JPH1045794A - Stabilization of protein and composition - Google Patents

Abstract

PROBLEM TO BE SOLVED: To stabilize various proteins used for clinical medicines, clinical diagnosis, medical devices, etc., without regard to states by the coexistence of a specific polymer having phosphorylcholine groups with a protein in the same system. SOLUTION: (A) A polymer, having phosphorylcholine group represented by formula II and obtained by polymerizing a phosphorylcholine group-containing monomer represented by formula I (X is H or methyl) [e.g. 2-(meth) acryloyloxyethyl-2'-(trimethylammonio)ethyl phosphate] and having the phosphorylchloine group represented by formula II in an amount of 1.0×10<-4> to 20wt.% is mixed with (B) a protein such as a blood plasma pharmaceutical preparation protein (e.g. an albumin pharmaceutical preparation) in an amount of 1.0×10<-14> to 20wt.% and (C) a buffer solution (e.g. a disodium hydrogenphosphate/sodium dihydrogenphosphate buffer solution) in an amount of 0-99.9wt.% to thereby stabilize various proteins used for clinical medicines, clinical diagnosis or medical devices, etc., without regard to a preservation state.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for stabilizing various proteins used in clinical, clinical diagnosis, medical devices and the like, and a composition using the method.
More specifically, stabilization of proteins contained in plasma preparations such as albumin preparations, blood coagulation factor preparations, immunoglobulin preparations, and the stability of enzymes, antibodies (or antigens), and labeled antibodies (or labeled antigens) used in clinical diagnosis The present invention relates to a method and a composition for stabilizing an enzyme used for washing a contact lens.

[0002]

2. Description of the Related Art Plasma preparations such as albumin preparations, blood coagulation factor preparations, and immunoglobulin preparations used in the clinic are:
Fresh frozen plasma or plasma circulated from expired stored blood is used as a raw material and stored in a solution state, a frozen state, or a lyophilized state, and the expiration date is 1 to 5 years. Among them, an intravenous immunoglobulin preparation is a preparation that allows only intravenous injection by extracting only IgG from human immunoglobulin. If the IgG is extracted as it is, it forms an aggregate and is difficult to function. Cause anaphylactic shock. Therefore, conventionally, a method of decomposing a molecule with pepsin or plasmin, which is a protease, a method of chemically changing a binding site to a V region while leaving a C region, a method of treating with a pH 4 solution, or a treatment with an ion exchange resin There are known ways to do this. However,
The enzyme treatment method has a problem that the half-life is shortened because the complement binding site (C region) is separated, and the chemical change method has a problem that the immunity is slightly lower than that of natural IgG immediately after injection.
The method of treating with an H4 solution or the method of treating with an ion exchange resin has a problem that it lacks versatility ('95 Pharmaceutical Pharmaceutical Data Book Vol.2, Fuji Keizai (1995)
Year)).

[0003] Antibodies (or antigens) or labeled antibodies (or labeled antigens) used for clinical diagnosis are used in the form of solution, frozen, or lyophilized (eg, antibody activity, antigenicity, enzyme activity). It is generally known to add saccharose, bovine serum albumin (abbreviated as BSA), or the like in order to maintain the protein concentration. In particular, a method is known in which saccharose, BSA, gelatin, or the like is added during lyophilization or to maintain its activity (antibody activity, antigenicity, enzyme activity, etc.) in the lyophilized state. However, the method using the above-mentioned various additives is 2
When the temperature is 5 ° C. or more and several months or more, there is a problem that its antibody activity, antigenicity or enzyme activity is reduced.

Further, a method for stabilizing an antigen or an antibody labeled with an acridinium derivative, characterized in that an antibody or an antigen labeled with an acridinium derivative is present in an aqueous solution in the presence of a cyclodextrin (JP-A-7-278)
184) and a method for stabilizing a physiologically active protein characterized by including crystallin in a solution of the physiologically active protein is known (Japanese Patent Application Laid-Open No. Hei 7-236483).

[0005] Furthermore, a method of adding 1) gelatin and / or casein, 2) polyhydric alcohol and / or saccharide, and 3) ethyl alcohol as a stabilizer to a hydrolase solution used for washing contact lenses is also available. Although it is known (Japanese Patent Laid-Open No. 41-152), its effect is not sufficient. Although a phosphorylcholine group-containing polymer is known to be excellent as an antithrombotic material, a method for stabilizing a protein using a phosphorylcholine group-containing polymer is not known. Further, a composition comprising a phosphorylcholine group-containing polymer, an antibody, a labeled immunologically active substance, a hydrolase and the like is not known.

[0006]

An object of the present invention is to provide various proteins used in clinical, clinical diagnosis or medical devices, for example, proteins contained in plasma preparations such as albumin preparations, blood coagulation factor preparations and immunoglobulin preparations; Enzymes, antibodies (or antigens) used in clinical diagnosis, labeled antibodies (or labeled antigens); enzymes used for washing contact lenses, regardless of their storage conditions, such as solution, frozen, or lyophilized An object of the present invention is to provide a method for stabilizing a protein which can be stabilized. It is another object of the present invention to provide a composition in which the protein of the plasma preparation is stabilized regardless of the storage condition. Another object of the present invention is to provide a composition wherein the labeled immunologically active substance is stabilized regardless of the storage condition. Still another object of the present invention is to provide a composition in which the enzyme is stabilized regardless of the storage condition.

