CN102807972A - Hepatitis A virus purification method - Google Patents
Hepatitis A virus purification method Download PDFInfo
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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
The present invention relates to the method for purifying virus field, particularly, relate to a kind of method of purification of hepatitis A virus.
Background technology
Hepatitis A is called for short hepatitis A, is the acute infectious disease of a kind of serious harm human health of being caused by hepatitis A virus (HAV).Hepatitis A is mainly propagated through " excrement-mouth " approach, or the propagation in the individual human world, thereby or causes that hepatitis A breaks out because of water or the food that has polluted HAV.After the big-age-child infects hepatitis A with the adult, be that clinical type infects more than 70%, case fatality rate is 0.3%～0.6%; Patient's case fatality rate is 1.8% more than 50 years old; After the chronic hepatopathy person infected hepatitis A, the danger that acute hepatic failure takes place raise.Along with the improvement of living condition, the grownup infects the hepatitis A number has the trend of increasing, and clinical type hepatitis A ratio rises, thereby hepatitis A becomes more serious public health problem.
China is the hepatitis A district occurred frequently, has at least every year 240000 people to suffer from hepatitis A, causes enormous economic loss and social danger, and therefore, development and exploitation effectively prevent and the vaccine and the medicine of treatment hepatitis A become problem demanding prompt solution.
The vaccine of two kinds of prevention and control hepatitis A is arranged at present in the world, and a kind of is Attenuated HAV; Another kind is a hav inactivated vaccine.Because the HAV purifying process that each manufacturer uses is different, quality product is uneven.
At present common HAV separation method is the sieve chromatography technology, and this technical characterstic is to operate simply relatively, and production application for a long time; But it is not should technology high to the purity of HAV purifying; The hav antigen purity that makes is lower than 50%, makes that total protein concentration is higher than 10 μ g/ml in every dose of hepatitis A inactivated vaccine finished product, and this has increased the unsafe factor of Hepatitis A Vaccine; Increase the risk that vaccine uses, and increased cost.Therefore it is a kind of good to the HAV purification effect to press for, and viral purity is higher, and unit volume chromatography glue purification efficient is high, and the hepatitis A virus method of purification is beneficial to extensive virus production purifying with the supply production of vaccine safely and effectively.
Summary of the invention
The object of the present invention is to provide a kind of method of purification of hepatitis A virus, to overcome above-mentioned deficiency.
The hepatitis A virus SH strain that the present invention uses, its deposit number is CGMCC No.4501, has been disclosed among the Chinese patent CN102174477A.
The present invention provides a kind of method of purification of hepatitis A virus, may further comprise the steps:
1) hepatitis A virus cell culture fluid ultrasonication, centrifugal removal cell debris is collected supernatant;
2) chloroform extracting supernatant obtains rough viral liquid;
3) with rough viral liquid ultrafiltration and concentration, buffer salt solution wash-out ultra-filtration membrane;
4) hydrophobic chromatography purifying concentrating virus liquid;
5) detection of viral liquid and collection.
Wherein, the ultrasonication of the said hepatitis A virus cell culture fluid of step 1) is the hepatitis A virus cell culture fluid ultrasonication under condition of ice bath that antigen titre is reached 1:256-1024.
Wherein, the Ultrasonic Cell Disruptor power that the said ultrasonication of step 1) is used is 500-1500w, and output rating is 50%-95%, and broken number of times is 4-6 time.
Wherein, the centrifugal method of said step 1) is 3500-4500r/min, centrifugal 5min-10min.
Wherein, said step 2) the chloroform extracting is to be that 1:1-3 adds the supernatant jolting by volume with chloroform, and the upper strata water is drawn in the centrifugal back of 3500-4500r/min then, so repeats extracting 4-6 time; With albumen mutually with chloroform again with the extraction buffer extracting and draw the upper strata water, merging upper strata water obtains rough viral liquid.
