US3723251A - Method for extracting urokinase - Google Patents

Method for extracting urokinase Download PDF

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US3723251A
US3723251A US00174204A US3723251DA US3723251A US 3723251 A US3723251 A US 3723251A US 00174204 A US00174204 A US 00174204A US 3723251D A US3723251D A US 3723251DA US 3723251 A US3723251 A US 3723251A
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urokinase
urine
fibers
solution
elution
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US00174204A
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Y Murayama
N Ogawa
N Koji
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Mochida Pharmaceutical Co Ltd
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    • 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
    • C12N9/6462—Plasminogen activators u-Plasminogen activator (3.4.21.73), i.e. urokinase
    • 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)
    • C12Y304/21073—Serine endopeptidases (3.4.21) u-Plasminogen activator (3.4.21.73), i.e. urokinase
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/814—Enzyme separation or purification
    • Y10S435/815—Enzyme separation or purification by sorption

Definitions

  • the present invention relates to a method for extracting a condensed, purified urokinase from human urine on an industrial scale.
  • Urokinase is known to be an enzyme occurring in small quantities in the human urine and found effective for treating various forms of thrombosis, because, when intravenously injected into the human body,
  • urokinase For the extraction of urokinase from human urine, various available media are known, such as: heavy metals, barium sulfate, silicic acid and its salts, and various ion exchangers.
  • the method according to the present invention is characterized by the step of adsorbing the urokinase in human urine on a material never utilized for extraction of urokinase, that is to say, an arcylonitrile synthetic fiber or its fabrics such as Bonnel (trade name of product by Mitsubishi Bonnel), Exlan (trade name of product by Japan Exlan) or Kashimiron (trade name of product by Asahi Kasei).
  • a material never utilized for extraction of urokinase that is to say, an arcylonitrile synthetic fiber or its fabrics
  • Bonnel trade name of product by Mitsubishi Bonnel
  • Exlan trade name of product by Japan Exlan
  • Kashimiron trade name of product by Asahi Kasei
  • the acrylonitrile synthetic fiber to be used in this invention is so cheap and easily accessible that it can be thrown away after use, and it needs no pretreatment before use. If, however, it is desirable to remove minor stains from the fiber, the purpose is simply attained by preliminarily dipping the fiber in water and stirring it therein. When air bubbles entrapped among fibers are to be eliminated before contacting the fibers with urine, a similar treatment will help.
  • Another feature of our new method resides in the fact that manpower for the collection of urine can be saved or even made unnecessary.
  • the enormous manpower required for collection of urine can be replaced by the simple collection of small, light acrylonitrile fibers overlying the urinal or filling a portion of the drain pipe connected to the bottom of the urinal.
  • the invented method will be highly useful for industrial extraction of urokinase.
  • the process according to our new method is roughly as follows: Human urine is brought into contact with acrylonitrile synthetic fibers, so as to adsorb urokinase from the urine into these fibers, which are picked out to be washed with water to remove the residual urine. Elution is then made using an alkaline solution. Since the urine composition is heavily influenced by' the intake of food or water, the movement, time or climate, it is difiicult to define a normal pH value for the urine, but it is supposed to range generally between 4.8 and 7.5 (Hidenobu MAJIMA: Physiology published by Bunkodo, 1968).
  • urokinase in this pH range of 4.8-7.5 urokinase can be adsorbed on acrylonitrile synthetic fibers, but for the sake of a higher yield the pH range of 5.5-6.5 would be preferable.
  • a 4% aqueous solution of ammonia is employed for the elution of adsorbed urokinase, an elution rate of 94% will be attained.
  • the contact between the urine and the acrylonitrile synthetic fibers is rendered more effective by following the above procedure after treating the urine at room temperature with its pH value adjusted to 7.5-9.5 or preferably to 8.5, and thereby removing the resultant sediments.
  • the urokinase-containing solution thus obtained is condensed or refined into a preparation suitable for medical purposes. It is well known that, for the purpose of condensation, the following methods are available: salting-o ut by means of ammonium sulfate, sedimentation by means of an organic solvent, Diafio (trade name of product by U.S. Amicon). For the purpose of refining, various ion exchangers may be utilized, but a high-purity urokinase will be obtained by another contact with acrylonitrile synthetic fibers.
  • Table 1 gives the yields of urokinase from various fibers when 2 g. of fibers was contacted with 3 l. of urine having a pH value of 6.0. The fibers were washed with water and the unokinase was then eluted using a 4% aqueous solution of ammonia.
  • Table 1 demonstrates the superiority of our new method with respect to the yield of urokinase.
  • Table 2 is a comparison between conventional methods and our new method.
  • EXAMPLE 1 A large volume of fresh urine collected from many healthy people was adjusted to pH 8.5 under agitation, using sodium hydroxide. The resulting sediment was filtered away or eliminated by centrifugal separation. Then, under violent agitation, 6 N hydrochloric acid was gradually added for adjustment of pH 5.5. This was immediately followed by the addition of Bonnel at a rate of 70 g. per 100 l. of urine and by violent agitation. The Bonnel" which had thus been brought into contact with urine was gathered in a large funnel and washed with water until the wash water turned colorless and clear. Then by compression the wash water was removed. Next using 2 l.
  • the urokinase adsorbed by the fibers was separated by elution.
  • Ammonium sulfate was added to the eluent under agitation to 60% saturation. After agitation had been continued further for one hour, the precipitate was collected for 30 minutes of centrifugal separation at 20,000 G, dissolved in 500 ml. of water, and then subjected to overnight dialysis, using 100 times as much water.
  • the solution thus obtained contained about 85% of the urokinase in the original urine, its specific activity being about 4,000 units/mg.
  • the solution finally obtained contained about 80% of urokinase in the crude urokinase solution, its specific activity being over 13,000 units/mg.
  • EXAMPLE 3 100 g. of kaolin was added to 100 l. of fresh urine from which sediment had been removed by filtration, and then the mixture was thoroughly stirred. The kaolin was collected by centrifugal separation and washed until the washing turned colorless and clear. Next, the kaolin-adsorbed urokinase was separated by elution using 3 l. of a 4% aqueous solution of ammonia and then subjected to overnight dialysis. The crude urokinase solution thus yielded was adjusted to pH 5.5 using 6 N hydrochloric acid. g. of Exlan (trade name of product of Japan Exlan) textile pieces was then added and well agitated. Thereafter the same process as in Example 1 was carried out. Namely, elution was performed using 1 l. of a 4% aqueous solution of ammonia. Thereafter the process was the same as in Example 1.
  • the solution thus obtained contained about of urokinase in the crude solution, or about 40% of it in the original urine, its specific activity being over 10,000 units/mg.
  • a method for extracting urokinase from a liquid containing said urokinase comprises the steps of bringing a material comprising synthetic acrylonitrile fibers into contact with said liquid until a substantial proportion of said urokinase has been adsorbed by said material, and then separating the adsorbed urokinase from said material by elution.

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

Abstract

ACRYLONITRILE MATERIAL IS USED TO ADSORB UROKINASE FROM A LIQUID, AND SAID UROKINASE IS SUBSEQUENTLY ELUTED FROM SAID MATERIAL.

