CN102600470A - Compound adjuvant, vaccine containing the same, and its preparation method - Google Patents
Compound adjuvant, vaccine containing the same, and its preparation method Download PDFInfo
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- CN102600470A CN102600470A CN2012100875663A CN201210087566A CN102600470A CN 102600470 A CN102600470 A CN 102600470A CN 2012100875663 A CN2012100875663 A CN 2012100875663A CN 201210087566 A CN201210087566 A CN 201210087566A CN 102600470 A CN102600470 A CN 102600470A
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Abstract
The invention relates a compound adjuvant, which comprises mixed oil (white oil and palm oil) and mixed surfactant (polyoxyethylene oleate, polyoxyethylene alkyl ether and mannitol oleate), specifically (by weight percentage) white oil 70-90, palm oil 1-15, polyoxyethylene oleate 1-10, polyoxyethylene alkyl ether 1-10, and mannitol oleate 1-10. The compound adjuvant is mixed with antigen aqueous solution, and emulsified under stirring to obtain W/O/W type vaccine. The vaccine has the advantages of good stability, little side effects, and high practical application value.
Description
Technical field
The present invention relates to the vaccine adjuvant field, be specifically related to a kind of compound adjuvant, contain vaccine of this compound adjuvant and preparation method thereof.
Background technology
But oil adjuvant vaccine is the vaccine of known efficient hardening immunity; Be one of type that product is maximum on the at present domestic and international vaccine market, containing oil-adjuvant vaccine has three kinds of dosage forms: Water-In-Oil (W/O) type, oil-in-water (O/W) type and W/O/W (W/O/W) type.
Water-In-Oil (W/O) type its vaccine application is the most general, and method for preparing is divided 3 steps, at first Si Ben-85 is added in the white oil, and tween 80 adds in the antigen aqueous solution, and then the two mixture is carried out emulsifying is prepared from.The remarkable advantage of this dosage form is to obtain high-level persistent immunoenhancement result; Shortcoming is than thickness; Injection is difficulty; While usually causes serious inoculation side reaction, forms aseptic suppuration infringement and inoculation position granuloma on every side owing to white oil content in the vaccine is higher relatively, can produce outside visible edema, swelling, scleroma and necrosis.The research report is arranged, and Water-In-Oil (W/O) dosage form vaccine has the possibility of retention at inoculation position, thereby has hindered the use of vaccine.Animals such as pig, dog, cat are especially responsive to this dosage form vaccine, and this has also limited its application in production reality.
For reducing the oil content in the Emulsion, reduce viscosity, be easy to injection, develop novel adjuvant, and set up oil-in-water (O/W) dosage form vaccine.But its vaccine application of this dosage form is not general, only in the vaccine for man application facet commercialization is arranged, and is used for the making of influenza vaccines like the MF59 adjuvant, on animal vaccine, does not apply as yet.Reason is a little less than the immunogenicity of oil-in-water (O/W) dosage form vaccine, only produces more weak immunne response in the induced animal body, still can not satisfy the prevention and control demand of aquaculture eqpidemic disease.
W/O/W (W/O/W) dosage form vaccine has the advantage of w/o type and O/W type simultaneously, and effect is fine.Conventional way is to prepare the w/o type vaccine earlier, is prepared from through being distributed to aqueous phase for the second time again.Purpose is to reduce viscosity, not only has been convenient to inject but also have good immunogenicity.But difficulty is bigger on the technology of preparing, and vaccine stability is poor, if can overcome this shortcoming, and it is optimal emulsion vaccine.The selection of adjuvant prescription, i.e. oil and surfactant selection and use is the key of making W/O/W (W/O/W) dosage form vaccine.Best this type of adjuvant of generally acknowledging both at home and abroad is ISA206 (French SEPPIC company produce).Adopt the self emulsifying principle, directly with ISA206 adjuvant and antigen aqueous solution, emulsifying, one-step method can be prepared W/O/W dosage form vaccine, has solved the problem of W/O/W type emulsion vaccine preparation difficulty.Its weak point is that the W/O/W dosage form vaccine stability for preparing is also also unsatisfactory, and price is expensive especially.Patent of invention " a kind of self-emulsifying vaccine adjuvant and preparation method thereof " (ZL 200510037639.8) has been announced the adjuvant compound method and the vaccine production method of a series of W/O/W dosage form vaccines, still has during (1) field test side reaction to take place; (2) stable less than is 12 months, can not satisfy the production actual needs; (3) do not see its commercialization use yet.Therefore, set up stable, inexpensive W/O/W dosage form technology and have extremely important realistic meaning and market value.
