SERUM-FREE CELL CULTURE MEDIUM

Abstract

The specification describes an improved serum-free animal cell culture medium, which can used for the production of a protein of interest. Ornithine, or a combination of ornithine and putrescine can be added to serum-free media or chemically defined media to improve viable cell density, to reduce cell doubling time, and to increase the production of a protein of interest.

Claims

1. A method for producing aflibercept comprising: (a) culturing Chinese hamster ovary (CHO) cells expressing aflibercept in a cell culture medium, wherein said cell culture medium is serum-free, (b) culturing said CHO cells in said cell culture medium at a temperature of 35° C. to 38° C. during a growth phase; and (c) culturing said CHO cells in said cell culture medium at a decreased temperature of 29° C. to 37° C. during a production phase when the cell density of the growth phase reaches between 1.6×10.sup.6 and 12.6×10.sup.6 viable cells per mL.

2. The method of claim 1, wherein step (c) comprises culturing said CHO cells at a decreased temperature of 30° C. to 34° C. during the production phase.

3. The method of claim 1, wherein the decreased temperature of step (c) of the production phase begins between day 3 and day 7 when the cell density of the growth phase reaches between 1.6×10.sup.6 and 12.6×10.sup.6 viable cells per mL.

4. The method of claim 2, wherein the decreased temperature of step (c) of the production phase begins between day 5 and day 7 when the cell density of the growth phase reaches between 2.0×10.sup.6 and 3.6×10.sup.6 viable cells per mL.

5. The method of claim 1, wherein said cell culture medium comprises ≥0.09 mM 0.014 mM ornithine and decreasing the temperature at step (c) of the production phase between day 3 and day 7 when the cell density of the growth phase reaches between 5.1×10.sup.6 and 12.6×10.sup.6 viable cells per mL.

6. The method of claim 2, wherein said cell culture medium comprises ≥0.09 mM 0.014 mM ornithine and decreasing the temperature at step (c) of the production phase between day 3 and day 7 when the cell density of the growth phase reaches between 5.1×10.sup.6 and 12.6×10.sup.6 viable cells per mL.

7. The method of claim 1, wherein said cell culture medium comprises ≥0.09 mM±0.014 mM ornithine and decreasing the temperature at step (c) of the production phase between day 5 and day 7 when the cell density of the growth phase reaches between 5.9×10.sup.6 and 12.6×10.sup.6 viable cells per mL.

8. The method of claim 1, wherein the cell culture medium comprises ornithine at a concentration ranging from 0.09±0.014 mM to 0.9±0.14 mM and decreasing the temperature at step (c) of the production phase between day 3 and day 7 when the cell density of the growth phase reaches between 5.1×10.sup.6 and 12.6×10.sup.6 viable cells per mL.

9. The method of claim 1, wherein the cell culture medium comprises ornithine at a concentration ranging from 0.09±0.014 mM to 0.9±0.14 mM and decreasing the temperature at step (c) of the production phase between day 5 and day 7 when the cell density of the growth phase reaches between 5.9×10.sup.6 and 12.6×10.sup.6 viable cells per mL.

10. The method of claim 1, wherein the cell culture medium comprises 0.6±0.09 mM ornithine and 0.714±0.11 mM putrescine and decreasing the temperature at step (c) of the production phase between day 3 and day 7 when the cell density of the growth phase reaches between 5.0×10.sup.6 and 12.4×10.sup.6 viable cells per mL.

11. The method of claim 2, wherein the cell culture medium comprises 0.6±0.09 mM ornithine and 0.714±0.11 mM putrescine and decreasing the temperature at step (c) of the production phase between day 5 and day 7 when the cell density of the growth phase reaches between 5.8×10.sup.6 and 12.4×10.sup.6 viable cells per mL.

12. The method of claim 1, wherein the cell culture medium comprises 0.6±0.09 mM ornithine and 0.714±0.11 mM putrescine and decreasing the temperature at step (c) of the production phase between day 5 and day 7 when the cell density of the growth phase reaches between 5.8×10.sup.6 and 12.4×10.sup.6 viable cells per mL.

13. The method of claim 2, wherein the cell culture medium comprises 0.6±0.09 mM ornithine and 0.714±0.11 mM putrescine and decreasing the temperature at step (c) of the production phase between day 3 and day 7 when the cell density of the growth phase reaches between 5.0×10.sup.6 and 12.4×10.sup.6 viable cells per mL.

