Multiplication of viruses in a cell culture
The present invention concerns methods for multiplication of viruses in cell culture in which cells are infected with a virus and after infection the cells are cultured in cell culture under conditions that permit multiplication of the viruses and at the same time targeted additional, at least two-fold, multiplication of the cells. The invention also concerns the use of the viruses so obtained or proteins expressed by them for production of drugs and diagnostic agents.
1 . Method for multiplication of viruses in cell culture, comprising steps in which
(a) cells are infected with a virus;
(b) after infection the cells are cultured in cell culture under conditions that permit multiplication of the viruses and at the same time targeted additional, at least two-fold, multiplication of the cells.
2 . Method according to claim 1 , characterized in that after infection the cells are cultured under conditions that cause at least five-fold multiplication of the cells.
3 . Method according to claim 2 , characterized in that after infection the cells are cultured at least 7 days in cell culture.
4 . Method according to claim 1 , characterized in that during multiplication of the viruses and multiplication of the cells, fresh medium, medium concentrate or media components are added at least once or at least part of the viruses and cells are transferred to a culture vessel that contains fresh medium, medium concentrate or media components.
5 . Method according to claim 4 , characterized in that the addition of the medium, medium concentrate or media components or transfer of the cells and viruses to another culture vessel is repeated at least once.
6 . Method according to claim 4 , characterized in that addition of the medium, medium concentrate or media components or transfer of the cells and viruses to another culture vessel is repeated several times.
7 . Method according to claim 4 , characterized in that medium is removed on multiplication of the viruses and cells.
8 . Method according to claim 1 , characterized in that during multiplication of the viruses and cells, medium is continuously removed and fresh medium, medium concentrate or media components are added.
9 . Method according to claim 1 , characterized in that the cells are MDCK cells.
10 . Method according to claim 1 , characterized in that before infection the cells are cultured adherently or as a suspension culture.
11 . Method according to claim 1 , characterized in that after infection the cells are cultured adherently or as a suspension culture.
12 . Method according to claim 9 , characterized in that the MDCK cells originate from the cell line MDCK 33016.
13 . Method according to claim 1 , characterized in that the virus is a ssDNA, dsDNA, RNA(+), RNA(−) or dsRNA virus.
14 . Method according to claim 1 , characterized in that the virus is chosen from adenoviruses, ortho- or paramyxoviruses, reoviruses, picornaviruses, enteroviruses, flaviviruses, arenaviruses, herpes viruses or pox viruses.
15 . Method according to claim 14 , in which the cells are infected with an adenovirus, polio virus, hepatitis A virus, Japanese encephalitis virus, Central European encelphalitis viruses and the related eastern (Russian or other) forms, dengue virus, yellow fever virus, hepatitis C virus, rubella virus, mumps virus, measles virus, respiratory syncytial virus, vaccinia virus, influenza virus, rotavirus, rhabdovirus, pneumovirus, reovirus, herpes simplex virus 1 or 2, cytomegalovirus, varicella zoster virus, canine adenovirus, Epstein-Barr virus, bovine or porcine herpes viruses, BHV-1 virus, pseudorabies virus, or rabies virus.
16 . Method according to claim 1 , characterized in that the virus has a viral genome comprising a sequence that codes for a heterologous functional protein with a size of at least 10 kd.
17 . Method according to claim 4 , characterized in that the medium is serum-free.
18 . Method according to claim 4 , characterized in that the medium is a chemically defined medium.
19 . Method according to claim 4 , characterized in that the medium is protein-free.
20 . Method according to claim 1 , characterized in that multiplication of the viruses is carried in a perfusion system.
21 . Method according to claim 1 , characterized in that multiplication of the viruses is carried in a batch system.
22 . Method according to claim 9 , characterized in that the MDCK cells are cultured at temperatures between 30 and 40° C. for multiplication of the viruses.
23 . Method according to claim 9 , characterized in that the MDCK cells are cultured at an oxygen partial pressure between 35 and 60% for multiplication of the viruses.
24 . Method according to claim 4 , characterized in that the pH value of the medium lies between pH 6.8 and pH 7.8 for multiplication of the viruses.
25 . Method according to claim 9 , characterized in that the virus is introduced to the MDCK cells by infection with a MOI value between 10 −8 and 10.
26 . Method according to claim 1 , characterized in that the viruses or a protein expressed by them are purified from the culture supernatant or the harvested cells.
27 . Method according to claim 26 , characterized in that the cells are MDCK cells and at least part of the culture medium is separated from at least part of the MDCK cells for purification of the viruses or protein.
28 . Method according to claim 27 , characterized in that the separation occurs by means of a deep bed filter or a separator.
29 . Method according to claim 26 , characterized in that the purification includes ultracentrifugation for concentration of the viruses.
30 . Method according to claim 26 , characterized in that the purification includes chromatography.
31 . Method according to claim 26 , characterized in that the viruses are inactive during purification.
32 . Method for production of a drug or diagnostic agent, characterized in that it includes a method according to claim 1 .
33 . Method for production of a drug or diagnostic agent according to claim 32 , characterized in that that the viruses or the protein are mixed with an appropriate adjuvant, auxiliary, buffer, diluent or drug carrier.
34 . The method of claim 2 , characterized in that after infection the cells are cultured under conditions that cause at least ten-fold multiplication of the cells.
35 . The method of claim 3 , characterized in that after infection the cells are cultured at least 21 days in cell culture.
36 . The method of claim 35 , characterized in that after infection the cells are cultured at least 28 days in cell culture.
37 . The method of claim 36 , characterized in that after infection the cells are cultured at least 35 days in cell culture.