JPH0779693B2 - Blood coagulation factor IX adsorbent and method for purifying blood coagulation factor IX using the same - Google Patents

Description

TECHNICAL FIELD The present invention relates to a blood coagulation factor IX adsorbent and a method for purifying blood coagulation factor IX using the adsorbent.

[Problems to be Solved by Conventional Techniques and Inventions] Blood coagulation factor IX, which is also called CHRISTMAS factor, has been conventionally lacking in the treatment of bleeding in hemophilia B patients. The method of administering the factor is generally practiced.

However, since blood coagulation factor IX exists only in a trace amount in plasma and is unstable, it is not easy to recover and purify blood coagulation factor IX from human plasma.

At present, factor IX concentrated preparations are used for supplementing blood coagulation factor IX to hemophilia B patients.

Usually, factor IX concentrated preparations are produced by a complex method that combines adsorption with barium citrate or barium sulfate, ammonium sulfate fractionation, DEAE-Sephadex chromatography, affinity chromatography with heparin agarose or benzamidine sepharose, etc. (SP Bajaj et al., "Preparative Biochemistry" 11
(4), 397-412, 1981 ", B. Osterud et al.," Journal of Biological chemistry, The ", 253 (17).
Vol., Pp. 5946-5952, 1978 ")
The loss of protein due to it is inevitable.

[Means for Solving Problems] As a result of intensive studies conducted by the present inventors, the water-insoluble porous gel has an exclusion limit molecular weight of 50,000 or more and has a sulfate ester group on at least a part of its surface. It has been found that the use of an adsorbent solves the above-mentioned problems, and that blood coagulation factor IX in body fluid can be adsorbed and separated efficiently, selectively and in high yield without complicated operations. It was

That is, the present invention is a blood coagulation factor IX adsorbent characterized by having a water-insoluble porous gel having an exclusion limit molecular weight of 50,000 or more and having a sulfate ester group on at least a part of its surface, and a blood coagulation factor IX. A method for purifying blood coagulation factor IX, which comprises treating a solution containing factor IX with the adsorbent to adsorb the blood coagulation factor IX and then eluting and collecting the blood coagulation factor IX .

[Examples] The term "body fluid" as used herein refers to any of blood, plasma, fractionated components obtained from these, and other biologically derived liquid components containing blood coagulation factor IX. Good.

The water-insoluble porous gel used in the present invention preferably has a large number of continuous pores having an appropriate diameter. That is, blood coagulation factor IX is a small molecule having a molecular weight of 57,000, and it is necessary that factor IX can easily enter the gel in order to adsorb it.

There are various methods for measuring the pore size, and the mercury intrusion method is most often used, but it is difficult to apply it to hydrophilic gels. The exclusion limit molecular weight is often used as a measure of the pore size of hydrophilic gels. Exclusion limit molecular weight is, as described in the publications (for example, Hiroyuki Hatano, Toshihiko Hanai, Experimental High Performance Liquid Chromatography, Kagaku Dojin, etc.) The molecular weight of the molecule having the smallest molecular weight. It is known that the exclusion limit molecular weight differs depending on the target compound, and in general, globular proteins, dextran, polyethylene glycol, etc. have been well investigated, and obtained using the most similar globular proteins (including virus). It is appropriate to use different values.

As a result of examination using various water-insoluble porous gels having different exclusion limit molecular weights, unexpectedly, even those having an exclusion limit molecular weight of about 50,000, which is smaller than the molecular weight of factor IX, show some adsorption capacity, and the pore size It was clarified that the larger the size, the larger the adsorption capacity, but rather the decreased capacity or the adsorption of proteins other than factor IX, that is, the optimum pore size range exists. That is, when a water-insoluble porous gel having an exclusion limit molecular weight of less than 50,000 is used, the adsorption amount of factor IX is small and it cannot be used practically, but the exclusion limit molecular weight is 50,000 to 120,000 and the molecular weight of factor IX is Even if a water-insoluble porous gel close to the above was used, an adsorbent that could be put to practical use was obtained to some extent. On the other hand, as the exclusion limit molecular weight increases, the adsorption amount of factor IX increases, but eventually reaches a ceiling, and when the exclusion limit molecular weight exceeds 100 million, the surface area is too small and the adsorption amount remarkably decreases.

Therefore, the exclusion limit molecular weight of the water-insoluble porous gel used in the present invention is 50,000 or more, and more preferably
It is 100,000 or more and 2 million or less.

