Factor VIII Molecules With Reduced VWF Binding

a technology molecule, applied in the field of recombinant factor viii (fviii) molecules, can solve the problems of affecting the quality of iv, unstable clot, and significant inconvenience and/or pain of many people, especially children and young peopl

Inactive Publication Date: 2015-12-31
NOVO NORDISK AS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0006]The present invention relates to a recombinant Factor VIII molecule, wherein said molecule has reduced vWF binding capacity, and wherein said molecule is covalently conjugated with at least one side group. The invention furthermore relates to methods for making such molecules as well as use of such molecules. Such molecules are having a modified circulatory half life.

Problems solved by technology

The clinical manifestation is not on primary haemostasis—formation of the blood clot occurs normally—but the clot is unstable due to a lack of secondary thrombin formation.
IV administration is for many, especially children and young persons, associated with significant inconvenience and / or pain.

Method used

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  • Factor VIII Molecules With Reduced VWF Binding
  • Factor VIII Molecules With Reduced VWF Binding
  • Factor VIII Molecules With Reduced VWF Binding

Examples

Experimental program
Comparison scheme
Effect test

example 1

Production of Recombinant B Domain Truncated O-Glycosylated Factor VIII and Variants Thereof, e.g., Factor VIII (Y1680F) or Factor VIII (Y1680C)

[0091]Cell Line and Culture Process

[0092]Using Factor VIII cDNA, a mammalian expression plasmid was constructed. The plasmids encodes a B-domain deleted Factor VIII comprising the Y1680F mutation, the Factor VIII heavy chain comprising amino acid 1-740 of full length human Factor VIII, and Factor VIII light chain comprising amino acid 1649-2332 of full length human Factor VIII. The heavy and light chain sequences are connected by a 21 amino acid linker (SFSQNSRHPSQNPPVLKRHQR—SEQ ID NO 4) comprising the sequence of amino acid 741-750 and 1638-1648 of full length human Factor VIII. FVIII variants comprising the linker defined in SEQ ID NO 4 may also herein be referred to as “N8”. The Factor VIII amino acid sequence encoded by this plasmid is as set forth in SEQ ID NO 1 (wt), SEQ ID NO 2 (Y1680F), SEQ ID NO 3 (Y1680C)

[0093]Chinese hamster ovary...

example 2

Procedure for PEGylation of Recombinant O-Glycosylated Factor VIII

[0102]The recombinant Factor VIII molecules obtained in Example 1 are conjugated with poly-ethyleneglycol (PEG) using the following procedure:

[0103]For the glycoPEGylation reaction to be efficient a FVIII concentration >5 mg / ml is required. Since FVIII is not normally soluble at the concentration a screening of selected buffer compositions was conducted (see table 1). Based on these considerations a buffer containing 50 mM MES, 50 mM CaCl2, 150 mM NaCl, 20% glycerol, pH 6.0 was found to be a suitable reaction buffer.

TABLE 1Evaluation of impact of reaction conditionson FVIII solubility and aggregation.Reaction buffer compositionPrecipitate% Aggregate10 mM Histidine, 260 mM Glycine, 1%YESn.d.Sucrose, 10 mM CaCl250 mM HEPES, 10 mM CaCl2, 150 mMYESn.d.NaCl, pH 7;50 mM MES, 10 mM CaCl2, 150 mM NaCl,YESn.d.pH 6.050 MM MES, 50 mM CaCl2, 150 mM NaCl,NO8pH 6.050 mM MES, 50 mM CaCl2, 150 mM NaCl,NO510% glycerol, pH 6.050 mM MES...

example 3

Pegylation of Y1680C with Peg-30K-Maleimide (Ref. US2006 / 0115876 A1)

Reagents:

[0114]1) BDD-FVIII N8-Y1680C (MW 178,000), 1200 μl, conc. 80 μg / ml, 96 μg, 0.54 nmol in buffer 20 mM imidazole, +10 mM CaCl2, +0.02% Tween 80, +1 M NaCl, 1 M gGlycerol, pH 7.3[0115]2) Triscarboxyethylphosphine (TCEP, MW 287): 700 eq; 0.0315 μmMol; 9 μg. 1 mg TCEP was dissolved in 1 ml of buffer 20 mM Imidazol, 10 mM CaCl2, 0.02% Tween 80, 1 M Glycerol, pH 7.3, 1 M NaCl. 109 ul of this solution was used.[0116]3) 30 kDa PEG-maleimid (Sunbright Me-300Ma from NOF Corp., MW 29300), 10 eq., 180 μg. 4.8 mg 30 kDa PEG-maleimid was dissolved in 2.4 ml of buffer 20 mM Imidazol, 10 mM CaCl2, 0.02% Tween 80, 1 M Glycerol, pH 7.3, 1 M NaCl. 90 uμl of this solution was used.

Buffers used for VivaP pure spin column (strong anion exchange) and Pro-spin (spin columns):[0117]Buffer A: 20 mM Imidazol, 10 mM CaCl2CaCl2, 0.02% Tween 80, 1M Glycerol, pH 7.3[0118]Buffer B: 20 mM Imidazol, 10 mM CaCl2CaCl2, 0.02% Tween 80, 1M Glyce...

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Abstract

The present invention relates to a recombinant Factor VIII molecule, wherein said molecule has reduced vWF binding capacity, and wherein said molecule is covalently conjugated with at least one side group.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application is a continuation of U.S. application Ser. No. 13 / 574,142, filed Oct. 22, 2012, which is a 35 U.S.C. §371 national stage application of International Patent Application PCT / EP2011 / 051438 (published as WO 2011 / 101242 A1), filed Feb. 2, 2011, which claimed priority of European Patent Application 10153718.1, filed Feb. 16, 2010; this application further claims priority under 35 U.S.C. §119 of U.S. Provisional Application 61 / 305,608, filed Feb. 18, 2010; the contents of which are incorporated herein by reference.FIELD OF THE INVENTION[0002]The present invention relates to recombinant factor VIII (FVIII) molecules. In particular, the present invention relates to FVIII molecules having reduced von Willebrand factor (vWF) binding compared to endogenous FVIII. The invention furthermore relates to use of such molecules as well as methods for obtaining such molecules.SEQUENCE LISTING[0003]The instant application contains a Sequence...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C07K14/755A61K38/37A61K39/395A61K47/48
CPCA61K38/00C07K14/755A61K38/37A61K47/6849A61K47/60A61K47/64A61K47/6811A61K39/3955C07K2319/30C07K2319/31A61P7/04
Inventor PESCHKE, BERNDKOFOD-HANSEN, MIKAELBUCHARDT, JENSSTENNICKE, HENNING RALFOESTERGAARD, HENRIKKJALKE, MARIANNEOLSEN, OLSEN, EVA H. NORLINGHANSEN, JENS JACOB
Owner NOVO NORDISK AS
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