WO1993009869A1 - Adsorbent for blood coagulation factor viii - Google Patents

Abstract

An adsorbent for blood coagulation factor VIII comprising a composite prepared by immobilizing a polycationic molecule onto a water-insoluble carrier, wherein the molecular weight of said molecule is at least 15,000 and the positive charge density is 1-200 νeq per milliliter of the composite. The adsorbent has such a remarkably specific adsorption behavior against the factor VIII that it can adsorb and recover the same from a solution containing the same at a high selectivity in a high yield.

Description

 Specification

 Adsorbent for blood coagulation factor V II I

 The present invention relates to an adsorbent for blood coagulation factor VIII (hereinafter often simply referred to as "factor VIII"). More specifically, molecules having a polycation suitable for efficiently adsorbing and recovering factor VIII from a solution containing various plasma proteins including factor VIII are bound and immobilized on a water-insoluble carrier. And an adsorbent made of a composite.

Background art

 Blood coagulation is a phenomenon in which various types of factors are involved in a complex manner. The qualitative or quantitative deficiency of these factors is observed as a hemorrhagic disease. Hemophilia A is an example of a congenital bleeding disorder. Hemophilia A is caused by a deficiency of a protein called factor VIII.In order for a person with hemophilia A to live as a healthy person, it is necessary to replace the lacking factor VIII. is there.

 In order to supplement factor VIII, a plasma fractionation product called factor VIII concentrate is widely used. Factor V II concentrates are usually prepared by using a precipitate from plasma called cryoprecipitate as a raw material and removing contaminating proteins such as fibrinogen and fibronectin.

[S.J.S lichtereta 1.: T ransf usion, Vol. 16, PP. 6 16-62 (1976)]].

 The recipe recipe can be obtained by centrifuging the precipitate generated by thawing fresh frozen plasma at a low temperature. However, assuming that the factor V III contained in plasma is 100%, the recovery of factor V III in cryoprecipitate is as low as 40 to 50%. Furthermore, since cryoprecipitate contains a large amount of contaminating proteins, it is necessary to remove those contaminating proteins.In general, adsorption is performed by immobilizing a low-molecular-weight ligand such as a getylaminoethyl group on a water-insoluble carrier. Michalskieta 1.: Vox Sang, Vo 1.55, pp. 202-210 (19988) is often used, but this adsorbent is However, when blood coagulation factor IX (hereinafter often simply referred to as “factor IX”) coexists with factor VIII, the factor VIII-enriched preparation is adsorbed because more factor IX is adsorbed. A more complicated purification process is required before preparation, and the factor VIII recovery in the final preparation is reduced to about 20%.

 In order to solve the above problems, it is desirable to develop an adsorbent capable of efficiently adsorbing and recovering factor VIII directly from plasma or from the supernatant after fractionation of cryoprecipitate. Had been.

Factor VIII is a protein with a molecular weight of about 300,000. It is known from the amino acid sequence analysis that there is a region rich in acidic amino acids [G.A.V ehareta 1.: Natur'e, Vol. 3.12, pp. 3 3 7-3 4 2 (1 998 4)]. Furthermore, it is known to exist in blood as a complex with a von Willebrand factor having a multimeric structure with a molecular weight of 1,000,000 to 2,000,000 (Z.M. R uggerieta 1.: Blood, Vol. 57, PP. 114-11 4 3 (1 98 1)).

 As a result of intensive studies, the present inventors have found that in order to efficiently adsorb such a protein having a huge molecular weight, it has a large number of anion-exchange groups and a sufficient length. However, they have found that an adsorbent having a substance capable of firmly adsorbing by interacting with the complex at a plurality of points is suitable. Furthermore, as a result of intensive studies to develop such an adsorbent, the adsorbent consisting of a complex in which a molecule having a polycation is bonded to and immobilized on a water-insoluble carrier has an extremely specific adsorption to factor VIII. — It showed that it was able to adsorb and recover Factor VIII with high selectivity and high yield from solutions containing various plasma proteins including Factor VIII. The present invention has been completed based on these findings.