Next, regarding the porous structure of the water-insoluble porous gel, the total porosity is preferable to the surface porosity, and the pore volume is preferably 20% or more. As the shape of the water-insoluble porous gel, any shape such as a granular shape, a fibrous shape, a film shape, or a hollow fiber shape can be selected. When a particulate water-insoluble porous gel is used, its particle size is preferably 1 to 5000 μm.

The water-insoluble porous gel used in the present invention may be either organic or inorganic, but it is desirable that the target blood component other than Factor IX is less adsorbed (so-called non-specific adsorption).

Typical examples of the water-insoluble porous gel used in the present invention include soft gels such as agarose, dextran and polyacrylamide, porous glass, inorganic porous materials such as porous silica gel, polymethylmethacrylate, polyvinyl alcohol, styrene-divinyl. Examples thereof include, but are not limited to, synthetic polymers such as benzene copolymers and porous polymer hard gels made from natural polymers such as cellulose.

Among them, porous cellulose gel is preferable because it has less nonspecific adsorption.

There are various methods of introducing a sulfate ester group into the water-insoluble porous gel, a method of fixing a sulfate ester group-containing compound to the water-insoluble porous gel, and a chlorosulfonic acid when the water-insoluble porous gel contains a hydroxyl group. A typical method is to introduce a sulfate ester group directly using a reagent such as concentrated sulfuric acid.

As a method for fixing the sulfate ester group-containing compound, a method via a covalent bond has high stability and is preferable.

Representative examples of the sulfuric acid ester group-containing compound used in the present invention include sulfuric acid esters of hydroxyl group-containing compounds such as alcohols, sugars, polyhydric alcohols and carbohydrates. A compound having a functional group that can be used for immobilization on a polymer gel is preferable. Among them, a partially sulfated ester of a polyhydric alcohol, particularly a sulfated ester of a saccharide contains both a sulfate ester group and a functional group necessary for immobilization and is preferable because of high biocompatibility and activity. Further, the sulfated polysaccharide is more preferable because it can be easily immobilized on the water-insoluble porous gel.

As a representative example of a sulfate ester group-containing compound, ethanolamine, ethylene glycol, glycerin, anisole, pentaerythritol, sorbitol, polyvinyl alcohol, alcohols such as polyhydroxyethyl methacrylate, sulfate ester of polyhydric alcohol, glucose, xylose, threose, Galactose, fucose, galactosamine, uronic acid, glucuronic acid, sugars such as ascorbic acid, sulfate ester of carbohydrates,
Heparin, dextran acid, chondroitin sulfate, chondroitin polysulfate, heparan sulfate, keratan sulfate, xylan sulfate, caronine sulfate, cellulose sulfate, chitin sulfate, chitosan sulfate, pectin sulfate, inulin sulfate, arginine sulfate, glycogen sulfate, polylactose sulfate,
Examples thereof include sulfate esterified polysaccharides such as carrageenan sulfate, sulfated starch, polyglucose sulfate, laminarin sulfate, galactan sulfate, levan sulfate, and mepesulfate, but are not limited thereto.

The amount of sulfate ion introduced is 0.01 μmol-10 ml / ml.
mmol is preferred. If it is 0.1 μmol or less, the adsorption capacity is not sufficient, and if it is 10 mmol or more, nonspecific adsorption is too much to make practical use difficult.

From the solution containing Factor IX, using the adsorbent according to the present invention,
In order to separate Factor IX, a solution containing Factor IX is contacted with an adsorbent to adsorb Factor IX, and then unadsorbed components are washed and then Factor IX is eluted.

As a method for eluting the adsorbed Factor IX, there are various methods such as changing the pH, but a method of desorbing with an aqueous solution having high ionic strength is preferable because the post-treatment is simple and easy. When components other than Factor IX are adsorbed depending on the type of adsorbent, Factor IX can be separated by a so-called gradient method in which ionic strength, pH, etc. are changed continuously or stepwise.

Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these.

Example 1 CK gel A-3 which is a porous cellulose gel (Chisso Corporation)
Made, globular protein exclusion limit molecular weight 5 × 10 ⁷ , particle size 45-105
μm) 10 ml was taken and dried in ethanol by critical point drying. Each dried gel was suspended in 10 ml of well dehydrated pyridine and cooled with ice. Chlorosulfonic acid 2m
l was added dropwise with stirring, and stirring was continued for 10 minutes after the addition was completed.
After completion of the reaction, the gel was filtered off and washed with pyridine and water to obtain a cellulose gel having sulfate ions introduced on its surface.

