SK659090A3 - Serumfree cell cultures useful for antigens preparation and process for producing flavivirus/virus antigen or arenavirus/virus antigen - Google Patents

Abstract

A matrix or substrate bears adherently bound human or animal cells infected with a virus. It has been shown that surface dependent cells useful for virus multiplication remain adherently bound to a matrix even when infected with a virus, produce virus antigens over a relatively long period of time and release them in the culture medium. In order to produce early Summer meningo-encephalitis (FSME) virus antigens by cultivating the FSME virus in cell cultures, a surface dependent permanent cell line, preferably the vero-cell line ATTC CCL 81, is inoculated with the FSME virus and the cells are bound to substrates and kept in a non lytic serum free system in conditions that ensure cellular growth, so that antigens are produced. The antigen-containing medium is then separated from the substrate bound cells and processed in a known manner by concentration, inactivation and purification until a galenically acceptable composition is obtained.

Description

SERUM-FREE CELL CULTURES FOR THE PRODUCTION OF ANTIGENES AND METHOD OF MANUFACTURE OF FLAVI-VIRUS / VIRUS ANTIGEN OR ARENAVIR / VIRUS ANTIGEN

Technical field

The invention relates to a serum-free cell culture for the production of antigens and to a method for producing a flavivirus / viral antigen or arena-virus / viral antigen using said culture.

BACKGROUND OF THE INVENTION

Infections caused by early summer meningoencephalitis virus (FSME) have been observed in Europe since World War II. In Austria, southern Germany and Czechoslovakia, several hundred patients are treated every year due to a transmissible FSME infection.

The FSME virus is classified in the Flavivirus family, the former serological group B of the arbovirus, which represents the family of viruses / Togaviridae.

There has been a long time vaccine against one of the most important and most common agents of encephalitis in humans, the Japanese B-virus encephalitis. These vaccines are obtained from the brain of infected mice, purified and are considered safe and safe [Hoke et al., N. Engl. J. Med., 319, 608 (1988).

Since r. In 1976, an FSME vaccine is available and authorized by the health authorities. To produce this vaccine, the virus is cultured in the brain of infected mouse pups, propagated in chick embryonic cells, inactivated with formalin, and then subjected to effective purification (Heinz et al., J. Med. Virol., 6, 103 (1980) //.

A number of possibilities of arbovirus propagation with respect to the possible production of a vaccine are described in the literature. Today, the most commonly used method is to vaccinate chicken embryonic fibroblasts with a virus grown

FSME obtained from mouse brain and by culturing the inoculated cells. This method requires costly purification of the antigen in order to remove complex, heterologous biological material and to avoid a sensitive effect in vaccinated persons when repeated doses of the vaccine obtained therefrom.

For the production of embryonic cells of chickens, the SPF / = specific pathogen free = egg must be based on. These SPF eggs must undergo a number of long-term tests to maintain their SPF status before each use.

Furthermore, chicken embryonic cell cultures exhibit only a small number of generations in further cultivation, thereby limiting batch sizes, culturing the primary culture with difficulty maintaining sterile, and there is no sustained quality of primary cells with respect to virus propagation and antigen production.

These drawbacks do not lie solely in the method of producing FSME virus for antigen production.

It is an object of the present invention to improve the production of virus / virus antigen, in particular FSME-virus / virus antigen, by eliminating the above-mentioned deficiencies and providing a method for culturing virus / virus antigen in cell cultures which allows large scale production to be carried out. while keeping the culture sterile in a simple manner. Further, the release of unwanted cellular proteins into the grown culture should be minimized.

SUMMARY OF THE INVENTION

An object of the present invention is a serum-free cell culture comprising a matrix with adherent human or animal cells adhering thereto for the production of flavivirus / viral antigen or arenavirus / viral antigen, wherein the cells are infected with flavivirus or arena-virus.

