Method for culturing Chinese hamster ovary cells
A biochemically defined culture medium for culturing engineered Chinese hamster ovary (CHO) cell lines, which is essentially free from protein, lipid and carbohydrate isolated from an animal source, having water, an osmolality regulator, a buffer, an energy source, amino acids including L-glutamine, an inorganic or recombinant iron source, and a synthetic or recombinant growth factor, and optionally non-ferrous metal ions vitamins and cofactors. Also cells adapted to grow in such a culture medium.
1. A method for growing genetically engineered CHO cells which comprises in suspension comprising the step of:
culturing genetically engineered CHO cells in suspension under cell growing conditions effective to support secretion of a product from the genetically engineered CHO cells over multiple passages for at least 20 days, wherein the cell growing conditions are characterized as:
in the absence being free of both serum and transferrin; and
in comprising a medium comprising water, an osmolality regulator, a buffer, an energy source. L-glutamine and at least one additional amino acid, an inorganic, organic or recombinant iron source and a recombinant or synthetic growth factor wherein each component of said medium is obtained from a source other than directly from an animal source.
2. A The method for culturing growing CHO cells in accordance with claim 1 , wherein the medium further comprises at least one component selected from the group consisting of: non-ferrous metals, vitamins or and cofactors.
3. A The method for culturing growing CHO cells in accordance with claim 1 , wherein the osmolality regulator maintains the medium at 200-350 mOsm.
4. A The method for culturing growing CHO cells in accordance with claim 1 , wherein the medium is maintained at a pH in the range of about 6.5 to about 7.5 by the buffer.
5. A The method for culturing growing CHO cells in accordance with claim 1 , wherein the concentration of the energy source is within the range of 1000-10,000 mg/liter.
6. A The method for culturing growing CHO cells in accordance with claim 5 , wherein the energy source is a monosaccharide.
7. A The method for culturing growing CHO cells in accordance with claim 1 , wherein the additional amino acids are selected from the group consisting of L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cystine cysteine, L-glutamic acid, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine and L-valine.
8. A The method for culturing growing CHO cells in accordance with claim 1 , wherein the concentration of L-glutamine is within the range of 400-600 mg/liter.
9. A The method for culturing growing CHO cells in accordance with claim 2 , wherein the medium further comprises a lipid factor in an amount of 0.05-10 mg/liter.
10. A The method for culturing growing CHO cells in accordance with claim 1 , wherein the iron source is an inorganic ferric or ferrous salt which is provided in a concentration of from 0.25-5 mg/liter.
11. A The method for culturing growing CHO cells in accordance with claim 1 , wherein the growth factor comprises is selected from the group consisting of: recombinant or synthetic insulin, platelet derived growth factor, thyroxine T 3 , thrombin, interleukin, progesterone, hydrocortisone or and vitamin E.
12. A The method for culturing growing CHO cells in accordance with claim 11 1 , wherein the growth factor is recombinant or synthetic insulin.
13. A The method for culturing growing CHO cells in accordance with claim 1 , wherein the medium further comprises at least one component selected from the group consisting of: a peptide digest, peptide hydrolysate or and peptide extract.
14. A method for culturing cells in accordance with claim 1 , A method for growing genetically engineered CHO cells in suspension, comprising the step of:
culturing genetically engineered CHO cells in suspension under cell growing conditions effective to support secretion of a product from the genetically engineered CHO cells over multiple passages for at least 20 days, wherein the cell growing conditions are characterized as:
being free of both serum and transferring; and
comprising a medium comprising water, an osmolality regulator, a buffer, an energy source, L-glutamine, and at least one additional amino acid, an inorganic, organic or recombinant iron source and a recombinant or synthetic growth factor, wherein each component of said medium is obtained from a source other than directly from an animal source, wherein the medium is essentially free of hypoxanthine and thymidine.
15. A The method for culturing growing CHO cells in accordance with claim 14 , wherein the medium further comprises methotrexate.
