CN101274097B

CN101274097B - Atomizing grippe vaccine composition and preparation thereof

Info

Publication number: CN101274097B
Application number: CN2008100980521A
Authority: CN
Inventors: 崔栋
Original assignee: Individual
Current assignee: Individual
Priority date: 2008-05-23
Filing date: 2008-05-23
Publication date: 2011-02-09
Anticipated expiration: 2028-05-23
Also published as: CN101274097A

Abstract

The invention relates to a preparation method of a spraying composition of influenza vaccine. The composition contains influenza virus antigens and drug-acceptable carriers which are chosen from interferon, ion exchange resins, human serum albumin or phosphate buffer.

Description

Atomizing grippe vaccine composition and preparation method thereof

Technical field:

The present invention relates to a kind of preparation method of atomizing grippe vaccine composition, belong to the bio-pharmaceutical formulation art.

Background technology:

Influenza is called for short influenza (influenza), mainly is the acute respiratory communicate illness that is caused respectively by first (A), second (B) two type influenza virus.Influenza A virus often occurs with popular form, causes worldwide flu outbreak, and Influenza B virus usually causes the local outburst of influenza, does not cause worldwide flu outbreak.Still lack effective medicine at present clinically the influenza patient is treated, influenza virus vaccine is to prevent that influenza disease from taking place and the most effective popular a kind of means.

Influenza Virus orthomyxoviridae family (Orthomyxovindae), Influenza Virus.Typical influenza virus particles is a ball-type, diameter 80～120nm.The influenza virus gene group is the strand of branch multi-segmental, the rna gene group of minus strand, is made of 7 on third type 8 sections that relative molecular mass does not wait.Easy producer reprovision.Wherein influenza A virus antigen often morphs.There are hemagglutinin (HA) albumen, neuraminidase (NA) and three kinds of structural protein of M2 in its virion surface, these two kinds of main causes that proteic variation is an influenza antigen generation antigenic drift of hemagglutinin (HA) and neuraminidase (NA).If a series of point mutation appears in aminoacid sequence among antigen HA and the NA, make NA and HA antigenic determinant that some change take place, can cause medium and small popularly, claim antigenic drift; If the antigenic variation amplitude is bigger, form a new hypotype, claim antigenic shift, usually cause bigger popularly, even cause world's flu outbreak.Nucleoprotein (NP) is soluble protein, and its antigenicity is stable.Memebrane protein (MP) is the major protein of virion, also is specific antigen, and they are main foundations of influenza virus typing, but their antibody can not in and influenza virus, so there is not protective effect.Because influenza virus often morphs, so people's the viral repeated infection that will experience repeatedly different shaped, hypotype and mutation in life.The variation of influenza virus has produced new epidemic isolates, so the strain of vaccine also must change, deals with new epidemic isolates.For this reason, the annual virus stain that is recommended in the influenza pandemic in winter (November～April next year) in the Northern Hemisphere February of the Committee of Experts of WHO, annual JIUYUE is recommended in the virus stain of the influenza pandemic in next winter (May～October) in the Southern Hemisphere.Influenza vaccines comprise three main hypotype: A (H1N1), A (H3N2) and Type B at present.

Nineteen thirty-seven Embryo Gallus domesticus cultivation influenza virus is succeedd, and makes the mass production influenza vaccines become possibility.The influenza all-virus inactivated vaccine goes through to use in the U.S. in nineteen forty-one first, and confirms effectively.Be widely used in the U.S. in 1945, the chick embryo allantoic liquid of this results is through erythrocyte absorption and discharge limited purified vaccine, belongs to early stage rough vaccine, and untoward reaction local and whole body is all very strong behind the inoculation human body.At the end of the sixties in last century, the application of supercentrifuge and chromatography chromatographic technique has improved the viral purification quality greatly, has made the totivirus inactivated vaccine.It comprises all albumen of virus, lipid and nucleic acid.The manufacture method of this vaccine is with fertilization chick embryo allantoic cavity inoculation influenza virus on the 9th～11, cultivation, results, purification and deactivation and get.This type of immune effect of vaccine is good, but untoward reaction has appearred when using in the child.Its one of the main reasons is because the cytotoxicity of virion itself causes.Nineteen sixty-eight goes through to use cracking purified stream influenza vaccine first in the U.S..The influenza split vaccine contains hemagglutinin (HA) and neuraminidase (NA) surface antigen and other antigens.Elder generation's purified virus, the back adds decomposition agent, as ether, 3-N-butyl phosphoric acid salt (tri-N-butylphosphate), polysorbate 80 (polysorbate80), NaTDC (deoxycholate sodium), trinitrotoluene decomposition agents such as (Tritonx-100).Make after removing viral lipid, the after birth of virus is destroyed, thus the virus protein mixture that obtains having hyperimmunization originality.Though can obtain the virus protein mixture of hyperimmunization originality, but be injection type all the time, injection inoculation immunity side reaction is heavier, be unfavorable on a large scale especially child, old man and weakling's inoculation, and the nasal spray inoculation has good tolerability, lower side reaction, can be used for child, adult and old people, and easy to use, can inoculate voluntarily, can use among the large tracts of land crowd, be the improved directions of present influenza vaccines.

