US3140984A - Production of high activity fibrinolytic agents - Google Patents

Production of high activity fibrinolytic agents Download PDF

Info

Publication number
US3140984A
US3140984A US156143A US15614361A US3140984A US 3140984 A US3140984 A US 3140984A US 156143 A US156143 A US 156143A US 15614361 A US15614361 A US 15614361A US 3140984 A US3140984 A US 3140984A
Authority
US
United States
Prior art keywords
fibrinolytic
blood pressure
pressure lowering
lowering agents
cross
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
Application number
US156143A
Inventor
Anthony L Tosoni
David A J Ives
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of Alberta
University of Toronto
Astra USA Inc
Original Assignee
University of Alberta
Astra Pharmaceutical Products Inc
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 University of Alberta, Astra Pharmaceutical Products Inc filed Critical University of Alberta
Priority to US156143A priority Critical patent/US3140984A/en
Priority to GB44626/62A priority patent/GB1022026A/en
Priority to DK515262AA priority patent/DK104292C/en
Application granted granted Critical
Publication of US3140984A publication Critical patent/US3140984A/en
Anticipated expiration legal-status Critical
Expired - Lifetime legal-status Critical Current

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)
    • C12N9/58—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from fungi
    • C12N9/62—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from fungi from Aspergillus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/06—Fungi, e.g. yeasts
    • A61K36/062—Ascomycota
    • 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

  • fibrinolytic material produced up to the present has been found to have the above-referred-to beneficial lytic action on fibrin clots, it has also been found to contain blood pressure lowering agents and it will be appreciated the value of such material would be materially enhanced if such blood pressure lowering agents were removed.
  • Another important object is to provide not only a fibrinolytic material free from such blood pressure reducing material but a material having high fibrinolytic activity.
  • Another important object is to enable the removal of such blood pressure lowering agents with a relatively simple procedure so that such enhanced fibrinolytic material may be produced in substantial quantities.
  • the principal feature of the invention resides in filtering the fibrinolytic material through a cross-linked dextran known in the trade as Sephadex G50, which has a cross linked dextran having a water regain value of 5.7 as defined in the Journal Acta histochemica Bd. 11, 1961, page 306.
  • the starting fibrinolytic material employed is a relatively crude preparation of a substance produced by growing strains of the mould Aspergillus oryzae, strain B1273, for example, by the deep culture techniques as set out in said co-pending application Serial No. 156,142.
  • This strain B1273 of Aspergillus oryzae is available from the Quartermaster Research and Development Center, Dept. of Army, Natick, Mass. (see Proc. Soc. Exper. Biol. & Med. 99, 505, 1958 and 102, 203, 1959).
  • the starting material may be a more purified material such as, for example, the purified fibrinolytic materials disclosed in said co-pending application Serial No. 156,144.
  • such fibrinolytic material in either the crude or more purified form is filtered through a column of cross-linked dextran described un- 3,140,984 Patented July 14, 1964 Ice der the trademark Sephadex G50.
  • the filter is then developed with an eluting agent and the eluate collected in fractions and the fractions of high fibrinolytic activity may then be pooled and dried in vacuo from the frozen state to produce fibrinolytic product which not only has a high potency but also is substantially free from blood pressure lowering agents.
