JP4186460B2 - Process for producing purified heparin, salt thereof or heparin derivative and process for producing purified low molecular weight heparin, salt or heparin derivative - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a purified method for producing heparin, a salt thereof or a heparin derivative, which comprises treating heparin, a salt thereof or a heparin derivative with a specific anion exchanger, and reducing the molecular weight of heparin or a salt thereof. The present invention relates to a method for producing a purified low molecular weight heparin, a salt thereof, or a heparin derivative, which comprises treating the resulting low molecular weight heparin or a salt thereof with a specific anion exchanger.
[0002]
[Prior art]
Heparin has strong blood anticoagulant activity. In addition to the treatment of generalized intravascular blood coagulation syndrome, the treatment and prevention of various thromboembolisms such as myocardial infarction and cerebral embolism, It is used for the prevention of blood coagulation when using an extracorporeal circulation device such as an oxygenator, inserting a blood vessel catheter, transfusion, and blood test. When heparin is used for the above treatment or prevention, there is a problem of increased bleeding tendency associated with the prolongation of blood coagulation time. Therefore, low molecular weight of heparin has been studied. Among them, the use of an anion adsorbent is considered. Investigation has been made (Japanese Patent Publication No. 63-44764).
[0003]
However, once heparin, its salts or heparin derivatives (hereinafter sometimes collectively referred to as heparins) are once adsorbed on an anion adsorbent and then released, the activity of the resulting heparin is reduced. There is a problem.
[0004]
Specific examples of the method for purifying heparin and the method for producing low molecular weight heparin include the production method described in Japanese Patent Publication No. 63-44764. The method still has problems in terms of obtaining heparin that retains high anti-factor Xa activity.
[0005]
[Problems to be solved by the invention]
The present inventors have intensively studied to solve the above problems. As a result, after the heparin is adsorbed to the anion exchanger in which the anion is replaced with the conjugated base ion, the treatment with the anion exchanger that liberates the heparin is performed, preferably a liquid chromatography method The purified heparin can be easily produced by carrying out the treatment using the above, and the molecular weight lowering treatment of heparin or a salt thereof, followed by treatment with an anion exchanger, preferably liquid chromatography It was found that purified low molecular weight heparin, a salt thereof or a heparin derivative can be easily produced by carrying out the treatment using a method, and the present invention was completed based on these findings. As is clear from the above description, the object of the present invention is to provide a method for producing purified heparins in which the activity of heparins is unlikely to decrease, and a purified low molecular weight heparin, a salt thereof or a heparin derivative having high anti-factor Xa activity. It is to provide a manufacturing method.
[0006]
[Means for Solving the Problems]
In the present invention, after the heparins are adsorbed to the anion exchanger in which the anions are replaced with conjugated base ions, the treatment with the anion exchanger that liberates the heparins again is performed, preferably the treatment A method for producing purified heparins characterized by using a liquid chromatography method and subjecting heparin or a salt thereof to a molecular weight reduction treatment, and then coupling the resulting low molecular weight heparin or a salt thereof with an anion Purified, low molecular weight heparin, characterized in that treatment with an anion exchanger is carried out using an anion exchanger substituted with a base ion, preferably the treatment is carried out using a liquid chromatography method , A method for producing the salt or heparin derivative thereof.
[0007]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, the present invention will be described in detail.
The heparin used in the present invention is not particularly limited. For example, the heparin may be obtained by warm digestion with a sodium hydroxide solution saturated with ammonium sulfate after self-digestion of the liver or lung. After neutralization, the heparin-protein complex is precipitated with alcohol, washed with alcohol, and then dissolved in an alkaline solution. The protein is digested and removed with trypsin, and then precipitated with alcohol to obtain a crude product. Mention may be made of those obtained by treatment with cadmium chloride to remove impurities and precipitation with acetone.
[0008]
The heparin salt used in the present invention is not particularly limited, and examples thereof include a heparin metal salt, and examples of the metal include sodium, potassium, calcium and potassium. Among these, heparin sodium can be preferably used in the present invention because it is a product in the Japanese Pharmacopoeia. The metal salt of heparin can be obtained by separating the heparin obtained by the above-mentioned method as a benzidine hydrochloride salt or a brucine salt and converting it to a metal salt. Moreover, as heparin sodium, use of heparin sodium described in the 13th revision Japanese Pharmacopoeia can be exemplified.
