CN100333793C - Diploid-cell rabies vaccine and purified rabies vaccine, freeze-drying preparation and water injection thereof - Google Patents
Diploid-cell rabies vaccine and purified rabies vaccine, freeze-drying preparation and water injection thereof Download PDFInfo
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Abstract
The present invention relates to purified diploid cell rabies vaccine in the dosage form of frozen-dried injection or liquid injection. The purified rabies vaccine is obtained by inoculating rabies virus on diploid cell used by a human body.
Description
Technical field:
The present invention relates to a kind of purified rabies vaccine, particularly the purified rabies vaccine that inoculation rabies virus seed culture of viruses obtains on the human diploid cell.
Background technology:
The mass-produced rabies vaccine in the world has four kinds at present: first kind is the diploid cell of representative with the U.S., through concentrating super rabies vaccine from preparation; Second kind is that the Vero cell of representative is a culture matrix with France, production lyophilizing inactivated vaccine, but can not guarantee the DNA of cytostromatic tumorigenesis and cell.The third and the 4th kind are that Embryo Gallus domesticus, these several vaccine bebcells of duck embryo of the ground Ren Mus of representative and Japan derive from conventional animal and can't guarantee that cell does not carry the DNA that exogenous factor can not guarantee cytostromatic oncogenicity and cell with China.
HDCV (HDCV):
Wiktor described HDCV first at the Wistar institute in 1964, and further tested by degree relatively and finally will derive from the Semple production of vaccine and adapt to WI-38 human diploid cell strain (adapting to the MRC-5 cell strain afterwards again) with PM rabies virus strain.Cultivate virus and be prepared into vaccine through clarification, heating and beta-propiolactone deactivation, lyophilizing.This vaccine was got permission in 1974 to produce first, and in beginning commercialization in 1978.HDCV does not contain the malicious factor of any nerve, does not contain any external source animal impurity, thereby can explain its tolerance preferably after duplicate injection.The stability that adopts the NIH method to detect vaccine confirms, vaccine is 4 ℃ and 37 ℃ of placements after one month, no significant difference.Further 5 batches of vaccines of 4.3～5.6 of tiring were deposited 3 years half for 4 ℃, all lot number titres are all greater than the 2.5IU/ agent.
Early stage investigation finds that January or March reach antibody peak value (about 10IU) after the HDCV prophylactic immunization, just reduce gradually subsequently, but titre is all the time greater than 0.5IU in 1～2 year, behind booster immunization in 1～3 year, antibody titer increases sharply 10～15 times, and intramuscular injection is close with the subcutaneous injection approach.
The appearance of HDCV makes people, and immunogenic evaluation has had basis to traditional vaccine.The antibody response that obtains behind confirmations such as Shah 2～4 pin HDCV inoculation human body is suitable with 14 pin Semple vaccines, and Cost-Berger injects 3 pin HDCV and SMBV respectively by the volunteer, finds that antibody response that the former induces is 5 times of the latter.Confirm more also that by HDCV and DEV the latter only has the effectiveness of limit.Bahmanyar is that the immunogenicity of employed any vaccine all can't be compared with HDCV in human trial in the conclusion that drew at that time.Human diploid cell (HDC) is the normal karyotype cell, non-carcinogenesis, and because high immunogenicity and good tolerability that HDCV had, use in the U.S., Canada, most of European countries and several Asian countries at present, this makes it become the standard vaccine of estimating any human novel vaccine.
The shortcoming of HDCV is that HDC cultivates not too easily, and the virus titer that rabies virus is cultivated on HDC is relatively low, only can cultivate in the limited cell bottle in space, and this makes that the price of vaccine is very expensive.In the U.S., expose a post processing expense course of treatment up to more than 1,000 dollars with HDCV; And in Pakistan, carrying out the full processing course of treatment with the Semple vaccine only needs 2.5 dollars, has so just limited the use of this vaccine in developing country.The detailed description for preparing rabies vaccine with this method is not appeared in the newspapers simultaneously.Because particularity and the complexity of rabies vaccine prepare this vaccine and have suitable difficulty, particularly aspect separation and purification.The primitive cell culture vaccine:
Hamster kidney cell vaccine (PHKCV): at first propose and further develop by Fenje by Kissling with hamster kidney cell tissue culture rabies virus development inactivated vaccine, the SAD fixed virus is adapted to the vaccine that hamster kidney cell (PHKC) is gone up the production deactivation, and succeed.The inoculation before Canada's approval is used for the human body reinforcement and exposes of this vaccine of nineteen sixty-eight.
