Anti-VEGF protein compositions and methods for producing the same

Patent metadata

Field	Value
Publication number	US-11505594-B2
Application number	US-202217711723-A
Country	US
Kind code	B2
Filing date	Apr 1, 2022
Priority date	Dec 6, 2019
Publication date	Nov 22, 2022
Grant date	Nov 22, 2022

Abstract

The present disclosure pertains to compositions comprising anti-VEGF proteins and methods for producing such compositions.

First claim

What is claimed is:

1. A method of producing MiniTrap from a host cell cultured in a chemically defined medium (CDM), comprising:
(a) culturing said host cell in said CDM under suitable conditions in which said host cell expresses aflibercept wherein (i) the cumulative concentration of nickel in said CDM is less than or equal to 0.4 μM, (ii) the cumulative concentration of iron in said CDM is less than or equal to 55.0 μM, (iii) the cumulative concentration of copper in said CDM is less than or equal to 0.8 μM, (iv) the cumulative concentration of zinc in said CDM is less than or equal to 56.0 μM, (v) the cumulative concentration of cysteine in said CDM is less than or equal to 10.0 mM, or (vi) the cumulative concentration of an antioxidant in said CDM is 0.001 mM to 10.0 mM for any single anti-oxidant and the cumulative concentration of all anti-oxidants is less than or equal to 30.0 mM;
(b) binding said aflibercept from said host cell cultured in said CDM to a first capture chromatography;
(c) eluting said aflibercept of step (b) and subjecting said aflibercept in eluate to enzymatic cleavage to remove its Fc domain thereby forming said MiniTrap;
(d) subjecting said MiniTrap of step (c) to a second capture chromatography, wherein said second capture chromatography step is subjected to one or more washes, wherein a flowthrough fraction comprises MiniTrap and has a first color, wherein said first color has a b* value ranging from 1.5 to 15.0 when protein concentration is normalized to 5.0 g/L, when characterized in the CIE L*, a*, b* color space, such that L* is about 70 to about 99, a* is about 0;
(e) subjecting said flowthrough fraction of step (d) to anion exchange chromatography (AEX) with an AEX column; and
(f) washing said AEX column of step (e), wherein said MiniTrap is collected in a flowthrough fraction and has a second color, wherein said second color has a b* value ranging from 0.4 to 15.0 when protein concentration is normalized to 5.0 g/L and wherein said first color is a more intense yellow brown color than said second color.

2. The method of claim 1 , wherein the cumulative concentration of nickel in said CDM is less than or equal to 0.4 μM, the cumulative concentration of iron in said CDM is less than or equal to 55.0 μM, the cumulative concentration of copper in said CDM is less than or equal to 0.8 μM, the cumulative concentration of zinc in said CDM is less than or equal to 56.0 μM, the cumulative concentration of cysteine in said CDM is less than or equal to 10.0 mM, and the cumulative concentration of an antioxidant in said CDM is about 0.001 mM to about 10.0 mM for any single anti-oxidant and the cumulative concentration of all anti-oxidants is less than or equal to 30.0 mM.

3. The method of claim 1 , wherein the cumulative concentration of nickel in said CDM is less than or equal to 0.4 μM and the cumulative concentration of cysteine in said CDM is less than or equal to 10.0 mM.

4. The method of claim 1 , wherein the cumulative concentration of nickel in said CDM is less than or equal to 0.4 μM and the cumulative concentration of an antioxidant in said CDM is about 0.001 mM to about 10.0 mM for any single anti-oxidant and the cumulative concentration of all anti-oxidants is less than or equal to 30.0 mM.

5. The method of claim 1 , wherein the cumulative concentration of nickel in said CDM is less than or equal to 0.4 μM and the cumulative concentration of iron in said CDM is less than or equal to 55.0 μM.

6. The method of claim 1 , wherein the cumulative concentration of copper in said CDM is less than or equal to 0.8 μM and the cumulative concentration of cysteine in said CDM is less than or equal to 10.0 mM.

7. The method of claim 1 , wherein the cumulative concentration of iron in said CDM is less than or equal to 55.0 μM and the cumulative concentration of cysteine in said CDM is less than or equal to 10.0 mM.

