JP2000189792A - Adsorbent of lipopolysaccharide and adsorbing and removing method of lipopolysaccharide - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an adsorbent of lipopolysaccharides which has high adsorption activity and which is stable against high pressure vapor sterilization and alkali treatment by fixing a chemotherapeutic to an insoluble carrier through a hydrophilic spacer arm having a specified number average mol.wt. SOLUTION: The adsorbent is prepared by fixing a chemotherapeutic to an insoluble carrier through a hydrophilic spacer arm having 300 to 5×106 number average mol.wt. The insoluble carrier used is, for example, a polysaccharide resin, plastics, glass, ceramics, fiber, paper, synthetic paper, hollow fiber, metal or the like. The hydrophilic spacer arm is, for example, polyethylene glycol and its derivs., polypropylene glycol and its deriv., polyacrylamide and its derivs., or the like. Further, the chemotherapeutic is preferably selected from aminoglycoside antibiotics, aminocyclitol antibiotics and peptide antibiotics.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a lipopolysaccharide adsorbent having high adsorption activity for lipopolysaccharide mixed in a solution, stable to 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 method for removing lipopolysaccharide by adsorption.

[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 applied for the 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.). Further, in the invention, when the chain length of the spacer arm bound to the carrier is short (the molecular weight of the spacer arm is about 100 to 200), a large amount of the ligand bound to the carrier due to steric hindrance, on the contrary, There is a problem that the adsorption activity of polysaccharide is significantly reduced.

[0005]

An object of the present invention is to solve the above-mentioned problems. That is, (1) to provide an adsorbent having a high lipopolysaccharide adsorption activity. (2) To provide a stable adsorbent for high-pressure steam sterilization. (3) To provide an adsorbent without desorption of a ligand in an acidic region, a neutral region and a basic region (pH 4.0 to pH 9.0). (4) To provide an adsorbent having excellent storage stability at a low temperature (4 ° C.). (5) To provide an adsorbent that satisfactorily adsorbs and removes lipopolysaccharide in a solution even when the ionic strength is in the range of 0.1 to 0.6. (6) An object of the present invention is to provide an adsorbent that can be easily distinguished from a carrier such as an artificially fertilized egg cell by coloring.

[0006]

In order to achieve the above object,
The inventors of the present invention have conducted various studies and found that a chemotherapeutic agent selected from the group consisting of aminoglycoside antibiotics, aminocyclitol antibiotics and peptide antibiotics has a number average molecular weight of 300. ~ 5,0
A carrier immobilized on an insoluble carrier via a 000 hydrophilic spacer arm provides (1) an adsorbent having high lipopolysaccharide adsorption activity. (2) Stable for high-pressure steam sterilization. (3) No elimination of the ligand in the acidic region, neutral region and basic region (pH 4.0 to pH 9.0). (4) Excellent storage stability at low temperature (4 ° C.). (5) To satisfactorily adsorb and remove lipopolysaccharide in a solution even when the ionic strength is in the range of 0.1 to 0.6. (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 compound having a number average molecular weight of 3
The present invention relates to a lipopolysaccharide adsorbent which is stable to high-pressure steam sterilization and alkali treatment, wherein a chemotherapeutic agent is immobilized on an insoluble carrier via a hydrophilic spacer arm of from 00 to 5,000,000.

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. ,
The present invention relates to the lipopolysaccharide adsorbent according to (1).

The present invention further relates to (4) the lipopolysaccharide adsorbent according to (1), wherein the chemotherapeutic agent is a peptide antibiotic.

The present invention further provides (5) a method of adsorbing a lipopolysaccharide, which comprises contacting the lipopolysaccharide adsorbent according to the above (1) with a liquid containing the lipopolysaccharide and then separating the two. It relates to the removal method.

Further, the present invention provides (6) the above (1),
After making the lipopolysaccharide adsorbent described in (2), (3) and (4) pyrogen-free, further sterilize it by high-pressure steam or heat it at a temperature of 56 ° C. or more and 90 ° C. or less for 10 hours or more and store it. About the method.

[0013] The present invention further relates to (7) the lipopolysaccharide adsorbent according to the above (1), (2), (3) or (4), which is colored.