[0009]
In the present invention, low molecular weight heparin or a salt thereof refers to heparin or a salt thereof having an average molecular weight smaller than that of standard heparin or a salt thereof. Since the average molecular weight of standard heparin or a salt thereof varies depending on the extraction source, the average molecular weight of the low molecular weight heparin or a salt thereof obtained by the production method of the present invention is not specified, but specifically 1000 to 7000. Of the range.
[0010]
The heparin derivative used in the present invention can be exemplified by a chemical structure changed from the heparin or its salt as a raw material. Specifically, sodium nitrite or potassium nitrite or the like is added to the heparin or its salt, and the molecular weight And those obtained by reducing the molecular weight using a reducing agent. Examples of the reducing agent include sodium borohydride and sodium cyanoborohydride.
[0011]
The anion exchanger used in the present invention is a cellulose carrier, epichlorohydrin or the like as a spacer, aminoethyl group, diethylaminoethyl group, triethylaminoethyl group, guanidoethyl group, p-aminobenzyl group or the like as a ligand. Can be illustrated. In addition to cellulose, agarose, styrene polymer or the like can be used as the carrier.
[0012]
More specifically, as the anion exchanger, DEAE Sephadex (trademark) A-25, DEAE Sephadex (trademark) A-50, QAE Sephadex (trademark) A-25, QAE Sephadex (trademark) A-50, DEAE Sephadex (Sephadex), DEAE Sephadex (trademark) CL-6B, Q-Sepharose Fastflow (trademark), DEAE Sepharose Fastflow (Sepharose Fast) flow, trademark), MonoQ (above, Amersham Pharmacia Biotech),
[0013]
DEAE-Toyopearl (trademark) 650, QAE-Toyopearl (trademark) 550, SuperQ-Toyopearl 650 (trademark, manufactured by Tosoh Corp.), DEAE-Cellulofine (trademark) A-200-m, DEAE-Cellulofine (trademark) Examples include commercially available anion exchangers such as A-500-m, DEAE-Cellulofine ™ A-500-sf and DEAE-Cellulofine ™ A-800-m (manufactured by Seikagaku Corporation). be able to.
[0014]
Examples of the anion exchanger in which the anion used in the present invention is substituted with a conjugated base ion include those obtained by permeating hydrochloric acid, sulfuric acid, acetic acid and the like into the above anion exchanger. Examples in which the counter ion is substituted with a conjugate base such as a chloride ion, a sulfate ion, and an acetate ion can be exemplified. By treating heparin and its salts with the anion exchanger, heparins have a pharmacological action and suppress elimination of O- or N-sulfate groups in the heparin molecule due to hydrolysis. Heparins with high anti-factor Xa activity can be obtained. Specifically, a method of shaking an anion exchanger with 0.05 to 6.0 mol / l hydrochloric acid can be exemplified, and an anion exchanger substituted with a conjugated base ion using a liquid chromatography apparatus in terms of economy. It is preferable to convert.
[0015]
The method for producing a purified heparin according to the present invention is characterized in that after the above-mentioned anion exchanger is adsorbed with the above-mentioned heparin, the treatment with the anion exchanger for liberating the heparin is performed again. This is a manufacturing method. Here, the adsorption means that the above-described anion exchanger is infiltrated and adsorbed with a solution in which heparins are dissolved in water or the like, and specifically, the above-described anion exchanger and heparins can be adsorbed. A method of filtering the solution after shaking with the solution can be exemplified, and it is preferable to use a liquid chromatography method in terms of economy. The liberation means that the adsorbed anion exchanger is made to penetrate an aqueous solution of sodium chloride, potassium chloride, sodium sulfate, potassium sulfate, sodium acetate, potassium acetate, etc. from the anion exchanger into the aqueous solution. It is to transfer heparin. Specifically, a method of separating the aqueous solution by filtration after shaking the above-described anion exchanger and the aqueous solution can be exemplified, and it is preferable to use a liquid chromatography method in terms of economy. The separated aqueous solution can be crystallized in a crystallization solvent to obtain solid heparins.
