DE2525733A1 - METHOD FOR MANUFACTURING VIRUS VACCINES - Google Patents

Description

The invention relates to a method for producing virus vaccines with the aid of permanent cell cultures.

In the known processes for the production of virus vaccines one works with stationary cell cultures, (Monolayer process, with BHR cells also suspension cultures) The cells multiplied in a nutrient medium are made by a physical method, e.g. by sedimentation, centrifugation and / or filtration separated from the nutrient medium, suspended in new medium, infected with the virus and after its growth separated.

These methods have the disadvantage that the isolation of the cells from the nutrient medium is tedious and expensive, as well as requiring personnel and is complex in terms of equipment. Special laboratories must be set up to carry out the measures described will. The cells can be damaged by physical methods when they are obtained. The proceedings cannot be carried out quantitatively, but are always associated with cell losses. Besides that

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the cell cultures can be contaminated by foreign microorganisms. These also multiply in the Culture and then appear in the end product - namely in the vaccine.

A method for producing virus vaccines has now been found, which is characterized in that the cells are multiplied in a nutrient medium which contains 2-10%, preferably 2-3 %, serum, and the medium is diluted with serum-free nutrient medium after the multiplication , infected with the virus, the virus reproduces in a known way and processed into a vaccine.

The method is also characterized by the proliferation of the cells and the dilution of the cell suspension can be carried out as often as desired and that the virus synthesis is carried out in the cell suspension without having to do so beforehand the cells are separated from the suspension medium.

According to the invention, all permanent cells can be cultured if they can be increased in suspension culture. So far, only the BHK cells have been of economic importance (Baby Hamster Kidney Cell) obtained from MacPherson and Stoker (Virology_14, 359-370, 1961) as well as the IBRS 2 cell described by Ribeiro et al in Arquivos do Instituto Biologico 38, 279-281, 1971 and a permanent one Pig kidney cell. However, the invention is not limited to these cell systems.

In the cell suspensions obtained according to the invention, all types of virus that are grown in permanent cells, be brought to increase. The FMD virus (foot and mouth disease virus), the reoviruses type I,

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II and III, porcine adenovirus and porcine enteroviruses, and herpes viruses. But the invention is not limited to these viruses.

All common, 2.T. commercially available Media into consideration like. Eagle's Medium, TCM 199 and Hanks or Earle's Medium. If desired, you can add to these media known and commercially available substances are added, e.g. tryptose phosphate, lactalbumin hydrolyzate, Casein peptone, glucose, galactose, lactose and yeast extract.

The following solution is particularly suitable as a nutrient medium:

Eagle ^! s medium 59 2.95% solution 89.0 Weight% Tryp to s epho sphat 5.0% 5.0 1! Sodium bicarbonate 20.0% 2.5 1! glucose 5.0 % ± g 2.0 ; it Yeast extract 1.0 ti Neomycin 0.5 dd

Eagle's medium is a mixture of amino acids and vitamins in Earle ^f shear brine. Its composition was published by Eagle in Science 150, 432, 1959. Tryptose phosphate is commercially available (Oxoid or Difco) and consists of the peptone tryptose, disodium phosphate, glucose and table salt. Neomycin is an antibiotic and is contained in the medium used in a concentration of 100 U / ml.

The method on which the present invention is based is particularly characterized in that the serum content is 2-10%, preferably 2-3%. Bovine or calf serum is used as serum in the ester line, but other serums Anv. For ^r ending. In the

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In practice, serum supplements of 10% and above have been used so far. It is e ± n object of the present invention, that this amount can be significantly reduced. In any case, according to the invention, the serum addition can be less than.

In the following table, the cell numbers from ten experiments are compared with one another. The initial concentration was 0.2-0.3 million BHK cells per ml that were in suspension below Use of 2.5% and 10.0% calf serum for 48 hours were brought to increase. The cell numbers were determined after vital staining with iypan blue in the counting chamber.

Experimental
No. 2.5% calf serum 10% calf serum 1 1.8 million 1.7 million 2 1.6 million 1.5 million 3 1.55 million 1.85 million 4th 2.5 million 1.7 million 5 3.0 million 2.0 million 6th 1.65 million 1.8 million 7th 1.08 million 0.98 million 8th 2.05 million 1.5 million 9 2.1 million 1.6 million 10 1.6 million 1.6 million Average Growth 1.898 million 1.323 million

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The ten tests with 2.5% calf serum resulted in an average growth of around 1.9 million cells was half higher than if according to the procedure of Prior art 10% calf serum was used.

For the virus multiplication, the cell suspension with serum-free Diluted nutrient medium. A cell-virus ratio of 10: 1 to 500 is recommended for the subsequent infection:

According to the suspension process mentioned in accordance with the prior art

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Technique, virus titers of 10 * to 10 * per ml are obtained. The virus titers obtained according to the invention are between 10 '' ^ and 10 ', thus about ten times higher than at the known method.

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example 1

100 ml of a BHK cell culture prepared in a known manner in the nutrient medium described in more detail on page 2 with an addition of 2.5% bovine serum, which has a cell content of 2.5 million per ml are diluted in 4 stages in a ratio of 1: 8 with the same nutrient medium and each Propagated for 48 hours at 37 ° C.