Disclosure of the invention

Accordingly, one and primary object of the present invention is to provide a solution containing various plasma proteins, including factor VIII, from a solution containing factor VIII. An adsorbent forest capable of directly and specifically adsorbing factor I, and capable of easily desorbing and recovering adsorbed factor VIII.

 The above and other objects, features, and advantages of the present invention will become apparent from the following detailed description of the present invention and the appended claims.

 According to the present invention, the molecular weight of a polycation-containing molecule is at least 15,500, unlike a complex formed by binding and immobilizing a molecule having a polycation to a water-insoluble carrier. Further, there is provided an adsorbent for blood coagulation factor VIII, wherein the positive charge density of the complex of 1 milliliter is from leq to 200 eq.

 The molecule having a polycation immobilized on the adsorbent of the present invention refers to a molecule having a molecular weight of at least 150000 and having a large number of anion exchange groups in the molecule. Examples of the anion exchange group include a primary amino group, a secondary amino group, a tertiary amino group, a primary ammonium base and the like. One of these anion exchange groups may be present alone in the molecule having a polycation, or two or more may be present.

Examples of such a molecule include a homopolymer of a basic vinyl monomer having an anion exchange group in a side chain, or a vinyl copolymer which can be copolymerized with the basic vinyl monomer. Copolymer with monomer Can be mentioned. Further, instead of the basic vinyl monomer, a vinyl monomer having a side chain to which an anion exchange group can be added after polymerization may be used. Further, there may be mentioned polypeptides containing basic amino acids such as lysine and arginine.

Examples of the basic vinyl monomer having an anion exchange group in the side chain include a vinyl monomer having a primary amino group, a secondary amino group, a tertiary amino group, and a quaternary ammonium base. Body included. Examples of such a biel monomer include acrylic acid derivatives, methacrylic acid derivatives, acrylic acid amide derivatives, and metaacrylic acid amines each having an amino group. Examples thereof include vinyl derivatives having a nitrogen-containing aromatic ring group such as a pyridyl group and an imidazolyl group in the side chain, and styrene derivatives substituted with an amino group. Specific examples include dimethylaminoethyl acrylate, dimethylaminoethyl acrylate, dimethylaminopropyl acrylate, 3-dimethylaminopropyl 2—hydroxypropylamine Rate, 3—Jethylamino2—Hydroxypropylacrylate, N—Dimethylaminoethylacrylate, N—Nethylethylaminoethylacrylate, Dimethylaminoethylmethacrylate Rate, getylaminoethyl methacrylate, getylaminopropyl methacrylate, 3—dimethylamine 2—hydroxypropyl propylmethacrylate, 3—getylamine 1—2— histamine Dro kiss Mouth pill methacrylate, N-dimethylamino methacrylate amide, N-ethylaminoethyl methacrylate, and the following formula:

(However, R is one CH ₃ or one C ₂ H ₅ , X is (CH ₂ ) n is an integer of 3)

Styrene derivatives represented by

のよ うな、 重合後にァミ ノ化のできる側鎖を有するビニル単— 量体を用いることもできる。 Also, for example, _CHz -CH

Such a vinyl monomer having a side chain that can be aminated after polymerization can also be used.

Examples of vinyl monomers that can be copolymerized with the basic vinyl monomers described above include N-bierpyrrolidone, acrylates, meta-atalylates, and N-alkyl acrylates. And amides of N-alkyl methacrylate. Specific preferred examples of such a vinyl monomer include hydroxyxethyl acrylate and hydroxypropynoleacrylate. And hydroxypropyl methacrylate, and hydroxypropyl methacrylate.

