CN1586622A - Diploid cell cerebritis B vaccine and purified cerebritis B vaccine, dosage form freeze-drying and water injection - Google Patents
Diploid cell cerebritis B vaccine and purified cerebritis B vaccine, dosage form freeze-drying and water injection Download PDFInfo
- Publication number
- CN1586622A CN1586622A CN 200410058112 CN200410058112A CN1586622A CN 1586622 A CN1586622 A CN 1586622A CN 200410058112 CN200410058112 CN 200410058112 CN 200410058112 A CN200410058112 A CN 200410058112A CN 1586622 A CN1586622 A CN 1586622A
- Authority
- CN
- China
- Prior art keywords
- vaccine
- cell
- encephalitis
- preparation
- diploid cell
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 229960005486 vaccine Drugs 0.000 title claims abstract description 102
- 206010014599 encephalitis Diseases 0.000 title claims abstract description 51
- 210000001840 diploid cell Anatomy 0.000 title claims abstract description 45
- 238000002347 injection Methods 0.000 title claims abstract description 16
- 239000007924 injection Substances 0.000 title claims abstract description 16
- 201000008191 cerebritis Diseases 0.000 title abstract 6
- 239000002552 dosage form Substances 0.000 title description 5
- 238000004108 freeze drying Methods 0.000 title 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 title 1
- 239000007788 liquid Substances 0.000 claims abstract description 27
- 238000002360 preparation method Methods 0.000 claims abstract description 23
- 241000700605 Viruses Species 0.000 claims description 75
- 238000000746 purification Methods 0.000 claims description 37
- 210000004027 cell Anatomy 0.000 claims description 35
- 238000011218 seed culture Methods 0.000 claims description 31
- 210000004556 brain Anatomy 0.000 claims description 18
- 230000003612 virological effect Effects 0.000 claims description 17
- 238000011081 inoculation Methods 0.000 claims description 15
- 210000002966 serum Anatomy 0.000 claims description 14
- 239000000243 solution Substances 0.000 claims description 14
- 235000015097 nutrients Nutrition 0.000 claims description 13
- 238000000108 ultra-filtration Methods 0.000 claims description 12
- 230000003321 amplification Effects 0.000 claims description 11
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 11
- 239000002671 adjuvant Substances 0.000 claims description 9
- 239000012531 culture fluid Substances 0.000 claims description 9
- 102000008100 Human Serum Albumin Human genes 0.000 claims description 8
- 108091006905 Human Serum Albumin Proteins 0.000 claims description 8
- 239000003223 protective agent Substances 0.000 claims description 6
- 102000004169 proteins and genes Human genes 0.000 claims description 6
- 108090000623 proteins and genes Proteins 0.000 claims description 6
- 229930182566 Gentamicin Natural products 0.000 claims description 5
- CEAZRRDELHUEMR-URQXQFDESA-N Gentamicin Chemical compound O1[C@H](C(C)NC)CC[C@@H](N)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](NC)[C@@](C)(O)CO2)O)[C@H](N)C[C@@H]1N CEAZRRDELHUEMR-URQXQFDESA-N 0.000 claims description 5
- 229930027917 kanamycin Natural products 0.000 claims description 5
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 claims description 5
- 229960000318 kanamycin Drugs 0.000 claims description 5
- 229930182823 kanamycin A Natural products 0.000 claims description 5
- 238000012856 packing Methods 0.000 claims description 5
- 238000005096 rolling process Methods 0.000 claims description 5
- 238000011084 recovery Methods 0.000 claims description 4
- 238000005199 ultracentrifugation Methods 0.000 claims description 4
- 238000005349 anion exchange Methods 0.000 claims description 3
- 238000005352 clarification Methods 0.000 claims description 3
- 230000009849 deactivation Effects 0.000 claims description 3
- 238000010790 dilution Methods 0.000 claims description 3
- 239000012895 dilution Substances 0.000 claims description 3
- 238000001641 gel filtration chromatography Methods 0.000 claims description 3
- 238000000926 separation method Methods 0.000 claims description 3
- 210000001082 somatic cell Anatomy 0.000 claims description 3
- 238000001179 sorption measurement Methods 0.000 claims description 2
- 241000701076 Macacine alphaherpesvirus 1 Species 0.000 abstract 1
- 239000000843 powder Substances 0.000 abstract 1
- 206010014596 Encephalitis Japanese B Diseases 0.000 description 15
- 201000005807 Japanese encephalitis Diseases 0.000 description 15
- 241000710842 Japanese encephalitis virus Species 0.000 description 15
- 238000005516 engineering process Methods 0.000 description 13
- 238000004519 manufacturing process Methods 0.000 description 12
- 238000000034 method Methods 0.000 description 11
- 241000699666 Mus <mouse, genus> Species 0.000 description 9
- 238000012360 testing method Methods 0.000 description 7
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 6
