CA1108051A - Method for purification of hbs antigen - Google Patents
Method for purification of hbs antigenInfo
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- CA1108051A CA1108051A CA312,397A CA312397A CA1108051A CA 1108051 A CA1108051 A CA 1108051A CA 312397 A CA312397 A CA 312397A CA 1108051 A CA1108051 A CA 1108051A
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Abstract
METHOD FOR PURIFICATION OF HBs ANTIGEN
Abstract of the Disclosure The specification discloses an improved method for the purification of HBS antigen comprising passing a partially purified HBs antigen prepared from blood plasma or serum through a column filled with an anion exchanger which may be equilibrated with a buffer solution having a specific ionic strength and pH level, and further passing the resulting effluent through a column filled with a cation exchanger which may be equilibrated with a buffer solution having a specific ionic strength and pH level. The HBs antigen obtained by the present invention is highly pur-ified and is useful for preparing vaccine for preventing Viral Hepatitis Type B.
Abstract of the Disclosure The specification discloses an improved method for the purification of HBS antigen comprising passing a partially purified HBs antigen prepared from blood plasma or serum through a column filled with an anion exchanger which may be equilibrated with a buffer solution having a specific ionic strength and pH level, and further passing the resulting effluent through a column filled with a cation exchanger which may be equilibrated with a buffer solution having a specific ionic strength and pH level. The HBs antigen obtained by the present invention is highly pur-ified and is useful for preparing vaccine for preventing Viral Hepatitis Type B.
Description
~8~
The present invention relates to an improved method for the purification of a surface antigen of heptatitis type B virus which is usually called "Hepatitis B surface antigen" and abbreviated to "HBs antigen".
So-called serum hepatitis or posttransfusion hepatitis is a disease induced by infection caused by a virus having a size of 42 nm and containing a DNA type nucleic acid, which is called "Hepatitis Type B virus" (hereinafter referred to as "HB virus"), and this hepatitis was designated ''V;ral Hepatitis Type B" by the World Health Organization (WHO) in 1973.
Viral hepatitis type B has not yet been adequately studied, but attention has been paid to this disease because it tends to reach chronic state and it has a close relation with chronic hepatitis, liver cirrhosis and hepatoma, and hence, it would be desirable to exterminate this disease. For this purpose, it is recommended that a vaccine prepared from a surface antigen of the HB virus be used.
Antigens of the HB virus include HBs antigen which is a surface antigen of HB virus: hepatitis type B core antigen which is a core antigen of the virus containing a nucleic acid and is usually abbreviated to "HBc antigen";
and an antigen which is still unclear but has a close relation with infectivity of HB virus and is usually abbreviated as "HBe antigen".
The blood of patients and carriers infected with HB
virus contains a large number of spherical particles and tubular structures of HBs antigen (size: 22 nm), which has no nucleic acid and may be a surface component of the virus, as well as particles of HB virus (size: A2 nm,
The present invention relates to an improved method for the purification of a surface antigen of heptatitis type B virus which is usually called "Hepatitis B surface antigen" and abbreviated to "HBs antigen".
So-called serum hepatitis or posttransfusion hepatitis is a disease induced by infection caused by a virus having a size of 42 nm and containing a DNA type nucleic acid, which is called "Hepatitis Type B virus" (hereinafter referred to as "HB virus"), and this hepatitis was designated ''V;ral Hepatitis Type B" by the World Health Organization (WHO) in 1973.
Viral hepatitis type B has not yet been adequately studied, but attention has been paid to this disease because it tends to reach chronic state and it has a close relation with chronic hepatitis, liver cirrhosis and hepatoma, and hence, it would be desirable to exterminate this disease. For this purpose, it is recommended that a vaccine prepared from a surface antigen of the HB virus be used.
Antigens of the HB virus include HBs antigen which is a surface antigen of HB virus: hepatitis type B core antigen which is a core antigen of the virus containing a nucleic acid and is usually abbreviated to "HBc antigen";
and an antigen which is still unclear but has a close relation with infectivity of HB virus and is usually abbreviated as "HBe antigen".
The blood of patients and carriers infected with HB
virus contains a large number of spherical particles and tubular structures of HBs antigen (size: 22 nm), which has no nucleic acid and may be a surface component of the virus, as well as particles of HB virus (size: A2 nm,
- 2 -'5~L
which are called "Dane particles"), and hence, an antibody against the HBs antigen may play a role in neutralizing the virus infectivity.
Thus, HBs antigen is useful as a material for a vaccine for preventing viral hepatitis type B. However, since a tissue culture of HB virus has never been com-pletely achieved, HBs antigen must be prepared Erom the blood of patients and carriers infected with HB virus, and hence, it is necessary to remove all components in such blood plasma other than HBs antigen such as plasma proteins, HBe antigen as well as HB virus.
Purification of HBs antigen has hitherto been done by various methods, such an ultracentrifugation, gel filtra-tion, affinity chromotography, or a combination thereof.
For instance, Gerin et al disclose a method for the purification of HBs antigen by twice repeating an isopycnic centriEugation using cesium chloride by a zonal rotor, subjecting the product to a cushion centriEugation with sucrose and further subjecting the product to an isopycnic centrifugation [cf. Gerin et al, J. Virol. Vol.
7, 569 (1971) and J. Immunol. Vol. 115 (1), 100 11975)1.
Blumberg et al disclose a purification of HBs antigen by a combination of centrifugation, treatment with an enzyme, column gel filtration, and ultracentrifugation under a density gradient with sucrose and cesium chloride (cf.
