[go: up one dir, main page]

WO2012036140A1 - Vecteur d'affinité pour la purification ou l'élimination d'une protéine ou d'un peptide ayant une séquence kringle, procédé de purification et procédé d'élimination dans lesquels le vecteur d'affinité est utilisé - Google Patents

Vecteur d'affinité pour la purification ou l'élimination d'une protéine ou d'un peptide ayant une séquence kringle, procédé de purification et procédé d'élimination dans lesquels le vecteur d'affinité est utilisé Download PDF

Info

Publication number
WO2012036140A1
WO2012036140A1 PCT/JP2011/070784 JP2011070784W WO2012036140A1 WO 2012036140 A1 WO2012036140 A1 WO 2012036140A1 JP 2011070784 W JP2011070784 W JP 2011070784W WO 2012036140 A1 WO2012036140 A1 WO 2012036140A1
Authority
WO
WIPO (PCT)
Prior art keywords
peptide
protein
affinity carrier
kringle sequence
solution
Prior art date
Application number
PCT/JP2011/070784
Other languages
English (en)
Japanese (ja)
Inventor
伸彦 佐藤
芳文 板垣
Original Assignee
株式会社カネカ
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 株式会社カネカ filed Critical 株式会社カネカ
Priority to JP2012534006A priority Critical patent/JPWO2012036140A1/ja
Publication of WO2012036140A1 publication Critical patent/WO2012036140A1/fr

