CN103599530A - Method for preparation of rubella attenuated live vaccine by serum-free culture medium - Google Patents
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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 of preparing attenuated rubella live vaccine with serum-free medium, particularly on human diploid cell MRC-5, 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 avoid the pollution of other pathogenic microorganisms of crazy heifer disease virus, mycoplasma and cattle susceptible; Utilize production of vaccine technique to remove serum completely and possible pollution is impossible; Therefore State Food and Drug Administration and even WHO avoid using animal derived raw material in advocating production of vaccine process.Exploitation serum-free production of vaccine technique, meets national policy requirement, meets global biological product development trend, meets the people's healthy and safe requirement.Cell cultivation process of the present invention is not used the culture fluid containing serum, has avoided the pollution causing because adding serum.
The present invention uses the attenuated rubella live vaccine of being licensed by U.S.'s WISTAR institute to produce and uses seed culture of viruses RA27/3.This seed culture of viruses is separated by WISTAR institute, and by continuous passage attenuation in WI-38 cell, in 25-33 generation, carried out clinical trial, proof 25-33 loses pathogenicity for virus to human body, but immunogenicity is good, can stimulate body to produce the immunity that enough rubella virus infect, Institute Pasteur and U.S. Merck company all use this seed culture of viruses to develop attenuated live vaccine afterwards.Used nearly many decades safely.
Gelatin is a kind of protective agent and the excipient that vaccine for man is conventional, derives from animal, and its production process cannot be controlled virulent pollution completely, and gelatin is relevant with anaphylaxis after vaccination.WHO advises global production of vaccine enterprise on production of vaccine associated guideline repeatedly, adopts as far as possible gelatine replacement as the freeze-dried excipient of vaccine.In vaccine excipient of the present invention, thoroughly broken away from gelatin, convenient sources, quality controllable.Outward appearance and dissolution time meet existing Chinese Pharmacopoeia requirement, and stablizing effect meets vaccine quality requirement.
China women's at reproduction age rubella positive rate 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 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, therefore, these countries 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 is annual just needs 3,200 ten thousand, 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 of preparing human diploid cell attenuated rubella live vaccine with serum-free medium.
It is human diploid cell MRC-5 that the present invention adopts cell, and, with this cell inoculation rubella virus RA27/3 attenuated strain, results virus liquid, adds after viral protective agent and excipient, and packing 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, specifically by following steps, realizes:
(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, inoculate rubella virus RA27/3 attenuated strain;
(4), results infection cell culture supernatant;
(5), virus liquid clarification filtration;
(6), add freeze drying protectant;
(7), packing 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 proteinum.If cell and Virus culture serum-free medium used can be MP-MRC-5 SFM, this culture medium does not contain the proteins and peptides of animal origin, applicable equally other serum-free mediums of the present invention.
Through calibrating, while there is pathological changes in cell, Multiple harvests virus liquid, through frozen, thaw, merge, detect after titre, obtain producing the rubella virus liquid of vaccine.The present invention uses serum-free medium to cultivate human embryonic lung diploid fibroblast and can access with there being blood serum medium and cultivate same effect, and the rubella virus titre obtaining is without significant difference (in Table 1), and whole process is not used antibiotic.The present invention's serum-free medium used is general serum-free medium.
Table 1, rubella virus has the titre comparison (logCCID of serum free culture system and serum-free culture
50/ ml)
| Kinds of culture medium | The 1st results | The 2nd results | The 3rd results | The 4th results |
| 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 (the National Institute for Biological Standards and Control of quality controling research institute, NIBSC), three grades of cell banks of model: master cell bank, master cell bank and working cell storehouse, and cell bank has been carried out to comprehensive calibrating, cell bank is examined institute's qualification test report in obtaining.
The proteins and peptides that does not contain animal origin in the amplification procedure that goes down to posterity of cell in the culture medium of using, whole process adapts to domestication without progressively reducing serum content.Peptic cell is used the trypsin in non-animal proteinum source, has digested the pancreatin inhibitor of rear interpolation 0.01%-0.1%, by the centrifugal removal pancreatin inhibitor of 232g-334g.In cell cultivation process, it is generally 1:2-1:4 that passage divides kind of rate.When Growth of Cells is to after fine and close monolayer, can inoculate rubella virus RA27/3 attenuated strain, now pH maintains 7.2 left and right, and condition of culture is 5%-10% carbon dioxide, 37 ℃.
The present invention sets up viral main seed lot and work storehouse seed lot, after antibacterial, mycete, mycoplasma inspection and exogenous factor passed examination, for vaccine, prepares.
The generation of virus work seed lot was no more than for 29 generations.Virus inoculation human diploid cell, condition of culture is 5%-10% carbon dioxide, 37 ℃.After cell infection virus, according to cytopathogenetic degree, carry out Multiple harvests virus liquid, generally 2,4,6,8,10,12 days results after infection.Frozen below-20 ℃ immediately after virus harvest.If cytopathy is serious, can carry out clarification filtration, then frozen.The virus liquid of results is used the membrane filtration clarification of 0.45/1.0 micron.Gather in the crops after measured virus liquid titre 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 titration of virus method that the present invention adopts is traditional few cells pathological changes method: suitable dilution rubella virus and RK-13 cell miscegenation are cultivated in 96 orifice plates, planted rear the 10th day observation port inner cell pathological changes situation of poison, with Karber method, calculate vaccine titre.