The invention is based on the discovery that surface-dependent cells which are suitable for virus multiplication remain adherently bound to the matrix even in an infected state, produce a continuous virus antigen for a relatively long time and pass it on to the culture medium.

It is possible to store the composition of the invention with the infected cells absorbed for a few days at a temperature between 0 ° C to 8 ° C, that is to say, under conditions in which the metabolism of the cell and hence the production of the virus is inhibited. The composition stored in this manner can then be used without difficulty by introducing it into the culture medium and adjusting the culture conditions to produce the virus antigen. Thus, the composition of the invention is a starting culture that can be produced with constant quality and activity in stock and whose condition can be easily tested for sterility and can be used at any time to produce a virus antigen.

Furthermore, the binding of antigen-producing cells to a carrier allows quite simple manipulation of virus-infected cells and capable of production. Thus, for example, the production of virus antigen can be carried out continuously in a perfusion reactor. The separation of the cells from the antigen-containing medium is substantially facilitated by their binding to the matrix, thereby facilitating large-scale production of the virus antigen.

A preferred embodiment of the composition according to the invention consists in using ATTC CCL 81 vero-cells, which are preferably used for the production of early summer meningoencephalitis (FSME) antigen, and therefore are infected with FSME virus as adherently bound cells.

However, cells bound adherently to the matrix may also be infected with flavivirus or arenavirus.

Glass, transverse crosslinked dextran, gelatin or plastics have proven to be a good material for the matrix, the matrix being best formed as a microcarrier, preferably having a particle diameter in the region of 100 μιτι to 300 μητ. porous structure.

Another preferred embodiment of the cell culture according to the invention is characterized in that between 1 x 10 ⁵ and 4 x 10 ⁵ cells are adherently bound on cm ^{2 of} the matrix surface.

The present invention further provides a method of producing a flavivirus / viral antigen or an arena-virus / viral antigen using said cell culture, comprising the step of inoculating a surface-dependent permanent cell, preferably vero-cells of ATTC CCL 81, with a flavivirus / viral antigen. virus and cells are maintained in the serum-free medium with their viability adherently bound to the matrix to maintain antigen formation in the cells and delivery of the antigen to the medium, then the antigen-containing medium is separated from the cells bound to the carrier and processed in a known manner , by inactivation and purification to a galenically acceptable preparation.

The vertebrate cell line ATTC CCL 81 is obtained from green vermin kidney tissue (cercopithecus aethiops) and can be stored metabolically active in serum-free medium. For such permanent cell lines, a parent cell bank and a parent cell bank are set up once and all contaminant examinations are performed. Thus, this permanent cell line is precisely characterized not only with respect to the absence of contaminating microorganisms, but also with regard to growth, culture, reproduction behavior and - once optimized - can be regarded as constant.

In the method according to the invention, the cells which are bound to the microcarrier are preferably used. This makes it possible to achieve high cell density, which can not be achieved with previously used primary cell structures or in Roux-flasks or in suspension and further reaches of ^r to increase the amount of virus antigen fermentation.

A preferred embodiment of the invention consists in that virus propagation and antigen production is carried out in a continuously operated perfusion reactor for at least 5 days, at a temperature between 34-37 ° C, wherein perfusion can be carried out with a perfusion yield of 0.3 to 5 ° C. /in day. In a perfusion reactor, a cell density of 2 x 10 ⁹ to 2 x 10 ¹ ° cells per liter of fermentation volume can also be achieved, the last reading for a fluidized bed fermenter.

The multiplication of the virus according to the invention in the perfusion culture allows a substantial reduction in the persistence of the virus and the antigen in the medium, which is predetermined by the perfusion yield, compared to the batch culture. Due to the shorter residence time, the thermal inactivation is considerably lower and thus the productivity according to the invention is higher. Thus, an antigen concentration of 1 to 10 µg / ml can be obtained and maintained in the perfusion medium.

