CN85101017A - Freeze dried hepatitis B vaccine - Google Patents

Abstract

The freeze-dried products of hepatitis B vaccine comprises that these goods are adsorbed on the alumina gel with lyophilised state in the presence of stabilizing agent by the hepatitis virus B surface antigen of the purification that can produce the antigenic recombinant organisms generation of HBs; The step of said lyophilized formulations preparation comprises adding alumina gel and stabilizing agent in the reorganization body source HBs of purification antigen, and this mixture of lyophilizing.Said lyophilized formulations can be preserved steadily in the long term and not lose its antigen valence; And can be used for the prevention that hepatitis virus B infects.

Description

Freeze dried hepatitis B vaccine

The present invention is the lyophilized formulations of relevant hepatitis B vaccine.The lyophilized formulations of particularly relevant hepatitis B vaccine, it is the product that obtains purification by HBs antigen, the lyophilized formulations of the hepatitis B vaccine that in the presence of alumina gel and a kind of stabilizing agent, prepares through lyophilizing, above-mentioned HBs antigen is to express by producing the antigenic inverting biological body of HBs, and this antigen prepares with conventional DNA recombinant technique.

Hepatitis B is a kind of eqpidemic disease that is caused by hepatitis virus B (hereinafter using " HBV " expression), and includes on the immunology and very important clinically problem.But never find any effective Therapeutic Method.For this reason, main research is prevention method.It is that the vaccine of active ingredient is given the people that probably will infect HBV with HBs antigen that suitable prevention method provides a kind of.A kind of vaccine uses in practice, and the preparation method of this vaccine is high-purity HBs antigen preparation of obtaining in the blood plasma of one group of latent virus carrier that only is called " bacillicarrier ".

Yet vaccine from blood is to obtain in the blood plasma with HBs antigen positive person, so its preparation method has various problems, and is just very difficult such as obtaining enough primitive plasmas; For must in the chimpanzee body, doing safety test, so that proof does not leave infectant in preparation, as hepatitis virus B or any other blood source virus; In addition, it is also very difficult with chimpanzee to obtain enough tests.

In order to address these problems, many researcheres are going in for the study and are obtaining the antigenic technology of a large amount of initial HBs, the way that adopts is to use the DNA recombinant technique, by with the HBV DNA of a coding HBs antigen protein in escherichia coli or yeast, and with the conversion of acquisition like this microorganism express HBs antigen.Recently, succeed with these recombinant microorganisms express HBs antigen.Particularly, thereafter, tested the HBS antigen that purification obtains like this in commercial scale, finishing with recombinant yeast production HBs antigen, and the preparation of preparation hepatitis B vaccine.

The hepatitis B vaccine is very effective to prevention medical personnel's viral infection, also effective for the researcher prevention that contacts with hepatitis B patient and potential viral carrier and patient, bacillicarrier and baby's family members once in a while, this moment, virus was by blood infection.It is said that in Japan, the bacillicarrier who finds is the 2-3% of total population usually, and reaches 10-15% in Southeast Asia and Africa.Therefore, extracting the hepatitis B vaccine worldwide all needs.According to this argument, need worldwide comprise Japan, make extensive available hepatitis B vaccine, and a kind of stable and preparation energy long preservation importantly will be provided.

Commercial hepatitis B vaccine is a kind of liquid formulation that obtains from the HBs antigenic product of blood source, and can lose its antigen valence gradually at storage period.

In the case, in the inventor has studied and has prepared over a long time on commercial scale, the method of the hepatitis B vaccine of bigger storage stability is arranged, but also found and to have replaced blood source HBs antigen with reorganization body source HBs antigen, and lyophilizing HBs antigen obtains desired vaccine under given conditions.

Known a kind of non-activated vaccine is by mixing adjuvant, preparing as alumina gel, in order that improve the intravital antibody production rate of people with vaccine the time.The hepatitis B bacterin preparation has generally also been admixed alumina gel.For lyophilizing hepatitis B vaccine, if the hepatitis B vaccine is just made by freeze dried HBs antigen, it must be by in advance HBs antigen (hereinafter calling " HBsAg " once in a while) being dissolved in injection saline and the distilled water for injection so, and this solution and alumina gel is mixed before using and preparation again.In such method, in the container of each minimum applying unit, the absorption of HBsAg on alumina gel has different, this depend on preparation process order, temperature, shake factor such as blended condition, so the production of antibodies rate just may change to some extent according to each batch vaccine.In addition, when a kind of body source HBs antigen of recombinating adopted the condition lyophilizing identical with the common liq preparation, in the freeze-drying process, the antigen valence of vaccine can undesirably reduce.

The inventor has deeply and carefully studied the lyophilisation condition of the reorganization body source HBsAg that is adsorbed on the alumina gel.The result of research can't illustrate some such problems, as the reduction of antigen valence or the deterioration of character, but can provide the desired lyophilized formulations that bigger storage stability is arranged than common liq preparation that has.Also further studied the composition of suitable stabilization formulations.Found that the preparation of desired lyophilized formulations can be by the reorganization of adsorption and purification on suspended state alumina gel body source HBsAg, steady dissolution agent and optional suspension protective agent, and lyophilizing is by the prepared mixture of above-mentioned steps.

An object of the present invention is to use to produce the antigenic recombinant microorganism of HBs, a kind of lyophilized formulations of improveing system hepatitis B vaccine is provided.Another object of the present invention is in order to obtain a kind of lyophilizing hepatitis B vaccine that fabulous storage stability arranged, and an antigenic freeze drying process of reorganization body source HBs is provided.These and other purpose and advantage of the present invention will make those of skill in the art be understood according to following narration.

Lyophilizing hepatitis B vaccine of the present invention can almost be in the aqueous solution of neutral buffer solution by alumina gel being added to a kind of body source HBs antigen of recombinating, HBs antigen is adsorbed onto on the alumina gel, again toward wherein adding a kind of stabilizing agent of aminoacid and/or saccharide and optional colloidal substance of being selected from, another optional common preservatives, isotonic agent etc., to prepare a kind of vaccine solution, with its lyophilizing, preferably lyophilizing again be distributed into bottle by minimum applying unit after.The hepatitis B vaccine freeze-drying preparation of Huo Deing can keep very high antigen valence in long-time in this way, and the every batch of preparation each contain the bottled preparation of minimum applying unit, all have identical antigen valence, it is very easily that tired this uses.

