WO2000043412A1 - Compositions containing highly purified heparin cofactor ii and method for separating the same - Google Patents

Compositions containing highly purified heparin cofactor ii and method for separating the same Download PDF

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Publication number
WO2000043412A1
WO2000043412A1 PCT/JP2000/000236 JP0000236W WO0043412A1 WO 2000043412 A1 WO2000043412 A1 WO 2000043412A1 JP 0000236 W JP0000236 W JP 0000236W WO 0043412 A1 WO0043412 A1 WO 0043412A1
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Prior art keywords
hcii
heparin cofactor
buffer
heparin
protein
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PCT/JP2000/000236
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French (fr)
Japanese (ja)
Inventor
Naomi Asahara
Akimasa Omizu
Takashi Gotoh
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Welfide Corporation
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Priority to AU30740/00A priority Critical patent/AU3074000A/en
Publication of WO2000043412A1 publication Critical patent/WO2000043412A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
    • C07K14/8121—Serpins
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/30—Partition chromatography
    • B01D15/305—Hydrophilic interaction chromatography [HILIC]
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 - B01D15/36
    • B01D15/3804—Affinity chromatography

Definitions

  • the present invention relates to an HCII-containing composition containing substantially no contaminating protein derived from heparin cofactor-1 II (hereinafter referred to as “HCII”), a HCII-containing compound having a short processing time, and a high recovery rate.
  • HCII heparin cofactor-1 II
  • the present invention relates to a method for separating contaminating proteins from the origin. Background art
  • HCII like antithrombin III (hereinafter referred to as " ⁇ ⁇ "), is an important anticoagulant protein existing in vivo (Matsuo et al .: Biomedical Perspectives, 2, 269-274 (1993)), and has a molecular weight of According to SDS polyacrylamide gel electrophoresis (SDS-PAGE) analysis, it is a 72 kDa single-chain plasma glycoprotein (Dougls. Tollfsen et al .: J. Biol. Chemistry, 257, 2162-2169 (1982)).
  • HCII is also effective for the treatment of a disease caused by enhanced dysfunction of cells in a living body such as macrophages and the like, and is effective in treating the disease.
  • Patent application PCT / JP99 / 03646).
  • a preparation containing HCII as an active ingredient is considered to be useful as a pharmaceutical.
  • HCII-containing composition contaminants to be removed in the purification process
  • proteins include contaminating proteins derived from HCII such as fragmented (low molecular weight) HCII and / or polymerized HCII, low molecular weight factors of HCII, and other proteins other than HCII.
  • HCII with reduced f content may show antigenicity or its effect may not last for a long time due to its short half-life.
  • polymerized HCII may cause side effects such as a decrease in blood pressure along with the possibility of showing antigenicity.
  • HCII can be found in whole plasma, in fractionated plasma, such as corn fractionated supernatant I, supernatant 11 + III or fractionated IV or de-cryoplasma, or in cultures of transformed cells into which the HCII gene has been incorporated. It is known to be contained. It has been reported in JP-A-9-186797 that the HCII can be purified by subjecting the above raw material to anion exchange chromatography, cation exchange chromatography or solid-phase valine treatment. ing.
  • the HCII depolymerizing factor mixed in the HCII-containing raw material is also removed.
  • the present inventors have proposed that the low molecular weight factor can be removed by hydrophobic chromatography, fractionation with a water-soluble polymer, salting out or affinity chromatography using a basic amino acid as a ligand. (International Publication No. W099 / 22753). It was also reported that a highly purified HCII composition having a purity of 98% or more could be obtained by performing gel filtration after removing the depolymerizing factor.
  • the sample must be highly concentrated in order to be subjected to the gel filtration treatment, but there is a problem that HCII concentrated in such a manner is likely to aggregate.
  • the aggregation tends to cause deterioration of the gel carrier, leading to a decrease in separation ability.
  • the gel filtration method is problematic in that it requires a long time for the treatment and that the recovery rate of HCII is low. Therefore, the gel filtration method is not practical to be performed on an industrial scale. I could't say it.
  • HCII crude products which may contain contaminating proteins derived from HCII such as low molecular weight HCII and Z or polymerized HCII
  • HCII crude products which may contain contaminating proteins derived from HCII such as low molecular weight HCII and Z or polymerized HCII
  • contaminating proteins derived from HCII such as low molecular weight HCII and Z or polymerized HCII
  • contacting the crude product with an insoluble carrier having a hydrophobic group as a ligand contacting the crude product with an insoluble carrier having a hydrophobic group as a ligand, and treating the crude product with a buffer solution having a different salt concentration.
  • a carrier treatment method was found, and the present invention was completed.
  • the present invention is as follows.
  • HCII in the crude product is eluted into the buffer by contacting the insoluble carrier with a sui buffer having a lower salt concentration than the buffer used in the step b), and HCII is recovered.
  • HCII-containing composition specific activity of HCII per unit amount of protein is equal to or is at least 18 units ZA 28 ".
  • FIG. 1 shows the results before the treatment of the HC II-containing composition obtained by the hydrophobic chromatography treatment of the present invention.
  • FIG. 2 shows the results of HPLC analysis of (FIG. 1A) and after treatment (FIG. 1B).
  • an insoluble carrier having a hydrophobic group as a ligand is also referred to as “hydrophobic chromatographic ffl carrier”.
  • the treatment using the carrier for hydrophobic chromatography is also referred to as “hydrophobic chromatography treatment”.
  • the desired HCII in the HCII-containing composition of the present invention is a single band visually observed at a position of a molecular weight of about 72 kDa by SDS-PAGE, has an isoelectric point of about 5, and has thrombin, chymotrypsin and A protein that exhibits cathepsin G inhibitory activity.
  • polymerized HCII means a polymer of HCII molecules having no physiological activity of HCII and low affinity for heparin. More specifically, it is a dimer or higher polymer.
  • low molecular weight HCII is defined as “a molecular weight low enough to be separated from intact (full-length) HCII molecules by SDS-PAGE and gel filtration high performance liquid chromatography (G3000 SWXL). (The conditions for gel filtration high performance liquid chromatography will be described later.) Therefore, in the present invention, HCII (hereinafter sometimes referred to as “effective HCIIj” in the sense of being distinguished from polymerized or low-molecularized HCII) is not necessarily a full-length molecule as long as it satisfies the above-mentioned properties.
  • the HCII-derived contaminant protein refers to HCII that has been reduced in molecular weight and / or HCII that has been polymerized.
  • the term "low-molecularizing factor” refers to a chemical or chemical that causes fragmentation of HCII. And enzymological factors, and do not include physical factors such as high temperature, low temperature, and physical shearing force. Specifically, for example, protease, sugar chain or sialic acid degrading enzyme, strong acid, strong alcohol, etc. can be mentioned, but considering the coming of HCII-containing solution as a starting material, the main depolymerizing factor is Proteases.
  • proteins not derived from HCII include, for example, albumin,
  • Antichymotrypsin hi 1—antitrypsin, hi 1—acid glycoprotein, antithrombin III, C 1—esterase inhibitor, C 1 q, C 3 c, C 4 c, C 5 c, cellulo Brasmin, cholinesterase, blood coagulation factor VII, factor VIII, factor IX, factor X, hypoprotein, fibrinogen, fibronectin, Gc-globulin, hemopoxin, haptoglobin, hemoglobin, IgA, Refers to proteins such as IgG, IgE, IgM, lactoferrin,? -Lipoprotein, 22-macroglobulin, plasminogen, prothrombin, 22-plasmin inhibitor and transferrin.
  • the HC11 crude product to be subjected to the hydrophobic gelation treatment of the present invention is not particularly limited as long as it contains an appropriate amount of the effective HCII specified by the above properties. All those derived from natural plasma or from recombinant hosts genetically engineered to produce HCII are included.
  • HCII crude product derived from plasma can be obtained, for example, from whole plasma, fractionated plasma, for example, supernatant I, supernatant ⁇ + ⁇ or fraction IV by corn fractionation, decryoplasma, or the like.
  • the HCII crude product derived from the recombinant host is, for example, a culture solution or cell extract obtained by culturing a host cell transformed with the gene encoding HCII, or a gene encoding HCII is incorporated. It can also be obtained from body fluids or milk of transgenic animals.
  • the crude product to be subjected to the hydrophobic chromatography treatment of the present invention is not particularly limited, but a partially purified HCII crude product is preferred, and more preferably, the purity of effective HCII with respect to the total protein amount in the crude product.
  • HCII crude product having at least 50%, especially HCII crudes of at least 80% are particularly preferred.
  • HCII crude product is not particularly limited, and examples thereof include a crude product obtained by the method described in JP-A-9-1286797.
  • HCII crude products from which low molecular weight factors (such as plasma or host cell-derived protease) have been removed include, for example, those obtained by the method described in International Application Publication No. TO 99/22753. .
  • the solution containing HCII and the depolymerizing factor is selected from hydrophobic chromatography, fractionation with a water-soluble polymer, salting out, and affinity chromatography using a basic amino acid as a ligand 1
  • hydrophobic chromatography treatment is performed, for example, by contacting a solution containing HCII and a molecular weight-reducing factor with a carrier for hydrophobic chromatography under conditions of pH 6 to 9. By this treatment, HCII is recovered in the non-adsorbed fraction, and the low molecular weight factor is adsorbed on the carrier.
  • Examples of the carrier for hydrophobic chromatography include butyl-agarose, butylpolyvinyl, octyl-agarose, octyldecyl-agarose, phenyl-agarose, and phenyl-cellulose.
  • the fractionation treatment with the water-soluble polymer is performed so that the water-soluble polymer has a concentration of 1 to 30% (wZv), preferably 3 to 20% (wZv), and more preferably 6 to 12% (w / v). Then, it is performed by adding to a solution containing HCI1 and a molecular weight-decreasing factor. By this treatment, HCII remains in the solution, and the depolymerizing factor precipitates.
  • the low molecular weight factor By collecting the supernatant (filtrate) by centrifuging or filtering the treatment solution, the low molecular weight factor can be separated from HCII.
  • the water-soluble polymer include polyethylene glycol (for example, polyethylene glycol having an average molecular weight of 4000 to 6000) or a nonionic surfactant (polyoxyethylene (160) polyoxypropylene (30) glycol, polyoxyethylene) (20) Sorbi monomonoate).
  • a solution containing HCII and a low molecular weight factor is added to a salt such as sodium, potassium, calcium, ammonium, etc., for example, chloride, sulfate, phosphate, etc.
  • soybean soup it is carried out by adding soybean soup to a concentration of 0.5 to 0.25M.
  • it is performed by adding barium chloride to a concentration of 0.05 to 2M.
  • This treatment leaves HC II in solution and precipitates the depolymerizing factor.
  • the low molecular weight factor can be separated from HCII.
  • affinity chromatography using a basic amino acid as a ligand a solution containing HCII and a molecular weight-reducing factor is converted to a basic amino acid such as lysine or arginine, preferably at pH 6 to 9.
  • HC11 is recovered in the unadsorbed fraction, and the depolymerizing factor is adsorbed on the carrier.
  • virus inactivation treatment such as heat treatment, trialkyl phosphate treatment, preferably treatment with tri-n-butyl phosphate (hereinafter referred to as “TNBP”) or ultraviolet irradiation treatment, and removal of nucleic acid of virus or virus are performed.
  • TNBP tri-n-butyl phosphate
  • a virus removal membrane treatment or the like can be incorporated into any step before or after the hydrophobic chromatography treatment, if necessary, alone or in any combination.
  • the carrier for hydrophobic chromatography used in the present invention includes an alkyl group having 2 to 11 carbon atoms (for example, butyl group, propyl group, ethyl group, hexyl group, octyl group, etc.), and a phenyl group.
  • an insoluble carrier having a functional group as a ligand examples include cellulose, agarose, dextran, polyacrylamide, polyvinyl, polystyrene, silica, and glass.
  • hydrophobic chromatographic carrier examples include Phenyl-agarose (trade name: Phenyl-sepharose 6FF (low sub), Phenyl-Sepharose 6FF (high sub), Phenyl-Sepharose).
