# US 10,119,117 B2 · Universal, glycosylation enhancer, completely chemically defined medium formulation

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- Number: US 10,119,117 B2
- Document ID: US10119117B2
- Status: Granted
- Kind: B2
- Application number: 15/404,007
- Filed: 2017-01-11
- Published: 2017-08-03
- Granted: 2018-11-06
- Earliest claimed: 2016-01-28
- Assignees: Nanogen Pharmaceutical Biotechnology
- Applicants: Nanogen Pharmaceutical Biotechnology Co., Ltd
- Inventors: Nhan Ho; Praveen Gupta; Si Minh Do; Phuong Thi Bich Ho; Ngoc-Thuy Bui; Huy Quang Nguyen; Trang Thi Thuy Nguyen; Ha Thi Hong Truong; Tuan Anh Le
- Web page: https://patentgrep.com/patent/US10119117B2

## CPC

- C12N 5/0018: {Culture media for cell or tissue culture (media for specific animal cell type C12N5/06)}
- C12N 2500/32: Amino acids
- C12N 2500/34: Sugars
- C12N 2500/38: Vitamins
- C12N 2500/40: Nucleotides, nucleosides or bases
- C12N 2500/60: Buffer, e.g. pH regulation, osmotic pressure
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

In one embodiment, the present application discloses a cell culture medium for culturing cell lines suitable for producing a therapeutic protein, comprising an amino acid selected from a group consisting of L-arginine, L-asparagine, L-proline, L leucine and L hydroxyproline and a mixture thereof; a vitamin selected from a group consisting of ascorbic acid Mg2+ salt, biotin, pyridoxine HCL, folic acid, riboflavin and D-calcium pantothenate, and a mixture thereof; an element selected from a group consisting of ammonium meta vanadate, sodium meta vanadate, germanium dioxide, barium acetate, aluminum chloride, rubidium chloride, cadmium chloride, ammonium molybedate, stannous chloride, cobalt chloride, chromium sulfate, silver nitrate, sodium metasilicate, zinc sulfate, manganese sulfate H2O, manganous chloride, ferric nitrate 9H2O, ferrous sulfate 7H2O, ferric ammonium citrate, magnesium chloride anhydrous, and magnesium sulfate anhydrous, and a mixture thereof; a nucleoside selected from a group consisting of uridine and cystidine; a sugar selected from a group consisting of galactose, mannose and N-Acetyl-D-Mannosamine; and a triple buffering system comprising sodium carbonate, sodium bicarbonate and HEPES; wherein the cell culture medium is animal component-free, plant component-free, serum-free, growth factors-free, recombinant protein-free, lipid-free, steroid-free, and free of plant or animal hydrolysates and/or extracts.

## Claims

### Claim 1

A cell culture medium for culturing cell lines suitable for producing a therapeutic protein, comprising:
a) an amino acid selected from a group consisting of L-arginine, L-asparagine, L-proline, L-leucine and L-hydroxyproline, or a mixture thereof;
b) a vitamin selected from a group consisting of ascorbic acid Mg2+ salt, biotin, pyridoxine HCL, folic acid, riboflavin and D-calcium pantothenate, or a mixture thereof;
c) an element selected from a group consisting of ammonium meta vanadate, sodium meta vanadate, germanium dioxide, barium acetate, aluminum chloride, rubidium chloride, cadmium chloride, ammonium molybedate, stannous chloride, cobalt chloride, chromium sulfate, silver nitrate, sodium metasilicate, zinc sulfate, manganese sulfate H2O, manganous chloride, ferric nitrate 9H2O, ferrous sulfate 7H2O, ferric ammonium citrate, magnesium chloride anhydrous, and magnesium sulfate anhydrous, or a mixture thereof;
d) a nucleoside selected from a group consisting of uridine and cystidine, or a mixture thereof;
e) a sugar selected from a group consisting of galactose, mannose and N-acetyl-D-mannosamine, or a mixture thereof; and
f) a triple buffering system comprising sodium carbonate, sodium bicarbonate and HEPES;
wherein the cell culture medium is animal component-free, plant component-free, serum-free, growth factors-free, recombinant protein-free, lipids-free, steroids-free, and free of plant or animal hydrolysates and/or extracts.