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes

Definitions

  • 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 / removing a protein or peptide comprising
  • 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. Inhibits angiogenesis, promotes cell proliferation, inactivates cell proliferation, and exhibits various physiological activities in vivo.
  • Plasminogen is a single-chain glycoprotein having a molecular weight of about 92 kDa composed of 791 amino acid residues, and has a serine protease region 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 a serine protease region 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 a serine protease region 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 ⁇ -thrombin. ⁇ -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 has an action of activating protein C and suppressing 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.
  • factor XII comes into contact with a foreign material surface (negatively charged membrane surface) such as glass, kaolin, basement membrane, or collagen, it changes its steric structure, binds to the foreign material surface near the end of the N group, and is limited to kallikrein and the like.
  • a foreign material surface negatively charged membrane surface
  • Factor XII activates pre-kallikrein and converts it into kallikrein, and kallikrein has a mutual activation effect of activating 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 the coagulation reaction, but also in many biological reactions, including cell mitogenic activity, activation of the fibrinolytic system, activation of complement, production of kinin from macromolecular kininogen via kallikrein It has been reported that
  • 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.
  • 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.
  • proteins and peptides having a kringle sequence 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 concentration in the body and removal of impurities in the pharmaceutical production process.
  • an affinity carrier on which lysine, which is an amino acid, is immobilized. This is based on the fact that the kringle sequence has lysine binding properties. However, lysine has two amino groups that are reactive sites.
  • Non-Patent Document 1 lysine is selectively used because it has a low affinity for the kringle sequence unless it is immobilized with an ⁇ -amino group. Need to be fixed. However, in order to selectively immobilize lysine, the reaction must be strictly controlled, which is not easy.
  • aminocaproic acid known as a lysine analog has one amino group as a reactive site, and it is not necessary to strictly control the reaction when it is immobilized on a carrier, and can be easily immobilized.
  • aminocaproic acid has an affinity for plasminogen as a lysine analogue, but agarose immobilized with aminocaproic acid has no affinity for lysine or has extremely low affinity.
  • Patent Document 1 Non-Patent Document 2, etc.
  • the amino caproic acid alkyl moiety, the free amino group at the end of epsilon, and the carboxyl group at the terminal make a large contribution.
  • aminocaproic acid When aminocaproic acid is immobilized, the amino group is not free and has no affinity for plasminogen. It is assumed that As described above, it has been conventionally difficult to remove or purify a protein or peptide having a kringle sequence such as plasminogen from an aminocaproic acid-immobilized carrier that is easier to immobilize than lysine.
  • An object of the present invention is to provide an affinity carrier for removing or purifying a protein or peptide having a kringle sequence such as plasminogen, and a method for removing or purifying a protein or peptide having a kringle sequence using the affinity carrier. Is to provide.
  • the inventor has intensively studied a method for preparing an affinity carrier for removing or purifying a protein or peptide having a kringle sequence such as plasminogen, which was originally difficult to produce. As a result, it has been found that proteins and peptides having a kringle sequence can be efficiently removed and purified in the case of a carrier on which aminocaproic acid is immobilized, particularly a polyvinyl alcohol carrier, and the present invention has been completed.
  • the present invention firstly relates to a method for producing an affinity carrier for removing or purifying a protein or peptide having a kringle sequence including plasminogen.
  • 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.
  • An affinity carrier on which aminocaproic acid is immobilized for purifying or removing a protein or peptide having a kringle sequence.
  • 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 affinity carrier according to any one of (1) to (7) above, which is at least one protein or peptide selected from the group consisting of:
  • a kringle sequence comprising the step of bringing the affinity carrier according to any one of (1) to (10) into contact with a protein solution or a peptide solution having a kringle sequence mixed with contaminating components.
  • the affinity carrier according to any one of (1) to (10) above is contacted with a protein solution or peptide solution having a kringle sequence mixed with contaminating components to selectively adsorb a protein or peptide having a kringle sequence.
  • a method for purifying a protein or peptide having a kringle sequence comprising the steps of: dissociating and recovering a protein or peptide having an adsorbed kringle sequence from the carrier.
  • At least one elution solution 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 was used as an elution solution.