14. The method of claim 1, wherein the aflibercept is produced at a titer of at least 100 mg/L.

15. The method of claim 1, wherein the aflibercept is produced at a titer of at least 1 g/L.

16. The method of claim 1, wherein the CHO cell is selected from the group of CHO K1 cells, CHO DUX B-11 cells, Veggie-CHO cells, GS-CHO cells, D-CHO cells, and CHO lec mutant cells.

17. The method of claim 1, wherein the culture medium comprises less than or equal to 7.5 g/L soy hydrolysate.

18. The method of claim 1, wherein the cell culture medium comprises: (a) ≥40±6 mM of a mixture of amino acids or salts thereof; (b) one or more fatty acids; (c) a mixture of nucleosides; or (d) one or more divalent cations.

19. A method for producing aflibercept comprising: (a) culturing cells capable of producing aflibercept at a temperature range of 35° C. to 38° C.; and then (b) culturing said cells at a decreased temperature of 29° C. to 34° C. when the cell density reaches between 1.6×10.sup.6 and 12.6×10.sup.6 viable cells per mL, wherein said cells are cultured in a serum-free cell culture medium.

20. The method of claim 19, wherein the temperature of step (b) is decreased between day 3 and day 7 when the cell density of the growth phase reaches between 1.6×10.sup.6 and 12.6×10.sup.6 viable cells per mL.

21. The method of claim 19, wherein said cell culture medium comprises ≥0.09 mM 0.014 mM ornithine and the temperature at step (b) is decreased between day 3 and day 7 when the cell density reaches between 5.1×10.sup.6 and 12.6×10.sup.6 viable cells per mL.

22. The method of claim 19, wherein said cell culture medium comprises ≥0.09 mM 0.014 mM omithine and the temperature at step (b) is decreased between day 5 and day 7 when the cell density reaches between 5.9×10.sup.6 and 12.6×10.sup.6 viable cells per mL.

23. The method of claim 21, wherein the cell culture medium comprises ornithine at a concentration ranging from 0.09±0.014 mM to 0.9±0.14 mM omithine and the temperature at step (b) is decreased when between day 3 and day 7 when the cell density reaches between 5.1×10.sup.6 and 12.6×10.sup.6 viable cells per mL.

24. The method of claim 19, wherein said cells capable of producing aflibercept are selected from the group of CHO K1 cells, CHO DUX B-11 cells, Veggie-CHO cells, GS-CHO cells, D-CHO cells, and CHO lec mutant cells.

25. A method for producing aflibercept comprising: (a) culturing Chinese hamster ovary (CHO) cells capable of producing aflibercept at a temperature range of 35° C. to 38° C., wherein said cell culture medium is serum-free and comprises hydrolysates and polyamines; and then (b) culturing said CHO cells at a decreased temperature of 29° C. to 34° C. when the cell density reaches between 1.6×10.sup.6 and 12.6×10.sup.6 viable cells per mL.

26. The method of claim 25, wherein said CHO cells capable of producing aflibercept are selected from the group of CHO K1 cells, CHO DUX B-11 cells, Veggie-CHO cells, GS-CHO cells, D-CHO cells, and CHO lec mutant cells.

27. The method of claim 25, wherein the decreased temperature of step (b) begins between day 5 and day 7 when the cell density of the growth phase reaches between 2.0×10.sup.6 and 3.6×10.sup.6 viable cells per mL.

28. The method of claim 27, wherein the aflibercept protein is produced at a titer of at least 100 mg/L.

29. The method of claim 28, wherein the cell culture medium comprises: (a) ≥40±6 mM of a mixture of amino acids or salts thereof; (b) one or more fatty acids; (c) a mixture of nucleosides; or (d) one or more divalent cations.

30. The method of claim 26, wherein said culture medium comprises one or more supplements selected form the group of: (i) about 29.8 mM NaHCO.sub.3, (ii) about 2mM glutamine, (iii) about 0.86 μM insulin, (iv) about 11.1 mM glucose, (v) about 6.54 μM zinc sulfate, (vi) copper sulfate, (vii) ferric chloride, (viii) nickel sulfate, (ix) about 85 μM EDTA, and (x) about 50 μM citrate.