  • the filtering of the starting fibrinolytic material through the cross-linked dextran, Sephadex G50 is carried out at low temperatures, for example, at temperatures slightly above the freezing point of water to provide increased yields of the desired blood-pressure-lowering-agent-free fibrinolytic material.
  • Example I 1.0 gram of fibrinolytic material having an activity of approximately 5000 units per mg. (as determined by a fibrin plate method of assay as is well understood in the art) (see proceedings of the Society for Experimental Biology and Medicine, volume 102, 1959, pages 201 to 203) but also having the characteristic of reducing the blood pressure when administered was dissolved in 15 ml. of water and the acidity adjusted to pH 4.8 with hydrochloric acid. This solution was filtered through crosslinked dextran (medium Sephadex G50) in a column approximately 2" in diameter and 22" in height and having a temperature slightly above the freezing point of water, about 3 C. to 6 C.
  • crosslinked dextran medium Sephadex G50
  • the filter was developed with an eluting agent consisting of distilled water adjusted to pH 4.8 by the careful addition of hydrochloric acid.
  • the eluate was collected in 15-ml fractions.
  • the fractions with high activity (fractions 33 to 41) were pooled and dried in vacuo from the frozen state.
  • the yield was mg. of material having a potency of approximately 12,000 units per mg. This product was tested and found to be substantially completely free of any blood pressure lowering agents.
  • the eluting agent was adjusted to pH 4.8, in similar examples the pH varied from about ph 3.5 to about pH 5.5 and similar yields obtained.
  • Example II Although the material obtained from Example I has remarkably high activity in addition to being free from blood pressure lowering agents and is suitable for clinical use, its activity may be further increased as follows according to these examples.
  • Example I A quantity of the material of Example I having a potency of 12,000 units per mg. was dialysed against running tap water yielding a fibrinolytic product containing about 18,000 units per mg.
  • Example III 50 mg. of a relatively cruder fibrinolytic material having a potency of between 3,000 and 4,000 units per mg. were dissolved in 10 ml. of 8 molar urea solution and the solution filtered through a column of cross-linked dextran, Sephadex G50, as in Example I. Water was then run through the column and approximately thirty 3 ml. fractions collected. Four of the fractions (fractions 13-16 inclusive) containing most of the activity were freeze-dried to give 11 mg. of a product having fibrinolytic activity of approximately 8,000 units per mg. Again, the use of the cross-linked dextran, Sephadex G50, was found to not only materially increase the potency of the fibrinolytic material but also to remove the blood pressure lowering agents present in the starting material.
  • the filtering the material through the cross-linked dextran is best accomplished by using the column in a refrigerator as it appears increased yields are obtained when work is carried out at low temperature, that is, a temperature slightly above the freezing point of water.
  • a process for purifying fibrinolytic material produced from Aspergillus oryzae B127 3 and removing blood pressure lowering agents therefrom consisting in filtering an aqueous solution of said fibrinolytic material through cross-linked dextran known as Sephadex G50, developing the filter with an eluting agent and collecting the eluate which contains the desired resulting fibrinolytic material.