  • the hydrophobic chromatography treatment is performed by bringing a crude HCII into contact with a carrier for hydrophobic chromatography and treating with a buffer solution having a different salt concentration.
  • the method may be either a column method or a batch method.
  • the salt used for adjusting the salt concentration is not particularly limited, but ammonium sulfate and sodium chloride are preferably used, and ammonium sulfate is particularly preferably used.
  • the process of the hydrophobic chromatography treatment will be described by taking as an example a case where effective HCII is separated from HCII in which the molecular weight has been reduced to low molecular weight and HC 11 which has been polymerized.
  • Ammonium sulfate concentration 0.95M or more and 1.5M or less, preferably 1.0M or more and 1.2M or more.
  • Examples of the buffer satisfying such conditions include a Tris buffer (pH 8.0) containing 1 M ammonium sulfate, a phosphate buffer (pH 6.0) containing 1 M ammonium sulfate, and the like.
  • the carrier on which the effective HCII and contaminating proteins (low molecular weight HCII and polymerized HCII) are adsorbed is successively contacted with a buffer having a low salt concentration, so that the low molecular weight HCII and effective HCII are Eluted.
  • the conditions for eluting the low molecular weight HCII are, for example, pH 5 to 9.5, preferably pH 6 to 8.5, and ammonium sulfate concentration of 0.91 ⁇ to less than 1.0M, preferably 0.95M.
  • Buffers satisfying such conditions include Tris buffer (pH 8.0) containing 0.95 M ammonium sulfate, 0.95 M ammonium sulfate, and the like.
  • a phosphate buffer solution (pH 6.0) containing monium is exemplified.
  • Examples of conditions for eluting effective HCII include pH 5 to 9.5, preferably pH 6 to 8.5, and ammonium sulfate concentration of 0.7 M or more and less than 0.9 M, and preferably 0.8 M.
  • Examples of the buffer satisfying such conditions include a Tris buffer (pH 8.0) containing 0.8 M ammonium sulfate.
  • contaminating proteins other than the polymerized HCII and HCII can be eluted.
  • the conditions for eluting the polymerized HCII include pH 5 to 9.5, preferably pH 6 to 9, an ammonium sulfate concentration of less than 0.7 M, and preferably no ammonium sulfate (0 M).
  • An example of a buffer satisfying such conditions is a Tris buffer (PH8.5).
  • the step of eluting the reduced molecular weight H C II can be omitted.
  • HCII crude product adjusted to the contact conditions where the above effective HC11 and HCII-derived contaminant proteins are adsorbed onto the hydrophobic chromatography support is equilibrated under the same conditions and used for hydrophobic chromatography packed in a column.
  • Apply to carrier Elute the low molecular weight H C II from the column using a buffer solution that elutes the low molecular weight H C II.
  • the effective HCII is eluted from the column using a buffer having a low salt concentration and under conditions that allow the effective HCII to be eluted, to obtain effective HCII containing no reduced HCII.
  • Other contaminating proteins such as polymerized HC11 are eluted with lower salt buffer.
  • the above treatment is preferably performed at a low temperature, for example, at 4 to 10 ° C, and more preferably at 4 ° C.
  • HCII-containing composition from which low-molecular-weight HC11 and / or polymerized HCII have been removed as impurities.
  • other proteins that are not derived from small molecules and HCII such as albumin, a 1—Antichymotrypsin, a 1—Antitribine, HI-acid glycoprotein, Anti-mouthlet tombin III, C 1-esterase inhibitor, C1q, C3c, C4c, C5c , Cellulobrasmin, cholinesterase, blood coagulation factor VII, factor VIII, factor IX, factor X, hyphen protein, fibrinogen, fibronectin, Gc-globulin, hemopoxin, haptoglobin, hemoglobin, I Proteins such as gA, IgG, IgE, IgM, lactoferrin,?
  • an effective HCII-containing composition can be obtained.
  • This effective HC II is the specific activity per unit protein content of at least 14 units / A 2BU, preferably at least 18 units ZA 28. Has a high specific activity.
  • the activity of HCII can be measured by the synthetic substrate method using the substrate attached to Test Team AT-III-2 kit (manufactured by KABI) as follows. Add 100 dL of thrombin (1 unit, ZmL) to 50 of the sample or standard solution (0.0031 to 0.025 unit / mL), and incubate at 37 ° C for 5 minutes. Further color development After adding 100 L of synthetic substrate solution, incubate at 37 for 5 minutes, and stop the reaction by adding 1. OmL of reaction stop solution containing citric acid. The solution is measured at an absorbance of 405 nm, and the HCII activity is calculated and calculated from a standard curve.
  • the A 28 " shows the protein concentration in the HCII-containing solution LML, have a numerical value is obtained by measuring the absorbance at a wavelength of 280 nm for the measurement specimen LML la.
  • a CII concentration of 0.1 mgZmL was defined as 1 unit / mL.
  • the effect of removing contaminating proteins from the composition obtained by performing the hydrophobic chromatography treatment can be confirmed, for example, by the following method. If the molecular weight can be distinguished from HC11, such as low molecular weight HC11 or polymerized HCII, analyze the analysis pattern by high performance liquid chromatography (hereinafter referred to as “HPLC”). It is confirmed by In addition, "substantially free from a low molecular weight factor” means that even after incubation in 0.1 M sodium phosphate buffer at pH 8, 25 ° C for 48 to 120 hours. This means that low molecular weight H CII is not detected by HPLC (G3000 SW XL ). Furthermore, the presence or absence of contamination of other proteins not derived from HCII is confirmed by a technique such as ochterlony double immunodiffusion based on their immunological properties.
  • “contains substantially no HCII-derived contaminating protein A” means that no beak of HC11-derived contaminating proteins (low-molecular-weight HC II, polymerized HC II) is detected by HP LC analysis. Means that. The conditions of HP LC are as follows. Temperature: room temperature
  • the purity of HCII with respect to the total protein means the ratio of effective HCII to the total protein. Usually, it can be measured as the ratio of the effective HC II peak area to the total area in HP LC analysis.
  • the thus obtained effective HCII-containing composition is prepared by a method known per se. Can be At this time, if necessary, heating, sterilization, sterilization filtration, freeze-drying, etc. are performed, and pharmaceutically acceptable carriers, additives, dissolution aids, etc. are added in each processing step. Lyophilized preparations, solutions, granules, etc. can be obtained.
  • Production Example 1 will be described in detail with reference to the following production examples, examples and experimental examples, but the present invention is not limited to these examples. Production Example 1
  • Heno, 'Raffinity Chromatography One Carrier (trade name: Heparinto Panoret, manufactured by Tosoh Corporation) at 16 ° C 21% ethanol in 10 mM sodium citrate buffer (pH 6.8) And packed into a 10 OmL column.
  • the corn fraction supernatant II + III obtained according to the method of Kohn et al. J. Am. Chem. Soc, 72, 465 (1950) was passed through the above column, and then 40 OmM sodium chloride at 2-10 ° C.
  • the heparin eluate obtained above was diluted with 40 mM phosphate buffer (pH 8), and this was diluted with an anion exchanger (trade name: QAE Toyopearl 550 C; After passing through a column packed with), a fraction eluted with a 4 OmM phosphate buffer containing 25 OmM sodium chloride was obtained.
  • anion eluate was dialyzed against a 0.02 M Tris-HCl buffer (pH 8.5) (this solution is hereinafter referred to as “anion eluate”). ⁇ ⁇ After passing the above anion eluate through a column packed with a heparin affinity chromatography carrier (trade name: Heparinto Yopearl, manufactured by Tosoh Corporation) equilibrated with a buffer solution, add 10 OmM sodium chloride. A fraction eluted with 2 OmM Tris-HCl buffer (pH 8.5) containing
  • a column (5 xl 00 cm, column volume 2 L) packed with gel filtration gel (trade name: Superdex 200pg: manufactured by Amersham Pharmacia) contains 0.15M NaC1 and ImM018 201111 ⁇ phosphate buffer solution, 117 (developing solution).
  • the HC11 crude product obtained in Production Example 1 was concentrated so that the A28n value became 11.5, and 2 OmL corresponding to 1% of the column volume was applied to the column.
  • the developing solution was flowed at a flow rate of 3.4 mLZ, and chromatography was performed. At this time, the protein amounts (recoveries of A and HCII activities were as shown in Table 1.
  • Table 1 the protein amounts (recoveries of A and HCII activities were as shown in Table 1.
  • a column ( ⁇ 1.6 x 5 cm, column volume 1 OmL) packed with a carrier for hydrophobic chromatography (trade name: Phenyl Sepharose, manufactured by Amersham Pharmaceuticals) is placed in a 2 OmM column containing 1 M ammonium sulfate. Equilibrated with squirrel-hydrochloric acid buffer, pH 8 (developing solution).
  • the HCII crude product obtained in Production Example 1 was prepared so that the A value was 2.5 and the concentration of ammonium sulfate was 1 M, and 5 mL of the crude product was applied to a column.
  • a developing solution having a volume of 6 to 8 times the column volume and a solution having a column volume of 2 times the amount of ammonium sulfate only reduced to 0.95 M are sequentially flowed at a flow rate of 2 mL / min.
  • the degraded HCII was separated into elution fractions.
  • a developing solution having a column volume of 5 times the amount of ammonium sulfate alone reduced to 0.8 M was flowed through the column at the same speed to obtain a HCII fraction containing no HCII and having a reduced molecular weight.
  • the protein amount (A 2M ) and the recovery rate of HC II activity were as shown in Table 1.
  • Hydrophobic chromatography treatment was performed according to Example 1, and the H CII-containing composition before and after the treatment was analyzed using HP CL.
  • Figure 1 shows the results.
  • P2 indicates the peak of HCII
  • P3 indicates the peak of low molecularized HCII
  • P1 indicates the peak of polymerized HCII.
  • the conditions of HPLC are as follows.
  • the time required for the treatment can be shortened, and an HCII-containing composition substantially free of low-molecular-weight or polymerized HCII-derived contaminating proteins such as HCII can be recovered at a high level. Can be obtained at a rate.
  • the specific activity of HCII per unit protein amount is at least 14-position ZA 2H . It is possible to obtain a high specific activity HCII- containing composition, preferably at least 18 units ZA 28n . Therefore, the method of the present invention is extremely useful for producing HCII as a pharmaceutical preparation on an industrial scale.
  • the present application the day has a 1999-year patent application No.

Abstract

A method for purifying HCII whereby an HCII-containing composition substantially free from any contaminant HCII-origin proteins (degraded or polymerized HCII, etc.) can be obtained within a short time at a high yield. This method comprises bringing a crude HCII product containing HCII and contaminant HCII-origin proteins into contact with an insoluble carrier having a hydrophobic group as a ligand and treating with buffer solutions at various concentrations to thereby separate HCII from the contaminant proteins. HCII-containing compositions having a high specific activity of HCII of at least 14 U/A280.

Description

明 細 書  Specification
高度に精製されたへパリンコフアクター II含有組成物およびその分離 法 技術分野 Highly purified composition containing heparin cofactor II and its separation method
本発明は、 へパリンコファクタ一 II (以下 「H CII」 という） 由来の夾雑蛋 を実質的に含有しない HC II含有組成物、 および処理に要する時間が短く、 かつ 高い回収率で HCII と HCII由来の夾雑蛋白とを分離する方法に関する。 背景技術  The present invention relates to an HCII-containing composition containing substantially no contaminating protein derived from heparin cofactor-1 II (hereinafter referred to as “HCII”), a HCII-containing compound having a short processing time, and a high recovery rate. The present invention relates to a method for separating contaminating proteins from the origin. Background art
HCIIは、 アンチトロンビン III (以下 「ΑΤΙΠ」 という） と同様に、 生体内 に存在する重要な抗凝固活性蛋白であり （松尾ら ： Biomedical Perspectives, 2, 269-274 (1993))、 その分子量は、 S D Sポリアクリルアミ ドゲル電気泳動 (SDS-PAGE)分析によると、 72kD aの一本鎖血漿糖蛋白である （Dougls . Tollfsenら ： J. Biol. Chemistry, 257, 2162-2169 (1982) )。  HCII, like antithrombin III (hereinafter referred to as "生 体"), is an important anticoagulant protein existing in vivo (Matsuo et al .: Biomedical Perspectives, 2, 269-274 (1993)), and has a molecular weight of According to SDS polyacrylamide gel electrophoresis (SDS-PAGE) analysis, it is a 72 kDa single-chain plasma glycoprotein (Dougls. Tollfsen et al .: J. Biol. Chemistry, 257, 2162-2169 (1982)).
