Medium for the protein-free and serum-free cultivation of cells

Abstract

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.

What is claimed is: 
     
         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.