CN106668854A - Quadrivalent subunit influenza vaccine and preparation method thereof - Google Patents
Quadrivalent subunit influenza vaccine and preparation method thereof Download PDFInfo
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/12—Viral antigens
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/70—Multivalent vaccine
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
- C12N2760/00011—Details
- C12N2760/16011—Orthomyxoviridae
- C12N2760/16111—Influenzavirus A, i.e. influenza A virus
- C12N2760/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
- C12N2760/00011—Details
- C12N2760/16011—Orthomyxoviridae
- C12N2760/16211—Influenzavirus B, i.e. influenza B virus
- C12N2760/16234—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Microbiology (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Medicinal Chemistry (AREA)
- Virology (AREA)
- Mycology (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The invention relates to the field of biological pharmacy, and particularly discloses a quadrivalent subunit influenza vaccine and a preparation method thereof. Used virus seeds are respectively of an H1N1 type (NYMC X-179A), an H3N2 type (NIB-88), a BY type (B/Phuket), and a BV type (NYMC BX-35). The preparation method comprises the following steps: performing chick embryo cultivation on the virus seeds; preparing a mono-valent virus stock solution by the steps of allantoic fluid collection, clarification, concentration, purification and the like; and proportioning four mono-valent virus stock solutions with different serotypes according to certain dilution points to prepare a quadrivalent subunit influenza vaccine sample. A relatively low content of ovalbumin and relatively low residual quantities of formaldehyde and a cracking agent in the quadrivalent subunit influenza vaccine sample ensure that adverse reactions of a quadrivalent subunit vaccine are lower, and the safety of the vaccine is improved. Meanwhile, compared with an existing trivalent vaccine, the quadrivalent subunit vaccine additionally has a BV serotype, thereby having a broader protection scope for popularity of influenzas with different serotypes.
Description
Technical field
The present invention relates to field of biological pharmacy, specifically, it is related to a kind of tetravalence subunit influenza vaccine.
Background technology
Influenza is caused by influenza virus, and the acute respiratory of extensive prevalence can be caused to spread disease.Influenza can be
Occur suddenly in a short time, spread rapidly, cause different degrees of prevalence, including pandemics, local outbreak and distribute.
People at highest risk is old and weak or with patients with chronic diseases.Always cause different degrees of in crowd after each influenza pandemic
Excess is dead.Therefore, the prevention of influenza is constantly subjected to the great attention of countries in the world.The maximally effective measure of flu-prevention is inoculation
Influenza vaccines.
The influenza vaccines that the current whole world is used all are trivalents, including two kinds of A types (H1N1, H3N2) and one kind it is B-mode
(Type B) influenza antigen composition, present most widely used vaccine includes being used for the inactivated vaccine of June above crowd and is used for
The attenuated live vaccine of 2-49 Sui.
Inactivated vaccine includes split vaccine and subunit vaccine.Split vaccine is to cultivate in chicken embryo virus to prepare disease
Venom, after harvesting allantoic fluid, split vaccine semi-finished product is prepared after clarified, concentration, purifying, inactivation, cracking, by trivalent virus
Stoste matches into split vaccine finished product by a certain percentage.Subunit vaccine is on the basis of split vaccine, to be split by suitable
Solution agent, cracking condition, memebrane protein HA and NA are cleaved, and select appropriate purification process to obtain HA and NA albumen, prepare and
Into.
Prevention of the trivalent vaccine to influenza is substantially limited.The trivalent vaccine that in the market is used is containing only the type of first 1 (H1N1), first 3
Type (H3N3) and B-mode (Type B) influenza antigen for pedigree.Trivalent vaccine is only provided with to another pedigree B virus
Limit protection.
In sum, in order to strengthen the security of product, expand protection domain of the crowd to the different prevalence types of influenza, be
Full crowd provides immunoprotection barrier, and particularly to the protection of June Infants Below and the elderly, influenza vaccines market needs four badly
The development and application of valency subunit influenza vaccine.
The content of the invention
In order to solve problems of the prior art, it is an object of the invention to provide a kind of tetravalence subunit influenza vaccine
And preparation method thereof.
In order to realize the object of the invention, technical scheme is as follows：
The invention provides a kind of tetravalence subunit influenza vaccine, its by H1N1 types, H3N2 types, BY types and BV types virus
Univalent stoste is formulated by certain dilution point；The hemagglutinin content of H1N1 types, H3N2 types, BY types and BV types is equal in the vaccine
It is 26.4 μ g-39.6 μ g/mL.
Compound method is specially：According to the hemagglutinin content of each univalent stoste, various influenza virus is pressed into same hemagglutinin
Content carries out semi-finished product preparation, and (hemagglutinin amount of preparation can be in the range of 30~36 μ g/mL, and annual various strains of influenza viruses should be by same
One hemagglutinin content is prepared), as semi-finished product.Volume according to required univalent dilution of stock is V and univalent stoste blood
Solidifying cellulose content C0, the volume V0=V/ (C0/ hemagglutinin amount of preparation) of unit price stoste needed for calculating, 0.01mol/L phosphate delays
The volume VP=V-V0 of fliud flushing.By taking a collection of univalent stoste as an example：If the volume of unit price dilution of stock is needed for certain batch
500mL, the hemagglutinin content of univalent stoste is 350 μ g/mL, and hemagglutinin amount of preparation is 35 μ g/mL, then univalent stoste volume is
The volume 500mL-50mL=450mL of 500/ (350/35)=50mL, 0.01mol/L phosphate buffer.