[0016]
The molecular weight reduction treatment in the present invention can be exemplified by adding sodium nitrite or potassium nitrite or the like or heparin degrading enzyme or the like to the heparin or a salt thereof. Moreover, it is a preferable processing method to use acidic solutions, such as hydrochloric acid, a sulfuric acid, an acetic acid, after adding the above-mentioned sodium nitrite in the point of a yield. More preferred is a method using hydrochloric acid. In addition, after using an acidic solution in terms of yield, it is neutralized with an inorganic base such as sodium hydroxide, potassium hydroxide or sodium carbonate. In terms of yield, the neutralization is preferably followed by a treatment using a reducing agent. Examples of the reducing agent include sodium borohydride and sodium cyanoborohydride.
[0017]
【Example】
Hereinafter, the present invention will be described in more detail with reference to examples.
The evaluation methods used in the examples are shown below.
[0018]
(1) Dissolve the sample for average molecular weight calibration and the sample so as to be 10 mg / mL in the mobile phase. Test 25 μL of these solutions by liquid chromatography. The detector was connected in the order of the UV-visible spectrophotometer and the differential refractometer, and the UV-visible spectrophotometer and the differential refractive index were obtained in advance using the UV-visible spectrophotometer and the differential refractometer, and obtained from the UV-visible spectrophotometer. The chromatogram retention time is corrected to the differential refractometer peak retention time. The standard solution is obtained by dividing the chromatogram obtained from the UV-visible spectrophotometer and the differential refractometer, and the sample solution is obtained by dividing the chromatogram obtained from the differential refractometer at regular intervals, and the peak height at each retention time (i). I ask for it. The relative average molecular weight was determined by the following formula, and the value was defined as the average molecular weight.
A coefficient used for calculation of the average molecular weight is obtained from two chromatograms obtained from the UV-visible spectrophotometer and the differential refractometer of the standard solution.
r = ΣRI _S i / ΣUV _S i
ΣRI _S i: Total peak height in retention time (i) of main peak obtained from differential refractometer of standard solution ΣUV _S i: Retention time of main peak obtained from UV-visible spectrophotometer of standard solution ( Total peak height in i) f = Mn / r
Mn: number average molecular weight Mi (molecular weight with respect to retention time (i)) of standard product for measuring average molecular weight = f × RI _S i / UV _S i
RI _S i: Peak height at retention time (i) of peak obtained from differential refractometer of standard solution
UV _S i: Peak height average relative molecular weight at retention time (i) of peak obtained from UV-visible spectrophotometer of standard solution = ΣRI _T i / Σ (RI _T i / Mi)
RI _T i: Peak height of sample solution obtained from differential refractometer at peak retention time (i) Note that low molecular weight heparin standard for molecular weight calibration (90/686) (National Institute for Biological Standards) and Control).
[0019]
(2) Anti-factor Xa activity test team Heparin S kit (Daiichi Kagaku Yakuhin Co., Ltd.) was used, and heparin concentration (IU / ml) was measured based on the attached text of the kit. .
[0020]
Example 1
An anion substituted with conjugated base ions by flowing 750 ml of 0.3 mol / l hydrochloric acid through a column (44 mmφ × 100 mm) packed with 150 ml of DEAE-Cellulofine (trademark) A-500 m and then 900 ml of ion-exchanged water. Exchangers were prepared. Next, a total amount of a solution prepared by dissolving 2.0 g of heparin sodium derived from porcine intestinal mucosa in 20 ml of ion-exchanged water was passed through the column and adsorbed onto the anion exchanger. Subsequently, the adsorbed sodium heparin was released and eluted using 150 ml of 0.28 mol / l sodium chloride aqueous solution, and the aqueous heparin sodium solution was fractionated. Ethanol was added to this aqueous solution for crystallization, followed by filtration to obtain 1.26 g of purified sodium heparin. The average molecular weight of this heparin sodium was 3050, and the anti-factor Xa activity was 72 IU / mg.
