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Preparation method of blood coagulation factor VIII affinity chromatographic resin

A blood coagulation factor and affinity technology, which is applied to the preparation method of peptides, blood coagulation/fibrinolytic factors, chemical instruments and methods, etc., can solve the problems of rodent protein contamination, etc., achieve efficient purification, enrich purification methods, and improve utilization rate Effect

Active Publication Date: 2016-10-26
XINXIANG MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

By the 1990s, two high-purity FVIII preparations prepared by monoclonal antibody affinity chromatography were available. Contamination with mouse proteins

Method used

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  • Preparation method of blood coagulation factor VIII affinity chromatographic resin
  • Preparation method of blood coagulation factor VIII affinity chromatographic resin

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Effect test

Embodiment 1

[0032] The preparation method of coagulation factor VIII affinity chromatography resin of the present invention, the detailed steps of this preparation method are as follows:

[0033] a, first take by weighing the Sepharose CL-6B that is preserved in the 20% aqueous ethanol solution in the refrigerator, namely the Sepharose CL-6B, use excessive distilled water to wash repeatedly, remove the ethanol on the surface of the Sepharose, and obtain wet Agarose gel;

[0034] b. Epichlorohydrin activation method activates the gel:

[0035] Weigh the wet agarose gel obtained in step a, place it in 0.3M NaOH solution, the volume of the NaOH solution added is 4 times the weight of the wet agarose gel; then shake at a constant temperature at 30°C for 1.5h, When shaking at constant temperature, the vibration speed is 150 rpm;

[0036] After shaking, take out the gel and wash it fully. After washing, carry out vacuum filtration (until there is no liquid dripping), and then add it to the Na...

Embodiment 2

[0045] The preparation method of coagulation factor VIII affinity chromatography resin of the present invention, the detailed steps of this preparation method are as follows:

[0046] a, first take by weighing the Sepharose CL-6B that is preserved in the 20% aqueous ethanol solution in the refrigerator, namely the Sepharose CL-6B, use excessive distilled water to wash repeatedly, remove the ethanol on the surface of the Sepharose, and obtain wet Agarose gel;

[0047] b. Epichlorohydrin activation method activates the gel:

[0048] Weigh the wet agarose gel obtained in step a, place it in 0.4M NaOH solution, the volume of the NaOH solution added is 5 times the weight of the wet agarose gel; then shake at a constant temperature at 28°C for 1.8h, When shaking at constant temperature, the vibration speed is 120 rpm;

[0049] After shaking, take out the gel and wash it fully. After washing, carry out vacuum filtration (filter until there is no liquid dripping), and then add it to...

Embodiment 3

[0058] The preparation method of coagulation factor VIII affinity chromatography resin of the present invention, the detailed steps of this preparation method are as follows:

[0059] a, first take by weighing the Sepharose CL-6B that is preserved in the 20% aqueous ethanol solution in the refrigerator, namely the Sepharose CL-6B, use excessive distilled water to wash repeatedly, remove the ethanol on the surface of the Sepharose, and obtain wet Agarose gel;

[0060] b. Epichlorohydrin activation method activates the gel:

[0061] Weigh the wet agarose gel obtained in step a, place it in 0.2M NaOH solution, and the volume of the NaOH solution added is 3 times the weight of the wet agarose gel; then shake at a constant temperature at 35°C for 0.8h, When shaking at constant temperature, the vibration speed is 100 rpm;

[0062] After shaking, take out the gel and wash it fully. After washing, carry out vacuum filtration (until there is no liquid dripping), and then add it to th...

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Abstract

The invention discloses a preparation method of blood coagulation factor VIII affinity chromatographic resin. The preparation method comprises the following steps: first weighing stored sepharose gel, washing the weighed sepharose gel, and activating the gel by adopting an epichlorohydrin activation method to obtain activated gel; performing an arm connection reaction for the obtained activated gel and spacing arm molecule N-hydroxysuccinimide to obtain arm-connected gel; performing a ligand coupling reaction for the arm-connected gel and peptide ligand; processing a coupling reaction product to obtain blood coagulation factor VIII affinity chromatographic resin; and washing the resin, and storing the resin in a 20 percent ethanol solution with three times volume of the gel under the condition of 4 DEG C. The product chromatographic resin is used for extracting blood coagulation factor VIII from blood plasma and can rapidly, specifically and efficiently purify the blood coagulation factor VIII.

Description

1. Technical field: [0001] The invention belongs to the technical field of biopharmaceuticals, and in particular relates to a preparation method of coagulation factor VIII affinity chromatography resin. 2. Background technology: [0002] Coagulation factor VIII can be very effective in replacement therapy for patients with hemophilia A. With the development and application of various chromatographic techniques, pure coagulation factor VIII products with a specific activity of 10 IU / mg protein have begun to be used in clinical treatment. In 1992, Baxter successfully launched the first genetically recombined coagulation factor VIII product. [0003] Although genetically recombined coagulation factor VIII products have achieved good clinical application effects, it does not mean that we have to give up plasma-derived coagulation factor VIII. On the one hand, the cost of plasma-derived coagulation factor VIII is lower than that of recombinant coagulation factor VIII, and becau...

Claims

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Application Information

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IPC IPC(8): B01J20/26B01J20/30B01D15/38C07K14/755C07K1/22C08B37/12
CPCB01D15/3804B01J20/26C07K14/755C08B37/0039
Inventor 陈正跃詹合琴陈美光王璐靳玫高夏欢
Owner XINXIANG MEDICAL UNIV
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