JPH07258291A - Method for purifying influenza HA protein - Google Patents

Abstract

(57) [Summary] [Composition] Recombinant DN using insects or cultured insect cells
The influenza HA protein-containing solution obtained by the A technique has NeuAcα2,3 (6) Galβ1,3 with free ends
(4) -A compound having at least 3 sugar molecules is immobilized on a carrier such as a polysaccharide gel or a porous polymer, and the influenza HA protein is selectively adsorbed on the immobilized compound, Purified influenza HA characterized by eluting and collecting influenza HA protein
Protein manufacturing method. [Effects] According to the present invention, purified influenza HA is prepared from an influenza HA protein-containing solution containing various contaminants.
The protein can be produced in high purity and can be obtained with a high recovery rate. Further, the purified influenza HA protein produced according to the present invention has very little contamination with nucleic acid, and it can be said that it is safer than conventional ones in consideration of problems such as oncogenes derived from contaminating heterologous or homologous DNA. . Therefore, according to the production method of the present invention, a highly safe and effective influenza vaccine can be economically obtained.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing a purified influenza HA protein obtained by expressing it in an insect or an insect culture cell using recombinant DNA technology and used as an influenza vaccine, and a column for producing the protein. .

[0002]

2. Description of the Related Art Influenza is a disease caused by the influenza virus. At present, however, no drug that directly acts on the virus has been found, and there is no effective preventive or therapeutic method other than vaccination with a vaccine. The influenza vaccine currently used is influenza HA (hemagglutinin), which is the outer coat of the virus.
Proteins and influenza NA (neuraminidase) proteins (hereinafter abbreviated as “HA proteins” and “NA proteins”, respectively) are widely used.

Recently, attempts have been made to express HA proteins and NA proteins in insects such as silkworms, armyworms and their cultured cells using recombinant DNA technology (Japanese Patent Laid-Open No. 3-108480, Kazumichi Kuroda). et al. Th
e EMBO Jounal 5, (6), PP 1359--1365, 1986.). According to this method, it is known that HA proteins and the like can be efficiently produced, but there is a problem in the separation and purification of these, and HA proteins with a high degree of purification, that is, purified HA proteins could not be obtained. That is, in the above method, it is necessary to separate HA proteins and the like from insect body fluids and insect cell liquids, but impurities such as nucleic acids originating in insect cells and the like are difficult to separate from HA proteins and the like, and purified HA proteins cannot be obtained. However, it has been a problem in terms of safety such as economy and antigenicity.

Regarding purification of HA protein and the like, for example, a method using a cellulose sulfate or a crosslinked polysaccharide is already known (Japanese Patent Publication No. 62-30752).
issue). However, when this method is applied to an HA protein obtained by recombinant DNA technology, a compound that binds to a sulfonyl group also remains in the column, so that the HA protein and the like are not specifically obtained, and various contaminants are obtained. May be included, and safety was not without problems in terms of antigenicity and the like.

[0005]

Therefore, in order to provide an economical and highly safe influenza vaccine by recombinant DNA technology utilizing insects or cells thereof, HA protein and NA protein are highly purified. It was necessary to provide a method for the above, and in particular, there was a strong demand for the HA protein, which is a major protective antigen of influenza virus.

[0006]

DISCLOSURE OF THE INVENTION The present inventors have conducted diligent research on the production of purified HA protein in view of the above-mentioned circumstances, and as a result, HA protein was specifically adsorbed on a sugar chain having a specific sequence, and The inventors have completed the present invention by finding that they can be easily released from this sugar chain by a simple elution treatment and that contaminants such as DNA can be efficiently removed to produce a purified HA protein.

That is, according to the present invention, a solution containing an influenza HA protein obtained by a recombinant DNA technique using insects or cultured insect cells has NeuAcα2,3 (6) Galβ1,3 (4) whose free ends are immobilized on a carrier. The present invention provides a method for producing a purified influenza HA protein, which comprises adsorbing to a compound having at least three sugar molecules which is-, and then eluting and collecting the influenza HA protein.