[0021]
Comparative Example 1
In a column (44 mmφ × 100 mm) packed with 150 ml of DEAE-Cellulofine ™ A-500 m, a total amount of a solution prepared by dissolving 2.0 g of heparin sodium derived from porcine intestinal mucosa in 20 ml of ion-exchanged water was applied to the column. did. Subsequently, the adsorbed sodium heparin was released and eluted using 150 ml of 0.28 mol / l sodium chloride aqueous solution, and the aqueous heparin sodium solution was fractionated. Ethanol was added to this aqueous solution for crystallization, followed by filtration to obtain 1.21 g of sodium heparin. This heparin sodium had an average molecular weight of 3100 and an anti-factor Xa activity of 58 IU / mg.
[0022]
Example 2
15.0 g of heparin sodium derived from porcine intestinal mucosa was dissolved in 220 ml of ion-exchanged water and cooled to 5 ° C. After cooling, an aqueous solution in which 0.33 g of sodium nitrite was dissolved in 6 ml of ion-exchanged water was added, and 45.8 g of 1 mol / l hydrochloric acid cooled to about 5 ° C. was added, followed by stirring for 15 minutes to carry out a decomposition reaction. Subsequently, 47.4 g of 1 mol / l sodium hydroxide aqueous solution was added to stop the reaction. Next, the reaction solution was heated to 25 ° C., 0.075 g of sodium borohydride was added, and the mixture was stirred for 4 hours to perform a reduction reaction. Thereafter, 22.3 g of a 1 mol / l acetic acid aqueous solution was added to bring the pH to 4 to decompose excess sodium borohydride, and 28.1 g of a 1 mol / l sodium hydroxide aqueous solution was added to adjust the pH to 7. Next, 6500 g of ethanol was added to the reaction solution for crystallization, followed by filtration to obtain 15.1 g of decomposed and reduced low molecular weight sodium heparin.
Next, 750 ml of 0.3 mol / l hydrochloric acid was passed through a column (44 mmφ × 100 mm) packed with 150 ml of DEAE-Cellulofine ™ A-500 m, and then 900 ml of ion-exchanged water was added to the conjugated base ions prepared. A column in which the substituted anion exchanger was packed was flushed with a solution of 2.0 g of this decomposed and reduced low molecular weight sodium heparin dissolved in 20 ml of ion-exchanged water and adsorbed on the anion exchanger. . Thereafter, 93 ml of ion-exchanged water and 225 ml of a 0.19 N aqueous sodium chloride solution were flowed, and then separated and eluted with 150 ml of a 0.28 N aqueous sodium chloride solution to fractionate a heparin sodium fraction. Ethanol was added to this fraction (0.28 N sodium chloride aqueous solution) for crystallization, followed by filtration to obtain 0.58 g of low molecular weight sodium heparin. The average molecular weight of this low molecular weight heparin sodium was 4800, and the anti-factor Xa activity was 126 IU / mg.
[0023]
Example 3
15.0 g of heparin sodium derived from porcine intestinal mucosa was dissolved in 220 ml of ion-exchanged water and cooled to 5 ° C. After cooling, an aqueous solution in which 0.33 g of sodium nitrite was dissolved in 6 ml of ion-exchanged water was added, and 45.8 g of 1 mol / l hydrochloric acid cooled to about 5 ° C. was added, followed by stirring for 15 minutes to carry out a decomposition reaction. Then, 47.4 g of 1 mol / l sodium hydroxide aqueous solution was added to stop the decomposition reaction. Next, the reaction solution was heated to 25 ° C., 0.075 g of sodium borohydride was added and stirred for 4 hours to carry out a reduction reaction, then 22.3 g of 1 mol / l acetic acid aqueous solution was added to adjust the pH to 4, and excess hydrogen was added. Sodium borohydride was decomposed, and 28.1 g of 1 mol / l sodium hydroxide aqueous solution was added to adjust the pH to 7. Subsequently, 6500 g of ethanol was added to the reaction solution for crystallization, followed by filtration to obtain 15.1 g of low molecular weight heparin sodium decomposed and reduced.
