# US 2021/0332402 A1 · Serum-free cell culture medium

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- Number: US 2021/0332402 A1
- Document ID: US20210332402A1
- Status: Published application
- Kind: A1
- Application number: 17/370,389
- Filed: 2021-07-08
- Published: 2021-10-28
- Earliest claimed: 2013-03-15
- Assignees: Regeneron Pharmaceuticals, Inc.
- Applicants: Regeneron Pharmaceuticals, Inc.
- Inventors: Shadia Abike OSHODI; Amy S. JOHNSON; Shawn M. LAWRENCE
- Web page: https://patentgrep.com/patent/US20210332402A1

## CPC

- C12P 21/00: Preparation of peptides or proteins (single cell protein C12N1/00)
- C07K 16/00: Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
- C07K 16/18: against material from animals or humans
- C12N 5/0031: {Serum-free culture media}
- 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 5/0682: {Cells of the female genital tract, e.g. endometrium; Non-germinal cells from ovaries, e.g. ovarian follicle cells (oocytes C12N5/0609)}
- C12N 5/10: Cells modified by introduction of foreign genetic material
- C12N 2500/22: Zinc; Zn chelators
- C12N 2500/24: Iron; Fe chelators; Transferrin
- C12N 2500/32: Amino acids
- C12N 2500/34: Sugars
- C12N 2500/46: Amines, e.g. putrescine
- C12N 2510/02: Cells for production
- C12N 2510/04: Immortalised cells
- C12N 2511/00: Cells for large scale production
- C12N 2523/00: Culture process characterised by temperature
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}
- 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)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## 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

### Claim 1

A method for cultivating a CHO cell expressing a receptor-Fc-fusion protein, comprising:
(a) culturing the CHO cell in a cell culture medium at a temperature of about 35° C. to 38° C. during a growth phase to form a cell culture, wherein said cell culture medium is serum-free and comprises plant hydrolysates and polyamines; and
(b) culturing the cell culture in the cell culture medium at a temperature of about 29° C. to 37° C. during a production phase.

### Claim 2 (depends on 1)

The method of claim 1, wherein step (b) comprises culturing the cell culture at a temperature of about 30° C. to 34° C. during the production phase.

### Claim 3 (depends on 1)

The method of claim 1, wherein the receptor-Fc-fusion protein is produced at a titer of at least 100 mg/L, at least 0.54 g/L or at least 1 g/L.

### Claim 4 (depends on 1)

The method of claim 1, wherein the polyamines comprise 0.09±0.014 mM to 0.9±0.14 mM ornithine.

### Claim 5 (depends on 1)

The method of claim 1, wherein the polyamines comprise 0.6±0.09 mM ornithine and 0.714±0.11 mM putrescine.

### Claim 6 (depends on 1)

The method of claim 1, wherein the plant hydrolysates comprise less than or equal to 7.5 g/L soy hydrolysate.

### Claim 7 (depends on 1)

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.

### Claim 8 (depends on 7)

The method of claim 7, wherein:
(a) the mixture of amino acids or salts thereof comprises one or more of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine or valine;
(b) the one or more fatty acids are selected from the group consisting of linoleic acid, linoleic acid, thioctic acid, oleic acid, palmitic acid, stearic acid, arachidic acid, arachidonic acid, lauric acid, behenic acid, decanoic acid, dodecanoic acid, hexanoic acid, lignoceric acid, myristic acid and octanoic acid;
(c) the mixture of nucleosides comprises one or more of adenosine, guanosine, cytidine, uridine, thymidine, or hypoxanthine; or
(d) the divalent cation is Ca2+, Mg2+, or both.

### Claim 9 (depends on 1)

The method of claim 1, wherein the CHO cell is a CHO DUX B-11 cell, a CHO DG-44 cell, a Veggie-CHO cell, a GS-CHO cell, an S-CHO cell, a CHO lec mutant cell, or CHO-K1 cell.

### Claim 10 (depends on 1)

The method of claim 1, wherein the method comprises a fed batch, continuous, semi-continuous or perfusion cell culture method.

### Claim 11

A method for cultivating a CHO cell expressing a VEGF trap protein, comprising:
(a) culturing the CHO cell in a cell culture medium at a temperature of about 35° C. to 38° C. during a growth phase to form a cell culture, wherein said cell culture medium is serum-free and comprises plant hydrolysates and polyamines; and
(b) culturing the cell culture in the cell culture medium at a temperature of about 29° C. to 37° C. during a production phase.