Japanese Patent Publication No. 19603jl975). Furthermore, Houwen et al disclose a purification of HBs antigen by affinity chromatography which comprises conjugating an anti-HBs antibody to Sepharose 4B gel, treating a plasma, which is positive oE HBs antigen by the resultant gel, and thereby eluting the HBs antigen adsorbed onto the gel.
s~ ~
The eluate fraction of this method still contains blood serum components ~e.g. prealbumin, albumin, transferrin, ~-lipoprotein, ~2-macroglobulin~ y-globulin), and hence, the fraction is futher passed through a Sepharose 4B gel column which is bonded with antihodies to normal human serum in order to remove these blood serum components.
These methods have some advantages but have also many drawbacks. For instance, the purification by ultracentri-ugation requires the density gradient centrifugation using cesium chloride and sucrose to be repeated several times and hence requires a large amount of cesium chloride and sucrose. Besides, the ultra-centrifuging machine should be provided with various rotors in accordance with the degree of purification and the steps therefor. The gel filtration method is poor in capacity and is not suitable for purification on a large scale. Moreover, this gel filtration method is also inferior in the degree of purification, and when the HBs antigen is prepared from blood plasma, there can not be obtained HBs antigen of high purity. The affinity chromatography method requires anti-HBs antibody, and hence, when it is carried out on a large scale, an enormous amount of anti-HBs antibody (human origin or immunized animal origin) must be prepared all the time.
The present inventors have intensively studied the purification of HBs antigen and have found that the desired HBs antigen having a high purity can comparatively simply be obtained by an ion exchange method.
An object of the present invention is to provide an improved method for the purification of HBs antigen.
Another object of the invention is to provide a method for preparing an acceptably pure HBs antigen From blood plasma ., or serum According to the present invention, there is provided a method Eor the purification of HBs antigen, which comprises passing an HBs antigen-containing solution prepared from blood plasma or serum through an anion exchanger and then passing the resulting effluent through a cation exchanger.
That is, a partially purified HBs antigen obtained from blood plasma or serum of patients and carriers infected wi~h HB virus by a conventional method is passed through an anion exchanger, whereby most of the blood plasma components other than HBs antigen and certain plasma proteins comprising mainly Y-globulin are adsorbed, and then the effluent thus passed through the anion exchanger, which contains HBs antigen a~d certain plasma proteins, is further passed through a cation exchanger, whereby the plasma proteins comprising mainly y-globulin are adsorbed and only the desired HBs antigen is contained in the effluent.
The present invention has been accomplished after extensive study of the behavior of the HBs antigen in anion and cation exchangers under various conditions. The puri~ication of HBs antigen is preferably carried out by passing an HBs antigen-containing solutibn through an anion exchanger and a cation exchanger whereby the impurities are adsorbed onto the exchangers~
Various methods have been reported for the purifi-cation of viruses using ion exchangers, for example, the purification of E. coli phage lCreaser and Taussing, Virol, Vol. 4, 20 (1957)1; the purification of tobacco mosaic virus [Cochram et al, Nature, Vol. 180, 1281 (1957)]; the purification of polio virus [Hoyer et al, Science, Vol. l27, 859 (1958)]; and the purification of vaccinia virus [McCrea and O'Loughlin, Virol, Vol. 8, 1~7 11959)]. ~owever, the pu~ification of antigens of viruses such as HBs antigen originating in blood plasma or serum without adsorption onto an ion exchanger has not been accomplished.
The ion exchanger used in the present invention is preferably a repeatedly usable column without specific activation and has a large binding capacity and is pre-erably an autoclavable gel having good stability under various conditions, e.g. at various pH levels, ionic strengths, etc.
Suitable examples of the anion exchanger are agarose gel introduced with an anionic substituent (e.g.
DEAE-Sepharose CL-6B), dextran gel introduced wîth an anionic substituent (e.g. DEAE-Sephadex, QAE-Sephadex~, cellulose introduced with an anionic substituent (e.g.
DEAE-cellulose, TEAE-cellulose), and the like. The anionic substituent includes diethyl-aminoethyl (DEAE), triethylaminoethyl (TEAE) and diethyl-(2-hydroxypropyl)-aminoethyl (QAE).
Suitable examples of the cation exchanger are agarose gel introduced with a cationic substituent (e.g.
CM-Sepharose CL-6B), dextran gel introduced with a cationic substituent (e.g. CM-Sephadex, CP-Sephadex), cellulose introduced with a cationic substituent (e.g.
CM-cellulosel, or the like. The cationic substituent includes carboxymethyl (CM), sulfopropyl (SP) and sulfoethyl (SE).
The partially purified HBs antigen is preferably prepared from blood plasma or serum of patients and carriers infected with HB virus by a conventional method, for instance, by treating the blood plasma with calcium chloride, salting out with ammonium sulfate, centrifuging, and adding ammonium sulfate to the resulting supernatant fluid, and thereby precipitating HBs antigen.
The partially purified HBs antigen thus prepared is applied onto a column filled with an anion exchanger which is previously equilibriated with an apropriate buffer solution having an ionic strength (~) of 0.02 to 0.1, preferably 0.04 to 0.06, and a pH level of 5.0 to 7.0, preferably 5.4 to 5.6, (e.g. acetate buffer, citrate buffer, phosphate buffer, succinate buffer, phthalate buffer, etc.). By this treatment, the HBs antigen and plasma protein comprising mainly Y-globulin pass through the column without being adsorbed threon and other plasma proteins such as albumin, ~- and ~-globulins, etc. are adsorbed thereon.
