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1. WO2020160133 - ATTRIBUTS DE L'AFLIBERCEPT ET LEURS PROCÉDÉS DE CARACTÉRISATION ET DE MODIFICATION

Note: Texte fondé sur des processus automatiques de reconnaissance optique de caractères. Seule la version PDF a une valeur juridique

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CLAIMS

What is claimed is:

1. A Y92L clipped species of aflibercept comprising SEQ ID NO: 3.

2. A composition comprising a mixture of aflibercept species, wherein the amount of Y92L clipped species in the composition is less than 5.0%, as determined by rCE-SDS.

3. The composition of claim 2, wherein the amount of Y92L clipped species in the composition is less than 0.8%.

4. The composition of claim 3, wherein the amount of Y92L clipped species in the composition is about 0.4%.

5. A composition comprising a mixture of aflibercept species, wherein at least 30% of the aflibercept species is occupied at position N68, as determined by rCE-SDS.

6. The composition of claim 5, wherein at least 50% of the aflibercept species is occupied at position N68.

7. The composition of claim 6, wherein between 50-60% of the aflibercept species is occupied at position N68.

8. The composition of claim 5, wherein about 39%, 53%, 54%, or 55% of the aflibercept species is occupied at position N68.

9. The composition of claim 8, wherein the amount of Y92L clipped species in the mixture is between 1% and 3%.

10. The composition of claim 8, wherein the amount of Y92L clipped species in the mixture is less than 0.8%.

11. The composition of claim 8, wherein the amount of Y92L clipped species in the mixture is about 0.4%.

12. The composition of claim 9, wherein between 54-55% of the aflibercept species is occupied at position N68 and wherein the amount of Y92L clipped species in the mixture is about 1.1%.

13. The composition of claim 11, wherein between 54-55% of the aflibercept species is occupied at position N68 and wherein the amount of Y92L clipped species in the mixture is about 0.4%.

14. A composition comprising a mixture of aflibercept species, wherein the total sialic acid content of aflibercept species is between 6.0 and 10.0 mol/mol protein, as determined by LC-MS based peptide mapping method.

15. The composition of claim 14, wherein the total sialic acid content is about 6.8, 8.5 or 9.5 (mol/mol protein).

16. The composition of claim 15, wherein the total sialic acid content is about 9.5 mol/mol protein, and wherein between 54-55% of the aflibercept species is occupied at position N68, and wherein the amount of Y92L clipped species in the mixture is about 1.1%.

17. The composition of claim 15, wherein the total sialic acid content is about 9.5 mol/mol protein, and wherein between 54-55% of the aflibercept species is occupied at position N68, and wherein the amount of Y92L clipped species in the mixture is about 0.4%.

18. A composition comprising a mixture of aflibercept species, wherein the

aflibercept species comprise between 1.0-12% afucosylation in the Fc domain.

19. The composition of claim 18, wherein the aflibercept species comprise less than 10% afucosylation in the Fc domain.

20. The composition of claim 19, wherein the aflibercept species comprises about 6% afucosylation in the Fc domain.

21. The composition of claim 19, wherein the aflibercept species comprises less than 5% afucosylation in the Fc domain.

22. The composition of claim 19, wherein the aflibercept species comprises about 4% afucosylation in the Fc domain.

23. The composition of any one of claims 18-22, wherein the aflibercept species comprise:

a) between 0.1-2.0 (mol/mol protein) of sialic acid at N68;

b) between 1.0-25.0% O-glycosylation at T33;

c) between 70%-99% N-glycosylation at N36;

d) between 20%-75% of N-glycosylation at N68;

e) between 0.1-1.0% high mannose in the Fc domain;

1) between 26% galactosylation in the Fc domain;

g) about 1% sialylation in the Fc domain; or

h) any combination thereof.

24. The composition comprising of claim 23, wherein the aflibercept species comprise:

a) about 0.7 (mol/mol protein) of sialic acid at N68;

b) about 4% O-glycosylation at T33;

c) about 95% N-glycosylation at N36;

d) about 26% of N-glycosylation at N68;

e) about 1% high mannose in the Fc domain;

f) about 26% galactosylation in the Fc domain;

g) about 1% sialylation in the Fc domain; or

h) any combination thereof.

25. The composition of claim 23, wherein the aflibercept species comprise: a) about 0.8 (mol/mol protein) of sialic acid at N68;

b) about 8.3% O-glycosylation at T33;

c) about 90.9% N-glycosylation at N36;

d) about 51.9% of N-glycosylation at N68;

e) about 0.4% high mannose in the Fc domain;

f) about 24.4% galactosylation in the Fc domain;

g) about 3.8% sialylation in the Fc domain; or

h) any combination thereof.

26. The composition of any one of claims 18-25, wherein the amount of Y92L clipped species in the mixture is about 0.4%.

27. A method of increasing the binding of a composition comprising a mixture of aflibercept species to P1GF and/or VEGF-A comprising reducing the amount of Y92L clipped species in the mixture.

28. A method of increasing the binding of a composition comprising a mixture of aflibercept species to P1GF comprising reducing the N68 occupancy of aflibercept.

29. The method of claim 27 or 28, wherein the P1GF is PlGF-1.

30. The method of claim 27 or 28, wherein the P1GF is P1GF-2.

31. The method of claim 27, wherein the method comprises purifying aflibercept with an anion exchange chromatography step followed by a hydrophobic interaction chromatography step.

32. A method of producing a composition comprising a mixture of aflibercept species comprising:

subjecting a cell culture fluid comprising a mixture of aflibercept species to a purification process comprising an anion exchange

chromatography (AEX) step followed by a hydrophobic interaction chromatography (HIC) step,

wherein a lower amount of Y92L clipped species is produced as compared to a purification process comprising an AEX step followed by a cation exchange chromatography (CEX) step.

33. The method of claim 32, wherein the purification process does not include a CEX step.

34. A method of producing a composition comprising a mixture of aflibercept species comprising:

a) harvesting cells producing aflibercept;

b) subjecting the harvested cell culture fluid to a Protein A column; and c) performing anion exchange chromatography (AEX) followed by cation exchange chromatography (CEX) or hydrophobic interaction chromatography (HIC).

35. The method of claim 34, wherein AEX is followed by HIC.

36. The method of claim 35, wherein the method does not include CEX.

37. The method of claim 36, wherein AEX is followed by CEX.

38. The method of any one of claims 34-37, wherein the cells are Chinese

Hamster Ovary (CHO) cells.

39. The method of any one of claims 34-38, wherein the cells are grown by

perfusion or fed-batch.

40. The method of any one of claims 34-39, wherein the cells are harvested by acid precipitation, centrifugation, depth filtration or any combination thereof.

41. The method of any one of claims 34-40, further comprising one or more viral inactivation and/or viral filtration step(s).

42. The method of any one of claims 34-41, further comprising a

ultrafiltration/diafiltration (UF/DF) step.

43. The method of any one of claims 34-42, wherein the method does not

comprise performing CEX prior to performing AEX.

44. The method of any one of claims 34-43, wherein the method does not

comprise performing size exclusion chromatography (SEC).