New medium for high performance mammalian fed-batch cultures

Patent metadata

Field	Value
Publication number	US-2016264940-A1
Application number	US-201415028989-A
Country	US
Kind code	A1
Filing date	Apr 3, 2014
Priority date	Oct 14, 2013
Publication date	Sep 15, 2016
Grant date	—

Abstract

The present invention relates to new serum- and protein-free culture media. These media are high performance culture media, which notably improve mammalian fed-batch cultures. The present invention also relates to methods for preparing and/or designing the medium, and methods of use thereof.

First claim

1 . A cell culture medium comprising NaH 2 PO 4 , L-Leucine, L-Lysine, L-Methionine, L-Glutamic acid, L-phenylalanine, L-proline, L-threonine, L-tryptophan, L-Valine, magnesium sulfate, calcium chloride, myo-inositol, sodium pyruvate, D-Biotin, choline chloride, L-Aspargine, folic acid, niacinamide (B3), D-pantothenic acid×½Ca, L-Serine, potassium chloride, pyridoxine, L-Aspartic acid, riboflavin, thiamine, ferric ammonium citrate, vitamin B12, hypoxanthine, thymidine, putrescine, ethanolamine, zinc sulfate, cupric sulfate, pluronic, L-tyrosine, sodium selenite, L-arginine, L-Cysteine, L-Histidine and L-Isoleucine, wherein the medium is serum and protein free.

2 . The medium according to claim 1 comprising: 1.7 to 10 mM of NaH 2 PO 4 , 2 to 9 mM of L-Leucine, 1 to 6 mM of L-Lysine, 0 to 3 mM of Glycine, 0.4 to 2 mM of L-Methionine, 1 to 4 mM of L-Glutamic acid, 0.5 to 3 mM of L-phenylalanine, 0.7 to 6 mM of L-proline, 0.7 to 6 mM of L-threonine, 0.5 to 2 mM of L-tryptophan, 1 to 7 mM L-Valine, 0.1 to 1.5 mM of Magnesium Sulfate, 0.1 to 1.05 mM of Calcium Chloride, 0.07 to 0.7 mM of myo-Inositol, 0.8 to 4 mM of Sodium pyruvate, 0.0008 to 0.01 mM of D-Biotin, 0.1 to 1 mM of Choline Chloride, 3 to 9 mM of L-Aspargine, 0.006 to 0.04 mM of Folic acid, 0.03 to 0.15 mM of Niacinamide (B3), 0.015 to 0.15 mM of D-pantothenic acid×½Ca, 1 to 8 mM of L-Serine, 1 to 10 mM of Potassium Chloride, 0.005 to 0.05 mM of Pyridoxine, 0.8 to 2.4 mM of L-Aspartic acid, 0.0003 to 0.003 mM of Riboflavin, 0.008 to 0.04 mM of Thiamine, 1 to 10 mg/L of Ferric ammonium citrate, 0.0003 to 0.004 mM of Vitamin B12, 0.008 to 0.04 mM of Hypoxanthine, 0.0015 to 0.006 mM of Thymidine, 0.006 to 0.03 mM of Putrescine, 0.1 to 0.5 mM of Ethanolamine, 0.004 to 0.02 mM of Zinc Sulfate, 0.00004 to 0.0008 mM of Cupric sulfate, 0.5 to 2.0 g/L of Pluronic, 0.7 to 3 mM of L-tyrosine, 0.00001 to 0.00006 mM of Sodium Selenite, 0 to 3 mM of L-Alanine, 1 to 3 mM of L-Arginine, 1 to 3 mM of L-Cysteine, 0.4 to 3 mM of L-Histidine, and 1 to 6 mM of L-Isoleucine.

3 . The medium according to claim 1 , further comprising glucose, NaHCO 3 , NaCl, NaOH, or combinations thereof.

4 . The medium according to claim 3 , wherein the glucose is at a concentration of about 6 g/L and NaHCO 3 is at a concentration of about 2 g/L.

5 . The medium according to claim 1 , wherein the osmolality ranges from 300 to 330 mOsm/kg.

6 . The medium according to claim 1 , wherein the pH ranges from 6.0-8.0.

7 . The medium according to claim 1 , further comprising Glycine or L-Alanine or both.

8 . An isolated mammalian cell line adapted to grow in a culture medium according to claim 1 .

9 . The mammalian cell line according to claim 8 , which is a CHO cell line or a recombinant CHO cell line.

10 . A method for culturing mammalian cells to obtain a product comprising growing the cells in the medium of claim 1 .

11 . A method of developing and/or optimizing a high-throughput media blend, comprising:
a) selecting media components or selecting a medium for optimization; wherein the medium for optimization comprises well characterized components; b) selecting 3 levels of concentrations for each of the components; c) preparing a set of media formulations with selected components at different concentrations; d) mixing the different formulations, at different concentrations, to obtain different media blends; e) evaluating each blend for cell culture performance, wherein the cell culture is a mammalian cell culture; f) monitoring the performance of each blend on cell culture; g) analyzing the data obtained in step f); and h) determining one or more final culture medium.

12 . The method according to claim 11 , wherein in the step (h) the final culture medium is optimized compared to other media or compared to the medium from which it has been optimized.

13 . The method according to claim 11 , wherein the culture medium is for cell expansion and/or fed-batch culture.

14 . The method according to claim 11 , wherein the media blends of step (c) further comprise glucose, NaHCO 3 , NaCl, NaOH, or combinations thereof.

15 . A high-throughput media blending method for developing and/or optimizing a culture medium, comprising the steps of:
a) selecting media components or selecting a medium for optimization, wherein the medium for optimization comprises well characterized components; b) selecting 3 levels of concentrations for each of the components; c) preparing a set of media formulations with the selected components at different concentrations; d) mixing the different formulations, at different concentrations, to obtain different media blends; e) evaluating each blend for cell culture performance, wherein the cell culture is a mammalian cell culture; monitoring the performance of each blend on cell culture; g) analyzing the data obtained in step f); h) determining the key component(s) for further optimization; i) repeat steps b) to g) based on the information obtained in step h); j) optionally repeat steps h) and i); and k) determining one or more final culture medium.

16 . The method according to claim 15 , wherein in the step (k) the final culture medium is optimized compared to other media or compared to the medium from which it has been optimized.

17 . The method according to claim 15 , wherein the culture medium is for cell expansion fed-batch culture, or a combination thereof.

18 . The method according to claim 15 , wherein step (d) further comprises glucose, NaHCO 3 , NaCl, NaOH, or combinations thereof.

19 . The method according to claim 15 , wherein the key component(s) of step h) comprise at least one component of ferric ammonium citrate, pantothenic acid, valine, methionine, arginine, biotin, serine, aspartic acid, asparagine, cupric sulfate, cysteine, Vitamin B12 or sodium selenite.

20 . A method of producing a protein comprising growing a recombinant CHO cell line in the medium of claim 1 and recovering the protein expressed by said cell line from said medium.

Assignees

• Ares Trading Sa

Inventors

• Broly Herve
• Stettler Matthieu
• Jordan Martin
• Perilleux Arnaud
• Rouiller Yolande

Classifications

• C12N2500/32

  Amino acids · CPC title

• C12N2500/46

  Amines, e.g. putrescine · CPC title

• C12N5/0682 Primary

  Cells of the female genital tract, e.g. endometrium; Non-germinal cells from ovaries, e.g. ovarian follicle cells (oocytes C12N5/0609) · CPC title

• C12N2500/38

  Vitamins · CPC title

• C12N5/005

  Protein-free medium · CPC title