JP2000051689A - High pressure steam-sterilizable adsorbent for lipopolysaccharide and method for removing lipopolysaccharide by adsorption - Google Patents

Abstract

PROBLEM TO BE SOLVED: To ensure the effective action regardless of a region, acidic, neutral or basic and a specified ionic strength without the elimination of a ligand and also the stable high pressure steam sterilization and the high conservation stability at low temperature by fixing a chemotherapeutic agent in an insoluble carrier. SOLUTION: A chemotherapeutic agent selected from amipacin and a derivative thereof selected from aminoglycoside antibiotics, hyglomycin B and a derivative thereof selected from aminocyclitol antibiotics and peptide antibiotics with many functional groups such as amikacin and bekamycin which take part in bonding with an insoluble carrier in a single molecule, is fixed in the insoluble carrier such as a polysaccharide resin, e.g. agarose, a plastic, glass or a fiber. The covalent bonding of the carrier with the chemotherapeutic agent is performed by a bromcyan activation process, a cyanuric chloride process or the like. Thus it is possible to obtain the lipopolysaccharide adsorbent which removes effectively a lipopolysaccharide from a solution by adsorption in each of the regions such as acidic, neutral and basic and without the elimination of a ligand with a specified ionic strength and is stable in high pressure steam sterilization and further shows its superior conservation stability at a low temperature.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for efficiently removing adsorption of lipopolysaccharide mixed in a solution, stable in high-pressure steam sterilization and alkali treatment, and capable of being stored for a long time at a low temperature (4 ° C.).
The present invention relates to a lipopolysaccharide adsorbent and a method for adsorbing and removing lipopolysaccharide.

[0002]

2. Description of the Related Art Lipopolysaccharide (hereinafter also referred to as lipopolysaccharide: endotoxin) is a physiologically active substance constituting the cell wall of Gram-negative bacteria, and activates lipid A having a molecular weight of about 2,000 bound to a protein. Is the center of Many studies have been made on the adsorption and removal method of the lipopolysaccharide, and there have been applications such as the adsorption and removal method using chitosan (JP-A-3-109397, JP-A-3-109940, etc.). These adsorption and removal methods have a problem that it is difficult to use them universally because it is difficult to set conditions for adsorption and removal.

[0003] That is, the method of adsorbing and removing nucleic acids and lipopolysaccharides with chitosan is based on ion exchange. However, since the primary amino group of chitosan is weakly basic and dissociates only in the acidic region, in order to remove nucleic acids and lipopolysaccharide from the protein solution by ion adsorption, the pH of the solution must be weakly acidic. I needed to. For this reason, it was not possible to adsorb and remove nucleic acids and lipopolysaccharide from a protein solution that was inactivated in a weakly acidic region.

In order to solve the above problems, the present inventors have invented a lipopolysaccharide adsorbent having an aminoglycoside antibiotic as a ligand and have filed an application (Patent No. 1).
865284). Further, the inventors of the present invention have also invented a method for quantifying lipopolysaccharide, and have filed an application (Patent No. 2).
690415). However, the invention (Patent No. 1)
865284) has a problem that some ligands are desorbed by alkali treatment, high-pressure steam sterilization, or storage at low temperature (4 ° C.).

[0005]

An object of the present invention is to solve the above-mentioned problems. That is, (1) to provide an adsorbent which satisfactorily adsorbs and removes lipopolysaccharide in a solution in an acidic region, a neutral region and a basic region (pH 4.0 to pH 9.0). (2) To provide a stable adsorbent for high-pressure steam sterilization. (3) acidic region, neutral region and basic region (pH 4.
(0 to pH 9.0) with no desorption of ligand. (4) To provide an adsorbent excellent in storage stability at low temperature (4 ° C.). (5) The ionic strength is 0.1 to
It is an object of the present invention to provide an adsorbent that satisfactorily adsorbs and removes lipopolysaccharide in a solution even in the range of 0.6.

[0006]

In order to achieve the above object,
The inventors of the present invention have conducted various studies and found that a carrier on which a chemotherapeutic agent selected from the group consisting of aminoglycoside antibiotics, aminocyclitol antibiotics and peptide antibiotics is immobilized. , (1) acidic region, neutral and basic region (pH 4.0 to pH 9.
In step 0), the lipopolysaccharide in the solution is adsorbed and removed well.
(2) Stable for high-pressure steam sterilization. (3) acidic region, neutral region and basic region (pH 4.0 to pH 9.0)
No elimination of ligand. (4) Excellent storage stability at low temperature (4 ° C.). (5) The ionic strength is 0.1 to
Even in the range of 0.6, the lipopolysaccharide in the solution is adsorbed and removed well. (6) Since the cells are colored, it is easy to distinguish cells such as artificially fertilized egg cells from the carrier, and it has been found that the cells can be easily collected, and the present invention has been completed.

