Serum-Free Cell Culture Medium

Abstract

The specification describes an improved serum-free animal cell culture medium, which can used for the production of a protein of interest. Ornithine, or a combination of ornithine and putrescine can be added to serum-free media or chemically defined media to improve viable cell density, to reduce cell doubling time, and to increase the production of a protein of interest.

What is claimed is: 
     
         1 . A cell culture medium, which is serum-free, comprising ≧0.09 mM±0.014 mM ornithine. 
     
     
         2 . The cell culture medium of  claim 1  comprising ≧0.20±0.03 mM putrescine. 
     
     
         3 . The cell culture medium of  claim 1  comprising 0.09±0.014 mM to 0.9±0.14 mM ornithine. 
     
     
         4 . The cell culture medium of  claim 3  comprising ornithine at 0.09±0.014 mM, 0.3±0.05 mM, 0.6±0.09 mM, or 0.9±0.14 mM. 
     
     
         5 . The cell culture medium of  claim 2  comprising 0.20±0.03 mM to 0.714±0.11 mM putrescine. 
     
     
         6 . The cell culture of  claim 5  comprising putrescine at 0.20±0.03 mM, 0.35±0.06, or 0.714±0.11 mM. 
     
     
         7 . The cell culture medium of  claim 1 , wherein the medium is hydrolysate-free. 
     
     
         8 . The cell culture medium of  claim 1 , wherein the medium is chemically defined. 
     
     
         9 . The cell culture medium of  claim 1  comprising ≧40±6 mM of a mixture of amino acids or salts thereof. 
     
     
         10 . The cell culture medium of  claim 9 , wherein the mixture of amino acids consists of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. 
     
     
         11 . The cell culture medium of  claim 1  comprising one or more fatty acids. 
     
     
         12 . The cell culture medium of  claim 11 , wherein the one or more fatty acids are selected from the group consisting of linoleic acid, linolenic acid, thioctic acid, oleic acid, palmitic acid, stearic acid, arachidic acid, arachidonic acid, lauric acid, behenic acid, decanoic acid, dodecanoic acid, hexanoic acid, lignoceric acid, myristic acid, and octanoic acid. 
     
     
         13 . The cell culture medium of  claim 1  comprising a mixture of nucleosides. 
     
     
         14 . The cell culture medium of  claim 13 , wherein the mixture of nucleosides comprises one or more of adenosine, guanosine, cytidine, uridine, thymidine, and hypoxanthine. 
     
     
         15 . The cell culture medium of  claim 14  comprising adenosine, guanosine, cytidine, uridine, thymidine, and hypoxanthine. 
     
     
         16 . The cell culture medium of  claim 1  comprising one or more divalent cations. 
     
     
         17 . The cell culture medium of  claim 16 , wherein the divalent cation is magnesium, calcium, or both. 
     
     
         18 . The cell culture medium of  claim 17  comprising Ca 2+  and Mg 2+ . 
     
     
         19 . A method for cultivating cells, comprising the steps of: (a) providing a cell culture medium according to  claim 1 , and (b) propagating or maintaining a cell in the cell culture medium to form a cell culture. 
     
     
         20 . The method of  claim 19 , wherein the cell is selected from the group consisting of mammalian cell, avian cell, insect cell, bacterial cell, and yeast cell. 
     
     
         21 . The method of  claim 20 , wherein the cell is a CHO cell. 
     
     
         22 . The method of  claim 21 , wherein the cell expresses a protein of interest. 
     
     
         23 . The method of  claim 22 , wherein the protein of interest is an antigen binding protein. 
     
     
         24 . The method of  claim 23 , wherein the protein of interest comprises an Fc domain. 
     
     
         25 . The method of  claim 24 , wherein the protein of interest is a receptor-Fc-fusion protein. 
     
     
         26 . The method of  claim 25 , wherein the receptor-Fc-fusion protein is a trap protein. 
     
     
         27 . The method of  claim 26 , wherein the trap protein is an IL-1 antagonist or a VEGF antagonist. 
     
     
         28 . The method of  claim 23 , wherein the protein of interest is an antibody or an antibody fragment. 
     
     
         29 . The method of  claim 28 , wherein the antibody or the antibody fragment is a recombinant human antibody or fragment thereof. 
     
