JP5836577B2 - Affinity carrier production method for purifying or removing a protein or peptide having a kringle sequence, its purification method, and removal method - Google Patents

Description

  The present invention relates to an affinity carrier for specifically purifying and removing a protein or peptide having a kringle sequence from a solution of a protein or peptide having a kringle sequence mixed with contaminating components, and a kringle sequence using the affinity carrier. The present invention relates to a method for purifying and removing a protein or peptide possessed. The present invention also relates to a technique for producing a protein or peptide having a highly purified kringle sequence.

  The kringle sequence is an amino acid sequence having a unique folding structure formed by three pairs of SS bridges, and is a structural domain named because the structure resembles Danish confectionery bread. Kringle sequences are frequently found in proteins involved in blood coagulation and fibrinolysis, and proteins and protein fragments containing kringle sequences promote not only blood coagulation and blood fibrinolysis but also angiogenesis. It exhibits various physiological activities in vivo, such as inhibiting angiogenesis, promoting cell proliferation, inactivating cell proliferation, and apoptosis.

  Plasminogen is a single-chain glycoprotein having a molecular weight of about 92 kDa composed of 791 amino acid residues, and has serine protease and five kringle sequences in the molecule. Plasminogen undergoes limited degradation by tissue plasminogen activator and urokinase plasminogen activator and is converted to an active plasmin having enzyme activity. Plasmin dissolves fibrin fibers, the main component of thrombi. Thus, plasminogen and active plasmin are in vivo proteins having a kringle sequence involved in the fibrinolytic reaction of blood.

  Tissue plasminogen activator is a glycoprotein having a molecular weight of about 70 kDa consisting of 527 amino acid residues produced in vascular endothelial cells, and has serine protease and two kringle sequences in the molecule. Tissue plasminogen activator binds to fibrin, degrades plasminogen that is also bound to fibrin, and promotes blood fibrinolysis as described above. It is also used as a treatment for stroke and cerebral infarction.

  Urokinase is a protease produced in kidney cells and present in urine or blood. It is a protein with a single kringle sequence in the molecule. As described above, plasminogen is limitedly decomposed and converted to active plasmin. Promotes the fibrinolytic cascade.

  Prothrombin is a glycoprotein having a molecular weight of about 72 kDa, and has serine protease and two kringle sequences in the molecule. It is a precursor of the serine protease thrombin. Prothrombin is degraded by factor Xa that forms a prothrombinase complex to α-thrombin. Alpha thrombin polymerizes fibrinogen into fibrin, activates factor XIII to strongly crosslink fibrin, activates platelets, activates factor VIII and factor V to promote coagulation Enzyme action such as. On the other hand, it is supplemented by thrombomodulin and activates protein C to suppress coagulation.

  Factor XII is a single-chain glycoprotein consisting of 596 amino acids and having a molecular weight of about 80 kDa, and has one kringle sequence in the molecule. When factor XII comes into contact with a foreign surface (negatively charged membrane surface) such as glass, kaolin, basement membrane, or collagen, it changes its steric structure, binds to the foreign surface near the end of the N group, and is limited to kallikrein. When activated, it becomes double-stranded factor XII. Factor XII has a reciprocal action of activating pre-kallikrein and converting it into kallikrein, which activates factor XII. Factor XII activates factor XI on the foreign body surface and initiates the intrinsic clotting reaction. Factor XIIa is involved not only in coagulation but also in many biological reactions, including mitogenic effects, fibrinolytic activation, complement activation, kinin production from high-molecular-weight kininogen via kallikrein. Has been reported.

  Angiostatin is a protein of an N-terminal fragment of plasminogen found by Professor Folkman of Harvard University in the United States, and has a plurality of kringle sequences in the molecule. Angiostatin has an angiogenesis inhibitory effect and has been reported to have a cancer regression effect and an antitumor metastasis effect.

  Hepatocyte growth factor is a protein discovered as a hepatocyte growth factor by Professor Toshikazu Nakamura, Osaka University. Heterodimer in which a heavy chain with a molecular weight of about 60 kDa and a light chain with a molecular weight of about 35 kDa having four kringle sequences are disulfide bonded. It is. Hepatocyte growth factor not only for hepatocytes but also for various cells such as lung, heart / vasculature, nervous system, cell proliferation promotion, cell movement promotion, anti-apoptosis, morphogenesis induction, angiogenesis, etc. It has been reported to have a versatile physiological activity responsible for protection. On the other hand, it has also been reported that in chronic fibrotic diseases represented by cirrhosis and chronic renal failure (renal sclerosis), its expression is reduced. In addition, therapeutic effects on patients with obstructive arteriosclerosis have been found by gene therapy of hepatocyte growth factor.