A compound having at least three sugar molecules having a terminal of NeuAcα2,3 (6) Galβ1,3 (4)-, which is used in the production method of the present invention (hereinafter, referred to as
As a “compound having a sialo sugar chain”, a sialylacto-type sugar chain (NeuAcα2,3 (6) Galβ1,
A compound having 3 (4) GluNAcβ1,3Galβ1-), a sialylganglio-based sugar chain (NeuAcα2,
Compound having 3Galβ1,3GalNAcβ1,4Galβ1-), hemacid sugar chain (NeuAcα2,3G)
Examples thereof include compounds having alβ1,4Gluβ1-), which may be sugars, glycoproteins or glycolipids. Among these, specific examples of the sialo-oligosaccharide-containing compound preferably used include fetuin, glycophorin, ganglioside, and the like, and particularly preferably fetuin.

On the other hand, the carrier used for immobilizing the compound having a sialo-oligosaccharide includes polysaccharide gels such as agarose gel, cellulose gel and dextrin gel, silica gel,
Porous polymers such as polyacrylamide, acrylic acid polymer, styrenedivinylbenzene polymer, and polymethacrylate are used, and of these, it is more preferable to use agarose gel sold under the trade name of Sepharose.

The immobilization of the compound having a sialo-oligosaccharide is carried out according to a conventional method (for example, "Shinsei Chemistry Laboratory Course, Protein 1" P227-237, issued by Tokyo Kagaku Dojin, etc.) depending on the carrier to be used. When a polysaccharide is used as a carrier, it is carried out by binding a functional group of a compound having a sialo-oligosaccharide as a ligand to a polysaccharide gel introduction group.

In binding the polysaccharide gel-containing compound with the sialo-oligosaccharide, it is necessary to select a polysaccharide gel having an introduction group corresponding to the functional group of the sialo-oligosaccharide-containing compound. That is, when the functional group is an amino group, an imino group activated with cyanogen bromide, a ω-carboxyalkyl group,
Activated carboxyl group (carboxy phthalate group),
Polysaccharide gel having an epoxy group or the like as an introduction group, when the functional group is a carboxyl group, ω-aminoalkyl group,
It is necessary to select a polysaccharide gel having a thiol group or the like as an introduction group and a polysaccharide gel having an epoxy group as an introduction group when the functional group is an alcoholic hydroxyl group.

When a porous polymer is used,
The compound having a sialo-oligosaccharide can be immobilized by adsorbing it to the porous material.

The carrier (hereinafter referred to as "sialo-glycan-binding carrier") on which the sialo-glycan-containing compound prepared as described above was immobilized was used as a column for producing influenza HA. In order to obtain a purified HA protein, the HA protein-containing solution may be applied to a sialo-glycan binding carrier, the HA protein may be selectively adsorbed, then washed, and finally the HA protein may be eluted.

As the HA protein-containing liquid used in the present invention,
After infecting insects such as silkworms and armyworms with nuclear polyhedrosis virus recombined with HA protein, body fluids obtained from these,
These insect cultured cells were infected with the nuclear polyhedrosis virus for HA protein expression, and the cultivated homogenate and the like were treated with an organic solvent such as ether or ethanol or a surfactant to remove the influenza outer coat or cell membrane. A solution in which HA protein is solubilized can be used.

The HA protein-containing solution may be applied to the sialo-glycan-bonding carrier according to a conventional column chromatography technique (or batch method). That is, for example, first, a column with a sialo-oligosaccharide-bonding carrier is packed and equilibrated with a buffer solution such as a phosphate buffer solution (pH 7.0).

Then, the HA protein-containing solution from which solid matters have been removed by filtration, centrifugation, etc. is added to a p-ion using an appropriate buffer solution or the like.
After adjusting the pH to about H6 to 11, preferably about pH 6.5 to 10, apply 1 to the equilibrated sialo-glycan binding carrier column.
Injection is performed at a flow rate (linear flow rate) of about 0 cm / h to adsorb HA protein. The ionic strength of the HA protein-containing solution at this time is as follows when the surfactant is removed by means such as dialysis.
It is about 0.001 to 2, preferably about 0.001 to 0.5, and when the HA protein-containing solution contains a surfactant, the preferable ionic strength is 0.001 to 0.1. . As a specific example of the buffer solution used here, 150
10 mM phosphate buffer (pH 7.
0) and 20 mM Tris-hydrochloric acid buffer solution (pH 8.2) containing 0.1% Triton X-100.

