# US 11,299,532 B2 · Anti-VEGF protein compositions and methods for producing the same

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- Number: US 11,299,532 B2
- Document ID: US11299532B2
- Status: Granted
- Kind: B2
- Application number: 17/489,495
- Filed: 2021-09-29
- Published: 2022-01-13
- Granted: 2022-04-12
- 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
- Web page: https://patentgrep.com/patent/US11299532B2

## 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 in chemically defined media to reduce amounts of certain Aflibercept variants.

## Claims

### Claim 1

A method of producing aflibercept, comprising:
(a) binding aflibercept from a clarified harvest to a Protein A resin, wherein said aflibercept is expressed by cells cultured in a chemically defined medium (CDM), and wherein said aflibercept includes variants that have at least one oxidized amino acid residue selected from the group consisting of tryptophan, histidine, phenylalanine, tyrosine and a combination thereof;
(b) eluting said aflibercept of step (a) forming an affinity eluate, wherein said affinity eluate has a first color,
wherein said first color is no more yellow-brown than European Color Standard BY1;
(c) subjecting said affinity eluate comprising aflibercept to anion exchange chromatography (AEX) column; and
(d) collecting a flowthrough fraction, wherein said flowthrough fraction has a second color,
wherein said second color has a European Color Standard BY2 or greater, and
wherein said first color of said affinity eluate is a more intense yellow brown color than said second color of said flowthrough fraction when said affinity eluate and flowthrough fraction protein concentrations are normalized.

### Claim 2 (depends on 1)

The method of claim 1, wherein said cell is selected from the group consisting of CHO, NS0, Sp2/0, embryonic kidney cells and BHK.

### Claim 3 (depends on 1)

The method of claim 1, wherein said oxidized amino acid residue is histidine.

### Claim 4 (depends on 1)

The method of claim 1, wherein said oxidized amino acid residue is tryptophan.

### Claim 5 (depends on 1)

The method of claim 1, wherein said AEX column comprises an anionic exchange substituent including diethylaminoethyl (DEAE), quaternary aminoethyl (QAE) and quaternary amine (Q) groups.

### Claim 6 (depends on 1)

The method of claim 1, wherein said aflibercept variant is selected from an amino acid residue on a polypeptide having an amino acid sequence as set forth in 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, SEQ ID NO.: 56, SEQ ID NO.: 69, SEQ ID NO.: 70, SEQ ID NO.: 71 and combinations thereof.

### Claim 7 (depends on 1)

The method of claim 1, further comprising after binding aflibercept from said clarified harvest, subjecting said aflibercept to one or more further chromatographic steps selected from the group consisting of: cation exchange chromatography, hydrophobic interactive chromatography, size exclusion chromatography and a combination thereof.

### Claim 8 (depends on 6)

The method of claim 6, wherein said aflibercept variant is selected from an amino acid residue on a polypeptide having an amino acid sequence comprising SEQ ID NO.: 17.

### Claim 9 (depends on 6)

The method of claim 6, wherein said aflibercept variant is selected from an amino acid residue on a polypeptide having an amino acid sequence comprising SEQ ID NO.: 18.

### Claim 10 (depends on 6)

The method of claim 6, wherein said aflibercept variant is selected from an amino acid residue on a polypeptide having an amino acid sequence comprising SEQ ID NO.: 19.

### Claim 11 (depends on 6)

The method of claim 6, wherein said aflibercept variant is selected from an amino acid residue on a polypeptide having an amino acid sequence comprising SEQ ID NO.: 20.

### Claim 12 (depends on 6)

The method of claim 6, wherein said aflibercept variant is selected from an amino acid residue on a polypeptide having an amino acid sequence comprising SEQ ID NO.: 21.

### Claim 13 (depends on 6)

The method of claim 6, wherein said aflibercept variant is selected from an amino acid residue on a polypeptide having an amino acid sequence comprising SEQ ID NO.: 22.

### Claim 14 (depends on 6)

The method of claim 6, wherein said aflibercept variant is selected from an amino acid residue on a polypeptide having an amino acid sequence comprising SEQ ID NO.: 23.

### Claim 15 (depends on 6)

The method of claim 6, wherein said aflibercept variant is selected from an amino acid residue on a polypeptide having an amino acid sequence comprising SEQ ID NO.: 56.

### Claim 16 (depends on 6)

The method of claim 6, wherein said aflibercept variant is selected from an amino acid residue on a polypeptide having an amino acid sequence comprising SEQ ID NO.: 69.

### Claim 17 (depends on 6)

The method of claim 6, wherein said aflibercept variant is selected from an amino acid residue on a polypeptide having an amino acid sequence comprising SEQ ID NO.: 70.

### Claim 18 (depends on 6)

The method of claim 6, wherein said aflibercept variant is selected from an amino acid residue on a polypeptide having an amino acid sequence comprising SEQ ID NO.: 71.

### Claim 19

A method of producing aflibercept, comprising:
(a) binding aflibercept from a clarified harvest cultured (CDM) to a Protein A resin, wherein said aflibercept is expressed by cells cultured in a chemically defined medium (CDM), and wherein said aflibercept includes oxidized species of aflibercept that have at least one oxidized amino acid residue selected from the group consisting of tryptophan, histidine, phenylalanine, tyrosine and a combination thereof;
(b) eluting said aflibercept of step (a) forming an affinity eluate, wherein said affinity eluate comprises oxidized species of aflibercept;
(c) subjecting said eluted aflibercept to anion exchange (AEX) chromatography column; and
(d) collecting a flowthrough fraction, wherein the percent of oxidized species of aflibercept in said affinity eluate is greater than the percent of oxidized species of aflibercept in said flowthrough fraction when the concentrations of protein in said affinity eluate and flowthrough fraction are normalized,
wherein said oxidized species of aflibercept is measured by subjecting said affinity eluate and said flowthrough fractions to digestion, followed by their analysis using reverse-phase ultra-performance chromatography (UPLC), detection at wavelengths of 280 nm, 320 nm and 350 nm and mass spectrometry analysis using a first mobile phase of 0.1% v/v formic acid in water and a second mobile phase of 0.1% v/v formic acid in acetonitrile and
wherein the percent of oxidized species of aflibercept in said flowthrough fraction is reduced by at least about 10% compared to the percent of oxidized species of aflibercept in said affinity eluate.

### Claim 20 (depends on 19)

The method of claim 19, wherein said cell is selected from theft group consisting of CHO, NS0, Sp2/0, embryonic kidney cells and BHK.

### Claim 21 (depends on 19)

The method of claim 19, wherein said oxidized amino acid residue is histidine.

### Claim 22 (depends on 19)

The method of claim 19, wherein said oxidized amino acid residue is tryptophan.