JPH04261196A - Separation of blood coagulation factor - Google Patents

Abstract

PURPOSE:To separate the title factor such as blood coagulation factor VIII efficiently and in high purity by bringing a separating agent prepared by bonding a material having affinity for blood coagulation factor to crosslinked glucomannan spherical particles into contact with a deprotenized blood coagulation factor-containing raw material. CONSTITUTION:A separating agent obtained by using a substance having affinity for blood coagulation factor such as blood coagulation factor VIII or complex of blood coagulation factor VIII-von Willebrand factor as a ligand, one or more of a group shown by the formula (R<1> and R<2> are H or lower alkyl; q is 0 or 1; m is 3-8 integer; n is 0 or 1; r is 0-5 integer), collagen, monoclonal antibody, etc., and bonding the ligand to crosslinked glucomannan spherical particles is brought into contact with a previously deprotenized blood coagulation factor-containing raw material, the blood coagulation factor is adsorbed on the separating agent, separated and eluted to efficiently separate and recover the high-purity blood coagulation factor.

Description

[Detailed description of the invention]

0001

[Field of Industrial Application] The present invention relates to a method for separating blood coagulation factors, and more specifically to a method for efficiently separating and recovering blood coagulation factors, particularly blood coagulation factor VIII, from plasma or raw materials containing blood coagulation factors with high purity. This invention relates to a method for separating blood coagulation factors.

0002

BACKGROUND OF THE INVENTION Human and animal blood contains various substances (blood coagulation factors) related to the process of blood coagulation. Various diseases are known to be caused by deficiencies in blood coagulation factors, especially blood coagulation factor VII.
Hemophilia A, due to deficiency of factor I, accounts for 80% of all these diseases. There is no known radical treatment for hemophilia A, and blood coagulation factor VIII replacement therapy is performed as needed.

Blood coagulation factor VIII has a molecular weight of approximately 26
10,000 proteins, but only 0.1 μg/kg in normal plasma.
Contains only ml. Blood coagulation factor VIII is a different blood coagulation factor in plasma with a molecular weight of 50.
The von Willebrand factor (von Willebrand factor) consists of 10,000 to 20 million proteins.
tor (hereinafter sometimes abbreviated as vWf)) (in the present invention, when simply referred to as a blood coagulation factor, such a complex is also included). Blood coagulation in plasma V
Since the concentration of factor III is low, it is not possible to replenish a sufficient amount of blood coagulation factor VIII for hemophilia A patients by using whole blood or plasma as is. Therefore, intravenous injection of concentrated preparations of blood coagulation factor VIII is widely practiced.

[0004] To concentrate blood coagulation factor VIII,
Cryoprecipitate is prepared from plasma and purified by heating and concentration or liquid chromatography.

Conventionally, the separation of blood coagulation factor VIII by liquid chromatography has been carried out using substances that have an affinity for blood coagulation factor VIII or vWf, such as aminoalkyl groups (Japanese Patent Application Laid-open No. 136518/1983, Ligands such as monoclonal antibodies (Japanese Patent Application Laid-open No. 1-157000), monoclonal antibodies (Japanese Patent Application Laid-Open No. 1986-13099, Japanese Patent Application Laid-Open No. 1-221396), collagen (Japanese Patent Application Laid-Open No. 63-198632, etc.) are bound to a support. This is carried out by bringing a separating agent into contact with cryoprecipitate, and causing blood coagulation factor VIII in the cryoprecipitate to be adsorbed to the separating agent. However, in such conventional methods, the amount of blood coagulation factor VIII adsorbed to the separation agent,
The relationship between the content of coexisting proteins such as fibrinogen (blood coagulation factor I) and fibronectin contained in cryoprecipitate has not been quantitatively investigated, and therefore blood coagulation factor VIII that is separated and collected The purity of the factor is low, and separation and recovery is performed inefficiently.

[0006]

SUMMARY OF THE INVENTION An object of the present invention is to provide a blood coagulation factor that can efficiently and quantitatively separate and recover blood coagulation factors, particularly blood coagulation factor VIII, with high purity, and that can reduce the size of the separation device. The purpose is to propose a separation method for

[0007]

[Means for Solving the Problems] The present inventors have developed a separation agent in which blood coagulation factor VIII and coexisting proteins such as fibrinogen and fibronectin in a blood coagulation-containing raw material have a ligand bound to crosslinked glucomannan spherical particles. The phenomenon of adsorption to the separating agent is competitive adsorption, and therefore, in order to increase the amount of blood coagulation factor VIII adsorbed to the separating agent, the inventors discovered that it is necessary to remove coexisting proteins as much as possible, and completed the present invention. .