Wherein, described extraction buffer is 120mM NaCl or 6.2mM PBS.
Wherein, the multiple of said step 3) ultrafiltration and concentration is 10-30 times, and the molecular weight cut-off of ultra-filtration membrane is 50-100KD.
Wherein, the PBS solution that said step 3) buffer salt solution is 2-4mol/L, pH7.2.
Wherein, said step 4) chromatography method is: each applied sample amount is 1000-2000ml, and sample-loading buffer is 2mol/L PBS, and pH7.2, elution buffer are 0.5mol/L PBS, and pH7.2, flow velocity are 15ml/min.
Further, with the 280nm wavelength wash-out virus liquid on the good post of balance is detected.
The invention provides the application of above-mentioned hepatitis A virus method of purification in the preparation Hepatitis A Vaccine.
Will through the hydrophobic chromatography collection step to viral liquid carry out deactivation, according to volume ratio 1:4000-5000 formaldehyde is added in the viral liquid, 37 ℃ of constant temperature rotate and accomplished inactivation process in 12-15 days.
The deactivation hepatitis A virus is according to tiring rare joining after be the Hepatitis A virus vaccine goods after the inspections such as aseptic, effectiveness, undue toxicity, bacterial endotoxin.
The present invention has following useful effect: the hepatitis A virus recovery has been improved in (1), and high density buffering salt wash-out ultra-filtration membrane effectively reduces the absorption of ultra-filtration membrane to virus; (2) improved viral purity; The cytopathy venom can effectively be removed foreign protein through ultrasonication, chloroform extracting, ultra-filtration membrane ultrafiltration and concentration and gel permeation chromatography; Improve viral purity, the purity of the hepatitis A virus that use the inventive method purifying obtains is greater than 95%; (3) hepatitis A virus that obtains of the inventive method purifying is used for the preparation of hepatitis A or vaccine; Total protein concentration is lower than 3 μ g/ml in the hepatitis A inactivated vaccine finished product of every dose of effective dose, and it is more safe and reliable to be higher than 10 μ g/ml than total protein concentration in the prior art.Therefore the inventive method virus of purifying is used to prepare Hepatitis A virus vaccine and has safe and reliablely, and immunogenicity is good, high specificity.
Description of drawings
Fig. 1 is VP1, VP2 and the VP3 content detection HPLC color atlas of the hepatitis A virus that purifying obtains among the embodiment 1.
Fig. 2 is VP1, VP2 and the VP3 content detection HPLC color atlas of the hepatitis A virus that purifying obtains among the embodiment 2.
Fig. 3 is VP1, VP2 and the VP3 content detection HPLC color atlas of the hepatitis A virus that purifying obtains among the embodiment 3.
Embodiment
Following examples further specify content of the present invention, but should not be construed as limitation of the present invention.Under the situation that does not deviate from the present invention's spirit and essence, modification or replacement to the inventive method, step or condition are done all belong to scope of the present invention.
If do not specialize the conventional means that used technique means is well known to those skilled in the art among the embodiment.
The method of purification of embodiment 1 hepatitis A virus and the application (1) for preparing vaccine
1, gets 1000ml hepatitis A virus results liquid (HAV SH strain preserving number: CGMCC No.4501).Hepatitis A virus is gathered in the crops liquid (aseptic antigen titre reaches 1:512) under condition of ice bath; Use ultrasonic grinding appearance (available from Ningbo Xin Zhi company, rated output 1500w, output rating 1200w) smudge cells temperature; Cell crashing ratio is reached more than 99%, ultrasonication 4 times;
2, with the enchylema after the ultrasonication with the centrifugal 10min of the rotating speed of 3500r/min, collect supernatant;
3, jolting in the chloroform 1:3 adding by volume supernatant, the centrifugal absorption of 3500r/min upper strata water; Albumen is mutually used extraction buffer (6.2mM PBS) extracting with chloroform again, adds chloroform and jolts the back to draw supernatant be 1 extracting, repeats extracting 5 times, and merging upper strata water gets rough viral liquid;
4, the rough hepatitis A venom that step 3 is made is through the ultra-filtration membrane ultrafiltration and concentration, and 10 times of ultrafiltration and concentration, the molecular weight cut-off of ultra-filtration membrane are 50KD;
5, with the PBS solution of high density buffer salt solution 2mol/L, pH7.2 wash-out ultra-filtration membrane;
6, spissated viral liquid is through gel chromatography, and the used chromatography column of gel chromatography is Phenyl Sepharose 6FF.Chromatography method is: each applied sample amount is 1000ml, and sample-loading buffer is 2mol/LPBS, and pH7.2, elution buffer are 0.5mol/L PBS, and pH7.2, flow velocity are 15ml/min.
