JP2019142787A - Protein stabilizer - Google Patents

Abstract

To provide a high-performance protein stabilizer by chemical synthesis, useful for maintaining activity of an antibody and the like used as a clinical diagnostic agent.SOLUTION: A protein stabilizer contains a hydrophilic repeating unit composed of a repeating unit represented by the following formula or the like, and a copolymer having a hydrophobic repeating unit. [In a formula (1), Ris a hydrogen atom or a methyl group, Rand Rindependently represent a hydrogen atom, or a C1-6 alkyl group or hydroxyalkyl group].SELECTED DRAWING: None

Description

The present invention relates to a stabilizer for various proteins used in, for example, clinical diagnostics, clinical diagnostic devices, biochips and the like.

  Proteins such as labeled antibodies, labeled antigens, enzymes, primary antibodies, and primary antigens used as clinical diagnostic agents are generally added with bovine serum albumin (BSA) in order to maintain their activity in a solution state. . However, even when BSA is added, the activity of the protein still decreases, and when a biologically derived stabilizer is used, there is a problem of biological contamination represented by BSE. The development of protein stabilizers is desired.

  As protein stabilizers by chemical synthesis, JP-A-10-45794 discloses a polymer having phosphorylcholine, JP-A-10-279594 discloses a fatty acid ester film typified by triacylglycerol, and JP-A-11-69973. Polyols typified by glycerol and polymers containing glycoside derivatives as monomer units have been proposed in JP-A-7-255477, but the protein activity maintaining effect of these stabilizers was not sufficient.

Japanese Patent Laid-Open No. 10-45794 Japanese Patent Laid-Open No. 10-279594 JP-A-11-69973 JP 7-255477 A

  An object of the present invention is to provide a high-performance protein stabilizer by chemical synthesis that is useful for maintaining the activity of antibodies used as clinical diagnostic agents.

  In order to solve this problem, the present inventors have found that a copolymer having a specific composition has a high protein stabilizing effect, and have completed the present invention.

  The protein stabilizer which concerns on 1 aspect of this invention is at least 1 chosen from the repeating unit represented by following formula (1), Formula (2), Formula (3), Formula (4), and Formula (5). A hydrophilic repeating unit (A) consisting of two repeating units and a hydrophobic repeating unit (B) consisting of at least one repeating unit selected from the repeating units represented by the following formula (6) and the following formula (7) Contains a copolymer.

[In Formula (1),
R ¹ represents a hydrogen atom or a methyl group,
R ² and R ³ each independently represent a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a hydroxyalkyl group. ]

[In Formula (2),
R ⁴ represents a hydrogen atom or a methyl group,
R ⁵ and R ⁶ each independently represent a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a hydroxyalkyl group. ]

[In Formula (3),
R ⁷ represents a hydrogen atom or a methyl group,
R ⁸ represents —O—, * — (C═O) —O—, * —NR ¹¹ — (C═O) — (R ¹¹ represents a hydrogen atom or an organic group having 1 to 10 carbon atoms, * Represents a position bonded to the carbon atom to which R ⁸ in Formula (2) is bonded) or a phenylene group.
R ⁹ represents an alkanediyl group having 2 to 4 carbon atoms, and n represents an average value of 2 to 100.
R ¹⁰ represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. ]

[In Formula (4),
R ¹² represents a hydrogen atom or a methyl group,
R ¹³ represents an alkanediyl group having 2 to 4 carbon atoms,
R ¹⁴ represents an alkanediyl group having 1 to 10 carbon atoms,
R <^15> , R <^16> and R < ¹⁷ > show a hydrogen atom or a C1-C8 hydrocarbon group each independently, and m shows 1-10 by an average value. ]

[In Formula (5),
X is-(C = O) O-,-(O = S = O) O-, -O (O = S = O) O-,-(S = O) O-, -O (S = O). ) O -, - OP (= O) (oR 23) O -, - OP (= O) (R 23) O -, - P (= O) (oR 23) O-, or -P (= O) (R ²³ ) O— (R ²³ represents an alkyl group having 1 to 3 carbon atoms),
R ¹⁸ represents a hydrogen atom or a methyl group,
R ¹⁹ and R ²² each independently represent a divalent organic group having 1 to 10 carbon atoms,
R ²⁰ and R ²¹ each independently represent a hydrocarbon group having 1 to 10 carbon atoms. ]

[In Formula (6),
R ²⁴ represents a hydrogen atom or a methyl group,
R ²⁵ is —O—, * — (C═O) —O—, * — (C═O) —NR ²⁷ —, * —NR ²⁷ — (C═O) — (R ²⁷ is a hydrogen atom or An organic group having 1 to 10 carbon atoms, * represents a position bonded to the carbon atom to which R ²⁵ in formula (7) is bonded) or a phenylene group;
R ²⁶ represents a hydrocarbon group. ]

[In Formula (7),
R ²⁸ represents a hydrogen atom or a methyl group,
Y represents an organic group having a formyl group or a keto group. ]
In the above protein stabilizer, the copolymer preferably has a weight average molecular weight of 3,000 to 1,000,000.

  In the above protein stabilizer, the copolymer is preferably water-soluble.

