CN102600468A - Preparation process of avian influenza virus split vaccine containing MF59 adjuvant - Google Patents
Preparation process of avian influenza virus split vaccine containing MF59 adjuvant Download PDFInfo
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- CN102600468A CN102600468A CN2012100512138A CN201210051213A CN102600468A CN 102600468 A CN102600468 A CN 102600468A CN 2012100512138 A CN2012100512138 A CN 2012100512138A CN 201210051213 A CN201210051213 A CN 201210051213A CN 102600468 A CN102600468 A CN 102600468A
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Abstract
The invention discloses a preparation process of an avian influenza virus split vaccine containing an MF59 adjuvant. The preparation process comprises the following steps of: inoculating an R1203 avian influenza virus strain onto a chicken embryo; inoculating viruses and culturing to obtain a virus liquid; inactivating viruses and purifying; cracking; purifying once again; removing bacteria and filtering, and the like. The preparation process has the beneficial effects that: the vaccine prepared from the MF59 adjuvant has high immunogenicity, and can generate higher antibody response; the immunity of children and the old who cannot generate immune response or have low immune response after inoculation of the conventional vaccine can be enhanced; cross immunity can be generated for different influenza virus subtypes; and on the premise of keeping constant immunity, the antigen content can be lowered, the content of foreign proteins capable of causing side reactions is lowered simultaneously, the side reaction of the vaccine is lower, higher safety is achieved, and the risks of virulence returning strength and variation are avoided.
Description
Technical field
The invention belongs to the vaccine production field, especially a kind of preparation technology who contains MF59 adjuvant bird flu virus split vaccine.
Background technology
Bird flu is the abbreviation of avian influenza, and it is the infectious disease that a kind of a kind of hypotype by influenza A virus (also claiming bird flu virus) causes, is decided to be category A infectious disease by International Office of Epizootics, claims fowl plague or European fowl plague again.It is a kind of acute infectious disease that causes by bird flu virus; Also can infect the mankind, the metainfective symptom of people mainly shows as hyperpyrexia, cough, watery nasal discharge, myalgia etc., and is most with serious pneumonia; Multiple organ failures such as the severe patient heart, kidney cause death, and case fatality rate is very high.
Aluminium adjuvant was used for vaccine existing more than 80 year, but immunosuppressant and intoxicating phenomenon occurred after having data to be presented at recently to use aluminium adjuvant in the vaccine production, and the too small absorption of use amount is incomplete, and excessive absorption fully but have safety.
Summary of the invention
The objective of the invention is to overcome the deficiency of prior art; And a kind of preparation technology of the MF59 of containing adjuvant bird flu virus split vaccine is provided; R1203 avian influenza strain is inoculated in Embryo Gallus domesticus, through cultivating, gathering in the crops production technologies such as viral liquid, inactivation of viruses and purification, cracking, repurity and process.The present invention adopts the MF59 adjuvant to produce the bird flu split vaccine, add this adjuvant than do not add adjuvant with add vaccine that aluminum hydroxide adjuvant produces more safety with effective.
The present invention solves the technical scheme that its technical problem adopts: the preparation technology of this MF59 of containing adjuvant bird flu virus split vaccine, and this method step is following:
(1), virus inoculation and cultivation: inoculate diluted bird flu work seed lot seed culture of viruses in chick embryo allantoic cavity, put 33～35 ℃ and cultivated 48～72 hours;
(2), virus results: screening live chickens embryo, put 2～8 ℃ of cold embryos of certain hour after, the results allantoic fluid in container, behind the clarification filtration by the calibrating of container sampling carrying out allantois results liquid;
(3), inactivation of virus: sampling and measuring protein content before the inactivation of virus; The control protein content carries out inactivation of virus in being no more than the scope of 5mg/ml; It is the formaldehyde of 200 μ g/ml that the allantoic fluid that contains virus of results adds final concentration; Put 2-8 ℃ of deactivation 15～24 hours, deactivation finishes respectively each inactivation of virus container to be sampled, and carries out inactivation of virus demonstration test and bacteria endotoxin content and measures;
(4) virus results liquid concentrates: the results liquid after the deactivation uses molecular cut off to be no more than 10 times as the ultrafilter membrane ultrafiltration and concentration of 1000KD through clarifying treatment then;
(5), viral purification:
(5.1) sucrose rate zonal centrifugation: when static, pump into 50～60% sucrose solution, regulate machine rotational speed simultaneously, be continuously pumped into viral liquid to 35000r/min～40000r/min with the amount that is no more than centrifugal chamber volume 50%; Sample introduction finishes; After pumping into buffer, regulate rotating speed, treat that machine comes to a complete stop to 0r/min; Under the 280nm wavelength monitoring, collect viral peak;
(5.2) column chromatography: with Sephrose-4FF gel column chromatography purification; Applied sample amount is no more than 8% of gel volume, and eluent is the PBS of 0.01mol/L, pH7.2; Last appearance and eluting linear flow rate are 5～6mm/min; The detection wavelength is 280nm, collects first peak, and protein content determination is carried out in sampling behind the purification;
(6), virolysis: it is 0.3～0.5%Triton X-100 and sodium deoxycholate that the viral liquid behind the purification adds final concentration, and mixing acts on 90～120 minutes down for rearmounted 20～30 ℃, and gradient centrifugation is 2 hours then, under the 280nm wavelength monitoring, collects first peak;
(7), remove sugar and remove decomposition agent: the lysate after centrifugal is carried out ultrafiltration and concentration with aseptic 0.01M pH7.2PBS solution; To remove sucrose and further to remove decomposition agent; Finally be concentrated into suitable liquid measure; Sampling bacterial detection endotoxin content and microbial limit, the control bacterial endotoxin is not higher than 50EU/ml, and microbial limit detects less demanding in 10cfu/ml;
(8), aseptic filtration: the lysate behind the purification is unit price stock solution through the membrane filtration in 0.22 μ m aperture, and unit price stock solution should place 2-8 ℃ of preservation.
