SEPARATION MATERIAL FOR BLOOD COAGULATION FACTOR, PREPARATION AND USE THEREOF

Abstract: A separation material capable of realizing efficient separation and recovery of a blood coagulation factor, especially blood coagulation factor VIII or complex thereof with von Willebrand factor, from plasma or from samples containing blood coagulation factors. The separation material for separating and recovering a blood coagulation factor comprises a porous matrix having linked thereon one or more ligands each consisting of a radical exhibiting an affinity to the blood coagulation factor to be recovered, said matrix having a specific surface area of at least 1. 5 m² per milliliter of the separation material with respect to pores having diameters of at least 0.1 µ m and being derived from a porous particulate material having an exclusion limit molecular weight of at least 1.5 X 10⁶ as determined with polyethylene glycol. The separation material is prepared by the process steps of subjecting a porous particulate material having an exclusion limit molecular weight of at least 1.5 X 10⁶ to an activation by l,l' -carbonyldiimidazolating, tresylating, carbodiimidizing, thiopropylating, epoxidizing, bromocyanizing or formylating using corresponding activating agent and reacting the so activated porous particulate material with one or more substances which exihit after being linked as ligand on the porous particulate material an affinity to the blood coagulation factor to be recovered, so as to form a porous matrix having a specific surface area of at least 1.5 m² per milliliter of the porous particulate material with respect to pores having diameters of at least 0.1 µ m.

Description: FIELD OF THE INVENTION The present invention relates to a separation material for separating and recovering a blood coagulation factor comprising a porous matrix having one or more ligands introduced therein and preparation and use thereof. More particularly, it relates to a separation material for separating and recovering a blood coagulation factor, especially for collecting blood coagulation factor VIII efficiently from plasma or samples containing such coagulation factor and to preparation thereof as well as to a process for recovering a blood coagulation factor using the separation material. BACKGROUND OF THE INVENTION In the blood of humans and other animals, various substances participating in the coagulation of blood (blood coagulation factors) are contained. Various disorders or diseases induced from the shortage or lack of blood coagulation factors have been known. In particular, hemophilia A, due to lack of blood coagulation factor VIII, occupies almost 80 % of the whole of such diseases. There has hitherto been proposed no effective therapy for radical cure of hemophilia A and, up to date, a therapeutic measure of replenishing the patient with blood coagulation factor VIII on requirement has currently been employed. Blood coagulation factor VIII consists of a protein having a molecular weight of about 260,000, which is contained in the normal blood plasma only in an amount of about 0.1 µg/ml. Blood coagulation factor VIII is present in blood plasma in the form of a complex with another blood coagulation factor, namely, von Willebrand factor (abbreviated hereinafter as vWf) composed of proteins having molecular weights of 500,000 - 20,000,000. In the context of this application, by the word "blood coagulation factor" is meant to include such a complex. In the therapy of hemophilia A, no sufficient replenishment of blood coagulation factor VIII can be achieved by transfusion of an intact whole blood or plasma as such to the patient, since the concentration of blood coagulation factor VIII in the plasma is quite low. It has therefore been a widespread practice to employ an intravenous injection of a concentrated preparation of blood coagulation factor VIII for such patients. The practice has been, for example, to carry out concentration of blood coagulation factor VIII by forming first a cryoprecipitate from blood plasma, separating it and then thawing it to obtain a concentrate, or purification by liquid chromatography. However, the yield of recovery of blood coagulation factor VIII in these prior techniques has been able to reach a figure of only about a little lower than 10 %, based on the weight of the plasma, and even by the technique using liquid chromatography, a value of about 20 %. In the technique employing liquid chromatography, separation materials have been employed, in which ligands, such as substances having affinity to blood coagulation factor VIII or to vWf, for example, substances having aminoalkyl groups (Japanese Patent Kokai Nos. 136518/1985 and 157000/1989), monoclonal antibodies (Japanese Patent Kokai Nos. 13099/1989 and 221396/1989) or collagens (Japanese Patent Kokai No. 198632/1988), are caused to link to the substrate. For a separation material for use for recovering blood coagulation factor VIII in liquid chromatography, the following properties are required: 1) It should not cause any non-specific adsorption of plasma proteins. 2) It should have a pore diameter capable of permeating a complex of blood coagulation factor VIII with vWf and a sufficiently wide internal surface area for making available a large adsorption capacity. 3) The substrate to be combined with the ligand should have high stability, not only physical but also chemical. In the conventional techniques mentioned above, separation materials prepared from natural high molecular weight substances, such as agarose, dextran or cellulose, are employed. These separation materials are superior in so far as they employ substrates consisting of natural substances, and non-specific adsorption will scarcely occur. For these prior art separation materials no quantitative assay as to the above property 2) has been made, so that it might have hitherto been probable that separation material having low adsorption capacity would give poor recovery of blood coagulation factors. EP-A-0233620 discloses an adsorbent for blood coagulation factor VIII that is a water-insoluble porous gel having an exclusion limit of from 8x10⁵ to 1x10⁸ and having a sulphate group on at least part of its surface. EP-A-0303329 discloses the use of liquid chromatography for isolating coagulation factors such as factor VIII and vWf, using an adsorbent material comprising a polymeric carrier to which amino groups are linked as ligands via spacers. SUMMARY OF THE INVENTION A novel separation material comprises a porous glucomannan matrix having linked thereon one or more ligands each comprising a radical exhibiting an affinity to a blood coagulation factor, said matrix having a specific surface area of at least 1.5 m² per ml of the separation material with respect to pores having diameters of at least 0.1 µm, and wherein the glucomannan has an exclusion limit molecular weight of at least 1.5 x 10⁶ as determined with polyethylene glycol. Such a separation material may be prepared by the steps of: subjecting the porous particulate glucomannan to activation by 1,1'-carbonyldiimidazolating, tresylating, carbodiimidising, thiopropylating, epoxidising, bromocyanising or formylating using a corresponding activating agent; and reacting the thus-activated porous particulate glucomannan with one or more substances providing the or each ligand. According to a further aspect of the invention, a process for recovering a blood coagulation factor from raw material containing it, comprises the steps of: bringing the raw material into contact with a separation material as defined above in the presence of an adsorption buffer solution; and subjecting the resulting separation material containing the blood coagulation factor adsorbed thereon to elution by contacting it with an eluent buffer solution. By means of this process, a blood coagulation factor such as blood coagulation factor VIII or a complex thereof with vWf can be recovered. DESCRIPTION OF THE INVENTION The glucomannan is preferably in the form of cross-linked spherical beads exhibiting little non-specific adsorption of plasma proteins, having chemical and physical stability, and having high compressive strength, thus permitting high velocity liquid flow therethrough. Such cross-linked glucomannan spherical beads are hydrophilic gel particles in which glucomannan composed mainly of D-glucose and D-mannose has been cross-linked using a cross-linking agent. As the glucomannan raw product, such products as those having molecular weights in the range from 9 × 10⁵ to 2.4 × 10⁶, preferably in the range from 1 × 10⁶ to 1.3 × 10⁶, are desirable. As a commercially available product, konjakmannan