CN106492215B - A kind of preparation method of load-carrying group viral hepatitis type E protein microsphere suspension formulations - Google Patents
A kind of preparation method of load-carrying group viral hepatitis type E protein microsphere suspension formulations Download PDFInfo
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Abstract
The present invention relates to a kind of preparation method of load-carrying group viral hepatitis type E protein microsphere suspension formulations, microsphere supported in suspension formulations be internal layer is alginate core, and the outer double-deck microballoon by chitosan layer is called alginate-chitosan microball carrier.For the average particle size distribution of load-carrying group viral hepatitis type E protein microsphere in prepared suspension formulations between 6.73 microns~8.39 microns, form is uniform.It is 60 mcg/mls (recombination viral hepatitis type E albumen and unadsorbed free recombination viral hepatitis type E albumen including microsphere supported absorption) that viral hepatitis type E final concentration of protein is recombinated in suspension formulations, by the measurement of the recombination viral hepatitis type E albumen to suspension external solution, calculates the microsphere supported adsorption rate to recombination viral hepatitis type E albumen and be greater than 90%.Recombination viral hepatitis type E vaccine of the load-carrying group viral hepatitis type E protein microsphere suspension compared with Isodose using aluminium hydroxide as adjuvant preferably can generate the immune response for being directed to viral hepatitis type E by stimulation test animal, this has greater significance in practical applications.
Description
Technical field
The present invention relates to a kind of preparation methods of load-carrying group viral hepatitis type E protein microsphere suspension formulations, belong to biological agent preparation
Technical field.
Background technique
Hepatitis E virus (Hepatitis E virus, HEV) full-length genome 7.2kb, it is partly overlapping comprising 3
ORF, wherein ORF2 contains important Neutralization and crystallization.The existing many segments of ORF2 are successful in different expression systems at present
It is expressed.HEV there is no suitable cell culture system.Now listing or in the HEV vaccine ground be gene engineering expression weight
The subunit vaccine of group HEV ORF2 antigen protein.China carries out only two companies of HEV vaccine large-scale production research,
(Xiamen ten thousand is safe, recombinates HEV P239 albumen) of listing and carry out clinical test (Changchun Biological Products Institute's Limited Liability public affairs
Department, recombinate HEV P179 albumen) recombined hepatitis E hepatitis vaccine all use aluminium hydroxide as the adjuvant of recombinant protein, but with
Aluminium hydroxide is limited to the immunological enhancement of Recombinant protein vaccine as adjuvant, and body cannot be stimulated to generate stronger needle
Immune response to HEV.And since domestic HEV vaccine product is less, to HEV vaccine novel form, new subsequent product research
Also less.
Nanosphere medicine carrier is that medicinal application new process, a protrusion in new technology represent in recent years.Existing 30 multiclass medicines
Object is using microballoon or microencapsulation technology in recent years, such as analgesic-antipyretic, antibiotic, polypeptide, contraceptive, vitamin, anticarcinogen
And diagnostic agent etc..This drug delivery technology Technical comparing in Chinese medicine or chemical drug is mature, but in albumen such as biological products
The application of matter drug is still in conceptual phase.Microballoon drug delivery technologies also attract attention in research and development prevention and treatment property vaccines arts.
Distribution is also different in vivo for the particle of different-grain diameter size, and usual partial size is most of at 2.5 ~ 10 microns
Productive set is in macrophage；It is generally absorbed by the macrophage in liver, spleen when less than 7 microns；0.2 ~ 0.4 micron of collection of particles in
It is removed by liver rapidly after liver；Particle less than 0.01 micron then slow productive set in marrow.It can be seen that when particle size range is distributed in
At 2.5 ~ 10 microns, particle is easier to be absorbed by phagocyte, and stimulation body generates immune response, but general recombinant expression preparation
Antigen protein partial size be nanometer range, can not directly cause effective specific immune response, after aluminium adjuvant is added, aluminium salt
The condensate of formation cannot form the particle with regular particle diameter distribution of consolidation, especially in shearing force and body itself fluid force
It learns its loose condensate under environment to be easier to be broken up, forms more wide in range particle size distribution range, limit its absorption
Distribution of the antigen in machine affects macrophage to the uptake ratio of antigen, and then influences body fight and originate in raw specificity
Immune response.And it is microsphere supported with relatively regular particle size distribution range, use smaller on shearing force and hydromechanical influence
Its change of size amplitude is little afterwards, easier can be swallowed by macrophage, thus cause the specific immune response of body, and
And transmitting and stocking system as vaccine, the microsphere supported slow release that can ease up the local concentration of antigen protein progress, in office
Portion's high concentration and continuing generates body in the case where stimulation stronger than standard adjuvant (Alum adjuvant), more special immune anti-
It answers.