  • the protein solution or peptide solution having a kringle sequence mixed with contaminating components is a body fluid, an animal tissue extract, a culture solution, or a solution obtained by pretreating them, (11) to (15 Or a method for purifying or removing the protein or peptide having the kringle sequence according to any one of the above.
  • the polyvinyl alcohol carrier on which aminocaproic acid is immobilized according to the present invention can efficiently remove and purify proteins and peptides having a kringle sequence. Since this affinity carrier does not require selective immobilization of an amino group like a lysine immobilization carrier, it is very easy to produce.
  • the protein or peptide having a kringle sequence in the present invention is not particularly limited as long as it has at least one kringle sequence in its structure.
  • proteins such as plasminogen, plasmin, angiostatin, apolipoprotein (a), tissue plasminogen activator, urokinase, hepatocyte growth factor, NK4, prothrombin, factor XII, and partial peptides of the above proteins
  • sequence can be mentioned.
  • the affinity carrier according to the present invention is one in which aminocaproic acid is immobilized on a water-insoluble carrier.
  • the water-insoluble carrier means a material that is solid at normal temperature and pressure and has a very low solubility in water.
  • water-insoluble corresponds to “Very slightly soluble” and “Practically insoluble, or Insoluble” in the definitions of the Japanese Pharmacopoeia and the United States Pharmacopoeia (The United States Pharmacopea).
  • the shape of the water-insoluble carrier is not particularly limited, and the size thereof is not particularly limited.
  • the water-insoluble carrier may be spherical, particulate, thread-like, hollow, or flat membrane.
  • the water-insoluble carrier is preferably used in a spherical or particulate form because the larger the specific surface area, the better the removal performance and the purification efficiency.
  • the average particle diameter of the spherical or particulate carrier is generally 0.5 ⁇ m or more and 10 mm or less. Further, the smaller the particle size, the larger the specific surface area and the better the performance as an affinity carrier. However, the physical and mechanical strength of the carrier is weakened, and those having a size of 10 ⁇ m or more and 3 mm or less are preferable.
  • the average particle size of the carrier may be referred to the catalog value, or if there is no catalog value, the particle size distribution may be measured on a volume basis with a particle size distribution meter and obtained from the obtained particle size distribution. Good.
  • the carrier is preferably a carrier having a large number of pores having an appropriate size, that is, a carrier having a porous structure.
  • the pore size and porosity of the porous structure are not particularly limited.
  • a porous carrier having a porous structure has a large surface area, which improves the separation efficiency and removal efficiency of proteins and peptides having a kringle sequence.
  • 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.
  • a carrier having pores (micropores) present when the coalescence is swollen with an affinity organic solvent is also included.
  • 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 material of the water-insoluble carrier in the present invention is not particularly limited, but the characteristics required for general affinity carriers and the characteristics required for medical materials, that is, resistance to organic solvents, resistance to abnormal pH and heat, physical mechanical strength Characteristics such as non-specific adsorption, interaction with blood cell components, suppression of thrombus formation and blood coagulation are required. From this viewpoint, natural polymer carriers containing sugars such as dextran and cellulose, and synthetic polymer carriers such as polyvinyl alcohol, polyglycidyl methacrylate, and polyhydroxy methacrylate are preferable. Also preferred are organic-organic, organic-inorganic composite carriers, etc. obtained by a combination thereof.
  • the water-insoluble carrier according to the present invention is preferably cross-linked.
  • the cross-linking method is not particularly limited, and examples thereof include a method using a cross-linking agent having a plurality of functional groups that react with a functional group present on the surface or terminal of a water-insoluble carrier.
  • a polymerizable monomer capable of reacting with both the functional group of the water-insoluble carrier and the functional group of the crosslinking agent may be used.
  • the cross-linking agent may be triallyl isocyanurate (TAIC), ethylene glycol dimethacrylate (EGDMA), glycerin di- Although methacrylate (GDMA) etc. are mentioned, this invention is not limited to these.
  • TAIC triallyl isocyanurate
  • ELDMA ethylene glycol dimethacrylate
  • GDMA glycerin di- Although methacrylate
  • the polymerizable monomer include vinyl acetate, vinyl alcohol, styrene, (meth) acrylic acid esters such as (meth) acrylic acid and methyl methacrylate, and acrylonitrile.
  • the water-insoluble carrier according to the present invention desirably has a hydroxyl group because it has low non-specific adsorption and high hydrophilicity and requires a functional group for introducing an active group.
  • 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.
  • a carrier made of a synthetic polymer carrier such as polyvinyl alcohol, polyglycidyl methacrylate, polyhydroxymethacrylate, etc. is excellent in removal performance and purification efficiency. More preferred is polyvinyl alcohol.
