US4569790A
Expired / Lapsed
US
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Process for recovering microbially produced interleukin-2 and purified recombinant interleukin-2 compositions
Assignee: Novartis Vaccines and Diagnostics Inc
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Filing: 1984-03-28
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Granted: 1986-02-11
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Expires: 2004-03-28
Abstract
Abstract A process for recovering microbially produced IL-2 in a highly pure form from the cellular material of the microorganisms that produced it comprising: disrupting the cell membranes of the microorganisms; extracting the disruptate with a chaotropic agent, such as urea, that selectively extracts microbial proteins from the cellular material; solubilizing the IL-2 in the solid phase of the extraction mixture with an aqueous solution of a solubilizing agent, such as SDS, containing a reducing agent; and separating the IL-2 from the resulting solution by an optional extraction with 2-butanol or 2-methyl-2-butanol followed by gel filtration chromatography, oxidizing the IL-2 and purifying the oxidized IL-2 by RP-HPLC.
Inventors
- 👤 Kirston Koths
- 👤 James Thomson
- 👤 Michael Kunitani
- 👤 Kenneth Wilson
- 👤 Wolf Hanisch
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-27 05:19
CPC Classifications
C; C12; C12N; C12N15/00; C07; C07K; C07K1/00; C07K1/107; C07K1/113; C07K1/1133; A; A61; A61P; A61P35/00; C07K14/00; C07K14/435; C07K14/52; C07K14/54; C07K14/55; A61K; A61K38/00
Claims Analysis
Total Claims: 23
Claim 1
Independent Claim
193 words
A process for recovering purified, oxidized human IL-2 from a transformed microorganism containing the IL-2 comprising: (a) disrupting the cell membrane of the microorganism; (b) extracting the disruptate with an aqueous solution of a chaotropic agent that extracts non IL-2 proteins selectively from the cellular material and from the IL-2; (c) solubilizing the IL-2 in the solid phase of the extraction mixture with an aqueous solution of a solubilizing agent that forms a water soluble complex with the IL-2, said solution containing a reducing agent to thereby fully reduce the IL-2 in the complex; (d) separating the reduced IL-2 from the resulting solution in the presence of the reducing agent; (e) oxidizing the reduced IL-2; (f) further purifying the resulting oxidized IL-2 product by gel filtration or reverse-phase high performance liquid chromatography as individual or combined steps; and (g) recovering a purified oxidized recombinant human IL-2 composition having an IL-2 content of at least about 95% as determined by reducing SDS-PAGE analysis, an endotoxin content of less than about 0.1 nanograms/mg of IL-2 and substantially free of pyrogens as determined by the U.S.P. rabbit pyrogen test at a dosage of 3.3×10 5 U/kg.
Claim 2
Dependent Claim
193 words
A process for recovering purified, oxidized human IL-2 from a transformed microorganism containing the IL-2 comprising: (a) disrupting the cell membrane of the microorganism; (b) extracting the disruptate with an aqueous solution of a chaotropic agent that extracts non IL-2 proteins selectively from the cellular material and from the IL-2; (c) solubilizing the IL-2 in the solid phase of the extraction mixture with an aqueous solution of a solubilizing agent that forms a water soluble complex with the IL-2, said solution containing a reducing agent to thereby fully reduce the IL-2 in the complex; (d) separating the reduced IL-2 from the resulting solution in the presence of the reducing agent; (e) oxidizing the reduced IL-2; (f) further purifying the resulting oxidized IL-2 product by gel filtration or reverse-phase high performance liquid chromatography as individual or combined steps; and (g) recovering a purified oxidized recombinant human IL-2 composition having an IL-2 content of at least about 95% as determined by reducing SDS-PAGE analysis, an endotoxin content of less than about 0.1 nanograms/mg of IL-2 and substantially free of pyrogens as determined by the U.S.P. rabbit pyrogen test at a dosage of 3.3×10 5 U/kg.
Claim 3
Dependent Claim
11 words
The process of claim 1 wherein the chaotropic agent is urea.
Claim 4
Dependent Claim
25 words
The process of claim 2 wherein the concentration of urea in the extraction mixture is in the range of about 3.5 M to 4.5 M.
Claim 5
Dependent Claim
15 words
The process of claim 1 wherein step (b) is carried out at a basic pH.
