# US 2022/0033472 A1 · Anti-vegf protein compositions and methods for producing the same

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- Number: US 2022/0033472 A1
- Document ID: US20220033472A1
- Status: Published application
- Kind: A1
- Application number: 17/492,440
- Filed: 2021-10-01
- Published: 2022-02-03
- Earliest claimed: 2019-12-06
- Assignees: Regeneron Pharmaceuticals, Inc.
- Applicants: Regeneron Pharmaceuticals, Inc.
- Inventors: Andrew Tustian; Ankit Vartak; Thomas Daly; Erica Pyles; Nisha Palackal; Shunhai Wang; Ning Li; Matthew Franklin
- Web page: https://patentgrep.com/patent/US20220033472A1

## 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 compositions comprising anti-VEGF proteins and methods for producing such compositions.

## Claims

### Claim 1

A method of producing aflibercept MiniTrap, comprising:
a) producing aflibercept using cells cultured in a chemically defined medium (CDM);
b) binding said aflibercept to a first capture chromatography;
c) eluting said aflibercept of step b) and subjecting said aflibercept to enzymatic cleavage to remove its Fc domain thereby forming MiniTrap;
d) subjecting said formed MiniTrap of step c) to a second capture chromatography, wherein said second capture chromatography step is subjected to one or more washes to obtain a first flowthrough fraction;
e) subjecting said first flowthrough fraction of step d) to an anion exchange chromatography (AEX) using an AEX column; and
f) washing said AEX column of step e) to obtain a second flowthrough fraction having MiniTrap, wherein said MiniTrap has an amount of glycans selected from the group consisting of about 40% to about 60% total fucosylated glycans, about 30% to about 55% total sialylated glycans, about 0% to about 20% mannose-5, and about 70% to about 95% galactosylated glycans,
wherein the amount of glycans are determined using hydrophilic interaction liquid chromatography with post-column fluorescence detection, and
wherein the glycans are separated and eluted using acetonitrile and aqueous 50 mM ammonium formate (pH 4.4) and detected using a fluorescence detector with an excitation wavelength of 265 nm and an emission wavelength of 425 nm.

### Claim 2 (depends on 1)

The method of claim 1, wherein said MiniTrap of step f) has about 40% to about 50% total fucosylated glycans.

### Claim 3 (depends on 1)

The method of claim 1, wherein said MiniTrap of step f) has about 30% to about 55% total sialylated glycans.

### Claim 4 (depends on 1)

The method of claim 1, wherein said MiniTrap of step f) has about 6% to about 15% mannose-5.

### Claim 5 (depends on 1)

The method of claim 1, wherein said MiniTrap of step f) has about 40% to about 50% total fucosylated glycans.

### Claim 6 (depends on 1)

The method of claim 1, wherein said MiniTrap of step f) has about 60% to about 79% galactosylated glycans

### Claim 7 (depends on 1)

The method of claim 1, wherein said MiniTrap of step f) has of about 40% to about 50% total fucosylated glycans, about 30% to about 55% total sialylated glycans, about 6% to about 15% mannose-5, and about 60% to about 79% galactosylated glycans.

### Claim 8 (depends on 1)

The method of claim 1, wherein MiniTrap of step f) is glycosylated at one or more Asparagines as follows: G0-GlcNAc glycosylation; G1-GlcNAc glycosylation; G1S-GlcNAc glycosylation; G0 glycosylation; G1 glycosylation; G1S glycosylation; G2 glycosylation; G2S glycosylation; G2S2 glycosylation; G0F glycosylation; G2F2S glycosylation; G2F2S2 glycosylation; G1F glycosylation; G1FS glycosylation; G2F glycosylation; G2FS glycosylation; G2FS2 glycosylation; G3FS glycosylation; G3FS3 glycosylation; G0-2GlcNAc glycosylation; Man4 glycosylation; Man4_A1G1 glycosylation; Man4_A1G1S1 glycosylation; Man5 glycosylation; Man5_A1G1 glycosylation; Man5A1G1S1 glycosylation; Man6 glycosylation; Man6 G0+Phosphate glycosylation; Man6+Phosphate glycosylation; and/or Man7 glycosylation.

### Claim 9 (depends on 1)

The method of claim 1, wherein said MiniTrap of step f) is xylosylated.

### Claim 10 (depends on 1)

The method of claim 1, wherein said MiniTrap of step f) comprises a trisulfide bridge.

### Claim 11 (depends on 1)

The method of claim 1, wherein said MiniTrap of step f) comprises a intrachain disulfide bridge.

