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Method for separating and purifying blood coagulation factor VIII

A coagulation factor, separation and purification technology, applied in the field of separation and purification of coagulation factor VIII, can solve problems such as affecting the activity of the final product, easy dissociation, and short half-life.

Inactive Publication Date: 2012-07-18
INST OF PROCESS ENG CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, in the existing separation and purification process, especially in ion-exchange chromatography, the macromolecular protein complex is easily dissociated, thereby affecting the activity of the final product (Zhou WB, et al, Ion-exchange chromatography of hepatitis B virus surface antigen from a recombinant Chinese hamster ovary cell line, Journal of Chromatography A 2005, 1095, 119-125); in addition, ion-exchange chromatography is a key step in purifying FVIII. Due to the short half-life of FVIII, the existing ion-exchange purification operation takes too long, This step results in a substantial loss of product activity

Method used

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  • Method for separating and purifying blood coagulation factor VIII
  • Method for separating and purifying blood coagulation factor VIII
  • Method for separating and purifying blood coagulation factor VIII

Examples

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Effect test

Embodiment 1

[0031] Take frozen healthy human plasma and melt it in a water bath at 37°C, and shake it continuously to fully dissolve FVIII, etc. After completely dissolving, inject 40mL into a 20mM, pH 7.5 citrate buffer containing 120mM glycine and 1mM calcium chloride. POROS 50HQ ion-exchange chromatography column, column inner diameter 2.6 cm, column bed height 10 cm, chromatography equipment GE AKTA Explorer 100, ultraviolet 280nm wavelength absorption detection, dimensionless flow rate 0.02S -1 After the sample injection, continue to wash with the above citrate buffer until the 280nm UV absorption signal returns to the baseline, and then use 20mM, pH 7.5 citrate buffer containing 120mM glycine, 1mM calcium chloride and 1M sodium chloride for gradient Elution, the concentration gradients are 20%, 30%, 50% and 100%, respectively, two column volumes are eluted for each gradient, and 35mL of the elution peak of the 50% gradient is collected, the protein concentration is 0.73g / L, and the s...

Embodiment 2

[0033] Component I obtained from Cohn precipitation of healthy human plasma was melted in a 37°C water bath, and continuously oscillated to fully dissolve FVIII, etc. After complete dissolution, inject 20mL of citrate containing 120mM glycine and 1mM calcium chloride at pH 7.5 to use 20mM Diethylamine-poly(glycidyl methacrylate-divinylbenzene-triallyl trimeric isocyanurate) type anion exchange chromatography column with well-balanced buffer solution, the inner diameter of the column is 2.6 cm, and the height of the column bed is 5 cm, chromatography equipment GE AKTA Explorer 100, ultraviolet 280nm wavelength absorption detection, dimensionless flow rate 0.01S -1 After loading the sample, continue to wash with the above citrate buffer until the 280nm UV absorption signal returns to the baseline, and then use 20mM, pH 7.5 citrate buffer containing 120mM glycine, 1mM calcium chloride and 1M sodium chloride for gradient Elution, the concentration gradients were 25%, 60% and 100%,...

Embodiment 3

[0035] Take 3g of healthy human plasma cryoprecipitate and dissolve it in 50ml of 20mM, pH7.5 citrate buffer containing 120mM glycine and 1mM calcium chloride, and take 40mL of the dissolved solution and inject it into the POROS50Q ion exchange that has been balanced with the above citrate buffer. In the chromatographic column, the inner diameter of the column is 2.6 cm, the height of the column bed is 10 cm, the chromatographic equipment is GE AKTA Explorer 100, the ultraviolet 280nm wavelength absorption detection, the dimensionless flow rate 0.005S -1 After loading the sample, continue to wash with the above citrate buffer until the 280nm UV absorption signal returns to the baseline, and then use 20mM, pH 7.5 citrate buffer containing 120mM glycine, 1mM calcium chloride and 1M sodium chloride for gradient Elution, the concentration gradients were 35%, 55% and 100%, respectively, each gradient eluted two column volumes, and collected 33mL of 50% gradient elution peak, the pro...

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Abstract

The invention relates to the field of separation and purification of blood products, in particular to a method for separating and purifying blood coagulation factor VIII. According to the method, a chromatographic raw material containing the blood coagulation factor VIII is separated by using super-macroporous ion exchange chromatography. The method also comprises a step of performing coarse separation treatment before separation of the super-macroporous ion exchange chromatography. According to the method, F VIII chromatography recovery rate stably reaches up to about 85 percent, the specific activity reaches up to 154 IU / mg protein, and the process is stable and easy to amplify; while in the prior art, the F VIII chromatography recovery rate is normally stabilized at about 25 percent, and the specific activity is 20-50 IU / mg protein; and thus, compared with the prior art, the method provided by the invention has the advantage of achieving an unexpected technical effect.

Description

technical field [0001] The invention relates to the field of separation and purification of blood products, in particular, the invention relates to a method for separation and purification of coagulation factor VIII. Background technique [0002] VIII (FVIII) is an important endogenous coagulation factor, the absence of VIII (FVIII) will lead to incurable hemophilia A (HA). The disease is prevalent worldwide, with an average of 1 in 5,000 men suffering from it (Singleton T, et al, Emergency Department Care for Patients with Hemophilia and Von Willebrand Disease, The Journal of Emergency Medicine, 2010, 39(2):158-165). The traditional treatment of HA is to infuse patients with fresh blood or supplement with FVIII-rich plasma products. This method has a certain effect, but there are risks such as large infusion volume and virus contamination (HIV, HBV), and has been gradually eliminated. Researchers have used genetic engineering to recombine and express FVIII for the treatme...

Claims

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Application Information

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IPC IPC(8): C07K14/755C07K1/18C07K1/36C07K1/34C07K1/30C07K1/16
Inventor 张焱苏志国罗坚康丽梅马光辉
Owner INST OF PROCESS ENG CHINESE ACAD OF SCI
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