JPH084503B2 - Method for recovering urokinase and urokinase-containing fraction obtained thereby - Google Patents
Method for recovering urokinase and urokinase-containing fraction obtained therebyInfo
- Publication number
- JPH084503B2 JPH084503B2 JP62297831A JP29783187A JPH084503B2 JP H084503 B2 JPH084503 B2 JP H084503B2 JP 62297831 A JP62297831 A JP 62297831A JP 29783187 A JP29783187 A JP 29783187A JP H084503 B2 JPH084503 B2 JP H084503B2
- Authority
- JP
- Japan
- Prior art keywords
- urokinase
- membrane
- urine
- concentrated
- containing fraction
- Prior art date
- Legal status (The legal status 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 status listed.)
- Expired - Lifetime
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)
- C12N9/6456—Plasminogen activators
Landscapes
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Enzymes And Modification Thereof (AREA)
- Separation Using Semi-Permeable Membranes (AREA)
- Peptides Or Proteins (AREA)
Description
【発明の詳細な説明】 イ．利用分野 本発明はウロキナーゼの回収方法及びそれにより得ら
れるウロキナーゼ含有画分に関する。DETAILED DESCRIPTION OF THE INVENTION Field of Application The present invention relates to a method for recovering urokinase and a urokinase-containing fraction obtained thereby.
ロ．従来技術 ウロキナーゼは人尿および腎細胞の組織培養液中の存
在する酵素であり、人血漿中に含まれるプラスミノーゲ
ンを賦活化してフィブリン溶解能を有するプラスミンを
生成する機能がある。この故にウロキナーゼは人尿等か
ら捕集して精製され、各種血栓症や心筋梗塞症等の治療
に広く臨床使用されている。のみならず、最近において
は制癌剤と併用することにより、制癌剤の薬効を向上せ
しめることが知られている。しかも、ウロキナーゼは人
間の尿または腎細胞の組織培養液からの抽出精製物であ
るから、抗原性を有さないため、抗原抗体反応による副
作用がなく、広く賞用されている。B. BACKGROUND ART Urokinase is an enzyme that is present in tissue culture fluid of human urine and kidney cells, and has a function of activating plasminogen contained in human plasma to produce plasmin having fibrinolytic ability. Therefore, urokinase is collected and purified from human urine and the like, and is widely clinically used for the treatment of various thrombosis, myocardial infarction and the like. In addition, recently, it has been known that the combined use of anticancer agents improves the efficacy of the anticancer agents. Moreover, since urokinase is an extracted and purified product from human urine or tissue culture medium of kidney cells, it does not have antigenicity and therefore has no side effect due to antigen-antibody reaction and is widely used.
ところで、ヒト尿などにはウロキナーゼがごく微量に
しか含まれていないので、効率よくウロキナーゼを分
離、濃縮、精製することが困難である。By the way, since human urine contains only a very small amount of urokinase, it is difficult to efficiently separate, concentrate and purify urokinase.
たとえば、従来から繁用されている硅酸塩、硅藻土な
どの鉱物を尿自体に接触させてウロキナーゼを吸着、溶
出する方法、種々のイオン交換クロマトグラフィーによ
って回収する方法においては操作性が繁雑であり、一度
に処理出来る尿も限られているため、微量成分であるウ
ロキナーゼを多量に回収するにはコスト高になるという
問題点がある。For example, in the method of adsorbing and eluting urokinase by contacting conventionally used minerals such as silicate and diatomaceous earth with urine itself, and recovering by various ion exchange chromatography, operability is complicated. However, since the amount of urine that can be treated at one time is limited, there is a problem in that it costs too much to collect a large amount of urokinase, which is a trace component.
ハ．本発明が解決しようとする問題点 通常の限外濾過膜による濃縮回収では、ウロキナーゼ
の一部が膜に取り込まれ、回収率が悪く（40−50％）、
精製は伴わない。C. Problems to be Solved by the Present Invention In concentrated recovery using a normal ultrafiltration membrane, a part of urokinase is incorporated into the membrane and the recovery rate is poor (40-50%).
No purification involved.
すなわち、具体的には以下のような問題が挙げられ
る。That is, the following problems are specifically mentioned.