The HBs antigen-containing ef1uent passed through the above anion exchanger column, which still contains some impurities comprising predominantly y-globulin, is then applied onto a column filled with a cation exchanger, which is also previously equilibriated with an appropriate buf~er solution having an ionic strength (~) of 0.02 to 0~14, preferably 0.06 to 0.09, and a pH level of 4~5 to 6.0, preferably 5.0 to 5.3, (e.g. acetate buffer, citrate buffer, phosphate buffer, succinate buffer, and phthalate buffer, etc.). By this treatment, the HBs antigen passes through the column without being adsorbed thereon and the plasma proteins such as Y-globulin contaminated into the HBs antigen are almost completely removed. Moreover, the HBs antigen obtaned by the present invention does not 8a~s~
contain substantial amounts of Dane particles of the HB
virus and has a high purity.
Thus/ a highly purified HBs antigen can be obtained from blood plasma or serum by slmple procedures which can be carried out on an industrial scale without any difficulty, and the HBs antigen thus obtained is useful or preparing a source of safe and stable vaccine for preventing viral hepatitis type B.
The present invention is illustrated by the following Examples but is not limited thereto, and also by the accompanying drawin~s, in which:
Figure 1 is a graph of optical density versus fraction number for the product of one stage of Example l; and Figure 2 is a similar graph of the product of another stage of Example 1.
Example 1 Step 1: Blood plasma, which was positive in HBe antibodyr was treated with calcium chloride and dextran sulfate. The supernatant fluid was separated and salted out with 1.2 M ammonium sulfate. After centrifugation, 1.8 M ammonium sulfate was added to the supernatant fluid to precipitate HBs antigen. By this salting out, about 80~ by weight of the components of human blood plasma were removed, and the HBs antigen-containing solution was con-centrated to about 1~10 by volume.
Step 2: DEAE-Sepharose CL-6B gel (made by Pharmacia &
Co.) was sufficiently equilibrated with acetate buffer (ionic strength: 0.05, pH: 5.5) and then the DEAE
Sepharose CL-6B gel thus treated was packed into a column. The HBs ant;gen-containing solution obtained in the ahove step 1 was sufficiently dialyzed against the same acetate 15~
buffer as above, and the resulting precipitate was removed by centrifugation, and the HBs antigen-containing solution thus obtained was applied onto the above column fi11ed With DEAE-Sepharose CL-6B gel. The column was eluted ~ith acetate buffer oÇ which the ionic strength was varied stepwise by adding sodium chloride.
Each fraction of tlle effluent and of the eluate with the acetate buEfer, was measured in terms of the content of HBs antigen and other proteins. The content of HBs antigen was estimated by immuno-electrosyneresis (abbre~
viation: IES) and shown by titer [cf. Bussard, A. r B~B.A~, Vol. 34, page 258 (1959)], and the content of proteins was measured by optical density at 280 nm (OD280). The results are shown in the accompanying Figure 1, wherein the HBs antigen is shown by a solid line and other proteins are shown by a dotted line. As is clear from Figure 1, HBs antigen passed through the column o~ an anion exchanger and was contained in the effluent, and proteins other than HBs antigen and y-globulin were adsorbed onto the column and eluted with the eluting acetate buffer (ionic strength (~): 0.15, 0.3 and 0.6).
The proteins contaminated in the effluent ~ionic strength ( ): Q.05) were mainly y-globulin. Moreover, about 80%
by weight of HBs antigen passed through the column, and the degree of purification of HBs antigen was about 14 times in comparison with the starting material.
Step 3: CM-Sepharose CL-6B gel (made by Pharmacia Co.) was sufficiently equilibrated with acetate buffer (ionic strentho 0.08, pH: 5.1) and then was packed into a column. The effluent obtained in the above step 2 was sufficiently dialyzed against the same acetate buffer as _ g _ B~
above and then applied onto the above column filled with CM-Sepharose CL-6B gel. The column was eluted with acetate buffer of which the ionic strength was varied stepwise by adding sodium chloride.
Each fraction of the effluent and of the eluate with acetate buffer was measured in terms of the content of HBs antigen and other proteins in the same manner as in the above step 2. The results are shown in Figure 2, wherein the HBs antigen is shown by a solid line and other proteins are shown by a dotted line. As is clear from Figure 2, HBs antigen again passed through the column of a cation exchanger and was contained in the effluent, and the remaining plasma proteins comprising mainly ~-globulin were adsorbed onto the column and eluted with eluting acetate buffer (ionic strength (~): 0.2, 0.4 and 0.6).
That is, the effluent consisted essentially of HBs antigen and contained almost no other proteins. Moreover, about 24 to 48~ by weight of HBs antigen was recovered in the effluent and the degree of puriEication was increased to 1500 times in comparison with the starting material.
In the following Table 1, there are shown the degree of purification and recovery of HBs antigen in each step of the above procedure.
s~
_ . , . . I
O ~ ,1 Q ~,~ o ~ o I ~J
u~ a~ ~l ~ u 0 ~ .. ,.. ~ , ~ .
. ~ , ~~ ~ ~g ~
J ' : ~ m ~ .. .. .. .. .
H O (~ - ~ ~1 ~1 ~ r~
. ~D U~
~ ~ o o ~ ~
. . ~ ~ . o ~ 7 o O
. E~ ~ O ~, ~ ~ , __ _ __ ~ h I * ,_ :
n - a) o U
OQ~ ~ ~)u~ O . 1~
U~ 1 . . . . O
~: ~O O 1~ ~ ~ Q
1~ ~ h ~,~ ~:
t~ h O ~--~ ,1 . ~ 'O o ~ ~) OD
O
v ~ 'e r~~t _, Ln ~~I
~ _ ~ .
. V ~ ~ m I I a:~
r~ O ~ ~ O ~: ~D ..
