JPH03167194A - Purification of lecithin - Google Patents

Abstract

PURPOSE:To obtain lecithin of endotoxin-free utilizable to injection agent having high safety by dissolving lecithin in alcohol, etc., and treating with immobilized histidine. CONSTITUTION:Lecithin is dissolved in alcohol or water/alcohol mixed solution and treated with immobilized histidine.

Description

[Detailed description of the invention]

[Industrial Application Field] The present invention relates to a purification method for obtaining endotoxin-free lecithin suitable for use in the pharmaceutical field, particularly for injections. [Prior art] Lecithin is a typical phospholipid that forms liposomes (lipid bilayer membrane packages), so it can be used as capsules for drugs. Since lecithin is a major component of liposomes and is highly safe for the human body, there have been many attempts in recent years to apply lecithin to liposome preparations as a new drug delivery system. There are various methods of administering liposome preparations.
Currently, intravenous administration is the mainstream, but when administering intravenous injections, it is essential that the injection does not contain pyrogens (pyrogenic substances). Pyrogen is a polymeric substance that exhibits pyrogenicity, and when it enters the blood vessels of wheeze mammals, it causes a temporary fever and is a harmful substance that can sometimes cause the animal to die from shock. Among the pyrogens, lipopolysaccharide (L
Endotoxin (endotoxin), whose main component is PS), is particularly highly thermogenic among pyrogienes and is ubiquitous. For this reason, the Japanese Pharmacopoeia has established test items for pyrogenic substances and regulates the amount of pyrogen in preparations. Therefore, lecithin used as a base material for liposome preparations must have a negative pyrogen test result. Methods for removing endotoxin include ■heat sterilization,
■Chemical treatment, ■Adsorbent treatment, ■Membrane filtration treatment, ■Distillation treatment, etc. are known, but among these, ■Heat sterilization treatment is
Because endotoxin is relatively stable against heat, 25
It is necessary to heat at 0℃ for one hour, and the chemical treatment in (■) uses acids, alkalis, and peroxides, which also decomposes the lecithin itself, making it difficult to process. Processing is also physically extremely difficult. Also, ■
It is known that activated carbon or ion exchange resins are used as adsorbent treatments, but both have the problem of low effectiveness and large loss of sample components. In addition, as membrane filtration treatment (■), sterilization filter (0.2 micron filter) filtration or ultrafiltration is known, but the sterilization filter has little effect, and ultrafiltration is usually performed with an aqueous solution. Therefore, there was a problem in that it was difficult to process lecithin, which is poorly soluble in water. That is, existing endotoxin removal methods generally use water, amino acids, sugars,
There was no known effective method for removing endotoxins from lecithin, which is poorly soluble in water and is used for water-soluble substances such as antibiotics and vitamins. In addition, when quantifying endotoxin in oil-soluble substances, the test substance is usually dissolved in an organic solvent and the aqueous solution extracted with endotoxin-free water is quantified using a Limulus test, etc.; however, when the test substance is lecithin, it is Due to the compatibility of endotoxins, extraction with endotoxin-free water was difficult, making accurate quantification impossible. [Problems to be Solved by the Invention] The present invention aims to solve the above-mentioned problems and obtain endotoxin-free lecithin without affecting lecithin.

[Means to solve the problem]

