine, urea, thiourea, formamidine, methylamidine, biguanide and the like.
Examples of these salts (A-4) include salts of the compound (a) represented by the following general formula (2). Examples of the salt include inorganic acid salts such as hydrochloride, phosphate, and sulfate, and organic acid salts such as carbonate, citrate, and acetate.

The content (% by weight) of the organic compound (A) contained in the stabilizer of the present invention is preferably 10 to 100, more preferably 20 to 90, based on the weight of the stabilizer from the viewpoint of protein stabilization. Next, it is more preferably 30 to 80, particularly preferably 30 to 70.
From the viewpoint of protein stabilization, the content (% by weight) of the compound (A) contained in the stabilizer of the present invention is 2 to 6000% by weight relative to the weight of the protein when the stabilizer is used. It is preferable to contain so that it may become, More preferably, it is contained so that it may become 5 to 500 weight%, and it is still more preferably contained so that it may become 10 to 100 weight% next.

  In the protein aqueous solution stabilizer of the present invention, the organic compound (A) is preferably a compound (a) represented by the following general formula (2) and a salt of the compound (a) from the viewpoint of sustained activity. .

  In the general formula (2), X represents an imino group, an oxygen atom or a sulfur atom.

  Specific examples of the compound (a) represented by the general formula (2) include guanidine, urea and thiourea.

Examples of the salt of the compound (a) represented by the general formula (2) include guanidine salts.
Examples of the salt include hydrochloride, carbonate, borate, sulfate and phosphate.

  The compound (a) and the salt of the compound (a) are preferably a guanidine salt and urea, more preferably a guanidine salt, and still more preferably a guanidine hydrochloride from the viewpoint of protein stabilization.

  The stabilizer of the present invention can further contain a compound (B) represented by the following general formula (3).

  In general formula (3), Q represents an alkyl group, and a part of the hydrogen atoms in the alkyl group may be substituted with a group other than a hydrogen atom.

Examples of the alkyl group of Q include an alkyl group having 1 to 22 carbon atoms, specifically a methyl group, an ethyl group, a propyl group, a butyl group, a pentyl group, a hexyl group, an octyl group, a nonyl group, a decyl group, and a dodecyl group. , A cetyl group, a stearyl group, a behenyl group, and the like. Some of the hydrogen atoms in these alkyl groups may be substituted with groups other than hydrogen atoms.
Examples of substituents other than hydrogen atoms include amino groups, carboxyl groups, amide groups, ester groups, imino groups, and hydroxyl groups. The number of substituents is preferably 1 to 3, more preferably 2 to 3. For example, when two hydrogen atoms at the terminal of the butyl group are substituted with one amino group and one carboxyl group, the compound (B) represents arginine.

  Examples of the compound (B) include arginine or a salt thereof (B-1) and an arginine derivative or a salt thereof (B-2).

  Arginine, its salt (B-1), arginine, arginine inorganic acid salt (hydrochloride, borate, phosphate, pyrophosphate, sulfate, silicate, etc.) and arginine organic acid salt (formic acid) Salt, acetate, oxalate, lactate, citrate, trimellitic acid salt and pyromellitic acid salt).

In the arginine derivative or a salt thereof (B-2), the arginine derivative is a derivative in which the α-amino group or α-carboxyl group of arginine represented by the following general formula (4) or both of these groups are substituted.
The substitution of the α-amino group is performed on the N-alkylcarbonyl-amide group (Y-1) represented by the following general formula (5) or the imino group (Y-2) represented by the following general formula (6). The substitution of the α-carboxyl group is to the ester group (Z-1) represented by the following general formula (7) or the N-alkylamide group (Z-2) represented by the following general formula (8). Is a replacement.

  In other words, in the arginine derivative or a salt thereof (B-2), at least one of an α-amino group and an α-carboxyl group is substituted. That is, when Y is an amino group, Z is an ester group (Z-1) represented by the following general formula (7) or an N-alkylamide group (Z-2) represented by the following general formula (8). Yes, when Z is a carboxyl group, Y is an N-alkylcarbonyl-amide group (Y-1) represented by the following general formula (5) or an imino group (Y-2) represented by the following general formula is there.