[0014]

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 binding the chemotherapeutic agent of the present invention to a carrier 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 overcome the problem of steric hindrance, which inhibits the adsorption of the lipopolysaccharide and the chemotherapeutic agent bound to the carrier, the number average molecular weight between the carrier and the chemotherapeutic agent is 300 to 5, It is preferred to have between 1,000,000 hydrophilic spacer arms. Epoxylation, reductive amination, thiol activation, and the like can be considered as methods for binding the ligand via the spacer arm.

The number average molecular weight is from 300 to 5,000,000.
As the hydrophilic spacer arm of No. 00, polyethylene glycol and its derivatives, polypropylene glycol and its derivatives, polyacrylamide and its derivatives, polyethyleneimine and its derivatives, poly (ethylene oxide) and its derivatives, poly (ethylene terephthalic acid) and its Derivatives, poly (ethylene vinyl acetic acid) and its derivatives, poly (3-hydroxybutyric acid) and its derivatives, poly L-amino acids and its derivatives, polyvinyl pyrrolidone and its derivatives, polysiloxane and its derivatives, and the like, Crosslinked products are also included. Number average molecular weight is 300-5,000,
However, the present invention is not limited to these as long as there are 000 hydrophilic compounds.

At present, Affigel 102 is a commercially available carrier for affinity chromatography in Japan.
(Manufactured by BIO-RAD), Affigel 601 (BIO-RAD).
RAD), IMMOBILIZED DIAMIN
ODIPROPYLAMINEGEL (PIERCE
CHEMICAL COMPANY), Immun
opure EPOXY-ACTIVATED AGA
ROSE (PIERCE CHEMICAL COMP
ANY), IMMOBILIZED TNB TH
IOL (PIERCE CHEMICAL COMPA
NY company), POLYSTYRENE HYDRAID
E BEADS (PIERCE CHEMICAL C
OMPANY), TRESYL ACTIVAT
ED AGAROSE (PIERCE CHEMICA
L Company), REACT GEL (PI
ERCE CHEMICAL COMPANY),
UltraLink Biosupport Medi
um (PIERCE CHEMICAL COMPAN
Y company), Eupergit C (EPCELASTIN)
PRODUCTS COMPANY, INC.), T
There are SK gel AFC type (manufactured by Tosoh Corporation), Cellulofine (manufactured by Chisso), Sepharose (manufactured by Pharmacia) and the like. It cannot be overcome and high adsorption activity cannot be realized.

The inventors of the present invention devised that a spacer arm of an appropriate length should be interposed between a carrier and a chemotherapeutic agent in order to overcome the problem of steric hindrance. The inventors have found that a hydrophilic spacer arm having a molecular weight of 300 to 5,000,000 can easily solve the problem, and have completed the present invention. The number average molecular weight of the spacer arm is preferably from 300 to 5,000,000, and more preferably from 300 to 8,000, which can be made into an aqueous solution.

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, geneticesin and its derivatives, netermicin and its derivatives, (2) hygromycin B selected from 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 Number; for holding a large number), (a) a high adsorption activity of lipopolysaccharide. (B) It is stable to high-pressure steam sterilization. (C) acidic region, neutral region and basic region (pH 4.0 to pH 9.
There is no elimination of ligand in 0). (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 further subjected to high-pressure steam sterilization is to prevent the lipopolysaccharide adsorbent from becoming pyrogen-free due to bacterial contamination that may occur after being made pyrogen-free. In addition, at least
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 non-specific 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 tubes, plastic pieces, etc., by coloring the carrier,
It is easy to discriminate cells such as artificial fertilized egg cells and the like for the purpose of adsorption and removal of lipopolysaccharide from the carrier, and cells can be easily collected.

