US4382027A
Expired / Lapsed
US
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Purification of human immune interferon
Assignee: Rorer Biotechnology Inc
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Filing: 1981-08-18
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Granted: 1983-05-03
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Expires: 2001-08-18
Abstract
Abstract Disclosed is a process for the purification of crude immune interferon to a near homogeneous preparation which comprises: (a) adsorbing the crude interferon onto a column containing Controlled Pore Glass beads and eluting with ammonium sulfate, (b) adsorbing the interferon containing eluant onto a column containing either Concanavalin A-Sepharose, lentil lectin-Sepharose or pea lectin-agarose and eluting with a buffer containing a sugar, (c) adsorbing the interferon containing eluant onto a column containing Heparin-Sepharose or Procian Red-agarose and eluting with a high salt content buffer, and (d) treating the interferon containing eluant in a gel-filtration column equilibrated in high salt to obtain a solution of immune interferon that is nearly homogeneous.
Inventors
- 👤 Irwin A. Braude
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-23 11:32
CPC Classifications
C; C07; C07K; C07K14/00; C07K14/435; C07K14/52; C07K14/555; C07K14/57; A; A61; A61K; A61K38/00; Y; Y10; Y10S; Y10S530/00; Y10S530/827; Y10S530/829
Claims Analysis
Total Claims: 20
Claim 1
Independent Claim
402 words
A process for purifying crude human immune interferon which comprises: (a) contacting a solution of crude interferon with Controlled Pore Glass beads for a period of time sufficient to accomplish equilibrated adsorption of the crude interferon to the beads in the presence of a neutral pH buffer, washing the glass beads having interferon adsorbed thereon with a chemically compatable buffer to remove the unbound contaminant until the optical density of the eluate at 280 nm is about 0, washing the glass beads having interferon adsorbed thereon with a neutral pH buffer solution until the optical density at 280 nm is about 0, eluting the adsorbed immune interferon from the glass beads with an ammonium sulfate solution; (b) contacting the solution of interferon with an absorbent selected from the group consisting of Concanavalin A-Sepharose, lentil lectin-Sepharose and pea lectin-agarose for the period of time sufficient to accomplish equilibrated adsorption of the interferon to the absorbent in the presence of a neutral pH buffer, washing the absorbent having interferon adsorbed thereon from the soluble fraction with ammonium sulfate until the optical density of the eluate at 280 nm is about 0, washing the absorbent with a neutral pH buffer until the optical density of the eluate at 280 nm is about 0, eluting the adsorbed interferon with a buffer containing a member of the group consisting of alpha-methyl D-mannoside and 1-methyl D-glucoside; (c) contacting the interferon with a member selected from the group consisting of Heparin-Sepharose or Procian Red-agarose for a period of time sufficient to accomplish equilibrated adsorption of the interferon to the member equilibrated in a neutral pH buffered solution, washing the member containing adsorbed interferon thereon with first a neutral pH buffered solution containing a sugar selected from the group consisting of alpha-methyl D-mannoside and 1-methyl D-glucoside to remove the unbound material until the optical density of the eluate at 280 nm is about 0, washing with a neutral pH buffered solution until the optical density is about 0 to remove the bound material eluting the adsorbed interferon with a second neutral pH buffered solution containing a highly concentrated salt, and; (d) further purifying by subjecting the elution fraction to gel-filtration treatment equilibrated in a neutral pH buffered solution containing a highly concentrated salt, and eluting whereby the solutes within the elution fractions are separated according to their molecular weights; and collecting the solute fraction containing a near homogeneous interferon preparation.