[0007]

Means for Solving the Problems The present inventors have found that a specific phosphorylcholine group-containing polymer stabilizes a protein, and completed the present invention. That is, according to the present invention, in the same system, in the same system, a polymer having a phosphorylcholine group represented by the formula (1) (hereinafter abbreviated as a PC polymer), for example, a formula (2) (where X is a hydrogen atom or A method for stabilizing a protein, comprising coexisting a protein or the like obtained by polymerizing a polymerization component containing a phosphorylcholine group-containing monomer represented by a methyl group) with a protein.

Embedded image According to the present invention, (A1) a polymer 1.0 obtained by polymerizing the phosphorylcholine group-containing monomer represented by the formula (2)
× 10 ⁻⁴ to 80% by weight, and (B1) plasma preparation protein 1.0 ×
There is provided a stabilized plasma preparation protein composition comprising 10 ^{-14 to} 20% by weight and (C1) 0 to 99.9% by weight of a buffer solution. Furthermore, according to the present invention, (A2) a polymer obtained by polymerizing the phosphorylcholine group-containing monomer represented by the above formula (2), 1.0 × 10 ⁻⁴ to 80% by weight, and (B2) labeled immunology A stable labeled immunologically active substance composition comprising 1.0 × 10 ^{−14 to} 20% by weight of the active substance and (C2) 0 to 99.9% by weight of the buffer solution. Further, according to the present invention, (A3) 1.0 × 10 ⁻⁴ to 80% by weight of a polymer obtained by polymerizing the phosphorylcholine group-containing monomer represented by the formula (2); 0 × 10 ^{-14 to} 20% by weight, (C
3) A stabilized enzyme composition comprising 0 to 99.9% by weight of a buffer solution is provided.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION In the stabilizing method of the present invention, a protein to be stabilized and the PC polymer are allowed to coexist in the same system. The PC polymer is a monomer having at least one phosphorylcholine group represented by the formula (1) in a molecule such as a phosphorylcholine group-containing monomer represented by the formula (2) (hereinafter abbreviated as PC monomer). Is a polymer of a polymerization component containing

The mixing ratio of the PC monomer in the PC polymer is as follows:
The amount is preferably 1 to 100 mol%, particularly preferably 5 mol% or more, based on all monomers constituting the PC polymer. If the mixing ratio is less than 1 mol%, it is difficult to obtain a protein stabilizing effect, which is not preferable. The molecular weight of the PC polymer is
The number average molecular weight is preferably from 1,000 to 2,000,000, particularly preferably from 2,000 to 1, although it varies depending on the polymerization temperature during preparation, the amount of polymerization initiator used, the presence or absence of a polymerization degree regulator, and the like. 1,000,000. If the number average molecular weight is less than 1,000, it is difficult to obtain a stabilizing effect on the protein, and if the number average molecular weight exceeds 1,000,000, the viscosity of the polymer becomes too high, and it is difficult to handle.

In order to prepare a PC polymer, the PC monomer is homopolymerized by a usual method such as radical polymerization, or other vinyl copolymerizable with a monomer such as a PC monomer. It can be easily obtained by a method of copolymerizing with a system monomer.

Specific examples of the PC monomer include, for example, 2- (meth) acryloyloxyethyl-2'-
(Trimethylammonio) ethyl phosphate, 2-
(Meth) acryloyloxypropyl-2 ′-(trimethylammonio) ethyl phosphate, 2- (meth) acryloyloxyethoxyethyl-2 ′-(trimethylammonio) ethyl phosphate, 2- (meth) acryloyloxydiethoxyethyl- 2 '-(trimethylammonio) ethyl phosphate, 2- (meth) acryloyloxytriethoxyethyl-2'-(trimethylammonio) ethyl phosphate and the like can be mentioned. In particular, 2-methacryloyloxyethyl-
2 ′-(trimethylammonio) ethyl phosphate (hereinafter abbreviated as MPC) = 2-methacryloyloxyethyl phosphorylcholine is preferred.

Other vinyl monomers (hereinafter abbreviated as monomer A) copolymerizable with the PC monomer include, for example, n-butyl (meth) acrylate and methyl (meth) acrylate. , Ethyl (meth) acrylate, butyl (meth) acrylate, pentyl (meth) acrylate, hexyl (meth) acrylate, heptyl (meth) acrylate, octyl (meth) acrylate, tridecyl (meth) acrylate, (Meth) acrylic monomers such as 2-hydroxyethyl methacrylate and (meth) acrylate; styrene monomers such as styrene, α-methylstyrene, methyl-substituted styrene, and chloro-substituted styrene; vinyl chloride;
Substituted hydrocarbon monomers such as vinylidene chloride, ethylene, propylene and isobutylene; vinyl ester monomers such as vinyl acetate and vinyl propionate; vinyl ether monomers such as ethyl vinyl ether and n-butyl vinyl ether; And divalent carboxylic acid monomers such as di-n-butyl itaconate.
Particularly, methacrylic acid ester, styrene and the like can be preferably mentioned.

