CN101306199A - Method for producing and purifying hepatitis a inactivated vaccine - Google Patents
Method for producing and purifying hepatitis a inactivated vaccine Download PDFInfo
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- CN101306199A CN101306199A CNA2007100224233A CN200710022423A CN101306199A CN 101306199 A CN101306199 A CN 101306199A CN A2007100224233 A CNA2007100224233 A CN A2007100224233A CN 200710022423 A CN200710022423 A CN 200710022423A CN 101306199 A CN101306199 A CN 101306199A
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
The invention relates to a method for producing and purifying a hepatitis A inactivated vaccine. The method comprises the following steps: a Vero cell is resuscitated from a work cell tank; the Vero cell is cultured by inoculation in a two to five-liter spinner bottle provided with two to five layers and used for holding culture solution for dense culture; the Vero cell is inoculated with a working seed virus seed; the virus seed is cultured until a virus multiplication peak time after being absorbed; a cell, which is cultured through the Vero cell and contains HAV virus, is obtained after being digested; and hepatitis A virus stock solution is prepared through adding phosphate buffer solution in a floating way through removing supernatant after the cell is centrifuged; after the hepatitis A virus stock solution is broken, chloroform is added, water phase on the upper layer is taken after being vibrated and centrifuged;then the phosphate buffer solution is added; rough hepatitis A virus stock solution is prepared, ultra-filtered and concentrated, purified in two steps of water column and gel column, filtered and sterilized;filtered fluid is collected into the purified hepatitis A virus stock solution to be inactivated, and then is absorbed through adjuvant to prepare the semi finished product of hepatitis A, and to obtain the hepatitis A inactivated vaccine through sub-package. The method can improve the density of the cell, enhance the output of the virus, reduce the production cost, and realize the batch production.
Description
Technical field
The present invention relates to the method for a kind of production and purification Hepatitis A Vaccine, belong to medicine bioengineering goods field.
Background technology
Hepatitis A be by hepatitis A virus (HAV) cause a kind of be the acute infectious disease of the high incidence of principal character with the liver injury.China is hepatitis A the most serious popular country in the world, in known all viral hepatitis, and the sickness rate tool first place of hepatitis A.And the inoculation hepatitis A vaccine is the popular effective method of prevention hepatitis A.
China succeeds in developing Live Attenuated HAV in the early 1990s, successfully developed hepatitis A inactivated vaccine (Vero cell) after 10 years again, succeeding in developing of these two kinds of vaccines with widely-used, for reducing the sickness rate of China's hepatitis A, the eruption and prevalence of control hepatitis A has played important role.The cellular matrix of existing Hepatitis A Vaccine is human diploid cell, and all adopt the cell monolayer flask culture, cell density is low, reproductive efficiency is not high is its common drawback, and the preparation hepatitis A vaccine needs a large amount of virus antigens, and this is the basic reason that hepatitis A vaccine production cost height, output difficulty have enlarged.
Summary of the invention
The method that the purpose of this invention is to provide the production purification Hepatitis A Vaccine that a kind ofly can improve cell density, improve viral yield, reduce production costs.
The technical scheme that realizes above-mentioned purpose is: a kind of method of producing the purification hepatitis A inactivated vaccine is characterized in that its processing step is:
1) recovery Vero cell from the working cell storehouse, carry out the amplification of going down to posterity of cell, the Vero cell inoculation in 2～5 layers 2～5 liters the rolling bottle that holds culture fluid, is carried out High Density Cultivation, culture fluid is 199 culture medium or the MEM culture medium that contains the 2-10% newborn calf serum, transfers PH to 7.2～7.4;
2) get work seed seed culture of viruses and be inoculated in the Vero cell, after adsorbing, be cultured to the virus multiplication peak period in 33 ℃～37 ℃, change culture fluid therebetween 3～5 times;
3) will gather in the crops behind the cell dissociation that contains HAV virus through the Vero cell culture, abandon supernatant, and add the phosphate buffer suspension and make hepatitis A virus (HAV) stock solution by after centrifugal;
4) hepatitis A virus (HAV) stock solution adds chloroform through after the fragmentation, jolting centrifuging and taking upper strata water, and then add phosphate buffer, and repeat extracting 3～5 times, make rough hepatitis A venom;
5) rough hepatitis A venom is behind ultrafiltration and concentration, again through drainage column, gel column two-step purifying, and last filtration sterilization, collecting filtrate is purification hepatitis A venom;
6) purification hepatitis A venom is carried out deactivation;
7) after the hepatitis A venom after the deactivation adsorbs through adjuvant, make the hepatitis A semi-finished product, packing gets hepatitis A inactivated vaccine.