  The protein stabilizer is at least one repeating unit selected from repeating units represented by the following formula (1), the following formula (2), the following formula (3), the following formula (4), and the following formula (5). Copolymer having a hydrophilic repeating unit (A) comprising units and a hydrophobic repeating unit (B) comprising at least one repeating unit selected from the repeating units represented by the following formula (6) and the following formula (7) By containing the coalescence, it has a high protein stabilizing effect.

[In Formula (1),
R ¹ represents a hydrogen atom or a methyl group,
R ² and R ³ each independently represent a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a hydroxyalkyl group. ]

[In Formula (2),
R ⁴ represents a hydrogen atom or a methyl group,
R ⁵ and R ⁶ each independently represent a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a hydroxyalkyl group. ]

[In Formula (3),
R ⁷ represents a hydrogen atom or a methyl group,
R ⁸ represents —O—, * — (C═O) —O—, * —NR ¹¹ — (C═O) — (R ¹¹ represents a hydrogen atom or an organic group having 1 to 10 carbon atoms, * Represents a position bonded to the carbon atom to which R ⁸ in Formula (2) is bonded) or a phenylene group.
R ⁹ represents an alkanediyl group having 2 to 4 carbon atoms, and n represents an average value of 2 to 100.
R ¹⁰ represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. ]

[In Formula (4),
R ¹² represents a hydrogen atom or a methyl group,
R ¹³ represents an alkanediyl group having 2 to 4 carbon atoms,
R ¹⁴ represents an alkanediyl group having 1 to 10 carbon atoms,
R <^15> , R <^16> and R < ¹⁷ > show a hydrogen atom or a C1-C8 hydrocarbon group each independently, and m shows 1-10 by an average value. ]

[In Formula (5),
X is-(C = O) O-,-(O = S = O) O-, -O (O = S = O) O-,-(S = O) O-, -O (S = O). ) O -, - OP (= O) (oR 23) O -, - OP (= O) (R 23) O -, - P (= O) (oR 23) O-, or -P (= O) (R ²³ ) O— (R ²³ represents an alkyl group having 1 to 3 carbon atoms),
R ¹⁸ represents a hydrogen atom or a methyl group,
R ¹⁹ and R ²² each independently represent a divalent organic group having 1 to 10 carbon atoms,
R ²⁰ and R ²¹ each independently represent a hydrocarbon group having 1 to 10 carbon atoms. ]

[In Formula (6),
R ²⁴ represents a hydrogen atom or a methyl group,
R ²⁵ is —O—, * — (C═O) —O—, * — (C═O) —NR ²⁷ —, * —NR ²⁷ — (C═O) — (R ²⁷ is a hydrogen atom or An organic group having 1 to 10 carbon atoms, * represents a position bonded to the carbon atom to which R ²⁵ in formula (7) is bonded) or a phenylene group;
R ²⁶ represents a hydrocarbon group. ]

[In Formula (7),
R ²⁸ represents a hydrogen atom or a methyl group,
Y represents an organic group having a formyl group or a keto group. ]

Hereinafter, the protein stabilizer which concerns on one Embodiment of this invention is demonstrated concretely.
1. Protein stabilizer 1.1. Constitution of protein stabilizer The protein stabilizer according to one embodiment of the present invention is:
The hydrophilic repeating unit (A) comprising at least one repeating unit selected from the following formula (1), the following formula (2), the following formula (3), the following formula (4), and the following formula (5); A copolymer having a hydrophobic repeating unit (B) composed of at least one repeating unit selected from the formula (6) and the following formula (7) is contained.

[In Formula (1),
R ¹ represents a hydrogen atom or a methyl group,
R ² and R ³ each independently represent a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a hydroxyalkyl group. ]

[In Formula (2),
R ⁴ represents a hydrogen atom or a methyl group,
R ⁵ and R ⁶ each independently represent a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a hydroxyalkyl group. ]

[In Formula (3),
R ⁷ represents a hydrogen atom or a methyl group,
R ⁸ represents —O—, * — (C═O) —O—, * —NR ¹¹ — (C═O) — (R ¹¹ represents a hydrogen atom or an organic group having 1 to 10 carbon atoms, * Represents a position bonded to the carbon atom to which R ⁸ in Formula (2) is bonded) or a phenylene group.
R ⁹ represents an alkanediyl group having 2 to 4 carbon atoms, and n represents an average value of 2 to 100.
R ¹⁰ represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. ]

[In Formula (4),
R ¹² represents a hydrogen atom or a methyl group,
R ¹³ represents an alkanediyl group having 2 to 4 carbon atoms,
R ¹⁴ represents an alkanediyl group having 1 to 10 carbon atoms,
R <^15> , R <^16> and R < ¹⁷ > show a hydrogen atom or a C1-C8 hydrocarbon group each independently, and m shows 1-10 by an average value. ]

[In Formula (5),
X is-(C = O) O-,-(O = S = O) O-, -O (O = S = O) O-,-(S = O) O-, -O (S = O). ) O -, - OP (= O) (oR 23) O -, - OP (= O) (R 23) O -, - P (= O) (oR 23) O-, or -P (= O) (R ²³ ) O— (R ²³ represents an alkyl group having 1 to 3 carbon atoms),
R ¹⁸ represents a hydrogen atom or a methyl group,
R ¹⁹ and R ²² each independently represent a divalent organic group having 1 to 10 carbon atoms,
R ²⁰ and R ²¹ each independently represent a hydrocarbon group having 1 to 10 carbon atoms. ]