Further, the semi-finished product preparation: according to the semi-finished product total stock solution consumption of being prepared, making hemagglutinin content is 30 μ g/ml; Amount by 50% adds the MF59 adjuvant; Add Buffer solution again, mixing is after the membrane filtration in 0.22 μ m aperture is semi-finished product.
The effect that the present invention is useful is:
1. the vaccine immunogenicity with the preparation of MF59 adjuvant is strong, can produce higher antibody response;
2. can not produce immunne response to inoculation behind the conventional vaccine or lower child and the old people of immunne response can strengthen its immunity;
3. can produce cross immunity to different influenza virus sub-strains;
4. keeping to reduce antigenic content under the constant prerequisite of immunity, reduced the foreign protein content that can cause side reaction simultaneously, thereby the side reaction of vaccine is lower, safety is better, virulence can occur and return danger strong and variation.
The specific embodiment
Below in conjunction with embodiment the present invention is described further:
The present invention adopts the MF59 adjuvant to prepare the bird flu split vaccine.MF59 is the best adjuvant that generally acknowledge in the present whole world, is a kind of oil-in-water Emulsion, comprises 1% zamene, and 0.5%Tween80 and 0.5% 3 oleic acid gather the O/w emulsion of Pyrusussuriensis fat.It can excite the immunne response that has failed; Strengthen the immunne response of body to virus; Make body produce more powerful immune protection effectiveness; MF59 has been applied to abroad prove the immune efficacy that can strengthen child and old people in the influenza vaccines, and can have produced cross immunity to different influenza virus sub-strains.
This preparation technology who contains MF59 adjuvant bird flu virus split vaccine of the present invention, this method step is following:
(1), virus inoculation and cultivation: inoculate diluted bird flu work seed lot seed culture of viruses in chick embryo allantoic cavity, put 33～35 ℃ and cultivated 48～72 hours;
(2), virus results: screening live chickens embryo, put 2～8 ℃ of cold embryos of certain hour after, the results allantoic fluid in container, behind the clarification filtration by the calibrating of container sampling carrying out allantois results liquid;
(3), inactivation of virus: sampling and measuring protein content before the inactivation of virus; The control protein content carries out inactivation of virus in being no more than the scope of 5mg/ml; It is the formaldehyde of 200 μ g/ml that the allantoic fluid that contains virus of results adds final concentration; Put 2-8 ℃ of deactivation 15～24 hours, deactivation finishes respectively each inactivation of virus container to be sampled, and carries out inactivation of virus demonstration test and bacteria endotoxin content and measures;
(4) virus results liquid concentrates: the results liquid after the deactivation uses molecular cut off to be no more than 10 times as the ultrafilter membrane ultrafiltration and concentration of 1000KD through clarifying treatment then;
(5), viral purification:
(5.1) sucrose rate zonal centrifugation: when static, pump into 50～60% sucrose solution, regulate machine rotational speed simultaneously, be continuously pumped into viral liquid to 35000r/min～40000r/min with the amount that is no more than centrifugal chamber volume 50%; Sample introduction finishes; After pumping into buffer, regulate rotating speed, treat that machine comes to a complete stop to 0r/min; Under the 280nm wavelength monitoring, collect viral peak;
(5.2) column chromatography: with Sephrose-4FF gel column chromatography purification; Applied sample amount is no more than 8% of gel volume, and eluent is the PBS of 0.01mol/L, pH7.2; Last appearance and eluting linear flow rate are 5～6mm/min; The detection wavelength is 280nm, collects first peak, and protein content determination is carried out in sampling behind the purification;
(6), virolysis: it is 0.3～0.5%Triton X-100 and sodium deoxycholate that the viral liquid behind the purification adds final concentration, and mixing acts on 90～120 minutes down for rearmounted 20～30 ℃, and gradient centrifugation is 2 hours then, under the 280nm wavelength monitoring, collects first peak;
(7), remove sugar and remove decomposition agent: the lysate after centrifugal is carried out ultrafiltration and concentration with aseptic 0.01M pH7.2PBS solution; To remove sucrose and further to remove decomposition agent; Finally be concentrated into suitable liquid measure; Sampling bacterial detection endotoxin content and microbial limit, the control bacterial endotoxin is not higher than 50EU/ml, and microbial limit detects less demanding in 10cfu/ml;
(8), aseptic filtration: the lysate behind the purification is unit price stock solution through the membrane filtration in 0.22 μ m aperture, and unit price stock solution should place 2-8 ℃ of preservation.