  NK4 is a partial protein of hepatocyte growth factor cleaved from hepatocyte growth factor, and similarly has four kringle sequences. NK4 works as an antagonist of hepatocyte growth factor and suppresses the invasion and metastasis of cancer, while it can suppress the action of angiogenic factors such as VEGF and bFGF, so it has a strong angiogenesis inhibitory activity. ing.

  Apolipoprotein (a) is a constituent protein of LDL cholesterol and has a repeating structure of a plurality of 12 to 51 kringle sequences in the molecule, although there are individual differences. Increased blood levels have been reported during acute myocardial infarction and acute inflammation.

  Thus, proteins and peptides having a kringle sequence, including plasminogen, exhibit various physiological activities in vivo. Therefore, removing or purifying a protein or peptide having a kringle sequence is very useful from a medical point of view, such as a therapeutic effect due to a decrease in the body concentration and purification from a solution containing contaminating components. There is an affinity carrier on which lysine, which is an amino acid, is immobilized as a material or method for achieving the object. This is based on the fact that the kringle sequence has lysine binding properties. However, lysine has two amino groups that are reactive sites. According to Non-Patent Document 1, lysine is selectively immobilized because it has a low affinity for the kringle sequence unless it is immobilized with an α-amino group. It is necessary to make it. However, in order to selectively immobilize lysine, the reaction must be strictly controlled, which is not easy. In order to selectively immobilize lysine, there is a method of deprotecting after immobilizing lysine protected with ε-amino group on a carrier, but the protected lysine is expensive and the process is increased. There are various problems.

Journal of Biomedical Materials Research Part A, Vol.49, Issue.3, 409-414 (1999)

  An object of the present invention is to use an affinity carrier to which lysine for removing or purifying a protein or peptide having a kringle sequence such as plasminogen is immobilized, by using a simple process without using a protecting group. It is to provide a method for removing or purifying a protein or peptide having a kringle sequence using the affinity carrier.

  The present inventor has intensively studied a method for producing an affinity carrier for removing or purifying a protein or peptide having a kringle sequence such as plasminogen, which has been difficult to produce originally. As a result, the lysine-immobilized affinity carrier obtained by bringing the activated carrier and lysine into contact with each other under a pH of 9 or less can efficiently remove and purify proteins and peptides having a kringle sequence. As a result, the present invention has been completed.

  That is, the present invention first relates to a method for producing an affinity carrier for removing or purifying a protein or peptide having a kringle sequence including plasminogen. Second, the present invention relates to a method for removing and purifying proteins and peptides having a kringle sequence including plasminogen using an affinity carrier.

The present invention specifically includes the following inventions.
(1) An affinity carrier for purifying or removing a protein or peptide having a kringle sequence, wherein the pH in the solution at the time of contacting the activated carrier and lysine in the production of the carrier is 9 or less An affinity carrier on which lysine is immobilized.
(2) The affinity carrier according to (1) above, wherein lysine is immobilized by a covalent bond.
(3) The affinity carrier according to (1) or (2) above, wherein N-hydroxysuccinimide is introduced into the activated carrier.
(4) The affinity carrier according to (3) above, wherein N-hydroxysuccinimide is introduced through a linker.
(5) The carrier according to any one of (1) to (4), wherein the affinity carrier comprises polyvinyl alcohol.
(6) The carrier according to (5), wherein polyvinyl alcohol is crosslinked.
(7) The carrier according to any one of (1) to (6), wherein the affinity carrier is a porous water-insoluble carrier.
(8) The carrier according to (7), wherein the porous water-insoluble carrier is in the form of particles.
(9) The protein or peptide having a kringle sequence is plasminogen, plasmin, angiostatin, apolipoprotein (a), tissue plasminogen activator, urokinase, hepatocyte growth factor, NK4, prothrombin, factor XII or a partial peptide thereof The carrier according to any one of (1) to (8) above, which is at least one protein or peptide selected from the group consisting of:
(10) The carrier according to (9), wherein the protein or peptide having a kringle sequence is plasminogen, angiostatin, or a partial peptide thereof.
(11) The carrier according to (10), wherein the protein or peptide having a kringle sequence is plasminogen.
(12) The kringle using the carrier, wherein the affinity carrier according to any one of (1) to (11) is brought into contact with a protein solution or a peptide solution having a kringle sequence in which contaminant components are mixed. A method for purifying a protein or peptide having a sequence, and a method for removing a protein or peptide having a kringle sequence.
(13) After the affinity carrier according to any one of (1) to (11) is brought into contact with a protein solution or peptide solution having a kringle sequence mixed with contaminating components and a protein having a kringle sequence is selectively adsorbed A method for purifying a protein or peptide having a kringle sequence, which comprises dissociating and recovering the adsorbed protein or peptide having a kringle sequence from the carrier.
(14) The purification method according to (13), wherein the protein or peptide having a selectively adsorbed kringle sequence is dissociated and recovered using an eluate.
(15) The eluent used was at least one eluent selected from a solution containing aminocaproic acid, a solution containing lysine, a solution containing arginic acid, a solution containing tranexamic acid, and a solution containing p-aminobenzamidine. The purification method according to the above (14).
(16) The protein solution or peptide having a kringle sequence mixed with contaminating components is a body fluid such as blood or plasma, an animal tissue extract, a culture solution, or a solution obtained by pretreating them (12) ) To (15). A method for purifying or removing a protein or peptide having the kringle sequence according to any one of (15) to (15).
(17) A protein or peptide having a kringle sequence purified by the method according to any one of (13) to (16).