may preferably be employed, though other products may of course be used. For the exclusion limit molecular weight of the cross-liked glucomannan spherical beads, there may be chosen adequate ranges in accordance with each specific purpose. The porous glucomannan matrix has an exclusion limit molecular weight of at least 1.5 × 10⁶, preferably in the range of 2 × 10⁶ - 1 × 10⁷, and even more preferably in the range of 2 × 10⁶ - 5 × 10⁶, before combination with the ligand, determined using a series of polyethylene glycol standards. The glucomannan also has a specific surface area (namely, "pore inner surface area"), with respect to pores having diameters of at least 0.1µm, of 1.5 m²/ml of the separation material after combination with the ligand. The exclusion limit molecular weight of the porous particulate material is determined in such a manner, that aqueous solutions of polyethylene glycol products each having a known molecular weight are passed through a column filled with the porous particulate material and each eluted amount of the polyethylene glycol determined using, for example, a differential refractometer or the like, is plotted on a graph against the molecular weight thereof and the molecular weight value at the break of the curve is assumed to be the exclusion limit molecular weight of the porous particulate material. The porous particulate material is then subjected to a treatment for linking the ligand thereto. Thus, separation material for recovering a blood coagulation factor according to the present invention contains one or more ligands each having an affinity to the blood coagulation factor to be recovered and has a specific surface area with respect to pores having diameters of at least 0.1 µm of 1.5 m²/ml of separation material, preferably at least 2 m²/ml of separation material, and even more preferably at least 3 m²/ml of separation material. The specific surface area is determined in such a manner that the pore size distribution of a porous separation material is measured by mercury compression technique after drying the separation material under vacuum and, from the thus obtained pore size distribution the specific surface area per g of the dried separation material in terms of m²/g is calculated and, parallel thereto, a wet volume per dry weight (volume upon immersion in water) in terms of ml/g is determined, whereupon the specific surface area per wet volume is calculated from these two values. For the ligand to be combined with the separation material according to the present invention, there is no special limitation and every material can be employed so long as they exhibit specific interaction (affinity) with the blood coagulation factor to be recovered. Thus, for example, collagens, monoclonal antibodies and substances having a group expressed by the general formula (I) given below may be employed: in which R¹ and R² represent each a hydrogen atom or a lower alkyl group and may be identical with or different from each other, ℓ is 0 or 1, m is an integer of 3 - 8, n is 0 or 1 and p is an integer of 0 - 5. They may be used solely or in combination of two or more of them. For the group represented by the general formula (I), in particular, the groups represented by the following formulae (II) and (III) may be given as examples : in which R¹ and R² have the same meaning as in the general formula (I), ℓ denotes 0 or 1, m represents an integer of 3 - 8 and p is an integer of 2 - 5. Concrete examples of the group represented by the general formula (II) include 4-aminobutyl [-CH₂(CH₂)₃NH₂], 6-aminohexyl [-CH₂(CH₂)₅NH₂], 4-aminobutylamino [-NH(CH₂)₄NH₂] and 6-aminohexylamino [-NH(CH₂)₆NH₂]. Concrete examples of the group represented by the general formula (III) include 4-aminobutylcarbamylbutyl [-CH₂(CH₂)₃C(=O)NH(CH₂)₄NH₂], 3-aminopropylcarbamylbutyl [-CH₂(CH₂)₃C(=O)NH(CH₂)₃NH₂], dimethylaminobutylcarbamylbutyl [-CH₂(CH₂)₃C(=O)NH(CH₂)₄N(CH₃)₂], dimethylaminopropylcarbamylbutyl [-CH₂(CH₂)₃C(=O)NH(CH₂)₃N(CH₃)₂], dimethylaminopropylcarbamylpentyl [-CH₂(CH₂)₄C(=O)NH(CH₂)₃N(CH₃)₂], 4-aminobutylcarbamylbutylamino [-NH(CH₂)₄C(=O)NH(CH₂)₄NH₂], 3-aminopropylcarbamylbutylamino [-NH(CH₂)₄C(=O)NH(CH₂)₃NH₂], dimethylaminobutylcarbamylbutylamino [-NH(CH₂)₄C(=O)NH(CH₂)₄N(CH₃)₂], dimethylaminopropylcarbamylbutylamino [-NH(CH₂)₄C(=O)NH(CH₂)₃N(CH₃)₂], and dimethylaminopropylcarbamylpentylamino [-NH(CH₂)₅C(=O)NH(CH₂)₃N(CH₃)₂]. For the collagen, there may be employed preferably a limited decomposition product of a collage IV by treating it with, for example, an enzyme, such as pepsin, or collagen III. The ligand may be combined directly to the porous matrix or through a suitable spacer. The ligand can be introduced into the separation material according to the present invention after specific portions have been activated by 1,1'-carbonyldiimidazolating, tresylating, carbodiimidizing, thiopropylating, epoxidizing, bromocyanizing or formylating using corresponding activating agent. For the activating agent, there may be employed, for example, 1,1'-carbonyldiimidazole, tresyl chloride (namely, 2,2,2-trifluoroethanesulfonyl chloride), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, 1,4-butanediol glycidyl ether, glutaraldehyde, hexamethylenediisocyanate, cyanogen bromide, bisoxirane or 1,4-bis(2,3-epoxypropoxy)butane. Now, description will be directed to the cross-linked glucomannan spherical beads to be employed as the porous particulate material for the separation material according to the present invention. Cross-linked glucomannan spherical beads before being combined with the ligand can be obtained by subjecting spherical glucomannan to a cross-linking reaction. Below, one concrete example of the production of crows-linked glucomannan spherical beads is explained: A commercially available glucomannan product is dissolved, after it has been purified using alcohol or the like, in a solvent such as formamide or dimethylformamide. Using this glucomannan solution, an ester of glucomannan is prepared using a catalyst, for example pyridine, by adding to the solution an acid such as acetic acid, acetic anhydride, propionic acid, butyric acid or nitric acid. The thus obtained glucomannan ester is dissolved in a solvent together with a porosifying agent, namely, an agent for producing porous structure. For the solvent, those which have a lower boiling point as compared with the aqueous medium explained below and which do not or only poorly dissolve in the aqueous medium may be employed. Concrete examples of such solvents include organic solvents based on chlorinated hydrocarbons, such as dichloromethane, chloroform, carbon tetrachloride or trichloroethylene, which may be employed alone or in mixtures. As the glucomannan ester, there may be employed those which have viscosity values of 8 - 20 cP, preferably 9 - 12 cP, determined at 30°C in a chloroform solution at a concentration of about 0.3 % by weight. The concentration of the glucomannan ester on preparing its solution may be 0.5 - 10 g, preferably 0.5 - 2 g, per 100 ml of the solvent. The porosifying agent is employed for producing a porous structure in the spherical beads of glucomannan ester by being included in the liquid droplets of the glucomannan ester solution. Most of the porosifying agent will be removed upon the saponification of the particles resulting from the glucomannan ester droplet, thus leaving micro pores and channels inside the gelled particle. Concrete examples of the porosifying agent include decalin (decahydronaphthalene), methyl n-caprinate, tetrahydronaphthalene, ethylbenzene, diethybenzene, methyl dedocanoate, toluene, hexyl alcohol, heptyl alcohol and octyl alcohol, all of which may have higher boiling points than that of the above-mentioned organic solvent and do not dissolve the glucomannan ester. The concentration of the porosifying agent may be in the range from 1 to 5 ml, preferably in the range from 2 to 4 ml per g of the glucomannan ester. The thus obtained glucomannan ester solution as explained above is suspended in an aqueous medium, in order to cause formation of liquid droplets of the glucomannan solution. For the aqueous medium, water or an aqueous solution having added there to a hydrophilic protective colloid such as polyvinyl alcohol, partially-saponified polyvinyl alcohol, carboxymethyl cellulose, ethyl cellulose, methyl cellulose, soluble starch or gelatine, may be employed. Hydrophilic protective colloids may be employed as an aqueous solution at a concentration in the range from 0.1 to 10 % by weight, preferably from 1 to 5 % by weight. The amount of the aqueous medium employed should be at least 2, preferably 10 - 50, times the volume of glucomannan