Abstract

Fibrinolytic material produced from Aspergillus Oryzal B1273 has blood pressure lowering agents removed therefrom by filtering the material through a cross-linked dextran having a water regain value of substantially 5 grams per gram and particle size 50-270 mesh dry basis, U.S. Standard Sieves. The filter may be developed with an eluting agent such as hydrochloride acid of pH 3.5 to 5.5 and the fractions of high fibrinolytic activity pooled and dried in vacuo from the frozen state.ALSO:Fibrinolytic material produced from Aspergillus oryz B1273 has blood pressure lowering agents removed therefrom by filtering the material through a cross-linked dextran having a water regain value of substantially 5 grams per gram and particle size 50-270 mesh dry basis, U.S. standard sieves. The filter may be developed with an eluting agent such as hydrochloric acid of pH 3.5 to 5.5 and the fractions of high fibrinolytic activity pooled and dried in vacuo from the frozen state.

Description

United States Patent M 3,140,984 PRODUCTION OF HIGH ACTIVITY FIBRINO- LYTIC AGENTS Anthony L. Tosoni, Toronto, Ontario, and David A. J. Ives, Weston, Ontario, Canada, assignors of one-half to The Governors of the University of Toronto and onehalf to Astra Pharmaceutical Products, Inc., Worcester, Mass., a corporation of New York No Drawing. Filed Nov. 30, 1961, Ser. No. 156,143 6 Claims. (Cl. 19566) This invention relates to the production of high activity fibrinolytic agents of microbiological origin substantially free of blood pressure lowering agents and to the fibrinolytic agents so produced.
It is now known that certain micro-organisms are the source of a product which has a lytic action on fibrin clots of human origin and that for example Aspergillus oryzae is a source of such product. The growing of certain strains of Aspergillus oryzae by deep culture techniques to provide an important source of such fibrinolytic product forms the subject matter of one co-pending application Serial No. 156,142, now abandoned. Another co-pending application Serial No. 156,144, now abandoned, relates to the separation of fibrinolytic material of microbiological origin from the medium in which it is produced and to the purification of such separated fibrinolytic material.
While such fibrinolytic material produced up to the present has been found to have the above-referred-to beneficial lytic action on fibrin clots, it has also been found to contain blood pressure lowering agents and it will be appreciated the value of such material would be materially enhanced if such blood pressure lowering agents were removed.
It is therefore the object of this invention to enable the production of fibrinolytic material substantially free from blood pressure lowering agents. Further, it is the object to enable the removal of such pressure lowering agents to be effected from fibrinolytic material whether of relative crude form or of highly purified form.
Another important object is to provide not only a fibrinolytic material free from such blood pressure reducing material but a material having high fibrinolytic activity.
Another important object is to enable the removal of such blood pressure lowering agents with a relatively simple procedure so that such enhanced fibrinolytic material may be produced in substantial quantities.
The principal feature of the invention resides in filtering the fibrinolytic material through a cross-linked dextran known in the trade as Sephadex G50, which has a cross linked dextran having a water regain value of 5.7 as defined in the Journal Acta histochemica Bd. 11, 1961, page 306.
According to the invention, the starting fibrinolytic material employed is a relatively crude preparation of a substance produced by growing strains of the mould Aspergillus oryzae, strain B1273, for example, by the deep culture techniques as set out in said co-pending application Serial No. 156,142. This strain B1273 of Aspergillus oryzae is available from the Quartermaster Research and Development Center, Dept. of Army, Natick, Mass. (see Proc. Soc. Exper. Biol. & Med. 99, 505, 1958 and 102, 203, 1959). Alternatively, the starting material may be a more purified material such as, for example, the purified fibrinolytic materials disclosed in said co-pending application Serial No. 156,144.
According to the invention such fibrinolytic material in either the crude or more purified form is filtered through a column of cross-linked dextran described un- 3,140,984 Patented July 14, 1964 Ice der the trademark Sephadex G50. The filter is then developed with an eluting agent and the eluate collected in fractions and the fractions of high fibrinolytic activity may then be pooled and dried in vacuo from the frozen state to produce fibrinolytic product which not only has a high potency but also is substantially free from blood pressure lowering agents.
Preferably, according to the invention, the filtering of the starting fibrinolytic material through the cross-linked dextran, Sephadex G50, is carried out at low temperatures, for example, at temperatures slightly above the freezing point of water to provide increased yields of the desired blood-pressure-lowering-agent-free fibrinolytic material.