Claim 2
Dependent Claim
402 words
A process for purifying crude human immune interferon which comprises: (a) contacting a solution of crude interferon with Controlled Pore Glass beads for a period of time sufficient to accomplish equilibrated adsorption of the crude interferon to the beads in the presence of a neutral pH buffer, washing the glass beads having interferon adsorbed thereon with a chemically compatable buffer to remove the unbound contaminant until the optical density of the eluate at 280 nm is about 0, washing the glass beads having interferon adsorbed thereon with a neutral pH buffer solution until the optical density at 280 nm is about 0, eluting the adsorbed immune interferon from the glass beads with an ammonium sulfate solution; (b) contacting the solution of interferon with an absorbent selected from the group consisting of Concanavalin A-Sepharose, lentil lectin-Sepharose and pea lectin-agarose for the period of time sufficient to accomplish equilibrated adsorption of the interferon to the absorbent in the presence of a neutral pH buffer, washing the absorbent having interferon adsorbed thereon from the soluble fraction with ammonium sulfate until the optical density of the eluate at 280 nm is about 0, washing the absorbent with a neutral pH buffer until the optical density of the eluate at 280 nm is about 0, eluting the adsorbed interferon with a buffer containing a member of the group consisting of alpha-methyl D-mannoside and 1-methyl D-glucoside; (c) contacting the interferon with a member selected from the group consisting of Heparin-Sepharose or Procian Red-agarose for a period of time sufficient to accomplish equilibrated adsorption of the interferon to the member equilibrated in a neutral pH buffered solution, washing the member containing adsorbed interferon thereon with first a neutral pH buffered solution containing a sugar selected from the group consisting of alpha-methyl D-mannoside and 1-methyl D-glucoside to remove the unbound material until the optical density of the eluate at 280 nm is about 0, washing with a neutral pH buffered solution until the optical density is about 0 to remove the bound material eluting the adsorbed interferon with a second neutral pH buffered solution containing a highly concentrated salt, and; (d) further purifying by subjecting the elution fraction to gel-filtration treatment equilibrated in a neutral pH buffered solution containing a highly concentrated salt, and eluting whereby the solutes within the elution fractions are separated according to their molecular weights; and collecting the solute fraction containing a near homogeneous interferon preparation.
Claim 3
Dependent Claim
13 words
The process according to claim 1, wherein the steps are carried out continuously.
Claim 4
Dependent Claim
13 words
The process according to claim 1, wherein the steps are carried out sequentially.
Claim 5
Dependent Claim
16 words
The process according to claim 1, wherein step (b) is carried out prior to step (a).
Claim 6
Dependent Claim
24 words
The process according to claim 1, wherein the sequence of steps is from step (c) to step (b) to step (a) to step (d).
Claim 7
Dependent Claim
50 words
The process according to claim 1, wherein the crude interferon is produced from cell cultures containing unlysed red blood cells and added thereto is sufficient tris (hydroxymethyl) amino methane to bring the concentration to 500 mM, the solution is then buffered to pH 9.5 to 9.7 prior to beginning purification.
Claim 8
Dependent Claim
21 words
The process according to claim 1, wherein the Controlled Pore Glass beads have a mesh size designated as 120/200 or 200/400.
Claim 9
Dependent Claim
23 words
The process according to claim 7, wherein the crude interferon solution is passed through a chromatographic column packed with Controlled Pore Glass beads.
Claim 10
Dependent Claim
96 words
The process according to claim 1, wherein the adsorbed interferon in step (a) is eluted from the glass beads by first washing the beads with tris (hydroxymethyl) amino methane until the optical density of the eluate at 280 nm is about 0; further treating the beads to sequential passes with a volume of neutral pH buffered solution to wash the beads of contaminants; treating the beads to a final pass volume of ammonium sulfate which selectively dissociates the immune interferon from its bead adsorption site, and collecting the final pass volume comprising the interferon-containing elution fraction.
Claim 11
Dependent Claim
48 words
The process according to claim 1, wherein the chemically compatible buffer is tris (hydroxymethyl) amino methane at a concentration of 500 mM and pH 9.5 and the neutral pH buffer is phosphate buffered saline and the final pass volume is 2 M ammonium sulfate at about pH 9.0.