The polymerization initiator used for preparing the PC polymer is not particularly limited as long as it is a usual radical polymerization initiator. For example, 2,2'-azobisisobutyronitrile, benzoyl peroxide, diisopropylperoxydioxy Examples include carbonate, t-butylperoxy-2-ethylhexanoate, t-butylperoxypivalate, t-butylperoxydiisobutyrate, and persulfates such as potassium persulfate and ammonium persulfate. These polymerization initiators may be used alone or in combination of two or more. A redox radical accelerator may be used in combination with the polymerization initiator. The amount of the polymerization initiator is 0.01 to 10 parts by weight based on 100 parts by weight of all monomers to be polymerized.
Part by weight is preferred, and particularly preferably 0.1 to 5 parts by weight.

The polymerization conditions for preparing the PC polymer are preferably at 30 to 80 ° C., particularly preferably at 40 to 70 ° C., for 2 to 72 hours. On this occasion,
A solvent may be used to carry out the polymerization reaction more smoothly, and examples of the solvent include water, methanol, ethanol, propanol, t-butanol, benzene, toluene, dimethylformamide, tetrahydrofuran, chloroform, and mixtures thereof. be able to.

The form of the PC polymer used in the stabilizing method of the present invention may be any of a powder, a suspension, and a solution, and is preferably a phosphate buffer, an acetate buffer which does not denature proteins. , A carbonate solution, a citrate buffer, a solution or a suspension of various physiological saline and the like. An organic solvent such as dimethylsulfoxide, tetrahydrofuran or N, N-dimethylformamide may be added to these liquids in an amount of 0.01 to 20% by weight.
Particularly preferably, a dissolving solution such as a phosphate buffer solution and various physiological salines can be used.

The protein used in the stabilizing method of the present invention is not particularly limited, and examples thereof include a plasma preparation protein, a labeled immunologically active substance, and an enzyme.

In the stabilizing method of the present invention, the coexistence of the PC polymer and the protein in the same system means that:
For example, the method can be carried out by a method in which the liquid PC polymer solution or the PC polymer powder is added to the protein solution in a solution state and coexist in the same solution. After the addition of the PC polymer powder, it may be frozen or freeze-dried after the addition. When the PC polymer and the protein coexist in the same liquid, the concentration of the PC polymer depends on the concentration and the state of the protein, but the lower limit is preferably 1.0 × 10 ⁻⁸ wt%, It is particularly preferably 1.0 × 10 ⁻⁵ wt%, more preferably 1.0 × 10 ⁻⁴ , and the upper limit is preferably 80 wt%, particularly preferably 70 wt%, and more preferably 50 wt%.
% By weight. The most preferable range is 1.0 × 10 ⁻³ to 40% by weight, which does not increase the liquid viscosity and is easy to handle. If the concentration of the PC polymer exceeds 80% by weight, the viscosity of the liquid becomes high, and the handleability is undesirably reduced. On the other hand, the concentration of the protein in the same liquid varies depending on its type and use, but is preferably 1.0 × 10 ^{−8 to} 20% by weight, particularly preferably 1.0 × 10 ⁻⁷ to 10% by weight.

The plasma product protein composition of the present invention, (A1) wherein among the PC monomer, formula (2) polymerizing a phosphorylcholine group-containing monomer represented by comprising polymer 1.0 × 10 ^{- Four}
８０80% by weight, and (B1) 1.0 × 10 ⁻¹⁴ plasma preparation protein
-20% by weight and (C1) buffer 0-99.9% by weight. As the polymer (A1), a polymer obtained by polymerizing a phosphorylcholine group-containing monomer represented by the formula (2) among the PC polymers described in the above stabilization method is used. If the content of the polymer is less than 1.0 × 10 ⁻⁴ , the effect of stabilizing the plasma preparation protein is not sufficient, and if it exceeds 80% by weight, the liquid viscosity at the time of preparation becomes high and handling becomes difficult. become. The plasma preparation protein (B1) refers to a protein contained in an albumin preparation, a blood coagulation factor preparation, an immunoglobulin preparation, and the like. Blood coagulation factor preparations include Factor VIII preparation, Factor IX preparation, Factor I preparation, Antithrombin II
I preparations, Factor II, Factor III IV, Factor V, Factor VII, Factor X and the like. Examples of the immunoglobulin preparation include an intramuscular preparation, an intravenous preparation, and the like. When the content ratio of the plasma preparation protein is less than 1.0 × 10 ⁻¹⁴ wt%, the activity as the plasma preparation protein may decrease, which is not preferable. If it exceeds 20% by weight, it is necessary to increase the content of the PC polymer, and at that time, the viscosity is so high that it is difficult to handle. As the buffer (C1), those listed in the above stabilization method can be preferably mentioned, and may be an aqueous solution or may contain a small amount of a water-soluble solvent. This buffer is not necessarily contained because it is added to facilitate the handling of the plasma preparation protein in the form of an aqueous solution. When the content of the buffer exceeds 99.9% by weight, the concentrations of the plasma preparation protein and the polymer become low, and the function and the effect are hardly exhibited.