In order to remove lipopolysaccharide in a lipopolysaccharide-containing solution or lipopolysaccharide in a cell solution such as an artificially fertilized egg cell, the lipopolysaccharide adsorbent according to the present invention is brought into contact with the solution and then solidified. By separating the liquid, a lipopolysaccharide-free solution or cells such as lipopolysaccharide-free artificial fertilized egg cells can be obtained. The contacting method may be such that the lipopolysaccharide adsorbent according to the present invention is suspended and mixed in a lipopolysaccharide-containing solution and then filtered to obtain a lipopolysaccharide-free solution, or the lipopolysaccharide adsorbent according to the present invention is applied to a column. By packing and preparing a lipopolysaccharide adsorption column and passing the lipopolysaccharide-containing solution through the column, a lipopolysaccharide-free solution can be obtained as an eluate. Further, to remove lipopolysaccharide in a cell solution such as an artificially fertilized egg cell, the lipopolysaccharide adsorbent according to the present invention is suspended and mixed in a lipopolysaccharide-containing cell suspension solution, and then filtered to remove lipopolysaccharide-free cells. Obtain a suspension solution, or
A lipopolysaccharide-free cell is placed in a well of a lipopolysaccharide-adsorbing microplate according to the present invention, and a lipopolysaccharide-containing cell suspension is put into the well, left for a certain period of time, and then transferred to another well. A suspension solution can be obtained.

[0024]

The present invention will be described in more detail with reference to examples.
The present invention is not limited at all by the examples. << Embodiment 1. Preparation of Amikacin-Sepharose 50 ml of a cross-linked agarose carrier (Sepharose CL-4B: manufactured by Pharmacia) and 4.6 g of bromocyan /
A 100 ml aqueous solution was mixed and kept in a 4 ° C. water bath.
Next, the solution was adjusted to pH 1 while maintaining the temperature at 10 ° C. ± 2 ° C.
100 ml of 1.0 ± 0.2 5N NaOH solution 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 1,000 ml of sterilized purified water, and sufficiently drained. 50 ml of the activated crosslinked agarose carrier is coupled with a coupling buffer (0.2 M sodium bicarbonate, 0.5 M sodium chloride, pH 9.0).
After suspending in 100 ml, a solution of polyethylene glycol bisamine (number average molecular weight: 2,000: Wako Pure Chemical Industries, Ltd .: 1.0 g / 100 ml) dissolved in the coupling buffer was added, and reacted at 4 ° C. for 24 hours. Was. After 24 hours, the suspension was transferred onto a Buchner funnel and the above-mentioned Couplinda buffer (0.2 M sodium bicarbonate, 0.15 M
The pH was returned to neutral by washing with 1,000 ml of sodium chloride (pH 9.0) and 2,000 ml of distilled water for injection. Next, after suspending the polyethylene glycol bisamine-bonded cross-linked agarose carrier in 50 ml of distilled water for injection, 60 ml of dimethylformamide and 20 ml of succinic anhydride 20
g, and reacted at room temperature for 12 hours.
After 12 hours, the suspension was transferred onto a Buchner funnel, and 200 ml of dimethylformamide and 1,1 of distilled water for injection were used.
Washed with 000 ml. Next, 0.1 mol of the polyethylene glycol bisamine-bonded cross-linked agarose carrier was added.
/ L of 4-morpholinoethanesulfonic acid solution (pH 4.
8) After suspending in 100 ml, 2 g of water-soluble 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride was added and reacted at room temperature for 30 minutes to activate the carboxyl group. Next, the polyethylene glycol bisamine-bonded carboxyl group-activated crosslinked agarose carrier was transferred onto a Buchner funnel, and ice-cooled 0.1 mol / L of 4 mol / L was added.
Morpholinoethanesulfonic acid solution (pH 4.8) 20
Washing with 0 ml removed excess carbodiimide. Next, the polyethylene glycol bisamine-bonded carboxyl group-activated crosslinked agarose carrier was added to an amikacin-containing (concentration: 10 mg / ml) coupling buffer (0.2%).
M sodium bicarbonate, 0.15M sodium chloride pH
8.3) Suspended in 200 ml and reacted at room temperature for 24 hours. After 24 hours, the suspension was transferred onto a Buchner funnel and a 0.15 mol / L sodium chloride solution
Washed with 0 ml and 1,000 ml of distilled water for injection.
The amikacin-bonded cross-linked agarose carrier was suspended in 100 ml of a 0.2 M glycine / tris-hydrochloric acid buffer (pH 8.0) and reacted at room temperature for 2 hours to inactivate unreacted active groups. The suspension was transferred onto a Buchner funnel, and the crosslinked agarose carrier was sufficiently washed with 1,000 ml of an acetate buffer (pH 4.0).
The resulting solution was sufficiently washed using the resulting solution to obtain amikacin-sepharose CL-4B using polyethylene glycol bisamine as a spacer arm. The amount of amikacin bound to the Sepharose measured by the Kjeldahl method was 3 m
g / ml. Also, lipopolysaccharide Met.

