CN103599530B - Method for preparation of rubella attenuated live vaccine by serum-free culture medium - Google Patents
Method for preparation of rubella attenuated live vaccine by serum-free culture medium Download PDFInfo
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- CN103599530B CN103599530B CN201310523326.8A CN201310523326A CN103599530B CN 103599530 B CN103599530 B CN 103599530B CN 201310523326 A CN201310523326 A CN 201310523326A CN 103599530 B CN103599530 B CN 103599530B
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Abstract
The invention discloses a method for preparation of a human diploid cell rubella attenuated live vaccine by a serum-free culture medium, and belongs to the field of preventive biological product. The serum-free culture medium is used in human diploid cell MRC-5 recovery, amplification and virus infection processes, the technological processes and the product are not added with antibiotics, and a traditional glass spinner bottle is replaced with a disposable cell engineering bottle. A more safe rubella virus RA27/3 attenuated strain is adopted, the strain loses pathogenicity to human bodies and has good immunity in 25th-33th generation, and the vaccine safety is greatly increased. A lyophilized agent adopts a formula without gelatin, and thus the shortcoming that the gelatin has uncertain pathogenicity on the human bodies due to complex composition is fundamentally solved.
Description
Technical field
The present invention relates to a kind of method that serum-free medium prepares attenuated rubella live vaccine, on human diploid cell MRC-5, particularly inoculate the preparation method of the rubella vaccine that rubella virus RA27/3 attenuated strain obtains.
Background technology
The vaccine of current domestic listing has been Ox blood serum and has cultivated.As everyone knows, the production process of Ox blood serum is difficult to the pollution of other pathogenic microorganisms avoiding crazy heifer disease virus, mycoplasma and cattle susceptible; Production of vaccine technique is utilized to remove serum completely and possible pollution is impossible; Therefore State Food and Drug Administration and even WHO avoid using animal derived raw material advocating in vaccine production process.Exploitation serum-free production of vaccine technique, meets national policy requirement, meets global biological product development trend, meet the healthy and safe requirement of the people.Cell cultivation process of the present invention does not use the culture fluid containing serum, avoids the pollution caused because adding serum.
The present invention uses the attenuated rubella live vaccine production seed culture of viruses RA27/3 licensed by U.S.'s WISTAR institute.This seed culture of viruses is separated by WISTAR institute, and by continuous passage attenuation in WI-38 cell, clinical trial has been carried out in 25-33 generation, prove that 25-33 generation virus loses pathogenicity to human body, but immunogenicity is good, body can be stimulated to produce the immunity of enough rubella virus infection, and Institute Pasteur and Merck company of the U.S. all develop attenuated live vaccine with this seed culture of viruses afterwards.Use nearly many decades safely.
Gelatin is a kind of protective agent and excipient that vaccine for man is conventional, and derive from animal, its production process cannot control virulent pollution completely, and gelatin is relevant with anaphylaxis after vaccination.WHO advises global production of vaccine enterprise on repeatedly production of vaccine associated guideline, adopts gelatine replacement as the freeze-dried excipient of vaccine as far as possible.Gelatin has thoroughly been broken away from, convenient sources in vaccine excipients of the present invention, quality controllable.Outward appearance and dissolution time meet existing Chinese Pharmacopoeia requirement, and stablizing effect meets vaccine quality requirement.
The rubella positive rate of China's Women of childbearing age is up to 80-95%, infection of pregnant women rubella may cause infants with congenital rubella syndrome, comprises cataract, deafness, congenital heart disease, mental retardation, pneumonia, hepatitis, thrombocytopenia, diabetes, thyroiditis etc.American-European wait developed country namely to come into effect Rubella vaccine immunization the eighties to plan, infant, the women of child-bearing age, adult women, new recruit, medical personnel all carry out compulsory immunization, and therefore, these countries are Eradication rubella.China's rubella immunological comparison is weak, whole nation manufacturer adds up to annual 3000 ten thousand doses of production capacity, and only neonatal immunity just needs 3,200 ten thousand every year, and this external demand immunity crowd also has the women of child-bearing age, new recruit and medical personnel, obviously, production capacity also far can not meet the basic immune needs of China.
