CN104208029B - A kind of nose vaccine combination powder preparation and preparation method thereof - Google Patents
A kind of nose vaccine combination powder preparation and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a kind of nose vaccine combination powder preparation and preparation method thereof.Described nose includes the following raw material component with vaccine combination powder preparation：（1）0.05%~5% antigen；（2）0.3~45% oil phase；（3）0.3~45% surfactant；（4）2~20% mucosa adhesion agent；With（5）4~40% freeze-dried excipients；The percentage is the mass percent that each composition accounts for raw material total amount.The preparation method comprises the following steps：（1）Oil phase and surfactant are mixed, water is stirred and add, adjuvant is obtained；（2）By step（1）After obtained adjuvant mixes with antigen, freeze-drying obtains powder, obtains final product.Nose vaccine combination powder preparation of the invention is intranasal mucosa-immune, and its preparation method is simple, and stability is high, simple operation in actual application, can greatly improve rate of vaccination, with extraordinary application prospect.
Description
Technical field
The invention belongs to pharmaceutical arts, and in particular to a kind of nose vaccine combination powder preparation and preparation method thereof.
Background technology
Vaccine is prevention and treatment virus infection class disease means the most cost-effective.By taking influenza vaccines as an example, in hair
Up to country, if the antigen of influenza vaccines and popular influenza virus matching are good, it becomes possible to protect the health of 70-90%
Adult is from there is clinical disease.At present, domestic main immune means are still traditional injection inoculation, but intramuscular injection
Administering mode there is many defects, including injection inoculation is inconvenient, obvious pain（Particularly with infant and children）, note
Penetrate unsafe factor that may be present etc..
Oral influenza vaccines need to overcome the sour environment of intestines and stomach, and sensitive antigen protein delivered intact to intestines and stomach are sticked
Microfold cell on film（M cells）.Due to gastrointestinal tract environment be not suitable for and influenza antigens albumen unstability, oral influenza
The research of vaccine rarely has breakthrough.
The appearance of nose spray influenza vaccines improves patient poor compliance of the intramuscular immunity caused by pain, and avoids
Blood infection or transmission risk that intramuscular injection may cause.But the medicine in nasal spray is still deposited in liquid form
, Cord blood and transport are needed, stability is not high enough.
Patent document CN1562364A discloses a kind of nose of fatty newborn adjuvant with influenza vaccines, and formulation process is needed
High-speed stirred and nanometer knock-on process are experienced, i.e., by high-speed stirred, mixes influenza all-virus suspension and nanosizing Fat Emulsion
Suspension；Clashed by nanometer, influenza virus Fat Emulsion mixed liquor is cracked into 10-12nm particulates.Whole preparation process control is difficult
Degree is high, and is easily caused antigen valence reduction.
Patent document CN101405027A discloses a kind of influenza virus particles combination comprising influenza virus reconstituted envelopes
Thing, and without the adjuvant of individually addition, for intranasal immunizations；Its preparation technology is complicated, it is difficult to be applied to big production.
Therefore, in order to improve convenience, the security of method of administration, the stabilization of raising immune formulation that immune formulation is administered
Property, medical expense is reduced, this area is at present in the urgent need to the nose bacterin preparation of Development of Novel.
The content of the invention
The technical problem to be solved in the present invention is poor in order to overcome existing nose bacterin preparation to prepare cumbersome, stability
Defect, and a kind of new nose vaccine combination powder preparation is provided.It is applied to the immune of different age people and animal
Inoculation, improves mucous membrane response and the systems response of immune system, improves the compliance of inoculator.Nose vaccine combination of the invention
Powder preparation, its preparation process is not required to the cracking of high-pressure homogeneous or nanometer, simplifies production stage, antigenic property in production process
Keep stabilization.
One of technical scheme that the present invention is provided is：A kind of nose vaccine combination powder preparation, it includes the following raw material
Component：
（1）0.05%~5% antigen；
（2）0.3~45% oil phase；
（3）0.3~45% surfactant；
（4）2~20% mucosa adhesion agent；With
（5）4~40% freeze-dried excipients；
The percentage is the mass percent that each composition accounts for raw material total amount.
It is preferred that described nose includes the following raw material component with vaccine combination powder preparation：
（1）0.15%~4% antigen；
（2）4~40% oil phases；
（3）4~40% surfactants；
（4）4~16% mucosa adhesion agent；With
（5）8~36% freeze-dried excipients；
The percentage is the mass percent that each composition accounts for raw material total amount.
More preferably, described nose includes the following raw material component with vaccine combination powder preparation：
（1）0.25%~3% antigen；
（2）20~30% oil phases；
（3）20~30% surfactants；
（4）10~15% mucosa adhesion agent；With
（5）25~32% freeze-dried excipients；
The percentage is the mass percent that each composition accounts for raw material total amount.
In the present invention, the antigen can be all kinds of antigens described in the routine of this area.
From for the species of antigen, the antigen can include but is not limited to influenza antigen, SARS virus antigen,
Measles virus antigens, Subclinical papillomavirus infection antigen, hepatitis B virus antigen, rabies virus antigen and polio disease
Any one in malicious antigen, more preferably influenza antigen, hepatitis B virus antigen and tetanus virus antigen；Described stream
Influenza Virus antigen includes seasonal influenza viral antigen and pandemic influenza viral antigen（As influenza antigen H1, H2,
H3, H5, H7, H9, subtype B and HA, NA, M1, M2 subunit antigen etc.）.
From for the source of antigen, the antigen can may originate from one kind comprising one or more separate sources, i.e. antigen
Or various different pathogen or different pathogen serotype.
From for the existence form and preparation method of antigen, the antigen can be inactivated vaccine, attenuated live vaccine, cracking epidemic disease
One or more in seedling, DNA vaccination, recombinant vaccine, subunit vaccine and polypeptide protein vaccine.
In the present invention, the oil phase is this area routinely oil phase, it is preferred that the oil phase is selected from squalene, spiny dogfish
One or more in alkane, median chain triglyceride oil, fish oil, olive oil and soybean oil, more preferably squalene, saualane and middle chain
One or more in triglyceride.
In the present invention, the surfactant is this area routinely surfactant, it is preferred that the surface-active
Agent is selected from tween, HS15（Solutol HS15）, HS15
（Solutol HS15）With one or more in the mixture of phosphatide, and the mixture of tween and phosphatide.The phosphatide is
This area routine, preferably pharmaceutically useful natural phospholipid or synthetic phospholipid, or phosphatide derivative, more preferably from soybean lecithin, ovum
One or more in phosphatide, egg yolk lecithin and hydrogenated soya phosphatide.
In the present invention, the mucosa adhesion agent is this area routinely mucosa adhesion agent, it is preferred that the mucosa adhesion
Agent is selected from one or more in shitosan, sodium carboxymethylcellulose, sodium alginate, Carbomer and Hydroxypropyl methylcellulose.
In the present invention, the freeze-dried excipient is this area routinely freeze-dried excipient, it is preferred that the lyophilized figuration
Agent is selected from the one kind or many in lactose, sucrose, trehalose, synanthrin, dextran, glucan, amino acid, mannitol and sorbierite
Kind, more preferably one or more in trehalose, lactose, synanthrin and mannitol.The amino acid is described in the routine of this area
Each amino acid, such as arginine, alanine, glutamic acid, glycine, proline.
In the present invention, it is preferred that described raw material also includes being no more than 8%（It is preferred that 2~5%）Buffer, the percentage
Than the mass percent that raw material total amount is accounted for for buffer.Described buffer is preferably selected from phosphate, 4- hydroxyethyl piperazine second
Sulfonic acid（HEPES）, one or more in acetate and citric acid.
In the present invention, it is preferred that described raw material also includes being no more than 60%（It is preferred that 5~30%）Carrier, the percentage
Than the mass percent that raw material total amount is accounted for for carrier.The carrier is preferably the mixture of sodium carboxymethylcellulose and lactose, more
Preferred mass ratio is 1: 50 sodium carboxymethylcellulose and the mixture of lactose.
Technical scheme that the present invention is provided two is：The foregoing nose preparation method of vaccine combination powder preparation.