Summary of the invention
The purpose of this invention is to provide a kind of compound adjuvant, contain the vaccine of this adjuvant, said compound adjuvant, contain the vaccine stable in properties of this adjuvant, side reaction is little, good immune effect.
The present invention provides a kind of compound adjuvant, is made up of miscella and mixed surfactant, and said miscella is that white oil and Petiolus Trachycarpi oil are formed, and said mixed surfactant is made up of polyoxyethylene oleic acid ether-ether, polyoxyethylene alkyl ether and mannitol oleate; The quality percentage composition of each component in compound adjuvant is:
White oil 70%-90%,
Petiolus Trachycarpi oil 1%-15%,
Polyoxyethylene oleic acid ether-ether 1%-10%,
Polyoxyethylene alkyl ether 1%-10%,
Mannitol oleate 1%-10%.
The better mass content of each component in compound adjuvant is:
White oil 75 ~ 85%,
Petiolus Trachycarpi oil 5 ~ 10%,
Polyoxyethylene oleic acid ether-ether 5 ~ 8%,
Polyoxyethylene alkyl ether 2 ~ 9%,
Mannitol oleate 1 ~ 5%.
The present invention also provides the method for preparing of said compound adjuvant, and is specific as follows: take by weighing white oil, Petiolus Trachycarpi oil, polyoxyethylene oleic acid ether-ether, polyoxyethylene alkyl ether and the mannitol oleate of recipe quantity respectively, mix homogeneously is sterilized then, promptly obtains compound adjuvant.
In addition, the present invention also provides the vaccine that contains said compound adjuvant, and this vaccine comprises the antigen aqueous solution, and the mass ratio of said antigen aqueous solution and compound adjuvant is 3:2～3.
The method for preparing of said vaccine is: compound adjuvant is preheated to 25～35 ℃, and antigenic solution is preheated to 15～35 ℃, then the two is mixed, and warp stirring or mechanical presses are processed the emulsification system of homogeneous, promptly get said vaccine.
The present invention utilizes polyoxyethylene oleic acid ether-ether, polyoxyethylene alkyl ether and 3 kinds of surfactants of mannitol oleate, uses the self emulsifying principle, processes the vaccine compound adjuvant of W/O/W dosage form.This adjuvant and antigen aqueous solution adopt methods such as hand, magnetic agitation or mechanical presses fully emulsified, process stable W/O/W dosage form vaccine.Successfully solved W/O/W dosage form vaccine production problem of difficult.In addition; (white oil comes out from Petroleum refining with the alternative part white oil of the Petiolus Trachycarpi oil of plant origin in the present invention; Can not be by organism metabolism); With novel low toxicity surfactant substitute tween, take charge of this etc. conventional surfactants, the toxicity of animal is significantly reduced, efficiently solve the problem of vaccine side reaction.Should method the emulsion vaccine of preparation not only be convenient to inject but also have good immunogenicity, compare with the W/O/W type emulsion vaccine of ISA206 made, its stability is better.
The specific embodiment
Further explain the present invention through embodiment below, but the present invention does not receive the restriction of these embodiment.
The preparation of embodiment 1 adjuvant
Prescription 1
White oil 80 g,
Petiolus Trachycarpi oil 10 g,
Polyoxyethylene oleic acid ether-ether 5 g,
Polyoxyethylene alkyl ether 2 g,
Mannitol oleate 3 g.
Prescription 2:
White oil 79 g,
Petiolus Trachycarpi oil 8 g,
Polyoxyethylene oleic acid ether-ether 7 g,
Polyoxyethylene alkyl ether 4 g,
Mannitol oleate 2 g.
Prescription 3:
White oil 77 g,
Petiolus Trachycarpi oil 5 g,
Polyoxyethylene oleic acid ether-ether 8 g,
Polyoxyethylene alkyl ether 9g,
Mannitol oleate 1 g.