ester solution. For preparing the suspension of glucomannan ester solution droplets in the aqueous medium, it is possible to carry out in such a manner, that the glucomannan ester solution is introduced into the aqueous medium all at once and the mixture is dispersed under agitation to form a liquid droplet suspension, or in such a manner, that the aqueous medium is held under agitation and the glucomannan ester solution is introduced therein all at once or dripped thereinto. The glucomannan ester solution will suspend in the aqueous medium in a finely dispersed form, since it is insoluble in water. Granulation from the droplets of the glucomannan ester solution occurs with accompaniment of vaporization of the organic solvent. In this manner, spherical beads of the glucomannan ester are formed, from which the organic solvent has substantially been removed. The temperature for formation of spherical beads of the glucomannan ester in the aqueous medium is selected at a point lower than the boiling point of the organic solvent and higher than the freezing point of the aqueous medium. Here, it is preferable to choose the temperature during the course of formation of the glucomannan ester beads, while evaporating off the organic solvent, at a point which is 1 - 5°C lower than the boiling point of the organic solvent, in order to facilitate vaporization of the organic solvent and to preserve the spherical shape of the liquid droplet. The contemplated spherical beads will be obtained by agitating the aqueous medium, wherein a desired particle size can be realized by adjusting the intensity of the agitation. It is enough to choose the agitation conditions in the process according to the present invention, so as to form beads having a particle size adapted for use as the separator medium, namely, a particle size in a range from 1 to 500µm. Thereafter, the so formed spherical beads of glucomannan ester are subjected to saponification. Here, it is necessary to employ a saponification bath capable of effecting saponification, while maintaining the original spherical form of the beads without deterioration of their spherical form. Examples of such saponification bath include methanolic solutions of sodium hydroxide or potassium hydroxide, solutions of sodium hydroxide or potassium hydroxide in an aqueous solution of salts, such as, sodium sulfate and so on. By bringing the glucomannan ester beads into contact with the saponification bath, the beads will be saponified, while most of the porosifying agent included in the beads is removed therefrom leaving fine pores therein. An almost 100 % saponification of the glucomannan ester beads will be attained by employing a reaction condition of a temperature of 10 - 50 °C and a reaction time of 0.5 - 24 hours. The resulting saponified spherical beads of glucomannan is subjected to cross linking. As the cross-linking agent, there may be employed, for example, bifunctional compounds such as epichlorohydrin, diepoxybutane, tolylene diisocyanate or hexamethylene diisocyanate. The cross-linking agent is employed in a form of a solution dissolved in an organic medium. As the organic solvent for the cross-linking agent, there may be employed, for example, kerosene, liquid paraffin and mixtures of them (in a volume ratio of, for example, 7 : 3), to which a surfactant (a nonionic surfactant such as an aliphatic ester of sorbitan) is added in an amount of 1 - 2 % by weight, or a mixture of acetone and dimethyl sulfoxide (in a volume ratio of, for example, 6 : 4), or further, a mixture of acetone with dimethylformamide (in a volume ratio of, for example, 2 : 3). The concentration of the cross-linking agent is preferably in the range of from 0.01 to 15 moles per liter of the organic solvent. The spherical beads of saponified glucomannan are then cross-linked by introducing 1 - 5 parts by weight of the saponified glucomannan spherical beads into 100 parts by volume of the cross-linking agent solution and holding the mixture under agitation for 24 - 36 hours at a temperature from room temperature to 70 °C. The degree of cross-linking can be adjusted by selecting the concentration of the cross-linking agent. Cross-linked spherical beads of glucomannan having an exclusion limit molecular weight of at least 1.5×10⁶ will be obtained by filtering off, washing with acetone and then with a neutral detergent, followed by water wash, of the so-reacted beads. The thus-obtained cross-linked spherical beads of glucomannan exhibit a high resistance to compression and considerable stability, both mechanical and chemical. They are almost insusceptible to various organic solvents and salt solutions, showing almost no swelling or contraction. Thus, they do not suffer from alteration of the cell structure, deformation and fusion of beads, even when the ligands are introduced with an organic solvent with simultaneous presence of salts, as described afterwards. Therefore, a separation material according to the present invention having sufficient pore size, specific surface area, ligand concentration and adsorption capacity can be prepared from such beads. Moreover, an autoclave pasteurization of the beads can be employed, since glucomannan beads thus obtained have superior thermal stability. Now, the description is directed to the concrete manner of introducing the ligand into the thus obtained porous particulate material or beads. The porous particulate material or beads produced as above are first soaked in a medium for effecting the reaction of the beads with an activating agent for pretreatment for the reaction with the ligand, in order to cause them to swell sufficiently by the medium and, at the same time, to exclude any air bubble entrained therein. For the reaction medium, those which will adapt to the subsequent pretreatment reaction of the porous beads with the activating agent are used. As the suitable medium, there may be employed, for example, dimethylformamide, a mixed liquid composed of acetone and pyridine, or an aqueous solution of sodium hydrogen borate with sodium hydoxide. To the resulting swollen porous beads soaked in the reaction medium, an activating agent is added in an amount sufficient to realize the pretreatment reaction, in order to cause activation of the inner cellular surfaces of the porous beads. The activation is incorporated, in order to cause the ligand molecules to link onto certain sites over the inner surfaces of the porous beads. The activation is carried out practically by, for example, 1,1'-carbonyldiimidazolating, tresylating, carbodiimidizing, epoxidizing, thiopropylating, bromocyanizing or formylating the above mentioned sites inside the cellular matrix of the bead. The amount of the activating agent employed depends on each specific activating agent employed and, in general, it ranges from 2 to 200 parts per 100 parts by weight of the dry beads. For the condition of activation, a reaction temperature in the range from 0 to 50°C and a reaction duration in the range from 5 minutes to 3 hours may be applicable. The followings are exemplifications of the concrete ways for carrying out the activation of the porous beads according to the present invention: In the 1,1'-carbonyldiimidazolation, the porous beads are reacted with 1,1'-carbonyldiimidazole in a reaction medium of dimethylformamide. In the tresylation, the porous beads are reacted with tresyl chloride in a reaction medium of a mixed liquid composed of acetone and pyridine. In the thiopropylation, the porous beads are first reacted with epichlorohydrin, then, with thiosulfuric acid, whereupon the resulting beads are subjected to a reduction with dithiothreitol, followed by disulfidation using 2,2'-dipyridyldisulfide. In the epoxidation, the porous beads are reacted with a bisoxirane, such as, 1,4-butanediol diglycidyl ether, in a 0.3 M aqueous solution of sodium hydroxide containing sodium hydrogen borate. In the bromocyanidation, the porous beads are reacted with cyanogen bromide in an aqueous solution of sodium hydroxide of a pH of 11 - 12. After the activation reaction has been completed, the remaining excess amount of the activating agent is removed by separating the thus activated porous beads from the reaction medium by, for example, filtering them out of the medium and then subjecting them to sufficient washing. While there can be selected for washing the reacted beads various adequate liquids, it is preferable, in the case of employing tresyl chloride as the activating agent and a mixed solvent composed of acetone and pyridine as the reaction medium, to choose a mixed solution composed of acetone and a low concentration aqueous solution of