The initial antigenic preparation procedure of purification of Recombinant body source HBs is: a coding is introduced organisms such as escherichia coli, yeast, cultured animals cell from the antigenic gene of the isolating HBs of hepatitis virus B DNA, thereby by gene transformed microbe, and HBs antigen is expressed in the activity by the HBs antigen gene.The antigenic method of preparation reorganization body source HBs is known already.For example, Valenzuela reported the antigenic method of preparation recombinant yeast source HBs (referring to Valenzuela, Nature, 298,347(1982) and Japan Patent (application) for the first time disclose 77823/1983), method comprises preparation a kind of shuttle vector (PHB516), a HBS gene is arranged therein, it be incorporated into yeast ethanol deoxygenase in the shuttle vector (PMA56) in a replication initiation zone with PBR322 plasmid start in, one 2 μ plasmid Trp1 replication initiation zone, Trp1 and yeast ethanol deoxygenase promoter region; This shuttle vector is introduced yeast, to prepare the yeast that transforms and to cultivate transformed yeast, to produce desired HBs antigen.

The antigenic method of another known preparation yeast source HBs is by people such as Miyanohara (referring to proc.Natl.Acad.Sci., USA.80 1(1983) discloses 31799/1984 for the first time with a day patent (application)) report.This method comprises a preparation shuttle vector (PAH203), there is a HBs gene to be connected in the transcribed acid phosphatase promoter in the shuttle vector with 2 μ plasmid replication initiation regions therein, the replication initiation zone of a pBR322 plasmid, the replication initiation zone of a yeast chromosomal, participate in the synthetic yeast leu2 of leucine gene, an anti-ampicillin gene of escherichia coli and the zymic acid phosphatase promoter region that suppresses; Shuttle vector is introduced yeast (AH22(a, leu2, his4, Canl, Cir ⁺)) with the preparation transformed yeast; And cultivate transformed yeast to produce desired HBs antigen.

Report according to another Hitzeman, once prepare a HBs gene wherein and be connected to shuttle vector on yeast glycerol 3-phosphate acid kinase (PGK) promoter, be used to preparation afterwards and can produce the antigenic transformed yeast of HBs (referring to Hitzeman, Nucleic Acids Research, 11(9), 2745(1983) and Japan Patent (application) for the first time disclose 109427/1983).

Report according to people such as Nozaki again in addition, by the HBs gene is originated from Coll E1 with having to be inserted into, the replication initiation zone of SV4O DNA in the escherichia coli plasmid of pMB1 or p15A, and can not in mammalian cell, duplicate the carrier reorganization in the zone of inhibition, and make a recombinant DNA.And by transform this mammalian cell with these recombinant DNies, it is mice LTK cell, cultivate the cell that this has transformed afterwards again, thereby obtain desired HBs antigen (referring to 30th Plenary Session of The Society of Virology, Japan, Abstracts, P-1069(1982) and Japan Patent (application) for the first time disclose 36698/1984).Also have some to utilize the DNA recombinant technique, produce the antigenic report of HBs, disclose 56685/1983,995/1983 and 39784/1982 for the first time as Japan Patent (application) by zooblast.

With the HBs antigen that so obtains with conventional purification process in addition high-purity purify.These methods all are used to separate and the purifying biological active substance usually, as smudge cells, extracting cell debris, with ammonium sulfate precipitation, gel filtration, ion-exchange chromatography, with Polyethylene Glycol fractionated, affinity chromatography, with the ultracentrifugation of sucrose and aluminum chloride etc., and highly purified HBs antigen is used as lyophilizing hepatitis B bacterin preparation of the present invention.

From the purification HBs antigen that above-mentioned recombinant microorganism obtains, lyophilizing as follows.HBs antigen is dissolved in the water or near in the buffer of neutral suitable concn, as 0.01 M phosphate buffer, 0.01M citrate buffer, or 0.005M McIlvaineShi buffer, so that HBs antigen and other additive have following concentration: promptly be contained in the no more than 0.1W/V% of HBs antigen in the protein concentrate, preferably less than 0.02W/V%.As the alumina gel that adjuvant is added, its content is 3 to 10 times of HBs antigen weight by weight.

HBsAg is adsorbed on the alumina gel, mix with alumina gel and carry out by containing the antigenic solution of HBs, perhaps, the liquor alumini chloridi of proper content contains the antigenic solution 1 of HBs and to the sodium hydrate aqueous solution that wherein adds debita spissitudo by being added, so as to having produced gel aluminum hydroxide, HBs antigen is adsorbed thereon simultaneously.As when using sodium phosphate aqueous solution to replace sodium hydrate aqueous solution, then produce Fosfalugel (Yamanouchi), just and HBs antigen is adsorbed on this gel.Therefore the used alumina gel of the present invention comprises gel aluminum hydroxide and Fosfalugel (Yamanouchi).

So the antigenic alumina gel suspension of absorption HBs that obtains mixes mutually with stabilizing agent, a kind of optional antiseptic and isotonic agent, afterwards with the mixture lyophilizing.

Stabilizing agent comprises aminoacid and polysaccharide.Aminoacid and polysaccharide can use separately, but preferably both use together.Colloidal substance can be chosen any one kind of them and add as a kind of stabilizing agent.

The aminoacid that is suitable for has glycine, alanine, glutamic acid, arginine, lysine etc. or its salt (as monosodium glutamate), and they can use separately or two or more merge use, and common use amount is 0.1-2.0W/V%.The saccharide that is suitable for has monosaccharide, as glucose, xylose, galactose, fructose etc., and disaccharide such as lactose, maltose, sucrose etc., and sugar alcohol such as mannitol, sorbitol, xylitol etc., they can be used alone, but also two or more merge and use.Its consumption is generally 0.1-15W/V%.The colloidal substance such as the gelatin that are suitable for, human albumin, dextrane etc., its consumption is generally 0.01-0.1W/V%.Optional antiseptic such as the sour sodium of ethyl hydrargyrum sulfo-water sun, its use amount is 0.05-0.1W/V%.

When using freeze dried vaccine, can be isoosmotic on physiology in order to make it, best and a kind of neutral salt of preparation mixes.Neutral salt comprises sodium chloride, potassium chloride, magnesium chloride, and best neutral salt is a sodium chloride, in the mixture of other neutral salt that it was mentioned above can being used to.The concentration of common contained neutral salt is 0.1-3W/V%, preferably 0.5-2W/V%.

