CN103735521A - Animal lyophilized inactivated vaccine and preparation process thereof - Google Patents
Animal lyophilized inactivated vaccine and preparation process thereof Download PDFInfo
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- CN103735521A CN103735521A CN201310548699.0A CN201310548699A CN103735521A CN 103735521 A CN103735521 A CN 103735521A CN 201310548699 A CN201310548699 A CN 201310548699A CN 103735521 A CN103735521 A CN 103735521A
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Abstract
The invention relates to an animal lyophilized inactivated vaccine and a preparation process thereof. The animal lyophilized inactivated vaccine comprises a separately-packaged immune adjuvant or diluent and a separately-packaged inactivated vaccine produced through vacuum freeze-drying, wherein vaccine antigen is inactivated and then is subjected to a vacuum freeze-drying process to obtain the inactivated vaccine, and the immune adjuvant or diluent has a good emulsifying performance. Compared with the animal lyophilized inactivated vaccine and the preparation process in the prior art, the animal lyophilized inactivated vaccine and the preparation process of the present invention have the following characteristics that: the vaccine can be subjected to freeze-drying to obtain the powdery or massive vaccine according to the immunizing dose of the single variety vaccine, the corresponding immune adjuvant is adopted to carry out preparation and use on site during use, and the fixed formulation is formed in a short time; farmers can selectively combine vaccines according to the requirements so as to well solve the problem of difficultly-achieved combined vaccine antigen content and further solve the defects of multiple immunization and too high immunizing dose of the traditional inactivated vaccine; and the difficult problem of short storage time of the inactivated vaccine is solved.
Description
Technical field
The present invention relates to belong to animal vaccines and manufacture field, especially relate to lyophilizing inactivated vaccine and preparation technology thereof for a kind of animal.
Background technology
The play vital effect of animal vaccine to prevention Animal diseases, the kind of animal vaccine comprises lyophilizing attenuated live vaccine, inactivated vaccine (liquid) now.
At present, animal is after antigen deactivation, to mix according to certain ratio with immunological adjuvant with inactivated vaccine, enter Over emulsfication (high speed shear, grinding), make the suspendible liquid (oil-in-water, Water-In-Oil, W/O/W, hydrophilic etc.) with certain dosage form, be finally divided in bottle.Emulsifying good in order to keep the particular dosage form of vaccine, require to preserve, during transportation 2～8 ℃ of conditions, if emulsifying process is bad, or storage temperature changes, vaccine dosage form easily changes, and vaccine quality is impaired, therefore, this type of vaccine shelf-life often only has 12 months.The cost that has so not only increased transportation and preserved, and enterprise is when dull sale, and vaccine easily produces expired scrapping, cause economic loss.In addition, once form fixed dosage form, be difficult to combine again use with other vaccines, when animal needs prevention of various diseases, need spininess immunity, be not only difficult to operation, and bring and repeatedly stress to animal.
Along with the continuous appearance of animal pathogenic, need the epidemic disease of prevention more and more, the kind of vaccine is more and more, and complicated immune programme for children makes raiser at a loss as to what to do, is difficult to arrange the immune time, and bigeminy vaccine and multiple vaccines arise at the historic moment like this.But the content of every kind of vaccine antigen has the bad assurance of certain limit in multiple vaccines, and a multiple vaccines of research and development need to drop into huge human and material resources, financial resources, energy.The compound market demand not necessarily again after researching and developing successfully.
Single for the production of the adjuvant dosage form of animal inactivated vaccine in the past, mainly take Water-In-Oil as main, main material is mineral white oil, department's basis 80, Tween 80, aluminium stearate etc.Emulsifying process is loaded down with trivial details, need to be by shearing equipment at a high speed, inactivated vaccine after emulsifying, be referred to as oil emulsion inactivated vaccine, this inactivated vaccine, easily layering, affects product quality and immune effect, have during immune animal simultaneously larger stress, such as side reactions such as vomitting, tremble, spit out white foams.