That is, the present invention relates to (1) a lipopolysaccharide adsorbent which is stable to high-pressure steam sterilization and alkali treatment, wherein a chemotherapeutic agent is immobilized on an insoluble carrier.

Further, the present invention provides (2) a chemotherapeutic agent selected from aminoglycoside antibiotics: amikacin and its derivatives, bekanamycin and its derivatives, dibekacin and its derivatives, fradiomycin and its derivatives, kanamycin and its derivatives (1) The derivative selected from the group consisting of derivatives, paromomycin and its derivatives, ribostamycin and its derivatives, sisomycin and its derivatives, tobramycin and its derivatives, geneticin and its derivatives, netermicin and its derivatives. Lipopolysaccharide adsorbent.

Further, the present invention is characterized in that (3) the chemotherapeutic agent is selected from the group consisting of hygromycin B and its derivatives selected from aminocyclitol antibiotics, and spectinomycin and its derivatives. ,
A lipopolysaccharide adsorbent according to (1).

Further, the present invention is characterized in that (4) the chemotherapeutic agent is selected from peptide antibiotics.
A lipopolysaccharide adsorbent according to (1).

The present invention further provides (5) the above (1),
The lipopolysaccharide adsorbent described in (2), (3) and (4) is made pyrogen-free, and further subjected to high-pressure steam sterilization (or 5).
Heating at a temperature of 6 ° C. or more and 90 ° C. or less for 10 hours or more) and then storing at a temperature of 3 ° C. to 10 ° C.

Further, the present invention is characterized in that (6) it is made pyrogen-free and further subjected to high-pressure steam sterilization (or heating at a temperature of 56 ° C. or more and 90 ° C. or less for 10 hours or more) (1), (2), ( A lipopolysaccharide adsorbent according to 3) and (4).

The present invention further provides (7) a method for adsorbing a lipopolysaccharide, which comprises contacting the lipopolysaccharide adsorbent according to the above (6) with a liquid containing the lipopolysaccharide and then separating the two. It relates to the removal method.

Further, the present invention provides (8) adjusting the ionic strength of the lipopolysaccharide adsorbent and the liquid containing the lipopolysaccharide according to the above (6) to 0.1 to 0.6, and then contacting and separating them. Thereby preventing non-specific adsorption of proteins in a liquid containing lipopolysaccharide to the lipopolysaccharide adsorbent.

Further, the present invention relates to (9) the lipopolysaccharide adsorbent described in (1), (2), (3) or (4), which is colored.

[0016]

BEST MODE FOR CARRYING OUT THE INVENTION The lipopolysaccharide adsorbent according to the present invention utilizes the affinity between the chemotherapeutic agent according to the present invention and lipopolysaccharide, and the insoluble carrier used includes:
Polysaccharide type resin (agarose, crosslinked agarose, cellulose, crosslinked cellulose, molded cellulose derivative, dextran gel, etc.), plastic (polyvinyl alcohol, cellulose, polyacrylamide, polyacrylic acid, methacrylic acid, polymethyl methacrylate, polyisopropylacrylamide, polyester) , Polyamide, polyethylene, polypropylene, polystyrene, polysulfone, etc.), glass, ceramic, fiber, paper, synthetic paper, hollow fiber, metal, and the like, but are not limited thereto, and their shapes, There are no restrictions on the surface condition or the like.

The method of covalently bonding to the carrier with the chemotherapeutic agent according to the present invention used in the present invention includes a bromocyan activation method, a cyanuric chloride method, an oxysilane cleavage reaction, a method using the same reactive bivalent reagent, A known method such as a method using a heteroreactive divalent reagent, a method using a photochemical reaction, an amide bond method, an ester bond method, an ether bond method, an amino bond method, an imino bond method, a sulfide bond method, and a disulfide bond method is used. be able to. Further, in order to prevent non-specific adsorption of the protein to the carrier, it is preferable to interpose a spacer such as hexamethylenediamine between the carrier and the chemotherapeutic agent. Epoxylation, reductive amination, thiol activation, and the like can be considered as methods for binding the ligand via the spacer.