So the vaccine solution branch of preparation installs in the container of a dosage unit packing, so that the HBs antigen amount that each container contains is 20 to 1000 μ g.Divide the solution that installs to each container by conventional quick freezing dry method or lyophilization lyophilizing at a slow speed, and obtain desired lyophilized formulations.Lyophilizing is normally being carried out under the following condition: be about to this solution under low temperature (as-40 ℃ or lower, preferably-50 ℃ or lower), under atmospheric pressure, give lyophilizing several hrs (as 3-10 hour), then at fixed higher temperature (as 0 °-8 ℃), (as 0.01 millimetres of mercury) first phase lyophilizing to tens hour under reduced pressure, this work-in-process temperature will be lower than-35 ℃ (as approximately-38 ℃), afterwards, (as the 0.001-0.005 millimetres of mercury) the second stage of lyophilizing several hours to tens hours (as 6-10 hour, preferably 7-9 hour) under the pressure that product will more reduce in the temperature (being 25 °-30 ℃) of fixed rising.

Lyophilized formulations contains reorganization body source HBs antigen, alumina gel, a kind of stabilizing agent and a kind of neutral salt at least.

Lyophilized formulations with the hepatitis B vaccine of method for preparing has good storage stability, and does not reduce antigen valence, when using, can be dissolved in the injection solution rapidly in addition.

When using lyophilized formulations of the present invention, preparation is dissolved in distilled water for injection or injection normal saline so that the concentration of HBs antigen protein is adjusted to 5 μ g/ml to 50 μ g/ml, and with the concentration of salt be transferred near etc. ooze.Physiological isosmotic solution uses during for subcutaneous medication.The dosage of vaccine is counted 5 μ g to 50 μ g by the HBs antigen protein usually to adult's a drug amount.

Preparation of the present invention, by what stipulate among " biological product minimum requirements " (Minimum Requirement of Biological Products) (No. 287 announcement of Japanese health and Department of Welfare (Health and human services department) issue in 1981), test with Cavia porcellus according to common experimental technique, do not manifest the undue toxicity.

The present invention can be illustrated with following reference example, but these examples should not regarded the restriction to said method as.

Reference example 1

Derive from the antigenic preparation of the zymic purification HBs of recombinant

According to people's such as Miyanohara method (disclosing 31799/1984 for the first time referring to Japan Patent (application)), preparation is also cultivated at group body yeast, to produce HBs antigen, separates and purification HBs antigen according to following method:

(1), the preparation of HBV DNA

(ⅰ), the preparation of viral DNA

The normal plasma (700ml) that to be gathered by 10 HBsAg and HBeAg positive person's (Subtype adr), with 5, centrifugal 20 minutes of 000r.p.m is to remove insoluble matter.In 4 ℃, centrifugal 8 hours of 18.000r.p.m, and resulting precipitate is dissolved in 10ml again contains 10mM Tris-Hcl is in the buffer (PH7.5) of 0.1M Nacl and 1mM EDTA with resulting solution.This solution is added to the top of the centrifuge tube that contains 30% sucrose, in 4 ℃, centrifugal 4 hours of 39.000r.p.m.Resulting precipitation is dissolved in once more as in the above-mentioned same buffer.

This buffer needs in a reactant mixture, and in 37 ℃, old HBV polymerase was handled 30 minutes.This mixture (500 μ l) contains 67mM Tris-Hcl(PH7.5), 80mM NH ₄Cl, 25mM Mgcl ₂, 0.5%(W/V%, down together) NP40(tergitol, Sigma company makes), 0.1%2-mercaptoethanol, 330 μ M dcTP(triphosphoric acid desoamine glycosides esters), the dGTP(deoxyguanosine triphosphate) and the dATP(deoxyadenosine triphosphate), 0.5 μ M α-( ³²P) dTTP(triphosphoric acid deoxyribosylthymine).Adding final concentration in reactant mixture is the dTTP of 330 μ M, and places 37 ℃ further to react 3 hours in this mixture.And then in reactant mixture, add the 100mM EDTA solution of same volume.By above-mentioned DNA polyreaction, dna single thigh zone all is repaired to intact bifilar, with obtain ( ³²P) material of labelling.This thing is added to the top of centrifuge tube, gives in the pipe and put into earlier 30%, 20% and 10% aqueous sucrose solution in turn, in 4 ℃, 39, centrifugal 4.5 hours of 000r.p.m.

For hydrolysis combine with DNA very tight protein, in (200 μ l) in the mixture of the pronase e that is deposited in 1mg/ml (manufacturing of Kaken chemical company) of above-mentioned acquisition and 0.2% sodium dodecyl sulfate aqueous solution, handled 2 hours in 37 ℃.With phenol (the 200 μ l) extracting twice of resulting mixture, the reuse ether is washed the resulting DNA of containing extract afterwards, obtains the HBV dna solution to remove phenolic solvent.The DNA that obtains thus have 2.5 * 10 ⁶CPm/ μ g specific radioactivity, and can be used to the digestion of being limited property restriction endonuclease.

(ⅱ), the cloning of HBV DNA

By using a phage Sharon 16A DNA,, pass through with known plasmid pACYC177 as carrier cloning once more then the bifilar ring-type HBV dna cloneization that said method obtains as carrier.Concrete grammar is as follows:

(A), going into-phage Sharon 16A host-carrier system in first Longhua:

At 10mM Tris-Hcl(PH7.4), 7mM Mgcl ₂, in the mixture of 100mM Nacl and 7mM 2 mercapto ethanol (20 μ l), handle HBV DNA(20ng with Cobra venom endonuclease XhoI in 37 ℃) and 2 hours.The mixture that obtains phenol (20 μ l) extracting, and the extracting of reuse ether add the cold ethanol of two volumes with deposit D NA toward water layer again.In-70 ℃ of placements 1 hour, afterwards with 10, centrifugal 5 minutes of 000r.P.m reclaimed sedimentary DNA with mixture.Isolating precipitate like this is dissolved in 10mM Tris-Hcl(PH7.4) and the mixture (5 μ l) of 1mM EDTA in.The lambda phage Sharon 16A DNA(that obtains HBV DNA and equimolar amounts through Cobra venom endonuclease XhoI cracking by above-mentioned same quadrat method has the recognition site of an Xho I), with T ₄Dna ligase (by 50mM Tris-Hcl(PH7.4), 10mM Mgcl ₂, 10mM dithiothreitol, DTT, 100 μ g/ml bovine serum albumin, 0.5mM ATP and 0.5 μ l enzyme preparation (T ₄Ligase, Takara biologics company makes, 1-5 * 10 ³Unit/ml)) mixture of forming (10 μ l).T ₄Ligase was in 4 ℃ of reactions 18 hours.With extracting with ether after the phenol, reuse ethanol precipitates according to said method afterwards with reactant mixture.This precipitate is dissolved in 10mM Tris-Hcl(PH7.4) and the mixture (10 μ l) of 1mM DETA in.