With this area routine, it is only needed to antigen the nose of the invention preparation method of vaccine combination powder preparation
（Such as influenza antigen stoste）It is after freeze-dried mixed with oil phase and surfactant then mixed with mucomembranous adhesion agent and freeze-dried excipient
Close uniform.According to this area routine, typically can last well mixed gained finished product it is filling conventional in this area
It is standby as dry powder inhalant for nose in doser.
It is preferred that the preparation method of described nose vaccine combination powder preparation comprises the following steps：
（1）Oil phase and surfactant are mixed, water is stirred and add, adjuvant is obtained；The addition of the water is uniform with energy
Emulsification oil phase and surfactant are defined；
（2）By step（1）After obtained adjuvant mixes with antigen, freeze-drying obtains powder, obtains final product.
Wherein, the consumption of the oil phase, surfactant and antigen is as previously described.
In the present invention, in step（2）Preferably also include step afterwards（3）：By step（2）The powder sieving of gained,
Then packing is preserved.
Preferably, in the present invention, in step（1）Also include step before（a）, and step（3）Replace with step（3’）；Institute
The step of stating（a）For：Sodium carboxymethylcellulose is dissolved in water adhesive is obtained, then by lactose addition described adhesive, mixed
Uniformly, granulation, whole grain and dry, obtain carrier, wherein, the sodium carboxymethylcellulose of the amount of the water described in dissolve is defined；
Described step（3’）For：By step（2）The powder and step of gained（a）Obtained carrier is well mixed, and obtains final product.The present invention is more
It is preferred that in step（3’）Afterwards by step（3’）In be well mixed products therefrom sieving, then packing preserve.
In the present invention, described sieving was preferably 100~400 mesh sieves.
In the present invention, step（1）The stirring is the stirring described in the routine of this area；Step（2）The freeze-drying is
This area is conventional described；Step（a）Described in granulation, whole grain and drying be this area routine operation.
On the basis of common sense in the field is met, above-mentioned each optimum condition can be combined, and obtain final product each preferable reality of the present invention
Example.
Agents useful for same of the present invention and raw material are commercially available.
Positive effect of the invention is：Nose vaccine combination powder preparation of the invention is to exempt from through schneiderian membrance
Epidemic disease, compared to conventional immune formulation, can be obviously improved immune effect, and it can be induced including humoral immunity and cellular immunity simultaneously
In interior systemic immunity response and mucosal immune response, the pathogen of disease can be also triggered via respiratory mucosa invasion human body
It is made instantly available in infection site and is effective against；It can cause the distal end of alimentary canal and genital tract to stick by common mucosal immune system
Film immune response, can also make antigen through the distinctive veil cells of mucous membrane（M cells）It is transported to BMDC（DC）Carried Deng antigen
In cell（APC）, APC is improved to the intake of antigen and offers ability.Additionally, nose of the invention vaccine combination powder preparation
Preparation method it is simple, stability is high, simple operation in actual application, can greatly improve rate of vaccination.
Specific embodiment
The present invention is further illustrated below by the mode of embodiment, but does not therefore limit the present invention to described reality
Apply among a scope.The experimental technique of unreceipted actual conditions in the following example, conventionally and condition, or according to
Catalogue is selected.
Embodiment 1
0.64g sodium carboxymethylcelluloses, plus 64ml water are weighed, 1% adhesive is prepared；30g lactose is weighed, with adhesive system
Softwood；Cross 20 mesh sieves granulation, 60 °C drying 30 minutes after respectively cross 60 mesh and 80 mesh sieve whole grains, 60 °C dry 120 minutes, sieve takes
100~400 mesh particles, obtain final product dust carrier.
Squalene 0.6g, median chain triglyceride oil 0.6g, Solutol HS150.9g, egg yolk lecithin 0.3g are weighed, is stirred
Mix, obtain oil phase/surfactant mixture；Citric acid 1.6g is weighed, sucrose 8g is added in 15mL waters for injection, and stirring makes molten
Solution, pH to 7 is adjusted as water phase with sodium hydroxide solution；Under agitation, sterile water for injection is slowly added into oil phase/surface to live
In property agent composition, the solution with opalescence is obtained；Add the influenza antigens of 150mg HA（B/Brisbane/60/2008）, add water
To 20mL, stir evenly, 0.22 μm of filter membrane filtration；It is dried using Labconco freeze drying box（Lyophilized program such as table 1）.
Sodium alginate 6g is weighed, is mixed with obtained freeze-drying pastille powder and carrier, make gross weight for 20g, according to 2mg/ agent（I.e.
15 μ g/ agent）It is filling in powder nose inhalant device, obtain final product vaccine combination.
Table 1 freezes program setting
Embodiment 2
0.64g sodium carboxymethylcelluloses, plus 64ml water are weighed, 1% adhesive is prepared；30g lactose is weighed, with adhesive system
Softwood；Cross 20 mesh sieves granulation, 60 °C drying 30 minutes after respectively cross 60 mesh and 80 mesh sieve whole grains, 60 °C dry 120 minutes, sieve takes
100-400 mesh particles, obtain final product dust carrier.
Squalene 2.4g, median chain triglyceride oil 2.4g, Solutol HS153.6g, egg yolk lecithin 1.2g are weighed, is stirred
Mix, obtain oil phase/surfactant mixture；Potassium dihydrogen phosphate 0.14g is weighed, lactose 4g is added in 15mL waters for injection, stirred
Mixing makes dissolving, and pH to 7 is adjusted as water phase with sodium hydroxide solution；Under agitation, by sterile water for injection be slowly added to oil phase/
In surfactant mixture, the solution with opalescence is obtained；Add the influenza antigens of 150mg HA（B/Brisbane/60/
2008）, 20mL is added water to, stir evenly, 0.22 μm of filter membrane filtration；It is dried using Labconco freeze drying box（Lyophilized program
Such as table 1）.
Shitosan 3g is weighed, is mixed with obtained freeze-drying pastille powder and carrier, make gross weight for 20g, according to 2mg/ agent（I.e. 15
μ g/ agent）It is filling in powder nose inhalant device, obtain final product vaccine combination.
Embodiment 3
0.64g sodium carboxymethylcelluloses, plus 64ml water are weighed, 1% adhesive is prepared；30g lactose is weighed, with adhesive system
Softwood；Cross 20 mesh sieves granulation, 60 °C drying 30 minutes after respectively cross 60 mesh and 80 mesh sieve whole grains, 60 °C dry 120 minutes, sieve takes
100-400 mesh particles, obtain final product dust carrier.
Squalene 1.8g, median chain triglyceride oil 4.3g, Solutol HS159g are weighed, is stirred, obtained oil phase/surface and live
Property agent composition；Citric acid 0.4g, lactose 2g are weighed, is added in 15mL waters for injection, be stirred to dissolve, use sodium hydroxide solution
PH to 7 is used as water phase for regulation；Under agitation, sterile water for injection is slowly added in oil phase/surfactant mixture, is obtained
Solution with opalescence；Add the influenza antigens of 150mg HA（B/Brisbane/60/2008）, 20mL is added water to, stir evenly, 0.22 μ
M filter membranes are filtered；It is dried using Labconco freeze drying box（Lyophilized program such as table 1）.
Shitosan 1.5g is weighed, is mixed with obtained freeze-drying pastille powder and carrier, make gross weight for 20g, according to 4mg/ agent（I.e.
30 μ g/ agent）It is filling in powder nose inhalant device, obtain final product vaccine combination.
Embodiment 4
0.64g sodium carboxymethylcelluloses, plus 64ml water are weighed, 1% adhesive is prepared；30g lactose is weighed, with adhesive system
Softwood；Cross 20 mesh sieves granulation, 60 °C drying 30 minutes after respectively cross 60 mesh and 80 mesh sieve whole grains, 60 °C dry 120 minutes, sieve takes
100-400 mesh particles, obtain final product dust carrier.
Saualane 0.3g, median chain triglyceride oil 0.3g, Solutol HS151.8g, soybean lecithin 0.6g are weighed, is stirred
Mix, obtain oil phase/surfactant mixture；Citric acid 0.3g, synanthrin 4g are weighed, is added in 15mL waters for injection, stirring makes
Dissolving, pH to 7 is adjusted as water phase with sodium hydroxide solution；Under agitation, sterile water for injection is slowly added to oil phase/surface
In active agent intermixture, the solution with opalescence is obtained；Add the influenza antigens of 150mg HA（B/Brisbane/60/2008）, plus
Water is stirred evenly to 20mL, 0.22 μm of filter membrane filtration；It is dried using Labconco freeze drying box（Lyophilized program such as table 1）.
