Factor viia complex using an immunoglobulin fragment

A technology of immunoglobulin and compound, which is applied in anti-animal/human immunoglobulin, coagulation/fibrinolytic factors, animal/human protein, etc., can solve the problems of insufficient prevention and treatment of hemophilia, and achieve prolonged serum Half-life, compliance-enhancing effects

Inactive Publication Date: 2013-04-03
HANMI SCI CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0008] MAXY-VII (Bayer / Maxygen) and NN7128 (Novo / Neose) are factor VII products, which prolong the serum half-life through Gla domain mutation and hyperglycosylation and 40K pegylation, respectively, but in phase I and During the phase II study, their respective serum half-lives were 5 times longer than native FacVII, which is still insufficient for the prevention and treatment of hemophilia

Method used

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  • Factor viia complex using an immunoglobulin fragment
  • Factor viia complex using an immunoglobulin fragment
  • Factor viia complex using an immunoglobulin fragment

Examples

Experimental program
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Effect test

Embodiment 1

[0090] Example 1: Preparation of Immunoglobulin Fc-PEG-FacVIIa Complex

[0091] Immunoglobulin Fc was pegylated at the N-terminus with 5K propionaldehyde (3) PEG (PEG with three terminal propionaldehyde groups, NOF, Japan). In this regard, 6 mg / mL of immunoglobulin Fc was reacted with PEG at 4°C for 4.5 hours, and the molar ratio of immunoglobulin Fc to PEG was set at 1:2. The reaction was performed in 100 mM potassium phosphate buffer at pH 6.0 in 20 mM SCB (NaCNBH 3 ) in the presence of a reducing agent. The reaction mixture was loaded onto SOURCEQ (LRC 2585ml, Pall Corporation) to purify mono-PEGylated immunoglobulin Fc. Then, FVII was coupled to immunoglobulin Fc-5K PEG at a molar ratio of 1:10 (FVII:immunoglobulin Fc-5K PEG) at 4° C. for 18 hours, and the total protein concentration was set at 20 mg / mL. Coupling reactions were performed in 100 mM potassium phosphate, pH 6.0, in the presence of 20 mM SCB as reducing agent. The coupling reaction mixture was purified by ...

Embodiment 2

[0102] Example 2: Preparation of 20k PEG-FacVIIa(N) Conjugate

[0103]FVII (FacVII) was pegylated at the N-terminus with 20K mPEG butyraldehyde (Nektar, USA). In this regard, 5 mg / mL of FVII was reacted with PEG at 4° C. for 10 hours, and the molar ratio of FVII to 20K PEG was set at 1:5. The reaction was performed in 100 mM sodium acetate buffer at pH 5.0 in 20 mM SCB (NaCNBH 3 ) in the presence of a reducing agent. Mono-PEGylated FVII was purified by RESOURCE Q (1 ml, prepackaged, GE Healthcare). Given a salt gradient of 1 M NaCl in 20 mM Tris (pH 7.5), the column eluted polyPEGylated FVII, monoPEGylated FVII and FVII sequentially. Then, a second purification was performed using a Superdex_200 (Hiroad 16 / 60, GE Healthcare) column to separate mono-PEGylated FVII from FVII and FVII multimer impurities. For activation, mono-PEGylated FVII was reloaded onto SOURCE Q, followed by pouring into a mobile phase containing 1.75 mM calcium ions for 1 hour. Elution was performed wi...

Embodiment 3

[0113] Example 3: Preparation of 20K PEG-FacVIIa(Lys) Conjugate

[0114] FVII was pegylated at lysine residues with 20k mPEG SPA (Nektar, USA). In this regard, 3 mg / mL of FVII was reacted with 20k PEG for 3 hours at room temperature, and the molar ratio of FVII to 20k PEG was set at 1:5. The reaction was performed in 100 mM sodium phosphate buffer, pH 8.0. Mono-PEGylated FVII was purified by RESOURCE Q (1 ml, prepackaged, GE Healthcare). Given a salt gradient of 1 M NaCl in 20 mM Tris (pH 7.5), the column eluted polyPEGylated FVII, monoPEGylated FVII and FVII sequentially. Then, a second purification was performed with a Superdex_200 (Hiroad 16 / 60, GE Healthcare) column to separate monoPEGylated FVII from FVII and FVII multimer impurities. For activation, purified mono-PEGylated FVII was reloaded onto SOURCE Q, followed by pouring on the column with a mobile phase containing 1.75 mM calcium ions for 1 hour. Elution was performed with 35 mM calcium ions to obtain monoPEGyla...

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Abstract

Disclosed are a blood coagulation factor complex in which FacVIIa, a non-peptidyl polymer and an immunoglobulin Fc region are bonded by covalent bonds, and the uses thereof. The FacVIIa complex guarantees the in vivo activity of FacVIIa and significantly enhances the serum half life of FacVIIa, so that it is useful for developing long-acting FacVIIa formulations which can improve the compliance of role behavior of patients whose blood does not coagulate.

Description

technical field [0001] The present invention relates to coagulation complexes for factor VIIa (factor VIIa, FacVIIa) long-acting preparations. More specifically, the present invention relates to coagulation complexes in which FacVIIa, a non-peptidyl polymer, and an immunoglobulin Fc region are covalently bonded together to significantly prolong serum half-life, maintain coagulation function, and significantly improve patient role behavior (role behavior) compliance. The invention also relates to methods for the preparation of coagulation factor complexes. Background technique [0002] An estimated 140,000 people worldwide suffer from hemophilia, and its incidence is increasing by 20% each year. Genetically, hemophilia occurs in 1 in 10,000 births, yet only about 25 percent of all hemophilia cases are diagnosed or treated. One of the biggest problems that occurs after treatment with clotting factors is the development of antibodies against conventional drugs. Hemophilia A...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K47/48A61K47/30C07K14/745C07K16/18
CPCA61K47/48415A61K47/48369A61K47/48715C07K14/745C12N9/96C07K2319/30A61K9/0019A61K47/68A61K47/6811A61K47/6889A61P7/04A61K47/30A61K47/50C07K16/18
Inventor 宋大海林圣仁金昌焕洪性甲任大成权世昌
Owner HANMI SCI CO LTD
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