Alginate-chitosan microball preparation condition is mild, needs less organic solvent, to activated protein in preparation process
The destruction of matter is less, low in cost particularly suitable for having the protein drug of bioactivity, and its abundance, is to have very much
The recombinant antigen carrier of future.Alginate is polysaccharide compound, and common diluted alkaline extracts preparation from brown alga.Sodium alginate can
It is dissolved in the water of different temperatures, does not dissolve in ethyl alcohol, ether and other organic solvents.It is multiple for can sharing with chitin or polylysine
Condensation material.Because calcium alginate is not soluble in water, therefore sodium alginate can solidify encystation with calcium chloride.It can be dissolved in after chitin deacetylation
Water is referred to as chitosan, and chitin and chitosan is the cationic animal origin uniquely found so far.Chitosan has one
Fixed biological degradability, decomposition product is nontoxic, has enhancing drug absorption, antibacterial anti-inflammatory, promotes wound healing, reduction gallbladder solid
Alcohol, the effect for inhibiting tumour cell.
Usually using edible oil when emulsion process prepares alginate microsphere core, but since edible oil presence is more apparent
Differences between batches form core and core Size Distribution are larger, and use atoleine instead and then avoid batch because of oily phase
Between the influence that core is formed of difference and oil soluble impurity.
What is now studied is to wrap up protein drug into inside microballoon in protein microsphere preparation, and because protein is unlike changing
It learns drug and equally there is apparent oil and water zonation property, so preparing the alginate-chitosan system for carrying albumen with emulsion process
Partially protein is dissolved in oil and mutually to carry protein quantity reduction in microballoon when agent, seriously affects the encapsulation rate of microball preparation, but by
In there is a large amount of primary amino group in chitosan molecule chain, there is a large amount of carboxyl on the strand of sodium alginate, so chitosan and sea
Alginates can attract to form polyelectrolyte film by positive and negative charge.It, can since the viral hepatitis type E albumen of recombination has negative electrical charge mostly
It to attract each other with alginate-chitosan microball carrier outer layer with the chitosan layer of positive charge, and adsorbs thereon, obtains
Alginate-chitosan microball of load-carrying group viral hepatitis type E albumen, and the load viral hepatitis type E albumen alginate-chitosan microball prepared is mixed
Suspension formulation is to mix albumen stoste with microballoon, the absorption of protein and microsphere surface in suspension there are dynamic equilibrium,
So that microballoon has a higher adsorption rate to albumen, and and protein-free loss, preparation method is simple, and more passes through
Ji.
Summary of the invention:
The present invention provides a kind of preparation method of load-carrying group viral hepatitis type E protein microsphere suspension formulations, uses alginate-shell
Carrier of the glycan microballoon as recombined hepatitis E hepatitis virus antigen protein belongs in the research of Hepatitis E vaccine at home first
Wound has filled up the blank of viral hepatitis type E vaccine dosage research.
Microsphere supported in suspension formulations be internal layer is alginate core, and the outer double-deck microballoon by chitosan layer claims it
For alginate-chitosan microball carrier.The average grain diameter of load-carrying group viral hepatitis type E protein microsphere in prepared suspension formulations point
For cloth between 6.73 microns~8.39 microns, form is uniform.It is 60 micrograms/milli that viral hepatitis type E final concentration of protein is recombinated in suspension formulations
It rises (recombination viral hepatitis type E albumen and unadsorbed free recombination viral hepatitis type E albumen including microsphere supported absorption), by suspension external solution
Recombination viral hepatitis type E albumen measurement, calculate it is microsphere supported to recombination viral hepatitis type E albumen adsorption rate be greater than 90%.