  • the production method of the water-insoluble carrier described in the present invention may be suspension polymerization or uniform droplet formation, but the present invention is not limited to these.
  • aminocaproic acid is immobilized on a water-insoluble carrier.
  • the method for immobilizing aminocaproic acid includes covalent bond and ionic bond, but since the possibility of elution of aminocaproic acid is extremely low, the affinity carrier to which aminocaproic acid is immobilized in the present invention is preferably aminocaproic acid.
  • a covalently bonded carrier As a method for immobilizing aminocaproic acid, a method in which an active group is introduced into a carrier and aminocaproic acid is reacted with the active group is generally used.
  • Active groups often used are epoxy groups, cyanogen halides, halogenated triazines, bromoacetyl bromides, aldehydes and the like.
  • the type of active group is not limited, but the active group can be easily introduced, the ligand or linker can be immobilized under mild conditions, the immobilized ligand or linker is stable and difficult to elute,
  • the active group is preferably an epoxy group because it can be handled industrially in large quantities and the amount of active group introduced can be adjusted according to the purpose.
  • Epoxy groups easily react with hydroxyl groups, amino groups, and thiol groups, and have few side reactions and quantitatively form ether bonds, alkylamine bonds, and thioether bonds.
  • the epoxy group when the epoxy group is decomposed in an aqueous solution, it becomes glycol and has an advantage of low toxicity.
  • the amino group of aminocaproic acid and the active group of the water-insoluble carrier are preferably covalently bonded.
  • aminocaproic acid is not covalently bonded to the water-insoluble carrier by covalent bond, for example, when it is indirectly immobilized by electrostatic interaction or the like, aminocaproic acid may be leaked.
  • aminocaproic acid is immobilized on a water-insoluble carrier through its amino group, there is an advantage that the original affinity of aminocaproic acid for the kringle sequence is not lost.
  • the epoxidizing agent for introducing the epoxy group into the carrier epichlorohydrin, bisepoxide, and polyepoxide are easy to introduce the epoxy group into the carrier, and the epoxy group and ligand introduced by the epoxidizing agent are used. This is preferable because it is easy to control the reaction.
  • the bisepoxides and polyepoxides mentioned here are 1 such as ethylene glycol diglycidyl ether, 1,4-butanediol diglycidyl ether, 1,6-hexanediol diglycidyl ether, diethylene glycol diglycidyl ether, cyclohexanedimethanol diglycidyl ether, etc. It refers to a compound having two or more epoxy groups in the molecule.
  • the reaction temperature for introducing the active group is preferably in the range of 10 ° C. to 60 ° C., since the reaction rate is low at low temperatures and side reaction crosslinking is likely to occur at high temperatures.
  • the reaction time can be selected from several minutes to several hours in consideration of the desired amount of active groups.
  • an active group can be introduced into the support under the same conditions as in epichlorohydrin, but a small amount of a reducing agent such as sodium borohydride may be added to the reaction system as a promoter.
  • the amount of active groups is not limited, but a carrier swollen in water, that is, 1 ⁇ mol or more and 10,000 ⁇ mol or less per mL of a water-insoluble carrier in a wet state is preferable for use of the affinity carrier. From the viewpoint of non-specific adsorption by a non-target substance, the amount is more preferably 10 ⁇ mol or more and 5,000 ⁇ mol or less per mL of carrier swollen in water. Furthermore, 15 ⁇ mol or more is preferable, 20 ⁇ mol or more is more preferable, 1,000 ⁇ mol or less is preferable, 500 ⁇ mol or less is more preferable, 100 ⁇ mol or less is further preferable, and 50 ⁇ mol or less is particularly 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 epoxy group introduced according to the present invention is determined by the following method.
  • a fixed amount of, for example, 6 mL of a carrier into which an epoxy group has been introduced is weighed, and water is removed on a glass filter under reduced pressure for 15 minutes.
  • About 1.5 g is weighed and added with 4.5 mL of 1.3 M sodium thiosulfate aqueous solution and reacted at 45 ° C. for 30 minutes.
  • the OH ion generated by the reaction of the epoxy group with sodium thiosulfate is titrated with phenolphthalein as an indicator until the phenolphthalein is no longer colored with 0.01N hydrochloric acid or 0.1N hydrochloric acid. Find the amount.
  • the reaction is preferably carried out in an aqueous solution under a basic condition of pH 7 to 13.
  • the pH may be adjusted using a buffer solution such as carbonic acid, boric acid, or phosphoric acid.
  • the reaction temperature may be selected as appropriate, but is usually about 0 ° C. or higher and 80 ° C. or lower, and the reaction time is preferably 1 hour to 48 hours.
  • the amount of aminocaproic acid immobilized can also be determined by titration or elemental analysis. Excess epoxy groups can be treated by blocking with a reagent such as glycine, ethanolamine, tris- (hydroxymethyl) -aminomethane, or by hydrolysis with an alkali or acid.
  • the immobilization ratio of aminocaproic acid in the affinity carrier according to the present invention is the same as the amount of active groups for introducing aminocaproic acid, the adsorption efficiency of a protein or peptide having a kringle sequence, and nonspecific adsorption of other proteins.
