JP2003310752A - Method of manufacturing biologically active substance fixed molding article - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an expensive, simple, and safe method of removing impurities contained in a amino group-containing biologically active substance fixed molding article. <P>SOLUTION: A method of manufacturing the biologically active substance fixed molding article in which the impurities contained in an insoluble carrier is removed by washing after an amino group-containing biologically active substance is treated to be fixed to the insoluble carrier. <P>COPYRIGHT: (C)2004,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for producing a physiologically active substance-immobilized molded article.

[0002]

2. Description of the Related Art Molded articles on which physiologically active substances are immobilized are used as selective adsorbents for removing harmful substances in blood or as cell function modifiers. However, during immobilization treatment of physiologically active substances, unreacted physiologically active substances and coexisting substances (hereinafter abbreviated as impurities) have large molecular weights and often have low solubility in water. , Easily remains inside the carrier.

[0003]

When a physiologically active substance-immobilized molded article is used as a medical material, it is necessary to remove these impurities to a level without problems for safety.
When an organic solvent is used for removing impurities, the remaining solvent used becomes a problem, so it is preferable to use water. However, since many impurities are difficult to dissolve in water, a large amount of ultrapure water is required for cleaning, which requires a large amount of cost and time. In addition, there are drawbacks such as discharging a large amount of drainage. On the other hand, if the temperature of the ultrapure water used for cleaning is increased in order to improve the cleaning efficiency, there arises a problem that the activity of the physiologically active substance immobilized on the molded product is impaired.

[0004]

[Means for Solving the Problems] In order to solve the above-mentioned problems, the inventors made extensive studies and found a method for producing a physiologically active substance-immobilized molded article by the following impurity removal method. That is, it is a method for producing a physiologically active substance-immobilized molded article, which comprises removing impurities contained in the molded article after the physiologically active substance-immobilized treatment with an organic acid. (1) After immobilizing an amino group-containing physiologically active substance on an insoluble carrier, washing the insoluble carrier with a solution of an organic acid or an aqueous solution of an alkali metal salt thereof,
A method for producing a physiologically active substance-immobilized molded article, which comprises removing impurities contained in the insoluble carrier to obtain a physiologically active substance-immobilized molded article. (2) The method for producing a physiologically active substance-immobilized molded article according to (1), wherein the amino group-containing physiologically active substance is a cyclic peptide antibiotic. (3) The method for producing a physiologically active substance-immobilized molded article according to (1) or (2), wherein the organic acid is dissolved in an aqueous solvent or an organic solvent. (4) The method for producing a physiologically active substance-immobilized molded article according to any one of (1) to (3), wherein the organic acid is a carboxylic acid having 8 or more carbon atoms. (5) The method for producing a physiologically active substance-immobilized molded article according to any one of (1) to (4), wherein the organic acid is a carboxylic acid having a steroid skeleton. (6) The method for producing a physiologically active substance-immobilized molded article according to any one of (1) to (5), wherein the impurities are antibiotics that are not immobilized on an insoluble carrier.

[0005]

BEST MODE FOR CARRYING OUT THE INVENTION Examples of the amino group-containing physiologically active substance according to the present invention include polymyxin B, colistin,
A cyclic or non-cyclic peptide consisting of 4 or more and 50 or less amino acids, represented by bacitracin, gramicidin, etc., having 1 to 10 or less free amino groups in its side chain, and its side chain There are one or more peptide compounds, alkaloids represented by reserpine, morphine, etc., aminoglucoside antibiotics such as gentamicin, streptomycin, etc., and their alkyl or acyl derivatives can be mentioned.

The impurities referred to in the present invention are cyclic or non-cyclic peptide antibiotics that have not been immobilized on an insoluble carrier and impurities that have been mixed in from the manufacturing process thereof. The cyclic peptide antibiotics referred to in the present invention means polymyxin B, colistin, bacitracin, gramicidin, etc., which have 1 to 10 free amino groups in their side chains. The immobilization referred to in the present invention means binding to an insoluble carrier by a chemical bond. The term "molded product" as used in the present invention means a product molded into a specific shape such as a fiber, a film, a semipermeable membrane, or a granular material.