Further, the virus unit price stoste is harvested through chick embryo culture, by allantoic fluid by seed culture of viruses, clarified, concentrated, split
Solve, obtain after purification；
Seed culture of viruses is to recommend after being assessed through World Health Organization (WHO), is provided by biological product determination research institute of Britain (NIBSC)
People's influenza virus strain, is respectively H1N1 types (NYMCX-179A), H3N2 types (NIB-88), BY types (B/Phuket), BV
Type (NYMCBX-35).
Wherein, concentration step is specially：
1) by the allantois harvest liquid of monotype influenza virus through centrifugal clarification after, with 1,000,000 molecular cut off milipore filters to it
It is concentrated by ultrafiltration, 20~60 times of cycles of concentration.
The cleavage step is specially：
1) virus liquid after concentration is placed in into concentration to be not higher than in 1.5% decomposition agent Triton N101/ sucrose solutions,
Under the conditions of 15~25 DEG C, 3.5~4.5h is cracked；SDGC is carried out while cracking, it is bent according to UV absorption
Line collects destination protein peak, and it is the protein peak between 9%~20% to collect sucrose concentration；When carrying out SDGC,
Centrifugal rotational speed is 25000 revs/min, 3.5~4.5h of centrifugation time；
Or 2)：
Virus liquid after concentration is placed in into concentration to be not higher than in 1.5% decomposition agent Triton N101, in 15~25 DEG C of bars
Under part, 0.5~2h is cracked；Virus liquid after cracking is separated in hydrophobic chromatography post Hi Trap PhenylFF (high sub)
Purifying, sample-loading buffer is 50mmol/L PBS+1.5mol/L (NH4)2SO4(pH7.0), liquid containing ammonium sulfate 100%, separately sets
Liquid containing ammonium sulfate is 0 in 50mmol/L PBS, after appropriate loading with ensure the flow velocity of post pressure from the ammonium sulphate content 100% of setting to
0 quick depletion is eluted, and is monitored with 280nm wavelength, and collect protein peak.
Further, the present invention is set out with seed culture of viruses, there is provided the preparation method of the complete virus unit price stoste, including such as
Lower step：
S1, virus inoculation and culture
Monotype working seed lots seed culture of viruses is diluted in the range of final concentration of 2.0lgEID50/mL~5.0lgEID50/mL
Afterwards, with the dose inoculation of 0.2mL/ embryos in chick embryo allantoic cavity, 33~35 DEG C are put and is cultivated 48~72 hours；
S2, virus harvest
Screening live chickens embryo, put 2~8 DEG C of cold embryos after 12~24 hours, harvested allantoic fluid；
S3, centrifugal clarification merge
The allantois harvest liquid of monotype influenza virus is merged into monovalent virus amalgamation liquid after centrifugal clarification；
S4, concentration, virolysis and purifying
S41, concentration
Monovalent virus amalgamation liquid is concentrated by ultrafiltration with 1,000,000 molecular cut off milipore filters, 20-60 times of cycles of concentration；
S42, virolysis
Virus liquid after concentration is placed in the decomposition agent Triton N101/ sucrose solutions that concentration is not higher than 1.5%, is placed in
15~25 DEG C, 3.5~4.5 hours virolysis time, 3.5~4.5 hours SDGC time, centrifugal rotational speed is
25000 revs/min, destination protein peak is collected according to ultraviolet absorption curve, it is the albumen between 9%~20% to collect sucrose concentration
Peak；
S43, purifying
The virus liquid after density gradient centrifugation is collected, Sephadex-G25 carries out gel chromatography for medium, and eluent is
0.01mol/L phosphate buffers, collect according to ultraviolet absorption curve, through dilution, aseptic filtration, to chromatographic solution after aseptic filtration
Protein content detected, 2500 μ g/mL should be not higher than, should be diluted to the concentration if above 2500 μ g/mL, add eventually
Concentration be 200 μ g/mL formaldehyde put 2~8 DEG C inactivate 2 days, obtain inactivation of virus liquid；
S44, repurity
Filter wash is carried out to inactivation of virus liquid, formaldehyde is removed, filter wash restrovirus liquid should be sampled and carry out bacteria endotoxin content survey
Fixed and limit test of microbe, limit test of microbe bacterium number should be less than 10cfu/mL, then virus list is after aseptic filtration
Valency stoste.