As the following diagram shows, the 100 ml used can be added to 400 liters of a cell suspension in 4 χ hours with 2.5 million cells / ml.

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ml
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Scheme

Dilute 1 ι 8

2.5 million cells / ml

300 000 cells / ml

after 48 hours at 37 ° C. 800 ml 2.5 million cells / ml

Stage II

6.4 I 6.4 I.

Dilute 1: 8

300,000 cells / ml .o.

after 48 hours at 37 C

2.5 million cells / ml

Stage III dilution 1: 8

51.2 I 300,000 cells / ml

after 48 hours at 37 ° C 51.2 I. 2.5 million cells / ml

Stage IV

400 I.

4OO I.

10OO I.

Dilute 1: 8

after 48 hours at 37 ° C '

300,000 cells / ml 7 ° C

2.5 million cells / ml

Dilute 1: 2.5

Infect with virus

1 million cells / ml

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For virus infection, the 400 liters obtained are diluted in a ratio of 1: 2.5 with the same nutrient medium, but this time without the addition of serum, thus making 1,000 liters with a cell content of 1 million per ml. The dilution is infected with FMD virus of one of the types A 5 Hannover, C Tölz or 0.1 Lausanne in a cell-virus ratio of 100: in which a suspension containing the virus is added to the cell suspension under sterile conditions. 40 hours after infection, the whole is cooled to 4 ° C., centrifuged to remove the cell debris, mixed with 2% chloroform for purification and then the chloroform, which forms a lower phase with the protein sediment, is separated off and discarded. Finally, the product is adsorbed on aluminum hydroxide and inactivated by ethyleneimine at 4 ° C. This product represents the FMD vaccine. It is well tolerated, effective against foot-and-mouth disease and can be kept for 24 months ^{at 4-6 ° C.}

The tables below show the infection titers of 6 and 7 experiments with vaccines of the three mentioned Virus types communicated.

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'Infectivity titre of FMD Vifus type 0 after infection / of JBHK suspension cell cultures.

■ ■ 1 2 attempt s-Kr.
4th VJI r 6 7th Cell count / ml in
Million if infected o, 9 o95 o, 925 ,, ίο ' 1.175 o, 95 1st, 4th Harvest in hours pi 44 2o 2o 27 24 21 22nd Virus titre / ml I ο V ⁵ 1o ⁸ > ° -i K, ⁸ ' ⁵

As the numbers in the third line show, the virus titers are up to almost ten times higher than those of the virus suspensions obtained according to the state of the art

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Example 2

450 ml of an IBRS-2 cell suspension with 670,000 cells / ml are diluted to 1.5 liters (200,000 cells / ml) according to Example 1 with the nutrient medium mentioned there and kept at 37.degree. After 72 hours, the cell number is 670 000 per ml, the resulting cell suspension is 1:. 3.35 diluted with the same culture medium to 5 liters and brought the cells (200 OOO / ml) at 37 ⁰ C again for propagation. After 72 hours, 680,000 cells / ml are counted. The amount of cells obtained is brought to 10 liters 1: 2 with the same, but this time serum-free, nutrient medium and infected with type C FMD virus. After 21 hours, the suspension is brought to 4 ° C. and the infectivity titer is measured. It is 10 ^{7> 6} KID ₅₀ / ml. The MKS vaccine is produced as described in Example 1.

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Example 3

According to Example 2, a volume of 8 of a cell suspension with a cell count of 650,000 / ml is obtained in two stages from 500 ml of an IBRS 2-cell suspension with a dilution of 1: 4 and an increase time of 72 hours at 37 ° C. The cell suspension is diluted 1: 2 with the serum-free nutrient medium, infected with a pig adenovirus and incubated at 37 ° C. for 72 hours. Then it is again diluted 1: 2 with the serum-free nutrient medium and kept at 37 ° C. for 48 hours. A total of 120 hours after infection is cooled to 4 ° C. and the infectivity titer is determined. It is 10 ¹ ^ KID ^ / ml. A vaccine against pig adenovirus is produced from the virus suspension obtained.

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Example 4

According to Example 1, 6 liters of a BHK cell suspension are used won. The cells, the number of which is 2.1 million cells / ml, become 1: 3 with serum-free nutrient medium to 700,000 cells / ml diluted, infected with Reovirus serotype III and stored at 37 ° C incubated. 48 hours after infection, the mixture is again diluted 1: 2 with the medium mentioned. 96 hours after the Infection - the yirus-containing cell suspension is cooled to 4 ° C and the infectivity titer was measured. He is

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10 '' KID ₅₀ per ml. The cell suspension obtained is used to produce a vaccine against the reovirus serotype III.

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Claims (1)

Claim A method for producing virus vaccines in cell cultures by multiplying cells in a nutrient medium and infecting the cells with a virus, characterized in that the cells are propagated in a nutrient medium which contains 2 to 10 %, preferably 2 to 3% serum, and this is diluted with further nutrient medium in the course of cell growth and the last dilution is carried out before the infection of the cells without isolating them with a serum-free nutrient medium. 609851/09 2 * 2