In the process according to the invention, the optimum culture conditions can be set in a simple manner. In addition, there are considerably fewer manipulations in the method of implementation than all other known methods, which provides greater safety in the handling of infectious material and allows continuous rapid processing of virus and virus antigen from the culture medium.

The production of the virus inoculum, the cultivation of the cells for the production of the virus or the virus antigen and the actual production of the virus or the virus antigen will be described in more detail.

DETAILED DESCRIPTION OF THE INVENTION

1. Virus inoculum

Cells (e.g. Vero ATTC CCL 81) are cultured in cylindrical flasks at 37 ° C until confluency and infected with 1 ml of virus suspension.On the day after infection, half of the medium is replaced daily with serum-free medium. Excess medium from day 4 through day 8 contains 2-5 x 10 ⁷ pfu per ml and stored at -20 ° C as virus inoculum until use.

2. Culturing the cells to produce virus / virus antigen

Starting from ATTC CCL 81 working parent cells embedded in liquid nitrogen, propagation of these cells in flasks on culture tissues is performed until a sufficient number of cells are allowed to inoculate the ferment. Further cultivation of the cells takes place in fermentation vessels at 37 ° C, whereby as much surface as possible for adhering to growing working cells should be available for adhesion, adhesion. Such large surfaces are available when using cylindrical bottles of glass or polystyrene or in use

I microcarriers (MC). Preferably, the crosslinked dextran MCs are between 170 and 250 µm in size.

MC with adsorbed basal cells is cultured at 37 ° C up to a cell density of 1.10-4.10 cells per cm. This cell density is generally reached after six days. During cultivation, the microcarrier will grow perfectly by the cells, and ultimately the individual microcarriers may grow together with their cell lawns adhering to the surface together to form groups.

3. virus / virus antigen production

Once said cell density has been reached, cells bound to the MC by virus inoculum (1 - 0.01 pfu / cell, preferably 0.1 pfu / cell) are infected to produce the matrix of the invention. The matrix according to the invention can be stored for several days at a temperature between 0 ° C to 8 ° C and used immediately for the production of the virus antigen.

For antigen production, MCs with adsorbed infected cells are introduced into a perfusion reactor. Since this time of viral infection, only serum-free medium is used in the culture, which is pumped continuously through the perfusion reactor, while the cells grown on microcarriers are retained by means of a retention device in the reactor. It is present in the effluent culture medium from day 2 after infection of the virus antigen at high concentration and can be obtained therefrom continuously for at least 10 days.

The method according to the invention will be further explained by the following examples. Virus antigen determination was carried out in all examples by ELISA for antigen determination.

Example 1

Vero-cells of ATTC CCL 81 were cultured in a 6-liter microcarrier fermentor (Cytodex 3 from Pharmacia) at 37 ° C up to 2 x 10 ⁶ cells / ml culture medium (DMEM = Dulbecco's Eagle medium) and infected with FSME virus (0). , 1 pfu / cell / and the virus is propagated by batch

Table 1

days after infection pg / ml virus antigen 2 0.20 3 0.70 4 1.60 5 2.70 6 4.00 7 3.80 8 2.90

Productivity of reagent per 1 fermentation volume of 4 mg virus antigen.

b) is infected with FSME virus (0.1 pfu) / cell and the culture medium (DMEM) is perfused continuously with a volume of 0.5 / volume of fermenter / day.

Table 2

days after infection virus antigen pg / ml 2 0.30 3 1.60 4 4.50 5 4.50 6 2.50 7 3.20 8 2.90 9 2.50 10 2.30

Productivity of reagent per 1 fermentation volume of 13.7 mg of virus antigen.

c) is infected with FSME virus (0.1 pfu) / cell and the culture medium (DMEM) is continuously perfused with 1 volume / volume fermenter / day.

Table 3

days after infection virus antigen pg / mi 2 0.45 3 1.40 4 2.00 5 2.00 6 1.70 7 1.60 8 1.10 9 1.10 10 0.90

Productivity of reagent per fermentation volume of 12.4 mg of virus antigen.