hydrochloric acid as the washing liquid. The so activated porous beads are then combined with one or more suitable ligands having affinity to the blood coagulation factor to be recovered, by contacting the so activated porous beads with a solution containing such one or more ligands under sufficient mixing to cause intimate contact of the porous beads with the solution. Here, as for the reaction condition, a temperature usually in the range from 0 to 40°C and a reaction duration usually in the range from 15 minutes to 24 hours may be employed. The solution containing the ligand may be prepared by dissolving each selected ligand or ligands in an adequate buffer solution. Usually, the ligand solution is used preferably in such an amount that the ligand or ligands in the solution will be present at least in a stoichiometric amount for reacting with the activated sites in the beads. The porous beads now having the selected one or more ligands linked thereto are separated from the reaction medium by filtration or other suitable procedure, and washed sufficiently. The washing liquid can satisfactorily be selected and, for example, an aqueous solution of sodium chloride, an acetate buffer solution containing sodium chloride, a bicarbonate buffer solution containing sodium chloride, pure water and various organic solvents may be chosen therefor. The unreacted active sites in the porous beads, namely, the vacant activated sites not combined with any ligand, are then blocked up with an adequate blocking agent. The blocking can be effected by immersing the porous beads in a solution of an adequate blocking agent at a temperature of 0 - 40°C for a duration in the range from 30 minutes to 24 hours. For the blocking agent, there may be exemplified monoethanolamine, a tris-hydrochloric acid buffer solution containing sodium chloride and so on. In the case of activating the porous beads with 1,1'-carbonyldiimidazole and the thus activated beads are combined with a ligand having a group expressed by the formula (I) given previously, the resulting separation material has a structure represented by the following general formula in which A represents the porous particulate material and R¹, R², m, n and p have the same meaning as given previously in explaining the general formula (I). While there is no restriction for the amount to one or more ligands (denoted hereinafter as ligand concentration) linked in the porous beads, it is preferable to choose such a ligand concentration that it suffices the requisite amount of blood coagulation factor(s) to be adsorbed on the separation material. When cross-linked glucomannan beads have one or more ligands linked thereto, as described above, the resulting separation material will have nearly the same exclusion limit molecular weight as that of the intact starting cross-linked glucomannan beads. The separation material prepared by introducing one or more ligands into cross-linked spherical glucamannan beads has excellent resistance to compression, so that it permits operation at high flow velocity, resulting in an increase in the hourly treatment output. There is no limitation in the manner of employment of the separation material for recovering a blood separation factor according to the present invention. The separation material according to the present invention is directed for use for recovery of a blood coagulation factor, especially, blood coagulation factor VIII and complex of the blood coagulation factor VIII with vWf. The separation material according to the present invention may be employed for recovering a blood coagulation factor from various substances (hereinafter denoted as raw substances), for example, blood plasma, cryoprecipitates, Cohn fractions or blood coagulation factor-containing material resulting from culture of cells secreting blood coagulation factors. The separation of a blood coagulation factor from the raw substances using the separation material according to the present invention may be realized by known methods, such as, by using an elution column (column method) and step-wise equilibrium on batches (batch method), in which the raw substances and the separation material according to the present invention are brought into contact with each other within a suitable adsorption buffer solution, so as to cause the contemplated blood coagulation factor to adsorb on the separation material. The resulting separation material on which the blood coagulation factor has been adsorbed is then subjected to desorption by immersing the separation material in an adequate desorption buffer solution to recover the desorbed blood coagulation factor. As the adsorption buffer solution to be employed in the column method, there may be employed those which develop a pH within the range from 4.5 to 9.5, preferably from 5.5 to 8.8. As the typical adsorption buffer solution, there may be exemplified bis-tris-hydrochloric acid (pH range: 5.5 - 7.3), triethanolamine-hydrochloric acid (pH range: 7.3 - 7.3), diethanolamine-hydrochloric acid (pH range: 8.4 - 8.8) and buffer solutions which are obtained by adding to the above-mentioned buffer solutions about 1 mM of calcium chloride and about 100 mM of sodium chloride. For the typical desorption buffer solution, there may be employed those buffer solutions in which the buffer solutions mentioned above for the typical adsorption buffer solution are modified by adding about 0.5 - 3 M of sodium chloride and about 500 mM of calcium chloride. For practicing the separation treatment, the buffer solution for the adsorption should preferably be selected among the above-mentioned buffer solutions one that develops a pH value as near as possible to that of the raw substance to be treated. For effecting practical adsorption, usually a linear flow velocity (LV) of 5 - 500 cm/hr, preferably 50 - 300 cm/hr of the adsorption buffer solution containing the raw substance through the column filled with the separation material according to the present invention may be employed. For desorption, the desorption buffer solution may be passed through the column after the adsorption step at a linear flow velocity usually of 5 - 500 cm/hr, preferably from 50 to 300 cm/hr. In a batch method, the separation material according to the present invention and the raw substance containing the blood coagulation factor are brought together at room temperature and the mixture should preferably be sufficiently shaken, for a period of at least 10 minutes, to give sufficient mixing of the separation material with the raw substance. Also in the batch method, the same buffer solutions as used in the column method can be employed. Since the separation material for a blood coagulation factor according to the present invention has been derived from a porous particulate material having an exclusion limit molecular weight of at least 1.5×10⁶, a blood coagulation factor or vWf complex can penetrate therethrough smoothly. Due to the large specific surface area of at least 1.5 m²/ml separation material, the raw substance can be entrapped by the ligand radicals linked on the inner surface quite efficiently. The greatly increased capacity of the separation material according to the present invention for adsorbing a blood coagulation factor or vWf complex in the raw substance is believed to be due to the above reasons. Cross-linking of beads of glucomannan may cause the specific surface area and the inner pore size to tend to increase, and nevertheless, the resistance or persistency against compression becomes improved. This is believed to be due to a possible increase in the cell wall thickness by the cross-linking. It is assumed that the increase in the pore size by the cross-linking provides smooth passage of the blood coagulation factors, especially blood coagulation factor VIII and the complex of blood coagulation factor VIII with von Willebrand factor. As described above, a separation material for a blood coagulation factor can be obtained according to the present invention, which permits separation and recovery of a blood coagulation factor, especially blood coagulation factor VIII in a high yield efficiently. In particular, when porous beads of cross-linked glucomannan are employed for the starting porous particulate material, a separation material capable of separating and recovering a blood coagulation factor more efficiently can be obtained, since they exhibit high resistance to compression and thus permit high flow velocity for the column method. Below, the present invention is further described specifically by way of Examples. By "Unit" as used herein for expressing the concentration of blood coagulation factor VIII, is meant "unit of coagulant activity of blood coagulation factor VIII". PRODUCTION EXAMPLES FOR CROSS-LINKED GLUCOMANNAN BEADS Porous spherical beads of cross-linked glucomannan for use as the matrix or substrate of the seapration material according to the present invention are produced by the following procedures: I. Production of Spherical Bead Product A₁ of Cross-linked Glucomannan 60 g of konjakumannan powder were dissolved in 6 liters of tap water at a temperature of about 80°C with agitation. The resulting solution was dropped slowly into 6 liters of ethanol so as to form precipitate of glucomannan. The precipitate was separated by filtration and dried in air. The air-dried precipitate was then dried under vacuum to obtain purified glucomannan product (hereinafter GM is used sometime for glucomannan). 