The insoluble carrier referred to in the present invention may be any one that is insoluble in water and is capable of immobilizing a physiologically active substance having an amino group by a covalent bond, and is an aromatic polyvinyl typified by polystyrene or polyvinyltoluene. compound, poly (p- phenylene ether sulfone): - {(p-C 6
_{H 4) -SO 2 - (p} -C 6 H 4) -O-} n- ( hereinafter abbreviated as U del polysulfone) polysulfone-based polymer represented by like, polyetherimide, polyimide, polyamide, polyether Polyphenylene sulfide and the like, which have a reactive functional group capable of immobilizing an amino group-containing physiologically active substance by a covalent bond, can be used, but are not limited thereto. Examples of reactive functional groups include halomethyl groups, haloacetyl groups, haloacetamidomethyl groups, active halogen groups such as halogenated alkyl groups, epoxide groups, carboxyl groups, isocyanic acid groups, thioisocyanic acid groups, and acid anhydride groups. In particular, an active halogen group, especially a haloacetyl group, is easy to produce and has a reasonably high reactivity, and the immobilization reaction can be carried out under mild conditions, and at the same time, the covalent bond generated at this time is It is chemically stable, which is preferable. Further specific examples of the insoluble carrier include chloroacetamidomethylated polystyrene, chloroacetamidomethylated Udel polysulfone, and chloroacetamidomethylated polyetherimide.

In the immobilization treatment of the present invention, the solution containing the amino group-containing physiologically active substance and the insoluble carrier are 0-1 under basic conditions.
Achieved by mixing at a temperature of 00 ° C.

The organic acid referred to in the present invention is glutamic acid, gluconic acid, lauric acid, deoxycholic acid, cholic acid and the like, and one or more of them can be used. In particular, those having less toxicity such as glutamic acid, gluconic acid, and cholic acid are preferably used, and of these, cholic acid and the like, which have a better cleaning effect, are more preferably used. Further, as the organic acid in the present invention,
Those having 8 to 24 carbon atoms are preferably used because of their solubility in a solvent.

The organic acid referred to in the present invention may be a general definition of "organic compounds having acidity". For example, carboxylic acid having a steroid skeleton represented by deoxycholic acid and cholic acid. Since the acid has a highly hydrophobic steroid skeleton and a highly hydrophilic carboxyl group,
Highly effective in cleaning impurities that are difficult to dissolve in water, such as cyclic peptides. As the organic acid used in the present invention, those having a high cleaning effect and a low toxicity are desirable. From two viewpoints, glutamic acid, gluconic acid and cholic acid are preferable, and cholic acid is particularly preferable.

These organic acids may be used alone or in combination of two or more. The organic acid of the present invention is preferably used by dissolving it in a solvent. As a solvent for dissolving the organic acid, water or a solvent having low toxicity such as ethanol is preferably used, and water is more preferably used from the viewpoint of toxicity and less trouble after washing. Further, an aqueous solution of an alkali metal salt of an organic acid is preferably used instead of the organic acid. It is not particularly limited as long as it is used as an alkali metal salt, but sodium salt, potassium salt and calcium salt can be preferably used.

The term "removal of impurities" in the present invention means to reduce the residual amount of impurities such as cyclic peptide antibiotics which are not immobilized on an insoluble carrier to a level harmless to the human body. In the present invention, This is achieved by washing the insoluble carrier with an organic acid or its alkali metal salt. As the cleaning mode of the present invention, a method of circulating an organic acid solution while forcibly flowing through the material, a method of stirring the material in the organic acid solution, and the like are preferably used. In particular, in the case of a knitted or woven fabric, a method of forcibly flowing an organic acid solution through the material and circulating it is more preferably used because impurities can be removed more efficiently.

The temperature at the time of washing of the present invention is not particularly limited, but from the viewpoint of the washing effect and the effect on the physiologically active substance-immobilized molded article, it is preferably 30 ° C to 60 ° C, and 40 ° C to 5 ° C.
0 ° C is more preferable. The concentration of the organic acid used in the present invention is not particularly limited, but the thinner the better as long as the cleaning effect is obtained, the better. Therefore, 0.0001 mol / L to 0.5 mol
/ L, especially from 0.001 mol / L to 0.1 mol / L in view of the effect of removing impurities and the ease of replacement with water after washing.
L is more preferred.