[In Formula (6),
R ²⁴ represents a hydrogen atom or a methyl group,
R ²⁵ is —O—, * — (C═O) —O—, * — (C═O) —NR ²⁷ —, * —NR ²⁷ — (C═O) — (R ²⁷ is a hydrogen atom or An organic group having 1 to 10 carbon atoms, * represents a position bonded to the carbon atom to which R ²⁵ in formula (7) is bonded) or a phenylene group;
R ²⁶ represents a hydrocarbon group. ]

[In Formula (7),
R ²⁸ represents a hydrogen atom or a methyl group,
Y represents an organic group having a formyl group or a keto group. ]
The protein stabilizer according to the present embodiment may contain the copolymer in a part thereof, or may be composed only of the copolymer.
1.1.1. Repeating unit (A)
In the copolymer, the hydrophilic repeating unit (A) is a main component that contributes to the expression of a high protein stabilizing effect. “Hydrophilic” means having a high affinity with water. Specifically, when it is a homopolymer consisting of only one type of repeating unit (having a number average molecular weight of about 10,000 by the measurement method of the example), it is 1 g or more per 100 g of pure water at room temperature (25 ° C.). When dissolved, the repeating unit is hydrophilic.

In the general formula (1), the carbon number of the alkyl group represented by R ^2, R ³ is preferably 1 to 3.

The alkyl group represented by R ² or R ³ may be linear or branched, and preferred specific examples include a methyl group, an ethyl group, an n-propyl group, and an isopropyl group.

The number of carbon atoms of the hydroxyalkyl group represented by R ^2, R ³ is preferably 1 to 6, more preferably 1 to 3. The alkyl group contained in the hydroxyalkyl group may be linear or branched, and preferred specific examples of the hydroxyalkyl group include a hydroxymethyl group, a hydroxyethyl group, a hydroxypropyl group, and a hydroxyisopropyl group. In addition, the substitution position of the hydroxy group in the hydroxyalkyl group is arbitrary.

  As monomers constituting the repeating unit represented by the general formula (1), dimethyl (meth) acrylamide, diethyl (meth) acrylamide, N-isopropyl (meth) acrylamide, N- (hydroxymethyl) (meth) acrylamide, N- (2-hydroxyethyl) (meth) acrylamide etc. are mentioned, You may use these individually or in combination of 2 or more types.

In the general formula (2), R ⁵ and R ⁶ each independently represent an alkanediyl group having 1 to 3 carbon atoms. The carbon number of the alkanediyl group is preferably 1 to 2.

  The alkanediyl group may be linear or branched, but is preferably linear. Preferable specific examples include methane-1,1-diyl group and ethane-1,2-diyl group.

  Examples of the monomer constituting the repeating unit represented by the general formula (2) include 4- (meth) acryloylmorpholine.

In the general formula (3), R ¹⁰ represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. The number of carbon atoms of the alkyl group represented by R10 is preferably 1 to 3, more preferably 1 or 2, and still more preferably 1, from the viewpoints of the desired effect and availability of the present invention. The alkyl group represented by R ¹⁰ may be linear or branched, and specifically includes a methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, sec-butyl group. , And a tert-butyl group.

Among such R ¹⁰ , from the viewpoints of the desired effect and availability of the present invention, a hydrogen atom and an alkyl group having 1 to 3 carbon atoms are preferable, and a hydrogen atom and an alkyl group having 1 or 2 carbon atoms are more preferable. A hydrogen atom or a methyl group is more preferable, and a methyl group is particularly preferable.

R ⁸ represents —O—, * — (C═O) —O—, * —NR ¹¹ — (C═O) — or a phenylene group. Examples of the phenylene group include a 1,2-phenylene group, a 1,3-phenylene group, and a 1,4-phenylene group.

The number of carbon atoms of the organic group represented by the ^{R 11} is 1 to 10, preferably 1 to 6. Examples of the organic group include hydrocarbon groups. Such a hydrocarbon group is a concept including an aliphatic hydrocarbon group, an alicyclic hydrocarbon group, and an aromatic hydrocarbon group.

The aliphatic hydrocarbon group in R ¹¹ may be linear or branched, and specifically includes a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group, and a sec-butyl group. Alkyl groups such as tert-butyl group, pentyl group, hexyl group, heptyl group, octyl group, nonyl group, decyl group, and the like.

  The alicyclic hydrocarbon group is roughly classified into a monocyclic alicyclic hydrocarbon group and a bridged ring hydrocarbon group. Examples of the monocyclic alicyclic hydrocarbon group include cycloalkyl groups such as a cyclopropyl group and a cyclohexyl group. Examples of the bridged ring hydrocarbon group include an isobornyl group.

  Examples of the aromatic hydrocarbon group include aryl groups such as a phenyl group.

Among R ⁸ as described above, * — (C═O) —O— is particularly preferable.

  Examples of the monomer constituting the repeating unit represented by the general formula (3) include polyethylene glycol (meth) acrylate, polypropylene glycol (meth) acrylate, polyethylene glycol polypropylene glycol (meth) acrylate, polyethylene glycol polytetramethylene glycol ( (Meth) acrylate, polypropylene glycol polytetramethylene glycol (meth) acrylate, methoxypolyethylene glycol (meth) acrylate, ethoxypolyethylene glycol (meth) acrylate, and the like, and these may be used alone or in combination of two or more. Good.

