Cell culture improvements

Abstract

The invention describes improved methods and compositions for producing a recombinant protein, e.g., an antibody, in mammalian cell culture. In addition, the invention provides improved cell culture media, including improved production media, feed solutions, and combination feeds, which may be used to improve protein productivity in mammalian cell culture.

1 . A serum-free cell culture medium comprising Part A, Part B, and Part C, wherein
 a) Part A consists essentially of a modified basal medium which excludes the following components: sodium bicarbonate, a buffer, mono-basic sodium phosphate, di-basic sodium phosphate, an osmolarity regulator, a surfactant, and monosaccharide glucose;   b) Part B consists essentially of an inorganic iron source; and   c) Part C comprises a recombinant growth factor; a buffer; an osmolarity regulator; an energy source; and at least two different non-animal hydrolysates.   
     
     
         2 - 34 . (canceled) 
     
     
         35 . A serum-free cell culture medium comprising:
 a) a basal medium;   b) about 8-12 ml/kg or 116-126 mg/L ferric citrate;   c) about 2-6 mg/kg recombinant human insulin;   d) about 2-5 g/kg anhydrous glucose;   e) about 0.1-0.5 g/kg L-glutamine;   f) about 1-3 g/kg sodium bicarbonate;   g) about 0.01-0.05 g/kg NaH 2 PO 4 .H 2 O;   h) about 0.4 to 0.5 g/kg of Na 2 HPO 4 .7H 2 O; and   i) about 1.0-3.0 g/kg yeast-based hydrolysate.   
     
     
         36 . The cell culture medium of  claim 35 , comprising
 a) a basal medium;   b) about 10.0 ml/kg or 122 mg/L ferric citrate;   c) about 4.0 mg/kg recombinant human insulin;   d) about 3.5 g/kg anhydrous glucose;   e) about 0.29 g/kg L-glutamine;   f) about 1.6 g/kg sodium bicarbonate;   g) about 0.03 g/kg NaH 2 PO 4 .H 2 O;   h) about 0.43 to 0.44 g/kg of Na 2 HPO 4 .7H 2 O; and   i) about 2.0 g/kg yeast-based hydrolysate.   
     
     
         37 - 41 . (canceled) 
     
     
         42 . A serum-free cell culture production medium comprising:
 a) a modified basal medium which excludes the following components sodium bicarbonate, buffer, mono-basic sodium phosphate, di-basic sodium phosphate, an osmolality regulator, a surfactant, and monosaccharide glucose;   b) about 8 to 12 ml/kg or 122.45 mg/L ferric citrate;   c) about 4 to 8 mL/kg or 10 to 14 mg/kg recombinant human insulin;   d) about 5 to 9 g/kg anhydrous glucose;   e) about 0.5 to 0.7 g/kg L-glutamine;   f) about 1 to 2 g/kg sodium bicarbonate;   g) about 1 to 2 g/kg HEPES;   h) about 2 to 3 g/kg NaCl;   i) about 0.5 to 2 g/kg Pluronic F-68;   j) about 0.01 to 0.1 g/kg NaH 2 PO 4 .H 2 O;   k) about 0.4 to 0.5 g/kg Na 2 HPO 4 .7H 2 O;   l) about 8 to 12 g/kg yeast-based hydrolysate; and   m) about 60 to 70 g/kg plant-based hydrolysate.   
     
     
         43 . The cell culture production medium of  claim 42 , wherein the cell culture medium comprises
 a) about 10.0 ml/kg or 122.45 mg/L ferric citrate;   b) about 6.0 mL/kg or 12 mg/kg recombinant human insulin;   c) about 7.0 g/kg anhydrous glucose;   d) about 0.58 to 0.59 g/kg L-glutamine;   e) about 1.6 g/kg sodium bicarbonate;   f) about 1.8 g/kg HEPES;   g) about 2.4 to 2.5 g/kg NaCl;   h) about 1.0 g/kg Pluronic F-68;   i) about 0.03 to 0.04 g/kg NaH 2 PO 4 .H 2 O;   j) about 0.43 to 0.44 g/kg Na 2 HPO 4 .7H 2 O;   k) about 10.7 g/kg yeast-based hydrolysate; and   l) about 6.9 to 7.0 g/kg plant-based hydrolysate.   
     