  According to the present invention, an affinity carrier on which lysine is immobilized is provided by a simple process without using a protecting group by contacting the activated carrier with lysine at pH 9 or less. The lysine-immobilized affinity carrier obtained by the present invention can efficiently remove and purify proteins and peptides having a kringle sequence.

  Embodiments of the present invention will be described in detail below, but the present invention is not limited to the following descriptions.

  The protein or peptide having a kringle sequence in the present invention specifically includes plasminogen, plasmin, angiostatin, apolipoprotein (a), tissue plasminogen activator, urokinase, hepatocyte growth factor, NK4, prothrombin, factor XII Any of the above proteins and partial peptides of the above-described proteins, which have a kringle sequence, may be used.

  The water-insoluble carrier in the present invention means a solid at room temperature and normal pressure and water-insoluble. The shape of the water-insoluble carrier in the present invention is not particularly limited, and the size thereof is not particularly limited, such as a spherical shape, a granular shape, a thread shape, a hollow shape, and a flat membrane shape. As the water-insoluble carrier, the larger the specific surface area, the better the removal performance and the purification efficiency. The spherical or granular carrier is preferably a carrier having a large number of pores of an appropriate size, that is, a porous structure. A carrier having a porous structure is naturally a carrier having a space (macropore) formed by agglomeration of microspheres when the base polymer matrix forms one spherical particle by agglomeration of microspheres. However, in the case of a carrier having pores formed between agglomerates of nuclei in one microsphere constituting the basic polymer matrix, or a carrier having a three-dimensional structure (polymer network). The case of a carrier having pores (micropores) existing when the polymer is swollen with an affinity organic solvent is also included.

  In view of the removal performance per unit volume of the carrier and the purification efficiency, the water-insoluble carrier having a porous structure is preferably more porous than the surface porosity, and the pore volume and specific surface area impair the adsorptivity. It is preferably as large as possible.

  The average particle size of the spherical or granular carrier is generally 0.5 μm to 10 mm, but the smaller the particle size, the larger the specific surface area and the better the performance as an affinity carrier. Since the mechanical strength is weakened, a thickness of 10 μm to 3 mm is preferable.

  As the water-insoluble carrier in the present invention, characteristics required for general affinity carriers and characteristics required for medical materials, that is, resistance to organic solvents, resistance to pH and heat, physical mechanical strength, non-specific adsorption, blood cells From the viewpoints of interaction with components, thrombus formation, blood coagulation, etc., natural polymer carriers containing sugars such as dextran, agarose and cellulose and synthetic polymer carriers such as polyvinyl alcohol, polyglycidyl methacrylate and polyhydroxy methacrylate are preferred. . Also preferred are organic-organic and organic-inorganic composite carriers obtained by these combinations. Further, they may be those that are carrier-crosslinked. Examples of the crosslinking agent include triallyl isocyanurate (TAIC), ethylene glycol dimethacrylate (EGDMA), and glycerin dimethacrylate (GDMA), but the present invention is not limited thereto. The carrier preferably has a hydroxyl group because it has little nonspecific adsorption and high hydrophilicity, and a functional group for introducing an active group is necessary. Polyglycidyl methacrylate is low in hydrophilicity because it does not have a hydroxyl group as it is, but it is treated with an alkali such as sodium hydroxide or potassium hydroxide, or an epoxy such as dilute sulfuric acid, perchloric acid, benzenesulfonic acid, or toluenesulfonic acid. When treated with an acid that does not react with, the epoxy group is ring-opened, converted into a diol and hydrophilized, making it a suitable material for the affinity carrier. As described above, various materials can be considered as the water-insoluble carrier in the present invention, but a carrier made of polyvinyl alcohol is preferred from the viewpoint of removal performance and purification efficiency.