In the above general formula (4), R ¹³ represents an alkanediyl group having 2 to 4 carbon atoms. In ^the case ^{where R 13} there are a ^{plurality, R 13} may be the same or different.

The number of carbon atoms of the alkanediyl group represented by R ¹³ is preferably 2 or 3, and more preferably 2.

The alkanediyl group represented by R ¹³ may be linear or branched, and specifically includes ethane-1,2-diyl group, propane-1,2-diyl group, propane-1,3-diyl. Group, propane-2,2-diyl group, butane-1,2-diyl group, butane-1,3-diyl group, butane-1,4-diyl group and the like. Among these, an ethane-1,2-diyl group is preferable from the viewpoints of the desired effect and availability of the present invention.

R ¹⁴ represents an alkanediyl group having 1 to 10 carbon atoms.

The carbon number of the alkanediyl group represented by R ¹⁴ is preferably 1 to 6, more preferably 1 to 4, still more preferably 2 or 3, and particularly preferably 2.

Further, the alkanediyl group represented by R ¹⁴ may be linear or branched, and preferred specific examples include the same alkanediyl groups represented by R ¹³ .

^Also, R 15, ^{R 1} 6 and R ¹⁷ are each independently represent a hydrogen atom or a hydrocarbon group having 1 to 8 carbon atoms, preferably a hydrocarbon group having 1 to 8 carbon atoms. The number of carbon atoms of the hydrocarbon group is preferably 1 to 4, more preferably 1 or 2, and particularly preferably 1.

  Examples of the hydrocarbon group include an alkyl group; an aryl group such as a phenyl group; and an aralkyl group such as a benzyl group, with an alkyl group being preferred.

  The alkyl group may be linear or branched, and preferred specific examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl. Groups.

  Moreover, m shows 1-10 by an average value, Preferably it is 1-7 by an average value, More preferably, it is 1-4 by an average value, More preferably, it is 1.

  As the monomer constituting the repeating unit represented by the general formula (4), 2- (meth) acryloyloxyethyl-2 ′-(trimethylammonio) ethyl phosphate, (2- (meth) acryloyloxyethyl phosphorylcholine) 3- (meth) acryloyloxypropyl-2 ′-(trimethylammonio) ethyl phosphate, 4- (meth) acryloyloxybutyl-2 ′-(trimethylammonio) ethyl phosphate, 2- (meth) acryloyloxyethoxyethyl -2 '-(trimethylammonio) ethyl phosphate, 2- (meth) acryloyloxydiethoxyethyl-2'-(trimethylammonio) ethyl phosphate, 2- (meth) acryloyloxyethyl-2 '-(triethylammonio) ) Ethyl phosphate, 2 (Meth) acryloyloxyethyl-2 '- (tributyl ammonio) ethyl phosphate, and the like, it may be those used in combination of these alone, or two or more kinds.

In the general formula (5), X is preferably — (C═O) O—. As the alkyl group represented by R ^23, a methyl group, an ethyl group, n- propyl group, an isopropyl group.

Further, in the above general formula ^{(5), R 19} and ^{R 22} each independently represents a divalent organic group having 1 to 10 carbon atoms. The carbon number of such a divalent organic group is preferably 1-8, more preferably 1-6.

  The divalent organic group is preferably a divalent hydrocarbon group, and more preferably a divalent aliphatic hydrocarbon group. The divalent aliphatic hydrocarbon group may be linear or branched. Moreover, as a bivalent aliphatic hydrocarbon group, an alkanediyl group is preferable. For example, methane-1,1-diyl group, ethane-1,2-diyl group, propane-1,1-diyl group, propane-1,2-diyl group, propane-1,3-diyl group, propane-2 , 2-diyl group, butane-1,4-diyl group, pentane-1,5-diyl group, hexane-1,6-diyl group and the like.

Further, in the above general formula ^{(5), R 20} and ^{R 21} each independently represent a hydrocarbon group having 1 to 10 carbon atoms. Carbon number of the said hydrocarbon group becomes like this. Preferably it is 1-6, More preferably, it is 1-4.

Examples of the hydrocarbon group represented by R ²⁰ and R ²¹ include an alkyl group; an aryl group such as a phenyl group; and an aralkyl group such as a benzyl group, with an alkyl group being preferred. The alkyl group may be linear or branched, and examples thereof include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group, a sec-butyl group, and a tert-butyl group. .

Examples of the monomer constituting the repeating unit represented by the general formula (5) include N- (meth) acryloyloxyethyl-N, N-dimethylammonium-α-N-methylcarboxybetaine, N- (meth) acryloyloxy. Examples thereof include ethyl-N, N-dimethylammonium-α-N-propylsulfobetaine, and these may be used alone or in combination of two or more.
1.1.2. Repeating unit (B)
In the copolymer, the hydrophobic repeating unit (B) contributes to the expression of a higher protein stabilizing effect by shifting the hydrophilic / hydrophobic balance of the copolymer to the hydrophobic side.

  “Hydrophobic” means having a low affinity with water. Specifically, when it is a homopolymer consisting of only one type of repeating unit (having a number average molecular weight of about 10,000 by the measurement method of the example), it is less than 1 g with respect to 100 g of pure water at room temperature (25 ° C.). If it only dissolves, the repeating unit is hydrophobic.

