Chemistry: molecular biology and microbiology – Spore forming or isolating process
Patent
1992-07-06
1995-02-21
Knode, Marian
Chemistry: molecular biology and microbiology
Spore forming or isolating process
43524025, 435 701, 435 703, 4352351, 435 1, C12N 500, C12N 502, C12N 700, A01N 102, C12P 2102
Patent
active
053914910
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
The invention relates to a biomass and to a method of producing virus/virus antigen.
DESCRIPTION OF RELATED ART
Methods for producing virus/virus antigen are known. Starting materials frequently comprise so-called primary cell cultures obtained from human or animal tissues. These primary cells are infected with virus ("seed virus") and virus antigen is formed by virus propagation.
A method of propagating, for instance, tick-borne encephalitis virus (TBE-virus) is described in AT-B 358,167: chick embryo cells are suspended in cell culture medium, are infected with the virus and are used as a biomass for the production of TBE-virus antigen. To this end, the biomass is kept in suspension between one and five days under aerobic conditions at a temperature of between 25.degree. and 38.degree. C. Then, the cells and cell fragments are separated by centrifugation, the virus suspension obtained is inactivated by means of formalin or .beta.-propiolacton and the virus antigen is concentrated by ultrafiltration, purified and further processed to vaccines in the usual manner.
With common preparations of primary cell cultures, it is particularly taken care that individual cells and cell aggregates as small as possible will be obtained. To reach this, the tissue must be disintegrated to the utmost extent mechanically or enzymatically. However, this treatment involves the decay of many cells. If such cell preparations are settled on the surface of suitable carrier materials, dead cells remain in the supernatant and can be removed. When using such cell preparations in suspension cultures for the production of TBE-virus antigen, there is, however, no way of separating living cells from dead or damaged cells. Consequently, the gradually occurring cell lysis results in a high degree of contamination of cell proteins in the medium, which are difficult to separate from the desired product.
Also the reproducibility of the virus/virus antigen production is low when using individual cells or small cell aggregates in suspension, because the cells may become heavily damaged, e.g., by the shearing forces created in stirring.
SUMMARY OF THE INVENTION
The invention has as its object to eliminate these disadvantages and to provide a biomass for producing virus/virus antigen, which leads to a high production output of virus/virus antigen in cultivation, is easy to handle and may be used on a commercial scale for the production of virus/virus antigen, wherein the virus/virus antigen is recoverable from the culture medium with a high purity.
The biomass according to the invention, which meets the demands pointed out above, is comprised of cell aggregates having diameters of between 100 .mu.m and 1,000 .mu.m, which biomass is infected with virus.
DETAILED DESCRIPTION OF THE INVENTION
The cell aggregates of the biomass according to the invention are obtained by mechanic and enzymatic treatment of human or animal tissues, wherein the tissue disintegrated in a mechanical way can be further communited to the desired size of the cell aggregates by means of a protease, such as trypsin, chymotrypsin or elastase, to further dissolve the cell aggregates.
The cell aggregates of the biomass according to the invention also may be obtained from human or animal single cells by treating the same with cell aggregating substances, such as agglutinin.
The separation of cell aggregates and single cells having diamaters larger than 1,000 .mu.m or smaller than 100 .mu.m may be effected by screening or, preferably, by sedimentation. Compared to screening, sedimentation is simple to carry out, because sieves having pore sizes of 100 .mu.m easily get obstructed. Moreover, no complex and expensive separators are required for sedimentation, the latter also offering advantages in terms of sterility. It has proved that the cell aggregates having diameters of between 100 .mu.m and 1,000 .mu.m deposit at a velocity faster than 1 cm/min, while the smaller cell aggregates deposit at a velocity of less than 1 cm/min. The separation of part
REFERENCES:
patent: 4059485 (1977-11-01), Tolbert et al.
patent: 4195130 (1980-03-01), Hoshino et al.
patent: 4335215 (1982-06-01), Tolbert et al.
patent: 5114855 (1992-05-01), Hu et al.
Slavik et al., "Optimalized conditions of Tick-borne encephalitis virus production in vitro," ACTA Virologica, vol. 27, Mar. 1983.
Dorner Friedrich
Eibl Johann
Mundt Wolfgang
Woehrer Wilfried
Dadio Susan M.
Immuno Aktiengesellschaft
Knode Marian
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