7, detect and collect virus.The viral liquid of going up wash-out with the 280nm wavelength XK50/30 post good to balance (available from GE company) detects (the observation electricity is led); Lead observed result according to electricity; Lead 30 from electricity and begin to collect viral liquid, collect electricity always and lead and drop to 2.4, merge the viral liquid of collecting and promptly accomplish the purifying technique process.
8, HPLC purity check
It is in the mixing solutions of 4mol/L hydrochloric acid arc (GuHCL) and lmmol/L WR 34678 (DTT) that purifying gained HAV liquid in the step 7 is added final concentration.50 ℃ of water bath with thermostatic control l0min cracking HAV particles.The HPLC chromatographic column is TSK-GEL G5000PW, and moving phase is 6.2mmol/L pH6.8PBS, and flow velocity is 0.6ml/min; The UV-detector wavelength is 280nm; Temperature is 25 ℃, and each sample size is 50 μ l, goes out behind the peak VP1, VP2 and VP3 protein peak from the high collection of half-peak hepatitis A virus.Find that through the high performance liquid chromatograph system software analysis VP1, VP2 and VP3 purity reach 97%, wherein VP1 purity 7%, VP2 purity 64%, VP3 purity 26%.The HPLC color atlas is seen Fig. 1.
9, deactivation.The hepatitis A virus liquid of collecting in the step 7 is added formaldehyde by 1/4000 of its TV, put again in 37 ℃ of thermostatic chambers, rotate and promptly accomplished inactivation process in 12 days.
10, calibrating.The deactivation hepatitis A virus is according to tiring rare joining after be the Hepatitis A virus vaccine goods after the inspections such as aseptic, effectiveness, undue toxicity, bacterial endotoxin.
(1) vaccine preparation:
The HAV antigen valence is 6400EU/ml after the deactivation of purifying hepatitis A venom, is the hepatitis A inactivated vaccine for preparing with 10 times of 2mM PBS (pH7.2) dilutions to HAV antigen valence 640EU/ml.
(2) sterility test
" Chinese people republic pharmacopeia " 2010 editions the 3rd appendix XII A sterility test method is carried out in pressing.With vaccine with 0.22 μ m membrane filtration after; With 0.1% peptone solution cleaning filter membranes, filter membrane is divided into three parts, puts into 2 sulphur glycollate culture mediums and 1 improvement Martin substratum respectively; Wherein 1 sulphur glycollate culture medium is put 30 ℃, and all the other are put 20 ℃ and cultivated 14 days.The sterility test result is qualified.
(3) render a service inspection
Undertaken by " Chinese people republic pharmacopeia " 2010 editions the 3rd appendix X S method.Vaccine is diluted to identical a series of extent of dilution with HAV antigen with reference to article, uses ELISA reagent, measure vaccine and,, calculate vaccine with respect to relative effectivenes with reference to article according to the parallel lines statistical technique with reference to each dilution absorbancy of article.The vaccine potency that present embodiment makes is 1.12.
