CN106492215A - A kind of preparation method of load-carrying group hepatitis E protein microsphere suspension formulations - Google Patents
A kind of preparation method of load-carrying group hepatitis E protein microsphere suspension formulations Download PDFInfo
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- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
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Abstract
The present invention relates to a kind of preparation method of load-carrying group hepatitis E protein microsphere suspension formulations, microsphere supported in suspension formulations is alginate core for internal layer, and alginate chitosan microball carrier is called by the outer double-deck microsphere by chitosan layer.Between 6.73 microns～8.39 microns, form is homogeneous for the average particle size distribution of the load-carrying group hepatitis E protein microsphere in prepared suspension formulations.In suspension formulations, restructuring hepatitis E final concentration of protein is 60 mcg/mls（Restructuring hepatitis E albumen and unadsorbed free restructuring hepatitis E albumen including microsphere supported absorption）, by the measure of the restructuring hepatitis E albumen to the outer liquid of suspension, calculate the microsphere supported adsorption rate to hepatitis E albumen of recombinating and be more than 90%.Restructuring hepatitis E vaccine of the load-carrying group hepatitis E protein microsphere suspensoid compared with Isodose using aluminium hydroxide as adjuvant preferably can produce the immunoreation for being directed to hepatitis E by stimulation test animal, and this has greater significance in actual applications.
Description
Technical field
The present invention relates to a kind of preparation method of load-carrying group hepatitis E protein microsphere suspension formulations, belongs to biological preparation preparation
Technical field.
Background technology
Hepatitis E viruss（Hepatitis E virus, HEV）Full-length genome 7.2kb, partly overlapping comprising 3
ORF, wherein ORF2 contain important Neutralization and crystallization.It is successful in different expression systems that ORF2 many fragment is had at present
Expressed.HEV there is no suitable cell culture system.Now list or be in the HEV vaccines for grinding the weight of gene engineering expression
The subunit vaccine of group HEV ORF2 antigen proteins.China carries out only two companies of HEV vaccines large-scale production research,
Listing（Xiamen ten thousand is safe, HEV P239 albumen of recombinating）With carry out clinical trial（Changchun Biological Products Institute's Limited Liability is public
Department, HEV P179 albumen of recombinating）Recombined hepatitis E hepatitis vaccine all use aluminium hydroxide as the adjuvant of recombiant protein, but with
, used as adjuvant, the immunological enhancement to Recombinant protein vaccine is limited for aluminium hydroxide, it is impossible to stimulate body to produce higher pin
Immunoreation to HEV.And as domestic HEV vaccine products are less, to HEV vaccine novel forms, new subsequent product is studied
Also less.
Nanosphere medicine carrier is that a protrusion in recent years in medicinal application new technology, new technique is represented.Existing 30 multiclass medicines
Thing is in recent years using microsphere or microencapsulation technology, such as antipyretic analgesic, antibiotic, polypeptide, contraceptive, vitamin, anticarcinogen
And diagnostic agent etc..This drug delivery technology Technical comparing in Chinese medicine or chemical medicine is ripe, but in albumen such as biological product
The application of matter medicine is still in conceptual phase.Microsphere drug delivery technologies also receive publicity in research and development prevention and treatment property vaccines arts.
Distribution is also different in vivo for the microgranule of different-grain diameter size, and usual particle diameter is at 2.5 ~ 10 microns, most of
Productive set is in macrophage；Typically absorbed by the macrophage in liver, spleen during less than 7 microns；0.2 ~ 0.4 micron of collection of particles in
Removed by liver after liver rapidly；Microgranule less than 0.01 micron then slow productive set in bone marrow.As can be seen here, when particle size range is distributed in
When 2.5 ~ 10 microns, microgranule is easier to be absorbed by phagocyte, stimulates body to produce immunoreation, but typically recombinant expressed preparation
Antigen protein particle diameter be nanometer range, it is impossible to directly cause effective specific immune response, after adding aluminium adjuvant, aluminium salt
The polymer of formation can not form the microgranule with regular particle diameter distribution of consolidation, especially in shearing force and body itself fluid force
Learn its loose polymer under environment to be easier to be broken up, form more wide in range particle size distribution range, limit what which adsorbed
Distribution of the antigen in machine, have impact on uptake ratio of the macrophage to antigen, and then affects body fight to originate in raw specificity
Immunne response.And microsphere supported with relatively regular particle size distribution range, less on shearing force and hydromechanical impact, use
Its change of size amplitude is little afterwards, can be easier by macrophage phagocytic, so as to cause the specific immune response of body, and
And the transmission as vaccine and stocking system, microsphere supported can carry out local concentration to antigen protein and ease up slow release, in office
Body producing ratio standard adjuvant is made in the case of portion's high concentration persistently stimulation（Alum adjuvant）Stronger, more special immunity is anti-
Should.