  In the general formula (4), Y is an amino group, an N-alkylcarbonyl-amide group (Y-1) represented by the following general formula (5) or an imino group (Y-) represented by the following general formula (6). 2). Z represents a carboxyl group, an ester group (Z-1) represented by the following general formula (7) or an N-alkylamide group (Z-2) represented by the following general formula (8).

In the general formula (5), R ¹ represents a hydrogen atom or a monovalent hydrocarbon group having 1 to 36 carbon atoms, and this hydrocarbon group has a part of the hydrogen atom as a functional group other than a hydrogen atom. May be substituted.

The hydrocarbon group of R ^{1 in} the N-alkylcarbonyl-amide group (Y-1) represented by the general formula (5) is a monovalent hydrocarbon group having 1 to 36 carbon atoms, and is linear or branched Aliphatic hydrocarbon groups, alicyclic hydrocarbon groups and aromatic hydrocarbon groups.
Linear aliphatic hydrocarbon groups include methyl, ethyl, propyl, butyl, pentyl, hexyl, octyl, nonyl, decyl, lauryl, palmityl, stearyl, oleyl and Examples include a behenyl group.
Examples of the branched aliphatic hydrocarbon group include an isopropyl group and a t-butyl group.
Examples of the alicyclic hydrocarbon group include a cyclohexyl group, a methylcyclohexyl group, and a cyclohexylmethyl group.
Examples of the aromatic hydrocarbon group include a phenyl group, a methylphenyl group, a benzyl group, a phenylethyl group, and a methylbenzyl group.
Of these hydrocarbon groups, from the viewpoint of stabilization of the protein-containing aqueous solution, a linear aliphatic hydrocarbon group is preferable, a methyl group and an ethyl group are more preferable, and a methyl group is most preferable.
Examples of substituents other than hydrogen atoms include amino groups, carboxyl groups, amide groups, ester groups, imino groups, and hydroxyl groups.

  Specific examples of the N-alkylcarbonyl-amide group (Y-1) represented by the general formula (5) include formamide group, acetylamide group, propionic acid amide group, butyric acid amide group, hexylic acid amide group, and cyclohexyl. Examples include an acid amide group, an octylic acid amide group, and a benzoylamide group.

In the general formula (6), R ² and R ³ each independently represent a hydrogen atom or a hydrocarbon group having 1 to 36 carbon atoms, and in these hydrocarbon groups, a part of the hydrogen atom is other than the original hydrogen atom. The functional group may be substituted.

In the imino group (Y-2) represented by the general formula (6), R ² and R ³ include the same hydrocarbon group as R ^1, and these hydrocarbon groups are the same as R ¹ , Some may be substituted with other functional groups.

  Examples of the imino group (Y-2) represented by the general formula (6) include a methylimino group.

In the general formula (7), R ⁴ represents a residue obtained by removing one hydroxyl group from a hydrocarbon group or a polyhydric alcohol or sugar having 1 to 36 carbon atoms.
In this hydrocarbon group, part of the hydrogen atoms may be substituted with another functional group such as a hydroxyl group, a methoxyl group, an ethoxyl group, a nitro group, or a hydroxyphenyl group.

In the ester group (Z-1) represented by the general formula (7), when R ⁴ is a hydrocarbon group having 1 to 36 carbon atoms, the hydrocarbon group includes the same hydrocarbon group as R ^1. It is.
In the case where R ⁴ is a hydrocarbon group having 1 to 36 carbon atoms, among these hydrocarbon groups, a linear aliphatic hydrocarbon group is preferable from the viewpoint of stabilization of the protein-containing aqueous solution, more preferably a methyl group and An ethyl group, most preferably an ethyl group.

Examples of the polyhydric alcohol include divalent to trivalent alcohols, and examples thereof include ethylene glycol, propylene glycol, diethylene glycol, and glycerin.
Examples of the sugar include glucose, sucrose, sorbitol, mannitol and trehalose.

In General Formula (8), R ⁵ represents a hydrogen atom or a hydrocarbon group having 1 to 36 carbon atoms, and in this hydrocarbon group, a part of the hydrogen atom is substituted with another functional group other than a hydrogen atom. May be.

