US4960702A
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US
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Methods for recovery of tissue plasminogen activator
Assignee: Bayer Healthcare Pharmaceuticals Inc
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Filing: 1988-03-11
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Granted: 1990-10-02
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Expires: 2008-03-11
Abstract
Abstract Methods for recovering t-PA from a liquid medium are disclosed. The methods comprise contacting a liquid medium with at least one substrate capable of effecting a separation of intact t-PA from degraded t-PA thereafter recovering the intact t-PA free from other unrelated protein. The present invention also provides compounds produced by this method, compounds comprising intact one-chain t-PA and pharmaceutical compositions containing them and methods for using such compositions.
Inventors
- 👤 Craig Rice
- 👤 Michael J. Morser
- 👤 Charles Glaser
- 👤 Peter A. Donner
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-28 08:00
CPC Classifications
C; C12; C12N; C12N9/00; C12N9/14; C12N9/48; C12N9/50; C12N9/64; C12N9/6421; C12N9/6424; C12N9/6456; C12N9/6459; C12Y; C12Y304/00; C12Y304/21; C12Y304/21069; Y; Y10; Y10S; Y10S435/00; Y10S435/814; Y10S435/815
Claims Analysis
Total Claims: 51
Claim 1
Independent Claim
92 words
A method for recovering intact tissue plasminogen activator (t-PA) form a liquid medium containing said intact t-PA and at least one of degraded t-PA and unrelated proteins comprising the steps of: (a) contacting said liquid medium with a metal chelate adsorbent substrate selected from divalent cation chelates; (b) subjecting said metal chelate adsorbent substrate to a first solution which selectively dissociates therefrom degraded t-PA but not said intact t-PA; and (c) subjecting said metal chelate adsorbent of step b to at least one second solution which selectively dissociates therefrom the intact t-PA.
Claim 2
Independent Claim
92 words
A method for recovering intact tissue plasminogen activator (t-PA) form a liquid medium containing said intact t-PA and at least one of degraded t-PA and unrelated proteins comprising the steps of: (a) contacting said liquid medium with a metal chelate adsorbent substrate selected from divalent cation chelates; (b) subjecting said metal chelate adsorbent substrate to a first solution which selectively dissociates therefrom degraded t-PA but not said intact t-PA; and (c) subjecting said metal chelate adsorbent of step b to at least one second solution which selectively dissociates therefrom the intact t-PA.
Claim 3
Dependent Claim
14 words
A method according to claim 1 wherein the second solution comprises a chaotropic agent.
Claim 4
Dependent Claim
21 words
The method of claim 2 wherein the choatropic agent is urea at a molar concentration of between about 1.0 and 3.0.
Claim 5
Dependent Claim
21 words
The method of claim 1 wherein the liquid medium comprises intact t-PA at a concentration of about 0.01 to 15.0 mg/ml.
Claim 6
Dependent Claim
55 words
The method as recited in claim 1 wherein said first solution comprises a low ionic strength solution having a salt concentration in the range of about 25 millimolar to 100 millimolar and said second solution comprises a high ionic strength solution having a salt concentration in the range of about 100 millimolar to 4 molar.
Claim 7
Dependent Claim
13 words
The method as recited in claim 5 wherein said salt is sodium chloride.
Claim 8
Dependent Claim
27 words
The method as recited in claim 6 wherein said first solution comprises about 25 mM sodium chloride and said second solution comprises about 1 molar sodium chloride.
Claim 9
Dependent Claim
34 words
The method as recited in claim 1 wherein said first solution further comprises at least one first disrupting agent capable of selectively disrupting the interaction between said degraded t-PA and said metal chelate adsorbent.
Claim 10
Dependent Claim
34 words
The method as recited in claim 1 wherein said second solution further comprises at least one second disrupting agent capable of selectively disrupting the interaction between said intact t-PA and said metal chelate adsorbent.
Claim 11
Dependent Claim
17 words
The method as recited in claim 8 wherein said first disrupting agent comprises about 25-250 mM imidazole.
Claim 12
Dependent Claim
22 words
The method as recited in claim 8 wherein said first solution comprises about 25 mM sodium chloride and about 100 mM imidazole.
Claim 13
Dependent Claim
26 words
The method as recited in claim 9 wherein said second disrupting agent is selected from the group consisting of imidazole, zinc, ethylenediaminetetraacetic acid and salts thereof.