Adopt following method to prepare compound adjuvant with prescription 1,2,3 respectively: to take by weighing white oil, Petiolus Trachycarpi oil, polyoxyethylene oleic acid ether-ether, polyoxyethylene alkyl ether and mannitol oleate respectively by formula ratio; Mix homogeneously then; Behind 115 ℃, 20min sterilization; Obtain compound adjuvant 1, compound adjuvant 2 and compound adjuvant 3 respectively by above-mentioned 3 kinds of prescriptions, room temperature preservation is subsequent use.
Embodiment 2The application of foot and mouth disease vaccine
(1) preparation of compound adjuvant
Make compound adjuvant 1, compound adjuvant 2 and compound adjuvant 3 by embodiment 1 method.
(2) preparation of Schweineseuche virus liquid
(2006-2008), related request prepares the Schweineseuche antigen aqueous solution of deactivation according to " biological quality quality standard compilation for animals ", and per 0.2 milliliter of viral liquid viral level answers>=10 before the deactivation
7.0LD
50
(3) preparation of foot and mouth disease vaccine
Under sterile working's situation, the compound adjuvant that above-mentioned (1) is made is heated to 30 ℃, gets the beaker that 15g puts into 50ml; The Schweineseuche inactivation of viruses liquid 15g of preparation in (2) is preheated to 25 ℃; Then with the two mixing, add the magnetic force rotor simultaneously and put on the magnetic stirring apparatus (containing heating function) and carry out stirring and emulsifying, rotating speed is 2000 rpms, temperature keeps 30 ℃, stirs 30 minutes, is emulsified into the homogeneous system.The vaccine that contains compound adjuvant 1, name vaccine 1-1; The vaccine that contains compound adjuvant 2, called after vaccine 1-2; Contain the vaccine called after vaccine 1-3 of compound adjuvant 3, be generically and collectively referred to as vaccine 1 of the present invention.
Same method substitutes the present invention's " compound adjuvant " with the ISA206 adjuvant, preparation control vaccine, name control vaccine A.
(4) vaccine physical behavior and immunocompetent comparison
The comparison of table 1 vaccine 1 of the present invention and control vaccine A
| Project | Vaccine 1-1 | Vaccine 1-2 | Vaccine 1-3 | Control vaccine A |
| Outward appearance | Peach even emulsion | Peach even emulsion | Peach even emulsion | Peach even emulsion |
| Dosage form | The W/O/W type drips a little vaccine in cleaning cold water surface, is the cloud diffusion | The W/O/W type drips a little vaccine in cleaning cold water surface, is the cloud diffusion | The W/O/W type drips a little vaccine in cleaning cold water surface, is the cloud diffusion | The W/O/W type drips a little vaccine in cleaning cold water surface, is the cloud diffusion |
| Centrifugal stability | 3000 rev/mins, 15 minutes, no water was separated out | 3000 rev/mins, 15 minutes, no water was separated out | 3000 rev/mins, 15 minutes, there is the water of 0.1ml to separate out | 3000 rev/mins, 15 minutes, there is the water of 0.2ml to separate out |
| 2-8 ℃ of stability | 13 months stable | 14 months stable | 13 months stable | 12 months stable |
| Viscosity | 3.0 second/0.4ml | 2.6 second/0.4ml | 2.9 second/0.4ml | 2.7 second/0.4ml |
| TPPA | 4 all antibody positive rate 80% behind the mouse immune | 4 all antibody positive rate 80% behind the mouse immune | 4 all antibody positive rate 80% behind the mouse immune | 4 all antibody positive rate 70% behind the mouse immune |
| Side reaction | Behind 10 mouse immunes, have 2 injection sites that lump is arranged, 1 week disappeared | Behind 10 mouse immunes, the injection site does not all have lump to take place | Behind 10 mouse immunes, the injection site does not all have lump to take place | Behind 10 mouse immunes, have 2 injection sites that lump is arranged, 1 week disappeared |
Can find out that through table 1 adopt the vaccine of compound adjuvant preparation of the present invention, stability was greater than 12 months, comparison is stable according to vaccine A, and simultaneously, its immune effect comparison is good according to vaccine A, and the side reaction comparison is few according to vaccine A, has extraordinary actual use value.