Summary of the invention:

The object of the invention provides a kind of atomizing grippe vaccine composition and preparation method thereof.

The invention provides a kind of influenza vaccine compositions, this vaccine is a spray-type, and described compositions contains influenza antigen and medicine acceptable carrier.Described carrier is selected from: interferon, ion exchange resin, human albumin or phosphate buffer.Described interferon is IFN-α, IFN-β or IFN-γ; Ion exchange resin is kayexalate, and described phosphate buffer is that pH is 7.0 phosphate buffer.Described influenza antigen is totivirus antigen, lytic virus antigen or subunit antigen.

The present invention preferably fills a prescription composed as follows:

Influenza antigen (purification)

60% (percent by volume)

Or

Influenza antigen (purification)	Be made into 2.15mg/ml (1 part)
		Kayexalate	Be made into 0.5g/ml (8 parts).
Water	In right amount

Wherein said kayexalate particle diameter is 20～40 μ m.

Preparation method of the present invention may further comprise the steps:

(1) obtains cell

(2) cell preparation

(3) virus inoculation

(4) gather in the crops viral liquid

(5) deactivation of viral liquid

(6) clarification filtration, concentrated

(7) purification

(8) cracking repurity

(9) filter removal Triton x-100

(10) preparation

The cell that the present invention uses is nephrocyte, comprises hamster kidney cell and Vero cell, and preferred Vero cell is at first set up Vero cell work storehouse, and the pair cell storehouse examines and determine comprehensively, and qualified rear can be used as cell bank.Before the preparation influenza vaccines, need carry out recovery, cultivation and the amplification of cell earlier, to reach the needs of production lot.The various mammalian cells that are used to prepare vaccine are and adhere to dependent cell, and cell is grown on certain carrier, and the training method of cell is that rolling bottle is cultivated in this technology.In the incubation, cell divides kind of rate to determine according to the needs of production lot, after cell grows up to fine and close monolayer, can inoculate influenza virus, each type is inoculated respectively, and the first type begins results about 46 hours, began results in B-mode about 70 hours, the viral liquid hemagglutinative titer of results can reach 1: 256.

The viral liquid of results obtains monotype virus results liquid with the formalin deactivation.Results liquid is unit price virus amalgamation liquid after merging.

Unit price virus merging liquid need concentrate to purify and be prepared into vaccine product, select for use tangential flow filtration system of process using GE company that virus results liquid is clarified in this technology, virus concentrates uses 750KD molecular weight ultrafiltration post ultrafiltration and concentration, make the viral purity of purification higher, improve the effectiveness of vaccine and reduced the side reaction generation, the Sepharose6FF column chromatography carries out purification to the influenza concentrated solution, the cracking purification adopts Triton x-100 that influenza virus purification is carried out cracking, removal Triton x-100 employing molecular weight is 10000 filter, filter the removal decomposition agent, clarification through four steps, concentrate, purification, the cracking purification, the vaccine total protein content has reduced about more than 99%.

The present invention adopts tangential flow filtration system of GE company that virus results liquid is clarified, and has substituted conventional centrifuging.Advantage: simple to operate, the unit interval treating capacity is 2～3 times of centrifuging, can reach clarification degerming double effects.Because degerming immediately after the results can at utmost reduce thermal source matter.Virus concentrates uses 750KD molecular weight ultrafiltration post, has substituted present domestic 100KD and the 300KD ultrafilter membrane bag that generally uses.Ultrafiltration can be shortened 1～2 times with a collection of virus results liquid duration, and the concentrated solution that obtains removed most of foreign protein, guarantees for purification provides in earlier stage, has prolonged purification and has used the gel life-span.Make the viral purity of purification higher, improved the effectiveness of vaccine and reduced the side reaction generation.We replace gradient density centrifugal method method of purification with the column chromatography purification method, are more suitable for industrialized great production.Adopt Triton x-100 that influenza virus purification is carried out cracking, lytic effect is better than sodium deoxycholate.Remove decomposition agent by ultrafiltration, that the method for removing decomposition agent than gradient density centrifugal method in the past has is simple to operate, cost reduction, energy savings, quality surpass advantages such as domestic like product.