Next, a conjugated ion prepared by flowing 750 ml of 0.3 mol / l hydrochloric acid through a column (44 mmφ × 100 mm) packed with 150 ml of DEAE-Cellulofine ™ A-500 m and then 900 ml of ion-exchanged water. In the column packed with the anion exchanger substituted with the above, a whole amount of a solution prepared by dissolving 2.0 g of this decomposed / reduced sodium heparin in 20 ml of ion-exchanged water was flowed and adsorbed on the anion exchanger. Next, 93 ml of ion-exchanged water and 225 ml of 0.19 N sodium chloride aqueous solution were flowed, and then heparin sodium adsorbed with 150 ml of 0.28 N sodium chloride aqueous solution was released and eluted to fractionate a heparin sodium fraction. Ethanol was added to this fraction (0.28 N sodium chloride aqueous solution) for crystallization, followed by filtration to obtain 0.50 g of low molecular weight heparin sodium. The average molecular weight of this low molecular weight sodium heparin was 5100, and the anti-factor Xa activity was 145 IU / mg.
[0024]
Example 4
15.0 g of heparin sodium derived from porcine intestinal mucosa was dissolved in 220 ml of ion-exchanged water and cooled to 5 ° C. After cooling, a solution prepared by dissolving 0.33 g of sodium nitrite in 6 ml of ion-exchanged water was added, and 45.8 g of 1 mol / l hydrochloric acid cooled to about 5 ° C. was added, followed by stirring for 15 minutes to carry out a decomposition reaction. Then, 47.4 g of 1 mol / l sodium hydroxide aqueous solution was added to stop the decomposition reaction. Next, the reaction solution was heated to 25 ° C., 0.075 g of sodium borohydride was added and stirred for 4 hours to carry out the reduction reaction, then 22.3 g of 1 mol / l acetic acid aqueous solution was added to adjust the pH to 4, and excess hydrogen was added. Sodium borohydride was decomposed, and then 28.1 g of 1 mol / l sodium hydroxide aqueous solution was added to adjust the pH to 7. Next, 6500 g of ethanol was added to the reaction solution for crystallization, followed by filtration to obtain 15.1 g of decomposed and reduced low molecular weight sodium heparin.
Next, 750 ml of 0.3 mol / l hydrochloric acid was passed through a column (44 mmφ × 100 mm) packed with 150 ml of DEAE-Cellulofine ™ A-500 m, and then 900 ml of ion-exchanged water was added to the conjugated base ions prepared. A total amount of a solution in which 2.0 g of this decomposed / reduced sodium heparin was dissolved in 20 ml of ion-exchanged water was passed through a column packed with a substituted anion exchanger. Next, after 93 ml of ion-exchanged water is flowed and adsorbed on the anion exchanger, low-molecular-weight heparin sodium adsorbed with 150 ml of 0.28 normal sodium chloride aqueous solution is released and eluted to separate the heparin sodium fraction. I took it. Ethanol was added to this fraction (0.28 N sodium chloride aqueous solution) for crystallization, followed by filtration to obtain 1.12 g of low molecular weight sodium heparin. This low molecular weight heparin sodium had an average molecular weight of 3200 and an anti-factor Xa activity of 86 IU / mg.
[0025]
【The invention's effect】
The method for producing purified heparins of the present invention is excellent in economic efficiency, and can easily produce purified heparins while preventing a decrease in the anti-factor Xa activity of heparin. Moreover, the method for producing low molecular weight heparin or a salt thereof of the present invention can easily produce a low molecular weight heparin or a salt thereof that is excellent in economy and has high anti-factor Xa activity.

Claims (4)

Anion exchange in which heparin, its salt or heparin derivative is adsorbed to an anion exchanger in which an anion is substituted with a conjugated base ion, and then heparin, its salt or heparin derivative is released from the anion exchanger A method for producing purified heparin, a salt thereof or a heparin derivative, characterized by performing treatment with a body. The method for producing purified heparin, a salt thereof, or a heparin derivative according to claim 1, wherein the treatment with an anion exchanger is performed using a liquid chromatography method. Heparin or a salt thereof is subjected to molecular weight lowering treatment, and then the obtained low molecular weight heparin or a salt thereof is adsorbed to an anion exchanger in which an anion is replaced with a conjugated base ion, and then the anion exchanger A method for producing a purified low-molecular-weight heparin, a salt thereof, or a heparin derivative, characterized in that the treatment is performed with an anion exchanger that is liberated from water. The method for producing a purified low-molecular-weight heparin, a salt thereof, or a heparin derivative according to claim 3, wherein the treatment with an anion exchanger is performed using a liquid chromatography method.