Further, the semi-finished product preparation: according to the semi-finished product total stock solution consumption of being prepared, making hemagglutinin content is 30 μ g/ml; Amount by 50% adds the MF59 adjuvant; Add Buffer solution again, mixing is after the membrane filtration in 0.22 μ m aperture is semi-finished product.
Each item inspection item are after detecting, and result's demonstration all meets the NF standard.
Annotate: the percent concentration of mentioning among the present invention, except that what mark especially, other are concentration of volume percent.
Except that the foregoing description, the present invention can also have other embodiments.All employings are equal to the technical scheme of replacement or equivalent transformation formation, all drop on the protection domain of requirement of the present invention.
Claims (2)
1. preparation technology who contains MF59 adjuvant bird flu virus split vaccine, it is characterized in that: this method step is following:
(1), virus inoculation and cultivation: inoculate diluted bird flu work seed lot seed culture of viruses in chick embryo allantoic cavity, put 33～35 ℃ and cultivated 48～72 hours;
(2), virus results: screening live chickens embryo, put 2～8 ℃ of cold embryos of certain hour after, the results allantoic fluid in container, behind the clarification filtration by the calibrating of container sampling carrying out allantois results liquid;
(3), inactivation of virus: sampling and measuring protein content before the inactivation of virus; The control protein content carries out inactivation of virus in being no more than the scope of 5mg/ml; It is the formaldehyde of 200 μ g/ml that the allantoic fluid that contains virus of results adds final concentration; Put 2-8 ℃ of deactivation 15～24 hours, deactivation finishes respectively each inactivation of virus container to be sampled, and carries out inactivation of virus demonstration test and bacteria endotoxin content and measures;
(4) virus results liquid concentrates: the results liquid after the deactivation uses molecular cut off to be no more than 10 times as the ultrafilter membrane ultrafiltration and concentration of 1000KD through clarifying treatment then;
(5), viral purification:
(5.1) sucrose rate zonal centrifugation: when static, pump into 50～60% sucrose solution, regulate machine rotational speed simultaneously, be continuously pumped into viral liquid to 35000r/min～40000r/min with the amount that is no more than centrifugal chamber volume 50%; Sample introduction finishes; After pumping into buffer, regulate rotating speed, treat that machine comes to a complete stop to 0r/min; Under the 280nm wavelength monitoring, collect viral peak;
(5.2) column chromatography: with Sephrose-4FF gel column chromatography purification; Applied sample amount is no more than 8% of gel volume, and eluent is the PBS of 0.01mol/L, pH7.2; Last appearance and eluting linear flow rate are 5～6mm/min; The detection wavelength is 280nm, collects first peak, and protein content determination is carried out in sampling behind the purification;
(6), virolysis: it is 0.3～0.5%Triton X-100 and sodium deoxycholate that the viral liquid behind the purification adds final concentration, and mixing acts on 90～120 minutes down for rearmounted 20～30 ℃, and gradient centrifugation is 2 hours then, under the 280nm wavelength monitoring, collects first peak;
(7), remove sugar and remove decomposition agent: the lysate after centrifugal is carried out ultrafiltration and concentration with aseptic 0.01M pH7.2PBS solution; To remove sucrose and further to remove decomposition agent; Finally be concentrated into suitable liquid measure; Sampling bacterial detection endotoxin content and microbial limit, the control bacterial endotoxin is not higher than 50EU/ml, and microbial limit detects less demanding in 10cfu/ml;
(8), aseptic filtration: the lysate behind the purification is unit price stock solution through the membrane filtration in 0.22 μ m aperture, and unit price stock solution should place 2-8 ℃ of preservation.
