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• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/06—Lysis of microorganisms
  • C12N1/063—Lysis of microorganisms of yeast
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2730/00—Reverse transcribing DNA viruses
  • C12N2730/00011—Details
  • C12N2730/10011—Hepadnaviridae
  • C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
  • C12N2730/10122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

• the present invention relates in general to methods for extraction of Hepatitis B surface Antigen (HBsAg) and in particular to methods for extracting HBsAg which employ nonionic detergents.
• HBsAg Hepatitis B surface Antigen
• the hepatitis B virus causes a disease now known as hepatitis B, but formerly known as "serum hepatitis". It has been estimated that there are more . than 200,000,000 people who persistently have hepatitis B virus in their blood. Infection with the virus is a major cause of acute liver disease. Carriers of hepatitis B virus have a high risk of contracting cirrhosis and hepatocellular carcinoma.
• the human hepatitis B virus has been identified with the "Dane" particle which is found in the serum of carriers and which is the causative agent of clinical hepatitis B infection.
• the Dane particle is a 42 nanometer membrane structure which includes lipids, DNA, and at least four proteins: hepatitis B surface antigen (HBsAg), hepatitis B core antigen (HBcAg), hepatitis B e antigen (HBeAg), and a DNA polymerase.
• HBsAg hepatitis B surface antigen
• HBcAg hepatitis B core antigen
• HBeAg hepatitis B e antigen
• a DNA polymerase hepatitis B surface antigen
• carriers also have 22 nanometer lipid particles which contain HBsAg but not DNA, HBcAg, HBeAg or DNA polymerase.
• Hepatitis B vaccines currently in use employ 22 nanometer particles obtained from human plasma.
• Human plasma used in the manufacture of hepatitis B vaccine has an antigen concentration of about 400 micrograms per milliliter. Because the total serum concentration is about 60 milligrams per liter, only a 150-fold purification is required.
• plasma-derived hepatitis B vaccines are limited in supply and extreme caution must be exercised to insure that they are free 10 of all harmful contaminating material, including infectious viruses.
• hepatitis B vaccine involves infecting cultured malignant cells, specifically hepatoma cells, with hepatitis B virus.
• Monkey kidney cells transfected with - c recombinant plasmids containing the gene for HBsAg liberate HBsAg by lysis or by secretion.
• Levinson, et al. European Patent Application No. 73,656.
• production of hepatitis B vaccine from monkey kidney fibroblasts shares the drawbacks inherent in the production of hepatitis B vaccine from hepatoma cells.
• the gene coding for HBsAg may be inserted into a bacterial plas id and may be amplified and expressed in several Escherichia coli host organisms.
• Rutter, et al. European Patent Application No. 020,251.
• this technique results in low yields because HBsAg is easily degraded within E. coli and the growth of E. coli is inhibited by HBsAg.
• Miyanohara, et al. European Patent Application No. 105,049.
• certain bacterial cell components for example lipopolysaccharides, are highly toxic to humans and pose purification problems.
• bacteria being prokaryotes (i.e., members -of the group of organisms which lack in nucleus) may provide inefficient translation of the genes of eukaryotes (i.e., members of the group of organisms which possess a nucleus) inasmuch as: bacteria cannot perform certain processes, such as splicing out of introns or proteolytic clevage of precursor proteins; bacteria do not glycosylate, phosphorylate or methylate proteins, all of which are post-translational modifications which may be important for the immunogenicity of proteins; bacteria do not recognize the so-called signal peptide which is important for secretion of gene products in eukaryotes; and codon preference (i.e.
• yeast offers several advantages for the production of eukaryotic gene products: yeast is readily grown in culture in large quantities; the technology of yeast culture on a large scale is well understood; because yeast cells are eukaryotic, they contain processing machinery for glycosylation, phosphorylation and methylation; and yeast cells better tolerate the HBsAg protein.
• yeast-derived particles have an ED 5Q (a measure of the effective dose necessary to elicit an immune response) of 112 ng which is only slightly higher than the ED 5Q of 98 ng for mammalian cell-derived particles.
• Burnette, et al. in "Modern Approaches to Vaccines", Chanock, et al., eds., Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 245-250 (1984).
• Protein micelles are aggregations of protein molecules having hydrophobic portions, which avoid contact with water molecules, and hydrophilic portions, which readily associate with water molecules. In a solution, the hydrophobic portions of the proteins in micelles become oriented toward the center of the micelle while the hydrophilic portions of the proteins are oriented toward the surface of the micelle.