Claim 14
Dependent Claim
19 words
The method as recited in claim 12 wherein said second disrupting agent comprises about 10-250 mM sodium ethylenediaminetetraacetic acid.
Claim 15
Dependent Claim
24 words
The method as recited in claim 9 wherein said second solution comprises about 1 molar sodium chloride and about 50 mM sodium ethylenediaminetetraacetic acid.
Claim 16
Dependent Claim
24 words
The method as recited in claim 1 wherein said metal chelate adsorbent substrate comprises molecules having a anionic ligand able to chelate metal ions.
Claim 17
Dependent Claim
15 words
The method as recited in claim 15 wherein said anionic ligand is a dicarboxylic acid.
Claim 18
Dependent Claim
12 words
The method as recited in claim 15 wherein said metal is zinc.
Claim 19
Dependent Claim
16 words
The method as recited in claim 1 further comprising concentrating said intact t-PA after step C.
Claim 20
Dependent Claim
19 words
The method of claim 18 wherein said intact t-PA is concentrated in the presence of non-ionic or zwitterionic detergent.
Claim 21
Dependent Claim
22 words
The method of claim 19 wherein said intact t-PA is concentrated by lyophilization in the presence of at least one stabilizing agent.
Claim 22
Dependent Claim
11 words
The method of claim 20 wherein said stabilizing agent is mannitol.
Claim 23
Independent Claim
207 words
A method for recovering intact tissue plasminogen activator (t-PA) from a cell culture medium comprising the steps of: (a) providing a liquid medium selected from the group consisting of serum-free medium, serum-supplemented medium, serum-fraction supplemented medium and albumin-supplemented medium; (b) pretreating said serum-supplemented or serum fraction supplemented medium with a first adsorbent substrate capable of removing substantially all plasminogen-like contaminants present in the serum-supplemented or serum-fractions supplemented medium; (c) adding to said liquid medium a plasmin inhibitor; (d) contacting said liquid medium with a metal chelate adsorbent substrate selected from divalent cation chelates; (e) subjecting said metal chelate adsorbent substrate to a first solution which selectively dissociates therefrom degraded t-PA but not said intact to-PA; (f) subjecting said metal chelate adsorbent of step e to at least one second solution which selectively dissociates therefrom the intact t-PA; (g) contacting the intact t-PA-containing solution eluted in step with A with a substrate comprising an immobilized aminocarboxylic acid; (h) subjecting said immobilized aminocarboxylic acid substrate to at least, one third solution that dissociates from said substrate other containing proteins but not said intact t-PA; and (i) subjecting said immobilized aminocarboxylic acid substrate of step h to at least one fourth solution that dissociates from said substrate said intact t-PA.
Claim 24
Independent Claim
207 words
A method for recovering intact tissue plasminogen activator (t-PA) from a cell culture medium comprising the steps of: (a) providing a liquid medium selected from the group consisting of serum-free medium, serum-supplemented medium, serum-fraction supplemented medium and albumin-supplemented medium; (b) pretreating said serum-supplemented or serum fraction supplemented medium with a first adsorbent substrate capable of removing substantially all plasminogen-like contaminants present in the serum-supplemented or serum-fractions supplemented medium; (c) adding to said liquid medium a plasmin inhibitor; (d) contacting said liquid medium with a metal chelate adsorbent substrate selected from divalent cation chelates; (e) subjecting said metal chelate adsorbent substrate to a first solution which selectively dissociates therefrom degraded t-PA but not said intact to-PA; (f) subjecting said metal chelate adsorbent of step e to at least one second solution which selectively dissociates therefrom the intact t-PA; (g) contacting the intact t-PA-containing solution eluted in step with A with a substrate comprising an immobilized aminocarboxylic acid; (h) subjecting said immobilized aminocarboxylic acid substrate to at least, one third solution that dissociates from said substrate other containing proteins but not said intact t-PA; and (i) subjecting said immobilized aminocarboxylic acid substrate of step h to at least one fourth solution that dissociates from said substrate said intact t-PA.
Claim 25
Dependent Claim
17 words
A method according to claim 22 wherein the second solution comprises about 0.5 to 3.5 M urea.
Claim 26
Dependent Claim
17 words
The method of claim 23 wherein the molar concentration of urea is about 1.0 to 3.0 M.
Claim 27
Dependent Claim
14 words
The method of claim 22 wherein the first adsorbent substrate comprises an aminocarboxylic acid.