The preparation method of load-carrying group viral hepatitis type E protein microsphere suspension formulations of the present invention, includes the following steps:
1, the preparation of microsphere supported alginate core
Preparation method is emulsion process, and concentration is 1%~1.5% through 115 DEG C of high pressure sterilizations, 30 minutes sodium alginate aqueous solutions
It is oily phase with atoleine for water phase, water phase and oil phase volume ratio are 1:2, using single emulsifier or blended emulsifier as newborn
Agent, emulsifier additive amount are 1% ~ 4%(emulsifier volume/oil phase volume), oil-water interfaces equilibrium valve (HLB value) control 4 ~ 6,
At 50 revs/min ~ 500 revs/min, the pre-emulsification time controlled at 5 ~ 30 minutes the control of pre-emulsification revolving speed, and emulsification revolving speed control exists
500 revs/min ~ 2000 revs/min, emulsification times control at 5 ~ 30 minutes, curing agent be through 115 DEG C high pressure sterilization 30 minutes
Calcium chloride water, concentration are 5% ~ 20%, and solidify liquid and lotion volume ratio are 1:1 ~ 1:3, and it is 3 millis that solidify liquid, which instills speed,
Liter/min ~ 20 ml/mins, continuing curing time is 10 ~ 120 minutes.Calcium alginate microsphere is after oily aqueous phase separation with sterile
Water for injection or sterile distilled water rinse 3 ~ 5 times, and centrifuge separation obtains microsphere supported alginate core；
2, the alginate core coating chitosan layer obtained prepares alginate-chitosan microball carrier
The alginate core obtained in step 1) is suspended in concentration to divide for 0.1% ~ 0.8% through 115 DEG C of high pressure sterilizations 30
In the chitosan aqueous solution of clock, at 25 DEG C ~ 42 DEG C, revolving speed is to incubate 10 ~ 60 minutes in 50 revs/min ~ 200 revs/min shaking tables,
It is centrifuged 1 ~ 30 minute through 1000 revs/min ~ 5000 revs/min, sterile water for injection or sterile distilled water rinse 3 ~ 5 times, obtain
Alginate-chitosan microball carrier；
3, the preparation of viral hepatitis type E protein microsphere preparation is recombinated
Recombination viral hepatitis type E protein solution is added in the alginate obtained into step 2-chitosan microball carrier, and uses nothing
Bacterium water for injection be settled to 1 used in the identical volume of water phase, make last antigen protein content in 60 mcg/mls,
25 DEG C ~ 42 DEG C, revolving speed is to incubate 10 ~ 60 minutes in 50 revs/min ~ 200 revs/min shaking tables, prepares and carries viral hepatitis type E protein microsphere
Suspension formulations, aseptic subpackaged after mixing, 2 DEG C ~ 8 DEG C preservations are even using front overhang.
In prepared suspension load-carrying group viral hepatitis type E protein microsphere average particle size distribution 6.73 microns~8.39 microns it
Between, form is uniform.By calculating microsphere supported to contained to viral hepatitis type E determining the protein quantity is recombinated in suspension formulations external solution
The adsorption rate of viral hepatitis type E albumen is greater than 90%, and adsorption rate calculation method is as follows:
Adsorption rate=(Tot Prot-suspension external solution Tot Prot)/Tot Prot × 100%
Unadsorbed antigen protein remains in suspension in the load-carrying group viral hepatitis type E protein suspension preparation of this method preparation,
Meet Drug absorbability dynamic equilibrium rule, realizes the maximization of drug utilization efficiency.
