Anti-VEGF protein composition and method for producing the same

Abstract

The present invention provides a composition containing an anti-VEGF protein and a method for producing the composition. [Solution] A method for producing aflibercept, comprising: (a) producing a clarified recovery product of cells cultured in a synthetic medium (CDM); (b) binding aflibercept from the clarified recovery product to a protein A resin; (c) eluting the aflibercept from step (b) to form an affinity eluate, wherein the eluate has a first color; (d) subjecting the eluate containing aflibercept to anion exchange chromatography; and (e) collecting a flow-through fraction, wherein the flow-through fraction has a second color, and when the protein concentrations of the eluate and the flow-through are normalized, the first color of the affinity eluate is a darker yellowish-brown than the second color of the flow-through fraction.

Claims

1. A method for producing aflibercept, (a) A step of producing a clarified recovery product of cells cultured in synthetic medium (CDM), (b) A step of binding aflibercept from the clarified recovered material to a protein A resin, (c) A step of eluting the aflibercept of step (b) to form an affinity eluate, wherein the eluate has a first color, (d) The step of subjecting the eluate containing aflibercept to anion exchange chromatography (AEX), (e) A step of collecting a flow-through fraction, wherein the flow-through fraction has a second color, and when the protein concentrations of the eluate and the flow-through are normalized, the first color of the affinity eluate is a darker yellowish-brown than the second color of the flow-through fraction. Methods that include...

2. When the protein concentration is normalized to 10.0 g / L, the first color is in the range of approximately 2.0 to approximately 20.0 b ＊ The method according to claim 1, wherein the value is...

3. When the protein concentration is normalized to 10.0 g / L, the second color is in the range of approximately 0.5 to approximately 5.0 b ＊ The method according to claim 1, wherein the value is...

4. The method according to claim 1, wherein the cells are selected from the group consisting of CHO, NS0, Sp2 / 0, fetal kidney cells, and BHK.

5. The method according to claim 1, wherein the clarified recovered material contains one or more aflibercept variants, and the variant has at least one oxidized amino acid residue.

6. The method according to claim 5, wherein the oxidized amino acid residue is selected from the group consisting of methionine, tryptophan, histidine, phenylalanine, tyrosine, and combinations thereof.

7. The method according to claim 6, wherein the oxidized amino acid residue is histidine.

8. The method according to claim 6, wherein the oxidized amino acid residue is tryptophan.

9. The method according to claim 1, wherein the AEX column comprises an anion exchange substituent containing diethylaminoethyl (DEAE), quaternary aminoethyl (QAE), and a quaternary amine (Q) group.

10. The method according to claim 5, wherein the aflibercept variant is selected from amino acid residues on a polypeptide having an amino acid sequence described in 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: 56, 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.

11. The method according to claim 1, further comprising the step of binding aflibercept from the clarified recovered product, followed by subjecting the aflibercept to one or more further chromatographic steps selected from the group consisting of cation exchange chromatography, hydrophobic interaction chromatography, size exclusion chromatography, and combinations thereof.

12. A method for producing aflibercept from a clarified recovery of cells cultured in a synthetic medium (CDM), (a) A step of binding aflibercept from the clarified recovered material to a protein A resin, (b) A step of eluting the aflibercept of step (a) to form an affinity eluate, wherein the eluate contains an acidic species of aflibercept, (c) The step of subjecting the eluted aflibercept to anion exchange chromatography (AEX), (d) A step of collecting one or more flow-through fractions, wherein when the concentrations of proteins in the eluate and the flow-through fractions are normalized, the percentage of acidic species of aflibercept in the affinity eluate is greater than the percentage of acidic species of aflibercept in the one or more flow-through fractions, the acidic species of aflibercept corresponds to a peak that elutes earlier than the main peak in the cation exchange chromatography (CEX) chromatogram of aflibercept, the 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 and 100 mM sodium chloride (pH 9.0) (mobile phase B), and the chromatogram is generated using detection at 280 nm. Methods that include...

13. The method according to claim 12, wherein, when the concentrations of the affinity eluate and the flow-through protein are normalized, the acidic species of aflibercept in the affinity eluate is reduced by at least 10 percent compared to the flow-through fraction.

14. The method according to claim 12, wherein the aflibercept from one or more flow-through fractions comprises less than 20% of the total acidic species of aflibercept.