### Claim 12

A method of producing aflibercept MiniTrap preparation, comprising:
a) producing aflibercept using cells cultured in a chemically defined medium (CDM);
b) binding aflibercept to a first capture chromatography;
c) eluting said aflibercept of step b) and subjecting said aflibercept to enzymatic cleavage to remove its Fc domain thereby forming MiniTrap;
d) subjecting MiniTrap of step c) to a second capture chromatography, wherein said second capture chromatography step is subjected to one or more washes to obtain a first flowthrough fraction;
e) subjecting said first flowthrough fraction of step d) to an anion exchange chromatography (AEX) using an AEX column;
f) washing said AEX column of step e) to obtain a second flowthrough fraction having MiniTrap, wherein said MiniTrap has an amount of glycans selected from the group consisting of about 40% to about 60% total fucosylated glycans, about 30% to about 55% total sialylated glycans, about 0% to about 20% mannose-5, and about 70% to about 95% galactosylated glycans,
wherein the amount of glycans are determined using hydrophilic interaction liquid chromatography with post-column fluorescence detection, and
wherein the glycans are separated and eluted using acetonitrile and aqueous 50 mM ammonium formate (pH 4.4) and detected using a fluorescence detector with an excitation wavelength of 265 nm and an emission wavelength of 425 nm;
g) formulating said MiniTrap of step f) into a drug product.

### Claim 13 (depends on 12)

The method of claim 12, wherein said MiniTrap of step g) has about 40% to about 50% total fucosylated glycans.

### Claim 14 (depends on 12)

The method of claim 12, wherein said MiniTrap of step g) has about 30% to about 55% total sialylated glycans.

### Claim 15 (depends on 12)

The method of claim 12, wherein said MiniTrap of step g) has about 6% to about 15% mannose-5.

### Claim 16 (depends on 12)

The method of claim 12, wherein said MiniTrap of step g) has about 40% to about 50% total fucosylated glycans.

### Claim 17 (depends on 12)

The method of claim 12, wherein said MiniTrap of step g) has about 60% to about 79% galactosylated glycans

### Claim 18 (depends on 12)

The method of claim 12, wherein said MiniTrap of step g) has of about 40% to about 50% total fucosylated glycans, about 30% to about 55% total sialylated glycans, about 6% to about 15% mannose-5, and about 60% to about 79% galactosylated glycans.

### Claim 19 (depends on 12)

The method of claim 12, wherein MiniTrap of step g) is glycosylated at one or more Asparagines as follows: G0-GlcNAc glycosylation; G1-GlcNAc glycosylation; G1S-GlcNAc glycosylation; G0 glycosylation; G1 glycosylation; G1S glycosylation; G2 glycosylation; G2S glycosylation; G2S2 glycosylation; G0F glycosylation; G2F2S glycosylation; G2F2S2 glycosylation; G1F glycosylation; G1FS glycosylation; G2F glycosylation; G2FS glycosylation; G2FS2 glycosylation; G3FS glycosylation; G3FS3 glycosylation; G0-2GlcNAc glycosylation; Man4 glycosylation; Man4_AlG1 glycosylation; Man4_AlG1S1 glycosylation; Man5 glycosylation; Man5_A1G1 glycosylation; Man5_A1G1S1 glycosylation; Man6 glycosylation; Man6_G0+Phosphate glycosylation; Man6+Phosphate glycosylation; and/or Man7 glycosylation.

### Claim 20 (depends on 12)

The method of claim 12, wherein said MiniTrap of step g) is xylosylated.

### Claim 21 (depends on 12)

The method of claim 12, wherein said MiniTrap of step g) comprises a trisulfide bridge.

### Claim 22 (depends on 12)

The method of claim 12, wherein said MiniTrap of step g) comprises a intrachain disulfide bridge.

### Claim 23

An aflibercept MiniTrap preparation, comprising aflibercept MiniTrap having about 40% to about 60% total fucosylated glycans, about 30% to about 55% total sialylated glycans, about 0% to about 20% mannose-5, and about 70% to about 95% galactosylated glycans,
wherein glycan analysis in carried out using hydrophilic interaction liquid chromatography with post-column fluorescence detection, and
wherein labeled glycans are separated and eluted using acetonitrile and aqueous 50 mM ammonium formate (pH 4.4) and detected using a fluorescence detector with an excitation wavelength of 265 nm and an emission wavelength of 425 nm.

### Claim 24

An aflibercept MiniTrap composition comprising aflibercept MiniTrap with a glycosylation profile including one or more glycan(s) selected from the group consisting of about 40% to about 60% total fucosylated glycans, about 30% to about 55% total sialylated glycans, about 0% to about 20% mannose-5, and about 70% to about 95% galactosylated glycans.

### Claim 25 (depends on 24)

The method of claim 24, wherein said aflibercept MiniTrap has about 40% to about 50% total fucosylated glycans.

### Claim 26 (depends on 24)

The method of claim 24, wherein said aflibercept MiniTrap has about 30% to about 55% total sialylated glycans.

### Claim 27 (depends on 24)

The method of claim 24, wherein said aflibercept MiniTrap has about 6% to about 15% mannose-5.

### Claim 28 (depends on 24)

The method of claim 24, wherein said aflibercept MiniTrap has about 40% to about 50% total fucosylated glycans.

### Claim 29 (depends on 24)

The method of claim 24, wherein said aflibercept MiniTrap has about 60% to about 79% galactosylated glycans

### Claim 30 (depends on 24)

The method of claim 24, wherein said aflibercept MiniTrap of has of about 40% to about 50% total fucosylated glycans, about 30% to about 55% total sialylated glycans, about 6% to about 15% mannose-5, and about 60% to about 79% galactosylated glycans