Sodium alginate 4g is weighed, is mixed with obtained freeze-drying pastille powder and carrier, make gross weight for 20g, according to 4mg/ agent（I.e.
30 μ g/ agent）It is filling in powder nose inhalant device, obtain final product vaccine combination.
Embodiment 5
0.64g sodium carboxymethylcelluloses, plus 64ml water are weighed, 1% adhesive is prepared；30g lactose is weighed, with adhesive system
Softwood；Cross 20 mesh sieves granulation, 60 °C drying 30 minutes after respectively cross 60 mesh and 80 mesh sieve whole grains, 60 °C dry 120 minutes, sieve takes
100-400 mesh particles, obtain final product dust carrier.
Weigh saualane 0.12g, median chain triglyceride oil 0.12g, Solutol HS150.3g, hydrogenated yolk lecithin
0.06g, stirring, obtains oil phase/surfactant mixture；Potassium dihydrogen phosphate 0.6g, mannitol 8g are weighed, 15mL injections are added
With in water, it is stirred to dissolve, pH to 7 is adjusted as water phase with sodium hydroxide solution；Under agitation, it is sterile water for injection is slow
Add in oil phase/surfactant mixture, obtain the solution with opalescence；Add the influenza antigens of 150mg HA（B/
Brisbane/60/2008）, 20mL is added water to, stir evenly, 0.22 μm of filter membrane filtration；Done using Labconco freeze drying box
It is dry（Lyophilized program such as table 1）.
Carbomer 1.5g, Hydroxypropyl methylcellulose 2g are weighed, is mixed with obtained freeze-drying pastille powder and carrier, make the gross weight be
20g, according to 6mg/ agent（That is 45 μ g/ agent）It is filling in powder nose inhalant device, obtain final product vaccine combination.
Embodiment 6
0.64g sodium carboxymethylcelluloses, plus 64ml water are weighed, 1% adhesive is prepared；30g lactose is weighed, with adhesive system
Softwood；Cross 20 mesh sieves granulation, 60 °C drying 30 minutes after respectively cross 60 mesh and 80 mesh sieve whole grains, 60 °C dry 120 minutes, sieve takes
100-400 mesh particles, obtain final product dust carrier.
Squalene 1.2g, median chain triglyceride oil 1.2g, tween 2.7g, hydrogenated soy phosphatidyl choline 0.3g are weighed, is stirred, obtained
To oil phase/surfactant mixture；Citric acid 0.8g, lactose 4g, mannitol 4g are weighed, is added in 15mL waters for injection, stirred
Mixing makes dissolving, and pH to 7 is adjusted as water phase with sodium hydroxide solution；Under agitation, by sterile water for injection be slowly added to oil phase/
In surfactant mixture, the solution with opalescence is obtained；Add the influenza antigens of 150mg HA（B/Brisbane/60/
2008）, 20mL is added water to, stir evenly, 0.22 μm of filter membrane filtration；It is dried using Labconco freeze drying box（Lyophilized program
Such as table 1）.
Sodium alginate 3g is weighed, is mixed with obtained freeze-drying pastille powder and carrier, make gross weight for 20g, according to 6mg/ agent（I.e.
45 μ g/ agent）It is filling in powder nose inhalant device, obtain final product vaccine combination.
Embodiment 7
0.64g sodium carboxymethylcelluloses, plus 64ml water are weighed, 1% adhesive is prepared；30g lactose is weighed, with adhesive system
Softwood；Cross 20 mesh sieves granulation, 60 °C drying 30 minutes after respectively cross 60 mesh and 80 mesh sieve whole grains, 60 °C dry 120 minutes, sieve takes
100-400 mesh particles, obtain final product dust carrier.
Weigh squalene 2g, median chain triglyceride oil 4g, tween 4.8g, egg yolk lecithin 1.8g, stir, obtain oil phase/
Surfactant mixture；HEPES0.8g is weighed, synanthrin 4g is added in 15mL waters for injection, is stirred to dissolve, and uses NaOH
Solution adjusts pH to 7 as water phase；Under agitation, sterile water for injection is slowly added in oil phase/surfactant mixture,
Obtain the solution with opalescence；Add the influenza antigens of 150mg HA（A/California/7/2009）, 20mL is added water to, stir evenly,
0.22 μm of filter membrane filtration；It is dried using Labconco freeze drying box（Lyophilized program such as table 1）.
Shitosan 1g is weighed, is mixed with obtained freeze-drying pastille powder and carrier, make gross weight for 20g, according to 2mg/ agent（I.e. 15
μ g/ agent）It is filling in powder nose inhalant device, obtain final product vaccine combination.
Embodiment 8
0.64g sodium carboxymethylcelluloses, plus 64ml water are weighed, 1% adhesive is prepared；30g lactose is weighed, with adhesive system
Softwood；Cross 20 mesh sieves granulation, 60 °C drying 30 minutes after respectively cross 60 mesh and 80 mesh sieve whole grains, 60 °C dry 120 minutes, sieve takes
100-400 mesh particles, obtain final product dust carrier.
Median chain triglyceride oil 0.06g, tween 0.03g, egg yolk lecithin 0.15g are weighed, is stirred, obtain oil phase/surface
Active agent intermixture；HEPES1.2g, lactose 8g are weighed, is added in 15mL waters for injection, be stirred to dissolve, it is molten with NaOH
Liquid adjusts pH to 7 as water phase；Under agitation, sterile water for injection is slowly added in oil phase/surfactant mixture, is obtained
To the solution with opalescence；The recombination hepatitis B antigen of 50000 μ g is added, 20mL is added water to, stirred evenly, 0.22 μm of filter membrane filtration；Use
Labconco freeze drying box is dried（Lyophilized program such as table 1）.
Shitosan 1g, sodium carboxymethylcellulose 2g are weighed, is mixed with obtained freeze-drying pastille powder and carrier, make the gross weight be
20g, according to 4mg/ agent（That is 10 μ g/ agent）It is filling in powder nose inhalant device, obtain final product vaccine combination.
Embodiment 9
0.64g sodium carboxymethylcelluloses, plus 64ml water are weighed, 1% adhesive is prepared；30g lactose is weighed, with adhesive system
Softwood；Cross 20 mesh sieves granulation, 60 °C drying 30 minutes after respectively cross 60 mesh and 80 mesh sieve whole grains, 60 °C dry 120 minutes, sieve takes
100-400 mesh particles, obtain final product dust carrier.
Squalene 0.6g, median chain triglyceride oil 0.6g, Solutol HS151.2g, egg yolk lecithin 0.3g are weighed, is stirred
Mix, obtain oil phase/surfactant mixture；Citric acid 0.08g, lactose 2g, mannitol 2g are weighed, 15mL waters for injection are added
In, it is stirred to dissolve, pH to 7 is adjusted as water phase with sodium hydroxide solution；Under agitation, sterile water for injection is slowly added to
In oil phase/surfactant mixture, the solution with opalescence is obtained；Add the influenza antigens of 150mg HA（B/Brisbane/
60/2008）, 20mL is added water to, stir evenly, 0.22 μm of filter membrane filtration；It is dried using Labconco freeze drying box（Lyophilized journey
Sequence such as table 1）.
Shitosan 3g is weighed, is mixed with obtained freeze-drying pastille powder and carrier, make gross weight for 20g, according to 2mg/ agent（I.e. 15
μ g/ agent）It is filling in powder nose inhalant device, obtain final product vaccine combination.
Embodiment 10
0.64g sodium carboxymethylcelluloses, plus 64ml water are weighed, 1% adhesive is prepared；30g lactose is weighed, with adhesive system
Softwood；Cross 20 mesh sieves granulation, 60 °C drying 30 minutes after respectively cross 60 mesh and 80 mesh sieve whole grains, 60 °C dry 120 minutes, sieve takes
100-400 mesh particles, obtain final product dust carrier.