The labeled immunologically active substance composition of the present invention comprises:
(A2) Among the PC polymers, a polymer 1.0 × obtained by polymerizing a phosphorylcholine group-containing monomer represented by the formula (2):
^10-4 to 80% by weight, and (B2) labeled immunologically active substance
0 × 10 ^{−14 to} 20% by weight, and (C2) buffer solution 0 to 99.9.
% By weight. As the polymer (A2), a polymer obtained by polymerizing a phosphorylcholine group-containing monomer represented by the formula (2) among the PC polymers described in the above stabilization method is used. When the content of the polymer is less than 1.0 × 10 ⁻⁴ , the effect of stabilizing the immunologically active substance is not sufficient,
If it exceeds 0% by weight, the viscosity of the aqueous solution is high and it is difficult to handle.

The immunologically active substances include, for example, antibodies to plasma proteins such as C-reactive protein (CRP), rheumatoid factor (RF) and transferrin, thyroid-stimulating hormone, excluding the immunoglobulins contained in the above-mentioned plasma preparations. (TSH), triiodothyronine (T3), thyroxine (T4), thyroxine binding protein (TBG), thyroglobulin, insulin, estriol (E3),
Antibodies against hormones such as chorionic gonadotropin (HCG) and human placental lactogen (HPL); antibodies against tumor-related substances such as carcinoembryonic antigen (CEA), β2-microglobulin and α-fetoprotein (AFP);
Antibodies to antigens and antibodies against viral hepatitis such as HBS antigen, HBS antibody, HBe antigen, HBe antibody or antigens, viruses such as mumps, herpes, measles, rubella, cytomegalo, and various biological components such as anti-AIDS antibody (HIV) Examples include antibodies to various drugs such as antibodies or antigens, phenobarbital, acetaminophenone, salicylic acid, and cyclosporin. Further, an antigen against the above-mentioned antibody or an antibody against the above-mentioned antigen is also included. Furthermore, the antibody may be a Fab fragment, an F (ab) ′ 2 fragment or a reduced antibody. The labeled immunologically active substance of (B2) is an enzyme,
It shows a substance in which a fluorescent substance, a luminescent substance, and the like are chemically bonded. Although the enzyme to be labeled is not particularly limited, acetylcholinesterase, alkaline phosphatase, β
-D-galactosidase, glucoamylase, glucose oxidase, glucose-6-phosphate dehydrogenase,
Hexokinase, penicillinase, peroxidase,
Lysozyme and the like, preferably, alkaline phosphatase generally used in enzyme immunoassay,
β-D-galactosidase, peroxidase and the like. The fluorescent substance to be labeled is not particularly limited, and fluorescein isothiocyanate (FITC), 4-chloro-7-nitrobenzofurazan, 4-fluoro-7-nitrobenzofurazan, sulforhodamine 101 acid chloride, 4-chloro- 7-sulfobenzofurazan, ammonium salts, N- (9-acridinyl) maleimide and the like. Preferably, fluorescein isothiocyanate (FITC), 4-chloro-7-nitrobenzofurazan, 4-fluoro-7-nitrobenzofurazan, which reacts only by mixing with the free amino group of the immunologically active substance,
And sulforhodamine 101 acid chloride.
Also, such as 10-methyl-9- (4- (2- (succinimidyloxycarbonyl) ethyl) phenyloxycarbonyl) acridinium fluorosulfate which easily reacts with the free amino group of the immunologically active substance. Chemiluminescent materials can also be used. When the content ratio of the labeled immunologically active substance is less than 1.0 × 10 ⁻¹⁴ wt%, the activity of the labeled immunologically active substance becomes low, which is not preferable when used. If it exceeds 20% by weight, it is necessary to increase the content of the PC polymer, and at that time, the viscosity becomes high and it becomes difficult to handle.

As the buffer (C2), those listed in the above-mentioned stabilization method can be preferably mentioned. The buffer may be an aqueous solution or may contain a small amount of a water-soluble solvent. This buffer is not necessarily contained because it is added to facilitate handling of the labeled immunologically active substance in the form of an aqueous solution. If the content of the buffer exceeds 99.9% by weight, the concentrations of the labeled immunologically active substance and the polymer will be low, and it will be difficult to exhibit functions and effects.