That is, the present invention provides the following method for separating blood coagulation factors.

(1) A separation agent in which a substance with affinity for blood coagulation factors is bound to crosslinked glucomannan spherical particles as a ligand is brought into contact with a blood coagulation factor-containing raw material from which proteins have been removed in advance, and blood coagulation factors are removed. A method for separating blood coagulation factors, which comprises adsorbing them to the separation agent. (2) The method according to (1) above, wherein the blood coagulation factor is blood coagulation factor VIII or a complex of blood coagulation factor VIII and von Willebrand factor. (3) The method according to (1) or (2) above, wherein the ligand is one or more selected from the group consisting of a group represented by the following formula 2, collagen, and a monoclonal antibody.

(In the formula, R1 and R2 represent a hydrogen atom or a lower alkyl group. R1 and R2 may be the same or different. q is 0 or 1, m is an integer of 3 to 8, and n is 0 or 1
, r represents an integer of 0 to 5. )

In the separation method of the present invention, coexisting proteins coexisting with blood coagulation factors are removed in advance from a raw material containing blood coagulation factors.

[0011] As the raw material containing blood coagulation factors, any material containing blood coagulation factors can be used, such as plasma, cryoprecipitate, Cohn's fraction,
Examples include blood coagulation factor-containing products obtained by culturing cells that secrete blood coagulation factors. Among these, those containing blood coagulation factor VIII or a complex of blood coagulation factor VIII and vWf (hereinafter referred to as vWf complex) are preferred.

[0012] In order to remove coexisting proteins from such blood coagulation factor-containing raw materials, the concentration of blood coagulation factors must be high relative to the protein concentration in the blood coagulation factor-containing raw materials, and moreover, the blood coagulation factors must be physiologically active due to protein denaturation, etc. Any method can be used as long as it does not cause deactivation of the enzyme. Examples of such methods include: (1) gel filtration chromatography;
Membrane methods, ion exchange chromatography methods, centrifugation methods, methods that use reagents to improve the degree of purification of blood coagulation factors during the preparation of blood coagulation factor-containing raw materials, and methods that combine these methods. I can give it to you.

[0013] The gel filtration chromatography method (■) is a method of separating blood coagulation factors and coexisting proteins based on the difference in their molecular weights, and is a method that separates blood coagulation factors and coexisting proteins based on the difference in molecular weight. Coexisting proteins can be removed by collecting only that fraction. There are no particular restrictions on the gel used.

[0014] The method (2) using a membrane is a method of separating blood coagulation factors and coexisting proteins based on the difference in their molecular weights, as in (2) above, and the coexisting proteins can be removed according to a conventional method. There are no particular restrictions on the material of the membrane used.

The ion-exchange chromatography method described in (2) above is a method for separating blood coagulation factors and coexisting proteins based on the difference in their exchange-adsorption properties with respect to the ion exchanger. Coexisting proteins can be removed by collecting only the fraction containing blood coagulation factors eluted from the blood coagulation factor. There are no particular limitations on the ion exchanger used, and for example, ion exchangers into which diethylaminoethyl (DEAE) groups, quaternary ammonium ethyl (QAE) groups, etc. have been introduced can be used.

[0016] In the centrifugation method (2) above, coexisting proteins can be removed by ordinary centrifugation or ultracentrifugation.

[0017] In the method using the reagent (①),

As typified by the literature [Example 1], polyethylene glycol (
Coexisting proteins can be removed using PEG), glycine, sodium chloride, etc.

Blood coagulation VII can be obtained by the separation method of the present invention.
When separating and recovering factor I, blood coagulation factor VIII 1
It is desirable to remove protein so that the coexisting protein concentration is 2 mg/ml or less, preferably 1 mg/ml or less. Here, U represents "Unit blood coagulation factor VIII: C".

In the separation method of the present invention, a blood coagulation factor-containing raw material from which coexisting proteins have been removed by the method described above,
A separation agent (hereinafter referred to as
(simply referred to as a separating agent). In this case, a method such as a column method or a batch method can be employed, and the blood coagulation factor-containing raw material and the separation agent are brought into contact in the presence of an appropriate adsorption buffer to adsorb the blood coagulation factor to be separated. Next, the separation agent adsorbed with blood coagulation factors is brought into contact with an appropriate desorption buffer,
Blood coagulation factors are desorbed and recovered.

