CN106676076A - Method for preparing rotavirus vaccine stock solution by using serum-free Vero cells and serum-free rotavirus vaccine product - Google Patents
Method for preparing rotavirus vaccine stock solution by using serum-free Vero cells and serum-free rotavirus vaccine product Download PDFInfo
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- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/12—Viral antigens
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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- C12N2720/00011—Details
- C12N2720/12011—Reoviridae
- C12N2720/12311—Rotavirus, e.g. rotavirus A
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- C12N2720/00011—Details
- C12N2720/12011—Reoviridae
- C12N2720/12311—Rotavirus, e.g. rotavirus A
- C12N2720/12351—Methods of production or purification of viral material
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Abstract
The invention provides a method for preparing a rotavirus vaccine stock solution by using serum-free Vero cells. The method comprises the following steps: culturing Vero cells by using a serum-free culture medium, thus obtaining serum-free culture medium adapted cell strains; establishing a serum-free Vero cell seed bank by utilizing the obtained serum-free culture medium adapted cell strains; establishing a serum-free rotavirus strain working seed bank by utilizing the obtained serum-free culture medium adapted cell strains; carrying out reviving, culturing, passage and amplification on cells in a Vero cell working seed bank by utilizing the serum-free culture medium, using the cells in the Vero cell working seed bank as basic cells cultured in a bioreactor, and carrying out continuous perfusion culture on high-density Vero cells by applying the bioreactor and a microcarrier and using the serum-free culture medium after cell amplification; after inoculating virus seeds in the rotavirus strain working seed bank, carrying out bioreactor-microcarrier serum-free culture, obtaining a virus solution when virus is amplified to the summit, obtaining liquid virus titer, and carrying out clarification and ultra-filtration concentration, thus obtaining a serum-free rotavirus stock solution for human.
Description
Technical field
The present invention relates to the preparation method of Rotavirus Vaccine stoste, more particularly to the culture of bioreactor microcarrier without
Serum Vero cells prepare the method and serum-free Rotavirus Vaccine product of Rotavirus Vaccine stoste.
Background technology
At present, the cellular matrix that a large amount of production people's Rotavirus Vaccines are utilized includes primary cell, and such as calf kidney is thin
Born of the same parents；Continuous passage cell, such as African green monkey kidney cell (Vero).Vero cells are that the production of vaccine of World Health Organization's accreditation is thin
Born of the same parents system.Compared with the primary cell as production of vaccine, diploid cell and some other passage cell matrix, Vero cells
With can continuous passage, fast growth, stabilization characteristics of genetics, vicious transformation low degree, biological safety are higher, condition of culture
It is required that it is not harsh, be easy to implement the advantage such as large-scale culture in bioreactor.
Used as adherent fibroblasts, Vero cells have dependence to serum, because serum can be the in vitro culture of cell
Necessary growth factor, hormone, the cell attachment factor and nutritional ingredient are provided.
The culture medium complementary element prepared as Vero cell culture and vaccine by the use of cow's serum, with it is many it is unfavorable because
Element：The complexity and uncertainty of serum component, cause the mass discrepancy between serum production batch, increased viral vaccine production
Unstability and control of product quality difficulty, affect the production technology and vaccine quality of viral vaccine；Exist in serum
The potential risk of viral (such as prion) microbiological contamination, the safety that can not be ignored is brought to the use of vaccine hidden
Suffer from.
Serum has certain inhibitory action, rotavirus, hepatitis A virus, varicella virus etc. to virus, it is necessary to pancreatin is activated
The virus of the fine infection cell of ability, because the serum in blood serum medium has neutralized pancreatin, prevents virus from fully activating, from
And reduce the titre for harvesting virus.
Due to case above, the developed country such as the U.S., European Union and Japan has stopped at application blood in bio-pharmaceuticals comprehensively
Clearly, FDA will stop cell culture process containing serum produce declaring for biotechnology new drug accept.Free serum culture has turned into bag
Include the general trend that vaccine is produced in interior biotech drug.