The enzyme composition of the present invention comprises (A3) a polymer obtained by polymerizing a phosphorylcholine group-containing monomer represented by the formula (2) among the above-mentioned PC polymers: 1.0 × 10 ^-4 to 80 × 10 ^-4 (B3) 1.0 × 10 ^{−14 to} 20% by weight of the enzyme (B3) and (C3)
Buffer solution from 0 to 99.9% by weight. As the polymer (A3), a polymer obtained by polymerizing the phosphorylcholine group-containing monomer represented by the formula (2) among the PC polymers described in the above stabilization method is used. When the content ratio of the polymer is 1.
If the amount is less than 0 × 10 ⁻⁴ , the effect of stabilizing the enzyme is not sufficient. The enzyme of (B3) is not particularly limited, and includes oxidoreductase, transferase, hydrolase, lyase, isomerase, and synthase. Particularly preferably, β-D-galactosidase of glycosyl hydrolase (glycolytic enzyme) which is a kind of hydrolase used for clinical diagnosis or medical devices, and cholesterol esterase of ester hydrolase which is also a kind of hydrolase And alkaline phosphatase, and horseradish peroxidase, a peroxidase that is a kind of oxidoreductase. Among them, when used for clinical diagnosis, preferably, cholesterol esterase, β-D-galactosidase, alkaline phosphatase, horseradish peroxidase and the like are mentioned. Also,
Examples of the hydrolase include carbohydrase, esterase, protease, amidase, halogenase,
Phosphatase and the like. Generally, commercially available products can be used. For example, biopulase (manufactured by Nagase Seikagaku Co., Ltd., trademark, peptide degrading enzyme), lipase seiken (Nagase Seikagaku Co., Ltd., trademark, ester hydrolase) and the like can be mentioned. The origin of the enzyme is not limited,
Any source, such as Bacillus, can be used. Among them, when used for washing contact lenses or skin whitening, it is preferable to use a proteolytic enzyme such as bioprase (Nagase Seikagaku Corporation)
Products, trademarks, peptidases) and the like. (B3)
If the content of the enzyme is less than 1.0 × 10 ^-14 % by weight, the activity of the enzyme is lowered, which is not preferable when used. 20
If the content is more than 10% by weight, it is necessary to increase the content of the PC polymer. As the buffer (C3), those exemplified in the above-mentioned stabilization method can be preferably mentioned, and may be an aqueous solution or may contain a small amount of a water-soluble solvent. This buffer is not necessarily contained because it is added to facilitate the handling of the enzyme in the form of an aqueous solution. If the content of the buffer exceeds 99.9% by weight, the concentrations of the enzyme and the polymer will be low, and it will be difficult to exhibit functions and effects.

[0023]

According to the method for stabilizing a protein of the present invention, protein activity can be easily maintained and long-term stabilization can be achieved simply by allowing a PC polymer to coexist in the same system. In addition, since the composition containing the plasma preparation, the labeled immunologically active substance or the enzyme of the present invention contains a specific PC polymer, each activity is kept high, and long-term stabilization is possible. .

[0024]

The present invention will be described in more detail with reference to the following Examples, but it should not be construed that the invention is limited thereto. Synthesis Example 1-1: Synthesis of Polymer B MPC 5.9 so that the total monomer concentration would be 1.0 mol / l and the amount of polymerization initiator would be 1 mol% with respect to the monomer.
05 g (0.02 mol) was weighed into a polymerization glass reaction tube, and 2,2′-azobisisobutyronitrile (hereinafter abbreviated as AIBN) 0.0328 was added thereto as a polymerization initiator.
g (0.2 mmol) and methanol 2 as a polymerization solvent.
0 ml was added. After sufficiently replacing the inside of the reaction tube with argon, it was sealed. Next, the polymerization reaction was performed by heating to 50 ° C. for 24 hours. After cooling the reaction mixture on ice,
The polymer was precipitated by dropwise addition to 400 ml of diethyl ether. The precipitate was separated by filtration, washed sufficiently with diethyl ether, and dried under reduced pressure to obtain 3.691 g of a white powdery polymer B. The yield was 62.5%.
The molecular weight was measured by analyzing the phosphate buffer solution of the polymer B using gel permeation chromatography (GPC), and as a result, it was 68,000 in terms of polyethylene glycol.

Synthesis Example 1-2: Synthesis of Polymer C The charged molar ratio of the monomer between MPC and n-butyl methacrylate (hereinafter abbreviated as BMA) is MPC / BMA =
40/60, the total monomer concentration was 1.0 mol / l, and the amount of the polymerization initiator was 1 mol% with respect to the monomer.
1.435 g (4.9 mmol) of PC and 2.1 of BMA
53 g (15.1 mmol) was weighed into a glass reactor for polymerization, and 0.0328 g of AIBN was added thereto as a polymerization initiator.
(0.2 mmol) and methanol 20 as a polymerization solvent.
ml was added. After sufficiently replacing the inside of the reaction tube with argon,
Sealed. Next, the polymerization reaction was performed by heating to 60 ° C. for 24 hours. After cooling the reaction mixture on ice, 40
The polymer was precipitated by dropwise addition to 0 ml of diethyl ether. The precipitate was separated by filtration, washed sufficiently with diethyl ether, and dried under reduced pressure to obtain 2.019 g of a polymer C as a white powder. The yield was 56.3%. The molecular weight was measured by analyzing a tetrahydrofuran solution of the polymer C using GPC, and as a result, it was 32,000 in terms of polystyrene. From the result of elemental analysis, the molar composition ratio was MPC / BMA = 38.5 / 61.5.