 Among the above examples of molecules having a polycation, the basic butyl monomer may be an acrylic acid derivative, a metaacrylic acid derivative, an acrylic acid amide derivative or a methacrylic acid derivative having an amino group. Using vinyl acrylamide derivatives, vinyl monomers that can be copolymerized with basic vinyl monomers include acrylic esters, metaacrylic esters, and N-alkylacrylic acids. Copolymers obtained using amides or N-alkyl methacrylates are preferred. '

The molecular weight of the molecule having a polycation of the present invention is at least 15,000. When the molecular weight is smaller than this, sometimes factor VIII is an extremely large protein, so that it is difficult to expect a sufficient interaction between the molecule and factor VIII. In addition, in the adsorbent of the present invention, the positive charge density of the complex (1 mil / liter) composed of a molecule having a polycation and a water-insoluble carrier constituting the adsorbent is leq to 200 eq. . If the positive charge density of the complex per milliliter is less than 1 μeq, factor VIII is not sufficiently adsorbed, and if it exceeds 200 μeq, the factor Problems such as difficulty in elution due to strong adsorption of factor VIII and increase in adsorption of other proteins will arise. The positive charge density of the composite constituting the adsorbent of the present invention is determined by the following method. Swollen with water Put 1 milliliter of adsorbed material into a plastic column, wash with 1N hydrochloric acid, water, and 1N aqueous sodium hydroxide in that order, and adjust the pH of the liquid falling from the cam to 6.5. Wash with water until it is about Then, transfer the adsorbent to a test tube of 50 milliliters, add 20 milliliters of 0.01N hydrochloric acid, and shake gently for 24 hours. Transfer 5 milliliters of the supernatant to a 20 milliliter Erlenmeyer flask, add phenolphthalein aqueous solution as an indicator, and use a 0.01N aqueous sodium hydroxide solution to add Determine the positive charge density by performing titration using a pellet.

 As the water-insoluble carrier used in the present invention, any of a hydrophilic carrier and a hydrophobic carrier can be used, but the hydrophobic carrier causes non-specific adsorption of proteins other than factor VIII to the carrier. Gives better results.

 The water-insoluble carrier is not particularly limited as long as it has a functional group that can be used for immobilizing a molecule having a polycation by chemical bonding. The chemical bond is preferably a covalent bond. Even when the water-insoluble carrier itself does not have such a functional group, one that can generate a functional group that can be used for immobilizing a molecule having a rebolication by appropriate chemical modification is used in the present invention. be able to.

As the shape of the water-insoluble carrier, any known shape such as a particle shape, a fibrous shape, a hollow fiber shape, and a membrane shape can be used, but handling as an adsorbent In view of the above, particles and fibrous materials are preferred.

 Among such water-insoluble carriers, examples of the particulate carrier include agarose ^, dextran-based, cellulose-based and other natural polymer-based carriers, polyacrylamide-based, polyamide-based, and poly- amide-based carriers. Synthetic polymer-based carriers such as ester-based, polyurethane-based, and vinyl compound polymers can be mentioned. When a carrier having a porous structure is used, the surface area of the carrier is increased, so that the molecule having immobilized polythione and the factor VIII can be easily brought into contact with each other. It gives good results with increased volume. In particular, a polymer of a vinyl compound hydrophilized by one unit of a hydrophilic monomer can easily form a carrier having a porous structure, and can easily obtain a carrier having high physical strength. Gives favorable results when incorporated into the purification process of Among them, a crosslinked copolymer having a bul alcohol unit often reduces the non-specific adsorption of a protein to a carrier, and gives a favorable result in many cases. In addition, as the fibrous carrier, known fibers such as a cellulose-based fiber and a polyester-based fiber can be used. When a fibrous carrier is used, the fiber diameter is preferably in the range of 0.02 denier or 10 denier. '' The particulate support having a porous structure has an exclusion limit molecular weight in the range of 1,000 to 100,000,000, and an average particle size of 5 to 1,000 μm. πι is preferred.

Binding and immobilization of polycation-containing molecules to water-insoluble carriers It is better to use a chemical bond, especially a covalent bond, for the conversion. For this purpose, a known method for activating a water-insoluble carrier and a method for immobilizing a molecule having a polycation, which are generally used in immobilized enzymes and affinity chromatography, can be used [Kenichi Kasai et al .: “Affinity mouth mouth one”, published by Tokyo Chemical Industry Co., Ltd. (1991):]. Further, if necessary, a molecule (spacer) having an arbitrary length can be introduced between the water-insoluble carrier and the molecule having a polycation.

