CN1248471A - Vero cell encephalitis B inactivated vaccine and preparation process thereof - Google Patents
Vero cell encephalitis B inactivated vaccine and preparation process thereof Download PDFInfo
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Abstract
The present invention relates to a vero cell encephalitis B inactivated vaccine and its preparation method. Said vaccine is a purified encephalitis B inactivated vaccine obtained by inoculating vero cell encephalitis B viral strain on the vero cell, and its preparation method includes the following steps: culturing and amplifying Vero cell, inoculating Vero cell encephalitis B viral strain on the Vero cell, collecting virus, inactivating, concentrating and purifying, etc.. Because it utilizes Vero cell culture and vero cell viral strain, so that it contains no contaminative factor and tumorigenicity, and possesses the advantages of high purity, good immune effect and high use safety, ad is favourable for implementing large-scale industrial production of encephalitis B vaccine.
Description
The present invention relates to bioengineering field, relate to a kind of Vero cell encephalitis B inactivated vaccine and preparation method thereof specifically.
Mass-produced in the world at present Vaccinum Encephalitidis Epidemicae has two kinds, a kind of be with Japan be representative with the white mice cerebral tissue that infects encephalitis b virus through grinding, emulsifying, concentrate, the purification Vaccinum Encephalitidis Epidemicae of ultracentrifugation preparation, the Japanese encephalitis inactivated vaccine that a kind of former generation hamster kidney cell that is China produces is cultivated and former generation ground Ren Mus Live Japanese Encephalitis, the cellular matrix of these vaccines all derives from the regular grade animal, can't guarantee not carry exogenous factor.
Mus brain purification Vaccinum Encephalitidis Epidemicae is made with cerebral tissue, and severe anaphylactic reaction once took place the cerebral tissue vaccine in history, though thereby the purified processing of the vaccine of the tissue preparation of requiring mental skill still have this melancholy to consider unavoidably; Secondly the preparation of Mus brain purification Vaccinum Encephalitidis Epidemicae must be through the encephalocoele inoculation of a white mice, the results of infected mice brain one by one, its operating process is loaded down with trivial details, can't form the suitability for industrialized production scale, so if make large batch of homogenizing vaccine, cost is very high, cost an arm and a leg, mainly by the country of import Vaccinum Encephalitidis Epidemicae,, be difficult to satisfy supply for some owing to price.
An investigation of initiating according to U.S. CDC shows the antibody male rotary rate (〉=1: 8) 77% after the injection of Mus brain purification Japanese encephalitis inactivated vaccine two pins, positive rate of rotation (1: 16) 100% behind 6～12 months reinforcement one pins, but rate of side effects is higher, comprises that local response and general reaction reach 41%.Also there are the report that serious anaphylactic reaction takes place in Denmark and Australia after using Japanese Mus brain purification Vaccinum Encephalitidis Epidemicae, use this vaccine to measure antibody response in India, and the result is unsatisfactory.
The ground Ren Mus Japanese encephalitis inactivated vaccine of the current production of China has played obvious effect really for reducing the encephalitis b sickness rate, and the encephalitis b sickness rate is descended steadily.But its serum neutralizing antibody sun rate of rotation is unsatisfactory, child's neutralizing antibody sun rate of rotation has only about 60% in 1 years old in non-popular district school age population or popular district, strengthen reaching about 90% behind the pin, and the side reaction after the vaccine injection mainly is anaphylaxis, though generally smaller and still can accept, but Tong Ji result according to investigations, in Beijing, its rate of side effects occupies the second in the vaccine that several children generally inoculate, be only second to whooping cough.Also has a kind of AS14-14-2 strain Live Japanese Encephalitis, owing to only need injection one pin that serum antibody response is preferably just arranged, and production technology is simply with low cost, so be subjected to the welcome of manufacturer and user, but the possibility that still can not get rid of former generation hamster kidney cell external source pollution factor, and there are some researches show that encephalitis b live virus immunoprophylaxis suppresses can to cause under the animal skins that virus goes into the brain breeding, so in the applying of live vaccine, also need suitably note, the plan of safety can be taked inactivated vaccine initial immunity within 1 years old, afterwards the way of strengthening with live vaccine.