I ~ ~ r_ ~ ~ o ~ ~ o ~ ~ ~t ~Ih~-l rV h ~ ) e rd ~1 rd ~ Ei .~:: 3 rn ~:: 3 rn rv U ~ Q, rn ~ ~ rj O U O p:;
~,1 ~ h ~ h ._ ~ ~ ~ ~1 3 ~ S~ .C r~ S~rC rr) ~ Q- h O rV o rD ~.C rV Q,.~::
t~ rV 1~ 0 ~ ~ E~ ~) r~t Q, ~ rd Q.
5 ~ ~ ~ ~ h a) ~ h rv u~ ~ o -,a fC ~u~g~
,_ . . . .. .. , _ ., .. , _ . _ _ __ _ _ 5~
When the HBs antigen-containing effluent finally obtained above was tested by reversed passive hemo-agglutination (abbreviation: r-PHA) [cf. Juji, T and Yokochi, T, Jap. J. Expo Med., Vol. 39, Page 6l5 (1969)1, the agglutination occurred till 1024 times dilution and this diluted mixture had such a small proteîn concentra-tion as 6 ~g/ml (measured by Kjeldahl method). Besides, even when the effluent was concentrated to 1/100 by volume, no serum component was detected by an immuno-diffusion method and immuno-electrophoresis.
Moreover, in order to prove the purity of HBs antigen-containing effluent obtained by the above ion exchange chromatography the following test was done. Each fraction at puriication steps was analyzed by immunoelectropho-retically using antibodies to normal human plasma components. The results o~ the test revealed that the effluent of an anion exchanger (DEAE-Sepharose CL-6B) contained a little of plasma components comprising predominantly ~globulin as a contaminant, but the final effluent o an cation exchanger (CM-~epharose CL 6~) contained no impurity other than HBs antigen. On the contrary, the eluates of both ion exchangers showed that they contained all o~ the impurities originated in plasma components. Besides, to confirm whether the finally purified HBs antigen contained any impurity such as serum proteins, it was injected into rabbits, and no antibody was observed in those animals except that to ~Bs.
Furthermore, the finally purified product (the degree of purification: 1500 times, recovery: 24-48% by weight) was analysed by a density gradient ultracentrifugation using sucrose and cesium chloride. As a result, the HBs 5~
antigen had a peak at the concentation of sucrose of 37%
by weight and had also a peak at the p = 1.207 g/cm~ of cesium chloride, and further showed a single sharp peak by r-PHA method, which means that the HBs antigen obtained is pure and homogeneous. Besides, it was also confirmed by an electron microscopic analysis that the final prepa-ration of HBs antigen consisted of dispersed spherical particles alone having a size of 22 nm.
Example 2 A blood plasma, which was positive of HBe antigen, was treated in the same manner as described in Example 1 except that a citrate buffer (ionic strength (~): 0.05, pH: 5.5) was used instead of the acetate buffer. The behavior of Dane particles was observed by surveying the HBc antigen. The survey of HBc antigen was carried out at a fixed titer of HBs antigen (16,000 times by r-PHa method). The results are shown in Table 2.
-- ~ -~1 ~ ~ C~ N N N N N
m ~ ~ ~ V V ~ V \/
5~ ~
~ ~ ~
~ ~ O O O O O O O O ' ..
~ ~ O O O O O O O O
. P~ ~ ~ O O O O O O O O
hm ~ ~ ~ ~ D ~ ~ N
_ ~
E~ - . : , ,~ o o o o o o o ~n ~D ~r o ~ ~r ~
g~ ~ . '':~, ~ `
U~
r~
,~ o o m~
~ O ~ ~
~ ,~ m ~ ~
u~ ~ ~ ~
which are called "Dane particles"), and hence, an antibody against the HBs antigen may play a role in neutralizing the virus infectivity.
Thus, HBs antigen is useful as a material for a vaccine for preventing viral hepatitis type B. However, since a tissue culture of HB virus has never been com-pletely achieved, HBs antigen must be prepared Erom the blood of patients and carriers infected with HB virus, and hence, it is necessary to remove all components in such blood plasma other than HBs antigen such as plasma proteins, HBe antigen as well as HB virus.
Purification of HBs antigen has hitherto been done by various methods, such an ultracentrifugation, gel filtra-tion, affinity chromotography, or a combination thereof.
For instance, Gerin et al disclose a method for the purification of HBs antigen by twice repeating an isopycnic centriEugation using cesium chloride by a zonal rotor, subjecting the product to a cushion centriEugation with sucrose and further subjecting the product to an isopycnic centrifugation [cf. Gerin et al, J. Virol. Vol.
7, 569 (1971) and J. Immunol. Vol. 115 (1), 100 11975)1.
Blumberg et al disclose a purification of HBs antigen by a combination of centrifugation, treatment with an enzyme, column gel filtration, and ultracentrifugation under a density gradient with sucrose and cesium chloride (cf.
Japanese Patent Publication No. 19603jl975). Furthermore, Houwen et al disclose a purification of HBs antigen by affinity chromatography which comprises conjugating an anti-HBs antibody to Sepharose 4B gel, treating a plasma, which is positive oE HBs antigen by the resultant gel, and thereby eluting the HBs antigen adsorbed onto the gel.
s~ ~
The eluate fraction of this method still contains blood serum components ~e.g. prealbumin, albumin, transferrin, ~-lipoprotein, ~2-macroglobulin~ y-globulin), and hence, the fraction is futher passed through a Sepharose 4B gel column which is bonded with antihodies to normal human serum in order to remove these blood serum components.
These methods have some advantages but have also many drawbacks. For instance, the purification by ultracentri-ugation requires the density gradient centrifugation using cesium chloride and sucrose to be repeated several times and hence requires a large amount of cesium chloride and sucrose. Besides, the ultra-centrifuging machine should be provided with various rotors in accordance with the degree of purification and the steps therefor. The gel filtration method is poor in capacity and is not suitable for purification on a large scale. Moreover, this gel filtration method is also inferior in the degree of purification, and when the HBs antigen is prepared from blood plasma, there can not be obtained HBs antigen of high purity. The affinity chromatography method requires anti-HBs antibody, and hence, when it is carried out on a large scale, an enormous amount of anti-HBs antibody (human origin or immunized animal origin) must be prepared all the time.