The present inventors conducted intensive studies to achieve the above objective, and after dissolving lecithin in alcohol or a mixed water/alcohol solution, immobilized histidine, which has an affinity for endotoxin, as a ligand. The present invention was achieved by discovering that endotoxin can be selectively adsorbed and removed by contacting with immobilized histidine, and that this method has no adverse effect on lecithin and is extremely safe. The lecithin to be treated in the present invention can be any existing natural or synthetic lecithin, but in order to be used in the pharmaceutical field, which is the purpose of the present invention, it is recommended to use highly purified lecithin. preferable. Here, the lecithin purification method may be any existing purification method. For example, a purification method such as solvent extraction, acetone reprecipitation, column chromatography, etc. may be used alone, or a combination of several of these methods may be used. The immobilized histidine used in the present invention is preferably one in which histidine, which has an affinity for endotoxin, is immobilized as a ligand on a carrier via a substrate, and the carrier may be a polymer polysaccharide, a synthetic polymer carrier, or an inorganic carrier. However, high molecular weight polysaccharides such as cellulose and agarose are preferred. Examples of commercially available immobilized histidine include "Pyrosetsub" manufactured by Daicel Chemical Industries, Ltd., in which histidine is covalently bonded to Sepharose (cross-linked agarose gel) using hexamethylene diamine as a spacer. Methods for treatment with immobilized histidine include column chromatography, in which immobilized histidine is packed into a column as a packing material and the lecithin solution is eluted, or it is added to the lecithin solution as an adsorbent, stirred, filtered, and fixed. A method such as removing oxidized histidine can be used, and endotoxin is selectively adsorbed and removed in a column or solution.
The used immobilized histidine can be regenerated by washing and other treatments and used repeatedly. As the alcohol used in the present invention, methanol, ethanol, and inbrobanol can be used, and each may be used alone or in a mixture. The composition of the water/alcohol mixed solution used in the present invention may be any composition as long as it completely dissolves the lecithin to be treated, but if the proportion of water is high, it will be difficult to remove the solvent. /ethanol=O~10/90
-100% by volume is desirable. Furthermore, the lecithin concentration in the water/alcohol mixed solution may be of any composition as long as it completely dissolves the lecithin to be treated, but it is preferably 5 to 20% by weight in order to improve workability. desirable. [Effect of the invention] The method for producing endotoxin-free lecithin of the present invention includes the following steps:
By dissolving lecithin in alcohol or a water/alcohol mixed solution and then contacting it with immobilized histidine, endotoxin in lecithin can be effectively adsorbed and removed. Because of its high safety, it is widely used in the pharmaceutical field, especially for injections. This will be of great industrial benefit as it will be possible to use it as a drug. In addition, the endotoxin removal treatment method of the present invention includes a method of filling a column with immobilized histidine and passing a lecithin water/ethanol mixture solution, or adding immobilized histidine to a lecithin water/ethanol mixture solution and stirring. Filtration is a relatively simple method that allows mass production, and the immobilized histidine used as an adsorbent can be reused repeatedly, making it extremely useful industrially.