Summary of the invention
The invention provides a kind of method that serum-free medium prepares human diploid cell attenuated rubella live vaccine.
The present invention adopts cell to be human diploid cell MRC-5, and namely with this cell inoculation rubella virus RA27/3 attenuated strain, results virus liquid, after adding viral protective agent and excipient, subpackage lyophilizing, makes vaccine.Vaccine of the present invention is freeze dried injection.
Of the present invention is a kind of preparation method of attenuated rubella live vaccine, realizes especially by following steps:
(1), the recovery of MRC-5 human diploid cell;
(2), the amplification of going down to posterity of MRC-5;
(3), on MRC-5 cell, rubella virus RA27/3 attenuated strain is inoculated;
(4), infection cell culture supernatant is gathered in the crops;
(5), virus liquid clarification filtration;
(6), freeze drying protectant is added;
(7), subpackage lyophilizing.
In the preparation technology of attenuated rubella live vaccine of the present invention, cultured cell culture medium used is serum-free medium, and pancreatin is non-animal proteins.If cell and Virus culture serum-free medium used can be MP-MRC-5 SFM, the proteins and peptides of this culture medium containing animal origin, the present invention is suitable for other serum-free mediums equally.
Through calibrating, when there is pathological changes in cell, Multiple harvests virus liquid, through frozen, thaw, merge, detect titre after, obtain the rubella venom of production vaccine.The present invention uses serum-free medium to cultivate human embryonic lung diploid fibroblast and can obtain cultivating same effect with there being blood serum medium, and the rubella virus titre obtained is without significant difference (be shown in table 1), and whole process does not use antibiotic.The present invention's serum-free medium used is general serum-free medium.
Table 1, rubella virus has serum free culture system to compare (logCCID with the titre of serum-free culture
50/ ml)
| Kinds of culture medium | 1st results | 2nd results | 3rd results | The 4th is gathered in the crops |
| There is serum free culture system | 5.64 | 6.24 | 5.74 | 5.45 |
| Serum-free culture | 6.00 | 6.00 | 6.48 | 5.21 |
Described human diploid cell MRC-5 is from Britain's biological reference standard and quality controling research institute (National Institute for Biological Standards and Control, NIBSC), first three grades of cell banks are set up: master cell bank, master cell bank and working cell storehouse, and comprehensive calibrating has been carried out to cell bank, cell bank examines institute's qualification test report in obtaining.
Proteins and peptides not containing animal origin in the culture medium used in the amplification procedure that goes down to posterity of cell, whole process carries out adaptation domestication without the need to progressively reducing serum content.Peptic cell uses the trypsin in non-animal proteins source, has digested the pancreatin inhibitor of rear interpolation 0.01%-0.1%, by 232g-334g centrifugal segregation pancreatin inhibitor.In cell cultivation process, passage divides kind of rate to be generally 1:2-1:4.When Growth of Cells is to after fine and close monolayer, can inoculate rubella virus RA27/3 attenuated strain, now pH maintains about 7.2, condition of culture be 5%-10% carbon dioxide, 37 DEG C.
The present invention sets up viral main seed lot and work storehouse seed lot, after antibacterial, mycete, mycoplasma inspection and exogenous factor passed examination, prepares for vaccine.
The generation of Working viral seed lot was no more than for 29 generations.Virus inoculation human diploid cell, condition of culture be 5%-10% carbon dioxide, 37 DEG C.Carry out Multiple harvests virus liquid according to cytopathogenetic degree after cell infection virus, generally within 2,4,6,8,10,12 days, gather in the crops after infection.Frozen below-20 DEG C immediately after virus harvest.If cytopathy is serious, can clarification filtration be carried out, then frozen.The virus liquid of results uses the membrane filtration clarification of 0.45/1.0 micron.Gather in the crops virus liquid titre after measured and be not less than 6.0 logCCID
50/ ml, vaccine semi-finished product titre is not less than 4.8 logCCID
50/ ml.Cell culture does not add antibiotic with connecing in the rear culture fluid of poison.
The virus titration method that the present invention adopts is traditional Microdose cytopathic effect assay: the rubella virus of acceptable diluent degree and RK-13 cell miscegenation are cultivated in 96 orifice plates, plants the rear 10th day observation port inner cell pathological changes situation of poison, calculates vaccine titre with Karber method.
