JPH0585527B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to the surface antigen of hepatitis B virus (hereinafter referred to as
The present invention relates to a method for purifying HBs antigen (abbreviated as HBs antigen), and more specifically to a method for highly purifying HBs antigen by column chromatography using cross-linked polysaccharide sulfate obtained by sulfuric esterification of cross-linked polysaccharide. Technical background of the invention and industrial fields of application Hepatitis B is primarily transmitted through blood.
It is caused by a virus with a diameter of 42 nm that contains a DNA-type nucleic acid. This hepatitis B virus not only causes acute hepatitis, but also chronic hepatitis, liver cirrhosis, and possibly hepatocellular carcinoma due to persistent viral infection. Hepatitis B is widely distributed throughout the world, and there are a large number of latent virus carriers (hereinafter simply referred to as carriers) who carry the virus for a long time without exhibiting any symptoms. The carrier of the virus is 2-3% of the total Japanese population, i.e.
It is said that there are 2-3 million people, and in Southeast Asia and Africa, it accounts for 10-15% of the population, and it is estimated that around 200 million people around the world are carriers of this virus. To prevent hepatitis B, usually highly purified
An effective method is to inactivate HBs antigen and use it as a hepatitis B vaccine, and this type of hepatitis B vaccine protects people at high risk of being infected with hepatitis B, such as medical workers, from hepatitis B infection. It is expected that it will not only protect the population, but also prevent new carrier outbreaks and ultimately eliminate hepatitis B from the earth. By the way, this HBs antigen is derived from three types of hepatitis B virus-related particles: Dane particles, which are the hepatitis B virus itself and have a core of 42 nm in diameter and 27 nm in diameter that contains nucleic acids, and Dane particles, which are 22 nm in diameter and do not contain nucleic acids. Antibodies against this HBs antigen act as protective antibodies against the hepatitis B virus, and utilize this mechanism of action. It is used as a hepatitis B vaccine. Prior Art To produce such a hepatitis B vaccine,
It is necessary to highly purify HBs antigen from plasma of hepatitis B virus carriers, cultures of recombinant organisms using genetic engineering techniques, culture supernatants, etc. this
Purification of HBs antigen generally requires a high degree of operational skill, which is a major cause of hindering the production of vaccines on an industrial scale. Density gradient centrifugation is currently the most common purification method for HBs antigen [Vyas.GN
et al., J.Immunology, 108 , 1114 (1972);
Hirshman SZ, Proceeding of National
Academy of Science USA, 71 , 3345 (1974)]
However, this method requires a large amount of cesium chloride and sucrose, as well as an ultracentrifuge and various types of rotors depending on the purification stage and its scale, and has the disadvantage of being expensive. In addition, a method for purifying HBs antigen by affinity chromatography using antigen-antibody reaction [Houwem.B et al., Journal of Immunological
Method, 8 , 185 (1975)] has also been proposed,
Although this method has extremely good purification efficiency,
When performing on an industrial scale, a large amount of HBs antibody-positive human serum is required to obtain HBs antibody for chromatography e-gel, and the HBs antibody must be further purified and bound to the gel matrix using cyanogen bromide etc. I have to create an affinity gel. In addition, in column chromatography using this affinity gel,
Because the binding between HBs antibody and gel is relatively weak
HBs antibody and HBs antibody-antigen reaction product may be detached during elution. If these substances are contaminated with vaccines, they may cause autoimmune diseases or kidney disease. Another method is to combine polysaccharides with sulfone groups such as dextran sulfate, chondroitin sulfate, heparin, etc. with cyanogen bromide to Cephalose CL-4B (manufactured by Pharmacia, Sweden) or Cephalose CL-6B (same as above). A method has been proposed for purifying HBs antigen using affinity gel [M. Andersson et al., Vox Sang, 41 , 91-97].
(1981); U.S. Patent No. 4138287;
114018], such affinity gels have a complicated manufacturing process, and the handling of cyanogen bromide is dangerous, making production on an industrial scale problematic. Furthermore, dextran sulfate, chondroitin sulfate, heparin, etc. are expensive, and there are limits to the ability to bind these compounds to carrier gels such as sepharose, making it difficult to obtain affinity gels of uniform quality and gels that provide a high degree of purification. . Furthermore, since the bond using cyanogen bromide is relatively weak, the polysaccharide having a sulfone group is removed and purified.
