JPH0611331B2 - Method for removing endotoxin from immobilized polymyxin moldings - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for removing endotoxin from an immobilized polymyxin molded article.

[Prior Art] For example, when an immobilized polymyxin molded product is used to detoxify endotoxin in blood or an aqueous solution, it is mixed with the immobilized polymyxin molded product in the manufacturing process, and the adhered endotoxin elutes. On the contrary, there was a problem of contaminating blood or aqueous solution.

However, since a method for quantifying endotoxin has not been established, an attempt to positively remove the endotoxin attached to the immobilized polymyxin molded product has not been studied.

For example, as an immobilized polymyxin molded article, one in which polymyxin is immobilized on agarose has been reported.
No aggressive endotoxin removal method is mentioned.

[Problems to be Solved by the Invention] The present invention provides a method for efficiently removing endotoxin attached to an immobilized polymyxin molded product without losing the activity of the immobilized polymyxin.

[Means for Solving Problems] A method for removing endotoxin from an immobilized polymyxin molded article, which comprises contacting the immobilized polymyxin molded article with an alkaline solution.

The immobilized polymyxin molded product in the present invention is a product in which polymyxin is immobilized on a carrier.

Polymyxin is a peptide antibiotic produced by Bacillus Polymyxa. Polymyxins include polymyxin A, polymyxin B ₁ , polymyxin B ₂ , polymyxin D ₁ , polymyxin E ₁ , polymyxin E _{2 and the} like, which have an antibacterial action against Gram-negative bacteria.

The alkaline solution referred to in the present invention may be a solution having a pH of 9 or more, more preferably a pH of 12 or more, and there is no particular limitation on its type.

Examples of the alkaline solution include alkali metal represented by sodium, potassium, lithium, cesium, etc., hydroxide of alkaline earth metal represented by calcium, barium, magnesium etc., or an aqueous solution of a weak acid salt thereof. Further, a hydroxide of a quaternary ammonium salt such as trimethylbenzylammonium hydroxide can be used.

Especially, sodium hydroxide, potassium hydroxide and the like are preferable. Further, its concentration is preferably 1N or more.

As the solvent, water, saline, an organic solvent or the like can be used.

Further, a mixed solution of water and a water-soluble organic solvent, for example, water and alcohol is adopted. If the concentration of alcohol at that time is too low, the effect is small, and if it is too high, the solubility of the alkali decreases, so it is 5 to 50%, preferably 10 to 30.
% Concentrations are applied.

As an example of the contact means, shaking, stirring, a method of continuously flowing through a column or a means such as dipping is adopted. Among them, stirring of the solution is preferable, and for example, ultrasonic stirring can wash the inside of the molded product. It is more preferable because it is possible.

The temperature of the solution is usually in the range of 0 ° C. or higher and 100 ° C. or lower, but room temperature is preferably applied.

The molded article referred to in the present invention includes, for example, fibers, hollow fibers,
There are membranes, beads, etc., but not limited to these.

Further, the molded product needs to have excellent chemical resistance, particularly alkali resistance, and is preferably an insoluble polymer. For example, a molded product obtained by immobilizing polymyxin on an insoluble vinyl polymer is a specific example.

Examples of insoluble polymers include styrene, vinyl chloride, acrylic acid amide, acrylonitrile, maleic anhydride,
A homopolymer of a monovinyl compound represented by methacrylic acid ester or a copolymer with a polyvinyl compound represented by divinylbenzene or methylenebisacrylamide, etc. Examples thereof include those cross-linked with an acid or the like.

In order to immobilize a polymyxin molecule to these inactive polymers by covalent bond, a functional group such as α-halogenated alkyl group or carboxylic acid ester group is directly attached to the polymer or a spacer. It is necessary that the spacers are bound via a spacer, and the length of the spacer is preferably 4 or more in terms of methylene group.

Example 1 Example 1 50 parts of polypropylene (Mitsui "Noblen" J3HG) was used as an island component, and polystyrene ("Stylon" 666) was used.
46 parts, polypropylene (Sumitomo "Noblen" WF-7
27-F) Sea-island type composite fiber containing 4 parts of the mixture as a sea component (16 islands, single yarn fineness 2.6 denier, tensile strength 2.9).
g / d, elongation 50%, filament number 42) 50 g N
It was immersed in a mixed solution of 60 g of methylol-α-chloroacetamide, 400 g of nitrobenzene, 400 g of 98% sulfuric acid, and 0.85 g of paraformaldehyde, and reacted at 20 ° C. for 1 hour.

The fiber was taken out of the reaction solution, poured into ice water 5 at 0 ° C. to stop the reaction, washed with water, and then nitrobenzene attached to the fiber was extracted and removed with methanol.
This fiber (fiber A) was vacuum dried at 50 ° C. to obtain 71 g of chloracetamidomethylated fiber.

Polymyxin B Sulfate (Sigma; 8000 units / mg)
Dissolve 4.8g in 600ml water and add fiber A2
After adding 2 g and shaking for 5 hours, magnesium oxide 1.
5 g was added and shaken for 12 hours at room temperature.

Next, this fiber was taken out of the reaction mother liquor, packed in a chromatographic column, and washed with 4800 ml of 0.1N hydrochloric acid to obtain a polymyxin-immobilized fiber (fiber B). The amount of polymyxin in the washing liquid and the reaction mother liquor was measured by the microburette method, and the difference between the amount and the charged amount was regarded as the amount of polymyxin immobilized on the fiber. The immobilized amount was 1.5 mg / g.

Add 1N NaOH20 to a sterile test tube containing 1g of fiber B.
30 ml of a% ethyl alcohol solution was added, the fiber and the solution were brought into contact with each other for 30 minutes with an ultrasonic cleaner, the liquid was exchanged, and the ultrasonic cleaner was again applied. The temperature of the solution at this time is 37
The temperature was adjusted to ° C.

After removing the endotoxin, the fiber was transferred to a sterile column, and sterile distilled water was flushed until the outflow water became neutral.

Next, 60 ml of sterile Tris-hydrochloric acid buffer was poured, and 120 ml of sterile distilled water was poured.

After removing the endotoxin, put the neutralized fiber (Fiber C) in a sterilized Erlenmeyer flask, add 30 ml of physiological saline, stopper the mixture, and shake at 90 ° C for 90 minutes. Was eluted into 30 ml of saline.

As a result, the amount was 2.9 ng / g fiber C, which was an extremely low amount.

Comparative Example Fiber B was placed in a sterilized Erlenmeyer flask and 30 ml of physiological saline was added.
Was added, the mixture was stoppered and shaken at 37 ° C. for 90 minutes to elute the endotoxin adhering to the surface of the fiber B, and the endotoxin was quantified. It was 56 ng / g fiber B.

[Effect of the Invention] The endotoxin attached to the immobilized polymyxin molded article can be efficiently removed without losing the activity of the polymyxin.

Claims (1)

[Claims] 1. A method for removing endotoxin from an immobilized polymyxin molded article, which comprises contacting the immobilized polymyxin molded article with an alkaline solution.