The method for producing a physiologically active substance-immobilized molded article provided by the present invention comprises treatment steps of preparation of an insoluble carrier, physiologically active substance immobilization treatment, washing and sterilization treatment. Sterilization treatment is immobilization treatment. Alternatively, the cleaning may be performed at an intermediate stage. The method for preparing the insoluble carrier is explained by using an aromatic polysulfone polymer as an example. The polymer is dissolved in heat, a solvent or the like and then molded into fibers, films, hollow fibers or granules. The chemical treatment will be described using an example of an aromatic polysulfone polymer. The main chain aromatic polysulfone is provided with an active halogen-containing substituent as a side chain functional group at a density of 0.001 or more and 0.5 or less per repeating unit. The solution of the polymer possessed by 1. and the solution of the physiologically active substance are mixed and reacted. To give a specific example, the corresponding physiologically active substance is added to the solution of the haloacetamidomethylated polysulfone, and the mixture is added at 0 to 100 ° C. It can be easily produced by reacting at a temperature of, and as a reaction solvent, in the case of reacting in a homogeneous system, tetrahydrofuran or dimethyl sulfoxide is used. De, N, N- dimethylformamide, N, N- dimethylacetamide and N- methylpyrrolidone polysulfone derivative and physiologically active solvents are preferred Mochiiraru to dissolve both substances, such as Don. The sterilization treatment is heating steam sterilization, gamma irradiation sterilization, or the like, and the physiologically active substance-immobilized molded article according to the present invention can be manufactured by the above steps.

The physiologically active substance-immobilized molded article of the present invention is used for extracorporeal circulation treatment for the purpose of removing toxic substances such as endotoxin in blood. It can also be used for the purpose of removing toxic substances such as endotoxin from blood for transfusion, serum, and plasma, but the present invention is not limited to these applications.

The present invention will be specifically described below based on examples.

[0017]

EXAMPLES The methods for preparing and evaluating the samples in the examples and comparative examples were as follows.

1. Amino Acid Analysis 0.1 g of the polymer was sealed in a glass sealed tube together with 10 mL of 6N hydrochloric acid, heated at 115 ° C. for 15 hours to hydrolyze, and then concentrated under reduced pressure. An amino acid analyzer (Hitachi 835: ninhydrin color development, special amino acid analysis method) is performed on this solution, and the contents of leucine and phenylalanine are compared with the contents in the raw material polymyxin B to determine the amount of peptide contained. It was

2. Analysis of polymyxin B concentration 2.1 Preparation of monoclonal antibody 2.1.1 Keyhole limpet hemocyanin-
Preparation of polymyxin B complex and immunokeyhole limpet hemocyanin (manufactured by Pierce Chemical Co.) 1 ml (5 mg / ml) aqueous solution of polymyxin B 200 μl (5 mg / ml phosphate buffer solution (manufactured by Nissui Pharmaceutical Co., Ltd.)) , BS ³ (Pierce Chemical Co., Ltd.) 1
00 μl (5 mg / ml phosphate buffer solution) was added and reacted for 1 hr at room temperature. 100 μl of 1M-TrisHCl buffer (pH 8.0) was added to the reaction solution, followed by desalting with a PD-10 column to obtain a keyhole limpet hemocyanin-polymyxin B complex (5 mg / 3 ml).

Immunization was carried out by intraperitoneally administering 100 μg of keyhole limpet hemocyanin-polymyxin B complex to mice four times. The first immunization is keyhole, limpet, hemocyanin-polymyxin B
Aluminium potassium sulfate (Wako Pure Chemical Industries, Ltd.) adjuvant (CytRx) 150 µl containing 10 µ 10 pertussis killed bacteria (Wako Pure Chemical Industries, Ltd.) containing 100 µg of the complex
(Phosphate buffer solution) was mixed and administered. At the second time, 100 μg of the complex was mixed with an aluminum potassium sulfate adjuvant (phosphate buffer solution), and at the third time and thereafter, 100 μg of the complex was dissolved in 130 μl of a phosphate buffer solution for immunization.

2.1.2 Preparation of anti-monoclonal antibody-producing hybridoma The spleen was excised from the mouse 3 to 4 days after the final immunization.
Splenocytes were isolated. This cell and myeloma cell (SP
2) (University of Heidelberg) was mixed at a cell number ratio of 10: 1, and cell fusion was performed with a 50% polyethylene glycol (Boehringer) solution containing Hanks balanced salt (Gibco). The cells after fusion were D-MEM (Sigma) (1
/ 100 amount of penicillin streptomycin (Gibco), 1/100 amount of 2-mercaptoethanol (Boehringer), 1/1000 amount of MEM non-essential amino acid solution (Boehringer), 2 ng / ml human IL-
6 added) to fetal calf serum (Gibco) (15
%), HAT (medium supplemented with pipoxanthin, aminopterin, and thymidine) (Gibco) and hybridoma fusion / cloning additive (Boehringer), and suspended in a 96-well microplate at 200 μl / well. (Corning Co., Ltd.), and the cells were cultured at 37 ° C. in the presence of 5% carbon dioxide gas.