- 229920002684 Sepharose Polymers 0.000 description 6
- 229930006000 Sucrose Natural products 0.000 description 6
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 6
- 230000000694 effects Effects 0.000 description 6
- 229940031551 inactivated vaccine Drugs 0.000 description 6
- 238000012423 maintenance Methods 0.000 description 6
- 239000002609 medium Substances 0.000 description 6
- 239000002356 single layer Substances 0.000 description 6
- 239000005720 sucrose Substances 0.000 description 6
- 241000699800 Cricetinae Species 0.000 description 5
- 241001529936 Murinae Species 0.000 description 5
- 238000000432 density-gradient centrifugation Methods 0.000 description 5
- 238000011010 flushing procedure Methods 0.000 description 5
- 210000003292 kidney cell Anatomy 0.000 description 5
- 235000018102 proteins Nutrition 0.000 description 5
- 210000003501 vero cell Anatomy 0.000 description 5
- 241000699670 Mus sp. Species 0.000 description 4
- WNROFYMDJYEPJX-UHFFFAOYSA-K aluminium hydroxide Chemical compound [OH-].[OH-].[OH-].[Al+3] WNROFYMDJYEPJX-UHFFFAOYSA-K 0.000 description 4
- 230000002490 cerebral effect Effects 0.000 description 4
- 229940079593 drug Drugs 0.000 description 4
- 239000003814 drug Substances 0.000 description 4
- 239000001963 growth medium Substances 0.000 description 4
- 230000002779 inactivation Effects 0.000 description 4
- 239000012925 reference material Substances 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 239000000758 substrate Substances 0.000 description 4
- 206010002198 Anaphylactic reaction Diseases 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 3
- 238000010521 absorption reaction Methods 0.000 description 3
- 208000003455 anaphylaxis Diseases 0.000 description 3
- 230000005875 antibody response Effects 0.000 description 3
- 238000009395 breeding Methods 0.000 description 3
- 230000001488 breeding effect Effects 0.000 description 3
- 238000004113 cell culture Methods 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 230000005847 immunogenicity Effects 0.000 description 3
- 230000003472 neutralizing effect Effects 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 238000007086 side reaction Methods 0.000 description 3
- 239000002904 solvent Substances 0.000 description 3
- 102000004506 Blood Proteins Human genes 0.000 description 2
- 108010017384 Blood Proteins Proteins 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 2
- 230000000890 antigenic effect Effects 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 238000004440 column chromatography Methods 0.000 description 2
- 239000012141 concentrate Substances 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000005227 gel permeation chromatography Methods 0.000 description 2
- 230000001900 immune effect Effects 0.000 description 2
- 238000009776 industrial production Methods 0.000 description 2
- 230000036512 infertility Effects 0.000 description 2
- 238000011835 investigation Methods 0.000 description 2
- 206010025482 malaise Diseases 0.000 description 2
- 239000011159 matrix material Substances 0.000 description 2
- 238000004448 titration Methods 0.000 description 2
- 238000012549 training Methods 0.000 description 2
- 201000005702 Pertussis Diseases 0.000 description 1
- 241000227425 Pieris rapae crucivora Species 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000036783 anaphylactic response Effects 0.000 description 1
- 210000004102 animal cell Anatomy 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 230000002238 attenuated effect Effects 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 229940030156 cell vaccine Drugs 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000001804 emulsifying effect Effects 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 229940124724 hepatitis-A vaccine Drugs 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000008676 import Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 210000004962 mammalian cell Anatomy 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000003340 mental effect Effects 0.000 description 1
- 238000000053 physical method Methods 0.000 description 1
- 229960001539 poliomyelitis vaccine Drugs 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000002787 reinforcement Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 239000004575 stone Substances 0.000 description 1
- 238000005728 strengthening Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000002255 vaccination Methods 0.000 description 1
Images
Landscapes
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The present invention relates to cerebritis B vaccine of diploid cell and purified cerebritis B vaccine in the preparation form of freeze dried powder for injection and injection liquid. The purified cerebritis B vaccine is obtained through inoculating cerebritis B virus strain onto diploid cell for human body.