In the N-alkylamide group (Z-2) represented by the general formula (8), when R ⁵ is a hydrocarbon group having 1 to 36 carbon atoms, the hydrocarbon group is the same as R ^1. A hydrogen group is included, and these hydrocarbon groups may be partially substituted with other functional groups in the same manner as R ¹ .
In the case where R ⁵ is a hydrocarbon group having 1 to 36 carbon atoms, among these hydrocarbon groups, a linear aliphatic hydrocarbon group is preferable from the viewpoint of stabilization of the protein-containing aqueous solution, and more preferably a methyl group and An ethyl group, most preferably a methyl group.

  When the arginine derivative or a salt thereof (B-2) is a salt of an arginine derivative, the salt includes inorganic acid salts (hydrochloride, borate, phosphate, pyrophosphate, sulfate, silicate, etc.) and organic Acid salts (formate, acetate, oxalate, lactate, citrate, trimellitic acid, pyromellitic acid, etc.).

  Specific examples of the arginine derivative or a salt thereof (B-2) include N-α-acetylarginine ethyl ester hydrochloride.

  The compound (B) contained in the stabilizer of the present invention is preferably an arginine derivative or a salt thereof (B-2), more preferably an α-amino group and α-amino group of arginine, from the viewpoint of sustained activity. It is a derivative in which both groups of the carboxyl group are substituted, and N-α-acetylarginine ethyl ester hydrochloride is particularly preferable.

The content (% by weight) of the compound (B) contained in the stabilizer of the present invention is preferably 10 to 90, more preferably 20 to 80, based on the weight of the stabilizer from the viewpoint of protein stabilization. More preferably, it is 30-70.
The content (% by weight) of the compound (B) contained in the stabilizer of the present invention is 2 to 500% by weight with respect to the weight of the protein when the stabilizer is used from the viewpoint of protein stabilization. It is preferable to contain so that it may become, More preferably, it is contained so that it may become 5 to 200 weight%, and it is still more preferably contained so that it may become 10 to 100 weight% next.

  Although the stabilizer of this invention should contain only an organic compound (A), it is preferable to contain an organic compound (A) and a compound (B) from a viewpoint of protein stabilization.

  When the stabilizer of the present invention contains the organic compound (A) and the compound (B), the weight ratio of (A) to (B) (weight of the organic compound (A) / weight of the compound (B)) is 0.1-9 are preferable, More preferably, it is 0.2-8, Most preferably, it is 0.5-5.

  In addition to the organic compound (A) and compound (B), the stabilizer of the present invention is a surfactant (E), inorganic salt (F), polyhydric alcohol (G), sugar (H), amino acid other than arginine. (I) and other organic compounds (J) can be used in combination.

  Surfactant (E) includes anionic surfactants, cationic surfactants, amphoteric surfactants and nonionic surfactants, polyethylene glycol, polyoxypropylene / polyoxyethylene copolymer, sorbitan Examples include an alkyl ester ethylene oxide adduct, an aliphatic alcohol ethylene oxide adduct, a fatty acid ethylene oxide adduct, and an alkylamine ethylene oxide adduct.

  Examples of the inorganic salt (F) include sodium chloride, sodium borate, calcium chloride, magnesium chloride, sodium formate, magnesium sulfate, and ammonium sulfate.

  Examples of the polyhydric alcohol (G) include ethylene glycol, propylene glycol, and glycerin.

  Examples of the sugar (H) include trehalose, sucrose, dextrin, cyclodextrin, maltose, fructose, hyaluronic acid and chondroitin sulfate.

  Examples of amino acids (I) other than arginine include glycine, alanine, aspartic acid, asparagine, phenylalanine, tryptophan, tyrosine, leucine, lysine, histidine, and salts thereof.

  Although it does not specifically limit as another organic compound (J), Serum albumin, collagen, casein, gelatin, and a silk peptide are mentioned.