In the general formula (6),
R ²⁵ represents —O—, * — (C═O) —O—, * — (C═O) —NR ²⁷ —, * —NR ²⁷ — (C═O) — or a phenylene group. Examples of the phenylene group include a 1,2-phenylene group, a 1,3-phenylene group, and a 1,4-phenylene group.

Among R ²⁵ , *-(C═O) —O—, * — (C═O) —NR ²⁷ —, and a phenylene group are preferable from the viewpoint of the desired effect and availability of the present invention, and * — (C═O) —O— and * — (C═O) —NR ²⁷ — are more preferred, and * — (C═O) —O— and * — (C═O) —NH— are particularly preferred.

R ²⁶ represents a hydrocarbon group. Carbon number of the said hydrocarbon group becomes like this. Preferably it is 1-30, More preferably, it is 1-24, More preferably, it is 1-18, Most preferably, it is 1-14.

^Although the carbon number of the organic group represented by ^{R 27} is 1 to 10, preferably 1 to 6. Examples of the organic group include hydrocarbon groups.

Here, the hydrocarbon group in R ²⁶ and R ²⁷ is a concept including an aliphatic hydrocarbon group, an alicyclic hydrocarbon group, and an aromatic hydrocarbon group. Further, it may be either linear or branched and may contain a ring structure.

The aliphatic hydrocarbon group in R ²⁶ and R ²⁷ may be linear or branched, and specifically includes a methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, sec Examples include alkyl groups such as -butyl group, tert-butyl group, pentyl group, hexyl group, heptyl group, octyl group, nonyl group, decyl group.

  The alicyclic hydrocarbon group is roughly classified into a monocyclic alicyclic hydrocarbon group and a bridged ring hydrocarbon group. Examples of the monocyclic alicyclic hydrocarbon group include cycloalkyl groups such as a cyclopropyl group and a cyclohexyl group. Examples of the bridged ring hydrocarbon group include an isobornyl group.

  Examples of the aromatic hydrocarbon group include aryl groups such as a phenyl group.

Among these, as R ²⁶ , an alkyl group is preferable. The carbon number is preferably 1-30, more preferably 1-24, even more preferably 1-18, and particularly preferably 1-14, as described above.

  As the monomer constituting the repeating unit represented by the general formula (6), alkyl (meth) acrylate is preferable. Specific examples include methyl (meth) acrylate, 2-ethylhexyl (meth) acrylate, lauryl (meth) acrylate, stearyl (meth) acrylate, and these may be used alone or in combination of two or more. .

Next, in the general formula (7), R ²⁸ represents a hydrogen atom or a methyl group, preferably a hydrogen atom.

  Y represents an organic group having a formyl group or a keto group. As a total carbon number of the said organic group, 4 or more are preferable, 4-13 are more preferable, 5-11 are still more preferable, and 6-9 are especially preferable. The number of formyl groups or keto groups is not particularly limited and may be one or more, but one is preferable.

  Moreover, as Y, what has an ester bond or an amide bond is preferable. The number of ester bonds or amide bonds is not particularly limited, but one is preferable.

  Y is preferably an organic group having one formyl group or keto group and one ester bond or amide bond, and more preferably a group represented by the following formula (8).

[In Formula (8),
X ¹ represents —O— or —NH—,
X ² represents an organic group having 3 to 12 carbon atoms (preferably 4 to 10 carbon atoms, more preferably 5 to 8 carbon atoms) having a formyl group or a keto group,
* Indicates a bond. ]
X ¹ is preferably —NH—.

X ² is preferably a group represented by the following formula (9).

[In Formula (9),
R ²⁹ represents a linear or branched alkanediyl group having 2 to 10 carbon atoms (preferably 3 to 8 carbon atoms, more preferably 4 to 6 carbon atoms),
X ³ represents a formyl group or — (C═O) —CH 3;
* Indicates a bond. ]
Preferable specific examples of X ² include groups represented by the following formula (10) or (11). Among these, a group represented by the formula (10) is more preferable, and a group represented by the formula (12) is particularly preferable.

[In Formula (10),
R ^{30 to} R ³³ each independently represent a hydrogen atom, a methyl group or an ethyl group,
* Indicates a bond. ]

[In Formula (11),
R ^{34 to} R ³⁷ each independently represent a hydrogen atom, a methyl group or an ethyl group,
* Indicates a bond. ]

[In formula (12), * indicates a bond. ]
Examples of the monomer constituting the repeating unit represented by the general formula (7) include diacetone (meth) acrylamide.

Further, the copolymer may contain at least one or more repeating units (A) and repeating units (B).
1.2. Manufacture of a protein stabilizer Next, the composition of the monomer used in order to manufacture the manufacturing method of the said copolymer is demonstrated.
1.2.1. Monomer (a)
The repeating unit (A) has a structure derived from at least one monomer (a).

The monomer (a) includes a (meth) acrylamide monomer represented by the above formulas (1) and (2), a monomer containing a polyalkylene glycol chain represented by the above formula (3), the above formula (4), It is preferable that it is at least 1 type of monomer chosen from the monomer containing the zwitterionic structure shown by (5). More preferably, at least one selected from N, N-dimethylacrylamide, 4- (meth) acryloylmorpholine, methoxypolyethylene glycol methacrylate, N-methacryloyloxyethyl-N, N-dimethylammonium-α-N-methylcarboxybetaine. is there.
1.2.2. Monomer (b)
The repeating unit (B) has a structure derived from at least one monomer (b).