Description

Patented Mar. 27, 1973 Int. C1. C05,; 7/026 US. Cl. 195-66 B 4 Claims ABSTRACT OF THE DISCLOSURE Acrylonitrile material is used to adsorb urokinase from a liquid, and said urokinase is subsequently eluted from said material.
SUMMARY OF THE INVENTION The present invention relates to a method for extracting a condensed, purified urokinase from human urine on an industrial scale. Urokinase is known to be an enzyme occurring in small quantities in the human urine and found effective for treating various forms of thrombosis, because, when intravenously injected into the human body,
it can dissolve pathologically formed fibrin or fibrinous coagulations clogging the blood vessels.
For the extraction of urokinase from human urine, various available media are known, such as: heavy metals, barium sulfate, silicic acid and its salts, and various ion exchangers.
Having sought for a method more eflective than the known methods, the present inventors discovered one which is more eflicient than any of the known methods and simple to carry out.
The method according to the present invention is characterized by the step of adsorbing the urokinase in human urine on a material never utilized for extraction of urokinase, that is to say, an arcylonitrile synthetic fiber or its fabrics such as Bonnel (trade name of product by Mitsubishi Bonnel), Exlan (trade name of product by Japan Exlan) or Kashimiron (trade name of product by Asahi Kasei). The following isa description of the fiber but it applies also for its fabrics.
The acrylonitrile synthetic fiber to be used in this invention is so cheap and easily accessible that it can be thrown away after use, and it needs no pretreatment before use. If, however, it is desirable to remove minor stains from the fiber, the purpose is simply attained by preliminarily dipping the fiber in water and stirring it therein. When air bubbles entrapped among fibers are to be eliminated before contacting the fibers with urine, a similar treatment will help.
Since the fibers are sufficiently effective in small quantities and the urokinase adsorbed thereon can be easily separated, our new method is economically superior to any conventional method.
Another feature of our new method resides in the fact that manpower for the collection of urine can be saved or even made unnecessary.
Specifically, the enormous manpower required for collection of urine can be replaced by the simple collection of small, light acrylonitrile fibers overlying the urinal or filling a portion of the drain pipe connected to the bottom of the urinal. Thus the invented method will be highly useful for industrial extraction of urokinase.
The process according to our new method is roughly as follows: Human urine is brought into contact with acrylonitrile synthetic fibers, so as to adsorb urokinase from the urine into these fibers, which are picked out to be washed with water to remove the residual urine. Elution is then made using an alkaline solution. Since the urine composition is heavily influenced by' the intake of food or water, the movement, time or climate, it is difiicult to define a normal pH value for the urine, but it is supposed to range generally between 4.8 and 7.5 (Hidenobu MAJIMA: Physiology published by Bunkodo, 1968). In this pH range of 4.8-7.5 urokinase can be adsorbed on acrylonitrile synthetic fibers, but for the sake of a higher yield the pH range of 5.5-6.5 would be preferable. When, for example, a 4% aqueous solution of ammonia is employed for the elution of adsorbed urokinase, an elution rate of 94% will be attained.
The contact between the urine and the acrylonitrile synthetic fibers is rendered more effective by following the above procedure after treating the urine at room temperature with its pH value adjusted to 7.5-9.5 or preferably to 8.5, and thereby removing the resultant sediments.
The urokinase-containing solution thus obtained is condensed or refined into a preparation suitable for medical purposes. It is well known that, for the purpose of condensation, the following methods are available: salting-o ut by means of ammonium sulfate, sedimentation by means of an organic solvent, Diafio (trade name of product by U.S. Amicon). For the purpose of refining, various ion exchangers may be utilized, but a high-purity urokinase will be obtained by another contact with acrylonitrile synthetic fibers.
Next, the selective adsorption of urokinase on acrylonitrile synthetic fibers will be specifically demonstrated. Table 1 gives the yields of urokinase from various fibers when 2 g. of fibers was contacted with 3 l. of urine having a pH value of 6.0. The fibers were washed with water and the unokinase was then eluted using a 4% aqueous solution of ammonia.
TABLE 1.-YIELDS OF UROKINASE Main compo- Yield Fibers sitlou (percent) Absorbent cotton Cellulose 8 Tevrronfl (trade name of product by Vinylchloride 15 Terjm, vinyl chloride synthetic fiber.)
Bonnel Acrylonitrile.... 89
