Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

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: 2013-02-14
NOVO NORDISK AS
View PDF4 Cites 7 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The patent is about a new type of Factor VIII molecule that has reduced binding capacity for vWF, a protein that plays a role in blood clotting. This molecule is also modified to have a longer half-life in the body. The technical effect of this invention is that it provides a safer and more stable Factor VIII molecule that can be used to treat hemophilia.

Problems solved by technology

The clinical manifestation is not on primary haemo-stasis—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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • 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 Varints Thereof, e.g., Factor VIII (Y1680F) or Factor VIII (Y1680C)

Cell Line and Culture Process

[0081]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)

[0082]Chinese hamster ovary (CHO) ...

example 2

Procedure for PEGylation of Recombinant O-glycosylated Factor VIII

[0090]The recombinant Factor VIII molecules obtained in Example 1 are conjugated with polyethylenglycol (PEG) using the following procedure:

[0091]For the glycoPEGylation reaction to be efficient a FVIII concentration >5mg / 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 conditions on FVIIIsolubility 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, 5...

example 3

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

Reagents:

[0104]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[0105]2) Triscarboxyethylphosphine (TCEP, MW 287): 700 eq; 0.0315 μmMol; 9 μg. 1 mg TCEP was dissolved in 1 ml of buffer 20 mM limidazol, 10 mM CaCl2, 0.02% Tween 80, 1 M Gglycerol, pH 7.3, 1 M NaCl. 109 ul of this solution was used.[0106]3) 30 kDa PEG-maleimid (Sunbright Me-300Ma from NOF Corp., MW 29300), 10 eq., 180 pg. 4.8 mg 30 kDa PEG-maleimid was dissolved in 2.4 ml of buffer 20 mM limidazol, 10 mM CaCl2, 0.02% Tween 80, 1 M Gglycerol, pH 7.3, 1 M NaCl. 90 uμl of this solution was used.

[0107]Buffers used for VivaP pure spin column (strong anion exchange) and Pro-spin (spin columns):

Buffer A: 20 mM Imidazol, 10 mM CaCl2CaCl2, 0.02% Tween 80, 1 M Glycerol, pH 7.3

Buffer B: 20 mM Imidazol, 10 mM CaCl2CaCl2, 0.02% Tween 80, 1 M Gly...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

PropertyMeasurementUnit
Massaaaaaaaaaa
Massaaaaaaaaaa
Massaaaaaaaaaa
Login to View More

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

FIELD OF THE INVENTION[0001]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.BACKGROUND OF THE INVENTION[0002]Haemophilia A is an inherited bleeding disorder caused by deficiency or dysfunction of coagulation factor VIII (FVIII) activity. The clinical manifestation is not on primary haemo-stasis—formation of the blood clot occurs normally—but the clot is unstable due to a lack of secondary thrombin formation. The disease is treated by intravenously injection of coagulation factor FVIII which is either isolated from blood or produced recombinantly.[0003]Current treatment recommendations are moving from traditional on-demand treatment towards prophylaxis. The circulatory half life of endogenous FVIII bound to v...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): C07K14/755A61P7/04A61K38/37C07K19/00C07K1/107
CPCA61K38/00C07K14/755A61K47/48561A61K47/48215A61K38/37C07K2319/30C07K2319/31A61K47/60A61K47/64A61K47/6811A61K47/6849A61P7/04A61K39/3955
Inventor PESCHKE, BERNDKOFOD-HANSEN, MIKAELBUCHARDT, JENSSTENNICKE, HENNING RALFOESTERGAARD, HENRIKKJALKE, MARIANNEOLSEN, EVA H. NORLINGHANSEN, JENS JACOB
Owner NOVO NORDISK AS
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com