Synthesis Example 1-3 Synthesis of Polymer D Instead of 2.153 g (15.1 mmol) of BMA, 1.573 g of styrene (hereinafter abbreviated as St) (15.
Except for using 1 mmol), the same procedure as in Synthesis Example 1-2 was carried out to obtain 1.972 g of a white powdery polymer D. The yield was 52.9%, the molecular weight was 42000 in terms of polystyrene,
The molar composition ratio was MPC / St = 39.5 / 60.5. Table 1 shows the monomers of Synthesis Examples 1-1 to 1-3 and their results.

[0027]

[Table 1]

Reference Example 1: Preparation of antibody-immobilized plate Titer plate (Maxisorp F96; NUN)
C) (5.0 μg / ml) dissolved in 100 mM disodium hydrogen phosphate / sodium dihydrogen phosphate buffer (pH 7.5).
00 μL was added and incubated at 4 ° C. for 12 hours. At the end of the incubation, add each well to 150 mM
10 mM disodium hydrogen phosphate with added NaCl /
The plate was washed three times with a sodium dihydrogen phosphate buffer (pH 7.5). 10 mM disodium hydrogen phosphate / sodium dihydrogen phosphate buffer (p.
H7.5) Add 300 μl of solution to each well and add 25
Incubated for 2 hours. After the incubation, the solution in each well was removed by decantation. Thereafter, the plate was frozen in a freezer at -80 ° C and dried to prepare a freeze-dried antibody-immobilized plate.

Example 1-1: Stabilization test of plasma preparation protein
Experiment 1 5 μg / ml dissolved in 100 mM disodium hydrogen phosphate / sodium dihydrogen phosphate buffer (pH 7.5)
Human immunoglobulin G solution (5.0 × 10 ^-4 % by weight)
The polymer B synthesized in Synthesis Example 1-1 was added to the mixture so as to have a concentration of 2.0% by weight. Thereafter, incubation was performed at 40 ° C. The day of addition was set to 0 days later.

0 days, 2 weeks, 1 month, 2 months,
Each human immunoglobulin G solution after 3 months and 4 months was added to 8 wells of the antibody-immobilized plate prepared in Reference Example 1 so as to have a concentration of 100 μL / well.
Incubated for 1 hour at ° C. At the end of the incubation, each well was filled with 10 mM supplemented with 150 mM NaCl.
Washed 3 times with mM disodium hydrogen phosphate / sodium dihydrogen phosphate buffer (pH 7.5). Then, 5.0
10% with weight% BSA and 150 mM NaCl
Horserad diluted 10000-fold with mM disodium hydrogen phosphate / sodium dihydrogen phosphate buffer (pH 7.5)
100 μL of ish Peroxidase-labeled anti-human immunoglobulin G was added to each well and incubated at 25 ° C. for 1 hour. At the end of the incubation, add each well to 150 mM
10 mM disodium hydrogen phosphate with added NaCl /
The plate was washed three times with a sodium dihydrogen phosphate buffer (pH 7.5). Use one tablet (OPD tablet) manufactured by Wako Pure Chemical Co., Ltd.
Phosphate / citrate buffer 1 containing 006% hydrogen peroxide 1
After adding 100 μL / well of the solution dissolved in 2 ml, the mixture was incubated at 25 ° C. for 10 minutes. Then, 2
After adding 50 μL / well of a sulfuric acid solution of N, the absorbance at 492 nm of each well was measured using a microplate reader “MPR-A4i” manufactured by Tosoh Corporation. I asked. In addition,
The composition is shown in Table 2, and the measurement results are shown in Table 3.

[0031]

[Table 2]

[0032]

[Table 3]

Examples 1-2 and 1-3: Plasma preparation proteins
Stabilization Tests 2 and 3 Synthesis Example 1 was replaced with Polymer B used in Example 1-1.
2 and Polymer C synthesized in Synthesis Example 1-3 (Example 1-
The procedure was performed in the same manner as in Example 1-1 except that 2) and polymer D (Example 1-3) were used. Table 3 shows the measurement results.

Comparative Example 1-1 In place of the polymer B synthesized in Synthesis Example 1-1, 2% by weight was used.
Was performed in the same manner as in Example 1-1, except that BSA was added. Table 3 shows the measurement results.

Comparative Example 1-2 5 μg / ml dissolved in 100 mM disodium hydrogen phosphate / sodium dihydrogen phosphate buffer (pH 7.5)
The procedure was performed in the same manner as in Example 1-1, except that nothing was added to the human immunoglobulin G solution. Table 3 shows the measurement results.

Example 2-1: Labeled antibody immunologically active substance
Stabilization test 1 Hor diluted 20,000-fold with 10 mM disodium hydrogen phosphate / sodium dihydrogen phosphate buffer (pH 7.5)
To the seradish peroxidase-labeled anti-human immunoglobulin G solution (about 1.0 × 10 ⁻⁷ wt%), the polymer B synthesized in Synthesis Example 1-1 was added so as to be 2.0 wt%. Thereafter, incubation was performed at 40 ° C. The date of addition is 0
Days later.