Further, a washing solution having a pH equal to or higher than the pH used for adsorption of HA protein in this column, or higher ionic strength, for example, about 0.001 to 2, preferably 0.0.
The column is washed with a detergent having an ionic strength of about 01 to 0.5, more preferably about 0.001 to 0.2 to remove impurities. An example of the cleaning agent used is 10 mM borate buffer (pH 1) containing 0.1% Triton X-100.
0.0) and a 10 mM phosphate buffer solution (pH 7.0) containing 1 M NaCl.

Finally, the HA protein is eluted from the column by using an appropriate buffer solution having a higher ionic strength than the buffer solution used for equilibration and washing of the column. It is preferable that the buffer solution contains about 0.05 to 5% of a surfactant, for example, a nonionic surfactant such as polyoxyethylene alkylphenyl ether known as Triton X series or Triton N series. The ionic strength thereof is preferably about 0.1 to 8, and more preferably about 0.2 to 2. An example of such a buffer is 4M NaCl.
Phosphate buffer containing 0.1% Triton X-100 and 2
10 mM borate buffer containing 0.1OmM NaCl, 0.1
Examples include 100 mM borate buffer containing% Triton X-100.

Instead of the above buffer, a buffer containing 50 mM or more of N-acetylneuraminic acid (NANA) or a buffer containing an ionic surfactant such as sodium deoxycholate (DOC) is used. You can also
The pH of the buffer solution in these cases is preferably about 6 to 11, particularly about 6.5 to 10. The ionic surfactant such as DOC is preferably 0.2% or more, and particularly preferably 0.5% or more.

The eluted HA protein fraction is considerably purified as it is, but may be further purified by the same column chromatography again, and if necessary, further purified by means such as dialysis and ion exchange chromatography. You may. In particular, it is advantageous to carry out purification using anion exchange chromatography such as DEAE-Cephalofine, since a purified HA protein having an extremely high purity can be produced.

The purified HA protein thus obtained is
Since it has a high degree of purification and contains almost no contaminants such as DNA, it can be used as a safe influenza vaccine. When the purified influenza HA protein of the present invention was administered to mice, sufficient antibodies against influenza to be used as an influenza vaccine could be produced.

[0022]

The present invention utilizes the fact that the HA protein specifically binds to the sialo-oligosaccharide, and this binding is easily dissociated under high salt concentration and high pH conditions (and optionally in the presence of a surfactant). Is.

That is, first, when the HA protein-containing liquid is applied to a column packed with a sialo-glycan binding carrier under substantially neutral conditions, the HA protein specifically binds to the sialo-glycan. On the other hand, among the contaminants, the positively charged protein binds nonspecifically to the negatively charged sialo-oligosaccharide, and the negatively charged protein cannot bind and elutes. Then high pH
When the column bound with HA protein was washed with the washing solution of 1., most of the bound contaminant proteins changed from negative to positive, were eluted from the column, and were strongly negatively charged, and bound specifically. Only HA protein remains on the column. Furthermore, when a high salt concentration solution having a high ionic strength is injected into the column at the same pH as the washing solution, purified influenza HA protein and a small amount of contaminant proteins are eluted from the column (purified influenza HA protein eluent).

The degree of purification of the HA protein is high even in the eluent in this state. However, when the HA protein is further subjected to anion exchange chromatography or the like, the HA protein is adsorbed on the column and the strongly negatively charged contaminating protein is eluted. By recovering the adsorbed HA protein, a highly purified purified influenza HA protein can be obtained (purified influenza HA protein recovery liquid).

[0025]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples.

