JPS63209750A - Adsorbent for purifying viii th blood coagulation factor and process for purifying said factor using the adsorbate - Google Patents

Abstract

PURPOSE:To enhance selective adsorptivity for a VIII th blood coagulation factor by forming an adsorbate for purifying the VIII th blood coagulation factor from water-insoluble porous gel having sulfuric ester groups on a part of its surface and 800,000-100,000,000 exclusion limit mol.wt. CONSTITUTION:An adsorbate for purifying the VIII th blood coagulation factor is formed from water-insoluble porous gel having sulfuric ester groups on at least a part of its surface and 800,000-100,000,000 exclusion limit mol.wt. The water-insoluble porous gel is preferably constituted of a compd. contg. OH groups, and the sulfuric ester groups are preferably introduced by the esterification of the OH groups of the water-insoluble porous gel having OH groups with sulfuric acid. After adsorbing the VII th blood coagulation factor by treating a soln. contg. the VIII th blood coagulation factor with the above described adsorbate, the blood coagulation factor is recovered by eluting the blood coagulation factor.

Description

Detailed Description of the Invention [Industrial Application Field] The present invention relates to an adsorbent for purifying blood coagulation factor (I) for adsorbing and separating blood coagulation factor (I) from body fluids, and a blood coagulation factor (I) purification method using the adsorbent. ■Regarding methods for purifying factors.

[Problems to be solved by conventional technology and invention]

Blood coagulation factor (I) is also called anti-hemophilia factor A. Traditionally, the method of administering blood coagulation factor (I), which is lacking in hemophilia A patients, has been effective and generally used to treat bleeding. It is being said.

However, since only a small amount of blood coagulation factor (I) exists in plasma and is unstable, recovery and purification of blood coagulation factor (I) from human plasma is not easy.

At present, cryoprecipitate and factor (2) concentrates are used to supplement blood coagulation factor (2) to hemophilia A patients.

Cryoprecipitate uses a crude fraction of plasma. Therefore, although it has the advantage of a high recovery rate of factor II, its administration tends to cause allergic reactions, and since it contains a large amount of fibrinogen, the concentration of fibrinogen in plasma increases, and the concentration of factor II There are disadvantages such as the need to inject a large amount of the preparation due to the low concentration.

Factor Ⅰ concentrates do not have these drawbacks, so they are not suitable for hemophilia A.
Although excellent for patient supplementation, factor II concentrates are usually made from crude factor II concentrates such as Cohn's fraction 11 or cryoprecipitate, as shown in U.S. Pat. No. 3,831,018. Since it is produced from fractions by a complicated method that combines polyethylene glycol precipitation fractionation, glycine precipitation fractionation, etc., there is a problem that the recovery rate of factor ① during concentration is very low at about 2096.

In addition, attempts have been made to purify factor II through adsorption, but no adsorbent has been found to be of practical use due to poor adsorption selectivity and low recovery rate of adsorbed factor II. .

In view of these circumstances, the present inventors have conducted extensive research and have discovered an adsorbent that can efficiently, selectively, and highly yield blood coagulation factor adsorbent without using complicated operations, and an adsorbent using the same. We discovered a method for purifying blood coagulation factor Ⅰ.

[Means for solving problems]

That is, the present invention provides an adsorbent for purifying blood coagulation factor (I), which is a water-insoluble porous gel with an exclusion limit molecular weight of 800,000 to 100 million, and has a sulfate ester group on at least a part of its surface. and purification of blood coagulation factor (I), characterized in that a solution containing blood coagulation factor (I) is treated with the adsorbent to adsorb blood coagulation factor (I), and then blood coagulation factor (I) is eluted and recovered. Regarding the law.

〔Example〕

In this specification, the body fluid may be blood, plasma, fractionated components obtained from these, or any other fluid component derived from a living body as long as it contains blood coagulation factor. .

The water-insoluble porous gel used in the present invention preferably has continuous pores of an appropriate size for adsorbing blood coagulation factor (I). In other words, Factor II is a giant molecule with a molecular weight of at least 1 million or more, and in order to adsorb it, it is necessary to create pores with a diameter large enough to allow Factor II to easily penetrate into the gel. is necessary.