Table 1 demonstrates the superiority of our new method with respect to the yield of urokinase. Table 2 is a comparison between conventional methods and our new method.
TABLE 2.COMPARISON BETWEEN CONVENTIONAL AND INVENTED METHODS It is clear from Table 2 that the invented method gives a higher yield of urokinase than conventional methods. Meanwhile it should be noted that the so-called absorbents hitherto known adsorb not only urokinase but also great masses of impurities, so that the purity of the product of elution is low, and the value of these adsorbents lies simply in the condensation of urokinase out of urine.
It is clear from Tables 1 and 2 that the invented method is an excellent one characterized not only by the possibility of elution in a condensed state but also b" high selectivity in urokinase adsorption.
Units of urokinase have been determined by the fibrin plate method advocated by Ploug et a1 [1. Ploug: Biochem. Biophys. Acta, vol. 24, p. 278 (1957)].
The following is a description of the present invention, with some examples of its execution. This will make the present invention more clearly understandable, but the invention is not limited to the cited examples only.
EXAMPLE 1 A large volume of fresh urine collected from many healthy people was adjusted to pH 8.5 under agitation, using sodium hydroxide. The resulting sediment was filtered away or eliminated by centrifugal separation. Then, under violent agitation, 6 N hydrochloric acid was gradually added for adjustment of pH 5.5. This was immediately followed by the addition of Bonnel at a rate of 70 g. per 100 l. of urine and by violent agitation. The Bonnel" which had thus been brought into contact with urine was gathered in a large funnel and washed with water until the wash water turned colorless and clear. Then by compression the wash water was removed. Next using 2 l. of a 4% aqueous solution of ammonia, the urokinase adsorbed by the fibers was separated by elution. Ammonium sulfate was added to the eluent under agitation to 60% saturation. After agitation had been continued further for one hour, the precipitate was collected for 30 minutes of centrifugal separation at 20,000 G, dissolved in 500 ml. of water, and then subjected to overnight dialysis, using 100 times as much water. The solution thus obtained contained about 85% of the urokinase in the original urine, its specific activity being about 4,000 units/mg.
EXAMPLE 2 Under vigorous agitation, 6 N hydrochloric acid was gradually added to a crude urokinase solution obtained as in Example 1 for adjustment to pH 5.5. Immediately thereafter, Bonnel was added to the crude urokinase solution at a rate of 100 g. per 1 l. of said solution and thoroughly stirred. Subsequent treatment was the same as in Example 1 and elution was carried out using 1 l. of a 4% aqueous solution of ammonia. Thereafter the process was the same as in Example 1.
The solution finally obtained contained about 80% of urokinase in the crude urokinase solution, its specific activity being over 13,000 units/mg.
EXAMPLE 3 100 g. of kaolin was added to 100 l. of fresh urine from which sediment had been removed by filtration, and then the mixture was thoroughly stirred. The kaolin was collected by centrifugal separation and washed until the washing turned colorless and clear. Next, the kaolin-adsorbed urokinase was separated by elution using 3 l. of a 4% aqueous solution of ammonia and then subjected to overnight dialysis. The crude urokinase solution thus yielded was adjusted to pH 5.5 using 6 N hydrochloric acid. g. of Exlan (trade name of product of Japan Exlan) textile pieces was then added and well agitated. Thereafter the same process as in Example 1 was carried out. Namely, elution was performed using 1 l. of a 4% aqueous solution of ammonia. Thereafter the process was the same as in Example 1.
The solution thus obtained contained about of urokinase in the crude solution, or about 40% of it in the original urine, its specific activity being over 10,000 units/mg.
What is claimed is:
1. A method for extracting urokinase from a liquid containing said urokinase, which method comprises the steps of bringing a material comprising synthetic acrylonitrile fibers into contact with said liquid until a substantial proportion of said urokinase has been adsorbed by said material, and then separating the adsorbed urokinase from said material by elution.
2. The method claimed in claim 1 in which said urokinase is eluted with an alkaline solution.
3. The method claimed in claim 1 in which the pH value of said liquid is adjusted to from 5.5 to 6.5 before said urokinase is adsorbed.
4. The method claimed in claim 1 in which said fibers are in fabric form.
References Cited UNITED STATES PATENTS 3,542,646 11/1970 Aokietal -66B 3,650,903 3/1972 Sloane 195-66 B LIONEL M. SHAPIRO, Primary Examiner
US00174204A 1970-09-04 1971-08-23 Method for extracting urokinase Expired - Lifetime US3723251A (en)