It is further preferable that the preparation method of the virus unit price stoste comprises the following steps：
S1, virus inoculation and culture
Monotype working seed lots seed culture of viruses is diluted in the range of final concentration of 2.0lgEID50/mL~5.0lgEID50/mL
Afterwards, with the dose inoculation of 0.2mL/ embryos in chick embryo allantoic cavity, 33~35 DEG C are put and is cultivated 48~72 hours；
S2, virus harvest
Screening live chickens embryo, put 2~8 DEG C of cold embryos after 12~24 hours, harvested allantoic fluid；
S3, centrifugal clarification merge
The allantois harvest liquid of monotype influenza virus is merged into monovalent virus amalgamation liquid after centrifugal clarification；
S4, concentration, virolysis and purifying
S41, concentration
Monovalent virus amalgamation liquid is concentrated by ultrafiltration with 1,000,000 molecular cut off milipore filters, 20-60 times of cycles of concentration；
S42, virolysis
Virus liquid after concentration is placed in into concentration to be not higher than in 1.5% decomposition agent Triton N101,15~25 are placed in
DEG C, 0.5~2 hour virolysis time；Afterwards, different serotypes sample is isolated and purified using hydrophobic chromatography；
S43, purifying
The virus liquid after density gradient centrifugation is collected, Sephadex-G25 carries out gel chromatography for medium, and eluent is
0.01mol/L phosphate buffers, collect according to ultraviolet absorption curve, through dilution, aseptic filtration, to chromatographic solution after aseptic filtration
Protein content detected, 2500 μ g/mL should be not higher than, should be diluted to the concentration if above 2500 μ g/mL, add eventually
Concentration be 200 μ g/mL formaldehyde put 2~8 DEG C inactivate 2 days, obtain inactivation of virus liquid；
S44, repurity
Filter wash is carried out to inactivation of virus liquid with 10,000 molecular weight milipore filters, formaldehyde is removed, filter wash restrovirus liquid should be sampled and carried out
Bacteria endotoxin content is determined and limit test of microbe, and limit test of microbe bacterium number should be less than 10cfu/mL, then through degerming
Virus unit price stoste is after filtering.
Wherein, in above-mentioned S42, using hydrophobic chromatography post Butyl-S-Sepharose 6ff (C4) to different serotypes sample
Product are isolated and purified；Chromatography media is butylff, Octyl ff or PhenylF, and sample-loading buffer is 50mmol/L PBS+
1.5mol/L(NH4)2SO4(pH7.0), liquid containing ammonium sulfate 100%, liquid containing ammonium sulfate is 0 in separately setting 50mmol/L PBS, appropriate loading
At the uniform velocity successively decreased wash-out from the ammonium sulphate content 100% to 0 of setting with ensureing the flow velocity of post pressure afterwards, be monitored with 280nm wavelength,
And collect protein peak.
The beneficial effects of the present invention are：
The invention provides a kind of tetravalence subunit influenza vaccine and preparation method thereof, using the tetravalence prepared by the present invention
The egg white protein content average of subunit vaccine sample is 115.4ng/mL, far below trivalent split vaccine (not higher than 500ng/
ML) and trivalent subunit vaccine (not higher than 400ng/mL) standard.The content of formaldehyde of tetravalence subunit influenza vaccine sample is
23.16 μ g/mL, influenza vaccines quality standard (not higher than 50 μ g/mL) and trivalent subunit influenza vaccines are cracked far below trivalent
Quality standard (not higher than 40 μ g/mL).The decomposition agent residual quantity verification result of tetravalence subunit vaccine is 120.5 μ g/mL, trivalent
Split vaccine quality standard is that, less than 300 μ g/mL, trivalent subunit vaccine quality standard is less than 350 μ g/mL.Tetravalence is sub- single
Relatively low egg white protein content, relatively low formaldehyde and decomposition agent residual quantity in the vaccine sample of position, it is ensured that tetravalence subunit vaccine
Adverse reaction it is lower, the security of vaccine is improved.Tetravalence subunit vaccine is increased on the basis of trivalent vaccine
One BV serotype, tetravalence subunit vaccine has wider range of defence protection model for the prevalence of different serotypes influenza
Enclose.
Brief description of the drawings
Fig. 1 is the polyacrylamide gel electrophoresis figure of tetravalence subunit vaccine and trivalent subunit vaccine, wherein, 1. albumen
Molecular weight standard, 2. trivalent subunit vaccine control, 3. tetravalence subunit vaccine sample.
Specific embodiment
The preferred embodiment of the present invention is described in detail below in conjunction with embodiment.It will be appreciated that following real
Being given merely to play descriptive purpose for example is applied, is not used to limit the scope of the present invention.The skill of this area
Art personnel can carry out various modifications and replacement in the case of without departing substantially from spirit of the invention and spirit to the present invention.
Experimental technique used in following embodiments is conventional method unless otherwise specified.
Material used, reagent etc. in following embodiments, unless otherwise specified, commercially obtain.
Embodiment 1
A kind of tetravalence subunit influenza vaccine, every dose of tetravalence Infuenza subunit vaccine contains first 1 (H1N1) type, first 3
(two pedigrees of (H1N3) type and second (B) type), are 30~36 μ g/mL per valency antigenic content.
1. virus inoculation and culture
It is (each after working seed lots seed culture of viruses is diluted in the range of final concentration of 2.0lgEID50/mL~5.0lgEID50/mL
Type influenza strain should be inoculated with by same virus titer respectively), with the dose inoculation of 0.2mL/ embryos in chick embryo allantoic cavity, put 33
~35 DEG C are cultivated 48~72 hours, and the working seed lots seed culture of viruses being once not used must not again return jelly and be continuing with.
2. virus harvest
Screening live chickens embryo, put 2~8 DEG C of cold embryos after 12~24 hours, harvests allantoic fluid and sampling is examined and determine：Microorganism limits
Degree, Hemagglutination titer.
3. centrifugal clarification merges
The allantois harvest liquid of monotype influenza virus merges into monovalent virus amalgamation liquid after centrifugal clarification.