<< Comparative Example 1. Preparation of Amikacin-Sepharose Cross-linked agarose carrier (Sepharose CL-4)
B: Pharmacia Co., Ltd.) (50 ml) and 2.3 g of an aqueous solution of bromocyan / 50 ml were mixed and kept in a 4 ° C. water bath. Next, a 5N NaOH solution having a pH of 11.0 ± 0.2 and keeping the temperature at 10 ° C. ± 2 ° C. was slowly added dropwise (over 10 minutes) to the solution 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 cross-linked agarose carrier is added to a coupling buffer (0.1 M sodium bicarbonate, 0.5 g).
M sodium chloride, pH 8.3)
Amikacin (manufactured by Wako Pure Chemical Industries, Ltd .: 1.0 g / 20 ml) dissolved in the coupling buffer was added, 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. -4
B was obtained. The amount of amikacin bound to the sepharose measured by the Kjeldahl method was 3 mg / ml. The lipopolysaccharide adsorption activity is 200 ng / 0.1
gwet ( E. coli 0111: B4).

<< Embodiment 2. ———————————————————————————————————————————————————————————————————————————————————————————————————————————————————————————————————————————————————————————————————————————————————————————————————— It 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 total protein 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. Up to the 10th high-pressure steam sterilization operation, almost no decrease was observed in the lipopolysaccharide adsorption capacity of the gel. Further, when the high-pressure steam sterilized gel was refrigerated at 4 ° C.,
For at least one year, almost no ligand was desorbed from the autoclaved gel.