     
         44 - 45 . (canceled) 
     
     
         46 . A serum-free cell culture medium comprising:
 a) a modified basal medium, which excludes the following components sodium bicarbonate, buffer, mono-basic sodium phosphate, di-basic sodium phosphate, an osmolality regulator, a surfactant, and monosaccharide glucose;   b) about 8 to 12 ml/kg or 122.45 mg/L ferric citrate;   c) about 3 to 5 mL/kg or 6 to 8 mg/kg recombinant human insulin;   d) about 5 to 9 g/kg anhydrous glucose;   e) about 0.1 to 2 g/kg L-glutamine;   f) about 1 to 2 g/kg sodium bicarbonate;   g) about 1 to 2 g/kg HEPES;   h) about 2 to 3 g/kg NaCl;   i) about 0.1 to 2 g/kg Pluronic F-68;   j) about 0.01 to 0.1 g/kg NaH 2 PO 4 .H 2 O;   k) about 0.4 to 0.5 g/kg Na 2 HPO 4 .7H 2 O;   l) about 0.4 to 0.5 g/kg L-asparagine monohydrate;   m) about 2 to 6 g/kg yeast-based hydrolysate; and   n) about 2 to 4 g/kg plant-based hydrolysate.   
     
     
         47 . The cell culture medium of  claim 46 , wherein the cell culture medium comprises
 a) about 10.0 ml/kg or 122.45 mg/kg ferric citrate;   b) about 3.8 to 3.9 mL/kg or 7.8 mg/kg recombinant human insulin;   c) about 7.0 g/kg anhydrous glucose;   d) about 0.8 to 0.9 g/kg L-glutamine;   e) about 1.6 g/kg sodium bicarbonate;   f) about 1.8 g/kg HEPES;   g) about 2.6 to 2.7 g/kg NaCl;   h) about 1.0 g/kg Pluronic F-68;   i) about 0.03 to 0.04 g/kg NaH 2 PO 4 .H 2 O;   j) about 0.43 to 0.44 g/kg Na 2 HPO 4 .7H 2 O;   k) about 0.45 g/kg L-asparagine monohydrate;   l) about 4.0 g/kg yeast-based hydrolysate; and   m) about 2.6 g/kg plant-based hydrolysate.   
     
     
         48 - 54 . (canceled) 
     
     
         55 . A serum-free cell culture medium comprising:
 a) a modified basal medium, which excludes the following components sodium bicarbonate, buffer, mono-basic sodium phosphate, di-basic sodium phosphate, an osmolality regulator, a surfactant, and monosaccharide glucose;   b) about 8 to 10 ml/kg or 120 to 130 mg/L ferric citrate;   c) about 3 to 5 mL/kg or 7.8 mg/kg recombinant human insulin;   d) about 5 to 9 g/kg anhydrous glucose;   e) about 0.8 to 0.9 g/kg L-glutamine;   f) about 0.3 to 0.5 g/kg L-asparagine monohydrate;   g) about 1 to 2 g/kg sodium bicarbonate;   h) about 1 to 2 g/kg HEPES;   i) about 2 to 3 g/kg NaCl;   j) about 0.5 to 2 g/kg Pluronic F-68;   k) about 0.01 to 0.1 g/kg NaH 2 PO 4 .H 2 O;   l) about 0.1 to 1.0 g/kg Na 2 HPO 4 .7H 2 O;   m) about 2 to 6 g/kg yeast-based hydrolysate; and   n) about 2 to 4 g/kg plant-based hydrolysate.   
     
     
         56 . The cell culture medium of  claim 55 , comprising
 a) about 10 ml/kg or 122 mg/L ferric citrate;   b) about 3.8 to 3.9 mL/kg or 7.8 mg/kg recombinant human insulin;   c) about 7.0 g/kg anhydrous glucose;   d) about 0.87 to 0.88 g/kg L-glutamine;   e) about 0.45 g/kg L-asparagine monohydrate;   f) about 1.6 g/kg sodium bicarbonate;   g) about 1.8 g/kg HEPES;   h) about 2.67 to 2.68 g/kg NaCl;   i) about 1.0 g/kg Pluronic F-68;   j) about 0.03 to 0.04 g/kg NaH 2 PO 4 .H 2 O;   k) about 0.43 to 0.44 g/kg Na 2 HPO 4 .7H 2 O;   l) about 4.0 g/kg yeast-based hydrolysate; and   m) about 2.6 g/kg plant-based hydrolysate.   
     