In the freeze-dry process that the present invention adopts, virus liquid adds mannitol by a certain percentage, dextran, sodium glutamate, carbamide, arginine hydrochloride, and people's blood cell albumin makes semi-finished product, obtains finished product through lyophilizing.Freeze-dried excipient formula is suitable for general freeze-dry process.The moisture of freeze dried vaccine is between 0.8%-1.5%.Freeze dried vaccine be placed in 37 DEG C 7 days, titre decline average out to 0.3 logCCID
50/ ml.The excipient formulation of gelatin-free still ensure that good apparent condition and good titre stability.
Above determination of water adopts and takes Xiu Shi method, uses Switzerland Wan Tongkashi coulomb meter to measure, after difference heavy method calculation sample consumption, sample is added in Ka Shi coulomb meter, speed to be electrolysed is down to 20ug/min and after numerical stability, read data, repeated measure is averaged for three times.
detailed description of the invention:
Below in conjunction with specific embodiment, the present invention is further elaborated.These embodiments are only for explaining instead of limitation of the present invention the present invention.
Embodiment 1
Diploid cell MRC-5 is from work seed bank, and 1ml/ draws 1, and 37 DEG C of water-baths are thawed, and are inoculated into T
25in Tissue Culture Flask, add 8mlMP-MRC-5 SFM serum-free medium (illustratively add 1%L-Gln with front, and 37 DEG C of preheatings), medium pH is 7.2, puts 37 DEG C, 5%CO
2cultivate in incubator.This process is without the need to carrying out the progressively domestication of serum-free culture to cell, cell culture 3 days can adherent and fine and close monolayer, does not add antibiotic.
Embodiment 2
With the Cell Stripper not containing animal proteinum
tMcell in non-enzymatic cell pyrolysis liquid digestion embodiment 1, collect the pancreatin inhibitor 0.1ml-1ml that the cell suspension digested adds 0.01%-0.2% concentration, 232g-334g centrifugal segregation supernatant is to remove pancreatin inhibitor.After cell is resuspended, suspension divides kind of an amplification in 1:2 ratio, cultivate the cell quantity that can reach production requirement for 3 days, and cell state is good.
Embodiment 3
Diploid cell MRC-5 is from work seed bank, and 1ml/ draws 1, and 37 DEG C of water-baths are thawed, and are inoculated into T
25in Tissue Culture Flask, add 8mlMP-MRC-5 SFM serum-free medium (with front adding 1%L-Gln, and 37 DEG C of preheatings), medium pH is 7.2, puts 37 DEG C, 5%CO
2cultivate in incubator.Through being cultured to cell attachment and after fine and close monolayer, using not containing the Cell Stripper of animal proteinum
tMnon-enzymatic cell pyrolysis liquid peptic cell, collect pancreatin inhibitor 0.1 ~ 1ml that the cell suspension digested adds 0.01%-0.2% concentration, then add fresh serum-free media, 232g-334g centrifugal segregation supernatant is to remove pancreatin inhibitor.After resuspended, suspension divides kind of an amplification in 1:2 ratio, cultivates the cell quantity and the state that within 3 days, reach production requirement.When cell grows up to fine and close monolayer, discard cell conditioned medium liquid, inoculation rubella virus RA27/3 attenuated strain, viral generation was 29 generations, and after inoculation, 72h renews fresh serum-free medium, 2,4,6,8,10,12 days results virus liquids after kind poison.The virus liquid of results is clarified with the membrane filtration of 0.45/1.0 micron.
Embodiment 4
Virus liquid after filtering in Example 3; add 2%(W/W) mannitol; 3%(W/W) dextran; 2%(W/W) sodium glutamate; 1%(W/W) carbamide, 0.2%(W/W) arginine hydrochloride, 0.2%(W/W) the protective agent composition such as people's blood cell albumin; make lyophilizing semi-finished product, these semi-finished product are not containing gelatin or gelatin.Divide to be filled in cillin bottle and carry out lyophilizing, every cillin bottle subpackage 0.5ml, carry out lyophilizing through following program:
Pre-freeze :-45 DEG C, 2h;
Baking temperature :-45 DEG C ~ 30 DEG C;
Dry run: maintain-45 DEG C, 0.02 Mpa 4h, then rising per hour 2 DEG C.