Further, the 5th) adopt ultrafilter membrane to carry out ultrafiltration and concentration in the step, the aperture of the fenestra of this ultrafilter membrane is 50,000～100,000 molecular weight.
After adopting technique scheme, the present invention adopts 2～5 layers of 2～5 liters of rolling bottle to carry out cell culture according to the condition of culture that has screened, cell density reaches 0.8～1.2 * 107/ml, compare cell density with original technology and improved 3～5 times, reduced production cost significantly, improved product quality, and can realize producing in batches.
Description of drawings
Accompanying drawing is a process chart of the present invention.
The specific embodiment
The present invention is further detailed explanation below in conjunction with drawings and Examples.
As shown in the figure, the method of a kind of production and purification hepatitis A inactivated vaccine, processing step is: take out the Vero cell (cell is the Vero cell strain for the ATCC of national drug administrative authority approval) in the work storehouse in liquid nitrogen container, be inoculated in the cell bottle, (culture fluid is 199 culture medium that contain 8% newborn calf serum in 37 ℃ of cultivations to add culture fluid, transfer pH value to 7.2～7.4), use pancreatin after growing up to monolayer---EDTA digestion, add above-mentioned culture fluid, with 1: 3 minute kind, passed 1 time in per 3 days, in batches certain until obtaining.
With Vero cell pancreatin---EDTA digestion was with 1: 50 ratio inoculation hepatitis A JS-4 strain, and 37 ℃ adsorbed 40 minutes, were inoculated in 3 layers of rolling bottle, added culture fluid.Put 35 ℃ of thermostatic chambers and carry out High Density Cultivation.Change 4 times during this time and keep liquid, receive poison to after date, add phosphate buffer again with trypsin digestion cell and suspend and be collected in the Centrifuge Cup, abandon supernatant after centrifugal, (cell density is 1 * 10 with the phosphate buffer suspension cell
7Individual/ml), be virus stock solution used, put-20 ℃ frozen.
Take out virus stock solution used thaw the back with the Ultrasonic Cell Disruptor smudge cells to percentage of damage more than 95%, add the equal-volume chloroform, through chloroform extracting 5 times, centrifugal 30 minutes results of 3000rpm upper strata water, and then add the equivalent phosphate buffer, repeat extracting 3～5 times, make rough hepatitis A venom, after rough hepatitis A venom adopts the ultrafilter membrane of 100,000 molecular weight to carry out ultrafiltration and concentration earlier, again through drainage column, 2 step of gel column purification, use 0.22 μ m filter membrane aseptic filtration at last, collect filtrate.Purification of samples is added final concentration under aseptic condition be 1: 4000 formalin, puts 37 ℃ of thermostatic chambers, and deactivation adds the NaHSO3 neutralization after 12 days.
Behind the hepatitis A venom assay approval after the deactivation, can do the appropriateness dilution according to virus antigen titre level, adding aluminium hydroxide preparation semi-finished product, is the 1.1ml/ bottle with the semi-finished product that prepare by adult's dosage, and child dose is made the finished product vaccine in the aseptic cillin bottle for the 0.6ml/ bottle is sub-packed in.
