# US 8,785,194 B2 · Serum-free mammalian cell culture medium, and uses thereof

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- Number: US 8,785,194 B2
- Document ID: US8785194B2
- Status: Granted
- Kind: B2
- Application number: 12/463,371
- Filed: 2009-05-08
- Published: 2009-11-12
- Granted: 2014-07-22
- Earliest claimed: 1996-08-30
- Assignees: Life Technologies Corporation
- Applicants: Stephen Gorfien; Richard Fike; Glenn Godwin; Joyce Dzimian; David A. Epstein; Dale Gruber; Don McClure; Paul Price
- Inventors: Stephen Gorfien; Richard Fike; Glenn Godwin; Joyce Dzimian; David A. Epstein; Dale Gruber; Don McClure; Paul Price
- Web page: https://patentgrep.com/patent/US8785194B2

## CPC

- C12N 5/0682: {Cells of the female genital tract, e.g. endometrium; Non-germinal cells from ovaries, e.g. ovarian follicle cells (oocytes C12N5/0609)}
- C07K 14/61: Growth hormone [GH], i.e. somatotropin
- C12N 5/0037: {Serum-free medium, which may still contain naturally-sourced components}
- C12N 5/0043: {Medium free of human- or animal-derived components}
- C12N 5/005: {Protein-free medium}
- C12N 5/0603: {Embryonic cells (production of embryos, nuclear transfer A01K67/027); Embryoid bodies}
- C12N 7/00: Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof (preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00)
- C12N 9/2402: {hydrolysing O- and S- glycosyl compounds (3.2.1)}
- C12N 9/2471: {Beta-galactosidase (3.2.1.23), i.e. exo-(1-->4)-beta-D-galactanase}
- C12N 15/85: for animal cells
- C12Y 302/01023: Beta-galactosidase (3.2.1.23), i.e. exo-(1-->4)-beta-D-galactanase
- C12N 2500/14: Calcium; Ca chelators; Calcitonin
- C12N 2500/16: Magnesium; Mg chelators
- C12N 2500/22: Zinc; Zn chelators
- C12N 2500/24: Iron; Fe chelators; Transferrin
- C12N 2500/32: Amino acids
- C12N 2500/34: Sugars
- C12N 2500/35: Polyols, e.g. glycerin, inositol
- C12N 2500/38: Vitamins
- C12N 2500/74: from fungi, e.g. yeasts
- C12N 2500/76: from plants
- C12N 2500/90: Serum-free medium, which may still contain naturally-sourced components
- C12N 2500/95: Protein-free medium and culture conditions
- C12N 2501/90: Polysaccharides
- C12N 2501/91: Heparin
- C12N 2710/10051: Methods of production or purification of viral material
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- C12Y: ENZYMES

## Abstract

The present invention provides a cell culture medium formulation that supports the in vitro cultivation, particularly in suspension, of mammalian cells, particularly epithelial cells and fibroblast cells, and methods for cultivating mammalian cells in suspension in vitro using these media. The media comprise a basal medium and a polyanionic or polyanionic compound, preferably a polysulfonated or polysulfated compound, and more preferably dextran sulfate. The present invention also provides chemically defined, protein-free eukaryotic cell culture media comprising an iron chelate and zinc, which is capable of supporting the growth (and particularly the high-density growth of mammalian cells) in suspension culture, increasing the level of expression of recombinant protein in cultured cells, and/or increasing virus production in cultured cells.

## Claims

### Claim 1

A suspension cell culture comprising a serum-free and protein-free cell culture medium and a Chinese Hamster Ovary (CHO) cell, wherein said culture medium comprises a transferrin substitute and an insulin substitute, said transferrin substitute comprising an iron or an iron containing compound and said insulin substitute comprising a zinc or a zinc containing compound, wherein said culture medium supports increased growth of the CHO cell in suspension as compared to cultivation in FMX-8 medium without supplementation of serum, and without supplementation of protein, and without expression of an exogenous recombinant protein in the CHO cell.

### Claim 2 (depends on 1)

The suspension cell culture of claim 1, wherein said CHO cell expresses a recombinant protein or a virus.

### Claim 3 (depends on 2)

The suspension cell culture of claim 2, wherein said recombinant protein is a secreted protein.

### Claim 4 (depends on 2)

The suspension cell culture of claim 2, wherein said recombinant protein is harvested from the used cell culture medium.

### Claim 5 (depends on 1)

The suspension cell culture of claim 1, wherein said cell is cultured in a bioreactor.

### Claim 6 (depends on 1)

The suspension cell culture of claim 1, wherein said increased growth produces higher concentrations of recombinant protein.

### Claim 7 (depends on 1)

The suspension cell culture of claim 1, wherein said medium further comprises glucose at a final concentration of about 1.000 g/L to about 12.60 g/L.

### Claim 8 (depends on 1)

The suspension cell culture of claim 1, wherein said medium comprises a trace element selected from the group consisting of barium, potassium, cobalt, manganese, chromium, copper, nickel, molybdenum, fluorine, silver, rubidium, tin, zirconium, cadmium, aluminum, germanium, titanium, vanadium, silicon and magnesium.

### Claim 9 (depends on 1)

The suspension cell culture of claim 1, wherein said medium comprises a total amino acid concentration of about 1.404 g/L to about 12.42 g/L.

### Claim 10 (depends on 1)

The suspension cell culture of claim 1, wherein said increased growth is about 1.5×106 to 2×107 cells/ml.

### Claim 11 (depends on 1)

The suspension cell culture of claim 1, wherein the medium further comprises one or more ingredients selected from the group consisting of L-arginine, L-asparagine.H2O, L-aspartic acid, L-glutamic acid, L-histidine, hydroxyl-L-proline, L-isoleucine, L-leucine, L-lysine.HCl, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, L-cystine.2HCl, Na2HPO4, pyridoxine.HCl, thiamine.HCl, glutathione, cupric sulfate.7H2O, cadmium chloride.5H2O, cobalt chloride.2H2O, stannous chloride.2H2O, manganous sulfate.H2O, nickel sulfate.6H2O, sodium metavanadate, ammonium molybdate.4H2O, barium acetate, potassium bromide, potassium iodide, chromium sulfate, sodium fluoride, silver nitrate, rubidium chloride, zirconyl chloride, aluminium chloride, germanium dioxide, titanium tetrachloride, sodium metasilicate, magnesium chloride (anhydrous), D-calcium pantothenate, calcium nitrate.4H2O, potassium chloride, ascorbic acid magnesium salt phosphate, pluronic F68 10% solution, Na2HPO4, D-glucose, folic acid, riboflavin, biotin, choline chloride, niacinamide, inositol, sodium pyruvate, vitamin B-12, β-mercaptoethanol, para-amino benzoic acid, β-glycerophosphate, sodium selenite, ethanolamine.HCl, spermine, putrescine.2HCl, monothioglycerol, and sodium bicarbonate, wherein each of said ingredients is present in an amount which supports the high-density growth of Chinese hamster ovary cells in suspension culture and/or the expression of recombinant protein.

### Claim 12 (depends on 2)

The suspension cell culture of claim 2, wherein said recombinant protein or said virus is isolated.