Methods for producing aflibercept in chemically defined media having reduced aflibercept variants

1. A method of producing aflibercept harvested from a host cell cultured in a chemically defined medium (CDM), comprising:

(a) providing a host cell genetically engineered to express aflibercept;

(b) culturing said host cell in said CDM under conditions suitable in which said host cell expresses said aflibercept wherein the cumulative concentration of nickel in said CDM is less than or equal to 0.4 μM or about 0.4 μM and one or more of the following:

i. the cumulative concentration of iron in said CDM is less than or equal to 55.0 μM;

ii. the cumulative concentration of copper in said CDM is less than or equal to 0.8 μM;

iii. the cumulative concentration of zinc in said CDM is less than or equal to 56.0 μM;

iv. the cumulative concentration of cysteine in said CDM is less than or equal to 10.0 mM, and

v. said CDM includes anti-oxidants where the cumulative concentration of an antioxidant is about 0.001 mM to about 10.0 mM for any single anti-oxidant; and

(c) harvesting aflibercept produced by said host cell.

2. The method of claim 1 , wherein said harvest has a color no more yellow-brown than European Color Standard BY2, wherein the aflibercept concentration is 5.0 g/L.

3. The method of claim 2 , wherein said anti-oxidants are taurine, hypotaurine, glycine, thioctic acid, glutathione, choline, hydrocortisone, Vitamin C, Vitamin E or combinations thereof.

4. The method of claim 2 , wherein said cumulative concentration of an anti-oxidant 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 about 30.0 mM or less than 30.0 mM.

5. The method of claim 1 , wherein aflibercept titer, viable cell concentration, viability, ammonia or osmolality is substantially unchanged.

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

7. The method of claim 1 , wherein said harvest comprises one or more aflibercept variants, wherein said variants have at least one oxidized amino acid residue.

8. The method of claim 7 , wherein said oxidized amino acid residue is selected from the group consisting of methionine, tryptophan, histidine, phenylalanine, tyrosine and a combination thereof.

9. The method of claim 8 , wherein said oxidized amino acid residue is histidine.

10. The method of claim 8 , wherein said oxidized amino acid residue is tryptophan.

11. The method of claim 7 , wherein said aflibercept variant comprises a polypeptide having an amino acid sequence selected from 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: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, 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 1 , wherein said anti-oxidants are taurine, hypotaurine, glycine, thioctic acid, glutathione, choline, hydrocortisone, Vitamin C, Vitamin E or combinations thereof.

13. The method of claim 1 , wherein the harvest has a color that is:

a. no more yellow-brown than European Color Standard BY2;

b. no more yellow-brown than European Color Standard BY3;

c. no more yellow-brown than European Color Standard BY4;

d. no more yellow-brown than European Color Standard BY5;

e. between European Color Standard BY2 and BY3; or

f. between European Color Standard BY2 and BY4, and

wherein the aflibercept concentration in the harvest is 5.0 g/L.

14. The method of claim 13 , wherein the color of harvest is characterized in the CIE L*, a*, b* color space, where L* is about 70 to about 99, a* is about 0 and b* is about 20 or less than 20 when the concentration of aflibercept is 5.0 g/L.

15. A method of producing aflibercept harvested from a host cell cultured in a chemically defined medium (CDM), comprising:

(a) providing a host cell genetically engineered to express aflibercept;

(b) culturing said host cell in said CDM under conditions suitable in which said host cell expresses said aflibercept wherein the cumulative concentration of nickel in said CDM is about 0.4 μM and one or more of the following:

i. the cumulative concentration of iron in said CDM is less than or equal to 55.0 μM;

ii. the cumulative concentration of copper in said CDM is less than or equal to 0.8 μM;

iii. the cumulative concentration of zinc in said CDM is less than or equal to 56.0 μM;

iv. the cumulative concentration of cysteine in said CDM is less than or equal to 10.0 mM; and

v. said CDM includes anti-oxidants where the cumulative concentration of an antioxidant is about 0.001 mM to about 10.0 mM for any single anti-oxidant; and

(c) harvesting aflibercept produced by said host cell, wherein the color of said harvest is:

a. no more yellow-brown than European Color Standard BY2;

b. no more yellow-brown than European Color Standard BY3;

c. no more yellow-brown than European Color Standard BY4;

d. no more yellow-brown than European Color Standard BY5;

e. between European Color Standard BY2 and BY3; or

f. between European Color Standard BY2 and BY4,

wherein the aflibercept concentration is 5.0 g/L.

16. A method of producing aflibercept harvested 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 the cumulative concentration of nickel in said CDM is less than or equal to 0.4 μM or about 0.4 μM and one or more of the following:

i. the cumulative concentration of iron in said CDM is less than or equal to 55.0 μM;

ii. the cumulative concentration of copper in said CDM is less than or equal to 0.8 μM;

iii. the cumulative concentration of zinc in said CDM is less than or equal to 56.0 μM;

iv. the cumulative concentration of cysteine in said CDM is less than or equal to 10.0 mM; and

v. said CDM includes anti-oxidants where the cumulative concentration of an antioxidant is about 0.001 mM to about 10.0 mM for any single anti-oxidant; and

(b) harvesting aflibercept produced by said host cell.