2. the preparation technology who contains MF59 adjuvant bird flu virus split vaccine according to claim 1; It is characterized in that: semi-finished product preparation: according to the semi-finished product total stock solution consumption of being prepared; Making hemagglutinin content is 30 μ g/ml, and the amount by 50% adds the MF59 adjuvant, adds Buffer solution again; Mixing is after the membrane filtration in 0.22 μ m aperture is semi-finished product.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| RU2584594C1 (en) * | 2015-07-02 | 2016-05-20 | Федеральное государственное унитарное предприятие "Санкт-Петербургский научно-исследовательский институт вакцин и сывороток и предприятие по производству бактерийных препаратов" Федерального медико-биологического агентства (ФГУП СПбНИИВС ФМБА России) | Inactivated split influenza vaccine and preparation method thereof |
| CN107779441A (en) * | 2017-11-10 | 2018-03-09 | 河南后羿生物工程股份有限公司 | A kind of concentrating and purifying process of avian influenza antigen, avian influenza antigen |
| CN108676781A (en) * | 2018-05-18 | 2018-10-19 | 山东信得动物疫苗有限公司 | A kind of industrialized purification method of recombinant fowl influenza virus |
| CN110664749A (en) * | 2019-10-21 | 2020-01-10 | 上海市皮肤病医院 | MF 59-based entrapped antigen nanoemulsion and preparation method and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006113214A2 (en) * | 2005-04-11 | 2006-10-26 | The Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services, Centers For Disease Control And Prevention | Vaccine against pandemic strains of influenza viruses |
| CN101623500A (en) * | 2009-07-28 | 2010-01-13 | 中国农业科学院上海兽医研究所 | Application of oil emulsion adjuvant |
| CN102133399A (en) * | 2011-03-09 | 2011-07-27 | 浙江天元生物药业有限公司 | Novel process for preparing influenza virus split vaccine |
-
2012
- 2012-03-01 CN CN2012100512138A patent/CN102600468A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006113214A2 (en) * | 2005-04-11 | 2006-10-26 | The Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services, Centers For Disease Control And Prevention | Vaccine against pandemic strains of influenza viruses |
| CN101623500A (en) * | 2009-07-28 | 2010-01-13 | 中国农业科学院上海兽医研究所 | Application of oil emulsion adjuvant |
| CN102133399A (en) * | 2011-03-09 | 2011-07-27 | 浙江天元生物药业有限公司 | Novel process for preparing influenza virus split vaccine |
Non-Patent Citations (3)
| Title |
|---|
| SURENDER KHURANA ET AL: "Vaccines with MF59 Adjuvant Expand the Antibody Repertoire to Target Protective Sites of Pandemic Avian H5N1 Influenza Virus", 《SCI TRANSL MED》 * |
| 何蕊等: "高致病性人禽流感H5N1 疫苗滴鼻免疫应答效果的初步研究", 《免疫学杂志》 * |
| 杨忠东等: "禽流感H5N1灭活疫苗与不同纳米化佐剂联合免疫研究", 《2011年中国生物制品年会暨第十一次全国生物制品学术研讨会》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| RU2584594C1 (en) * | 2015-07-02 | 2016-05-20 | Федеральное государственное унитарное предприятие "Санкт-Петербургский научно-исследовательский институт вакцин и сывороток и предприятие по производству бактерийных препаратов" Федерального медико-биологического агентства (ФГУП СПбНИИВС ФМБА России) | Inactivated split influenza vaccine and preparation method thereof |
| CN107779441A (en) * | 2017-11-10 | 2018-03-09 | 河南后羿生物工程股份有限公司 | A kind of concentrating and purifying process of avian influenza antigen, avian influenza antigen |
| CN107779441B (en) * | 2017-11-10 | 2021-04-16 | 河南后羿生物工程股份有限公司 | Concentration and purification method of avian influenza antigen and avian influenza antigen |
| CN108676781A (en) * | 2018-05-18 | 2018-10-19 | 山东信得动物疫苗有限公司 | A kind of industrialized purification method of recombinant fowl influenza virus |
| CN110664749A (en) * | 2019-10-21 | 2020-01-10 | 上海市皮肤病医院 | MF 59-based entrapped antigen nanoemulsion and preparation method and application thereof |
| CN110664749B (en) * | 2019-10-21 | 2021-07-30 | 上海市皮肤病医院 | MF 59-based entrapped antigen nanoemulsion and preparation method and application thereof |
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Application publication date: 20120725 |