The positive effect of the present invention is:
Microsphere supported alginate-the chitosan microball obtained for emulsion process that viral hepatitis type E albumen is carried in preparation, in preparation
It uses atoleine instead of edible oil as oily phase, effectively avoids because oily phase quality and oily phase differences between batches are to acquisition microballoon
The influence of carrier.It is combined and viral hepatitis type E albumen is microsphere supported by being adsorbed in, rather than viral hepatitis type E albumen is packed in microballoon, be prepared for
Viral hepatitis type E final concentration of protein is the load viral hepatitis type E protein microsphere suspension formulations of 60 mcg/mls, by suspension external solution viral hepatitis type E egg
White content detection, it was demonstrated that the microsphere supported absorption to viral hepatitis type E albumen is preferable, and adsorption rate can reach 90% or more, successfully avoids
Be packed in that method encapsulation rate low and a large amount of viral hepatitis type E albumen because caused by albumen is mutually distributed in grease of formula is wasted shows
As.The load-carrying group viral hepatitis type E protein microsphere of acquisition, uniform particle diameter, particle size distribution range is narrow, and average grain diameter is less than 10 microns, form
It is uniform.Zoopery comparison, recombination of the load-carrying group viral hepatitis type E protein microsphere suspension compared with Isodose using aluminium hydroxide as adjuvant
Viral hepatitis type E vaccine preferably can generate the immune response for being directed to viral hepatitis type E by stimulation test animal, this has more careless in practical applications
Justice.
Detailed description of the invention
Load-carrying group viral hepatitis type E protein microsphere particle 400 in five batches of suspension formulations prepared by embodiment 1 ~ 6 in Fig. 1, the present invention
Times light microscopic micrograph (the microballoon particle of preparation is prepared under the conditions of the corresponding embodiment 1 ~ 5 of number 1 ~ 5)；
Load-carrying group viral hepatitis type E protein microsphere in five batches of suspension formulations prepared by embodiment 1 ~ 6 in Fig. 2 .1 ~ Fig. 2 .5, the present invention
Particle size distribution figure (prepares the microballoon particle of preparation) under the conditions of the corresponding embodiment 1 ~ 5 of number 1 ~ 5；
Load-carrying group viral hepatitis type E protein microsphere particle is average in five batches of suspension formulations prepared by embodiment 1 ~ 6 in Fig. 3, the present invention
Partial size compares (the microballoon particle of preparation is prepared under the conditions of the corresponding embodiment 1 ~ 5 of number 1 ~ 5)；
Five batches of suspension formulations prepared by embodiment 1 ~ 6 compare the adsorption rate of recombination viral hepatitis type E albumen in Fig. 4, the present invention
(the microballoon particle of preparation is prepared under the conditions of the corresponding embodiment 1 ~ 5 of number 1 ~ 5)；
The used two batches time of test example 1 edible oil (soybean oil) is the alginate core of oil phase preparation in Fig. 5, the present invention
It is compared with average grain diameter of the two batches time atoleine as the oily mutually alginate core of preparation is used
It is prepared by the load viral hepatitis type E protein microsphere suspension formulations and absorption method of the preparation of pack described in Fig. 6, test example of the present invention 2
Load viral hepatitis type E protein microsphere suspension formulations encapsulation rate (adsorption rate) comparison
Five batches of suspensions and aluminium adjuvant vaccine experiments animal blood serum specific IgG described in test example 3 in Fig. 7, the present invention
Positive seroconversion rate compares (the microballoon particle of preparation is prepared under the conditions of the corresponding embodiment 1 ~ 5 of number 1 ~ 5)
Five batches of suspensions and aluminium adjuvant vaccine experiments animal blood serum specific IgG described in test example 3 in Fig. 8, the present invention
Antibody OD mean value compares (the microballoon particle of preparation is prepared under the conditions of the corresponding embodiment 1 ~ 5 of number 1 ~ 5).
Specific embodiment
As described below is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Embodiment 1
It through 100 milliliters of 30 minutes sodium alginate aqueous solutions of 115 DEG C of high pressure sterilizations is water that the present embodiment preferred concentration, which is 1%,
Phase is oily phase with 200 milliliters of atoleine, and water phase and oil phase volume ratio are 1:2, is made with Tween 80 and span 85 blended emulsifier
For emulsifier, emulsifier additive amount is 2.02 milliliters (emulsifier volume/oil phase volume=1%), oil-water interfaces equilibrium valve (HLB value)
Control is computed in 4 [HLB=(1.8* span 85 volume+15.0* Tween 80 volume)/(span 85 volume+Tween 80 volume)],
Blended emulsifier be configured to 1.68 milliliters span 85 and 0.34 milliliter of Tween 80.Said mixture is through 200 revs/min of pre-emulsifications
10 minutes, 1000 revs/min emulsified 30 minutes.200 milliliters of solidifications are instilled in the lotion into stirring with 8 ml/mins drop speed
Agent (115 DEG C of high pressure sterilization 30 minutes 15% calcium chloride waters) solidify liquid and lotion volume ratio are 1:1.5, and solidify liquid drips off
Afterwards, continue solidification 90 minutes.It is rinsed 3 ~ 5 times after oily aqueous phase separation with sterile water for injection, centrifuge separation obtains microsphere supported sea
Alginates core.