The adsorption buffer used in the column method may have a pH of 4.5 to 9.5, preferably 5.5 to 8.8. Typical adsorption buffers include, for example, bis-tris-hydrochloric acid (pH 5.5-7.3).
), triethanolamine-hydrochloric acid (pH 7.3-7.7
), diethanolamine-hydrochloric acid (pH 8.4-8.8)
, citric acid-sodium citrate, and these buffers to which about 1 mM calcium chloride and about 100 mM sodium chloride are added. In addition, a typical desorption buffer is the adsorption buffer mentioned above plus about 0.5 to 3M sodium chloride and 50%
Examples include those to which about 0mM calcium chloride is added. For the separation treatment, it is preferable to select and use a buffer solution as close as possible to the pH of the raw material from among the above-mentioned buffer solutions.

[0021] The flow velocity LV during adsorption is usually 5 to 500 cm/
hr, preferably 50 to 300 cm/hr, and the flow rate LV during desorption is usually 5 to 500 cm/hr, preferably 50 to 300 cm/hr.
300cm/hr is desirable.

[0022] In the batch method, shaking at a shaking intensity of 10°C is sufficient to mix the blood coagulation factor-containing raw material and the separation agent at room temperature.
It is preferable to shake for at least a minute. In the batch method, the same buffer solution as in the column method can be used.

[0023] By separating blood coagulation factors using the method described above, blood coagulation factors can be separated and recovered with high purity, efficiently, and quantitatively. Furthermore, by removing coexisting proteins, the separation device can be downsized.

Next, the separation agent used in the separation method of the present invention will be explained.

The cross-linked glucomannan spherical particles before binding with a ligand are hydrophilic gel particles in which glucomannan containing D-glucose and D-mannose as main components is cross-linked with a cross-linking agent. Glucomannan has a molecular weight of 9 x 105 to 2.4 x 106, preferably 1
x106 to 1.3 x106 is desirable. Such glucomannan is preferably konjac mannan, but is not limited thereto.

The ligand to be bound to such crosslinked glucomannan spherical particles is not particularly limited as long as it has a specific interaction (affinity) with the blood coagulation factor to be separated. Examples include a group represented by , collagen, and a monoclonal antibody, and one or more of these can be used.

Examples of the group represented by the above formula 2 include groups represented by the following formula 3 or 4.

[0028]

[Chemical formula 3]

[0029]

embedded image (In the formula, R1 and R2 are the same as in the above formula 2. q is 0 or 1, m is an integer of 3 to 8, and r is an integer of 2 to 5.)

Specifically, the group represented by the above formula 3 is 4-aminobutyl group [-CH2(CH2)3NH2
], 6-aminohexyl group [-CH2(CH2)5NH
2], 4-aminobutylamino group [-NH(CH2)4
NH2], 6-aminohexylamino group [-NH(CH
2) 6NH2].

Specifically, the group represented by the above formula 4 is 4-aminobutylcarbamylbutyl group [-CH2(
CH2)3C(=O)NH(CH2)4NH2], 3-
Aminopropylcarbamylbutyl group [-CH2(CH2
)3C(=O)NH(CH2)3NH2], dimethylaminobutylcarbamylbutyl group [-CH2(CH2)3
C(=O)NH(CH2)4N(CH3)2], dimethylaminopropylcarbamylbutyl group [-CH2(CH
2) 3C(=O)NH(CH2)3N(CH3)2],
dimethylaminopropylcarbamylpentyl group [-CH
2(CH2)4C(=O)NH(CH2)3N(CH3
)2], 4-aminobutylcarbamylbutylamino group [
-NH(CH2)4C(=O)NH(CH2)4NH2
], 3-aminopropylcarbamylbutylamino group [-
NH(-CH2)4C(=O)NH(CH2)3NH2
], dimethylaminobutylcarbamylbutylamino group [
-NH(CH2)4C(=O)NH(CH2)4N(C
H3)2], dimethylaminopropylcarbamylbutylamino group [-NH(-CH2)4C(=O)NH(CH
2) 3N(CH3)2], dimethylaminopropylcarbamylpentylamino group [-NH(CH2)5C(=O
)NH(CH2)3N(CH3)2].

As collagen, limited degradation products obtained by enzymatically treating type IV collagen with pepsin or the like, type III collagen, etc. can be preferably used.