To 8 wells of the antibody-immobilized plate prepared in Reference Example 1, 1.0 μg / ml human immunoglobulin was added at 100 μL / well, and then added at 25 ° C.
Incubated for 1 hour. After the incubation, each well was washed three times with a 10 mM disodium hydrogen phosphate / sodium dihydrogen phosphate buffer (pH 7.5) supplemented with 150 mM NaCl. After completion of washing, 2 days after 0 day, 3 days, 1 week, 2 weeks, 3 weeks, 4 weeks
A Horseradish Peroxidase-labeled anti-human immunoglobulin G solution diluted 0000-fold was added to 8 wells at 100 µL / well, and then incubated at 25 ° C for 2 hours. At the end of the incubation, each well is
10 mM disodium hydrogen phosphate / sodium dihydrogen phosphate buffer (pH 7.0) supplemented with 50 mM NaCl.
Washed 3 times in 5). Product name "OPD" manufactured by Wako Pure Chemical
One tablet ”is treated with phosphoric acid containing 0.006% hydrogen peroxide /
100 μL of the solution dissolved in 12 ml of citrate buffer
/ Well, and then incubated at 25 ° C. for 10 minutes. Subsequently, after adding 50 μL / well of a 2N sulfuric acid solution, a microplate reader “MPR manufactured by Tosoh Corporation” was added.
-A4i ”, the absorbance at 492 nm of each well was measured, and the absorbance at day 0 was defined as 100%,
I asked. Table 2 shows the composition and Table 4 shows the measurement results.

Examples 2-2 and 2-3: Labeled antibody immunology
Example 2 Stabilization Test of Active Substances Synthesis Example 1 Instead of Polymer B Synthesized in Synthesis Example 1-1
-2 and Polymer C synthesized in Synthesis Example 1-3 (Example 2-
The procedure was performed in the same manner as in Example 2-1 except that 2) and polymer D (Example 2-3) were used. Table 4 shows the measurement results.

Comparative Example 2-1 In place of the polymer B synthesized in Synthesis Example 1-1, 2% by weight was used.
Example 2-1 was performed except that BSA was added. Table 4 shows the measurement results.

Comparative Example 2-2 5 μg / ml dissolved in 100 mM disodium hydrogen phosphate / sodium dihydrogen phosphate buffer (pH 7.5)
The procedure was performed in the same manner as in Example 2-1 except that nothing was added to the human immunoglobulin G solution. Table 4 shows the measurement results.

[0041]

[Table 4]

Example 3-1: Stabilization of enzyme 1-1 5 fmol / m in 10 mM disodium hydrogen phosphate / sodium dihydrogen phosphate buffer (pH 7.0) to which 100 mM NaCl and 1 mM MgCl ₂ were added.
1 (10 ⁻¹⁵ mol / ml) of β-D-galactosidase solution (1.0 × 10 ⁻¹³ wt%) was added with the polymer synthesized in Synthesis Example 1-1 so as to be 2.0 wt%. . Thereafter, incubation was performed at 40 ° C. The day of addition was set to 0 days later.

At 0, 3, 1, 2, 3 and 4 weeks, 0.4 ml of each β-D-galactosidase solution was added with 100 mM NaCl and 1 mM magnesium chloride, and 10 mM hydrogen phosphate was added. 50 mmol / L 2-nitrophenyl β-D- dissolved in disodium / sodium dihydrogen phosphate buffer (pH 7.0)
0.2 ml of a galactoside solution was added, and the mixture was incubated at 30 ° C. for 10 minutes. After the incubation is completed, 0.1
After adding 2 ml of mol / L sodium carbonate, absorbance at 420 nm was measured using a UV / VIS measuring device “Ubest-50” manufactured by JASCO Corporation. The absorbance at day 0 was defined as 100%, and the% at each day was determined. Table 5 shows the measurement results.
Shown in

Examples 3-2 and 3-3: Stabilization of enzyme 1
-2, 1-3 Instead of the polymer B of the synthesis example 1-1, the polymer C (Example 3-2) and the polymer D (Example 3) synthesized in the synthesis examples 1-2 and 1-3. Example 3-1 except that 3-3) was used.
The same was done. Table 5 shows the measurement results.

Comparative Example 3-1 In place of the polymer B synthesized in Synthesis Example 1-1, 2% by weight was used.
The procedure was performed in the same manner as in Example 3-1 except that BSA was added. Table 5 shows the measurement results.

Comparative Example 3-2 5 fmol / m dissolved in 10 mM disodium hydrogen phosphate / sodium dihydrogen phosphate buffer (pH 7.0) to which 100 mM NaCl and 1 mM MgCl ₂ were added.
The procedure was performed in the same manner as in Example 3-1 except that nothing was added to the 1β-D-galactosidase solution. Table 5 shows the measurement results.

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[Table 5]

Reference Example 2 Preparation of Protein-Adhered Lens Artificial tear (0.6 g of albumin, 0.3 g of globulin,
A contact lens (contact lens “Super EX1” manufactured by Seiko) was immersed in 0.2 g of lysozyme and 0.1 g of mucin dissolved in physiological saline to make 100 ml, and heated to 65 ° C. to attach the protein.