4. concentration, virolysis and purifying
1) concentrate
Monovalent virus amalgamation liquid is concentrated by ultrafiltration with 1,000,000 molecular cut off milipore filters, 20-60 times of cycles of concentration.
Concentrate should be sampled carries out bacteria endotoxin content measure.
2) virolysis
Compound concentration is not higher than 1.5% decomposition agent Triton N101/ sucrose solutions, is placed in 15~25 DEG C, virolysis
3.5~4.5 hours time, 3.5~4.5 hours SDGC time, centrifugal rotational speed is 25000 revs/min, according to
Ultraviolet absorption curve collects destination protein peak, and it is the protein peak between 9%~20% to collect sucrose concentration.
3) purify
Gel chromatography is carried out by medium of Sephadex-G25, eluent is 0.01mol/L phosphate buffers, according to purple
Outer absorption curve is collected, and through dilution, aseptic filtration, the protein content to chromatographic solution after aseptic filtration is detected, Ying Bugao
In 2500 μ g/mL, the concentration should be diluted to if above 2500 μ g/mL, add the formaldehyde of final concentration of 200 μ g/mL to put 2~8
DEG C inactivation 2 days.
4) repurity
Filter wash is carried out to inactivation of virus liquid, formaldehyde is removed, filter wash restrovirus liquid should be sampled and carry out bacteria endotoxin content survey
Fixed and limit test of microbe, limit test of microbe bacterium number should be less than 10cfu/mL, then unit price original is after aseptic filtration
Liquid.
5. prepare
According to the hemagglutinin content of each univalent stoste, various influenza virus is carried out into semi-finished product by same hemagglutinin content and is matched somebody with somebody
(hemagglutinin amount of preparation can be in the range of 30~36 μ g/mL, and annual various strains of influenza viruses should be carried out by same hemagglutinin content for system
Prepare), as semi-finished product.
The tetravalence influenza virus subunit vaccine semi-finished product technical study of experimental example 1
First, material
Four type influenza virus unit price stostes
2nd, method
According to the hemagglutinin content of each univalent stoste, various influenza virus is carried out into semi-finished product by same hemagglutinin content and is matched somebody with somebody
(hemagglutinin amount of preparation can be in the range of 30~36 μ g/mL, and annual various strains of influenza viruses should be carried out by same hemagglutinin content for system
Prepare), as semi-finished product.Volume according to required univalent dilution of stock is V and univalent stoste hemagglutinin content C0, is calculated
The volume V0=V/ (C0/ hemagglutinin amount of preparation) of required univalent stoste, the volume VP=V- of 0.01mol/L phosphate buffers
V0.By taking a collection of univalent stoste as an example：If the volume of unit price dilution of stock is 500mL, the blood clotting of univalent stoste needed for certain batch
Cellulose content is 350 μ g/mL, and hemagglutinin amount of preparation is 35 μ g/mL, then univalent stoste volume is 500/ (350/35)=50mL,
The volume 500mL-50mL=450mL of 0.01mol/L phosphate buffers.It is 33.0 μ g/mL with point that this experiment is dilute.
3rd, situation is prepared
Its preparing materials：Four type influenza virus unit price stostes
Compound method：
According to the hemagglutinin content of each univalent stoste, various influenza virus is carried out into semi-finished product by same hemagglutinin content and is matched somebody with somebody
(hemagglutinin amount of preparation can be in the range of 30~36 μ g/mL, and annual various strains of influenza viruses should be carried out by same hemagglutinin content for system
Prepare), as semi-finished product.Volume according to required univalent dilution of stock is V and univalent stoste hemagglutinin content C0, is calculated
The volume V0=V/ (C0/ hemagglutinin amount of preparation) of required univalent stoste, the volume VP=V- of 0.01mol/L phosphate buffers
V0.By taking a collection of univalent stoste as an example：If the volume of unit price dilution of stock is 500mL, the blood clotting of univalent stoste needed for certain batch
Cellulose content is 350 μ g/mL, and hemagglutinin amount of preparation is 35 μ g/mL, then univalent stoste volume is 500/ (350/35)=50mL,
The volume 500mL-50mL=450mL of 0.01mol/L phosphate buffers.
It is 33.0 μ g/mL (four types are consistent) with point that this experiment is dilute, and nine batches of tetravalence influenza viruses of trial-production are sub- single
Position antigenic components of vaccines and specificity are errorless, and hemagglutinin content complies fully with the 80%-120% of amount of preparation.
4th, semi-finished product and the calibrating of finished product main project and analysis
1st, hemagglutinin content
1.1 material
Nine batches, the tetravalence subunit influenza vaccine sample of trial-production：20160301；20160302；20160303；20160504；
20160505；20160506；20160507；20160508；20160509.
1.2 method
Using uni-directional diffusion method.Standard antigen and stoste are added separately into 1% agarose containing standard antibody to coagulate
On offset plate, aperture is 4mm, per the μ L of hole 6, stands and is no less than 18 hours.After being soaked 1 hour with PBS, dry, dye, decolourize.It is accurate
The deposit ring diameter that true measurement standard antigen and stoste are formed, the diameter of the precipitation ring formed with standard antigen is to its corresponding antigens
Trial carries out linear regression, obtains linear regression equation, substitutes into the precipitation diameter of stoste, you can the hemagglutinin for obtaining stoste contains
Amount.