HCIIの生理機能としては、 トロンビン等のプロテア一ゼを阻害する事が知ら れているが、 A Till とは作用部位が異なり血管内よりも血管外における卜ロン ビン阻害物質として機能していると思われる。 そこで、 HCIIが特定の臓器、 組 織または細胞で抗凝固作用を示すことから、 本発明者らは、 HCIIの静脈内、 動 脈内等の全身投与が、 敗血症、 血流異常を生じる疾患、 肝、 肺、 腎、 脳等の臓器 障害等の疾患の予防および治療に有効であることを見出した （EP 0781558 A2)。 さらに、 本発明者らは、 HCIIをマクロファージ等の生体内細胞の機能異常亢進 に起因する疾患に関して局所投与した場合にも該疾患の治療に有効であることを 見出し、 特許出願 （PCT/JP99/03646) を行った。  It is known that the physiological function of HCII is to inhibit proteases such as thrombin, but it has a different site of action from A Till and functions as a thrombin inhibitor outside the blood vessel rather than inside the blood vessel. Seem. Therefore, since HCII exhibits an anticoagulant effect in specific organs, tissues or cells, the present inventors have found that systemic administration of HCII to veins, arteries and the like may cause sepsis, diseases causing abnormal blood flow, It has been found that it is effective for the prevention and treatment of diseases such as organ disorders such as liver, lung, kidney, and brain (EP 0781558 A2). Furthermore, the present inventors have found that HCII is also effective for the treatment of a disease caused by enhanced dysfunction of cells in a living body such as macrophages and the like, and is effective in treating the disease. Patent application (PCT / JP99 / 03646).
このように、 HCIIを有効成分とする製剤は医薬品としての有用性が考えられ る。  Thus, a preparation containing HCII as an active ingredient is considered to be useful as a pharmaceutical.
HCII含有組成物を得るにあたり、その精製工程において除去されるべき夾雑 蛋白として断片化（低分子化） した HCIIおよび/またはポリマー化した HCII のような HC II由来の夾雑蛋白、 HCIIの低分子化因子や HCII以外の他の蛋 白等が挙げられる。低分 f化した HCIIは抗原性を示す可能性や、 半減期が短い ために効果が長時間持続しない可能性がある。 一方、 ポリマー化した HCIIは抗 原性を示す可能性と並んで血圧低下等の副作用を生じさせる可能性がある。 In obtaining an HCII-containing composition, contaminants to be removed in the purification process Examples of proteins include contaminating proteins derived from HCII such as fragmented (low molecular weight) HCII and / or polymerized HCII, low molecular weight factors of HCII, and other proteins other than HCII. HCII with reduced f content may show antigenicity or its effect may not last for a long time due to its short half-life. On the other hand, polymerized HCII may cause side effects such as a decrease in blood pressure along with the possibility of showing antigenicity.
HCIIは、 全血漿、 分画血漿、 例えばコーンの分画法による上清 I、 上清 11 + III若しくは分画 IVまたは脱クリオ血漿、あるいは HCIIの遺伝子を組み込んだ 形質転換細胞の培養物などに含有されていることが知られている。 該 HCII は、 上記の原料に陰イオン交換クロマトグラフィ一処理、 陽イオン交換クロマトグラ フィ一処理または固相化へバリン処理を施すことにより精製され得ることが特開 平 9一 286797号公報に報告されている。  HCII can be found in whole plasma, in fractionated plasma, such as corn fractionated supernatant I, supernatant 11 + III or fractionated IV or de-cryoplasma, or in cultures of transformed cells into which the HCII gene has been incorporated. It is known to be contained. It has been reported in JP-A-9-186797 that the HCII can be purified by subjecting the above raw material to anion exchange chromatography, cation exchange chromatography or solid-phase valine treatment. ing.
さらに、 HCIIの保存安定性を向上させるためには、 HCII含有原料に混入し ている HCII の低分子化因子も除去されていることが好ましい。 本発明者らは、 疎水性クロマトグラフィー、 水溶性ポリマーによる分画、 塩析または塩基性アミ ノ酸をリガンドとするァフィ二ティ一クロマトグラフィー等により、 該低分子ィ匕 因子を除去し得ることを見出した （国際出願公開 W099/22753号公報）。 また、 該 低分子化因子の除去後にゲル濾過処理を施せば、 純度 98%以上の高度精製され た H C II組成物が得られることも報告した。  Furthermore, in order to improve the storage stability of HCII, it is preferable that the HCII depolymerizing factor mixed in the HCII-containing raw material is also removed. The present inventors have proposed that the low molecular weight factor can be removed by hydrophobic chromatography, fractionation with a water-soluble polymer, salting out or affinity chromatography using a basic amino acid as a ligand. (International Publication No. W099 / 22753). It was also reported that a highly purified HCII composition having a purity of 98% or more could be obtained by performing gel filtration after removing the depolymerizing factor.
しかしながら、 卜.記ゲル濾過処理に付すには試料を高度に濃縮する必要がある が、 そのように濃縮された HCIIは凝集しやすいという問題がある。 さらにこの 凝集によりゲル担体の劣化が生じ易く、 分離能の低下につながった。 すなわち、 ゲル濾過法においては、処理に時間を要すること並びに HCIIの回収率が低いこ とが問題となっており、 そのため、 ゲル濾過処理法は工業的な規模で実施するに は実用的とはいえなかった。  However, the sample must be highly concentrated in order to be subjected to the gel filtration treatment, but there is a problem that HCII concentrated in such a manner is likely to aggregate. In addition, the aggregation tends to cause deterioration of the gel carrier, leading to a decrease in separation ability. In other words, the gel filtration method is problematic in that it requires a long time for the treatment and that the recovery rate of HCII is low. Therefore, the gel filtration method is not practical to be performed on an industrial scale. I couldn't say it.
したがって、 本発明の目的は、 低分子化した HCIIおよび Zまたはポリマー化 した HCIIのような HCII由来の夾雑蛋白を実質的に含有しない、高度に精製さ れた HCII含有組成物を、 より卨ぃ回収率で且つより短い処理時間で得ることが できる、 HC IIの精製方法を提供することである。 また、 本発明の 的は、 上記 精製方法により得られる、低分子化した HCIIおよび Zまたはボリマー化した H CIIのような HCII由来の夾雑蛋白を実質的に含有しない、高度に精製された H CII含有組成物を提供することであり、 あるいは、 比活性が少なくとも 14単位 /A28fl、 好ましくは少なくとも 18単位 ZA28Uである高度に精製された HCII含 有組成物を提供することである。 発明の開示 Accordingly, an object of the present invention is to provide highly purified, substantially free of HCII-derived contaminating proteins such as low molecular weight HCII and Z or polymerized HCII. An object of the present invention is to provide a method for purifying HCII, which can obtain an HCII-containing composition with a lower recovery rate and a shorter treatment time. Further, the object of the present invention is to provide a highly purified HCII which is substantially free of HCII-derived contaminating proteins, such as low molecular weight HCII and Z or volimerized HCII, obtained by the above-mentioned purification method. To provide a highly purified HCII-containing composition having a specific activity of at least 14 units / A 28 fl , preferably at least 18 units ZA 28U . Disclosure of the invention
本発明者らは、 上記課題を解決するために種々検討を行った結果、 低分子化し た HCIIおよび Zまたはポリマー化した HCIIのような HCII由来の夾雑蛋白 を含有する可能性のある HCII粗製物から、 HCIIと HCII由来の該夾雑蛋白 を分離するために、該粗製物を疎水性基をリガンドとする不溶性担体に接触させ、 塩濃度を変えた緩衝液で処理することを特徴とする疎水性担体処理法を見出し、 本発明を完成するに至った。  The present inventors have conducted various studies in order to solve the above problems, and as a result, have found that HCII crude products which may contain contaminating proteins derived from HCII such as low molecular weight HCII and Z or polymerized HCII To separate HCII from the contaminating proteins derived from HCII, contacting the crude product with an insoluble carrier having a hydrophobic group as a ligand, and treating the crude product with a buffer solution having a different salt concentration. A carrier treatment method was found, and the present invention was completed.
すなわち、 本発明は以下の通りである。  That is, the present invention is as follows.
(1) HCIIおよび HCII由来の夾雑蛋白を含有する HCII粗製物を、 疎水性 基をリガンドとする不溶性担体に接触させ、 塩濃度を変えた緩衝液で処理するこ とにより、 HCIIと該夾雑蛋白とを分離する方法。  (1) HCII and the contaminating proteins containing HCII and the contaminating proteins derived from HCII are brought into contact with an insoluble carrier having a hydrophobic group as a ligand, and treated with a buffer having a different salt concentration. And how to separate.
( 2)HCII由来の夾雑蛋白が低分子化した HCIIおよび Zまたはポリマー化し た HCIIである上記 （ 1) の方法。  (2) The method according to (1) above, wherein the contaminating proteins derived from HCII are HCII and Z of low molecular weight or HCII of polymerized HCII.
(3) HCII由来の夾雑蛋白が低分子化した HCIIである上記 （1) の方法。 (3) The method according to the above (1), wherein the contaminating protein derived from HCII is low molecular weight HCII.
(4) HCII由来の夾雑蛋白が低分子化した HCII、 または低分子化した HCII とポリマー化した HCIIの混合物である上記 （1) の方法。 (4) The method according to (1) above, wherein the contaminating protein derived from HCII is HCII in a low molecular weight, or a mixture of HCII in a low molecular weight and a polymerized HCII.
(5) a)該粗製物を該不溶性担体に、 HCIIおよび該夾雑蛋白が不溶性担体上 に吸着される塩濃度を有する緩衝液中で接触させて、 HCIIおよび該夾雑蛋白を 不溶性担体上に吸着させ、 (5) a) bringing the crude product into contact with the insoluble carrier in a buffer having a salt concentration at which HCII and the contaminating protein are adsorbed on the insoluble carrier, to remove HCII and the contaminating protein; Adsorb on an insoluble carrier,
b) 該不溶性担体を前記 a) の工程で用いた緩衝液より低い塩濃度の緩衝液と接 触させて、 該粗製物中の低分子化した HC IIを該緩衝液中に溶出させ、 b) contacting the insoluble carrier with a buffer having a lower salt concentration than the buffer used in the step a) to elute the low molecular weight HC II in the crude product into the buffer;
c) 該不溶性担体を前記 b) の工程で用いた緩衝液よりさらに低い塩濃度の綏衝 液と接触させて、該粗製物中の HCIIを該緩衝液中に溶出させ、 HCIIを回収す ることにより、 HCIIと該夾雑蛋白とを分離する上記（3)または（4)の方法。 ( 6 ) HCII由来の夾雑蛋白がポリマー化した HCIIである上記 （ 1) の方法。c) The HCII in the crude product is eluted into the buffer by contacting the insoluble carrier with a sui buffer having a lower salt concentration than the buffer used in the step b), and HCII is recovered. The method of (3) or (4) above, wherein HCII and the contaminating protein are separated. (6) The method according to (1), wherein the contaminating protein derived from HCII is polymerized HCII.