  As the polymerization method of the water-insoluble carrier described in the present invention, suspension polymerization, uniform droplet formation, and the like can be considered, but the present invention is not limited to these.

  The affinity carrier on which lysine is immobilized in the present invention is preferably a carrier on which lysine is covalently bound. The lysine may be L, D, DL, or a mixture thereof. As a method for immobilizing lysine, a method in which an active group is introduced into a carrier and lysine is reacted with the active group is generally used. Examples of frequently used active groups include epoxy groups, cyanogen halides, halogenated triazines, bromoacetyl bromides, aldehydes, and N-hydroxysuccinimide. In the present invention, the type of the active group is not limited, but the active group can be easily introduced, the ligand or linker can be immobilized under mild conditions, the activity is high, and the immobilized ligand or linker is stable. The active group is preferably N-hydroxysuccinimide because it is difficult to elute and the amount of active group introduced can be adjusted according to the purpose. N-hydroxysuccinimide easily reacts with amino groups and hydroxyl groups, has few side reactions, and quantitatively forms amide bonds and ester bonds. N-hydroxysuccinimide also has the advantage of low toxicity when decomposed in aqueous solution.

  As a method of introducing N-hydroxysuccinimide into a carrier, a method of activating N-hydroxysuccinimide by introducing a carboxylic acid into the carrier and then mixing it with a suitable condensing agent and N-hydroxysuccinimide. Can be considered. Although the kind of condensing agent is not limited, various condensing agents such as dicyclohexylcarbodiimide, diisopropylcarbodiimide, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide can be used. Since the reaction does not easily proceed in a 100% aqueous solution, the solvent is preferably an organic solvent, and more preferably an aprotic organic solvent.

  Examples of a method for introducing a carboxylic acid include a method for immobilizing a molecule having a carboxylic acid on a carrier and a method for hydrolyzing polymethyl methacrylate (pMMA). In order to immobilize a molecule having a carboxylic acid, for example, an molecule having an amino group and a carboxylic acid in the molecule is introduced into an activated carrier into which an epoxy group has been introduced using epichlorohydrin or bisepoxide via the amino group. Carboxylic acid can be introduced by immobilization. A molecule having an amino group and a carboxylic acid may be various molecules such as amino acids, but a molecule having two or more amino groups and two or more carboxylic acids in the molecule is a carrier during the reaction for introducing N-hydroxysuccinimide. This is undesirable because there is a possibility of cross-linking between each other. A linear molecule such as aminocaproic acid is preferred because it also serves as a linker away from the carrier surface. Since polymethyl methacrylate is a polymer having a carboxylic acid ester in the side chain, hydrolysis of the carboxylic acid methyl ester with alkali results in a form in which the carboxylic acid is presented on the carrier side, and the carboxylic acid can be introduced into the carrier. . The method for introducing a carboxylic acid in the present invention is not limited, and the amount of epoxy introduced in the present invention is such that N-hydroxysuccinimide is introduced regardless of the method in which the carboxylic acid is introduced. Is possible.

  In the present invention, the amount of active groups is not limited, but an amount of active groups of 1 to 10,000 μmol per mL of carrier swollen in water is preferable for use of the affinity carrier. From the viewpoint of non-specific adsorption by a non-target substance, an amount of 10 to 5,000 μmol active groups per mL of carrier swollen in water is more preferable. The amount of the active group can be appropriately adjusted by optimizing various conditions of the active group introduction reaction (addition amount of active group introduction agent, alkali amount, reaction temperature, reaction time, etc.) according to the use.