1.3 pilot sample finished product hemagglutinin content verification results
Table 1 H1N1 types hemagglutinin content (μ g/mL) data statistic
Table 2 H3N2 types hemagglutinin content (μ g/mL) data statistic
Table 3 BY types hemagglutinin content (μ g/mL) data statistic
Table 4 BV types hemagglutinin content (μ g/mL) data statistic
2nd, free formaldehyde content
The measure of free formaldehyde content uses colorimetric method, can be generated with formaldehyde in an acidic solution according to magenta sulphurous acid purple
Color compound.9 batches of finished products are tested, is summarized as follows：
2.1. material：Tetravalence influenza virus subunit vaccine sample finished product lot number：20160301、20160302、
20160303、20160504、20160505、20160506、20160507、20160508、20160509。
Reagent：0.005% formaldehyde reference substance solution, magenta sulphurous acid solution, mixed acid solution.
2.2. method：Using the content of colorimetric method for determining tetravalence influenza virus subunit vaccine finished product free formaldehyde, first
Standard curve is made, equation of linear regression is drawn, the content of test sample free formaldehyde is then drawn according to equation of linear regression.To
It is separately added into formaldehyde reference substance solution pipe (concentration is 25 μ g/mL, 50 μ g/mL, 75 μ g/mL, 100 μ g/mL) and test sample pipe
The magenta sulphurous acid solution and the mixed acid solution of 10mL of 10mL, place 3 hours in 25 DEG C, determine the absorbance at 590nm, root
Equation of linear regression and coefficient correlation are drawn according to the absorbance of formaldehyde reference substance solution, test sample free formaldehyde is drawn according to this
Content.
2.3. nine batches of finished product formaldehyde examine and determine content results
The free formaldehyde content of table 5 (μ g/mL) data statistic
3rd, egg white protein content
3.1 material：Tetravalence influenza virus subunit vaccine finished product lot number 20160301,20160302,20160303,
20160504、20160505、20160506、20160507、20160508、20160509。
Reagent：Ovalbumin ELISA detection kit (Seramun), purified water.
3.2 method：Using double crush syndrome method, how anti-with antiovalbumin does capture antibody, HRP marks
Ovalbumin antibody does detection antibody.By white of an egg standard items and the test sample addition how anti-micropore enzyme being coated with of antiovalbumin
In target, HRP conjugates, 37 DEG C of incubation 60min are added.Tmb substrate lucifuge colour developing 10min is added after board-washing, is added and is terminated
Detection in liquid, 30min.Detection wavelength 450nm, reference wavelength 620nm.It is (or flat with the ovalbumin concentration and OD values of standard items
Equal OD values) standard curve is done, regression equation is tried to achieve, the OD values of test sample are brought into regression equation, try to achieve its ovalbumin dense
Degree.
3.3 9 batches of Product eggs albumin assay results
Table 6 egg white protein content (ng/mL) data statistic
4th, decomposition agent Triton N-101 residual quantities (semi-finished product calibrating)
The measure of decomposition agent Triton N-101 residual quantities uses colorimetric method, and its principle is：Contain in Triton N-101
Polyethoxy, polyethoxy and ammonium cobalt thiocyanate react to form blue complex, and the compound dissolves in dichloromethane, uses colorimetric
Method can determine Triton N-101 residual quantities.We are tested 9 batches of semi-finished product, are summarized as follows：
4.1 material：Tetravalence influenza virus subunit vaccine semi-finished product lot number：20160301、20160302、20160303、
20160504、20160505、20160506、20160507、20160508、20160509。
Reagent：Ethanol-sodium chloride saturated solution, sulphur cyanocobalamin solution, Triton N-101 reference substance solutions, dichloromethane
Solution.
4.2 method：Using the residual quantity of decomposition agent in colorimetric method for determining tetravalence influenza virus subunit vaccine semi-finished product.It is first
First, standard curve is made, the decomposition agent residual for then being calculated in test sample according to the equation of linear regression for obtaining.To Triton
N-101 reference substance solutions pipe (concentration is respectively 40 μ g/mL, 100 μ g/mL, 200 μ g/mL, 300 μ g/mL, 400 μ g/mL) and confession
Respectively add methylene chloride solution 2mL, sulphur cyanocobalamin solution 3mL in test product (concentrated treatment) pipe, jumps a queue, and mixes, and room temperature places 1.5
Hour, concussion in every 15 minutes is once.30 minutes are stood before determining, upper liquid is abandoned.Lower floor's dichloromethane is determined at wavelength 620nm
The absorbance of liquid, the absorbance according to Triton N-101 reference substance solution pipes draws equation of linear regression, and confession is drawn according to this
The decomposition agent residual quantity of test product.
4.3 9 batches of semi-finished product decomposition agent residual quantity verification results
The tetravalence influenza virus subunit vaccine semi-finished product decomposition agent residual quantity result of table 7
5. Polyacrylamide Gel Electrophoresis
5.1 method
5.1.1 glue
5.1.1.1 separation gel：
The 3 μ L of μ L, TEMED of Resolver A liquid 3mL, Resolver B liquid 3mL, 10%APS 30, fill after mixing immediately
Note between two blocks of glass of electrophoresis tank both sides, and reserve space needed for perfusion concentrates glue, add water and bind, be polymerized at room temperature, point
After glue polymerization (about 50 minutes) completely, incline coating liquid, with filter paper bar suck dry moisture.
