US3904480A
Expired / Lapsed
US
⏱️ 0.0 years remaining
Enhanced production of plasminogen activator
Assignee: Eli Lilly and Co
•
Filing: 1974-07-03
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Granted: 1975-09-09
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Expires: 1994-07-03
Abstract
Abstract Enhanced plasminogen activator yields are obtained by culturing plasminogen activator producing mammalian tissue cells in an aqueous nutrient culture medium until cells have attained development to confluency or maximum population density; continuing incubation in the presence of an antimitotic agent, e.g., podophylotoxin, colchicine and derivatives thereof, vincristine, vinblastine and derivatives thereof; recovering the activator from the separated aqueous culture medium by adsorption on hydroxyapatite and elution therefrom; and purifying the activator via salt precipitation followed by DEAE cellulose chromatography and gel filtration chromatography. Plasminogen activator is provided in clinically useful amounts for treatment of thromboembolic conditions.
Inventors
- 👤 Robert N Hull
- 👤 Rolf M Huseby
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-02 07:05
CPC Classifications
C; C12; C12N; C12N9/00; C12N9/14; C12N9/48; C12N9/50; C12N9/64; C12N9/6421; C12N9/6424; C12N9/6456; Y; Y10; Y10S; Y10S435/00; Y10S435/814; Y10S435/815
Claims Analysis
Total Claims: 25
Claim 1
Independent Claim
95 words
THE METHOD OF PRODUCING PLASMINOGEN ACTIVATOR WHICH COMPRISES: A. CULTURING PLASMINOGEN ACTIVATOR PRODUCING MAMMALIAN CELLS IN AN AQUEOUS NUTRIENT TISSUE CULTURE MEDIUM CONTAINING ASSIMILABLE SOURCES OF NITROGEN, CARBON AND INORGANIC SALTS, B. FURTHER CULTURING SAID CELLS IN AN AQUEOUS NUTRIENT TISSUE CULTURE MEDIUM CONTAINING BETWEEN 0.1 AND 10 MCG/ML. OF AN ANTIMITOTIC AGENT SELECTED FROM THE GROUP CONSISTING OF PODOPHYLOTOXIN, COLCHICINE, DESACETYLCOLCHICINE, DESACETAMIDOCOLCHICINE, N-DESACETYL-N-METHYLCOLCHICINE, 4-CYANOCOLCHICINE, VINCRISTINE, VINBLASTINE, VINBLASTINE N-METHYLAMIDE AND DESACETYLVINBLASTINE AMIDE UNTIL A SUBSTANTIAL AMOUNT OF PLASMINOGEN ACTIVATOR IS PRESENT IN SAID MEDIUM, AND C. RECOVERING THE PLASMINOGEN ACTIVATOR FROM SAID TISSUE CULTURE MEDIUM.
Claim 2
Independent Claim
95 words
THE METHOD OF PRODUCING PLASMINOGEN ACTIVATOR WHICH COMPRISES: A. CULTURING PLASMINOGEN ACTIVATOR PRODUCING MAMMALIAN CELLS IN AN AQUEOUS NUTRIENT TISSUE CULTURE MEDIUM CONTAINING ASSIMILABLE SOURCES OF NITROGEN, CARBON AND INORGANIC SALTS, B. FURTHER CULTURING SAID CELLS IN AN AQUEOUS NUTRIENT TISSUE CULTURE MEDIUM CONTAINING BETWEEN 0.1 AND 10 MCG/ML. OF AN ANTIMITOTIC AGENT SELECTED FROM THE GROUP CONSISTING OF PODOPHYLOTOXIN, COLCHICINE, DESACETYLCOLCHICINE, DESACETAMIDOCOLCHICINE, N-DESACETYL-N-METHYLCOLCHICINE, 4-CYANOCOLCHICINE, VINCRISTINE, VINBLASTINE, VINBLASTINE N-METHYLAMIDE AND DESACETYLVINBLASTINE AMIDE UNTIL A SUBSTANTIAL AMOUNT OF PLASMINOGEN ACTIVATOR IS PRESENT IN SAID MEDIUM, AND C. RECOVERING THE PLASMINOGEN ACTIVATOR FROM SAID TISSUE CULTURE MEDIUM.
Claim 3
Dependent Claim
10 words
The method of claim 1 wherein the antimitotic is colchicine.
Claim 4
Dependent Claim
10 words
The method of claim 1 wherein the antimitotic is N-desacetyl-N-methylcolchicine.
Claim 5
Dependent Claim
10 words
The method of claim 1 wherein the antimitotic is vinblastine.
Claim 6
Dependent Claim
10 words
The method of claim 1 wherein the antimitotic is vincristine.
Claim 7
Dependent Claim
29 words
The method of claim 1 wherein the mammalian cells are cultured in the presence of an inert solid support to form a culture of confluent cells on said support.