The group that the PHKCV of China is put the great waves leader by Wuhan Biological Products Inst., Ministry of Public Health woods develops.Beginning to test used seed culture of viruses is Beijing strain Medulla Leporis seu Oryctolagi fixed virus, adapts on PHKC through " cell mixing culture method ", passes for 50～60 generations continuously to adapt to successfully; The strain that alternately goes down to posterity for 3 times through guinea pig brain and PHKC is called the aG strain again.The aG strain virus is cultivated results on PHKC, the formalin deactivation adds Al (OH) 3 adjuvants.It is 1.3～2.5IU that vaccine regulation is tired, behind ultrafiltration and concentration, and the concentrated vaccine palpus 〉=2.5IU that tires.Prove that through clinical trial the antibody response of the PHKCV of various dosage forms in human body all is better than Medulla caprae seuovis Semple vaccine.
The production code that this vaccine obtained the health ministry approval in 1980 in China replaces the Semple vaccine.Ten years in the past, the PHKCV that China produces is the rabies vaccine that adds up the volume of production maximum in the world, the volume of production and marketing of annual this type of vaccine of peak time surpasses 5,000,000 person-portions.At present, each production unit of China just progressively adopts improved concentrating-refining PHKCV.
Chick embryo cell vaccine: Kando1965 adapts to chick-embryo cell with Flary HEP strain, cultivate virus through the beta-propiolactone deactivation, concentrate, lyophilizing (back employing band centrifugation purification is improved) is prepared into vaccine and in Japanese goods production.Nineteen eighty-three Barth etc. differentiate from above-mentioned Japanese vaccine and a kind ofly adapt to purification chick embryo cell vaccine (PCECV) on the chick-embryo cell with Flary LEP strain.Cultivate that virus adopts the beta-propiolactone deactivation and through saccharose gradient band centrifugation purification with concentrate.This vaccine is produced by German Chiro Behring GmbH company at present.Clinical test results before and after human body exposes shows that the immune effect and the HDCV of vaccine are suitable, and can not induce at the proteic antibody of chicken cell, uses this vaccine only to produce slight local response.PCECV now in the Europe, Asia and Africa, more than 20 country in South America get permission to use.FDA ratified this vaccine and entered American market in October, 1997.
The advantage that above-mentioned primitive cell culture vaccine has is needn't freezing preservation cell kind.But the impurity (antibacterial, mycoplasma, virus) in organ source is cultivated in every batch of vaccine inspection, and animal organ's available quantity is the deindustrialization factor of production.
The continuous cell line vaccine:
The Vero cell vaccine: 1984 by French Merieun Research Institute success.Adopted in the preparation process and made cell be attached to the microcarrier of suspension culture on the microcarrier so that carry out big jar of cultivation of industrialization.The Strain that this vaccine uses is identical with HDCV, is PM1503-3M.The virus of results is made freeze dried vaccine through ultrafiltration and concentration, density gradient centrifugation, beta-propiolactone deactivation.Studies confirm that do not have tumor and transfer in early days in the experimental animals of 100 inoculations, the residual cells DNA of every vaccinating agent measures less than 50pg, and the excellent stability of vaccine is similar with HDCV.Human immunity or exposure post processing before no matter the eighties and nearest a large amount of experiment confirms are used as and expose, the Vero vaccine all obtains good immune effect.
Because this vaccine import price is higher relatively, therefore, China carries out the development of the human Vero cell vaccine of chromatogram purification since nineteen ninety-five.The Strain of using in the preparation process is the rabies virus (CTN-1V) that adapts to the Vero cell.Present having comprises that the Duo Jia institute of biological products of Shanghai Biological Products Inst., Ministry of Public Health succeeds in developing, and obtains producing code, progressively to replace the existing PHKCV of China.
The Vero cell can be produced higher rabies virus titre than HDC, and can utilize microcarrier to carry out industrialization and cultivate for big jar, thereby its price is cheap than HDCV.Use Vero cell 100,000,000 doses of poliomyelitis vaccine of cumulative production at present, 2,000 ten thousand doses of rabies vaccine and 1,000,000 doses of oral polio vaccine confirm the safety of this cell vaccine.But must use guaranteeing not having oncogenicity at low cell algebraically with Vero cell preparation vaccine, and residual cells DNA amount must be less than the 100pg/ agent.
