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Modified proteins

A glycoprotein modification technology, applied in the field of modified glycoproteins, can solve problems such as accelerated clearance, hindering therapeutic effect, antibody interference, etc.

Inactive Publication Date: 2014-11-12
NOVO NORDISK HEALTH CARE AG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The immune response elicited by a therapeutic protein can have various side effects in addition to its accelerated clearance from circulation: steric hindrance to the binding site on the therapeutic protein makes it possible for the antibody to interfere or block the therapeutic effect, artificial antibodies can Cross-reacts with self-proteins, resulting in autoimmune reactions, etc.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0328] 10K PEG-ONH 2 : 10K-SMB-PEG (1,00 g; 0.1 mmol; Nektar Inc) was dissolved in DCM (10 ml). 4-(N-tert-butoxycarbonylaminooxy)butylamine (0.20 g, 1 mmol, prepared according to WO 2005014049A2) was added, and the mixture was stirred at room temperature for 16 h. Diethyl ether (90ml) was added and the white precipitate was filtered off. The steps of dissolving the precipitate with DCM (10 ml) and adding diethyl ether (90 ml) were repeated. The precipitated material was then reconstituted with DCM (6 ml) and Amberlyst 15 ion exchange resin (2,0 g; washed first with DCM and 10% EtOH in DCM) was added. The mixture was stirred at room temperature for 30 minutes, then the resin was filtered off and washed well with DCM. The combined DCM solution was concentrated to a small volume with a rotary evaporator, and then diethyl ether (90ml) was added to precipitate the product. The product was dissolved in DCM (6ml) and TFA (6ml) was added. The mixture was stirred at room temperatu...

Embodiment 2

[0331] Single 10K-PEG-FVIIa (0128-0000-1018-1A):

[0332] Dissolve in 10ml 25mM Gly-Gly, 50mM NaCl, 25mM CaCl 2 , Factor VIIa (14mg, 0.28umol) of pH 6.0 (GlyGly buffer) was added to 100ul CaCl 2 20 mM NaIO in free GlyGly buffer 4 solution and 1000ul of 10K-PEG-ONH in GlyGly buffer 2 Solution (Example 1, 42 mg; 4.2 umol; 15 equivalents of Factor Vila): The mixture was placed in an ice bath for 2 h with occasional shaking. Then add 500ul MeONH in GlyGly buffer (1M NaOH to adjust pH to 6.0) in the reaction mixture 2 . HCl solution (17 mg; 0,21 mmol). The mixture was placed in an ice bath for another 10 min. A cold solution of 100 mM second generation EDTA (4,5 ml) was then added to the reaction mixture, maintaining the pH below 9.0. Then adjust the pH to 8.0 with 100ul 1N HCl solution.

[0333] Ion Exchange Chromatography:

[0334] Removal of excess PEG-ONH by ion exchange chromatography 2 . The cooled reaction mixture was loaded onto a 5 ml HiTrap Q ion exchange column...

Embodiment 3

[0339] Preparation of highly substituted (HS) and low substituted (LS) 10K-PEG-FVIIa

[0340] Dissolve in 10ml 25mM Gly-Gly, 50mM NaCl, 25mM CaCl 2 100ul CaCl was added to factor VIIa (14mg, 0.28umol) of pH 6.0 (GlyGly buffer) 2 20 mM NaIO in free GlyGly buffer 4 solution and 1000ul of 10K-PEG-ONH in GlyGly buffer 2 Solution (Example 1, 42 mg; 4.2 umol; 15 equivalents to Factor Vila): The mixture was placed at 4° C. for 24 h with occasional shaking. Then add MeONH in 500ul GlyGly buffer (adjust pH to 6.0 with 1M NaOH) to the reaction mixture 2 . HCl solution (17 mg; 0,21 mmol). The mixture was placed in an ice bath for 10 min. A cold 100 mM second generation EDTA solution (4,5 ml) was then added to the reaction mixture to maintain the pH below 9.0. Then adjust the pH to 8.0 with 60ul 1N HCl solution.

[0341] Ion Exchange Chromatography:

[0342] Removal of excess PEG-ONH by ion exchange chromatography 2 . The cooled reaction mixture was loaded onto a 5 ml HiTrap Q i...

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Abstract

Method of conjugating glycoproteins by means of chemical modification is provided as well as new modified glycoproteins.

Description

field of invention [0001] The present invention relates to improved preparation of pharmaceuticals, in particular to the preparation of modified glycoproteins with improved pharmacodynamic and pharmacokinetic properties. Background of the invention [0002] Since proteins of biological origin are often highly potent and selective for their natural ligands, they hold great promise as therapeutic drugs. Since the organism already has a very clear metabolic pathway and clearance mechanism available, the nature of the biological source increases the possibility that the biological source protein is non-toxic, which is safer than conventional small molecule drugs. Combining it with reality, proteins can now be prepared using recombinant DNA technology in many different expression systems, achieving large-scale production, making proteins ideal drug candidates. However, therapeutic proteins of interest such as hormones, soluble receptors, cytokines, enzymes, etc. often have short...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/435A61K47/48A61K38/00A61K47/51
CPCA61K47/48A61K38/00C07K14/435A61K47/50
Inventor C·贝伦斯
Owner NOVO NORDISK HEALTH CARE AG
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