Anti-VEGF protein compositions and methods for producing the same

Patent metadata

Field	Value
Publication number	US-11098112-B2
Application number	US-202016996042-A
Country	US
Kind code	B2
Filing date	Aug 18, 2020
Priority date	Dec 6, 2019
Publication date	Aug 24, 2021
Grant date	Aug 24, 2021

Abstract

The present disclosure pertains to compositions comprising aflibercept and methods for producing such compositions in chemically defined media and using chromatography to reduce amounts of certain aflibercept variants.

First claim

What is claimed is:

1. A method of producing aflibercept, comprising:
(a) producing a clarified harvest of cells cultured in a chemically defined medium (CDM);
(b) binding aflibercept from said clarified harvest using an affinity chromatography column comprising a polypeptide capable of binding to or interacting with said aflibercept, wherein said polypeptide is an antibody, a fusion protein, a ScFv or a fragment thereof and wherein said polypeptide comprises an isolated amino acid sequence selected from a SEQ ID NO.: 72, SEQ ID NO.: 73, SEQ ID NO.: 74, SEQ ID NO.: 75, SEQ ID NO.: 76, SEQ ID NO.: 77, SEQ ID NO.: 78, SEQ ID NO.: 79 and SEQ ID NO.: 80;
(c) eluting said aflibercept of step (b) forming an affinity eluate; optionally,
(d) subjecting said eluted aflibercept of (c) to a second chromatography capture step; and
(e) collecting a flowthrough fraction, wherein said flowthrough fraction has aflibercept.

2. The method of claim 1 , further comprising equilibrating said affinity chromatography column of (b) using an equilibration buffer.

3. The method of claim 2 , wherein said equilibration buffer is Dulbecco's Phosphate-Buffered Saline or Tris hydrochloride.

4. The method of claim 3 , wherein said equilibration buffer has a pH of about 8.3 to about 8.6.

5. The method of claim 1 , further comprising washing column (d) with an equilibration buffer to obtain one or more flowthrough fractions.

6. The method of claim 5 , wherein said equilibration buffer is Dulbecco's Phosphate-Buffered Saline and has a pH of about 7.0 to about 8.6.

7. The method of claim 1 , further comprising subjecting column of (b) with an elution buffer to obtain one or more eluted fractions.

8. The method of claim 7 , wherein said elution buffer comprises 100 mM glycine buffer having a pH of about 2.5.

9. The method of claim 7 , wherein pH of said elution buffer is between about 2.0 to about 3.5.

10. The method of claim 7 , further comprising neutralizing said eluted fractions with the addition of a neutralizing buffer.

11. The method of claim 10 , wherein said neutralizing buffer is Tris hydrochloride.

12. The method of claim 1 , wherein an amount of host-cell proteins in (c) is significantly reduced by about 90%, about 95%, about 98%, or about 99% as compared to the amount of host-cell proteins in said clarified harvest.

13. The method of claim 1 , wherein said second capture chromatography of (d) comprises anion exchange chromatography (AEX).

14. The method of claim 13 , wherein the conductivity of both said equilibration buffer and wash buffer for said AEX column can be from about 1.50 to about 3.0 mS/cm.

15. The method of claim 13 , wherein aflibercept from said one or more flowthrough fractions of (e) comprises less than 20% total acidic species of aflibercept, wherein acidic species correspond to peaks that elute earlier than the main peak in a cation exchange chromatography (CEX) chromatogram of aflibercept, and wherein a chromatogram is generated using a first mobile phase of 20 mM 2-(N-morpholino)ethanesulfonic acid (MES), pH 5.7 and a second mobile phase of 40 mM sodium phosphate, 100 mM sodium chloride pH 9.0 (Mobile phase B), and wherein a chromatogram is generated using detection at 280 nm.

16. A method of producing aflibercept, comprising:
(a) providing a host cell genetically engineered to express aflibercept;
(b) culturing said host cell under conditions suitable in which said aflibercept is expressed;
(c) harvesting a preparation comprising aflibercept and at least one impurity produced by said host cell; and
(d) subjecting said preparation to affinity chromatography under suitable conditions, wherein said affinity chromatography comprises a polypeptide capable of binding to or interacting with said aflibercept, wherein said polypeptide comprises an isolated amino acid sequence selected from a SEQ ID NO.: 72, SEQ ID NO.: 73, SEQ ID NO.: 74, SEQ ID NO.: 75, SEQ ID NO.: 76, SEQ ID NO.: 77, SEQ ID NO.: 78, SEQ ID NO.: 79 and SEQ ID NO.: 80.

17. The method of claim 16 , wherein said polypeptide capable of binding to or interacting with said aflibercept comprises an isolated amino acid sequence selected from a SEQ ID NO.: 72.

18. The method of claim 16 , wherein said polypeptide capable of binding to or interacting with said aflibercept comprises an isolated amino acid sequence selected from a SEQ ID NO.: 73.

19. The method of claim 16 , wherein said polypeptide capable of binding to or interacting with said aflibercept comprises an isolated amino acid sequence selected from SEQ ID NO.: 74.

20. The method of claim 16 , wherein said polypeptide capable of binding to or interacting with said aflibercept comprises an isolated amino acid sequence selected from a SEQ ID NO.: 75.

21. The method of claim 16 , wherein said polypeptide capable of binding to or interacting with said aflibercept comprises an isolated amino acid sequence selected from a SEQ ID NO.: 76.

22. The method of claim 16 , wherein said polypeptide capable of binding to or interacting with said aflibercept comprises an isolated amino acid sequence selected from a SEQ ID NO.: 77.

23. The method of claim 16 , wherein said polypeptide capable of binding to or interacting with said aflibercept comprises an isolated amino acid sequence selected from a SEQ ID NO.: 78.

24. The method of claim 16 , wherein said polypeptide capable of binding to or interacting with said aflibercept comprises an isolated amino acid sequence selected from a SEQ ID NO.: 79.

25. The method of claim 16 , wherein said polypeptide capable of binding to or interacting with said aflibercept comprises an isolated amino acid sequence selected from a SEQ ID NO.: 80.

26. The method of claim 16 , wherein the amount of host-cell proteins in said eluted fractions is significantly reduced by about 90%, about 95%, about 98%, or about 99% as compared to the amount of host-cell proteins in (c).

Assignees

• Regeneron Pharma
• Regeneron Pharmnaceuticals Inc

Inventors

• Tustian Andrew
• Vartak Ankit
• Daly Thomas
• Pyles Erica
• Palackal Nisha

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