[0033] The ligand may be bonded directly to the crosslinked glucomannan spherical particles or may be bonded via a suitable spacer. The ligand is produced by 1,1'-carbonyldiimidazolization, tresylation, carbodiimidization, thiopropylation, epoxidation, bromocyanation, or formylation of a certain site in the crosslinked glucomannan spherical particles using an activating agent. After activation, it can be introduced.

[0034] Examples of the above activator include 1,1'
-carbonyldiimidazole, tresyl chloride (2,
2,2-trifluoroethanesulfonyl chloride), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, 1,4-butanediol glycidyl ether, glutaraldehyde, hexamethylene diisocyanate, cyanogen bromide, bisoxirane, 1,4-bis(
Examples include 2,3-epoxypropoxy)butane.

[0035] Crosslinked glucomannan spherical particles before introduction of a ligand can be produced by crosslinking glucomannan. Specific examples of manufacturing methods include the following methods.

First, a purified product of glucomannan obtained by refining commercially available glucomannan with alcohol or the like is dissolved in a solvent such as formamide or dimethylformamide, and using pyridine or the like as a catalyst, acetic acid, acetic anhydride, propionic acid,
Glucomannan esters are produced by adding acids such as butyric acid and nitric acid.

Next, the glucomannan ester and the porosity-forming agent are dissolved in a solvent. As a solvent, it has a boiling point lower than the aqueous medium described below and does not dissolve in the aqueous medium at all, or
Or use one that dissolves only slightly. Specifically, chlorinated hydrocarbon organic solvents such as dichloromethane, chloroform, carbon tetrachloride, and trichloroethylene are used as such solvents, and these can be used alone or in combination. The dissolved concentration of glucomannan ester is 0.5 to 10 g per 100 ml of solvent.
Preferably the proportion is 0.5 to 2 g.

Porosifying agents are used to make spherical particles porous after they are removed, and specifically, decalin (decahydronaphthalene), methyl n-caprate, tetrahydrocarbon naphthalene, ethylbenzene, diethylbenzene, methyl dodecanoate, toluene,
Used are hexyl alcohol, heptyl alcohol, octyl alcohol, and other solvents that have a higher boiling point than the above-mentioned organic solvents and do not dissolve the glucomannan ester. The concentration of the porosity agent is ester of glucomannan 1
The ratio is 1 to 5 ml, preferably 2 to 4 ml, per g.

Next, the solution of glucomannan ester obtained as described above is suspended in an aqueous medium to form droplets. As the aqueous medium, water or water to which hydrophilic protective colloids such as polyvinyl alcohol, partially saponified polyvinyl alcohol, carboxymethyl cellulose, ethyl cellulose, methyl cellulose, soluble starch or gelatin are added can be used. The hydrophilic protective colloid is preferably used as an aqueous solution of 0.1 to 10% by weight, preferably 1 to 5% by weight. Further, the amount of the aqueous medium used is at least twice the volume of the glucomannan ester solution, preferably 10 to 50 times the volume.

[0040] Methods for suspending the glucomannan ester solution in an aqueous medium include adding the entire amount of the glucomannan ester solution to the aqueous medium and stirring to disperse and suspend it; Examples include a method in which a solution of glucomannan ester is added all at once or dropwise to the stirred state. Glucomannan esters are water-insoluble, so they can be finely dispersed in aqueous media.
suspend At this time, evaporation of the organic solvent begins simultaneously with granulation, and finally, the solvent is substantially removed and spherical particles of glucomannan ester with pores formed are obtained. The temperature used to evaporate the organic solvent in the droplets is above the freezing point of the aqueous medium and below the boiling point of the organic solvent. The temperature is preferably 1 to 5°C lower than the boiling point of the organic solvent.

[0041] Spherical particles are formed by stirring the aqueous medium, and particles of any desired size can be obtained by adjusting the stirring speed. In the case of the present invention, it is sufficient to stir the separation agent so as to form particles having a suitable particle size in the range of 1 to 500 μm.

Next, the glucomannan ester spherical particles are saponified. In this case, it is necessary to use a saponification bath that can saponify the spherical particles while maintaining their shape without destroying it. Saponification is carried out by contacting spherical particles of glucomannan ester with a saponification bath. Saponification removes most of the porogen from the pores of the spherical particles. Examples of saponification baths include methanol solutions of sodium hydroxide or potassium hydroxide, and solutions in which sodium hydroxide or potassium hydroxide is dissolved in an aqueous salt solution such as sodium sulfate.

