IE43380B1

IE43380B1 - Process for preparing viruses

Info

Publication number: IE43380B1
Application number: IE123476A
Authority: IE
Original assignee: Behringwerke Ag
Priority date: 1975-06-10
Filing date: 1976-06-09
Publication date: 1981-02-11
Also published as: LU75106A1; BE842796A; DE2525733A1; AT362495B; FR2314252B1; DK254876A; ATA420076A; IT1061406B; FR2314252A1; AR208012A1; NL7606074A; GB1548258A; ES448573A1; IE43380L; BR7603715A; IN141155B

Abstract

[IN141155]

Description

The invention relates to a process for preparing viruses with the aid of permanent cell cultures.

In the known processes for preparing virus vaccines, stationary cell cultures are used, for example, in the monolayer process and, with BHK cells also suspension cultures.

The cells proliferating in a culture medium are separated by a physical method, for example by sedimentation, centrifugation and/or filtration, from the culture medium, suspended in a new medium, infected with the virus and isolated after the latter has grown.

These processes have the disadvantage that the separation of the cells from the culture medium is very time-consuming and expensive and requires great expenditure with regard to personnel and apparatus. During isolation, the cells may be damaged by the physical methods used. Furthermore, these processes cannot be carried out quantitatively, but rather always involve losses of cells. In addition, the cell cultures can be contaminated by foreign micro-organisms as these, too, proliferate in the culture medium and appear then in the final product- i e. in the vaccine.

The present invention provides a Droeess for producing viruses in a cell culture, which comprises allowing the cells to proliferate in a culture medium which comprises less than 100%, preferably from 2 to less than 10%, and advantageously from 2 to 3%, (volume by volume) of serum, diluting the culture with a serum-free culture medium, infecting the cells 4338 - 3 with the virus, allowing the virus to proliferate and, if desired, working the virus up to a vaccine.

In the process of the invention, the proliferation of the cells and the dilution of the cell suspension can be carried out any desired time, and the growth of the virus is carried out in the cell suspension without previously separating the cells from the suspension medium.

According to the invention, any permanent cells can be cultivated, provided they can be grown in a suspension culture Until now, the only cells to have economic importance as cell cultures are the BHK-cell (Baby Hamster Kidney cell), which was isolated by MacPherson and Stoker (Virology 14, 359—3*70, 1961), and the IBRS 2-cell described by Ribeiro et al. in Arguivos do Instituto Biologico 38, 279—281, 1971, which re15 presents a permanent pig's kidney cell. The invention, however, is not limited to these cell systems.

In the cell suspensions obtained according to the invention, any type of virus which can be cultivated in permanent cells can be brought to proliferation. Particularly suitable are the foot-and-mouth disease virus, the reoviruses type I, II and III, the adenovirus of pigs and the entero-virus of pigs as well as herpes viruses. Again, the invention is not limited to these viruses.

As the culture medium there may be used any of the usual partly commercial, media, for example, Eagle's Medium, TCM 199, Hanks' or Earle's medium. Such a medium may be combined, if desired, with known and commercial substances, for example tryptose phosphate, lactalbumin hydrolysate, caseine peptone, glucose, galactose, lactose and yeast extract. - 4 43380 As the culture medium, the following solution is particularly suitable: Culture Medium A Eagle's medium 59 tryptose phosphate sodium bicarbonate glucose yeast extract Neomycin 2.95% solution 5,0% strength 20.0% strength 5.0% strength 89.5% by weight 5.0% by weight 2.5% by weight 2.0% by weight 1.0% by weight 0.5% by weight ) Eagle's medium is a mixture of amino-acids and vitamins in an Earle's solution. Its composition was published by Eagle in Science 130, 432, 1959. Tryptose phosphate is commercially available (Oxoid or Difco) and consists of the peptone tryptose, disodium phosphate, glucose and sodium chloride. Neomycin is an antibiotic, which is generally added to the medium in a concentration of 100 U/ml.

The process of the present invention is characterized by the use of serum in an amount of less than 10% (v/v), preferably to 2 to 3% (v/v). As the serum, cattle or calf serum is generally used, but other sera may also be used with equal success.

The following Table shows a comparison of the cell numbers of 10 tests. The starting concentration was 0.2 to 0.3 million BHK cells which were brought to proliferation during a period of 48 hours in a suspension using 2.5% (v/v) and 10.0% (v/v) calf serum. The cell numbers were determined in vivo in a counting chamber, by coloration with typan blue. - 5 43380 Test number 2.5% calf serum 10% calf serum 1 1.8 millions 1.7 millions 2 1.6 millions 1.5 millions 3 1.55 millions 1.85 millions 4 2.5 millions 1.7 millions 5 3.0 millions 2.0 millions e 1.65 millions 1.8 millions 7 1.08 millions 0.98 million 8 2.05 millions 1.5 millions 9 2.1 millions 1.6 millions 10 1.6 millions 1.6 millions Average growth 1.898 millions 1.323 millions The 10 tests with the 2.5% calf serum showed a medium growth of about 1,9 millions of cells, which was approximately 50% higher than when a 10% calf serum according to the state of the art was used.