Alginate-chitosan microball preparation condition is gentle, needs less organic solvent, to activated protein in preparation process
The destruction of matter is less, is particularly suitable for the protein drug for having biological activity, and its abundance, with low cost, is have very much
The recombinant antigen carrier of future.Alginate is polysaccharide compound, and conventional diluted alkaline extracts preparation from Brown algae.Sodium alginate can
It is dissolved in the water of different temperatures, insoluble in ethanol, ether and other organic solvents.It is multiple that can share with chitin or polylysine
Condensation material.Because calcium alginate water insoluble, therefore sodium alginate can with calcium chloride solidify encystation.Can be dissolved in after chitin deacetylase base
Water is referred to as shitosan, and chitin and chitosan is unique so far cation animal fiber for finding.Shitosan has one
Fixed biological degradability, its catabolite are nontoxic, and with strengthening, drug absorption, anti-inflammation, promotion wound healing, reduction gallbladder are solid
Alcohol, the effect for suppressing tumor cell.
Emulsion process is usually used edible oil when preparing alginate microsphere core, but due to edible oil exist more obvious
Differences between batches, form to core and core Size Distribution are larger, and use liquid paraffin instead and then avoid criticizing because of oil phase
Between the impact that core is formed of difference and oil soluble impurity.
It is to wrap up protein drug into inside microsphere in protein microsphere preparation now to study, and because being not as of protein
Learn medicine and equally there is obvious oil and water zonation property, so in alginate-shitosan system that load albumen is prepared with emulsion process
During agent, partially protein is dissolved in oil phase and causes to carry protein quantity in microsphere and reduces, and has a strong impact on the envelop rate of microball preparation, but by
There is substantial amounts of primary amino radical in chitosan molecule chain, on the strand of sodium alginate, have substantial amounts of carboxyl, so shitosan and sea
Alginate can attract to form polyelectrolyte film by positive and negative charge.Hepatitis E albumen due to recombinating carries negative charge mostly, can
Attracted each other with the chitosan layer with the outer layer of alginate-chitosan microball carrier with positive charge, and adsorbed thereon, obtained
Alginate-the chitosan microball of load-carrying group hepatitis E albumen, and the load hepatitis E albumen alginate-chitosan microball for preparing is mixed
Suspension formulation is to mix albumen stock solution with microsphere, and the protein in suspension has dynamic equilibrium with the absorption of microsphere surface,
So that microsphere has a higher adsorption rate to albumen, and and protein-free loss, preparation method is simple, and more passes through
Ji.
Content of the invention：
The present invention provides a kind of preparation method of load-carrying group hepatitis E protein microsphere suspension formulations, using alginate-shitosan
Carrier of the microsphere as recombined hepatitis E hepatitis virus antigen protein, belongs to pioneering, fills out in the research of hepatitis E vaccine at home
The blank of hepatitis E vaccine dosage research is mended.
Microsphere supported in suspension formulations is alginate core for internal layer, and the outer double-deck microsphere by chitosan layer claims which
For alginate-chitosan microball carrier.The mean diameter of the load-carrying group hepatitis E protein microsphere in prepared suspension formulations point
Between 6.73 microns～8.39 microns, form is homogeneous for cloth.In suspension formulations, restructuring hepatitis E final concentration of protein is 60 micrograms/in the least
Rise（Restructuring hepatitis E albumen and unadsorbed free restructuring hepatitis E albumen including microsphere supported absorption）, by the outer liquid of suspension
Restructuring hepatitis E albumen measure, calculate microsphere supported to recombinate hepatitis E albumen adsorption rate be more than 90%.