### Claim 2 (depends on 1)

The cell culture medium according to claim 1, wherein the cell culture medium comprises:
about 350 to about 500 mg/L L-arginine;
about 700 to about 900 mg/L L-asparagine;
about 350 to about 500 mg/L L-proline;
about 500 to about 650 mg/L leucine; and
about 90 to about 110 mg/L hydroxyproline.

### Claim 3 (depends on 1)

The cell culture medium according to claim 1, wherein the cell culture medium comprises:
about 8 to about 14mg/L ascorbic acid Mg2+ salt;
about 1.0 to about 1.5 mg/L biotin;
about 2.5 to about 4 mg/L pyridoxine HCL;
about 19 to about 23 mg/L folic acid;
about 4 to about 6 mg/L riboflavin; and
about 3 to about 4 mg/L D-calcium pantothenate.

### Claim 4 (depends on 1)

The cell culture medium according to claim 1, comprising:
about 1 to about 1.5 μg/L ammonium meta vanadate;
about 1 to about 1.8 μg/L sodium meta vanadate;
about 0.2 to about 0.8 vg/L germanium dioxide;
about 2 to about 3 μg/L barium acetate;
about 1 to about 1.6 μg/L aluminum chloride;
about 1.1 to about 1.7 μg/L rubidium chloride;
about 35 to about 75 μg/L cadmium chloride;
about 5 to about 20 μg/L ammonium molybedate;
about 0.1 to about 0.5 μg/L stannous chloride;
about 5 to about 15 μg/L cobalt chloride;
about 0.1 to about 1 μg/L chromium sulfate;
about 0.1 to about 0.2 μg/L silver nitrate;
about 200 to about 600 μg/L sodium metasilicate; and
about 100 to about 600 μg/L zinc sulfate.

### Claim 5 (depends on 1)

The cell culture medium according to claim 1, comprising:
about 15 to about 25 mg/L Uridine; and
about 18 to about 25 mg/L Cytidine.

### Claim 6 (depends on 1)

The cell culture medium according to claim 1, comprising:
about 0.1 to about 0.9 mg/L manganese sulfate H2O;
about 0.1 to about 0.5 mg/L manganous chloride;
about 0.3 to about 2 mg/L ferric nitrate 9H2O;
about 2 to about 5 mg/L ferrous sulfate 7H2O;
about 1 to about 2.5 mg/L ferric ammonium citrate;
about 40 to about 100 mg/L magnesium chloride anhydrous; and
about 10 to about 20 mg/L magnesium sulfate anhydrous.

### Claim 7 (depends on 1)

The cell culture medium according to claim 1, comprising:
about 100 mg to about 500 mg/L galactose;
about 100 to about 500 mg/L mannose;
about 50 to about 150 mg/L N-Acetyl-D-Mannosamine; and
about 100 to 300 mg/L glucosamine.

### Claim 8 (depends on 1)

The cell culture medium according to claim 1, wherein the cell culture medium comprises the triple buffering system comprising:
about 1.5 g/L sodium carbonate;
about 2.2 g/L sodium bi carbonate; and
about 2.38 g/L HEPES.

### Claim 9 (depends on 1)

The cell culture medium according to claim 1, wherein the cell culture medium is capable of growing the cell lines at a cell density of more than 20 million cells/mL.

### Claim 10 (depends on 1)

The cell culture medium according to claim 1, wherein the cell culture medium is used to grow the cell lines in a batch, fed-batch, or perfusion mode culture without the need of cell clone adaptation.

### Claim 11 (depends on 1)

The cell culture medium according to claim 1, wherein the cell culture medium enhances glycosylation of therapeutic protein by about 20% to about 200%.

### Claim 12 (depends on 1)

The cell culture medium according to claim 1, wherein the cell culture medium enhances glycosylation of therapeutic protein by about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 100%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190% and 200%.

### Claim 13 (depends on 1)

The cell culture medium according to claim 1, wherein the cell culture medium increases the expression of recombinant proteins in a fed-batch mode culture by about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190% and 200%.

### Claim 14 (depends on 1)

The cell culture medium according to claim 1, wherein the cell culture medium is useful in commercial manufacturing of therapeutic proteins.

### Claim 15 (depends on 1)

A kit for culturing CHO cell lines suitable for producing a therapeutic protein, comprising a cell culture medium according to claim 1.