Description

EXAMPLE 1

Improved Viable Cell Culture Density

[0090] A 250 mL shake flask was inoculated from a seed culture of a recombinant antibody producing cell line derived from CHO K1. The inoculated cells were grown at 36.5° C. for seven days and fed glucose on days three and five. Cells were grown in each of two separate chemically defined (hydrolysate-free and serum-free) media. The first medium contained about 75 mM amino acids (Medium 1), the second medium contained about 40 mM amino acids (Medium 2), and both formulations contained no more than 2.5 μM (0.4 mg/L) putrescine. Another group of medium conditions was generated by adding soy hydrolysate at a concentration of 7.5 g/L to Medium 2. To each of the three control media, about 593 μM ornithine (as 100 mg/L L-ornithine HCl), or a combination of about 593 μM ornithine (as 100 mg/L L-ornithine HCl) and about 714 μM putrescine (as 115 mg/L putrescine 2HCl) were added. Aliquots of 3 mL culture were removed on days 3, 5, and 7 and viable cell counts were conducted using trypan blue exclusion on a BioProfile FLEX™ instrument (Nova Biomedical). At day zero, all cultures contained 0.8×10.sup.6 viable cells per mL. For a given medium (Medium 1, Medium 2, or Medium 2+Soy), viable cell counts over a seven-day period revealed that CHO cells grown in media supplemented with ornitihine or ornithine plus putrescine had increased viable cell densities. The effect was especially pronounced in the hydrolysate free media (i.e., 2-fold to 4-fold or more increase in viable cell density) during the seven-day period. Hydrolysate free OS Medium 2 performed comparably to soy containing non-OS Medium 2 indicating that the cell growth benefit of soy hydrolysate can be replicated by ornithine replacement. Increased cell density by adding ornithine or ornithine and putrescine to Medium 2+soy was also observed. Results are presented in Table 1.

TABLE-US-00001 TABLE 1 AVERAGE VIABLE CELL CULTURE DENSITY (10.sup.6 CELLS PER MILLILITER) AND X FOLD INCREASE OVER BASELINE* Ornithine + Supplement: Time Unsupplemented Omitihine putrescine Medium 1 3 days 2.4/1X 6.1/2.5X 5.0/2.1X 5 days 3.4/1X 12.6/3.7X  12.4/3.6X  7 days 3.6/1X 7.0/1.9X 6.8/1.9X Medium 2 3 days 1.7/1X 5.1/3.0X 5.2/3.1X 5 days 2.0/1X 7.6/3.8X 8.0/4.0X 7 days 1.6/1X 5.9/3.7X 5.8/3.6X Medium 2 + 3 days 5.2/1X 5.4/1X.sup.  4.7/0.9X soy 5 days 7.7/1X 9.3/1.2X 9.3/1.2X hydrolysate 7 days ND 9.6/ND  9.1/ND  *Base line is unsupplemented media for a given medium formulation on a given day.

[0091] We also examined the effect of various amounts of ornithine HCl (i.e., 50 mg/mL, 100 mg/mL, and 150 mg/mL) on viable cell density in Medium 3, which contains about 75 mM of amino acids and 0.4 mg/L putrescine HCl (“Medium 3”). A single seed train culture of a recombinant antibody producing cell line derived from CHO K1 was used to inoculate 50 mL TubeSpin® Bioreactors (TPP) at 0.4×10.sup.6 cells/mL at a 15 mL working volume. The cells were grown in a 37° C. incubator for three days. Aliquots of 3 mL culture were removed on day 3 and viable cell counts were conducted using trypan blue exclusion on a BioProfile FLEX™ instrument (Nova Biomedical). All three levels of ornithine improved cell density on average (N=3) by slightly more than two fold. The results are depicted in Table 2.

TABLE-US-00002 TABLE 2 VIABLE CELL CULTURE DENSITY (10.sup.6 CELLS PER MILLILITER) AND X FOLD INCREASE OVER BASELINE* Concentration Control Ornithine HCl (mg/mL) 0 50 100 150 Viable cell 1.3 3.2 3.1 3.1 density (10.sup.6 cells/mL) Fold increase 1X 2.5X 2.4X 2.4X over control *Base line is unsupplemented Medium 3.

