N-terminal derivatisation of proteins with polysaccharides
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.
1. A method for producing 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, said method comprising:
(i) reacting a polysialic acid at the amine group of the N-terminus of a protein in aqueous acidic solution to produce an N-terminal derivative; and
(ii) purifying the resultant N-terminal derivative in aqueous solution of higher pH than in (i);
wherein the pH of the aqueous solution in (i) is in the range 4.0-6.0 and in step (ii) is in the range 6.5-8.5.
2. A method according to claim 1 , wherein the polysialic acid has a reactive aldehyde group and (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 polysialic acid.
4. A method according to claim 2 , wherein the reactive aldehyde is at the reducing end of the polysialic acid and the non-reducing end has been passivated such that it does not react with the N-terminus of a protein.
5. A method according to claim 1 , in which the protein is a therapeutic protein.
6. A method according to claim 1 , in which the protein is leptin, interferon, FSH, galactosidase or DNase.
7. A method according to claim 1 , wherein a formulation additive is added to the aqueous acidic solution in (i).
8. A method according to claim 7 , 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.
9. A method according to claim 8 , wherein the formulation additive is sorbitol, trehalose or sucrose.
10. A method according to claim 8 , wherein the formulation additive is a non-ionic surfactant.
11. A method according to claim 8 , wherein the formulation additive is a polymer selected from PSA, PEG or hydroxy-beta-cyclodextrin.
12. A method according to claim 8 , wherein the formulation additive is a divalent metal ion.
13. A method according to claim 8 , wherein the formulation additive is sodium phosphate/acetate buffer.
14. A method according to claim 2 , wherein borohydride is present in the aqueous acidic solution in (i) to provide the reducing conditions.
15. 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.
16. A composition according to claim 15 , wherein the protein is leptin and the derivatives comprise 100-150 sialic acid units.
17. A composition according to claim 15 , wherein the protein is FSH and the derivatives comprise 75-200 sialic acid units.
18. A composition according to claim 15 , wherein the protein is alpha-galactosidase and the derivatives comprise 20-150 sialic acid units.
19. A composition according to claim 15 , wherein the protein is DNase and the derivatives comprise 2-120 sialic acid units.
20. A composition according to claim 15 , wherein the protein is IFN and the derivatives comprise 80-180 sialic acid units.
21. A composition according to claim 15 wherein the protein is obestatin and the derivatives comprise 20-100 sialic acid units.
22. A composition according to claim 15 , wherein the polysialic acid has a polydispersity of less than 1.3.
23. A composition according to claim 15 , which is a pharmaceutical composition and further comprises one or more pharmaceutically acceptable excipients.
24. An N-terminal derivative of a protein which is obtainable by a method according to claim 1 .