16. A method for culturing growing genetically engineered CHO cells which comprises in suspension comprising the step of:
culturing and growing Chinese hamster ovary genetically engineered CHO cells in suspension under cell growing conditions effective to support secretion of a product from the genetically engineered CHO cells over multiple passages for at least 20 days, wherein the cell growing conditions are characterized as:
in the absence being free of both serum and transferrin;
in comprising a medium comprising:
an osmolality regulator to maintain the osmolality of the medium within the range of about 200-350 mOsm,
a buffer to maintain the pH of the medium within the range of about 6.5 to 7.5,
about 1000-10,000 mg of a monosaccharide,
about 400-600 mg of L-glutamine,
about 10-200 mg of at least one amino acid selected from the group consisting of L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cystine cysteine, L-glutamic acid, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine and L-valine,
about 0.25-5 mg of an inorganic or recombinant iron source,
about 5 μg-5 mg of a recombinant or synthetic insulin,
and sufficient water to provide one liter of medium.
17. A method for culturing CHO cells which comprises culturing and growing Chinese hamster ovary cells in the absence of serum in a medium comprising
a base medium containing the amino acids, non-ferrous metal ions, vitamins and cofactors essentially as set forth in Table 1 ,
an osmolality regulator selected from NaCl, KCl, and KNO 3 in an amount sufficient to maintain the osmolality of the medium within the range of about 200 - 350 mOsm,
at least one buffer selected from CaCl 2 . 2 H 2 O, MgSO 4 . 7 H 2 O, NaH 2 PO 4.2H 2 O, sodium pyruvate, N-[ 2 -hydroxyethyl]piperazine-N′-[ 2 -ethanesulphonic acid (HEPES) and 3 -[N-morpholino]-propanesulfonic acid (MOPS) in an amount sufficient to maintain the medium within the pH range of about 6 . 5 - 7 . 5 ,
about 1000 - 10 , 000 mg of mannose, fructose, glucose or maltose,
about 5 ml of 200 mM L-glutamine,
about 50 mg each of L-proline, L-threonine, L-methionine, L-cysteine and L-tyrosine,
about 20 - 50 mg of L-ascorbic acid,
about 0 . 01 - 0 . 5 mg each of Vitamin B 6 and Vitamin B 12 ,
about 0 . 25 - 5 mg of a ferric or ferrous salt,
about 1 mg of zinc sulfate,
about 2 . 5 μg of copper sulfate,
about 10 , 000 - 100 , 000 IU of at least one antibiotic selected from the group consisting of polymyxin, neomycin, penicillin and streptomycin,
about 3 μl of ethanolamine,
about 0 . 01 - 1 . 0 mg of putrescine,
about 5 μg- 5 mg of recombinant insulin and sufficient water to comprise one liter of medium; wherein each component of said medium is obtained from a source other than directly from an animal source.
18. A The method for growing CHO cells in accordance with claim 1 , wherein each component of the medium is selected from at least one source selected from the group consisting of: an inorganic, synthetic, recombinant, plant or and bacterial source.
19. A method for growing genetically engineered CHO cells in suspension, comprising the step of:
culturing genetically engineered CHO cells in suspension at a density greater than 1 × 10 5 cells/mL under cell growing conditions effective to support secretion of a product from the genetically engineered CHO cells over multiple passages for at least 20 days, wherein the cell growing conditions are characterized as:
being free of both serum and transferrin; and
comprising a medium comprising water, an osmolality regulator, a buffer, an energy source, L-glutamine and at least one additional amino acid, an inorganic, organic or recombinant iron source and a recombinant or synthetic growth factor, wherein each component of said medium is obtained from a source other than directly from an animal source.
20. The method for growing CHO cells in accordance with any one of claims 1 to 16 , 18 , or 19 , wherein said culturing results in secretion of said product from the genetically engineered CHO cells into said medium.
21. The method according to any one of claims 1 to 16 , 18 or 19 , wherein said method results in growth of said genetically engineered CHO cells in suspension and secretion of said product over multiple passages for at least 30 days.
22. The method according to any one of claims 1 to 16 , 18 or 19 , wherein said method results in growth of said genetically engineered CHO cells in suspension and secretion of said product over multiple passages for at least 40 days.