8. The method of claim 1 , wherein said HIC mobile phase buffer has a pH between 4.5 and 8.5.

9. The method of claim 1 , wherein said AEX uses a mobile phase flow through wash having a pH between 7.7 and 8.5 and conductivity between 1.9 mS/cm and 4.1 mS/cm.

10. The method of claim 1 , wherein said AEX uses a mobile phase flow through wash having a pH between 7.9 and 8.1 and conductivity between 2.4 mS/cm and 2.6 mS/cm.

11. The method of claim 1 , wherein the b* after step (f) is between 0.7 and 2.0.

12. The method of claim 2 , wherein the b* after step (f) is between 0.4 and 0.7.

13. A method of producing aflibercept MiniTrap from a host cell cultured in a chemically defined medium (CDM), comprising: (a) culturing said host cell in said CDM under conditions suitable in which said host cell expresses said aflibercept wherein (i) the cumulative concentration of nickel in said CDM is less than or equal to 0.4 μM, (ii) the cumulative concentration of iron in said CDM is less than or equal to 55.0 μM, (iii) the cumulative concentration of copper in said CDM is less than or equal to 0.8 μM, (iv) the cumulative concentration of zinc in said CDM is less than or equal to 56.0 μM, (v) the cumulative concentration of cysteine in said CDM is less than or equal to 10.0 mM, or (vi) the cumulative concentration of an antioxidant in said CDM is 0.001 mM to 10.0 mM for any single anti-oxidant and the cumulative concentration of all anti-oxidants is less than or equal to 30.0 mM;
(b) binding said aflibercept from said host cell cultured in said CDM to a first capture chromatography;
(c) eluting said aflibercept of step (b) and subjecting said aflibercept in eluate to enzymatic cleavage to remove its Fc domain thereby forming MiniTrap, wherein said MiniTrap in eluate includes MiniTrap with one or more acidic species;
(d) subjecting said MiniTrap of step (c) to a second capture chromatography, wherein said second capture chromatography step is subjected to one or more washes, wherein a flowthrough fraction comprises MiniTrap;
(e) subjecting flowthrough fraction of step (d) to anion exchange chromatography (AEX) with an AEX column; and
(f) washing said AEX column of step (e) and collecting an AEX flowthrough fraction, wherein the percent of acidic species of MiniTrap in said eluate of step (c) is greater than the percent of acidic species of MiniTrap in said AEX flowthrough fraction when the concentration of MiniTrap in said eluate of step (c) and concentration of MiniTrap in said AEX flowthrough fractions are normalized, wherein said acidic species of MiniTrap correspond to the peaks that elute earlier than the main peak in a strong 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.

14. The method of claim 13 , wherein the cumulative concentration of nickel in said CDM is less than or equal to 0.4 μM, the cumulative concentration of cysteine in said CDM is less than or equal to 10.0 mM and the cumulative concentration of iron in said CDM is less than or equal to 55.0 μM.

15. The method of claim 13 , wherein the relative amount of oxidized His19 residue from step (d) is reduced by 15% after step (f).

16. The method of claim 13 , wherein the relative amount of oxidized His86 residue from step (d) is reduced by 30% after step (f).

17. The method of claim 13 , wherein the relative amount of oxidized His95 residue from step (d) is reduced by 25% after step (f).

Assignees

• Regeneron Pharma
• Regeneran Pharmaceuticals Inc

Inventors

• Tustian Andrew
• Vartak Ankit
• Daly Thomas
• Pyles Erica
• Palackal Nisha
• Wang Shunhai
• Li Ning
• Franklin Matthew
• Lawrence Shawn
• Johnson Amy
• Casey Meghan
• Grapel Jaimie

Classifications

• C07K1/18

  Ion-exchange chromatography · CPC title

• C12N5/0018

  Culture media for cell or tissue culture (media for specific animal cell type C12N5/06) · CPC title

• C07K2319/30

  Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto · CPC title

• C07K1/16

  by chromatography · CPC title

• C07K2319/33

  fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies · CPC title