Fed-batch method of making human anti-TNF-alpha antibody

• US 8,906,646 B2
• Filed: 03/26/2014
• Issued: 12/09/2014
• 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
• Primary Examiner(s): LANDSMAN, ROBERT S
• Application Number:
  US14/226,333
• Publication Number:
  US 20140206038A1
• Time in Patent Office: 258 Days
• US Class Current: 435/41
• 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 fed-batch method of making a human anti-TNFα

antibody comprising a light chain variable region (LCVR) comprising the sequence of SEQ ID NO;

1 and a heavy chain variable region (HCVR) comprising the sequence of SEQ ID NO;

2, said method comprising culturing Chinese Hamster Ovary (CHO) cells comprising a nucleic acid encoding said LCVR and HCVR of said anti-TNFα

antibody in a cell culture production medium in large-scale,

wherein glucose concentration in said cell production medium is monitored, the glucose concentration in said medium decreases to below 2 g/L, and glucose is added to said medium when the glucose concentration in said medium decreases to below 2 g/L, or the glucose concentration in said medium is monitored and glucose is added to said medium to maintain the glucose concentration in said medium at a concentration of at least 2 g/L but no greater than 7 g/L, such that said anti-TNFα

antibody is produced, and wherein said produced antibody is further affinity purified using a Protein A resin.

2. The method of claim 1, wherein said produced antibody has a titer of at least 1.5 g/L in said cell culture production medium.

3. The method of claim 2, wherein said culturing lasts 9-15 days.

4. The method of claim 1, wherein said produced antibody has a titer of at least 2 g/L in said cell culture production medium.

5. The method of claim 1, wherein said cell culture production medium is a serum-free medium.

6. The method of claim 5, wherein said serum-free medium comprises at least one non-animal-based hydrolysate selected from the group consisting of a yeast-based hydrolysate, a soy-based hydrolysate, and a combination thereof.

7. The method of claim 5, wherein said serum-free medium comprises galactose and manganese.

8. The method of claim 1, wherein said CHO cells are cultured at a temperature ranging from 32°

C. to 38°

C.

9. The method of claim 8, wherein said temperature is maintained as a constant temperature throughout said production method, said temperature being selected from a temperature range of 32°

C. to 38°

C.

10. A fed-batch production method of making adalimumab, comprising culturing Chinese Hamster Ovary (CHO) cells comprising a nucleic acid encoding adalimumab in a cell culture production medium in large-scale, wherein glucose concentration in said medium is monitored, the glucose concentration in said medium decreases to below 2 g/L, and glucose is added to said medium when the glucose concentration in said medium decreases to below 2 g/L, or the glucose concentration in said medium is monitored and glucose is added to said medium to maintain the glucose concentration in said medium at a concentration of at least 2 g/L but no greater than 7 g/L, such that adalimumab is produced, and wherein said produced adalimumab is further affinity purified using a Protein A resin.

11. The method of claim 10, wherein said produced antibody has a titer of at least 1.5 g/L in said cell culture production medium.

12. The method of claim 11, wherein said culturing lasts 9-15 days.

13. The method of claim 11, wherein said cell culture production medium is a serum-free medium.

14. The method of claim 13, wherein said serum-free medium comprises at least one non-animal-based hydrolysate selected from the group consisting of a yeast-based hydrolysate, a soy-based hydrolysate, and a combination thereof.

15. The method of claim 13, wherein said serum-free medium comprises galactose and manganese.

16. The method of claim 10, wherein said produced antibody has a titer of at least 2 g/L in said cell culture production medium.

17. The method of claim 16, wherein said culturing lasts 9-15 days.

18. The method of claim 16, wherein said cell culture production medium is a serum-free medium.

19. The method of claim 18, wherein said serum-free medium comprises at least one non-animal-based hydrolysate selected from the group consisting of a yeast-based hydrolysate, a soy-based hydrolysate, and a combination thereof.

20. The method of claim 18, wherein said serum-free medium comprises galactose and manganese.

21. The method of claim 11, wherein said produced antibody has a titer of at least 2.5 g/L in said cell culture production medium.

22. The method of claim 21, wherein said culturing lasts 9-15 days, and wherein (a) said cell culture production medium is a serum-free medium and comprises at least one non-animal-based hydrolysate, or (b) said cell culture production medium is a serum-free medium comprising galactose and manganese.

23. The method of claim 10, wherein said produced antibody has a titer of at least 4 g/L in said cell culture production medium and (a) said cell culture production medium is a serum-free medium and comprises at least one non-animal-based hydrolysate, or (b) said cell culture production medium is a serum-free medium comprising galactose and manganese.

24. The method of claim 10, wherein said CHO cells are cultured at a temperature ranging from 32°

C. to 38°

C.

25. A fed-batch method of producing adalimumab, comprising expressing adalimumab in Chinese Hamster Ovary (CHO) cells in a cell culture production medium in large -scale, wherein said CHO cells comprise an expression vector encoding adalimumab, wherein glucose concentration in said medium is monitored, and the glucose concentration in said medium drops to a level between 1 and 5 g/L, and glucose is added to said medium to maintain the glucose concentration in said medium at least 1-5g/L, and wherein adalimumab is further affinity purified using a Protein A resin.

26. The method of claim 25, wherein said produced antibody has a titer of at least 1.5 g/L in said cell culture production medium.

27. The method of claim 25, wherein said produced antibody has a titer of at least 2 g/L in said cell culture production medium.

28. The method of claim 25, wherein said produced antibody has a titer of at least 2.5 g/L in said cell culture production medium.