Claim 12
Dependent Claim
96 words
The process according to claim 1, wherein in step (b) the neutral pH buffer is phosphate buffered saline, the adsorbed interferon eluted by first washing with 2 M ammonium sulfate; further washed with sequential volumes of a neutral pH buffered solution to wash the adsorbent free of contaminants, until the optical density of the eluant at 280 nm is about 0; treating the absorbent to a final pass volume of phosphate buffered saline containing from 0.1 M to 2 M alpha-methyl D-mannoside of 1 methyl D-glycoside, and collecting the final pass volume containing the interferon fraction.
Claim 13
Dependent Claim
18 words
The process according to claim 11, wherein the interferon is passed through a chromatographic column packed with absorbent.
Claim 14
Dependent Claim
60 words
The process according to claim 1, wherein in step (c) said first neutral pH buffered solution is phosphate buffered saline containing sugar selected from the group consisting of alpha-methyl D-mannoside and 1 methyl D-glucoside, and said second neutral pH buffered solution is phosphate buffered saline containing about 2 M sodium chloride, and collecting the final pass volume containing the interferon.
Claim 15
Dependent Claim
46 words
The process according to claim 1, wherein in step (d) said gel-filtration treatment includes equilibrating the column with phosphate buffered saline containing 2 M sodium chloride, and eluting with about 2 M sodium chloride in phosphate buffered saline to achieve separation of interferon from the solutes.
Claim 16
Dependent Claim
42 words
The process according to claim 1, wherein the thus collected interferon is further contacted with phenyl-Sepharose for a period of time sufficient to accomplish equilibrated adsorption of the interferon to the phenyl-Sepharose, and eluting the thus treated interferon with low salt buffers.
Claim 17
Dependent Claim
107 words
A process for purifying crude human immune interferon which comprises, contacting a solution of crude interferon with a resin from the group consisting of Heparin-Sepharose or Procian-Red agarose for a period of time sufficient to accomplish equilibrated adsorption of the interferon to the resin, washing the resin interferon adsorbed thereon with phosphate buffered saline containing sugar selected from the group consisting of alpha-methyl D-mannoside, and 1-methyl D-glucoside until the optical density at 280 nm is about 0, washing with phosphate buffered saline solution until the optical density at 280 nm is about 0, and eluting the adsorbed interferon with phosphate buffered saline containing 2 M sodium chloride.
Claim 18
Dependent Claim
107 words
A process for purifying crude human immune interferon which comprises, contacting a solution of crude interferon with a resin from the group consisting of Heparin-Sepharose or Procian-Red agarose for a period of time sufficient to accomplish equilibrated adsorption of the interferon to the resin, washing the resin interferon adsorbed thereon with phosphate buffered saline containing sugar selected from the group consisting of alpha-methyl D-mannoside, and 1-methyl D-glucoside until the optical density at 280 nm is about 0, washing with phosphate buffered saline solution until the optical density at 280 nm is about 0, and eluting the adsorbed interferon with phosphate buffered saline containing 2 M sodium chloride.
Claim 19
Dependent Claim
66 words
In a process for purifying human immune interferon comprising contacting a solution of crude interferon with Controlled Pore Glass beads for a period of time sufficient to accomplish equilibrated adsorption of the crude interferon to the beads; separating the adsorbed interferon from the glass beads, the improvement comprising; eluting the adsorbed human immune interferon from the Controlled Pore Glass beads with a solution containing ammonium sulfate.
Claim 20
Dependent Claim
66 words
In a process for purifying human immune interferon comprising contacting a solution of crude interferon with Controlled Pore Glass beads for a period of time sufficient to accomplish equilibrated adsorption of the crude interferon to the beads; separating the adsorbed interferon from the glass beads, the improvement comprising; eluting the adsorbed human immune interferon from the Controlled Pore Glass beads with a solution containing ammonium sulfate.
References Cited (Backward Citations)
US4168261A
Cited
US4314935A
Cited
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1986-05-02 | 1986-11-01 | 1987-05-03 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1990-05-02 | 1990-11-01 | 1991-05-03 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1994-05-02 | 1994-11-01 | 1995-05-03 | $7700.00 | Window Closed / Lapsed |