The invention will be more fully understood from the following examples:
Example I 1.0 gram of fibrinolytic material having an activity of approximately 5000 units per mg. (as determined by a fibrin plate method of assay as is well understood in the art) (see proceedings of the Society for Experimental Biology and Medicine, volume 102, 1959, pages 201 to 203) but also having the characteristic of reducing the blood pressure when administered was dissolved in 15 ml. of water and the acidity adjusted to pH 4.8 with hydrochloric acid. This solution was filtered through crosslinked dextran (medium Sephadex G50) in a column approximately 2" in diameter and 22" in height and having a temperature slightly above the freezing point of water, about 3 C. to 6 C. The filter was developed with an eluting agent consisting of distilled water adjusted to pH 4.8 by the careful addition of hydrochloric acid. The eluate was collected in 15-ml fractions. The fractions with high activity (fractions 33 to 41) were pooled and dried in vacuo from the frozen state. The yield was mg. of material having a potency of approximately 12,000 units per mg. This product was tested and found to be substantially completely free of any blood pressure lowering agents.
While in the specific example, the eluting agent was adjusted to pH 4.8, in similar examples the pH varied from about ph 3.5 to about pH 5.5 and similar yields obtained.
Example II Although the material obtained from Example I has remarkably high activity in addition to being free from blood pressure lowering agents and is suitable for clinical use, its activity may be further increased as follows according to these examples.
A quantity of the material of Example I having a potency of 12,000 units per mg. was dialysed against running tap water yielding a fibrinolytic product containing about 18,000 units per mg.
Example III 50 mg. of a relatively cruder fibrinolytic material having a potency of between 3,000 and 4,000 units per mg. were dissolved in 10 ml. of 8 molar urea solution and the solution filtered through a column of cross-linked dextran, Sephadex G50, as in Example I. Water was then run through the column and approximately thirty 3 ml. fractions collected. Four of the fractions (fractions 13-16 inclusive) containing most of the activity were freeze-dried to give 11 mg. of a product having fibrinolytic activity of approximately 8,000 units per mg. Again, the use of the cross-linked dextran, Sephadex G50, was found to not only materially increase the potency of the fibrinolytic material but also to remove the blood pressure lowering agents present in the starting material.
It is to be pointed out that the filtering the material through the cross-linked dextran is best accomplished by using the column in a refrigerator as it appears increased yields are obtained when work is carried out at low temperature, that is, a temperature slightly above the freezing point of water.
The process of using cross-linked dextran, Sephadex G50, in accordance with the invention is thus shown to be a convenient and useful method for preparing a product having high fibrinolytic activity and a product from which all blood pressure lowering agents have been substantially completely eliminated. It has been found that for efiicient operation of these columns on a repetitive basis such as repeating Examples I and III, it is useful to wash the columns between successive uses with dilute acid, such for example, as 0.1 N hydrochloric acid where hydrochloric acid was used as the eluate. Subsequently, this acid may be removed with the eluant which is used for eluting the fibrinolytic material.
While the above examples are typical illustrations of the invention, it is not intended the invention be limited thereto and variations in such examples, as will be apparent to those skilled in the art, may be made without departing from the spirit of the invention or scope of the appended claims.
What we claim is:
1. A process for purifying fibrinolytic material produced from Aspergillus oryzae B127 3 and removing blood pressure lowering agents therefrom consisting in filtering an aqueous solution of said fibrinolytic material through cross-linked dextran known as Sephadex G50, developing the filter with an eluting agent and collecting the eluate which contains the desired resulting fibrinolytic material.
2. A process as claimed in claim 1 in which the solution is filtered through the cross-linked dextran in a column.
3. A process as claimed in claim 1 in which the solution is filtered through the cross-linked dextran while maintaining the temperature thereof at about from 3 C. to 6 C.
4. A process as claimed in claim 1 in which the eluting agent consists of distilled water adjusted to have a pH of about 3.5 to about 5.5.
5. A process as claimed in claim 1 in which said collected eluate is dried in vacuo from the frozen state.
6. A process as claimed in claim 5 in which the product resulting from said drying is dialysed.
References Cited in the file of this patent Proceedings of the Society for Experimental Biology and Medicine, volume 99 (1958), pages 504-507; volume 102 (1959), pages 201-203.
Porath et al.: Nature 191, 69-70, July 1961.
Flodin et al.: Nature 188, 493-494, November 5, 1960.

