The present invention relates to a process for purifying hepatitis B viral surface antigen containing a preS2 peptide; and, more specifically, to a process for purifying hepatitis B viral surface antigen containing a preS2 peptide from a recombinant yeast cell, which includes a cell disruption step carried out in the presence of a chaotropic salt to obtain a cell homogenate and the step of alkalifying the cell homogenate to enhance the solubilization of the surface antigen.
Hepatitis B is one of the worldwide public health problems and approximately 200 to 300 millions of the world population are said to carry hepatitis B virus(xe2x80x9cHBVxe2x80x9d). The HBV infection frequently progresses into cirrhosis and hepatocellular carcinoma, leading to the possible death of the patient.
Hitherto, no treating agent for hepatitis B has been developed, and, as such, the importance of vaccines has been emphasized.
Blumberg et al. discovered the so-called Australian antigen from the blood of hepatitis B patients in 1955; and Krugman et al. reported, in 1971, an active immunization method using a heat-treated human serum containing HBV, thereby offering the possibility of developing hepatitis B vaccines. Thereafter, the first generation hepatitis B vaccines, which are prepared by separating and purifying a hepatitis B viral surface antigen(HBsAg) from the blood plasma of hepatitis B patients, have been commercialized(M. R. Hilleman et al., Develop. Bio. Standard, 54, 3-12(1983)).
However, the vaccines derived from the blood plasma have the problems that: their purification and inactivation processes are cumbersome and require high costs; supply of human blood is limited; and an inoculated person may be infected with the pathogens from the blood source.
Accordingly, in order to solve the above problems, genetic engineering approaches have been tried in developing hepatitis B vaccines.
For instance, Valenzuela et al. have developed a process for producing HBsAg in yeast(Nature, 293, 347-350(1982)). The recombinant HBsAg(r-HBsAg) consists primarily of S protein(P25) having 226 amino acids, and when purified, it forms surface antigen particles which are almost identical to those of HBsAg separated from blood plasma.
K. H. Heermann et al. have reported that the hepatitis B viral envelope protein contains significant amounts of L-protein(preS1+preS2+S: p39) and M-protein(preS2+S: p31), as well as S-protein(J. Virol., Nov., 396-402 (1984)). The preS1 peptide has been known to play an important role with respect to the onslaught of hepatitis B virus on the liver after it infects human. The preS2 peptide, which consists of 55 amino acids, has been found to help the antibody formation against the surface antigen in animal experiments(D. R. Milich et al., Proc. Natl. Acad. Sci. U.S.A., 82, 8168-8172 (1985)).
Further, it has been known that antibodies formed against the preS2 peptide exhibit defensive activity against viral infection(Y. Ito et al., Proc. Natl. Acad. Sci. U.S.A., 83, 9174-9178(1986)). Therefore, a vaccine containing the preS2 peptide may be useful to a person who cannot form antibodies against a pre-existing surface antigen. The development of such a vaccine is also important for the protection against infection by recently discovered hepatitis B viral variants.
However, since the pres peptide is very sensitive to proteases present in a yeast cell, preparation of a hepatitis B viral surface antigen containing the pres peptide has met with various difficulties. In order to overcome the difficulties, Kobayashi et al. have produced a vaccine which is prepared by genetically modifying the protease-sensitive region between the preS2 and S peptides(J. Bacteriology, 8, 1-22(1988)); and U.S. Pat. No. 4,742,158 discloses a process for producing a vaccine containing the pres peptide, wherein the peptide is protected from protease attack by using a protease inhibitor in the cell disruption step. However, the effect of the genetic modification by Kobayashi et al. on the activity of the preS2 peptide has not been fully characterized, and the latter process is not practical because protease inhibitors are too expensive to be used in a mass purification process. Further, the level of preS2 peptide cannot be maintained beyond a certain amount regardless of the amount of protease inhibitors added when the purification time becomes longer as the purification scale or requirement increases.
European Patent No. 0 337 492 A1 provides a process for purifying HBsAg from the culture of Pichia sp. using potassium thiocyanate. Potassium thiocyanate is used for raising the yield of lipophilic proteins, and the entire process is aimed at the purification of the HBsAg containing the S peptide only. When the HBsAg further contains the preS2 peptide consisting of 55 amino acids in front of the S peptide consisting of 226 amino acids, it has immunological properties similar to those of the surface antigen comprising the S peptide only because the antigenicity and immunogenicity of the S peptide moiety are the same. However, the two surface antigens are inevitably different in their physicochemical properties. In particular, the preS2 peptide is sufficiently hydrophilic to be exposed on the surface of the antigen particle, and, accordingly, its purification requires a special procedure.
Therefore, various processes for purifying the hepatitis B viral surface antigen comprising the preS2 peptide have been developed.
European Patent No. 0 130 178 A1 describes a process for purifying HBsAg comprising the preS2 peptide, which is characterized by separating the surface antigen using two liquid phases which are prepared by adding a suitable amount of dextran and glycol to a yeast extract. However, this process has problems in that: the separated surface antigen is not sufficiently pure; it is not suitable for a large scale purification process because dextran and polyethylene glycol are expensive; and it is not economical due to the fact that an additional procedure is required for the removal of a surfactant used.
U.S. Pat. No. 4,742,158 teaches a process for purifying HBsAg containing the preS2 peptide, which comprises: preparing a yeast extract in the presence of various protease inhibitors and purifying the surface antigen therefrom by a series of column chromatographic separation steps using an affinity column prepared by attaching a human serum albumin polymer to a gel matrix as well as a hydrophobic column eluting with a surfactant. However, this process has such difficiencies as: the protease inhibitors used in the process are very expensive; the affinity column is not suitable for mass purification; and a special procedure is required for the removal of a surfactant used in the hydrophobic column chromatography.
M. Kobayashi et al. have also reported a process for purifying HBsAg comprising the preS2 peptide(J. of Biotechnology, 8, 1-22(1988). However, this process is not suitable for mass purification as it employs an immunoaffinity column which gives a low yield.
Korean Patent No. 065305 presents a process for purifying HBsAg, which comprises a pH precipitation, and silica and anion exchange column chromatography. This process has the disadvantage that it is difficult to maintain the preS2 peptide content at a suitable level because the preS2 peptide is digested by proteases during the initial stage of the process.
Therefore, there still exists a need for an efficient process for purifying HBsAg containing the preS2 peptide.
Accordingly, it is a primary object of the present invention to provide a process for purifying hepatitis B viral surface antigen containing a preS2 peptide from a yeast cell in a sufficiently pure state to be directly incorporated into a vaccine.
In accordance with one aspect of the present invention, there is provided a process for purifying hepatitis B viral surface antigen containing the preS2 peptide, which is expressed in recombinant organism cells, characterized in that the recombinant organism cells are disrupted using a buffer containing a chaotropic salt to obtain a cell homogenate, followed by alkalifying the homogenate to a pH ranging from 11.0 to 13.5 in the presence of a surfactant to enhance the solubilization of the surface antigen.