Hepatitis A virus purification method

A technology of hepatitis A virus and purification method, which is applied in the field of purification of hepatitis A virus, can solve the problems of increasing unsafe factors of hepatitis A vaccine, increasing the risk of vaccine use, and low purity of hepatitis A virus, so as to improve the quality of the virus Purity, reduced adsorption, good immunogenicity

Active Publication Date: 2012-12-05
SHENZHEN KANGTAI BIOLOGICAL PROD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, the common separation method of hepatitis A virus is molecular sieve chromatography technology, which is characterized by relatively simple operation and has been used in production for a long time, but the purity of hepatitis A virus purification by this technology is not high, and the purity of the prepared hepatitis A antigen is lower than 50%. The total protein concentration in the finished product of each dose of hepatitis A inactivated vaccine is higher than 10 μg / ml, which increases the unsafe factors of hepatitis A vaccine, increases the risk of vaccine use, and increases the cost

Method used

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Embodiment 1

[0032] The purification method of embodiment 1 hepatitis A virus and the application of preparation vaccine (1)

[0033] 1. Take 1000ml of Hepatitis A virus harvest liquid (Hepatitis A virus SH strain preservation number: CGMCC No.4501). The hepatitis A virus harvest solution (sterile antigen titer up to 1:512) was crushed in an ice bath using an ultrasonic pulverizer (purchased from Ningbo Xinzhi Company, rated power 1500w, output power 1200w) to break the cell temperature and make the cells The crushing rate is more than 99%, and the ultrasonic crushing is 4 times;

[0034] 2. Centrifuge the ultrasonically disrupted cell solution at a speed of 3500r / min for 10min, and collect the supernatant;

[0035] 3. Add chloroform to the supernatant at a volume ratio of 1:3 and shake, centrifuge at 3500r / min to absorb the upper aqueous phase; extract the protein phase and chloroform with extraction buffer (6.2mM PBS), add chloroform to shake and absorb The supernatant was extracted on...

Embodiment 2

[0054] The purification method of embodiment 2 hepatitis A virus and the application of preparation vaccine (2)

[0055] 1. Take 1000ml of Hepatitis A virus harvest liquid (Hepatitis A virus SH strain preservation number: CGMCC NO.4501). The hepatitis A virus harvest solution (sterile antigen titer up to 1:256) was crushed in an ice bath using an ultrasonic pulverizer (purchased from Ningbo Xinzhi Company, rated power 1500w, output power 1200w) to break the cell temperature and make the cells The crushing rate is above 99%, and the ultrasonic crushing is performed 5 times;

[0056] 2. Centrifuge the ultrasonically crushed cell solution at a speed of 4000r / min for 8min, and collect the supernatant;

[0057] 3. Add chloroform to the supernatant at a volume ratio of 1:2 and shake, centrifuge at 4000r / min to absorb the upper aqueous phase; then extract the protein phase and chloroform with extraction buffer (120mM NaCl), add chloroform and shake, and absorb the upper layer. Clea...

Embodiment 3

[0076] The purification method of embodiment 3 hepatitis A virus and the application of preparation vaccine (3)

[0077] 1. Take 1000ml of Hepatitis A virus harvest liquid (Hepatitis A virus strain preservation number: CGMCC NO.4501). The hepatitis A virus harvest solution (sterile antigen titer up to 1:1024) was crushed in an ice bath using an ultrasonic pulverizer (purchased from Ningbo Xinzhi Company, rated power 1500w, output power 1200w) to break the cell temperature and make the cells The crushing rate is above 99%, and the ultrasonic crushing is performed 5 times;

[0078] 2. Centrifuge the sonicated cell solution at a speed of 4500r / min for 5 minutes, and collect the supernatant;

[0079] 3. Add chloroform to the supernatant at a volume ratio of 1:1 and shake, centrifuge at 4500r / min to absorb the upper aqueous phase; then extract the protein phase and chloroform with extraction buffer (120mM NaCl), add chloroform and shake, and absorb the upper layer. Clear is 1 ext...

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Abstract

The invention provides a hepatitis A virus purification method. With hepatitis A virus cell culture fluid serving as raw materials, the hepatitis A virus purification technology is completed via the technological processes of ultrasonication, centrifugal removal of cell debris, chloroform extraction, ultra-filtering concentration, hydrophobic chromatography, detection, collection and the like. Inactivated hepatitis A vaccines prepared from the hepatitis A virus cell culture fluid are high in purity and valence, simple in technology, easy to magnify and safe and reliable to use.

Description

technical field [0001] The invention relates to the field of virus purification methods, in particular to a method for purifying hepatitis A virus. Background technique [0002] Hepatitis A, referred to as hepatitis A, is an acute infectious disease caused by hepatitis A virus (HAV), which seriously endangers human health. Hepatitis A is mainly transmitted through the "fecal-oral" route, or transmission among individuals, or outbreaks of hepatitis A are caused by water or food contaminated with hepatitis A virus. After older children and adults are infected with hepatitis A, more than 70% are clinical infections, and the case fatality rate is 0.3% to 0.6%; the case fatality rate of patients over 50 years old is 1.8%. After chronic liver disease is infected with hepatitis A, the risk of acute liver failure raised. With the improvement of living conditions, the number of adults infected with hepatitis A tends to increase, and the proportion of clinical hepatitis A increases,...

Claims

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Application Information

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IPC IPC(8): C12N7/02A61K39/29A61P31/14C12R1/93
CPCY02A50/30
Inventor 张现臣魏文进黄秋香刘雨包建伟钟汉斌王春雨孟红彦
Owner SHENZHEN KANGTAI BIOLOGICAL PROD
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