Description
Technical field:
The present invention relates to a kind of purified vaccine of encephalitis B, particularly the Japanese encephalitis purified vaccine that inoculation encephalitis b virus seed culture of viruses obtains on the human diploid cell.
Background technology:
Mass-produced in the world at present Vaccinum Encephalitidis Epidemicae has three kinds, first kind 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 second kind of former generation hamster kidney cell that is China produces cultivated and former generation ground Ren Mus Live Japanese Encephalitis, the cellular matrix of these two kinds of vaccines all derives from the regular grade animal, can't guarantee not carry exogenous factor; The third is that Beijing Biological Product Inst. is the lyophilizing encephalitis b purification inactivated vaccine that culture matrix is produced with the Vero cell, but can not guarantee the cytostromatic DNA that causes tumor and cell.
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 one one white mice, the results of infected mice brain one by one, and 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 sun rate (〉=1: be 77% 8) after the injection of Mus brain purification Japanese encephalitis inactivated vaccine two pins, positive rate of rotation (1: 16) is 100% behind 6-12 month reinforcement one pin, 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 the capable district of school age population base flow, non-popular district, strengthen reaching about 90% behind the pin, and the side reaction after the vaccine injection mainly is anaphylaxis, though general smallerly still can accept, but Tong Ji result according to investigations, in Beijing, 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 encephalitis b attenuated live vaccine, 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 live 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 effect thereof etc. are all than trouble, and it gives birth to the pollution that technology also is unfavorable for controlling exogenous factor, more is not suitable for large-scale industrial production.
In addition, also some progress of China's vaccine in recent years, Vero cell encephalitis B purified vaccine is also come out one after another, but Vero cell system tumor and DNA can not well remove, and causes the safety and the immunogenicity of Vero cell encephalitis B purified vaccine undesirable.
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; the expressing protein majority is an E albumen; PrM and NSI; all satisfied through testing calibrating and inducing antibody response; but through the protection test of virus attack all not as good as the ideal of whole virus vaccine. at present; domestic the development with diploid studied various vaccines; the diploid poliomyelitis vaccine is wherein arranged; hepatitis A vaccine etc. exempt from effective, better than the vaccine effect of other substrate, and safer after the inoculation.
This vaccine is a U.S. Wistar institute initiative, and personnel selection diploid WI-38 strain adapts to and cultivates virus, and later French Merierx institute is produced vaccine with the MRC-5 human diploid cell.Vaccine confirms inoculation back light side-reaction after the human vaccination of commensurate does not observe, good immune effect can subtract the pin injection, thinks the standard control vaccine that can be used as a kind of new development vaccine at present.But preparing Vaccinum Encephalitis B with this method does not appear in the newspapers.
Because the particularity and the complexity of Vaccinum Encephalitis B prepare this vaccine and have suitable difficulty, particularly aspect separation and purification.
The present invention has developed human diploid cell-WJ-38 and has studied Japanese encephalitis purified vaccine after deliberation.
Summary of the invention:
The invention provides a kind of human diploid cell encephalitis b purified vaccine, this vaccine is the Japanese encephalitis purified vaccine that inoculation encephalitis b virus seed culture of viruses obtains on the human diploid cell.
The present invention also provides the preparation method and the application thereof of encephalitis b purified vaccine of the present invention.
The present invention adopts human diploid cell-WJ-38 to prepare Japanese encephalitis purified vaccine, through comprehensively research evaluation, it is stable that WI-38 cell has karyogy, there be not exogenous factor pollution and the oncogenicity advantage responsive to virus, meet 2000 editions rules requirements of Chinese Pharmacopoeia fully, and be can be used as the substrate of production of vaccine by the WHO approval about the passage cell of biological product.
