Method for purifying type 71 enterovirus

A purification method and enterovirus technology, applied in the biological field, can solve the problems of high cost, residual additives, etc., and achieve the effects of easy process, good safety, and no contamination by exogenous factors.

Active Publication Date: 2013-12-25
ZHEJIANG PUKANG BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Traditional purification methods have become more and more difficult to meet the requirements of vaccine products for the limited amount of cellular DNA. Other meth...

Method used

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  • Method for purifying type 71 enterovirus
  • Method for purifying type 71 enterovirus
  • Method for purifying type 71 enterovirus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] 1) Centrifugal clarification: select a suitable centrifuge and rotor according to the volume of the cell culture, centrifuge at 4°C for 30 minutes with a centrifugal force of 9000g, and combine the centrifuged supernatant, which is the virus clarification solution;

[0038] 2) Concentration by ultrafiltration

[0039] Use 0.5M sodium hydroxide (NaOH) in the ultrafiltration system equipped with an appropriate number (depending on the number of samples to be processed) of 100KD ~ 500KD membrane packs at an appropriate pressure (inlet pressure 20 ~ 30psi, return pressure 8psi, through mouth pressure 0psi) and flow rate (1-3LPM) for 60 minutes, then wash the system with 5 times the volume of water for injection to remove residual NaOH, and then wash with PBS until the pH value is neutral; The ultrafiltration system concentrates to 1 / 30-1 / 50 of the original volume, washes and filters 2-3 times, and finally obtains the EV71 virus concentrate.

[0040] 3) Gel filtration chrom...

Embodiment 2

[0052] 1) Filtration clarification: The preferred method is to pre-filter through a surface filter with a pore size of 2.5 μM; then filter again through a filter with a pore size of 0.45 μM. The filtrate is the clarified liquid of EV71 virus.

[0053] 2) Concentration by ultrafiltration

[0054] Use 0.5M sodium hydroxide (NaOH) in the ultrafiltration system equipped with an appropriate number (depending on the number of samples to be processed) of 100KD ~ 500KD membrane packs at an appropriate pressure (inlet pressure 20 ~ 30psi, return pressure 8psi, through mouth pressure 0psi) and flow rate (1-3LPM) for 60 minutes, then wash the system with 5 times the volume of water for injection to remove residual NaOH, and then wash with PBS until the pH value is neutral; The ultrafiltration system concentrates to 1 / 30-1 / 50 of the original volume, washes and filters 2-3 times, and finally obtains the EV71 virus concentrate.

[0055] 3) Gel filtration chromatography

[0056] Put Sepha...

Embodiment 3

[0069] Various tests of EV71 virus purification solution:

[0070] The inactivated vaccine prepared by the EV71 virus purification solution of the present invention has been tested for the following indicators: free formaldehyde residue, bovine serum albumin residue, host cell protein residue, host cell DNA residue, purity, antigen identification test, bacterial endotoxin residue, Antibiotic Residue, Sterility Test, Abnormal Toxicity Check Inactivation Verification and pH Check. The verification method refers to the relevant method in the appendix of "Chinese Pharmacopoeia" (2010 edition). The test results are shown in Table 1.

[0071] Table I

[0072] Test items Verification standard test result Free formaldehyde residue ≤50ug / dose 6.5ug / dose bovine serum albumin residue ≤50ng / dose ≤30ng / dose host cell protein residue ≤50ug / dose ≤30ug / dose host cell DNA residue ≤100pg / dose ≤10pg / dose purity ≥95% ≥95% antigen ide...

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Abstract

The invention relates to the field of biotechnology, and particularly relates to a method for purifying type 71 enterovirus (EV71) from the cell culture harvest by a chromatographic process. The method comprises the following steps: clarifying the cell culture of the virus; performing ultrafiltration and concentration of the virus; purifying the virus through gel filtering chromatography and anion exchange chromatography. The continuous chromatography step has the advantages that the virus is purified and the replacement of chromatography buffer solution and adjustment of pH value and electrical conductivity are avoided; the purified virus liquid has the advantage of low residue of host protein and host desoxyribonucleic acid. The purified EV71 liquid prepared by the method can be used for preparing an inactivated vaccine against hand-foot-mouth diseases and can also be used for preparing reference antigen and antibody in a diagnostic reagent.

Description

field of invention [0001] The invention relates to the field of biotechnology, in particular to a method for purifying enterovirus type 71 (EV71) from cell culture harvests by chromatographic methods. Background technique [0002] Hand, foot and mouth disease (HFMD) is an acute infectious disease caused by enterovirus. It is mainly transmitted through close contact or digestive tract. It mostly occurs in infants under 10 years old. Rashes, herpes, and ulcers on the skin and mucous membranes of the mouth and other parts are typical manifestations, and individual patients may cause complications such as myocarditis, pulmonary edema, and aseptic meningoencephalitis. Since the disease was first reported in 1957, it has been popular in many countries for many times. Since the disease was discovered in Shanghai in 1981 in my country, more than a dozen provinces (cities) including Beijing, Hebei, Tianjin, Fujian, Jilin, Shandong, Hubei, and Guangdong Both are reported. Since the ...

Claims

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Application Information

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IPC IPC(8): C12N7/02C12R1/93
Inventor 姜云水高孟周康凤陈科达毛子安高丽美唐彩华朱莲王一虎庄昉成毛江森
Owner ZHEJIANG PUKANG BIOTECH
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