Power failure usually taking place or be difficult to guarantee the liquid nitrogen supply in developing country, preserves relatively difficulty of cell provenance.Thereby limited the use of this vaccine, developing novel Antirabic Vaccine to reduce cost, particularly find insoluble purifying process, is an important topic, the present invention is through research, found a kind of purified rabies vaccine that inoculation rabies virus seed culture of viruses obtains on the human diploid cell, these human diploid cells are WI-38, CCC-HPF-1P5, MRC-5, resulting vaccine, cheap, easy to use, the purity height is fit to large-scale production and large-scale application.
Summary of the invention:
The invention provides a kind of human diploid cell purified rabies vaccine, this vaccine is the purified rabies vaccine that inoculation rabies virus seed culture of viruses obtains on the human diploid cell.
The present invention also provides the preparation method and the application thereof of purified rabies vaccine of the present invention.
The human diploid cell that the present invention adopts is WI-38, CCC-HPF-1P5, MRC-5, preferably WI-38.Process comprehensively research identifies that it is stable that WI-38, CCC-HPF-1P5, MRC-5 cell have karyogy, does not have exogenous factor pollution and the oncogenicity advantage responsive to virus, meets 2005 editions rules requirements about the passage cell of biological product of Chinese Pharmacopoeia.
The invention provides a kind of human diploid cell purified rabies vaccine, this vaccine is an inoculation rabies seed culture of viruses on the human diploid cell, the rabies vaccine that obtains through separation and purification.Described vaccine virus seed culture of viruses is selected from mad dog seed culture of viruses SNK-CTN strain or SNK-aG strain, preferably SNK-CTN strain.Vaccine of the present invention is freeze dried injection or aqueous injection.
The preparation method of purified vaccine of the present invention may further comprise the steps:
(1), the recovery of human diploid cell;
(2), the cultivation of human diploid cell amplification;
(3), inoculation rabies virus seed culture of viruses on the human diploid cell;
(4), gather in the crops viral liquid;
(5), the deactivation of viral liquid;
(6), clarification ultrafiltration;
(7), purification;
(8), dilution packing.
Described purification can may further comprise the steps: viral liquid is removed the part foreign protein through ultrafiltration purification earlier, through DEAE-SepharoseFF anion exchange or district's band ultracentrifugation, passes through Sepharose4FF post or other gel filtration chromatographies then again.The cultivation amplification of described human diploid cell is carried out in cell nutrient solution, and cell nutrient solution is formulated by 199 culture fluid adding 2-10% Ox blood serum.Cell nutrient solution also can add an amount of kanamycin and gentamycin where necessary.The cultivation amplification of described human diploid cell is to cultivate in rolling bottle.Preparation method of the present invention also comprises the adjuvant adsorption step or/and with protective agent human albumin's step.It is 0.2-0.5% that the amount that wherein adds the human albumin preferably makes its concentration.
Among the preparation technology of purified rabies vaccine of the present invention, the used culture medium of cell culture is 199 culture medium, adopts the ultrafiltration purification preliminary purification, and sucrose density gradient centrifugation and Sepharose4FF gel permeation chromatography carry out purer purification.Test result shows, after three steps purification of the present invention, it is about more than 99% that the vaccine total protein content is reduced, the Ox blood serum residual volume all meets 2005 editions requirements about the biological product rules of Chinese Pharmacopoeia, make tiring of vaccine improve 20% after adding aluminum hydroxide adjuvant, stability improves simultaneously greatly, slows down the speed that virus discharges during inoculation, keeps good antibody horizontal.Add the persistency that adjuvant can increase vaccine by purified vaccine, vaccine can stimulate body to produce antibody lastingly.
Purifying process may further comprise the steps:
(1), makes thick vaccine behind the inactivation of viruses;
(2), clarification filtration;
(3), ultrafiltration purification;
(4), sucrose density gradient centrifugation; (or adopting DEAE-SepharoseFF) anion exchange)
(5), Sepharose4FF column chromatography purification;
(6), adjuvant absorption is with protective agent human albumin (or not adding adjuvant absorption);
(7), dilution packing.