30 g of the purified glucomannan product were introduced into 1 liter of formamide and allowed to stand for 7 hours at 55 °C to cause swelling of the product. Then, 300 ml of pyridine were added thereto. After 2 hours, 300 ml of acetic anhydride were added thereto and the reaction was conducted at 55 °C for 4 days to esterify the glucomannan product. This reaction mixture was poured into 7 liters of water with agitation. The precipitate formed was separated by filtration, washed with water, dried in air and then dried under vacuum. The so obtained raw acetate product was dissolved in 10 liters of acetone. After removal of the insoluble matter, the solution was introduced into about 20 liters of water with agitation. The precipitate formed was separated by filtration and dried in air, whereupon it was dried under vacuum to obtain glucomannan acetate product A. A chloroform solution of this acetate product A of a concentration of 0.29 wt. % exhibited a viscosity of 10 cP at 30°C. 10 g of the acetate product A were dissolved together with 35 milliliters of methyl n-caprinate as the porosifying agent into 560 milliliters of chloroform. This solution was dropped into 5 liters of 1 wt. % aqueous solution of a 90 %-saponified polyvinyl alcohol at 55°C with agitation at an agitation rate of 600 rpm. and the agitation was continued. After 24 hours, the solution was allowed to stand, with gradual cooling, and the thus formed spherical particles were then separated by filtration and washed with water. The spherical particles were then introduced into a mixed solution of 225 milliliters of methanol and 25 milliliters of 10 N aq. NaOH and the particles were subjected to saponification for 2 hours with agitation. The resulting saponified spherical particles were separated by filtration, added to a mixture composed of 200 milliliters of acetone, 300 milliliters of dimethylformamide and 70 milliliters of epichlorohydrin, and subjected to cross-linking for 24 hours at 60°C. The particles were then collected, washed with water and subjected to extraction with acetone to obtain spherical beads A₁ of cross-linked glucomannan (hereinafter abbreviated as GM-A₁). II. Production of Cross-linked Glucomannan Beads A₂ 10 g of the above acetate product A were dissolved together with 28 milliliters of decalin as the porosifying agent in 740 milliliters chloroform. This solution was dropped into 5 liters of a 1 wt. % aqueous solution of a 90%-saponified polyvinyl alcohol at 55°C, while agitating at an agitation rate of 600 rpm. and the agitation was continued. The vessel for particulation, namely particle formation, had baffle plates provided on its manner walls. Thereafter, the same procedures as in the production of GM-A₁ were followed to obtain a cross-linked glucomannan beads product A₂ (hereinafter abbreviated as GM-A₂). III. Production of Cross-linked Glucomannan Beads A₃ 10 g of the above acetate product A were dissolved together with 30 milliliters of decalin as the porosifying agent in 740 milliliters chloroform. This solution was dropped into 5 liters of 1 wt. % aqueous solution of a 90%-saponified polyvinyl alcohol at 55°C, while agitating at an agitation rate of 600 rpm. and the agitation was continued. The vessel for particulation has baffle plates provided on its inner walls. Thereafter, the same procedures as in the production of GM-A₁ were followed to obtain a cross-linked glucomannan beads product A₃ (hereinafter abbreviated as GM-A₃). IV. Production of Cross-linked Glucomannan Beads B₁ The procedures for the production of GM-A₁ were followed except that the porosifying agent methyl n-caprinate was replaced by decalin and the agitation rate was changed to 500 rpm., whereby a cross-linked glucomannan beads B₁ (hereinafter abbreviated as GM-B₁) was obtained. V. Production of Cross-linked Glucomannan Beads B₂ 15 g of the purified glucomannan product obtained in the course of production of GM-A₁ were introduced into 150 milliliters of 1 : 1 mixture (by volume) of formamide and pyridine and the mixture was allowed to stand for 12 hours at room temperature to cause swelling of the product. The thus swollen glucomannan product separated from the supernatant liquid was introduced into a kneader together with 85 milliliters of acetic anhydride to conduct esterification for 4 days at 50°C. The reaction mixture was processed in the same procedures as in the production of GM-A₁ to obtain glucomannan acetate product B. A 0.29 wt. % solution of this acetate product B exhibited a viscosity of 2.5 cP at 30°C. Thereafter, the same procedures as in the production of GM-A₁ were followed to obtain a cross-linked glucomannan beads product B₂ (hereinafter abbreviated as GM-B₂). VI. Material Properties of Cross-linked Glucomannan Beads Exclusion limit molecular weight was determined for the beads products GM-A₁ to GM-A₃, GM-B₁ and GM-B₂ using a series of standard polyethylene glycol products. The results are given in Table 1. Further, the relationship between the flow velocity and the pressure drop on passing water through a stainless column having an inner diameter of 6 mm and a length of 100 mm, and charged with each of the above-mentioned beads products was detected using a high pressure pump and observing the indications of pressure gauges. The results are also given in Table 1. From the data given in Table 1, it is seen that all the cross-linked glucomannan spherical beads products have a linear relationship between the flow rate and the pressure drop, even under the condition of high flow velocity observed, showing thus a high resistance to compression. VII. Preparation of Ligand-linked Separation Material 1) Cross-linked Glucomannan Beads Ligand of aminobutylamino radical, aminohexylamino, radical or aminobutylcarbamylbutylamino radical was introduced into each of the beads products GM-A₁ to GM-A₃ and GM-B₁ and GM-B₂ obtained as above, in the following manner: 1-a) Introduction of Aminohexylamino Radical Each of the cross-linked glucomannan beads products GM-A₁ to GM-A₃ and GM-B₁ and GM-B₂ was washed with water and then with acetone while filtering under suction, and then dried under vacuum. A predetermined amount of the dried beads of cross-linked glucomannan was introduced into a predetermined amount of dimethylformamide (occasionally abbreviated hereinafter as DMF) and thereto was added a predetermined amount of 1,1'-carbonyldiimidazole (sometimes abbreviated hereinafter as CDI) and the mixture was shaken for a predetermined period of time. The resulting CDI-activated beads were separated by suction filtration and washed with DMF. A predetermined amount of the so washed CDI-activated beads was introduced into a predetermined amount of DMF and thereto was added a predetermined amount of 1,6-diaminohexane (occasionally abbreviated as DAH) and the mixture was shaken for a predetermined period of time. The resulting beads having introduced therein aminohexylamino radical were separated by suction filtration and were washed with DMF and then with water to obtain each of separation material products A₁ to A₃, B₁ and B₄. These bead products having linked aminohexylamino radicals were stored in a 20 vol. % aq. ethanol solution. The above procedures were carried out at room temperature. The conditions for introducing the ligand radical are given in Table 2. 1-b) Introduction of Aminobutylamino Radical The same procedures as in the introduction of the aminohexylamino radical were followed except that 1,4-diaminobutane (occasionally abbreviated hereinafter as DAB) was employed instead of 1,6-diaminohexane, whereby a separation material product B₂ was obtained. The conditions for introducing the ligand are given in Table 2. 