Anti-VEGF protein compositions and methods for producing the same

Patent metadata

Field	Value
Publication number	US-11459374-B2
Application number	US-202217667330-A
Country	US
Kind code	B2
Filing date	Feb 8, 2022
Priority date	Dec 6, 2019
Publication date	Oct 4, 2022
Grant date	Oct 4, 2022

Abstract

The present disclosure pertains to methods for preparing a prefilled syringe and methods for preparing an auto injector prefilled syringe comprising aflibercept and methods for producing such aflibercept in chemically defined media.

First claim

What is claimed is:

1. A method for preparing a prefilled syringe comprising the step of loading a syringe with aflibercept, wherein a process of producing aflibercept comprises the steps of:
(a) binding aflibercept produced in a chemically defined media (CDM) to a first capture chromatography, 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, and a combination thereof;
(b) eluting said aflibercept of step (a) forming an affinity eluate, wherein said eluate has a first color;
(c) subjecting said affinity eluate to an ion exchange chromatography 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 affinity eluate and said flowthrough fraction protein concentrations are normalized to 5.0 g/L.

2. The method of claim 1 , wherein said first capture chromatography comprises Protein A resin.

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 oxidized species of aflibercept comprises an amino acid sequence selected from an amino acid residue on a polypeptide as set forth in the group consisting of: SEQ ID NO.: 17, 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.: 68, and combinations thereof.

6. The method of claim 1 , wherein said aflibercept includes one of an oxidized histidine selected from the group consisting of His86, His110, His145, His209, His95, His19, His203, and a combination thereof, and an oxidized tryptophan selected from the group consisting of Trp58, Trp138 and combinations thereof.

7. The method of claim 2 , further comprising at least one additional chromatographic step selected from the group consisting of: hydrophobic interaction chromatography, mixed mode chromatography, affinity chromatography, size exclusion chromatography and a combination thereof.

8. The method of claim 5 , wherein the b* value of aflibercept after step (d) is between 0.5 and 5, when the flowthrough fraction protein concentration is normalized to 5.0 g/L.

9. The method of claim 1 , wherein the b* value of aflibercept after step (d) is between 1.5 and 5, when the flowthrough fraction protein concentration is normalized to 5.0 g/L.

10. The method of claim 1 , wherein the syringe is glass.

11. The method of claim 1 , wherein the syringe is an auto injector.

12. The method of claim 1 , wherein said prefilled syringe has about 2 mg of aflibercept.

13. The method of claim 1 , wherein said prefilled syringe has about 2 mg/0.05 mL of aflibercept.

14. A method of preparing a prefilled auto injector syringe comprising aflibercept, wherein said aflibercept is produced in chemically defined media (CDM) by:
(a) binding aflibercept produced in a chemically defined media (CDM) to a first capture chromatography, 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, and a combination thereof;
(b) eluting said aflibercept of step (a) forming an affinity eluate, wherein said eluate has a first color;
(c) subjecting said affinity eluate to an ion exchange chromatography 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 affinity eluate and said flowthrough fraction protein concentrations are normalized to 5.0 g/L.

15. The method of claim 14 , wherein said aflibercept in said auto injector syringe has a b* value between 0.5 and 5 when the concentration of aflibercept is normalized to 5 g/L protein concentration.

16. The method of claim 14 , wherein said prefilled auto injector syringe has between 40 mg/mL of said aflibercept to about 250 mg/mL of said aflibercept.

17. The method of claim 14 , wherein the amino acid residues of said aflibercept of step (d) comprise between 0.1% and 1.5% oxidized histidine and tryptophan collectively.

18. The method of claim 14 , further comprising at least one additional chromatographic step selected from the group consisting of: hydrophobic interaction chromatography, mixed mode chromatography, affinity chromatography, size exclusion chromatography and a combination thereof.

19. The method of claim 14 , wherein the aflibercept loaded into said syringe is formulated with one or more pharmaceutically acceptable excipients.

20. A method of preparing a prefilled syringe comprising aflibercept,
(a) producing said aflibercept in a chemically defined media (CDM), wherein said aflibercept has a BY value of less than 5 when the concentration of aflibercept is normalized to 5 g/L protein concentration;
(b) purifying said aflibercept by a process that includes the following chromatography steps to obtain aflibercept having a BY value of at least 5;
(c) affinity chromatography; and
(d) ion exchange chromatography, wherein the purified aflibercept has a BY value of at least 5 and wherein said aflibercept includes one of an oxidized histidine selected from the group consisting of His86, His 110, His145, His209, His95, His19, His203, and a combination thereof, and an oxidized tryptophan selected from the group consisting of Trp58, Trp138, and combinations thereof.

21. The method of claim 20 , wherein said prefilled syringe has between 40 mg/mL to about 250 mg/mL of said aflibercept.

22. The method of claim 19 , wherein said aflibercept comprises more than 1% oxidized histidine and tryptophan before purification and between 0.1% and 1% oxidized histidine and tryptophan after it is purified.

23. The method of claim 19 , wherein said aflibercept comprises more than 1.5% oxidized histidine and tryptophan before purification and between 0.1% and 1.5% oxidized histidine and tryptophan after it is purified.

24. The method of claim 20 , wherein said prefilled syringe is glass.

25. The method of claim 20 , wherein said prefilled syringe is tinted.

26. The method of claim 20 , wherein said prefilled syringe is an auto injector.

Assignees

• Regeneron Pharma

Inventors

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

Classifications

• C07K1/18

  Ion-exchange chromatography · CPC title

• G01N2030/027

  Liquid chromatography · CPC title

• C12N5/0031

  Serum-free culture media · CPC title

• C07K14/71 Primary

  for growth factors; for growth regulators · CPC title

• A61K38/179 Primary

  for growth factors; for growth regulators · CPC title