CELL CULTURE IMPROVEMENTS

• US 20140206038A1
• Filed: 03/26/2014
• Published: 07/24/2014
• Est. Priority Date: 09/13/2006
• Status: Active Grant

• Current Assignee: Abbvie Incorporated
• Original Assignee: Abbvie Incorporated
• Inventors: Pla, Itzcoatl A., Matuck, Joseph G., Fann, John C., Schulz, Christof, Roy, Nichole A., Bruton, David F., McIntire, James, Chang, Yu-Hsiang David, Seewoester, Thomas
• Granted Patent: US 8,906,646 B2
• Time in Patent Office: Days
• US Class Current: 435/69.6
• CPC Class Codes: C07K 16/00 Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies antibodies with enzymatic activity, e.g. abzymes C12N9/0002
• CPC Class Codes: C07K 16/241 Tumor Necrosis Factors
• CPC Class Codes: C07K 16/244 Interleukins [IL]
• CPC Class Codes: C07K 16/2869 against hormone receptors for antibodies against neuromediator receptors C07K16/286
• CPC Class Codes: C07K 2317/14 Specific host cells or culture conditions, e.g. components, pH or temperature
• CPC Class Codes: C07K 2317/21 from primates, e.g. man
• CPC Class Codes: C12N 2500/24 Iron; Fe chelators; Transferrin
• CPC Class Codes: C12N 2500/25 Insulin-transferrin; Insulin-transferrin-selenium
• CPC Class Codes: C12N 2500/30 Organic components metal chelators C12N2500/10; calcitonin C12N2500/14; transferrin C12N2500/24
• CPC Class Codes: C12N 2500/32 Amino acids
• CPC Class Codes: C12N 2500/34 Sugars
• CPC Class Codes: C12N 2500/38 Vitamins
• CPC Class Codes: C12N 2500/50 Soluble polymers, e.g. polyethyleneglycol [PEG]
• CPC Class Codes: C12N 2500/60 Buffer, e.g. pH regulation, osmotic pressure
• CPC Class Codes: C12N 2500/74 from fungi, e.g. yeasts
• CPC Class Codes: C12N 2500/76 from plants
• CPC Class Codes: C12N 2500/90 Serum-free medium, which may still contain naturally-sourced components
• CPC Class Codes: C12N 2501/10 Growth factors
• CPC Class Codes: C12N 2501/33 Insulin together with transferrin C12N2500/25; Insulin-like growth factors C12N2501/105
• CPC Class Codes: C12N 2510/02 Cells for production
• CPC Class Codes: C12N 2511/00 : Cells for large scale production
• CPC Class Codes: C12N 5/0031 : Serum-free culture media
• CPC Class Codes: C12N 5/0037 : Serum-free medium, which may still contain naturally-sourced components
• CPC Class Codes: C12N 5/0603 : Embryonic cells production of embryos, nuclear transfer A01K67/027; Embryoid bodies

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.

Claims

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. A fed batch method of producing a protein comprising

a) culturing mammalian cells comprising a nucleic acid encoding the protein in a cell culture comprising a cell culture production medium;

and

b) feeding the mammalian cells by adding a hydrolysate enrichment solution and a basal enrichment solution to the cell culture during a time period,

wherein the hydrolysate enrichment solution comprises at least two different non-animal-based hydrolysates,

such that the protein is produced.

3. The method of claim 2, wherein the basal enrichment solution comprises a concentrated basal medium.

4. The method of claim 2, wherein the basal enrichment solution comprises a basal medium, asparagine, and glucose.

5. The method of claim 3, wherein the basal medium is PF CHO.

6. The method of claim 2, wherein the mammalian cell is a Chinese Hamster Ovary (CHO) cell.

7. The method of claim 2, wherein the protein is an antibody.

8. The method of claim 7, wherein the antibody is selected from the group consisting of an anti-TNFα

antibody, an anti-IL-12 antibody, an anti-IL-18 antibody, and an anti-EPO receptor (EPO-R) antibody.

9. The method of claim 2, wherein the hydrolysate enrichment solution comprises a first hydrolysate which is not derived from a plant or an animal and a second plant-based hydrolysate.

10. The method of claim 9, wherein the hydrolysate which is not derived from a plant or an animal and a plant-based hydrolysate is a yeast-based hydrolysate.

11. The method of claim 10, wherein the plant-based hydrolysate is a soy-based hydrolysate

12. A fed batch method of producing an anti-TNFα

antibody comprising

a) culturing Chinese Hamster Ovary (CHO) cells comprising a nucleic acid encoding the anti-TNFα

antibody in a cell culture comprising a cell culture production medium; and

b) feeding the CHO cells by adding a hydrolysate enrichment solution and a basal enrichment solution to the cell culture during a time period,

wherein the basal enrichment solution comprises a basal medium, asparagine, and glucose, and

wherein the hydrolysate enrichment solution comprises at least two different non-animal-based hydrolysates,

such that the anti-TNFα

antibody is produced.

13. The method of claim 12, further comprising recovering the anti-TNFα

antibody.

14. The method of claim 12, wherein the cell culture is cultured at a temperature ranging from about 32 to 38°

C.

15. The method of claim 14, wherein the culturing temperature is about 35°

C.

16. The method of claim 12, wherein the cell culture production medium is maintained between 20 and 65% dissolved oxygen.

17. The method of claim 16, wherein the cell culture production medium is maintained at about 30% dissolved oxygen.

18. The method of claim 12, wherein the osmolarity of the cell culture production medium is maintained throughout the culturing to no more than 500 mOsm.

19. The method of claim 12, wherein the hydrolysate enrichment solution comprises a first hydrolysate which is not derived from a plant or an animal and a second plant-based hydrolysate.

20. A method for producing at least about 1.5 g/L of an antibody from a mammalian cell culture comprising

a) culturing mammalian cells comprising a nucleic acid encoding the antibody in a cell culture production medium;

and

b) adding a combination feed solution having a pH of about 6.7 to 7.2 to the cell culture production medium, wherein the combination feed solution comprises glucose;

a basal cell medium;

an amino acid other than glutamine; and

at least two different non-animal based hydrolysates,

such that at least about 1.5 g/L of the antibody is produced.