RECOMBINANT CELL CLONES HAVING INCREASED STABILITY AND METHODS OF MAKING AND USING SAME
Disclosed are a stable recombinant cell clones which are stable in serum- and protein-free medium for at least 40 generations, a biomass obtained by multiplying the stable cell clone under serum- and protein-free culturing conditions, and a method of preparing recombinant proteins by means of the biomass. Furthermore, the invention relates to a method of recovering stable recombinant cell clones.
1 . A method for producing a stable recombinant mammalian cell clone, which is stable under production conditions in serum- and protein-free medium for at least 40 generations, the method comprising:
providing a recombinant original mammalian cell clone,
cultivating the recombinant original cell clone on serum-containing medium,
adapting the cells to serum- and protein-free medium in the absence of a selection pressure,
testing the cell culture after adaptation for stable product-producers in the absence of a selection pressure and
cloning a stable product-producer-cell clone in serum- and protein-free conditions in the absence of a selection pressure.
2 . The method according to claim 1 , wherein the stable cell clone obtained is present in isolated form after the step of cloning.
3 . The method according to claim 1 , wherein the recombinant cell clone comprises a nucleic acid encoding a recombinant polypeptide or protein.
4 . A stable recombinant mammalian cell clone, obtainable by a method according to of claim 1 , wherein the cell clone is stable for at least 40 generations and expresses a recombinant product under production conditions in serum- and protein-free medium, in the absence of a selection pressure.
5 . The cell clone according to claim 4 , wherein the clone is present in isolated form after the step of cloning.
6 . The cell clone according to claim 4 , wherein the cell comprises a nucleic acid encoding a recombinant polypeptide or protein.
7 . A cell culture obtainable by cultivating a stable recombinant cell clone according to claim 1 .
8 . The cell culture of claim 7 , wherein the cell culture contains at least 90% stable recombinant cell clones.
9 . The cell culture according to claim 7 , wherein the stable recombinant cells comprise a nucleic acid encoding a recombinant polypeptide or protein.
10 . The cell culture according to claim 7 , wherein the stable recombinant cells are immobilised on a microsupport.
11 . A method for industrial production of a recombinant product under serum- and protein-free conditions, the method comprising:
providing an isolated, stable recombinant cell clone according to claim 4 ,
propagating the stable cell clone in serum- and protein-free medium from the initial clone up to the cell culture in the absence of a selection pressure,
producing the cell culture containing stable cells in the bioreactor in the absence of a selection pressure, and
harvesting the recombinant product from the culture supernatant.
12 . The method according to claim 11 , wherein the serum- and protein-free medium is a synthetic minimal medium comprising an extract selected from the group consisting of: a yeast extract and a soy extract.
13 . The method according to claim 11 , wherein the medium contains cyclodextrin or a derivative thereof.
14 . The method according to claim 11 , wherein the serum- and protein-free medium contains a protease inhibitor.