Oligopeptide-free cell culture media
View Patent ↗The present invention relates to oligopeptide-free cell culture media comprising at least 0.5 mg/L of a polyamine and to methods for cultivating cells in said oligopeptide-free cell culture media comprising at least 0.5 mg/L of a polyamine. The invention also relates to methods for expressing at least one protein in a medium comprising at least 0.5 mg/L of a polyamine and to methods for producing at least one virus in a medium comprising at least 0.5 mg/L of a polyamine.
1. A method for expressing a coagulation factor VIII protein, comprising the steps of:
(a) providing a culture of CHO cells;
(b) introducing at least one nucleic acid sequence comprising a sequence coding for the coagulation factor VIII protein into the cells;
(c) selecting the cells carrying the nucleic acid sequence; and
(d) expressing the coagulation factor VIII protein in a chemically defined protein-free medium that does not comprise oligopeptides, the medium comprising DMEM:HAM's F12 (1:1) basal medium, putrescine at a concentration of at least 1 mg/L, and Fe(II) and Cu(II) at a level greater than the amount in basal DMEM:HAM's F12 (1:1), wherein the medium is supplemented with L-glutamine, ascorbic acid, ethanolamine, sodium selenite, and a non-ionic surfactant, wherein the cells are cultivated by chemostat cultivation.
2. The method of claim 1 , wherein the medium further comprises a polyamine selected from the group consisting of cadaverine, spermidine, spermine, agmatine, ornithine, or combinations thereof.
3. The method of claim 1 , wherein the medium comprises ornithine, spermine, or combinations thereof.
4. The method of claim 1 , wherein the putrescine is synthetically produced.
5. The method of claim 1 , wherein the putrescine originates from a source other than a protein hydrolysate.
6. The method of claim 1 , wherein the medium is further supplemented with L-Asparagine, L-Cysteine, L-Cystine, L-Proline, and L-Tryptophan.
7. The method of claim 1 , wherein the medium comprises putrescine at a concentration of from about 1.0 mg/L to about 20 mg/L.
8. The method of claim 1 , wherein the non-ionic surfactant comprises copolymers or mixtures of polyethylene glycols and polypropylene glycols.
9. The method of claim 1 , wherein the medium further comprises a buffer substance.
10. The method of claim 9 , wherein the buffer substance comprises one or more of sodium bicarbonate, antioxidants, stabilizers, or protease inhibitors.