WO1995004077A1 - Procede de purification du plasminogene - Google Patents

Procede de purification du plasminogene Download PDF

Info

Publication number
WO1995004077A1
WO1995004077A1 PCT/JP1994/001124 JP9401124W WO9504077A1 WO 1995004077 A1 WO1995004077 A1 WO 1995004077A1 JP 9401124 W JP9401124 W JP 9401124W WO 9504077 A1 WO9504077 A1 WO 9504077A1
Authority
WO
WIPO (PCT)
Prior art keywords
plasminogen
anion exchanger
containing composition
purifying
purification
Prior art date
Application number
PCT/JP1994/001124
Other languages
English (en)
Japanese (ja)
Inventor
Shinobu Mochizuki
Takashi Kobayashi
Original Assignee
The Green Cross Corporation
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 The Green Cross Corporation filed Critical The Green Cross Corporation
Publication of WO1995004077A1 publication Critical patent/WO1995004077A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/6435—Plasmin (3.4.21.7), i.e. fibrinolysin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21007—Plasmin (3.4.21.7), i.e. fibrinolysin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to a method for purifying plasminogen, and more particularly to a method for producing high-purity plasminogen from which contaminants in a plasminogen-containing composition have been removed.
  • Plasminogen is activated by perokinase, streptokinase and the like to form plasmin, which breaks down fibrin and causes fibrinolysis. It is attracting attention as a drug that can be applied clinically. In particular, the usefulness of lysyl-type plasminogen whose N-terminal is a lysyl residue is expected.
  • plasminogen has been purified by isoelectric point-acid extraction, ammonium sulfate fractionation, polyethylene glycol fractionation, etc., to obtain crude plasminogen with a specific activity of about 0.1 to 0.1 C UZnig. Methods are known.
  • an affinity chromatography method using lysine-agarose is known.
  • plasminogen is purified to a very high degree of purity, but still contains impurities. Some of these impurities have plasminogen antigenicity and cannot be removed by affinity chromatography.
  • the added volume of the sample is reduced (usually, the added sample volume is about 1 to 5 vZv% of the column volume).
  • Techniques such as increasing the length (increase the bet height) and (3) reducing the flow velocity are required.
  • it is necessary to perform a concentration operation of the sample to be added to the column beforehand.
  • the conventional ion exchange treatment is a method of once adsorbing and separating on an ion exchanger. According to this method, (1) it is necessary to optimize the conditions for adsorption and separation on the ion exchanger in detail. (2) Timely adjustment is necessary even after optimization. (3) A large amount of buffer is required for separation and elution. (4) A large amount of ion exchanger is required to adsorb the target substance. There is a problem.
  • the conventional method is time-consuming, labor-intensive, and involves problems that need to be solved in terms of cost, and is unsuitable for treating a large amount of a plasminogen-containing composition.
  • the present inventors have conducted various studies on an efficient method for purifying plasminogen in order to solve the problems in the conventional method, and as a result, have been able to remove contaminants from the plasminogen-containing composition in a short time to obtain a highly pure This has led to the establishment of a method that can obtain brassminogen in high yield and economically.
  • the present invention provides a method for purifying plasminogen, which comprises contacting a plasminogen-containing composition with an anion exchanger and collecting a non-adsorbed fraction thereof.
  • the purification is preferably carried out under the conditions of a salt concentration of 0.1 to 2 wZv%, more preferably a salt concentration of 0.4 to 0.8 wZv%, and more preferably a pH of 6 to 8 Under the conditions. That is, a preferred embodiment of the present invention is a method for purifying plasminogen, which comprises contacting a plasminogen-containing composition with an anion exchanger under the specific conditions and collecting a non-adsorbed fraction thereof.
  • the plasminogen which is the target product in the purification method of the present invention includes methionyl type (N-terminal amino acid is methionine), glutamyl type (N-terminal amino acid is Examples are glutamic acid) and lysyl type (the N-terminal amino acid is lysine). Further, a mixture thereof may be used. Preferably, it is lysyl-plus minogen.
  • the plasminogen-containing composition as a starting material in the present invention is subjected to the method of the present invention as an aqueous solution. Plasminogen is present in blood, body fluids, placenta and the like, and a plasminogen-containing composition can be prepared therefrom by a generally known method.
  • the degree of purification of the plasminogen-containing composition upon contact with the anion exchanger is not particularly limited, and the method of the present invention can be applied to any degree of purification. Therefore, contact with the anion exchanger can occur at any stage of plasminogen purification.
