JPS61148127A - Purification of hbc antigen - Google Patents

Abstract

PURPOSE:To purify the titled antigen useful as a diagnostic reagent for hepatitis B, in high purity, by adding an acid to the raw material of HBe antigen produced from a recombinant obtained by the use of especially a recombinant DNA technique, and subjecting the addition product to ion-exchange chromatography. CONSTITUTION:An HBe antigen, especially a raw material of HBe antigen expressed and produced by the recombinant imparted with the HBe antigen producing activity is treated with an acid (e.g. hydrochloric acid), and the addition product is subjected to the ion-exchange chromatography using an anion exchange material to effect the purification of said antigen. The pH of the system is shifted to the acidic side by the addition of an acid, and the most parts of lipids and contaminant proteins inhibiting the chromatographic treatment are separated from the HBe antigen in the form of precipitate. Preferably, the product is subjected to the purification by the salting-out with ammonium sulfate and to the concentration, the HBe antigen is adsorbed to an anion exchange material preferably containing diethylaminoethyl group as the functional group, the foreign materials are washed out, and the adsorbed antigen is eluted to obtain the purified HBe antigen.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a hepatitis B virus membrane antigen (hereinafter referred to as 1-I
A method for purifying Bc antigen (abbreviated as Bc antigen), especially recombinant DNA
1-IB produced by a recombinant using A technology
Concerning the method for purifying c-antigen.

Technical Background and Industrial Application Fields of the Invention Hepatitis B is a disease caused by the hepatitis B virus (hereinafter abbreviated as HBV), and it poses very serious epidemiological and clinical problems. However, no effective therapeutic measures have been found. This disease is widely distributed throughout the world, but is particularly prevalent in Asia and Africa, where it is becoming a problem.

Early detection of HBV infection is important for effective prevention of type 13 hepatitis, and as a clinical diagnostic method, current (Bs antigen, HBs antibody, HBc antibody, 1113e
Detection methods such as antigen and ll3e antibody have been adopted, and r
-1] ■lA method, PIA method, EIA method, RIA
Several methods are known, and diagnostic reagents used for these methods are commercially available. However, the antigens necessary for manufacturing these diagnostic reagents and their preventive vaccines must be obtained from the blood and liver of people with latent HBV infection, called carriers, and there are many manufacturing constraints. There is. In particular, I
-I B c antigen must be prepared directly from an infectious virus, and its preparation is extremely dangerous.

The present inventors used recombinant DNA technology to inject I-I encoding the HBs antigen protein into Escherichia coli and yeast.
We succeeded in introducing and expressing BV DNA (see Japanese Patent Application No. 142,460/1982), and also established an industrial purification method for I-Ins antigen, which is a raw material for vaccines (Japanese Patent Application No. 142,460/1983). 186187).

The present inventors further attempted to produce HBC by recombinant cells (Cell Engineering, Vol. 3, No. 4, 367-3
70, 1984), thereby producing HBc
We have succeeded in highly purifying antigens to the extent that they can be used as diagnostic reagent materials, and this has enabled us to treat HB, which is particularly difficult to handle.
A technology has been established that can provide c antigen in large quantities at low cost, and is expected to be applied to diagnostic reagents for hepatitis B.

Conventional techniques As a method for purifying liver-derived HBc antigen, a method combining density gradient centrifugation and gel filtration [Bud-Kowa et al.
ska, A. et al., J. Immunol
ogy, 118°1300 (1977), and 0
hori, H, et al, Inter-viro
logL 13.74 (1980)], density gradient centrifugation [Fei-telson, M.A. et at.
J. Virology, 43,687 (198
2)] has been reported.

1 [For the purification method of IIBC antigen derived from u fluid, HBV
After the infectious virus particles, which are the main body of the virus, are purified by centrifugation, a surfactant is added to remove the virus particles.)
Furthermore, a method of purification by density gradient centrifugation has been reported [Takahashi, K, etal, J.
, [mmunology, 122.275 (1
979) etc.].

Furthermore, density gradient centrifugation is a method for purifying the l1nc antigen expressed in E. coli using recombinant DNA technology.
Roggendorf, M., et al., J.
Vir.

Logical Methods, 6.61 (19
83)] has been reported.

