Glycopolysialylation of non-blood coagulation proteins

Patent metadata

Field	Value
Publication number	US-9795683-B2
Application number	US-84328410-A
Country	US
Kind code	B2
Filing date	Jul 26, 2010
Priority date	Jul 27, 2009
Publication date	Oct 24, 2017
Grant date	Oct 24, 2017

Abstract

A water soluble polymer, in particular polysialic acid (PSA) or a modified PSA (mPSA), is conjugated to an oxidized carbohydrate moiety of a glycoprotein other than a blood coagulation protein or to a ganglioside or drug delivery system by contacting the oxidized carbohydrate moiety with the water soluble polymer, wherein said water soluble polymer contains an aminooxy group and an oxime linkage is formed between the oxidized carbohydrate moiety and the aminooxy group on the water soluble polymer or wherein said water soluble polymer contains a hydrazide group and a hydrazone linkage is formed between the oxidized carbohydrate moiety and the hydrazide group on the water soluble polymer. Conjugates of aminooxy- or hydrazide-water soluble polymer, such as PSA and mPSA, are thus obtained in which the PSA or mPSA is attached via a carbohydrate moiety.

First claim

The invention claimed is:

1. A method of conjugating a polysialic acid (PSA) or a modified PSA (mPSA) to an oxidized carbohydrate moiety on a polypeptide with DNase activity, comprising contacting the oxidized carbohydrate moiety with the PSA or mPSA under conditions that allow conjugation,
wherein said PSA or mPSA contains an aminooxy group and an oxime linkage is formed between the oxidized carbohydrate moiety and the aminooxy group on the PSA or mPSA; and
wherein said polypeptide has a specific activity of at least 80% relative to an unmodified polypeptide.

2. The method of claim 1 wherein the PSA or mPSA is colominic acid or modified colominic acid.

3. The method of claim 1 wherein the PSA or mPSA comprises 10-300 sialic acid units.

4. The method of claim 1 , further comprising oxidizing the carbohydrate moiety by incubating said polypeptide with sodium periodate (NaIO 4 ).

5. The method of claim 1 , further comprising oxidizing the PSA or mPSA to form an aldehyde group on a terminal unit of the PSA or mPSA, and reacting the oxidized PSA or mPSA with an aminooxy linker.

6. The method of claim 5 comprising oxidizing the PSA or mPSA using NaIO 4 .

7. The method of claim 1 wherein said contacting the oxidized carbohydrate moiety with PSA or mPSA occurs in a buffer comprising a nucleophilic catalyst selected from the group consisting of aniline and aniline derivatives.

8. The method of claim 1 wherein the aminooxy group is formed by reacting oxidized PSA or mPSA with an aminooxy linker selected from the group consisting of 3- oxa-pentane-1,5 -dioxyamine and 3,6, 9-trioxa-undecane-1, 11-dioxyamine.

9. The method of claim 1 further comprising reducing an oxime linkage said polypeptide by incubation in the presence of a reducing compound.

10. The method according to claim 9 wherein the reducing compound is sodium cyanoborohydride (NaCNBH 3 ) or ascorbic acid (vitamin C).

11. A conjugated polypeptide with DNase activity, comprising:
(a) a polypeptide with DNase activity; and
(b) at least one aminooxy-PSA or aminooxy-mPSA bound to the polypeptide of (a), wherein said aminooxy-PSA or aminooxy-mPSA is attached to the polypeptide via one or more oxidized carbohydrate moieties; and
wherein said polypeptide has a specific activity of at least 80% relative to an unmodified polypeptide.

Assignees

• Jain Sanjay
• Gregoriadis Gregory
• Dwivedi Archana
• Nath Srijit
• Siekmann Juergen
• Haider Stefan
• Rottensteiner Hanspeter
• Turecek Peter
• Lipoxen Tech Ltd
• Baxalta Inc
• Baxalta GmbH

Inventors

• Jain Sanjay
• Gregoriadis Gregory
• Dwivedi Archana
• Nath Srijit
• Siekmann Juergen
• Haider Stefan
• Rottensteiner Hanspeter
• Turecek Peter

Classifications

• A61K47/65

  Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers · CPC title

• A61K47/6455 Primary

  Polycationic oligopeptides, polypeptides or polyamino acids, e.g. for complexing nucleic acids · CPC title

• A61K47/60 Primary

  the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol · CPC title

• A61K31/70

  Carbohydrates; Sugars; Derivatives thereof (sorbitol A61K31/047) · CPC title

• A61K47/61

  the organic macromolecular compound being a polysaccharide or a derivative thereof · CPC title