Along with the appearance of novel adjuvant, simplified the program of emulsifying, correspondingly expedited the emergence of new vaccine type and preparation technology.Immunologic adjuvant type has comprised Water-In-Oil, oil-in-water, W/O/W, hydrophilic etc. at present, and immunologic adjuvant not only kind has increased, and can shake up the emulsifying program that replaces high speed shear by low speed, realizes with freeze dried vaccine now with the current.
Novel lyophilizing inactivated vaccine, is first lyophilized into powdery or bulk according to the immunizing dose of single variety vaccine, in the time of use, carries out now with the currently with corresponding immunological adjuvant, forms at short notice fixing dosage form.Raiser, according to self-demand, combines use vaccine selectively, and one has well solved the implacable problem of connection Seedling antigenic content, and two have solved the puzzlement that traditional inactivated vaccine immune time is too much, immunizing dose is excessive.
Summary of the invention
Object of the present invention is exactly to provide a kind of arrangement that facilitates vaccine in order to overcome the defect of above-mentioned prior art existence, and arbitrarily combination connection Seedling has reduced lyophilizing inactivated vaccine and the preparation technology thereof for animal that transport and store cost simultaneously.
Object of the present invention can be achieved through the following technical solutions:
Animal lyophilizing inactivated vaccine, comprises the separately immunological adjuvant of packing or the inactivated vaccine of diluent and vacuum lyophilization production,
Described inactivated vaccine, then is obtained through vacuum freeze-drying technique by vaccine antigen after deactivation,
Described immunological adjuvant or diluent have good emulsifiability.
That described vaccine antigen comprises is bacillary, viral, the composition for animal vaccine of the pathogenic microorganism between antibacterial or virus and expression, and animal comprises all animal species except the mankind.
Described vaccine antigen is the viral antigens such as PCV-2, PRRS, epidemic diarrhea, influenza virus, the bacterial antigens such as the secondary haemophilus of pig, streptococcus, escherichia coli, other antigens such as mycoplasma, coccidiosis, Eperythrozoon.
Described vaccine antigen is through chemical ablation method or physical deactivation method.
Described physical deactivation method comprises electric deactivation, pulse deactivation, light deactivation, ultrasound wave deactivation or its combination.
Described vacuum lyophilization 1:1～5 mixes the vaccinogen liquid after deactivation and freeze drying protectant by volume, according to the freeze-drying curve of this kind of vaccine, carries out vacuum lyophilization.
The dosage form of described immunological adjuvant comprises oil-in-water type, W/O/W type, water-in-oil type or hydrophilic adjuvant, and described diluent is the diluent that immunological adjuvant configuration obtains.
In described immunological adjuvant, including immunity increases material, the material of this material for stimulating humoral immunization and cellular immunization to strengthen, the material of material, polymerization or the coupled antigen granule of storage or slow release antigenic action.
It is the material that meets deactivation freeze dried vaccine that aluminium hydroxide gel, white oil immunological adjuvant AD04 etc. have well emulsify characteristic for immunity increases material that described immunity increases material.
Animal adopts following steps by the preparation technology of lyophilizing inactivated vaccine:
Inactivated vaccine is carried out after deactivation through physical method or chemical method by vaccine antigen, again the vaccinogen liquid after deactivation and freeze drying protectant are mixed 1:1～5 by volume, according to the freeze-drying curve of this kind of vaccine, carry out vacuum lyophilization and through vacuum freeze-drying technique, obtain inactivated vaccine again;
Immunological adjuvant or the diluent with good emulsifiability are separated to packing with inactivated vaccine, obtain animal lyophilizing inactivated vaccine.
Described inactivated vaccine and immunological adjuvant or diluent are preserved under 2-8 ℃ of conditioned disjunction room temperature.
Compared with prior art, the present invention can first be lyophilized into powdery or bulk according to the immunizing dose of single variety vaccine, in the time of use, carries out now with the currently with corresponding immunological adjuvant, forms at short notice fixing dosage form.Raiser, according to self-demand, combines use vaccine selectively, and one has well solved the implacable problem of connection Seedling antigenic content, and two have solved the puzzlement that traditional inactivated vaccine immune time is too much, immunizing dose is excessive.