Preparation spray flow influenza vaccine is by adding kayexalate and influenza antigens preparation spray flow influenza vaccine in this technology.

Another kind method is to be mixed with the spray flow influenza vaccine behind interferon, influenza antigens, human albumin, the PBS phosphate buffer.

Each component variable concentrations is the best of breed of studying through repeatedly in two kinds the compound method, obtains through screening, can produce best immune effect.Below prove effect of the present invention by contrast test:

Immune mouse

With spray flow influenza vaccine embodiment 1 and 8 female adult BALB/c mouse of embodiment 2 collunariums (6～8 week) nostril of this prepared, every hole 50 microlitres, 8 female adult BALB/c mouse of positive controls intramuscular injection 100 microlitres (6～8 week).Embodiment 1 and embodiment 2 every mice equivalent nasal cavity immunity secondaries, 14 days at interval, a matched group intramuscular injection immunity once.

Negative control group adopts the PBS phosphate buffer.

Sampling method:

In eye socket blood sampling, centrifugal, separation of serum, serum is mixed on the same group, and 4 ℃ standby.

The HI method is measured antibody titer

In blood-coagulation-board, with 25 μ l normal saline doubling dilutions serum to be checked, add the virus of four HAUs of equivalent, shake up rearmounted room temperature 60min gently, add the red cell suspension of 25 μ l11%, shake up again and put observed result behind room temperature 30～60min.

The result

The antibody titer of different strain vaccines

Detect antibody through hemagglutination inhibition test (HI test) (HI) method, the spray flow influenza vaccine antigenicity of two kinds of prescriptions is similar to the positive control vaccine.

Goods are to the animal protection Journal of Sex Research

To distinguish collunarium immunity 11～13g white mice with embodiment 1 and embodiment 2 vaccines, 21 days posterulas of immunity are attacked first 1, first 3 and Influenza B virus, and each nostril infects 10 ^-1～10 ^-6Virus 0.05ml.Observe every day then, what infects death in 24 hours is non-specific death, not in statistics.Observed 14 days, and set up contrast simultaneously, not vaccination, direct counteracting toxic substances after 21 days.Calculate LD50 with Reed and Muench formula, measure the protection index.

White mice is divided into 4 groups, 90 every batch, 15 of matched groups, male and female half and half.Collunarium immunity white mice, every immunity 50 microlitre vaccines were observed 21 days.Attack influenza virus A 1, first 3 and B-mode, get 10 ^-1～10 ^-6The viral dilution degree, each dilution factor nasal cavity infects 5 white mice, each nostril influenza virus infection 0.05ml of white mice, infecting dead in 24 hours be non-specific death, observes 14 days, and trachea is got in dissection, and hemagglutination test is directly done in grinding.Calculate LD50 with Reed and Muench formula, measure the protection index.

Result of the test shows that vaccine has protective effect to white mice, and the protection index is all more than 100.The result is as follows:

The spray flow influenza vaccine is protected situation to white mice

Behind the spray flow influenza vaccine collunarium of this prepared immunity white mice, can effectively make body produce immunne response, the infringement of virus is had protective effect.

Be prepared the spray flow influenza vaccine according to this technology, prescription is unique, need not inject nonirritant, quality is controlled easily, product is stable, and good toleration, low side reaction are arranged, can be used for child, adult and old people, and easy to use, can inoculate voluntarily, can use among the large tracts of land crowd.

The specific embodiment:

Embodiment 1,

Carry out influenza antigens preparation according to the preparation method in this technology, also can buy from the market and obtain, prepare after getting influenza antigens.Get kayexalate, kayexalate is the medicine by State Food and Drug Administration's approval, be made into aqueous solution, making its content is 0.5g/ml, get influenza antigens and be made into aqueous solution, making its content is 2.15mg/ml, mixes according to 8: 1, stirs and makes the mixing aqueous suspension in 1 hour.With this suspension with the amount of each 0.6ml at human nasal intracavity spray delivery.

Embodiment 2:

The preparation method of using this technology prepares influenza antigens, also can buy from the market to obtain, and gets the influenza antigens for preparing and is mixed with 600ml, adds interferon 10 ^4.5Unit/L, human albumin 25ml, all the other make 1000ml spray flow influenza vaccine for the PBS phosphate buffer after mixing.

Claims

1. the preparation method of an atomizing grippe vaccine composition, described composite formula is:

1 part of influenza antigen 2.15mg/ml

8 parts of kayexalate 0.5g/ml

Described kayexalate particle diameter is 20～40 μ m;

It is characterized in that, may further comprise the steps:

(1) obtain cell:

(2) cell preparation;