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Method of purifying foot-and-mouth disease inactivated virus antigen through ion exchange chromatography

An ion-exchange chromatography and virus antigen technology, applied in the field of chromatographic separation for the separation and purification of foot-and-mouth disease inactivated virus vaccine antigens, can solve the problems of poor safety, death, and easy loss of immune activity of antigens, and achieves low cost, few operation steps, and large scale. The effect of practical application value

Inactive Publication Date: 2015-08-05
INST OF PROCESS ENG CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are two main problems with the foot-and-mouth vaccine: 1. The immune protection is unstable, and the antibody level is not detected or the antibody level is low after immunization, or the immunization period is short; 2. The safety is poor, and the side effects are strong. Food loss, food stoppage and even death after vaccination
The biggest problem with gel filtration is that the processing volume is small, the feeding volume of the feed liquid is only 1 / 10 of the volume of the gel filtration column, and the concentration of the antigen will further decrease after gel filtration, resulting in long processing time and material cost. High, and the antigen is also prone to lose immune activity during long-term processing

Method used

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  • Method of purifying foot-and-mouth disease inactivated virus antigen through ion exchange chromatography
  • Method of purifying foot-and-mouth disease inactivated virus antigen through ion exchange chromatography
  • Method of purifying foot-and-mouth disease inactivated virus antigen through ion exchange chromatography

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Get 100mL of cell culture supernatant containing FMD virus, the total protein concentration is 0.47g / L, and the FMD virus antigen concentration is 2.8 μg / mL.

[0031] Use a plate ultrafiltration membrane (Sartorius) with a molecular weight cut-off of 50kDa to concentrate the cell supernatant to about 10mL at one time, add 80mL 20mM pH 7.0 sodium phosphate buffer solution several times to dilute, and continue to concentrate to about 10mL until the conductivity is about 7.0mS / cm; during ultrafiltration, the membrane flow rate is 10cm / s, and the pressure is controlled below 0.3Mpa. The final concentration of FMD virus antigen was about 20 μg / mL.

[0032] The supernatant was fed into a DEAE Sepharose FF ion-exchange chromatographic column (GE Healthcare, 5cm×1.6cm I.D.) equilibrated with sodium phosphate buffer (pH 7.0) adjusted to conductivity 7mS / cm in advance with sodium chloride, after feeding After continuing to rinse, use sodium chloride to adjust the conductance to...

Embodiment 2

[0037] Take 100mL supernatant of cell culture medium containing FMD virus, the total protein concentration is 4.5g / L, the FMD virus antigen concentration is 21μg / mL, dilute 10 times with 20mM Tris-HCl buffer solution, pH 9.0 to a conductivity of 1mS / cm , the antigen concentration is about 2 μg / mL.

[0038]Mix the supernatant with the Q Sepharose FF ion-exchange packing pre-balanced with Tris-HCl buffer (pH 9.0) adjusted to conductance 1mS / cm with ammonium chloride, stir and absorb at 100-200rpm for 30min, then add to the chromatographic column ( GE Healthcare, 5 cm x 1.6 cm I.D.). After continuous washing, ammonium chloride was used to adjust the conductivity to Tris-HCl buffer (pH 9.0) of 10-40mS / cm for gradient elution, and the ultraviolet absorption peak containing the foot-and-mouth disease virus antigen with a recovery rate greater than 20% was collected. eluent. The chromatographic packing was regenerated with 1M sodium hydroxide.

[0039] The antigen concentration an...

Embodiment 3

[0041] Get 1000mL of cell culture supernatant containing FMD virus, the total protein concentration is 0.47g / L, and the FMD virus antigen concentration is 2.8 μg / mL.

[0042] Use a plate ultrafiltration membrane (Sartorius) with a molecular weight cut-off of 300kDa to concentrate the cell supernatant to about 20mL at one time, add 100mL 20mM pH 8.0 sodium phosphate buffer solution several times to dilute, and continue to concentrate to about 10mL until the conductivity is about 5.0mS / cm; during ultrafiltration, the membrane flow rate is 100cm / s, and the pressure is controlled below 0.3Mpa. The final concentration of FMD virus antigen is about 200 μg / mL.

[0043] The supernatant was fed to an ANX Sepharose FF (high sub) ion-exchange chromatographic column (GE Healthcare, 15cm×1.6cm I.D.) equilibrated with sodium phosphate buffer (pH 8.0) adjusted to conductance 7mS / cm with ammonium sulfate in advance, After feeding, after continuing to rinse, use ammonium sulfate to adjust th...

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Abstract

The invention relates to a method of purifying a foot-and-mouth disease inactivated virus antigen through ion exchange chromatography. In the method, by means of interaction between charged groups on a filling material of the ion exchange chromatography and different charged components in a solution of the foot-and-mouth disease inactivated virus antigen, the foot-and-mouth disease inactivated virus antigen can be separated from protein impurities and nucleic acids, thereby purifying the foot-and-mouth disease inactivated virus antigen. The method is less in steps, is stable in process, is high in purifying speed, is high in yield of the foot-and-mouth disease inactivated virus antigen, is easy to amplify and easy to achieve industrial large-scale production and has a huge practical application value.

Description

technical field [0001] The invention relates to the field of separation and purification of vaccine antigens, in particular to a chromatographic separation method for separation and purification of foot-and-mouth disease inactivated virus vaccine antigens from cell culture fluid. Background technique [0002] Foot and Mouth Disease (FMD) is a severe infectious disease caused by Foot-and-Mouth Disease Virus (FMDV), which mainly infects cloven-hoofed animals such as cattle, sheep, and pigs. Foot-and-mouth disease spreads quickly and spreads widely, and its epidemics often bring huge losses to the animal husbandry industry, and it has always been the focus of international health circles. [0003] The foot-and-mouth disease vaccine is currently the main way to control the foot-and-mouth disease epidemic. The foot-and-mouth disease vaccine currently in use is to cultivate baby hamster kidney cells (BHK-21 cells) on a large scale by shaking flasks or suspension methods, and then...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/02C12R1/93
Inventor 张松平苏志国杨延丽马光辉李浩张焱
Owner INST OF PROCESS ENG CHINESE ACAD OF SCI
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