  • the carrier swollen in water that is, the water-insoluble carrier in a wet state is preferably 1 ⁇ mol or more, more preferably 10 ⁇ mol or more, further preferably 15 ⁇ mol or more, particularly preferably 20 ⁇ mol or more, and preferably 10,000 ⁇ mol or less.
  • 5,000 ⁇ mol or less is more preferable, 1,000 ⁇ mol or less is further preferable, 500 ⁇ mol or less is further preferable, 100 ⁇ mol or less is further preferable, and 50 ⁇ mol or less is particularly preferable.
  • the affinity carrier on which aminocaproic acid thus obtained 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.
  • 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.
  • a protein or peptide having a kringle sequence when 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 affinity carrier according to the present invention is brought into contact with a protein solution or peptide solution having a kringle sequence mixed with contaminating components.
  • the solution to be brought into contact with the affinity carrier according to the present invention is not particularly limited as long as it is a liquid that may contain a protein or peptide having a kringle sequence.
  • a liquid that may contain a protein or peptide having a kringle sequence for example, bodily fluids, animal tissue extracts, culture solutions, and solutions obtained by pretreating them can be mentioned.
  • Examples of the body fluid include blood, cerebrospinal fluid, ascites, lymph fluid, intra-articular fluid, and bone marrow fluid.
  • Animal tissue extract refers to fluid extracted from organs of animals including humans.
  • Examples of the culture solution include a culture solution of a transformed bacterium having the ability to secrete a protein or peptide having a kringle sequence.
  • Examples of the pretreated solution include plasma from which blood cells have been removed by adding an anticoagulant to blood and then centrifuging, and serum from which clots formed by leaving the blood are removed. .
  • the method of bringing the affinity carrier according to the present invention into contact with a protein solution having a kringle sequence, etc. uses the affinity carrier of the present invention packed in a column. It is also possible to use a batch contact method, and the contact 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 amount of the affinity carrier according to the present invention may be appropriately adjusted depending on, for example, the concentration of a protein having a kringle sequence contained in the liquid to be treated. As mentioned above, it can be set to about 50 times or less. If the ratio is 0.01 volume times or more, the protein or the like contained in the liquid can be more reliably adsorbed. On the other hand, if the ratio is too large, the necessary component may be adsorbed. Therefore, the ratio is preferably 50 times or less. The ratio is preferably 0.05 volume times or more, more preferably 0.1 volume times or more, particularly preferably 0.15 volume times or more, and preferably 10 volume times or less, more preferably 5 volume times or less. 1 volume times or less is particularly preferable.
  • the affinity carrier according to the present invention is brought into contact with a protein solution or the like having a kringle sequence so that the protein or the like is adsorbed to the carrier, and then the carrier and the liquid are combined. Separate, and dissociate and recover the protein and the like from the carrier.
  • the residual liquid may be eluted by washing the carrier packed in the column with water or the like.
  • the carrier may be washed with water or the like after filtration or centrifugation.
  • an eluate may be used to dissociate and recover the protein or the like from the carrier adsorbing the protein or peptide having a kringle sequence.
  • 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.
  • the concentration of the eluate is not limited and may be selected according to the purpose.
  • the above is an example of a removal method or a purification method, and the usage method is not limited.
  • Example 1 (1) Synthesis of aminocaproic acid immobilization carrier 45 g vinyl acetate, 13.95 g triallyl isocyanurate, 23.85 g heptane, 38.79 g ethyl acetate, 4.32 g polyvinyl acetate (degree of polymerization 400), and polymerization initiator A homogeneous mixed solution composed of 2.25 g of 2,2′-azobis (2,4-dimethylvaleronitrile) (V-65) was added to 4 g of polyvinyl alcohol, 0.9 g of sodium alpha olefin sulfonate, and 62 g of particulate calcium triphosphate.
  • V-65 2,2′-azobis (2,4-dimethylvaleronitrile
  • 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
  • the introduced epoxy group may react with both an amino group and a carboxy group, whereas carboxylic acid under alkaline conditions is not reactive with the epoxy group, whereas the amino group with respect to the epoxy group. Since the reactivity of the group is very high, aminocaproic acid is considered to be bound to the carrier mainly through the amino group.
  • Example 2 Evaluation of plasminogen purification using column of aminocaproic acid-immobilized carrier In the same manner as in Example 1, an aminocaproic acid-immobilized carrier was obtained.
  • the recovery rate of plasminogen was calculated from the plasminogen concentration in plasma before treatment and the plasminogen concentration in the recovered solution, the recovery rate of purified plasminogen was 67%.
  • the affinity carrier according to the present invention can selectively adsorb plasminogen, which is a protein having a kringle sequence, from blood or the like, and can remove and purify plasminogen. .