There is also a risk of contamination with HBs antigen. Purpose of the Invention The present inventors have conducted various studies to find a simpler and cheaper method of purifying HBs antigen by column chromatography. It was discovered that Ride sulfate ester has a specific affinity for HBs antigen, and when this cross-linked polysaccharide sulfate ester was used to purify HBs antigen from biochemical substances such as serum and plasma, it was possible to obtain extremely high purity and high yield. The inventors found that the desired highly purified HBs antigen can be obtained easily and at a high rate of production, leading to the completion of the present invention. That is, the present invention provides an improved method for purifying HBs antigen useful for the industrial production of hepatitis B vaccines, which involves column chromatography using cross-linked polysaccharide sulfate.
This provides a method for highly purifying HBs antigen. Structure and Effects of the Invention The present invention separates HBs antigen from HBs antigen-containing liquids such as serum or plasma of hepatitis B virus carriers, cultures of recombinants using genetic engineering, and culture supernatants by column chromatography. The purification method is characterized by using a crosslinked polysaccharide sulfate obtained by sulfuric acid esterification of a crosslinked polysaccharide as a gel for chromatography. The crosslinked polysaccharide sulfate used in the present invention is obtained by crosslinking polysaccharide such as dextran, cellulose, agarose, etc. with a crosslinking agent such as epichlorohydrin, dichlorohydrin, dibromhydrin, or ethylene glycol bisepoxypropyl ether. It is obtained by sulfuric acid esterification of crosslinked polysaccharide. Cross-linked polysaccharides are already commercially available, and examples of cross-linked dextrans include Cephadex G-10, G-25, G-50, and G-100 (manufactured by Pharmacia), and cross-linked agaroses include Cepharose CL-2B and CL-. 4B, CL-6B (manufactured by Pharmacia), and crosslinked cellulose such as Cellulofine GCL-25 and GCL-90 (manufactured by Chitsuso). By treating these gels with chlorosulfonic acid, sulfuric anhydride, or the like in the presence of an organic solvent such as pyridine, the desired crosslinked polysaccharide sulfate ester can be obtained. In order to purify HBs antigen by column chromatography using this cross-linked polysaccharide sulfate ester, operations employed in ordinary column chromatography are applied. The ester (preferably a gel formed into spherical particles) is filled with a suitable buffer having an ionic strength of about 0.001 to 1.0, such as McIlvaine's buffer (PH5.0) or 0.01M phosphoric acid. Buffered saline (PH
7.2), citrate buffer containing 0.1M NaCl (PH7.2)
After equilibrating the column with a solution containing HBs antigen, the HBs antigen is adsorbed onto the column, and the column is washed with the buffer solution used for the above equilibration. After this, use a suitable buffer (PH5-10) with an ionic strength of about 0.1 to 5.0, which is higher than the ionic strength of the buffer used for the above-mentioned equilibration and washing, such as pine kirbene buffer containing 0.6M sodium chloride. Highly purified HBs antigen can be obtained by elution with a phosphate buffer solution (PH 4-8) containing 0.6M sodium chloride (PH 6-9). According to the purification method of the present invention, the crosslinked polysaccharide sulfate ester used has a sulfone group directly bonded to the matrix of the crosslinked polysaccharide,
For this reason, the content of sulfone groups is high, and the specific adsorption ability for HBs antigen is excellent, and the degree of purification of HBs antigen is several tens of times higher.Moreover, the recovery rate of HBs antigen by column chromatography reaches more than 90% and nearly 100%. . This method can be carried out with simple operations even on an industrial scale, does not require special expensive reagents, allows industrial purification of HBs antigen at extremely low cost, has excellent gel stability, and This method also has the advantage that there is no risk of contamination of the resulting product with impurities such as antigen-antibody reactants, unlike in conventional methods. The method of the present invention can also be combined with conventional techniques such as ultracentrifugation or ion exchange chromatography, and in this case, very superior results can be obtained compared to conventional results. EXAMPLES Next, the present invention will be described in more detail with reference to reference examples and examples. Reference Example 1 Add 11 ml of chlorosulfonic acid dropwise to 200 ml of pyridine at a temperature below 0°C and mix. After finishing dropping,
Heat the mixture to 65-70°C. Add 7.5 g of Cephadex G-50 (manufactured by Pharmacia), which is an epichlorohydrin cross-linked dextran, to this.