  The amount of N-hydroxysuccinimide introduced in the present invention is determined by the following method. A fixed amount, for example, 6 mL of a carrier into which N-hydroxysuccinimide has been introduced is measured, and water is removed on a glass filter under reduced pressure for 15 minutes. An appropriate amount is measured, an aqueous ammonia solution is added thereto, and the amount of N-hydroxysuccinimide can be determined from the absorbance of the supernatant.

  As conditions for immobilizing lysine on a carrier into which N-hydroxysuccinimide has been introduced, for example, it is preferable to carry out the reaction in an aqueous solution under a pH of 9 or less. When the pH is higher than 9, the rate of immobilization via the ε-amino group of lysine increases, and the deactivation rate of N-hydroxysuccinimide increases, so that the removal performance of a protein or peptide having a kringle sequence And purification efficiency is greatly reduced. If the pH is 9 or less, the rate of immobilization through the α-amino group of lysine is increased, the deactivation rate of N-hydroxysuccinimide is decreased, and the removal performance of a protein or peptide having a kringle sequence Since the purification efficiency becomes high, it is preferable to contact the lysine with the activated carrier into which N-hydroxysuccinimide is introduced under the condition of pH 9 or less. The pH may be adjusted using a buffer solution such as carbonic acid, boric acid, and phosphoric acid. The reaction temperature may be appropriately selected, but is usually about 0 ° C. to 80 ° C., and the reaction time is preferably 1 minute to 48 hours.

  The thus obtained affinity carrier on which lysine is immobilized is bound by a very stable covalent bond and is also thermally stable. High-pressure steam sterilization is possible, and the ligand elution after sterilization and the required performance degradation associated therewith do not occur. For methods other than heat sterilization, sterilization with ethylene oxide gas is also possible. From the above, it has excellent performance for medical use.

  The method for removing and purifying proteins and peptides having a kringle sequence such as plasminogen in the present invention is a protein having a kringle sequence from a protein solution or peptide solution having a kringle sequence mixed with undesired contaminant components. And removal or purification of peptides. For example, when a protein or peptide having a kringle sequence is a pathogenesis-related substance, the disease can be treated by removing it from a body fluid such as blood. In addition, when a protein or peptide having a kringle sequence is used as a drug, it is purified from a human body fluid and used as a high-purity natural protein preparation, or purified from a protein-producing culture medium using bacteria and used as a high-purity protein preparation. It can also be used in the purification step.

  The method for removing a protein or peptide having a kringle sequence can be realized by bringing a protein solution or peptide solution having a kringle sequence mixed with contaminating components into contact with the affinity carrier in the present invention. The contacting method may be used by filling the column with the affinity carrier of the present invention, or may be a batch contacting method, and the contacting method is not limited. The method of packing and contacting the column is to prepare an apparatus filled with the carrier in a container having an inlet / outlet of liquid and a device for preventing the carrier from flowing out of the container, and the inlet of the apparatus A solution from which the protein or peptide having the kringle sequence is removed is obtained by flowing a protein solution or peptide solution having a kringle sequence mixed with contaminating components and collecting the solution from the outlet. Batch contact means that a protein having a kringle sequence is collected by mixing only a solution component after mixing a protein solution or peptide solution having a kringle sequence in which the affinity carrier and contaminant components are mixed in a container without filling the column. Alternatively, a solution from which the peptide has been removed is obtained.

  The method for purifying a protein or peptide having a kringle sequence can also be realized by bringing a protein solution or peptide solution having a kringle sequence mixed with an affinity carrier in the present invention and a contaminating component, in the same manner as the removing method. The contact method is the same as above, but after washing the affinity carrier with an appropriate solution from the inlet side or the outlet side after contact, the protein or peptide having a kringle sequence bound to the affinity carrier is eluted with the eluate. By doing so, it can be purified. The eluent is a solution containing aminocaproic acid, a solution containing lysine, a solution containing arginic acid, a solution containing tranexamic acid, or a solution containing p-aminobenzamidine, which may be used in combination. Further, the concentration of the eluate is not limited and may be selected according to the purpose. This is an example of a removal method or a purification method, and the method of use is not limited.