  The monomer (b) is preferably at least one selected from alkyl (meth) acrylate monomers represented by the above formula (5) or (meth) acrylamide monomers represented by the above formulas (6) to (13). It is a seed. More preferably, it is at least one selected from methyl methacrylate and diacetone acrylamide.

  The monomer composition for producing the copolymer is preferably based on 100% by mass of all monomers, preferably 1 to 99% by mass of monomer (a), 1 to 99% by mass of monomer (b), more preferably monomer (a a) 10 to 90% by mass, monomer (b) 10 to 90% by mass.

  If the monomers (a) and (b) are out of the above range, the nonspecific adsorption preventing effect may be inferior.

  Further, the mass ratio of the amount of the monomer (a) used and the amount of the monomer (b) used is more preferably 1:99 to 99: 1, and further preferably 10:90 to 90:10.

  If the amount of the monomer (b) used is less than 99: 1 with respect to the amount of the monomer (a) used, the protein stabilizing effect may be inferior, whereas if it exceeds 1:99, it is insoluble in water. In some cases, the protein cannot be added to the aqueous solution containing the protein.

  The monomer to be used can be used for copolymerization after being purified as an industrial raw material or unpurified.

  The polymerization of the monomer can be performed by a known polymerization method such as radical polymerization, anionic polymerization, or cationic polymerization, and is preferably radical polymerization because of easy production.

  The polymerization of the monomer is carried out by stirring and heating together with a known solvent, initiator, chain transfer agent and the like. The polymerization time is usually 30 minutes to 24 hours, and the polymerization temperature is about 0 to 120 ° C.

The copolymer aqueous solution after polymerization is preferably purified by a dialysis membrane, a dialyzer, an acylizer, ultrafiltration or the like.
1.3. Physical Properties and Use of Protein Stabilizer In the protein stabilization according to the present embodiment, the copolymer has a mass average molecular weight of usually 1,000 to 1,000,000, preferably 5,000 to 500,000. More preferably, it is 8,000-200,000. In addition, the molecular weight distribution of the copolymer typically has a mass average molecular weight / number average molecular weight of 1.5 to 8. If the weight average molecular weight of the copolymer is less than the above range, the protein stabilizing effect may be insufficient. On the other hand, if the weight number average molecular weight of the copolymer is greater than the above range, the viscosity of the solution May increase and handling may be difficult.

  The copolymer contained in the protein stabilizer according to this embodiment is water-soluble. In the present invention, “water-soluble” means that when a copolymer is added to and mixed with water so as to have a polymer solid content of 1% at 25 ° C., it is transparently dissolved.

  In the protein stabilizer according to the present embodiment, since the repeating unit (B) of the copolymer interacts with the protein and the repeating unit (A) has a dispersing action on water, the protein has a conformation. It can be changed to prevent hydrophobicity and to suppress protein aggregation.

  The protein stabilizer according to the present embodiment may contain osmolytes such as sugar, sugar alcohol, amino acid, and betaine.

The protein stabilizer according to the present embodiment includes, for example, a labeled antibody, a labeled antigen, an enzyme, a primary antibody, a primary antigen stabilizer used as a clinical diagnostic agent; a protein stabilizer contained in a plasma preparation; a contact When added to a protein solution as a stabilizer such as an enzyme used for lens cleaning, protein activity can be maintained over a long period of time.
Furthermore, the protein stabilizer according to the present embodiment has an effect of suppressing non-specific adsorption of proteins and nucleic acids by coating on containers / equipment and the like, and an effect of suppressing signal of non-specific specimens when used as a diluent for immunodiagnostics. Have

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated further in detail, this invention is not restrict | limited by these.

In this example, the mass average molecular weight (Mw) and number average molecular weight (Mn) were measured using a TSK gel α-M column manufactured by Tosoh Corporation, the flow rate was 0.5 ml / min, and the elution solvent was 0.03M bromide. It was measured by gel permeation chromatography (GPC) using monodisperse polyethylene styrene as a standard under analytical conditions of lithium, 0.02M phosphoric acid / N-methylpyrrolidone solvent, and column temperature of 40 ° C. Absorbance was measured with an ARVO-X5 microplate reader manufactured by PerkinElmer.
2.1. Example 1
50 g of dimethylacrylamide (hereinafter referred to as DMAA) as the monomer (a) and 50 g of diacetone acrylamide (hereinafter referred to as DAAM) as the monomer (b) were mixed with 900 g of water and placed in a separable flask equipped with a stirrer. While nitrogen was blown into this, 0.5 g of potassium persulfate (hereinafter referred to as KPS) and 0.58 g of sodium pyrosulfite (hereinafter referred to as SMS) were added as initiators, followed by polymerization at room temperature for 8 hours. . The obtained copolymer solution was purified by ultrafiltration and then diluted with pure water to 1% TSC to obtain a protein stabilizer (S-1) of this example.

  The number average molecular weight by GPC of the protein stabilizer (S-1) was 42,000, and the weight average molecular weight was 91,000.