The content (% by weight) of the surfactant (E) contained in the stabilizer of the present invention is preferably 0 to 50, more preferably 0 to 30 with respect to the weight of the stabilizer from the viewpoint of protein stabilization. Next, it is more preferably 0-20.
The content (% by weight) of the inorganic salt (F) contained in the stabilizer of the present invention is preferably 0-20, more preferably 0-15, with respect to the weight of the stabilizer from the viewpoint of protein stabilization. More preferably, it is 0-10.
The content (% by weight) of the polyhydric alcohol (G) contained in the stabilizer of the present invention is preferably 0 to 70, more preferably 0 to 60, based on the weight of the stabilizer from the viewpoint of protein stabilization. Next, it is more preferably 0-50.
The content (% by weight) of the saccharide (H) contained in the stabilizer of the present invention is preferably 0 to 50, more preferably 0 to 30 with respect to the weight of the stabilizer from the viewpoint of protein stabilization. More preferably, it is 0-20.
The content (% by weight) of the amino acid (I) other than arginine contained in the stabilizer of the present invention is preferably 0 to 30 with respect to the weight of the stabilizer, more preferably 0 to 0, from the viewpoint of protein stabilization. 20, then more preferably 0-10.
The content (% by weight) of the other organic compound (J) contained in the stabilizer of the present invention is preferably 0 to 10, more preferably 0 to 0 with respect to the weight of the stabilizer from the viewpoint of protein stabilization. 5, then more preferably 0-3.

Another embodiment of the present invention is an aqueous protein solution containing the protein stabilizer, protein and water.
In the protein aqueous solution, the content (% by weight) of the stabilizer is preferably 0.1 to 50 based on the weight of the protein aqueous solution from the viewpoint of protein stabilization.
In the aqueous protein solution, the content (% by weight) of the protein is preferably 0.01 to 2 based on the weight of the aqueous protein solution from the viewpoint of long-term stability.
In the protein aqueous solution, the content (% by weight) of water is preferably 48 to 99.8 based on the weight of the protein aqueous solution from the viewpoint of protein stabilization.

In the protein aqueous solution of the present invention, the content (% by weight) of (A) is preferably 0.01 to 40 based on the weight of the protein aqueous solution from the viewpoint of protein stabilization.
In the protein aqueous solution, the weight ratio of the protein in the protein aqueous solution to (A) (weight of (A) / weight of protein) is preferably 2 to 6000, more preferably 5 to 500, from the viewpoint of protein stabilization. Then, more preferably, it is 10-100.
In the protein aqueous solution, the content (% by weight) of (B) is preferably 0.01 to 20 based on the weight of the protein aqueous solution from the viewpoint of protein stabilization.
In the protein aqueous solution, the weight ratio of the protein in the protein aqueous solution to (B) (weight of (B) / weight of protein) is preferably 2 to 500, more preferably 5 to 200, from the viewpoint of protein stabilization. Then, more preferably, it is 10-100.

  Although it does not specifically limit as protein which can apply the stabilizer of protein aqueous solution of this invention, An enzyme, recombinant protein, an antibody, a peptide, etc. are mentioned.

  Enzymes include oxidoreductases {cholesterol oxidase, glucose oxidase, ascorbate oxidase, peroxidase, etc.}, hydrolases {lysozyme, protease, serine protease, amylase, lipase, cellulase, glucoamylase, etc.}, isomerase {glucose isomerase, etc. Etc.}, transferase {acyltransferase, sulfotransferase, etc.}, synthase {fatty acid synthase, phosphate synthase, citrate synthase, etc.} and elimination enzyme {pectin lyase etc.}.

  As recombinant proteins, protein preparations {interferon α, interferon β, interleukins 1 to 12, growth hormone, erythropoietin, insulin, granular colony stimulating factor (G-CSF), tissue plasminogen activator (TPA), sodium And diuretic peptides, blood coagulation factor VIII, somatomedin, glucagon, growth hormone releasing factor, serum albumin, calcitonin and the like} and vaccines {hepatitis A vaccine, hepatitis B vaccine and hepatitis C vaccine} and the like.

  Examples of antibodies include monoclonal antibodies and polyclonal antibodies.

The peptide is not particularly limited in amino acid composition, and examples thereof include dipeptides and tripeptides.
Among these proteins, enzymes are preferable from the viewpoint of long-term stabilization of activity.