Next, the saponified glucomannan spherical particles are crosslinked. Examples of the crosslinking agent include difunctional compounds such as epichlorohydrin, diepoxybutane, tolylene diisocyanate, and hexamethylene diisocyanate. These crosslinking agents are dissolved in an organic medium and used as a crosslinking agent solution.

The organic medium for the crosslinking agent is, for example, kerosene or liquid paraffin or a mixture thereof (for example, in a volume ratio of 7:3) and a surfactant (nonionic surfactant, for example, sorbitan fatty acid ester) in an amount of 1 to 2 weight parts. % mixture, a mixed solution of acetone and dimethyl sulfoxide (eg, volume ratio 6:4), a mixed solution of acetone and dimethylformamide (eg, volume ratio 2:3), etc. are used. The concentration of the crosslinking agent is in the range of 0.01 to 15 mol/l based on the organic medium liquid of the crosslinking agent.

Adding 1 to 5 parts by weight of saponified glucomannan spherical particles to 100 parts by volume of the crosslinking agent solution,
By continuing stirring at room temperature to 70°C for 24 to 36 hours, the glucomannan spherical particles are crosslinked. The degree of crosslinking can be adjusted by adjusting the concentration of the crosslinking agent. The crosslinked particles are filtered, washed with acetone, then with a neutral detergent, and then with water to obtain crosslinked glucomannan spherical particles.

The cross-linked glucomannan spherical particles thus obtained have high pressure resistance and are extremely stable chemically and physically, for example, hardly swelling or shrinking in various organic solvents or salt solutions. . Therefore, even if a ligand is introduced using an organic solvent or salts by the method of introducing a ligand described below, the pore structure of the particles will not change and the particles will not be deformed or coalesced. Therefore, a separation agent having sufficient pore size, surface area, ligand density (amount of ligand introduced into cross-linked glucomannan spherical particles), and adsorption capacity can be prepared. Furthermore, since crosslinked glucomannan spherical particles have excellent heat resistance, they can be sterilized using an autoclave or the like.

Next, a method for introducing a ligand into crosslinked glucomannan spherical particles will be described in detail.

First, the crosslinked glucomannan spherical particles are sufficiently brought into contact with a reaction solvent to swell them, and at the same time, bubbles are degassed. The reaction solvent used here is preferably a solution that is a suitable solvent for reacting the crosslinked glucomannan spherical particles with the activator in the next step, such as dimethylformamide, a mixed solution of acetone and pyridine, Examples include an aqueous sodium hydrogen borate solution of sodium hydroxide.

Next, the above-mentioned activator is added, and the activator and the wet crosslinked glucomannan spherical particles are sufficiently mixed and reacted to activate the crosslinked glucomannan spherical particles. Activation is performed by activating a certain site in the cross-linked glucomannan spherical particles 1,1
′-Carbonyldiimidazolization, tresylation, carbodiimidation, thiopropylation, epoxidation, cyanogen bromide, or formylation, etc., to bind the ligand to be introduced to this activated site. .

The proportion of the activator used depends on the type of activator used, but usually crosslinked glucomannan spherical particles 1
It is used in a proportion of 2 to 200 parts by weight per 00 parts by weight. The reaction conditions for activation are usually a reaction temperature of 0 to 50°C and a reaction time of 5 minutes to 3 hours.

[0051] Specifically, the crosslinked glucomannan spherical particles can be activated by the following method.

To form 1,1'-carbonyldiimidazole, there is a method in which crosslinked glucomannan spherical particles are reacted with 1,1'-carbonyldiimidazole in dimethylformamide. Tresylation can be carried out by reacting crosslinked glucomannan spherical particles with tresyl chloride in a mixed solution of acetone and pyridine. To thiopropylate, crosslinked glucomannan spherical particles are reacted with epichlorohydrin, followed by thiosulfate, then reduced with dithiothreitol, and then 2
, 2'-dipyridyl disulfide, and the like. To epoxidize, cross-linked glucomannan spherical particles are mixed with sodium borohydride.
．． Examples include a method of reacting with bisoxirane (for example, 1,4-butanediol diglycidyl ether) in a 3M aqueous sodium hydroxide solution. In order to produce cyanogen bromide, a method may be used in which crosslinked glucomannan spherical particles are reacted with cyanogen bromide in an aqueous sodium hydroxide solution (pH 11 to 12).