For the proliferation of the viruses, the cell suspension is diluted with a serum-free culture medium. For infection, a cell-virus ratio of 10:1 to 500:1 (calculated as number of cells:numberof virus particles) is recommended.

After using the above process according to the state of the art, virus titers of 10®θ to IO7®3 per ml were obtained.

The virus titers obtained according to the invention are 6 75 8 5 between 10 and 10 ' and are thus about 10 times higher than those obtained by the known processes.

The following Examples illustrate the invention.

EXAMPLE 1. 100 ml of a BHK-cell culture prepared in known manner and having a cell content of 2.5 millions per ml were diluted in 4 steps, each time in a ratio of 1:8, with the culture - 6 medium A described above, with an addition of 2.5% (v/v) of cattle serum, and then brought to proliferation each time for 48 hours at 37°C.

As is shown in the following scheme, the 100 ml used initially and containing 2.5 million cells per ml was brought within 4 x 48 hours to 400 1 of a cell suspension containing 2.5 millions cells/ml. lOO ml ) Step I 800 ml 800 ml Step II 6.4 liters 6.4 liters Step III 51.2 liters 51.2 liters Step IV 400 liters 400 liters 1000 liters Scheme Dilution 1:8 after 48 hours Dilution 1:8 after 48 hours Dilution 1:8 after 48 hours Dilution 1:8 after 48 hours Dilution 1:2.5 Infection with at 37°C at 37°C at 37°C at 37°C 2.5 million cells/ml 300 000 cells/ml 2.5 million cells/ml 300 000 cells/ml 2.5 million cells/ml 300 000 cells/ml 2.5 million cells/ml 300 000 cells/ml 2.5 million cells/ml million cells/ml virus For the infection with virus, the 400 liters obtained were diluted at a ratio of 1:2.5 with the same culture medium, but without addition of serum, thus to 1000 liters with a cell content of 1 million per ml. The dilution was 3 3 8 0 - 7 then infected with foot-and-mouth disease virus of one of the types A5 Hannover, C Tolz or 0.1 Lausanne at a cell-virus particle ratio of 100:1 by adding a suspension containing the virus under sterile conditions to the cell suspension. 40 hours after the infection, the whole was cooled to 4 °C, centrifuged in order to eliminate any residual parts of damaged cells, mixed for purification with 2% of chloroform and then the chloroform which formed with the protein sediment a lower phase was separated and rejected. Finally, the product was adsorbed on aluminium hydroxide and inactivated by ethyleneimine at 4°c. This product represented the footand-mouth disease vaccine. It was found to be well tolerated, to be active against an infection with foot-and-mouth disease and to be stable for 24 months at 4—6°C.

The following Tables indicate the infectiosity titers of 6 and 7 tests with vaccines of the mentioned 3 virus types. Infectiosity titers of foot-and-mouth disease viruses Type A in BHK-cell suspension cultures Test No. 1 2 3 4 5 6 7 Cell number/ml in millions at infection 1.05 1.0 0.85 1.05 1.45 1,3 1.4 Harvest in hours p.i. (post infectionem) 20 20 18 22 22 20 22 Titer/ml IO7·75 io8·0 io8·25 io7·5 ., .8.25 10 ,„7.5 10 io8*5 - 8 Infectiosity titer of foot-and-mouth diseases viruses Type C in BHK-cell suspension cultures Test No. 1 2 3 4 5 6 Cell nuniber/ml in millions at infection 1.3 1.5 1.0 1.1 1.3 1.35 Harvest in hours p. i. 22 20 22 44 24 24 Titer/ml IO7’5 IO6·75 io8'5 io7’5 io7·25 , 7-° 10 Infectiosity titers of foot-and-mouth virus type o after infection of ΒίϊΚ-cell suspension cultures Test No. 1 2 3 4 5 6 7 Cell nuniber/ml in millions at infection 0.9 0.95 0.925 1.10 1. 175 0.95 1.4 Harvest in hours p.i. 44 20 20 27 24 21 22 Virus titer/ml. io7·05 io7·75 ,„8.25 10 io7·75 ,^7.75 io , 8.0 10 8.5 »10 As the numbers in the respective third lines show, the virus titers are almost ten times higher than those obtained with the virus suspensions according to the state of the art.