     
         57 . (canceled) 
     
     
         58 . A serum-free cell culture medium comprising:
 a) basal cell growth medium;   b) about 8 to 12 ml/kg or 120 to 130 mg/L ferric citrate;   c) about 2 to 6 mg/kg recombinant human insulin;   d) about 150 to 250 g/kg anhydrous glucose;   e) about 0.1 to 0.5 g/kg L-glutamine;   f) about 1 to 2 g/kg sodium bicarbonate; and   g) about 5 to 15 g/kg yeast-based hydrolysate.   
     
     
         59 . The cell culture medium of  claim 58 , comprising
 a) about 10 ml/kg or 122.45 mg/L ferric citrate;   b) about 4 mg/kg recombinant human insulin;   c) about 200 g/kg anhydrous glucose;   d) about 0.29 to 0.30 g/kg L-glutamine;   e) about 1.6 g/kg sodium bicarbonate; and   f) about 11 g/kg yeast-based hydrolysate.   
     
     
         60 - 65 . (canceled) 
     
     
         66 . A serum-free cell culture medium comprising:
 a) basal cell growth medium;   b) about 8 to 12 ml/kg or 120 to 130 mg/L ferric citrate;   c) about 2 to 6 mg/kg recombinant human insulin;   d) about 1 to 3 g/kg anhydrous glucose;   e) about 0.1 to 1 g/kg L-glutamine;   f) about 1 to 2 g/kg sodium bicarbonate; and   g) about 1 to 4 g/kg yeast-based hydrolysate.   
     
     
         67 . The cell culture medium of  claim 66 , comprising
 a) about 10 ml/kg or 122.45 mg/L ferric citrate;   b) about 4 mg/kg recombinant human insulin;   c) about 1.5 g/kg anhydrous glucose;   d) about 0.29 to 0.30 g/kg L-glutamine;   e) about 1.6 g/kg sodium bicarbonate; and   f) about 2 g/kg yeast-based hydrolysate.   
     
     
         68 - 76 . (canceled) 
     
     
         77 . A cell culture medium comprising:
 a) a modified basal medium which excludes the following components sodium bicarbonate, buffer, mono-basic sodium phosphate, di-basic sodium phosphate, an osmolality regulator, a surfactant, and monosaccharide glucose;   b) about 8 to 12 ml/kg or 120 to 130 mg/L ferric citrate;   c) about 2.5 to 4.5 mL/kg or 7.8 mg/kg recombinant human insulin;   d) about 5 to 9 g/kg anhydrous glucose;   e) about 0.5 to 1 g/kg L-glutamine;   f) about 0.1 to 1 g/kg L-asparagine monohydrate;   g) about 1 to 2 g/kg sodium bicarbonate;   h) about 1 to 2 g/kg HEPES;   i) about 1 to 4 g/kg NaCl;   j) about 0.1 to 2 g/kg Pluronic F-68;   k) about 0.01 to 0.1 g/kg NaH 2 PO 4 .H 2 O;   l) about 0.1 to 1 g/kg Na 2 HPO 4 .7H 2 O;   m) about 2 to 6 g/kg yeast-based hydrolysate; and   n) about 2 to 6 g/kg plant-based hydrolysate.   
     
     
         78 . The cell culture medium of  claim 77 , comprising
 a) about 10 ml/kg or 122.45 mg/L ferric citrate;   b) about 3.8 to 3.9 mL/kg or 7.8 mg/kg recombinant human insulin;   c) about 7.0 g/kg anhydrous glucose;   d) about 0.87 to 0.88 g/kg L-glutamine;   e) about 0.45 g/kg L-asparagine monohydrate;   f) about 1.6 g/kg sodium bicarbonate;   g) about 1.8 g/kg HEPES;   h) about 2.67 g/kg NaCl;   i) about 1.0 g/kg Pluronic F-68;   j) about 0.03 to 0.04 g/kg NaH 2 PO 4 .H 2 O;   k) about 0.43 to 0.44 g/kg Na 2 HPO 4 .7H 2 O;   l) about 4.0 g/kg yeast-based hydrolysate; and   m) about 2.6 g/kg plant-based hydrolysate.   
     