Example 1 (1) A recombinant virus for HA protein expression was diluted 10-fold, and 50 μl / head was inoculated into silkworm larvae (Akimitsu × Ryuhaku; 5th instar, 1st day). Those that did not move 5 to 6 days after the inoculation were collected and stored frozen, and used as HA protein-expressing virus-infected silkworms. 100 virus-infected silkworms for HA protein expression were homogenized to 1 at 39,800 xg.
It was centrifuged at high speed for 5 minutes. The obtained precipitate was solubilized with a 10 mM phosphate buffer (pH 7.0) containing 2% Triton X-100 (manufactured by Wako Pure Chemical Industries, Ltd.).
Ultracentrifugation was performed at 000 × g for 1 hour to obtain a supernatant (ultracentrifuged supernatant).

(2) CNBr-activated Sepharose 4B (manufactured by Pharmacia) and fetuin (NOF2379 manufactured by Sigma) were coupled to the obtained supernatant in accordance with the manual to prepare a fetuin-sepharose column (CNBr-activated Sepharose 4B). 10 mg of fetuin was bound to 1 ml). The column was first loaded with 10 volumes of 0.1% Triton X-100 in 10 mM phosphate buffer (pH
7.0) followed by 0.1% Triton X-100 in 10
It was washed with mM borate buffer (pH 10.0). 0.
The peaks eluted with 100 mM borate buffer containing 1% Triton X-100 were collected and purified influenza HA
It was used as a protein eluent.

(3) The purified influenza HA protein eluate thus obtained was added to 10 m containing 0.1% Triton X-100.
It was dialyzed against M phosphate buffer, and this dialysate was further applied to a Fetuin-Sepharose column.
The peak portion was collected in the same manner as above to obtain a dialysis collected product. The obtained dialysis recovery product is diluted 2-fold to obtain DEAE-Cellu.
Adsorbed on lofine A-800, Triton X-1
20 mM Tris-HCl (pH 8.
After washing with 2) to remove Triton X-100, 200
A purified influenza HA recovery solution was obtained by dropping mM NaCl.

(4) Next, the HA protein concentration, the liquid amount, the HA protein amount and the total protein amount in each of the above steps are shown in Table 1, and the recovery rate, the degree of purification and the purity obtained from them are shown in Table 2. .

[0030]

[0031]

As is clear from these results, the purified influenza HA protein having a high degree of purification was obtained at a high recovery rate by the method of the present invention. That is, a purified influenza HA protein eluent having a considerably high degree of purification can be obtained by a single treatment of the Fetuin-Sepharose column, and DE
By combining with an AE-cellulofine column, a purified influenza HA protein recovery liquid having an extremely high degree of purification was obtained.

Comparative Example (1) The free end immobilized on the carrier was NeuAcα2,
The results of testing a compound having 3 (6) Galβ1,3 (4)-and two sugar molecules are shown below. The free end is NeuAcα2,3 (6) Galβ1,3 (4)-,
As a compound of two sugar molecules, NeuAc-Gal / G
A mucin-type glycoprotein having a structural formula of alNAc was used. (2) In the same manner as in Example 1, 10 silkworms infected with HA protein-expressing virus were homogenized to give 39,800 ×
High speed centrifugation at g for 15 minutes. 20 mM Tris-HCl buffer containing 2% Triton × 100 (pH)
7.0) to solubilize the resulting solubilized product to 141,000 × g
Ultracentrifugation was performed for 1 hour to obtain a supernatant.

(3) The obtained supernatant was coupled to CNBr-activated Sepharose 4B (Pharmacia) and mucin-type glycoprotein (Sigma) according to the manual to prepare a mucin-sepharose column (CNBr).
10 mg of mucin-type glycoprotein was bound to 1 ml of activated Sepharose 4B). (4) 20 mM containing 0.1% Triton X-100
It was washed with Tris-HCl buffer and eluted with 2M NaCl buffer. Table 3 shows the results of measuring the HA protein in each step by the enzyme immunoassay method.

[0035]

(5) From the results shown in Table 3, H was detected in the column effluent.
It can be understood that the amount of A protein contained was 75% or more, and the mucin-sepharose column had almost no adsorptivity for HA protein. It should be noted that similar results were obtained when various buffer solutions such as 10 mM phosphate buffer solution were used as other solubilization conditions. (6) Therefore, the free end immobilized on the carrier is Neu
Even if Acα2,3 (6) Galβ1,3 (4)-,
Compounds with less than 3 sugar molecules are purified influenza HA
It can be said that it is not suitable for protein production.