 The active group generated by activating the water-insoluble carrier is a group capable of reacting with a nucleophilic reactive group having an active hydrogen such as an amino group, a carboxyl group, a hydroxyl group, or a thiol group of a molecule having a polycation. Typically, epoxy group, tresil group (tresy 1 gr

0 up) (— O— S 0 ₂ — CH ₂ — CF ₃ ), imidazolyl carbamate, imidcarbonate, carbonate, formyl, bromoacetyl, halogenated triazine, etc. Can be mentioned.

 The adsorbent of the present invention can be obtained by contacting a solution containing various plasma proteins containing Factor VII

1 Adsorb factor II. The solution containing various plasma proteins including factor VIII at this time may be plasma or a product from plasma. The products from plasma include the above-mentioned cryoprecipitate, The supernatant after separation and removal of the pithate, plasma from which some or all of the contaminant components have been removed by appropriate treatment with an ion exchange resin or the like can be mentioned.

 The adsorbent of the present invention is capable of adsorbing factor VIII in plasma under various conditions, but usually adsorbs at normal temperature and normal pressure. It is preferred in terms of sex. The temperature should be within the stable range of Factor V I I I, but is usually between 0 and 50 ° C. Although the adsorption time is not particularly limited, it is usually in the range of 10 minutes to 2 hours, and an appropriate time can be selected depending on the temperature and the method of adsorption.

 Regarding the method of adsorption, either a batch method or a column method can be used, but when a large amount of plasma is to be treated, the batch method is often preferred because it can be processed in a shorter time.

Regarding the amount of adsorbent to be used, the lower the value of the adsorbent / plasma ratio is, the more advantageous from the viewpoint of cost. It can be used in the range of Z20 to 110.

BEST MODE FOR CARRYING OUT THE INVENTION

 The present invention will be described in more detail in the following examples, but the present invention is not limited to the examples.

 Example 1

As a water-insoluble carrier, a crosslinked copolymer having a vinyl alcohol unit was synthesized as shown below. 100 g of vinyl acetate, 64.3 g of triarylsocyanate, 100 g of ethyl acetate, 100 g of heptane, 7.5 g of polyvinyl acetate (polymerization degree: 500) and 2 g , 2 '- Azobisui Sobuchironi DOO drill 3. 8 g and Yo Li becomes homogeneous mixture of polyvinyl alcohol 1 wt _^0/0, V phosphate 2 hydrogen isocyanatomethyl Li um dihydrate 0.0 5 wt. / ₀ and phosphoric acid dihydrogen sodium dihydrate 1 dihydrate 1.5 wt% dissolved in water 400 ml, mixed and stirred for + minutes, then 65 with 1 The suspension was heated and stirred at 75 ° C. for 8 hours and further at 75 ° C. for suspension polymerization to obtain a granular crosslinked copolymer. After washing the cross-linked copolymer, two liters of methanol containing 46.5 g of sodium hydroxide were stirred at 40 ° C for 18 hours to perform a saponification reaction of the cross-linked copolymer. Was carried out to obtain a water-insoluble carrier composed of a crosslinked copolymer having a vinyl alcohol unit. The average particle size of this water-insoluble carrier was 100 m, and the exclusion limit molecular weight was 2,000, 000.

Next, the following copolymer was synthesized as a molecule having a polycation. X 3

 2 — 1 liter of an ethanol solution containing 0.8 mol of hydroxyethyl methyl acrylate and 0.2 mol of acetylaminoethyl methacrylate, 2, 2′-one as a polymerization initiator A radical copolymerization reaction was carried out at 60 ° C. for 8 hours by adding 0.05 mol of azobisisobutyronitrile. The molecular weight of the polycation-containing molecule obtained in this manner was determined by gel permeation chromatography using dimethylformamide as an eluent (column: Shodex AD-80 MZS, Japan, It was 180,000 (polyethylene glycol equivalent) as determined by Showa Denko KK.