( 7) A) 該粗製物を該不溶性担体に、 H C 11および該夾雑蛋白が不溶性担体上 に吸着される塩濃度を有する緩衝液中で接触させて、 H C IIおよび該夾雑蛋白を 不溶性担体上に吸着させ、 (7) A) The crude product is brought into contact with the insoluble carrier in a buffer having a salt concentration at which HC 11 and the contaminating protein are adsorbed on the insoluble carrier, and HC II and the contaminating protein are brought into contact with the insoluble carrier. Adsorb to
B) 該不溶性担体を前記 A) の工程で用いた緩衝液より低い塩濃度の緩衝液と接 触させて、該粗製物中の HCIIを該緩衝液中に溶出させ、 HCIIを回収すること により、 HCIIと該夾雑蛋白とを分離する上記 (6) の方法。  B) By contacting the insoluble carrier with a buffer having a lower salt concentration than the buffer used in the step A), eluting HCII in the crude product into the buffer, and recovering HCII The method according to (6) above, wherein HCII and the contaminating protein are separated.
(8)疎水性基をリガンドとする不溶性担体に接触させる HCII粗製物において、 粗製物中の総蛋白量に対する HCIIの純度が少なくとも 50%である上記 （ 1) (8) The crude HCII to be brought into contact with an insoluble carrier having a hydrophobic group as a ligand, wherein the purity of HCII is at least 50% based on the total amount of protein in the crude.
〜 （7) のいずれかに記載の方法。 The method according to any one of (1) to (7).
(9) 上記 （ 1 ) 〜 （8) のいずれかに記載の方法により分離して得た、 HCII 由来の夾雑蛋白を実質的に含有しない H C II含有組成物。  (9) An HCII-containing composition substantially free of HCII-derived contaminating proteins, obtained by separation by the method according to any one of the above (1) to (8).
( 10) さらに低分子化因子を実質的に含有しない上記 （9) の HCII含有組成 物。  (10) The HCII-containing composition according to the above (9), further containing substantially no molecular-weight-decreasing factor.
( 1 1) さらに HCIIに由来しない他の蛋白を実質的に含有しない上記 （ 10) の HCII含有組成物。  (11) The HCII-containing composition according to (10), further containing substantially no other protein not derived from HCII.
( 12)単位蛋白量あたりの HCIIの比活性が少なくとも 14単位/ A21inである ことを特徴とする HC1I含有組成物。 (12) An HC1I-containing composition, wherein the specific activity of HCII per unit protein is at least 14 units / A 21 in.
( 13)単位蛋白量あたりの HCIIの比活性が少なくとも 18単位 ZA28„である ことを特徴とする HCII含有組成物。 ( 14)低分子化した HC IIおよびポリマー化した HC IIを実質的に含有しない 上記 （12) または （13) の HCII含有組成物。 図面の簡単な説明 (13) HCII-containing composition specific activity of HCII per unit amount of protein is equal to or is at least 18 units ZA 28 ". (14) The HCII-containing composition according to the above (12) or (13), which does not substantially contain low molecular weight HC II and polymerized HC II. BRIEF DESCRIPTION OF THE FIGURES
第 1図は、本発明の疎水性クロマト処理により得た HC II含有組成物の処理前 FIG. 1 shows the results before the treatment of the HC II-containing composition obtained by the hydrophobic chromatography treatment of the present invention.
(図 1A) と処理後 （図 1B) の HPLC分析の結果を示す図である。 発明を実施するための最良の形態 FIG. 2 shows the results of HPLC analysis of (FIG. 1A) and after treatment (FIG. 1B). BEST MODE FOR CARRYING OUT THE INVENTION
本明細書において、 疎水性基をリガンドとする不溶性担体を 「疎水性クロマ卜 ffl担体」 ともいう。 また該疎水性クロマト用担体を用いる処理を 「疎水性クロマ ト処理」 ともいう。  In the present specification, an insoluble carrier having a hydrophobic group as a ligand is also referred to as “hydrophobic chromatographic ffl carrier”. The treatment using the carrier for hydrophobic chromatography is also referred to as “hydrophobic chromatography treatment”.
本発明の HCII含有組成物で所望される HCIIとは、 SD S-PAGEにより 分子量約 72kD aの位置に肉眼上単一のバンドを示し、 等電点が約 5であり、 ト ロンビン、 キモトリブシンおよびカテブシン G阻害作用を冇する蛋臼をいう。 本 発明において、 「ポリマ一化した HCII」 とは、 HCII の生理活性がなくへパリ ンに対するァフィ二ティーが小さい、 HCII分子の重合体を意味する。 より具体 的には、二量体またはそれ以上の重合体である。本発明において、 「低分子化した HCII」とは、 SD S— PAGEおよびゲル濾過高速液体クロマトグラフィー（G 3000 SWXL) により 「インタク卜な （完全長） HCII分子から分離し得る程 度に低分子量の H CII断片化物」 を意味する （ゲル濾過高速液体クロマトグラフ ィ一の条件は後で記載する）。 したがって、 本発明において HCII (以下、 ポリマ 一化または低分子化した HCIIと区別する意味で、 特に 「有効 HCIIj という場 合もある） は、 卜-記の性質を満たす限り必ずしも完全長の分子に限定されない。 本発明において、 HCII由来の夾雑蛋 ΰとは、低分子化した HCIIおよび/ま たはポリマー化した HCIIをいう。  The desired HCII in the HCII-containing composition of the present invention is a single band visually observed at a position of a molecular weight of about 72 kDa by SDS-PAGE, has an isoelectric point of about 5, and has thrombin, chymotrypsin and A protein that exhibits cathepsin G inhibitory activity. In the present invention, the term "polymerized HCII" means a polymer of HCII molecules having no physiological activity of HCII and low affinity for heparin. More specifically, it is a dimer or higher polymer. In the present invention, “low molecular weight HCII” is defined as “a molecular weight low enough to be separated from intact (full-length) HCII molecules by SDS-PAGE and gel filtration high performance liquid chromatography (G3000 SWXL). (The conditions for gel filtration high performance liquid chromatography will be described later.) Therefore, in the present invention, HCII (hereinafter sometimes referred to as “effective HCIIj” in the sense of being distinguished from polymerized or low-molecularized HCII) is not necessarily a full-length molecule as long as it satisfies the above-mentioned properties. In the present invention, the HCII-derived contaminant protein refers to HCII that has been reduced in molecular weight and / or HCII that has been polymerized.
本発明において、 低分子化因子とは、 HCIIの断片化を引き起こす化学的およ び酵素学的要因をいい、 高温、 低温、 物理的な剪断力などの物理的要因は含まな レ、。 具体的には、 例えばプロテアーゼ、 糖鎖もしくはシアル酸分解酵素、 強酸、 強アル力リなどが挙げられるが、出発原料となる HCII含有溶液の 来を考慮す れば、 主要な低分子化因子はプロテア一ゼである。 In the present invention, the term "low-molecularizing factor" refers to a chemical or chemical that causes fragmentation of HCII. And enzymological factors, and do not include physical factors such as high temperature, low temperature, and physical shearing force. Specifically, for example, protease, sugar chain or sialic acid degrading enzyme, strong acid, strong alcohol, etc. can be mentioned, but considering the coming of HCII-containing solution as a starting material, the main depolymerizing factor is Proteases.
本発明において、 HCIIに由来しない他の蛋白とは、 例えばアルブミン、 ひ 1 In the present invention, other proteins not derived from HCII include, for example, albumin,
—アンチキモトリブシン、 ひ 1—アンチ卜リプシン、 ひ 1—酸性グリコプロティ ン、 アンチトロンビン III、 C 1—エステラーゼインヒビ夕一、 C 1 q、 C 3 c、 C4 c、 C 5 c、 セルロブラスミン、 コリンエステラーゼ、血液凝固第 VII因子、 第 VIII因子、 第 IX因子、 第 X因子、 ひフヱトプロテイン、 フイブリノ一ゲン、 フイブロネクチン、 Gc—グロブリン、 へモぺキシン、 ハプトグロビン、 へモグ ロビン、 I gA、 I gG、 I gE、 I gM、 ラク 卜フェリン、 ?—リポプロティ ン、 ひ 2—マクログロブリン、 プラスミノゲン、 プロ トロンビン、 ひ 2—プラス ミンインヒビ夕一、 トランスフェリン等の蛋白をいう。 —Antichymotrypsin, hi 1—antitrypsin, hi 1—acid glycoprotein, antithrombin III, C 1—esterase inhibitor, C 1 q, C 3 c, C 4 c, C 5 c, cellulo Brasmin, cholinesterase, blood coagulation factor VII, factor VIII, factor IX, factor X, hypoprotein, fibrinogen, fibronectin, Gc-globulin, hemopoxin, haptoglobin, hemoglobin, IgA, Refers to proteins such as IgG, IgE, IgM, lactoferrin,? -Lipoprotein, 22-macroglobulin, plasminogen, prothrombin, 22-plasmin inhibitor and transferrin.
本発明の疎水性ク口マト処理に付される H C 11粗製物は、上記の性質により特 定される有効 HCIIを適当な量含有するものであれば、その ώ来に特に限定はな く、天然血漿由来または HCIIを産生するように遺伝子操作された組換え宿主由 来のものもすベて包含される。  The HC11 crude product to be subjected to the hydrophobic gelation treatment of the present invention is not particularly limited as long as it contains an appropriate amount of the effective HCII specified by the above properties. All those derived from natural plasma or from recombinant hosts genetically engineered to produce HCII are included.
血漿由来の HCII粗製物は、 例えば全血漿、 分画血漿、 例えばコーンの分画法 による上清 I、上清 ΙΙ + ΙΠ若しくは分画 IVまたは脱クリオ血漿等から得ること ができる。 また、 組換え宿主由来の HCII粗製物は、例えば HCIIをコードする 遺伝子で形質転換された宿主細胞を培養して得られる培養液や細胞抽出液、 また は HCII をコ一ドする遺伝子を組み込んだ卜ランスジエニック動物の体液また は乳汁等からも得ることができる。  An HCII crude product derived from plasma can be obtained, for example, from whole plasma, fractionated plasma, for example, supernatant I, supernatant {+} or fraction IV by corn fractionation, decryoplasma, or the like. The HCII crude product derived from the recombinant host is, for example, a culture solution or cell extract obtained by culturing a host cell transformed with the gene encoding HCII, or a gene encoding HCII is incorporated. It can also be obtained from body fluids or milk of transgenic animals.
本発明の疎水性クロマト処理に付される粗製物は特に限定されるものではない が、 部分的に精製された HCII粗製物が好ましく、 より好ましくは粗製物中の総 蛋白量に対する有効 HCIIの純度が少なくとも 50%である HCII粗製物、就中 少なくとも 80%である HCII粗製物が特に好ましい。 The crude product to be subjected to the hydrophobic chromatography treatment of the present invention is not particularly limited, but a partially purified HCII crude product is preferred, and more preferably, the purity of effective HCII with respect to the total protein amount in the crude product. HCII crude product having at least 50%, especially HCII crudes of at least 80% are particularly preferred.