Annealed DNA is pressed " Method in Engymolgy ", 68, the described method of 299-309 generates lambda phage through external packaging operation, and with escherichia coli DP50 SupF(referring to Blattner, F.R.et al., Scieucl, 196,161,1977) (23Cm * 23Cm) goes up and further produces plaque (10 at the L-agar plate to make indicant ⁴).These plaques method for preparing ³²The HBV DNA of P labelling makes probe (referring to SCience, 196,180,1977) these plaques is done plaque hybridization, so that the plaque that screening is formed by the phage with HBV DNA.By this method, isolated the aggregation of desired phage.

(B), making carrier with plasmid pACYC177 clones again

What obtain from above-mentioned (A) has a HBV DNA phage, by using escherichia coli DP50-SuPF as the antibacterial that infects, and by above-mentioned " Methods in Enzymology ", 68,245-378,1979 method prepares phage DNA.The DNA that obtains like this digested 2 hours under condition same as described above with Xho I, and with the reactant mixture that obtains through using the electrophoretic separation of 0.75% agarose gel, obtain HBV DNA(3.2Kb).This HBV DNA is adsorbed in the DEAE(diethylaminoethyl cellulose) on the paper (Japanese Toyo Roshi company make), so that separate with carrier DNA, use the drip washing of 1M Nacl aqueous solution then, and obtain on two ends, having the HBV DNA of Xho I end.

In addition, the plasmid pACYC177(that an one Xho I cracking site is arranged in kanamycin-resistant gene is referring to Chang, A.C.Y., Cohen, S.N.; J.Bacteriol., 134,1141-1156,1978) according to above-mentioned with quadrat method phenol extracting, handle and ethanol precipitation by ether to product for available Xho I.

What obtain like this mixes with 1: 5 molar ratio with the terminal HBV DNA of cracked pACYC177 of Xho I and the Xho I-that obtains in said method, and by above-mentioned T ₄The catalytic reaction of dna ligase is reacted and was made it covalent bond in 18 hours.

(10 μ l) is added to by M.V.Norgard with reactant mixture, Gene, 3, the 0.1ml escherichia coli X1776(of method preparation is referring to R.III.Curtiss 279(1978), et al., " Molecular Cloning of recombinant DNA " esd.W.A.Scott and R.Werner, page99, Academic press(1877)) in,, and it was left standstill 25 minutes at 0 ℃ with the mixture mixing.Mixture moved on to contain ampicillin (20 μ g/ml), α-biotin (1 μ g/ml), on the L-agar plate of meso diaminopimelic acid (100 μ g/ml) and thymus pyrimidine (20 μ g/ml), and in 37 ℃ of overnight incubation.The bacterium colony that generates moves on to the agar plate that contains kanamycin (20 μ g/ml) and contains on the agar plate of ampicillin (20 μ g/ml), only filters out containing the bacterium colony of growing on the agar plate of ampicillin.Use Matsubara(J.Virol., 16,479,1975) described method prepares plasmid by the bacterium colony that screens.The plasmid that is obtained, promptly pACYC177-HBV DNA(names and is " pHBV ") recombinant DNA, handle under above-mentioned identical condition with Xho I, and obtain complete HBV dna fragmentation (3.2Kb).

(2), the preparation of shuttle vector pAM82

(RAP can obtain from yeast S2882C gene bank to contain about 8.000 nucleotide pair EcoRI fragments of 60.000 dalton polypeptides (P60) gene of the group quenchable acid phosphatase of structure (RAP), referring to Clarke, L.and Carbon, J.Cell, 9,91-99,1976) be inserted into the EcoRI site of known escherichia coli plasmid pBR322, thus obtain a kind of plasmid as parent material.

In order to remove the RAP coded sequence, initial being limited property of plasmid restriction endonuclease Sal I digestion and once more with T ₄The dna ligase covalent bond.Resulting plasmid pAT25, (said plasmid pAT25 is the segmental plasmid that the Sal I site from the EcoRI site to pBR322 is arranged by to acid phosphatase gene fragment 5.2Kb in disappearance Sal I site.PBR322 contains the fragment in an anti-ampicillin gene and a Sal I site from the EcoRI site to the yeast acid phosphatase gene, and these two fragments respectively link with its corresponding end therein).

An EcoRI fragment (1.4Kb) that contains arsl and Trpl gene is inserted in EcoRI site at above-mentioned pAT25, and the Trpl gene is to handle plasmid YRP7(referring to Struhl with EcoRI, K.et al., proc.Natl.Acad.Sci.U.S.A., 76,1035-1039,1979) obtain.After above-mentioned insertion, obtain plasmid pAT26.Said arsl-Trpl fragment has the single recognition site of a restriction endonuclease Hind III in the Trpl gene.

On the Hind of above-mentioned pAT26 III site, insert a Hind III fragment that contains Leu2 and 2 μ ori, obtain shuttle vector pAT77.2 μ ori are by handling plasmid pSLEl(referring to Tohe with Hind III, A.et al., J.Bacteriol., 141,413-416,1980) make.Be stated from beer yeast (being Saccharomyces Cerevisiae AH 22/pAT77) and be deposited at fermentation research institute under the Japanese industry science and technology Room according to budapest treaty, registration number is " FERMBP-324 ".