Square vase, rolling bottle and cell factory (the static bottle of multilayer) level are rested on existing free serum culture technology more.And side
There is technological parameter and be not easily controlled, it is impossible to realize the High Density Cultivation of cell in bottle, rolling bottle and cell factory as culture vessel
Virus liquid with acquisition compared with high titre, the homogeneity of product also cannot be guaranteed.Therefore, the biology that can be mass produced is developed
Reactor microcarrier free serum culture Vero cells people is very necessary with the preparation method of Rotavirus Vaccine virus stock solution used.
The defect of prior art can be generalized into it is following some.
1. sensitized vaccine produces unstable, quality and is difficult to control to；
2. potential viral micro-organisms can cause unsafe hidden danger in serum；
3. serum has certain inhibitory action to virus, and using having, blood serum medium Cultivation of Vero and preparation are viral
Liquid, makes virus harvest liquid titre relatively low.More particularly need pancreatin activate could very well infection cell virus (such as colyliform disease
Poison etc.), because the serum in blood serum medium has neutralized pancreatin, prevent virus from preferably activating, so as to be greatly reduced
Harvest the virus titer of virus liquid.
4. square vase, rolling bottle and cell factory are used as culture vessel, it is impossible to control technological parameter well, are unfavorable for that cell is given birth to
Gas exchanges and nutrition ample supply in growth process, it is impossible to control technical process well, it is impossible to realize the high density of cell
Cultivate and obtain the virus liquid compared with high titre, it is also difficult to realize the homogeneity of product.
The content of the invention
A kind of defect it is an object of the invention to solve above-mentioned prior art, there is provided bioreactor microcarrier serum-free
The preparation method and serum-free Rotavirus Vaccine product of Cultivation of Vero Rotavirus Vaccine virus stock solution used, for preparing
Go out that homogeneity is good, titre is high, serum-free composition and safe serum-free viral vaccine stoste, to mass produce without blood
The people of clear culture production Vero cellular matrixs is laid the foundation with Rotavirus Vaccine.
First technical scheme of the invention is that a kind of bioreactor microcarrier culture serum-free Vero cells prepare colyliform
The method of viral vaccine stoste, it is characterised in that comprise the steps of,
Step 1. serum free medium Cultivation of Vero, obtains serum free medium and adapts to cell line；
Step 2. adapts to cell line using the serum free medium for obtaining, and sets up serum-free Vero cell seed banks；
Step 3. adapts to cell line using the serum free medium for obtaining, and sets up the work of serum-free rotavirus strain
Seed bank；
Step 4. is recovered using serum free medium, cultivated, passed on, amplification Vero cell work seed bank cells, as
The basal cell of bioreactor culture, after cell amplification, using bioreactor and microcarrier, using serum free medium,
Continuously perfused culture high density Vero cells；
After step 5. inoculation rotavirus strain work seed bank seed culture of viruses, the training of bioreactor microcarrier serum-free is carried out
Support, at virus amplification to peak, harvest virus liquid, harvest liquid virus titer, at clarified, ultrafiltration concentration, inactivation and purifying
Reason, obtains serum-free people and uses rotavirus stoste.
Second technical scheme is based on the first technical scheme, it is characterised in that in the step 2, directly with free serum culture
Base recovery, culture and the passage amplification serum free medium adapt to cell line, set up the main seed bank of serum-free Vero cells and
Work seed bank.
3rd technical scheme is based on the second technical scheme, it is characterised in that in the step 3, using serum free medium,
Recovery, passage and amplification serum-free Vero cell work seed bank cells, virus inoculation vaccine strain are harvested after culture amplification
Freeze, set up the serum-free main seed bank of viral vaccine strain, then connect with the main seed bank seed culture of viruses of serum-free viral vaccine strain
Serum-free Vero cell work seed bank cells are planted, is harvested after culture amplification and frozen, set up serum-free viral vaccine strain work
Make seed bank.