  • the plasminogen-containing composition as a starting material is not particularly limited as long as it contains plasminogen.For example, it is obtained from serum, plasma, ascites, placental extract, placental tissue extract, and plasma. Composition or gene set consisting of fractions n + m or II of the low-temperature alcohol fractionation method of corn and other fractions obtained by treating plasma or tissue extract by various fractionation methods A culture solution obtained by a recombinant host or tissue culture and a commercially available plasminogen preparation are exemplified.
  • the plasminogen-containing composition is subjected to fractionation operations such as ammonium sulfate fractionation, polyethylene glycol fractionation or dextran fractionation, lysine-affinity chromatography, gel Various separation operations by a filtration method or a combination thereof may be performed. Preferably, lysine affinity chromatography has been performed.
  • the plasminogen-containing composition thus obtained is subjected to the following anion exchange treatment.
  • the anion exchanger is obtained by binding a strong or weakly basic functional group to a suitable support such as cellulose, dextran, a hydrophilic polymer, silica gel synthetic resin or agarose.
  • a strongly basic functional group refers to a basic residue having an ion-exchange ability that is completely dissociated in a wide range of PH.
  • getyl (2-hydroxy) examples thereof include quaternary aminoethylene (QAE-) such as cypropyl) aminoethyl and getylmethylaminoethyl, and quaternary aminomethyl (Q-) such as trimethylaminomethyl.
  • the weakly basic functional group refers to a basic residue having an ion exchange ability whose degree of dissociation, ie, exchange capacity, is significantly changed by PH. Specifically, getylaminoethyl (DEAE ), Aminoethyl (AE), etc.
  • the anion exchanger used in the present invention is not particularly limited, but is preferably an anion exchanger formed by bonding a strongly basic functional group.
  • a strongly basic functional group are those having a trimethylaminomethyl, trimethylaminoethyl, or getyl- (2-hydroxypropyl) aminoethyl group as a functional group.
  • QAE Toyopearl 550 C (manufactured by Toso Corporation) and Q—Sepharose Fast Flow (manufactured by Pharmacia) are preferred.
  • the salt concentration in the contacting environment is 0.1 to 2%, particularly 0.4 to 0.8 wZv%, and the pH is 6 to 8 It is.
  • the buffer used is not particularly limited as long as it has a buffering capacity in the above pH range. Preferred are a Tris-HCl buffer and a phosphate buffer.
  • the concentration of the buffer used is usually 0.005 to 0.2M, preferably 0.01 to 0.05M. Further, lysine or the like of about 0.2 wv% may be blended.
  • Examples of the salt to be used include sodium chloride, potassium chloride, sodium citrate and the like, and preferably sodium chloride.
  • anion exchanger be sufficiently equilibrated under the above conditions before use.
  • the plasminogen-containing composition to be brought into contact with the anion exchanger under the above conditions is preferably prepared under the same buffer conditions as the anion exchanger.
  • the preparation method include a dialysis method using the same buffer as an external solution as the buffer used for equilibrating the anion exchanger.
  • the thus-prepared solution containing plasminogen is brought into contact with the equilibrated anion exchanger, and the treatment is carried out at a temperature of 0 to 60 '(: preferably 4 to 25 ° C).
  • the gel capacity of the anion exchanger used depends on the plasminogen-containing solution to be contacted.
  • the total amount of protein contained in the solution is preferably not less than 1 Omg.
  • plasminogen and impurities can be separated by one operation, and high-purity plasminogen can be obtained from the plasminogen-containing composition.
  • the contaminants are included in the plasminogen-containing composition before being subjected to the anion exchange treatment under the above-described conditions, and are a comprehensive concept representing substances other than the monomeric plasminogen. .
  • Specific examples include high-molecular substances having plasminogen antigenicity (plasminogen dimers and polymers, riboprotein A, etc.), riboproteins, and the like.
  • the method of the present invention is particularly effective for removing these substances. It is.
  • the plasminogen thus obtained has a specific activity of 24 CUZmg and a high recovery, and the method of the present invention is particularly effective for removing these.
  • the plasminogen-containing composition to be subjected to the anion exchanger treatment may have been subjected to an SD (Solvent Detergent) treatment before that.
