Process for the production of preformed conjugates of albumin and a therapeutic agent

• US 20070269863A1
• Filed: 12/22/2006
• Published: 11/22/2007
• Est. Priority Date: 12/22/2005
• Status: Abandoned Application

• Current Assignee: ConjuChem LLC (Colabor Group, Inc.)
• Original Assignee: ConjuChem LLC (Colabor Group, Inc.)
• Inventors: Quraishi, Omar, Bridon, Dominique, Bousquet-Gagnon, Nathalie
• Application Number:
  US11/645,297
• Publication Number:
  US 20070269863A1
• Time in Patent Office: Days
• US Class Current: 435/69.100
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61K 47/643 Albumins, e.g. HSA, BSA, ovalbumin or a Keyhole Limpet Hemocyanin [KHL]
• CPC Class Codes: C07K 1/20 Partition-, reverse-phase or hydrophobic interaction chromatography
• CPC Class Codes: C07K 14/575 Hormones derived from pro-opiomelanocortin, pro-enkephalin or pro-dynorphin C07K14/665, e.g. corticotropin C07K14/695
• CPC Class Codes: C07K 14/57545 Neuropeptide Y
• CPC Class Codes: C07K 14/57563 Vasoactive intestinal peptide [VIP]; Related peptides
• CPC Class Codes: C07K 14/58 Atrial natriuretic factor complex; Atriopeptin; Atrial natriuretic peptide [ANP]; Cardionatrin; Cardiodilatin
• CPC Class Codes: C07K 14/60 Growth hormone-releasing factor [GH-RF], i.e. somatoliberin
• CPC Class Codes: C07K 14/605 Glucagons
• CPC Class Codes: C07K 14/76 Albumins
• CPC Class Codes: C07K 14/765 Serum albumin, e.g. HSA
• CPC Class Codes: Y02A 50/30 Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The present invention provides processes for the production of preformed albumin conjugates. In particular, the invention provides processes for the in-vitro conjugation of a therapeutic compound to recombinant albumin, wherein a therapeutic compound comprising a reactive group is contacted to recombinant albumin in solution to form a conjugate. The processes provide for conjugation to albumin species of increasing homogeneity. The resulting conjugate is purified by chromatography, in particular hydrophobic interaction chromatography comprising phenyl sepharose and butyl sepharose chromatography.

Claims

1. A process for the preparation of a conjugate, said conjugate comprising albumin covalently linked to a compound, the process comprising purifying the conjugate by a first hydrophobic interaction chromatography followed by a second hydrophobic interaction chromatography.

2. The process of claim 1, wherein the first hydrophobic interaction chromatography is phenyl sepharose chromatography.

3. The process of claim 1, wherein the second hydrophobic interaction chromatography is butyl sepharose chromatography.

4. The process of claim 3, wherein the butyl sepharose chromatography comprises:

a. equilibrating butyl sepharose resin in 750 mM ammonium sulfate;

b. contacting the butyl sepharose resin with a solution comprising the conjugate; and

c. applying a decreasing salt gradient from 750-0 mM ammonium sulfate to separate monomeric conjugated albumin species from non-monomeric albumin species.

5. The process of claim 1, wherein the first hydrophobic interaction chromatography is different than the second hydrophobic interaction chromatography.

6. The process of claim 1, further comprising the step of further purifying the conjugate by ultrafiltration.

7. The process of claim 1, further comprising the step of further purifying the conjugate by a method selected from ion exchange chromatography, affinity chromatography, and size exclusion chromatography.

8. The process of claim 1, wherein the conjugate is formed in a solution by contacting albumin contained in the solution with a compound, said compound comprising a reactive group, under reaction conditions wherein the reactive group is capable of covalently binding cysteine 34 thiol of the albumin to form a conjugate.

9. The process of claim 8 wherein the solution comprises a culture medium of a host organism secreting recombinant albumin therein.

10. The process of claim 9, wherein the culture medium is separated from the host organism prior to contacting the albumin with the compound.

11. The process of claim 8, wherein the solution is a lysate of a host organism producing recombinant albumin.

12. The process of claim 8, wherein the solution comprises recombinant albumin purified by hydrophobic interaction chromatography.

13. The process of claim 8, wherein the albumin is mercaptalbumin-enriched albumin.

14. The process of claim 13, wherein mercaptalbumin is enriched by contacting the albumin with thioglycolic acid.

15. The process of claim 13, wherein mercaptalbumin is enriched by contacting the albumin with dithiothreitol.

16. The process of claim 8, wherein the albumin is deglycated albumin.

17. The process of claim 8, wherein the albumin is deglycated albumin enriched for mercaptalbumin.

18. The process of claim 16 or 17, wherein the albumin is deglycated by aminophenylboronic acid agarose affinity chromatography.

19. The process of claim 16 or 17, wherein the albumin is deglycated by concanavalin A sepharose affinity chromatography.

20. The process of claim 8, wherein said reaction conditions comprise a reaction temperature of 20°

to 25°

C.

21. The process of claim 8, wherein said reaction conditions comprise a reaction time of at least 30 minutes.

22. The process of claim 8, wherein said reaction conditions comprise a final molar ratio of the compound to recombinant albumin of 0.1:

1 to 1;

1.

23. The process of claim 8, wherein said reaction conditions comprise a final molar ratio of the compound to albumin of 0.5:

1 to 0.9;

1.

24. The process of claim 8, wherein said reaction conditions comprise a final molar ratio of the compound to albumin of 0.7:

1.

25. The process of claim 1, wherein the compound comprises an amino acid, a peptide, a protein, an organic molecule, RNA, or DNA.

