# US 11,299,760 B2 · Use of monensin to regulate glycosylation of recombinant proteins

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- Number: US 11,299,760 B2
- Document ID: US11299760B2
- Status: Granted
- Kind: B2
- Application number: 17/464,115
- Filed: 2021-09-01
- Published: 2021-12-23
- Granted: 2022-04-12
- Earliest claimed: 2013-10-31
- Assignees: Amgen Inc.
- Applicants: Amgen Inc.
- Inventors: Sandhya Pande; Mirna Mujacic
- Web page: https://patentgrep.com/patent/US11299760B2

## CPC

- C12P 21/005: {Glycopeptides, glycoproteins}
- C07K 16/00: Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
- C07K 16/2875: {against the NGF/TNF superfamily, e.g. CD70, CD95L, CD153, CD154 (against NGF C07K16/22, against TNF C07K16/241)}
- C07K 2317/14: Specific host cells or culture conditions, e.g. components, pH or temperature
- C07K 2317/41: Glycosylation, sialylation, or fucosylation
- 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)

## Abstract

Methods of modulating the properties of a cell culture expressing a protein of interest are provided. In various embodiments the methods relate to the addition of cell-cycle inhibitors to growing cell cultures.

## Claims

### Claim 1

A method of regulating the high mannose denosumab glycoform content of a denosumab composition during a mammalian cell culture process, comprising:
establishing, in a serum-free culture medium in a bioreactor, a cell culture comprising genetically engineered CHO expressing denosumab,
maintaining the CHO cells during a production phase, and
adding monensin to said CHO cell culture during the production phase at a concentration of about 25 nM to about 500 nM,
wherein the addition of monensin increases the high mannose denosumab glycoform content of the denosumab composition.

### Claim 2 (depends on 1)

The method according to claim 1, wherein the amount of monensin is about 25 nM to about 200 nM.

### Claim 3 (depends on 2)

The method of claim 2, wherein the monensin is added within about 0 to about 6 days from the start of the production phase of the cell culture.

### Claim 4 (depends on 3)

The method of claim 3, wherein the monensin is added within about 0 to about 3 days from the start of the production phase of the cell culture.

### Claim 5 (depends on 1)

The method of claim 1, wherein the monensin is added to the cell culture by addition of a perfusion medium containing the monensin, and wherein the concentration of monensin in the cell culture is maintained from about 25 nM to about 500 nM.

### Claim 6 (depends on 4)

The method of claim 4, wherein the concentration of monensin maintained in the cell culture is from about 100 nM to about 500 nM.

### Claim 7 (depends on 2)

The method of claim 2, further comprising the step of purifying denosumab from a mammalian cell culture.

### Claim 8 (depends on 7)

The method of claim 7, wherein the purified denosumab is formulated into a pharmaceutically acceptable formulation.

### Claim 9 (depends on 4)

The method of claim 4, further comprising the step of purifying denosumab from a mammalian cell culture.

### Claim 10 (depends on 9)

The method according to claim 9, wherein the purified denosumab is formulated into a pharmaceutically acceptable formulation.