(4) undue toxicity inspection
Undertaken by " Chinese people republic pharmacopeia " 2010 editions the 3rd appendix XII F method.The abdominal injection vaccine, injected in mice 0.5ml, totally 5, cavy injection 5.0ml, totally 2.Observed 7 days.Detecting the undue toxicity of finding the present embodiment vaccine is 0.50.
(5) bacterial endotoxin inspection
Undertaken by " Chinese people republic pharmacopeia " 2010 editions the 3rd appendix XII E gel test method(s) of limiting the quantity of.With TAL bacterial detection intracellular toxin, vaccine should dilute 80 times, to eliminate the influence of interfering factors.The result finds that bacterial endotoxin that present embodiment makes hepatitis A vaccine is＜2.0EU/ml.
The method of purification of embodiment 2 hepatitis A virus and the application (2) for preparing vaccine
1, gets 1000ml hepatitis A virus results liquid (HAV virus SH strain preserving number: CGMCC NO.4501).Hepatitis A virus is gathered in the crops liquid (aseptic antigen titre reaches 1:256) under condition of ice bath; Use ultrasonic grinding appearance (available from Ningbo Xin Zhi company, rated output 1500w, output rating 1200w) smudge cells temperature; Cell crashing ratio is reached more than 99%, ultrasonication 5 times;
2, the enchylema after the ultrasonication is collected supernatant with the centrifugal 8min of the rotating speed of 4000r/min;
3, jolting in the chloroform 1:2 adding by volume supernatant, the centrifugal absorption of 4000r/min upper strata water; Albumen is mutually used extraction buffer (120mM NaCl) extracting with chloroform again, adds chloroform and jolts the back to draw supernatant be 1 extracting, repeats extracting 6 times, and merging upper strata water gets rough viral liquid;
4, the rough hepatitis A venom that step 3 is made is through the ultra-filtration membrane ultrafiltration and concentration, and 20 times of ultrafiltration and concentration, the molecular weight cut-off of ultra-filtration membrane are 50KD;
5, with the PBS solution of high density buffer salt solution 3mol/L, pH7.2 wash-out ultra-filtration membrane;
6, spissated viral liquid is through gel chromatography, and the used chromatography column of gel chromatography is Phenyl Sepharose 6FF.Chromatography method is: each applied sample amount is 1500ml, and sample-loading buffer is 2mol/LPBS, and pH7.2, elution buffer are 0.5mol/L PBS, and pH7.2, flow velocity are 15ml/min.
7, detect and collect virus.The viral liquid of going up wash-out with the 280nm wavelength XK50/30 post good to balance (available from GE company) detects (the observation electricity is led); Lead observed result according to electricity; Lead 30 from electricity and begin to collect viral liquid, collect electricity always and lead and drop to 2.4, merge the viral liquid of collecting and promptly accomplish the purifying technique process.
8, HPLC purity check
It is in the mixing solutions of 4mol/L hydrochloric acid arc (GuHCL) and lmmol/L WR 34678 (DTT) that purifying gained HAV liquid in the step 7 is added final concentration.50 ℃ of water bath with thermostatic control l0min cracking HAV particles.The HPLC chromatographic column is TSK-GEL G5000PW, and moving phase is 6.2mmol/L pH6.8PBS, and flow velocity is 0.6ml/min; The UV-detector wavelength is 280nm; Temperature is 25 ℃, and each sample size is 50 μ l, goes out behind the peak VP1, VP2 and VP3 protein peak from the high collection of half-peak hepatitis A virus.Find that through the high performance liquid chromatograph system software analysis VP1, VP2 and VP3 purity reach 98%, wherein VP1 purity 7%, VP2 purity 65%, VP3 purity 26%.The HPLC color atlas is seen Fig. 2.
9, deactivation.The hepatitis A virus liquid of collecting in the step 7 is added formaldehyde by 1/4500 of its TV, put in 37 ℃ of thermostatic chambers again, inactivation process is promptly accomplished in the ROT13 sky.