After activation, the activated crosslinked glucomannan spherical particles are collected by filtration or the like, and then thoroughly washed to remove the activator. The cleaning liquid can be selected as appropriate, but for example, when a mixture of acetone and pyridine is used as the reaction solvent and tresyl chloride is used as the activator, it is preferable to use a mixture of acetone and low concentration hydrochloric acid.

Next, a ligand is bound to the ligand-containing liquid and the activated crosslinked glucomannan spherical particles and introduced. The reaction conditions in this case are usually a reaction temperature of 0 to 40°C;
The reaction time is 15 minutes to 24 hours. The ligand-containing solution is prepared by dissolving the ligand in an appropriate buffer or the like. The amount of the ligand used is usually preferably at least the reaction equivalent with the activation site in the crosslinked glucomannan spherical particles.

[0055] Next, the crosslinked glucomannan spherical particles into which the ligand has been introduced are collected by filtration or the like, and then thoroughly washed. The washing liquid can be selected as appropriate, and includes, for example, an aqueous sodium chloride solution, an acetate buffer containing sodium chloride, a bicarbonate buffer containing sodium chloride, pure water, and an organic solvent.

Next, unreacted activated sites in the crosslinked glucomannan spherical particles are blocked. Blocking can be carried out by immersing unblocked crosslinked glucomannan spherical particles in a suitable blocking agent (solution) at 0 to 40°C for 30 minutes to 24 hours. As the blocking agent (solution), for example, a Tris-hydrochloric acid buffer containing sodium chloride,
Examples include monoethanolamine.

[0057] Crosslinked glucomannan spherical particles are 1,1'-
When activated with carbonyldiimidazole and using the group represented by the above formula 2 as a ligand, the resulting separating agent has the form shown in the following formula 5, for example.

[0058]

embedded image (Here, A represents crosslinked glucomannan spherical particles. R
1, R2, m, n and r are the same as in Chemical Formula 2 above. )

The amount of ligand introduced into the crosslinked glucomannan spherical particles (ligand density) is not particularly limited, but it is preferable to introduce a sufficient amount of ligand necessary for adsorption.

[0060] The separation agent obtained by introducing a ligand into crosslinked glucomannan spherical particles by such a method has excellent pressure resistance, so it can be used at a high flow rate and can be processed through a large amount per hour.

[0061]

As described above, according to the present invention, since the blood coagulation factor raw material from which proteins present in the blood coagulation factor-containing raw material have been removed in advance is brought into contact with a specific separation agent, the blood coagulation factor In particular, blood coagulation factor VIII can be separated and recovered with high purity, efficiently, and quantitatively, and the separation device can be downsized.

[0062]

[Example] Next, an example of the present invention will be described. The unit U indicating the concentration of blood coagulation factor VIII is “Uni
tBlood coagulation factor VIII:C.

Example 1 (1) Preparation of crosslinked glucomannan spherical particles 60 g of konjac mannan powder was added to 6 liters of tap water at about 80° C. and dissolved with stirring. This solution was dropped little by little into 6 liters of ethanol, and the resulting precipitate was separated by filtration, air-dried, and vacuum-dried to obtain purified glucomannan (hereinafter, glucomannan may be abbreviated as GM).

[0064] 30 g of the above purified glucomannan was placed in 1 liter of formamide and swollen at 55°C for 7 hours, then 300 ml of pyridine was added, and 2 hours later, 300 ml of acetic anhydride was added and reacted at 55°C for 4 days. Esterified. The reaction mixture was added to 7 liters of water with stirring. The resulting precipitate was filtered, washed with water, air-dried, and then vacuum-dried. The obtained crude acetate was dissolved in 10 liters of acetone to remove insoluble matter, and then added to about 20 liters of water with stirring. The resulting precipitate was filtered, air-dried, and then vacuum-dried to obtain glucomannan acetate A.

10 g of the above acetate A was added to 560 ml of chloroform together with 35 ml of methyl n-caprate as a pore-forming agent.
dissolved in This solution was poured into 5 liters of 1 wt% aqueous solution of 90% saponified polyvinyl alcohol at 55°C for 600 ml.
The mixture was added dropwise with stirring at a stirring speed of pm. 24
After a period of time, the mixture was slowly cooled, and the spherical particles were filtered off and washed with water. The spherical particles were added to a mixed solution of 225 ml of methanol and 25 ml of 10N sodium hydroxide, and the mixture was stirred for 2 hours to saponify the mixture.