• Micelles formed from HBsAg protein molecules produce consistently higher levels of antibodies than are induced by intact Hepatitis B particles. Skelly, et al., Nature, 290, 51-54 (1981).
• a micellar vaccine prepared from yeast HBsAg micelles has a slightly lower EDJ Q (22 ng) than micelles prepared from mammalian cells (25 ng). Burnette, et al., supra.
• the process of extraction of polypeptides from the host cell is of importance for the preparation of polypeptide vaccines in general, and for the formation of micelles in particular.
• Detergents may be employed for the extraction of membrane proteins. Different types of detergents have different effects upon the nature and activity of protein aggregates formed. Three detergents which may be employed for protein extraction are bile salts, ionic detergents and nonionic detergents.
• Membrane proteins solubilized by bile salts may be precipitated out of salt solutions over a wide range of salt concentrations, a property useful for the separation of protein fractions. Tzagoloff et al., Methods in Enzymology, 22, 219-230 (1971). However, bile salts may dissociate integral membrane protein complexes so that native properties of an extracted protein, such as the state of aggregation, may not be maintained. Helenius et al.. Methods in Enzymology, 56, 734-749 (1979).
• Ionic detergents are very effective solubilizing agents for membrane proteins. Tzagoloff et al., supra. However, ionic detergents nearly always denature proteins at the concentrations and temperatures required for complete solubilization of membranes. Helenius et al., supra.
• Nonionic detergents are effective in dissociating lipids from proteins, but are relatively ineffective at disrupting protein-protein interactions. Helenius et al., supra. Thus, nonionic detergents are especially useful in preserving the structure of protein micelles during the extraction process.
• Triton X-100 ® A nonionic detergent which may be employed to isolate viral membrane proteins is sold under the name Triton X-100 ® , a trademark of Rohm and Haas Co., Philadelphia, Pennsylvania.
• Triton X-100 ® is an example of a group of commercially available, nonionic detergents having polar groups containing p ⁇ lyoxyethylene. Members of this group are almost always heterogenous in composition in that they contain a wide distribution of polyoxyethylene chain lengths. The chain length for a member of the group is commonly specified as an average chain length.
• Triton X-100 ® may be -generically described as a polyethylene glycol p-isooctylphenyl ether having an average polyoxyethylene chain length of 9.6.
• Helenius et al., supra contains lists of polyoxyethylene- containing detergents and information regarding their properties. Helenius et al, supra, is incorporated by reference herein.
• Triton X-100 ® is used at various concentrations for the extraction of membrane proteins: at 0.05%, 0.3% and 2% for Semliki forest virus membrane proteins [Simons et al., J. Mol. Biol., 80,119-133 (1973); at 5% (v/v) for influenza virus envelope protein [Larin et al., J. Hy . Camb. , 69_, 35-46 (1971)]; at 2% with EDTA [Schnaitman, J. Bacteriol. ,108, 553-563 (1971)] or without EDTA [Schnaitman, J. Bacteriol., 108, 545-552 (1971)] for E. coli cytoplasmic membrane proteins and for E.
• Methods for production of recombinant HBsAg polypeptides generally employ 0.1% or 2% concentrations of Triton X-100 ® as in Skelly et al, J. gen. Virol. , 44, 679 (1979) [Rutter et al, European Patent Application No. 62,574; Miyanohara et al., European Patent Application No. 105,149; Valenzuela et al, in "Modern Approaches to Vaccines," Chanock et al. eds., 209-213, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (1984); and Valenzuela et al..
• a nonionic detergent may be used to contact a chloroform-aqueous solution containing a viral suspension in order to obtain heavy subunits of influenza virus. Nevertheless, no suggestion is made that the use of a different concentration of nonionic detergent might result in a high yield of subcellular particles.
• HBsAg may be isolated from serum as a mixture of particle types: small, spherical particles about 20 n in diameter; tubular particles about 20 nm in diameter; and large spherical particles about 42 nm in diameter with a core.
• a method for conversion of these particle types into a uniform spherical particle of 18 to 22 nm in diameter involves heating affinity-puri ied HBsAg for from 5 to 120 minutes at 40 to 80 degrees C. (preferably 60 degrees C.) in an isotonic.
• sodium chloride solution having' a pH ranging from 5 to 9 and prferably being 7.2; and containing 0.05% to 5% (preferably 0.5%) of a surfactant.
• Surfactants useful according to this method include alkalai metal salts of bile acids, alkalai metal salts of lauroyl sarcosinic acid, polyoxyethylene alkyl phenols containing an average of 7-10 molecules of oxyethylene and polyoxyethylenesorbitan monoalkylester containing an average of 20 molecules of oxyethylene capable of delipidation in isotonic sodium chloride solution at about neutral pH.