1-c) Introduction of Aminobutylcarbamylbutylamino Radical A predetermined amount of 5-aminovaleric acid was dissolved in a mixture of a predetermined amount of DMF with a small amount of aqueous NaOH solution containing an equivalent weight of NaOH. To this solution was added the washed CDI-activated beads obtained in a similar manner as in the introduction of aminohexylamino radical described above, whereupon the mixture was shaken for 17 hours to cause a reaction of 5-aminovaleric acid with the CDI-activated sites in the beads. The resulting beads were separated from the medium by filtration under suction and were then washed with water, 0.05 N hydrochloric acid, water and finally with DMF, in this sequence. The washed beads were introduced into a predetermined amount of DMF and thereto was added a predetermined amount of N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride, whereupon the mixture was shaken for 26 hours. The thus obtained carbodiimide-activated beads were separated from the reaction mixture by suction filtration and washed with DMF. The thus washed beads were introduced into a predetermined amount of an aqueous solution of 1,4-diaminobutane containing 0.1 M NaHCO₃ and 0.5 M NaCl and the mixture was shaken for 2.5 hours. The resulting beads having linked aminobutylcarbamylbutylamino radicals were separated from the reaction mixture by suction filtration and washed with water to obtain a separation material product B₃. The so obtained beads having linked aminobutylcarbamylbutylamino radicals were stored in a 20 vol. % aq. ethanol solution. The above procedures were carried out at room temperature. The conditions for introducing the ligand radical are given in Table 2. 2) Commercial Porous Beads Aminohexylamino radicals were introduced into four commercial bead products, i.e., Sephacryl S-1000 (Trademark; a product of Pharmacia; beads of cross-linked allyldextran obtained using as cross-linking agent N,N'-methylene-bis-acrylamide; occasionally abbreviated hereinafter as S-1000), Sepharose CL-4B (Trade mark; a product of Pharmacia; beads of agarose; occasionally abbreviated hereinafter as CL-4B), Sepharose CL-2B (Trade mark; a product of Pharmacia; beads of agarose; occasionally abbreviated hereinafter as CL-2B) and Toyopearl HW-75C (Trade mark; a product of Tosoh K.K.; beads of a hydrophilic vinyl polymer; occasionally abbreviated hereinafter as HW-75C). 2-a) Introduction of Aminohexylamino Radicals into Sephacryl S-1000 8 milliliters of wet Sephacryl S-1000 beads taken by a messcylinder were filtered under suction and were washed with DMF to replace the soaking medium. The beads were then introduced into 16 milliliters of DMF and thereto was added CDI in an amount of 500 µM/ml gel and the mixture was shaken for 30 minutes at 25°C. The resulting CDI-activated beads were separated from the medium by suction filtration and were then washed with DMF. The washed CDI-activated beads were introduced into 8 milliliters of DMF containing DAH in an amount of 500 µM/ml gel and the mixture was shaken at 25°C for 30 minutes. The resulting Sephacryl S-1000 having linked aminohexylamino radicals were collected by suction filtration and were washed with DMF and then with water to obtain a separation material product A₄. This separation material product A₄ was stored in a 20 vol. % aq. ethanol solution. The conditions of the introduction of ligand are given in Table 2. 2-b) Introduction of Aminohexylamino Radicals into Sepharoses CL-4B and CL-2B Commercial bead products of Sepharoses CL-4B and CL-2B were subjected to solvent replacement by the normal practice from "as sold" state without drying to DMF-soaked state and were activated with CDI. The so obtained CDI-activated beads were processed in the same procedures as in the introduction of aminohexylamino radicals into the beads of cross-linked glucomannan, whereby separation material products B₅ and B₆ were obtained. The conditions for introducing the ligand are given in Table 2. 2-c) Introduction of Aminohexylamino Radical into Toyopearl HW-75C. 7 milliliters of Toyopearl HW-75C separated from a fine particle fraction were washed with water and then with acetone while filtering under suction and were then dried under vacuum. This dried Toyopearl HW-75C was introduced into 14 milliliters of DMF and thereto was added CDI in an amount of 500 µM/ml gel and the mixture was shaken for 30 minutes at 25°C. The resulting CDI-activated beads were separated by filtration under suction and were washed with DMF. The washed CDI-activated beads were introduced into 7 milliliters of DMF containing DAH in an amount of 500 µM/ml gel and the mixture was shaken for 30 minutes at 25°C. The resulting Toyopearl HW-75C beads having linked aminohexylamino radicals were separated by filtration under suction and washed with DMF and then with water, whereby a separation material product B₇ was obtained. This separation material product B₇ was stored in a 20 vol. % aq. ethanol solution. The conditions for introducing the ligand are given in Table 2. 3) Analysis of the Separation Material The practical procedures for determining the wet apparent volumes of the beads of the separation materials before and after the introduction of ligand into the beads for the products of cross-linked glucomannan and for the commercial products, the ligand concentration and the specific surface area were as follows: Wet apparent volume of beads: An amount of wet beads is charged into a measuring cylinder together with an adequate amount of water and the measuring cylinder contents are allowed to stand for 1 day and the apparent volume of the sedimented layer of beads is observed. All the beads in the measuring cylinder are then dried and the dry weight thereof is determined. The quotient of the wet apparent volume of the beads measured as above divided by the dry weight thereof is given as "wet apparent volume of beads" (ml/g). Ligand concentration: A predetermined amount of beads is dried and introduced into 0.005 N aq. HCl. The mixture is allowed to stand overnight, whereupon the amount of HCl consumption is determined by titrating it with 0.01 N aq. NaOH, from which the ligand concentration is calculated. Specific surface area (pore inner surface area): Beads of each separation material are vacuum-dried and the distribution of sizes of inner pores is determined by the mercury compression technique using Poresizer Model 9310 of Micromeritix. The value of specific surface area per unit dry weight (m²/g) obtained from the determination of size distribution of inner pores and the value of wet apparent volume of the beads per dry weight of the beads (ml of beads immersed in water per g dry weight) determined separately are used for calculating the value (m²/ml separation material) of the specific surface area per unit wet volume of the beads. The results of analysis are given in Table 3. Here, the exclusion limit molecular weights for beads products S-1000, CL-4B, CL-2B and HW-75C were determined in the same manner as that for GM-A₁. As may be assessed from the data of Table 3, it is clear that the separation material products A₁ to A₄ are within the scope of the present invention. It is seen from the results of Table 3, that the separation material products B₅ and B₆ show a considerable contraction of the apparent volume of the beads after the introduction of ligand, whereas all the other separation material products show no change in the apparent volume of beads regardless of the material of the beads, alteration of ligand species and ligand concentration. By microscopic inspection, some deformation and fusion of beads of the products B₅ and B₆ were detected. Neither fusion nor deformation of beads was detected for the other separation material products. Examples 1-4 1) Preparation of Samples (Cryoprecipitates) containing Blood Coagulation Factor VIII To 1 liter of human blood plasma, trypsin inhibitor from soybean, benzamidine and diisopropyl fluorophosphate were added in this sequence over a period of about 15 minutes while maintaining the temperature within the range from 0 to 10°C so that the concentrations of these additives became 100 mg/l, 1.5 g/l and 1 mM respectively. Thereto was then added a polyethylene glycol product (molecular weight 4000) in an amount of 1 % by weight and the mixture was agitated for 30 - 40 minutes. Ethanol was dropped thereinto at a temperature of 0 - 2 °C over a period of 40 - 50 minutes gradually until a concentration of 3 vol. % was reached and the mixture was maintained at that temperature for about 1 hour with frequent agitation. After 1 hour, the mixture was centrifugated at 4°C. The precipitate was dissolved in 40 ml of a 55 mM citrate buffer (pH 7.4). This was subjected to centrifugation again and the supernatant was collected, which was warmed to 26°C. To this supernatant was added a 2.6 M glycine solution (including 0.3 M NaCl and 25 mM tris-hydrochloride buffer; pH 6.8) warmed to 26°C, so as to reach a glycine concentration of 2 M and the mixture was agitated for 30 minutes. Then, the mixture was subjected to centrifugation at 26°C and the supernatant was collected. To this supernatant, sodium chloride was added at a temperature of 20 - 23°C so as to reach a NaCl concentration of 90.6 g/l and the mixture was agitated. This was then subjected to centrifugation at room temperature and the resulting precipitate was dissolved in a 55 mM citrate buffer solution (pH 7.4) to obtain a cryoprecipitate solution. The quantitative analysis of blood coagualtion factor VIII was carried out using Testteam (Trademark) F VIII (a product of Kabi Vitrum AB, Sweden) by the endpoint method. The samples containing blood coagulation factor VIII served for the experiment were as given in Table 4. 