The present inventors have intensively studied the purification of HBs antigen and have found that the desired HBs antigen having a high purity can comparatively simply be obtained by an ion exchange method.
An object of the present invention is to provide an improved method for the purification of HBs antigen.
Another object of the invention is to provide a method for preparing an acceptably pure HBs antigen From blood plasma ., or serum According to the present invention, there is provided a method Eor the purification of HBs antigen, which comprises passing an HBs antigen-containing solution prepared from blood plasma or serum through an anion exchanger and then passing the resulting effluent through a cation exchanger.
That is, a partially purified HBs antigen obtained from blood plasma or serum of patients and carriers infected wi~h HB virus by a conventional method is passed through an anion exchanger, whereby most of the blood plasma components other than HBs antigen and certain plasma proteins comprising mainly Y-globulin are adsorbed, and then the effluent thus passed through the anion exchanger, which contains HBs antigen a~d certain plasma proteins, is further passed through a cation exchanger, whereby the plasma proteins comprising mainly y-globulin are adsorbed and only the desired HBs antigen is contained in the effluent.
The present invention has been accomplished after extensive study of the behavior of the HBs antigen in anion and cation exchangers under various conditions. The puri~ication of HBs antigen is preferably carried out by passing an HBs antigen-containing solutibn through an anion exchanger and a cation exchanger whereby the impurities are adsorbed onto the exchangers~
Various methods have been reported for the purifi-cation of viruses using ion exchangers, for example, the purification of E. coli phage lCreaser and Taussing, Virol, Vol. 4, 20 (1957)1; the purification of tobacco mosaic virus [Cochram et al, Nature, Vol. 180, 1281 (1957)]; the purification of polio virus [Hoyer et al, Science, Vol. l27, 859 (1958)]; and the purification of vaccinia virus [McCrea and O'Loughlin, Virol, Vol. 8, 1~7 11959)]. ~owever, the pu~ification of antigens of viruses such as HBs antigen originating in blood plasma or serum without adsorption onto an ion exchanger has not been accomplished.
The ion exchanger used in the present invention is preferably a repeatedly usable column without specific activation and has a large binding capacity and is pre-erably an autoclavable gel having good stability under various conditions, e.g. at various pH levels, ionic strengths, etc.
Suitable examples of the anion exchanger are agarose gel introduced with an anionic substituent (e.g.
DEAE-Sepharose CL-6B), dextran gel introduced wîth an anionic substituent (e.g. DEAE-Sephadex, QAE-Sephadex~, cellulose introduced with an anionic substituent (e.g.
DEAE-cellulose, TEAE-cellulose), and the like. The anionic substituent includes diethyl-aminoethyl (DEAE), triethylaminoethyl (TEAE) and diethyl-(2-hydroxypropyl)-aminoethyl (QAE).
Suitable examples of the cation exchanger are agarose gel introduced with a cationic substituent (e.g.
CM-Sepharose CL-6B), dextran gel introduced with a cationic substituent (e.g. CM-Sephadex, CP-Sephadex), cellulose introduced with a cationic substituent (e.g.
CM-cellulosel, or the like. The cationic substituent includes carboxymethyl (CM), sulfopropyl (SP) and sulfoethyl (SE).
The partially purified HBs antigen is preferably prepared from blood plasma or serum of patients and carriers infected with HB virus by a conventional method, for instance, by treating the blood plasma with calcium chloride, salting out with ammonium sulfate, centrifuging, and adding ammonium sulfate to the resulting supernatant fluid, and thereby precipitating HBs antigen.
The partially purified HBs antigen thus prepared is applied onto a column filled with an anion exchanger which is previously equilibriated with an apropriate buffer solution having an ionic strength (~) of 0.02 to 0.1, preferably 0.04 to 0.06, and a pH level of 5.0 to 7.0, preferably 5.4 to 5.6, (e.g. acetate buffer, citrate buffer, phosphate buffer, succinate buffer, phthalate buffer, etc.). By this treatment, the HBs antigen and plasma protein comprising mainly Y-globulin pass through the column without being adsorbed threon and other plasma proteins such as albumin, ~- and ~-globulins, etc. are adsorbed thereon.
The HBs antigen-containing ef1uent passed through the above anion exchanger column, which still contains some impurities comprising predominantly y-globulin, is then applied onto a column filled with a cation exchanger, which is also previously equilibriated with an appropriate buf~er solution having an ionic strength (~) of 0.02 to 0~14, preferably 0.06 to 0.09, and a pH level of 4~5 to 6.0, preferably 5.0 to 5.3, (e.g. acetate buffer, citrate buffer, phosphate buffer, succinate buffer, and phthalate buffer, etc.). By this treatment, the HBs antigen passes through the column without being adsorbed thereon and the plasma proteins such as Y-globulin contaminated into the HBs antigen are almost completely removed. Moreover, the HBs antigen obtaned by the present invention does not 8a~s~
contain substantial amounts of Dane particles of the HB
virus and has a high purity.
Thus/ a highly purified HBs antigen can be obtained from blood plasma or serum by slmple procedures which can be carried out on an industrial scale without any difficulty, and the HBs antigen thus obtained is useful or preparing a source of safe and stable vaccine for preventing viral hepatitis type B.