Hold at 65-70°C for 4 hours with stirring. After the reaction is completed, it is cooled and neutralized by adding an aqueous sodium hydroxide solution. The gel is overseparated and washed thoroughly with 0.01M phosphate buffered saline to obtain cross-linked dextran sulfate. Reference Example 2 Pyridine prepared in the same manner as in Reference Example 1 above.
Add Cellulofine, an epichlorohydrin cross-linked cellulose gel, to 210 ml of chlorsulfonic acid mixture.
Add 7.5g of dried GCL-25 (manufactured by Chitsuso) to 65
React for 4 hours at ~70°C. After the reaction is completed, it is cooled and neutralized by adding an aqueous sodium hydroxide solution.
The gel is hyperseparated and thoroughly washed with 0.01M phosphate buffered saline to obtain 7.2 g of crosslinked cellulose sulfate. Reference Example 3 Pyridine prepared in the same manner as in Reference Example 1 above.
Sepharose CL, which is an epichlorohydrin cross-linked agarose gel, is added to 210 ml of chlorsulfonic acid mixture.
-6B (manufactured by Pharmacia) pyridine inclusion 30ml
and react at 65-70°C for 4 hours. After the reaction is completed, it is cooled and neutralized by adding an aqueous sodium hydroxide solution. The gel is hyperseparated and thoroughly washed with 0.01M phosphate buffered saline to obtain 23 ml of cross-linked agarose sulfate. Example 1 Cross-linked dextran sulfate obtained in the same manner as in Reference Example 1 was applied to a column (26.4 mmφ x 182 mm).
containing 0.05M sodium chloride.
Equilibrate with 0.027M pine kilbene buffer (PH7.41). This column contains HBs antigen positive human serum [HBs
Antigen titer 1: 16000 RPHA (reverse passive hemagglutination reaction), protein content 70.0 mg/ml (Lowry method), specific activity (RPHA/protein content) 229 After passing 3.0 ml of the solution, wash thoroughly with the above buffer solution. Then 0.6M
Elute with 0.027M pine kilbene buffer (PH7.38) containing sodium chloride to obtain 43.2 ml of eluate fraction. The HBs antigen titer of this elution fraction was 1:
1024RPHA, specific activity with protein content 0.41mg/ml
It was 2.498. The recovery rate of HBs antigen was 92.2%,
Purity (specific activity of elution fraction/specific activity of raw serum)
reached 10.9 times. Example 2 A cross-linked agarose sulfate gel obtained in the same manner as in Reference Example 3 was applied to a column (26.4 mmφ x 47.0
mm) and contains 0.05M sodium chloride
After equilibration with 0.027M citrate buffer (PH7.36), HBs antigen positive human serum [HBs antigen titer 1:
32000RPHA, protein content 70.0mg/ml, specific activity
457] After passing 0.5 ml, wash thoroughly with the above buffer. of the flow through and wash effluent (total 10.75 ml).
HBs antigen titer is 32RPHA, protein content 1118mg/
ml, and the specific activity was 28.7. Then 0.027M citrate buffer containing 0.6M sodium chloride (PH7.23)
Elute to obtain an eluate fraction of 8.00 ml. The HBs antigen titer of this elution fraction was 2048 RPHA, and the protein content was 0.217.
mg/ml, specific activity 9437.8, recovery rate of HBs antigen is
It was 85.2%, and the degree of purification reached 16.0 times. Example 3 Cross-linked cellulose sulfate obtained in the same manner as in Reference Example 2 was applied to a column (26.4 mmφ x 105 mm).
containing 0.05M sodium chloride.