EXAMPLE 2. 450 ml of an IBRS—2 cell suspension containing 670,000 cells/ml were diluted according to Example 1 with the culture medium described in the said Example to a volume of 1,5 liters (200,000 cells/ml) and kept at 37°C. After 72 hours, - 9 the cell number was 670,000 per ml. The cell suspension was diluted in a ratio of 1:3.35 to a volume of 5 liters and the cells (200 000/ml) were again brought to proliferation at 37°C. After 72 hours, 680 000 cells/ml were counted. The quantity of cells was brought at a ratio of 1:2 with the same culture medium, which was this time free of serum, to a volume of 10 liters and infected with the foot-and-mouth disease virus of the type C. After 21 hours, the suspension was cooled to 4°C and the infectiosity titer was determined. 6 It was found to be 10 ' KID_ /ml. The foot-and-mouth 50 disease vaccine was prepared as in Example 1.

EXAMPLE 3.

In a manner analogous to Example 2, there was prepared in two steps from 500 ml of an IBRS—2 cell suspension with a dilution of 1:4 and a proliferation time of 72 h at 37°C, a volume of 8 liters of a cell suspension that had a cell number of 650 000/ml. This cell suspension was diluted at a ratio of 1:2 with the serum-free culture medium, infected with an adeno-virus of pigs and incubated for 72 hours at 37°C. It was then again diluted at a ratio of 1:2 with the serum-free culture medium and kept for 48 hours at 37°C.

After 120 hours after the infection, the whole was cooled to 4°C and the infectiosity titer was determined. It was found 5 5 to be 10 ' ΚΙΒ^θ/ιηΙ. From the virus suspension so obtained, a vaccine against pigs, adeno virus was prepared.

EXAMPLE 4.

Liters of a BHK-cell suspension were obtained according to Example 1. The cells, the number of which amounted to 2.1 million cells/ml, were diluted at a ratio of 1:3 with a serum-free culture medium to 700 000 cells/ml, infected with reo-virus Serotype III and incubated at 37°C. - lo 48 hours after the infection, the cell culture was diluted at a ratio of 1:2 with 'the mentioned medium. 96 hours after the infection, the virus-containing cell suspension was cooled to 4°C and the infectiosity titer was determined. It 7.7 was found to be 10 KID^^/ml. The cell suspension so obtained served for the preparation of a vaccine against the reo-virus Serotype III.

Claims

1. CLAIMS:1. A process for producing viruses in a cell culture, wherein cells are proliferated in a culture medium comprising less than 10% (volume by volume) of serum, the culture is diluted with serum-free culture medium, the cells are infected with the virus, and the virus is allowed to proliferate.

2. A process as claimed in claim 1, wherein the culture is diluted one or more times with serum-containing culture medium prior to dilution with serum-free culture medium.

3. A process as claimed in claim 1 or claim 2, wherein the culture medium comprises from 2 to 3% of serum.

4. A process as claimed in any one of claims 1 to 3, wherein the serum is bovine Serum.

5. A process as claimed in any one of claims 1 to 4, wherein the culture after infection with the virus is diluted one or more times with serum-free culture medium.

6. A process as claimed in any one of claims 1 to 5, wherein the culture medium is Eagle's medium, TCM 199, Hanks' medium or Earle's medium.

7. A process as claimed in claim 6, wherein the medium also comprises one or more of the following: tryptose phosphate, lactalbumin hydrolysate, casein peptone, glucose, galactose, lactose and yeast extract.

8. A process as claimed in claim 7, wherein-the culture medium comprises Eagle's medium 59 tryptose phosphate 2.95% solution sodium bicarbonate 5.0% strength glucose yeast extract Neomycin 20 0% strength 5.0% strength 89.5% by weight 5.0% by weight 2.5% by weight 2.0% by weight 1.0% by weight 0.5% by weight

9. A process as claimed in any one of claims 1 to 8, wherein the cells are BHK-cells or IBRS 2-cells.

10. A process as claimed in any one of claims 1 to 9, 0 wherein the virus is foot-and-mouth disease virus, reo-virus type I, II or III, the adeno-virus or entero-virus of pigs, or a herpes virus.

11. A process as claimed in any one of claims 1 to 10, wherein at infection a cell: virus particle ratio of 5 10:1 to 500:1 is used.

12. A process as claimed in claim 1, carried out substantially as described in any one of the Examples herein.

13. Virus whenever produced by a process as claimed in any one of claims 1 to 12. 3

14. A process as claimed in any one of claims 1 to 12, wherein the resulting viruses are worked up to a vaccine.

15. A process as claimed in Claim 14, carried out substantially as described in any one of the Examples herein.

16. A vaccine whenever prepared by a process as i claimed in claim 14 or claim 15.