The used production seed culture of viruses of hamster kidney cell Vaccinum Encephalitidis Epidemicae that China produces is still uses the common white mice brain of infection to prepare, though prove safely and effectively through use for many years, but antigenic content is lower, its cerebral tissue is not only one of anaphylactogen, and feeding and management animal and preparation seed culture of viruses and use thereof etc. all bother, its production technology also is unfavorable for controlling the pollution of exogenous factor, more is not suitable for large-scale industrial production.
In order to prepare better Vaccinum Encephalitidis Epidemicae; since the research report of the eighties relevant for the genetic engineering Vaccinum Encephalitidis Epidemicae; its carrier is varied; expressing protein is E albumen, PrM and NS1 mostly; through laboratory verification with to induce antibody response all satisfied, but through the protection test of virus attack all not as good as the whole virus vaccine ideal.
The Vero cell is the monkey-kidney cells system that Japanese scholar set up from cercopithecus aethiops (cercopithecus aethiops monkey) kidney in 1962.The origin of its title is that preceding two letters of VERDE and preceding two letters of " kidney " word of taking from " green " word in the Esperanto lump together, but does not allow VERE to exist in the Esperanto, and needs the ending with " O ", so called after " Vero ".In its 93rd generation, be brought to allergy and the infectious disease institute tropic virus research department of America NI H, and the 113rd generation was submitted to ATCC, was numbered ATCC NO.CCL-81.
Through comprehensively research evaluation, it is stable that the Vero cell has karyogy, do not have exogenous factor to pollute and the advantage of oncogenicity, meet the rules requirement of WHO in 1987 fully, and ratified can be used as the substrate of production of vaccine by WHO about the passage cell that is used for biological product.So, Vero cell poliomyelitis inactivated vaccine, Live Poliomyelitis Vaccine, Vero cell rabies are developed out and granted production in France in succession, and relevant research report with Vero cell preparation vaccine for man has report also the time, but yet there are no system's report about the research with Vero cell preparation Vaccinum Encephalitis B.
One of purpose of the present invention is to provide a kind of Vero cell encephalitis B inactivated vaccine, does not contain murine brain in this vaccine, the purity height, and good immune effect, safe to use.
Two of purpose of the present invention is to research and propose a kind of preparation method of Vero cell encephalitis B inactivated vaccine, replacing original hamster kidney cell with the Vero cell culture cultivates, replace present murine brain seed culture of viruses with Vero cell seed culture of viruses, and by concentrated and purified, improve vaccine quality and result of use, realize the large-scale industrial production of Vaccinum Encephalitidis Epidemicae.
Vero cell encephalitis B inactivated vaccine provided by the invention is meant the purified encephalitis B inactivated vaccine that inoculation Vero cell encephalitis b virus seed culture of viruses obtains on the Vero cell.
The preparation method that the present invention proposes may further comprise the steps:
(1), the cultivation of Vero cell amplification;
(2), inoculation Vero cell encephalitis b virus seed culture of viruses on the Vero cell;
(3), results virus;
(4), inactivation of viruses is made thick vaccine;
(5), ultrafiltration and concentration;
(6), the purification of vaccine.
The Vero cell kind that the present invention uses at first set up Vero cell seed bank and Vero cell work storehouse, and pair cell storehouse cell is carried out the calibrating of system from ATCC.Before the preparation vaccine, need carry out recovery, cultivation and the amplification of cell earlier, to reach the needs of production lot.Can use animal cell culture liquid to add the formulated cell nutrient solution of Ox blood serum, said culture fluid can be selected 199 culture fluid, MEM culture fluid etc., wherein to use 199 culture fluid effects comparatively desirable, preferably contain 2～10% Ox blood serum in the cell nutrient solution, amplification is cultivated at 37 ± 1 ℃ in PH7.0～8.0.