     
         30 . The method of  claim 19 , wherein the cells have an average doubling time of ≦30 hours. 
     
     
         31 . The method of  claim 19 , wherein the cells have an average doubling time of ≦24 hours. 
     
     
         32 . The method of  claim 19 , wherein the cells have an average doubling time that is at least one third that of cells grown in a cell culture medium that contains <0.3±0.045 mM ornithine and <0.2±0.03 mM putrescine. 
     
     
         33 . The method of  claim 19 , wherein the cell culture is capable of attaining a viable cell count density that is at least 15% greater than a similar cell culture in media that contains <0.09±0.014 mM ornithine and <0.2±0.03 mM putrescine. 
     
     
         34 . The method of  claim 19 , wherein the cell culture is capable of attaining a viable cell count density that is at least 3-fold greater than a similar cell culture in a similar cell culture medium that contains <0.09±0.014 mM ornithine and <0.2±0.03 mM putrescine. 
     
     
         35 . The method of  claim 19  comprising the step of adding one or more point-of-use additions to the cell culture medium. 
     
     
         36 . The method of  claim 35 , wherein the point-of-use additions comprise one of more of NaHCO 3 , glutamine, insulin, glucose, CuSO 4 , ZnSO 4 , FeCl 3 , NiSO 4 , Na 4  EDTA, and Na 3  Citrate. 
     
     
         37 . The method of  claim 36 , wherein each of NaHCO 3 , glutamine, insulin, glucose, CuSO 4 , ZnSO 4 , FeCl 3 , NiSO 4 , Na 4  EDTA, and Na 3  Citrate are added to the medium as point-of-use additions. 
     
     
         38 . A method for producing a protein comprising the steps of: (a) introducing into a cell a nucleic acid comprising a sequence encoding a protein of interest; (b) selecting a cell carrying the nucleic acid; (c) culturing the selected cell in a cell culture medium of  claim 1 ; and (d) expressing the protein of interest in the cell, wherein the protein of interest is secreted into the medium. 
     
     
         39 . The method of  claim 38 , wherein the cell is a CHO cell, 293 cell or BHK cell. 
     
     
         40 . The method of  claim 38 , wherein the protein of interest is an antigen-binding protein. 
     
     
         41 . The method of  claim 40 , wherein the protein of interest comprises an Fc domain. 
     
     
         42 . The method of  claim 41 , wherein the protein of interest is selected from the group consisting of receptor-Fc-fusion protein (TRAP), soluble TCR-Fc fusion protein, antibody, Fc-fusion protein, and ScFv protein. 
     
     
         43 . The method of  claim 38 , wherein the protein-of-interest is produced at an average 7-day titer that is at least 7% greater than the average 7-day titer produced by a similar cell in a cell culture medium that contains less than 0.09±0.014 mM ornithine and less than 0.2±0.03 mM putrescine. 
     
     
         44 . The method of  claim 38 , wherein the protein-of-interest is produced at an average 7-day titer that is at least 14% greater than the average 7-day titer produced by a similar cell in a cell culture medium that contains less than 0.09±0.014 mM ornithine and less than 0.2±0.03 mM putrescine. 
     
     
         45 . The method of  claim 38 , wherein the protein-of-interest is produced at an average 7-day titer that is at least 80% greater than the average 7-day titer produced by a similar cell in a cell culture medium that contains less than 0.09±0.014 mM ornithine and less than 0.2±0.03 mM putrescine. 
     
     
         46 . The method of  claim 38 , wherein the protein-of-interest is produced at an average 7-day titer that is at least 2-fold greater than the average 7-day titer produced by a similar cell in a cell culture medium that contains less than 0.09±0.014 mM ornithine and less than 0.2±0.03 mM putrescine. 
     
     
         47 . The method of  claim 38 , wherein the protein-of-interest is produced at an average 7-day titer that is at least 3-fold greater than the average 7-day titer produced by a similar cell in a cell culture medium that contains less than 0.09±0.014 mM ornithine and less than 0.2±0.03 mM putrescine. 
     
     
         48 . The method  claim 41 , wherein the protein-of-interest is a recombinant human antibody.