Embodiment 2
It through 100 milliliters of 30 minutes sodium alginate aqueous solutions of 115 DEG C of high pressure sterilizations is water that the present embodiment preferred concentration, which is 1%,
Phase is oily phase with 200 milliliters of atoleine, and water phase and oil phase volume ratio are 1:2, is made with Tween 80 and span 85 blended emulsifier
For emulsifier, emulsifier additive amount is 8.33 milliliters (emulsifier volume/oil phase volume=4%), oil-water interfaces equilibrium valve (HLB value)
Control is computed in 5 [HLB=(1.8* span 85 volume+15.0* Tween 80 volume)/(span 85 volume+Tween 80 volume)],
Blended emulsifier be configured to 6.31 milliliters span 85 and 2.02 milliliters of Tween 80s.Said mixture is through 200 revs/min of pre-emulsifications
10 minutes, 1000 revs/min emulsified 30 minutes.200 milliliters of solidifications are instilled in the lotion into stirring with 8 ml/mins drop speed
Agent (115 DEG C of high pressure sterilization 30 minutes 15% calcium chloride waters) solidify liquid and lotion volume ratio are 1:1.5, and solidify liquid drips off
Afterwards, continue solidification 90 minutes.It is rinsed 3 ~ 5 times after oily aqueous phase separation with sterile water for injection, centrifuge separation obtains microsphere supported sea
Alginates core.
Embodiment 3
It through 100 milliliters of 30 minutes sodium alginate aqueous solutions of 115 DEG C of high pressure sterilizations is water that the present embodiment preferred concentration, which is 1%,
Phase is oily phase with 200 milliliters of atoleine, and water phase and oil phase volume ratio are 1:2, is made with Tween 80 and span 85 blended emulsifier
For emulsifier, emulsifier additive amount is 4.08 milliliters (emulsifier volume/oil phase volume=2%), oil-water interfaces equilibrium valve (HLB value)
Control is computed in 6 [HLB=(1.8* span 85 volume+15.0* Tween 80 volume)/(span 85 volume+Tween 80 volume)],
Blended emulsifier be configured to 2.78 milliliters span 85 and 1.30 milliliters of Tween 80s.Said mixture is through 200 revs/min of pre-emulsifications
10 minutes, 1000 revs/min emulsified 30 minutes.200 milliliters of solidifications are instilled in the lotion into stirring with 8 ml/mins drop speed
Agent (115 DEG C of high pressure sterilization 30 minutes 15% calcium chloride waters) solidify liquid and lotion volume ratio are 1:1.5, and solidify liquid drips off
Afterwards, continue solidification 90 minutes.It is rinsed 3 ~ 5 times after oily aqueous phase separation with sterile water for injection, centrifuge separation obtains microsphere supported sea
Alginates core.