（１）膜は蛋白（ウロキナーゼ）を吸着する性質があ
り、そのままでは、収量が大幅に低下する。収量をあげ
るには、pHを10〜11に調整したり、吸着阻害剤を添加す
る等の前処理が必要。(1) The membrane has a property of adsorbing a protein (urokinase), and if it is left as it is, the yield is significantly reduced. To increase the yield, pretreatment such as adjusting the pH to 10-11 or adding an adsorption inhibitor is necessary.
（２）濃縮するにしたがって、蛋白の濁りを生じ、次工
程のトラブル原因となる為、遠心又は、ろ過工程をいれ
て、濁りをろ過してやる必要がある。(2) As the protein is concentrated, it becomes turbid and causes troubles in the next step. Therefore, it is necessary to add a centrifugation or filtration step to filter the turbidity.
（３）濁りの多い原料尿使用の時は、水酸化ナトリウム
処理等の前処理をおこない、前もって濁りを除去してや
らねばならない。(3) When using raw urine with a lot of turbidity, pretreatment such as sodium hydroxide treatment must be performed to remove turbidity in advance.
（４）尿から数種類の蛋白を製造する場合、分子量の接
近した蛋白分子の分離（ウロキナーゼとコロニー形成刺
激因子またはカリクレインなど）は、この工程では困難
で、次工程にクロマトを用いることで、始めて可能とな
る。しかし、それは液状のままで蛋白を長時間置くこと
となり、その間の失活等が考えられる。(4) When several kinds of proteins are produced from urine, separation of protein molecules having similar molecular weights (such as urokinase and colony formation stimulating factor or kallikrein) is difficult in this step. It will be possible. However, it means that the protein is left in a liquid state for a long time, and it is considered that the protein is deactivated during that time.
そこで、尿中から効率良くウロキナーゼを回収するた
めの処理方法を検討したところ、アルカリ処理したポリ
アクリロニトリル系限外濾過膜を用いることにより、原
尿からウロキナーゼの濃縮・精製が一度に行いうること
を見出し、本発明を完成した。Therefore, when a treatment method for efficiently collecting urokinase from urine was examined, it was found that the concentration and purification of urokinase from raw urine can be performed at once by using an alkali-treated polyacrylonitrile-based ultrafiltration membrane. Heading, completed the present invention.
ニ．問題点を解決するための手段 本発明の原料である尿（原尿中）には、ウロキナーゼ
は１〜10IU/ml程度含有されている。D. Means for Solving Problems The urine (raw urine) as a raw material of the present invention contains urokinase in an amount of about 1 to 10 IU / ml.
担体となる限外濾過膜はポリアクリロニトリル系で、
分画分子量１万〜５万程度であることが好ましい。以
下、本発明に使用できる商品名の具体例を挙げる。The ultrafiltration membrane used as a carrier is a polyacrylonitrile-based
The molecular weight cutoff is preferably about 10,000 to 50,000. Specific examples of trade names that can be used in the present invention will be given below.
ダイセル化学工業（株）:DUY−Ｈ（基礎分画分子量１
万）、DUY−Ｍ（同２万）、DUY−Ｍ（同４万）、PLS−H
A1090（同１万）、PLS−MA（同２万）、PLS−LA1090
（同４万） ミリポア社:PTGC（同１万）、PTTK（同３万） アミコン社:H1P10（同１万）、H1P30（同３万）、H1P50
（同５万）、H1X50（同５万）、H5P10（同１万）、H5P3
0（同３万）、H5P50（同５万）、H10P10（同１万）、H1
0X50（同５万） ロミコン社:PM10（同１万）、XM50（同５万）などが挙
げられる。Daicel Chemical Industries, Ltd .: DUY-H (basic molecular weight cutoff 1
10,000), DUY-M (20,000 same), DUY-M (40,000 same), PLS-H
A1090 (10,000 same), PLS-MA (20,000 same), PLS-LA1090
(40,000) Millipore: PTGC (10,000), PTTK (30,000) Amicon: H1P10 (10,000), H1P30 (30,000), H1P50
(50,000 same), H1X50 (50,000 same), H5P10 (10,000 same), H5P3
0 (30,000 same), H5P50 (50,000 same), H10P10 (10,000 same), H1
0X50 (50,000 same) Romicon: PM10 (10,000 same), XM50 (50,000 same), etc.