15. The method according to claim 12, wherein the acidic species of aflibercept comprises aflibercept having at least one oxidized amino acid residue selected from the group consisting of methionine, tryptophan, histidine, phenylalanine, tyrosine, and combinations thereof.

16. The method according to claim 12, wherein the pH of both the equilibration buffer and the wash buffer for the AEX column is approximately 8.30 to approximately 8.

60.

17. The method according to claim 12, wherein the conductivity of both the equilibration buffer and the washing buffer for the AEX column can be about 1.50 to about 3.0 mS / cm.

18. The method according to claim 12, further comprising the step of binding aflibercept from the clarified recovered product, followed by subjecting the aflibercept to one or more further chromatographic steps selected from the group consisting of cation exchange chromatography (CEX), hydrophobic interaction chromatography, size exclusion chromatography, and combinations thereof.

19. A method for producing aflibercept from a clarified recovery of cells cultured in a synthetic medium (CDM), (a) A step of binding aflibercept from the clarified recovered material to a protein A resin, (b) A step of eluting the aflibercept of step (a) to form an affinity eluate, wherein the eluate contains an oxidized species of aflibercept, (c) The step of subjecting the eluted aflibercept to anion exchange chromatography (AEX), (d) A step of collecting a flow-through fraction, wherein when the concentrations of protein in the eluate and the flow-through fraction are normalized, the percentage of oxidized species of aflibercept in the affinity eluate is greater than the percentage of acidic species of aflibercept in the flow-through fraction, and the oxidized species of aflibercept is measured by subjecting the affinity eluate and the flow-through fraction to digestion, followed by analysis using reversed-phase ultrafast chromatography (UPLC), detection at wavelengths of 280 nm, 320 nm, and 350 nm, and mass spectrometry using a first mobile phase of 0.1% formic acid in water and a second mobile phase of 0.1% formic acid in acetonitrile. Methods that include...

20. The method according to claim 19, wherein the percentage of oxidized aflibercept species in the flow-through fraction is reduced by at least about 10% compared to the percentage of oxidized aflibercept species in the affinity eluate.

21. The method according to claim 19, wherein the oxidized amino acid residue is selected from the group consisting of methionine, tryptophan, histidine, phenylalanine, tyrosine, and combinations thereof.

22. The method according to claim 19, wherein the oxidized amino acid residue is selected from amino acid residues on a polypeptide having an amino acid sequence described in 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, and SEQ ID NO:

67.

23. The method according to claim 19, wherein the protein further comprises one or more variant amino acid residues selected from polypeptides having amino acid sequences described in 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: 56, 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.

24. A method for producing aflibercept from a clarified recovery of cells cultured in a synthetic medium (CDM), (a) A step of collecting a protein sample from a clarified recovered material, wherein the protein sample contains aflibercept and at least one aflibercept variant, and when the concentration of the protein sample is normalized to 10.0 g / L, the protein sample is greater than 0.5 b ＊ A process of collecting values, (b) A step of binding aflibercept from the clarified recovered material of step (a) to a first capture chromatography and eluting the aflibercept, (c) A step of subjecting the aflibercept from (b) to an anion exchange chromatography (AEX) column and collecting at least one flow-through fraction, wherein when the protein concentration in the flow-through fraction is normalized to 10.0 g / L, the flow-through fraction has a lighter yellowish-brown color compared to the protein sample from the clarified recovery. Methods that include...

25. The method according to claim 24, wherein the capture chromatography comprises a protein A resin.

26. The method according to claim 24, wherein the clarified recovered product comprises one or more aflibercept variants, the variant having at least one oxidized amino acid residue selected from the group consisting of methionine, tryptophan, histidine, phenylalanine, tyrosine, and combinations thereof.

27. The method according to claim 26, wherein the oxidized amino acid residue is histidine.

28. The method according to claim 26, wherein the oxidized amino acid residue is tryptophan.

29. The method according to claim 24, wherein the aflibercept variant is selected from amino acid residues on a polypeptide having an amino acid sequence described in 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: 56, 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.

30. The method according to claim 24, further comprising the step of collecting aflibercept from the clarified recovered material, followed by subjecting the aflibercept to one or more further chromatographic steps selected from the group consisting of cation exchange chromatography, hydrophobic interaction chromatography, size exclusion chromatography, and combinations thereof.

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