23. The method according to any one of claims 1 to 16 , 18 or 19 , wherein said method results in growth of said genetically engineered CHO cells in suspension and secretion of said product over multiple passages for at least 50 days.
24. The method according to any one of claims 1 to 16 , 18 or 19 , wherein said method results in growth of said genetically engineered CHO cells in suspension and secretion of said product over multiple passages for at least 60 days.
25. The method according to any one of claims 1 to 16 , 18 or 19 , wherein said method results in growth of said genetically engineered CHO cells in suspension and secretion of said product over multiple passages for at least 70 days.
26. The method according to any one of claims 1 to 16 , 18 or 19 , wherein said method results in growth of said genetically engineered CHO cells in suspension and secretion of said product over multiple passages for at least 80 days.
27. The method according to any one of claims 1 to 16 , 18 or 19 , wherein said method results in growth of said genetically engineered CHO cells in suspension and secretion of said product over multiple passages for at least 6 months.
28. A method for growing genetically engineered CHO cells in suspension, comprising the step of:
culturing genetically engineered CHO cells in suspension at a density greater than 1 × 10 5 cells/mL under cell growing conditions effective to support secretion of a product from the genetically engineered CHO cells over multiple passages for at least 20 days, wherein the cell growing conditions are characterized as:
being free of both serum and transferrin; and
comprising a medium comprising water, an osmolality regulator, a buffer, an energy source, L-glutamine and at least one additional amino acid, an inorganic, organic or recombinant iron source and a recombinant or synthetic growth factor, wherein each component of said medium is obtained from a source other than directly from an animal source,
wherein said culturing results in secretion of at least 30 mg/L of said product from the genetically engineered CHO cells into said medium.
29. The method according to claim any one of claims 1 to 16 , 18 , 19 , or 28 , wherein said genetically engineered CHO cells are dhfr − CHO cells.
30. The method for growing CHO cells in accordance with any one of claims 14 , 16 , 19 , or 28 wherein the medium further comprises at least one component selected from the group consisting of: non-ferrous metals, vitamins and cofactors.
31. The method for growing CHO cells in accordance with any one of claims 14 , 19 , or 28 , wherein the osmolality regulator maintains the medium at 200 - 350 mOsm.
32. The method for growing CHO cells in accordance with any one of claims 14 , 19 , or 28 , wherein the medium is maintained at a pH in the range of about 6 . 5 to about 7 . 5 by the buffer.
33. The method for growing CHO cells in accordance with any one of claims 14 , 19 , or 28 , wherein the concentration of the energy source is within the range of 1000 - 10 , 000 mg/liter.
34. The method for growing CHO cells in accordance with claim 33 , wherein the energy source is a monosaccharide.
35. The method for growing CHO cells in accordance with any one of claims 14 , 19 , or 28 , wherein the additional amino acids are selected from the group consisting of L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-glutamic acid, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine and L-valine.
36. The method for growing CHO cells in accordance with any one of claims 14 , 19 , or 28 , wherein the concentration of L-glutamine is within the range of 400 - 600 mg/liter.
37. The method for growing CHO cells in accordance with any one of claims 14 , 16 , 19 , or 28 , wherein the medium further comprises a lipid factor in an amount of 0 . 05 - 10 mg/liter.
38. The method for growing CHO cells in accordance with any one of claims 14 , 19 , or 28 , wherein the iron source is an inorganic ferric or ferrous salt which is provided in a concentration of 0 . 25 - 5 mg/liter.
39. The method for growing CHO cells in accordance with any one of claims 14 , 19 , or 28 , wherein the growth factor is selected from the group consisting of: recombinant or synthetic insulin, platelet derived growth factor, thyroxine T 3 , thrombin, interleukin, progesterone, hydrocortisone and vitamin E.
40. The method for growing CHO cells in accordance with any one of claims 14 , 19 , or 28 , wherein the growth factor is recombinant or synthetic insulin.
41. The method for growing CHO cells in accordance with any one of claims 14 , 16 , 19 , or 28 , wherein the medium further comprises at least one component selected from the group consisting of: a peptide digest, peptide hydrolysate and peptide extract.