Squalene 1.5g, median chain triglyceride oil 1.5g, Solutol HS152.4g are weighed, is stirred, obtain oil phase/surface
Active agent intermixture；HEPES0.12g, sucrose 4g are weighed, is added in 15mL waters for injection, be stirred to dissolve, it is molten with NaOH
Liquid adjusts pH to 7 as water phase；Under agitation, sterile water for injection is slowly added in oil phase/surfactant mixture, is obtained
To the solution with opalescence；Add the influenza antigens of 150mg HA（A/California/7/2009）, 20mL is added water to, stir evenly,
0.22 μm of filter membrane filtration；It is dried using Labconco freeze drying box（Lyophilized program such as table 1）.
Shitosan 0.8g is weighed, is mixed with obtained freeze-drying pastille powder and carrier, make gross weight for 20g, according to 4mg/ agent（I.e.
30 μ g/ agent）It is filling in powder nose inhalant device, obtain final product vaccine combination.
Embodiment 11
0.64g sodium carboxymethylcelluloses, plus 64ml water are weighed, 1% adhesive is prepared；30g lactose is weighed, with adhesive system
Softwood；Cross 20 mesh sieves granulation, 60 °C drying 30 minutes after respectively cross 60 mesh and 80 mesh sieve whole grains, 60 °C dry 120 minutes, sieve takes
100-400 mesh particles, obtain final product dust carrier.
Squalene 0.3g, median chain triglyceride oil 0.3g, Solutol HS151.2g, egg yolk lecithin 0.3g are weighed, is stirred
Mix, obtain oil phase/surfactant mixture；Citric acid 0.16g, synanthrin 4g are weighed, is added in 15mL waters for injection, stirring makes
Dissolving, pH to 7 is adjusted as water phase with sodium hydroxide solution；Under agitation, sterile water for injection is slowly added to oil phase/surface
In active agent intermixture, the solution with opalescence is obtained；Add the influenza antigens of 150mg HA（A/California/7/2009）,
20mL is added water to, is stirred evenly, 0.22 μm of filter membrane filtration；It is dried using Labconco freeze drying box（Lyophilized program such as table 1）.
Sodium alginate 2g is weighed, is mixed with obtained freeze-drying pastille powder and carrier, make gross weight for 20g, according to 2mg/ agent（I.e.
15 μ g/ agent）It is filling in powder nose inhalant device, obtain final product vaccine combination.
Embodiment 12
0.64g sodium carboxymethylcelluloses, plus 64ml water are weighed, 1% adhesive is prepared；30g lactose is weighed, with adhesive system
Softwood；Cross 20 mesh sieves granulation, 60 °C drying 30 minutes after respectively cross 60 mesh and 80 mesh sieve whole grains, 60 °C dry 120 minutes, sieve takes
100-400 mesh particles, obtain final product dust carrier.
Squalene 0.3g, Solutol HS152.4g is weighed, is stirred, obtain oil phase/surfactant mixture；Weigh phosphorus
Acid dihydride potassium 0.08g, arginine 3g, are added in 15mL waters for injection, are stirred to dissolve, and pH to 7 is adjusted with sodium hydroxide solution
As water phase；Under agitation, sterile water for injection is slowly added in oil phase/surfactant mixture, is obtained with opalescence
Solution；Add the influenza antigens of 200mg HA（A/California/7/2009）, 20mL is added water to, stir evenly, 0.22 μm of filter membrane filter
Cross；It is dried using Labconco freeze drying box（Lyophilized program such as table 1）.
Sodium alginate 2g is weighed, is mixed with obtained freeze-drying pastille powder and carrier, make gross weight for 20g, according to 2mg/ agent（I.e.
20 μ g/ agent）It is filling in powder nose inhalant device, obtain final product vaccine combination.
Embodiment 13
0.64g sodium carboxymethylcelluloses, plus 64ml water are weighed, 1% adhesive is prepared；30g lactose is weighed, with adhesive system
Softwood；Cross 20 mesh sieves granulation, 60 °C drying 30 minutes after respectively cross 60 mesh and 80 mesh sieve whole grains, 60 °C dry 120 minutes, sieve takes
100-400 mesh particles, obtain final product dust carrier.
Squalene 0.18g, Solutol HS151.2g is weighed, is stirred, obtain oil phase/surfactant mixture；Weigh
Citric acid 0.3g, lactose 6g, are added in 15mL waters for injection, are stirred to dissolve, and pH to 7 is adjusted as water with sodium hydroxide solution
Phase；Under agitation, sterile water for injection is slowly added in oil phase/surfactant mixture, obtains the solution with opalescence；Plus
Enter the influenza antigens of 100mg HA（A/California/7/2009）, 20mL is added water to, stir evenly, 0.22 μm of filter membrane filtration；Use
Labconco freeze drying box is dried（Lyophilized program such as table 1）.
Sodium alginate 3g is weighed, is mixed with obtained freeze-drying pastille powder and carrier, make gross weight for 20g, according to 10mg/ agent
（That is 50 μ g/ agent）It is filling in powder nose inhalant device, obtain final product vaccine combination.
Embodiment 14
0.64g sodium carboxymethylcelluloses, plus 64ml water are weighed, 1% adhesive is prepared；30g lactose is weighed, with adhesive system
Softwood；Cross 20 mesh sieves granulation, 60 °C drying 30 minutes after respectively cross 60 mesh and 80 mesh sieve whole grains, 60 °C dry 120 minutes, sieve takes
100-400 mesh particles, obtain final product dust carrier.
Squalene 0.06g, Solutol HS150.06g is weighed, is stirred, obtain oil phase/surfactant mixture；Weigh
HEPES0.48g, lactose 0.8g, mannitol 0.8g, add in 15mL waters for injection, are stirred to dissolve, and are adjusted with sodium hydroxide solution
Section pH to 7 is used as water phase；Under agitation, sterile water for injection is slowly added in oil phase/surfactant mixture, obtains band
The solution of opalescence；2400000IU tetanus vaccines are added, 20mL is added water to, stirred evenly, 0.22 μm of filter membrane filtration；Use
Labconco freeze drying box is dried（Lyophilized program such as table 1）.
Sodium alginate 0.6g is weighed, is mixed with obtained freeze-drying pastille powder and carrier, make gross weight for 20g, according to 2mg/ agent
（That is 240IU/ agent）It is filling in powder nose inhalant device, obtain final product vaccine combination.
Embodiment 15
0.64g sodium carboxymethylcelluloses, plus 64ml water are weighed, 1% adhesive is prepared；30g lactose is weighed, with adhesive system
Softwood；Cross 20 mesh sieves granulation, 60 °C drying 30 minutes after respectively cross 60 mesh and 80 mesh sieve whole grains, 60 °C dry 120 minutes, sieve takes
100-400 mesh particles, obtain final product dust carrier.
Weigh squalene 1.8g, median chain triglyceride oil 3g, tween 3.6g, egg yolk lecithin 1.2g, HEPES0.9g, sugarcane
Sugared 4g, is added in 15mL waters for injection, is stirred to dissolve, and pH to 7 is adjusted as water phase with sodium hydroxide solution；Under agitation,
Sterile water for injection is slowly added in oil phase/surfactant mixture, the solution with opalescence is obtained；Add 1600000IU
Tetanus toxin, add water to 20mL, stir evenly, the filtration of 0.22 μm of filter membrane；It is dried using Labconco freeze drying box（Freeze
Dry program such as table 1）.
Shitosan 0.8g, sodium alginate 0.8g are weighed, are mixed with obtained freeze-drying pastille powder and carrier, make gross weight for 20g,
According to 3mg/ agent（That is 240IU/ agent）It is filling in powder nose inhalant device, obtain final product vaccine combination.
Embodiment 16
Squalene 5g, median chain triglyceride oil 4g, Solutol HS156.6g, egg yolk lecithin 2.4g are weighed, is stirred, obtained
To oil phase/surfactant mixture；HEPES0.08g, synanthrin 0.8g are weighed, is added in 15mL waters for injection, stirring makes molten
Solution, pH to 7 is adjusted as water phase with sodium hydroxide solution；Under agitation, sterile water for injection is slowly added into oil phase/surface to live
In property agent composition, the solution with opalescence is obtained；The recombination hepatitis B antigen of 50000 μ g is added, 20mL is added water to, stirred evenly, 0.22 μ
M filter membranes are filtered；It is dried using Labconco freeze drying box（Lyophilized program such as table 1）.