However, in these methods, recombinant-derived 1-
It has the disadvantage that various problems in the purification of II3c antigen cannot be adequately addressed.

In other words, an important issue in purifying recombinant-derived HBc antigens is that contaminant components in the raw materials, such as host-derived proteins and lipids, are qualitatively and quantitatively derived from liver or blood. Since it is completely different from the HB c antigen, sufficient purification cannot be achieved using the conventional techniques described above. Incidentally, in the above-mentioned n, an attempt was made to purify the recombinant-derived [-I B c antigen.

Even in the method of Ggendorr et al., the product was only purified to the extent that it could be analyzed, and the yield was not investigated, so it cannot be applied as an industrial method for purifying HBc antigen.

Purpose of the Invention The present inventors have conducted various studies in order to find a safe and practical purification method for HBc antigen derived from recombinants, and as a result, the present inventors have discovered host-derived components obtained from recombinant cultures. Add acid to the H 13 c antigen raw material solution containing pt ~
By bringing I to the acidic side, most of the lipids and contaminant proteins that interfere with chromatography can be separated and removed from the 1-I B c antigen as a precipitate, and in the next step, ions can be removed using an anion exchanger. The inventors have discovered that HBc antigen can be highly purified by exchange chromatography and density gradient centrifugation, leading to the completion of the present invention.

That is, the purpose of the present invention is to make it possible to stably supply safe and inexpensive HBC antigens to 9 people. The purpose of the present invention is to provide a method for industrially efficiently purifying a large amount of I-I B C antigen to extremely high purity.

The present invention relates to the purification of IIBc antigen, in particular to the purification of HBc antigen produced by a recombinant using recombinant DNA technology.
To the raw material solution containing B C antigen and host-derived components,
Add acid to shift the pH to the acidic side, remove most of the lipids and contaminant proteins that interfere with the chromatography method as precipitates, and then directly purify and concentrate by salting out ammonium sulfate. , is characterized by performing ion exchange chromatography using an anion exchanger.

The HBc antigen material solution in the present invention refers to a recombinant transformed to produce an HBC antigen, such as Escherichia coli or yeast, using recombinant DNA technology in an appropriate medium and culture conditions. After culturing to produce and accumulate H13C antigen, I
It is obtained by crudely extracting IBc antigen.

Examples of recombinants capable of producing such IJ E C antigens have already been announced by the present inventors (cell engineering,
Vol. 3, No. 4, pp. 367-370, 1984 and 31st General Meeting of the Japanese Society of Virology, Osaka, 1983, Abstract No. 2036), for example, as follows.

Plasmid ptl13■ containing the entire HBV DNA sequence (
The DNA sequence containing the I-1[3ca gene obtained by digesting the I-1[3ca gene (see Japanese Patent Application No. 145093/1983) with a restriction enzyme such as n5aE is extracted from the Xhol of plasmid pAcYc+77.
site to obtain recombinant plasmid pAHBc.

One DNA sequence containing the HBc gene obtained by digesting this pAtlBc with the restriction enzyme
(see No. 3) into the Xhol site to obtain HBc expression plasmid pAc301.

Alternatively, in order to remove the pre-C region at the 5' end, the above ptl13c was partially digested with the restriction enzyme Xhol, and T4D
cohesive end with NA synthase
) to a blunt end (rlush end), then E
Add a coRI linker to the Xhol site to EcoR1
Add it to your site. Next, Xh on the 5° side of the HBc gene
Select a plasmid that has changed to ol site or EcoR1 site. The thus obtained plasmid pHBcl was digested with Econl, further digested with BAI and 31, a 5all linker was attached, and this was digested with the restriction enzyme Xho I.
, Digest with Sal I to obtain an HBc gene fragment with approximately 600 bp of the pre-C region removed. This is shuttle vector pAM81 (see Japanese Patent Application No. 145093/1982).
5all site of HBc expression vector pA
Get c701.

The above expression vector was transformed into the yeast fungus Saccharomyces cerevisiae A I 22 [a 1eu2 bis4 cant
(C+r)] (Feikoken Jokyo No. 312) in the same manner as described in the above-mentioned Japanese patent application, the transformed yeast Saccharomyces cerevisiae pAc301 having the ability to produce 1-I BC antigen and Satucharomyces cerevine pA701 is obtained.