5.1.1.2 glue is concentrated：
The 2 μ L of μ L, TEMED of Stacker A liquid 1mL, Stacker B liquid 1mL, 10%APS 10, mix immediately, are gathering
After directly perfusion concentrates glue (about 1cm high) on the separation gel of conjunction, clean Teflon combs are inserted in sol solution is concentrated immediately,
Gel is disposed vertically at room temperature.After glue polymerization completely to be concentrated, gel is placed in electrophoresis tank, adds electrophoretic buffer,
Then it is careful to extract comb.
5.1.2 sample treatment：
5.1.2.1 glycosylation：
18 μ L protein samples are taken, 2 μ L glycoprotein denaturation buffers are added, 100 DEG C are boiled 10 minutes, sample is fully become
Property；It is subsequently adding 2.5 μ L 10 × G2 buffer solutions, 2.5 μ L 10%NP-40,1 μ L glycosidases F, 37 DEG C incubate 18 hours.Add
26 μ L 2 × Non sample-loading buffers, mix, and put in water-bath 100 DEG C and heat 3~5 minutes.
5.1.2.2 Non：
30 μ L protein samples are taken, 30 μ L 2 × Non sample-loading buffers are added, mixed, put 100 DEG C of heating 3 in water-bath
~5 minutes.
5.1.3 electrophoresis：
The gel that will be prepared is put into electrophoresis tank, adds 1 × electrode buffer, floods inside groove and at least 1/3rd
Water jacket, the sample (per the μ L of hole 20) and Marker (per the μ L of hole 10) that then will be handled well with injector is added in hole；Gel institute
Making alive is 80 volts, after Bromophenol Blue dye forward position enters separation gel (about 30 minutes), voltage is brought up to 150 as one sees fit~
200 volts, electrophoresis carefully takes out gel after terminating from glass plate, makes marks.
5.2 gels, fixed, dyeing and interpretation of result
Gel is put into the gel-colored liquid of appropriate protein electrophoresis, it is ensured that dyeing liquor can fully cover gel.It is placed in water
The slow shake of yawing bed, dyes 1~2 hour；Add appropriate protein electrophoresis gel destainer, it is ensured that destainer can be covered fully
Gel.It is placed in horizontal shaker slowly to shake, room temperature is decolourized.Period at least changes destainer 1~2 time, until blue background is basic
Upper whole is divested, and protein band Color reaches expection.
As shown in Figure 1, trivalent subunit vaccine and the prepared tetravalence subunit vaccine sample hemagglutinin molecule of this experiment
Amount meets the theoretical molecular of influenza virus hemagglutinin in 70KDa or so.Trivalent subunit vaccine and made in electrophoresis picture
Standby tetravalence subunit vaccine sample is all difficult to see obvious miscellaneous band, shows that the two hemagglutinin purity is all very high.Prepared
Tetravalence subunit vaccine sample is strong compared with trivalent subunit vaccine signal, illustrates the former hemagglutinin content apparently higher than the latter (see figure
1)。
5th, tetravalence subunit vaccine sample verification result and trivalent subunit vaccine quality standard and trivalent split vaccine matter
Amount standard comparing.
The verification result of table 8 compares
6th, the antibody titer of immunogenicity and animal
This research method is that the antibody level of the resisiting influenza virus for producing in vivo will be detected after vaccine immune mouse.By one
It is that 20160504, trivalent flu subunit vaccine (is used after inactivating to criticize trial-production tetravalence influenza virus subunit vaccine finished product lot number
H1N1 unit prices stoste, H3N2 unit price stostes and BY unit price stostes are configured to vaccine sample) difference immune mouse, before immune, just
Exempt from 28 days, just exempt from 42 days after be grouped blood sampling, separate serum, with hemagglutination-inhibition test method determine antibody titer.
The Mouse immunogenicity trial result of table 9
Result of the test shows：After the immune animal of tetravalence influenza virus subunit vaccine, NAT is significantly higher than exempts from
Before, the antibody level of generation is higher, illustrates that the vaccine sample has good immunogenicity and stronger stimulation mouse body product
The ability of raw neutralizing antibody, tetravalence influenza virus subunit vaccine sample and trivalent subunit vaccine sample NAT,
The antibody level of generation is close, and different blood are not made because of increased monovalence on the basis of trivalent subunit vaccine in sample
There is the situation of mutual antagonism or mutual enhancing effect between clear type antigen.Tetravalence subunit vaccine sample is compared with trivalent vaccine
The many BV serotype antigens of sample, so as to expand vaccine during for human immunity in future prevent different serotypes influenza pandemic
Shield scope lays the foundation.
The egg white protein content average of tetravalence subunit vaccine sample is 115.4ng/mL, far below trivalent split vaccine
The standard of (the not higher than 400ng/mL) of (not higher than 500ng/mL) and trivalent subunit vaccine, it ensure that in tetravalence in future Asia
During unit influenza vaccines human immunity, tetravalence subunit vaccine has lower adverse reaction.Tetravalence subunit influenza vaccine sample
Content of formaldehyde be 23.16 μ g/mL, crack influenza vaccines (not higher than 50 μ g/mL) and trivalent subunit influenza far below trivalent
The quality standard of vaccine (not higher than 40 μ g/mL).The decomposition agent residual quantity verification result of tetravalence subunit vaccine is 120.5 μ g/
ML, trivalent split vaccine quality standard is that, less than 300 μ g/mL, trivalent subunit vaccine quality standard is less than 350 μ g/mL.Compared with
Low formaldehyde and decomposition agent residual quantity so that the non-effective component content in tetravalence subunit influenza vaccine sample is lower, so that
Substantially increase the security of tetravalence subunit influenza vaccine.