The preparation method of load-carrying group hepatitis E protein microsphere suspension formulations of the present invention, comprises the steps：
The preparation of the 1st, microsphere supported alginate core
Preparation method is emulsion process, and it 1%～1.5% is water through 115 DEG C of autoclavings sodium alginate aqueous solution of 30 minutes that concentration is
Phase, with liquid paraffin as oil phase, water is 1 with oil phase volume ratio:2, using single emulsifier or blended emulsifier as emulsifying agent,
Emulsifying agent addition is 1% ~ 4%（Emulsifying agent volume/oil phase volume）, oil-water interfaces equilibrium valve（HLB value）Control 4 ~ 6, pre- breast
Change rotating speed control at 50 revs/min ~ 500 revs/min, at 5 ~ 30 minutes, emulsifying rotating speed was controlled 500 pre-emulsification time control
Rev/min ~ 2000 revs/min, emulsification times were controlled at 5 ~ 30 minutes, and firming agent is through 115 DEG C of autoclavings chlorine of 30 minutes
Change calcium aqueous solution, its concentration is 5% ~ 20%, and solidify liquid is 1 with emulsion volume ratio:1~1:3, solidify liquid instill speed be 3 milliliters/
Minute ~ 20 ml/mins, continued hardening time for 10 ~ 120 minutes.Calcium alginate microsphere uses aseptic note after profit phase separation
Penetrate and rinsed 3 ~ 5 times with water or sterile distilled water, centrifugation obtains microsphere supported alginate core；
2nd, the alginate core coating chitosan layer for obtaining prepares alginate-chitosan microball carrier
By step 1）The alginate core of middle acquisition is suspended in concentration for 0.1% ~ 0.8% through 115 DEG C of autoclavings 30 minutes
In chitosan aqueous solution, at 25 DEG C ~ 42 DEG C, rotating speed is to incubate 10 ~ 60 minutes in 50 revs/min ~ 200 revs/min shaking tables, warp
1000 revs/min ~ 5000 revs/min are centrifuged 1 ~ 30 minute, and sterile water for injection or sterile distilled water are rinsed 3 ~ 5 times, obtain sea
Alginate-chitosan microball carrier；
3rd, the preparation of restructuring hepatitis E protein microsphere preparation
To step 2）The alginate of middle acquisition-chitosan microball carrier adds restructuring hepatitis E protein solution, and uses aseptic note
Penetrate with water be settled to 1 in used water phase identical volume, make last antigen protein content in 60 mcg/mls, 25 DEG C ~
42 DEG C, rotating speed is to incubate 10 ~ 60 minutes in 50 revs/min ~ 200 revs/min shaking tables, prepares load hepatitis E protein microsphere suspension
Preparation, aseptic subpackaged after mixing, 2 DEG C ~ 8 DEG C preservations are even using front overhang.
In prepared suspension load-carrying group hepatitis E protein microsphere average particle size distribution 6.73 microns～8.39 microns it
Between, form is homogeneous.By to hepatitis E determining the protein quantity of recombinating in the outer liquid of suspension formulations, calculating microsphere supported to contained
The adsorption rate of hepatitis E albumen is more than 90%, and adsorption rate computational methods are as follows：
Adsorption rate=（The outer liquid Tot Prot of Tot Prot-suspension）/ Tot Prot × 100%
In load-carrying group hepatitis E protein suspension preparation prepared by this method, unadsorbed antigen protein is remained in suspension, is met
Drug absorbability dynamic equilibrium rule, it is achieved that drug utilization efficiency is maximized.