The various mammalian cells that are used to prepare vaccine are and adhere to dependent cell, and cell need be attached to growing on certain carrier in incubation, and the training method of cell can have rolling bottle to cultivate among the present invention, also can use the microcarrier reactor to cultivate.
When the Vero cell is cultivated in the microcarrier reactor, microcarrier use amount 2～7 grams per liters, 30～55 rev/mins of rotating speeds, wherein the microcarrier of available use have multiple, as Cytodex1,2,3 types (pharmacia), CT
1, CT
2, CT
3(Huadong Chemical College) and available from Chinese medical courses in general institute blood grind and the microcarrier of east wind biochemical reagents factory of the Chinese Academy of Sciences etc., can both meet the demands through evidence.
For preventing germ contamination, in the preparation cell nutrient solution, can add an amount of gentamycin.
In the incubation, cell divides kind of rate to determine according to the needs of production lot, it generally can be 1: 2～1: 10, after cell grows up to fine and close monolayer, can inoculate encephalitis b virus, preferably add the above human albumin of 0.1% (w/w) in the cell maintenance medium, adjust PH7.2～8.0 simultaneously, 32～37 ℃ of cultivation temperature, and can add an amount of aminoacid and antibiotic, operational culture fluid comprises 199 liquid, MEM liquid etc., seeded process comprise earlier with flushing cell surfaces such as culture fluid such as EarleShi liquid, remove residual Ox blood serum, inoculate encephalitis b virus seed culture of viruses and above-mentioned cell maintenance medium then on the Vero cell of growing in blocks, MOI 0.1～0.00001, cultivates and changes fresh cell maintenance medium after about 24 hours, continue to cultivate about 48 hours, can gather in the crops virus.
The encephalitis b virus kind of the encephalitis b virus that the present invention inoculates on the Vero cell on the Vero cell, going down to posterity, there is experimental result to show, encephalitis b virus growth and breeding well on the Vero cell, and in 10 generations of Vero passage encephalitis b virus, are with interior very stable, and aspect preparation Vero cell Vaccinum Encephalitidis Epidemicae, in 10 generations, are with the not obviously change of immunogenicity of interior passage seed culture of viruses, the immunogenicity of seed culture of viruses then significantly decreases after 10 generations, that is to say, the preparation vaccine preferably selected for use for 10 generations with the interior seed culture of viruses that goes down to posterity, and the seed culture of viruses after 10 generations should not be used to prepare inactivated vaccine.As for the then preferred encephalitis b Mus brain seed culture of viruses P of seed culture of viruses
3Strain is at the seed culture of viruses that goes down to posterity of Vero cell.
The growth and breeding of encephalitis b virus on the Vero cell can be kept the long period, therefore the results of virus can be taked the mode repeatedly collected, preferably per 1～2 day results are once in time measured virus titer to the viral liquid of results, require the virus titer of the every batch of viral liquid of gathering in the crops all should be 10
7LD
50More than/the ml, because have only the viral liquid of high titre could guarantee the efficient of vaccine.
What the viral liquid of results obtained with the formalin solution inactivation of viruses is thick vaccine.
Thick vaccine after the deactivation need concentrate to purify and be prepared into pure vaccine product, the present invention selects for use the method for ultrafiltration and concentration that the thick vaccine that obtains is concentrated, generally be with the ultrafilter concentrated vaccine of holding back 100,000～300,000 molecular weight, be concentrated into more than 10 times, then purified vaccine.