  The water contained in the protein aqueous solution of the present invention is not particularly limited, and examples thereof include tap water, ion exchange water, distilled water, and reverse osmosis water.

A buffering agent may be added to the protein-containing aqueous solution of the present invention as long as the stabilizing effect is not impaired.
Examples of the buffer include known buffers, and specific examples include Tris buffer (Tris buffer), HEPES buffer, and MOPS buffer. Among these, an alkaline buffer is preferable, and a tris buffer is particularly preferable from the viewpoint of availability.

  An example of a method for producing an aqueous protein solution using the stabilizer of the present invention will be described below. For production of an aqueous protein solution, the stabilizer may be added to the aqueous protein solution as it is or dissolved in water. May be added to the aqueous solution of the stabilizer, or the protein, stabilizer and water may be mixed together.

As an example of a method for producing an aqueous protein solution using the stabilizer of the present invention, an example in the case of producing a stabilized aqueous solution of an enzyme separated in a separation and purification step will be given below.
(1) A stabilizer is added to water to prepare an aqueous solution.
(2) The enzyme aqueous solution after separation and purification is added to the aqueous solution.
(3) Store sealed at 25 ° C. or refrigerator.

  The protein stabilization method of the present invention is a protein stabilization method in which the protein stabilizer, the protein and water are mixed and stirred for 1 minute to 2 hours to stabilize the protein.

  In the protein stabilization method of the present invention, the amount of stabilizer added, the amount of protein, the amount of water, the weight ratio of protein to organic compound (A) in the stabilizer, and the amount of protein in stabilizer The weight ratio of the compound (B) is the same as described in the protein aqueous solution, and the preferred range is also the same.

  In the protein stabilization method of the present invention, when the stabilizer, protein and water are mixed, the stabilizer may be added as it is or dissolved in water to the protein aqueous solution, or the protein may be added to the stabilizer aqueous solution. You may add, and you may mix protein, a stabilizer, and water together.

  In the protein stabilization method of the present invention, the temperature (° C.) of the solution when mixing the protein stabilizer, protein and water is preferably 4 ° C. to 40 ° C. from the viewpoint of protein stability. More preferably, it is 4 degreeC-25 degreeC.

  The following examples further illustrate the invention, but the invention is not limited thereto.

<Production Example 1>
N-α-acetylarginine {Arginine acetamide, MP Bio Japan Co., Ltd.} 12.6 parts by weight (0.05 mole part), 1 part of methanesulfonic acid and 92 parts by weight of ethanol (2 mole part) were mixed uniformly. The mixture was heated and stirred at 5 ° C. for 5 hours, concentrated with an evaporator, and then neutralized by adding 5.2 parts by weight (0.05 parts by mole) of hydrochloric acid (35% by weight). Then, it recrystallized from water, dried under reduced pressure {60 degreeC, 20Pa}, and obtained N- (alpha) -acetylarginine ethyl ester hydrochloride which is a compound (B).

<Example 1>
100 mg of guanidine hydrochloride was used as a stabilizer (N-1).

<Examples 2 to 15>
In Example 1, the stabilizers (N-2) to (N-15) were the same as in Example 1 except that the organic compounds (A) and the compounds (B) were changed to the compositions and amounts shown in Table 1. Used as.

<Comparative Example 1>
A conventional stabilizer, 100 mg of propylene glycol, was used as a stabilizer (HN-1).

<Comparative Examples 2-4>
In Comparative Example 1, it was used as stabilizers (HN-2) to (HN-4) in the same manner as Comparative Example 1 except that propylene glycol was changed to the composition and amount shown in Table 2.
In Comparative Example 4, nothing was used as the stabilizer (0 mg), but for convenience, the stabilizer (HN-4) was used.

In the above Examples and Comparative Examples, the following reagents were used for Compound (A) and Compound (B).
Guanidine hydrochloride: Wako Pure Chemical Industries, Ltd. Urea: Wako Pure Chemical Industries, Ltd. Pyridine: Wako Pure Chemical Industries, Ltd. Arginine hydrochloride: Wako Pure Chemical Industries, Ltd. Arginine ethyl ester: Wako Pure Acetyl arginine hydrochloride manufactured by Yakuhin Kogyo Co., Ltd .: Wako Pure Chemical Industries, Ltd.