<< Comparative Example 2. --- High pressure steam sterilization of streptomycin-Sepharose Streptomycin-Sepharose CL-4B (hereinafter, gel) prepared according to the method described in Comparative Example 1 was made pyrogen-free, and then filled into a vial.
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,
After refrigerated storage at ℃, one month after storage, about 5% of the amount of protein bound to the gel (the amount of streptomycin) was released. After storage for 3 months, 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 3. Gentamicin-Sepharose CL-4B (hereinafter, gel) prepared according to the method described in Comparative 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 3 Preparation of Kanamycin-Cellulose Cellulose beads (manufactured by Chisso: Cellulofine GC-
700) and kanamycin (manufactured by Wako Pure Chemical Industries), except that kanamycin-cellulose was prepared according to the method described in Example 1. The amount of kanamycin introduced was 3 mg /
ml · gel. The kanamycin-cellulose (hereinafter, gel) was filled in a vial, and kept at 121 ° C for 20 minutes.
Autoclaved for 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 are performed in one step.
Repeated 0 times. As a result, the high-pressure steam sterilization operation 10
The amount of released protein (amount of kanamycin) bound to the gel by the first round 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 4 --- Adsorption test of lipopolysaccharide with kanamycin-cellulose --- After filling a column with 10 ml of kanamycin-cellulose prepared by the method described in Example 3 to make it pyrogen-free, an adsorption test of lipopolysaccharide was carried out. Was. 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 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 5 FIG. --- Adsorption test of lipopolysaccharide with kanamycin-cellulose and streptomycin-cellulose (effect of pH fluctuation) After filling a column with 10 ml of kanamycin-cellulose prepared L by the method described in Example 3, after making the column pyrogen-free Lipopolysaccharide adsorption test. 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% in each case).
Next, a similar test was performed using streptomycin-cellulose prepared according to the method described in Comparative Example 1. 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%; both 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 6 FIG. Preparation of Colistin Covalently Bonded Micropute 120 mg of 1-ethyl-3 (3-diethylaminopropyl) carbodiimide hydrochloride (ECD hydrochloride) was added at pH 4.0.
5 in purified water (reagent grade). The ECD solution was dispensed into each well of a carboxyl group-immobilized micropout (manufactured by Sumibe Medical) in a volume of 200 ul and allowed to react at room temperature for 2 hours. Next, each hole of the microplate was washed five times with purified water. Next, each hole of the micropute was washed with an ice-cooled 0.1 mol / L 4-morpholinoethanesulfonic acid solution (pH 4.8) to remove excess carbodiimide. Next, the coupling buffer (0.
2M sodium bicarbonate, 0.5M sodium chloride pH
9.0) dissolved in polyethylene glycol bisamine (number average molecular weight: 2,000: manufactured by Wako Pure Chemical Industries, Ltd .: 1.0 g) Polyethylene glycol bisamine was introduced into each well of the icropod. Next, a mixed solution of dimethylformamide and succinic anhydride was added to each well of the microplate and reacted to introduce a carboxyl group. Next, 0.1 mol was added to each hole of the micropout.
/ L of 4-morpholinoethanesulfonic acid solution (pH 4.
8) dissolved in water-soluble 1- (3-dimethylaminopropyl) -3-d To activate the carboxyl group. Next, each hole of the micropute was washed with an ice-cooled 0.1 mol / L 4-morpholinoethanesulfonic acid solution (pH 4.8),
Excess carbodiimide was removed. Next, colistin-containing (concentration: 10 mg / m 2)
l) Coupling buffer (0.2 M carbonate) The mixture was dispensed and reacted at room temperature for 24 hours. After blocking the colistin-immobilized microput with bovine serum albumin (manufactured by Wako Pure Chemical Industries, Ltd.) solution to make it pyrogen-free,
Filled in sterile bags. 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. ----Adsorption removal of lipopolysaccharide from artificial fertilized egg cells using colistin covalently attached microplates All holes of colistin covalently attached microplates, which were prepared according to the method of Example 6, and were colored pale blue in their entirety,
Pyrogen-free phosphate buffer (pH 7. (Lipopolysaccharide content of the suspension: 10 pg / ml), and incubated for 5 minutes. After 5 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 10th transfer of the artificial fertilized egg cells (lipopolysaccharide content: 0.1 pg / ml or less).

Embodiment 8 FIG. Preparation of Kanamycin-Binding Hollow Fiber Membrane Kanamycin is covalently bonded to the inner surface of a cellulose hollow fiber membrane (cuprophan hollow fiber membrane) using polyethylene glycol bisamine as a spacer arm according to the method of Example 1. did. Next, the dialyzer volume of 10
Twice the volume of pyrogen-free water 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 9 FIG. Preparation of spectinomycin-bound flat membrane Cuprophan membrane (EN) regenerated by cuprammonium method
Spectinomycin-bound flat membrane was prepared according to the method of Example 1 except that KA product was used. Next, the cuprofan film was modularized. Next, 0.2M NaO
The interior of the module was filled with H / 40% ethanol aqueous solution and circulated at room temperature for 16 hours. Sixteen hours later,
10 times the volume of the module was circulated with pyrogen-free water. Next, 0.2 times as much as three times the volume of the module
The M acetic acid solution was circulated. Finally, 10 times the volume of the module was circulated with pyrogen-free water. next,
The module was put in a bag for high-pressure steam sterilization and sterilized by high-pressure steam 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 was concentrated 10-fold and the total protein was quantified by the Kjeldahl method (according to a comparative example). 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.

Embodiment 10 Preparation of Polymyxin B-Binding Hollow Fiber Membrane The regenerated cellulose membrane (AM-SD 15H) was kept in a 4 ° C water bath for 1 hour. Next, while keeping the regenerated cellulose membrane in the water bath, 46 g of a bromocyan / liter acetone solution was circulated for 1 hour inside the regenerated cellulose membrane to activate the inside of the regenerated cellulose membrane. Further, the inside of the regenerated cellulose membrane was aminated by circulating a triethylamine solution for one hour inside the regenerated cellulose membrane. Next, the inside of the regenerated cellulose membrane was washed by circulating a 50% distilled water / acetone solution and further distilled water. Next, the inside of the regenerated cellulose membrane was filled with a buffer solution (0.2 mol / L sodium bicarbonate-0.5 mol).
/ L sodium chloride: pH 9.0)
Circulating lysine hydrobromide at room temperature for 4 hours,
Poly-L-lysine (PO
LY-ε-CBZ-L-LYSIN: MW1,000)
Was combined. Next, the inside of the regenerated cellulose membrane,
0.25 containing 0.5 mol / L sodium chloride
Washing was performed by circulating a mol / L phosphate buffer (pH 9.0). Furthermore, after the inside of the poly-L-lysine-bonded regenerated cellulose membrane was activated with bromocyan by the method described above, polymyxin B was bound to prepare a polymyxin B-bound hollow fiber membrane.