上記の部分的に精製された HCII粗製物は、 特に限定されないが、 例えば特開 平 9一 286797号公報に記載の方法により得た粗製物が挙げられる。 また、 低分子化因子 （血漿または宿主細胞由来のプロテア一ゼ等） が除去された HCII 粗製物は、 例えば、 国際出願公開 TO 99/22753号公報に記載の方法により得たも のが挙げられる。 詳細には、 HCIIおよび低分子化因子を含有する溶液を、 疎水 性クロマトグラフィー、 水溶性ポリマ一による分画、 塩析および塩基性アミノ酸 をリガンドとするァフィ二ティ一クロマトグラフィ一から選択される 1または 2 以上の処理に付すことにより、低分子化因子が除去された HCII含有組成物が得 られる。 疎水性クロマトグラフィー処理は、 例えば、 pH 6〜9の条件下で、 H CIIおよび低分子化因子を含有する溶液を、疎水性クロマト用担体と接触させる ことにより行われる。該処理により HCIIは未吸着画分に回収され、 低分子化因 子は担体に吸着する。 疎水性クロマト用担体としては、 ブチル—ァガロース、 ブ チルポリビニル、 ォクチルーァガロース、 ォクチルデシルーァガロース、 フエ二 ル一ァガロース、 フヱニル—セルロース等が例示される。 水溶性ポリマーによる 分画処理は、 水溶性ボリマ一を 1〜30% (wZv)、 好ましくは 3〜20% (w Zv)、 より好ましくは 6〜 12% (w/v) の濃度となるようにして、 HCI1 および低分子化因子を含有する溶液に添加することにより行われる。 該処理によ り HCIIは溶液中に残り、 低分子化因子は沈殿する。処理液を遠心分離または濾 過して上淸 （濾液） を回収することにより、 HCIIから低分子化因子を分離する ことができる。 水溶性ポリマ一としては、 ポリエチレングリコール （例えば、 平 均分子翳 4000〜 6000のポリエチレングリコール） または非イオン性界面 活性剤 （ポリオキシエチレン （ 160) ポリオキシプロピレン （30) グリコ一 ル、 ポリオキシエチレン （20) ソルビ夕ンモノォレエート等） が例示される。 塩析処理は、 HCIIおよび低分子化因子を含 する溶液に、 ナトリウム、 力リウ ム、 ノ Jゥム、 アンモニゥム等の塩、 例えば、塩化物、 硫酸塩、 リン酸塩等を 0. 0 5〜0 . 2 5 Mの濃度となるように添カ卩することにより行われる。好ましくは、 塩化バリゥムを 0 . 0 5〜2 Mの濃度となるように添加することにより行われる。 該処理により H C IIは溶液中に残り、 低分子化因子は沈殿する。処理液を遠心分 離または濾過して上清 （濾液） を回収することにより、 H C I Iから低分子化因子 を分離することができる。 塩基性アミノ酸をリガンドとするァフィ二ティ一クロ マトグラフィ一処理は、 p H 6 ~ 9の条件下で、 H C I Iおよび低分子化因子を含 有する溶液を、 リジン、 アルギニン等の塩基性アミノ酸、 好ましくはリジンをリ ガンドとして固相化したクロマト用担体に接触させることにより行われる。 担休 としては、 寒天、 ァガロース、 架橋ァガロース、 セルロース、 シリカ、 ナイロン、 親水性のビニルポリマー等が竽げられる。該処理により H C 11は未吸着画分に回 収され、 低分子化因子は担体に吸着する。 The above partially purified HCII crude product is not particularly limited, and examples thereof include a crude product obtained by the method described in JP-A-9-1286797. HCII crude products from which low molecular weight factors (such as plasma or host cell-derived protease) have been removed include, for example, those obtained by the method described in International Application Publication No. TO 99/22753. . Specifically, the solution containing HCII and the depolymerizing factor is selected from hydrophobic chromatography, fractionation with a water-soluble polymer, salting out, and affinity chromatography using a basic amino acid as a ligand 1 Alternatively, by subjecting the composition to two or more treatments, an HCII-containing composition from which low molecular weight reducing factors have been removed can be obtained. The hydrophobic chromatography treatment is performed, for example, by contacting a solution containing HCII and a molecular weight-reducing factor with a carrier for hydrophobic chromatography under conditions of pH 6 to 9. By this treatment, HCII is recovered in the non-adsorbed fraction, and the low molecular weight factor is adsorbed on the carrier. Examples of the carrier for hydrophobic chromatography include butyl-agarose, butylpolyvinyl, octyl-agarose, octyldecyl-agarose, phenyl-agarose, and phenyl-cellulose. The fractionation treatment with the water-soluble polymer is performed so that the water-soluble polymer has a concentration of 1 to 30% (wZv), preferably 3 to 20% (wZv), and more preferably 6 to 12% (w / v). Then, it is performed by adding to a solution containing HCI1 and a molecular weight-decreasing factor. By this treatment, HCII remains in the solution, and the depolymerizing factor precipitates. By collecting the supernatant (filtrate) by centrifuging or filtering the treatment solution, the low molecular weight factor can be separated from HCII. Examples of the water-soluble polymer include polyethylene glycol (for example, polyethylene glycol having an average molecular weight of 4000 to 6000) or a nonionic surfactant (polyoxyethylene (160) polyoxypropylene (30) glycol, polyoxyethylene) (20) Sorbi monomonoate). In the salting-out treatment, a solution containing HCII and a low molecular weight factor is added to a salt such as sodium, potassium, calcium, ammonium, etc., for example, chloride, sulfate, phosphate, etc. It is carried out by adding soybean soup to a concentration of 0.5 to 0.25M. Preferably, it is performed by adding barium chloride to a concentration of 0.05 to 2M. This treatment leaves HC II in solution and precipitates the depolymerizing factor. By collecting the supernatant (filtrate) by centrifuging or filtering the treated solution, the low molecular weight factor can be separated from HCII. In affinity chromatography using a basic amino acid as a ligand, a solution containing HCII and a molecular weight-reducing factor is converted to a basic amino acid such as lysine or arginine, preferably at pH 6 to 9. It is carried out by bringing lysine into contact with a solid-phased chromatographic carrier as a ligand. Abortions include agar, agarose, cross-linked agarose, cellulose, silica, nylon, hydrophilic vinyl polymers, and the like. By this treatment, HC11 is recovered in the unadsorbed fraction, and the depolymerizing factor is adsorbed on the carrier.
さらに、 加熱処理、 卜リアルキルフォスフエ一卜処理、 好ましくは卜リー n— ブチルフォスフェート（ 以下 「T N B P」 という） 処理または紫外線照射処理等 のウイルス不活化処理およびノまたはウイルスの核酸を除去するためのウィルス 除去膜処理等を、 必要に応じて疎水性クロマト処理の前または後のどの工程にも 単独または任意の組合せによって組み込むことができる。  Furthermore, virus inactivation treatment such as heat treatment, trialkyl phosphate treatment, preferably treatment with tri-n-butyl phosphate (hereinafter referred to as “TNBP”) or ultraviolet irradiation treatment, and removal of nucleic acid of virus or virus are performed. A virus removal membrane treatment or the like can be incorporated into any step before or after the hydrophobic chromatography treatment, if necessary, alone or in any combination.
本発明に使用される疎水性クロマト用担体としては、 炭素数 2〜 1 1のアルキ ル基 （例えば、 ブチル基、 プロピル基、 ェチル基、 へキシル基、 ォクチル基等）、 フエニル基等の疎水性基をリガンドとして有する不溶性担体が挙げられる。また、 不溶性担体としては、 セルロース、 ァガロース、 デキストラン、 ボリアクリルァ ミ ド、 ポリビニル系、 ポリスチレン系、 シリカ系、 ガラス系等が挙げられる。 本 発明に使用される疎水性クロマト用担体の例としては、フエ二ルーァガロース（商 品名：フエ二ソレセファロ一ス [Phenyl— Sepharose 6FF ( low sub)， Phenyl -Sepharose 6FF (high sub) , Phenyl- Sepharose HP 等]、 アマシャムフアルマシア社製）、 フ ヱニル—セルロース （商品名：フエニルセル口ファイン、 生化学工業社製)、 フエ ニルポリビニル （商品名：フエニルトヨパール、 東ソ一社製）、 プチルーァガ口一 ス （商品名：ブチルーセファロ一ス、 アマシャムフアルマシア社製）、 ブチルポリ ビニル（商品名：ブチルトヨパール、 朿ソ一社製）、 フエ二ルポリスチレン架橋ジ ビニルベンゼン （商品名 ： SOURCE 15 PHE、 アマシャムフアルマシア社製） 等が挙 げられる。 好適にはフエ二ルーァガロースが用いられる。 The carrier for hydrophobic chromatography used in the present invention includes an alkyl group having 2 to 11 carbon atoms (for example, butyl group, propyl group, ethyl group, hexyl group, octyl group, etc.), and a phenyl group. And an insoluble carrier having a functional group as a ligand. Examples of the insoluble carrier include cellulose, agarose, dextran, polyacrylamide, polyvinyl, polystyrene, silica, and glass. Examples of the hydrophobic chromatographic carrier used in the present invention include Phenyl-agarose (trade name: Phenyl-sepharose 6FF (low sub), Phenyl-Sepharose 6FF (high sub), Phenyl-Sepharose). HP, etc.), Amersham Pharmacia), phenyl-cellulose (trade name: Phenyl Cell Mouth Fine, Seikagaku Corporation), Hue Nylpolyvinyl (trade name: Phenyl Toyopearl, manufactured by Tohso Ichisha Co., Ltd.), Puchirugagaguchi (tradename: butyl-cephalos, Amersham Fulmasia Co., Ltd.), butylpolyvinyl (tradename: butyltoyopearl, Shirazo Ichisha Co., Ltd.), phenyl polystyrene cross-linked divinylbenzene (trade name: SOURCE 15 PHE, Amersham Fulmasia Co., Ltd.) and the like. Preferably, fenyl agarose is used.
疎水性クロマト処理は、 一般に、 HCII粗製物を、 疎水性クロマト用担体に接 触させ、 塩濃度を変えた緩衝液で処理することにより行われる。 その方法として はカラム法、 バッチ法の何れにて行ってもよい。  In general, the hydrophobic chromatography treatment is performed by bringing a crude HCII into contact with a carrier for hydrophobic chromatography and treating with a buffer solution having a different salt concentration. The method may be either a column method or a batch method.
塩濃度の調整に用いる塩は特に限定されないが、好ましくは硫酸アンモニゥム、 塩化ナトリウムが用いられ、 特に好ましくは硫酸アンモニゥムが用いられる。 疎水性クロマト処理の工程を、有効 HCIIを低分子化した HCIIおよびポリマ ーィ匕した H C 11と分離する場合を例にして説明する。  The salt used for adjusting the salt concentration is not particularly limited, but ammonium sulfate and sodium chloride are preferably used, and ammonium sulfate is particularly preferably used. The process of the hydrophobic chromatography treatment will be described by taking as an example a case where effective HCII is separated from HCII in which the molecular weight has been reduced to low molecular weight and HC 11 which has been polymerized.
HCII粗製物を、 有効 HCIIおよび HCII由来の夾雑蛋白が疎水性クロマ卜 用担体上に吸着される塩濃度を有する緩衝液中で疎水性クロマト用担体に接触さ せる。 かかる接触条件としては、 pH5〜9. 5程度、 好ましくは pH6〜8. The crude HCII is brought into contact with the hydrophobic chromatography carrier in a buffer having a salt concentration at which effective HCII and contaminating proteins derived from HCII are adsorbed onto the hydrophobic chromatography carrier. Such contact conditions include a pH of about 5 to 9.5, preferably a pH of 6 to 8.
5、 硫酸アンモニゥム濃度 0. 95M以上 1. 5M以下、 好ましくは 1. 0 M以 上 1. 2M以ドが例示される。 このような条件を具備する緩衝液としては、 1M 硫酸アンモニゥムを含有する トリス緩衝液（pH8. 0)、 1M硫酸アンモニゥム を含有するリン酸緩衝液 （pH 6. 0) 等が例示される。 5. Ammonium sulfate concentration 0.95M or more and 1.5M or less, preferably 1.0M or more and 1.2M or more. Examples of the buffer satisfying such conditions include a Tris buffer (pH 8.0) containing 1 M ammonium sulfate, a phosphate buffer (pH 6.0) containing 1 M ammonium sulfate, and the like.
次いで、有効 HCIIおよび夾雑蛋白（低分子化した HCIIおよびポリマー化し た HCII) が吸着した担体を、 塩濃度を低くした緩衝液に順次接触させることに より、 低分子化した HCII、 有効 HCIIの順に溶出される。  Next, the carrier on which the effective HCII and contaminating proteins (low molecular weight HCII and polymerized HCII) are adsorbed is successively contacted with a buffer having a low salt concentration, so that the low molecular weight HCII and effective HCII are Eluted.