【Example】

The present invention will be specifically explained using examples and comparative examples. All instruments used in the Examples and Comparative Examples were dry heat sterilized at 250°C for 2 hours or more, or immersed in a 0.2M NaOH aqueous solution (containing 20 vol% ethanol) for 12 hours or more. and washed with endotoxin-free water (distilled water for injection) before use. In addition, all of the treated lecithins used were tested in the Limulus test (
Reagent used: Seikagaku Kogyo ■ Virodip) was used after confirming that it was positive. In addition, when performing the Limulus test, since it is difficult to extract endotoxin in lecithin with distilled water for injection, all test lecithin is
After accurately weighing mg and adding lmA of distilled water for injection, 50 to 60
A water suspension solution subjected to ultrasonic dispersion at ℃ for 30 minutes was used as the test sample. All treatments were performed on a class 100 clean bench. In a 50mI2 beaker, lecithin purity 99% by weight
2.06 g of purified egg yolk lecithin was weighed out, and then a mixed solution of water/methanol = 5/9 5% by volume was added so that the lecithin concentration was 10% by weight to completely dissolve it. Next, this lecithin solution was passed through an oven glass force ram with an inner diameter ICm filled with 10 mβ of immobilized histidine (Pyrosep J manufactured by Daicel Chemical Industries, Ltd.) at an elution rate of 1.
．． After eluting with Omllll minutes (SV = 6), 30 ml of water/methanol = 5/9 5% by volume mixed solution was added to 1
．． It was eluted with OmAl. All eluates were 100mβ
It was collected in an eggplant flask, and the solvent was removed using an evaporator to obtain a solid content of 1.98 g (C conversion rate: 96.1%). The purity of lecithin in the obtained solid content was 99.0% by weight, and the result was negative (N.D.) in the Limulus test (reagent used: Viroditub, manufactured by Seikagaku Corporation). Weigh out 2.12g of hydrogenated soybean lecithin with a lecithin purity of 99.1% by weight into a 50mg beaker, and then add a mixed solution of water/ethanol = 5/95% by volume to a 50mg beaker with a lecithin concentration of 5%.
% by weight and completely dissolved. Next, this lecithin solution was placed in an oven glass force ram with an inner diameter of 1 cm and filled with 10 ml of immobilized histidine ("Pyrosep J" manufactured by Daicel Chemical Industries, Ltd.) in advance at an elution rate of 0. .5
After elution at mβ/min (SV=3), 30 mI2 of a mixed solution of water/ethanol = 5/95% by volume was added at 0.0 mI2. 5r
r+j! /min. 100ml of all eluate
Collect in an eggplant flask, remove solvent with an evaporator,
A solid content of 2.05 g (recovery rate 96.7%) was obtained. The lecithin purity in the obtained solid content was 99.1% by weight,
The Limulus test (wiping agent used: Baiguchi Datsubu manufactured by Seikagaku Kogyo ■) was negative (N.D.). Weigh out 2.10g of synthetic DMPC (dimyristoylphosphatidylcholine) with lecithin purity of 99.8% by weight into a 50mQ beaker, then add a mixed solution of water/isobrobanol = 5/9 5% by volume. This lecithin solution was added to a lecithin concentration of 5% by weight and completely dissolved. Next, this lecithin solution was added to a 1cm tube with an inner diameter filled with immobilized histidine ("Virosep J" manufactured by Daicel Chemical Industries, Ltd.) in advance.
m oven glass column, elution rate 0.5 mβ/
After elution at a rate of 0.5 mβ/min (SV=3), 30 mβ of a mixed solution of water/isobrobanol = 5/95% by volume was eluted at a rate of 0.5 mβ/min. All the eluate was collected in a 100 ml eggplant flask, and the solvent was removed using an evaporator to reduce the solid content to 2.0.
5 g (recovery rate: 97.6%). The lecithin purity in the obtained solid content was 99.8% by weight, and the Limulus test (reagent used: Virodip, manufactured by Seikagaku Corporation) was negative (
N. D. )Met. Weigh out 10.04g of synthetic DPPC (divalmitoylphosphatidylcholine) with lecithin purity of 99.6% by weight into a 100mI2 beaker, and then add water/ethanol = 0.2/99.8% by volume. The mixed solution was added so that the lecithin concentration was 20% by weight and completely dissolved. Next, this lecithin solution was placed in an oven glass oven with an inner diameter of 1 cm filled with 10 mA of immobilized histidine (Vyrosep J manufactured by Daicel Chemical Industries, Ltd.). In the ram, elution rate 2.
After elution at 0 mI2/min (SV=12), 30 mβ of a mixed solution of water/ethanol==Q, 2/99.8% by volume
was eluted at 2.0 mβ/min. All eluates are 100%
Collected in a ml eggplant flask, the solvent was removed using an evaporator to obtain a solid content of 9.87 g (recovery rate 98.3%). The lecithin purity in the solid content was 99.6% by weight,
Limulus wiping test (reagent used: Seikagaku Kogyo Co., Ltd. PyroDip) showed stagnation (N.D.). Lecithin purity 99.6% by weight was placed in a 100 mβ beaker with one back opening.
Weigh out 10.11g of synthesized DPPC (divalmitoylphosphatidylcholine), then water/ethanol =
A mixed solution of 0.2/99.8% by volume with a lecithin concentration of 2
Add Pyrosep to this lecithin solution to give a total concentration of 0% by weight and dissolve completely. Next, add Pyrosep to this lecithin solution in advance with a mixed solution of water/ethanol = 0.2/99.8% by volume at 10 ml2.
The mixture was moistened so that the mixture was wetted and stirred at 25°C for 1 hour. Next, a PTFE 0.2 micron sterilization filter was used to elute at a rate of 2. After filtering with Omitl, a mixed solution of water/ethanol = 0.2/99.8% by volume 30
2.0m for ml! Washed and filtered for 1 minute. All filtrate 1
Collected in a 00rr + 42 eggplant flask and removed the solvent using an evaporator, solid content 9.83g (recovery rate 97.2%)
I got it. The lecithin purity in the obtained solid content was 99.6% by weight, and the Limulus test (reagent used: Virodip, manufactured by Seikagaku Corporation) was negative (ND). Stop 1t
Yu50n+j! Weigh out 10.09 g of synthetic DPPC (divalmitoylphosphatidylcholine) with lecithin purity of 99.6% by weight into a beaker, then add a mixed solution of water/ethanol = Q, 2/99.8% by volume to lecithin. It was added to a concentration of 20% by weight and completely dissolved. Next, this lecithin solution was filtered through a 0.2 micron sterilization filter made of PTFE at an elution rate of 2.0 mj2/min, and then a mixed solution of water/ethanol = 0.2/99.8% by volume (30 mβ) was added. was washed and filtered at 2.0 mβ/min. All of the filtrate was collected in a 100 mI2 eggplant flask, and the solvent was removed using an evaporator to obtain a solid content of 9.91 g (recovery rate 98.2%). The purity of lecithin in the obtained solid content was 99.6% by weight, but the purity of lecithin in the obtained solid content was 99.6% by weight.
Pyro-dip) was positive. As is clear from the results of these Examples and Comparative Examples, when lecithin is purified by the method of the present invention in which it is treated with immobilized histidine, endotoxin can be efficiently removed without adversely affecting lecithin. It can be seen that endotoxin cannot be efficiently removed simply by filtering with a sterilization filter without treatment with immobilized histidine.

Claims (1)

[Claims] 1. A method for purifying lecithin, which comprises dissolving lecithin in alcohol or a mixed water/alcohol solution and treating the solution with immobilized histidine.