Embodiment 4
The present embodiment preferred concentration is 1.5% to be through 100 milliliters of 30 minutes sodium alginate aqueous solutions of 115 DEG C of high pressure sterilizations
Water phase is oily phase with 200 milliliters of atoleine, and water phase and oil phase volume ratio are 1:2, with Tween 80 and span 85 blended emulsifier
As emulsifier, emulsifier additive amount is 4.08 milliliters (emulsifier volume/oil phase volume=2%), oil-water interfaces equilibrium valve (HLB
Value) control is in 4 [HLB=(1.8* span 85 volume+15.0* Tween 80 volume)/(span 85 volume+Tween 80 volume)], through counting
Calculate, blended emulsifier be configured to 3.40 milliliters span 85 and 0.68 milliliter of Tween 80.Said mixture is pre- through 200 revs/min
Emulsification 10 minutes, 1000 revs/min emulsify 30 minutes.200 milliliters are instilled in the lotion into stirring with 8 ml/mins drop speed
Curing agent (115 DEG C of high pressure sterilization 30 minutes 15% calcium chloride waters) solidify liquid and lotion volume ratio are 1:1.5, solidify liquid
After dripping off, continue solidification 90 minutes.It is rinsed 3 ~ 5 times after oily aqueous phase separation with sterile water for injection, centrifuge separation, obtains microballoon load
Body alginate core.
Embodiment 5
It through 100 milliliters of 30 minutes sodium alginate aqueous solutions of 115 DEG C of high pressure sterilizations is water that the present embodiment preferred concentration, which is 1%,
Phase is oily phase with 200 milliliters of atoleine, and water phase and oil phase volume ratio are 1:2, is made with Tween 80 and span 85 blended emulsifier
For emulsifier, emulsifier additive amount is 4.08 milliliters (emulsifier volume/oil phase volume=2%), oil-water interfaces equilibrium valve (HLB value)
Control is computed in 4 [HLB=(1.8* span 85 volume+15.0* Tween 80 volume)/(span 85 volume+Tween 80 volume)],
Blended emulsifier be configured to 3.40 milliliters span 85 and 0.68 milliliter of Tween 80.Said mixture is through 200 revs/min of pre-emulsifications
10 minutes, 1000 revs/min emulsified 30 minutes.200 milliliters of solidifications are instilled in the lotion into stirring with 8 ml/mins drop speed
Agent (115 DEG C of high pressure sterilization 30 minutes 15% calcium chloride waters) solidify liquid and lotion volume ratio are 1:1.5, and solidify liquid drips off
Afterwards, continue solidification 90 minutes.It is rinsed 3 ~ 5 times after oily aqueous phase separation with sterile water for injection, centrifuge separation obtains microsphere supported sea
Alginates core.
Embodiment 6
The alginate microsphere core prepared in embodiment 1 ~ 5 precipitating is hung into 100 milliliters of 115 DEG C of high pressure sterilizations 30 respectively
In 0.3% chitosan solution of minute, incubated 30 minutes in 37 DEG C of shaking tables with 100 revs/min of revolving speeds.3000 revs/min of centrifugations
It 5 minutes, discards supernatant, collects alginate-chitosan microball precipitating.Alginate-chitosan is cleaned with sterile water for injection
Microballoon precipitates 3 times, is centrifuged 5 minutes with 3000 revs/min of revolving speed every time, discards supernatant cleaning solution, and 2.00 milliliters of addition is dense
Degree is the recombination viral hepatitis type E albumen stoste of 3 mg/mls, and is settled to 100 milliliters with sterile water for injection, makes to recombinate viral hepatitis type E albumen
Final concentration of 60 mcg/ml, at 37 DEG C, revolving speed is to incubate 30 minutes in 100 revs/min of shaking tables, obtains five batches of different parameters
The load-carrying group viral hepatitis type E protein microsphere suspension formulations of preparation, are shown in Fig. 1.
Every batch of preparation is mixed, 100 pieces of particles is randomly selected in every batch of preparation and is measured with micro- micrometer, through computer SPSS
Software statistics microspherulite diameter and its distribution, between 6.73 microns~8.39 microns of average grain diameter, form is uniform, sees Fig. 2 .1 ~ figure
2.5, Fig. 3.
It takes 10 milliliters of samples to be centrifuged 5 minutes through 6000 revs/min every batch of suspension formulations, collects supernatant (external solution),
External solution volume is measured, and measures and wherein recombinates viral hepatitis type E protein content, calculates the adsorption rate of recombination viral hepatitis type E albumen:
Adsorption rate=(protein content * external solution volume in 60 10 milliliters-external solutions of mcg/ml *)/600 microgram * 100%
Through detecting, the recombination viral hepatitis type E protein adsorption rate of 1 ~ embodiment of embodiment, 5 five batches of suspension formulations is all larger than 90%, sees
Fig. 4.