The key for preparing the biological product safety with passage cell is the content of cell rests DNA.By the requirement of WHO rules, the substrate DNA of vaccine must be less than 100pg/ dosage.To comprise that chemical method and physical method etc. can have multiple about the purification process of vaccine, through repetition test and research, the present invention proposes two kinds of preferred purification process: a kind of is to use protamine sulfate that the residual DNA in the vaccine is precipitated earlier, through high speed centrifugation, get supernatant, utilize the method for sucrose density gradient band centrifugation to purify again, collect the merging component that albumen is low, antigen active is high.Wherein, the working concentration of protamine sulfate and vaccine whether concentrate and cycles of concentration relevant, under the process conditions that the present invention proposes, concentrated vaccine is under the effect of the protamine sulfate of 0.8～2.0mg/ml, not only can effectively remove residual DNA, and antigen active do not have obviously and lose, also find in the research, protamine sulfate has also been removed part foreign protein in the vaccine (about 20～60%) in removing the DNA process, played the effect of preliminary purification.Then can measure as for the DNA residual volume by the isotopic labeling Vero cell DNA probe or the photobiotin labelling Vero cell DNA detecting probe method of existing report.
In the concrete operations, protamine sulfate can be dissolved into the solution of 50mg/ml earlier with phosphate-buffered saline (PBS), in vaccine, drip and constantly shake, under 2～8 ℃, left standstill 2～3 hours then, through 6000～10000 rev/mins of high speed centrifugations 5～30 minutes, DNA and protamine co-precipitation are come out.
The above-mentioned vaccine supernatant of removing behind the DNA with protamine sulfate precipitation adopts the method for sucrose density gradient band centrifugation to purify again, operating condition: sucrose liquid concentration 36～60% (w/w), 2～10 ℃, 23000～28000 rev/mins centrifugal 4～6 hours, the fraction collection component to its sampling and measuring albumen, antigen and sucrose, merges the component that albumen is low, antigen active is high, its assay method belongs to known technology, no longer describes in detail.
The purification of concentrated vaccine can also be realized by column chromatography among the present invention: promptly concentrated vaccine is removed part foreign protein and cell DNA through ion-exchange chromatography earlier, further removes residual impurity through gel filtration chromatography again.Ion exchange column such as DEAE-Sepharose FF etc. optionally adsorb foreign protein and DNA in the concentrated vaccine liquid; And gel permeation chromatography is the simplest in the chromatographic technique, condition is the gentleest, to keeping the active best method of biomacromolecule, the material that is fit to isolated molecule amount great disparity, the molecular weight of encephalitis b virus is more than 4,000,000, differ bigger with the foreign protein molecular weight in the vaccine, can very conveniently remove small molecular weight impurity residual in the concentrated vaccine effectively so use gel filtration chromatography, this solvent resistant column can be Sepharose CL-6B, Sepharose 4FF, Sphacryl S-500 HR post or other gels, and testing result shows, behind two step chromatographic column purification, the vaccine total protein content reduces about more than 99%, and the residual volume of Ox blood serum and cell DNA all meets the relevant rules requirement of WHO.
Purified vaccine needed to add protective agent before finished product is made in final lyophilizing, and this protective agent can have multiple choices, as human albumin protective agent and maltose, human albumin protective agent and sucrose, gelatin, or the like.
In a word, the present invention proposes a kind of new way for preparing Vaccinum Encephalitidis Epidemicae, promptly use the production substrate of Vero cell, and replace existing murine brain seed culture of viruses, prepare high-quality Vaccinum Encephalitidis Epidemicae by corresponding process production with the seed culture of viruses that goes down to posterity of Vero cell as vaccine.