2) Experiments for Separating Blood Coagulation Factor VIII 2-a) Adsorption Experiments in Batch Method In a 1. 5 ml Eppendorf sample vial, a predetermined amount of beads of each separation material was charged and the beads were washed with buffer solution B (20 mM tris-hydrochloride, 500 mM CaCl₂, 2 M NaCl; pH 7.4) and then with buffer solution A (20 mM tris-hydrochloride, 1 mM CaCl₂, 100 mM NaCl; pH 7.4). To this vial, a predetermined amount of each of the cryoprecipitate solutions was added and the mixture was agitated at room temperature for 1 hour. Then, the mixture was subjected to centrifugation at 10,000 rpm. for 3 minutes. The supernatant was collected and the blood coagulation factor VIII thereof was quantitatively determined in the same manner as that in the preparation of the sample. With respect to the separation material product A₁ and A₂, the relationship between the concentration of blood coagulation factor VIII determined as above and the amount of adsorption of blood coagulation factor VIII per milliliter of the separation material was detected. On the other hand, the difference between the concentration of blood coagulation factor VIII in the loaded cryoprecipitate and the concentration of blood coagulation factor VIII in the supernatant after the adsorption was detected and the proportion in % of this difference to the concentration of the blood coagulation factor VIII in the loaded cryoprecipitate is denoted as adsorption rate of blood coagulation factor VIII. The results are summarized in Table 5. The relationship between the concentration of blood coagulation factor VIII in the supernatant and the amount of adsorption thereof is shown in the graph of Fig. 1. 2-b) Adsorption-Desorption Experiment by Column Method The separation material product A₁ was charged in a glass column having an inner diameter of 3 mm and a length of 40 mm according to normal practice. This column was loaded with 500 µl of the cryoprecipitate solution A using an injector at room temperature. For this, a flow velocity of 25.5 cm/hr was employed. While monitoring the effluent liquid by UV (280 nm), the buffer solution A was passed therethrough until no protein had been detected. Then, the column was loaded with 1 ml of the buffer solution B at room temperature using an injector to cause desorption. For this, a flow velocity of 85 cm/hr was chosen. The amount of blood coagulation factor VIII in the effluent passing through the column without being adsorbed on the beads and the amount of blood coagulation factor VIII desorbed from the beads were determined in the same manner as in the preparation of the sample. The results of the determinations show that 98 % of blood coagulation factor VIII had been adsorbed on the separation material A₁ and that 99 % of the thus adsorbed blood coagulation factor VIII had been recovered (Example 3). The adsorption rate and the recovery yield of blood coagulation factor VIII were not influenced by incorporating a flow velocity of 170 cm/hr upon adsorption and desorption of blood coagulation factor VIII (Example 4). From the results of Examples 3 and 4, it is seen that the separation material A₁ allows an efficient separation of blood coagulation factor VIII in cryoprecipitate solutions at a high flow rate. Comparison Examples 1 - 7 1) Experiments for the Separation of Blood Coagulation Factor VIII Adsorption experiments were carried out in batch method in the same manner as in Examples 1 and 2 using each of the separation material products B₁ - B₆ and B₈ instead of using the separation material products A₁ and A₂. The results are summarized also in Table 5. As for Comparison Examples 5 and 7, the relationship between the concentration of blood coagulation factor VIII in the supernatant liquid and the amount of adsorption thereof is plotted in the graph of Fig. 1. The separation material product B₈ was a commercial product EAH-Sepharose 4B (Trademark, a product of an epoxy-activated aminohexylamino-Sepharose of Pharmacia) as such. As seen from Table 5, the separation material product A₁, which is obtained by introducing aminohexylamino radicals up to a ligand concentration of 53µM/ml separation material into a cross-linked glucomannan bead product GM-A₁ having an exclusion limit molecular weight of 1,700,000 and a specific surface area of 3.4 m²/ml separation material with respect to the pores that have diameters of at least 0.1µm, exhibits an adsorption capacity which becomes greater as the concentration of blood coagulation factor VIII is increased, wherein an adsorption capacity of 35.4 Units per ml of separation material was observed at a blood coagulation factor VIII concentration in the supernatant of 0.868 Units per ml of the solution (Example 1). Thus, using the separation material product A₁, blood coagulation factor VIII contained in raw substances can easily and efficiently be recovered by first causing blood coagulation factor VIII to adsorb on this separation material and then eluting the thus adsorbed coagulation factor from the separation material using a desorption buffer solution having no content of blood coagulation factor VIII to effect desorption thereof. It is also seen that the separation material product A₂, which is obtained by introducing aminohexylamino radicals into porous cross-linked glucomannan product GM-A₂ having an exclusion limit molecular weight of 3,000,000, up to a ligand concentration of 71 µM/ml separation material, and a specific surface area, with respect to the pores having diameters of at least 0.1 µm, of 4.3 m²/ml separation material, exhibited a greater adsorption capacity than the separation material product A₁ (Example 2). On the other hand, the separation material products B₁ - B₆ and B₈ showed lower adsorption capacities for blood coagulation factor VIII (Comparison Examples 1 -7). Examples 5 - 8, Comparison Examples 8 - 12 Using the separation material products A₁ (Example 5), A₂ (Example 6), A₃ (Example 7) and A₄ (Example 8) and the separation material products B₁ (Comparison Example 8), B₄ (Comparison Example 9), B₅ (Comparison Example 10), B₆ (Comparison Example 11) and B₇ (Comparison Example 12), a series of adsorption experiments by the batch method were carried out in the same manner as in Examples 1 and 2, in order to find out the relationship between the concentration of blood coagulation factor VIII in the supernatant and the amount of adsorption of blood coagulation factor per ml of separation material. The typical example of the relationship between the blood coagulation factor concentration in the supernatant and the amount of adsorption thereof is shown in Fig. 2. Examples 9 - 12, Comparison Examples 13 - 15 Comparison of the amount of adsorption of blood coagulation factor VIII was carried out for each separation material product. The amount of adsorption of blood coagulation factor VIII for each separation material at a concentration of the blood coagulation factor of 0.5 Units per milliliter was calculated from the graph of Fig. 2. The values for Example 8 and for Comparison Example 12 were obtained from curves in the graph by extrapolation. The relationship between the amount of adsorption of blood coagulation factor VIII for each of the separation materials and the specific surface area with respect to pores having diameters of at least 0.1µm for the separation material is shown in the graph of Fig. 3. In Fig. 3, the specific surface area is denoted on the abscissa as "pore inner surface area". Example 9 provides for use of the separation material A₁, Example 10 for the separation material A₂, Example 11 for the separation material A₃, Example 12 for the separation material A₄, Comparison Example 13 for the separation material B₁, Comparison