There are various methods for measuring pore diameter, and mercury intrusion method is the most commonly used, but it is difficult to apply to hydrophilic gels. Therefore, the exclusion limit molecular weight is often used as a guideline for the pore size of a hydrophilic gel. The exclusion limit molecular weight is the molecular weight that cannot enter the pores (is excluded) in gel permeation chromatography, as stated in books such as "Experimental High Performance Liquid Chromatography" (written by Hiroyuki Hatano and Toshihiko Hana, published by Imosa Kagaku Dojin). It refers to the molecular weight of the molecule that has the smallest molecular weight among molecules. It is known that the exclusion limit molecular weight varies depending on the target compound, and in general, globular proteins, dextran, polyethylene glycol, etc. have been well investigated. It is appropriate to use the values obtained using

As a result of studies using various water-insoluble porous gels with different exclusion limit molecular weights, we found that, contrary to expectations, even gels with exclusion limit molecular weights of around 800,000, which are smaller than the molecular weight of factor It has been shown that the larger the pore size, the greater the adsorption capacity; if the pore size exceeds a certain size, the capacity decreases and proteins other than factor II are adsorbed, so there is an optimal pore size range. It became clear that it would. In other words, if a water-insoluble porous gel with an exclusion limit molecular weight of less than 800,000 is used, the adsorption amount of factor (I) is too small to be practical; or 200
Even if a water-insoluble porous gel was used, an adsorbent that could be put to practical use to some extent was created. As the exclusion limit molecular weight of the water-insoluble porous gel increases, the adsorption amount of factor (2) increases, but eventually reaches a plateau, and when the exclusion limit molecular weight exceeds 100 million, the surface area is too small, so the adsorption amount decreases noticeably.

Therefore, the exclusion limit molecular weight of the water-insoluble porous gel used in the present invention is preferably 800,000 to 100 million, more preferably 2 million to 50 million.

Further, regarding the porous structure of the water-insoluble porous gel, total porosity is preferable to surface porosity, and the pore volume is preferably 20% or more in order to increase the adsorption surface area. The shape of the water-insoluble porous gel can be selected from any shape such as granules, fibers, membranes, and hollow fibers. When using particulate water-insoluble porous gel, the particle size should be 1 to 5,000.
It is desirable that it be can.

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

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

Soft gels such as agarose have the advantage of less nonspecific adsorption compared to gels made of synthetic polymers or inorganic substances, but hard gels (
Polymer hard gel) is more preferable because adsorption and desorption operations can be performed at a faster flow rate.

Porous cellulose gels are particularly preferred because they have the characteristics of both soft and hard gels, and sulfate ester groups can be easily introduced.

There are various methods for introducing a sulfate ester group into a water-insoluble porous gel. Typical methods include a method of directly introducing sulfate ester groups by converting the hydroxyl groups of a water-insoluble porous gel containing hydroxyl groups into sulfate esters using a reagent such as concentrated sulfuric acid.

As a method for fixing a sulfate ester group-containing compound to a water-insoluble porous gel, a method via covalent bonding is preferred because of its high stability.

Examples of the sulfate group-containing compound used in the present invention include sulfate 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 is preferred.

Among these, partially sulfated esters of polyhydric alcohols, particularly sulfated saccharides, are preferred because they contain both sulfate ester groups and functional groups necessary for fixation, and have high biocompatibility and activity. Furthermore, sulfate-esterified polysaccharides are particularly preferred because they can be easily fixed in water-insoluble porous gels.

Examples of compounds containing sulfate ester groups include alcohols such as ethanolamine, ethylene glycol, glycerin, anisole, pentaerythritol, sorbitol, polyvinyl alcohol, and polyhydroxyethyl methacrylate, sulfate esters of polyhydric alcohols, glucose, xylose, threose, galactose, fucose,
Saccharides such as galactosamine, uronic acid, glucuronic acid, ascorbic acid, sulfuric acid esters of carbohydrates, heparin, dextran acid, chondroitin sulfate, chondroitin polysulfate, heparan sulfate, keratan sulfate, xylan sulfate, caronine sulfate, cellulose sulfate, chitin sulfate, Sulfated polysaccharides such as chitosan sulfate, pectin sulfate, inulin sulfate, arginine sulfate, glycogen sulfate, polylactose sulfate, carrageenan sulfate, sulfated starch, polyglucose sulfate, laminarin sulfate, galata sulfate, levan sulfate, mebesulfate, etc. Examples include, but are not limited to.

Among the sulfate-esterified polysaccharides, those having a molecular weight of 100,000 or less are particularly preferable because they hardly adsorb fibrinogen or the like. Further, among the sulfated polysaccharides, those having a sulfur content of 5 to 20% are preferred because of their high adsorption activity.

The number of sulfate groups introduced is preferably 0.19 to loa+nol per ml of adsorbent. 0.1μs
If it is less than +o1, the adsorption capacity is not sufficient, and if it is less than 10 mm
If it exceeds 1, non-specific adsorption, especially fibrinogen adsorption, will be too large, making it difficult to put it to practical use. More preferably, it is 1 to 500 μll1O1.

