# US 10,308,970 B2 · Methods for controlling fucosylation levels in proteins

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- Number: US 10,308,970 B2
- Document ID: US10308970B2
- Status: Granted
- Kind: B2
- Application number: 15/819,351
- Filed: 2017-11-21
- Published: 2018-03-29
- Granted: 2019-06-04
- Earliest claimed: 2013-07-23
- Assignees: Biocon Limited
- Applicants: Biocon Limited
- Inventors: Pradip Nair; Ramakrishnan Melarkode; Rasika Venkataraman; Laxmi Adhikary; Ankur Bhatnagar; Sunaina Prabhu; Kriti Shukla; Dinesh Baskar; Saravanan Desan; Harish Venkatraman Pai; Jose Enrique Montero Casimiro
- Web page: https://patentgrep.com/patent/US10308970B2

## CPC

- C12P 21/005: {Glycopeptides, glycoproteins}
- A61P 31/04: Antibacterial agents
- A61P 31/10: Antimycotics
- A61P 31/18: for HIV
- A61P 33/02: Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
- C07K 16/00: Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
- C07K 16/2896: {against molecules with a "CD"-designation, not provided for elsewhere}
- A61K 2039/505: {comprising antibodies}
- C07K 2317/24: containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
- C07K 2317/41: Glycosylation, sialylation, or fucosylation
- C07K 2317/54: F(ab')2
- C07K 2317/72: Increased effector function due to an Fc-modification
- C07K 2317/73: Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
- C07K 2317/732: Antibody-dependent cellular cytotoxicity [ADCC]
- C07K 2317/92: Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
- C07K 2319/30: Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
- Y02A 50/30: Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
- 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)}
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- 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)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE

## Abstract

The present invention relates to a method or process for controlling, inhibiting or reducing protein fucosylation in a eukaryote and/or eukaryotic protein expression system. Said method comprises carrying out the protein expression and/or post-translational modification in the presence of an elevated total concentration of manganese or manganese ions.

## Claims

### Claim 1

A method for decreasing fucosylation and increasing Mannose 5 and GlcNAc2Man3GlcNAc2 (G0) in the glycosylation pattern of an expressed antibody in an eukaryotic protein expression system, the method comprising:
providing a culturing medium;
introducing into the culturing medium the eukaryotic protein expression system, wherein the eukaryotic protein expression system comprises an expression cassette encoding the expressed antibody, wherein the expressed antibody is Itolizumab, wherein the eukaryotic protein expression system comprises mammalian cells, wherein the mammalian cells are CHO hamster cells;
adding manganese or manganese ions into the culture medium to form a manganese containing culture medium, wherein the manganese or manganese ions is in a concentration range of ≥0.075 mM-≤0.5 mM;
culturing the eukaryotic protein expression system in the manganese containing medium to obtain the expressed antibody; and
recovering the expressed antibody from the manganese containing medium, wherein the expressed antibody exhibits decreased fucose and increased levels of Mannose 5 and GlcNAc2Man3GlcNAc2 (G0) in the glycosylation pattern compared to an expressed antibody cultured in a medium wherein no manganese or manganese ions were introduced into the culture medium.

### Claim 2 (depends on 1)

The method according to claim 1, wherein the antibody expressed in claim 1 demonstrates an increased ADCC activity compared to an antibody expressed in the absence of the manganese or manganese ions added to the culture medium.

### Claim 3 (depends on 1)

The method according to claim 1, wherein the antibody expressed in claim 1 binds to one or more cellular surface antigen involved in cell-mediated immune defense.

### Claim 4 (depends on 1)

The method according to claim 1, further comprising introducing amino acids to the culturing medium to maintain cell growth and expression of the antibody expressed in claim 1.

### Claim 5 (depends on 1)

The method according to claim 1, wherein the manganese or manganese ions are in a concentration range of ≥0.1 mM-≤0.25 mM.