Embodiment 2
On the basis of embodiment 1, density gradient centrifugation is replaced with hydrophobic chromatography technology.
Specially：Compound concentration is not higher than in 1.5% decomposition agent Triton N101, is placed in 15~25 DEG C, virolysis
0.5~2 hour time.Afterwards, different serotypes sample is entered using hydrophobic chromatography post Butyl-S-Sepharose 6ff (C4)
Row is isolated and purified.Chromatography media is butylff, Octyl ff or PhenylF, and sample-loading buffer is 50mmol/L PBS+
1.5mol/L(NH4)2SO4(pH7.0), liquid containing ammonium sulfate 100%, liquid containing ammonium sulfate is 0 in separately setting 50mmol/L PBS, appropriate loading
At the uniform velocity successively decreased wash-out from the ammonium sulphate content 100% to 0 of setting with ensureing the flow velocity of post pressure afterwards, be monitored with 280nm wavelength,
And collect protein peak.
Sample after hydrophobic chromatography is analyzed, sample and the sample of density gradient centrifugation treatment after hydrophobic chromatography is found,
The hemagglutinin yield and purity of sample do not have notable difference.Hydrophobic chromatography more operated quickly and conveniently, only can at 30 minutes or so
To complete, and density gradient centrifugation then need it is several or more than more than ten hour.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (7)
1. a kind of tetravalence subunit influenza vaccine, it is characterised in that by the virus unit price of H1N1 types, H3N2 types, BY types and BV types
Stoste is prepared from；
The hemagglutinin content of H1N1 types, H3N2 types, BY types and BV types is 26.4 μ g-39.6 μ g/mL in the vaccine.
2. tetravalence subunit influenza vaccine according to claim 1, it is characterised in that the virus unit price stoste is by seed culture of viruses
Harvested through chick embryo culture, by allantoic fluid, clarified, concentrated, crack, obtained after purification；
Wherein, concentration step is specially：
1) by the allantois harvest liquid of monotype influenza virus through centrifugal clarification after, it is carried out with 1,000,000 molecular cut off milipore filters
It is concentrated by ultrafiltration, 20~60 times of cycles of concentration.
3. tetravalence subunit influenza vaccine according to claim 2, it is characterised in that the cleavage step is specially：
1) virus liquid after concentration is placed in into concentration to be not higher than in 1.5% decomposition agent Triton N101/ sucrose solutions, in 15
Under the conditions of~25 DEG C, 3.5~4.5h is cracked；SDGC is carried out while cracking, is received according to ultraviolet absorption curve
Ji Mudanbaifeng, it is the protein peak between 9%~20% to collect sucrose concentration；
Or 2)：
Virus liquid after concentration is placed in into concentration to be not higher than in 1.5% decomposition agent Triton N101, in 15~25 DEG C of conditions
Under, crack 0.5~2h；It is pure that virus liquid after cracking carries out separation in hydrophobic chromatography post Hi Trap PhenylFF (high sub)
Change, sample-loading buffer is 50mmol/L PBS+1.5mol/L (NH4)2SO4(pH7.0), liquid containing ammonium sulfate 100%, separately sets 50mmol/
Liquid containing ammonium sulfate is 0 in L PBS, ensureing the flow velocity of post pressure from the quick depletion of ammonium sulphate content 100% to 0 of setting after appropriate loading
Wash-out, is monitored, and collect protein peak with 280nm wavelength.
4. tetravalence subunit influenza vaccine according to claim 3, it is characterised in that carry out SDGC
When, centrifugal rotational speed is 25000 revs/min, 3.5~4.5h of centrifugation time.
5. tetravalence subunit influenza vaccine according to claim 1, it is characterised in that the preparation of the virus unit price stoste
Method comprises the following steps：
S1, virus inoculation and culture
After monotype working seed lots seed culture of viruses is diluted in the range of final concentration of 2.0lgEID50/mL~5.0lgEID50/mL, with
The dose inoculation of 0.2mL/ embryos is put 33~35 DEG C and is cultivated 48~72 hours in chick embryo allantoic cavity；
S2, virus harvest
Screening live chickens embryo, put 2~8 DEG C of cold embryos after 12~24 hours, harvested allantoic fluid；
S3, centrifugal clarification merge
The allantois harvest liquid of monotype influenza virus is merged into monovalent virus amalgamation liquid after centrifugal clarification；
S4, concentration, virolysis and purifying
S41, concentration
Monovalent virus amalgamation liquid is concentrated by ultrafiltration with 1,000,000 molecular cut off milipore filters, 20-60 times of cycles of concentration；
S42, virolysis
Virus liquid after concentration is placed in the decomposition agent Triton N101/ sucrose solutions that concentration is not higher than 1.5%, it is placed in 15~
25 DEG C, 3.5~4.5 hours virolysis time, 3.5~4.5 hours SDGC time, centrifugal rotational speed is
25000 revs/min, destination protein peak is collected according to ultraviolet absorption curve, it is the albumen between 9%~20% to collect sucrose concentration
Peak；
S43, purifying
The virus liquid after density gradient centrifugation is collected, Sephadex-G25 carries out gel chromatography for medium, and eluent is
0.01mol/L phosphate buffers, collect according to ultraviolet absorption curve, through dilution, aseptic filtration, to chromatographic solution after aseptic filtration
Protein content detected, 2500 μ g/mL should be not higher than, should be diluted to the concentration if above 2500 μ g/mL, add eventually
Concentration be 200 μ g/mL formaldehyde put 2~8 DEG C inactivate 2 days, obtain inactivation of virus liquid；
S44, repurity
Filter wash is carried out to inactivation of virus liquid with 10,000 molecular weight milipore filters, formaldehyde is removed, filter wash restrovirus liquid should be sampled and carry out bacterium
Endotoxin assay and limit test of microbe, limit test of microbe bacterium number should be less than 10cfu/mL, then through aseptic filtration
Virus unit price stoste is afterwards.