In the above Examples and Comparative Examples, the following reagents were used as conventional stabilizers.
Propylene glycol: Wako Pure Chemical Industries, Ltd. Glycerin: Wako Pure Chemical Industries, Ltd. Trehalose: Wako Pure Chemical Industries, Ltd.

<Measurement of enzyme activity of cellulase>
Stabilizers (N-1) to (N-15) of Examples 1 to 15 and Comparative Examples 1 to 4 and 25 of stabilizers (HN-1) to (HN-4) of Comparative Examples 1 to 4 were each used. Added to 1.0 mL of 50 mmol / L Tris buffer (pH = 7) at 50 ° C., and mixed uniformly. Further, 5.0 mg of cellulase {Wako Pure Chemical Industries, Ltd.} was added and mixed uniformly for 1 minute. S-1) to (S-15) and (HS-1) to (HS-4) were obtained, respectively. The obtained cellulase aqueous solution was sealed in a thermostat at 25 ° C. for 2 days, 5 days, 2 weeks, and 1 month, and the enzyme activity was measured by the following method.
200 μL of each cellulase aqueous solution was added to 800 μL of a 5 wt% cellulose suspension (5 g of cellulose manufactured by Wako Pure Chemical Industries, Ltd. added to 95 g of Tris buffer with pH = 7 and suspended), and tumbled at 37 ° C. for 2 hours. Mixed. 100 μL of the supernatant was added to 100 μL of glucose determination kit solution (“Test Wako” manufactured by Wako Pure Chemical Industries, Ltd.). Immediately after that, the absorbance (A ₀ ) at 492 nm was measured at 25 ° C. with a microplate reader (manufactured by TECAN Austria GmbH, model: Sunrise Thermo), and the measurement solution was allowed to stand at 25 ° C. for 15 minutes and then again. The absorbance (A ₁₅ ) was measured at 25 ° C., and the difference (A ₁₅ −A ₀ ) (ΔA) was calculated.
On the other hand, immediately after preparing the cellulase aqueous solution (HS-4), the absorbance difference (A ₁₅ -A ₀ ) (ΔA _b ) was calculated in the same manner as described above, and the “enzyme activity remaining ratio” was calculated from the following equation. did.

Enzyme activity remaining rate (%) = (ΔA / ΔA _b ) × 100

The results are shown in Tables 1 and 2.

<Measurement of enzyme activity of protease>
Stabilizers (N-1) to (N-15) of Examples 1 to 15 and Comparative Examples 1 to 4 and 25 of stabilizers (HN-1) to (HN-4) of Comparative Examples 1 to 4 were each used. Add to 1.0 mL of 50 mmol / L Tris buffer (pH = 7) at 50 ° C. and mix uniformly. Further, add 5.0 mg of protease {Wako Pure Chemical Industries, Ltd.} and mix uniformly for 1 minute. P-1) to (P-15) and (HP-1) to (HP-4) were obtained, respectively. The obtained protease aqueous solution was sealed in a thermostatic chamber at 25 ° C. for 2 days, 5 days, 2 weeks, and 1 month, and the enzyme activity was measured by the following method.
1 mL of each protease solution is weighed into a 15 mL test tube, and 5 mL of 0.5 wt% casein solution (0.5 g of casein manufactured by Wako Pure Chemical Industries, Ltd. dissolved in 100 mL of 0.05 mol / L Tris buffer) In addition, it was left at 25 ° C. for 10 minutes. Thereafter, 5 mL of a 5% TCA solution [trichloroacetic acid (manufactured by Wako Pure Chemical Industries, Ltd.) dissolved in ion-exchanged water] was added and left at 25 ° C. for 20 minutes. Processing was performed at 2500 rpm for 20 minutes with a centrifuge (manufactured by KUBOTA, model: cooling centrifuge 3922), and 2 mL of the remaining supernatant was measured into another 15 mL test tube. To this test tube, 5 mL of a 0.5 M aqueous sodium carbonate solution and 1 mL of a foreign reagent (manufactured by Wako Pure Chemical Industries) diluted 3-fold with ion-exchanged water were added and left for 20 minutes.
The absorbance (A) of this solution at 660 nm at 25 ° C. was measured using a spectrophotometer (manufactured by Shimadzu Corporation, model: UV-1700), and the enzyme activity of the protease was calculated.
For the blank, a measurement solution was prepared in the same manner as described above using the protease aqueous solution (HP-4) immediately after preparation, and the absorbance (A _b ) was measured. The “enzyme activity remaining rate” was calculated from the following equation.