The desired recombinant culture can be obtained by culturing these transformed yeast using conventional methods.

As a method for extracting HBc antigen from the above culture,
First, bacterial cells are separated from the culture by centrifugation and disrupted in an appropriate buffer. The crushing methods include ultrasonic crushing,
Use various methods such as glass bead crushing, Manton-Gaulin crushing, or enzymatic dissolution of cell walls to form spheroplasts, which are then destroyed by the action of a surfactant, or a combination of these methods. be able to. This cell destruction product is subjected to low-speed centrifugation to separate cell wall debris and the like, and if necessary, it is filtered through a membrane filter to provide l-I BC antigen raw material.

In the present invention, examples of acids used in the acid addition treatment include inorganic acids such as hydrochloric acid, sulfuric acid, and phosphoric acid, and organic acids such as acetic acid and oxalic acid.

The method of acid addition treatment is to add acid so that the pI of the raw material liquid is 6 or less, preferably in the range of 50 to 5.5.
Iro): Perform a adjustment. The temperature of the acid treatment is preferably 20°C or lower, preferably 4 to 10°C.

After pII adjustment, the precipitate was centrifuged to obtain a two-separate solution containing HBc antigen.

Next, if necessary, purification and concentration operations are performed by salting out ammonium sulfate. Aqueous ammonia at an appropriate concentration was added to the IIBc antigen-containing supernatant obtained by the acid addition treatment, and ammonium sulfate was added while maintaining the ptI of the supernatant in the range of 6.0 to 8.0 to remove the HI3c antigen. The precipitate is separated from the supernatant by centrifugation.

The obtained precipitate is dissolved in a suitable near-neutral buffer with an ionic strength of about 0.001 to 1.0, such as 0.1M phosphate buffer, and this is dialyzed against the same buffer. It is used as a material for chromatography using an anion exchanger.

If ammonium sulfate precipitation is not performed, add aqueous ammonia to the HBc antigen-containing supernatant to adjust I)H to approximately 7.0, and then dilute with the same buffer solution or dialyze to prepare a chromatography material. As the anion exchanger used in the present invention, an anion exchanger having diethylaminoethyl as a functional group is most effective, and DEAE-Sephadex A.25. DEAE-Sephadex A-5
0゜DEAE-Sepharose, DEAE-Sephacel (
Pharmacia Fine Chemicals), DEAE
Bio Gel A, Selex D (manufactured by Bio-Rad)
, DEAE-Cellulofine (manufactured by Seikagaku Corporation)
They are commercially available under the names of , and all of these can be used. Ion exchange chromatography using this anion exchanger is carried out in the following manner.

Adsorption of 1-[B c antigen to an anion exchanger can be carried out by both a column method and a batch method.

In the column method, a column packed with an anion exchanger is
After equilibration was carried out by passing the same buffer solution used in the preparation of the chromatography material, the material was passed through it, and H
Contaminant substances derived from the host are separated by adsorbing the Bc antigen. After passing through the column, an equilibration buffer is passed through the column, the column is washed to remove impurities, and the column is subjected to the elution procedure described below.

In the case of a batch method, an anion exchanger equilibrated with a buffer solution is added to the material solution, and the material is stirred at a slow speed for about 0.5 to 2 hours to adsorb the HBc antigen. After adsorption, the anion exchanger is collected on a filter, an equilibration buffer is added to the gel, and washing and filtration separation are repeated several times, followed by an elution operation. In addition, in the case of adsorption by this batch method, upon elution, it is preferable to load the anion exchanger adsorbed with the IBc antigen into column I:+ and elute with a column.

For elution, the ionic strength is 0. Stepwise elution or concentration gradient elution is performed using a near-neutral buffer solution that is about O1 to 0.5 and has a higher ionic strength than the equilibration buffer to separate adsorbed contaminants and I-I B c The antigen is separated and eluted, and a fraction containing the HBc antigen is collected. Concentration gradient elution is, for example, o.

IM-0.5M phosphate buffer concentration gradient can be used, or 0.01M phosphate buffer with saline addition 0.01 → 0.5
M concentration gradient may be used, and other suitable concentration gradient buffers that provide good separation between I]Bc antigen and contaminants may also be used. In the case of stepwise elution, a buffer with an ionic strength is passed through which only either the HBc antigen or the contaminants are eluted, followed by a buffer with a high ionic strength to ensure highly purified HBc antigens and contaminants. A fraction containing the HBc antigen is collected.