Claim 28
Dependent Claim
11 words
The method of claim 25 wherein the aminocarboxylic acid is L-lysine.
Claim 29
Dependent Claim
13 words
The method as recited in claim 22 further comprising concentrating said intact t-PA.
Claim 30
Dependent Claim
19 words
The method of claim 27 wherein said intact t-PA is concentrated in the presence of non-ionic or zwitterionic detergent.
Claim 31
Dependent Claim
22 words
The method of claim 28 wherein said intact t-PA is concentrated by lyophilization in the presence of at least one stabilizing agent.
Claim 32
Dependent Claim
11 words
The method of claim 29 wherein said stabilizing agent is mannitol.
Claim 33
Dependent Claim
21 words
A biologically active compound produced by the method of claim 6 or 22 comprising intact t-PA substantially free from degraded t-PA.
Claim 34
Dependent Claim
17 words
A composition having thrombolytic activity comprising intact t-PA substantially free from degraded t-PA proteins and unrelated proteins.
Claim 35
Dependent Claim
17 words
A composition having thrombolytic activity comprising intact t-PA substantially free from degraded t-PA proteins and unrelated proteins.
Claim 36
Dependent Claim
17 words
A composition of claim 32 comprising intact one-chain t-PA substantially free from two-chain t-PA and degraded t-PA.
Claim 37
Dependent Claim
24 words
A composition of claim 33 wherein the percent one-chain form of t-PA is about or in excess of 90% (w/w) of the total t-PA.
Claim 38
Dependent Claim
20 words
A composition of claim 34 wherein the specific activity is between about 700,000 and approximately 800,000 international units per milligram.
Claim 39
Dependent Claim
13 words
A composition of claim 34 wherein the composition is substantially free of detergent.
Claim 40
Dependent Claim
14 words
A composition of claim 33 wherein the t-PA is substantially in a nonaggregated form.
Claim 41
Dependent Claim
20 words
A composition of claim 37 wherein the nonaggregated form of t-PA is at least about 99% of the total t-PA.
Claim 42
Dependent Claim
20 words
A composition of claim 32 having a sialic acid content less than about 1.0% of the total composition by weight.
Claim 43
Dependent Claim
27 words
A composition of intact one-chain t-PA substantially free of degraded t-PA and having a specific activity approximately at or in excess of 650,000 international units per milligram.
Claim 44
Dependent Claim
27 words
A composition of intact one-chain t-PA substantially free of degraded t-PA and having a specific activity approximately at or in excess of 650,000 international units per milligram.
Claim 45
Dependent Claim
20 words
A composition of claim 40 wherein the percent one-chain t-PA is at least about 95% (w/w) of the total t-PA.
Claim 46
Dependent Claim
16 words
A composition of claim 40 wherein the t-PA is a t-PA free of non-human eukaryote proteins.
Claim 47
Dependent Claim
20 words
A composition of claim 42 having a sialic acid content less than about 0.5% of the total composition by weight.
Claim 48
Dependent Claim
13 words
A composition of claim 40 wherein the t-PA is substantially free of detergent.
Claim 49
Dependent Claim
79 words
A composition comprising t-PA that is characterized by a specific activity above about 700,000 international units per milligram; that is substantially free of detergent; that is composed of one-chain t-PA in excess of about 95% (w/w) of the total t-PA; that is substantially free of non-human proteins and proteins not related to t-PA; that is composed of sialic acid that is less than about 0.5% of the total composition by weight; and that is substantially free of degraded t-PA.
Claim 50
Dependent Claim
79 words
A composition comprising t-PA that is characterized by a specific activity above about 700,000 international units per milligram; that is substantially free of detergent; that is composed of one-chain t-PA in excess of about 95% (w/w) of the total t-PA; that is substantially free of non-human proteins and proteins not related to t-PA; that is composed of sialic acid that is less than about 0.5% of the total composition by weight; and that is substantially free of degraded t-PA.
Claim 51
Dependent Claim
29 words
A method of treating a host in need of thrombolytic therapy comprising administering to said host an effective amount of the composition of any of claim 31 or 32.
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USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1993-10-01 | 1994-04-02 | 1994-10-02 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1997-10-01 | 1998-04-02 | 1998-10-02 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2001-10-01 | 2002-04-02 | 2002-10-02 | $7700.00 | Window Closed / Lapsed |