2.1.3 Antibody Production from Hybridomas and Purification of Antibodies Culture supernatants of hybridomas cultured for about 10 days in serum-free medium (SFM) were collected, and 1/20 amount of 1M-Tri was collected.
sHCl buffer (pH 8.0) was added. This solution was injected into a protein G column, washed with a phosphate buffer solution, and eluted with 50 mM glycine-hydrochloric acid buffer solution (pH 2.4) in 0.5 ml fractions, and 3,4,5 fractions were diluted with 1 M-TrisHCl buffer solution. (PH 8.0) 3
Collected in 00 μl. The collected antibody was replaced with a phosphate buffer solution using a PD-10 column, and 0.02% sodium azide was added to give a purified anti-polymyxin B monoclonal antibody.

2.2 Preparation of Polyclonal Antibody Immunization was performed by subcutaneously administering 300 μg of the keyhole limpet hemocyanin-polymyxin B complex to a rabbit four times. The first immunization is 100 μg of keyhole limpet hemocyanin-polymyxin B complex.
300 μl (PBS solution) of potassium aluminum sulphate adjuvant containing 10 to the power of 10 pertussis killed. The second time, 300 μg of the complex was mixed with aluminum potassium sulfate adjuvant (phosphate buffer solution), and the third time and thereafter, 300 μg of the complex was mixed with 1 ml of PBS.
It was dissolved in a solution and immunized.

Blood was collected from a rabbit 10 days after the final immunization and centrifuged at 3000 rpm for 15 minutes to obtain plasma 4
ml was obtained. Plasma was diluted with the same amount of PBS and purified with a protein G column to obtain an anti-polymyxin B polyclonal antibody. 2.3 Preparation of biotinylated antibody Mouse anti-polymyxin B monoclonal antibody 100μ
g, rabbit anti-polymyxin B polyclonal antibody 200
μg was replaced with a 50 mM sodium hydrogen carbonate aqueous solution on a PD-10 column, and centrifugal concentration (using Centricut ultra mini with a cut-off molecular weight of 10,000, 7500 rpm, 7 min) was carried out to obtain a solution of anti-polymyxin B monoclonal antibody 100.
μl, anti-polymyxin B polyclonal antibody 180 μl
The amount of Add 1/10 volume of Sulfo-NH to this solution
S-LC-biotin (4 mg / ml of 50 mM sodium hydrogen carbonate aqueous solution) was added, and the mixture was reacted at 25 ° C. for 4 hours. The reaction solution was replaced with a phosphate buffer solution using a PD-10 column, and 0.02% sodium azide was added to obtain a biotinylated anti-polymyxin B monoclonal antibody and an anti-polymyxin B polyclonal antibody.

