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Pegylated factor VII glycoforms

a technology of glycoforms and pegylated factor, which is applied in the field of compositions, can solve the problems of difficult to obtain adequate dose regulation and restrict the patient's way of living, and achieve the effect of improving functional properties

Inactive Publication Date: 2005-05-26
KLAUSEN NIELS +3
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0017] It has been found by the present investigators that preparations of Factor VII polypeptides having glycoform patterns which contain at least one oligosaccharide group covalently linked to at least one polymeric group exhibit improved functional properties. Accordingly, the present invention relates to methods and compositions that provide these conjugate protein preparations.

Problems solved by technology

As a consequence adequate dose regulation is difficult to obtain and the need of frequent intravenous administrations imposes restrictions on the patient's way of living.

Method used

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  • Pegylated factor VII glycoforms
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  • Pegylated factor VII glycoforms

Examples

Experimental program
Comparison scheme
Effect test

example 1

Glycopegylation of Product with High Sialic Content

[0183] Polyethylene glycol-CMP-sialic acid (PEG-CMPSA) is prepared by covalently attaching PEG with mw 10.000 Da to sialic acid.

[0184] Factor VIIa with 87-99% content of sialic acid is treated with sialidase, e.g., as described in U.S. Pat. No. 5,272,066, and re-sialylated with sialyltransferase using PEG-CMPSA as donor molecule (e.g., as described in U.S. Pat. No. 6,399,336). After the PEGylation reaction has reached maximal incorporation, CMPSA is added to the reaction mixture to cap any exposed terminal galactose.

[0185] Incorporation of PEGylated sialic acid is analyzed by SDS-PAGE, CE-PAGE, isoelectric focusing gels, and CE-IEF.

[0186] 94-100% sialic acid is incorporated; a mean of 1-4 PEG groups are incorporated.

example 2

Glycopegylation of Product with Medium Sialic Content

[0187] Polyethylene glycol-CMP sialic acid (PEG-CMPSA) is prepared by covalently attaching PEG with mw 10.000 Da to sialic acid.

[0188] Factor VIIa with 87-93% content of sialic acid is treated with sialyltransferase using PEG-CMPSA as donor molecule (e.g., as described in U.S. Pat. No. 6,399,336). After the PEGylation reaction has reached maximal incorporation, CMPSA is added to the reaction mixture to cap any exposed terminal galactose.

[0189] Incorporation of PEGylated sialic acid is analyzed by SDS-PAGE, CE-PAGE, isoelectric focusing gels, and CE-IEF.

[0190] 87-100% sialic acid is incorporated; a mean of 0.1-0.5 PEG groups are incorporated.

example 3

[0191] Pegylated cytidine 5′-monophospho-sialic acid derivative (CMP-SA-PEG): N-Acetyl-O2-methyl-9-amino-9-deoxy-neuraminic acid methyl ester (10 mg, 0.031 mmol, prepared according to Isecke, R.; Brossmer, R., Tetrahedron 1994, 50(25), 7445-7460) is dissolved in water (2 ml), and mPEG-SBA (170 mg, 0.03 mmol, 5 kDa, Shearwater 2M450H01)) is added. The mixture is stirred at ambient temperature until completion according to TLC. The solvent is removed by lyophilization, and the residue redissolved in a 1:1 mixture of methanol and 0.1 M NaOH solution (5 ml). The mixture is stirred at room temperature for 1 h, then passed through a column of Dowex 50W-X8 (H+) resin at 4° C. and lyophilized. The residue is then redissolved in water (5 ml), Dowex 50W-X8 (H+) resin is added and the mixture is stirred until completed by TLC.

[0192] Cytidine 5′-monophospho analogues of sialic acid derivatives of general formula I is in general prepared according to E. S. Simon, M. D. Bednarski and G. M. White...

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Abstract

The invention concerns a preparation comprising a plurality of Factor VII polypeptides or Factor VII-related polypeptides, wherein the polypeptides comprise asparagine-linked and / or serine-linked oligosaccharide chains, and wherein at least one oligosaccharide group is covalently attached to at least one polymeric group.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application is a continuation of International Application no. PCT / DK03 / 00420 filed Jun. 20, 2003 and claims priority under 35 U.S.C. 119 of Danish application no. PA 2002 00964 filed Jun. 21, 2002 and U.S. application Ser. No. 60 / 394,778 filed Jul. 1, 2002, and claims priority under 35 U.S.C. 120 of international application no. PCT / DK03 / 00420 filed Jun. 20, 2003, the contents of which are fully incorporated herein by reference.FIELD OF THE INVENTION [0002] The present invention relates to compositions comprising Factor VII conjugates having predetermined patterns of glycosylation. BACKGROUND OF THE INVENTION [0003] Factor VII is a vitamin K-dependent plasma protein synthesized in the liver and secreted into the blood as a single chain glycoprotein with a molecular weight of approximately 50 kDa. The FVII zymogen is converted into an activated form (FVIIa) by proteolytic cleavage. FVIIa in complex with tissue factor (TF) is able t...

Claims

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Application Information

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IPC IPC(8): A61KC12N9/64A61K38/00A61K38/36A61K38/48A61K47/48A61PA61P7/02A61P7/04C07KC07K14/745C07K14/755C12N
CPCA61K38/4846C07K14/755A61K47/48215A61K47/60A61P7/00A61P7/02A61P7/04
Inventor KLAUSEN, NIELSBJORN, SORENBEHRENS, CARSTENGARIBAY, PATRICK
Owner KLAUSEN NIELS
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