Embodiment 11 FIG. << Adsorption of Lipopolysaccharide by Polymyxin B-Binding Hollow Fiber Membrane-- A pyrogen-free water was circulated inside the polymyxin B-binding hollow fiber membrane prepared by the method of Example 10.
Next, a distilled aqueous solution for injection (pH 7.0) containing 10 ng / ml of lipopolysaccharide was applied to the inside of the hollow fiber membrane, and allowed to flow at a flow rate of 100 ml / min. 1 effluent
One liter was pooled, and the lipopolysaccharide concentration of the pool solution was quantified using Endospecy (manufactured by Seikagaku Corporation). As a result, no lipopolysaccharide was detected from the pool solution. Further, the polymyxin B-bound hollow fiber membrane was resistant to high-pressure steam sterilization. Furthermore, the polymyxin B
The bonded hollow fiber membrane adsorbed about 10 times as much lipopolysaccharide as the conventional hollow fiber membrane for adsorbing lipopolysaccharide having a short chain length of the spacer arm.

[0038]

The lipopolysaccharide adsorbent according to the method of the present invention comprises:
(1) Compared with a conventional lipopolysaccharide adsorption carrier having a short spacer arm chain length, the lipopolysaccharide adsorption activity is remarkably high. (2) acidic region, neutral region and basic region (pH5.
(0 to pH 9.0), it is possible to satisfactorily adsorb and remove lipopolysaccharide in the solution. (3) Stable adsorbent for high-pressure steam sterilization. (4) A lipopolysaccharide adsorbent having no ligand detachment in an acidic region, a neutral region and a basic region (pH 4.0 to pH 9.0). (5) A lipopolysaccharide adsorbent having excellent storage stability at low temperature (4 ° C.). (6) A lipopolysaccharide adsorbent capable of satisfactorily adsorbing and removing lipopolysaccharide in a solution even when the ionic strength is in the range of 0.1 to 0.6.

Continued on the front page F-term (reference) 4C087 AA01 AA10 BB61 BB63 CA04 NA06 ZA81 4G066 AA13D AA31D AA34D AA43D AB01D AB06A AB06B AB07D AB09D AB15D AB26A AB26B AB27A AB27B AB27D AC01AAC ACA BAB AC02BAO ACB AC02A ACB BA62 EA34 FA81

Claims (7)

[Claims] (1) a number average molecular weight of 300 to 5,000,000
A lipopolysaccharide adsorbent that is stable to high-pressure steam sterilization and alkali treatment, wherein a chemotherapeutic agent is immobilized on an insoluble carrier via a hydrophilic spacer arm of No. 00. 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, paromomycin and its derivatives Ribostamycin and its derivatives, sisomycin and its derivatives, tobramycin and its derivatives,
2. The lipopolysaccharide adsorbent according to claim 1, wherein the lipopolysaccharide adsorbent is selected from the group consisting of genenetin 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 a peptide antibiotic. 5. A method for adsorbing and removing lipopolysaccharide, comprising bringing the lipopolysaccharide adsorbent according to claim 1 into contact with a lipopolysaccharide-containing liquid, and then separating the two. 6. After making the lipopolysaccharide adsorbent according to claim 1, 2, 3, or 4 pyrogen-free, it is further subjected to high-pressure steam sterilization or 56 ° C. or more and 90 ° C.
A method of storing after heating at the following temperature for 10 hours or more. 7. The lipopolysaccharide adsorbent according to claim 1, wherein the adsorbent is colored.