EXAMPLE 2

Improved Cell Culture Doubling Time

[0092] The doubling time of a recombinant antibody producing cell line derived from CHO K1 cells in logarithmic growth phase was determined under various cell culture media conditions. Seed train cultures were passaged at 36.5° C. in 250 mL shaker flasks over a period of 14 days in each of three separate media: Medium 1, Medium 2, and Medium 2 containing soy hydrolysate (Medium 2+Soy). Aliquots of 1 mL were removed from each condition on Day 0 and at the time of seed train passage (every 2 or 3 days), and viable cell counts were conducted using trypan blue exclusion on a CDV™ instrument (Nova Biomedical). Medium 1 was tested unsupplemented or supplemented with ornithine HCl at 100 mg/L or both putrescine⋅2HCl at 115 mg/L and ornithine HCl at 100 mg/L. Medium 2 with low putrescine⋅2HCl (0.4 mg/L) was tested unsupplemented or supplemented with ornithine⋅HCl at 100 mg/L or both putrescine⋅2HCl at 115 mg/L and ornithine⋅HCl at 100 mg/L. The results are depicted in Tables 3 and 4. Ornithine supplementation, either with or without putrescine, to Medium 1 was required to achieve significant growth. Supplementing hydrolysate free Medium 2 with ornithine or ornithine +putrescine decreased the cell doubling time by about 25% to 30%. Doubling times were also reduced to a lesser extent upon the addition of ornithine or ornithine+putrescine to hydrolysate containing Medium 2.

TABLE-US-00003 TABLE 3 CELL DOUBLING TIME (HOURS) AND APPROXIMATE PERCENT DOUBLING TIME DECREASE RELATIVE TO BASELINE* IN MEDIUM 1 Supplement Medium 1 *None 75 Ornithine 23 69% Putrescine + ornithine 21 72% *Baseline is unsupplemented Medium1.

TABLE-US-00004 TABLE 4 CELL DOUBLING TIME (HOURS) AND APPROXIMATE PERCENT DOUBLING TIME DECREASE RELATIVE TO BASELINE* IN MEDIUM 2 Supplement Medium 2 Medium 2 + Soy *Unsupplemented 27 22.5 Ornithine 21 22% 20.5 8.9% Putrescine + 19.5 28% 21 6.7% ornithine *Baseline is unsupplemented Medium 2.

EXAMPLE 3

Improved Antibody Titers

[0093] Having established that the inclusion of ornithine or ornitihine+putrescine improves cell proliferation and viable cell density in culture, we further investigated the effect of those conditions on recombinant protein production titers. We examined the expression and secretion of recombinant IgG by a CHO-K1 derived cell line. In this experiment, the average antibody titer was determined at day seven in culture under various media formats. As above, Medium 1 with low putrescine (0.4 mg/L putrescine 2HCl), ornithine (100 mg/L ornithine HCl), and both ornithine and putrescine (100 mg/L ornithine HCl/115 mg/L putrescine 2HCl) were tested. Medium 2 and Medium 2+Soy with low putrescine (0.4 mg/L putrescine 2HCl), ornithine (100 mg/L ornithine HCl), and both ornithine and putrescine (100 mg/L ornithine HCl/115 mg/L putrescine 2HCl) were also tested. In all cases, the inclusion of ornithine or ornithine and putrescine at a level above 0.4 mg/L resulted in a significantly larger protein titer, i.e., at least about two-fold higher titers. The results are depicted in Table 5.

TABLE-US-00005 TABLE 5 AVERAGE SEVEN-DAY ANTIBODY TITERS AND APPROXIMATE FOLD INCREASE (X) IN TITER RELATIVE TO BASELINE* Supplement Medium 1 Medium 2 Medium 2 + Soy Unsupple- 0.31 g/L .sup. 1X 0.29 g/L .sup. 1X 0.54 g/L 1X mented* Ornithine 0.94 g/L 3.0X 0.65 g/L 2.2X 0.98 g/L 1.8X.sup.  Putrescine + 0.95 g/L 3.1X 0.64 g/L 2.2X 1.07 g/L 2X ornithine *Baseline is set at titer in for unsupplemeted media of each type.

[0094] We also examined the effect of various amounts of ornithine HCl (i.e., 50 mg/mL, 100 mg/mL, and 150 mg/mL) in Medium 3 on antibody production. A single seed train culture of a recombinant antibody producing cell line derived from CHO K1 was used to seed 50 mL TubeSpin® Bioreactors (TPP) at 0.4×10.sup.6 cells/mL at a 15 mL working volume. The cells were grown in a 37° C. incubator for three days. All three levels of ornithine supplementation improved antibody titer on the average (N=3) by slightly more than 50%. The results are depicted in Table 6.

TABLE-US-00006 TABLE 6 ANTIBODY TITERS (GRAMS PER LITER) AND X FOLD INCREASE IN TITER OVER BASELINE* Supplement concentration Control Ornithine HCl (mg/mL) 0 50 100 150 Antibody titer (mg/mL) 79 120 127 124 Fold increase over control  1X 1.5X 1.6X 1.6X *Base line is unsupplemented Medium 3.