 The water-insoluble carrier obtained in the above step was used as a water-insoluble carrier (swelled with acetate). 3 Milliliters were suspended in acetonitrile 2.1 milliliters. To the suspension, add Tresyl Mouth Ride 45 microliters and pyridine 135 microliters and shake at room temperature for 10 minutes to obtain the active group. An activated carrier having a tresyl group was obtained. (1) above, 2-copolymer of hydroxyethyl methyl acrylate and methylaminoethyl methacrylate 30 Omg 5

To the solution dissolved in 0% dimethylformamide aqueous solution in 6 milliliters was added 3 milliliters of the above-mentioned activated carrier, and the pH was adjusted to 8 with an aqueous sodium hydroxide solution. By shaking at room temperature for 24 hours, an adsorbent was obtained, which was a complex in which the above-mentioned polycation-containing molecule was bound and immobilized on the water-insoluble carrier. The positive charge density of the adsorbent, which was determined by titration with a sodium hydroxide aqueous solution, was 40 / ^ eq. 0.2 milliliters of the adsorbent thus obtained and 4 milliliters of plasma from a healthy subject were placed in a plastic test tube and stirred at room temperature with gentle rotation for 1 hour to perform an adsorption experiment. Went. Using a commercially available kit for measuring blood coagulation factor VIII activity (Test Team FVIII, manufactured by Daiichi Kagaku, Japan), measure blood coagulation factor VIII activity before and after the adsorption experiment As a result, the factor VIII activity in the plasma after the adsorption experiment was 7 before the adsorption experiment. /. It was.

 In order to confirm that the result of the factor VIII activity measurement was not due to the inactivation of factor VIII but to the result of the factor VIII being adsorbed to the adsorbent of the present invention, blood coagulation factor VIII was used. Using a commercial kit (Aceracrom FVIIIR: Ag, manufactured by Boehringer Mannheim Yamanouchi Co., Ltd., Japan), the amount of blood coagulation factor VIII antigen in the plasma before and after the above adsorption experiment was measured. The amount of factor VIII antigen after the adsorption experiment was 10% before the adsorption experiment.

Since the decrease in the factor VIII activity and the amount of the factor VIII antigen after the adsorption experiment corresponded, it was confirmed that factor VIII in plasma was adsorbed by the adsorbent of the present invention. Incidentally, the correspondence between this activity and the amount of antigen was the same in the following Examples. Next, in order to confirm the usefulness of using the adsorbent of the present invention to obtain factor VIII, an elution experiment of factor VIII from the adsorbent after the above-mentioned adsorption experiment was performed.

 The adsorbent after the adsorption experiment was transferred to a plastic column, and washed three times with 2 milliliters of a 10 mM aqueous sodium citrate solution (pH 7.4). Then, an aqueous solution containing 2 OmM sodium phosphate, 1% lysine, 1 M sodium chloride, 20% ethylene glycol, and 1% triton X100 ( Factor VIII was eluted using two PH 6.0) 2 milliliters. When the factor VIII activity contained in the eluate was measured, 69% of the factor VIII in the plasma used in the adsorption experiment was recovered.

 As described above, it was confirmed that Factor VIII was recovered at a high recovery rate. In the following examples, similarly, a high recovery rate was obtained.

Example 2 (1) 10 milliliters of the water-insoluble carrier obtained in Example 1 was suspended in 12 milliliters of dimethyl sulfoxide, and 8 milliliters of the suspension was added to the suspension. Add a little epichlorohydrin and 1 milliliter of 50% aqueous sodium hydroxide solution and shake at 30 ° C for 5 hours to activate the active group. As a result, an activated carrier having an epoxy group was obtained.

Molecule having polycation synthesized in Example 1 135 mg Was dissolved in 50% aqueous solution of dimethylformamide in 13.5 milliliters of aqueous solution, and 9 milliliters of the activated carrier obtained as described above was added thereto. Adjust the pH to 12 with, shake for 20 hours at 45 ° C, and fix the copolymer of 2-hydroxymethyl methacrylate and getylaminoethyl methacrylate. Thus, a modified adsorbent was obtained. The positive charge density per milliliter of the adsorbent determined by titration with an aqueous sodium hydroxide solution was 110 / eq.

 In the same manner as in Example 1, when an adsorption experiment was performed using plasma of a healthy subject, the factor VII I activity in the plasma after the adsorption experiment was 16% of that before the adsorption experiment.