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CA (1) CA952457A (en)
DE (1) DE2143816C3 (en)
DK (1) DK128568B (en)
FR (1) FR2105243B1 (en)
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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4010074A (en) * 1974-01-22 1977-03-01 The Green Cross Corporation Method for purification and recovery of urokinase
US4025390A (en) * 1975-04-18 1977-05-24 Hitachi Chemical Company, Ltd. Method for recovering urokinase from a urokinase-containing solution
US4028187A (en) * 1975-07-04 1977-06-07 Asahi Kasei Kogyo Kabushiki Kaisha Method of obtaining urokinase
USRE29980E (en) * 1975-07-04 1979-05-01 Asahi Kasei Kogyo Kabushiki Kaisha Method of obtaining urokinase
US4169764A (en) * 1975-08-13 1979-10-02 Ajinomoto Co., Inc. Process for production of urokinase
WO1981001417A1 (en) * 1979-11-13 1981-05-28 S Husain Isolation of plasminogen activators useful as therapeutic and diagnostic agents
USRE32271E (en) * 1979-11-13 1986-10-28 Isolation of plasminogen activators useful as therapeutic and diagnostic agents
US5300490A (en) * 1988-12-27 1994-04-05 Mochida Pharmaceutical Co., Ltd. Anticoagulant substance obtained from urine
CN105238772B (en) * 2015-11-16 2016-07-13 江苏尤里卡生物科技有限公司 A kind of urokinase isolation and purification method

