MEDIUM FOR THE PROTEIN-FREE AND SERUM-FREE CULTIVATION OF CELLS
A medium is described for the protein-free and serum-free cultivation of cells, especially mammalian cells, whereby the medium contains a proportion of soy hydrolysate.
1 . An animal protein-free and serum-free cell culture medium, the medium comprising a soy hydrolysate having a total nitrogen content of between 7.6% and 11.4%, and the medium comprising
0.001-1 g/L L-asparagine,
0.001-1 g/L L-cysteine,
0.001-1 g/L L-cystine,
0.001-1.5 g/L L-proline,
0.001-1 g/L L-tryptophan, and
0.05-1 g/L L-glutamine.
2 . The animal protein-free and serum-free cell culture medium of claim 1 , wherein the medium comprises an endotoxin content of <500 U/g.
3 . The animal protein-free and serum-free cell culture medium of claim 1 , wherein the medium comprises more than 10 wt. % ultrafiltered soy hydrolysate based on the total dry weight of the medium, and wherein at least 40% of the soy hydrolysate has a molecular weight of ≦500 daltons.
4 . The animal protein-free and serum-free cell culture medium of claim 3 , wherein at least 50% of the soy hydrolysate has a molecular weight of ≦500 daltons.
5 . The animal protein-free and serum-free cell culture medium of claim 3 , wherein at least 55% of the soy hydrolysate has a molecular weight of ≦500 daltons.
6 . The animal protein-free and serum-free cell culture medium of claim 1 , wherein the medium contains ultrafiltered soy hydrolysate.
7 . The animal protein-free and serum-free cell culture medium of claim 1 , wherein the medium comprises an amino acid.
8 . The animal protein-free and serum-free cell culture medium of claim 7 , wherein the amino acid is selected from the group consisting of L-asparagine, L-cysteine, L-cystine, L-proline, L-tryptophan, L-glutamine and mixtures thereof.
9 . The animal protein-free and serum-free cell culture medium of claim 1 , wherein the animal protein-free and serum-free cell culture medium further comprises: 1 to 100 g/L synthetic minimal medium; 0.05-1 g/L glutamine; 0.0005-0.05 g/L ascorbic acid; 0.1-10 g/L NaHCO 3 ; 0.0005-0.05 g/L ethanolamine; and 1-15 μg/L sodium selenite.
10 . A method of preparing a recombinant protein, the method comprising:
culturing mammalian cells clone that produce a recombinant protein and expressing the recombinant protein in the medium of claim 1 so as to obtain a cell culture comprising the recombinant protein; and
recovering the expressed recombinant protein from the cell culture.
11 . The method of claim 10 , wherein the recombinant protein is selected from the group consisting of: Factor II, Factor V, Factor VII, Factor VIII, Factor IX, Factor X, Factor XI, Protein S, Protein C, activated forms of these factors, and vWF.
12 . The method of claim 11 , wherein the recombinant protein is Factor VIII.
13 . A method for adapting mammalian cells that express a recombinant protein to a serum-free and animal protein-free medium, the method comprising:
culturing recombinant cells in serum-containing medium,
transferring the cells into a serum-free and animal protein free medium, and
culturing the cells in the serum- free and animal protein-free medium, wherein the serum-free and animal protein free medium comprises more than 10 wt. % ultrafiltered soy hydrolysate based on the total dry weight of the medium, and wherein at least 40% of the soy hydrolysate has a molecular weight of ≦500 daltons.
14 . The method of claim 13 , wherein at least 50% of the soy hydrolysate has a molecular weight of ≦500 daltons.
15 . The method of claim 13 , wherein at least 55% of the soy hydrolysate has a molecular weight of <500 daltons.
16 . The method of claim 13 , wherein the soy hydrolysate has an endotoxin content of <500 U/g.
17 . The method of claim 13 , wherein the mammalian cells are CHO cells or BHK cells.
18 . The method of claim 13 , wherein the recombinant protein is selected from the group consisting of: Factor II, Factor V, Factor VII, Factor VIII, Factor IX, Factor X, Factor XI, Protein S, Protein C, activated forms of these factors, and vWF.
19 . The method of claim 18 , wherein the recombinant protein is Factor VIII.
20 . The method of claim 13 , wherein the serum- free and animal protein-free medium comprises an amino acid.
21 . The method of claim 20 , wherein the amino acid is selected from the group consisting of L-asparagine, L-cysteine, L-cystine, L-proline, L-tryptophan, L-glutamine and mixtures thereof
22 . The method of claim 13 , wherein the animal protein-free and serum-free cell culture medium further comprises: 1 to 100 g/L synthetic minimal medium; 0.05-1 g/L glutamine; 0.0005-0.05 g/L ascorbic acid; 0.1-10 g/L NaHCO 3 ; 0.0005-0.05 g/L ethanolamine; and 1-15 μg/L sodium selenite.