In order to separate factor ■ from a solution containing factor ■ using the adsorbent according to the present invention, after bringing the solution containing factor ■ into contact with the adsorbent to adsorb factor ■, unadsorbed components After washing, factor ① may be eluted.

There are various methods for eluting the adsorbed factor ①, such as increasing the temperature and changing the pH, but the method of eluting with an aqueous solution with high ionic strength is preferred because post-treatment is simple. If components other than factor ① are adsorbed depending on the type of adsorbent, factor ① may be adsorbed using the so-called gradient-end method in which the ionic strength, pH, etc. are continuously changed, or by the stepwise method in which the ionic strength, pH, etc. are changed stepwise.
Factors can also be separated.

The present invention will be explained in more detail below using Examples, but the present invention is not limited to these Examples.

Example 1 CK gel A-3 (trade name, porous cellulose gel)
Made by Chisso ■, exclusion limit molecular weight of globular protein 50 million,
A sample of 10 ml (particle size 45-105 μts) was taken and dried by critical point drying in ethanol. The dried gel was suspended in 10 ml of well-dehydrated pyridine and cooled on ice. 2 ml of chlorosulfonic acid was added to the solution while stirring, and after the dropwise addition was completed, stirring was continued for another 10 minutes. After the reaction was completed, the gel was filtered and washed with pyridine and water to obtain a cellulose gel having sulfate groups introduced onto its surface. The amount of sulfate ester group introduced was 110 μmol per ml of adsorbent.

Example 2 A cellulose gel having sulfate ester groups introduced onto the surface was obtained in the same manner as in Example 1, except that the amount of chlorosulfonic acid was changed to 3 ml and the stirring time after dropping was changed to 60 minutes. The amount of sulfate ester group introduced is 750 per ml of adsorbent.
It was μlll01.

Example 3 Cellulofine GCL-2, a porous cellulose gel
000 (Product name, manufactured by Chisso ■, exclusion limit molecule ff13 million for globular proteins, particle size 45-1057M) LOml
After washing with water and suction filtering, add dimethyl sulfoxide 6 to this.
ml, 2N NaOH2,6ml and 1.5ml of 1-epichlorohydrin were added and stirred at 40°C for 2 hours. After the reaction, the gel was filtered and washed with water to obtain a cellulose gel into which epoxy groups were introduced.

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

2g of the gel obtained has a molecular weight of about 5000. Sulfur content 1
A solution prepared by dissolving 4 g of 5% sodium dextran sulfate in 0.1 ml of phosphate buffer (pH 8.0) was added, and the mixture was shaken at room temperature for 16 hours. After reaction NaCNBHs20
After adding B and stirring at room temperature for 30 minutes, the mixture was heated at 40°C for 4 hours, and the gel was filtered and washed with water to obtain a cellulose gel on which dextran sulfate was fixed. The amount of dextran sulfate introduced was 3.4 mg per ml of adsorbent.

Example 4 A cellulose gel on which dextran sulfate was fixed was obtained in the same manner as in Example 3, except that dextran sulfate having a molecular weight of about 500,000 and a sulfur content of 4.5% was used. The amount of dextran sulfate introduced was 5.4 m per ml of adsorbent.
It was g.

Comparative example cellulose gel was Cellulofine GC70G (trade name, manufactured by Chisso ■, exclusion limit molecular weight of globular protein 400,000,
A cellulose gel with sulfate ions introduced onto the surface was obtained in the same manner as in Example 1, except that a layer with a particle size of 45 to 105 μm was used. - The amount of sulfate ester group introduced was 250 μsol per ml of adsorbent.

Example 5 1 ml of each gel synthesized in Examples 1 to 4 and Comparative Example was placed in a test tube, 6 ml of citrated human plasma was added thereto, and the gel was incubated at 37° C. for 1 hour with stirring.

Table 1 shows the activity of factor ① and the concentration of fibrinogen in the plasma after adsorption. Factor Ⅰ activity was measured by the APTT method.

Margin below C].

Table 1 As is clear from the results shown in Table 1, the adsorbent according to the present invention (Examples 1 to 4) was obtained using cellulose gel with an exclusion limit molecular weight of 400,000, and the large adsorbent (comparative example) ) shows superior adsorption ability for factor ①.

Furthermore, among the adsorbents of the present invention, the adsorbents obtained in Examples 1 to 3 are the adsorbents obtained using dextran sulfate with a molecular weight of 500,000 and a sulfur content of 4.5% (Example 4).
The adsorbent (Example 2), which exhibits superior selectivity compared to the adsorbents obtained in Examples 1 and 3, and in which the amount of sulfate ester groups introduced is 750 μsol per ml of adsorbent, is selective compared to the adsorbents obtained in Examples 1 and 3. It can be seen that they are inferior in gender.