The pAT77(1 μ g that so uses Sal I cracking to obtain), at 20mM Tris-Hcl(PH8.2), 12mM Cacl ₂, 12mM Mgcl ₂, in the solution of 0.2M Nacl and 1mM EDTA (50 μ l), with exonuclease BAL 31(0.1 unit) handled 30 seconds to 1 minute.This reactant mixture is used phenol extracting, ethanol precipitation as stated above.Resulting precipitation and Xho I are connect thing (linker, 1 Pmol) mixing, and be connected with the T4 dna ligase by above-mentioned condition, reaction was carried out 12 hours.Adopt the described method of people " Molecular Cloning of recombinaut DNA " eds.W.A.Scott and R.Werner such as R.III.Curtiss, Page99, Academic Press(1977) with above-mentioned reactant mixture transformed into escherichia coli.Press K.Matsubara(J.Virol., 16,479,1975) described method, preparation plasmid DNA s in the resulting transformed bacteria.According to the Maxam-Gilbert method (referring to Maxam, A.﹠amp; Gilbert, W., Pro.Natl.Acad.Sci., 74,560-564), measured the order of the nucleotide of gained DNA, further cut away the acid phosphatase gene zone with BAL31.In these DNA, screen and separate desired plasmid pAM82.

" A " among first aminoacid (protein acid) codon ATG of the product P 60 of coding phosphoric acid enzymatic structure gene among the carrier pAM82 is decided to be "+1 ", cuts away zone up to till-33.The PAM82 that is stated from beer yeast (being Saccharomyces Cerevisiae AH 22/PAM82) is deposited at fermentation research institute under the Japanese industry science and technology Room according to budapest treaty, and registration number is " FERM BP-313 ".

(3), the preparation of HBsAg gene expression plasmid

Handle HBV DNA and the Xho I cracking shuttle vector pAM82 that plasmid pHBV obtains with Xho I, under above-mentioned identical condition,, pass through T with 5: 1 molar ratios ₄Dna ligase is recombinated.

With reactant mixture transformed into escherichia coli α 1776, and in the transformed bacteria of resulting anti-ampicillin, prepare plasmid DNA.With various restriction endonucleases such as Xho I, Xba I and Hind III analyze prepared plasmid DNA.Be inserted into HBV DNA and direction of insertion thereof in the carrier so as to mensuration.

So the HBsAg gene expression plasmid (name and be PAH203) that obtains on the order of phosphatase promoter downstream direction, has HBs gene and HBc gene.

(4), the preparation of transformed yeast

Initial yeast is beer yeast AH22 (a, Leu2, his4, Can1(Cir ⁺)), being deposited at fermentation research institute under the Japanese industry science and technology Room according to budapest treaty, registration number is " FERM BP-312 ".Initial yeast-inoculated on the YPD culture medium (100ml) of 1% yeast extract and 2% glucose, and in 30 ℃ of following overnight incubation, afterwards, is used the centrifuging collecting cell in containing peptone more than 2% (Polypeptone).(20ml) washes the cell of collection with aquesterilisa, being suspended in 1.2M sorbitol and 100 μ g/ml enzymolysis enzymes (zymolyase)-60.000(Japan Seikagaku Kogyo company makes) solution (5ml) in, suspension left standstill under 30 ℃ 30 minutes and obtain spheroid.Wash spheroid three times with the 1.2M sorbitol solution, be suspended in 2M sorbitol, 10mM Cacl then ₂With 10mM Tris-Hcl(PH7.5) solution (0.6ml) in.Is in the volume packing small test tube of 60 μ l with prepared suspension with every part.The plasmid recombinant pAH203(30 μ l of above-mentioned (3) preparation) solution is added in the suspension.Behind the mix homogeneously, add 0.1M Cacl by the 10mM final concentration ₂(3 μ l) places mixture and left standstill under the room temperature 5-10 minute.20% Polyethylene Glycol, 4.000 solution that add 1ml in the resulting mixture, 10mM Cacl ₂With 10mM Tris-Hcl(PH7.5) each 1ml, and place room temperature to leave standstill 20 minutes the mixture.The mixture (every part of 0.2ml) that obtains is added in the culture medium (10ml) that contains 22% sorbitol, 2% glucose, 0.7% yeast nitrilo aminoacid, 2%YPD, 20 μ g/ml histidine and 3% agar, is placed on 45 ℃ the constant temperature.After mixing gently, containing previously prepared 1.2M sorbitol, and containing on the bottom line culture plate of 0.7% yeast nitrogen base aminoacid, 2% glucose, 20 μ g/ml histidine and 2% agar and add this mixture of one deck, and it is being left standstill.Cultivate under culture plate places 30 ℃ and do not needed leucic zymic bacterium colony.Bacterium colony places interpolation leucine (20 μ g/ml) BurkHolder minimal medium (minimal medium) to go up again and cultivates (referring to Tohe, A, et al., J.Bachterol., 113,727-738,1973) thus obtain desired transformed yeast: beer yeast (Saccharomyces Cerevisiae) PAH 203.

(5), produce HBsAg with transformed yeast

The transformed yeast that is obtained in above-mentioned (4) is inoculated in the BurkHolder minimal medium (10ml) that has added histidine (20 μ g/ml), and in 30 ℃ of cultivations.The gained culture further is inoculated into interpolation histidine (20 μ g/ml) BurkHolder minimal medium (10l) and cultivates and add stirring 48 hours in 30 ℃.Collect the cell that is in exponential phase with centrifuging, be suspended in not phosphorous hydrochlorate and (in the BurkHolder minimal medium, substitute KH with Kcl ₂Po4 makes, and that continues adds 20 μ g/ml histidine) minimal medium in (10l), cell concentration is approximately 4 * 10 ⁶Cell/ml.In 30 ℃ cultivate 24 hours after, culture medium centrifugal 10 minutes, collecting cell (approximately 120g) with 4.000r.p.m.

(6), the preparation of HBs antigen purification product

In the cell of the method preparation of reusing above-mentioned (5) (about 1Kg), add 0.1M phosphate buffer (PH7.2) (5l), and 600 to 700Kg/Cm ²Pressure is handled said mixture to smash cell to pieces with the Manton-Gaulin disintegrating machine down.The cell of smashing to pieces is removed thick broken cell sheet through centrifugal, obtains the antigenic crude extract of HBs.By dripping 10% aqueous acetic acid the pH value of crude extract is transferred to 5.2.Mixture is used the centrifuging disgorging after stirring 30 minutes under 4 ℃.

Add ammonia in the supernatant that is obtained and be about 6.5, and slowly to add its final concentration of ammonium sulfate to supernatant be 2.5M, but will keep above-mentioned pH value to regulate PH.After making it leave standstill 30 minutes, mixture contains the antigenic precipitate of HBs through centrifugal taking-up.The precipitate that obtains like this is suspended among the about 300ml of 0.1M phosphate buffer (PH7.2), and mixture is to above-mentioned used same buffer dialysis.