26. The process of claim 1, wherein the compound is less than 30 kDa.

27. The process claim 1, wherein the compound is insulin, atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), peptide YY (PYY), growth hormone releasing factor (GRF), glucagon-like peptide-1 (GLP-1), exendin-3, or exendin-4.

28. The process of claim 1, wherein the compound is GLP-1.

29. The process of claim 1, wherein the compound is exendin-3.

30. The process of claim 1, wherein the compound is exendin-4.

31. The process of claim 1, wherein the compound comprises a reactive group.

33. The process of claim 31, wherein the reactive group is a maleimido-containing group.

34. The process of claim 31, wherein the reactive group is maleimid-propionic acid (MPA).

35. The process of claim 31, wherein the reactive group is a cysteine residue.

36. The process of claim 8, wherein the albumin is recombinant serum albumin.

37. The process of claim 8, wherein the albumin is recombinant human serum albumin.

38. The process of claim 8, wherein the albumin is fused to a peptide.

39. The process of claim 38, wherein the peptide is glucagon-like peptide 1, exendin 3, or exendin-4.

40. The process of claim 1, wherein the conjugate is according to the following:

41. The process of claim 1, wherein the conjugate is according to the following:

42. The process of claim 8, wherein the albumin is produced by a host organism.

43. The process of claim 42, wherein the host is a yeast strain transformed to express recombinant albumin.

44. The process of claim 43, wherein the yeast is selected from the group comprising Saccharomyces cerevisiae, Pichia pastoris, Kluyveromyces lactis, Arxula adeninivorans, and Hansenula polymorpha.

45. The process of claim 42, wherein the host is a bacterium transformed to express recombinant albumin.

46. The process of claim 45, wherein the bacterium is Escherichia coli.

47. The process of any claim 42, wherein the host is a transgenic plant expressing recombinant albumin.

48. The process of claim 42, wherein the host is a transgenic animal expressing recombinant albumin.

49. The process of claim 8, wherein the recombinant albumin is produced by a mammalian cell transformed with a vector encoding albumin, or a variant or derivative thereof.

32. The process of 31, wherein the reactive group is a Michael acceptor, a succinimidyl-containing group, a maleimido-containing group or an electrophilic thiol acceptor.

50. A process for the preparation of a conjugate, the conjugate comprising recombinant albumin and a compound having less than 30 kDa that is selected from the group consisting of an amino acid, a peptide, a protein, an organic molecule, RNA and DNA, where the compound is modified by coupling a reactive group thereto and the conjugate is formed by the reaction of the modified compound and recombinant albumin, the process comprising the steps of:

a. producing recombinant albumin by culturing a host organism in a culture media, such that the recombinant albumin is secreted in the culture media;

b. concurrently with step (a), adding the modified compound to the culture media and allowing the modified compound to react with recombinant albumin secreted in the culture media; and

c. purifying the conjugate resulting from the reaction of step (b).

54. A process according to claim 50, 51, or 52, wherein the host organism is a yeast.

56. A process according to claim 50, having an additional step (b-1) of purification of the recombinant albumin obtained in step (b) prior to its reaction with the modified compound of step (c).

57. A process according to claim 56, wherein the recombinant albumin obtained by the purification step (b-1) comprises capped albumin and mercaptalbumin, and the process further comprises a step (b-2) of enrichment of mercaptalbumin, and the process further comprises a step (b-2) of enrichment of mercaptalbumin prior to the reaction with the modified compound of step (c).

51. A process for the preparation of a conjugate, the conjugate comprising recombinant albumin and a compound having less than 30 kDA that is selected from the group consisting of an amino acid, a peptide, a protein, an organic molecule, RNA, and DNA, where the compound is modified by coupling a reactive group thereto and the conjugate is formed by the reaction of the modified compound and recombinant albumin, the process comprising the steps of:

a. producing recombinant albumin by culturing a host organism in a culture media, such that the recombinant albumin is secreted in the culture media;

b. collecting the culture media containing the recombinant albumin;

c. adding the modified compound to the collected culture media obtained at step (b) and allowing the modified compound to react with recombinant albumin; and

d. purifying the conjugate resulting from the reaction of step (b).

52. A process for the preparation of a conjugate, the conjugate comprising recombinant albumin and a compound having less than 30 kDA that is selected from the group consisting of an amino acid, a peptide, a protein, an organic molecule, RNA, and DNA, where the compound is modified by coupling a reactive group thereto and the conjugate is formed by the reaction of the modified compound and recombinant albumin, the process comprising the steps of:

a. producing recombinant albumin by culturing a host organism in a culture media, such that the recombinant albumin is secreted in the culture media;

b. purifying the secreted recombinant albumin;

c. adding the modified compound recombinant albumin purified at step (b) and allowing the modified compound to react with recombinant albumin; and

d. purifying the conjugate resulting from the reaction of step (c).

53. A process according to claim 52, wherein the purified recombinant albumin of step (b) comprises capped albumin and mercaptalbumin, and the process further comprises a step of enrichment of mercaptalbumin prior to the reaction with the modified compound of step (c).

55. A process for the preparation of a conjugate, the conjugate comprising recombinant albumin and a compound having less than 30 kDA, that is selected from the group consisting of an amino acid, a protein, an organic molecule, RNA, and DNA, where the compound is modified by coupling a reactive group thereto and the conjugate is formed by the reaction of the modified compound and recombinant albumin, the process comprising the steps of:

a. producing recombinant albumin by culturing a host organism in a culture media, such that recombinant albumin is stored intracellularly;

b. physically separating recombinant albumin from the cell of the host organism;

c. adding the modified compound to the recombinant albumin obtained in step (b) and allowing the modified compound to react with recombinant albumin; and

d. purifying the conjugate resulting from the reaction of step (b).

58. A process according to claim 55, 56, or 57 wherein the host organism is a bacteria.