Enzyme activity remaining rate (%) = (A / A _b ) × 100

The results are shown in Tables 1 and 2.

<Measurement of enzyme activity of lipase>
Stabilizers (N-1) to (N-15) of Examples 1 to 15 and Comparative Examples 1 to 4 and 25 of stabilizers (HN-1) to (HN-4) of Comparative Examples 1 to 4 were each used. 50 mL / L Tris buffer solution (manufactured by Wako Pure Chemical Industries, Ltd.) (pH = 7) at 1.0 ° C. and uniformly mixed, and 5.0 mg of lipase {Wako Pure Chemical Industries, Ltd.} is added for 1 minute. It mixed uniformly and obtained lipase aqueous solution (L-1)-(L-15) and (HL-1)-(HL-4), respectively. The obtained lipase aqueous solution was sealed in a thermostatic chamber at 25 ° C. for 2 days, 5 days, 2 weeks and 1 month, and the enzyme activity was measured by the following method.
100 μL of each lipase aqueous solution is weighed into a 5 mL test tube, 2 mL of 50 mmol / L Tris buffer (pH = 7.0) is added, and 1 mL of 5 μM p-nitrophenyl acetate (manufactured by Wako Pure Chemical Industries, Ltd.) is added. It was.
Absorbance change (ΔA) (absorption of the product of trinitrophenyl acetate decomposed by enzyme) at 400 nm immediately after preparation of the measurement solution and after standing at 25 ° C. for 10 minutes was measured with a spectrophotometer (manufactured by Shimadzu Corporation, model: UV -1700).
As a blank, a measurement solution was prepared in the same manner as described above using the (HL-4) lipase aqueous solution immediately after preparation, and the change in absorbance (ΔA _b ) was measured.
When the lipase (enzyme) is not denatured, the activity is maintained and p-nitriphenyl acetate is efficiently decomposed, the absorbance increases (the absorbance is close to that of a blank).
The “enzyme activity remaining rate” was calculated from the following equation.

Enzyme activity remaining rate (%) = (ΔA / ΔA _b ) × 100

The results are shown in Tables 1 and 2.

<Measurement of enzyme activity of amylase>
Stabilizers (N-1) to (N-15) of Examples 1 to 15 and Comparative Examples 1 to 4 and 25 of stabilizers (HN-1) to (HN-4) of Comparative Examples 1 to 4 were each used. Add 50 mL of 50 mmol / L Tris buffer (pH = 7) at 1.0 ° C. and mix uniformly. Add 5.0 mg of lipase {Wako Pure Chemical Industries, Ltd.} and mix uniformly for 1 minute. AM-1) to (AM-15) and (HAM-1) to (HAM-4) were obtained. The obtained amylase aqueous solution was sealed in a thermostat at 25 ° C. for 2 days, 5 days, 2 weeks, and 1 month, and the enzyme activity was measured by the following method.
To 10 μL of each amylase aqueous solution, 5 mL of 0.5% by weight starch suspension (0.5 g of starch manufactured by Wako Pure Chemical Industries, Ltd. dissolved in 100 mL of 0.05 mol / L Tris buffer) was added, and at 60 ° C. Shake for 20 minutes.
After shaking, the mixture was filtered through Whatman's filter paper (grade No. 1, 9 cm) to obtain a filtrate. To a 96-well microplate, 100 μL of the filtrate and 100 μL of glucose determination reagent (“Test Wako” manufactured by Wako Pure Chemical Industries, Ltd.) were added and allowed to stand at 25 ° C. for 10 minutes. Absorbance (A ₂₀ ) at 492 nm was measured with a microplate reader (manufactured by TECAN Austria GmbH, model: Sunrise Thermo).
In addition, a measurement solution was prepared in the same manner as described above using an aqueous amylase solution of (HAM-4) just prepared as a blank, and absorbance (A _20b ) was measured.
When the amylase (enzyme) is not denatured, the activity is maintained and the starch is efficiently decomposed, the absorbance increases (the absorbance is close to that of a blank).
The “enzyme activity remaining rate” was calculated from the following equation.