Equilibrate through 0.027M citrate buffer (PH7.39). 20 ml of HBs antigen positive human plasma diluted with the above buffer solution is passed through this column. Then, wash thoroughly with the above buffer solution. Then
Elute with 0.027M citrate buffer (PH7.39) containing 0.6M sodium chloride. The results are shown in Table 1. As shown in Table 1, most of the HBs antigen was recovered in the elution fraction, and the specific activity and degree of purification were both increased approximately 17 times.

[Table] A portion of the purified HBs antigen was analyzed by ultracentrifugation. Specifically, a cesium chloride density gradient was created using a Hitachi 70P-72 ultracentrifuge and an RPS40T rotor, a sample was placed on it, and the sample was loaded at 40,000 rpm.
When centrifuged for 15 hours, this HBs antigen had ρ=1.2.
A sharp peak was observed in the vicinity. this is refined
This shows that the HBs antigen is a single entity. Comparative Example 1 Cellulofine, a crosslinked cellulose gel
Fill a column (26.4mmφ x 120mm) with GCL-25,
This is equilibrated with 0.027M citrate buffer (PH7.40) containing 0.05M sodium chloride. 20.0 ml of plasma from the same lot as the HBs antigen positive human plasma used in Example 3 was diluted twice with the above buffer solution and passed through this column, and the column was thoroughly washed with the same buffer solution.
It was then eluted with 0.027M citrate buffer (PH7.40) containing 0.6M sodium chloride. HBsAg 98.2
% was recovered in the flow-through fraction, and almost completely (96.7%) of the protein was recovered in the flow-through. No absorbance peak was observed in the eluted fraction, and the HBs antigen titer was less than 1:2 (RPHA), meaning that HBs antigen could not be purified at all. Example 4 A column (26.4 mmφ x 125 mm) was filled with cross-linked cellulose sulfate prepared in the same manner as in Reference Example 3, and equilibrated with 0.01M phosphate buffered saline (PH7.20). After salting out HBs antigen positive human serum with ammonium sulfate, it was purified using a density gradient zonal centrifuge (Hitachi 70P-72) (using a Hitachi RPZ-35-T rotor, sucrose density gradient 10-45% w/w, 30000 rpm, 20 time and Hitachi RPZ-48T rotor, sucrose density 20-50
%w/w, 42000rpm, 20 hours) after
40 ml of an HBs antigen-containing solution (HBs antigen titer 1:32000 RPHA, normal human seropositive by immunoelectrocross method) dialyzed against 0.01M phosphate buffered saline is passed through the column. After washing with 0.01M phosphate buffered saline (PH7.20), elution was performed with 0.01M phosphate buffered saline (PH7.20) containing 0.6M sodium chloride to obtain 12.0 ml of eluted fraction. This eluate had an HBs antigen titer of 1:102,400, and no plasma proteins other than HBs antigen were detected by immunoelectric cross-section, and the HBs antigen was almost completely purified, with a recovery rate of 96.0%.

Claims (1)

[Claims] 1. A method for obtaining purified hepatitis B virus surface antigen by subjecting a hepatitis B virus surface antigen-containing solution to column chromatography, in which a polysaccharide is used as a gel for chromatography in combination with epichlorohydrin, dichlorohydrin, dibromohydrin, and A crosslinked polysaccharide gel obtained by reacting a crosslinked polysaccharide gel obtained by crosslinking with a crosslinking agent selected from ethylene glycol bisepoxypropyl ether with a sulfating agent in an organic solvent is used. Method for purifying hepatitis B virus surface antigen. 2. The crosslinked polysaccharide sulfate is one selected from crosslinked cellulose sulfate, crosslinked agarose sulfate, and crosslinked dextran sulfate, and these are, respectively,
Crosslinked cellulose gel, crosslinked agarose gel, and crosslinked dextran gel obtained by crosslinking cellulose, agarose, and dextran with a crosslinking agent selected from epichlorohydrin, dichlorohydrin, dibromhydrin, and ethylene glycol bisepoxypropyl ether are reacted with a sulfating agent in an organic solvent. The method according to item 1 above, which is obtained by