Patent US4569790A successfully refreshed from Google Patents.
US4569790A
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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 11:05
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.
References Cited (Backward Citations)
JPS55148098A
Cited
US4315852A
Cited
US4464355A
Cited
US4450103A
Cited
EP0092163A2
Cited
EP0094317A2
Cited
US4490289A
Cited
US4462940A
Cited
US4512922A
Cited
US4511502A
Cited
US4511503A
Cited
EP0118977A1
Cited
EP0119621A1
Cited
EP0121352A1
Cited
EP0128467A1
Cited
US4464295A
Cited
US4476049A
Cited
EP0147819A2
Cited
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 |