低分子化した H CIIを溶出させる条件としては、 pH5〜9. 5、 好ましくは pH6〜8. 5、 硫酸アンモニゥム濃度 0. 91^以上1. 0M未満、 好ましくは 0. 95 Mが例示される。 このような条件を具備する緩衝液としては、 0. 95 M硫酸アンモニゥムを含有するトリス緩衝液（pH 8. 0)、 0. 95 M硫酸アン モニゥムを含有するリン酸緩衝液 （pH6. 0) 等が例 くされる。 The conditions for eluting the low molecular weight HCII are, for example, pH 5 to 9.5, preferably pH 6 to 8.5, and ammonium sulfate concentration of 0.91 ^ to less than 1.0M, preferably 0.95M. . Buffers satisfying such conditions include Tris buffer (pH 8.0) containing 0.95 M ammonium sulfate, 0.95 M ammonium sulfate, and the like. A phosphate buffer solution (pH 6.0) containing monium is exemplified.
有効 HCIIを溶出させる条件としては、 pH5〜9. 5、 好ましくは pH6〜 8. 5、 硫酸アンモニゥム濃度 0. 7M以上 0. 9M未満、 好ましくは 0. 8M が例示される。 このような条件を具備する緩衝液としては、 0. 8 M硫酸アンモ 二ゥムを含有するトリス緩衝液 （pH8. 0) 等が例示される。  Examples of conditions for eluting effective HCII include pH 5 to 9.5, preferably pH 6 to 8.5, and ammonium sulfate concentration of 0.7 M or more and less than 0.9 M, and preferably 0.8 M. Examples of the buffer satisfying such conditions include a Tris buffer (pH 8.0) containing 0.8 M ammonium sulfate.
担体をさらに低い塩濃度の緩衝液と接触させることにより、 ポリマ一化した H CIIや HCII以外の夾雑蛋白を溶出させることもできる。ポリマ一化した HCII を溶出させる条件としては、 pH5〜9. 5、 好ましくは pH6〜9、 硫酸アン モニゥム濃度 0. 7M未満、 好ましくは硫酸アンモニゥムを含有しない （0M) 条件が例示される。このような条件を具備する緩衝液としては、 トリス緩衝液（P H8. 5) が例示される。  By contacting the carrier with a buffer having a lower salt concentration, contaminating proteins other than the polymerized HCII and HCII can be eluted. Examples of the conditions for eluting the polymerized HCII include pH 5 to 9.5, preferably pH 6 to 9, an ammonium sulfate concentration of less than 0.7 M, and preferably no ammonium sulfate (0 M). An example of a buffer satisfying such conditions is a Tris buffer (PH8.5).
HCI1粗製物が低分子化した H C 11を含有しない場 は、低分子化した H C II を溶出する工程を省略することができる。  If the HCI1 crude does not contain the reduced molecular weight H C11, the step of eluting the reduced molecular weight H C II can be omitted.
上記の処理工程をカラム法を例にして具体的に説明する。上記の有効 H C 11お よび HCII 由来の夾雑蛋白が疎水性クロマト用担体上に吸着される接触条件に 調整した HCII粗製物を、 同じ条件で平衡化され、 かつカラムに充填された疎水 性クロマト用担体にアプライする。低分子化した H C IIが溶出される条件の緩衝 液を用いて低分子化した H C IIをカラムから溶出させる。次に、 塩濃度を低くし た、有効 HCIIが溶出される条件の緩衝液を用いて有効 HCIIをカラムから溶出 させ、低分子化した HCIIを含有しない有効 HCIIを得る。ポリマ一化した HC 11等の他の夾雑蛋白はより低い塩濃度の緩衝液で溶出される。  The above-mentioned processing steps will be specifically described using a column method as an example. HCII crude product adjusted to the contact conditions where the above effective HC11 and HCII-derived contaminant proteins are adsorbed onto the hydrophobic chromatography support is equilibrated under the same conditions and used for hydrophobic chromatography packed in a column. Apply to carrier. Elute the low molecular weight H C II from the column using a buffer solution that elutes the low molecular weight H C II. Next, the effective HCII is eluted from the column using a buffer having a low salt concentration and under conditions that allow the effective HCII to be eluted, to obtain effective HCII containing no reduced HCII. Other contaminating proteins such as polymerized HC11 are eluted with lower salt buffer.
また、 上記の処理は低温で行われることが好ましく、 例えば、 4〜10°C、 好 ましくは 4°Cで行われる。  Further, the above treatment is preferably performed at a low temperature, for example, at 4 to 10 ° C, and more preferably at 4 ° C.
上記の処理を行うことにより、低分子化した H C 11および/またはポリマ一化 した HCIIが '夾質的に除去された有効 HCII含有組成物を得ることができる。さ らに、同時に、低分子化因子や H CIIに由来しない他の蛋白、例えばアルブミン、 a 1—アンチキモトリブシン、 a 1—アンチ卜 リブシン、 ひ 1一酸性グリコプロ ティン、 アンチ口 トンビン III、 C 1一エステラーゼインヒビ夕一、 C lq、 C 3 c、 C 4 c、 C 5 c、 セルロブラスミン、 コリンエステラーゼ、 血液凝固第 VII 子、 第 VIII因子、 第 IX因子、 第 X因子、 ひフエトプロテイン、 フイブリノ一 ゲン、 フイブロネクチン、 Gc—グロブリン、 へモぺキシン、 ハプトグロビン、 ヘモグロビン、 I gA、 I gG、 I gE、 I gM、 ラク トフエリン、 ?—リポプ 口ティン、 ひ 2—マクログロブリン、 プラスミノゲン、 プロ トロンビン、 ひ 2— プラスミンィンヒビ夕一、 卜ランスフェリン等の蛋白が突質的に除去された有効 HCII含有組成物を得ることができる。この有効 HC IIは単位蛋白量あたりの比 活性が少なくとも 14単位/ A2BU、 好ましくは少なくとも 18単位 Z A28。である 高比活性を有する。 By performing the above treatment, it is possible to obtain an effective HCII-containing composition from which low-molecular-weight HC11 and / or polymerized HCII have been removed as impurities. In addition, at the same time, other proteins that are not derived from small molecules and HCII, such as albumin, a 1—Antichymotrypsin, a 1—Antitribine, HI-acid glycoprotein, Anti-mouthlet tombin III, C 1-esterase inhibitor, C1q, C3c, C4c, C5c , Cellulobrasmin, cholinesterase, blood coagulation factor VII, factor VIII, factor IX, factor X, hyphen protein, fibrinogen, fibronectin, Gc-globulin, hemopoxin, haptoglobin, hemoglobin, I Proteins such as gA, IgG, IgE, IgM, lactoferrin,? -lipop, tin-2, macroglobulin, plasminogen, prothrombin, hi-2, plasmin-inhibi, and transranferin Thus, an effective HCII-containing composition can be obtained. This effective HC II is the specific activity per unit protein content of at least 14 units / A 2BU, preferably at least 18 units ZA 28. Has a high specific activity.
HCII の活性は、 テス トチーム AT— III · 2キヅ ト （KABI社製） に添付 の基質を用いた合成基質法により、 次のようにして測定できる。 試料または標準 液 （0. 0031〜0. 025単位/ mL ) 50 に トロンビン （1単位 Zm L) を 100 dL添加後、 37°Cで 5分間インキュベーションする。 さらに発色 合成基質溶液 100 L添加後、 37でで 5分間インキュベーションし、 クェン 酸を含む反応停止液 1. OmLを添加して反応を停止させる。 該溶液を波良 40 5 nmの吸光度で測定し、 標準曲線から HCII活性を算出して求める。  The activity of HCII can be measured by the synthetic substrate method using the substrate attached to Test Team AT-III-2 kit (manufactured by KABI) as follows. Add 100 dL of thrombin (1 unit, ZmL) to 50 of the sample or standard solution (0.0031 to 0.025 unit / mL), and incubate at 37 ° C for 5 minutes. Further color development After adding 100 L of synthetic substrate solution, incubate at 37 for 5 minutes, and stop the reaction by adding 1. OmL of reaction stop solution containing citric acid. The solution is measured at an absorbance of 405 nm, and the HCII activity is calculated and calculated from a standard curve.
A28„とは、 HCII含有溶液 lmLに含まれる蛋白濃度を示し、 その値は測定試 料 lmLについて波長 280 nmにおいて吸光度を測定して求められる数値をい ラ。 The A 28 ", shows the protein concentration in the HCII-containing solution LML, have a numerical value is obtained by measuring the absorbance at a wavelength of 280 nm for the measurement specimen LML la.
HCIIの比活性 （単位/ A28。） とは、 測定試料について、 上記の方法により求 めた H C 11活性および蛋白濃度（ A28n)から換算した値であって、単位蛋 A量（ A 280= 1 ) あたりの HCII活性をいう。 HCII specific activity (Units / A 28.) And, for the measurement sample, a value converted from the calculated meth HC 11 activity and protein concentration (A 28n) by the method described above, the unit蛋A quantity (A 280 = 1) HCII activity.
また、 HCIIの合成基質 （S— 2238) を用いたトロンビン活性阻害は、 H In addition, inhibition of thrombin activity using HCII synthetic substrate (S-2238)
CII濃度 0. lmgZmLを 1単位/ mLと定義した。 疎水性クロマ卜処理を施して得られた組成物についての夾雑蛋白の除去効果は、 例えば以下の方法により確認できる。例えば低分子化した H C 11やポリマー化し た H C II等のように分子量の違 Wこより有効 H C 11と区別できる場合には、高速 液体クロマトグラフィー（以下 「HPLC」 という）による分析パターンを解析す ることにより確認される。 また、 「低分子化因子を実質的に含まない」 とは、 0. 1 Mリン酸ナトリゥム緩衝液中、 p H 8、 25 °Cの条件下で 48〜 120時間ィ ンキュベ一卜した後も、 HPLC (G 3000 SWXL) により低分子化された H CIIが検出されないことを意味する。 さらに、 HCIIに由来しない他の蛋白は、 その免疫学的特性からォクタロニー二重免疫拡散法 （Ochterlony double immunodiffusion) 等の手法により夾雑の有無が確認される。 A CII concentration of 0.1 mgZmL was defined as 1 unit / mL. The effect of removing contaminating proteins from the composition obtained by performing the hydrophobic chromatography treatment can be confirmed, for example, by the following method. If the molecular weight can be distinguished from HC11, such as low molecular weight HC11 or polymerized HCII, analyze the analysis pattern by high performance liquid chromatography (hereinafter referred to as “HPLC”). It is confirmed by In addition, "substantially free from a low molecular weight factor" means that even after incubation in 0.1 M sodium phosphate buffer at pH 8, 25 ° C for 48 to 120 hours. This means that low molecular weight H CII is not detected by HPLC (G3000 SW XL ). Furthermore, the presence or absence of contamination of other proteins not derived from HCII is confirmed by a technique such as ochterlony double immunodiffusion based on their immunological properties.
本発明において、 「HCII 由来の夾雑蛋 Aを実質的に含有しない」 とは、 HP L C分析で H C 11由来の夾雑蛋白（低分子化した H C II、ポリマー化した H C II ) のビークが検出されないことを意味する。 HP LCの条件は下記の通りである。 温度：室温  In the present invention, “contains substantially no HCII-derived contaminating protein A” means that no beak of HC11-derived contaminating proteins (low-molecular-weight HC II, polymerized HC II) is detected by HP LC analysis. Means that. The conditions of HP LC are as follows. Temperature: room temperature
カラム ： G 3000 SWXL (ø 7. 8 mm x 30 c m, 東ソ一社製） Column: G 3000 SW XL (ø7.8 mm x 30 cm, manufactured by Tosoh Corporation)
展開液： 0. 1M酢酸ナトリウム、 0. 3M塩化ナトリウム、 0. 1%アジ化ナ トリウム、 pH 6. 8 Developing solution: 0.1 M sodium acetate, 0.3 M sodium chloride, 0.1% sodium azide, pH 6.8
流速： 1. OmL/分 Flow rate: 1. OmL / min
検出波長： 280 nm Detection wavelength: 280 nm
検体濃度： AMn= 3 Sample concentration: A Mn = 3
検体量： 30〃 L Sample volume: 30〃 L
分析時間： 12分 Analysis time: 12 minutes
本発明において、 総蛋白量に対する H CIIの純度とは、 総蛋白量屮に占める有 効 HCIIの割合をいう。通常、 HP LC分析における有効 HC IIのピーク面積の 全面積に対する割合として測定することができる。  In the present invention, the purity of HCII with respect to the total protein means the ratio of effective HCII to the total protein. Usually, it can be measured as the ratio of the effective HC II peak area to the total area in HP LC analysis.