After the dialysis, mixture dilutes about 3 times with the 0.1M phosphate buffer, and (gel content: about 1l), this post has been used above-mentioned same buffer balance in advance, just is adsorbed on the gel by above step HBs antigen to make it pass through the apatite hydroxide packed column then.This post during with balance used buffer carefully wash, and then with 0.2M kaliumphosphate buffer (PH7.2, about 3l) by post, to remove pollutant.After this, pass through post with 0.5M kaliumphosphate buffer (PH7.2, about 3l), with eluting HBs antigen.After the dialysis of 0.1M kaliumphosphate buffer, contain the antigenic part of the HBs apatite hydroxide post (gel content: about 500ml), just be attracted on the gel by having crossed with above-mentioned same buffer balance in advance once more by above-mentioned steps HBs antigen.It is that the kaliumphosphate buffer (about 2l) of 0.2 → 0.5M contains the antigenic part of HBs by post with collection that reuse has a Concentraton gradient.

What obtain like this contains the antigenic part of HBs (about 1000ml), to the dialysis of 0.01M phosphate buffer, uses hollow fiber membrane ultrafiltration device (Minimodule, the manufacturing of Japanese Asahi chemical industrial company) to be concentrated into 100ml then.

A kind of 50% sucrose solution, a kind of 20% sucrose solution and containing in the centrifuge tube of HBs antigen part with three layers of adding Hitachi Rp-42 supercentrifuge of being obtained, in 4 ℃, with 27.000r.p.m centrifugal 16 hours, be concentrated in around the sucrose solution interface by this step HBs antigen.

Like this purification contain the antigenic part of HBs, to having added the 0.01M phosphate buffer dialysis of 0.14M Nacl, and to wherein adding the cesium chloride that concentration is 1.2g/ml.With Hitachi RP-42 supercentrifuge with 25.000r.p.m, in 10 ℃ with centrifugal 60 hours of mixture, obtain the HBs antigen of purification.Purified like this contains the antigenic part of HBs to having added the 0.01M phosphate buffer dialysis of 0.14M Nacl, makes with Minimodule(Japan Asahi company afterwards) hollow fiber membrane ultrafiltration device concentrates.Gains have been to having added the 0.01M phosphate buffer dialysis of 0.14M Nacl, then after filtration degerming to obtain the antigenic pure product of HBs (HBs antigen protein content: 106 μ g/ml; 12ml).

According to the fine gel electrophoresis of SDS-polypropylene, the antigenic pure product of the HBs of above-mentioned acquisition manifest protein subunit matter (molecular weight: about 25.000) single band.In addition, use the immunodiffusion method of anti-reorganization source HBs antigen one Cavia porcellus antibody and a kind of anti-people source HBs antigen Cavia porcellus antibody, compare with the antigenic standard substance of people source HBs, two kinds of pure product of HBs antigen have showed same antigenicity.

Reference example 2

Derive from the antigenic preparation of purification HBs of the mammalian cell of reorganization

According to people's such as Nozaki method (disclosing 36689/1984 for the first time referring to Japan Patent (application)), preparation is also cultivated recombinant mammalian cells, and to produce HBs antigen, antigenic separation of HBs and purification are as follows:

(1) the segmental preparation of HBV DNA BamRI

The plasmid PHBV of preparation in the above-mentioned reference example 1, (1) adopts conventional method to handle with BamHI, and resulting reactant mixture reuse 0.75% agarose gel carries out electrophoresis, to obtain the BamHI fragment of HBV DNA.

(2) preparation of carrier (PXRIIG BamHI fragment)

Shuttle vector pXRIIG(is derived from U.S. Harvard university) (1 μ g) add contain 10mM Tris-Hcl(PH8.0), 7mM Mgcl ₂, 100mM Nacl and the alcoholic acid mixture of 2mM alpha-mercapto (20 μ l), and add unit of BamHI(of 1 unit inward: the enzymatic activity of energy catapepsis in per 1 hour 1 μ g λ DNA), this mixture is placed 30 ℃ of reactions 1 hour down.Reactant mixture phenol extracting, ethanol precipitation is used in water layer ether extracting then.Precipitate is dissolved in the water, and this solution is used to prepare recombinant DNA.

(3), the preparation of HBV DNA pXRIIG recombinant DNA

The solution (50 μ l) that contains HBV DNA BamHI fragment (150ng) and pXRIIG BamHI fragment (50ng) reacted 4 hours with the T4 dna ligase in 16 ℃.

Reactant mixture transformed into escherichia coli α 1776 with above-mentioned method acquisition of the same race.Again by above-mentioned (1) (B) described method with resulting transformed bacteria after cultivating 12 hours on the L-agar culture medium, filter out the bacterium colony of on agar culture medium, growing, again the bacterium colony that screens is added to respectively and contains tetracycline (TC) (10 μ g/ml) agar culture medium and containing on the agar culture medium of ampicillin (Ap) (40 μ g/ml), will in containing the agar culture medium of TC, can not grow and containing the bacterium colony that to grow on the agar culture medium of AP and screen.Cultivate in the culture fluid of the escherichia coli α 1776 that each bacterium colony had all been mentioned in the above, and extract plasmid with above-mentioned method of the same race.By the cleavage map analysis of using various restriction endonucleases (as BamHI, XhoI, HindIII, SalI), filter out three BamHI fragments containing HBV DNA and the described recombinant DNA of pXR-IIG(name be pSHB3) a segmental recombinant DNA.

(4), mice LTK ^-Transformation

Prepare following liquid A and liquid B:

Liquid A: containing 50mM Hepes(is the N-2-ethoxy

Piperazine-N-2-ethylsulfonic acid), 280mM Nacl and 15mM Na ₂HPO412H ₂The solution of O (1.25ml, PH7.1).

Liquid B: contain pSHB3(50 μ g), PTK(2.5 μ g) (referring to Colbere-Garapin, F., Proc.Natl.Acad.Scic.USA, 76,3755,1979), salmon sperm dna (carries gene DNA, Carrier DNA) (50 μ g) and 2M Cacl ₂Dna solution (0.15ml) (1.1ml) mixture.

