# US 11,306,135 B2 · Anti-VEGF protein compositions and methods for producing the same

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- Number: US 11,306,135 B2
- Document ID: US11306135B2
- Status: Granted
- Kind: B2
- Application number: 17/460,578
- Filed: 2021-08-30
- Published: 2022-01-13
- Granted: 2022-04-19
- Earliest claimed: 2019-12-06
- Assignees: Regeneron Pharmaceuticals, Inc.
- Applicants: Regeneron Pharmaceuticals, Inc.
- Inventors: Shunhai Wang; Ning Li; Hunter Chen; Amardeep Singh Bhupender Bhalla
- Web page: https://patentgrep.com/patent/US11306135B2

## CPC

- A61K 38/179: {for growth factors; for growth regulators}
- C07K 14/71: for growth factors; for growth regulators
- A61K 38/1866: {Vascular endothelial growth factor [VEGF]}
- A61P 17/06: Antipsoriatics
- A61P 19/02: for joint disorders, e.g. arthritis, arthrosis
- A61P 27/02: Ophthalmic agents
- A61P 35/00: Antineoplastic agents
- C07K 1/06: using protecting groups or activating agents {(C07K1/003, C07K1/006 take precedence)}
- C07K 1/113: without change of the primary structure
- C07K 1/16: by chromatography
- C07K 1/18: Ion-exchange chromatography
- C07K 1/22: Affinity chromatography or related techniques based upon selective absorption processes
- C07K 1/36: by a combination of two or more processes of different types
- C07K 16/22: against growth factors {; against growth regulators}
- C07K 16/283: {against Fc-receptors, e.g. CD16, CD32, CD64 (CD23 C07K16/2851)}
- C12N 5/0018: {Culture media for cell or tissue culture (media for specific animal cell type C12N5/06)}
- C12N 5/0031: {Serum-free culture media}
- C12N 15/66: General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
- C12P 21/02: having a known sequence of two or more amino acids, e.g. glutathione
- C12P 21/06: produced by the hydrolysis of a peptide bond, e.g. hydrolysate products (preparing foodstuffs by protein hydrolysis A23J3/00)
- C12Y 304/21004: Trypsin (3.4.21.4)
- G01N 30/80: Fraction collectors
- C07K 2317/622: Single chain antibody (scFv)
- C07K 2319/30: Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
- C07K 2319/33: fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
- C12N 2800/10: Plasmid DNA
- G01N 2030/027: {Liquid chromatography}
- 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)
- 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)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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)}
- C12Y: ENZYMES
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

The present disclosure pertains to compositions comprising anti-VEGF proteins and methods for producing such compositions.

## Claims

### Claim 1

A composition comprising oxo-aflibercept, wherein one or more amino acid residues of aflibercept is oxidized and wherein said one or more amino acid residues is histidine.

### Claim 2 (depends on 1)

The composition of claim 1 further comprising a pharmaceutically acceptable carrier.

### Claim 3 (depends on 1)

The composition of claim 1, wherein said oxo-aflibercept is freeze-dried.

### Claim 4 (depends on 1)

The composition of claim 1, wherein said composition is sterile.

### Claim 5 (depends on 1)

The composition of claim 1, wherein said oxo-aflibercept is enzymatically digested resulting in one or more oligopeptides, and wherein said one or more oligopeptides is selected from the group consisting of: SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 22, SEQ ID NO. 23, and combinations thereof.

### Claim 6 (depends on 5)

The composition of claim 5, wherein said enzymatic digestion is performed using trypsin.

### Claim 7

A composition comprising oxo-aflibercept, wherein one or more amino acid residues of aflibercept is oxidized and wherein said one or more amino acid residues is tryptophan.

### Claim 8 (depends on 7)

The composition of claim 7 further comprising a pharmaceutically acceptable carrier.

### Claim 9 (depends on 7)

The composition of claim 7, wherein said oxo-aflibercept is freeze-dried.

### Claim 10 (depends on 7)

The composition of claim 7, wherein said composition is sterile.

### Claim 11 (depends on 7)

The composition of claim 7 further comprising said oxo-aflibercept, wherein one or more amino acid residues of aflibercept is oxidized and wherein said one or more amino acid residues are tryptophan and histidine.

### Claim 12 (depends on 7)

The composition of claim 7, wherein said oxo-aflibercept is enzymatically digested resulting in one or more oligopeptides, and wherein said one or more oligopeptides is selected from the group consisting of: SEQ ID NO. 28, SEQ ID NO. 29, SEQ ID NO. 114, SEQ ID NO. 115, and combinations thereof.

### Claim 13 (depends on 12)

The composition of claim 12, wherein said enzymatic digestion is performed using trypsin.

### Claim 14

An oxo-aflibercept composition produced in a cell culture media comprising a CDM, wherein one or more amino acid residues of aflibercept is oxidized and wherein said one or more amino acid residues is histidine and/or tryptophan.

### Claim 15 (depends on 14)

An oxo-aflibercept composition produced in a cell culture media comprising a CDM of claim 14, wherein said one or more amino acid residues of aflibercept is oxidized and wherein said one or more amino acid residues is histidine.

### Claim 16 (depends on 14)

An oxo-aflibercept composition produced in a cell culture media comprising a CDM of claim 14, wherein said one or more amino acid residues of aflibercept is oxidized and wherein said one or more amino acid residues is tryptophan.