The chemotherapeutic agent according to the present invention comprises (1) amikacin and its derivatives, bekanamycin and its derivatives, dibekacin and its derivatives, fradiomycin and its derivatives, kanamycin and its derivatives selected from aminoglycoside antibiotics , Paromomycin and its derivatives, ribostamycin and its derivatives, sisomycin and its derivatives, tobramycin and its derivatives, geneticin and its derivatives, netermicin and its derivatives, (2) hygromycin B selected from among aminocyclitol antibiotics and Derivatives thereof, spectinomycin and its derivatives,
(3) It is preferable to select from peptide antibiotics. The chemotherapeutic agent according to the present invention contains a large number of functional groups (amikacin: NH) involved in binding to a carrier in one molecule.
₂ Number; 4, Bekamaishin: NH ₂ Number; 5, dibekacin: NH ₂ Number; 5, fradiomycin: NH ₂ Number; 5,
Kanamycin: NH ₂ Number; 4, paromomycin: NH ₂
Number; 5, ribostamycin: NH ₂ Number; 4, sisomicin: NH ₂ Number; 4, tobramycin: NH ₂ Number; 5, Polymyxin B: NH ₂ Number; number, colistin: NH
₍₂ ); (a) is stable to high-pressure steam sterilization. (B) acidic regions, neutral and basic regions (pH
(4.0 to pH 9.0) with no elimination of ligand. (C)
There is a feature that the storage stability at low temperature (4 ° C.) is excellent.

The lipopolysaccharide adsorbent according to the present invention is preferably pyrogen-free and then used.
The pyrogen-free condition is 250 ° C or 121 ° C.
Known methods such as a method of heating for a long time at ℃ and a method of washing with alkali can be used. The reason why the lipopolysaccharide adsorbent according to the present invention is made pyrogen-free and then subjected to high-pressure steam sterilization is to prevent the lipopolysaccharide adsorbent from becoming pyrogen-free due to bacterial contamination that may occur after the pyrogen-free. In addition, at least 56 ° C and 90
The reason for heating at a temperature of not more than 10 ° C. for 10 hours or more is to apply to plastics that are weak to heating at high temperature such as polystyrene.

In the lipopolysaccharide adsorbent according to the present invention, the ionic strength of the lipopolysaccharide adsorbent and the liquid containing the lipopolysaccharide is adjusted to 0.1 to 0.6, more preferably 0.3 to 0.6. Thereafter, by contacting and separating the two, it is possible to adsorb the lipopolysaccharide while preventing nonspecific adsorption of the protein in the liquid containing the lipopolysaccharide to the lipopolysaccharide adsorbent. Under the ionic strength conditions, chitosan cannot adsorb lipopolysaccharide at all.

The lipopolysaccharide adsorbent according to the present invention is also characterized by being colored. The carrier of the lipopolysaccharide adsorbent,
In the case of gel, polystyrene Petri dishes, centrifuge tubes, test tube plastic pieces, etc., coloring the carrier makes it easy to distinguish the carrier from cells such as artificially fertilized egg cells for the purpose of adsorbing and removing lipopolysaccharide. In addition, cells can be easily collected.

[0022]

The present invention will be described in more detail with reference to examples.
The present invention is not limited at all by the examples. << Example 1 >> Preparation of Amikacin-Sepharose Cross-linked agarose carrier (Sepharose CL-4)
B: Pharmacia) 50 ml and 2.3 g of BrC
N / 50 ml of an aqueous solution and kept in a 4 ° C. water bath. Next, the solution was added at a temperature of 10 ° C. ± 2 ° C.
A 5N NaOH solution of H11.0 ± 0.2 was slowly added dropwise (over 10 minutes) to activate the cross-linked agarose carrier. Next, on the Buchner funnel, the activated cross-linked agarose carrier was sufficiently washed with sterilized purified water, and sufficiently drained. 10 g of the activated crosslinked agarose carrier is added to a coupling buffer (0.1 M NaHCO ₃ , 0.5 MN
Amicacin (manufactured by Wako Pure Chemical Industries, Ltd .: 1.aCl pH8.3) was dissolved in 20 ml of the coupling buffer.
08/20 ml) solution and reacted at room temperature for 5 hours. After 5 hours, 0.2 M glycine / Tris-HCl buffer (pH 8.0) was added to the suspension to inactivate unreacted active groups. The suspension is transferred onto a Buchner funnel, and the cross-linked agarose carrier is thoroughly washed with an acetate buffer (pH 4.0), and further thoroughly washed with sterile purified water, and then purified with Amikacin-Sepharose CL. -4B was obtained.

<< Embodiment 2. --- High pressure steam sterilization of Amikacin-Sepharose Amikacin-Sepharose CL prepared in Example 1
After making -4B (hereinafter, gel) pyrogen-free,
The vial was filled and autoclaved at 121 ° C. for 20 minutes. After the gel returned to room temperature, the gel was suction filtered and dried. A part of the dried gel was taken, and the total protein was quantified by the Kjeldahl method. Similarly, the operation of high-pressure steam sterilization and the operation of quantification of the total protein amount were repeated 10 times. As a result, until the tenth high-pressure steam sterilization operation, almost no decrease in the total protein amount of the gel was observed. Also,
Up to the tenth high-pressure steam sterilization operation, almost no decrease was observed in the lipopolysaccharide adsorption capacity of the gel. Further, when the autoclaved gel was stored refrigerated at 4 ° C., almost no ligand was detached from the autoclaved gel for at least one year.