The finished product verification result is as follows:
| Sequence number | The calibrating project | Criterion | Verification result |
| 1 | The relative effectivenes value | ≥1.0 | 1.14 |
| 2 | The DNA residual quantity detects | ≤ 100pg/ agent | ＜100pg/ agent |
| 3 | The Ox blood serum residual volume is measured | ≤ 50ng/1600EU | 10ng/1600EU |
| 4 | Endotoxin content is measured | ＜10EU/ml | ＜10EU/ml |
| 5 | Free formaldehyde content is measured | ≤0.10mg/ml | 0.02mg/ml |
| 6 | 2-phenoxyethanol assay | 4-6mg/ml | 5.11mg/ml |
| 7 | Al (OH) 3 assays | 0.50-1.25mg/ml | 0.89mg/ml |
| 8 | The undue toxicity | Animal is strong deposits and weight increase | Qualified |
Claims (3)
1, the method for a kind of production and purification hepatitis A inactivated vaccine is characterized in that its processing step is:
1) recovery Vero cell from the working cell storehouse, carry out the amplification of going down to posterity of cell, with the Vero cell inoculation in 2～5 layers 2～5 liters the rolling bottle that holds culture fluid, carry out High Density Cultivation, culture fluid is 199 culture medium or the MEM culture medium that contains the 2-10% newborn calf serum, transfers pH value to 7.2～7.4;
2) get work seed seed culture of viruses and be inoculated in the Vero cell, after adsorbing, be cultured to the virus multiplication peak period in 33 ℃～37 ℃, change culture fluid therebetween 3～5 times;
3) will gather in the crops behind the cell dissociation that contains HAV virus through the Vero cell culture, abandon supernatant, and add the phosphate buffer suspension and make hepatitis A virus (HAV) stock solution by after centrifugal;
4) hepatitis A virus (HAV) stock solution adds chloroform through after the fragmentation, jolting centrifuging and taking upper strata water, and then add phosphate buffer, and repeat extracting 3～5 times, make rough hepatitis A venom;
5) rough hepatitis A venom is behind ultrafiltration and concentration, again through drainage column, gel column two-step purifying, and last filtration sterilization, collecting filtrate is purification hepatitis A venom;
6) purification hepatitis A venom is carried out deactivation;
7) after the hepatitis A venom after the deactivation adsorbs through adjuvant, make the hepatitis A semi-finished product, packing gets hepatitis A inactivated vaccine.
2, the method for production according to claim 1 and purification hepatitis A inactivated vaccine is characterized in that: the described the 5th) adopt ultrafilter membrane to carry out ultrafiltration and concentration in the step, the aperture of the fenestra of this ultrafilter membrane is 50,000～100,000 molecular weight.
3, the method for production according to claim 1 and purification hepatitis A inactivated vaccine is characterized in that: the described the 7th) adjuvant that adopts in the step is an aluminium hydroxide.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2007100224233A CN101306199A (en) | 2007-05-18 | 2007-05-18 | Method for producing and purifying hepatitis a inactivated vaccine |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2007100224233A CN101306199A (en) | 2007-05-18 | 2007-05-18 | Method for producing and purifying hepatitis a inactivated vaccine |
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| Publication Number | Publication Date |
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| CN101306199A true CN101306199A (en) | 2008-11-19 |
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| Application Number | Title | Priority Date | Filing Date |
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| CNA2007100224233A Pending CN101306199A (en) | 2007-05-18 | 2007-05-18 | Method for producing and purifying hepatitis a inactivated vaccine |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101816786A (en) * | 2010-04-30 | 2010-09-01 | 长春生物制品研究所 | Inactivated hepatitis A vaccine and preparation method thereof |
| CN102807972A (en) * | 2012-08-08 | 2012-12-05 | 深圳康泰生物制品股份有限公司 | Hepatitis A virus purification method |
| CN110669739A (en) * | 2019-09-30 | 2020-01-10 | 长春生物制品研究所有限责任公司 | Preparation method of novel hepatitis A virus antigen |
-
2007
- 2007-05-18 CN CNA2007100224233A patent/CN101306199A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101816786A (en) * | 2010-04-30 | 2010-09-01 | 长春生物制品研究所 | Inactivated hepatitis A vaccine and preparation method thereof |
| CN101816786B (en) * | 2010-04-30 | 2012-09-05 | 长春生物制品研究所有限责任公司 | Inactivated hepatitis A vaccine and preparation method thereof |
| CN102807972A (en) * | 2012-08-08 | 2012-12-05 | 深圳康泰生物制品股份有限公司 | Hepatitis A virus purification method |
| CN110669739A (en) * | 2019-09-30 | 2020-01-10 | 长春生物制品研究所有限责任公司 | Preparation method of novel hepatitis A virus antigen |
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Open date: 20081119 |