KR20000052109A - A process for producing antigens of hepatitis B vaccine - Google Patents

Abstract

PURPOSE: A method for preparing vaccinia virus of hepatitis A is provided for simplifying steps of preparation of a large scale of virus antigen using a suspension media which does not contain Ca ion and glutamine. CONSTITUTION: A method for preparing vaccinia virus comprises: inactivating and proliferating by a cell culture of virus; recovering a virus from the proliferated cell; concentrating and then purifying the recovered virus by concentration gradient-ultracentrifugation using solution for concentration gradient which is harmless to human; and preparing the vaccinia virus of hepatitis A by performing dialysis to remove the residual solution.

Description

A process for producing antigens of hepatitis B vaccine

The present invention relates to a method of culturing the hepatitis A virus using cell culture and to isolate and purify the virus from the cultured cells to produce an antigen of the hepatitis A vaccine.

The proliferation of hepatitis A virus through cell culture was first confirmed by Provot and Hilleman in 1979 (Provost, PJ, Hilleman, MR, Proc. Soc. Exp. Biol. Med., 1979, 160, p.213). The proliferation of hepatitis A virus was then confirmed in various cell cultures. Proliferation of hepatitis A virus is mostly done using monkey kidney cells, BS-C-1 cells, BGM, VERO, FRhK-4, and human lung cells, MRC-5 cells (Journal of General Virology, 1991, 72). , p. 2159-2166).

When hepatitis A virus is infected with cultured cells, it proliferates in the cell and exists as two types of virus in the cell. The incomplete virus form includes only the complete virus including the RNA of the hepatitis A virus and the viral envelope without RNA. It's in the cell. Both types of virus in cultured cells can be used as antigens for the hepatitis A vaccine (Summary of 35th Meeting of the Society of Japanese Virologists, 1987, p.257), and the virus can be broken down to obtain free virus. Freeze-thaw methods of cultured cells were mainly used (Journal of Virology, 1986, p. 307-313, vol 58, No. 2). Conventional techniques for obtaining free viruses have concentrated the virus by polyethylene glycol (PEG) precipitation and centrifugation to fractionate the virus (Virology, 155, 1986, p.732-736).

Most of the proliferative cells of hepatitis A virus are adherent cells, which are cells growing on the culture vessel. In order to recover the virus present in the cell, a process of detaching adherent cells from the container is required. Until now, most of them have been freeze-thawed or trypsin was used and then ultrasonic crushing was used. However, the conventional method has a disadvantage in that it is difficult to use industrially when culturing a virus using a large amount of culture vessel. In addition, polyethylene glycol and cesium chloride (CsCl), which are used in the concentration and purification of viruses, are inadequate materials for human use and require a lot of time when a large amount of virus stock is concentrated.

As a drawback in the above-described manufacturing process of the hepatitis A vaccine antigen, the freeze-thaw process and the enzyme-ultrasonic treatment technology for virus recovery through mass cell culture are limited in industrial applications in the mass virus recovery. In addition, virus concentration and purification using polyethylene glycol or cesium chloride solution has disadvantages that are not suitable for vaccine production by using a substance harmful to the human body. Therefore, in the present invention, in order to effectively recover the virus from the cultured cells in which the virus has grown, the virus could be effectively separated by simply changing the medium by using a suspension medium for cell culture. Concentration of the viral stock solution could achieve time and device savings by using ultrafiltration instead of PEG concentration. The solution used for the purification of the virus used a concentration gradient prepared directly, this material can be used as a medical contrast agent was used to increase the safety of the vaccine. The concentration gradient remaining in the vaccine stock was removed by dialysis. The suspended media used in the process of separating viruses from virus cultured cells is a medium that does not contain calcium ions and glutamine, and when it is added and cultured for 3 days, the cells attached to the culture vessels fall off and the cells in the cells are broken down. Spill out of the cell. When the virus was recovered by adding a suspension medium, the virus could be separated out of the cell simply by replacing the suspension medium with the cultured cells in which the virus had grown. The cells were simply removed by centrifugation or a filter, and only the virus stock solution was separated. Could. After the virus stock solution was concentrated by ultrafiltration, a concentration gradient consisting of a concentration gradient from 30% to 76% was prepared, and the concentrated virus was centrifuged on the upper layer to obtain a virus fraction according to the density gradient. Depending on the type of virus, two types of virus were obtained, one containing the virus in its full form and one without RNA, and both of them were antigenic to the hepatitis A vaccine.