Shitosan 0.8g is weighed, is mixed with obtained freeze-drying pastille powder, make gross weight for 20g, according to 2mg/ agent（That is 5 μ g/
Agent）It is filling in powder nose inhalant device, obtain final product vaccine combination.This example does not include carrier.
Embodiment 17
0.64g sodium carboxymethylcelluloses, plus 64ml water are weighed, 1% adhesive is prepared；30g lactose is weighed, with adhesive system
Softwood；Cross 20 mesh sieves granulation, 60 °C drying 30 minutes after respectively cross 60 mesh and 80 mesh sieve whole grains, 60 °C dry 120 minutes, sieve takes
100-400 mesh particles, obtain final product dust carrier.
Squalene 1.2g, median chain triglyceride oil 1.2g, tween 2.7g, hydrogenated soy phosphatidyl choline 0.3g are weighed, is stirred, obtained
To oil phase/surfactant mixture；Citric acid 1.6g, lactose 4g, mannitol 4g are weighed, is added in 15mL waters for injection, stirred
Mixing makes dissolving, and pH to 7 is adjusted as water phase with sodium hydroxide solution；Under agitation, by sterile water for injection be slowly added to oil phase/
In surfactant mixture, the solution with opalescence is obtained；Add the influenza antigens of 150mg HA（A/Perth/16/2009）,
20mL is added water to, is stirred evenly, 0.22 μm of filter membrane filtration；It is dried using Labconco freeze drying box（Lyophilized program such as table 1）.
Hydroxypropyl methylcellulose 1.5g, mixes with obtained freeze-drying pastille powder and carrier, makes gross weight for 20g, according to 4mg/ agent
（That is 30 μ g/ agent）It is filling in powder nose inhalant device, obtain final product vaccine combination.
Embodiment 18
0.64g sodium carboxymethylcelluloses, plus 64ml water are weighed, 1% adhesive is prepared；30g lactose is weighed, with adhesive system
Softwood；Cross 20 mesh sieves granulation, 60 °C drying 30 minutes after respectively cross 60 mesh and 80 mesh sieve whole grains, 60 °C dry 120 minutes, sieve takes
100-400 mesh particles, obtain final product dust carrier.
Median chain triglyceride oil 3g, Solutol HS154.2g is weighed, is stirred, obtain oil phase/surfactant mixture；
Sucrose 0.6g, arginine 0.9g are weighed, is added in 15mL waters for injection, be stirred to dissolve, pH to 7 is adjusted with sodium hydroxide solution
As water phase；Under agitation, sterile water for injection is slowly added in oil phase/surfactant mixture, is obtained with opalescence
Solution；50000 μ g tetanus vaccines are added, 20mL is added water to, stirred evenly, 0.22 μm of filter membrane filtration；Freezed using Labconco dry
Dry case is dried（Lyophilized program such as table 1）.
Shitosan 0.5g, Carbomer 1g are weighed, is mixed with obtained freeze-drying pastille powder and carrier, make gross weight for 20g, according to
2mg/ agent（That is 5 μ g/ agent）It is filling in powder nose inhalant device, obtain final product vaccine combination.
Embodiment 19
0.64g sodium carboxymethylcelluloses, plus 64ml water are weighed, 1% adhesive is prepared；30g lactose is weighed, with adhesive system
Softwood；Cross 20 mesh sieves granulation, 60 °C drying 30 minutes after respectively cross 60 mesh and 80 mesh sieve whole grains, 60 °C dry 120 minutes, sieve takes
100-400 mesh particles, obtain final product dust carrier.
Squalene 0.12g, median chain triglyceride oil 0.6g, Solutol HS153g are weighed, is stirred, obtain oil phase/surface
Active agent intermixture；Sucrose 2g, mannitol 2g are weighed, is added in 15mL waters for injection, be stirred to dissolve, use sodium hydroxide solution
PH to 7 is used as water phase for regulation；Under agitation, sterile water for injection is slowly added in oil phase/surfactant mixture, is obtained
Solution with opalescence；2500000IU tetanus vaccines are added, 20mL is added water to, stirred evenly, 0.22 μm of filter membrane filtration；Use
Labconco freeze drying box is dried（Lyophilized program such as table 1）.
Sodium alginate 0.6g, sodium carboxymethylcellulose 2g are weighed, is mixed with obtained freeze-drying pastille powder and carrier, make gross weight
It is 20g, according to 2mg/ agent（That is 250IU/ agent）It is filling in powder nose inhalant device, obtain final product vaccine combination.
Embodiment 20
0.64g sodium carboxymethylcelluloses, plus 64ml water are weighed, 1% adhesive is prepared；30g lactose is weighed, with adhesive system
Softwood；Cross 20 mesh sieves granulation, 60 °C drying 30 minutes after respectively cross 60 mesh and 80 mesh sieve whole grains, 60 °C dry 120 minutes, sieve takes
100-400 mesh particles, obtain final product dust carrier.
Squalene 3g, Solutol HS153.6g, egg yolk lecithin 1.8g are weighed, is stirred, obtain oil phase/surfactant
Mixture；Potassium dihydrogen phosphate 1.2g, lactose 2g, mannitol 2g are weighed, is added in 15mL waters for injection, be stirred to dissolve, use hydrogen
Sodium hydroxide solution adjusts pH to 7 as water phase；Under agitation, sterile water for injection is slowly added into oil phase/surfactant to mix
In compound, the solution with opalescence is obtained；Add the influenza antigens of 150mg HA（A/Perth/16/2009）, 20mL is added water to, stir
It is even, 0.22 μm of filter membrane filtration；It is dried using Labconco freeze drying box（Lyophilized program such as table 1）.
Carbomer 3g is weighed, is mixed with obtained freeze-drying pastille powder and carrier, make gross weight for 20g, according to 2mg/ agent（I.e. 15
μ g/ agent）It is filling in powder nose inhalant device, obtain final product vaccine combination.
Experimental example 21
0.64g sodium carboxymethylcelluloses, plus 64ml water are weighed, 1% adhesive is prepared；30g lactose is weighed, with adhesive system
Softwood；Cross 20 mesh sieves granulation, 60 °C drying 30 minutes after respectively cross 60 mesh and 80 mesh sieve whole grains, 60 °C dry 120 minutes, sieve takes
100-400 mesh particles, obtain final product dust carrier.
Fish oil 2.4g, median chain triglyceride oil 2.4g, Solutol HS153.6g, egg yolk lecithin 1.2g are weighed, are stirred,
Obtain oil phase/surfactant mixture；Potassium dihydrogen phosphate 0.08g is weighed, lactose 4g is added in 15mL waters for injection, and stirring makes
Dissolving, pH to 7 is adjusted as water phase with sodium hydroxide solution；Under agitation, sterile water for injection is slowly added to oil phase/surface
In active agent intermixture, the solution with opalescence is obtained；Add the influenza antigens of 150mg HA（B/Brisbane/60/2008）, plus
Water is stirred evenly to 20mL, 0.22 μm of filter membrane filtration；It is dried using Labconco freeze drying box（Lyophilized program such as table 1）.
Shitosan 1.5g is weighed, is mixed with obtained freeze-drying pastille powder and carrier, make gross weight for 20g, according to 2mg/ agent（I.e.
15 μ g/ agent）It is filling in powder nose inhalant device, obtain final product vaccine combination.
Embodiment 22
0.64g sodium carboxymethylcelluloses, plus 64ml water are weighed, 1% adhesive is prepared；30g lactose is weighed, with adhesive system
Softwood；Cross 20 mesh sieves granulation, 60 °C drying 30 minutes after respectively cross 60 mesh and 80 mesh sieve whole grains, 60 °C dry 120 minutes, sieve takes
100-400 mesh particles, obtain final product dust carrier.
Soybean oil 1.8g, median chain triglyceride oil 4.2g, Solutol HS156g are weighed, is stirred, obtained oil phase/surface and live
Property agent composition；Citric acid 1.6g, lactose 2g are weighed, is added in 15mL waters for injection, be stirred to dissolve, use sodium hydroxide solution
PH to 7 is used as water phase for regulation；Under agitation, sterile water for injection is slowly added in oil phase/surfactant mixture, is obtained
Solution with opalescence；Add the influenza antigens of 150mg HA（B/Brisbane/60/2008）, 20mL is added water to, stir evenly, 0.22 μ
M filter membranes are filtered；It is dried using Labconco freeze drying box（Lyophilized program such as table 1）.
