Patent US4612283A successfully refreshed from Google Patents.
US4612283A
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Method for purification of HBs antigen
Assignee: Chemo Sero Therapeutic Research Institute Kaketsuken
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Filing: 1984-10-01
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Granted: 1986-09-16
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Expires: 2004-10-01
Abstract
Abstract A method for purification of HBs antigen, which comprises treating an HBs antigen-containing material with an acid, optionally subjecting to salting out with ammonium sulfate, and subjecting the material to a chromatography with hydroxyapatite, by which a highly purified HBs antigen suitable for the preparation of HBV vaccine or diagnostic agent can be obtained in an industrial scale. This method is particularly useful for the purification of HBs antigen-containing material produced by recombinants obtained by DNA recombination technique.
Inventors
- 👤 Keishin Sugahara
- 👤 Chikateru Nozaki
- 👤 Fukusaburo Hamada
- 👤 Fumio Miake
- 👤 Nobuya Ohtomo
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-31 12:24
CPC Classifications
C; C07; C07K; C07K14/00; C07K14/005; C12; C12N; C12N15/00; C12N15/09; C12N15/11; A; A61; A61K; A61K39/00; C12N2730/00; C12N2730/00011; C12N2730/10011; C12N2730/10111; C12N2730/10122
Claims Analysis
Total Claims: 16
Claim 1
Independent Claim
44 words
A method for purification of HBs antigen, which comprises treating an HBs antigen-containing material with an acid to adjust the pH value to a range of 5 to 6, removing the resulting precipitates, and subjecting the HBs antigen-containing solution to a chromatography with hydroxyapatite.
Claim 2
Independent Claim
44 words
A method for purification of HBs antigen, which comprises treating an HBs antigen-containing material with an acid to adjust the pH value to a range of 5 to 6, removing the resulting precipitates, and subjecting the HBs antigen-containing solution to a chromatography with hydroxyapatite.
Claim 3
Dependent Claim
22 words
The method according to claim 1, wherein the HBs antigen-containing solution after acid treatment is subjected to salting out with ammonium sulfate.
Claim 4
Dependent Claim
34 words
The method according to claim 1, wherein the starting HBs antigen-containing material is a material produced by a recombinant being capable of producing HBs antigen which is obtained by a conventional DNA recombination technique.
Claim 5
Dependent Claim
22 words
The method according to claim 3, wherein the recombinant is a member selected from a recombinant Escherichia coli and a recombinant yeast.
Claim 6
Dependent Claim
39 words
The method according to claim 1, wherein the staring HBs antigen-containing material is a crude extract of HBs antigen which is obtained by subjecting recombinant cells to ultrasonic fracture, glass beads fracture, Manton-Gaulin fracture or treatment with a detergent.
Claim 7
Independent Claim
29 words
The method according to clam 1, wherein the acid is a member selected from the group consisting of hydrochloric acid, sulfuric acid, phosphoric acid, acetic acid and oxalic acid.
Claim 8
Independent Claim
29 words
The method according to clam 1, wherein the acid is a member selected from the group consisting of hydrochloric acid, sulfuric acid, phosphoric acid, acetic acid and oxalic acid.
Claim 9
Dependent Claim
31 words
The method according to claim 1, wherein the acid treatment is carried out at a pH range of 5 to 6 and at a temperature of 4° C. to 20° C.
Claim 10
Dependent Claim
22 words
The method according to claim 1, wherein the chromatography with hydroxyapatite is carried out by a column method or a batch method.
Claim 11
Dependent Claim
74 words
The method according to claim 8, wherein the chromatography with hydroxyapatite is carried out by a column method which comprises equilibrating a column packed with hydroxyapatite with a buffer having a pH range of 6 to 8, passing through the equilibrated column the HBs antigen-containing solution, washing the column with the same buffer as used for equilibration, and eluting the adsorbed HBs antigen with a buffer having a pH range of 6 to 8.
Claim 12
Dependent Claim
65 words
The method according to claim 9, wherein the elution is carried out stepwise by first passing through a buffer having a pH range of 6 to 8 and a concentration of 0.1M to 0.2M to remove the contaminants and secondly passing through a buffer having a pH range of 6 to 8 and a concentration of 0.5M to 1.0M to elute out the HBs antigen.
Claim 13
Dependent Claim
45 words
The method according to claim 9, wherein the elution is carried out with a concentration gradient of an eluent using a buffer selected from a phosphate buffer having a concentration gradient of 0.1M→0.5M and a sodium chloride-added phosphate buffer having a concentration gradient of 0.1M→1.0M.
Claim 14
Dependent Claim
70 words
The method according to claim 8, wherein the chromatography with hydroxyapatite is carried out by a batch method which comprises equilibrating hydroxyapatite with a buffer having a pH range of 6 to 8 and a concentration of 0.1M to 0.2M, mixing the equilibrated hydroxyapatite with the HBs antigen-containing material, washing the hydroxyapatite with the same buffer as used for equilibration, and then eluting the HBs antigen adsorbed onto the hydroxyapatite.
Claim 15
Dependent Claim
26 words
The method according to claim 12, wherein the elution of HBs antigen is carried out with a buffer having a pH range of 6 to 8.
Claim 16
Dependent Claim
48 words
The method according to claim 12, wherein the elution of HBs antigen is carried out with a concentration gradient of an eluent using a buffer selected from a phosphate buffer having a concentration gradient of 0.1M→0.5M and a sodium chloride-added phosphate buffer having a concentration gradient of 0.1M→1.0M.
References Cited (Backward Citations)
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1989-09-15 | 1990-03-17 | 1990-09-16 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1993-09-15 | 1994-03-17 | 1994-09-16 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1997-09-15 | 1998-03-17 | 1998-09-16 | $7700.00 | Window Closed / Lapsed |