The protein stabilizer (S-1) was diluted 10-fold with PBS buffer, and horseradish peroxidase (hereinafter referred to as HRP, Oriental Yeast) was added to this solution at 250 ng / mL. 10 μl of this HRP solution and 100 μl of 1-Step ^™ ABTS Substrate Solution (hereinafter referred to as “ABST”, manufactured by Thermo Fisher SCIENTIFIC) were mixed to develop color. Ten minutes later, 100 μl of 1% sodium dodecyl sulfate aqueous solution was added to stop the reaction, and the absorbance at 405 nm was measured (measurement of absorbance before storage). The remaining HRP solution was stored at 40 ° C. for 7 days, and then the absorbance was measured in the same manner (measurement of absorbance after storage). As a result, when the absorbance of ABTS color development by HRP before storage was taken as 100%, the absorbance after storage, which is an index of the protein activity maintenance rate, was 83%.
2.2. Example 2
60 g of DMAA as the monomer (a) and 40 g of DAAM as the monomer (b) were mixed with 900 g of water and put into a separable flask equipped with a stirrer. While nitrogen was blown into this, 0.5 g of KPS and 0.58 g of SMS were added as initiators, and then polymerization was performed at room temperature for 8 hours. The obtained copolymer solution was purified by ultrafiltration and then diluted with pure water to 1% of TSC to obtain a protein stabilizer (S-2) of this example.

  The number average molecular weight by GPC of the protein stabilizer (S-2) was 33,000, and the weight average molecular weight was 83,000.

Moreover, as a result of measuring the absorbance before storage and the absorbance after storage in the same manner as in Example 1 except that the protein stabilizer (S-2) was used, the absorbance before storage (100%) was measured. The absorbance after storage was 75%.
2.3. Example 3
As a monomer (a), 70 g of DMAA and 30 g of DAAM as a monomer (b) were mixed with 900 g of water and put into a separable flask equipped with a stirrer. While nitrogen was blown into this, 0.5 g of KPS and 0.58 g of SMS were added as initiators, and then polymerization was performed at room temperature for 8 hours. The obtained copolymer solution was purified by ultrafiltration and then diluted with pure water to 1% TSC to obtain the protein stabilizer (S-3) of this example.

  The number average molecular weight by GPC of the protein stabilizer (S-3) was 28,000, and the weight average molecular weight was 90,000.

Moreover, as a result of measuring the absorbance before storage and the absorbance after storage in the same manner as in Example 1 except that the protein stabilizer (S-3) was used, the absorbance before storage (100%) was measured. The absorbance after storage was 62%.
2.4. Example 4
25 g of DMAA as monomer (a), 25 g of methoxypolyethylene glycol methacrylate (hereinafter referred to as M90G (manufactured by Shin-Nakamura Chemical Co., Ltd.)) having an average addition mole number of ethylene oxide of 9 are mixed with 900 g of water as monomer (b). And placed in a separable flask with a stirrer. While nitrogen was blown into this, 0.5 g of KPS and 0.58 g of SMS were added as initiators, and then polymerization was performed at room temperature for 8 hours. The obtained copolymer solution was purified by ultrafiltration and then diluted with pure water to 1% TSC to obtain a protein stabilizer (S-4) of this example.

  The number average molecular weight by GPC of the protein stabilizer (S-4) was 34,000, and the weight average molecular weight was 86,000.

Further, the absorbance before storage and the absorbance after storage were measured in the same manner as in Example 1 except that the protein stabilizer (S-4) was used. As a result, the absorbance before storage (100%) was measured. The absorbance after storage was 83%.
2.5. Example 5
As monomer (a), 25 g of DMAA, 25 g of N-methacryloyloxyethyl-N, N-dimethylammonium-α-N-methylcarboxybetaine (hereinafter referred to as GLBT (produced by Osaka Organic Chemical Industry)), as monomer (b) 50 g of DAAM was mixed with 900 g of water and placed in a separable flask equipped with a stirrer. While nitrogen was blown into this, 0.5 g of KPS and 0.58 g of SMS were added as initiators, and then polymerization was performed at room temperature for 8 hours. The obtained copolymer solution was purified by ultrafiltration and then diluted with pure water to 1% TSC to obtain a protein stabilizer (S-5) of this example.

  The number average molecular weight by GPC of the protein stabilizer (S-5) was 25,000, and the weight average molecular weight was 67,000.

Moreover, as a result of measuring the absorbance before storage and the absorbance after storage in the same manner as in Example 1 except that the protein stabilizer (S-5) was used, the absorbance before storage (100%) was measured. The absorbance after storage was 92%.
2.6. Example 6
As monomer (a) 4-acryloylmorpholine (hereinafter referred to as ACMO (manufactured by Kojin Film & Chemicals)) 60 g, M90G 75 g, monomer (b) as methyl methacrylate (hereinafter referred to as MMA) 15 g, as chain transfer agent 4.5 g of α-thioglycerol was mixed with 850 g of water and put into a separable flask equipped with a stirrer. The temperature was raised to 60 ° C. while blowing nitrogen, and 3 g of 2,2′-azobis (2-methylpropionamidine) dihydrochloride (hereinafter referred to as V-50) was added as an initiator for 3 hours. After polymerization, it was cooled to room temperature. The obtained copolymer solution was purified by ultrafiltration and then diluted with pure water to 1% TSC to obtain a protein stabilizer (S-6) of this example.

  The number average molecular weight by GPC of the protein stabilizer (S-6) was 3,000, and the weight average molecular weight was 11,000.