23 . A method for obtaining an isolated stable recombinant mammalian cell clone that produces a recombinant product and is stable under production conditions in serum-free and animal protein free medium for at least 40 generations, the method comprising:
providing a recombinant original mammalian cell clone, wherein the recombinant original mammalian cell clone has a selection marker and an amplification marker,
cultivating the recombinant original cell clone on serum-containing medium,
adapting the cells to serum-free and animal protein free medium with neither selection pressure for the selection marker nor selection pressure for the amplification marker, wherein the serum-free and animal protein free medium comprises more than 10 wt. % ultrafiltered soy hydrolysate based on the total dry weight of the medium, and wherein at least 40% of the soy hydrolysate has a molecular weight of ≦500 daltons,
testing the cell culture after adaptation for stable product-producers with neither selection pressure for the selection marker nor selection pressure for the amplification marker, and
isolating a stable product-producer-cell clone from the serum-free and animal protein free medium with neither selection pressure for the selection marker nor selection pressure for the amplification marker.
24 . The method of claim 23 , wherein at least 50% of the soy hydrolysate has a molecular weight of ≦500 daltons.
25 . The method of claim 23 , wherein at least 55% of the soy hydrolysate has a molecular weight of ≦500 daltons.
26 . The method of claim 23 , wherein the soy hydrolysate has an endotoxin content of <500 U/g.
27 . The method of claim 23 , wherein the recombinant original cell clone is a CHO cell or a BHK cell.
28 . The method of claim 23 , wherein the recombinant product is selected from the group consisting of: Factor II, Factor V, Factor VII, Factor VIII, Factor IX, Factor X, Factor XI, Protein S, Protein C, activated forms of these factors, and vWF.
29 . The method of claim 28 , wherein the recombinant product is Factor VIII.
30 . The method of claim 23 , wherein the serum-free and animal protein free medium comprises an amino acid.
31 . The method of claim 30 , wherein the amino acid is selected from the group consisting of L-asparagine, L-cysteine, L-cystine, L-proline, L-tryptophan, L-glutamine and mixtures thereof
32 . The method of claim 23 , wherein the animal protein-free and serum-free cell culture medium further comprises: 1 to 100 g/L synthetic minimal medium; 0.05-1 g/L glutamine; 0.0005-0.05 g/L ascorbic acid; 0.1-10 g/L NaHCO 3 ; 0.0005-0.05 g/L ethanolamine;
and 1-15 μg/L sodium selenite.
33 . A method for preparing a recombinant protein, the method comprising:
culturing a stable recombinant mammalian cell clone that produces a recombinant protein, wherein the cell clone is stable in serum- and protein-free medium for at least 40 generations with neither selection pressure for a selection marker nor selection pressure for an amplification marker in the cell clone, and expressing the recombinant protein in serum- and protein-free medium so as to obtain a cell culture; and
recovering the expressed recombinant protein from the cell culture, wherein the medium comprises more than 10 wt. % ultrafiltered soy hydrolysate based on the total dry weight of the medium, and wherein at least 40% of the soy hydrolysate has a molecular weight of ≦500 daltons.
34 . The method of claim 33 , wherein at least 50% of the soy hydrolysate has a molecular weight of ≦500 daltons.
35 . The method of claim 33 , wherein at least 55% of the soy hydrolysate has a molecular weight of ≦500 daltons.
36 . The method of claim 33 , wherein the soy hydrolysate has an endotoxin content of <500 U/g.
37 . The method of claim 33 , wherein the stable recombinant mammalian cell clone is a CHO cell or a BHK cell.
38 . The method of claim 33 , wherein the recombinant protein is selected from the group consisting of: Factor II, Factor V, Factor VII, Factor VIII, Factor IX, Factor X, Factor XI, Protein S, Protein C, activated forms of these factors, and vWF.
39 . The method of claim 38 , wherein the recombinant protein is Factor VIII.
40 . The method of claim 33 , wherein the serum- free and animal protein-free medium comprises an amino acid.
41 . The method of claim 40 , wherein the amino acid is selected from the group consisting of L-asparagine, L-cysteine, L-cystine, L-proline, L-tryptophan, L-glutamine and mixtures thereof.
42 . The method of claim 33 , wherein the animal protein-free and serum-free cell culture medium further comprises: 1 to 100 g/L synthetic minimal medium; 0.05-1 g/L glutamine; 0.0005-0.05 g/L ascorbic acid; 0.1-10 g/L NaHCO 3 ; 0.0005-0.05 g/L ethanolamine; and 1-15 μg/L sodium selenite.