6. tetravalence subunit influenza vaccine according to claim 1, it is characterised in that the preparation of the virus unit price stoste
Method comprises the following steps：
S1, virus inoculation and culture
After monotype working seed lots seed culture of viruses is diluted in the range of final concentration of 2.0lgEID50/mL~5.0lgEID50/mL, with
The dose inoculation of 0.2mL/ embryos is put 33~35 DEG C and is cultivated 48~72 hours in chick embryo allantoic cavity；
S2, virus harvest
Screening live chickens embryo, put 2~8 DEG C of cold embryos after 12~24 hours, harvested allantoic fluid；
S3, centrifugal clarification merge
The allantois harvest liquid of monotype influenza virus is merged into monovalent virus amalgamation liquid after centrifugal clarification；
S4, concentration, virolysis and purifying
S41, concentration
Monovalent virus amalgamation liquid is concentrated by ultrafiltration with 1,000,000 molecular cut off milipore filters, 20-60 times of cycles of concentration；
S42, virolysis
Virus liquid after concentration is placed in into concentration to be not higher than in 1.5% decomposition agent Triton N101,15~25 DEG C, disease are placed in
Malicious pyrolysis time 0.5~2 hour；Afterwards, different serotypes sample is isolated and purified using hydrophobic chromatography；
S43, purifying
The virus liquid after density gradient centrifugation is collected, Sephadex-G25 carries out gel chromatography for medium, and eluent is
0.01mol/L phosphate buffers, collect according to ultraviolet absorption curve, through dilution, aseptic filtration, to chromatographic solution after aseptic filtration
Protein content detected, 2500 μ g/mL should be not higher than, should be diluted to the concentration if above 2500 μ g/mL, add eventually
Concentration be 200 μ g/mL formaldehyde put 2~8 DEG C inactivate 2 days, obtain inactivation of virus liquid；
S44, repurity
Filter wash is carried out to inactivation of virus liquid with 10,000 molecular weight milipore filters, formaldehyde is removed, filter wash restrovirus liquid should be sampled and carry out bacterium
Endotoxin assay and limit test of microbe, limit test of microbe bacterium number should be less than 10cfu/mL, then through aseptic filtration
Virus unit price stoste is afterwards.
7. tetravalence subunit influenza vaccine according to claim 6, it is characterised in that using hydrophobic chromatography post Butyl-S-
Sepharose 6ff (C4) are isolated and purified to different serotypes sample；Chromatography media is butylff, Octyl ff or
PhenylF, sample-loading buffer is 50mmol/L PBS+1.5mol/L (NH4)2SO4(pH7.0), liquid containing ammonium sulfate 100%, separately sets
Liquid containing ammonium sulfate is 0 in 50mmol/L PBS, after appropriate loading with ensure the flow velocity of post pressure from the ammonium sulphate content 100% of setting to
0 at the uniform velocity successively decreases wash-out, is monitored with 280nm wavelength, and collect protein peak.
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| CN108159411A (en) * | 2018-01-11 | 2018-06-15 | 江苏中慧元通生物科技有限公司 | A kind of influenza virus subunit vaccine purification process and its application |
| RU2710239C1 (en) * | 2019-06-14 | 2019-12-25 | Федеральное государственное унитарное предприятие "Санкт-Петербургский научно-исследовательский институт вакцин и сывороток и предприятие по производству бактерийных препаратов" Федерального медико-биологического агентства | Method of producing antigen or antigens for production of influenza vaccine and a vaccine based on it |
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| CN112125960A (en) * | 2020-08-27 | 2020-12-25 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | General method for removing endotoxin suitable for large-scale industrial production operation |
| CN111803625B (en) * | 2020-09-09 | 2020-12-18 | 天津中逸安健生物科技有限公司 | Subunit influenza vaccine cracking agent and application thereof |
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| CN115992101A (en) * | 2023-03-22 | 2023-04-21 | 深圳市卫光生物制品股份有限公司 | Preparation method of influenza virus split vaccine stock solution |
| CN115992101B (en) * | 2023-03-22 | 2023-07-28 | 深圳市卫光生物制品股份有限公司 | Preparation method of influenza virus split vaccine stock solution |
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