The positive effect of the present invention is：
Microsphere supported alginate-the chitosan microball obtained for emulsion process of hepatitis E albumen is carried in preparation, uses liquid in preparation
Body paraffin replaces edible oil as oil phase, effectively avoids because oil phase quality and oil phase differences between batches are microsphere supported to obtaining
Impact.Hepatitis E albumen is combined by being adsorbed in microsphere supported, rather than hepatitis E albumen is wrapped into microsphere, is prepared for hepatitis E
Final concentration of protein is the load hepatitis E protein microsphere suspension formulations of 60 mcg/mls, by containing to the outer liquid hepatitis E albumen of suspension
Amount detection, it was demonstrated that preferably, adsorption rate can reach more than 90%, successfully avoid bag for the microsphere supported absorption to hepatitis E albumen
Enter the phenomenon that the low and a large amount of hepatitis E albumen of envelop rate that the method for formula causes because albumen is mutually distributed is wasted in profit.Obtain
The load-carrying group hepatitis E protein microsphere for obtaining, its uniform particle diameter, particle size distribution range are narrow, and mean diameter is less than 10 microns, and form is homogeneous.
Zoopery is contrasted, restructuring hepatitis E epidemic disease of the load-carrying group hepatitis E protein microsphere suspensoid compared with Isodose using aluminium hydroxide as adjuvant
Seedling preferably can produce the immunoreation for being directed to hepatitis E by stimulation test animal, and this has greater significance in actual applications.
Description of the drawings
Load-carrying group hepatitis E protein microsphere microgranule 400 in 1 ~ 6 prepared five batch of suspension formulations of embodiment in Fig. 1, the present invention
Times light microscopic microgram（The microsphere microgranule of preparation of preparation under the conditions of 1 ~ 5 corresponding embodiment 1 ~ 5 of numbering）；
Load-carrying group hepatitis E protein microsphere microgranule in 1 ~ 6 prepared five batch of suspension formulations of embodiment in Fig. 2 .1 ~ Fig. 2 .5, the present invention
Grain size distribution（The microsphere microgranule of preparation of preparation under the conditions of 1 ~ 5 corresponding embodiment 1 ~ 5 of numbering）；
Load-carrying group hepatitis E protein microsphere microgranule mean diameter in 1 ~ 6 prepared five batch of suspension formulations of embodiment in Fig. 3, the present invention
Contrast（The microsphere microgranule of preparation of preparation under the conditions of 1 ~ 5 corresponding embodiment 1 ~ 5 of numbering）；
Adsorption rate contrast of 1 ~ 6 prepared five batch of suspension formulations of embodiment to hepatitis E albumen of recombinating in Fig. 4, the present invention（Numbering 1
The microsphere microgranule of preparation of preparation under the conditions of ~ 5 corresponding embodiments 1 ~ 5）；
1 used two batches of test example time edible oil in Fig. 5, the present invention（Soybean oil）For oil phase prepare alginate core with make
The mean diameter contrast of the alginate core prepared as oil phase with two batches time liquid paraffin
Load prepared by load hepatitis E protein microsphere suspension formulations and absorption method prepared by Fig. 6, pack described in test example of the present invention 2
Hepatitis E protein microsphere suspension formulations envelop rate（Adsorption rate）Contrast
Five batches of suspensions and aluminium adjuvant vaccine experiments animal serum specific IgG antibodies described in test example 3 in Fig. 7, the present invention
Conversion rate is contrasted（The microsphere microgranule of preparation of preparation under the conditions of 1 ~ 5 corresponding embodiment 1 ~ 5 of numbering）
Five batches of suspensions and aluminium adjuvant vaccine experiments animal serum specific IgG antibodies described in test example 3 in Fig. 8, the present invention
OD averages are contrasted（The microsphere microgranule of preparation of preparation under the conditions of 1 ~ 5 corresponding embodiment 1 ~ 5 of numbering）.
Specific embodiment
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, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications also should
It is considered as protection scope of the present invention.