The present invention compares with the Vaccinum Encephalitidis Epidemicae from murine brain virus of present use, topmost advantage is as follows: (1) Vero cell has been proved to be and has not contained any pollution factor and oncogenicity, as the substrate of producing vaccine, obviously be better than the hamster kidney cell and the murine brain of existing vaccine; (2) seed culture of viruses of Vero cell Vaccinum Encephalitidis Epidemicae is the cell culture seed culture of viruses, has fundamentally got rid of the murine brain that Mus brain purification vaccine and ground Ren Mus Vaccinum Encephalitidis Epidemicae use; (3) with the Vero cells produce vaccine technology of passage cell, be suitable for suitability for industrialized production homogenizing vaccine in batches, this is that existing hamster kidney cell vaccine and Mus brain purification vaccine is out of the question; (4) vaccine of the present invention antigen active after purifying obviously improves, and foreign protein reduces more than 99%, and the purity of vaccine improves greatly; (5) people's anthroposcopy result shows surplus 1000, vaccine of the present invention serum neutralizing antibody sun rate of rotation behind injection two pins in the 1 years old child in non-popular district school age population and popular district reaches more than 90%, the seroconversion rate of serum antibody of strengthening behind the pin reaches 100%, except that idol has one to cross the heat pyrexia, do not find local response and general reaction, we can say reliable safety.
Accompanying drawing provides the block flow diagram of preparation method of the present invention.
Embodiment 1
The Vero cell is from ATCC, used for 138～150 generations, cell nutrient solution is to contain 4% Ox blood serum and 25Iu/ml gentamycin in 199 culture fluid, and adjusts PH to 7.2, cultivate into behind the fine and close monolayer according to kind rate amplification in 1: 5 minute with 37 ℃ of 15L rolling bottles, when increasing production lot, through about 4 days, when cell grows up to fine and close monolayer, discard cell nutrient solution, with the EarlShi liquid flushing cell face of PH7.2, inoculation Vero cell encephalitis b virus uses encephalitis b Mus brain seed culture of viruses P
3The second filial generation work seed culture of viruses (P that strain is gone down to posterity on the Vero cell
3V
2), seed culture of viruses concentration MOI0.05, cell maintenance medium is for containing the MEM liquid of 0.1% (w/w) human albumin, and PH7.6 cultivates down for 35 ℃, change after 24 hours with fresh cell maintenance medium and continue to cultivate 2 days, begin results virus, per 2 days results are once received 5 times altogether, every batch of viral liquid is all taken a sample and is carried out titration of virus and sterility test, requires the virus titer of the viral liquid of results to reach 10
7LD
50More than/the ml; Merge viral liquid adding formalin (final concentration 1/2000) and place 26 ± 1 ℃, 8 days inactivation of viruses obtain thick vaccine, are condensed into 20 times of concentrated vaccines with the ultrafilter of holding back 300,000 molecular weight.
Get the solution of protamine sulfate with PBS melt into 50mg/ml; be added dropwise to then in the concentrated vaccine liquid and constantly shake simultaneously; the final concentration 1.0mg/ml that adds protamine sulfate; left standstill under 4 ℃ 2 hours; 7000rpm centrifugal 30 minutes fast; get supernatant and carry out the sucrose density gradient band centrifugation; last sample PBS 100ml; concentrated vaccine 1000ml after protamine sulfate is handled, 36-60% (w/w) sucrose 590ml, 4 ℃; use the 55P-72 of Hitachi type centrifuge; the rotary head of RP-50T dihedral, centrifugal 4 hours of 23000rpm collects component; every 25ml one pipe; difference sampling and measuring albumen; antigen and sucrose, it is low to merge albumen; the component that antigen is high is removed sucrose with ultrafiltration; add 0.2% human albumin protective agent and 5% maltose and according to the antigen titration result; be diluted to enzyme-labelled antigen 320 units with phosphate-buffered saline, be distributed into the dosage form of 0.5ml/ vaccinating agent, make the vaccine finished product through lyophilizing.