EXAMPLES Hereinafter, although an Example demonstrates in detail regarding this invention, this invention is not limited only to a following example.
Example 1
45 g vinyl acetate, 13.95 g triallylic isocyanurate, 23.85 g heptane, 38.79 g ethyl acetate, 4.32 g polyvinyl acetate (degree of polymerization 400), and polymerization initiator 2,2′-azobis (2, 4-dimethylvaleronitrile) (V-65) 2.25 g of a homogeneous mixed solution dissolves 4 g of polyvinyl alcohol, 0.9 g of sodium alpha olefin sulfonate, 62 g of fine calcium triphosphate and 0.8 g of sodium nitrite. The mixture was added to a 2 L separable flask having a flat stirring blade and two baffle plates in which an aqueous phase containing 773 mL of water was charged in advance, and reacted at 65 ° C. for 5 hours.

  Next, hydrochloric acid was added to the contents of the separable flask to adjust the pH to 2 or less to dissolve the tribasic calcium phosphate, and then washed well with water. After confirming that the pH of the cleaning liquid was close to neutral, water was replaced with acetone, and the polymer was thoroughly washed with acetone. Next, acetone was replaced with water, and then an amount of sodium hydroxide (NaOH) of the following formula was added as an aqueous solution so as to be an excess amount with respect to the vinyl acetate unit.

NaOH (solid weight) = particle dry weight / 86.09 × 40 × 1.5
The amount of water was adjusted so that the NaOH concentration relative to water was 4% by weight. This was saponified with stirring at a reaction temperature of 40 ° C. for 6 hours. Then, it washed with water until pH of the washing | cleaning liquid became neutrality, and also wash | cleaned fully with 80 degreeC warm water. Next, autoclave treatment at 121 ° C. for 20 minutes was performed to obtain a clean crosslinked polymer carrier.

  To 151 g of this crosslinked polymer carrier, 233 mL of dimethyl sulfoxide (DMSO), 19.4 mL of 30% NaOH, and 156 mL of epichlorohydrin were added and reacted at 40 ° C. for 5 hours. The epoxy activated carrier was obtained by washing with 0.6 L of DMSO and 3.0 L of water. The epoxy amount of this epoxy activated carrier was measured and found to be 32 μmol / mL.

  To 160 mL of the epoxy activated carrier obtained above, 94 mL of water, 40.0 mL of 2M aqueous sodium carbonate solution and 25.6 mL of 2M aqueous aminocaproic acid solution were added and reacted at 50 ° C. for 5 hours. The slurry was washed with 3.2 L of water to obtain an aminocaproic acid immobilized carrier. When the amount of aminocaproic acid immobilized on the aminocaproic acid-immobilized carrier was measured by titration, it was 42 μmol / mL.

  98 mL of this aminocaproic acid-immobilized support was washed with 245 mL of DMSO, and 94 mL of DMSO, 12.6 g of NHS and 21.0 g of EDC were added to the washed support and reacted at 40 ° C. for 10 hours. This slurry was washed with 2.0 L of DMSO and 0.3 L of IPA to obtain an NHS activated carrier. The NHS amount of this NHS activated carrier was 19 μmol / mL.

  4.0 mL of the NHS-activated carrier obtained above was washed with 20 mL of ice-cold 1 mM hydrochloric acid, and 3.6 mL of 1 mM hydrochloric acid, 0.44 mL of IPA, (0.4 M sodium bicarbonate + 1 M sodium chloride) aqueous solution (pH 7. 5) 4.0 mL, 2 M lysine aqueous solution 0.1 mL, and water 0.66 mL were added, and it was made to react at 5 degreeC for 3.5 hours. The slurry was washed with 40 mL of water to obtain a lysine-immobilized carrier (Carrier A). Based on the lysine concentration in the supernatant after the immobilization reaction, the amount of carrier A immobilized on lysine was 17 μmol / mL.

  After substituting 0.5 mL of the carrier A obtained above with physiological saline, 2.5 mL of plasma coagulated with citric acid was added and stirred at room temperature for 10 minutes. Next, 2.5 mL of stirred plasma was collected so that the aminocaproic acid-immobilized carrier was not mixed. When the plasminogen removal rate was calculated from the plasminogen concentration in the plasma before treatment and the plasminogen concentration in the plasma after stirring, it was confirmed that 76% before the stirring was removed. Next, after thoroughly washing the carrier with a phosphate buffer, the supernatant was removed as much as possible, a 0.01 M aminocaproic acid solution was added, and the mixture was stirred at room temperature for 6 minutes. The supernatant was collected so as not to contaminate the carrier.