Embodiment 1
It 1% is water phase through 100 milliliters of 115 DEG C of autoclavings sodium alginate aqueous solution of 30 minutes that the present embodiment preferred concentration is, with
200 milliliters of liquid paraffin is oil phase, and water is 1 with oil phase volume ratio:2, using Tween 80 and sorbester p37 blended emulsifier as breast
Agent, emulsifying agent addition are 2.02 milliliters（Emulsifying agent volume/oil phase volume=1%）, oil-water interfaces equilibrium valve（HLB value）Control
In 4 [HLB=（1.8* sorbester p37 volume+15.0* Tween 80 volumes）/（Sorbester p37 volume+Tween 80 volume）], it is computed, mixes
Emulsifying agent is configured to 1.68 milliliters of sorbester p37 and 0.34 milliliter of Tween 80.Said mixture is through 10 points of 200 revs/min of pre-emulsifications
Clock, 1000 revs/min of emulsifying 30 minutes.200 milliliters of firming agent are instilled in the emulsion in stirring with 8 ml/mins drop speed
（115 DEG C of autoclavings, 15% calcium chloride water of 30 minutes）Solidify liquid is 1 with emulsion volume ratio:1.5, after solidify liquid is dripped off,
Continue solidification 90 minutes.Rinsed 3 ~ 5 times with sterile water for injection after being separated through profit, centrifugation obtains microsphere supported Sargassum
Hydrochlorate core.
Embodiment 2
It 1% is water phase through 100 milliliters of 115 DEG C of autoclavings sodium alginate aqueous solution of 30 minutes that the present embodiment preferred concentration is, with
200 milliliters of liquid paraffin is oil phase, and water is 1 with oil phase volume ratio:2, using Tween 80 and sorbester p37 blended emulsifier as breast
Agent, emulsifying agent addition are 8.33 milliliters（Emulsifying agent volume/oil phase volume=4%）, oil-water interfaces equilibrium valve（HLB value）Control
In 5 [HLB=（1.8* sorbester p37 volume+15.0* Tween 80 volumes）/（Sorbester p37 volume+Tween 80 volume）], it is computed, mixes
Emulsifying agent is configured to 6.31 milliliters of sorbester p37 and 2.02 milliliters of Tween 80s.Said mixture is through 10 points of 200 revs/min of pre-emulsifications
Clock, 1000 revs/min of emulsifying 30 minutes.200 milliliters of firming agent are instilled in the emulsion in stirring with 8 ml/mins drop speed
（115 DEG C of autoclavings, 15% calcium chloride water of 30 minutes）Solidify liquid is 1 with emulsion volume ratio:1.5, after solidify liquid is dripped off,
Continue solidification 90 minutes.Rinsed 3 ~ 5 times with sterile water for injection after being separated through profit, centrifugation obtains microsphere supported Sargassum
Hydrochlorate core.
Embodiment 3
It 1% is water phase through 100 milliliters of 115 DEG C of autoclavings sodium alginate aqueous solution of 30 minutes that the present embodiment preferred concentration is, with
200 milliliters of liquid paraffin is oil phase, and water is 1 with oil phase volume ratio:2, using Tween 80 and sorbester p37 blended emulsifier as breast
Agent, emulsifying agent addition are 4.08 milliliters（Emulsifying agent volume/oil phase volume=2%）, oil-water interfaces equilibrium valve（HLB value）Control
In 6 [HLB=（1.8* sorbester p37 volume+15.0* Tween 80 volumes）/（Sorbester p37 volume+Tween 80 volume）], it is computed, mixes
Emulsifying agent is configured to 2.78 milliliters of sorbester p37 and 1.30 milliliters of Tween 80s.Said mixture is through 10 points of 200 revs/min of pre-emulsifications
Clock, 1000 revs/min of emulsifying 30 minutes.200 milliliters of firming agent are instilled in the emulsion in stirring with 8 ml/mins drop speed
（115 DEG C of autoclavings, 15% calcium chloride water of 30 minutes）Solidify liquid is 1 with emulsion volume ratio:1.5, after solidify liquid is dripped off,
Continue solidification 90 minutes.Rinsed 3 ~ 5 times with sterile water for injection after being separated through profit, centrifugation obtains microsphere supported Sargassum
Hydrochlorate core.