The HBc antigen thus obtained is further subjected to sucrose step gradient centrifugation or sucrose linear gradient centrifugation, then sedimentation equilibrium centrifugation or linear gradient centrifugation using cesium chloride,
It is possible to obtain highly purified I-I I3cQ source.

The present inventors used chromatography gels such as 0M cellulose, 0M Sepharose, phosphocellulose, and hydroxyapatite in place of the anion exchanger under various conditions to purify recombinant HBc antigen. I tried to use it, but I couldn't get much of a purification effect.

Next, the present invention will be explained in more detail with reference to Examples.

Example 1 Recombinant yeast Saccharomyces cerevisiae AI (22ph
After culturing o80/pΔC701, about 600 g of bacterial cells were collected by centrifugation and added with 50 mM phosphate buffer (pH 7,2) 3
(!) and put it through a Manton-Gorlin crusher at a pressure of 600 kg/cm2 for about 3 hours to destroy the bacterial cells.

The destroyed bacterial cells are centrifuged, coarse bacterial fragments are separated, and H
Be antigen activity approximately 30,000 cpm [400-fold dilution, R
IA kit: HBeR [A kit (manufactured by Apobut)] 1 (obtain Bc antigen progenitor extract).

Next, check with a pH meter and add acetic acid dropwise to the crude extract to adjust the pH to 5.4. After stirring at 4°C for about 30 minutes, remove the precipitate by centrifugation. Add aqueous ammonia to the centrifuged supernatant, and gradually add ammonium sulfate to a final concentration of 2.5M while keeping ptoI at about 6.5. Stir at 4°C for 30 minutes, leave overnight, then centrifuge to remove HB.
The precipitate containing the cBe antigen is separated and collected.

The collected precipitate is made cloudy with 10mM phosphate buffer, 50mM potassium chloride (pH 7,3) at about 300 mQ (knee u), and dialyzed against about 100 times the same buffer using a dialysis tube. After dialysis. , 10mM phosphate buffer, 50mM
It is diluted approximately 3 times with potassium chloride (p[-17,3) and added to a DEAE-cellulose column (gel volume: 112) that has been equilibrated with the same buffer to adsorb the HBC antigen. After thoroughly washing the column with equilibration buffer, about 4.
Elute by passing 0g of 50mM to 500mM potassium chloride gradient phosphate buffer and collect the fraction containing HBcBe antigen.

The collected fractions were pooled and concentrated using an ultrafiltration concentrator Amicon TCP-10 (manufactured by Amicon), and 60% sucrose solution, 20% sucrose solution, and HBc antigen concentrate were placed in an ultracentrifugal duplex. 35mσ
, 35m (layered, 30,0 10σ
Ultracentrifugation is performed at rpm and 4°C for 19 hours to concentrate and purify the H13c antigen at the sucrose liquid layer interface.

The obtained HBc antigen fraction was diluted with 50 mM Tris-I.
C (1°150mM sodium chloride, ImMEDTA (
After dialyzing to pH 7.5), senlum chloride was 1.32
g/mQ concentration, and 30.00Orp
m.

Ultracentrifugation is performed at 4°C for 60 hours to obtain concentrated and purified IIBc antigen.

The recovery rate and degree of purification of IBc antigen were evaluated for up to 6 days.
Shown in the table.

Table 1 Example 2 Recombinant yeast Satucharomyces cerevisiae Al22 ph
After culturing o80'/pA C701, the cells (100 g) collected by centrifugation were added to 50 mM phosphate buffer (pH 7,
2) Add 500ml of water and subject it to ultrasonication at 46C for about 90 minutes to destroy the bacterial cells. This is carried out in the same manner as in Example 1 to remove bacterial cell debris by centrifugation to obtain a crude extract.

This crude extract was purified in the same manner as in Example 1.
(Obtain the Be antigen. The recovery rate and degree of purification of the H[3c antigen up to each step are shown in Table 2.