<< Comparative Example 1. ————————————————————————————————————————————————————————————————————————————————————————————————————————————————————————————————————————————————————————————————————
The solution was autoclaved at 1 ° C. for 20 minutes. After the gel returned to room temperature, the gel was suction filtered and dried. A part of the dried gel was taken, and the total protein was quantified by the Kjeldahl method. Similarly, the operation of high-pressure steam sterilization and the operation of quantification of the total protein amount were repeated four times. As a result, 13% of the amount of protein bound to the gel (amount of streptomycin) was released in the first operation of the high-pressure steam sterilization, and 4% of the amount of protein bound to the gel (amount of streptomycin) was released in the second operation of the high-pressure steam sterilization. At the third autoclaving operation, 9% of the amount of protein bound to the gel (streptomycin amount) was released, and at the fourth autoclaving operation, 4% of the protein amount (streptomycin amount) bound to the gel was released. From the above results, it was shown that streptomycin-sepharose does not withstand high-pressure steam sterilization. In addition, for streptomycin-sepharose, which is not subjected to autoclaving operation,
When stored refrigerated at ℃, one month after storage, about 5% of the amount of protein (the amount of streptomycin) bound to the gel was released. After 3 months of storage, about 10% of the amount of streptomycin bound to the gel was released. Therefore, it was shown that streptomycin-Sepharose does not withstand refrigerated storage at 4 ° C.

<< Comparative Example 2. Gentamicin-Sepharose CL-4B (hereinafter, gel) prepared according to the method described in Example 1 was filled in a vial, and high-pressure steam was applied at 121 ° C. for 20 minutes. Sterilized.
After the gel returned to room temperature, the gel was suction filtered and dried. A part of the dried gel was taken, and the total protein was quantified by the Kjeldahl method. Similarly, the operation of high-pressure steam sterilization and the operation of quantification of the total protein amount were repeated four times. As a result, 8% of the amount of protein (amount of gentamicin) bound to the gel during the first high-pressure steam sterilization operation was released, and the amount of the protein (amount of gentamicin) bound to the gel during the second high-pressure steam sterilization operation was released.
Was released, and 6% of the amount of protein (amount of gentamicin) bound to the gel in the third autoclaving operation was released, and 2% of the amount of protein (amount of gentamicin) bound to the gel in the fourth autoclaving operation. % Was released. From the above results, it was shown that gentamicin-sepharose does not withstand high-pressure steam sterilization.

<< Embodiment 2. Preparation of Kanamycin-Cellulose Cellulose beads (manufactured by Chisso: Cellulofine GC-
700) was sufficiently washed on a glass filter using sterile purified water. By suction filtration, 10 ml of 1,4-butanediol glycidyl ether (manufactured by Aldrich) and 10 ml of a 0.6 M NaOH solution containing 10 mg of NaBH ₄ were added to 10 g of the cellulose beads which had been sufficiently drained. Shaking, stirring,
Cellulofine into which 1.5 mmol of epoxy groups had been introduced was obtained. After 10 g of the epoxy group-introduced Cellulofine, which had been sufficiently drained by suction filtration, was suspended in 10 ml of a coupling buffer (1 M Na ₂ CO ₃ solution),
A solution of kanamycin (manufactured by Wako Pure Chemical Industries, Ltd .: 2.0 g / 20 ml) dissolved in the coupling buffer was added, and the solution was added at room temperature.
The reaction was performed for 4 hours. After 24 hours, a 1 M ethanolamine solution was added to the suspension to inactivate unreacted active groups. The amount of kanamycin introduced was 3 mg / ml · gel
Met. The kanamycin-cellulose (hereinafter, gel) was filled in a vial and sterilized by high pressure steam at 121 ° C. for 20 minutes. After the gel has returned to room temperature, the gel is suction filtered,
Dried. A part of the dried gel was taken, and the total protein was quantified by the Kjeldahl method. Similarly, the operation of high-pressure steam sterilization and the operation of quantification of the total protein amount were repeated 10 times. As a result, the liberated amount of protein (amount of kanamycin) bound to the gel by the 10th high pressure steam sterilization operation was 1% or less. From the above results, kanamycin-sepharose was shown to be resistant up to the 10th autoclaving. Further, when the autoclaved gel was stored refrigerated at 4 ° C., almost no ligand was detached from the autoclaved gel for at least one year.