Sodium carboxymethylcellulose 2g is weighed, is mixed with obtained freeze-drying pastille powder and carrier, make gross weight for 20g, according to
4mg/ agent（That is 30 μ g/ agent）It is filling in powder nose inhalant device, obtain final product vaccine combination.
Embodiment 23
0.64g sodium carboxymethylcelluloses, plus 64ml water are weighed, 1% adhesive is prepared；30g lactose is weighed, with adhesive system
Softwood；Cross 20 mesh sieves granulation, 60 °C drying 30 minutes after respectively cross 60 mesh and 80 mesh sieve whole grains, 60 °C dry 120 minutes, sieve takes
100-400 mesh particles, obtain final product dust carrier.
Olive oil 0.3g, median chain triglyceride oil 0.3g, Solutol HS151.8g, soybean lecithin 0.6g are weighed, is stirred
Mix, obtain oil phase/surfactant mixture；Citric acid 0.9g, synanthrin 8g are weighed, is added in 15mL waters for injection, stirring makes
Dissolving, pH to 7 is adjusted as water phase with sodium hydroxide solution；Under agitation, sterile water for injection is slowly added to oil phase/surface
In active agent intermixture, the solution with opalescence is obtained；The recombination hepatitis B antigen of 50000 μ g is added, 20mL is added water to, stirred evenly,
0.22 μm of filter membrane filtration；It is dried using Labconco freeze drying box（Lyophilized program such as table 1）.
Sodium alginate 4g is weighed, is mixed with obtained freeze-drying pastille powder and carrier, make gross weight for 20g, according to 2mg/ agent（I.e.
5 μ g/ agent）It is filling in powder nose inhalant device, obtain final product vaccine combination.
Embodiment 24
0.64g sodium carboxymethylcelluloses, plus 64ml water are weighed, 1% adhesive is prepared；30g lactose is weighed, with adhesive system
Softwood；Cross 20 mesh sieves granulation, 60 °C drying 30 minutes after respectively cross 60 mesh and 80 mesh sieve whole grains, 60 °C dry 120 minutes, sieve takes
100-400 mesh particles, obtain final product dust carrier.
Fish oil 3g, Solutol HS154.2g is weighed, is stirred, obtain oil phase/surfactant mixture；Weigh sucrose
0.8g, arginine 0.6g, are added in 15mL waters for injection, are stirred to dissolve, and pH to 7 is adjusted as water with sodium hydroxide solution
Phase；Under agitation, sterile water for injection is slowly added in oil phase/surfactant mixture, obtains the solution with opalescence；Plus
Enter the tetanus toxin of 2500000IU, add water to 20mL, stir evenly, 0.22 μm of filter membrane filtration；Use Labconco freeze drying box
It is dried（Lyophilized program such as table 1）.
Shitosan 0.4g, Carbomer 0.4g are weighed, is mixed with obtained freeze-drying pastille powder and carrier, make gross weight for 20g, pressed
According to 2mg/ agent（That is 250IU/ agent）It is filling in powder nose inhalant device, obtain final product vaccine combination.
Embodiment 25
0.64g sodium carboxymethylcelluloses, plus 64ml water are weighed, 1% adhesive is prepared；30g lactose is weighed, with adhesive system
Softwood；Cross 20 mesh sieves granulation, 60 °C drying 30 minutes after respectively cross 60 mesh and 80 mesh sieve whole grains, 60 °C dry 120 minutes, sieve takes
100-400 mesh particles, obtain final product dust carrier.
Soybean oil 0.12g, median chain triglyceride oil 0.6g, Solutol HS153g are weighed, is stirred, obtain oil phase/surface
Active agent intermixture；Sucrose 2g, mannitol 2g are weighed, is added in 15mL waters for injection, be stirred to dissolve, use sodium hydroxide solution
PH to 7 is used as water phase for regulation；Under agitation, sterile water for injection is slowly added in oil phase/surfactant mixture, is obtained
Solution with opalescence；The tetanus toxin of 2500000IU is added, 20mL is added water to, stirred evenly, 0.22 μm of filter membrane filtration；Use
Labconco freeze drying box is dried（Lyophilized program such as table 1）.
Sodium alginate 1g, sodium carboxymethylcellulose 1g are weighed, is mixed with obtained freeze-drying pastille powder and carrier, make the gross weight be
20g, according to 2mg/ agent（That is 250IU/ agent）It is filling in powder nose inhalant device, obtain final product vaccine combination.
The measure of the powder preparation flu vaccine hemagglutinin potency of effect example 1
The vaccine combination powder preparation containing influenza antigens prepared in embodiment 1-25 is redissolved through water and processes laggard
Row hemagglutinin titration, and compare with the hemagglutinin measurement result of antigen stock, it is as a result as follows（Table 2）：
The hemagglutinin titration of table 2
As shown in Table 2, the hemagglutinin potency of Inflenza vaccine composition powder preparation of the invention and lytic virus
Antigen stock is consistent.
The antigenic structure stability of effect example 2 is verified
Using SDS- polyacrylamide gel electrophoresises（SDS-PAGE）Method is to the vaccine combination prepared by embodiment 1-25
Powder preparation carries out antigenic structure study on the stability.The electrophoresis strip that antigen in result display composition is formed with antigen stock
Band is consistent, shows that the antigenic structure in vaccine combination powder preparation does not change.
The measure of particle diameter after the redissolution of the powder preparation of effect example 3
During vaccine combination powder preparation obtained by embodiment 1-25 added into a small amount of physiological saline, gentle agitation or shake
Shake it is several under, the solution with opalescence can be formed in a few minutes, surveyed using Malvern Nano-ZS90 dynamic light scattering particle diameters current potential
Determine instrument and determine its particle diameter, it is as a result as shown in the table（Table 3）.
Particle size determination result after the redissolution of the powder preparation of table 3
As shown in Table 3, after obtained vaccine combination powder preparation is contacted with suitable quantity of water, it is through slight oscillatory
Can quickly redissolve, form the solution with opalescence, particle diameter is in the range of 20-100nm, it is adaptable to nose administration.
The animal vivo test of the Inflenza vaccine composition powder preparation of effect example 4
Vaccinogen liquid：A type influenza vaccines（A/Perth/16/2009）.
Vaccine formulation：
（1）Vaccine immunity control group：This group is removed outside oil phase and surfactant, and remaining component is with embodiment 12.Claim
0.64g sodium carboxymethylcelluloses, plus 64ml water are taken, 1% adhesive is prepared；30g lactose is weighed, with adhesive softwood；Cross 20 mesh
Sieve series grain, 60 °C drying 30 minutes after respectively cross 60 mesh and 80 mesh sieve whole grains, 60 °C dry 120 minutes, sieve take 100-400 mesh
Grain, obtains final product dust carrier.Potassium dihydrogen phosphate 0.08g, lactose 2g, mannitol 2g are weighed, is added in 15mL waters for injection, stirring makes
Dissolving；Add the influenza antigens of 50mg HA（A/Perth/16/2009）, 20mL is added water to, stir evenly, 0.22 μm of filter membrane filtration；Make
It is dried with Labconco freeze drying box（Lyophilized program such as table 1）.
Lyophilized pastille powder and carrier are mixed, makes gross weight for 20g, according to 2mg/ agent（That is 5 μ g/ agent）It is filling in nose powder
In mist agent device, control group vaccine combination is obtained final product.
（2）Vaccine combination powder preparation of the present invention：Vaccine combination powder preparation prepared by embodiment 12, regulator
Measure as 5 μ g/2mg carry out rat immunity experiment.
（3）Blank control group：0.64g sodium carboxymethylcelluloses, plus 64ml water are weighed, 1% adhesive is prepared；Weigh 30g breasts
Sugar, with adhesive softwood；Cross 20 mesh sieves granulation, 60 °C drying 30 minutes after respectively cross 60 mesh and 80 mesh sieve whole grains, 60 °C baking
Dry 120 minutes, sieve takes 100-400 mesh particles, obtains final product dust carrier.It is administered the dust carrier as blank powder.