Example 3

Sepharose (Pharmacia, Sweden), which is an agarose-based carrier, was used as the water-insoluble carrier. A solution prepared by dissolving 6 O mg of a copolymer of 2-hydroxylethylmethacrylate and getylaminoethylmethacrylate synthesized in Example 1 in 50% aqueous solution of 60% dimethylformamide in 6 milliliters In addition, as an activating carrier having a tresyl group as an active group, commercially available tresyl-activated Sepharose 4B (Pharmacia, Sweden) 3 milliliters In addition, the pH was adjusted to 8 with an aqueous sodium hydroxide solution, and the mixture was shaken at room temperature for 24 hours to obtain an adsorbent. The positive charge density of the adsorbent per milliliter determined by titration with an aqueous solution of sodium hydroxide was 12 μeq. I got it.

 As in Example 1, when an adsorption experiment was performed using plasma from a healthy subject, the factor VII I activity in the plasma after the adsorption experiment was 10% of that before the adsorption experiment.

Example 4

As a water-insoluble carrier, Toray Pearl (Tosoichi Japan, Ltd.), a hydrophilic bullpolymer carrier, was used. As an activating carrier having a tresyl group as an active group, commercially available AF-tresyl toyopearl 6.50 (manufactured by Tosoh Corporation B, Japan) 3 milliliters was used in Example 1. 30 mg of a copolymer of 2-hydroxyhydroxymethyl methacrylate and getylaminoethyl methacrylate, which was synthesized in step 2, was dissolved in 6 milliliter of 50% aqueous dimethylformamide solution. In addition to the solution, the pH was adjusted to 8 with an aqueous solution of sodium hydroxide, followed by shaking at room temperature for 20 hours to obtain an adsorbent. Positive charge density of Ri per adsorber material 1 ml was determined from the titration by hydroxide Na Application Benefits um aqueous solution 2 0 mu mediation 7 this in eq ₀

 In the same manner as in Example 1, when an adsorption experiment was performed using plasma of a healthy individual, the factor VII I activity in the plasma after the adsorption experiment was 28% of that before the adsorption experiment.

Example 5

Using a crosslinked copolymer having a butyl alcohol unit synthesized in Example 1 as a water-insoluble carrier, in the same manner as in Example 1, An activated carrier having an epoxy group as an active group was obtained using epichlorohydrin. As a polycation-containing molecule, poly-L-lysine (molecular weight: 20,000, ICN Immunobiological, America) was added to a concentration of 0.2 mg / 100 mg Z-milliliter. To 6 milliliters of a solution of an aqueous solution of sodium phosphate (PH10), add 3 milliliters of the activated carrier described above, and shake at 45 ° C for 20 hours. Thus, an adsorbent was obtained. The positive charge density of the adsorbent, determined by titration with an aqueous sodium hydroxide solution, was 92 μeq.

 As in Example 1, when an adsorption experiment was performed using plasma from a healthy individual, the factor VIII activity in the plasma after the adsorption experiment was 28% of that before the adsorption experiment.

Example 6

 Using the adsorbent used in Example 1, adsorption of factor VIII and factor IX on 3 milliliters of healthy human plasma by changing the amount of adsorbent from 0.6 lg to 0.6 g The behavior was investigated. -The adsorption rates of Factor V and Factor IX are as follows:

 3% 6% at 0 g of adsorbent (factor VI, factor IX, and so on)

 32%, 29% at 0.2 g of adsorbent

 68%, 45% at 0.3 g of adsorbent

90%, 52% at 0.4 g of adsorbent 0.5% of adsorbent 92%, 60%

 95 at 0.6 g of adsorbent. /. , 65%

 In addition, the adsorption rates of factor VIII and factor IX were determined by measuring the respective activities remaining in plasma after the adsorption experiment. The measurement of the factor VIII activity was carried out in the same manner as in Example 1. The activity of Factor IX is determined by the Hard i sty—step method [R.M.Had i s t y e a 1.: Thro m b os D a he s.H a e m o h., V o

1.7, p.p. 2 15 (1962)].