Embodiment 4
It 1.5% is water phase through 100 milliliters of 115 DEG C of autoclavings sodium alginate aqueous solution of 30 minutes that the present embodiment preferred concentration is,
With 200 milliliters of liquid paraffin as oil phase, water is 1 with oil phase volume ratio:2, using Tween 80 and sorbester p37 blended emulsifier as
Emulsifying agent, emulsifying agent addition are 4.08 milliliters（Emulsifying agent volume/oil phase volume=2%）, oil-water interfaces equilibrium valve（HLB value）Control
System is in 4 [HLB=（1.8* sorbester p37 volume+15.0* Tween 80 volumes）/（Sorbester p37 volume+Tween 80 volume）], it is computed, mixes
Co-emulsifier is configured to 3.40 milliliters of sorbester p37 and 0.68 milliliter of Tween 80.Said mixture is through 200 revs/min of pre-emulsifications 10
Minute, 1000 revs/min of emulsifying 30 minutes.200 milliliters of firming agent are instilled in the emulsion in stirring with 8 ml/mins drop speed
（115 DEG C of autoclavings, 15% calcium chloride water of 30 minutes）Solidify liquid is 1 with emulsion volume ratio:1.5, after solidify liquid is dripped off,
Continue solidification 90 minutes.Rinsed 3 ~ 5 times with sterile water for injection after being separated through profit, centrifugation obtains microsphere supported Sargassum
Hydrochlorate core.
Embodiment 5
It 1% is water phase through 100 milliliters of 115 DEG C of autoclavings sodium alginate aqueous solution of 30 minutes that the present embodiment preferred concentration is, with
200 milliliters of liquid paraffin is oil phase, and water is 1 with oil phase volume ratio:2, using Tween 80 and sorbester p37 blended emulsifier as breast
Agent, emulsifying agent addition are 4.08 milliliters（Emulsifying agent volume/oil phase volume=2%）, oil-water interfaces equilibrium valve（HLB value）Control
In 4 [HLB=（1.8* sorbester p37 volume+15.0* Tween 80 volumes）/（Sorbester p37 volume+Tween 80 volume）], it is computed, mixes
Emulsifying agent is configured to 3.40 milliliters of sorbester p37 and 0.68 milliliter of Tween 80.Said mixture is through 10 points of 200 revs/min of pre-emulsifications
Clock, 1000 revs/min of emulsifying 30 minutes.200 milliliters of firming agent are instilled in the emulsion in stirring with 8 ml/mins drop speed
（115 DEG C of autoclavings, 15% calcium chloride water of 30 minutes）Solidify liquid is 1 with emulsion volume ratio:1.5, after solidify liquid is dripped off,
Continue solidification 90 minutes.Rinsed 3 ~ 5 times with sterile water for injection after being separated through profit, centrifugation obtains microsphere supported Sargassum
Hydrochlorate core.
Embodiment 6
The alginate microsphere core precipitation prepared in embodiment 1 ~ 5 is hung into 100 milliliters of 115 DEG C of autoclavings 30 minutes respectively
0.3% chitosan solution in, incubated 30 minutes with 100 revs/min of rotating speeds in 37 DEG C of shaking tables.3000 revs/min are centrifuged 5 points
Alginate-chitosan microball precipitation collected by clock, supernatant discarded.Alginate-chitosan microball is cleaned with sterile water for injection
Precipitation 3 times, is centrifuged 5 minutes with 3000 revs/min of rotating speed, supernatant discarded cleanout fluid every time, and it is 3 to add 2.00 milliliters of concentration
The restructuring hepatitis E albumen stock solution of mg/ml, and 100 milliliters are settled to sterile water for injection, make restructuring hepatitis E albumen dense eventually
Spend for 60 mcg/mls, at 37 DEG C, rotating speed is to incubate 30 minutes in 100 revs/min of shaking tables, obtains five batches of different parameters and prepares
Load-carrying group hepatitis E protein microsphere suspension formulations, see Fig. 1.
Every batch of preparation is mixed, and 100 pieces of microgranules 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 its mean diameter, form is homogeneous, sees Fig. 2 .1 ~ figure
2.5th, Fig. 3.
Every batch of suspension formulations are taken with 10 milliliters of samples to be centrifuged 5 minutes through 6000 revs/min, supernatant is collected（Outer liquid）,
The outer liquid product of measurement, and wherein restructuring hepatitis E protein content is measured, calculate the adsorption rate of restructuring hepatitis E albumen：
Adsorption rate=（The outer liquid products of protein content * in 60 10 milliliters of mcg/mls *-outer liquid）/ 600 micrograms * 100%
After testing, the restructuring hepatitis E protein adsorption rate of 1 ~ embodiment of embodiment, 5 five batches of suspension formulations is all higher than 90%, sees Fig. 4.