When the recovered solution was electrophoresed, a single band was detected in the target molecular weight region, and the purity was confirmed to be almost 100%. Further, when the recovery rate of plasminogen was calculated from the plasminogen concentration in plasma before treatment and the plasminogen concentration in the recovery solution, the recovery rate of purified plasminogen was 29%.
(Example 2)
After obtaining an NHS-activated carrier in the same manner as in Example 1, lysine was immobilized at pH 8.0 by the same method to obtain a lysine-immobilized carrier (carrier B). Plasminogen was purified by the same method. The amount of lysine immobilized on carrier B was 14 μmol / mL. Similarly, when the recovered solution was electrophoresed, a single band was detected in the target molecular weight region, and the purity was confirmed to be almost 100%. The plasminogen removal rate was 75%, and the recovery rate was 31%.
(Example 3)
After obtaining an NHS-activated carrier in the same manner as in Example 1, lysine was immobilized at pH 8.5 by the same method to obtain a lysine-immobilized carrier (carrier C). Plasminogen was purified by the same method. The amount of lysine immobilized on carrier C was 17 μmol / mL. Similarly, when the recovered solution was electrophoresed, a single band was detected in the target molecular weight region, and the purity was confirmed to be almost 100%. The plasminogen removal rate was 73%, and the recovery rate was 27%.
(Comparative Example 1)
After obtaining an NHS-activated carrier in the same manner as in Example 1, lysine was immobilized at pH 9.5 in the same manner to obtain a lysine-immobilized carrier (carrier D). Plasminogen was purified by the same method. The amount of lysine immobilized on carrier D was 16 μmol / mL. Similarly, when the recovered solution was electrophoresed, a single band was detected in the target molecular weight region, and it was confirmed that the purity was almost 100%, but the plasminogen removal rate was 61% and the recovery rate was It was 9%.

  Table 1 shows the results of purification of plasminogen in Examples and Comparative Examples.

Claims (16)

A method for manufacturing an affinity carrier for purifying or removing protein or peptide having a kringle sequence, is contacted with lysine activated activated carrier with the introduction of N- hydroxysuccinimide in the preparation of the carrier method characterized by Te to the pH of the solution to 9 or less at the time of fixing the lysine. The method according to claim 1, wherein lysine is immobilized by a covalent bond. The method according to claim 1 or 2, wherein N-hydroxysuccinimide is introduced via a linker. The method according to claim 3, wherein the linker is a molecule having one amino group and one carboxylic acid in the molecule. The method according to claim 1, wherein the affinity carrier comprises polyvinyl alcohol. 6. The method of claim 5, wherein the polyvinyl alcohol is cross-linked. The method according to claim 1, wherein the affinity carrier is a porous water-insoluble carrier. 8. The method according to claim 7, wherein the porous water-insoluble carrier is in the form of particles. The protein or peptide having a kringle sequence is selected from plasminogen, plasmin, angiostatin, apolipoprotein (a), tissue plasminogen activator, urokinase, hepatocyte growth factor, NK4, prothrombin, factor XII or partial peptides thereof The method according to claim 1, wherein the method is at least one protein or peptide. The method according to claim 9, wherein the protein or peptide having a kringle sequence is plasminogen, angiostatin, or a partial peptide thereof. The method according to claim 10, wherein the protein or peptide having a kringle sequence is plasminogen. A kringle sequence using the carrier, wherein the affinity carrier produced by the method according to any one of claims 1 to 11 is brought into contact with a protein solution or a peptide solution having a kringle sequence mixed with contaminating components. And a method for removing a protein or peptide having a kringle sequence. After the affinity carrier produced by the method according to any one of claims 1 to 11 is brought into contact with a protein solution or peptide solution having a kringle sequence mixed with contaminating components and a protein having a kringle sequence is selectively adsorbed, A method for purifying a protein or peptide having a kringle sequence, which comprises dissociating and recovering the adsorbed protein or peptide having a kringle sequence from the carrier.   14. The purification method according to claim 13, wherein the protein or peptide having a selectively adsorbed kringle sequence is dissociated and recovered using an eluate.   15. The eluent used is at least one eluent selected from a solution containing aminocaproic acid, a solution containing lysine, a solution containing arginic acid, a solution containing tranexamic acid, and a solution containing p-aminobenzamidine. The purification method as described.   The protein solution or peptide having a kringle sequence mixed with contaminating components is a body fluid such as blood or plasma, an animal tissue extract, a culture solution, or a solution obtained by pretreating them. A method for purifying or removing a protein or peptide having any of the kringle sequences.