2.4 Measurement by ELISA method A 100 μL solution of mouse anti-polymyxin B monoclonal antibody was placed in a microplate for ELISA (manufactured by Nunc International) and allowed to stand overnight at 4 ° C. for immobilization. After washing with a washing solution (0.05% Tween 20 (manufactured by Sigma-Aldrich Japan Co., Ltd.) / Phosphate buffer (3 times), polymyxin B (Pfizer Pharmaceutical Co., Ltd.) solution 50
μL and dilution buffer (0.25% bovine serum albumin (manufactured by Bayer), 0.05% Tween 20, 0.02)
% Sodium azide (manufactured by Wako Pure Chemical Industries, Ltd.), 1M Tris-hydrochloric acid (manufactured by Life Technology Co., Ltd.) buffer (pH
8.0)) 50 μL was dispensed to a solid-phased plate and reacted for 1 hour at room temperature. Unreacted substances were removed and washed (3 times) with a washing solution (0.05% Tween 20 / phosphate buffer solution),
Biotinylated anti-polymyxin B polyclonal antibody solution 1
00 μL was dispensed on the same plate and reacted for 1 hour at room temperature. The unreacted material was removed and washed (three times) again as a washing solution (0.05% Tween 20 / phosphate buffer solution), and avidin HRP was dispensed on the same plate and reacted at room temperature for 20 minutes. Unreacted materials were removed and washed (3 times) with a washing solution (0.0
5% Tween20 / phosphate buffer solution was used to develop color solution (2 mg / ml TMB (Nacalai Tesque, Inc.) / D
MF, 0.1 M acetic acid (produced by Hayashi Pure Chemical Industries, Ltd.) / Sodium citrate (produced by Kishida Chemical Co., Ltd.) buffer (pH 3.
5), 100 μL of 0.006% hydrogen peroxide / 0.1 M sodium acetate (manufactured by Nacalai Tesque, Inc.) / Citric acid (manufactured by Nacalai Tesque, Inc.) (pH 4.5) was dispensed on the plate. Then, after color development for 10 to 15 minutes at room temperature, 1N-sulfuric acid (Sigma Aldrich Japan Co., Ltd.)
The reaction was stopped with 100 μL. 450 of this solution
Absorbance at the wavelength of nm is the microplate reader (Corona Electric Co., Ltd., Microplate reader MTP-12)
0)).

3. Analysis of endotoxin component in blood The endotoxin concentration was determined by using a Wako Pure Chemical Industries' rimlas ES test Wako reagent and a toxinometer (ET301).

4. Sample preparation <Preparation of insoluble carrier> Polypropylene (“Noblene”)
J3HG) 50 parts as an island component, a mixture of polystyrene ("Stylon" 666) 46 parts and polypropylene ("Nobren" J3HG) 4 parts as a sea component is a multi-core sea-island type composite fiber (16 islands, single yarn fineness 2 6.6 denier, tensile strength 2.9 g / d, elongation 50%, filament number 4
2) was prepared by the melt spinning method, and two yarns were combined to form a cylinder knit (polypropylene reinforced polystyrene fiber).

After dissolving 1.7 g of paraformaldehyde in a mixed solution of 800 g of nitrobenzene and 800 g of sulfuric acid at 20 ° C., the mixture was cooled to 0 ° C. and 100 g of N-methylol-
α-Chloracetamide was added and dissolved at 5 ° C or lower. 100 g of the polypropylene-reinforced polystyrene fiber prepared above was dipped in this and allowed to stand at room temperature for 2 hours. After that, take out the fiber and put it in a large excess of cold methanol,
Washed. After thoroughly washing the fibers with methanol, wash with water,
After drying, 140 g of α-chloroacetamide methylated polystyrene fiber (substitution rate: 100%) was obtained. <Physiologically active substance-immobilized treatment> Next, polymyxin B sulfate (Pfizer Pharmaceutical Co., Ltd.) aqueous solution (0.2 mg / mL)
20 g of the α-chloroacetamide methylated polystyrene fiber obtained above was immersed in 1 L, and a 1 M sodium hydroxide aqueous solution was added to adjust the pH of the solution to 9.5. After permeating the reaction mixture for 24 hours, the fibers were taken out and washed with 1 L of 0.1 M hydrochloric acid (manufactured by Nacalai Tesque, Inc.). The fibers were washed with water until the pH of the washing liquid reached 5. As a result of amino acid analysis of this fiber, the amount of polymyxin B bound was Polymer 1
It was 10 mg per gram. <Sterilization treatment of the physiologically active substance-immobilized molded article> The physiologically active substance-immobilized fiber was cut into a size of 0.5 g, placed in physiological saline and autoclaved at 120 ° C for 20 minutes. Then, it was washed 3 times with 30 mL of ultrapure water (Sample 1).

Example 1 1.22 g of cholic acid (manufactured by Wako Pure Chemical Industries, Ltd.) was dissolved in 30 mL of ultrapure water to prepare a 0.1 mol / L cholic acid aqueous solution. Sample 1 was added to 30 mL of cholic acid aqueous solution,
The mixture was stirred at 37 ° C for 2 hours. After 2 hours, it was washed 5 times with 30 mL of ultrapure water. The washed sample was put in 50 mL of bovine serum and stirred at 37 ° C. for 2 hours. Two hours later, the amount of polymyxin B in bovine serum was determined by the ELISA method.