Embodiment 3The application of pig circular ring virus vaccine
(1) preparation of compound recipe white-oil adjuvant
Method according to embodiment 1 prepares compound adjuvant 1,2,3 respectively.
(2) preparation of pig circular ring virus 2 venom
(2006-2008), related request prepares the pig circular ring virus 2 venom of deactivation according to " biological quality quality standard compilation for animals ", and every milliliter of viral liquid viral level answers>=10 before the deactivation
5.5TCID
50
(3) preparation of pig circular ring virus vaccine
Under sterile working's situation, the compound adjuvant for preparing in above-mentioned (1) is heated to 30 ℃, get the vial that 30g puts into 100ml; The Schweineseuche inactivation of viruses liquid 30g of preparation in (2) is preheated to 20 ℃; With the two mixing, use shaft-like homogenizer then, 12000 rev/mins, emulsifying 10 minutes forms the homogeneous system, gets final product.The vaccine that contains adjuvant 1, name vaccine 2-1; The vaccine that contains adjuvant 2, called after vaccine 2-2; Contain the vaccine called after vaccine 2-3 of adjuvant 3, be generically and collectively referred to as vaccine 2 of the present invention.
Substitute " compound recipe white-oil adjuvant " with method with the ISA206 adjuvant, preparation control vaccine, name control vaccine B.
(4) vaccine physical behavior and immunocompetent comparison
The comparison of table 2 vaccine 2 of the present invention and control vaccine B
| Project | Vaccine 2-1 | Vaccine 2-2 | Vaccine 2-3 | Control vaccine B |
| Outward appearance | The even emulsion of white | The even emulsion of white | The even emulsion of white | The even emulsion of white |
| Dosage form | The W/O/W type drips a little vaccine in cleaning cold water surface, is the cloud diffusion | The W/O/W type drips a little vaccine in cleaning cold water surface, is the cloud diffusion | The W/O/W type drips a little vaccine in cleaning cold water surface, is the cloud diffusion | The W/O/W type drips a little vaccine in cleaning cold water surface, is the cloud diffusion |
| Centrifugal stability | 3000 rev/mins, 15 minutes, no water was separated out | 3000 rev/mins, 15 minutes, no water was separated out | 3000 rev/mins, 15 minutes, there is the water of 0.1ml to separate out | 3000 rev/mins, 15 minutes, there is the water of 0.2ml to separate out |
| 2-8 ℃ of stability | 13 months stable | 14 months stable | 13 months stable | 12 months stable |
| Viscosity | 3.1 second/0.4ml | 2.8 second/0.4ml | 3.0 second/0.4ml | 3.0 second/0.4ml |
| TPPA | To be higher than 1:1600 be 60% to 5 all antibody titers behind the mouse immune | To be higher than 1:1600 be 70% to 5 all antibody titers behind the mouse immune | To be higher than 1:1600 be 70% to 5 all antibody titers behind the mouse immune | To be higher than 1:1600 be 60% to 5 all antibody titers behind the mouse immune |
| Side reaction | Behind 10 mouse immunes, have 2 injection sites that lump is arranged, 1 week disappeared | Behind 10 mouse immunes, the injection site does not all have lump to take place | Behind 10 mouse immunes, the injection site does not all have lump to take place | Behind 10 mouse immunes, have 3 injection sites that lump is arranged, 1 week disappeared |
Can find out that through table 2 adopt the vaccine of compound adjuvant preparation of the present invention, stability was greater than 12 months, comparison is stable according to vaccine B, and simultaneously, its immune effect comparison is good according to vaccine B, and the side reaction comparison is few according to vaccine B, has extraordinary actual use value.
Claims (5)
1. compound adjuvant; It is characterized in that this adjuvant is made up of miscella and mixed surfactant; Said miscella is made up of white oil and Petiolus Trachycarpi oil; Said mixed surfactant is made up of polyoxyethylene oleic acid ether-ether, polyoxyethylene alkyl ether and mannitol oleate, and the quality percentage composition of each component in compound adjuvant is:
White oil 70%-90%,
Petiolus Trachycarpi oil 1%-15%,
Polyoxyethylene oleic acid ether-ether 1%-10%,
Polyoxyethylene alkyl ether 1%-10%,
Mannitol oleate 1%-10%.