Embodiment 3 ------------------------------------------------------------------------------------------------------------------------------------------------------- That is, 0.1% bovine serum albumin (BSA) and 1%
A 0.1 N phosphate buffer (pH 7.2: ionic strength 0.3) containing 0 ng / ml of lipopolysaccharide was applied and allowed to flow at a flow rate of 1 ml / 5 min. The effluent was fractionated by 1 ml, and the concentration of lipopolysaccharide was quantified using Endospecy (manufactured by Seikagaku Corporation) for up to the 20th fraction. As a result, no lipopolysaccharide was detected from any of the fractions. Therefore, the kanamycin-cellulose gel exhibited a sufficient adsorption activity even under the condition of ionic strength 0.3. Also,
The amount of bovine serum albumin adsorbed on the gel under the ionic strength conditions (ionic strength 0.3) was 5% or less.

<< Embodiment 4. »−− Adsorption test of lipopolysaccharide by kanamycin-cellulose and streptomycin-cellulose (effect of pH fluctuation) After filling a column with 10 ml of kanamycin-cellulose prepared by the method described in Example 2 and making it pyrogen-free, A lipopolysaccharide adsorption test was performed. That is, an acetate buffer (pH 4.0) containing 10 ng / ml lipopolysaccharide was applied to the column, and the flow rate was 1 ml / 5 min.
Flowed down. The effluent was fractionated by 1 ml, and the concentration of lipopolysaccharide was quantified using Endospecy (manufactured by Seikagaku Corporation) for up to the 20th fraction. As a result, no lipopolysaccharide was detected from any of the fractions. For five separately packed kanamycin-cellulose packed columns, the pH of the buffer was adjusted to 5.0, 7.0, 7.8, 8.2, and 9.
The above-mentioned adsorption test was carried out by changing to 0. As a result, the adsorption rate of lipopolysaccharide on these columns was pH 5.0 (10
0%), pH 6.8 (90%), pH 7.4 (94
%), PH 8.0 (92%), and pH 9.0 (88%: the adsorption rate at pH 4.0 was 100% for all).
Next, a similar test was performed using streptomycin-cellulose prepared according to the method described in Example 2. As a result, the lipopolysaccharide adsorption rate of the streptomycin-cellulose packed column was adjusted to pH 5.0 (100
%), PH 6.8 (96%), pH 7.4 (94%),
pH 8.0 (85%), pH 9.0 (45%: p
H4.0 was taken as 100%). Therefore, the adsorption rate of the lipopolysaccharide of the column packed with streptomycin-cellulose was hardly different from that of the column packed with kanamycin-cellulose up to pH 8.0, but in the pH region higher than pH 9.0, the adsorption rate of lipopolysaccharide was lower. It was shown that the adsorptive activity of was greatly reduced.

Embodiment 5 FIG. Preparation of Bekamycin-Binding Petri Dish by Photochemical Reaction a) Synthesis of Azidostyrene 5 g of 3-nitrostyrene was suspended in 20 ml of a mixed solution of ethanol and concentrated hydrochloric acid, and SnC dissolved in ethanol was further dissolved.
l ₃ 2H ₂ O solution was added with vigorous stirring, at room temperature overnight, and allowed to react. The mixture was neutralized with NaOH, the solid component was separated by filtration, and the product was extracted with ether from the filtrate. After the ether layer was dried over MgSO ₄ , concentrated sulfuric acid was added to obtain an intermediate. The intermediate was dissolved in 10 ml of a 10% sulfuric acid solution, cooled with ice, and a 1N NaNO ₂ solution was added. After 2 hours, NaN
₃ aqueous solutions were added, and after returning to room temperature, it stirred for 3 hours. Extract with ethyl acetate and extract the solution with 0.1N NaHCO
Washed with ₃ aqueous solutions and purified water, added MgSO ₄ and dried. The solvent is distilled off, and chloroform / hexane (1/4)
It was dissolved in a mixed solvent and purified with a silica gel column. The solvent was distilled off to obtain 3-azidostyrene. b) Synthesis of azidostyrene-styrene copolymer One mole of azidostyrene and 4 moles of styrene were diluted 5-fold with benzene, and 0.01 equivalent of N, N'-azobisisobutyronitrile (AIBN) was added. , Degas, seal tube, 6
Polymerization was performed at 0 ° C. for 4 hours. After standing to cool, pour into methanol,
The precipitated azidostyrene-styrene copolymer was recovered. The number average molecular weight of the obtained copolymer was 100,000. c) Preparation of Petri Dish Covalently Linked with Bekamycin The azidostyrene-styrene copolymer synthesized by the above method was dissolved in acetone to prepare a 1% solution. This solution was applied to each well of an 8-well microplate for cell culture (manufactured by Becton Dickinson) and dried to form a film of an azidostyrene-styrene copolymer. Fill the wells of the treated microplate for cell culture with a 1% bekamycin solution in a phosphate buffer solution, allow to stand for 1 hour, adsorb the bekamycin, and emit 1 ultraviolet ray using a high pressure mercury lamp. Irradiated for minutes. Next, each well of the cell culture microplate was washed with purified water and dried. The entire microplate for cell culture (including the lid) is placed in a 0.
It was immersed in a 2M NaOH / 40% ethanol aqueous solution and allowed to stand at room temperature for 16 hours. Sixteen hours later, the cell culture microplate was taken out of the aqueous ethanol solution under a sterile environment, shaken, drained, and then immersed in pyrogen-free water. Subsequently, in an aseptic environment, the operation of immersing in pyrogen-free water, taking out, shaking, and draining water was repeated 10 times. Next, the microplate for cell culture was dried under a sterile environment, and the same procedure was followed to cover the cell with a pyrogen-free lid, and filled in a sterile bag. Finally, the sterile bag was stored in a dryer at 65 ° C. for 24 hours, cooled to room temperature, and refrigerated at 4 ° C.