Embodiment 2
The Vero cell goes down to posterity with conventional trypsinization from ATCC, uses for 138 generations, cell concentration 3 * l0
5/ mg uses Cytodexl 1, and 3g/L cultivates in the Celligen of 5L bioreactor (NBS Co.Inc), working volume 3.8L, 40～45 rev/mins of rotating speeds, dissolved oxygen 25～50%, 37 ℃ of cultivation temperature, cell growth medium is for containing 199 liquid of 3% (w/w) calf serum, PH7.0～7.2.Began to change liquid on the 3rd day, the growing state of observation of cell at any time changes liquid 1/3～1/2 according to the growing state of cell every day, and continuous culture 7 days grows up to fine and close monolayer up to cell, when cell density reaches 2.16 * 10
6The time, after EarleShi liquid washing 2 times, add and do not contain Ox blood serum, contain the MEM liquid (PH7.4～7.6) of 0.1% (w/w) human albumin, and inoculate Vero cell P
3The strain encephalitis b virus, MOI0.01～0.001,36 ℃ of Virus culture temperature, change with fresh cell maintenance medium after 24 hours, and began results virus in back 72 hours, viral suspension of per 36～48 hours results in kind of poison, till all cells pathological changes, virus titer reaches 8.20～9.20log LD
50/ ml.
The viral suspension of results is made thick vaccine with the formalin deactivation; the clarification back is condensed into 20 times of concentrated vaccines with the ultrafilter of holding back 300,000 molecular weight; concentrated vaccine after the ultrafiltration is crossed ion exchange column DEAE-SepharoseFF and solvent resistant column Sepharose 4FF; through two step column chromatographies; obtain purified vaccine; the purified vaccine finished product is made in lyophilizing after adding protective agent; surpassed Chinese Vaccinum Encephalitidis Epidemicae reference material and Japanese Vaccinum Encephalitidis Epidemicae reference material through the calibrating vaccine potency, other every calibratings all meet the requirement of relevant this vaccine of WHO.
Claims (9)
1, a kind of Vero cell encephalitis B inactivated vaccine is characterized in that: this vaccine is the purified encephalitis B inactivated vaccine that inoculation Vero cell encephalitis b virus seed culture of viruses obtains on the Vero cell.
2, a kind of preparation method of Vero cell encephalitis B inactivated vaccine is characterized in that may further comprise the steps:
(1), the cultivation of Vero cell amplification;
(2), inoculation Vero cell encephalitis b virus seed culture of viruses on the Vero cell;
(3), results virus;
(4), inactivation of viruses is made thick vaccine;
(5), ultrafiltration and concentration;
(6), the purification of vaccine.
3, the preparation method of Vero cell encephalitis B inactivated vaccine according to claim 2, it is characterized in that, the purification process of vaccine is to use protamine sulfate that the residual DNA in the vaccine is precipitated earlier, through high speed centrifugation, get supernatant, utilize the method for sucrose density gradient band centrifugation to purify again, collect the merging component that albumen is low, antigen active is high.
4, the preparation method of Vero cell encephalitis B inactivated vaccine according to claim 2, it is characterized in that, the purification of concentrated vaccine can also be realized by column chromatography: concentrated vaccine is removed part foreign protein and cell DNA through ion-exchange chromatography earlier, further removes residual impurity through gel filtration chromatography again.
5, the preparation method of Vero cell encephalitis B inactivated vaccine according to claim 2 is characterized in that, this cell nutrient solution can use 199 culture fluid adding, 2～10% Ox blood serums formulated.
According to the preparation method of claim 2 or 5 described Vero cell encephalitis B inactivated vaccines, it is characterized in that 6, the Vero cell is cultivated in rolling bottle.
According to the preparation method of claim 2 or 5 described Vero cell encephalitis B inactivated vaccines, it is characterized in that 7, the Vero cell is cultivated in the microcarrier reactor, microcarrier use amount 2～7 grams per liters.
8, according to the preparation method of claim 2 or 4 described Vero cell encephalitis B inactivated vaccines, it is characterized in that, in the preparation cell nutrient solution, can add an amount of gentamycin.
9, the preparation method of Vero cell encephalitis B inactivated vaccine according to claim 2 is characterized in that, adds the human albumin more than 0.1% in the cell maintenance medium.
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| CN 98119364 CN1248471A (en) | 1998-09-23 | 1998-09-23 | Vero cell encephalitis B inactivated vaccine and preparation process thereof |
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