このようにして、 得られた有効 HCII含有組成物は、 自体公知の手法にて製剤 化することができる。 この際、 必要に応じて、 加熱、 滅菌、 除菌瀘過処理、 凍結 乾燥処理等が行われ、 各処理工程において医薬上許容される担体、 添加剤、 溶解 補助剤などの添加が行われ、 凍結乾燥製剤、 液剤、 顆粒剤等の形態が得られる。 本発明を以下の製造例、 実施例および実験例により詳述するが、 本 明はこれ らの実施例によって限定されるものではない。 製造例 1 The thus obtained effective HCII-containing composition is prepared by a method known per se. Can be At this time, if necessary, heating, sterilization, sterilization filtration, freeze-drying, etc. are performed, and pharmaceutically acceptable carriers, additives, dissolution aids, etc. are added in each processing step. Lyophilized preparations, solutions, granules, etc. can be obtained. The present invention will be described in detail with reference to the following production examples, examples and experimental examples, but the present invention is not limited to these examples. Production Example 1
ヒ卜血漿画分からの H C 11粗製物の製造 Production of H C 11 crude from human plasma fraction
へノ、'リンァフィ二ティ一クロマトグラフィ一担体 （商品名 ：へパリントョパー ノレ、 東ソ一社製） を一 6°Cで 2 1 %エタノール含有 10 mMクェン酸ナト リウム 緩衝液 （pH 6. 8) で洗浄し、 1 0 OmLのカラムに充填した。 コーンらの方 法 （J. Am. Chem. Soc, 72, 465 (1950)) に従って得たコーン分画上清 II + III を上記カラムに通した後、 2〜 10°Cで 40 OmM塩化ナトリゥムを含有する 1 OmMクェン酸ナトリウム水溶液（pH 7. 0)を用いて溶出した画分（以 F「へ パリン溶出液」 という） を得た。  Heno, 'Raffinity Chromatography One Carrier (trade name: Heparinto Panoret, manufactured by Tosoh Corporation) at 16 ° C 21% ethanol in 10 mM sodium citrate buffer (pH 6.8) And packed into a 10 OmL column. The corn fraction supernatant II + III obtained according to the method of Kohn et al. (J. Am. Chem. Soc, 72, 465 (1950)) was passed through the above column, and then 40 OmM sodium chloride at 2-10 ° C. A fraction eluted with a 1 OmM aqueous solution of sodium citrate (pH 7.0) containing the following (hereinafter, referred to as "heparin eluate") was obtained.
上記で得たへパリン溶出液を 40 mMリン酸緩衝液 （p H 8 ) で希釈し、 これ を、 同緩衝液で洗浄した陰イオン交換体 （商品名 ： QAE トヨパール 5 50 C、 束ソ一社製） を充填したカラムに通した後、 2 5 OmM塩化ナトリウムを含有す る 4 OmMリン酸緩衝液を用いて溶出した画分を得た。 この溶出液を、 0. 02 M卜リス—塩酸緩衝液 （pH 8. 5) に対して透析した （この溶液を以下 「陰ィ オン溶出液」という）。问緩衝液で平衡化したへパリンァフィ二ティ一クロマトグ ラフィー担体（商品名：へパリントヨパール、東ソ一社製）を充填したカラムに、 上記陰イオン溶出液を通した後、 10 OmM塩化ナトリウムを含有する 2 OmM トリスー塩酸緩衝液 （pH 8. 5) を用いて溶出した画分を得た。  The heparin eluate obtained above was diluted with 40 mM phosphate buffer (pH 8), and this was diluted with an anion exchanger (trade name: QAE Toyopearl 550 C; After passing through a column packed with), a fraction eluted with a 4 OmM phosphate buffer containing 25 OmM sodium chloride was obtained. The eluate was dialyzed against a 0.02 M Tris-HCl buffer (pH 8.5) (this solution is hereinafter referred to as “anion eluate”).し た After passing the above anion eluate through a column packed with a heparin affinity chromatography carrier (trade name: Heparinto Yopearl, manufactured by Tosoh Corporation) equilibrated with a buffer solution, add 10 OmM sodium chloride. A fraction eluted with 2 OmM Tris-HCl buffer (pH 8.5) containing
得られた H C II粗製物について H PLCで分析したところ、総蛋白量に対する When the obtained H C II crude product was analyzed by HPLC,
HCII純度は 90%であった。 比較例 1 HCII purity was 90%. Comparative Example 1
ゲル濾過処理  Gel filtration treatment
ゲル濾過用枸体 （商品名 ： Superdex 200pg： アマシャムフアルマシア社製） を 充填したカラム （ 5 x l 00 cm、 カラム体積 2 L) を、 0. 15M N a C 1および ImM 0丁八を含有する201111^リン酸緩衝液、 117 (展開液） で 、ド衡化した。製造例 1で得られた H C 11粗製物を A28n値が 1 1. 5になるように 濃縮し、 カラム体積の 1 %に当たる 2 OmLをカラムに付した。 展開液を 3. 4 mLZ分の流速で流し、 クロマトグラフィーを行った。 この際の蛋白量 （A および HCII活性の回収率は表 1の通りであった。 表 1 A column (5 xl 00 cm, column volume 2 L) packed with gel filtration gel (trade name: Superdex 200pg: manufactured by Amersham Pharmacia) contains 0.15M NaC1 and ImM018 201111 ^ phosphate buffer solution, 117 (developing solution). The HC11 crude product obtained in Production Example 1 was concentrated so that the A28n value became 11.5, and 2 OmL corresponding to 1% of the column volume was applied to the column. The developing solution was flowed at a flow rate of 3.4 mLZ, and chromatography was performed. At this time, the protein amounts (recoveries of A and HCII activities were as shown in Table 1. Table 1
Figure imgf000016_0001
実施例 1
Figure imgf000016_0001
Example 1
疎水性クロマト処理  Hydrophobic chromatographic treatment
疎水性クロマト用担体 （商品名 フェニルセファロ一ス、 アマシャムフアルマ シァ社製） を充填したカラム （Φ 1. 6 X 5 cm、 カラム体積 1 OmL) を、 1 M硫酸アンモニゥムを含存する 2 OmM卜リス—塩酸緩衝液、 pH8 (展開液） で平衡化した。 製造例 1で得られた HCII粗製物を A 値が 2. 5、 硫酸アンモ ニゥム濃度が 1Mになるように調製したのち、 該粗製物の 5mLをカラムに付し た。 その後、 カラム体積の 6〜 8倍量の展開液および硫酸アンモニゥム濃度のみ 0.95 Mに下げたカラム体積 2倍量の展閧液を、順次 2 m L/分の流速で流し、 低分子化した HCIIを溶出画分に分離した。続いて、 硫酸アンモニゥム濃度のみ 0. 8 Mに下げたカラム体積 5倍量の展開液を同速度でカラムに流し、 低分子化 した HCIIを含有しない HCII画分を得た。 この際の蛋白量（A2M)および HC II活性の回収率は表 1の通りであった。 実験例 1 A column (Φ 1.6 x 5 cm, column volume 1 OmL) packed with a carrier for hydrophobic chromatography (trade name: Phenyl Sepharose, manufactured by Amersham Pharmaceuticals) is placed in a 2 OmM column containing 1 M ammonium sulfate. Equilibrated with squirrel-hydrochloric acid buffer, pH 8 (developing solution). The HCII crude product obtained in Production Example 1 was prepared so that the A value was 2.5 and the concentration of ammonium sulfate was 1 M, and 5 mL of the crude product was applied to a column. Thereafter, a developing solution having a volume of 6 to 8 times the column volume and a solution having a column volume of 2 times the amount of ammonium sulfate only reduced to 0.95 M are sequentially flowed at a flow rate of 2 mL / min. The degraded HCII was separated into elution fractions. Subsequently, a developing solution having a column volume of 5 times the amount of ammonium sulfate alone reduced to 0.8 M was flowed through the column at the same speed to obtain a HCII fraction containing no HCII and having a reduced molecular weight. At this time, the protein amount (A 2M ) and the recovery rate of HC II activity were as shown in Table 1. Experimental example 1
蛋白量および HCII活性回収率の比較 Comparison of protein amount and HCII activity recovery rate
ゲル濾過処理 （比較例 1 ) または疎水性クロマト処理 （実施例 1 ) の各処理後 の蛋白量 （A280 ) および H CII活性の回収率を比較し、 その結果を表 1に示し た。蛋白量回収率および HCII活性の回収率ともに、 疎水性クロマト処理のほう がゲル濾過処理に比べて高かった。 実験例 2  The protein amount (A280) and the recovery rate of HCII activity after each treatment of the gel filtration treatment (Comparative Example 1) or the hydrophobic chromatography treatment (Example 1) were compared, and the results are shown in Table 1. Both the protein recovery and the recovery of HCII activity were higher in the hydrophobic chromatography treatment than in the gel filtration treatment. Experimental example 2
処理時間の比較 Comparison of processing time
血漿量 2400 L相当のコーンの分画上清 II + IIIを製造例 1の方法で処理し たところ、 総蛋白量 36,000 (A280 xmL) の H C II粗製物が得られた。 該粗製物 についてゲル濾過処理 （比較例 1) または疎水性クロマト処理 （実施例 1) を行 つた。同量の H C 11含有組成物を各々冏量のゲル量で処理した場合に要した処理 時間を換算により求め、 表 2に示した。 尚、 ゲル濾過処理では、 連続処理すると 分離能力が低下するため、 毎回苛性ソーダ （0. 1M) でのゲルの処理が必要と なる。 従って、 該苛性ソーダ処理および再生一平'衡化処理の時間も組み入れて換 算した。 When the fraction supernatant II + III of corn corresponding to a plasma volume of 2400 L was treated by the method of Production Example 1, a crude HC II product having a total protein amount of 36,000 (A 280 × mL) was obtained. The crude product was subjected to gel filtration treatment (Comparative Example 1) or hydrophobic chromatography treatment (Example 1). The treatment time required when the same amount of the HC 11-containing composition was treated with the same amount of gel, was calculated and shown in Table 2. In the case of gel filtration, continuous separation reduces the separation capacity, so the gel must be treated with caustic soda (0.1 M) each time. Therefore, the time for the caustic soda treatment and the time for the regeneration / equilibrium treatment were also incorporated and converted.
その結果、 疎水性クロマト処理の所要時間は、 ゲル濾過処理の所要時間の約 1 00分の 1以下であった。 表 2 As a result, the time required for the hydrophobic chromatography treatment was about one hundredth or less of the time required for the gel filtration treatment. Table 2
Figure imgf000018_0001
実験例 3
Figure imgf000018_0001
Experiment 3
HPLC分析  HPLC analysis
実施例 1に準じて疎水性クロマト処理を行い、該処理前後の H CII含有組成物 について HP C Lを用いて分析した。その結果を図 1に 、した。 P 2は HCIIの、 P 3は低分子化した HCIIの、 P 1はポリマー化した HCIIのピークをそれぞれ 示す。 その結果、 疎水性クロマ卜処理後の組成物について、 低分子化した HCII およびポリマー化した H CIIのピークは検出されなかった。 H P L Cの条件は下 記の通りである。  Hydrophobic chromatography treatment was performed according to Example 1, and the H CII-containing composition before and after the treatment was analyzed using HP CL. Figure 1 shows the results. P2 indicates the peak of HCII, P3 indicates the peak of low molecularized HCII, and P1 indicates the peak of polymerized HCII. As a result, in the composition after the treatment with the hydrophobic chromatograph, the peaks of low molecular weight HCII and polymerized HCII were not detected. The conditions of HPLC are as follows.