The specific embodiment
Below in conjunction with specific embodiment, the present invention is described in detail.
Embodiment 1
The preparation of Streptococcus suis lyophilizing inactivated vaccine
1. antigen preparation is inoculated into Streptococcus suis seeding in the buffering improvement martin's bouillon of pH value 7.2-7.6 in 5-15% ratio.37 ℃ of temperature, stirring at low speed, obstructed vapour is cultivated.Cultivate and start, the ratio in 0.2% adds 50% glucose solution, adds the Ox blood serum of 2-5% to carry out feed supplement.Cultivate after 4-6 hour, the supplementary improvement of the buffering containing 0.2% glucose, 2-5% Ox blood serum martin's bouillon is appropriate, within 4 hours before cultivation finishes, stops feed supplement, and whole content glucose is no more than 0.2%.Cultivate after 12-18 hour, results culture fluid, obtains vaccine antigen bacterium liquid.
2. deactivation is deactivation 48 hours under 0.1～0.5%, 37 ℃ of conditions to adding formalin to make final concentration in the qualified antigen bacteria liquid of antigenic content, steriling test, safety examination, and results bacterium liquid is placed in-20 ℃ of freezers and saves backup.
3. lyophilizing is made homogenate by the aseptic inactivation antigen liquid of check in the freeze drying protectant of 1:1～5 ratio; add appropriate antibiotic; fully mix, after quantitative separating, by suitable freeze-dry process curve, carry out lyophilisation rapidly, be prepared into lyophilizing inactivated vaccine finished product.
Embodiment 2
The preparation of porcine circovirus 2 type lyophilizing inactivated vaccine
1, antigen preparation is inoculated in porcine circovirus 2 type (PCV-2) on well-grown PK15 cell, and 37 ℃ of condition low suspensions are cultivated, and cultivate after 24～72 hours harvesting culture fluid.
2, deactivation is to being deactivation 48 hours under 0.1～0.5%, 37 ℃ of conditions through adding suitable inactivator to make final concentration in the qualified cell culture fluid of antigenic content, steriling test, and results antigen liquid is placed in-20 ℃ of freezers and saves backup.
3, lyophilizing is made homogenate by the aseptic inactivation antigen liquid of check in the freeze drying protectant of 1:1～5 ratio; add appropriate antibiotic; fully mix, after quantitative separating, by suitable freeze-dry process curve, carry out lyophilisation rapidly, be prepared into lyophilizing inactivated vaccine finished product.
Embodiment 3
The preparation of pseudorabies lyophilizing inactivated vaccine
1. antigen preparation is inoculated in PRV (Pseudorabies virus) in well-grown monolayer chick embryo fibroblast, 37 ℃ of rotating and culturing, every day observation of cell pathological changes situation, when cytopathy large 75% culture fluid of harvesting when above.
2. deactivation is to being deactivation 24～48 hours under 0.1～1%, 25～37 ℃ of conditions through adding suitable inactivator to make final concentration in the qualified cell culture fluid of antigenic content, steriling test, and results antigen liquid is placed in-20 ℃ of freezers and saves backup.
3. lyophilizing is made homogenate by the aseptic inactivation antigen liquid of check in the freeze drying protectant of 1:1～5 ratio, adds appropriate antibiotic, fully mixes, and carries out rapidly lyophilisation after quantitative separating, is prepared into lyophilizing inactivated vaccine finished product.
Embodiment 4
The preparation of pig erysipelas-pig pasteurella multocida bigeminy lyophilizing inactivated vaccine
1. antigen preparation
1.1 bacillus rhusiopathiae suis antigen preparations add pig erysipelas seed liquor by 1%～3% of culture medium total amount, by 1%～5%, add cracking blood cell whole blood simultaneously, or 0.1% tween 80 and 0.3%～0.7% glucose, cultivate 28～48 hours for 37 ℃, results culture fluid, and carry out count plate, count results is as joining Seedling reference.