The invention provides a kind of human diploid somatic cell encephalitis B purified vaccine, this vaccine is an inoculation encephalitis b virus seed culture of viruses on the human diploid cell, the Vaccinum Encephalitis B that obtains through separation and purification.Described encephalitis b virus seed culture of viruses is selected from encephalitis b Mus brain seed culture of viruses P
3Strain or AS14-14-2 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 encephalitis b 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.Also can add an amount of kanamycin and gentamycin in case of necessity in the cell nutrient solution.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.
In the preparation technology of encephalitis b purified 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 Sepharose 4FF 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 2000 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.
The human diploid Japanese encephalitis purified vaccine that this technology provides is meant the Japanese encephalitis purified vaccine that inoculation encephalitis b virus seed culture of viruses obtains on the human diploid cell.The preparation method that this technology is concrete may further comprise the steps:
(1), the recovery of human diploid cell;
(2) cultivation of human diploid cell amplification;
(3), inoculation encephalitis b virus seed culture of viruses on the human diploid cell;
(4), results virus;
(5), inactivation of viruses is made thick vaccine;
(6) clarification filtration;
(7), ultrafiltration purification;
(8), sucrose density gradient centrifugation; (or DEAE-SepharoseFF anion exchange)
(9), Sepharose4FF column chromatography purification;
(10), adjuvant absorption is with protective agent human albumin (or not adding adjuvant absorption)
(11), dilution packing.
The human diploid cell kind that this technology is used derives from CDC, at first set up thin human diploid cell 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, preferably contain the Ox blood serum of 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 encephalitis b virus, the human albumin who preferably adds 0.4-0.55% (w/w) in the cell maintenance medium, adjust PH7.2-7.8 simultaneously, cultivation temperature 32-37 ℃, and can add an amount of aminoacid and antibiotic, operational training liquid 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 encephalitis b virus seed culture of viruses MOL0.1-0.00001 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 encephalitis b virus kind of the encephalitis b virus that this technology is inoculated on the human diploid cell for going down to posterity, there is experimental result to show, encephalitis b virus growth and breeding well on the human diploid cell, go down to posterity 10 generations of encephalitis b virus with interior very stable at the human diploid cell, and preparation human diploid cell Vaccinum Encephalitidis Epidemicae 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 encephalitis b sodoku kind P3 strain of seed culture of viruses at the human diploid cell.
The growth and breeding of encephalitis b 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 3-4 days 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
7More 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 formalin solution 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 2000 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 Sepharose 4FF column chromatography purification.
The ultrafiltration purification vaccine promptly is concentrated to certain multiple to ultrafiltration of vaccine, add an amount of PBS flushing then, be reduced to former multiple more deeply, add an amount of PBS flushing 5-6 time 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 Vero 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 Sepharose 4FF 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 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 4FF 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 white egg of protective agent human blood and aluminum hydroxide adjuvant.With the production substrate of human diploid 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 human diploid cell as vaccine.With not containing murine brain in the Japanese encephalitis purified 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 Vaccinum Encephalitidis Epidemicae.
This technology is compared with the Vaccinum Encephalitidis Epidemicae from murine brain virus of 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 murine brain of existing vaccine; (2) seed culture of viruses of human diploid cell Vaccinum Encephalitidis Epidemicae is the seed culture of viruses of cell culture, has fundamentally upgraded the murine brain that Mus brain purification vaccine and ground Ren Mus Vaccinum Encephalitidis Epidemicae use; (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 Mus brain purification 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, use 38-50 generation, 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 the Earl ＇ s liquid flushing cell face of PH7.2, inoculation diploid 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 diploid cell
3-2), 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 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
7More than the LD50/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.
The concentrated vaccine of ultrafiltration purification is crossed sucrose density gradient centrifugation and solvent resistant column Sepharose 4FF; 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 Japanese encephalitis purified vaccine is liquid drugs injection dosage form 5ml specification.
The calibrating vaccine potency has reached Chinese Vaccinum Encephalitidis Epidemicae reference material and the requirement of Japanese Vaccinum Encephalitidis Epidemicae reference material, the be combined vaccine requirement of 2000 editions biological product rules of Pharmacopoeia of People's Republic of China standard of other every calibratings.