Example 2

Vero-cells (ATTC CCL 81) were cultured in a 40 liter microcarrier fermenter (Cytodex 3 from Pharmacia) at 37 ° C up to 2 x ΙΟ / ml and after FSME-virus infection (0.1 pfu / cell) perfused continuously with medium (DMEM) / 0.33 vol./fermentora vol./day/.

Table 4

days after infection virus antigen su pg / ml 2 1.60 3 3.50 4 5.00 5 4.30 6 4.00 7 2.90 8 2.70 9 2.10 10 2.00

Productivity of reagent per fermentation volume of 10.7 IU virus antigen.

Example 3

Vero cells (ATTC CCL 81) were cultured in a 40 liter microcarrier fermentor (Cytodex 3 from Pharmacia) at 37 ° C up to 2 x ΙΟ / ml and infected with FSME virus (0.1 pfu) cell / perfused continuously with medium (DMEM) / 1 vol / fermentor vol / day /.

Table 5

days after infection pg / ml virus antigen 2 1.10 3 3.80 4 3.90 5 3.00 6 2.30 7 2.20 8 2.00 9 3.15 10 2.30

The yield was reduced to 0.5 V / fermenter volume / day.

The productivity per 1 fermentation volume was 13.7 mg virus antigen.

Vero cells (ATTC CCL 81) were cultured in a 150 liter microcarrier fermentor (Cytodex 3 from Pharmacia) at 37 ° C up to 2 x 10 ⁶ cells / ml and perfused with FSME virus (0.1 pfu) / cell / perfused continuously with medium (DMEM) (0.33 v / v fermenter v / day).

Table 6

days after infection virus antigen pg / ml 2 0.20 , 3 1.90 4 2.40 5 4.80 6 5.40 7 4.10 8 4.40 9 3.20 10 4.50

Productivity of reagent per 1 fermentation volume of 14.7 mg virus antigen.

Claims (10)

A serum-free cell culture comprising a matrix with human or animal cells adhering thereto for the production of flavivirus / viral antigen or arena-virus / viral antigen, wherein the cells are infected with flavivirus or arena-virus. Cell cultures according to claim 1, characterized in that they comprise ATCC CCL 81 vero-cells as adherently bound cells. Cell cultures according to claim 1 or 2, characterized in that the matrix consists of glass, cross-linked dextran, gelatin or plastic. Cell cultures according to claim 3, characterized in that the matrix is a microcarrier. Cell cultures according to one or more of Claims 1 to 4, characterized in that 1 x 10 ⁵ to 4 x 10 ⁵ cells are adherently bound to the surface of the matrix per cm ² . ^{6 * * *} Method for producing a flavi-virus / viral antigen or arenavirus / viral antigen using the matrix according to any one of claims 1 to 5, characterized in that surface-dependent permanent cells, preferably vero-cells of ATTC CCL 81, are inoculated with flavi-virus. virus or arenavirus and the cells are maintained in serum-free medium while maintaining their viability, adherently bound to the matrix in order to maintain the serum. preserving antigen production in cells and delivering the antigen to the medium, for which the antigen-containing medium is separated from the carrier-bound cells and processed in a known manner by concentration, inactivation and purification to a galenically acceptable preparation. Method according to claim 6, characterized in that virus propagation and antigen production are carried out continuously for at least 5 days at a temperature in the range of 34 to 37 ° C. Method according to claim 6, characterized in that the perfusion is carried out with a perfusion value of 0.3 to 10 v / v / day. Method according to claim 6, characterized in that a cell density of 2 x 10 ⁹ to 2 x 10 ¹⁰ per liter of fermentation volume, higher in a fluidized bed fermenter, is used. Method according to one or more of claims 6 to 9, characterized in that a virus / antigen concentration of 1 to 10 µg / ml is maintained in the medium.