4th technical scheme is based on the 3rd technical scheme, it is characterised in that in the step 4, using serum free medium,
The serum-free Vero cell works seed bank cell is recovered, passes on and expands, as the basal cell of bioreactor culture,
Appropriate microcarrier is weighed, serum free medium balance is added overnight, the cell of results is transferred in cell inoculation bottle, aseptic company
Cell inoculation bottle and bioreactor are connect, are seeded cells into tank using malleation, supplement serum-free medium to working volume,
Adjustment bioreactor culture parameter, after cell inoculation certain hour, adjusts rotating speed of agitator, starts perfusion cell amplification cultivation
Base, when cell quantity reaches ormal weight, carries out the preparation of virus inoculation.
5th technical scheme is based on the 4th technical scheme, it is characterised in that in the step 5, the full ball rate on microcarrier
Up to more than 80%, liquid is changed, sampling carries out cell count, and serum free medium is added by MOI0.001-0.01, carried out thin on carrier
Born of the same parents and the absorption of virus, after completing cell to the absorption of virus, when viral peak is arrived, harvest virus liquid.
6th technical scheme is based on the 3rd technical scheme, it is characterised in that in the step 3, is added in virus liquid and contained
The activator of 10-40 μ g/mL trypsase, 37 DEG C of water-baths act on 60min, take the Vero cells in good condition for covering with individual layer,
Virus liquid strain after activation is inoculated with Vero cells with MOI 0.005,37 DEG C of thermostatic chamber absorption 60min are placed in, serum-free is added
Viral maintaining liquid liquid is gently mixed to 30mL, cell bottle is continued to be placed in 37 DEG C of thermostatic chamber cultures, in the virus inoculation stipulated time
Afterwards, viral peak period harvests virus and samples, and cell bottle and the sampling seal of tube are placed in -20 DEG C, and continuous freeze thawing three times is placed in -80
DEG C freeze.
7th technical scheme is based on the 4th technical scheme, it is characterised in that in the step 4, using serum free medium,
Recovery, passage and amplification serum-free Vero cell work seed bank cells, according to bioreactor working volume, are put into by carrier
Amount 8-18g/L is calculated, and weighs appropriate microcarrier, is soaked with 0.05mol/L PBS (pH 7.2), liquid is changed 3~4 times, with PBS in 4
DEG C soaked overnight, after autoclaving, carrier is imported in tank body with fluid filling pump, adds serum free medium, 50 revs/min of rotating speed
Clock, 37 DEG C of temperature, PH7.2-7.6, DO20-60, balance overnight, the cell of results is transferred in cell inoculation bottle, and control is thin
The volume of born of the same parents' suspension, sampling carries out cell count, it is ensured that postvaccinal cell density is not less than 1.0-1.5 × 106Individual/ml, nothing
Bacterium connects cell inoculation bottle and biological reactor, is seeded cells into tank using malleation, supplement serum-free medium to work
Volume, whole biological respinse culture parameters：Temperature is 37 DEG C, DO20-60, and throughput is 0.5LPM, rotating speed of agitator 25-35 turns/
Minute, 37 DEG C of temperature, PH7.2-7.6, after cell is inoculated with 24 hours, adjustment rotating speed of agitator starts to fill to 50-60 revs/min
Stream cell amplification culture medium, the first to three day is 0.5-1 tank volume/perfusion flow, is within second day 2.5 tanks the 3rd day to the 7th
It is 1-3 tank volume/perfusion flow, and observation of cell growth conditions under daily sampling mirror become in combination with oxygen consumption and PH
Change situation adjusts perfusion flow, when the 6-7 days cell quantities reach 0.9-1.1 × 107During individual/ml, the preparation of virus inoculation is carried out.