Embodiment 2
Diploid cell is from CDC, use 38-50 generation, 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 the Earl ＇ s liquid flushing cell face of PH7.2, inoculation diploid cell encephalitis b virus, the second filial generation work seed culture of viruses (P that uses encephalitis b Mus brain seed culture of viruses AS14-14-2 strain on diploid cell, to go down to posterity
3-2), 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 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
7More than the LD50/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.
The concentrated vaccine of ultrafiltration purification is crossed sucrose density gradient centrifugation and solvent resistant column Sepharose 4FF; 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 Japanese encephalitis purified vaccine is liquid drugs injection dosage form 2ml specification.
The calibrating vaccine potency has reached Chinese Vaccinum Encephalitidis Epidemicae reference material and the requirement of Japanese Vaccinum Encephalitidis Epidemicae reference material, the be combined vaccine requirement of 2000 editions biological product rules of Pharmacopoeia of People's Republic of China standard of other every calibratings.
Embodiment 3
(comprise injection, do not inoculate the Vaccinum Encephalitidis Epidemicae person, inoculate 2 pin Japanese encephalitis inactivated vaccines, two pins 7-10 days at interval through experiment with the Japanese encephalitis inactivated vaccine of claim 1 method preparation to 12 4-6 year child.) proving that neutralizing antibody sun rate of rotation is 91.7%, no side reaction takes place.
Claims (10)
1, a kind of human diploid somatic cell encephalitis B purified vaccine is characterized in that, this vaccine is an inoculation encephalitis b virus seed culture of viruses on the human diploid cell, the Vaccinum Encephalitis B that obtains through separation and purification.
2, the purified vaccine of claim 1 is characterized in that, described encephalitis b virus seed culture of viruses is selected from seed culture of viruses P3 strain of encephalitis b Mus brain or AS14-14-2 strain.
3, the purified vaccine of claim 1 is characterized in that, is freeze dried 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 encephalitis b 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.
5, preparation method according to claim 4, it is characterized in that, described purification 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, pass through Sepharose4FF post or other gel filtration chromatographies then again.
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 nutrient solution.
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 3 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.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2004100581129A CN1299768C (en) | 2004-08-13 | 2004-08-13 | Diploid cell cerebritis B vaccine and purified cerebritis B vaccine, dosage form freeze-drying and water injection |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2004100581129A CN1299768C (en) | 2004-08-13 | 2004-08-13 | Diploid cell cerebritis B vaccine and purified cerebritis B vaccine, dosage form freeze-drying and water injection |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1586622A true CN1586622A (en) | 2005-03-02 |
| CN1299768C CN1299768C (en) | 2007-02-14 |
Family
ID=34603321
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB2004100581129A Expired - Lifetime CN1299768C (en) | 2004-08-13 | 2004-08-13 | Diploid cell cerebritis B vaccine and purified cerebritis B vaccine, dosage form freeze-drying and water injection |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1299768C (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100333793C (en) * | 2005-06-28 | 2007-08-29 | 崔栋 | Diploid-cell rabies vaccine and purified rabies vaccine, freeze-drying preparation and water injection thereof |
| CN101352569B (en) * | 2007-07-27 | 2011-07-27 | 崔栋 | Diploid somatic cell encephalitis B vaccine and method for preparing purified encephalitis B vaccine |
| CN102406927A (en) * | 2011-11-14 | 2012-04-11 | 成都康华生物制品有限公司 | Production method of human diploid cell encephalitis B inactivated vaccine |
| CN101524536B (en) * | 2009-03-26 | 2012-10-10 | 成都康华生物制品有限公司 | Japanese encephalitis vaccine prepared by human embryonic lung fibroblasts and preparation method thereof |
