Anti-VEGF protein compositions and methods for producing the same

Patent metadata

Field	Value
Publication number	US-12049489-B2
Application number	US-202318340707-A
Country	US
Kind code	B2
Filing date	Jun 23, 2023
Priority date	Dec 6, 2019
Publication date	Jul 30, 2024
Grant date	Jul 30, 2024

Abstract

The present disclosure pertains to methods for producing aflibercept from a host cell cultured in a chemically defined medium (CDM) including purification of aflibercept, wherein aflibercept following purification includes aflibercept variants that have at least one oxidized amino acid residue selected from the group consisting of tryptophan, histidine and a combination thereof.

First claim

What is claimed is:

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 cell culture media wherein chemically defined media (CDM) is added to said cell culture media, 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 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, and wherein said oxidized species of aflibercept is measured by subjecting said affinity eluate and said flowthrough fraction 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.

2. The method of claim 1 , wherein said oxidized amino acid residue 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, and SEQ ID NO.: 67.

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

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

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

6. The method of claim 1 further comprising at least one additional chromatographic step selected from the group consisting of: cation exchange chromatography (CEX), hydrophobic interactive chromatography, size exclusion chromatography and a combination thereof.

7. A method of producing aflibercept, comprising:
(a) binding aflibercept from a clarified harvest to a first affinity capture, wherein said aflibercept is expressed by cells cultured in a cell culture media wherein chemically defined media (CDM) is added to said cell culture media, and wherein said clarified harvest comprises one or more aflibercept variants having at least one oxidized amino acid residue selected from group consisting of tryptophan, histidine, phenylalanine, tyrosine and a combination thereof, wherein a protein sample from said clarified harvest comprises aflibercept and at least one aflibercept variant, and wherein said protein sample has a b* value of more than 0.5 when the concentration of said protein sample is normalized to 10.0 g/L;
(b) eluting said aflibercept of step (a) forming an eluate, wherein said eluate has a first color;
(c) subjecting said affinity eluate to an anion exchange chromatography (AEX) column; and
(d) collecting a flowthrough fraction, wherein said flowthrough fraction has a second color, and wherein said first color is a more intense yellow-brown color than said second color when said eluate and said flowthrough fraction protein concentrations are normalized.

8. The method of claim 7 , wherein said first capture chromatography comprises Protein A resin.

9. The method of claim 7 , wherein said oxidized amino acid residue is histidine.

10. The method of claim 7 , wherein said oxidized amino acid residue is tryptophan.

11. The method of claim 7 , wherein said oxidized amino acid residue 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.: 64, SEQ ID NO.: 65, SEQ ID NO.: 67, SEQ ID NO.: 68, SEQ ID NO.: 69, SEQ ID NO.: 70, SEQ ID NO.: 71, and combinations thereof.

12. The method of claim 7 , further comprising at least one additional chromatographic step selected from the group consisting of: cation exchange chromatography, hydrophobic interactive chromatography, size exclusion chromatography and a combination thereof.

13. 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 cell culture media wherein chemically defined media (CDM) is added to said cell culture media, 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.

14. The method of claim 13 , wherein said cell is selected from the group consisting of CHO, NS0, Sp2/0, embryonic kidney cells and BHK.

15. The method of claim 13 , wherein said oxidized amino acid residue is histidine.

16. The method of claim 13 , wherein said oxidized amino acid residue is tryptophan.

17. The method of claim 13 , wherein said AEX column comprises an anionic exchange substituent including diethylaminoethyl (DEAE), quaternary aminoethyl (QAE) and quaternary amine (Q) groups.

18. The method of claim 13 , 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.

19. The method of claim 13 , 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.

20. 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 cell culture media wherein chemically defined media (CDM) is added to said cell culture media, 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 afliberce

Assignees

• Regeneron Pharma

Inventors

• Tustian Andrew
• Vartak Ankit
• Daly Thomas
• Pyles Erica
• Palackal Nisha
• Wang Shunhai
• Li Ning

Classifications

• C12N2529/10

  Stimulation by light · CPC title

• C12N15/85

  for animal cells · CPC title

• C07K1/06

  using protecting groups or activating agents {(C07K1/003, C07K1/006 take precedence)} · CPC title

• C12N2510/02

  Cells for production · CPC title

• C07K1/165

  mixed-mode chromatography · CPC title