The present invention is illustrated by the following Examples but is not limited thereto, and also by the accompanying drawin~s, in which:
Figure 1 is a graph of optical density versus fraction number for the product of one stage of Example l; and Figure 2 is a similar graph of the product of another stage of Example 1.
Example 1 Step 1: Blood plasma, which was positive in HBe antibodyr was treated with calcium chloride and dextran sulfate. The supernatant fluid was separated and salted out with 1.2 M ammonium sulfate. After centrifugation, 1.8 M ammonium sulfate was added to the supernatant fluid to precipitate HBs antigen. By this salting out, about 80~ by weight of the components of human blood plasma were removed, and the HBs antigen-containing solution was con-centrated to about 1~10 by volume.
Step 2: DEAE-Sepharose CL-6B gel (made by Pharmacia &
Co.) was sufficiently equilibrated with acetate buffer (ionic strength: 0.05, pH: 5.5) and then the DEAE
Sepharose CL-6B gel thus treated was packed into a column. The HBs ant;gen-containing solution obtained in the ahove step 1 was sufficiently dialyzed against the same acetate 15~
buffer as above, and the resulting precipitate was removed by centrifugation, and the HBs antigen-containing solution thus obtained was applied onto the above column fi11ed With DEAE-Sepharose CL-6B gel. The column was eluted ~ith acetate buffer oÇ which the ionic strength was varied stepwise by adding sodium chloride.
Each fraction of tlle effluent and of the eluate with the acetate buEfer, was measured in terms of the content of HBs antigen and other proteins. The content of HBs antigen was estimated by immuno-electrosyneresis (abbre~
viation: IES) and shown by titer [cf. Bussard, A. r B~B.A~, Vol. 34, page 258 (1959)], and the content of proteins was measured by optical density at 280 nm (OD280). The results are shown in the accompanying Figure 1, wherein the HBs antigen is shown by a solid line and other proteins are shown by a dotted line. As is clear from Figure 1, HBs antigen passed through the column o~ an anion exchanger and was contained in the effluent, and proteins other than HBs antigen and y-globulin were adsorbed onto the column and eluted with the eluting acetate buffer (ionic strength (~): 0.15, 0.3 and 0.6).
The proteins contaminated in the effluent ~ionic strength ( ): Q.05) were mainly y-globulin. Moreover, about 80%
by weight of HBs antigen passed through the column, and the degree of purification of HBs antigen was about 14 times in comparison with the starting material.
Step 3: CM-Sepharose CL-6B gel (made by Pharmacia Co.) was sufficiently equilibrated with acetate buffer (ionic strentho 0.08, pH: 5.1) and then was packed into a column. The effluent obtained in the above step 2 was sufficiently dialyzed against the same acetate buffer as _ g _ B~
above and then applied onto the above column filled with CM-Sepharose CL-6B gel. The column was eluted with acetate buffer of which the ionic strength was varied stepwise by adding sodium chloride.
Each fraction of the effluent and of the eluate with acetate buffer was measured in terms of the content of HBs antigen and other proteins in the same manner as in the above step 2. The results are shown in Figure 2, wherein the HBs antigen is shown by a solid line and other proteins are shown by a dotted line. As is clear from Figure 2, HBs antigen again passed through the column of a cation exchanger and was contained in the effluent, and the remaining plasma proteins comprising mainly ~-globulin were adsorbed onto the column and eluted with eluting acetate buffer (ionic strength (~): 0.2, 0.4 and 0.6).
That is, the effluent consisted essentially of HBs antigen and contained almost no other proteins. Moreover, about 24 to 48~ by weight of HBs antigen was recovered in the effluent and the degree of puriEication was increased to 1500 times in comparison with the starting material.
In the following Table 1, there are shown the degree of purification and recovery of HBs antigen in each step of the above procedure.
s~
_ . , . . I
O ~ ,1 Q ~,~ o ~ o I ~J
u~ a~ ~l ~ u 0 ~ .. ,.. ~ , ~ .
. ~ , ~~ ~ ~g ~
J ' : ~ m ~ .. .. .. .. .
H O (~ - ~ ~1 ~1 ~ r~
. ~D U~
~ ~ o o ~ ~
. . ~ ~ . o ~ 7 o O
. E~ ~ O ~, ~ ~ , __ _ __ ~ h I * ,_ :
n - a) o U
OQ~ ~ ~)u~ O . 1~
U~ 1 . . . . O
~: ~O O 1~ ~ ~ Q
1~ ~ h ~,~ ~:
t~ h O ~--~ ,1 . ~ 'O o ~ ~) OD
O
v ~ 'e r~~t _, Ln ~~I
~ _ ~ .
. V ~ ~ m I I a:~
r~ O ~ ~ O ~: ~D ..
I ~ ~ r_ ~ ~ o ~ ~ o ~ ~ ~t ~Ih~-l rV h ~ ) e rd ~1 rd ~ Ei .~:: 3 rn ~:: 3 rn rv U ~ Q, rn ~ ~ rj O U O p:;
~,1 ~ h ~ h ._ ~ ~ ~ ~1 3 ~ S~ .C r~ S~rC rr) ~ Q- h O rV o rD ~.C rV Q,.~::
t~ rV 1~ 0 ~ ~ E~ ~) r~t Q, ~ rd Q.
5 ~ ~ ~ ~ h a) ~ h rv u~ ~ o -,a fC ~u~g~
,_ . . . .. .. , _ ., .. , _ . _ _ __ _ _ 5~
When the HBs antigen-containing effluent finally obtained above was tested by reversed passive hemo-agglutination (abbreviation: r-PHA) [cf. Juji, T and Yokochi, T, Jap. J. Expo Med., Vol. 39, Page 6l5 (1969)1, the agglutination occurred till 1024 times dilution and this diluted mixture had such a small proteîn concentra-tion as 6 ~g/ml (measured by Kjeldahl method). Besides, even when the effluent was concentrated to 1/100 by volume, no serum component was detected by an immuno-diffusion method and immuno-electrophoresis.