Claim 6
Dependent Claim
18 words
The process of claim 4 wherein the pH is in the range of about 8.1 to about 8.5.
Claim 7
Dependent Claim
17 words
The process of claim 1 wherein the solubilizing agent is sodium dodecyl sulfate or sodium lauryl sarcosine.
Claim 8
Dependent Claim
52 words
The process of claim 1 wherein step (f) is carried out by reverse phase high performance liquid chromatography at a pH in the range of 2.1 to 2.3 using a bonded phase wide pore silica gel and a gradient solvent system comprising an organic acid and an organic solvent for eluting IL-2.
Claim 9
Dependent Claim
17 words
The process of claim 7 wherein the solvent system is acetic acid-propanol, trifluoroacetic acid-propanol, or trifluoroacetic acid-acetonitrile.
Claim 10
Dependent Claim
36 words
The process of claim 1 wherein step (f) is carried out by isolating an IL-2-containing fraction from the solution by gel filtration and purifying the resulting IL-2 from the fraction by reverse-phase high performance liquid chromatography.
Claim 11
Dependent Claim
49 words
The process of claim 9 wherein the reverse phase high performance liquid chromatography is carried out at a pH in the range of 2.1 to 2.3 using a bonded phase wide pore silica gel and a gradient solvent system comprising an organic acid and an organic solvent for IL-2.
Claim 12
Dependent Claim
17 words
The process of claim 10 wherein the solvent system is acetic acid-propanol, trifluoroacetic acid-propanol, or trifluoroacetic acid-acetonitrile.
Claim 13
Dependent Claim
43 words
The process of claim 1 including the following additional steps of: (i) extracting the IL-2 from the aqueous solution of (c) with 2-butanol or 2-methyl-2-butanol; (ii) acid precipitating the IL-2 from the extract; and (iii) purifying the acid precipitated IL-2 by gel filtration.
Claim 14
Dependent Claim
41 words
The process of claim 12 wherein the chaotropic agent is urea, the concentration of urea in the extraction mixture is 3.5 M to 4.5 M, and the extraction of step (f) is carried out at a pH of 5 to 7.5.
Claim 15
Dependent Claim
19 words
The process of claim 12 including purifying the resulting oxidized gel filtered product by reverse-phase high performance liquid chromatography.
Claim 16
Dependent Claim
61 words
Purified recombinant human interleukin-2 (IL-2) composition wherein the IL-2 is unglycosylated and in oxidized form, having an IL-2 content of at least about 95% as determined by reducing SDS-PAGE analysis, an endotoxin content of less than about 0.1 nanogram/mg of IL-2 and substantially free of pyrogens as determined by the U.S.P. rabbit pyrogen test at a dosage of 3.3×10 5 U/kg.
Claim 17
Dependent Claim
61 words
Purified recombinant human interleukin-2 (IL-2) composition wherein the IL-2 is unglycosylated and in oxidized form, having an IL-2 content of at least about 95% as determined by reducing SDS-PAGE analysis, an endotoxin content of less than about 0.1 nanogram/mg of IL-2 and substantially free of pyrogens as determined by the U.S.P. rabbit pyrogen test at a dosage of 3.3×10 5 U/kg.
Claim 18
Dependent Claim
22 words
The purified recombinant IL-2 composition of claim 15, wherein the IL-2 content is greater than about 98% as determined by reducing SDS-PAGE.
Claim 19
Dependent Claim
22 words
The purified recombination human IL-2 composition of claim 15, wherein the IL-2 content is greater than about 98% as determined by RP-HPLC.
Claim 20
Dependent Claim
16 words
The purified recombinant human IL-2 of claim 15, wherein the IL-2 is des-alanyl, serine 125 IL-2.
Claim 21
Dependent Claim
16 words
The purified recombinant human IL-2 of claim 16, wherein the IL-2 is des-alanyl, serine 125 IL-2.
Claim 22
Dependent Claim
16 words
The purified recombinant human IL-2 of claim 17 wherein the IL-2 is des-alanyl, serine 125 IL-2.
Claim 23
Dependent Claim
12 words
The process of claim 7 wherein the solvent system is acetic acid-propanol.
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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) | 1989-02-10 | 1989-08-12 | 1990-02-11 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1993-02-10 | 1993-08-12 | 1994-02-11 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1997-02-10 | 1997-08-12 | 1998-02-11 | $7700.00 | Window Closed / Lapsed |