Example 2 0.44 g of glutamic acid (manufactured by Wako Pure Chemical Industries, Ltd.) was dissolved in 30 mL of ultrapure water to prepare a 0.1 mol / L glutamic acid aqueous solution. Sample 1 is glutamic acid aqueous solution 30
It was put in mL and stirred at 37 ° C. for 2 hours. After 2 hours, it was washed 5 times with 30 mL of ultrapure water. The washed sample was put in 50 mL of bovine serum and stirred at 37 ° C. for 2 hours. Two hours later, the amount of polymyxin B in bovine serum was determined by the ELISA method.

Example 3 0.59 g of gluconic acid (manufactured by Wako Pure Chemical Industries, Ltd.) was dissolved in 30 mL of ultrapure water to prepare a 0.1 mol / L gluconic acid aqueous solution. Sample 1 was placed in 30 mL of gluconic acid aqueous solution and stirred at 37 ° C. for 2 hours. 2 hours later, ultrapure water 30
Wash 5 times with mL. The washed sample was put in 50 mL of bovine serum and stirred at 37 ° C. for 2 hours. Two hours later, the amount of polymyxin B in bovine serum was determined by the ELISA method.

Comparative Example 1 Sample 1 was placed in 30 mL of ultrapure water and stirred at 37 ° C. for 2 hours. After 2 hours, it was washed 5 times with 30 mL of ultrapure water. The washed sample was put in 50 mL of bovine serum and stirred at 37 ° C. for 2 hours. After 2 hours, determine the amount of polymyxin B in bovine serum
It was determined by the ISA method.

Comparative Example 2 Sample 1 was placed in 30 mL of ultrapure water and stirred at 100 ° C. for 2 hours. After 2 hours, it was washed 5 times with 30 mL of ultrapure water. The washed sample was put in 50 mL of bovine serum and stirred at 37 ° C. for 2 hours. Two hours later, the amount of polymyxin B in bovine serum was changed to E.
It was determined by the LISA method.

Comparative Example 3 Sample 1 was placed in 50 mL of bovine serum without washing operation and stirred at 37 ° C. for 2 hours. After 2 hours, the amount of polymyxin B in bovine serum was determined by the ELISA method.

The results are shown in Table 1.

[0036]

[Table 1]

From Table 1, it can be seen that Examples 1, 2 and 3 of the present invention greatly reduce the amount of polymyxin B as an impurity, as compared with Comparative Example 1. In particular, it can be seen that the sample of Example 1 has a particularly low amount of polymyxin B. Also,
In Comparative Example 2, washing with the temperature of ultrapure water at 100 ° C. resulted in a reduction in the amount of polymyxin B remaining as compared with Comparative Example 1, but the endotoxin neutralizing performance of the product was significantly reduced. On the other hand, in Examples 1, 2, and 3, the cleaning effect equivalent to or higher than that of Comparative Example 2 was obtained without impairing the endotoxin neutralization performance of the products. That is, it was confirmed that by using the organic acid, the impurity polymyxin B can be efficiently washed without impairing the performance of the product.

[0038]

The amino group-containing physiologically active substance is immobilized on an insoluble carrier, and then impurities contained in the insoluble carrier are removed by using a solution of an organic acid or an aqueous solution of an alkali metal salt thereof, thereby reducing the cost. It can be removed easily and safely.

Claims (6)

[Claims] 1. An insoluble carrier is obtained by immobilizing an amino group-containing physiologically active substance on an insoluble carrier and then washing the insoluble carrier with a solution of an organic acid or an aqueous solution of an alkali metal salt thereof. A method for producing a physiologically active substance-immobilized molded article, which comprises removing impurities to obtain a physiologically active substance-immobilized molded article. 2. The method for producing a physiologically active substance-immobilized molded article according to claim 1, wherein the amino group-containing physiologically active substance is a cyclic peptide antibiotic. 3. The method for producing a physiologically active substance-immobilized molded article according to claim 1, wherein the organic acid is dissolved in an aqueous solvent or an organic solvent. 4. The method for producing a physiologically active substance-immobilized molded article according to claim 1, wherein the organic acid is a carboxylic acid having 8 or more carbon atoms. 5. The method for producing a physiologically active substance-immobilized molded article according to any one of claims 1 to 4, wherein the organic acid is a carboxylic acid having a steroid skeleton. 6. The method for producing a physiologically active substance-immobilized molded article according to claim 1, wherein the impurities are antibiotics that are not immobilized on an insoluble carrier.