The human diploid cell kind that this technology is used derives from CDC, at first set up human diploid born of the same parents' seed bank and human diploid cell work storehouse, and pair cell storehouse cell carries out the calibrating of system.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, flat permanent salt culture fluid etc., wherein to use 199 culture fluid effects comparatively desirable, the Ox blood serum that preferably contains 2-10% in the cell nutrient solution, PH7.0-7.6 cultivates amplification at 37 ± 0.5 ℃.
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.
For preventing germ contamination, in the preparation cell nutrient solution, can add an amount of kanamycin and gentamycin.
In the incubation, cell divides kind of rate to determine according to the needs of production lot, generally can be 1: 2～1: 4 after cell grows up to fine and close monolayer, can inoculate rabies virus, the human albumin who preferably adds 0.4～0.5% (W/W) in the cell maintenance medium, adjust PH7.2～7.8 simultaneously, 32～37 ℃ of cultivation temperature, and can add an amount of aminoacid and antibiotic, operational culture fluid comprises 199 liquid, flat permanent saline solution etc., seeded process comprises earlier with flushing cell surfaces such as culture fluid such as EarleShi liquid, remove residual Ox blood serum, growing into inoculation rabies virus seed culture of viruses MOL0.1～0.01 and above-mentioned cell maintenance medium on the human diploid cell of fine and close monolayer, cultivated about 72 hours then, can gather in the crops virus.
The human diploid rabies seed culture of viruses of the rabies virus that this technology is inoculated on the human diploid cell for going down to posterity, experimental result shows, rabies virus growth and breeding well on the human diploid cell, go down to posterity 10 generations of rabies virus with interior very stable at the human diploid cell, and preparation human diploid cell rabies vaccine aspect, in 10 generations, are with the not obviously change of immunogenicity of interior passage seed culture of viruses, the immunogenicity of the seed culture of viruses after 10 generations then significantly decreases, 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 purified vaccine.As for the go down to posterity seed culture of viruses of the then preferred mad dog seed culture of viruses SNK-CTN of seed culture of viruses strain at the human diploid cell.
The growth and breeding of rabies virus on the human diploid cell can be kept the long period, therefore the results of virus can be taked the mode repeatedly collected, preferably the 3rd～4 day results once, to results viral liquid in time measure virus titer, require every batch gather in the crops viral liquid virus titer all should be 10
5.0More than the LD50/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 beta-propiolactone inactivation of viruses is thick vaccine.
Thick vaccine after the deactivation need concentrate to purify and be prepared into pure vaccine product, this technology 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 20 times, then purified vaccine.
The key for preparing the biological product safety with the human diploid cell is the content of remaining Ox blood serum residual quantity.By the rules requirement of 2005 editions relevant biological product of Chinese Pharmacopoeia, the Ox blood serum residual quantity of vaccine must be less than 50ng/ml.To comprise that chemical method and physical method etc. can have multiple about the purification process of vaccine, and through repetition test and research, this technology proposes purification process: ultrafiltration purification, super from purification and Sepharose4FF column chromatography purification.Adopt serum-free medium to cultivate in addition and can directly get rid of the influence of serum goods.
The ultrafiltration purification vaccine promptly is concentrated to certain multiple to ultrafiltration of vaccine, add an amount of PBS flushing then, reconcentration is to former multiple, add an amount of PBS flushing 5～6 times so repeatedly again, the Ox blood serum residual quantity can be removed more than 93%, again through the method for sucrose density gradient centrifugation with reference to the method purification human diploid cell Vaccinum Encephalitidis Epidemicae of the intelligent extensive band centrifugation purification of delivering in clever 1998 of stone, sucrose density gradient with 36% and 45% (W/W), 23000rpm ultracentrifugation 4 hours, through antigenic content to ultraviolet absorption peak, the position at viral place has been determined in the analysis of protein concentration.And then through the Sepharose4FF purification, 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 rabies 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 Sepharose4FF post or other gels, and testing result shows, behind three step chromatographic column purification, the vaccine total protein content reduces about more than 99%, and the residual volume of Ox blood serum all meets the relevant rules requirement of Pharmacopoeia of People's Republic of China, and lower than the desired content of rules.Purified vaccine is made finished product (being the liquid drugs injection dosage form), adds protective agent human albumin and aluminum hydroxide adjuvant.With the production substrate of human diploid cell, and replace existing hamster kidney cell and Vero cell seed culture of viruses, prepare high-quality rabies vaccine by corresponding process production with the seed culture of viruses that goes down to posterity of human diploid cell as vaccine.With not containing exogenous factor in the purified rabies vaccine of this prepared, the purity height, immune effect improves greatly, and is safe to use, and can realize the large-scale industrial production of rabies vaccine.