Thereafter, the saponified spherical particles were filtered, added to a mixture of 200 ml of acetone, 300 ml of dimethylformamide, and 70 ml of epichlorohydrin, and incubated at 60° C. for 24 hours.
Crosslinking was achieved by reacting for a period of time. The particles were collected, washed with water and extracted with acetone to obtain crosslinked glucomannan spherical particles A (hereinafter referred to as GM
-A) was obtained.

(2) Introduction of aminohexylamino group The GM-A obtained in (1) above was washed with water and then with acetone while being suction-filtered, and then vacuum-dried. These dried crosslinked glucomannan spherical particles and dimethylformamide (hereinafter referred to as D
(sometimes abbreviated as MF) was placed in a measuring cylinder and allowed to stand for one day. Next, DM-DM 6.7ml of precipitated GM-A.
Add to F13.4ml and add 2.7mmol of 1,1
'-Carbonyldiimidazole (hereinafter sometimes abbreviated as CDI) was added, and the mixture was shaken and stirred for 0.5 hour. The CDI activated particles thus obtained were filtered off by suction filtration and washed with DMF.

The washed CDI activated particles were placed in 6.7 ml of DMF, 3.4 mmol of 1,6-diaminohexane was added, and the mixture was shaken and stirred for 0.5 hour. The aminohexylamino group-introduced particles obtained in this way are filtered by suction filtration,
Separating agent A was obtained by washing with DMF and then water. The aminohexylamino group-introduced particles were stored in a 20% by volume ethanol aqueous solution. Note that the series of operations were performed at room temperature.

■Preparation of raw material containing blood coagulation factor VIII (cryoprecipitate) In a low temperature laboratory (4°C), 1 liter of plasma was treated with 100 mg of soybean trypsin inhibitor (SBTI), 1.5 g of benzamidine, and 1M diisopropylfluorophosphate. (DFP) 1 ml was added and stirred for about 1 hour. Thereafter, 10 g of Macrogol 4000 (polyethylene glycol) was added and stirred again for about 1 hour. Next, 35 ml of ethanol was added dropwise over 2 to 3 hours, and the mixture was stirred overnight. The next day, this plasma was incubated at 4°C and 9500 rpm.
Centrifugation was performed at ×20 minutes to obtain cryoprecipitate. 55m of this cryoprecipitate
It was dissolved in 40 ml of M citrate buffer (pH 7.4) to obtain a crude cryoprecipitate solution. The concentration of blood coagulation factor VIII in this solution was 9.3 U/ml, and the protein concentration was 77 mg/ml (8.3 mg/ml per 1 U/ml of blood coagulation factor VIII).

Next, in order to remove protein, the unpurified cryoprecipitate solution was purified. That is, the above unpurified cryoprecipitate was heated at 26°C and 9500 rpm.
Centrifugation was performed at m×20 minutes to obtain 35 ml of supernatant. To this supernatant, 117 m
1 and stirred at 26°C for about 30 minutes. Next, this solution was centrifuged at 26° C. and 9500 rpm for 20 minutes to obtain 140 ml of supernatant. Add 11 sodium chloride to this supernatant.
2.7 g was added and stirred at room temperature for about 30 minutes. After that 2
Centrifugation was performed at 0° C. and 9500 rpm for 20 minutes to obtain purified cryoprecipitate. This purified cryoprecipitate was added again to 55mM citrate buffer (pH 7.4)2.
0 ml to obtain a purified cryoprecipitate solution. The concentration of blood coagulation factor VIII in this solution is 5 U/m
l, protein concentration is 5 mg/ml (blood coagulation VII
(1 mg/ml per 1 U/ml of factor I), and the protein concentration was reduced to about 1/8 per 1 U/ml of blood coagulation factor VIII compared to the unpurified cryoprecipitate solution.

Furthermore, by changing the conditions when centrifuging the unpurified cryoprecipitate solution, four types of purified cryoprecipitate solutions having different protein concentrations were obtained. The determination of blood coagulation factor VIII in each cryoprecipitate solution was conducted by Test Team FVII.
I (Kabi Vitrum AB, Sweden
Manufactured by Kabi Vitrum AB
The endpoint method was used to conduct the test using the end point method.

■ Adsorption test for blood coagulation factor VIII An adsorption test for separating agent A of blood coagulation factor VIII was carried out in a batch test using the six types of purified or unpurified cryoprecipitate solutions obtained in the above (■). I did it.