Example 6 1 ml of the adsorbent obtained in Example 1 was packed into a polypropylene column, and 3 ml of human plasma was poured into it. Next, 10 ml of physiological saline was poured to wash away unadsorbed components, and then a solution with the salt concentration changed continuously from O, 15M to 2M was poured to flow out of the column (gradient elution).
The resulting solution was collected using a fraction collector.

Total protein concentration was determined for each fraction. Furthermore, the elution pattern of factor Ⅰ and fibrinogen was examined by enzyme immunoassay.

The results are shown in FIG. In Figure 1, the elution patterns of factor Ⅰ and fibrinogen are shown as relative concentrations, which are expressed by the peak heights obtained by developing color using enzyme immunoassay and measuring the 60Qnm glue fraction (reflection) using a densitometer. .

From the results shown in FIG. 1, it can be seen that blood coagulation factor (2) is eluted at high salt concentrations and is eluted separately from other adsorbed proteins, especially fibrinogen.

Example 7 1 ml of the adsorbent synthesized in Example 3 was packed into a polypropylene column, and 0.05M) lis(hydroxymethyl)aminomethane-hydrochloric acid buffer (pH 7.4) containing 0.154M NaCj was added in advance. Equilibration was carried out.Next, Cryobulin (trade name, manufactured by Immuno AG), which is a blood coagulation factor II concentrate, was added to the same buffer solution at a concentration of factor II of 50%.
After adding 3 ml of the solution dissolved in the same buffer solution and washing the unadsorbed components with 5 ml of the same buffer solution, the same buffer solution with the salt concentration changed in two stages, 0.5M and 1.5M, was added. The liquid that flowed out from the column was separated into several fractions.

The flow of protein and each fraction (B (3 m
l), C (5.4ml), D-I (2ml)
, D-II (2 ml), E-I (2 ml), and E-II (2 ml)), the M1 activity determined by the A-PTT method is shown in FIG.

From the results shown in FIG. 2, it was shown that almost 100% of factor ① was adsorbed and could be separated and recovered by increasing the salt concentration. The recovery rate was 61%, specific activity (per total protein)
The specific activity (activity of the factor) was 145.12, which was about 10 times higher than the specific activity of the original solution, which was 14.03.

〔Effect of the invention〕

The adsorbent of the present invention and the method for purifying factor ① using the adsorbent have the effect of separating and recovering factor ① efficiently, selectively, and in high yield without using complicated operations.

[Brief explanation of the drawing]

Figure 1 shows that human plasma was poured into a column filled with the adsorbent of the present invention obtained in Example 1, and then 0.15M to 2M
2 is a graph showing the salt concentration, total protein concentration, and elution pattern of factor ① and fibrinogen in each fraction when gradient elution is performed at a salt concentration of . Figure 2 shows that after flowing a buffer solution in which cryobulin, which is a blood coagulation factor (I) concentrate, was passed through a column filled with the adsorbent of the present invention obtained in Example 3, 0.5M and 1.5M were dissolved.
FIG. 2 is a graph showing changes in salt concentration and protein concentration when eluted with a buffer solution with two levels of salt concentration in M and factor Ⅰ activity in each fraction. FIG. 1 figure

Claims (1)

[Scope of Claims] 1. A water-insoluble porous gel with an exclusion limit molecular weight of 800,000 to 100 million, for purification of blood coagulation factor VIII, characterized by having a sulfate ester group on at least a part of its surface. adsorbent. 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 a sulfate ester group is introduced by sulfuric acid esterification of the hydroxyl group of a water-insoluble porous gel containing a hydroxyl group. 4. The adsorbent according to claim 1, wherein the sulfate ester group-containing compound is fixed to a water-insoluble porous gel by covalent bonds. 5. The adsorbent according to claim 4, wherein the sulfate group-containing compound is a sulfate-esterified polysaccharide. 6 The content of sulfate ester groups is 0.1 per ml of adsorbent.
The adsorbent according to claim 1, which has a molecular weight of μ to 10 mmol. 7 The content of sulfate ester groups is 1 to 5 per ml of adsorbent.
The adsorbent according to claim 1, which has an amount of 00 μmol. 8. The adsorbent according to claim 5, wherein the sulfated polysaccharide has a molecular weight of 100,000 or less. 9. A solution containing blood coagulation factor VIII is made into a water-insoluble porous gel with an exclusion limit molecular weight of 800,000 to 100 million, and has a sulfate ester group on at least a part of its surface. A method for purifying blood coagulation factor VIII, which comprises treating it with a factor VIII adsorbent to adsorb blood coagulation factor VIII, and then eluting and recovering blood coagulation factor VIII.