Example 4-1 Synthetic Example 1-1: A solution (0.4% by weight) of 4.0 mg / ml bioprase (trade name, peptide degrading enzyme, manufactured by Nagase Seikagaku Corporation) dissolved in physiological saline was used. Was added so as to be 2.0% by weight. Thereafter, incubation was performed at 40 ° C. The date of addition is 0
Days later. 0 days, 1 month, 2 months, 3 months,
Four months later, the proteolytic enzyme activity of each bioprase solution was measured by the casein-forin method. The activity after each month was determined with the activity after 0 day as 100%. Table 2 shows the composition
Table 6 shows the measurement results.

Further, the protein-adhered lens prepared in Reference Example 2 was placed in each of the bioprase solutions on day 0, 1 month, 2 months, 3 months, and 4 months, and washed while standing. The cleaning effect was visually determined. A case where the attached protein was almost completely removed was judged as good, and the other cases were judged as bad. Table 7 shows the measurement results.

Examples 4-2 and 4-3 In place of the polymer B synthesized in Synthesis Example 1-1, Synthesis Example 1 was used.
-2 and the polymer C synthesized in Synthesis Example 1-3 (Example 4-
The procedure was performed in the same manner as in Example 4-1 except that 2) and the polymer D (Example 4-3) were used. Tables 6 and 7 show the measurement results.

Comparative Example 4-1 The procedure of Example 4-1 was repeated, except that 2.0% by weight of BSA was added instead of the polymer B synthesized in Synthesis Example 1-1. Tables 6 and 7 show the measurement results.

Comparative Example 4-2 Example 4 was repeated except that nothing was added to a 4.0 mg / ml bioprase (trademark, peptide degrading enzyme, manufactured by Nagase Seikagaku Corporation) dissolved in physiological saline. Performed similarly to 1. Tables 6 and 7 show the measurement results. As a result, it can be seen that the examples of the present invention have better stability than the comparative examples.

[0054]

[Table 6]

[0055]

[Table 7]

──────────────────────────────────────────────────続 き Continuation of the front page (51) Int.Cl. ⁶ Identification number Agency reference number FI Technical display location C12N 9/96 C12N 9/96 C12Q 1/25 9452-4B C12Q 1/25 // A61K 38/00 C08F 30/02 MNS 38/16 C08L 43/02 LKA 38/43 A61K 37/02 C08F 30/02 MNS 37/04 C08L 43/02 LKA 37/465 (72) Inventor Shujiro Sakaki 2-Kasuga, Tsukuba, Ibaraki 20-3 (72) Inventor Kenshiro Shuto 3-7-1 Hanahata, Tsukuba, Ibaraki (72) Inventor Satoshi Yamada 2-20-3, Kasuga, Tsukuba, Ibaraki (72) Inventor Ryota Ando, Kasuga, Tsukuba, Ibaraki 2-26-2 (72) Inventor Kazuo Matsuyama 2-17-14 Kasuga, Tsukuba City, Ibaraki Prefecture (72) Inventor Yasumi Koinuma 32-16 Higashi Arai, Tsukuba City, Ibaraki Prefecture (72) Inventor Norio Nakabayashi Matsudo, Chiba Prefecture Small Kinbara -6-20 (72) inventor Kazuhiko Ishihara, Tokyo Kodaira Josuihon-cho 3-16-37

Claims (5)

[Claims] 1. A method for stabilizing a protein, comprising coexisting a polymer having a phosphorylcholine group represented by the formula (1) and a protein in the same system. Embedded image 2. A polymer containing a phosphorylcholine group-containing monomer represented by the formula (2), wherein X is a hydrogen atom or a methyl group. The method for stabilizing a protein according to claim 1, which is a polymer obtained by polymerizing components. Embedded image Wherein (A1) (2) (wherein X represents a hydrogen atom or a methyl group) is polymerized phosphorylcholine group-containing monomer represented by comprising polymer 1.0 × 10 ^-4 ~ 80% by weight (B1) a plasma preparation protein of 1.0 × 10 ^{−14 to} 20% by weight;
(C1) A stabilized plasma preparation protein composition, comprising 0 to 99.9% by weight of a buffer solution. Wherein formula (A2) (2) (wherein X represents a hydrogen atom or a methyl group) is polymerized phosphorylcholine group-containing monomer represented by comprising polymer 1.0 × 10 ^-4 ~ 80% by weight (B2) a stabilized labeled immunologically active substance composition comprising 1.0 × 10 ^{−14 to} 20% by weight of a labeled immunologically active substance and (C2) 0 to 99.9% by weight of a buffer solution . Wherein (A3) formula (2) (wherein X represents a hydrogen atom or a methyl group) is polymerized phosphorylcholine group-containing monomer represented by comprising polymer 1.0 × 10 ^-4 ~ 80% by weight, (B3) A stabilized enzyme composition comprising 1.0 × 10 ^{−14 to} 20% by weight of an enzyme and (C3) 0 to 99.9% by weight of a buffer solution.