EP0135435A2
Expired / Lapsed
EP
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Immunogenic HBsAg derived from transformed yeast
Assignee: Merck and Co Inc
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Filing: 1984-08-21
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Granted: —
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Expires: 2004-08-21
Abstract
Abstract @ The surface antigen protein of human Hepatitis B virus is synthesized in Saccharomyces cerevisiae as a 23,000-26,000 dalton polypeptide, essentially free of intermolecular disulfide bonds. This antigen is a poor immunogen in animals and man. No prior precedent or method exists for efficiently converting the non-disulfide bonded antigen to a fully intermolecular disulfide bonded particle. We describe the first example of such a conversion in vitro and show that the act of this conversion enhances the immunogenicity of the antigen about 10-fold. The in vitro conversion makes practical the production of hepatitis B surface antigen from microorganisms using recombinant DNA methods.
Inventors
- 👤 Arthur Friedman
- 👤 E. Dale Lehman
- 👤 William J. Mcaleer
- 👤 Ted F. Schaefer
- 👤 Edward M. Scolnick
- 👤 Eugene D. Wampler
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-11-10 02:15
CPC Classifications
C; C07; C07K; C07K14/00; C07K14/005; C12; C12N; C12N15/00; A; A61; A61K; A61K39/00; C12N2730/00; C12N2730/00011; C12N2730/10011; C12N2730/10111; C12N2730/10122
Claims Analysis
Total Claims: 21
Claim 1
Independent Claim
54 words
An immunogenic form of HBsAg having a particle size of from about 16 to about 25nm characterized by the absence of glycosylation, by the presence of at least some intermolecular disulfide bonds, and by migration in a non-reducing denaturing polyacrylamide gel electrophoresis system as a polypeptide having a molecular weight of about 50,000 daltons.
Claim 2
Dependent Claim
54 words
An immunogenic form of HBsAg having a particle size of from about 16 to about 25nm characterized by the absence of glycosylation, by the presence of at least some intermolecular disulfide bonds, and by migration in a non-reducing denaturing polyacrylamide gel electrophoresis system as a polypeptide having a molecular weight of about 50,000 daltons.
Claim 3
Dependent Claim
10 words
A substantially purified form of HBsAg according to Claim 1.
Claim 4
Dependent Claim
73 words
A non-glycosylated immunogenic form of HBsAg particles wherein at least about 90% of the sulfur atoms have formed disulfide bonds at least part of which are intermolecular disulfide bonds and which form fails to migrate in a non-reducing denaturing polyacrylamide gel electrophoresis system as a polypeptide having a molecular weight greater than 100,000 daltons, the HBsAg being unstable at pH 2 and unstable to pepsin, and containing phosphatidylinositol but being free of cholesterol.
Claim 5
Dependent Claim
73 words
A non-glycosylated immunogenic form of HBsAg particles wherein at least about 90% of the sulfur atoms have formed disulfide bonds at least part of which are intermolecular disulfide bonds and which form fails to migrate in a non-reducing denaturing polyacrylamide gel electrophoresis system as a polypeptide having a molecular weight greater than 100,000 daltons, the HBsAg being unstable at pH 2 and unstable to pepsin, and containing phosphatidylinositol but being free of cholesterol.
Claim 6
Dependent Claim
10 words
A substantially purified form of HBsAg according to Claim 3.
Claim 7
Dependent Claim
31 words
An immunogenic form of HBsAg according to Claim 1 which is formed in vitro by subjecting HBsAg expressed by a transformed eukaryotic host to a pH of about 8.5 or above.
Claim 8
Dependent Claim
34 words
An immunogenic form of H B sAg according to Claim 3 which is formed in vitro by subjecting HBsAg expressed by a transformed eukaryotic host to chemical means adapted to form intermolecular disulfide bonds.
Claim 9
Dependent Claim
27 words
An immunogenic form of HBsAg according to Claim 6 wherein the chemical means comprises treatment with thiocyanate, urea, or pH of from about 11 to about 12.5.
Claim 10
Dependent Claim
33 words
A method of preparing an immunogenic form of HBsAg according to Claim 1 which comprises treating HBsAg expressed by a transformed eukaryotic host to a pH of about 8.5 or above in vitro.
Claim 11
Dependent Claim
34 words
A method of preparing an immunogenic form of HBsAg according to Claim 3 which comprised subjecting HBsAg expressed by a transformed eukaryotic host to chemical means adapted to form intermolecular disulfide bonds in vitro.
Claim 12
Dependent Claim
31 words
A method of preparing an immunogenic form of HBsAg according to Claim 9 wherein the chemical means comprises treatment with thiocyanate, urea, or pH of from about 11 to about 12.5.
Claim 13
Dependent Claim
36 words
A butyl agarose gel containing from about 4 to about 13 um/mL of butylamine covalently bonded to the gel, with less than half of the butylamine being bonded to the gel by positively charged covalent bonds.
Claim 14
Dependent Claim
36 words
A butyl agarose gel containing from about 4 to about 13 um/mL of butylamine covalently bonded to the gel, with less than half of the butylamine being bonded to the gel by positively charged covalent bonds.
Claim 15
Dependent Claim
36 words
A butyl agarose gel containing from about 4 to about 13 p m/mL of butylamine covalently bonded to the gel, with more than half of the butylamine being bonded to the gel by uncharged covalent bonds.
Claim 16
Dependent Claim
36 words
A butyl agarose gel containing from about 4 to about 13 p m/mL of butylamine covalently bonded to the gel, with more than half of the butylamine being bonded to the gel by uncharged covalent bonds.
Claim 17
Independent Claim
43 words
A method of removing pyrogenic substances and yeast protein from HBsAg produced in yeast cells which comprises passing the HBsAg across a hydrophobic substrate formed by bonding a phenyl or a hydrocarbon substituent to the substrate by a preponderance of uncharged covalent bonds.
Claim 18
Independent Claim
43 words
A method of removing pyrogenic substances and yeast protein from HBsAg produced in yeast cells which comprises passing the HBsAg across a hydrophobic substrate formed by bonding a phenyl or a hydrocarbon substituent to the substrate by a preponderance of uncharged covalent bonds.
Claim 19
Dependent Claim
19 words
A method according to Claim 13 wherein the substituent is an alkylamine of from 4 to 10 carbon atoms.
Claim 20
Dependent Claim
15 words
A method according to Claim 13 wherein the alkylamine is butylamine, hexylamine, octylamine or decylamine.
Claim 21
Dependent Claim
11 words
A method according to Claim 13 wherein the alkylamine is n-butylamine.
References Cited (Backward Citations)
No citation records indexed.
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