1.2 pig pasteurella multocida antigen preparations are inoculated in martin's bouillon culture medium by pig pasteurella multocida seed, inoculum density is that 1%～5%, 37 ℃ of total amount are cultivated 12～20 hours, results culture fluid, and carry out count plate, count results is as joining Seedling reference.
2. join shoot root according to the count plate result of two kinds of antigens, in the ratio of bacillus rhusiopathiae suis and pig pasteurella multocida 1～2:1, mix, shake up.Save backup to 2～8 ℃, the time is no more than 2 two days.
3. deactivation is in the bacillus rhusiopathiae suis through being up to the standards, pig pasteurella multocida antigen liquid, and the concentration by 0.1%～0.3% slowly adds suitable inactivator, and under 25～37 ℃ of conditions, deactivation is 48 hours, and results antigen liquid is placed in-20 ℃ of freezers and saves backup.
4. lyophilizing is made homogenate by the aseptic inactivation antigen liquid of check in the freeze drying protectant of 1:1～5 ratio, adds appropriate antibiotic, fully mixes, and carries out rapidly lyophilisation after quantitative separating, is prepared into lyophilizing inactivated vaccine finished product.
Embodiment 5
Aluminium hydroxide gel normal saline dilution agent preparation
1, preparation adds aluminium hydroxide gel 200g in the normal saline of 800 milliliters, after mixing, is divided in 100 milliliters of vials or plastic bottle.
2, autoclave cabinet is put in the aluminium hydroxide gel normal saline dilution agent that sterilizing subpackage will minute install, and 100～126 ℃ of sterilizings 1～3 hour, label after sterilizing, are stored in 2～8 ℃, 2 years shelf-lifves.The aluminium hydroxide gel normal saline dilution agent preparing.
Embodiment 6
The preparation of nanometer adjuvant AD04 diluent
1. preparation is by after Nano type AD04 adjuvant autoclaving, and 8%～15% ratio adds in sterile saline by volume, with non-foaming low speed, stirs 45～60 minutes.Then, divide and be filled in 20mL or 100mL bottle, label, to 2-8 ℃ of preservation, 3 years shelf-lifves, finished product.
Embodiment 7
Animal lyophilizing inactivated vaccine, comprise the separately immunological adjuvant of packing or the inactivated vaccine of diluent and vacuum lyophilization production, inactivated vaccine, then is obtained through vacuum freeze-drying technique by vaccine antigen after deactivation, and immunological adjuvant or diluent have good emulsifiability.
Vaccine antigen is the viral antigen of PCV-2, and vaccine antigen, through electric inactivation treatment, then, by the vaccinogen liquid after deactivation and freeze drying protectant 1:1 mixing by volume, carries out vacuum lyophilization according to the freeze-drying curve of this kind of vaccine.
The dosage form of immunological adjuvant is oil-in-water type adjuvant, comprising there being aluminium hydroxide gel, has well emulsify characteristic and meets deactivation freeze dried vaccine.
Animal adopts following steps by the preparation technology of lyophilizing inactivated vaccine:
By inactivated vaccine by vaccine antigen after physical method carries out deactivation, by the vaccinogen liquid after deactivation and freeze drying protectant 1:1 mixing by volume, according to the freeze-drying curve of this kind of vaccine, carry out vacuum lyophilization and through vacuum freeze-drying technique, obtain inactivated vaccine more again;
Immunological adjuvant or the diluent with good emulsifiability are separated to packing with inactivated vaccine, obtain animal lyophilizing inactivated vaccine, inactivated vaccine and immunological adjuvant or diluent are preserved under 2-8 ℃ of conditioned disjunction room temperature.
Embodiment 8
Animal lyophilizing inactivated vaccine, comprise the separately immunological adjuvant of packing or the inactivated vaccine of diluent and vacuum lyophilization production, inactivated vaccine, then is obtained through vacuum freeze-drying technique by vaccine antigen after deactivation, and immunological adjuvant or diluent have good emulsifiability.