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS5821691A (en) * 1981-07-29 1983-02-08 Mochida Pharmaceut Co Ltd Purifying method of interferon

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4010074A (en) * 1974-01-22 1977-03-01 The Green Cross Corporation Method for purification and recovery of urokinase
US4025390A (en) * 1975-04-18 1977-05-24 Hitachi Chemical Company, Ltd. Method for recovering urokinase from a urokinase-containing solution
US4028187A (en) * 1975-07-04 1977-06-07 Asahi Kasei Kogyo Kabushiki Kaisha Method of obtaining urokinase
USRE29980E (en) * 1975-07-04 1979-05-01 Asahi Kasei Kogyo Kabushiki Kaisha Method of obtaining urokinase
US4169764A (en) * 1975-08-13 1979-10-02 Ajinomoto Co., Inc. Process for production of urokinase
WO1981001417A1 (en) * 1979-11-13 1981-05-28 S Husain Isolation of plasminogen activators useful as therapeutic and diagnostic agents
USRE32271E (en) * 1979-11-13 1986-10-28 Isolation of plasminogen activators useful as therapeutic and diagnostic agents
US5300490A (en) * 1988-12-27 1994-04-05 Mochida Pharmaceutical Co., Ltd. Anticoagulant substance obtained from urine
CN105238772B (en) * 2015-11-16 2016-07-13 江苏尤里卡生物科技有限公司 A kind of urokinase isolation and purification method

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SE372538B (en) 1974-12-23
CA952457A (en) 1974-08-06
BR7105833D0 (en) 1973-05-10
NL164091B (en) 1980-06-16
DE2143816B2 (en) 1973-03-08
NL164091C (en) 1980-11-17
JPS4810232B1 (en) 1973-04-02
FR2105243A1 (en) 1972-04-28
DE2143816C3 (en) 1973-10-04
FR2105243B1 (en) 1975-04-18
DE2143816A1 (en) 1972-03-09
GB1305510A (en) 1973-02-07
NL7112068A (en) 1972-03-07
DK128568B (en) 1974-05-27

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