Claims (1)

1. A PROCESS FOR PURIFYING FIBRINOLYTIC MATERIAL PRODUCED FROM ASPERGILLUS ORYZAE B1273 AND REMOVING BLOOD PRESSURE LOWERING AGENTS THEREFROM CONSISTING IN FILTERING AN AQUEOUS SOLUTION OF SAID FIBRINOLYTIC MATERIAL THROUGH CROSS-LINKED DEXTRAN KNOWN AS SEPHADEX G50 DEVELOPING THE FILTER WITH AN ELUTING AGENT AND COLLECTING THE ELUATE WHEN CONTAINS THE DESIRED RESULTING FIBRINOLYTIC MATERIAL.
US156143A 1961-11-30 1961-11-30 Production of high activity fibrinolytic agents Expired - Lifetime US3140984A (en)

Priority Applications (3)

Application Number Priority Date Filing Date Title
US156143A US3140984A (en) 1961-11-30 1961-11-30 Production of high activity fibrinolytic agents
GB44626/62A GB1022026A (en) 1961-11-30 1962-11-26 Production of high activity fibrinolytic agents
DK515262AA DK104292C (en) 1961-11-30 1962-11-29 Process for purifying a fibrinolytic material of microbiological origin.

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
US156143A US3140984A (en) 1961-11-30 1961-11-30 Production of high activity fibrinolytic agents

Publications (1)

Publication Number Publication Date
US3140984A true US3140984A (en) 1964-07-14

Family

ID=22558289

Family Applications (1)

Application Number Title Priority Date Filing Date
US156143A Expired - Lifetime US3140984A (en) 1961-11-30 1961-11-30 Production of high activity fibrinolytic agents

Country Status (3)

Country Link
US (1) US3140984A (en)
DK (1) DK104292C (en)
GB (1) GB1022026A (en)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3256158A (en) * 1963-03-22 1966-06-14 Abbott Lab Purification of urokinase
US3281331A (en) * 1961-11-08 1966-10-25 Astra Ab Process of isolating and separating proteolytic enzymes
US3660239A (en) * 1968-11-22 1972-05-02 Bayer Ag Fibrinolysokinases from streptomyces
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
US11503674B2 (en) 2014-10-09 2022-11-15 Nvent Services Gmbh Voltage-leveling heater cable

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
None *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3281331A (en) * 1961-11-08 1966-10-25 Astra Ab Process of isolating and separating proteolytic enzymes
US3256158A (en) * 1963-03-22 1966-06-14 Abbott Lab Purification of urokinase
US3660239A (en) * 1968-11-22 1972-05-02 Bayer Ag Fibrinolysokinases from streptomyces
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
US11503674B2 (en) 2014-10-09 2022-11-15 Nvent Services Gmbh Voltage-leveling heater cable

Also Published As

Publication number Publication date
DK104292C (en) 1966-05-02
GB1022026A (en) 1966-03-09

Similar Documents

Publication Publication Date Title
EP0208215B1 (en) Purification of blood clotting factors and other blood proteins on non-carbohydrate sulfated matrices
US2461922A (en) Antibiotic extraction from solids of actinomyces griseus culture
US3743722A (en) Anti-coagulant isolation
Lindner et al. Gel filtration as a method for purification of protein-bound peptides exemplified by oxytocin and vasopressin
US3140984A (en) Production of high activity fibrinolytic agents
US3879369A (en) Anti-coagulant isolation from malayan pit viper using affinity chromatography
US3256158A (en) Purification of urokinase
US4698301A (en) Angiogenic factor production
US3657416A (en) Thrombin-like defibrinating enzyme from the venom ancistrodon rhodostoma
US3905870A (en) Purification of kallikrein
JPS645576B2 (en)
KR850001285A (en) Manufacturing method of natural human interleukin-2
US3634196A (en) Process for the separation of pyrogens from crude preparations of l-asparaginase
US4495096A (en) Process for producing and obtaining anaphylatoxin-and cocytotaxin-containing leucotaxine preparations and of anaphylatoxin and cocytotaxin proteins in molecularly homogeneous, biologically active form
US3042586A (en) Purification of streptokinase
Shifrine et al. On the cell wall composition of Saccharomycopsis guttulata
US3660237A (en) Process for obtaining kallekrein from pancreas or submandibularis glands of pigs
US3660238A (en) Extraction of asparaginase from bacterial culture
US3994782A (en) Methods for extracting and purifying kallidinogenase
US2957809A (en) Insulinase
RU2186848C1 (en) Method of isolation of superoxide dismutase
JPH0361440B2 (en)
SU1421348A1 (en) Method of producing trombolytic ferment from fungus trichotecium roseum strain d
US2594314A (en) Process for preparing an antipernicious anemia concentrate
Griffiths et al. The separation and purification of VI and O antigens of S. typhi using a column precipitation technique