特に、アクリロニトリルとビニルポロリドンの共重合
体からなるポリアクリロニトリル系限外濾過膜を用いる
ことが好ましい。In particular, it is preferable to use a polyacrylonitrile-based ultrafiltration membrane made of a copolymer of acrylonitrile and vinylporolidone.
このものは以下のような化学構造を有する。 This has the following chemical structure.
この限外濾過膜をアルカリで処理する。アルカリとし
ては、0.1〜1N程度の水酸化物（pH10〜13程度）が好ま
しく、より好ましくは水酸化ナトリウムを用いる。 The ultrafiltration membrane is treated with alkali. The alkali is preferably about 0.1 to 1 N hydroxide (pH about 10 to 13), more preferably sodium hydroxide.
その調製方法としては、限外濾過膜をアルカリ溶液で
10〜60分間循環させた後に10〜20時間静置し、次いで冷
精製水で洗浄した後に原尿を循環させる。これを数回く
り返すことが好ましい。The method of preparation is to filter the ultrafiltration membrane with an alkaline solution.
It is circulated for 10 to 60 minutes and then left to stand for 10 to 20 hours, then washed with cold purified water, and then circulated with raw urine. It is preferable to repeat this several times.
こうして調製した限外濾過膜を用いて原尿を処理す
る。Raw urine is treated using the ultrafiltration membrane thus prepared.
原尿をpH6〜８に調整し、限外濾過膜に接触させ、限
外濾過膜により濃縮する。その条件としては、プリーツ
型モジュールで操作圧１〜5kg/cm2程度が好ましい。こ
の時、原尿中のウロキナーゼが吸着する。The raw urine is adjusted to pH 6-8, brought into contact with the ultrafiltration membrane, and concentrated by the ultrafiltration membrane. As the condition, it is preferable that the pleated module has an operating pressure of about 1 to 5 kg / cm 2 . At this time, urokinase in the original urine is adsorbed.
濃縮液はpH6〜８、低イオン強度（例えば、0.01〜0.1
M程度）の条件下で押し出せば、UK以外の尿蛋白（例え
ば、コロニー形成刺激因子、カリクレインなど）を回収
することも可能である。The concentrated solution has a pH of 6 to 8 and low ionic strength (for example, 0.01 to 0.1).
It is also possible to recover urinary proteins other than UK (for example, colony-stimulating factor, kallikrein, etc.) by extruding under conditions of about M).
濃縮液（未吸着液）を除去した後、所望により当該膜
をpH6〜８、低イオン強度（例えば0.005〜0.05M程度）
の条件下で洗浄した後、pH6〜８、イオン強度0.05〜0.3
M程度、好ましくは0.2〜0.3M程度の条件下で、膜に吸着
していたウロキナーゼを前記した濃縮時の操作圧と同程
度で押し出しながら、または落差圧で透過側へ（すなわ
ち、膜を透過して）溶出させて、エンドトキシンおよび
菌を除去されたウロキナーゼ含有画分を得る。After removing the concentrated liquid (unadsorbed liquid), if necessary, the membrane is pH 6-8, low ionic strength (for example, 0.005-0.05M)
After washing under the conditions of pH6 ~ 8, ionic strength 0.05 ~ 0.3
Under conditions of about M, preferably about 0.2 to 0.3 M, while pushing out the urokinase adsorbed to the membrane at the same level as the operating pressure at the time of concentration described above, or to the permeate side by the drop pressure (that is, permeation through the membrane Elution) to obtain a urokinase-containing fraction free from endotoxin and fungi.
かくして精製された溶出液であるエンドトキシンおよ
び菌を除去されたウロキナーゼ含有画分は常法により脱
塩、濃縮、高度精製の処理を行い、医薬品として使用で
きるウロキナーゼとすることができる。The thus-purified eluate, the endotoxin- and bacterium-free fraction containing urokinase, can be desalted, concentrated, and highly purified by a conventional method to give urokinase usable as a pharmaceutical.