Embodiment 6 FIG. Preparation of non-woven fabric bound with fradiomycin by photochemical reaction A terpolymer of 2- (4-azidobenzoyloxy) ethyl methacrylate: styrene: O-nirobenzyl acrylate (charged molar ratio of 3: 6:
1) (hereinafter PASN) was synthesized. The PASN is applied to a non-woven fabric (Sonara 8005) manufactured by DuPont.
After forming the thin film, ultraviolet rays were irradiated. Next, the nonwoven fabric was immersed in a phosphate buffer solution containing 0.1% fradiomycin and water-soluble carbodiimide, and reacted at 37 ° C. for 16 hours. Next, the nonwoven fabric was washed and dried with a 1 M aqueous sodium chloride solution and a phosphate buffer. The nonwoven fabric was made pyrogen-free according to the method of Example 5 and filled in a sterile bag. Finally, the sterile bag was stored in a dryer at 65 ° C. for 24 hours, cooled to room temperature, and refrigerated at 4 ° C.

Embodiment 7 FIG. Preparation of colistin covalently bonded microput Glutaraldehyde solution (electron microscope grade)
It was dissolved in a 7.4 phosphate buffer and adjusted to a concentration of 2%. 200 μl of the glutaraldehyde solution was added to each well of an amino group-fixed micropout (manufactured by Sumibe Medical).
The mixture was dispensed one by one and reacted at room temperature for 2 hours. Next, each well of the microplate was washed twice with purified water. Next, 200 μl of a colistin solution (manufactured by Wako Pure Chemical Industries, Ltd.) dissolved in a phosphate buffer of pH 7.4 and adjusted to a concentration of 1% was dispensed into each well of the micropout at a temperature of 4 ° C. overnight. And reacted. Next, pH7.
A solution of bovine serum albumin (manufactured by Wako Pure Chemical Industries, Ltd.) dissolved in a phosphate buffer solution of No. 4 and adjusted to a concentration of 1% was dispensed at 200 μl each and reacted at room temperature for 4 hours. Next, each well of the microplate was washed twice with purified water. Next, the microplate was dried under a sterile environment to make it pyrogen-free, and then filled in a sterile bag. Finally, put the sterile bag in 6
It was stored in a dryer at 5 ° C. for 24 hours, cooled to room temperature, and refrigerated at 4 ° C.

Embodiment 8 FIG. << Adsorption Removal of Lipopolysaccharide from Artificial Fertilized Egg Cells Using Colistin Covalently-Coupled Micropute >> A pyrogen was added to all the holes of the colistin-covalently-coupled micropout prepared in accordance with the method of Example 7 and colored blue in its entirety. Free phosphate buffer (pH 7.2) for 20
The solution was dispensed by 0 μl. Artificial fertilized egg cells (lipopolysaccharide content of suspension: 10 pg / ml)
One was placed and incubated for 3 minutes. After 3 minutes, the artificially fertilized egg cells were transferred to another well of the microplate. This operation was performed 20 times in total. All the operations were performed in an ice water bath in a clean bench, and when transferring the artificially fertilized egg cells, the operation was performed while minimizing the contamination of the phosphate buffer. As a result, no lipopolysaccharide was detected from the phosphate buffer in the well (well) after the tenth transfer of the artificial fertilized egg cells (lipopolysaccharide content: 0.1 pg / m2).
l or less).