This technology is compared from hamster kidney cell or Vero cell vaccine with present use, topmost advantage is as follows: (1) human diploid 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 Vero cell of existing vaccine; (2) seed culture of viruses of human diploid cell rabies vaccine is the seed culture of viruses that the human diploid cell is cultivated, and has fundamentally upgraded ground Ren Mus purification vaccine and Vero cell vaccine; (3) produce vaccine technology with the human diploid cell, be applicable to suitability for industrialized production homogenizing vaccine in batches, this is that existing hamster kidney cell vaccine and Vero cell vaccine is out of the question, (4) vaccine of this prepared antigen active after purifying obviously improves, foreign protein reduces more than 99%, and the purity of vaccine improves greatly.
Description of drawings:
Fig. 1 is the block flow diagram of preparation method of the present invention.
The specific embodiment:
Further specify the present invention by the following examples.
Embodiment 1
Diploid cell is from CDC, cell nutrient solution is to contain 2-10% Ox blood serum and 25IU/ml gentamycin and 25IU/ml kanamycin in 199 culture medium, and adjustment PH to 7.2, cultivate into behind the fine and close monolayer according to kind rate amplification in 1: 2 minute with 37 ℃ of 3L rolling bottles, when increasing production lot, through about 4 days, when cell grows up to fine and close monolayer, discard cell nutrient solution, with Earl ' the s liquid flushing cell face of PH7.2, inoculation diploid cell rabies virus, the second filial generation work seed culture of viruses that uses mad dog seed culture of viruses SNK-CTN strain on diploid cell, to go down to posterity, seed culture of viruses concentration MOI0.05, cell maintenance medium is for containing 0.4-0.5% (W/W) human albumin's 199 culture fluid, 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, every 3-4 days results are once received 3 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 the viral liquid of results to reach 10
5.0More than the LD50/ml; Merge viral liquid adding beta-propiolactone (final concentration 1/4000) and place 4 ± 1 ℃, 24 hours inactivation of viruses obtain thick vaccine, are condensed into 60 times of concentrated vaccines with the ultrafilter of holding back 300,000 molecular weight.
The concentrated vaccine of ultrafiltration purification is crossed sucrose density gradient centrifugation and solvent resistant column Sepharose4FF; through three step purification, obtain purified vaccine, add human blood protein's protective agent and aluminum hydroxide adjuvant and make the purified vaccine finished product; packing, making purified rabies vaccine is liquid drugs injection dosage form 5ml specification.
The calibrating vaccine potency has reached Chinese rabies vaccine reference material and U.S.'s rabies vaccine reference material requirement, the be combined vaccine requirement of 2005 editions biological product rules of Pharmacopoeia of People's Republic of China of other every calibratings.
Embodiment 2
Diploid cell is from CDC, cell nutrient solution is to contain 2-10% Ox blood serum and 25IU/ml gentamycin and 25IU/ml kanamycin in 199 culture medium, and adjustment PH to 7.2, cultivate into behind the fine and close monolayer according to kind rate amplification in 1: 2 minute with 37 ℃ of 3L rolling bottles, when increasing production lot, through about 4 days, when cell grows up to fine and close monolayer, discard cell nutrient solution, with Earl ' the s liquid flushing cell face of PH7.2, inoculation diploid cell rabies virus, the second filial generation work seed culture of viruses that uses mad dog seed culture of viruses SNK-CTN strain on diploid cell, to go down to posterity, seed culture of viruses concentration MOI0.05, cell maintenance medium is for containing 199 culture fluid of 0.4-0.5% (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, every 3-4 days results are once received 3 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 the viral liquid of results to reach 10
5.0More than the LD50/ml; Merge viral liquid adding beta-propiolactone (final concentration 1/4000) and place 4 ± 1 ℃, 24 hours inactivation of viruses obtain thick vaccine, are condensed into 60 times of concentrated vaccines with the ultrafilter of holding back 300,000 molecular weight.