ホ．効果 本発明の方法により （１）ウロキナーゼ吸着のための尿処理は不要 （２）限外濾過膜濃縮液を押し出すだけで他の尿蛋白と
分離が簡単にできる （３）ウロキナーゼの濃縮と精製が一度にできる （４）限外濾過膜濃縮液は少量となっているので、精製
し、他の蛋白取得等多目的に使える （５）濁り、エンドトキシンおよび菌を除去できる （６）結果としてウロキナーゼの収量、比活性は向上
し、製造時間は短縮できる。E. Effect According to the method of the present invention, (1) no urine treatment is required for urokinase adsorption (2) it can be easily separated from other urinary proteins simply by pushing out the ultrafiltration membrane concentrate (3) the concentration and purification of urokinase It can be done at one time (4) The ultrafiltration membrane concentrate is small enough to be used for multiple purposes such as purification, etc. (5) Turbidity, endotoxin and fungi can be removed (6) As a result, urokinase yield The specific activity is improved and the manufacturing time can be shortened.
などの効果がある。And so on.
従って、簡単な操作によってウロキナーゼを微量しか
含有せず、かつ多量に不純物を含む尿から高能率、高純
度、低コストでエンドトキシンおよび菌を除去されたウ
ロキナーゼ含有画分を精製することができる。Therefore, it is possible to purify a urokinase-containing fraction in which endotoxin and fungi have been removed from urine containing only a trace amount of urokinase and containing a large amount of impurities by a simple operation, with high efficiency, high purity, and low cost.
ヘ．実験例・実施例 本発明をより詳細に説明するために実験例および実施
例を挙げるが、本発明はこれらによって何ら限定される
ものではない。F. Experimental Examples / Examples Experimental examples and examples are given to describe the present invention in more detail, but the present invention is not limited thereto.
実験例 本発明法と従来法により回収したウロキナーゼ画分中
の生菌数およびエンドトキシン含有の有無を比較した
（第１表）。Experimental Example The viable cell count and the presence / absence of endotoxin in the urokinase fractions collected by the method of the present invention and the conventional method were compared (Table 1).
本発明法は、実施例に準じて行った。 The method of the present invention was carried out according to the examples.
従来法は、実施例において、膜に吸着したウロキナー
ゼを押し出し操作を行うことなく（即ち、膜を透過させ
ずに）濃縮側へ溶出させた。In the conventional method, in Example, urokinase adsorbed on the membrane was eluted to the concentration side without performing an extrusion operation (that is, without permeating the membrane).
生菌数は、寒天培地（ニッスイ製又は栄研製）を使用
し、JIS規格の方法により、エンドトキシンはリムラス
反応により測定した。The viable cell count was determined by using the agar medium (made by Nissui or Eiken) and measuring the endotoxin by the Limulus reaction according to the JIS standard method.
実施例１ 原尿をpH6.0に調整し、参考例により調製したプリー
ツ型ポリアクリロニトリル系限外濾過膜（分画分子量２
万、ダイセル（株）製）を用いて濃縮した後、0.01Mリ
ン酸緩衝液（pH7.0）で押し出すことにより濃縮液を除
去した。膜を0.01Mリン酸緩衝液（pH7.0）で洗浄した後
に0.25M硫酸アンモニウムを用いて落差圧でウロキナー
ゼを透過側へ溶出した。第２表に示す値を得た。 Example 1 The urine was adjusted to pH 6.0, and the pleated polyacrylonitrile-based ultrafiltration membrane prepared according to the reference example (fraction molecular weight 2
After being concentrated using Daicel Co., Ltd., the concentrate was removed by extruding with 0.01M phosphate buffer (pH 7.0). After washing the membrane with 0.01 M phosphate buffer (pH 7.0), urokinase was eluted to the permeate side with 0.25 M ammonium sulfate at a head pressure. The values shown in Table 2 were obtained.