Test example 1
Edible soybean oil from two different batches is used as oil phase, and the liquid paraffin of two different batches is pressed as oil phase
The method provided in embodiment 5 prepares alginate core, using core mean diameter and particle diameter distribution as index to two kinds of sides
Microsphere core prepared by method is compared result and sees Fig. 5.
By contrast, edible soybean oil differences between batches have considerable influence to the preparation of microsphere core, such as use edible soybean oil
Will be unable to obtain stable preparation technology as emulsifying oil phase, and liquid paraffin is used as emulsifying oil phase, then that prepared is micro-
Ball core mean diameter is less, and particle size distribution range is narrower, can obtain stable preparation technology.
Test example 2
Two batches are prepared using embodiment 5 and 6 methods described of embodiment and carries hepatitis E albumen alginate-chitosan microball suspension
Preparation（Absorption method）, calculate the adsorption rate of microball preparation respectively.
Adsorption rate=（The outer liquid products of protein content * in 60 10 milliliters of mcg/mls *-outer liquid）/ 600 milligram * 100%
Mutually change the water in embodiment 5 into aqueous solution containing 6 milligrams of hepatitis E albumen, and prepare according to 5 methods described of embodiment
Going out to wrap up the alginate microsphere core of hepatitis E albumen, and chitosan layer being wrapped up with method described in embodiment 6, centrifugation is wrapped
Alginate-the chitosan microball of hepatitis E albumen is wrapped up in, plus sterile water for injection is settled to 100 milliliters, obtain prepared by pack
The alginate of hepatitis E albumen-chitosan microball mixed suspension preparation, shares the method and prepares two batch mixed suspension preparations.Extract per batch
10 milliliters of suspensoids, centrifugation microsphere crack microsphere with PH8.0 lysates, determine protein content in microsphere, computational envelope rate
* 100 milliliters of protein content/10 milliliter/6 milligram * 100% in envelop rate=microsphere
The hepatitis E albumen prepared by hepatitis E protein microsphere mixed suspension preparation prepared by two batch packs and two batch absorption methods
The envelop rate of microsphere mixed suspension preparation（Adsorption rate）Contrast, as a result see Fig. 6, it can be seen that absorption method prepare hepatitis E albumen micro-
Ball mixed suspension preparation is more to the absorption of albumen, and hepatitis E protein microsphere mixed suspension preparation hepatitis E albumen prepared by pack is in preparation process
Middle loss is more, finally so that envelop rate is low, so as to prove to replace the pack for now commonly using relatively inexpensive with absorption method
Prepare hepatitis E protein microsphere mixed suspension preparation.
Test example 3
Load-carrying group hepatitis E protein microsphere suspension formulations prepared by 1 ~ embodiment of embodiment, the 5 five batches of different parameters for obtaining and biography
The restructuring HEV vaccines of system aluminium hydroxide adjuvant carry out animal experiment contrast.
200 health SPF level NIH mices are chosen, male and female half and half are divided into 7 groups, matched group 20, male and female half and half, and remaining 6
Group be immune group, 30 per group, male and female half and half, organize 1 immunity medicine be traditional handicraft prepare containing aluminum hydroxide adjuvant
Restructuring HEV antigen protein vaccines（Restructuring hepatitis E final concentration of protein is 60 mcg/mls）, 2 ~ 6 immunity medicines of group are embodiment 1
Load-carrying group hepatitis E protein microsphere suspension formulations prepared by ~ 6 five batches of different parameters for being obtained（Restructuring hepatitis E final concentration of protein be
60 mcg/mls）.