Example 2 The amount of nucleic acid contained in the purified influenza HA recovery solution obtained in Example 1 was measured by a threshold system using an anti-DNA antibody (Molecular Device). Table 4 shows the result.

[0038]

The HA antibody titer that can be used as a vaccine is
Approximately 3-4p when converted into a single dose (1shot) that rises to the same level as or higher than that of a commercially available influenza HA vaccine
g of nucleic acid content.

[0040] Concerning the safety of nucleic acid contamination in pharmaceuticals made by recombinant DNA technology, there is the risk of transferring the oncogene to the recipient at the time of vaccination,
According to a summary of the World Health Organization (WHO) committee, a nucleic acid content of 100 pg / shot of heterologous nucleic acid is a level at which the risk of developing a tumor in the recipient is negligible (WHO Technical Report).
No. 747, 1987. and Developmen
tal Biology Standards, 68: 4
3-49, 1987).

On the other hand, according to the national test method of influenza vaccine according to the Japanese standard for biological products, 2 per 1 ml.
It should not contain more than 40 μg of protein (Tamura / Kurata “Infection / Inflammation / Immunity” 21-
(1), 1-10.1991), the purified influenza HA protein recovery solution obtained by the method of the present invention has a nucleic acid content of about 3-4 p in this maximum protein amount of 240 μg / ml.
g, which is considered to be sufficiently low. That is, it is understood that the purified influenza HA protein recovery solution obtained by the method of the present invention has a remarkably low nucleic acid content and is very excellent in safety.

[0042]

INDUSTRIAL APPLICABILITY According to the present invention, H containing various impurities
The purified HA protein can be purified to a high purity from the A protein-containing solution, and this can be obtained at a high recovery rate. In addition, as shown in the examples, the purified influenza HA protein produced by the present invention has very little contamination with nucleic acid, and was produced by recombinant DNA technology such as oncogene derived from heterologous or homologous DNA to be contaminated. It can be said that the drug is highly safe. Therefore, according to the production method of the present invention, it is possible to economically obtain a highly safe and effective influenza vaccine. that's all

Front page continuation (72) Inventor Kiyoshi Kiyoshi 4-5-25 Chuo, Matsumoto City, Nagano Katakura Industry Co., Ltd., Institute for Biological Sciences (72) Inventor Reiro Shimamura 1548 Miyago, Sayama, Saitama Prefecture Katakura Industrial Co., Ltd. Inside the Central Silkworm Laboratory (72) Inventor Yuu Nomura 1548 Miyago Shimookutomi, Sayama City, Saitama Prefecture Katakura Industrial Co., Ltd. Inside the Central Silkworm Laboratory (72) Inventor Sasaki Kinno 1-16-13 Kitakasai, Edogawa-ku, Tokyo Daiichi Pharmaceutical Co., Ltd. Tokyo Research and Development Center (72) Inventor Haruo Makino Kawabe Asahishi Village, Kikuchi District, Kumamoto Prefecture Chemistry and Serum Therapy Laboratory Kikuchi Laboratory

Claims (29)

