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Blood coagulation factor VIII separating and purifying medium and preparation method thereof

A blood coagulation factor, separation and purification technology, applied in the preparation method of peptide, blood coagulation/fibrinolysis factor, VII factor, etc., to achieve the effect of improving selectivity

Inactive Publication Date: 2014-01-15
汪志友
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to propose a medium for separating and purifying blood coagulation factor VIII and its preparation method in view of many problems of the macromolecular antibody ligand affinity separation medium currently used for the separation and purification of blood coagulation factor VIII

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Measure 20ml (sedimentation volume) of Sepharose FF medium stored in 20% ethanol aqueous solution, wash repeatedly with excess deionized water to remove ethanol, place it in a G3 sintered glass funnel and vacuum-dry it for 5min; %, 70% aqueous solution of dimethyl sulfoxide to clean the medium after suction filtration for 5 minutes; add a certain amount of dimethyl sulfoxide (30%), ethylene dichloride, epichlorohydrin (10 %), sodium hydroxide (0.8M), water; the medium suspension was reacted at 40° C. for 2 hours under shaking conditions. After the reaction, it was repeatedly washed with excess deionized water until no epoxy groups remained in the cleaning solution to obtain epichlorohydrin-activated agarose gel microspheres, and the agarose epoxy modification density was 60 μmol / ml gel.

Embodiment 2

[0030]Mix 8.5 volumes of 0.2M K2HPO4 solution with 1.5 volumes of 0.2M KH2PO4 solution to make the coupling buffer. Take 5 g of the epichlorohydrin-activated sepharose microspheres in Example 1 and suspend them in 10 ml of coupling buffer. Take a certain amount of small peptide ligand and dissolve it in the buffer solution for coupling to make it completely dissolved. The minimum concentration is 200 μmol ligand / ml gel. Mix the gel suspension and the coupling buffer solution at a ratio of 1:0.5 to 1:1 for suspension reaction, seal and keep stirring (or shaking, shaking) in a water bath at 20°C-45°C for 20 hours. Wash with coupling buffer to remove excess ligand. Suspend the reacted and washed gel into a solution of 1m0l / L ethanolamine (PH8), and react at 40°C-50°C for a minimum of 4 hours or overnight at room temperature to eliminate residual active groups on the surface of the medium. After the reaction, wash alternately with 0.1M acetate buffer (pH 4.0) containing 0.5M NaC...

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PUM

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Abstract

The invention discloses a simple and fast method for improving the separation efficiency of a blood coagulation factor VIII from its analogs, and the purity and biological specific activity of the blood coagulation factor VIII. The method comprises the following steps: processing hydrophilic gel microspheres in an activator as a matrix to prepare an activated matrix, and bonding the activated matrix with a blood coagulation factor VIII specificity affinity small peptide ligand through a coupling process to realize the covalent connection between the matrix and the blood coagulation factor VIII specificity affinity small peptide ligand. The separation medium is used for the industrialized large-scale production of the blood coagulation factor VIII through cooperating with affinity chromatography chromatogram or expanded bed adsorption, is highly cost-competitive, allows highly pure blood coagulation factor VIII products to be directly extracted from biological raw materials comprising blood, serum, blood plasma, blood plasma fractions, cell culturing solutions, cell homogenates, gene engineering cell culturing solutions and the like only through one step, and realizes time saving and convenience.

Description

technical field [0001] The invention relates to a medium for separating and purifying coagulation factor VIII and a preparation method thereof, belonging to the field of biological separation materials. Specifically, it relates to a novel biological method for separating and purifying coagulation factor VIII from biological raw materials such as blood, serum, plasma, plasma components, cell culture fluid, and cell homogenization fluid by affinity chromatography or extended bed adsorption. Separation materials and methods for their preparation. Background of the invention [0002] Bioseparation media are a class of materials specifically used for the purification and refinement of biologically related products. Separation media are widely used in the separation of pharmaceutical industry, fermentation industry, biological products, blood products, vaccines and genetic engineering products. When purifying products, the separation medium has good separation effect, high produ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): B01J20/24B01J20/30B01D15/08C07K14/755C07K1/22
Inventor 汪志友
Owner 汪志友
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