In the freeze-dry process that the present invention adopts, virus liquid adds mannitol by a certain percentage, dextran, and sodium glutamate, carbamide, arginine hydrochloride, people's blood cell albumin is made semi-finished product, through lyophilizing, obtains finished product.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 ℃ 7 days, titre decline average out to 0.3 logCCID
50/ ml.The excipient formula of gelatin-free has still guaranteed 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 poor heavy method calculation sample consumption, sample is added in Ka Shi coulomb meter, after electrolysis speed is down to 20ug/min and numerical stability, reading out data, repeated measure is averaged for three times.
the specific embodiment:
Below in conjunction with specific embodiment, the present invention is further elaborated.These embodiment are only for explaining rather than 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 ℃ of water-baths are thawed, and are inoculated into T
25in Tissue Culture Flask, add 8mlMP-MRC-5 SFM serum-free medium (add 1%L-Gln by front visible subsidy normally, and 37 ℃ of preheatings), medium pH is 7.2, puts 37 ℃, 5%CO
2in incubator, cultivate.This process is without cell being carried out to the progressively domestication of serum-free culture, and cell culture 3 days can adherent and fine and close monolayer, does not add antibiotic.
Embodiment 2
With not containing the Cell Stripper of animal proteinum
tMcell in non-enzyme cell pyrolysis liquid digestion embodiment 1, collects the pancreatin inhibitor 0.1ml-1ml that the cell suspension having digested adds 0.01%-0.2% concentration, and the centrifugal removal supernatant of 232g-334g 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 ℃ 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 ℃ of preheatings), medium pH is 7.2, puts 37 ℃, 5%CO
2in incubator, cultivate.After being cultured to cell attachment and fine and close monolayer, with not containing the Cell Stripper of animal proteinum
tMnon-enzyme cell pyrolysis liquid peptic cell, collects the cell suspension having digested and adds the pancreatin inhibitor 0.1～1ml of 0.01%-0.2% concentration, then add fresh serum-free medium, and the centrifugal removal supernatant of 232g-334g 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, after inoculation, 72h renews fresh serum-free medium, 2,4,6,8,10,12 days results virus liquids after kind poison.The membrane filtration clarification of 0.45/1.0 micron for the virus liquid of results.
Embodiment 4
Get the virus liquid after filtration in embodiment 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 do not contain gelatin or gelatin composition.Divide to be filled in cillin bottle and carry out lyophilizing, every cillin bottle packing 0.5ml, carries out lyophilizing through following program:
Pre-freeze :-45 ℃, 2h;
Baking temperature :-45 ℃～30 ℃;
Dry run: maintain-45 ℃, 0.02 Mpa 4h, then rising per hour is 2 ℃.
By three batches of above-mentioned condition lyophilizing, vaccine after lyophilizing, the 0.5ml sterilized water for injection of often drawing redissolves, suitable dilution rubella virus and RK-13 cell miscegenation are cultivated in 96 orifice plates, plant rear the 10th day observation port inner cell pathological changes situation of poison, with Karber method, calculate vaccine titre, measure and tire in Table 2.
Table 2, before and after lyophilizing, semi-finished product and finished product potency ratio are compared with (logCCID
50/ ml)
In lyophilizing formula, do not use gelatin, but finished product apparent condition and solubility are good.Determination of water adopts and takes Xiu Shi method, use Switzerland Wan Tongkashi coulomb meter to measure, after poor heavy method calculation sample consumption, sample is added in Ka Shi coulomb meter, after electrolysis speed is down to 20ug/min and numerical stability, reading out 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 ℃ 7 days, titre decline average out to 0.3 logCCID
50/ ml.Titre meets the requirement of 2010 editions pharmacopeia.
Claims (11)
1. with serum-free medium, prepare a method for attenuated rubella live vaccine, 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), results infection cell culture supernatant; (5), virus liquid clarification filtration; (6), add freeze drying protectant; (7), packing lyophilizing.
2. preparation method according to claim 1, in MRC-5 cell amplification and rubella virus infection cell processes, all cultivates with serum-free medium.
3. according to claim 2, in MRC-5 cell cultivation process, do not add antibiotic and without the progressively domestication of carrying out serum-free culture.
4. according to claim 2, serum-free medium does not contain protein or the polypeptide of animal origin.
5. preparation method according to claim 1, is characterized in that the trypsin that step (2) MRC-5 cell dissociation is used is non-animal source protein, as Cell Stripper
tMthe Digestive system in cell dissociation buffer or other plant source.
6. preparation method according to claim 1, is characterized in that with protease inhibitor, stopping digestion after step (2) MRC-5 cell dissociation, and the working concentration of pancreatin inhibitor is 0.01%-0.1%.
7. preparation method according to claim 1, is characterized in that step (2) adopts centrifugal method to remove trypsin inhibitor, and centrifugal speed is 50g-927g, more properly, and 83g-412g; Be more properly 232g-334g.
8. preparation method according to claim 1, after it is characterized in that step (2) cell is resuspended, cell divides kind of rate at 1:2-1:8; More properly, should be at 1:2-1:4.
9. preparation method according to claim 1, is characterized in that it is rubella virus RA27/3 attenuated strain that step (3) is used virus, and work seed lot was no more than for 29 generations.
10. preparation method according to claim 1, is characterized in that step (4) can Multiple harvests virus liquid, harvest time for kind of poison after 2,4,6,8,10,12 days, in step (5), virus liquid adopts the membrane filtration clarification of 0.45/1.0 micron.
11. preparation methoies according to claim 1, in step (6), 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) people's blood cell albumin, do not add gelatin or gelatin composition, make lyophilizing semi-finished product.
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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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