     
         79 - 80 . (canceled) 
     
     
         81 . A cell culture production medium comprising:
 a) a modified basal medium, which is modified to remove the following components sodium bicarbonate, HEPES buffer, mono-basic sodium phosphate, di-basic sodium phosphate, an osmolality regulator, a surfactant, and monosaccharide glucose;   b) about 8 to 12 ml/kg or 120 to 130 mg/L ferric citrate;   c) about 4 to 8 mL/kg or 10 to 14 mg/kg recombinant human insulin;   d) about 5 to 9 g/kg anhydrous glucose;   e) about 0.1 to 1 g/kg L-glutamine;   f) about 1 to 2 g/kg sodium bicarbonate;   g) about 1 to 2 g/kg HEPES;   h) about 1 to 3 g/kg NaCl;   i) about 0.5 to 2 g/kg Pluronic F-68;   j) about 0.01 to 0.1 g/kg NaH 2 PO 4 .H 2 O;   k) about 0.1 to 1 g/kg Na 2 HPO 4 .7H 2 O;   l) about 8 to 12 g/kg yeast-based hydrolysate; and   m) about 6 to 8 g/kg plant-based hydrolysate.   
     
     
         82 . The cell culture production medium of  claim 81 , comprising
 a) about 10 ml/kg or 122.45 mg/L ferric citrate;   b) about 6.0 mL/kg or 12 mg/kg recombinant human insulin;   c) about 7.0 g/kg anhydrous glucose;   d) about 0.58 to 0.59 g/kg L-glutamine;   e) about 1.6 g/kg sodium bicarbonate;   f) about 1.8 g/kg HEPES;   g) about 2.45 g/kg NaCl;   h) about 1.0 g/kg Pluronic F-68;   i) about 0.03 to 0.04 g/kg NaH 2 PO 4 .H 2 O;   j) about 0.43 to 0.44 g/kg Na 2 HPO 4 .7H 2 O;   k) about 10.7 g/kg yeast-based hydrolysate; and   l) about 6.9 to 7.0 g/kg plant-based hydrolysate.   
     
     
         83 - 84 . (canceled) 
     
     
         85 . A cell culture production medium comprising:
 a) a modified basal medium which excludes the following components sodium bicarbonate, buffer, mono-basic sodium phosphate, di-basic sodium phosphate, an osmolality regulator, a surfactant, and monosaccharide glucose;   b) about 8 to 12 ml/kg or 110 to 130 mg/L ferric citrate;   c) about 4 to 8 mL/kg or 11 to 15 mg/kg recombinant human insulin;   d) about 5 to 9 g/kg anhydrous glucose;   e) about 0.1 to 1 g/kg L-glutamine;   f) about 1 to 2 g/kg sodium bicarbonate;   g) about 1 to 2 g/kg HEPES;   h) about 1 to 3 g/kg NaCl;   i) about 0.1 to 2 g/kg Pluronic F-68;   j) about 0.01 to 0.1 g/kg NaH 2 PO 4 .H 2 O;   k) about 0.1 to 1 g/kg Na 2 HPO 4 .7H 2 O;   l) about 12 to 16 g/kg yeast-based hydrolysate; and   m) about 8 to 10 g/kg plant-based hydrolysate.   
     
     
         86 . The cell culture production medium of  claim 85 , comprising
 a) about 10 ml/kg or 122.45 mg/L ferric citrate;   b) about 6.5 mL/kg or 13 mg/kg recombinant human insulin;   c) about 7.0 g/kg anhydrous glucose;   d) about 0.58 to 0.59 g/kg L-glutamine;   e) about 1.6 g/kg sodium bicarbonate;   f) about 1.8 g/kg HEPES;   g) about 2.45 g/kg NaCl;   h) about 1.0 g/kg Pluronic F-68;   i) about 0.03 to 0.04 g/kg NaH 2 PO 4 .H 2 O;   j) about 0.43 to 0.44 g/kg Na 2 HPO 4 .7H 2 O;   k) about 14.2 to 14.3 g/kg yeast-based hydrolysate; and   l) about 9.2 to 9.3 g/kg plant-based hydrolysate.   
     
     
         87 . The cell culture production medium of  claim 86 , further comprising methotrexate. 
     
     
         88 - 91 . (canceled) 
     
     
         92 . A Chinese Hamster Ovary (CHO) cell in the cell culture medium of  claim 86 . 
     
     
         93 - 158 . (canceled) 
     
     
         159 . A combination feed solution comprising
 a) glucose;   b) a basal medium;   c) an amino acid other than glutamine; and   d) at least two different non-animal based hydrolysates;   wherein the feed solution has a pH of about 6.0 to 8.0.   
     
     
         160 - 233 . (canceled)