The concentrated vaccine of ultrafiltration purification is crossed sucrose density gradient centrifugation and solvent resistant column Sepharose4FF; through three step purification, obtain purified vaccine, add human blood protein's protective agent and aluminum hydroxide adjuvant and make the purified vaccine finished product; packing, making mad dog purified vaccine is liquid drugs injection dosage form 0.5ml specification.
The calibrating vaccine potency has reached Chinese rabies vaccine reference material and U.S.'s rabies vaccine reference material requirement, the be combined vaccine requirement of 2005 editions biological product rules of Pharmacopoeia of People's Republic of China of other every calibratings.
Embodiment 3
With the mad dog inactivated vaccine of embodiment 1 method preparation, do not inoculate purified rabies vaccine after 14 days to inoculating the rabies vaccine person, neutralizing antibody sun rate of rotation is higher than 95.0%, and does not have the side reaction generation.
Claims (10)
1, a kind of human diploid cell purified rabies vaccine is characterized in that, this vaccine is an inoculation SNK-CTN seed culture of viruses on the human diploid cell, the rabies vaccine that obtains through separation and purification.
2, the purified vaccine of claim 1 is characterized in that, described rabies virus seed culture of viruses is selected from mad dog seed culture of viruses CTN strain or aG strain, and described human diploid cell is WI-38, CCC-HPF-1P5, MRC-5.
3, the purified vaccine of claim 1 is characterized in that, is to freeze in injection or aqueous injection.
4, the preparation method of the purified vaccine of claim 1 is characterized in that, may further comprise the steps:
(1), the recovery of human diploid cell;
(2), the cultivation of human diploid cell amplification;
(3), inoculation rabies virus seed culture of viruses on the human diploid cell;
(4), gather in the crops viral liquid;
(5), the deactivation of viral liquid;
(6), purification.
5, preparation method according to claim 4 is characterized in that, described purification may further comprise the steps:
(1), makes thick vaccine behind the inactivation of viruses;
(2), clarification filtration;
(3), ultrafiltration purification;
(4), sucrose density gradient centrifugation or employing DEAE-SepharoseFF anion exchange;
(5), Sepharose4FF column chromatography purification;
(6), adjuvant absorption, with the protective agent human albumin.
6, preparation method according to claim 4 is characterized in that, the cultivation amplification of described human diploid cell is carried out in cell nutrient solution, and cell nutrient solution is formulated by 199 culture fluid adding 2-10% Ox blood serum.
7, preparation method according to claim 6 is characterized in that, also adds an amount of kanamycin and gentamycin in the cell culture fluid.
8, preparation method according to claim 4 is characterized in that, the cultivation amplification of described human diploid cell is to cultivate in rolling bottle.
9, preparation method according to claim 4 is characterized in that, also comprises the adjuvant adsorption step or/and with protective agent human albumin's step.
10, preparation method according to claim 9 is characterized in that, it is 0.2--0.5% that the adding human albumin makes its concentration.
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| CN101716341B (en) * | 2009-12-14 | 2012-03-21 | 成都康华生物制品有限公司 | Human diploid cell inactivated rabies vaccine and preparation method thereof |
| CN103052399B (en) | 2010-08-12 | 2016-06-29 | 依生生物制药控股有限公司 | For reducing the method for DNA impurity in virus composition |
| CN104357406A (en) * | 2014-09-30 | 2015-02-18 | 施耐克江苏生物制药有限公司 | Rabies virus SNK-CTN strain and application thereof |
| CN104353068A (en) * | 2014-11-18 | 2015-02-18 | 成都康华生物制品有限公司 | Large-scale production method of rabies vaccines by using human diploid cells |
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| CN102671192A (en) * | 2012-05-07 | 2012-09-19 | 成都康华生物制品有限公司 | Human diploid cell rabies vaccine and preparation method thereof |
| CN102671192B (en) * | 2012-05-07 | 2013-10-30 | 成都康华生物制品有限公司 | Human diploid cell rabies vaccine and preparation method thereof |
| CN106834237A (en) * | 2016-12-26 | 2017-06-13 | 成都康华生物制品有限公司 | A kind of technique of human diploid cell culture rabies viruses |
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| CN1712068A (en) | 2005-12-28 |
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