  • the SD treatment can be performed according to a known method (JP-A-60-511116, JP-A-3-218322). It is also preferable to carry out the reaction in the presence of a surfactant. More preferably, as a stabilizer during the SD treatment, benzamidines (such as benzamidine, i> -ryminobenzamidine) and basic amino acids (arginine, Lysine, etc.), abrotinin or ⁇ -aminocaproic acid (EACA). As the surfactant, Tween 80, Teen 20, TritonXlO0 or the like is used.
  • the amount of stabilizing agent added is about 0.0001 to 0.1 M for benzamidines, about 0.1 to 1 M for basic amino acids, and 10 to 100% for aprotinin, and 1 to 10% for KIZmL EACA ( w / v) degree.
  • the anion exchanger treatment may be performed by any method such as a batch method, a column method, and a membrane method.
  • the column method is used.
  • a series of operations such as equilibration of the anion exchanger (gel), purification of brassinogen, regeneration of the gel, etc. can be repeatedly and efficiently performed economically with the column packed. it can.
  • the anion exchanger (gel) can be regenerated under extremely mild conditions, preferably by increasing the salt concentration, and used repeatedly for the purification of plasminogen. Plasminogen obtained by anion exchanger treatment can be subjected to further purification treatment if necessary. For example, plasminogen which is more highly purified can be obtained by subjecting it to affinity chromatography again using lysine or aprotinin as a ligand, or by dialysis.
  • the plasminogen thus purified is used as a pharmaceutical. Therefore, the plasminogen is preferably subjected to a treatment for virus inactivation. Normal processing is applied as the virus inactivation processing. Specifically, for example, a method of heating a liquid composition of brassinogen at 50 to 100 ° C for 5 to 30 hours, and a method of heating a dried composition at 50 to 100 ° C for 10 to 1 hour. Examples include a method of heating for 50 hours, a method of contacting with a surfactant, and a combination of these treatments.
  • the mixture was applied to a lysin agarose (trade name: lysine sepharose 4B) column equilibrated with 0.9wZv 0.9wZv% glycine solution (pH 7.2) added with sodium chloride. After washing with 1 M sodium chloride-added 0.9% wZv glycine solution (pH 7.2), elution was carried out with 0.9 wZv% sodium chloride-added 0.25 M lysine solution ( ⁇ 7.2) to obtain eluate 35.
  • TTB P tri-W-butyl phosphate
  • Tween 80 tri-W-butyl phosphate
  • the plasminogen-containing solution obtained in Reference Example 1 was added to each of the anion exchangers shown in Table 1 ( ⁇ 5 mm x 5 cm; 1), and ion-exchange chromatography (flow rate: 0.5 ⁇ / min. Buffer: 0.9 wZv% glycine, 0.45 w / v% NaC £, pH 7.2)
  • the unadsorbed fraction that passed through the column as it was was analyzed by gel filtration (hereinafter referred to as GPC).
  • GPC gel filtration
  • Recovery rate of plasminogen by each column treatment [Recovery rate of recovered protein mass (activity) when protein amount before treatment is 100, same hereafter. ] And (when the sample was G PC analysis, A 28. Impurities relative nm A 28. Nm ratio of the sample, the same. Or less) amount of impurities shown in Table 1.
  • Get Table 3 shows the results of measurement by GPC of the unadsorbed fraction and the adsorbed fraction eluted with the elution solution (2 OmM Tris buffer, 0.2 wZv% lysine, 1 MNa C £, PH 7.2). Show.
  • the plasminogen-containing solution obtained in Reference Example 1 is subjected to ion-exchange chromatography (flow rate: ⁇ , ⁇ ⁇ ⁇ .) Using Q-Sepharose Fast Flow ( ⁇ 5 mm x 5 cm; 1 ⁇ &; manufactured by Pharmacia). At this time, change the ⁇ 1 of the equilibration buffer (2 OmM Tris buffer, 0.2 w / v% lysine, 0.45% ⁇ 3 ⁇ ⁇ ) to 6.2, 6.7, 7.2, 7.7, 8.3, The effect of pH on gen recovery and purification was investigated.
  • Figure 1 shows the results of analysis of plasminogen treated with ion-exchange chromatography on gel filtration chromatography.
  • 1 shows the peak of the polymer substance.
  • the addition of the gel imO 1Z5 amount (0.2 to 1Z10 amount (0.1) removed the high molecular substance completely).
  • FIG. 1 is a diagram showing the results of analysis of plasminogen treated with ion exchange chromatography in Example 4 by gel filtration chromatography.
  • untreated indicates a gel filtration chromatogram of a plasminogen-containing solution that has not been subjected to ion exchange chromatography.
  • the amounts shown at the upper left of each chromatogram (1.0 m £, 0.5 md, 0.5 li, 0.1 indicate the amount of the plasminogen-containing solution added to the ion exchanger.
  • the purification method of the present invention it is possible to easily remove substances contaminating the plasminogen-containing composition, especially high molecular substances having plasminogen antigenicity.
  • a high-purity plasminogen can be obtained from a gen-containing composition in a short time, efficiently, with good yield, and economically. Therefore, it is particularly advantageous in purification from a large amount of a plasminogen-containing composition, and is extremely useful as a process for producing plasminogen on an industrial scale.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)