Example 2 Cellulofine GCL-2000 which is a porous cellulose gel
(Chisso Corporation, exclusion limit molecular weight of globular protein 3 × 10
⁶ ) 10 ml was washed with water and suction filtered, ⁶ ml of dimethyl sulfoxide, 2.6 ml of 2N NaOH and 1.5 ml of epichlorohydrin were added thereto, and the mixture was stirred at 40 ° C. for 2 hours. After the reaction, the gel was separated by filtration and washed with water to obtain a cellulose gel having an epoxy group introduced therein.

6 ml of concentrated aqueous ammonia was added thereto, and the mixture was reacted at 40 ° C. for 2 hours to obtain an aminated cellulose gel.

A solution prepared by dissolving 4 g of dextran sulfate sodium having a molecular weight of about 5000 in 8 ml of 0.1 M phosphate buffer (pH 8.0) was added to 2 g of the obtained gel, and the mixture was shaken at room temperature for 16 hours. After the reaction NaCNBH ₃ 20 mg
Was added, and the mixture was stirred at room temperature for 30 minutes, heated at 40 ° C. for 4 hours, filtered and washed with water to give a dextran sulfate-immobilized cellulose gel.

Example 3 Cellulofine GC700 (Chisso Co., Ltd.)
Made, globular protein exclusion limit molecular weight 4 × 10 ⁵ , particle size 45 ~ 105
The same procedure as in Example 1 except that the cellulose gel having sulfate ion introduced on the surface was obtained.

Example 4 Cellulofine GC200m (Chisso Corporation)
Made, globular protein exclusion limit molecular weight 1.2 × 10 ⁵ , particle size 45-10
The same procedure as in Example 1 except that the cellulose gel having sulfate ions introduced on the surface was obtained.

Reference example Cellulofine GCL-90 (Chisso Corporation)
Made, globular protein exclusion limit molecular weight 3.5 × 10 ⁴ , particle size 45 ~ 10
The same procedure as in Example 1 except that the cellulose gel having sulfate ions introduced on the surface was obtained.

Example 5 Each of the gels synthesized in Examples 1 and 4 and Reference Example was placed in a 1 ml test tube, and 6 ml of citrate-added human plasma was added thereto to give 1
The plate was incubated at 25 ° C for 2 hours with stirring every 5 minutes. The activity of factor IX in plasma after adsorption was measured by the APTT method. The results are shown in Table 1.

Example 6 25 ml of the gel obtained in Example 1 was packed in a polycarbonate column, and 35 ml of human plasma was flown through the column. Next, 125 ml of physiological saline was poured to wash unadsorbed proteins, and then 0.38M
The adsorbed protein was eluted by flowing NaCl. 22% of the applied amount of factor IX was found in the major eluted protein. The specific activity (factor IX activity / protein amount) was increased about 5 times.

As is clear from the results shown in Table 1, the exclusion limit molecular weight of the adsorbents of the present invention (Examples 1 and 4) was 3.5.
Compared to the one using a lot of cellulose gel (reference example)
It can be seen that it exhibits excellent adsorption ability for factor IX.

[Advantages of the Invention] According to the adsorbent of the present invention and the method for purifying Factor IX using the adsorbent, it is possible to purify Factor IX easily and efficiently.

Claims (6)

[Claims] 1. A blood coagulation factor IX adsorbent, which is a water-insoluble porous gel having an exclusion limit molecular weight of 50,000 or more and having a sulfate ester group on at least a part of its surface. 2. The adsorbent according to claim 1, wherein the water-insoluble porous gel is composed of a hydroxyl group-containing compound. 3. The adsorbent according to claim 2, wherein the sulfuric acid ester group is introduced by converting the hydroxyl group of the hydroxyl group-containing water-insoluble porous gel into a sulfuric acid ester. 4. The adsorbent according to claim 1, wherein the sulfate ester group-containing compound is immobilized on the water-insoluble porous gel by a covalent bond. 5. The adsorbent according to claim 4, wherein the sulfate ester group-containing compound is a sulfated polysaccharide. 6. A solution containing blood coagulation factor IX, which is a water-insoluble porous gel having an exclusion limit molecular weight of 50,000 or more and having a sulfate ester group on at least a part of its surface. Blood coagulation by treatment with factor IX adsorbent
A method for purifying blood coagulation factor IX, which comprises adsorbing factor IX and then eluting and collecting blood coagulation factor IX.