That is, accurately measure 50 μl of the separation agent A obtained in step ① above into a microtube with an internal volume of 1.5 ml, and
Next, add 1 ml of each cryoprecipitate solution and
Shake at ℃ for 1 hour. Next, the separation agent A and the cryoprecipitate solution in the microtube were centrifuged at 10,000 rpm for 20 minutes. The concentration of blood coagulation factor VIII and protein in the obtained supernatant was quantified, and the difference from the concentration in the cryoprecipitate solution used in the test was determined. Quantity. 1 U/m in cryoprecipitate solution
FIG. 1 shows the relationship between the concentration of protein coexisting with blood coagulation factor VIII (mg/ml) and the amount of protein adsorption (mg/ml separation agent). Also, the amount of protein adsorption (m
g/ml separation agent) and blood coagulation factor VIII adsorption amount (U
/ml separation agent) is shown in Figure 2. In each figure,
○ is the result of using unpurified cryoprecipitate solution,
● is the result using purified cryoprecipitate solution.

From FIGS. 1 and 2, it can be seen that when the protein concentration in the cryoprecipitate solution decreases, the amount of protein adsorption decreases (FIG. 1), and as the amount of protein adsorption decreases, the amount of blood coagulation factor VIII adsorption decreases. It can be seen that the number is increasing (Figure 2). This means that by reducing the protein concentration in the cryoprecipitate solution, the adsorption amount of blood coagulation factor VIII can be increased, and the purity of blood coagulation factor VIII when recovered can be increased. It shows that it is possible. Furthermore, the adsorption amount of blood coagulation factor VIII can be quantitatively predicted from the protein concentration in the cryoprecipitate solution. Furthermore, it is found that blood coagulation factor VIII can be adsorbed at an adsorption amount of 20 U/ml or more by removing coexisting proteins from the cryoprecipitate solution.

Example 2 Biogel A-15m (particle size 80-150 μm)
Packed into a stainless steel column of m x 250 mm, N = 2
A performance of 800/m was obtained. Next, this column was attached to a commercially available high performance liquid chromatography device, and 20mM Tris-HCl, 100mM sodium chloride (pH 7.4) was used as the eluent at 157μl/min (LV=0.33m/hr).
) Aging was performed for a while at a flow rate of

Next, unpurified cryoprecipitate solution (
50 μl of blood coagulation factor VIII (10 U/ml, protein concentration 65 mg/ml) was injected into the column, and each fraction of the resulting chromatogram was fractionated. Note that detection was performed using UV 280 nm. Figure 3 shows the preparative chromatogram.
Shown below. As a result, peak A contained coexisting proteins with blood coagulation factor VIII, and peaks B and C contained only coexisting proteins. Peak A Blood Coagulation VII
Factor I concentration is 0.3 U/ml, protein concentration 0.2 m
g/ml, and the protein concentration is 1 U/ml of blood coagulation factor VIII compared to unpurified cryoprecipitate solution.
per ml was reduced to about 1/10.

[0077] An adsorption test for blood coagulation factor VIII was conducted in the same manner as in Example 1 using the unpurified and peak A cryoprecipitate solutions. As a result, in the case of the unpurified cryoprecipitate solution, the protein adsorption amount was 15 mg/ml, and the blood coagulation factor VIII adsorption amount was 10 U/ml, but the peak A cryoprecipitate solution was If the protein adsorption amount is 7mg/m
l Separation agent, blood coagulation factor VIII adsorption amount 30U/ml
It is a separating agent and the adsorption amount of blood coagulation factor VIII increased.

[Brief explanation of the drawing]

FIG. 1 is a graph showing the results of Example 1.

FIG. 2 is a graph showing the results of Example 1.

FIG. 3 is a graph showing the results of Example 2.

Claims (3)

[Claims] 1. A separating agent in which a substance having affinity for blood coagulation factors is bound to crosslinked glucomannan spherical particles as a ligand is brought into contact with a blood coagulation factor-containing raw material from which proteins have been removed in advance. A method for separating blood coagulation factors, which comprises adsorbing them to a separating agent. 2. The method according to claim 1, wherein the blood coagulation factor is blood coagulation factor VIII or a complex of blood coagulation factor VIII and von Willebrand factor. 3. The ligand is a group represented by the following formula 1,
3. The method according to claim 1, wherein the antibody is one or more selected from the group consisting of collagen and monoclonal antibodies. [Formula 1] (In the formula, R1 and R2 represent a hydrogen atom or a lower alkyl group. R1 and R2 may be the same or different. q is 0 or 1, m is an integer of 3 to 8, and n is 0 or 1
, r represents an integer of 0 to 5. )