Stir the companion and down liquid B is added drop-wise in the liquid A, mixture was placed under room temperature 30 minutes.After drawing q.s with dropper, mixed liquor (0.5ml) dropwise added fill monolayer mice LTK ^-The flask of cell interior (about 10 ⁵Cell/flask).For mixture is adsorbed on the cell, under the room temperature flask was placed 30 minutes, and toward the Eagle culture medium that wherein adds the Dulbecco improvement (hereinafter claim " DMEM ", 5ml), this culture medium contain 10% calf serum (referring to Dulbecco, R ﹠amp; Freeman, G.; Virology.8,396,1959), and with this mixture put at 5%Co ₂Under the environment,, cultivated about 5 hours in 37 ℃.Behind the DMEM that renews, mixture was further cultivated 24 hours.Then, culture medium changes the culture medium that contains hypoxanthine (15 μ g/ml), aminopterin (1 μ g/ml) and thymidine (5 μ g/ml) (hereinafter claiming " HAT " culture medium) into (referring to Littlefield; J.Proc.Natl.Acad.Sci.USA, 72,3961-3965, (1963)).The duration of cultivation, changed once new HAT culture medium in per 2 days to 3 days.After 4 week, collect TK ⁺The cell bacterium colony, thus desired transformant obtained.

(5), produce HBs antigen with the mammalian cell that transforms

To containing 10% calf serum, penicillin (cultivate this mixture 1 weeks down at 37 ℃ by 250 units/ml) and the DMEM culture medium of streptomycin (0.2 μ g/ml) with transformed mouse L cell inoculation prepared in above-mentioned (4).Contain the HBs antigen that concentration is approximately 400ng/ml in the supernatant of culture broth.

(6), the purification HBs antigen of the mammalian cell production that transforms

Repeat to cultivate by above-mentioned (5), obtain culture supernatants (10l), (Minimodule, Japanese ASahi chemical industrial company makes) is concentrated into 1000ml with it with hollow fiber membrane ultrafiltration device.Being 6.5 o'clock by adding ammonia maintenance solution pH value, slowly add ammonium sulfate, making final concentration is 2.5M.After leaving standstill 30 minutes, the antigenic precipitate of HBs is separated with centrifuging.With resulting precipitate be dissolved in added 0.14M Nacl 0.01M phosphate buffered solution (PH7.2) (100ml) in, this mixture is to above-mentioned used identical buffer dialysis.

After the dialysis, (gel content: 2l) post is made gel permeation chromatography by agarose (Sepharose CL 6B) (being made by Sweden pharmacia company) with this mixture.Collect and preserve and contain the antigenic part of HBs (, beginning to be equivalent to first peak part of void volume from eluting) by the assay determination of uv absorption monitor.To 0.1M kaliumphosphate buffer (PH7.2) dialysis, by having used the equilibrated apatite hydroxide of above-mentioned identical buffer (gel content is approximately 250ml) post in advance, HBs antigen just is adsorbed on the gel then with concentrated part.To remove pollutant, use the 0.5M kaliumphosphate buffer with the used same buffer flushing post of balance afterwards with eluting HBs antigen.Contain HBs antigen part (about 250ml) to 0.01M phosphate buffer (PH6.2) dialysis, the reuse hollow fiber membrane ultrafiltration device is concentrated into 50ml.

With 50% sucrose solution, 20% sucrose solution and above-mentioned preparation contain the centrifuge tube that the antigenic part of HBs adds Hitachi RP-42 type supercentrifuge respectively, form three layers, in 4 ℃, with 27.000 r.p.m ultracentrifugations 16 hours, HBs antigen was just concentrated on the interface of two sucrose solution layers whereby.

The purified antigenic part of HBs that contains, dialysis in the 0.01M phosphate buffer that has added 0.14M Nacl is made with Minimodule(Japan Asahi chemical industrial company then) concentrate.Use the Filtration degerming, and obtain a kind of HBs antigen product (HBs antigen protein content: 98 μ g/ml of purification; 14ml).

Example 1

Gel aluminum hydroxide is added in the solution of the reorganization body source HBs antigen purification product of pressing the production of reference example 1 method, gel addition (pressing aluminium hydroxide weight calculates) by weight is 8 times of the HBs antigen protein.Mixture is through the centrifugal supernatant of removing, thereby obtains having adsorbed the antigenic gel aluminum hydroxide of HBs.

Yet, when reusing said method, replace aluminium hydroxide with aluminum phosphate, can obtain having adsorbed the antigenic Fosfalugel (Yamanouchi) of HBs.

In every part of absorption HBs antigen alumina gel by above-mentioned acquisition, listed being dissolved in added the various stabilizing agents of the 0.01M phosphate buffer (PH6.0) of 0.14M Nacl as table 1, promptly obtains vaccine solution (HBs antigen protein concentration: 40 μ g/ml).

This solution of every part of 1ml is packed in the bottle of 2ml, and in-50 ℃, gives lyophilizing under the normal pressure 6 hours.Continue the pressure of 0.04 millimetres of mercury that reducing pressure after, in 5 ℃ of first phase lyophilizing 15 hours, afterwards under 0.04 millimetres of mercury, 30 ℃ through the second stage of lyophilizing 8 hours.Thereby just obtain desired lyophilized formulations.

In freeze dried product serial number 1-11, add physiological solt solution (each 2ml) respectively, and add sodium citrate with complete dissolved aluminum gel.Make by using AUSRIA II(Japan Dainabbott radioisotopic laboratory) radio immunoassay, measure the HBs antigen in the solution, then the data that obtain are compared with reference value (product before the lyophilizing).Antigenicity is as shown in table 2 relatively.

^*) antigenicity of freeze-drying prods is to the antigenic relative value of freeze-drying prods (latter's value is in 1) not.

Example 2

(HBs antigen protein concentration: 96 μ g/ml), add aluminum chloride (60mg) aqueous solution (5ml) in the antigenic pure product solution of reorganization body source HBs of pressing reference example 1 described method preparation.PH to 6.1 with the NaoH regulator solution of 1N.Mixture is through the centrifugal supernatant of removing, and obtains having adsorbed the antigenic alumina gel of HBs.With resulting gel with contain lactose (10W/V%), l-monosodium glutamate (0.4W/V%), arginine (0.4W/V%), gelatin (0.08W/V%) and ethyl hydrargyrum sulfo-water sun sour sodium (0.005W/V%), the 0.01M phosphate buffer (PH6.2,72ml) mixing that have added 0.14M Nacl.