10, calibrating.The deactivation hepatitis A virus is according to tiring rare joining after be the Hepatitis A virus vaccine goods after the inspections such as aseptic, effectiveness, undue toxicity, bacterial endotoxin.
(1) vaccine preparation:
The HAV antigen valence is 12800EU/ml after the hepatitis A venom deactivation of purifying, is the hepatitis A inactivated vaccine for preparing with 20 times of 2mM PBS (pH7.2) dilutions to HAV antigen valence 640EU/ml.
(2) sterility test
" Chinese people republic pharmacopeia " 2010 editions the 3rd appendix XII A sterility test method is carried out in pressing.With vaccine with 0.22 μ m membrane filtration after; With 0.1% peptone solution cleaning filter membranes, filter membrane is divided into three parts, puts into 2 sulphur glycollate culture mediums and 1 improvement Martin substratum respectively; Wherein 1 sulphur glycollate culture medium is put 32 ℃, and all the other are put 23 ℃ and cultivated 14 days.The sterility test result that present embodiment makes vaccine is qualified.
(3) render a service inspection
Undertaken by " Chinese people republic pharmacopeia " 2010 editions the 3rd appendix X S method.Vaccine is diluted to identical a series of extent of dilution with HAV antigen with reference to article, uses ELISA reagent, measure vaccine and,, calculate vaccine with respect to relative effectivenes with reference to article according to the parallel lines statistical technique with reference to each dilution absorbancy of article.The effectiveness that present embodiment makes vaccine is 1.05.
(4) undue toxicity inspection
Undertaken by " Chinese people republic pharmacopeia " 2010 editions the 3rd appendix XII F method.The abdominal injection vaccine, injected in mice 0.5ml, totally 5, cavy injection 5.0ml, totally 2.Observed 7 days.The undue toxicity that present embodiment makes vaccine is 0.50.
(5) bacterial endotoxin inspection
Undertaken by " Chinese people republic pharmacopeia " 2010 editions the 3rd appendix XII E gel test method(s) of limiting the quantity of.With TAL bacterial detection intracellular toxin, vaccine should dilute 80 times, to eliminate the influence of interfering factors.The bacterial endotoxin that present embodiment makes vaccine is＜2.0EU/ml.
The method of purification of embodiment 3 hepatitis A virus and the application (3) for preparing vaccine
1, gets 1000ml hepatitis A virus results liquid (HAV strain preserving number: CGMCC NO.4501).Hepatitis A virus is gathered in the crops liquid (aseptic antigen titre reaches 1:1024) under condition of ice bath; Use ultrasonic grinding appearance (available from Ningbo Xin Zhi company, rated output 1500w, output rating 1200w) smudge cells temperature; Cell crashing ratio is reached more than 99%, ultrasonication 5 times;
2, the enchylema after the ultrasonication is collected supernatant with the centrifugal 5min of the rotating speed of 4500r/min;
3, jolting in the chloroform 1:1 adding by volume supernatant, the centrifugal absorption of 4500r/min upper strata water; Albumen is mutually used extraction buffer (120mM NaCl) extracting with chloroform again, adds chloroform and jolts the back to draw supernatant be 1 extracting, repeats extracting 4 times, and merging upper strata water gets rough viral liquid;
4, the rough hepatitis A venom that step 3 is made is through the ultra-filtration membrane ultrafiltration and concentration, and 30 times of ultrafiltration and concentration, the molecular weight cut-off of ultra-filtration membrane are 100KD;
5, with the PBS solution of high density buffer salt solution 4mol/L, pH7.2 wash-out ultra-filtration membrane;
6, spissated viral liquid is through gel chromatography, and the used chromatography column of gel chromatography is Phenyl Sepharose 6FF.Chromatography method is: each applied sample amount is 2000ml, and sample-loading buffer is 2mol/LPBS, and pH7.2, elution buffer are 0.5mol/L PBS, and pH7.2, flow velocity are 15ml/min.