Animal immune scheme：
By 24 body weight for the female sd inbred rats of 125-175g are randomly divided into 4 groups, every group 6, powder spray nasal-cavity administration.Side
Case is as follows：
1st group：Vaccine immunity control group, 5 μ g HA/ are only；
2nd group：Vaccine combination powder preparation of the present invention, 5 μ g HA/ are only；
3rd group：Blank control group, gives merely the carrier of homogenous quantities.
Immune effect is detected：
14th day, 28 days after immune, rat eye socket takes blood, separates serum.Tested by HI, determine the blood clotting suppression in serum
Titre processed.Using indirect ELISA method, the total IgG titre in detection serum.Result is as follows（It is shown in Table 4 and table 5）：
Table 4HI result of the tests
Blank
Difference between different groups is compared using one-way analysis of variance, sets significance as p<0.05.From
Data analysis understands that this composition can dramatically increase antibody titer, be significantly higher than vaccine immunity group.
The animal immune research of the tetanus vaccine composition powder body preparation of effect example 5
Vaccinogen liquid：Tetanus toxoid
Vaccine formulation：
（1）Vaccine immunity control group：This group is removed outside oil phase and surfactant, and remaining component is with embodiment 14.Claim
0.64g sodium carboxymethylcelluloses, plus 64ml water are taken, 1% adhesive is prepared；30g lactose is weighed, with adhesive softwood；Cross 20 mesh
Sieve series grain, 60 °C drying 30 minutes after respectively cross 60 mesh and 80 mesh sieve whole grains, 60 °C dry 120 minutes, sieve take 100-400 mesh
Grain, obtains final product dust carrier.
HEPES0.48g, lactose 0.8g, mannitol 0.8g are weighed, in addition 15mL waters for injection；Add 2400000IU's
Tetanus toxoid, adds water to 20mL, stirs evenly, 0.22 μm of filter membrane filtration；It is dried using Labconco freeze drying box（Freeze
Dry program such as table 1）.Obtained freeze-drying pastille powder is mixed with carrier, makes gross weight for 20g, according to 2mg/ agent（That is 240IU/ agent）
It is filling in powder nose inhalant device, obtain final product.
（2）Vaccine combination powder preparation of the invention：Vaccine combination powder preparation is carried out prepared by Example 14
Experiment.
（3）Blank control group：0.64g sodium carboxymethylcelluloses, plus 64ml water are weighed, 1% adhesive is prepared；Weigh 30g breasts
Sugar, with adhesive softwood；Cross 20 mesh sieves granulation, 60 °C drying 30 minutes after respectively cross 60 mesh and 80 mesh sieve whole grains, 60 °C baking
Dry 120 minutes, sieve takes 100-400 mesh particles, obtains final product dust carrier.As blank administration group.
Animal immune scheme：
By 18 body weight for the female sd inbred rats of 125-175g are randomly divided into 3 groups, every group 6, powder spray nasal-cavity administration.Side
Case is as follows：
1st group：Vaccine immunity control group, gives merely tetanus toxoid, and 40IU/ is only；
2nd group：Vaccine combination powder preparation of the present invention, 40IU/ is only；
3rd group：Blank control group, gives merely homogenous quantities carrier.
Immune effect is detected：
28th day after immune, rat eye socket takes blood, separates serum.It is total in detection serum using indirect ELISA method
IgG titres.Result see the table below（Table 6）：
The ELISA experimental results of the tetanus vaccine composition powder body preparation of table 6
Difference between different groups is compared using one-way analysis of variance, sets significance as p<0.05.From
Data analysis understands that this composition can dramatically increase antibody titer, be significantly higher than simple vaccine immunity group.
The animal vivo test of the hepatitis B vaccine composition powder body preparation of effect example 6
Vaccinogen liquid：Recombinant hepatitis B virus antigen（By Yeast expression and purify）, concentration is 180 μ g/ml.
Vaccine formulation：
（1）Vaccine immunity control group：0.64g sodium carboxymethylcelluloses, plus 64ml water are weighed, 1% adhesive is prepared；Weigh
30g lactose, with adhesive softwood；Cross 20 mesh sieves granulation, 60 °C drying 30 minutes after respectively cross 60 mesh and 80 mesh sieve whole grains,
60 °C dry 120 minutes, and sieve takes 100-400 mesh particles, obtains final product dust carrier.
HEPES0.08g is weighed, synanthrin 0.8g is added in 15mL waters for injection, is stirred to dissolve, adjusted with sodium hydroxide solution
Section pH to 7 is used as water phase；The recombinant hepatitis B virus antigen of 50000 μ g is added, 20mL is added water to, stirred evenly, 0.22 μm of filter membrane filtration；
It is dried using Labconco freeze drying box（Lyophilized program such as table 1）.
Obtained freeze-drying pastille powder is mixed with carrier, makes gross weight for 20g, according to 2mg/ agent（That is 5 μ g/ agent）It is filling in nose
With in powder spray device, obtain final product.
（2）Vaccine combination powder preparation of the present invention：Vaccine combination powder preparation is tried prepared by Example 16
Test.
（3）Blank control group：0.64g sodium carboxymethylcelluloses, plus 64ml water are weighed, 1% adhesive is prepared；Weigh 30g breasts
Sugar, with adhesive softwood；Cross 20 mesh sieves granulation, 60 °C drying 30 minutes after respectively cross 60 mesh and 80 mesh sieve whole grains, 60 °C baking
Dry 120 minutes, sieve takes 100-400 mesh particles, obtains final product dust carrier.As blank administration group.
Animal immune scheme：
By 18 body weight for the female sd inbred rats of 125-175g are randomly divided into 3 groups, every group 6, powder spray nasal-cavity administration.Side
Case is as follows：
1st group：Vaccine immunity control group, gives merely recombinant hepatitis B virus antigen（By Yeast expression and purify）, 5 μ g/
Only；
2nd group：Vaccine combination powder preparation of the present invention, 5 μ g/ are only；
3rd group：Blank control group, gives merely homogenous quantities carrier.
Immune effect is detected
2nd week, 4 weeks, 6 weeks, 8 weeks after immune, rat eye socket takes blood, separates serum.Using indirect ELISA method, detection
Total IgG titre in serum.Result see the table below（Table 7）：
The ELISA experimental results of the hepatitis B vaccine composition powder body preparation of table 7
Difference between different groups is compared using one-way analysis of variance, sets significance as p<0.05.From
Data analysis understands that this composition can dramatically increase antibody titer, be significantly higher than simple vaccine immunity group.
The animal vivo test of the Inflenza vaccine composition powder preparation of effect example 7
Vaccinogen liquid：A type influenza vaccines（A/Perth/16/2009）.
Vaccine formulation：
（1）Vaccine immunity control group：0.64g sodium carboxymethylcelluloses, plus 64ml water are weighed, 1% adhesive is prepared；Weigh
30g lactose, with adhesive softwood；Cross 20 mesh sieves granulation, 60 °C drying 30 minutes after respectively cross 60 mesh and 80 mesh sieve whole grains,
60 °C dry 120 minutes, and sieve takes 100-400 mesh particles, obtains final product dust carrier.Potassium dihydrogen phosphate 0.14g is weighed, lactose 4g is added
In 15mL waters for injection, it is stirred to dissolve；Add the influenza antigens of 150mg HA（A/Perth/16/2009）, 20mL is added water to,
Stir evenly, 0.22 μm of filter membrane filtration；It is dried using Labconco freeze drying box（Lyophilized program such as table 1）.
Lyophilized pastille powder and carrier are mixed, makes gross weight for 20g, according to 2mg/ agent（That is 5 μ g/ agent）It is filling in nose powder
In mist agent device, control group vaccine combination is obtained final product.
（2）Vaccine combination powder preparation of the present invention：Vaccine combination powder preparation prepared by embodiment 2, regulator
Measure as 5 μ g/2mg carry out rat immunity experiment.
（3）Blank control group：0.64g sodium carboxymethylcelluloses, plus 64ml water are weighed, 1% adhesive is prepared；Weigh 30g breasts
Sugar, with adhesive softwood；Cross 20 mesh sieves granulation, 60 °C drying 30 minutes after respectively cross 60 mesh and 80 mesh sieve whole grains, 60 °C baking
Dry 120 minutes, sieve takes 100-400 mesh particles, obtains final product dust carrier.It is administered the dust carrier as blank powder.