Moreover, in order to prove the purity of HBs antigen-containing effluent obtained by the above ion exchange chromatography the following test was done. Each fraction at puriication steps was analyzed by immunoelectropho-retically using antibodies to normal human plasma components. The results o~ the test revealed that the effluent of an anion exchanger (DEAE-Sepharose CL-6B) contained a little of plasma components comprising predominantly ~globulin as a contaminant, but the final effluent o an cation exchanger (CM-~epharose CL 6~) contained no impurity other than HBs antigen. On the contrary, the eluates of both ion exchangers showed that they contained all o~ the impurities originated in plasma components. Besides, to confirm whether the finally purified HBs antigen contained any impurity such as serum proteins, it was injected into rabbits, and no antibody was observed in those animals except that to ~Bs.
Furthermore, the finally purified product (the degree of purification: 1500 times, recovery: 24-48% by weight) was analysed by a density gradient ultracentrifugation using sucrose and cesium chloride. As a result, the HBs 5~
antigen had a peak at the concentation of sucrose of 37%
by weight and had also a peak at the p = 1.207 g/cm~ of cesium chloride, and further showed a single sharp peak by r-PHA method, which means that the HBs antigen obtained is pure and homogeneous. Besides, it was also confirmed by an electron microscopic analysis that the final prepa-ration of HBs antigen consisted of dispersed spherical particles alone having a size of 22 nm.
Example 2 A blood plasma, which was positive of HBe antigen, was treated in the same manner as described in Example 1 except that a citrate buffer (ionic strength (~): 0.05, pH: 5.5) was used instead of the acetate buffer. The behavior of Dane particles was observed by surveying the HBc antigen. The survey of HBc antigen was carried out at a fixed titer of HBs antigen (16,000 times by r-PHa method). The results are shown in Table 2.
-- ~ -~1 ~ ~ C~ N N N N N
m ~ ~ ~ V V ~ V \/
5~ ~
~ ~ ~
~ ~ O O O O O O O O ' ..
~ ~ O O O O O O O O
. P~ ~ ~ O O O O O O O O
hm ~ ~ ~ ~ D ~ ~ N
_ ~
E~ - . : , ,~ o o o o o o o ~n ~D ~r o ~ ~r ~
g~ ~ . '':~, ~ `
U~
r~
,~ o o m~
~ O ~ ~
~ ,~ m ~ ~
u~ ~ ~ ~
3 ~ ~ 1 ~` o ,~ o .-1 o O ~,)U ~1 0 U
O
~,~ ~ ~
. h ~ ~ O E~~ ~1 ~ ~) E~ ~ -~ a.) ~ O O h o ~ o ~,1 o t~ U~ 3 1l 3 1l S~ 3 11 ~1 ~ ~1) ~ N ~) c~ Q~ ~ ..
~ ~ ~1 P~ ~.C U ~ ~ ~ 1 ~ 0 ~ ~ O :~ O
u~3 ~ 3 a ~ c~ o __ . ~
` `
s~
Example 3 A blood serum, which was positive of Hse antibody, was pretreated in the same manner as described in Example 1, step 1.
DEAE-cellulose (made by Serva ~ Co.) was activated and sufficiently equilibrated with citrate buffer (ionic strength (~): 0.04, pH: 5.5), and then was packed into a column. The above pretreated HBs antigen-containing solution was sufficiently dialyzed against the same buffer and then was applied onto the above column filled with DEAE-cellulose.
CM-Sephadex C-25 (made by Pharmacia & Co.) was suf-ficiently equilibrated with citrate buffer (ionic strength ~ 0.055, pH: 5.3) and then was packed into a column.
The effluent passed through the first column of DEAE-cellulose was sufficiently dialyzed against the second citrate buffer as above and then applied onto the second column of CM-Sephadex C-25. The results are shown in Table 3.
. - l5 -- -h `~ ~ o ~ ~ a) ~O
~m ~ O ~ O ~n o ~ o I U~
U ~ ~ ~ O
a~ o\ 3 c~
~; O ~
_ . __.
~1 O ~ . ,~
O Q) O JJ
r~
~rl O
aJ ~ h O
Q~ H
U~
S~
a) 00 r~
~m ~
~ rJ ,~ " ~, ,, U~
~o .
. ._ . ~ .
: ~ .
I ~ ~_ . ~ ~ O ~ ~ o ~r o . U r~ rl ~ ~ ~
a) , U~ o o ~ ~ 0\o :~
U ~ o--.
~ _~ :
o O
O ~ o O
o~ ~ ~ ~
~, .
.
~ ~ I
E~ ~
a) ~
Ul ~ I
0 3 ~ :
.,~
. O ~ 1 ~
,_1 O O~ .~(IS ~ 1~1 U ~ ~O hO ~ t.) ~ ~ ~1 ~ o Ul ~ o ~C
,1 ~ ~ O ~
h ~ rd 0~ ~ 0 ~ ~ / ~ a U~ ~ ~ U ~ I
~81~5~ "
As is clear from Table 3, 50 ~ by weight of HBs antigen was recovered into the effluent passed through the DE~E-cellulose column and the degree of pur.ification was increased to 9.3 times of that of the starting material.
This effluent was contaminated with serum proteins.which comprised mainly y-globulin. sesides, 25 ~ by weight of the ~IBs antigen was recovered into the effluent passed through the CM-Sephadex C-25 column, and the degree of purification was increased to 463 times to that of the starting material. In this effluent, no serum protein was observed by immuno-electrophoresis.