7, detect and collect virus.The viral liquid of going up wash-out with the 280nm wavelength XK50/30 post good to balance (available from GE company) detects (the observation electricity is led); Lead observed result according to electricity; Lead 30 from electricity and begin to collect viral liquid, collect electricity always and lead and drop to 2.4, merge the viral liquid of collecting and promptly accomplish the purifying technique process.
8, HPLC purity check
It is in the mixing solutions of 4mol/L hydrochloric acid arc (GuHCL) and lmmol/L WR 34678 (DTT) that purifying gained HAV liquid in the step 7 is added final concentration.50 ℃ of water bath with thermostatic control l0min cracking HAV particles.The HPLC chromatographic column is TSK-GEL G5000PW, and moving phase is 6.2mmol/L pH6.8PBS, and flow velocity is 0.6ml/min; The UV-detector wavelength is 280nm; Temperature is 25 ℃, and each sample size is 50 μ l, goes out behind the peak VP1, VP2 and VP3 protein peak from the high collection of half-peak hepatitis A virus.Find that through the high performance liquid chromatograph system software analysis VP1, VP2 and VP3 purity reach 97%, wherein VP1 purity 7%, VP2 purity 64%, VP3 purity 26%.The HPLC color atlas is seen Fig. 3.
9, deactivation.The hepatitis A virus liquid of collecting in the step 7 is added formaldehyde by 1/5000 of its TV, put again in 37 ℃ of thermostatic chambers, rotate and promptly accomplished inactivation process in 15 days.
10, calibrating.The deactivation hepatitis A virus is according to tiring rare joining after be the Hepatitis A virus vaccine goods after the inspections such as aseptic, effectiveness, undue toxicity, bacterial endotoxin.
(1) vaccine preparation:
The HAV antigen valence is 12800EU/ml after the hepatitis A venom deactivation of purifying, is the hepatitis A inactivated vaccine for preparing with 20 times of 2mM PBS (pH7.2) dilutions to HAV antigen valence 640EU/ml.
(2) sterility test
" Chinese people republic pharmacopeia " 2010 editions the 3rd appendix XII A sterility test method is carried out in pressing.With vaccine with 0.22 μ m membrane filtration after; With 0.1% peptone solution cleaning filter membranes, filter membrane is divided into three parts, puts into 2 sulphur glycollate culture mediums and 1 improvement Martin substratum respectively; Wherein 1 sulphur glycollate culture medium is put 35 ℃, and all the other are put 25 ℃ and cultivated 14 days.The sterility test result that present embodiment makes vaccine is qualified.
(3) render a service inspection
Undertaken by " Chinese people republic pharmacopeia " 2010 editions the 3rd appendix X S method.Vaccine is diluted to identical a series of extent of dilution with HAV antigen with reference to article, uses ELISA reagent, measure vaccine and,, calculate vaccine with respect to relative effectivenes with reference to article according to the parallel lines statistical technique with reference to each dilution absorbancy of article.The effectiveness that present embodiment makes vaccine is 1.10.
(4) undue toxicity inspection
Undertaken by " Chinese people republic pharmacopeia " 2010 editions the 3rd appendix XII F method.The abdominal injection vaccine, injected in mice 0.5ml, totally 5, cavy injection 5.0ml, totally 2.Observed 7 days.The undue toxicity that present embodiment makes vaccine is 0.50.
(5) bacterial endotoxin inspection
Undertaken by " Chinese people republic pharmacopeia " 2010 editions the 3rd appendix XII E gel test method(s) of limiting the quantity of.With TAL bacterial detection intracellular toxin, vaccine should dilute 80 times, to eliminate the influence of interfering factors.The bacterial endotoxin that present embodiment makes vaccine is＜2.0EU/ml.
