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Method for manufacturing high purified factor IX

A blood coagulation factor and factor technology, which is applied in the field of preparation of human coagulation factor IX, can solve the problems that the virus cannot be completely overcome, the virus infection cannot be ruled out, and the side effects cannot be ruled out.

Active Publication Date: 2012-07-04
THE GREEN CROSS CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0011] Although factor IX can be selectively isolated by monoclonal antibody, since animal cells are required to produce antibodies, virus infection derived from animal cells cannot be ruled out
In addition, side effects caused by immunoglobulin (IgG) eluted with antibodies during the chromatography process cannot be ruled out
Also, the virus problem in isolating Factor IX from recombinant cell culture media cannot be completely overcome

Method used

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  • Method for manufacturing high purified factor IX
  • Method for manufacturing high purified factor IX
  • Method for manufacturing high purified factor IX

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1: Purification by anion exchange chromatography (traditional procedure)

[0027] Using the plasma of the decondensed protein or the cell culture medium of recombinant factor IX as the raw material, the raw material is mixed with diethylamino-ethyl-glucose gel A-50 (DEAE-Sephadex A-50), and pre-washed with buffer (prewash), it stirred at 4 degreeC temperature conditions for 2 hours. Then, the gel adsorbed with factor IX is filtered and separated by centroids. After washing, Factor IX is eluted by buffers with high salt concentration. The pH of the washing solution was appropriately adjusted to about 7.5, followed by dialysis, concentration, and storage at -70°C. In addition, the concentrate was separately sampled (aliquot), and the concentration was measured according to the standards and experimental methods for blood preparations and reconstituted preparations prescribed by KFDA (Korea Food and Drug Administration). The virus purification process is complet...

Embodiment 2

[0028] Example 2: Purification using Heparin sepharose 6FF column chromatography

[0029] After adjusting 10 ml of the IX coagulation factor complex solution purified and prepared by the above Example 1 to an ionic strength of 20 mS / cm or less and a pH of 7.0, it was placed on a heparin sepharose 6FF column (column conditions: diameter 1 cm, height 18 cm, Flow rate (flow rate: 1.0 ml / min, normal temperature) was flowed into an equilibration buffer (20 mM sodium citrate, pH 7.0) to maintain equilibrium, and a loading sample was flown through to adsorb, by the same amount as the equilibration buffer. The solution was washed once with 0.1M NaCl, followed by a second wash with 0.25M NaCl. The protein concentration of the eluent was measured, followed by SDS-PAGE (BSA standard (2 mg / ml, PIERCE)), and the concentration was measured using Coagulation timer KC10 from Amelung. Comparing the specific activity of the second anion column washing solution of Example 1 and the eluent of th...

Embodiment 3

[0030] Example 3: Purification by Cation Exchange Chromatography (CM-sepharose FF)

[0031] After adjusting 10ml of the IX coagulation factor complex purified by the above Example 2 to an ionic strength of 20mS / cm or less and pH 4.0, it is suitable for equilibration buffer (0.30-0.4M sodium chloride in citrate accounts for 20mM). , pH 4.0) to maintain an equilibrium CM-sepharose FF column (column conditions: diameter 1 cm, height 5 cm, flow rate 1.0 ml / min), and the non-adsorbed liquid was collected. The protein concentration of the above-mentioned non-adsorbed liquid was measured and then SDS-PAGE was carried out, and the lowest specific activity was found to be 150 IU / mg or more.

[0032] image 3 It is a schematic diagram showing the results of SDS-PAGE for confirming the purity of the XI coagulation factor preparation prepared by the present invention and other products on the market.

[0033] like image 3 As shown, the IX coagulation factor preparation purified and pr...

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Abstract

The present invention relates to a method for manufacturing a highly purified human blood coagulation factor IX, including: subjecting objects containing human blood coagulation factor IX (derived from human plasma or recombinant cell culture medium) to anion exchange chromatography, cation exchange chromatography, heparin affinity chromatography, preparing the human blood coagulation factor IX, and including: subjecting the S / D treatment (Solvent / Detergent treatment) to inactive viruses or removing the viruses through nanofiltration, thereby obtaining a highly purified, safe coagulation factor IX having a specific activity of above 150 IU / mg, with substantially inactivation or removal of all impure proteins and viruses.

Description

technical field [0001] The present invention relates to a preparation method of high-purity human blood coagulation factor IX, in particular to a method for ion-exchange chromatography and affinity layer of a substance containing blood coagulation factor IX (taken from human plasma or recombinant cell culture medium). In the stage of inactivating or removing the virus through the analysis, a safe and high-purity coagulation factor IX with a specific activity of more than 150 IU / mg can be obtained with almost no protein contamination. Background technique [0002] Human coagulation factor IX is an indispensable glycoprotein in the coagulation process. In the absence of or insufficient coagulation factor IX in the blood due to genetic or pathological factors, the coagulation process in the blood will be incomplete. , resulting in hemophilia B. [0003] Hemophilia B mainly occurs through heredity, and 1 in 30,000 to 50,000 baby boys has hemophilia B, which is a rare genetic di...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K1/14
CPCC12Y304/21022C12N9/644C07K1/14
Inventor 姜镛崔龙云孙基桓李成来成学模金基镕许在旭
Owner THE GREEN CROSS CORP
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