[Claims] 1. An influenza HA protein-containing solution obtained by expression in insects or insect cultured cells using recombinant DNA technology, wherein the free end immobilized on a carrier is NeuAc.
Production of a purified influenza HA protein characterized by elution and recovery of influenza HA protein after adsorption to a compound having at least three sugar molecules which is α2,3 (6) Galβ1,3 (4)- Law. 2. The free end is NeuAcα2,3 (6) G.
The method for producing a purified influenza HA protein according to claim 1, wherein the compound having at least three sugar molecules which is alβ1,3 (4)-is a saccharide, a glycoprotein or a glycolipid. 3. The free end is NeuAcα2,3 (6) G.
The method for producing a purified influenza HA protein according to claim 1 or 2, wherein the compound having at least three sugar molecules which is alβ1,3 (4)-is selected from fetuin, glycophorin or ganglioside. 4. The free end is NeuAcα2,3 (6) G.
The method for producing a purified influenza HA protein according to any one of claims 1 to 3, wherein the compound having at least three sugar molecules which is alβ1,3 (4)-is fetuin. 5. The method for producing a purified influenza HA protein according to claim 1, wherein the carrier is a polysaccharide gel or a porous polymer. 6. The carrier polysaccharide gel is agarose gel,
The method for producing a purified influenza HA protein according to any one of claims 1 to 5, which is selected from cellulose gel and dextrin gel. 7. The carrier porous polymer is silica gel,
The method for producing a purified influenza HA protein according to any one of claims 1 to 5, which is selected from polyacrylamide, acrylic acid polymer, styrenedivinylbenzene polymer and polymethacrylate. 8. The carrier is an agarose gel.
7. The method for producing the purified influenza HA protein according to any one of items 6 to 6. 9. The method for producing a purified influenza HA protein according to claim 1, wherein the influenza HA protein is eluted at a higher ionic strength and a higher pH than when adsorbed. 10. The method for purifying influenza HA protein according to claim 1, wherein the influenza HA protein is eluted with a buffer containing a surfactant. 11. The influenza HA protein is eluted with a buffer containing a nonionic surfactant.
10. The method for purifying influenza HA protein according to any one of 10 above. 12. The method for purifying influenza HA protein according to claim 1, wherein the influenza HA protein is eluted with a buffer containing polyoxyethylene alkylphenyl ether. 13. The influenza HA protein is eluted with a buffer containing deoxycholate.
0. The method for producing the purified influenza HA protein according to any one of 0. 14. The influenza HA protein is eluted with N
-The method for producing a purified influenza HA protein according to any one of claims 1 to 10, which is carried out using a buffer solution containing acetylneuraminic acid. 15. The method for producing the purified influenza HA protein according to claim 1, wherein the influenza HA protein-containing solution is produced by a protein expression system using an insect nuclear polyhedrosis virus as a vector. . 16. The method for producing a purified influenza HA protein according to claim 1, wherein the influenza HA protein-containing solution is produced by a protein expression system using a silkworm nuclear polyhedrosis virus as a vector. . 17. The method for producing a purified influenza HA protein according to claim 1, wherein the influenza HA protein eluate is subjected to anion exchange column chromatography to recover the influenza HA protein. 18. The method for producing a purified influenza HA protein according to claim 17, wherein the anion exchange column chromatography is a carrier to which DEAE is bound. 19. The method for producing the purified influenza HA protein according to claim 17 or 18, wherein the anion exchange column chromatography is DEAE-cellulofine. 20. An eluent containing an influenza HA protein obtained by the method according to any one of claims 1 to 17. 21. A recovery solution containing influenza HA protein obtained by the method according to any one of claims 1 to 19. 22. NeuAcα2,3 (6) having a free end
A column for producing influenza HA protein, which comprises a Galβ1,3 (4) -compound having at least three sugar molecules immobilized on a carrier. 23. The free end is NeuAcα2,3 (6).
The compound having at least three sugar molecules which is Galβ1,3 (4)-is selected from fetuin, glycophorin or ganglioside.
A column for producing the purified influenza HA protein described. 24. The free end is NeuAcα2,3 (6).
The compound having at least three sugar molecules which is Galβ1,3 (4)-is fetuin.
Or a column for producing the purified influenza HA protein according to 23. 25. The column for producing a purified influenza HA protein according to any one of claims 22 to 24, wherein the carrier is a polysaccharide gel or a porous polymer. 26. The column for producing a purified influenza HA protein according to any one of claims 22 to 25, wherein the carrier polysaccharide gel is selected from agarose gel, cellulose gel and dextrin gel. 27. The carrier porous polymer is silica gel,
The column for producing a purified influenza HA protein according to any one of claims 22 to 25, which is selected from polyacrylamide, acrylic acid polymer, styrenedivinylbenzene polymer and polymethacrylate. 28. The carrier according to claim 2, which is an agarose gel.
A column for producing the purified influenza HA protein according to any one of 2 to 26. 29. The free end is NeuAcα2,3 (6).
The purified influenza HA protein according to any one of claims 22 to 24, wherein the compound having at least three sugar molecules, which is Galβ1,3 (4)-, is fetuin, and the carrier to be immobilized is agarose gel. Column.