The vaccine solution of gained is installed in the 2ml bottle with every part of 0.5ml branch, carry out lyophilizing by example 1 described method then and handle.

Freeze dried vaccine preparation that is obtained in the above-mentioned example 2 and initial backlog are relatively examined and determine the immunogenicity that it shows in the mice body.Just the freeze dried vaccine preparation is dissolved in distilled water, the guinea pig back subcutaneous injection, inoculum concentration is respectively 0.5 μ g, 1 μ g and 2 μ g HBsAg.After 5 weeks, in animal body, take a blood sample, survey its anti-HBs antibody with radioimmunoassay (use to detect the medicine box AUSAB of anti-HBs antibody, Japanese Dainabbott company makes).As reference, measured antibody titer equally with initial backlog inoculation.The results are shown in table 3.

^*The average antibody value and the initial backlog of corresponding every kind of freeze dried vaccine injection volume are average

The average relative value that value for antibody is compared (latter's value is in 1).

Freeze dried vaccine has also carried out following storage stability experiment.

Freeze dried vaccine after keeping a set time under the steady temperature, is added sodium citrate in vaccine sample,, survey its antigenicity with radioimmunoassay then with the dissolved aluminum gel.The relative value of lyophilized products calculates to compare calculating with the antigenic standard sample of HBs (promptly by people's albumen being added in the antigenic pure goods of people source HBs, divide and install in each bottle, hold it in-80 ℃ of lyophilised states, when measuring, liquefied sample is as a reference) (the reference antibody value is in 1).The relative value of each sample lists in the listed lyophilizing sample value of table 4(herein in 1).

Freeze-drying prods of the present invention even 37 ℃ preserve and to be still in 25 weeks stablely, and this product and aqueous vaccine have very big difference (disclosed part in the Comparative Examples that vide infra 1).

In addition, 37 ℃ of samples of preserving for 25 weeks have been carried out abnormal toxicity test, the result does not observe any undue toxicity.

Comparative Examples 1

With the suspension (2.9ml) of aluminium hydroxide gel be added to the used pure product of reorganization body source HBs antigen of example 2 solution (HBs antigen protein concentration: 96 μ g/ml) (50ml) in.This mixture is through the centrifugal supernatant of removing, and obtains a kind of antigenic alumina gel of HBs that adsorbed.Alumina gel and contain thiomersalate (50 μ g/ml) adding the 0.01M phosphate buffer (PH6.0) of 0.14M Nacl (60ml) mix, and draw vaccine solution.This vaccine solution (every part of 1ml) is dispensed into the 2ml bottle, obtains liquid vaccine preparation.

This bacterin preparation carries out the storage stability test by the method for example 2.The results are shown in table 5.

In addition, with the bacterin preparation of preparation in the example 2, do the storage stability test without lyophilizing (promptly leaving standstill).The results are shown in table 6.

Example 3

Press reorganization body source HBs antigen (the HBs antigen protein concentration: 105 μ g/ml) in the solution (80ml) of purified product, add aluminium hydroxide (50mg) aqueous solution (5ml) for preparing with quadrat method described in the reference example 2.This mixture is removed supernatant and is obtained having adsorbed the antigenic alumina gel of HBs through centrifugal.The alumina gel that so obtains and the 0.01M phosphate buffer liquid (PH6.0,105ml) mixing that contain lactose (10W/V%), glycine (1W/V%), bright acid (0.05W/V%), ethyl hydrargyrum sulfo-bigcatkin willow poplar acid sodium (0.005W/V%) and added 0.14M Nacl.

Divide the 2ml bottle of packing into the acquisition vaccine solution with every part of 1ml, carry out lyophilizing by the method for example 1 and handle.

Freeze-drying prods carries out the storage stability test by the method for example 2.The results are shown in table 7.

1) method of use-case 2 is measured

In 37 ℃ of samples of preserving for 25 weeks, carry out abnormal toxicity test as stated above, but do not observe any undue toxicity.

Claims (9)

1, the lyophilized formulations of hepatitis B vaccine, comprise the purification hepatitis virus B surface antigen that produces by the organism that can produce the antigenic reorganization of HBs, in the presence of stabilizing agent, it is adsorbed on the alumina gel with lyophilised state, said lyophilized formulations is by in the reorganization body source HBs of purification antigen, adds alumina gel and stabilizing agent and this mixture of lyophilizing and prepares.

2, by the preparation of claim 1, wherein alumina gel is one of to be selected from gel aluminum hydroxide and the Fosfalugel (Yamanouchi) person.

3, by the preparation of claim 1, wherein stabilizing agent is one of to be selected from aminoacid or its salt and the saccharide person.

4, by the preparation of claim 3, wherein contain a seed amino acid or its salt and at least a saccharide at least.

5, by the preparation of claim 3, stabilizing agent wherein is at least a aminoacid or its salt, the combination of at least a sugar and at least a colloidal substance.

6, by the preparation of claim 4, aminoacid wherein and salt thereof are selected from one of glycine, alanine, monosodium glutamate, arginine and lysine person; And sugar is one of to be selected from glucose, xylose, gala acid, fructose, lactose, maltose, sucrose, mannitol, sorbitol and the xylitol person.

7, by the preparation of claim 5, wherein aminoacid or its salt are one of to be selected from glycine, alanine, monosodium glutamate, arginine and the lysine person; Saccharide is one of to be selected from glucose, xylose, breast enlargement sugar, fructose, lactose, maltose, sucrose, mannitol, sorbitol and the xylitol person; Colloidal substance is to be selected from one of gelatin, human albumin and dextrane person.

8, the method for the lyophilized formulations of preparation hepatitis B vaccine, comprise by producing the antigenic recombinant organisms of HBs, in the aqueous solution of the hepatitis virus B surface antigen of the purification that produces, add a kind of alumina gel and a kind of stabilizing agent, the mixture that is adsorbed in the hepatitis virus B surface antigen on the alumina gel is divided to minimum applying unit, and the mixture of each applying unit of lyophilizing.

9, by the method for claim 8, step of freeze drying wherein comprises under the low temperature atmospheric pressure several hours pre-lyophilizing; The higher temperature decompression is ten to tens hours first phase lyophilizing down; Elevated temperature and three steps of the second stage of lyophilizing of more several hours to ten hours under the low-pressure again.