1 is a ELISA antigen of hepatitis A virus according to the fraction number after concentration gradient ultracentrifugation according to the present invention.

Figure 2 HPLC using a Toyo TSK 3000 SW column before dialysis of the virus stock solution to remove the concentration gradient.

Figure 3 HPLC using a Toyo TSK 3000 SW column after dialysis of the virus stock solution to remove the concentration gradient in accordance with the present invention.

The present invention is infected with a hepatitis A virus to the host cell and cultured, and added to the culture medium and added a culture medium containing calcium ions and glutamine, and then cultured to allow the virus to flow out of the cell, centrifugation or filtration To remove the virus stock solution, and concentrate and purify the virus stock solution to provide a hepatitis A vaccine antigen.

Hereinafter will be described in more detail with respect to the method for producing a hepatitis A vaccine antigen according to the present invention. In the method for preparing the hepatitis A vaccine antigen according to the present invention, first, MRC-5 cells (ATCC CCL-171), which are viral host cells, are cultured in a roller bottle for one week. After the cells were completely grown on the culture vessel, hepatitis A virus (ATCC VR-1357, strain PA21) was infected and replaced with a minimal medium, and cultured for 4 weeks, and the medium was changed once a week. After the incubation of the virus, the cultured cells were washed with phosphate buffer, suspended media, and cultured for 3 days to release the virus in the cells. The cells were centrifuged and removed, and the supernatant was taken as the virus stock solution. Inactivated at 37 ° C. for 12 days by addition to% (v / v). The virus stock was concentrated to 1/20 with a filter (Filtron, Ultrasette ^™ ) having a pore size of 100 KDa. Concentrated virus was put on a tube gradient gradient of 30%, 40%, 50%, 60%, 76%, and ultracentrifuged for 18 hours at 10 ° C and 40,000 rpm. After centrifugation, the bottom of the tube was drilled to obtain a fraction, and the antigenicity of the hepatitis A virus was measured for each fraction (FIG. 1). The antigenic fractions were collected and mixed with 3 times phosphate buffer and centrifuged at 10 ° C. and 30,000 rpm for 6 hours to obtain virus precipitates. Left to remove residual concentration gradient (FIG. 3). The hepatitis A virus solution obtained here was used as an antigen of the hepatitis A vaccine.

Example 1

Virus isolation from virus cultured cells

MRC-5 cells infected with hepatitis A virus were cultured in a roller bottle of 850 cm 2 for 4 weeks at 37 ° C. The composition of the medium used was as follows.

ESS: 9.4 g / L

Fetal bovine serum (FBS) 2%

Sodium bicarbonate2.2 g / L

Neomycin / Penicillin 100 U / ml

L-glutamine2 mM

Once inoculated with the virus, the medium was exchanged 200 ml per roller bottle once a week. When the virus proliferated, the cultured cells were washed three times with 100 ml of phosphate buffer, and the suspension medium was placed in one roller bottle at 100 ml to release the virus in the cells for 3 days. Suspension medium is a medium that does not contain calcium ions, L-glutamine and serum, and is a medium characterized by stopping the growth of cells and detaching adherent cells from the surface of the roller bottle to release the virus in the infected cells out of the cells. The recovered virus was centrifuged at 8,000 rpm for 20 minutes to remove cells and recover the supernatant. The composition of the suspension medium is as follows.

Minimum essential medium containing eagle salt 8-10 g / L

(Without CaCl ₂ and L-glutamine)

Sodium bicarbonate1.5-3 g / L

Example 2

Viral Antigen Comparison of Suspension and Freeze-thaw Methods

As a method of recovering the viral antigen from the culture cells, the amount of the antigen was compared by comparing the suspension medium and the freeze-thaw method. After culturing the virus in an 80 cm 2 flask, 20 ml of the suspension medium was added and recovered 3 days later. 20 ml of phosphate buffer was added and the mixture was recovered by freezing on an ethanol-dry ice mixture and thawing in a 37 ° C. water bath three times. Got it. Each recovered solution was centrifuged at 8,000 rpm for 20 minutes to obtain a supernatant to obtain virus suspension. The remaining cells were placed in 5 ml of phosphate buffer and sonicated at 100W for 1 minute to recover the remaining virus in the cells. Each sample was measured for antigen by ELISA method and the results are shown in Table 1 below.