Claim 8
Dependent Claim
21 words
The method of claim 1 wherein the mammalian cells are cultured to maximum population density in a submerged cell suspension culture.
Claim 9
Dependent Claim
22 words
The method of claim 6 in which the mammalian cells are porcine kidney cells PK (15), ATCC No. CCL 33 PK (15).
Claim 10
Dependent Claim
20 words
The method of claim 6 in which the mammalian cells are porcine kidney cells LLC-PK, (Hull), ATCC No. CL 101.
Claim 11
Dependent Claim
20 words
The method of claim 6 in which the mammalian cells are rabbit kidney cells LLC-RK1 (Hull), ATCC No. CCL 106.
Claim 12
Dependent Claim
21 words
The method of claim 6 in which the mammalian cells are rhesus monkey kidney cells LLC-MK2 (Hull), ATCC No. CL 7.
Claim 13
Dependent Claim
20 words
The method of claim 6 in which the mammalian cells are rhesus monkey kidney cells LLC-MK2, ATCC No. CCL 7.1.
Claim 14
Dependent Claim
19 words
The method of claim 6 in which the mammalian cells are porcine kidney cells LLC-PK1A, ATCC No. CL 101.1.
Claim 15
Dependent Claim
11 words
The method of claim 8 in which the antimitotic is colchicine.
Claim 16
Dependent Claim
11 words
The method of claim 9 in which the antimitotic is vincristine.
Claim 17
Dependent Claim
11 words
The method of claim 10 in which the antimitotic is vinblastine.
Claim 18
Dependent Claim
12 words
The method of claim 11 in which the antimitotic is vinblastine N-methylamide.
Claim 19
Dependent Claim
11 words
The method of claim 12 in which the antimitotic is desacetylcolchicine.
Claim 20
Dependent Claim
22 words
The method of claim 7 in which the mammalian cells are porcine kidney cells PK (15), ATCC No. CCL 33 PK (15).
Claim 21
Dependent Claim
19 words
The method of claim 7 in which the mammalian cells are porcine kidney cells LLC-PK1A, ATCC No. CL 101.1.
Claim 22
Dependent Claim
11 words
The method of claim 19 in which the antimitotic is colchicine.
Claim 23
Dependent Claim
11 words
The method of claim 20 in which the antimitotic is colchicine.
Claim 24
Dependent Claim
339 words
The method of recovering plasminogen activator in a purified form from the plasminogen activator containing culture medium of claim 1 which comprises; a. separating insolubles from the aqueous tissue medium containing plasminogen activator; b. diluting the separated medium with distilled water to adjust the specific conductance of said medium to 8 millimhos or less; c. contacting said plasminogen activator containing medium for between 1 and 24 hours with between 8 and 12 g of hydroxyapatite per liter of medium; d. separating the hydroxyapatite containing adsorbed plasminogen activator from said medium; e. washing said hydroxyapatite; f. eluting said plasminogen activator from the washed hydroxyapatite with 0.7 M phosphate buffer at pH 6.8; g. adding to the plasminogen activator containing eluate a water soluble inorganic salt in an amount sufficient to achieve a salt concentration of approximately 20 percent of saturation; h. separating the precipitated impurities from said eluate; i. adding to said eluate an additional amount of said salt to achieve a salt concentration of between approximately 45 and 90 percent of saturation; j. separating the precipitated plasminogen activator from said salt containing eluate; k. dissolving the plasminogen activator in phosphate buffer having a molarity of between approximately 0.001 and 0.005 and dialyzing said buffer solution; l. contacting the dialyzed solution of plasminogen activator with diethylaminoethyl cellulose adsorbent; m. eluting the adsorbed plasminogen activator from said adsorbent with 0.005 molar phosphate buffer; n. adding to the plasminogen activator containing eluate a water soluble inorganic salt in an amount sufficient to achieve a salt concentration of approximately 75 percent of saturation; o. separating the precipitate of plasminogen activator; p. dissolving the plasminogen activator in 0.1 M tris-hydroxymethylaminomethane: 0.1 M potassium chloride buffer, pH 8.0 and dialyzing the buffer against said buffer alone; q. contacting the dialyzed bufFer solution with a modified dextran gel; r. eluting the adsorbed plasminogen activator from said gel with 0.1 M tris-hydroxymethylaminomethane: 0.1 M potassium chloride, pH 8.0 buffer; and s. lyophilizing the plasminogen activator containing eluate to obtain in a purified solid form the plasminogen activator.
Claim 25
Dependent Claim
15 words
The method of claim 23 in which the water soluble inorganic salt is ammonium sulfate.
References Cited (Backward Citations)
No citation records indexed.
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1978-09-08 | 1979-03-10 | 1979-09-09 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1982-09-08 | 1983-03-10 | 1983-09-09 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1986-09-08 | 1987-03-10 | 1987-09-09 | $7700.00 | Window Closed / Lapsed |