2k Example 3 Recombinant yeast Zaccharomyces cerevisiae A H22ph
After culturing o80/pAC701, it was centrifuged and the collected bacterial cells (200 g) were treated with 100 μg/mσ of Zymolyase (
50mM potassium phosphate buffer (manufactured by Seikagaku Corporation) containing
Add 600 inches of pi-17,2), stir at 30°C for 30 minutes, and centrifuge to obtain spheroplasted yeast as a precipitate. This precipitate contains 0.1% Triton X100.
Add 200mQ of 0mM phosphate buffer (pH 7,2),
Stir at room temperature for 1 hour to obtain a lysate, which is centrifuged to remove bacterial cell debris to obtain a crude extract.

40% (W/W) polyethylene glycol 6000 was added to this crude extract to give a final polyethylene glycol concentration of 3%. After stirring at 4°C for 1 hour, remove the supernatant by centrifugation, add 80 mQ of 0 mM potassium phosphate buffer, 50 mM potassium chloride (pH 7,2) to the precipitate, and suspend. This suspension is solubilized by sonication for 5 minutes. This was mixed with 10mM potassium phosphate buffer, 50mM K C
(! After 1 night dialysis with 49C against (pH 7, 2), DEA
Add to E-cellulose. The 1-I B c antigen is then purified in the same manner as in Example 1 to obtain the 1-I B c antigen. Table 3 shows the recovery rate and degree of purification of HBc antigen up to each step.

Table 3 Patent Applicant Chemo- and Serum Therapy Research Foundation Agent Patent Attorney Written amendment by one person (voluntary) February 20, 1985 Commissioner of the Japan Patent Office - 1. Indication of the case ・
Patent Application No. 271432 of 1981 2 Name of the invention Method for purifying HBc antigen 3 Relationship with the case of the person making the amendment Notes to the patent applicant 668 Okubo, Shimizu-cho, Kumamoto City, Kumamoto Prefecture Name Chemo-Serotherapy Foundation Research Institute 4, Agent 5 Date of amendment order Voluntary 7, hli Masanori) Contents (1) Claims column appendix.

(11) Detailed Description of the Invention Column (i) Page 2, line 14, ``Correct the phrase ``membrane antigen 1'' to ``nuclear antigen.''

(2) On page 6, 3 lines from the bottom, amend the text ``Heading,'' to ``1 heading.''

(3) Insert the following text between lines 7 and 6 from directly below +4.

[In order to increase the degree of purification in ion exchange chromatography or to improve the degree of purification when fractions containing I-[Bc antigen are broadly pooled,
A combination of ion exchange chromatography and hydroxyapatite chromatography is useful. Hydroxyapatite is a gel for affinity chromatography whose component is calcium phosphate.
Hydroxyapatite (Seikagaku Kogyo Co., Ltd., ^L
BIOCRFM-BEHRING), IIydrox
3°, which is commercially available under the name of ylapatiLe (Bio-Rad), i.e., both parts containing the HI3c antigen, were prepared at an ionic strength of 0.
．． Dialyze against a suitable near-neutral buffer such as 0.1M phosphate buffer, or dilute with the same buffer, and then equilibrate with the same buffer. When the liquid was passed through the hydroxyapatite column, I-
The I B c antigen is recovered in the absorbed and passed-through fraction. ” (4) Delete “Hydroxyapatite” from the bottom four lines of page 14.

(5) Page 15, line 3, “Example 1” [Example 1
-N and correct it.

(6) Page 17, lines 7 and 8, replace “Table 1” with 1
Table 1-1”.

(7) The following Example 1-2 and Table 1-2 are inserted before Example 2 on page 18.

[Example 1-2 HBc antigen is concentrated and purified at the sucrose liquid layer interface under exactly the same conditions as in Example 1-1.

The obtained HBc antigen fraction was added to 0.1M phosphate buffer (pH
After dialyzing against 7,3), the solution is passed through a hydroxyapatite column equilibrated with the same buffer. After pooling the I-I B c antigen-containing fractions collected in the flow-through fractions, cesium chloride was added to a concentration of 1.32 g/ml, and the mixture was incubated at 30,000 rpm and 4°C for over 60 hours. Centrifuge to obtain concentrated and purified IIBc antigen.

The recovery rate and degree of purification of the I-I I3 c antigen at this time are as shown in Table 1-2.

Table 1-2” Claims as amended above (1) HB characterized in that in purifying the HB C antigen, the HB C antigen raw material is acid-added and then subjected to ion exchange chromatography using an anion exchanger.
c Antigen purification method.