Abstract

On purifie le plasminogène en mettant une composition contenant un plasminogène en contact avec un échangeur d'anions et en récupérant la partie non adsorbée. Pour ce faire, on dose la concentration d'un sel tel que du chlorure de sodium à 0,1-2v% en poids de préférence 0,4 à 0,8 v% en poids et, si nécessaire, on établit le pH entre 6 et 8. Ledit procédé est utile pour extraire les contaminants, en particulier une substance de masse molaire élevée présentant une antigénicité vis à vis du plasminogène, d'une composition contenant un plasminogène. Ledit procédé permet également de purifier le plasminogène efficacement, rapidement et à faible coût. Ce procédé est avantageux pour préparer un plasminogène pur à partir d'une grande quantité d'une composition contenant un plasminogène, et pour produire du plasminogène à l'échelle industrielle.
PCT/JP1994/001124 1993-07-29 1994-07-08 Procede de purification du plasminogene WO1995004077A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP18789693A JPH0739375A (ja) 1993-07-29 1993-07-29 プラスミノーゲンの精製方法
JP5/187896 1993-07-29

Publications (1)

Publication Number Publication Date
WO1995004077A1 true WO1995004077A1 (fr) 1995-02-09

Family

ID=16214096

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP1994/001124 WO1995004077A1 (fr) 1993-07-29 1994-07-08 Procede de purification du plasminogene

Country Status (2)

Country Link
JP (1) JPH0739375A (fr)
WO (1) WO1995004077A1 (fr)

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1232254A4 (fr) * 1999-11-13 2005-02-02 Bayer Healthcare Llc Procede de production d'une composition de plasmine acidifiee inactivee de maniere reversible
US6964764B2 (en) 1999-11-13 2005-11-15 Talecris Biotherapeutics, Inc. Method of thrombolysis by local delivery of reversibly inactivated acidified plasmin
US6969515B2 (en) 1999-11-13 2005-11-29 Talecris Biotherapeutics, Inc. Method of thrombolysis by local delivery of reversibly inactivated acidified plasmin
US7544500B2 (en) 1999-11-13 2009-06-09 Talecris Biotherapeutics, Inc. Process for the production of a reversibly inactive acidified plasmin composition
US8617863B2 (en) 2008-06-04 2013-12-31 Grifols Therapeutics Inc. Composition, method, and kit for preparing plasmin
US9206410B2 (en) 2009-03-03 2015-12-08 Grifols Therapeutics Inc. Compositions, methods and kits for preparing plasminogen and plasmin prepared therefrom
CN108138090A (zh) * 2015-08-31 2018-06-08 泰华施公司 用于稳定的液体四乙酰乙二胺组合物的方法和组合物

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0297386A (ja) * 1988-07-28 1990-04-09 Immuno Ag Chem Med Prod １ｙｓ―プラスミノーゲンの製造方法