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Analytical Chemistry (AREA)
  • Peptides Or Proteins (AREA)
  • Solid-Sorbent Or Filter-Aiding Compositions (AREA)

Abstract

La présente invention concerne un vecteur d'affinité pour la purification ou l'élimination d'une protéine ou d'un peptide ayant une séquence Kringle, tel que le plasminogène ; et un procédé d'élimination ou de purification d'une protéine ou d'un peptide ayant une séquence Kringle par l'utilisation du vecteur d'affinité. Cette présente invention, sur laquelle un acide aminocaproïque est immobilisé, permet à un peptide ou à une protéine ayant une séquence Kringle d'être éliminé ou purifié avec une efficacité élevée. La présente invention concerne également un procédé de purification et un procédé d'élimination d'une protéine ou d'un peptide ayant une séquence Kringle par l'utilisation du vecteur d'affinité.
PCT/JP2011/070784 2010-09-14 2011-09-13 Vecteur d'affinité pour la purification ou l'élimination d'une protéine ou d'un peptide ayant une séquence kringle, procédé de purification et procédé d'élimination dans lesquels le vecteur d'affinité est utilisé WO2012036140A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2012534006A JPWO2012036140A1 (ja) 2010-09-14 2011-09-13 クリングル配列を有する蛋白質またはペプチドを精製または除去するアフィニティ担体、及びそれを用いた精製方法、除去方法

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2010-206170 2010-09-14
JP2010206170 2010-09-14

Publications (1)

Publication Number Publication Date
WO2012036140A1 true WO2012036140A1 (fr) 2012-03-22

Family

ID=45831599

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2011/070784 WO2012036140A1 (fr) 2010-09-14 2011-09-13 Vecteur d'affinité pour la purification ou l'élimination d'une protéine ou d'un peptide ayant une séquence kringle, procédé de purification et procédé d'élimination dans lesquels le vecteur d'affinité est utilisé

Country Status (2)

Country Link
JP (1) JPWO2012036140A1 (fr)
WO (1) WO2012036140A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2021178321A (ja) * 2016-08-01 2021-11-18 株式会社カネカ カルシプロテインパーティクルの吸着材、および吸着除去システムとその利用方法
WO2022257560A1 (fr) * 2021-06-09 2022-12-15 深圳普门科技股份有限公司 Microsphère non poreuse de copolymère d'agent de réticulation polyvinylique-monomère vinylique, son procédé de préparation, et son application

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH01157000A (ja) * 1987-08-14 1989-06-20 Waander Riethorst 凝固因子の単離方法およびそれに適した吸着剤
JPH03128398A (ja) * 1989-07-12 1991-05-31 Green Cross Corp:The 血漿蛋白の分画方法
JPH0427865A (ja) * 1989-12-21 1992-01-30 Kurita Water Ind Ltd 血液凝固因子用分離剤およびその製造方法
JP2003514789A (ja) * 1999-11-13 2003-04-22 バイエル・コーポレーシヨン 可逆的不活性化酸性化プラスミン
JP2004528853A (ja) * 2001-05-21 2004-09-24 オムリックス・バイオファーマシューティカルズ・エス．アー． 蛋白質溶液からのプラスミノゲン（プラスミン）の除去
JP2008510724A (ja) * 2004-08-20 2008-04-10 プロメティック バイオサイエンシズ，リミテッド 親和性クロマトグラフィーによるタンパク質の逐次的単離および精製スキーム

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH01157000A (ja) * 1987-08-14 1989-06-20 Waander Riethorst 凝固因子の単離方法およびそれに適した吸着剤
JPH03128398A (ja) * 1989-07-12 1991-05-31 Green Cross Corp:The 血漿蛋白の分画方法
JPH0427865A (ja) * 1989-12-21 1992-01-30 Kurita Water Ind Ltd 血液凝固因子用分離剤およびその製造方法
JP2003514789A (ja) * 1999-11-13 2003-04-22 バイエル・コーポレーシヨン 可逆的不活性化酸性化プラスミン
JP2004528853A (ja) * 2001-05-21 2004-09-24 オムリックス・バイオファーマシューティカルズ・エス．アー． 蛋白質溶液からのプラスミノゲン（プラスミン）の除去
JP2008510724A (ja) * 2004-08-20 2008-04-10 プロメティック バイオサイエンシズ，リミテッド 親和性クロマトグラフィーによるタンパク質の逐次的単離および精製スキーム