Animal immune scheme：
By 24 body weight for the female sd inbred rats of 125-175g are randomly divided into 4 groups, every group 6, powder spray nasal-cavity administration.Side
Case is as follows：
1st group：Vaccine immunity control group, 5 μ g HA/ are only；
2nd group：Vaccine combination powder preparation of the present invention, 5 μ g HA/ are only；
3rd group：Blank control group, gives merely the carrier of homogenous quantities.
Immune effect is detected：
14th day, 28 days after immune, rat eye socket takes blood, separates serum.Tested by HI, determine the blood clotting suppression in serum
Titre processed.Using indirect ELISA method, the total IgG titre in detection serum.Result is as follows（It is shown in Table 8 and table 9）：
Table 8HI result of the tests
Difference between different groups is compared using one-way analysis of variance, sets significance as p<0.05.From
Data analysis understands that this composition can dramatically increase antibody titer, be significantly higher than vaccine immunity group.
The animal vivo test of the Inflenza vaccine composition powder preparation of effect example 8
Vaccinogen liquid：A type influenza vaccines（A/Perth/16/2009）.
Vaccine formulation：
（1）Vaccine immunity control group：0.64g sodium carboxymethylcelluloses, plus 64ml water are weighed, 1% adhesive is prepared；Weigh
30g lactose, with adhesive softwood；Cross 20 mesh sieves granulation, 60 °C drying 30 minutes after respectively cross 60 mesh and 80 mesh sieve whole grains,
60 °C dry 120 minutes, and sieve takes 100-400 mesh particles, obtains final product dust carrier.Citric acid 1.6g is weighed, sucrose 8g adds 15mL
In water for injection, it is stirred to dissolve；Add the influenza antigens of 50mg HA（A/Perth/16/2009）, 20mL is added water to, stir evenly,
0.22 μm of filter membrane filtration；It is dried using Labconco freeze drying box（Lyophilized program such as table 1）.
Lyophilized pastille powder and carrier are mixed, makes gross weight for 20g, according to 2mg/ agent（That is 5 μ g/ agent）It is filling in nose powder
In mist agent device, control group vaccine combination is obtained final product.
（2）Vaccine combination powder preparation of the present invention：Vaccine combination powder preparation prepared by embodiment 1, regulator
Measure as 5 μ g/2mg carry out rat immunity experiment.
（3）Blank control group：0.64g sodium carboxymethylcelluloses, plus 64ml water are weighed, 1% adhesive is prepared；Weigh 30g breasts
Sugar, with adhesive softwood；Cross 20 mesh sieves granulation, 60 °C drying 30 minutes after respectively cross 60 mesh and 80 mesh sieve whole grains, 60 °C baking
Dry 120 minutes, sieve takes 100-400 mesh particles, obtains final product dust carrier.It is administered the dust carrier as blank powder.
Animal immune scheme：
By 24 body weight for the female sd inbred rats of 125-175g are randomly divided into 4 groups, every group 6, powder spray nasal-cavity administration.Side
Case is as follows：
1st group：Vaccine immunity control group, 5 μ g HA/ are only；
2nd group：Vaccine combination powder preparation of the present invention, 5 μ g HA/ are only；
3rd group：Blank control group, gives merely the carrier of homogenous quantities.
Immune effect is detected：
14th day, 28 days after immune, rat eye socket takes blood, separates serum.Tested by HI, determine the blood clotting suppression in serum
Titre processed.Using indirect ELISA method, the total IgG titre in detection serum.Result is as follows（It is shown in Table 10 and table 11）：
Table 10HI result of the tests
Difference between different groups is compared using one-way analysis of variance, sets significance as p<0.05.From
Data analysis understands that this composition can dramatically increase antibody titer, be significantly higher than vaccine immunity group.
The vaccine combination powder preparation Detection of Stability of effect example 9
At 25 °C, relative humidity is to place three months under conditions of 25%, vaccine group prepared by the embodiment of the present invention 1~25
The antigen valence of compound powder preparation keeps constant, and antigen stock potency is down to 25%.Result shows, vaccine combination powder system
Agent has stability higher.
Claims (9)
1. a kind of nose vaccine combination powder preparation, it is characterised in that it includes the following raw material component：
(1) 0.05%~5% antigen；
(2) 0.3~45% oil phases；The oil phase is selected from one or more in squalene, saualane and median chain triglyceride oil；
(3) 0.3~45% surfactants；The surfactant is phosphatide；The phosphatide be selected from soybean lecithin, lecithin,
One or more in egg yolk lecithin and hydrogenated soya phosphatide；
(4) 2~20% mucosa adhesion agent；With
(5) 4~40% freeze-dried excipients；The freeze-dried excipient be selected from trehalose, lactose, synanthrin and mannitol in one kind or
It is various；
The percentage is the mass percent that each composition accounts for raw material total amount.
2. nose as claimed in claim 1 vaccine combination powder preparation, it is characterised in that the antigen is selected from influenza virus
Any one in antigen, hepatitis B virus antigen and tetanus virus antigen.
3. nose as claimed in claim 1 vaccine combination powder preparation, it is characterised in that the mucosa adhesion agent is selected from shell
One or more in glycan, sodium carboxymethylcellulose, sodium alginate, Carbomer and Hydroxypropyl methylcellulose.
4. nose as claimed in claim 1 vaccine combination powder preparation, it is characterised in that described raw material also includes not surpassing
8% buffer is crossed, the percentage accounts for the mass percent of raw material total amount for buffer；Described buffer is selected from phosphoric acid
One or more in salt, 4- HEPESs and citric acid.
5. nose as claimed in claim 1 vaccine combination powder preparation, it is characterised in that described raw material also includes not surpassing
60% carrier is crossed, the percentage accounts for the mass percent of raw material total amount for carrier；The carrier is sodium carboxymethylcellulose
With the mixture of lactose.
6. the preparation method of vaccine combination powder preparation of the nose as described in any one of Claims 1 to 5, it is characterised in that
It comprises the following steps：
(1) oil phase and surfactant are mixed, stirs and add water, obtain adjuvant；
(2) after adjuvant obtained in step (1) is mixed with antigen, freeze-drying obtains powder, obtains final product.
7. preparation method as claimed in claim 6, it is characterised in that also include step (3) after step (2)：By step
(2) the powder sieving obtained by, then packing is preserved.
8. preparation method as claimed in claim 7, it is characterised in that also included step (a), and step before step (1)
(3) step (3 ') is replaced with；Described step (a) is：Sodium carboxymethylcellulose is dissolved in water adhesive is obtained, then lactose is added
Enter in described adhesive, be well mixed, granulation, whole grain are simultaneously dried, and obtain carrier；Described step (3 ') is：By step (2) gained
Powder be well mixed with carrier obtained in step (a), obtain final product.
9. preparation method as claimed in claim 8, it is characterised in that will be well mixed in step (3 ') after step (3 ')
Products therefrom is sieved, and then packing is preserved.
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| CN107233217A (en) * | 2016-03-25 | 2017-10-10 | 李和伟 | A kind of lyophilized formulations containing surfactant and its wash shield product |
| CN107802832A (en) * | 2017-12-15 | 2018-03-16 | 山东农业大学 | A kind of injection-type inactivated vaccine adjuvant for animals |
| CN111346224B (en) * | 2018-12-24 | 2023-12-08 | 洛阳赛威生物科技有限公司 | Immunoadjuvant composition, preparation method and application thereof |
| EP3698773A1 (en) | 2019-02-21 | 2020-08-26 | Università degli Studi di Parma | Composition and manufacturing of powders containing nanoadjuvants for mucosal vaccination |
| WO2022168889A1 (en) * | 2021-02-03 | 2022-08-11 | 応用酵素医学研究所株式会社 | Fine particle powder-type mucosal vaccine |
| GB202205231D0 (en) * | 2022-04-09 | 2022-05-25 | Nasaleze Patents Ltd | Vaccine compositions |
| CN115590826B (en) * | 2022-10-21 | 2023-12-19 | 北京安必奇生物科技有限公司 | Active protein mucous membrane administration preparation and preparation method and application thereof |
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