Vaccine antigen is the bacterial antigen of the secondary haemophilus of pig, through pulse inactivation treatment, then, by the vaccinogen liquid after deactivation and freeze drying protectant 1:3 mixing by volume, according to the freeze-drying curve of this kind of vaccine, carries out vacuum lyophilization.The dosage form of immunological adjuvant is W/O/W type.In immunological adjuvant, including immunity increases material,, in the present embodiment, be white oil immunological adjuvant AD04, there is the material that meets deactivation freeze dried vaccine of well emulsify characteristic.
Animal adopts following steps by the preparation technology of lyophilizing inactivated vaccine:
By inactivated vaccine by vaccine antigen after physical method carries out deactivation, by the vaccinogen liquid after deactivation and freeze drying protectant 1:3 mixing by volume, according to the freeze-drying curve of this kind of vaccine, carry out vacuum lyophilization and through vacuum freeze-drying technique, obtain inactivated vaccine more again;
Immunological adjuvant or the diluent with good emulsifiability are separated to packing with inactivated vaccine, obtain animal lyophilizing inactivated vaccine.Inactivated vaccine and immunological adjuvant or diluent are preserved under 2-8 ℃ of conditioned disjunction room temperature.
Embodiment 9
Animal lyophilizing inactivated vaccine, comprise the separately immunological adjuvant of packing or the inactivated vaccine of diluent and vacuum lyophilization production, inactivated vaccine, then is obtained through vacuum freeze-drying technique by vaccine antigen after deactivation, and immunological adjuvant or diluent have good emulsifiability.
Vaccine antigen is mycoplasma antigen, through chemical ablation, processes, and then, by the vaccinogen liquid after deactivation and freeze drying protectant 1:5 mixing by volume, according to the freeze-drying curve of this kind of vaccine, carries out vacuum lyophilization.The dosage form of immunological adjuvant is water-in-oil type, and in immunological adjuvant, including immunity increases material, the material of this material for stimulating humoral immunization and cellular immunization to strengthen, the material of material, polymerization or the coupled antigen granule of storage or slow release antigenic action.
Animal adopts following steps by the preparation technology of lyophilizing inactivated vaccine:
By inactivated vaccine by vaccine antigen after chemical method is carried out deactivation, by the vaccinogen liquid after deactivation and freeze drying protectant 1:5 mixing by volume, according to the freeze-drying curve of this kind of vaccine, carry out vacuum lyophilization and through vacuum freeze-drying technique, obtain inactivated vaccine more again;
Immunological adjuvant or the diluent with good emulsifiability are separated to packing with inactivated vaccine, obtain animal lyophilizing inactivated vaccine.Inactivated vaccine and immunological adjuvant or diluent are preserved under 2-8 ℃ of conditioned disjunction room temperature.
Claims (10)
1. animal lyophilizing inactivated vaccine, is characterized in that, comprises the separately immunological adjuvant of packing or the inactivated vaccine of diluent and vacuum lyophilization production,
Described inactivated vaccine, then is obtained through vacuum freeze-drying technique by vaccine antigen after deactivation,
Described immunological adjuvant or diluent have good emulsifiability.
2. animal according to claim 1 lyophilizing inactivated vaccine, it is characterized in that, that described vaccine antigen comprises is bacillary, viral, the composition for animal vaccine of the pathogenic microorganism between antibacterial or virus and expression, and animal comprises all animal species except the mankind.
3. animal according to claim 2 lyophilizing inactivated vaccine, it is characterized in that, described vaccine antigen is the viral antigen of PCV-2, PRRS, epidemic diarrhea or influenza virus, or be the secondary haemophilus of pig, streptococcus or colibacillary bacterial antigen, or be mycoplasma, coccidiosis or eperytozoa isoantigen.
4. animal according to claim 1 lyophilizing inactivated vaccine, is characterized in that, described vaccine antigen is through chemical ablation method or physical deactivation method.
5. animal according to claim 4 lyophilizing inactivated vaccine, is characterized in that, described physical deactivation method comprises electric deactivation, pulse deactivation, light deactivation, ultrasound wave deactivation or its combination.
