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Method for purifying type 71 enterovirus

A technology of enterovirus and purification method, which is applied in the field of purification of enterovirus 71 (EV71), can solve the problems of high cost and residual additives, and achieve the effect of easy process, good safety, and no contamination by exogenous factors

Active Publication Date: 2015-06-03
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 methods, such as adding nuclease and protamine treatment, can remove DNA, but they are not only expensive, but also need to be detected in the final product. residue problem

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, in particular to a method for purifying enterovirus type 71 (EV71) from cell culture harvests by chromatographic methods. The method involves clarification of virus cell cultures followed by ultrafiltration to concentrate virus followed by purification of virus using gel filtration chromatography and anion exchange chromatography, with successive chromatography steps having the ability to purify virus while avoiding chromatography buffer exchange, avoiding pH and the advantages of conductivity adjustment, the prepared virus purification solution has the advantages of low host protein and host deoxyribonucleic acid residues. The EV71 purified liquid prepared by the method can be used for the preparation of the hand, foot and mouth disease inactivated vaccine, and can be used for the preparation of reference antigens and antibodies in diagnostic reagents.

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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Patent Type & Authority Patents(China)
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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