### Claim 12 (depends on 11)

The method of claim 11, wherein step (b) comprises culturing the cell culture at a temperature of about 30° C. to 34° C. during the production phase.

### Claim 13 (depends on 11)

The method of claim 11, wherein the VEGF trap protein is produced at a titer of at least 100 mg/L, at least 0.54 g/L or at least 1 g/L.

### Claim 14 (depends on 11)

The method of claim 11, wherein the polyamines comprise 0.09±0.014 mM to 0.9±0.14 mM ornithine.

### Claim 15 (depends on 11)

The method of claim 11, wherein the polyamines comprise 0.6±0.09 mM ornithine and 0.714±0.11 mM putrescine.

### Claim 16 (depends on 11)

The method of claim 11, wherein the plant hydrolysates comprise less than or equal to 7.5 g/L soy hydrolysate.

### Claim 17 (depends on 11)

The method of claim 11, 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.

### Claim 18 (depends on 17)

The method of claim 17, wherein:
(a) the mixture of amino acids or salts thereof comprises one or more of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine or valine;
(b) the one or more fatty acids are selected from the group consisting of linoleic acid, linoleic acid, thioctic acid, oleic acid, palmitic acid, stearic acid, arachidic acid, arachidonic acid, lauric acid, behenic acid, decanoic acid, dodecanoic acid, hexanoic acid, lignoceric acid, myristic acid and octanoic acid;
(c) the mixture of nucleosides comprises one or more of adenosine, guanosine, cytidine, uridine, thymidine, or hypoxanthine; or
(d) the divalent cation is Ca2+, Mg2+, or both

### Claim 19 (depends on 11)

The method of claim 11, wherein the CHO cell is a CHO DUX B-11 cell, a CHO DG-44 cell, a Veggie-CHO cell, a GS-CHO cell, an S-CHO cell, a CHO lec mutant cell, or CHO-K1 cell.

### Claim 20 (depends on 11)

The method of claim 11, wherein the method comprises a fed batch, continuous, semi-continuous or perfusion cell culture method.

### Claim 21

A method for cultivating a CHO cell expressing aflibercept, comprising:
(a) culturing the CHO cell in a cell culture medium at a temperature of about 35° C. to 38° C. during a growth phase to form a cell culture, wherein said cell culture medium is serum-free and comprises plant hydrolysates and polyamines; and
(b) culturing the cell culture in the cell culture medium at a temperature of about 29° C. to 37° C. during a production phase.

### Claim 22 (depends on 21)

The method of claim 21, wherein step (b) comprises culturing the cell culture at a temperature of about 30° C. to 34° C. during the production phase.

### Claim 23 (depends on 21)

The method of claim 21, wherein the aflibercept is produced at a titer of at least 100 mg/L, at least 0.54 g/L or at least 1 g/L.

### Claim 24 (depends on 21)

The method of claim 21, wherein the polyamines comprise 0.09±0.014 mM to 0.9±0.14 mM ornithine.

### Claim 25 (depends on 21)

The method of claim 21, wherein the polyamines comprise 0.6±0.09 mM ornithine and 0.714±0.11 mM putrescine.

### Claim 26 (depends on 21)

The method of claim 21, wherein the plant hydrolysates comprise less than or equal to 7.5 g/L soy hydrolysate.

### Claim 27 (depends on 21)

The method of claim 21, 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.

### Claim 28 (depends on 27)

The method of claim 27, wherein:
(a) the mixture of amino acids or salts thereof comprises one or more of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine or valine;
(b) the one or more fatty acids are selected from the group consisting of linoleic acid, linoleic acid, thioctic acid, oleic acid, palmitic acid, stearic acid, arachidic acid, arachidonic acid, lauric acid, behenic acid, decanoic acid, dodecanoic acid, hexanoic acid, lignoceric acid, myristic acid and octanoic acid;
(c) the mixture of nucleosides comprises one or more of adenosine, guanosine, cytidine, uridine, thymidine, or hypoxanthine; or
(d) the divalent cation is Ca2+, Mg2+, or both.

### Claim 29 (depends on 21)

The method of claim 21, wherein the CHO cell is a CHO DUX B-11 cell, a CHO DG-44 cell, a Veggie-CHO cell, a GS-CHO cell, an S-CHO cell, a CHO lec mutant cell, or CHO-K1 cell.

### Claim 30 (depends on 21)

The method of claim 21, wherein the method comprises a fed batch, continuous, semi-continuous or perfusion cell culture method.