Claims (10)
1. the method for purification of a hepatitis A virus may further comprise the steps:
1) hepatitis A virus cell culture fluid ultrasonication, centrifugal removal cell debris is collected supernatant;
2) chloroform extracting supernatant obtains rough viral liquid;
3) with rough viral liquid ultrafiltration and concentration, buffer salt solution wash-out ultra-filtration membrane;
4) hydrophobic chromatography purifying concentrating virus liquid;
5) detection of viral liquid and collection.
2. the method for claim 1 is characterized in that, the ultrasonication of the said hepatitis A virus cell culture fluid of step 1) is the hepatitis A virus cell culture fluid ultrasonication under condition of ice bath that antigen titre is reached 1:256-1024.
3. method as claimed in claim 2 is characterized in that, the Ultrasonic Cell Disruptor power that said ultrasonication is used is 500-1500w, and output rating is 50%-95%, and broken number of times is 4-6 time.
4. the method for claim 1 is characterized in that, the centrifugal method of said step 1) is 3500-4500r/min, centrifugal 5min-10min.
5. the method for claim 1 is characterized in that, said step 2) the chloroform extracting is that chloroform is that by volume 1:1-3 adds the supernatant jolting, the upper strata water is drawn in the centrifugal back of 3500-4500r/min then, so repeats extracting 4-6 time; With albumen mutually with chloroform again with the extraction buffer extracting and draw the upper strata water, merging upper strata water obtains rough viral liquid.
6. method as claimed in claim 5 is characterized in that, described extraction buffer is 120mM NaCl or 6.2mM PBS.
7. the method for claim 1 is characterized in that, the multiple of said step 3) ultrafiltration and concentration is 10-30 times, and the molecular weight cut-off of ultra-filtration membrane is 50-100KD.
8. the method for claim 1 is characterized in that, the PBS solution that said step 3) buffer salt solution is 2-4mol/L, pH7.2.
9. the method for claim 1 is characterized in that, said step 4) chromatography method is: each applied sample amount is 1000-2000ml, and sample-loading buffer is 2mol/L PBS, and pH7.2, elution buffer are 0.5mol/L PBS, and pH7.2, flow velocity are 15ml/min.
10. the application of the arbitrary described method of claim 1-9 in the preparation Hepatitis A Vaccine.
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| CN107384873A (en) * | 2017-09-05 | 2017-11-24 | 成都汇宇生物技术有限公司 | The purification process of recombined adhenovirus |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101306199A (en) * | 2007-05-18 | 2008-11-19 | 江苏延申生物科技股份有限公司 | Method for producing and purifying hepatitis a inactivated vaccine |
| CN102058882A (en) * | 2010-12-28 | 2011-05-18 | 北京民海生物科技有限公司 | Method of preparing hepatitis A inactivated vaccine |
| CN102174477A (en) * | 2010-12-28 | 2011-09-07 | 深圳康泰生物制品股份有限公司 | Hepatitis A virus strain SH and diploid cell adaptation method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101306199A (en) * | 2007-05-18 | 2008-11-19 | 江苏延申生物科技股份有限公司 | Method for producing and purifying hepatitis a inactivated vaccine |
| CN102058882A (en) * | 2010-12-28 | 2011-05-18 | 北京民海生物科技有限公司 | Method of preparing hepatitis A inactivated vaccine |
| CN102174477A (en) * | 2010-12-28 | 2011-09-07 | 深圳康泰生物制品股份有限公司 | Hepatitis A virus strain SH and diploid cell adaptation method thereof |
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| 马波 等: "应用凝胶过滤筛选纯化甲型肝炎病毒介质", 《中国生物制品学杂志》, vol. 17, no. 2, 31 March 2004 (2004-03-31) * |
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| CN107384873A (en) * | 2017-09-05 | 2017-11-24 | 成都汇宇生物技术有限公司 | The purification process of recombined adhenovirus |
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