Anti-VEGF protein compositions and methods for producing the same
The present disclosure pertains to compositions comprising anti-VEGF proteins and methods for producing such compositions.
1. A method of producing aflibercept 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);
(d) subjecting said aflibercept 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 aflibercept 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 aflibercept 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 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.
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 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.
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 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), wherein said aflibercept in eluate comprises aflibercept with one or more acidic species;
(d) subjecting said aflibercept 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 aflibercept;
(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 aflibercept in said eluate of step (c) is greater than the percent of acidic species of aflibercept in said AEX flowthrough fraction when the concentration of aflibercept in said eluate of step (c) and concentration of aflibercept in said AEX flowthrough fractions are normalized, wherein said acidic species of aflibercept 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 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).
18. The method of claim 13 , wherein the relative amount of oxidized His145 residue from step (d) is reduced by 25% after step (f).
19. The method of claim 13 , wherein the relative amount of oxidized His209 residue from step (d) is reduced by 30% after step (f).
20. The method of claim 13 , wherein the relative amount of oxidized Trp58 residue from step (d) is reduced by 30% after step (f).
21. The method of claim 13 , wherein the relative amount of oxidized Trp138 residue from step (d) is reduced by 10% after step (f).
22. 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 suitable conditions 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 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 zine 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, or
v. 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) harvesting aflibercept produced by said host cell, wherein said aflibercept comprises acidic species of aflibercept;
(c) subjecting said aflibercept and acidic species of aflibercept using anion exchange chromatography (AEX) and at least two additional chromatography steps, in any order, selected from the group consisting of Protein A, affinity chromatography, cation exchange chromatography (CEX), hydrophobic interaction chromatography (HIC), and mixed mode chromatography.
23. The method of claim of 22 , wherein said acidic species of aflibercept from step (b) is reduced by more than 10% following step (c).
24. The method of claim of 22 , wherein the chromatography steps of (c) include at least Protein A and AEX, wherein said AEX chromatography 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.
25. The method of claim of 22 , wherein the chromatography steps of (c) include at least Protein A, AEX, and HIC, wherein said AEX chromatography 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 and HIC is performed in flowthrough mode.
26. The method of claim 25 , wherein HIC uses a wash buffer having a pH between 4.0 and 5.5 and a total salt concentration greater than 70 mM.
27. 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 (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 aflibercept from said host cell cultured in said CDM to a Protein A resin;
(c) eluting said aflibercept of step (b), wherein said aflibercept in eluate comprises aflibercept with one or more acidic species;
(d) subjecting said aflibercept of step (c) to a second capture chromatography, wherein said second capture chromatography step is subjected to one or more washes, and wherein a flowthrough fraction comprises aflibercept;
(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) to obtain an AEX flowthrough fraction, wherein the percent of oxidized species of aflibercept in said eluate of step (c) is greater than the percent of oxidized species in said AEX flowthrough fraction when the concentration aflibercept in said eluate of step (c) and concentration of aflibercept in said AEX flowthrough fractions are normalized, and wherein said oxidized species of aflibercept is measured by subjecting said eluate and said flowthrough fractions 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% formic acid in water and a second mobile phase of 0.1% formic acid in acetonitrile.
28. The method of claim 27 , wherein a relative amount of oxidized Trp58 residue from step (c) is reduced by 10% after step (f).
29. The method of claim 27 , wherein a relative amount of oxidized Trp138 residue from step (c) is reduced by 10% after step (0.
30. The method of claim 27 , wherein said host cell is selected from the group consisting of CHO, NS0, Sp2/0, embryonic kidney cells and BHK.