(2) The method according to item (1) above, wherein after the acid addition treatment, ammonium sulfate salting out is performed and then ion exchange chromatography is performed.

(3) The method according to item (2) above, wherein the HBc antigen is expressed and produced by a recombinant transformed by recombinant DNA techniques and endowed with the ability to produce HBc antigen.

(4) I-IBc antigen raw material is crude extraction of I-IBc antigen obtained by disrupting recombinant cells by ultrasonic disruption, glass bead disruption, Manton-Gorlin disruption, surfactant treatment, or a combination thereof. The above-mentioned (1) which is a thing
) Method described in section.

(5) The method according to item (1) above, wherein the acid addition treatment comprises a step of adding an acid to the HBc antigen raw material to separate contaminant proteins and lipids as a precipitate.

(6) Filter acid, hydrochloric acid, sulfuric acid, phosphoric acid, used for acid addition treatment,
At least 01 species selected from acetic acid and oxalic acid +'
+The method described in paragraph (5) of ri.

(7) The method according to item (1) above, in which an anion exchanger having a noethylaminoedyl group as a functional group is used as an anion exchanger in ion exchange chromatography.

(8) The method according to item (1) above, wherein the ion exchange chromatography is performed by a column method or a batch method.

(9) The method according to item (1) above, wherein ion exchange chromatography and hydroxyapatite chromatography are carried out in combination.

Procedural amendment (Biao) 1 Indication of the case Patent application No. 271432 of 1982 2 Name of the invention Method for purifying HEc antigen 3 Relationship with the person making the amendment Patent applicant address 668 Okubo, Shimizu-cho, Kumamoto City, Kumamoto Prefecture Name Chemo and Serum Therapy Research Institute 4 Agent 5 Date of amendment order April 30, 1985 (shipment date) 7
, Ni positive contents (1) Specification page 4, line 16 to page 5, line 1, [[I3ud
kowaska, A, et al, (1
982)] is corrected as follows.

"[3udkowaska, A,
et al.), Japan Immunology (J.
I mn+unology), l l B.

+300 (1977), and Ohori et al.

Il, et al.), Interpyrology (I
inter-virology), l 3 , 74
(1980)], density gradient centrifugation [Feitelson et al.
PeiLelsonoM, A, etat, ), J, Virology, 4
3, 687 (19B 2)] J (2) Dokichi page 5 lines 6-7, r['l”akahash
i, K.

(!L al,...etc.].) is corrected as follows.

“[Takahashi, K. et al.
al,), Japan Immunology (J, Imm)
unology), 122, 275 (+979
)Such]. (3) Ibid., p. 5, lines 10-II, r[Roggendo
rf.

M, (1983)j is corrected as follows [
[Roggendorf et al.
et al.).

J. Pyrological Mesolas (J.

Virological Methods), 6
, 61 (1983)] Sand over J

Claims (8)

[Claims] (1) In purifying the HBc antigen, the HBc antigen raw material is acid-added and then subjected to ion exchange chromatography using an anion exchanger.
Antigen purification method. (2) The method according to item (1) above, wherein after the acid addition treatment, ammonium sulfate salting out is performed and then ion exchange chromatography is performed. (3) The method according to item (1) above, wherein the HBc antigen is expressed and produced by a recombinant transformed by recombinant DNA techniques and endowed with the ability to produce HBc antigen. (4) The HBc antigen raw material is an HBc antigen crude extract obtained by disrupting recombinant cells by ultrasonic disruption, glass bead disruption, Manton-Gaulin disruption, surfactant treatment, or a combination thereof. The method described in section 1). (5) The method according to item (1) above, wherein the acid addition treatment comprises a step of adding an acid to the HBc antigen raw material to separate contaminant proteins and lipids as a precipitate. (6) The acid used in the acid addition treatment is hydrochloric acid, sulfuric acid, phosphoric acid,
The method according to item (5) above, wherein at least one selected from acetic acid and oxalic acid is used. (7) The method according to item (1) above, in which an anion exchanger having a diethylaminoethyl group as a functional group is used as an anion exchanger in ion exchange chromatography. (8) The method according to item (1) above, wherein the ion exchange chromatography is performed by a column method or a batch method.