Enzyme activity remaining rate (%) = (A ₂₀ ) / (A _20b ) × 100

The results are shown in Tables 1 and 2.

From the results of Tables 1 and 2, when the stabilizers (N-1) to (N-15) of Examples 1 to 15 of the present invention were used, Comparative Example 4 (HN-) containing no stabilizer was used. Compared with 4), it can be seen that the residual enzyme activity after storage for 1 month is extremely large. Furthermore, even when compared with the conventional stabilizers (HN-1) to (HN-4) of Comparative Examples 1 to 3, the residual enzyme activity after storage for 1 month is extremely large, and the physiological activity of the protein is prolonged. It can be seen that the period can be maintained.
Moreover, when the compound (A) is guanidine hydrochloride and Examples 1, 4 to 7, 14 and 15 using the same amount of the compound (A) are compared, Example 1 using only the compound (A) In Examples 4 to 7, 14 and 15 in which the compounds (A) and (B) are used in combination, the residual rate of enzyme activity after storage for 1 month is larger, and the physiological activity of the protein is maintained high for a long time. I understand that I can do it.

  The protein aqueous solution stabilizer of the present invention can be effectively used in the fields of pharmaceuticals, foods, detergents and biochemistry because it stabilizes proteins and does not reduce activity for a long period of time. For example, it can be used for protein pharmaceutical liquid preparations, enzyme liquid preparations, industrial enzyme aqueous solutions, liquid detergents, beverages, measuring reagents for diagnostic agents, protein standard solutions, and the like.

Claims (10)

A protein aqueous solution stabilizer comprising an organic compound (A) that satisfies the following conditions (1) to (3):
(1) When a proton is added to the molecule of the organic compound (A), at least one atom (Z) in the molecule has an atom (Z) and an atom (Y) bonded to the atom (Z). The π bond formed by π electrons has a resonance structure,
An atom (Z) and an atom (Y) have,
There are 4 or more π electrons participating in the π bond of this resonance structure (2) The ionic strength of the 0.1 mol / dm ³ solution is 0.1 or more (3) The molecular weight is less than 300 thing. The protein aqueous solution stabilizer according to claim 1, wherein the organic compound (A) is the compound (a) represented by the following general formula (2).

[Wherein, X represents an imino group, an oxygen atom or a sulfur atom. ] The protein aqueous solution stabilizer according to claim 1, wherein the organic compound (A) is a salt of the compound (a) represented by the following general formula (2).

[Wherein, X represents an imino group, an oxygen atom or a sulfur atom. ] The protein aqueous solution stabilizer according to claim 1, wherein the organic compound (A) is guanidine hydrochloride. The protein aqueous solution stabilizer according to any one of claims 1 to 4, wherein the protein is an enzyme. Furthermore, the stabilizer of the protein aqueous solution in any one of Claims 1-5 containing the compound (B) represented by following General formula (3).

[Wherein, Q represents an alkyl group, and a part of hydrogen atoms in the alkyl group may be substituted with a group other than a hydrogen atom. ] The protein aqueous solution containing the stabilizer of protein aqueous solution in any one of Claims 1-6, protein, and water. The protein aqueous solution according to claim 7, wherein the content of the organic compound (A) is 0.01 to 40% by weight based on the weight of the protein aqueous solution. A method for stabilizing a protein, comprising mixing the protein aqueous solution stabilizer according to any one of claims 1 to 6, protein and water, and stirring the mixture for 1 minute to 2 hours. The method for stabilizing a protein according to claim 9, wherein a weight ratio of the protein to the organic compound (A) in the stabilizer (weight of the organic compound (A) / weight of the protein) is 2 to 6000.