Example 14 for the seapration material B₄ and Comparison Example 15 for the separation material B₇ respectively. Example 13 1) Preparation of a Solution of Limited Degradation Product of Collagen IV A collagen IV was dissolved in 0.1 M acetic acid (pH 2.9) to prepare a solution of a concentration of 4 mg/ml. Here, the dissolution was effected carefully with a revolving rotor for a period of 3 - 4 hours in a plastic sample tube so as not to cause bubbling. Thereto was added of a solution of pepsin of 1 mg per milliliter of sodium acetate buffer solution (pH 5.5) in an amount of 80µℓ per 1 milliliter of the collagen solution and digestion of the collagen was caused for a predetermined period of time at 37 °C. Then, the pH of the solution was adjusted to 7 - 8 by adding 140 µℓ of 1 M tris solution. Thereafter, in order to reduce the decomposed limited degradation product of collagen, 136µℓ of 0.1 M dithiothreitol solution (dissolved in 100 mM phosphate buffer solution containing 5 mM EDTA; pH 6.0) were added thereto to conduct the reaction for 30 minutes at 37°C. Then, 87µℓ of 0.5 M monoiodoacetamide (dissolved in 275 mM tris-hydrochloride solution; pH 8.8) were added thereto in order to prevent the re-combination by -SH group and the reaction was conducted at 30°C for 10 minutes. The resulting solution was dialyzed under the condition of 4°C against a buffer solution of 0.1 M NaHCO₃ - 0.5 M NaCl of pH 8.0, in order to remove the unreacted materials and low-molecular weight amino acids in the solution, whereby a solution of the limited degradation product of collagen IV was obtained. 2) Preparation of Tresylated Glucomannan Beads 0.4 g of dried cross-linked glucomannan beads having an exclusion limit molecular weight of 2× 10⁶ determined as in the case of GM-A₁ was introduced into a mixed liquid of 3 ml of acetone and 150µℓ of pyridine. A sample tube filled with this reaction liquid was briefly shaken and thereto were added 100 µℓ of tresyl chloride as the activating agent. The reaction was conducted for 10 minutes while shaking the sample tube. After the reaction, the tresylated glucomannan beads were separated from the reaction medium by filtration under suction. The beads were washed a mixed solution of acetone and 5 mM hydrochloric acid in a volume ratio of 70 : 30, a mixed solution of acetone and 5 mM hydrochloric acid in a volume ratio of 30 : 70 and finally with 1 mM hydrochloric acid in this sequence. The tresylated glucomannan beads so prepared were stored in 1 mM hydrochloric acid at 4°C (this is denoted hereinafter as tresylated GM/1 mM HCl). 3) Production of Separation Material The limited degradation product of collagen IV was introduced into the tresylated glucomannan beads in the manner given below to produce a separation material. An adequate amount of the suspension of tresylated GM/1 mM HCl was placed in a 10 ml sample tube and was subjected to a centrifugation. The supernatant of the centrifugation was pipetted out so as to adjust the volume ratio of the tresylated glucomannan beads to the supernatant to be 1 : 1. The tresylated GM/1 mM HCl held in a separated state by the centrifugation was shaken to form a uniform suspension of the beads and 1 milliliter of the so formed uniform suspension was quickly pipetted out and transferred into a 3 ml Serumtube (Trade mark, a product of Sumitomo Chemical Co.). After centrifugation, almost all the supernatant was removed by pipetting out using a Pasteur pipette. To 0.5 ml of the tresylated glucomannan beads remaining in the Serumtube, 1 ml of the solution of limited degradation product of collagen IV was added in a Vortex mixer while shaking. During the first few minutes, the shaking of the reaction mixture was continued as such. Then, the reaction of the mixture was conducted in an incubator for 3 hours at 30°C. After 3 hours, the reaction mixture was filtered on a 3G2 glass filter to separate the solids. The thus obtained separation material A₅ was washed with 0.1 M NaHCO₃ - 0.5 M NaCl buffer solution (pH 8.0) and with 1 M NaCl solution. In order to block up the tresyl groups remaining unreacted, the separation material was immersed in 0.1 M tris-hydrochloride - 0.5 M NaCl buffer solution (pH 8.0) at 4°C. The specific surface area with respect to pores with diameters of at least 0.1 µm as observed in the same manner as in the separation material A₁ was 3.0 m²/ml separation material. 4) Experiment of Adsorption of Blood Coagulation Factor VIII The separation material A₅ was charged by normal practice into a glass column having an inner diameter of 3 mm and a length of 40 mm. This column was loaded with 500 µℓ of the cryoprecipitate solution A using an injector and then buffer solution A (20 mM tris-hydochloride, 1 mM CaCl₂, 100 mM NaCl; pH 7.4) was passed through the column at a flow velocity of 0.01 ml/min., while monitoring the effluent liquid by a UV (280 nm), until no protein had been detected. The concentration of blood coagulation factor VIII in the effluent was determined using Testteam ® F VIII and the amount of blood coagulation factor VIII adsorbed on the separation material A₅ was calculated from the difference between this analysis value and the loaded amount thereof. It was determined that 1.33 Units of blood coagulation factor VIII were adsorbed per 1 ml of the separation material A₅. The ratio of the adsorbed amount of blood coagulation factor VIII on the separation material A₅ to the loaded amount thereof was determined to be 53 %.

Claims: A separation material comprising a porous glucomannan matrix having linked thereon one or more ligands each comprising a radical exhibiting an affinity to a blood coagulation factor, said matrix having a specific surface area of at least 1.5 m² per ml of the separation material with respect to pores having diameters of at least 0.1 µm, and wherein the glucomannan has an exclusion limit molecular weight of at least 1.5 x 10⁶ as determined with polyethylene glycol. A separation material as claimed in claim 1, wherein the ligand is selected from collagens, monoclonal antibodies and substances having a group of formula (I) -(NH) l -(CH₂) m -(CONH) n -(CH₂) p -NR¹R² (I) in which R¹ and R² are independently hydrogen or lower as alkyl, l is 0 or 1, m is an integer of 3 - 8, n is 0 or 1 and p is an integer of 0 - 5. A separation material as claimed in claim 2, wherein the group is selected from aminobutylamino, aminohexylamino and aminobutylcarbamylbutylamino. A separation material as claimed in any preceding claim, wherein the glucomannan has an exclusion limit molecular weight of 2 x 10⁶ to 5 x 10⁶. A separation material as claimed in any preceding claim, wherein the glucomannan is konjakmannan. A separation material as claimed in any preceding claim, wherein the matrix has a specific surface area of at least 3 m² per ml of separation material with respect to pores having diameters of at least 0.1 µm. A separation material as claimed in any preceding claim, wherein the blood coagulation factor is blood coagulation factor VIII or a complex thereof with von Willebrand factor. A process for preparing a separation material as claimed in any preceding claim, comprising the steps of: subjecting the porous particulate glucomannan to activation by 1,1'-carbonyldiimidazolating, tresylating, carbodiimidising, thiopropylating, epoxidising, bromocyanising or formylating using a corresponding activating agent; and reacting the thus-activated porous particulate glucomannan with one or more substances providing the or each ligand. A process for recovering a blood coagulation factor from raw material containing it, comprising the steps of: bringing the raw material into contact with a separation material as claimed in any of claims 1 to 6 in the presence of an adsorption buffer solution; and subjecting the resulting separation material containing the blood coagulation factor adsorbed thereon to elution by contacting it with an eluent buffer solution. A process as claimed in claim 9, wherein the blood coagulation factor to be recovered is blood coagulation factor VIII or a complex thereof with von Willebrand factor and the separation material is as claimed in claim 7.

Inventors: JOKO, ISAO, MORITA, HIROSHI, MOTOZATO, YOSHIAKI, TOMODA, TAKASHI, WAKITA, MASAAKI, YAMAGUCHI, MASATO, YAMAMOTO, TETSURO, JOKO, ISAO, C/O KURITA WATER, MORITA, HIROSHI, C/O KURITA WATER, TOMODA, TAKASHI, C/O KURITA WATER, WAKITA, MASAAKI, C/O KURITA WATER, YAMAGUCHI, MASATO, C/O KURITA WATER
Applicants: KURITA WATER INDUSTRIES LTD., MOTOZATO, YOSHIAKI, YAMAMOTO, TETSURO
Application year: 1990
Main classification: B01J20
IPC codes: G01N33, C07K1, B01D15, C07K19, C07K14, C07K17, B01J20