Test example 1
Select the edible soybean oil of two different batches as the atoleine of oily phase and two different batches as oil
Phase prepares alginate core by the method provided in embodiment 5, using core average grain diameter and particle diameter distribution as index to two
The microballoon core of kind method preparation is compared result and sees Fig. 5.
By contrast, edible soybean oil differences between batches are prepared with larger impact to microballoon core, such as use edible soybean oil
It mutually will be unable to obtain stable preparation process as oil emulsion, and use atoleine as oil emulsion phase, then for preparing is micro-
Ball core average grain diameter is smaller, and particle size distribution range is narrower, can get stable preparation process.
Test example 2
It is mixed that two batches load viral hepatitis type E albumen alginate-chitosan microball is prepared using embodiment 5 and 6 the method for embodiment
Suspension formulation (absorption method), calculates separately the adsorption rate of microball preparation.
Adsorption rate=(protein content * external solution volume in 60 10 milliliters-external solutions of mcg/ml *)/600 milligram * 100%
Change the water phase in embodiment 5 into aqueous solution containing 6 milligrams of viral hepatitis type E albumen, and according to 5 the method for embodiment
The alginate microsphere core of package viral hepatitis type E albumen is prepared, and the method described in embodiment 6 wraps up chitosan layer, centrifugation obtains
Alginate-chitosan microball that viral hepatitis type E albumen must be wrapped up adds sterile water for injection to be settled to 100 milliliters, obtains package legal system
The alginate of standby viral hepatitis type E albumen-chitosan microball mixed suspension preparation shares this method and prepares two batch mixed suspension preparations.Every batch of
10 milliliters of suspensions are extracted, microballoon is centrifugated, microballoon is cracked with PH8.0 lysate, measures protein content in microballoon, calculate packet
Envelope rate
* 100 milliliters of protein content/10 milliliter/6 milligram * 100% in encapsulation rate=microballoon
The viral hepatitis type E prepared by the viral hepatitis type E protein microsphere mixed suspension preparation prepared to two batch packs and two batch absorption methods
The comparison of the encapsulation rate (adsorption rate) of protein microsphere mixed suspension preparation, is as a result shown in Fig. 6, it can be seen that the viral hepatitis type E egg of absorption method preparation
Bai Weiqiu mixed suspension preparation is more to the absorption of albumen, prepared by the viral hepatitis type E protein microsphere mixed suspension preparation viral hepatitis type E albumen of pack preparation
It loses in the process more, finally makes encapsulation rate low, so that proof replaces the existing pack generally used can be more with absorption method
Economy preparation viral hepatitis type E protein microsphere mixed suspension preparation.
Test example 3
Load-carrying group viral hepatitis type E protein microsphere suspension formulations prepared by 1 ~ embodiment of embodiment, 5 five batches of different parameters of acquisition
Animal experiment comparison is carried out with the recombination HEV vaccine of traditional aluminium hydroxide adjuvant.
SPF grades of NIH mouse of 200 health are chosen, half male and half female is divided into 7 groups, and control group 20, half male and half female, remaining 6
Group is immune group, and every group 30, half male and half female, it is traditional handicraft preparation containing aluminum hydroxide adjuvant that group 1, which is immunized with drug,
HEV antigen protein vaccine (recombination viral hepatitis type E final concentration of protein is 60 mcg/mls) is recombinated, it is embodiment 1 that group 2 ~ 6, which is immunized with drug,
(recombinate viral hepatitis type E final concentration of protein is the load-carrying group viral hepatitis type E protein microsphere suspension formulations of ~ 6 five batches of different parameters preparations obtained
60 mcg/mls).
