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Method for separating and purifying recombinant human coagulation factor VIII from cell culture fluid

A cell culture, separation and purification technology, applied in the field of separation and purification of recombinant human coagulation factor eight, can solve the problems of easy degradation, poor stability and inactivation of eight factors, and achieve the goal of increasing specific activity, increasing stability and improving work efficiency Effect

Inactive Publication Date: 2014-01-29
上海泰龙生物医药科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The purification of recombinant human blood coagulation factor 8 is a difficult point in the preparation process of factor 8, mainly because factor 8 has poor stability and is prone to degradation and inactivation

Method used

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  • Method for separating and purifying recombinant human coagulation factor VIII from cell culture fluid
  • Method for separating and purifying recombinant human coagulation factor VIII from cell culture fluid
  • Method for separating and purifying recombinant human coagulation factor VIII from cell culture fluid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] 1. Purpose of the test:

[0042] The effect of divalent metal ions, especially calcium ions and zinc ions on the stability of the eight factors during the purification process of the eight factors was investigated.

[0043] The experimental design investigated the stability of the eight factors under the conditions of adding calcium ions alone, adding zinc ions alone and adding calcium ions and zinc ions at the purification stage of the eight factors.

[0044] 2. Main material:

[0045] Recombinant human blood coagulation factor eight cell culture medium: Recombinant human blood coagulation factor eight CHO engineered cells (prepared according to the prior art) were obtained by culturing in SFMII302 medium (provided by SIGMA).

[0046] Q Sepharose FF ion-exchange chromatography column: GE Company of the United States

[0047] Eight-factor affinity (V8 SELECT) chromatography column: GE Company

[0048] DEAE FF ion exchange column: American GE company

[0049] Sephacr...

Embodiment 2

[0074] 1. Purpose of the test: To investigate the effects of different concentrations of calcium and zinc ions on the stability of recombinant human coagulation factor 8

[0075] 2. Main material: same as embodiment 1

[0076] 3. Experimental steps:

[0077] The purification scheme is the same as Scheme A of Example 1, except that the concentrations of calcium chloride and zinc acetate added to all buffers are different, as shown in Table 3.

[0078] The impact of different calcium chloride and zinc acetate concentrations on the stability of recombinant factor eight in the chromatography buffer of table 3

[0079] Program

Calcium chloride concentration in buffer (mM)

Zinc acetate concentration in buffer (mM)

E

1

1

F

10

10

G

50

50

H

100

100

[0080] 4. Result inspection: same as embodiment 1

[0081] 5. The results are shown in Table 4:

[0082] The impact result of ...

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Abstract

The invention discloses a method for separating and purifying recombinant human coagulation factor VIII from cell culture fluid. The method comprises the following steps: removing cells in the cell culture fluid of recombinant human coagulation factor VIII; ultrafiltration concentrating the culture fluid with cells removed; purifying the ultrafiltration concentrated culture fluid through one or more of anion exchange chromatography, immunoaffinity chromatography or gel filtration chromatography to obtain purified recombinant human coagulation factor VIII, wherein equilibration buffer and elution buffer adopted in the anion exchange chromatography, immunoaffinity chromatography and the gel filtration chromatography contain calcium ions with mol concentration of 1-100 mM and zinc ions with mol concentration of 1-100 mM. According to the method disclosed by the invention, the calcium ions and the zinc ions are added in the buffer solutions adopted in the whole chromatography process to effectively solve the degradation problem of the factor VIII and carry out the whole purification operation under normal temperature, thereby greatly improving the working efficiency and improving the specific activity of the final product.

Description

technical field [0001] The present invention relates to the downstream technology of bioengineering, in particular to a method for separating and purifying recombinant human coagulation factor eight from cell culture fluid. Background technique [0002] Eight coagulation factors, also known as antihemophilic factors, play a very important role in the endogenous coagulation system and are cofactors to activate coagulation factor IX. Recombinant gene factor VIII (rFVIII), the recombinant human coagulation factor VIII, is the first recombinant coagulation factor product to be marketed. In 1992, the first recombinant human coagulation factor VIII product, Baxter's Recombinate, was approved by the FDA, and Bayer's Recombinate was successively launched. Kogenate FS, ReFacto of Genetics Institute, FDA approved Baxter's second-generation product Avate in 2003, and SFDA approved the first domestically marketed rFⅧ Baikeqi in 2007. The clinical application for 20 years shows that the...

Claims

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Application Information

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IPC IPC(8): C07K14/755C07K1/36C07K1/34C07K1/22C07K1/18C07K1/16
CPCC07K14/755
Inventor 郭颀然杨松峰许必雄
Owner 上海泰龙生物医药科技有限公司
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