6. animal according to claim 1 lyophilizing inactivated vaccine, is characterized in that, described vacuum lyophilization 1:1～5 mixes the vaccinogen liquid after deactivation and freeze drying protectant by volume, according to the freeze-drying curve of this kind of vaccine, carries out vacuum lyophilization.
7. animal according to claim 1 lyophilizing inactivated vaccine, it is characterized in that, the dosage form of described immunological adjuvant comprises oil-in-water type, W/O/W type, water-in-oil type or hydrophilic adjuvant, and described diluent is the diluent that immunological adjuvant configuration obtains.
8. according to the lyophilizing inactivated vaccine of the animal described in claim 1 or 7, it is characterized in that, in described immunological adjuvant, include immunity and increase material, the material of this material for stimulating humoral immunization and cellular immunization to strengthen, the material of material, polymerization or the coupled antigen granule of storage or slow release antigenic action.
9. animal according to claim 8 lyophilizing inactivated vaccine, is characterized in that, it is the material that meets deactivation freeze dried vaccine with well emulsify characteristic that described immunity increases material, comprises aluminium hydroxide gel or white oil immunological adjuvant.
10. according to the animal described in any one in claim 1-9, use the preparation technology of lyophilizing inactivated vaccine, it is characterized in that these process using following steps:
Inactivated vaccine is carried out after deactivation through physical method or chemical method by vaccine antigen, again the vaccinogen liquid after deactivation and freeze drying protectant are mixed 1:1～5 by volume, according to the freeze-drying curve of this kind of vaccine, carry out vacuum lyophilization and through vacuum freeze-drying technique, obtain inactivated vaccine again;
Immunological adjuvant or the diluent with good emulsifiability are separated to packing with inactivated vaccine, obtain animal lyophilizing inactivated vaccine, inactivated vaccine and immunological adjuvant or diluent are preserved under 2-8 ℃ of conditioned disjunction room temperature.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105462840A (en) * | 2015-12-10 | 2016-04-06 | 浙江卫信生物药业有限公司 | Method for preparing living freeze-dried body of mycoplasma orale |
| CN106109414A (en) * | 2016-07-28 | 2016-11-16 | 金宇保灵生物药品有限公司 | A kind of foot-and-mouth disease vaccine diluent and preparation method and application |
| CN113559274A (en) * | 2021-08-30 | 2021-10-29 | 河南兴华生物技术有限公司 | Freeze-drying protective agent for live vaccine of pig escherichia coli |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102065889A (en) * | 2008-04-16 | 2011-05-18 | 葛兰素史密丝克莱恩生物有限公司 | Vaccine |
| CN102099052A (en) * | 2008-04-16 | 2011-06-15 | 葛兰素史密丝克莱恩生物有限公司 | Vaccine |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102065889A (en) * | 2008-04-16 | 2011-05-18 | 葛兰素史密丝克莱恩生物有限公司 | Vaccine |
| CN102099052A (en) * | 2008-04-16 | 2011-06-15 | 葛兰素史密丝克莱恩生物有限公司 | Vaccine |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105462840A (en) * | 2015-12-10 | 2016-04-06 | 浙江卫信生物药业有限公司 | Method for preparing living freeze-dried body of mycoplasma orale |
| CN106109414A (en) * | 2016-07-28 | 2016-11-16 | 金宇保灵生物药品有限公司 | A kind of foot-and-mouth disease vaccine diluent and preparation method and application |
| CN106109414B (en) * | 2016-07-28 | 2019-07-16 | 金宇保灵生物药品有限公司 | A kind of aftosa vaccine dilution and the preparation method and application thereof |
| CN113559274A (en) * | 2021-08-30 | 2021-10-29 | 河南兴华生物技术有限公司 | Freeze-drying protective agent for live vaccine of pig escherichia coli |
| CN113559274B (en) * | 2021-08-30 | 2024-01-26 | 河南兴华生物技术有限公司 | Freeze-drying protective agent for live vaccine of escherichia coli |
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