Method for culturing Chinese hamster ovary cells

Abstract

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. REEXAMINATION RESULTS The questions raised in reexamination request no. 90/006656, filed Jun. 2, 2003 have been considered and the results thereof are reflected in this reissue patent which constitutes the reexamination certificate required by 35 U.S.C. 307 as provided in 37 CFR 1.570(e), for ex parte reexaminations, or the reexamination certificate required by 35 U.S.C. 316 as provided in 37 CFR 1.997(e) for inter partes reexaminations.

1. A method for growing CHO cells which comprises culturing CHO cells under cell growing conditions in the absence of serum in 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 method for culturing CHO cells in accordance with  claim 1  wherein the medium further comprises non-ferrous metals, vitamins or cofactors. 
     
     
       3. A method for culturing CHO cells in accordance with  claim 1 , wherein the osmolality regulator maintains the medium at 200-350 mOsm. 
     
     
       4. A method for culturing 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 method for culturing 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 method for culturing CHO cells in accordance with  claim 5 , wherein the energy source is a monosaccharide. 
     
     
       7. A method for culturing 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, 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 method for culturing CHO cells in accordance with  claim 1 , wherein the concentration of L-glutamine is within the range of 400-600 mg/liter. 
     
     
       9. A method for culturing CHO cells in accordance with  claim 2 , wherein the medium comprises a lipid factor in an amount of 0.05-10 mg/liter. 
     
     
       10. A method for culturing 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 method for culturing CHO cells in accordance with  claim 1 , wherein the growth factor comprises recombinant or synthetic insulin, platelet derived growth factor, thyroxine T 3 , thrombin, interleukin, progesterone, hydrocortisone or vitamin E. 
     
     
       12. A method for culturing CHO cells in accordance with  claim 11 , wherein the growth factor is recombinant or synthetic insulin. 
     
     
       13. A method for culturing cells in accordance with  claim 1 , wherein the medium further comprises a peptide digest, hydrolysate or extract. 
     
     
       14. A method for culturing cells in accordance with  claim 1 , wherein the medium is essentially free of hypoxanthine and thymidine. 
     
     
       15. A method for culturing cells in accordance with  claim 14 , wherein the medium further comprises methotrexate. 
     
     
       16. A method for culturing CHO cells which comprises culturing and growing Chinese hamster ovary cells in the absence of serum in 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, 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 genetically engineered CHO cells in suspension 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 .2H 2 O, MgSO 4 .7H 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,  or about  0 . 01 - 0 . 2  mg of sodium selenite; 
 about 0.01-0.5 mg each of Vitamin B6 and Vitamin B12, ; 
 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 insulinand 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. ; 
 a base medium containing                                       L-Alanine     20-50       mg/L             L-Arginine (HCl)       50-100       mg/L             L-Asparagine (H   2   O)       20-50       mg/L             L-Aspartic Acid       20-50       mg/L;             L-Cystine (disodium salt)       50-100       mg/L;             L-Glutamic acid       50-100       mg/L;             L-Glutamine       400-600       mg/L;             Glycine       20-50       mg/L;             L-Histidine (HCl•H   2   O)       30-60       mg/L;             L-Isoleucine       50-150       mg/L;             L-Leucine       50-150       mg/L;             L-Lysine (HCl)       100-200       mg/L;             L-Methionine       20-50       mg/L;             L-Phenylalanine       40-80       mg/L;             L-Proline       30-60       mg/L;             L-Serine       30-60       mg/L;             L-Threonine       50-120       mg/L;             L-Tryptophan       10-20       mg/L;             L-Tyrosine (disodium salt)       50-120       mg/L;             L-Valine       80-120       mg/L;             Biotin       0.01-0.5       mg/L;             D Calcium Pantothenate       0.2-8       mg/L;             Folic acid       0.2-8       mg/L;             i-Inositol       0.2-8       mg/L;             Nicotinamide       0.2-8       mg/L;             Pyridoxal HCL       about 4       mg/L;             Riboflavin       0.1-1       mg/L;             Thiamin HCl       0.2-8       mg/L;             Vitamin B12       0.1-0.5       mg/L;                                                               
 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     2 H   2   O, sodium pyruvate, N - [ 2   - hydroxyethyl]piperazine - N′ - [ 2   - ethanesulphonic acid]  ( HEPES ) ,  3   - [N - morpholino] - propanesulfonic acid  ( MOPS ) , and NaHCO   3    in an amount sufficient to maintain the medium within the pH range of about  6 . 5 - 7 . 5 ; 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 method in accordance with  claim 1 , wherein each component of the medium is selected from an inorganic, synthetic, recombinant, plant or bacterial source.