1 immune programme for children of table
Extract within 21st day eyeball of mouse collection blood and prepare serum, carry out 100 times of dilutions, and anti-by hepatitis E viruss IgG
The IgG antibody Conversion rate of the body euzymelinked immunosorbent assay (ELISA) diagnostic kit detection corresponding anti-HEV of mice serum, is shown in Fig. 7, and by corresponding anti-
The contrast of body OD values, is shown in Fig. 8, finds load-carrying group hepatitis E protein microsphere suspension formulations under the immune programme for children for reducing primary immune response
The restructuring hepatitis E protein vaccine immunological comparison of the aluminium hydroxide adjuvant prepared with traditional handicraft, is obtained identical or is preferably exempted from
Epidemic disease effect, it is seen that load-carrying group hepatitis E protein microsphere suspension formulations preferably can be directed to the special of HEV antigens by excitation experiment animal
Property immunoreation, and immune programme for children can be simplified, this has greater significance in actual 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 liquid paraffin
Exempt from because edible oil oil-soluble impurity and edible oil differences between batches are to preparing the impact of microsphere core so that preparation technology is more
Stable；Replace pack to prepare using absorption method and carry protein microsphere, it is to avoid protein is big because of the distribution in the profit phase
Amount loss so that the load protein microsphere of preparation is more economic, also so that microball preparation has higher adsorption rate；Tried by animal
The contrast that tests, also can be shown that the load hepatitis E albumen alginate-chitosan microball mixed suspension preparation of present invention preparation, does not only break
The immunogenicity of bad hepatitis E albumen, or even the immune effect of said preparation is also better than the same dosage using aluminium hydroxide as adjuvant and exempts from
Epidemic disease preparation.
Claims (2)
1. a kind of preparation method of load-carrying group hepatitis E protein microsphere suspension formulations, comprises the following steps：
1）Preparation method is emulsion process, and concentration is 1%～1.5% to be through 115 DEG C of autoclavings sodium alginate aqueous solution of 30 minutes
Water phase, with liquid paraffin as oil phase, water is 1 with oil phase volume ratio:2, using single emulsifier or blended emulsifier as emulsifying
Agent, emulsifying agent addition are 1% ~ 4%（Emulsifying agent volume/oil phase volume）, oil-water interfaces equilibrium valve（HLB value）Control 4 ~ 6, in advance
At 50 revs/min ~ 500 revs/min, at 5 ~ 30 minutes, emulsifying rotating speed was controlled 500 pre-emulsification time control the control of emulsifying rotating speed
Rev/min ~ 2000 revs/min, emulsification times were controlled at 5 ~ 30 minutes, and firming agent is through 115 DEG C of autoclavings chlorine of 30 minutes
Change calcium aqueous solution, its concentration is 5% ~ 20%, and solidify liquid is 1 with emulsion volume ratio:1~1:3, solidify liquid instill speed be 3 milliliters/
Minute ~ 20 ml/mins, continued hardening time for 10 ~ 120 minutes.
2. calcium alginate microsphere is rinsed 3 ~ 5 times with sterile water for injection or sterile distilled water after profit phase separation, centrifugation,
Obtain microsphere supported alginate core；
2）The alginate core coating chitosan layer of acquisition prepares alginate-chitosan microball carrier
By step 1）The alginate core of middle acquisition is suspended in concentration for 0.1% ~ 0.8% through 115 DEG C of autoclavings 30 minutes
In chitosan aqueous solution, at 25 DEG C ~ 42 DEG C, rotating speed is to incubate 10 ~ 60 minutes in 50 revs/min ~ 200 revs/min shaking tables, warp
1000 revs/min ~ 5000 revs/min are centrifuged 1 ~ 30 minute, and sterile water for injection or sterile distilled water are rinsed 3 ~ 5 times, obtain sea
Alginate-chitosan microball carrier；
3）The preparation of restructuring hepatitis E protein microsphere preparation
To step 2）The alginate of middle acquisition-chitosan microball carrier adds restructuring hepatitis E protein solution, and uses aseptic note
Penetrate with water be settled to 1 in used water phase identical volume, make last antigen protein content in 60 mcg/mls, 25 DEG C ~
42 DEG C, rotating speed is to incubate 10 ~ 60 minutes in 50 revs/min ~ 200 revs/min shaking tables, prepares load hepatitis E protein microsphere suspension
Preparation, aseptic subpackaged after mixing, 2 DEG C ~ 8 DEG C preservations, obtains final product target product.
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