Further, the absorbance before storage and the absorbance after storage were measured in the same manner as in Example 1 except that the protein stabilizer (S-6) was used. As a result, the absorbance before storage (100%) was measured. The absorbance after storage was 23%.
2.7. Comparative Example 1
In Example 1, instead of using 50 g of DMAA as monomer (a) and 50 g of DAAM as monomer (b), protein stabilization was performed in the same manner as in Example 1 except that only 100 g of DMAA was used as the monomer. Agent (X-1) was obtained.

  The number average molecular weight by GPC of the protein stabilizer (X-1) was 14,000, and the mass average molecular weight was 84,000.

Results of measuring the absorbance before and after storage in the same manner as in Example 1 except that the protein stabilizer (X-1) was used instead of the protein stabilizer (S-1) The absorbance after storage relative to the absorbance before storage (100%) was 5%.
2.8. Comparative Example 2
In Example 1, polymerization was carried out in the same manner as in Example 1, except that 50 g of DMAA as monomer (a) and 50 g of DAAM as monomer (b) were used, and only 100 g of DAAM was used as the monomer. However, since a white substance insoluble in water was generated and stirring became difficult, the polymerization was stopped.
2.9. Comparative Example 3
The absorbance before storage and the absorbance after storage were measured in the same manner as in Example 1 except that BSA was used instead of the protein stabilizer (S-1). As a result, the absorbance before storage (100 The absorbance after storage with respect to% was 11%.
2.10. Comparative Example 4
Except for using a commercially available surfactant (manufactured by Sigma-Aldrich, Tween 20) instead of the protein stabilizer (S-1), the absorbance before storage and the storage after storage were the same as in Example 1. As a result of measuring the absorbance, the absorbance after storage with respect to the absorbance before storage (100%) was 3%.
2.11. Comparative Example 5
The absorbance before storage and the absorbance after storage were measured in the same manner as in Example 1 except that sucrose was used instead of the protein stabilizer (S-1). As a result, the absorbance before storage (100 %) After storage was 3%.
3. Measurement Results Table 1 shows the measurement results of the protein stabilization effect in the above Examples and Comparative Examples.

Claims (3)

A hydrophilic repeating unit (A) comprising at least one repeating unit selected from repeating units represented by the following formula (1), formula (2), formula (3), formula (4), and formula (5); A protein stabilizer comprising a copolymer having a hydrophobic repeating unit (B) comprising at least one repeating unit selected from repeating units represented by the following formula (6) and the following formula (7).

[In Formula (1),
R ¹ represents a hydrogen atom or a methyl group,
R ² and R ³ each independently represent a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a hydroxyalkyl group. ]

[In Formula (2),
R ⁴ represents a hydrogen atom or a methyl group,
R ⁵ and R ⁶ each independently represent a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a hydroxyalkyl group. ]

[In Formula (3),
R ⁷ represents a hydrogen atom or a methyl group,
R ⁸ represents —O—, * — (C═O) —O—, * —NR ¹¹ — (C═O) — (R ¹¹ represents a hydrogen atom or an organic group having 1 to 10 carbon atoms, * Represents a position bonded to the carbon atom to which R ⁸ in Formula (2) is bonded) or a phenylene group.
R ⁹ represents an alkanediyl group having 2 to 4 carbon atoms, and n represents an average value of 2 to 100.
R ¹⁰ represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. ]

[In Formula (4),
R ¹² represents a hydrogen atom or a methyl group,
R ¹³ represents an alkanediyl group having 2 to 4 carbon atoms,
R ¹⁴ represents an alkanediyl group having 1 to 10 carbon atoms,
R <^15> , R <^16> and R < ¹⁷ > show a hydrogen atom or a C1-C8 hydrocarbon group each independently, and m shows 1-10 by an average value. ]

[In Formula (5),
X is-(C = O) O-,-(O = S = O) O-, -O (O = S = O) O-,-(S = O) O-, -O (S = O). ) O -, - OP (= O) (oR 23) O -, - OP (= O) (R 23) O -, - P (= O) (oR 23) O-, or -P (= O) (R ²³ ) O— (R ²³ represents an alkyl group having 1 to 3 carbon atoms),
R ¹⁸ represents a hydrogen atom or a methyl group,
R ¹⁹ and R ²² each independently represent a divalent organic group having 1 to 10 carbon atoms,
R ²⁰ and R ²¹ each independently represent a hydrocarbon group having 1 to 10 carbon atoms. ]

[In Formula (6),
R ²⁴ represents a hydrogen atom or a methyl group,
R ²⁵ is —O—, * — (C═O) —O—, * — (C═O) —NR ²⁷ —, * —NR ²⁷ — (C═O) — (R ²⁷ is a hydrogen atom or An organic group having 1 to 10 carbon atoms, * represents a position bonded to the carbon atom to which R ²⁵ in formula (7) is bonded) or a phenylene group;
R ²⁶ represents a hydrocarbon group. ]

[In Formula (7),
R ²⁸ represents a hydrogen atom or a methyl group,
Y represents an organic group having a formyl group or a keto group. ]   The protein stabilizer according to claim 1, wherein the copolymer has a weight average molecular weight of 3,000 to 1,000,000.   The protein stabilizer according to any one of claims 1 to 2, wherein the copolymer is water-soluble.