~ .. , .,, .. ,, ., , _ . __ ,, , _,
O
~,~ ~ ~
. h ~ ~ O E~~ ~1 ~ ~) E~ ~ -~ a.) ~ O O h o ~ o ~,1 o t~ U~ 3 1l 3 1l S~ 3 11 ~1 ~ ~1) ~ N ~) c~ Q~ ~ ..
~ ~ ~1 P~ ~.C U ~ ~ ~ 1 ~ 0 ~ ~ O :~ O
u~3 ~ 3 a ~ c~ o __ . ~
` `
s~
Example 3 A blood serum, which was positive of Hse antibody, was pretreated in the same manner as described in Example 1, step 1.
DEAE-cellulose (made by Serva ~ Co.) was activated and sufficiently equilibrated with citrate buffer (ionic strength (~): 0.04, pH: 5.5), and then was packed into a column. The above pretreated HBs antigen-containing solution was sufficiently dialyzed against the same buffer and then was applied onto the above column filled with DEAE-cellulose.
CM-Sephadex C-25 (made by Pharmacia & Co.) was suf-ficiently equilibrated with citrate buffer (ionic strength ~ 0.055, pH: 5.3) and then was packed into a column.
The effluent passed through the first column of DEAE-cellulose was sufficiently dialyzed against the second citrate buffer as above and then applied onto the second column of CM-Sephadex C-25. The results are shown in Table 3.
. - l5 -- -h `~ ~ o ~ ~ a) ~O
~m ~ O ~ O ~n o ~ o I U~
U ~ ~ ~ O
a~ o\ 3 c~
~; O ~
_ . __.
~1 O ~ . ,~
O Q) O JJ
r~
~rl O
aJ ~ h O
Q~ H
U~
S~
a) 00 r~
~m ~
~ rJ ,~ " ~, ,, U~
~o .
. ._ . ~ .
: ~ .
I ~ ~_ . ~ ~ O ~ ~ o ~r o . U r~ rl ~ ~ ~
a) , U~ o o ~ ~ 0\o :~
U ~ o--.
~ _~ :
o O
O ~ o O
o~ ~ ~ ~
~, .
.
~ ~ I
E~ ~
a) ~
Ul ~ I
0 3 ~ :
.,~
. O ~ 1 ~
,_1 O O~ .~(IS ~ 1~1 U ~ ~O hO ~ t.) ~ ~ ~1 ~ o Ul ~ o ~C
,1 ~ ~ O ~
h ~ rd 0~ ~ 0 ~ ~ / ~ a U~ ~ ~ U ~ I
~81~5~ "
As is clear from Table 3, 50 ~ by weight of HBs antigen was recovered into the effluent passed through the DE~E-cellulose column and the degree of pur.ification was increased to 9.3 times of that of the starting material.
This effluent was contaminated with serum proteins.which comprised mainly y-globulin. sesides, 25 ~ by weight of the ~IBs antigen was recovered into the effluent passed through the CM-Sephadex C-25 column, and the degree of purification was increased to 463 times to that of the starting material. In this effluent, no serum protein was observed by immuno-electrophoresis.
~ .. , .,, .. ,, ., , _ . __ ,, , _,
Claims (4)
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A method for the purification of HBs antigen, which comprises passing an HBs antigen-containing solution prepared from blood plasma or serum through an anion exchanger and then passing the resulting effluent through a cation exchanger.
2. A method according to claim 1, wherein the anion exchanger is previously equilibrated with a buffer solu-tion having an ionic strength of 0.02 to 0.1 and a pH
level of 5.0 to 7.0, and the cation exchanger is pre-viously equilibrated with a buffer solution having an ionic strength of 0.02 to 0.14 and a pH level of 4.5 to 6Ø
level of 5.0 to 7.0, and the cation exchanger is pre-viously equilibrated with a buffer solution having an ionic strength of 0.02 to 0.14 and a pH level of 4.5 to 6Ø
3. A method according to claim 2, wherein the buffer solution for treating the anion exchanger has an ionic strength of 0.4 to 0.06 and a pH level of 5.4 to 5.6, and the buffer solution for treating the cation exchanger has an ionic strength of 0.06 to 0.09 and a pH level of 5.0 to 5.3.
4. A method according to claim 2 or 3, wherein the HBs antigen-containing solution is dialyzed against the buffer solution to enable the anion exchanging optimal before passing through the anion exchanger and the effluent is dialyzed against the buffer solution to enable the cation exchanging optimal before passing through the cation exchanger.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP3655578A JPS55304A (en) | 1978-03-31 | 1978-03-31 | Purification of hbs antigen |
| JP36555/1978 | 1978-03-31 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1108051A true CA1108051A (en) | 1981-09-01 |
Family
ID=12472994
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA312,397A Expired CA1108051A (en) | 1978-03-31 | 1978-09-29 | Method for purification of hbs antigen |
Country Status (2)
| Country | Link |
|---|---|
| JP (1) | JPS55304A (en) |
| CA (1) | CA1108051A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4599231A (en) * | 1984-03-09 | 1986-07-08 | Scripps Clinic And Research Foundation | Synthetic hepatitis B virus vaccine including both T cell and B cell determinants |
-
1978
- 1978-03-31 JP JP3655578A patent/JPS55304A/en active Granted
- 1978-09-29 CA CA312,397A patent/CA1108051A/en not_active Expired
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4599231A (en) * | 1984-03-09 | 1986-07-08 | Scripps Clinic And Research Foundation | Synthetic hepatitis B virus vaccine including both T cell and B cell determinants |
Also Published As
| Publication number | Publication date |
|---|---|
| JPS6145610B2 (en) | 1986-10-08 |
| JPS55304A (en) | 1980-01-05 |
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