温度：室温  Temperature: room temperature
カラム： G3000 SWXL (Φ7. 8mm 30 cm, 東ソ一社製） Column: G3000 SW XL (Φ7.8 mm 30 cm, manufactured by Tosoh Corporation)
展開液： 0. 1M酢酸ナトリウム， 0· 3 M塩化ナトリウム， 0. 1%アジ化ナ トリゥム， pH 6. 8  Developing solution: 0.1 M sodium acetate, 0.3 M sodium chloride, 0.1% sodium azide, pH 6.8
流速： 1. OmLノ分  Flow rate: 1. OmL
検出波長： 280 nm  Detection wavelength: 280 nm
検体濃度： A28()= 3 Sample concentration: A 28 () = 3
検体量： 30 / L 分析時間： 12分 Sample volume: 30 / L Analysis time: 12 minutes
上記条件で HPLC分析を行ったとき、 有効 HCII のビークは保持時間 9分、 低分子化した HCIIのビークは保持時間 9. 6分、ポリマー化した HCIIのピ一 クは保持時間 6 ~ 8分でそれそれ検出される。 産業上の利用可能性  When HPLC analysis was carried out under the above conditions, the effective HCII beak had a retention time of 9 minutes, the low molecular weight HCII beak had a retention time of 9.6 minutes, and the polymerized HCII peak had a retention time of 6 to 8 minutes. In it it is detected. Industrial applicability
本発明の精製方法によれば、 処理に要する時間を短縮化でき、 且つ低分子化ま たはポリマー化した HCII等の HCII 由来の夾雑蛋白を実質的に含有しない H CII含有組成物を高い回収率で得ることができる。また、本発明の方法によれば、 単位蛋白量あたりの HCIIの比活性が少なくとも 14甲.位 ZA2H。、 好ましくは少 なくとも 18単位 ZA28nである高比活性の H CII 含有組成物を得ることができ る。 したがって、 本発明の方法は、 医薬製剤としての HCIIを工業的規模で製造 するのに極めて有用である。 本出願は、 日木国で出願された平成 1 1年特許願第 010911 1を墓礎とし ており、そこに開示される内容は本明細書にすべて包含されるものである。また、 ここで述べられた特許および特許出願明細書を含む全ての刊行物に記載された内 容は、 ここにその全てが明 7f、されたと同程度に本明細書に組み込まれるものであ る。 According to the purification method of the present invention, the time required for the treatment can be shortened, and an HCII-containing composition substantially free of low-molecular-weight or polymerized HCII-derived contaminating proteins such as HCII can be recovered at a high level. Can be obtained at a rate. According to the method of the present invention, the specific activity of HCII per unit protein amount is at least 14-position ZA 2H . It is possible to obtain a high specific activity HCII- containing composition, preferably at least 18 units ZA 28n . Therefore, the method of the present invention is extremely useful for producing HCII as a pharmaceutical preparation on an industrial scale. The present application, the day has a 1999-year patent application No. 010911 1, which was filed in the tree countries and Hakaishizue, what is disclosed therein is intended to be incorporated in full herein by this reference. Also, the contents described in all publications, including the patents and patent application specifications mentioned herein, are hereby incorporated by reference in their entirety, to the same extent as if all were explicitly stated 7f. .

Claims

請 求 の 範 M Range of claims M
1 .へパリンコフアクター I Iおよびへパリンコファクタ一 II由来の夾雑蛋白を含 有するへパリンコファクタ一 I I粗製物を、疎水性基をリガンドとする不溶性担体 に接触させ、 塩濃度を変えた緩衝液で処理することにより、 へパリンコファク夕 一 IIと該夾雑蛋白とを分離する方法。  1.A crude product containing heparin cofactor-1 II containing contaminating proteins derived from heparin cofactor II and heparin cofactor II is brought into contact with an insoluble carrier having a hydrophobic group as a ligand, and a buffer in which the salt concentration is changed. A method of separating heparin cofacient II and the contaminating protein by treating with a liquid.
2 .へパリンコフアクター II由来の夾雑蛋白が低分子化したへパリンコファクタ -I I および Zまたはポリマ一化したへパリンコファクタ一 11 である請求の範囲 1の方法。  2. The method according to claim 1, wherein the contaminant protein derived from heparin cofactor II is low molecular weight heparin cofactor-II and Z or polymerized heparin cofactor-1 11.
3 .へパリンコファクタ一 I I由来の夾雑蛋白が低分子化したへパリンコファクタ 一 I Iである請求の範囲 1の方法。  3. The method according to claim 1, wherein the contaminating protein derived from heparin cofactor III is low molecular weight heparin cofactor-1II.
4 .へパリンコファクター H由来の夾雑蛋 Aが低分子化したへパリンコファクタ — 11、 または低分子化したへバリンコフアクター I I とポリマ一化したへパリンコ ファクタ一 IIの混合物である請求の範囲 1の方法。  4. The contaminant protein A derived from heparin cofactor H is a low molecular weight heparin cofactor-11 or a mixture of a low molecular weight heparin cofactor II and a polymerized heparin cofactor-1 II. Range 1 way.
5 . a ) 該粗製物を該不溶性担体に、 へパリンコファクタ一 IIおよび該夾雑蛋 ΰ が不溶性担体上に吸着される塩濃度を有する緩衝液中で接触させて、 へパリンコ ファクター 11および該夾雑蛋白を不溶性担体上に吸着させ、 b ) 該不溶性担体を 前記 a ) の工程で用いた緩衝液より低い塩濃度の緩衝液と接触させて、 該粗製物 中の低分子化したへパリンコファクター IIを該緩衝液中に溶出させ、  5.a) bringing the crude product into contact with the insoluble carrier in a buffer having a salt concentration at which heparin cofactor-1 II and the contaminating protein are adsorbed on the insoluble carrier, to obtain heparin cofactor 11 and the heparin cofactor 11; The contaminating protein is adsorbed on the insoluble carrier, and b) the insoluble carrier is brought into contact with a buffer having a lower salt concentration than the buffer used in the step a) to reduce the molecular weight of heparinco in the crude product. Eluting Factor II in the buffer,
c ) 該不溶性担体を前記 b ) の工程で用いた緩衝液よりさらに低い塩濃度の緩衝 液と接触させて、該粗製物中のへパリンコファクタ一 IIを該緩衝液中に溶出させ、 へパリンコファクタ一 I Iを回収することにより、へパリンコファクタ一 II と該夾 雑蛋 ΰとを分離する請求の範囲 3または 4の方法。  c) contacting the insoluble carrier with a buffer having a lower salt concentration than the buffer used in the step b) to elute heparin cofactor-II in the crude product into the buffer; 5. The method according to claim 3 or 4, wherein heparin cofactor-II is separated from said contaminating protein by recovering palincofactor-II.
6 .へパリンコファクター I I由来の夾雑蛋白がポリマー化したへパリンコファク 夕一 IIである請求の範囲 1の方法。  6. The method according to claim 1, wherein the contaminating protein derived from heparin cofactor II is polymerized heparin cofactor Yuichi II.
7 . Α) 該粗製物を該不溶性担体に、 へパリンコファクタ一 IIおよび該夾雑蛋白 が不溶性担体上に吸着される塩濃度を有する緩衝液中で接触させて、 へパリンコ ファクター IIおよび該夾雑蛋白を不溶性担体上に吸着させ、 B )該不溶性担体を 前記 A) の工程で用いた緩衝液より低い塩濃度の緩衝液と接触させて、 該粗製物 中のへパリンコファクタ一 IIを該緩衝液中に溶出させ、へパリンコファクター II を回収することにより、へパリンコファクター IIと該夾雑蛋白とを分離する請求 の範 W 6の方法。 7. Α) Contacting the crude product with the insoluble carrier in a buffer having a salt concentration at which heparin cofactor II and the contaminating protein are adsorbed on the insoluble carrier, Factor II and the contaminating protein are adsorbed on an insoluble carrier, and B) the insoluble carrier is brought into contact with a buffer having a lower salt concentration than the buffer used in the above step A), whereby heparinco in the crude product is obtained. The method according to claim 6, wherein heparin cofactor II and said contaminating protein are separated by eluting factor-1 II into said buffer and recovering heparin cofactor II.
8. 疎水性基をリガンドとする不溶性担体に接触させるへパリンコファクタ一II 粗製物において、粗製物中の総蛋白量に対するへパリンコファクタ一 IIの純度が 少なくとも 50%である請求の範 1用 1〜7のいずれかの方法。  8. A heparin cofactor-II crude product which is contacted with an insoluble carrier having a hydrophobic group as a ligand, wherein the purity of heparin cofactor-II is at least 50% based on the total amount of protein in the crude product. For any of 1-7 methods.
9. 請求の範囲 1〜8のいずれかの方法により分離して得た、 へパリンコファク 夕一 II 由来の夾雑蛋白を実質的に含有しないへパリンコフアクター II 含有組成 物。  9. A heparin cofactor II-containing composition substantially free of contaminating proteins derived from heparin cofac Yuichi II obtained by the method according to any one of claims 1 to 8.
10. さらに低分子化因了-を実質的に含有しない請求の範囲 9のへパリンコファ クタ一 II含^組成物。  10. The heparin cofactor-II composition according to claim 9, further containing substantially no molecular weight reduction factor.
1 1. さらにへパリンコファクタ一IIに由来しない他の蛋白を実質的に含^しな い請求の範囲 10のへパリンコファクタ一 II含有組成物。  1 1. The heparin cofactor-II-containing composition according to claim 10, further comprising substantially no other protein not derived from heparin cofactor-II.
12.単位蛋白量あたりのへパリンコファクタ一 IIの比活性が少なくとも 14単 位 A28。であることを特徴とするへパリンコフアクター II含有組成物。 12. Specific activity of heparin cofactor-II per unit protein is at least 14 units A 28 . A composition containing heparin cofactor II, characterized in that:
13.単位蛋白量あたりのへパリンコフアクター Πの比活性が少なくとも 18単 位 ZA28。であることを特徴とするへパリンコファク夕一II含有組成物。 13. The specific activity of Heparin Coffactor II per unit protein is at least 18 units ZA 28 . A composition containing heparin cofac Yuichi II, characterized in that:
14.低分子化したへパリンコファクター IIおよびポリマー化したへパリンコフ ァク夕一 Π を実質的に含有しない請求の範囲 12または 13のへパリンコファ クタ一 II含有組成物。  14. The heparin cofactor-II-containing composition according to claim 12 or 13, wherein the composition does not substantially contain low-molecular-weight heparin cofactor II and polymerized heparin cofactor-1.
PCT/JP2000/000236 1999-01-19 2000-01-19 Compositions containing highly purified heparin cofactor ii and method for separating the same WO2000043412A1 (en)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH09286797A (en) * 1996-04-18 1997-11-04 Green Cross Corp:The Heparin cofactor ii and its purification
WO1999022753A1 (en) * 1997-11-05 1999-05-14 Yoshitomi Pharmaceutical Industries, Ltd. Heparin cofactor ii preparations and process for producing the same

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH09286797A (en) * 1996-04-18 1997-11-04 Green Cross Corp:The Heparin cofactor ii and its purification
WO1999022753A1 (en) * 1997-11-05 1999-05-14 Yoshitomi Pharmaceutical Industries, Ltd. Heparin cofactor ii preparations and process for producing the same

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
PETZELBAUER E. ET AL.: "Modulation of heparin cofactor II activity by glycosaminoglycans and adhesive glycoproteins", THROMBOSIS RESEARCH, vol. 66, no. 5, 1992, pages 559 - 567, XP002927649 *
TOULON P. ET AL.: "Purification of heparin cofactor II from human plasma", JOURNAL OF CHROMATOGRAPHY, vol. 539, no. 2, 1991, pages 493 - 500, XP002927648 *
YAMAGISHI R. ET AL.: "Purification and biological property of heparin cofactor II: activation of heparin cofactor II and antithrombin III by dextran sulfate and various glycosaminoglycans", THROMBOSIS RESEARCH, vol. 36, no. 6, 1984, pages 633 - 642, XP002927647 *

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