Example 9 Preparation of polymyxin B covalently bound microplot 1-ethyl-3 (3-diethylaminop)
ropyl) carbodiimide hydroc
120 mg of chloride (ECD) was dissolved in purified water of pH 4.5 (reagent grade). The ECD solution was dispensed into each well of a carboxyl group-immobilized micropout (manufactured by Sumibe Medical) in an amount of 200 μl, and reacted at room temperature for 2 hours. Next, each hole of the microplate was washed five times with purified water. Next, in each hole of the microput,
200 μl of a polymyxin B (manufactured by Wako Pure Chemical Industries, Ltd.) solution dissolved in a phosphate buffer of pH 7.4 and adjusted to a concentration of 1% was prepared.
The mixture was dispensed one at a time and reacted at 4 ° C. overnight. Next, 200 μl of a bovine serum albumin (manufactured by Wako Pure Chemical Industries, Ltd.) solution dissolved in a phosphate buffer of pH 7.4 and adjusted to a concentration of 1% was dispensed into each well of each of the microplates at room temperature. The reaction was performed for 4 hours. Next, each well of the microplate was washed twice with purified water. Next, the microplate was dried under a sterile environment to make it pyrogen-free, and then filled in a sterile bag. Finally, the sterile bag was stored in a dryer at 65 ° C. for 24 hours, cooled to room temperature, and refrigerated at 4 ° C.

Embodiment 10 Preparation of Tobramycin-Binding Beads Glutaraldehyde solution (electron microscope grade)
It was dissolved in a phosphate buffer solution of 7.4 and adjusted to a concentration of 12.5%. 10 ml of the glutaraldehyde solution and 50 hydrazide beads (manufactured by Pierce) were placed in a sterilized centrifuge tube having a volume of 50 ml, and the mixture was gently shaken at room temperature for 2 hours. The hydrazide beads were transferred to a Buchner funnel, washed with 200 ml of purified water, and furthermore, 0.1 M of pH 6.0.
Washed with 50 ml of phosphate buffer. Next, 5 mg of tobramysiso was added to 10 ml of a 0.1 M phosphate buffer at pH 6.0.
Was prepared. Fifty hydrazide beads thus treated were collected in a sterilized centrifuge tube having a volume of 50 ml,
Further, the above-mentioned tobramycin solution and 2 mg of sodium cyanoborohydride were added, and the mixture was gently shaken at room temperature overnight. Transfer the hydrazide beads to a Buchner funnel,
Wash with 200 ml of 0.1 M phosphate buffer of pH 6.0, and further add 10 M of 0.1 M sodium carbonate solution of pH 6.0.
Washed with 0 ml. The tobramycin-bound hydrazide beads were immersed in a 0.2 M NaOH / 40% ethanol aqueous solution and allowed to stand at room temperature for 16 hours. After 16 hours, the tobramycin-bound hydrazide beads were removed from the aqueous ethanol solution under a sterile environment, shaken and drained,
It was immersed in pyrogen-free water. Then, in a sterile environment, immerse in pyrogen-free water, remove, shake,
The operation of draining was repeated 10 times. Next, the tobramycin-bound hydrazide beads were immersed in a vial filled with a pyrogen-free 40% ethanol aqueous solution, stored in a constant-temperature water bath at 65 ° C for 24 hours, cooled to room temperature, and refrigerated at 4 ° C.

Embodiment 11 FIG. Preparation of Kanamycin-Binding Hollow Fiber Membrane A method such as a raft (Akio Kishida, Yoshito Raft: artificial organ,
17: 47-50, 1988), a dialyzer (MC120) to which kanamycin was bound via polyethylene glycol was prepared. 0.2M NaOH /
The inside of the dialyzer was filled with a 40% aqueous ethanol solution and circulated at room temperature for 16 hours. Sixteen hours later,
Pyrogen-free water was circulated in an amount 10 times the volume of the dialyzer. Next, a 0.2 M acetic acid solution was circulated three times the volume of the dialyzer. Next, 10 times as much pyrogen-free water as the dialyzer volume was circulated. Next, the dialyzer was placed in a high-pressure steam sterilization bag, and subjected to high-pressure steam sterilization at 121 ° C. for 20 minutes. After high-pressure steam sterilization, 10 times the volume of the dialyzer was circulated with pyrogen-free water. The pyrogen-free water was concentrated 10-fold and the total protein was quantified by the Kjeldahl method (according to a comparative example). As a result, no protein was detected from the pyrogen-free water. Therefore, kanamycin was not released from the kanamycin-bound hollow fiber membrane. In addition, the kanamycin-bound hollow fiber membrane did not show a decrease in lipopolysaccharide adsorption activity even after high-pressure steam sterilization. Therefore, the kanamycin-bound hollow fiber membrane was resistant to autoclaving.