1 immune programme of table
Excision eyeball of mouse acquisition blood prepares serum within 21st day, carries out 100 times of dilutions, and pass through Hepatitis E virus
IgG antibody enzyme-linked immunization diagnostic kit detects the IgG antibody Conversion rate of the corresponding anti-HEV of mice serum, sees Fig. 7, and pass through
The comparison of corresponding antibodies OD value is shown in that Fig. 8, discovery load-carrying group viral hepatitis type E protein microsphere suspension formulations are reducing the immune of primary immunization
With the recombination viral hepatitis type E protein vaccine immunological comparison of the aluminium hydroxide adjuvant of traditional handicraft preparation under program, obtain identical or more
Good immune effect, it is seen that load-carrying group viral hepatitis type E protein microsphere suspension formulations preferably can be directed to HEV antigen by excitation experiment animal
Specific immune response, and immune programme can be simplified, this has greater significance in practical applications.
According to test example 1 ~ 3, it can be seen that the present invention replaces edible oil mutually successfully to keep away as oil emulsion using atoleine
The influence because of edible oil oil-soluble impurity and edible oil differences between batches to microballoon core is prepared is exempted from, so that preparation process is more
Stablize；It replaces pack preparation to carry protein microsphere using absorption method, it is big because of the distribution in grease phase to avoid protein
Amount loss, so that the load protein microsphere of preparation is more economic, but also microball preparation adsorption rate with higher；It is tried by animal
The comparison tested also can be shown that load viral hepatitis type E albumen alginate-chitosan microball mixed suspension preparation prepared by the present invention, not only without broken
The immunogenicity of bad viral hepatitis type E albumen or even the immune effect of said preparation are also better than the same dosage using aluminium hydroxide as adjuvant and exempt from
Epidemic disease preparation.
Claims (1)
1. a kind of preparation method of load-carrying group viral hepatitis type E protein microsphere suspension formulations, comprising the following steps:
1) preparation method is emulsion process, and concentration is 1%~1.5% to be through 115 DEG C of high pressure sterilizations, 30 minutes sodium alginate aqueous solutions
Water phase is oily phase with atoleine, and water phase and oil phase volume ratio are 1:2, using single emulsifier or blended emulsifier as emulsifying
Agent, emulsifier additive amount are emulsifier volume/oil phase volume 1% ~ 4%, and the control of oil-water interfaces equilibrium valve is 4 ~ 6, pre-emulsification revolving speed
At 50 revs/min ~ 500 revs/min, the pre-emulsification time was controlled at 5 ~ 30 minutes for control, the control of emulsification revolving speed 500 revs/min ~
2000 revs/min, at 5 ~ 30 minutes, curing agent was water-soluble through 115 DEG C of high pressure sterilizations, 30 minutes calcium chloride for emulsification times control
Liquid, concentration are 5% ~ 20%, and solidify liquid and lotion volume ratio are 1:1 ~ 1:3, and it is 3 ml/mins ~ 20 millis that solidify liquid, which instills speed,
Liter/min, continuing curing time is 10 ~ 120 minutes；
Calcium alginate microsphere is rinsed 3 ~ 5 times after oily aqueous phase separation with sterile water for injection or sterile distilled water, and centrifuge separation obtains
Microsphere supported alginate core；
2) the alginate core coating chitosan layer obtained prepares alginate-chitosan microball carrier
By the alginate core obtained in step 1) be suspended in concentration be 0.1% ~ 0.8% through 115 DEG C high pressure sterilization 30 minutes
In chitosan aqueous solution, at 25 DEG C ~ 42 DEG C, revolving speed is to incubate 10 ~ 60 minutes in 50 revs/min ~ 200 revs/min shaking tables, is passed through
1000 revs/min ~ 5000 revs/min are centrifuged 1 ~ 30 minute, and sterile water for injection or sterile distilled water rinse 3 ~ 5 times, obtain sea
Alginates-chitosan microball carrier；
3) preparation of viral hepatitis type E protein microsphere preparation is recombinated
The alginate obtained into step 2-chitosan microball carrier is added recombination viral hepatitis type E protein solution, and with sterile note
Penetrate with water be settled to 1) used in the identical volume of water phase, make last antigen protein content in 60 mcg/mls, at 25 DEG C
~ 42 DEG C, revolving speed is to incubate 10 ~ 60 minutes in 50 revs/min ~ 200 revs/min shaking tables, prepares and carries the suspension of viral hepatitis type E protein microsphere
Liquid formulation, aseptic subpackaged after mixing, 2 DEG C ~ 8 DEG C save to get target product.
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