参考例 プリーツ型ポリアクリロニトリル系限外ろ過膜（分画
分子量２万、ダイセル（株）製）に0.5N水酸化ナトリウ
ムを30分間循環させた後に10時間静置し、冷精製水で洗
浄し、原尿を循環する。これを５回くり返した。 Reference Example 0.5N sodium hydroxide was circulated for 30 minutes in a pleated polyacrylonitrile-based ultrafiltration membrane (fraction molecular weight 20,000, manufactured by Daicel Corp.), then allowed to stand for 10 hours, washed with cold purified water, Circulate raw urine. This was repeated 5 times.
Claims (3)
限外濾過膜に原尿を接触させ、限外濾過による濃縮を行
うことにより尿中のウロキナーゼを膜に吸着させた後に
濃縮液（未吸着液）を除去し、次に膜に吸着したウロキ
ナーゼを膜を透過して溶出させることを特徴とするウロ
キナーゼの回収方法。1. An alkaline-treated polyacrylonitrile-based ultrafiltration membrane is brought into contact with raw urine and concentrated by ultrafiltration to adsorb urokinase in urine onto the membrane, and then the concentrated solution (unadsorbed solution) is removed. A method for recovering urokinase, which comprises removing and then eluting the urokinase adsorbed on the membrane through the membrane.
限外濾過膜に原尿を接触させ、限外濾過による濃縮を行
うことにより尿中のウロキナーゼを膜に吸着させた後に
濃縮液（未吸着液）を除去し、次に膜に吸着したウロキ
ナーゼを膜を透過して溶出させることにより得られるウ
ロキナーゼ含有画分。2. A polyacrylonitrile-based ultrafiltration membrane that has been treated with alkali is brought into contact with raw urine and concentrated by ultrafiltration to adsorb urokinase in urine onto the membrane, and then the concentrated solution (unadsorbed solution) is removed. A urokinase-containing fraction obtained by removing and then eluting the urokinase adsorbed on the membrane through the membrane.
求の範囲第２項記載のウロキナーゼ含有画分。3. The urokinase-containing fraction according to claim 2, from which endotoxin and fungi have been removed.
Priority Applications (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP62297831A JPH084503B2 (en) | 1987-11-27 | 1987-11-27 | Method for recovering urokinase and urokinase-containing fraction obtained thereby |
| KR1019880005182A KR890008315A (en) | 1987-11-27 | 1988-05-04 | Collection method of urokinase |
| CN88103076A CN1033279A (en) | 1987-11-27 | 1988-05-23 | The recovery method of urokinase |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP62297831A JPH084503B2 (en) | 1987-11-27 | 1987-11-27 | Method for recovering urokinase and urokinase-containing fraction obtained thereby |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH01141593A JPH01141593A (en) | 1989-06-02 |
| JPH084503B2 true JPH084503B2 (en) | 1996-01-24 |
Family
ID=17851717
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP62297831A Expired - Lifetime JPH084503B2 (en) | 1987-11-27 | 1987-11-27 | Method for recovering urokinase and urokinase-containing fraction obtained thereby |
Country Status (3)
| Country | Link |
|---|---|
| JP (1) | JPH084503B2 (en) |
| KR (1) | KR890008315A (en) |
| CN (1) | CN1033279A (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104666342A (en) * | 2015-01-12 | 2015-06-03 | 精秐生物科技（上海）有限公司 | Adsorbent capable of reducing in vivo urotoxin |
| CN107557271B (en) * | 2017-10-13 | 2018-10-16 | 义乌市绿美生物科技有限公司 | A kind of recycle device of medicinal urokinase raw material |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5929197B2 (en) * | 1977-01-12 | 1984-07-18 | 三菱レイヨン株式会社 | Protein collection method |
| JPS63102679A (en) * | 1986-10-17 | 1988-05-07 | Green Cross Corp:The | Recovery of urokinase |
-
1987
- 1987-11-27 JP JP62297831A patent/JPH084503B2/en not_active Expired - Lifetime
-
1988
- 1988-05-04 KR KR1019880005182A patent/KR890008315A/en not_active Application Discontinuation
- 1988-05-23 CN CN88103076A patent/CN1033279A/en active Pending
Also Published As
| Publication number | Publication date |
|---|---|
| JPH01141593A (en) | 1989-06-02 |
| KR890008315A (en) | 1989-07-10 |
| CN1033279A (en) | 1989-06-07 |
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