| CN114306587A (en) * | 2021-12-22 | 2022-04-12 | 辽宁成大生物股份有限公司 | Preparation method of low-serum Japanese encephalitis inactivated vaccine and Japanese encephalitis inactivated vaccine |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DK0604566T3 (en) * | 1991-09-19 | 2000-04-10 | Us Health | Chimeric and / or growth restricted flaviviruses |
| CN1029239C (en) * | 1992-12-29 | 1995-07-05 | 卫生部长春生物制品研究所 | method for producing hepatitis A vaccine |
| CN1248471A (en) * | 1998-09-23 | 2000-03-29 | 卫生部北京生物制品研究所 | Vero cell encephalitis B inactivated vaccine and preparation process thereof |
| CN100528227C (en) * | 2004-05-14 | 2009-08-19 | 上海荣盛生物药业有限公司 | Vaccine for virus of encephalitis B and preparation method |
-
2004
- 2004-08-13 CN CNB2004100581129A patent/CN1299768C/en not_active Expired - Lifetime
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100333793C (en) * | 2005-06-28 | 2007-08-29 | 崔栋 | Diploid-cell rabies vaccine and purified rabies vaccine, freeze-drying preparation and water injection thereof |
| CN101352569B (en) * | 2007-07-27 | 2011-07-27 | 崔栋 | Diploid somatic cell encephalitis B vaccine and method for preparing purified encephalitis B vaccine |
| CN101524536B (en) * | 2009-03-26 | 2012-10-10 | 成都康华生物制品有限公司 | Japanese encephalitis vaccine prepared by human embryonic lung fibroblasts and preparation method thereof |
| CN102406927A (en) * | 2011-11-14 | 2012-04-11 | 成都康华生物制品有限公司 | Production method of human diploid cell encephalitis B inactivated vaccine |
| CN114306587A (en) * | 2021-12-22 | 2022-04-12 | 辽宁成大生物股份有限公司 | Preparation method of low-serum Japanese encephalitis inactivated vaccine and Japanese encephalitis inactivated vaccine |
| CN114306587B (en) * | 2021-12-22 | 2023-12-29 | 辽宁成大生物股份有限公司 | Preparation method of low-serum Japanese encephalitis inactivated vaccine and Japanese encephalitis inactivated vaccine |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1299768C (en) | 2007-02-14 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101897963B (en) | Vaccine for hand-foot-and-mouth disease viruses | |
| JP5095685B2 (en) | Enhanced immunogen for inactivated vaccine against Japanese encephalitis virus group infection and method for producing the same | |
| CN101352570B (en) | Diploid cell rabies vaccine and method for preparing purified rabies vaccine | |
| CN107267466A (en) | A kind of method for mass producing swine pseudorabies vaccine | |
| CN101716341B (en) | Human diploid cell inactivated rabies vaccine and preparation method thereof | |
| CN103386126B (en) | Multivalent immunogenic composition containing enterovirus antigens | |
| CN101352569B (en) | Diploid somatic cell encephalitis B vaccine and method for preparing purified encephalitis B vaccine | |
| RU2447898C2 (en) | Ipv-dpt vaccine | |
| CN1248471A (en) | Vero cell encephalitis B inactivated vaccine and preparation process thereof | |
| CN101524536B (en) | Japanese encephalitis vaccine prepared by human embryonic lung fibroblasts and preparation method thereof | |
| CN1299768C (en) | Diploid cell cerebritis B vaccine and purified cerebritis B vaccine, dosage form freeze-drying and water injection | |
| CN102671192B (en) | Human diploid cell rabies vaccine and preparation method thereof | |
| Perez et al. | Production methods for rabies vaccine | |
| CN100333793C (en) | Diploid-cell rabies vaccine and purified rabies vaccine, freeze-drying preparation and water injection thereof | |
| KR20120027381A (en) | Japanese encephalitis vaccine and method of manufacturing the same | |
| CN106075423B (en) | Combined vaccine for preventing hand-foot-and-mouth disease | |
| JP2007068401A (en) | West nile virus vaccine | |
| CN1404874A (en) | Method for preparing thermal purifying-inactivating vaccine to bleeding of double-adicity vero cell kidney syndrome and use thereof | |
| CN102160892A (en) | Epidemic encephalitis B and enterovirus 71 (EV71) combined vaccine | |
| CN1112934C (en) | Epidemic hemorrhagic fever passage cell polyvalent purified vaccine | |
| CN1216985C (en) | Hepatitis A virus strain, method for preparing hepatitis A inactivated vaccine and obtained vaccine | |
| CN1695736A (en) | Vaccine for virus of encephalitis B and preparation method | |
| CN1297314C (en) | Method for cultivating attenuated strain pilio inactivated vaccine | |
| CN100413536C (en) | Hydrophobia split vaccine for human body | |
| CN102671194A (en) | Human vaccine for preventing hydrophobia and tetanus |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CX01 | Expiry of patent term |
Granted publication date: 20070214 |
|
| CX01 | Expiry of patent term |