Comparative Example 1

 The adsorption behavior of factor VIII and factor IX was examined using DEAE-Sepharose (Swedish), a commercial product, in the same manner as in Example 6, and the adsorption of factor VIII and factor IX was performed. The rates are listed below.

 3%, 63% at 0 g of adsorbent (factor V

IX factor order. The same applies to the following.) 1 0.2% of adsorbent 9%, 89%

 29%, 90% at 0.3 g of adsorbent

 Adsorbent 0.4 g, 59%, 90% 'Adsorbent 0.5 g, 82%, 90%

 95%, 90% at 0.6 g of adsorbent

The adsorption rates of factor VIII and factor IX were determined by the respective activities remaining in the plasma after the adsorption experiment, as in Example 6. It was determined by measuring.

 Example 7

 An activated carrier obtained by epoxy-activating the crosslinked copolymer having a bul alcohol unit synthesized in Example 1 in the same manner as in Example 2 was used.

 As a molecule having a polycation, the activated carrier 3 was added to a solution prepared by dissolving 150 mg of the copolymer synthesized in Example 1 in 6% of a 75% aqueous dimethyl sulfoxide solution. Milliliter was added, the pH was adjusted to 8 with an aqueous solution of sodium hydroxide, and the mixture was shaken at room temperature for 24 hours to obtain an adsorbent. The positive charge density of 1 milliliter of the adsorbent determined by titration with an aqueous sodium hydroxide solution was 43 μeq.

 As in Example 1, an adsorption experiment was carried out using plasma from a healthy subject. As a result, the factor VIII activity in the plasma after the adsorption experiment was 26% of that before the adsorption experiment. Further, factor VIII was eluted using the same eluate as in Example 1, and the activity of factor V factory II contained in the eluate was measured. Factor VIII had been recovered.

Example 8 'In order to examine the multi-molecular structure of the von Willebrand factor in the eluate in Example 1, SDS electrophoresis using a 2% agarose gel was performed. The existence of multimers was recognized up to the same high molecular weight region. Industrial applicability

 Since the blood coagulation factor VIII adsorbent of the present invention has a molecule having a polycation, it can be compared with a conventional adsorbent using a low-molecular-weight compound as a ligand. Interaction becomes more specific, and plasma can be used as it is without using the conventional method via cryoprecipitate.

The VI I I factor can be adsorbed at a high rate. As described above, the adsorbent for blood coagulation factor VIII of the present invention makes a remarkable contribution to the production of a blood coagulation factor VIII preparation.

Claims

The scope of the claims  1. The molecular weight of the polycation-containing molecule is at least 155,000, which is smaller than that of a complex in which a molecule having a polycation is bound and immobilized on a water-insoluble carrier. An adsorbent for blood coagulation factor VIII, characterized in that the positive charge density of the milliliter is 1 μe (! To 200 μeq).  2. A molecule having a polycation is a homopolymer of a basic vinyl monomer having an anion exchange group in a side chain or a copolymer of the basic vinyl monomer and a vinyl monomer copolymerizable therewith. 2. The adsorbent according to claim 1, wherein the adsorbent is a polymer.  3. The basic vinyl monomer is selected from an acrylic acid derivative, a metaacrylic acid derivative, an acrylic acid amide conductor and a methacrylic acid amide derivative each having an amino group. The adsorbent according to claim 2, characterized in that:  4. The vinyl monomers that can be copolymerized with the basic vinyl monomer are N-vinylinolepyrrolidone, estenole atalinoleate, methacrylyl ester, N-anolexcreacrylic acid amide and N-alkylmethataryl acid. 3. The adsorbent according to claim 2, wherein the adsorbent is selected from the following. - 5. The adsorbent according to claim 1, wherein the water-insoluble carrier is a particulate carrier made of a natural polymer or a synthetic polymer having a porous structure. 6. The water-insoluble carrier is a particulate carrier having a porous structure, 2. The method according to claim 1, wherein the exclusion limit molecular weight is 1,000 to 100,000, 0000, and the average particle diameter is 5 to 1, Ο Ο Ομπι. Adsorbent as described.  7. The adsorbent according to claim 5, wherein the particulate carrier is a cross-linked copolymer having a vinyl alcohol unit.