Virus Antigen Recovery Method Antigen (ELISA unit / 80 cm 2 flask) Suspension Virus suspension 3.85 After cell disruption 0.483 Freeze-thaw Virus suspension 3.5 After cell disruption 0.924

From the above results, there was no difference between the antigens recovered by adding the suspension medium and the freeze-thaw method, and the virus antigens in the cells were more freeze-thawed than the recovery method using the suspension medium. It can be seen that. Therefore, in the case of using a suspension medium, most of the virus antigens in the cells could be recovered by only one medium change.

Example 3

Virus enrichment

The virus recovery solution was concentrated to 1/20 by ultrafiltration using Ultrasette ^™ product of Filtron, to prepare a virus concentrate.

Example 4

Preparation of Concentration Gradient

In 1 ml of water for injection, 660 mg of diatrizoate meglumine and 100 mg of diatrizoate sodium were dissolved, and the pH was adjusted between 6.0 and 7.7 with sodium carbonate solution, sodium hydroxide solution or hydrochloric acid. This solution was diluted gradually with 76% of the stock solution and used as a filling solution for concentration gradient ultracentrifugation. The viscosity of the concentration gradient was 15 cps at 25 ° C., 9.1 cps at 37 ° C., and the osmotic pressure was 1870 mOsm / kg.

Example 5

Ultracentrifugation Process

Sucrose, cesium chloride, and the like are used as concentration gradient materials in the hepatitis A virus purification method, but the concentration gradient solution was used in the present invention. A tube of 100 ml capacity was prepared, and the concentration gradient was prepared by diluting the concentration gradient in phosphate buffer, followed by the following procedure.

1.76 ml concentration gradient 15 ml

2. 60% concentration gradient 15 ml

3. 50% concentration gradient 15 ml

4. 40% concentration gradient 15 ml

5. 30% concentration gradient 10 ml

6. Hepatitis A virus concentrate 30 ml

100 ml total capacity

Each tube filled with the above-mentioned method was put in a 45Ti rotor, centrifuged at 10 ° C. and 40,000 rpm for 18 hours, and then, each of the tubes was punched in the bottom of the tube. Only fractions containing viral antigens were recovered (FIG. 1).

Example 6

Concentration gradient removal process

In order to remove the concentration gradient contained in the fraction recovered in Example 3, diluted with three times the volume of phosphate buffer solution of the collected fractions and centrifuged for 6 hours at 30,000 rpm to obtain a virus precipitate. The supernatant was immediately removed after centrifugation and dissolved by injecting water into the virus precipitate. At this time, most of the concentration gradient was removed, but the trace amount remained, and dialysis was performed to remove it again. The virus solution was placed in a dialysis bag having a molecular weight size (MWCO) of 10,000 and placed in a phosphate buffer, and left at 4 ° C. for 12 hours to remove the residual concentration gradient. In order to confirm that the concentration gradient was removed, the fraction was analyzed at 280 nm by HPLC using a Toyo TSK 3000 SW column to confirm that the concentration gradient was significantly removed after dialysis (FIG. 3). As a control, the fractions before the concentration gradient was removed were HPLC, and the results are shown in FIG. 2.

The present invention can efficiently recover the virus present in the cells outside the cells by using a suspension medium in the method of producing the antigen of the hepatitis A vaccine, thereby simplifying the large-scale virus antigen production process. In the purification process, the virus was purified by concentration gradient ultracentrifugation using a concentration gradient solution that is harmless to the human body, and only a hepatitis A virus antigen was obtained by removing a trace concentration gradient remaining through dialysis.

Claims (3)

Hepatitis A virus is infected with a host cell and cultured, and the culture medium is added with a suspension containing calcium ions and glutamine and cultured to allow the virus to flow out of the cell, and centrifuged or filtered to remove the cells. A method of producing a hepatitis A vaccine antigen by obtaining a viral stock solution, which is concentrated and purified. The method of claim 1, wherein the concentration of the viral stock is carried out by ultrafiltration. The method according to claim 1, wherein the virus is purified by ultracentrifugation using a concentration gradient containing diazoate meglumine and diazotate sodium as a concentration gradient.