By above-mentioned condition lyophilizing three batches, vaccine after lyophilizing, 0.5ml sterilized water for injection of often drawing redissolves, the rubella virus of acceptable diluent degree and RK-13 cell miscegenation are cultivated in 96 orifice plates, plant the rear 10th day observation port inner cell pathological changes situation of poison, calculate vaccine titre with Karber method, measure and tire in table 2.
Table 2, semi-finished product and finished product potency ratio comparatively (logCCID before and after lyophilizing
50/ ml)
Do not use gelatin in lyophilizing formula, but finished appearance state and solubility good.Determination of water adopts and takes Xiu Shi method, Switzerland Wan Tongkashi coulomb meter is used to measure, after difference heavy method calculation sample consumption, sample is added in Ka Shi coulomb meter, speed to be electrolysed is down to 20ug/min and after numerical stability, read data, repeated measure is averaged for three times, and the moisture of freeze dried vaccine is between 0.8%-1.5%.Freeze dried vaccine be placed in 37 DEG C 7 days, titre decline average out to 0.3 logCCID
50/ ml.Titre meets the requirement of 2010 editions pharmacopeia.
Claims (1)
1. prepare a method for attenuated rubella live vaccine with MP-MRC-5 serum-free medium, comprising: the recovery of (1), MRC-5 human diploid cell, (2), the amplification of going down to posterity of MRC-5, (3) on MRC-5, inoculate rubella virus RA27/3 attenuated strain, (4), infection cell culture supernatant is gathered in the crops, (5), virus liquid clarification filtration, (6), freeze drying protectant is added, (7), subpackage lyophilizing, wherein in MRC-5 cell amplification and rubella virus infection cell processes, all cultivate with MP-MRC-5 serum-free medium, wherein point kind of a rate for step (2) passage is 1:2, the virus that step (3) uses is rubella virus RA27/3 attenuated strain, working seed lots was no more than for 29 generations, step (4) Multiple harvests virus liquid, harvest time is kind of poison rear 2, 4, 6, 8, 10, 12 days, in step (5), virus liquid adopts the membrane filtration clarification of 0.45/1.0 micron, 2%(W/W is added in step (6)) mannitol, 3%(W/W) dextran, 2%(W/W) sodium glutamate, 1%(W/W) carbamide, 0.2%(W/W) arginine hydrochloride, 0.2%(W/W) people's blood cell albumin, do not add gelatin or gelatin, make lyophilized formulations.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4985244A (en) * | 1987-06-08 | 1991-01-15 | The Kitasato Institute | Stabilized live attenuated vaccine and its production |
| 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 |
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- 2013-10-30 CN CN201310523326.8A patent/CN103599530B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4985244A (en) * | 1987-06-08 | 1991-01-15 | The Kitasato Institute | Stabilized live attenuated vaccine and its production |
| 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 |
Non-Patent Citations (2)
| Title |
|---|
| 无明胶保护剂在麻疹风疹联合减毒活疫苗中的应用;石金辉等;《中国生物制品学杂志》;20121031;第25卷(第10期);摘要，第1264页左栏第2段、右栏第1段，第1265页右栏第2节，第1266页第3节 * |
| 聚乙烯吡咯烷酮稳定剂风疹减毒活疫苗的研制;公殿力等;《中国生物制品学杂志》;20090131;第22卷(第1期);摘要，第59页第1节，第60页第3节 * |
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Effective date of registration: 20190708 Address after: 125000 No. 5, West Kunshan Road, Area A, Dongdaihe New District, Huludao City, Liaoning Province Co-patentee after: ZHONGYI ANKE BIOTECHNOLOGY Co.,Ltd. Patentee after: LIAONING MAOKANGYUAN BIOTECHNOLOGY Co.,Ltd. Address before: 300000 Tianjin Binhai New Area Tianjin Free Trade Test Zone (Airport Economic Zone) No. 99-1 Huanhan Road Factory Building Patentee before: TIANJIN JINSITE BIOTECHNOLOGY CO.,LTD. |
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