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FX ACTIVATION PROCESS AND USE THEREOF IN PREPARATION OF FXa COMPOSITION

An activation method and composition technology, applied in chemical instruments and methods, biochemical equipment and methods, peptide preparation methods, etc., can solve problems such as prolonged blood coagulation time and increased blood loss

Pending Publication Date: 2021-02-26
OCTAPHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] Prolonged clotting time and dramatically increased blood loss due to impairment of the amplification pathway in hemophilia A and hemophilia B patients

Method used

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  • FX ACTIVATION PROCESS AND USE THEREOF IN PREPARATION OF FXa COMPOSITION
  • FX ACTIVATION PROCESS AND USE THEREOF IN PREPARATION OF FXa COMPOSITION
  • FX ACTIVATION PROCESS AND USE THEREOF IN PREPARATION OF FXa COMPOSITION

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0147] Example 1 (Activation)

[0148] Mix 40 mg of synthetic phosphatidylserine (SPS) with 10 ml of reaction buffer (20 mM TRIS and 150 mM NaCl at pH = 7.5), and use Disperse for 30 minutes and cool to room temperature to obtain SPS dispersion.

[0149] 55ml of the initial protein mixture containing 2mg / ml total protein, about 40IU / ml FX and about 0.5U / ml FII was mixed with 27.5ml of SPS dispersion, 27.5ml of reaction buffer and 55ml of reaction buffer containing 75mM calcium ions. The pH 7.5 mixture was incubated at room temperature for 6 hours with gentle agitation before adding 55 ml of reaction buffer with 200 mM EDTA. The dispersed PS was dissolved by adding 1.5 mM Triton X-100, and the resulting clear solution was filtered through a 0.45 μm PES membrane filter to recover the FXa-containing intermediate.

[0150]The mixture thus prepared, as defined in the assay instructions, had a FXa-specific activity of about 9.6 IU / ml, a thrombin level of less than 50 IU / ml, and...

example 2

[0151] Example 2 (Activation)

[0152] 100 ml of the initial protein mixture containing 40 mg / ml total protein, about 60 IU / ml FX and about 60 U / ml FII was mixed with 90 ml of reaction buffer and 100 ml of reaction buffer containing 75 mM Ca ions. 10 ml of the PS dispersion were added dropwise in a continuous operation. The pH 7.0 mixture was incubated at 22° C. for 16 hours with gentle agitation before adding 100 ml of reaction buffer with 200 mM EDTA. The dispersed PS was dissolved by adding 1.5 mM Triton X-100, and the resulting clear solution was filtered through a 0.45 μm PES membrane filter to recover FXa-containing intermediates.

[0153] The FXa-specific activity of the mixture thus prepared was 8.2 IU / ml, the FIIa-specific activity was 619 IU / ml, and the total protein content was 9.9 mg / ml. The yield of FXa was 33% based on the FX content activated by PS and Ca ions.

example 3

[0154] Example 3 (Activation)

[0155] The initial protein mixture was diluted with reaction buffer, mixed with PS dispersion and Ca ions to obtain a solution containing 40 IU / ml FX, 0.33 mg / ml PS and 25 mM Ca ions. The mixture was incubated at 15°C for 10 hours with gentle agitation, then the reaction was terminated with 50 mM EDTA. The dispersed PS was dissolved by adding 1.5 mM Triton X-100, and the resulting clear solution was filtered through a 0.45 μm PES membrane filter to recover FXa-containing intermediates.

[0156] The FXa-specific activity of the mixture thus prepared was 4.1IU / ml, the FIIa-specific activity was 3.1IU / ml, and the total protein content was 0.4mg / ml. Based on the FX content activated by PS and calcium ions, the FXa yield was 41 %.

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Abstract

The invention relates to a high purity coagulation factor Xa (FXa or activated coagulation factor X) preparation and an activation and purification process to obtain the FXa of high purity and high degree of activation without the addition of proteinaceous activators during manufacturing.

Description

technical field [0001] The present invention relates to the preparation of high-purity blood coagulation factor Xa (FXa or activated blood coagulation factor X) and an activation and purification method for obtaining the high-purity and high-activation degree FXa, which does not need to add protein activators in the production process. Background technique [0002] Coagulation factor Xa (FXa) is a two-chain glycoprotein generated through the activation of its vitamin K-dependent zymogen factor X (FX) throughout the coagulation process. FXa in vivo can be produced by activation of the extrinsic pathway (ie, by Factor Vila activation) or by the intrinsic pathway (ie, by activated coagulation factors IX (FIXa) and VIII (FVIIIa)). The molecule undergoes enzymatic cleavage of the Arg15-Ile16 bond, releasing the 52 amino acid activation peptide. In the presence of its cofactors, coagulation factor Va (FVa), calcium (Ca) ions, and negatively charged phospholipid membranes, FXa ass...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/48C12N9/64
CPCA61K38/4846C12N9/6432A61K38/4833A61K2300/00C07K1/18C07K1/22C07K1/36C12Y304/21005C12Y304/21006
Inventor 朱尔根·罗兰·罗米什凯瑟琳·波克佩特拉·舒尔茨西尔维奥·乌施科迈克尔·安德斯纳安德烈·穆兰尼
Owner OCTAPHARMA
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