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
RYOKI TAKAHASHI ET AL.: "Affinity chromatography for purification of two urokinases from human urine.", J. CHROMATOGR. B, vol. 742, 2000, pages 71 - 78 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2021178321A (ja) * 2016-08-01 2021-11-18 株式会社カネカ カルシプロテインパーティクルの吸着材、および吸着除去システムとその利用方法
JP7116276B2 (ja) 2016-08-01 2022-08-10 株式会社カネカ カルシプロテインパーティクルの吸着材、および吸着除去システムとその利用方法
WO2022257560A1 (fr) * 2021-06-09 2022-12-15 深圳普门科技股份有限公司 Microsphère non poreuse de copolymère d'agent de réticulation polyvinylique-monomère vinylique, son procédé de préparation, et son application

Also Published As

Publication number Publication date
JPWO2012036140A1 (ja) 2014-02-03

Similar Documents

Publication Publication Date Title
JP5396933B2 (ja) 液体クロマトグラフィー用充填剤、及び生体高分子の分離精製方法
PL168353B1 (pl) Sposób wytwarzania zatezonego, standaryzowanego ludzkiego c z y n n i k a von Willebranda o wysokiej czystosci PL PL PL PL PL PL PL
US20150297820A1 (en) Adsorbent
KR20210042422A (ko) 치료학적 단백질의 정제 방법
JPH0724723B2 (ja) 硫酸化された非炭水化物ゲルマトリツクス吸着剤
US9422541B2 (en) Apparatus for the extracorporeal treatment of blood
US20180056271A1 (en) Method for producing porous cellulose beads, and adsorbent using same
JP6440320B2 (ja) 多孔質セルロースビーズの製造方法およびそれを用いた吸着体
CN109651502B (zh) 一种从人血浆中同时分离纯化凝血因子ix、x和ⅶ的方法
WO2012036140A1 (fr) Vecteur d'affinité pour la purification ou l'élimination d'une protéine ou d'un peptide ayant une séquence kringle, procédé de purification et procédé d'élimination dans lesquels le vecteur d'affinité est utilisé
CN106928344A (zh) 用于从含有凝血因子的溶液中减少和/或除去FXI和FXIa的方法
CN103933947A (zh) 用于清除类风湿因子的血液净化材料及其制备方法
JP3926573B2 (ja) フィブリノーゲンおよび／またはフィブリンの濃度を減少させるための吸着剤の製造法、吸着剤、および吸着装置の製造のための吸着剤の使用
JPH02149341A (ja) 血清アミロイドｐ蛋白用吸着体
JP5836577B2 (ja) クリングル配列を有する蛋白質またはペプチドを精製または除去するアフィニティ担体の製造方法、その精製方法、除去方法
JPH0436300A (ja) 変性ｌｄｌの吸着体およびそれを用いる変性ｌｄｌの除去装置
CA1283073C (fr) Absorbant et procede pour la purification du facteur viii de coagulation du sang
KR20220111307A (ko) 프로-트롬빈 정제
CN112011527B (zh) 一种凝血酶的制备方法
WO1999020655A1 (fr) Procede de purification de substrats de thrombine et/ou d'inhibiteurs de thrombine ou procede d'elimination de ceux-ci
CN105985940B (zh) 用于制备凝血酶的方法
US20160236171A1 (en) Method for producing porous cellulose beads
JP2002327001A (ja) 糖類、粒子、化合物、細胞接着因子の選択的吸着方法、及び癌転移抑制方法
FI62103B (fi) Foerfarande foer framstaellning av heparin med hoeg specifik aktivitet
JP2014135976A (ja) 塩基性線維芽細胞増殖因子の吸着材とその利用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 11825142

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: 2012534006

Country of ref document: JP

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 11825142

Country of ref document: EP

Kind code of ref document: A1