• HBsAg particles having a molecular weight of about 2,200,000 daltons are collected in an aqueous solution. The surfactant is removed from the aqueous solution of antigen particles by dialysis or gel filtration. Funakoshi, et al., U.S. Patent No. 4,113,712.
• a yeast cell-lysis buffer according to the present invention enhances the recovery of subcellular particles.
• the cell-lysis buffer includes an aqueous solution of a non-ionic detergent at a concentration within the range from about 0.5% to about 1.0% by volume.
• a method according to the present invention provides for the highly efficient extraction of subcellular particles from cells.
• the method involves suspending the cells in a lysis buffer comprising an aqueous solution of a non-ionic detergent at a concentration within the range from about 0.5% to about 1.0% by volume.
• the method also involves maintaining the cells in the lysis buffer at a temperature within a range from about 3°C. to about 8°C.
• the figure illustrates the effect of the concentration of Triton X-100 ® in a cell lysis buffer upon the recovery of HBsAg micellar particles from the lysis of yeast host cells.
• Cells of S_ ⁇ cerevisiae expressing HBsAg according to Bitter, et al., Gene, 32, 263-274 (1984) 25 were suspended in approximately 2 volumes of lysis buffer to obtain a suspension having an ODg Q0 (optical density at 600 nm) of 10.
• the operating chamber of the glass bead mill has a cooling jacket for controlling the temperature of the operating chamber containing the glass beads.
• the amount of HBsAg particles released into solution were measured by a radioimmunoassay as available under the name AusriaTM Assay from Abbott Laboratories, North Chicago, Illinois.
• the amount of protein was also measured by the Biuret method, as an indication of cell breakage.
• Example I the buffer and method are utilized in a test tube scale model.
• Example II the method and buffer are employed in a batch process.
• Example III describes a preferred composition for the buffer and applies the buffer and method to a continuous process.
• the concentration of Triton X-100 ® in the cell-lysis buffer described above was varied between 0% and 2% by volume and processed as described below.
• HBsAg-expressing S ⁇ cerevisiae cells were suspended in 300 ml of a fermentation medium at O.D.10 (pH 4.45) and were centrifuged in a Beckman J6B centrifuge, available from Beckman Instruments, Mountain
• the resulting pellet was resuspended in 30 ml of 10% glycerol, 50mM Tris, ImM EDTA, 150 mM NaCl, pH
• Example II frozen cell pellets from HB ' sAg S_ ⁇ cerevisiae cell paste prepared according to Bitter, et al., supra, were prepared as indicated in Table II.
• the cell pellet was resuspended in 4 volumes of 50 M Tris buffered containing lOmM EDTA, at pH 8.0 and at 4°C by using a mixer.
• the resulting cell suspension was centrifuged in a Beckman J6B Centrifuge,as above, at 5000g (i.e. at 4000 rpm in a JS 4.2 rotor available from Beckman
• Each pellet was resuspended in 1.5 volumes of 1% (v/v) Triton X-100 ® in the cell-lysis buffer as described above.
• the cells were dispersed with a mixer and the pH of the solution was adjusted to 8.0 ⁇ 0.1 with 50% NaOH as needed.
• recovery of 150% of the control values obtained in test tube runs may be obtained by the batchwise method of lysis.
• EXAMPLE III HBsAg cell suspensions were prepared as in Example II with two of five samples of cells being suspended in 1% (v/v) Triton X-100 ® and three of five samples being suspended 0.5% (v/v) Triton X-100 ® .
• a glass bead mill was primed by pumping 2 liters of cell-lysis buffer, described above,through the reaction chamber while chilling the mill 5° ⁇ 3°C. The mill was turned on and cold cell suspension was pumped into the mill. One retention volume of lysate was discarded while the remaining lysate was collected for further processing.
• Run X-100 ® Wet Control Lysate (mg/Kg of % of No. (v/v%) Kg/ml) ⁇ g/OD-L) ( ⁇ g/OD-L) wet cells)
• Control H- a Weight Control Lysate (mg/Kg of % of No. (v/v%) Kg/ml) ⁇ g/OD-L) ( ⁇ g/OD-L) wet cells)
• the currently preferred buffer for cell-lysis is:

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• 1986
  • 1986-08-15 JP JP50454086A patent/JPS63501121A/ja active Pending
  • 1986-08-15 EP EP19860905526 patent/EP0232410A4/de not_active Withdrawn
  • 1986-08-15 WO PCT/US1986/001704 patent/WO1987001128A1/en not_active Application Discontinuation

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