2. compound adjuvant according to claim 1 is characterized in that the mass percent of each component in compound adjuvant is:
White oil 75%-85%,
Petiolus Trachycarpi oil 5%-10%,
Polyoxyethylene oleic acid ether-ether 5%-8%,
Polyoxyethylene alkyl ether 2%-9%,
Mannitol oleate 1%-5%.
3. the method for preparing of the said compound adjuvant of claim 1; It is characterized in that this method is specific as follows: the white oil, Petiolus Trachycarpi oil, polyoxyethylene oleic acid ether-ether, polyoxyethylene alkyl ether and the mannitol oleate that take by weighing recipe quantity respectively; Mix homogeneously is sterilized then, promptly obtains compound adjuvant.
4. a vaccine that contains the said compound adjuvant of claim 1 is characterized in that this vaccine also comprises the antigen aqueous solution, and the mass ratio of said antigen aqueous solution and compound adjuvant is 3:2～3.
5. the method for preparing of the said vaccine of claim 4 is preheated to 25～35 ℃ with said compound adjuvant, and antigenic solution is preheated to 15～35 ℃, then the two is mixed, and warp stirring or mechanical presses are processed the emulsification system of homogeneous, promptly get said vaccine.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102813922A (en) * | 2012-09-11 | 2012-12-12 | 山东省农业科学院畜牧兽医研究所 | Compound oil adjuvant as well as preparation method and application thereof |
| CN103479997A (en) * | 2013-09-23 | 2014-01-01 | 天津瑞普生物技术股份有限公司 | Preparation method for avian reovirus virus water-in-oil-in-water type inactivated vaccines |
| CN106511996A (en) * | 2016-11-04 | 2017-03-22 | 江苏省农业科学院 | Multiple emulsion type adjuvant for foot-and-mouth disease vaccine and preparation method of multiple emulsion type adjuvant |
| CN106511995A (en) * | 2016-10-19 | 2017-03-22 | 天津佐沐生物科技有限公司 | Water-in-oil-in-water type vaccine adjuvant and application thereof |
| CN107812185A (en) * | 2016-09-14 | 2018-03-20 | 广东省肇庆市超能实业有限公司 | A kind of ultra tiny water-in-oil-in-water emulsion adjuvant |
-
2012
- 2012-03-29 CN CN2012100875663A patent/CN102600470A/en active Pending
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102813922A (en) * | 2012-09-11 | 2012-12-12 | 山东省农业科学院畜牧兽医研究所 | Compound oil adjuvant as well as preparation method and application thereof |
| CN102813922B (en) * | 2012-09-11 | 2013-12-11 | 山东省农业科学院畜牧兽医研究所 | Compound oil adjuvant as well as preparation method and application thereof |
| CN103479997A (en) * | 2013-09-23 | 2014-01-01 | 天津瑞普生物技术股份有限公司 | Preparation method for avian reovirus virus water-in-oil-in-water type inactivated vaccines |
| CN103479997B (en) * | 2013-09-23 | 2015-05-20 | 天津瑞普生物技术股份有限公司 | Preparation method for avian reovirus virus water-in-oil-in-water type inactivated vaccines |
| CN107812185A (en) * | 2016-09-14 | 2018-03-20 | 广东省肇庆市超能实业有限公司 | A kind of ultra tiny water-in-oil-in-water emulsion adjuvant |
| CN106511995A (en) * | 2016-10-19 | 2017-03-22 | 天津佐沐生物科技有限公司 | Water-in-oil-in-water type vaccine adjuvant and application thereof |
| CN106511996A (en) * | 2016-11-04 | 2017-03-22 | 江苏省农业科学院 | Multiple emulsion type adjuvant for foot-and-mouth disease vaccine and preparation method of multiple emulsion type adjuvant |
| CN106511996B (en) * | 2016-11-04 | 2019-09-10 | 江苏省农业科学院 | Emulsion type adjuvant and preparation method thereof for aftosa vaccine |
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