Embodiment 12 FIG. Preparation of spectinomycin-bound flat membrane Cuprophan membrane (EN) regenerated by cuprammonium method
KA product) into a test tube with a stopper, 20 ml of spectinomycin phosphate buffer (concentration: 10 mg / ml) and 0.1 ml.
mol of Ce ²⁺ nitric acid aqueous solution (1 ml) was added.
Next, argon gas was blown into the test tube with a stopper for 15 minutes. Next, the test tube with the stopper was placed in a thermostat at 40 ° C. for 1 hour.
Allowed to react for hours. One hour later, the membrane was immersed in 10 times the volume of the cuprophan membrane for 2 hours in pyrogen-free water. Next, the cuprofan film was modularized.
Next, the inside of the module was filled with a 0.2 M NaOH / 40% ethanol aqueous solution and circulated at room temperature for 16 hours. After 16 hours, 10 times the volume of the module was circulated with pyrogen-free water. Next, a 0.2 M acetic acid solution was circulated three times the volume of the module. Next, 10 times the volume of the module was circulated with pyrogen-free water. Next, the module was placed in a high-pressure steam sterilization bag and subjected to high-pressure steam sterilization at 121 ° C. for 20 minutes. After autoclaving, 10 times the volume of the module was circulated with pyrogen-free water. The pyrogen-free water is concentrated 10-fold and the Kjeldahl method (according to the comparative example)
Was used to determine the total protein amount. As a result, no protein amount was detected from the pyrogen-free water. Therefore, no spectinomycin was released from the spectinomycin-coupled flat membrane. The spectinomycin-bound flat membrane did not show a decrease in lipopolysaccharide adsorption activity even after high-pressure steam sterilization. Therefore, the spectinomycin-conjugated flat membrane was resistant to autoclaving.

[0037]

The lipopolysaccharide adsorbent according to the method of the present invention comprises:
(1) acidic region, neutral region and basic region (pH 5.0 to 5.0)
(pH 12.0), it is possible to satisfactorily adsorb and remove lipopolysaccharide in the solution. (2) It is a stable adsorbent for high-pressure steam sterilization. (3) acidic region, neutral and basic region (p
Lipopolysaccharide adsorbent which has no elimination of ligand at H4.0-pH9.0). (4) A lipopolysaccharide adsorbent having excellent storage stability at low temperatures (4 ° C.). (5) The ionic strength is 0.
It is a lipopolysaccharide adsorbent that can satisfactorily adsorb and remove lipopolysaccharide in a solution even in the range of 1 to 0.6.

 ──────────────────────────────────────────────────続 き Continued on the front page F term (reference) 4C077 AA09 AA26 BB03 KK09 KK13 KK21 MM06 NN20 PP02 PP08 PP09 PP10 PP12 PP13 PP15 PP24 PP29 4D017 AA11 BA07 CA11 CA14 CB03 CB05 DA01 DB03 DB10 EA01 EB10 AB03AB ABA AB26C AB27A AB27B AB30A AC01C AC02C AC06C AC11C AC14C AC17C AC35C AE19C AE20B CA56 FA07 FA34 FA35 FA40

Claims (9)

[Claims] 1. A lipopolysaccharide adsorbent which is stable to high-pressure steam sterilization and alkali treatment, wherein a chemotherapeutic agent is immobilized on an insoluble carrier. 2. Amikacin and its derivatives, bekanamycin and its derivatives, dibekacin and its derivatives, fradiomycin and its derivatives, kanamycin and its derivatives, paromomycin and its derivatives, wherein the chemotherapeutic agent is selected from aminoglycoside antibiotics Ribostamycin and its derivatives, sisomycin and its derivatives, tobramycin and its derivatives,
The lipopolysaccharide adsorbent according to claim 1, wherein the lipopolysaccharide adsorbent is selected from the group consisting of geneticesin and its derivatives, netermicin and its derivatives. 3. The method according to claim 1, wherein the chemotherapeutic agent is selected from the group consisting of hygromycin B and its derivatives, spectinomycin and its derivatives selected from aminocyclitol antibiotics. Lipopolysaccharide adsorbent. 4. The lipopolysaccharide adsorbent according to claim 1, wherein the chemotherapeutic agent is selected from peptide antibiotics. 5. The lipopolysaccharide adsorbent according to claim 1, 2, 3, or 4 is made pyrogen-free, and further subjected to high-pressure steam sterilization (or at a temperature of 56 ° C. to 90 ° C. for 10 hours or more). Heating) and then storing at a temperature of 3 ° C to 10 ° C. 6. The composition according to claim 1, wherein the composition is pyrogen-free and further subjected to high-pressure steam sterilization (or heating at a temperature of 56 ° C. or more and 90 ° C. or less for 10 hours or more).
The lipopolysaccharide adsorbent according to claim 3 or 4. 7. A method for adsorbing and removing lipopolysaccharide, comprising bringing a lipopolysaccharide adsorbent according to claim 6 into contact with a lipopolysaccharide-containing liquid and then separating the two. 8. The lipopolysaccharide adsorbent according to claim 6, and a lipopolysaccharide-containing liquid having an ionic strength of 0.1 to 0.6.
And then contacting and separating the two to prevent non-specific adsorption of proteins in the liquid containing lipopolysaccharide to the lipopolysaccharide adsorbent. 9. The method according to claim 1, wherein the coloring is performed.
The lipopolysaccharide adsorbent according to claim 2, 3, or 4.