N-TERMINAL DERIVATISATION OF PROTEINS WITH POLYSACCHARIDES

• US 20100056427A1
• Filed: 07/25/2007
• Published: 03/04/2010
• Est. Priority Date: 07/25/2006
• Status: Active Grant

• Current Assignee: Lipoxen Technologies Limited (Xenetic Biosciences, Inc.)
• Original Assignee: Lipoxen Technologies Limited London Bioscience Innovation Centre
• Inventors: Jain, Sanjay, Gregoriadis, Gregory, Laing, Peter
• Granted Patent: US 8,394,921 B2
• Time in Patent Office: Days
• US Class Current: 514/1.100
• 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 38/1816 Erythropoietin [EPO]
• CPC Class Codes: A61K 38/193 Colony stimulating factors [CSF]
• CPC Class Codes: A61K 38/465 acting on ester bonds (3.1), e.g. lipases, ribonucleases
• CPC Class Codes: A61K 47/61 the organic macromolecular compound being a polysaccharide or a derivative thereof
• CPC Class Codes: A61P 11/00 Drugs for disorders of the respiratory system
• CPC Class Codes: A61P 13/12 of the kidneys
• CPC Class Codes: A61P 3/04 Anorexiants; Antiobesity agents
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 37/06 Immunosuppressants, e.g. drugs for graft rejection
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 5/06 of the anterior pituitary hormones, e.g. TSH, ACTH, FSH, LH, PRL, GH
• CPC Class Codes: A61P 5/50 for increasing or potentiating the activity of insulin
• CPC Class Codes: A61P 7/00 Drugs for disorders of the blood or the extracellular fluid
• CPC Class Codes: A61P 7/06 Antianaemics
• CPC Class Codes: C07K 14/505 Erythropoietin [EPO]
• CPC Class Codes: C07K 14/52 Cytokines; Lymphokines; Interferons
• CPC Class Codes: C07K 14/535 Granulocyte CSF; Granulocyte-macrophage CSF
• 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: C12N 9/22 : Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113
• CPC Class Codes: C12Y 301/21001 : Deoxyribonuclease I (3.1.21.1)
• 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 relates to methods for producing N-terminal derivatives of proteins in which a polysaccharide, preferably having at least terminal sialic units and preferably consisting essentially only of sialic acid units, is reacted at the N-terminus of a protein or peptide under controlled conditions to produce an N-terminal derivative. The controlled conditions include use of acidic pH for the derivatisation step and a higher pH for purification. The derivatives are useful for improving pharmacokinetics and pharmacodynamics of proteins and peptides.

Claims

1. A method for producing a purified N-terminal derivative of a protein or peptide in which

(i) a polysaccharide is reacted at the amine group of the N-terminus of a protein or peptide in aqueous acidic solution to produce an N-terminal derivative;

and

(ii) the resultant N-terminal derivative is purified in aqueous solution of higher pH than in step (i).

2. A method according to claim 1, wherein the polysaccharide has a reactive aldehyde group and step (i) is carried out under reducing conditions.

3. A method according to claim 2, wherein the reactive aldehyde group is at the non-reducing end of the polysaccharide.

4. A method according to claim 1, wherein the polysaccharide comprises at least one sialic acid unit, or moiety derived from a sialic acid unit.

5. A method according to claim 4, wherein the polysaccharide comprises a sialic acid unit, or moiety derived from a sialic acid unit, at its non-reducing and/or reducing end.

6. A method according to claim 4, wherein the polysaccharide is a polysialic acid consisting substantially only of units of sialic acid.

7. A method according to claim 6, wherein the polysaccharide has at least 2 sialic acid units in the molecule.

8. A method according to claim 2, wherein the reactive aldehyde is at the reducing end of the polysaccharide and the non-reducing end has been passivated such that it does not react with the N-terminus of a protein.

9. A method according to claim 1, wherein the pH of the aqueous solution in step (i) is in the range 4.0-6.0 and in step (ii) is in the range 6.5-8.5.

10. A method according to claim 1, in which the protein is a therapeutic protein.

11. A method according to claim 1, in which the protein is leptin, interferon, FSH, galactosidase or DNase.

12. A method according to claim 1, wherein a formulation additive is added to the aqueous acidic solution in step (i).

13. A method according to claim 12, wherein the formulation additive is selected from the group consisting of one or more buffers, stabilisers, surfactants, salts, polymers, metal ions, sugars, polyols and amino acids.

14. A method according to claim 13, wherein the formulation additive is sorbitol, trehalose or sucrose.

15. A method according to claim 13, wherein the formulation additive is a non-ionic surfactant.

16. A method according to claim 13, wherein the formulation additive is a polymer selected from PSA, PEG or hydroxy-beta-cyclodextrin.

17. A method according to claim 13, wherein the formulation additive is a divalent metal ion.

18. A method according to claim 13, wherein the formulation additive is sodium phosphate/acetate buffer.

19. A method according to claim 2, wherein borohydride is present in the aqueous acidic solution in step (i) to provide the reducing conditions.

29. An N-terminal derivative of protein or peptide which is obtainable by a method according to claim 1.

20. A composition comprising a population of polysialic acid derivatives of a protein, wherein the derivatives comprise between 2 and 200 sialic acid units and wherein the population consists substantially only of N-terminal derivatives of the protein.

21. A composition according to claim 20, wherein the protein is leptin and the derivatives comprise 100-150 sialic acid units.

22. A composition according to claim 20, wherein the protein is FSH and the derivatives comprise 75-200 sialic acid units.

23. A composition according to claim 20, wherein the protein is alpha-galactosidase and the derivatives comprise 20-150 sialic acid units.

24. A composition according to claim 20, wherein the protein is DNase and the derivatives comprise 2-120 sialic acid units.

25. A composition according to claim 20, wherein the protein is IFN and the derivatives comprise 80-180 sialic acid units.

26. A composition according to claim 20 wherein the protein is obestatin and the derivatives comprise 20-100 sialic acid units.

27. A composition according to claim 20, wherein the polysialic acid has a polydispersity of less than 1.3.

28. A composition according to claim 20, which is a pharmaceutical composition and further comprises one or more pharmaceutically acceptable excipients.