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0297386A (ja) * 1988-07-28 1990-04-09 Immuno Ag Chem Med Prod １ｙｓ―プラスミノーゲンの製造方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
BIOCHIMICA ET BIOPHYSICA ACTA, Vol. 221 (1970), PER WALLEN and BJORN WIMAN, "Characterization of Human Plasminogen", pages 20-30. *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1232254A4 (fr) * 1999-11-13 2005-02-02 Bayer Healthcare Llc Procede de production d'une composition de plasmine acidifiee inactivee de maniere reversible
US6964764B2 (en) 1999-11-13 2005-11-15 Talecris Biotherapeutics, Inc. Method of thrombolysis by local delivery of reversibly inactivated acidified plasmin
US6969515B2 (en) 1999-11-13 2005-11-29 Talecris Biotherapeutics, Inc. Method of thrombolysis by local delivery of reversibly inactivated acidified plasmin
US7544500B2 (en) 1999-11-13 2009-06-09 Talecris Biotherapeutics, Inc. Process for the production of a reversibly inactive acidified plasmin composition
US7871608B2 (en) 1999-11-13 2011-01-18 Talecris Biotherapeutics, Inc. Reversibly inactivated acidified plasmin
US8268782B2 (en) 1999-11-13 2012-09-18 Grifols Therapeutics Inc. Composition and method for preparing plasminogen
US9879246B2 (en) 1999-11-13 2018-01-30 Grifols Therapeutics Inc. Reversibly inactivated acidified plasmin composition
US8617863B2 (en) 2008-06-04 2013-12-31 Grifols Therapeutics Inc. Composition, method, and kit for preparing plasmin
US9206410B2 (en) 2009-03-03 2015-12-08 Grifols Therapeutics Inc. Compositions, methods and kits for preparing plasminogen and plasmin prepared therefrom
CN108138090A (zh) * 2015-08-31 2018-06-08 泰华施公司 用于稳定的液体四乙酰乙二胺组合物的方法和组合物

Also Published As

Publication number Publication date
JPH0739375A (ja) 1995-02-10

Similar Documents

Publication Publication Date Title
RU2088590C1 (ru) Способ получения стандартизированного концентрата человеческого фактора виллебранда и концентрат, полученный этим способом
AU594325B2 (en) Method for purifying antihemophilic factor
JP2562192B2 (ja) 血清アルブミンの純化方法
CA2220501C (fr) Nouveaux procedes de purification du facteur ix
JPH04504720A (ja) タンパク質精製のための方法
JP2005500265A (ja) 治療的使用のためのヒト免疫グロブリン濃縮物の調製方法
JP4250770B2 (ja) カチオン交換クロマトグラフィーによるフォンビルブラント因子の精製
JPH082313B2 (ja) 不溶性封入体からの蛋白質の精製および活性化
KR20100058520A (ko) 인자 Ⅷ 및 폰 빌리브란트 인자(ｖＷＦ)의 정제 방법
NO167627B (no) Fremgangsmaate for rensing av faktor viii:c.
JPS6262153B2 (fr)
CZ277939B6 (en) Process for preparing stable concentrate of a complex factor viii-von willebrand factor exhibiting high specific activity
JPH0698019B2 (ja) タンパク質の精製
JP4660048B2 (ja) 血漿プロテアーゼからのフィブリノゲンの分離
JPH0545600B2 (fr)
WO1995004077A1 (fr) Procede de purification du plasminogene
JP3471379B2 (ja) ヒトアンチトロンビン−ｉｉｉの調製方法
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
US5919909A (en) Process for the preparation of factor IX from biological sources
NO178882B (no) Fremgangsmåte for separasjon av vitamin K-avhengige blodkoagulerende faktorer
WO1995011966A1 (fr) Preparation de proteine c humaine activee et son procede de production
ES2552337T3 (es) Procedimientos para la preparación de plasminógeno
JPS6034916A (ja) 第１ｘ因子および他のビタミンｋ依存性タンパク質の高純度精製
WO1999020655A1 (fr) Procede de purification de substrats de thrombine et/ou d'inhibiteurs de thrombine ou procede d'elimination de ceux-ci
JPS5810522A (ja) 不活化トロンビンゲルによるアンチトロンビン３の精製法

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): CA KR US

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IE IT LU MC NL PT SE

121 Ep: the epo has been informed by wipo that ep was designated in this application
122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: CA