8th technical scheme is based on the 5th technical scheme, it is characterised in that in the step 5, is added in virus liquid and contained
The activator of 10-40 μ g/mL trypsase, 37 DEG C of water-baths act on 60min, bioreactor microcarrier Cultivation of Vero 7 days
Left and right, treats that full ball rate is up to more than 80% on microcarrier, changes liquid, and sampling carries out cell count, and disease is added by MOI0.001-0.01
Poison, and trypsase to 2-4 μ g/mL is supplemented, cell and viral absorption on carrier then are carried out with 25-30 revs/min, about
30-60 minutes, rotating speed of agitator after the completion of absorption, was adjusted to 50 revs/min by 33-35 DEG C of temperature, completes suction of the cell to virus
After attached, rotating speed of agitator is carried to 50-60 revs/min, and 35 DEG C of temperature, observation of cell lesion situation is viral after three days or so
Peak is arrived, 3-7 days, results virus liquid, standby after continuous freeze thawing 3 times.
9th technical scheme is based on first to the 8th any technical scheme, it is characterised in that in the step 5, according to
The capacity of bioreactor, for the capacity of the bioreactor less than 14L, carries out perfusion culture.
Tenth technical scheme is serum-free Rotavirus Vaccine product, it is characterised in that the serum-free rotavirus system
Product are obtained by any one of claim 1 to 9.
Brief description of the drawings
Fig. 1 be with bioreactor microcarrier free serum culture Vero cells prepare Rotavirus Vaccine virus stock solution used and
The process chart of finished product；
Fig. 2 is the figure of serum-free Vero cell works storehouse cell state；
Fig. 3 serum-free Vero cell works storehouse verification of mycoplasma result and positive control figure (DNA decoration methods)；
Fig. 4 is Vero cells generation lesion photo (T225 square vases) after the virus of inoculation rotavirus work storehouse；
Fig. 5 is the figure that biological anti-master answers device microcarrier free serum culture Vero cell attachments and growing state；
Fig. 6 is the culture of bioreactor microcarrier without blood culture Vero cells rotavirus people-ox reassortant G1 types virus
The figure of situation before and after virus inoculation.
Specific embodiment
Embodiments of the present invention are illustrated below.Specific embodiment only conduct described in following implementation methods
Exemplary illustration, is not meant to limit the scope of the invention.
First, technical scheme is illustrated.
Obtain serum free medium and adapt to cell line.Free serum culture Vero cells seed bank and nothing are set up on the basis of this again
Serum sickness toxicity vaccine strain seed bank.
Using serum free medium VP-SFM to Vero cell work seed banks cell recovery, culture, passage, amplification.
After cell amplification, using NBS (New Brunswich Scientific) company's 7.5L-50L bioreactors
(Celligen 310 or the Bioreactor System With Cell Lift Impeller of Celligen 310) and by GE
The microcarrier cytodex-1 of company (General Electric Company) production, with 8-20g/L high density microcarrier cultures
Technology, using serum free medium, extensive continuously perfused culture high density Vero cells.By culture, can reach
1.5x107The cell density of/milliliter, after about 6-8 days, full ball rate is more than 85%.
Virus inoculation vaccine strain work seed bank seed culture of viruses, such as rotavirus people-cow genome reassortant G1 (D × UK),
G2 (DS-1 × UK), G3 (P × UK), G4 (ST-3 × UK) and CDC-9 (G1P8), varicella virus Oka attenuated vaccine strains, hepatitis A
Toxic vaccine strain etc., carries out bioreactor microcarrier free serum culture, after about 3-5 days, during virus amplification to peak, and thawing
The Virus culture of cell-released virus particle, harvest liquid virus titer reaches more than 8.0LD50/ milliliters, and clarified, ultrafiltration is dense
The steps such as contracting, inactivation and purifying, preparing serum-free people's Rotavirus Vaccine etc. needs the viral of freeze thawing ability releasing virus
Vaccinogen liquid.
Fig. 1 is the technique stream that Rotavirus Vaccine stoste is prepared with bioreactor microcarrier culture serum-free Vero cells
Cheng Tu.
Hereinafter, according to the process chart of Fig. 1, preparation method of the invention is illustrated.
1. obtain serum free medium and adapt to cell line
Using the CCL-81Vero cells introduced from ATCC, the serum free medium VP- for directly being produced with GIBCO companies
The culture of SFM, obtains serum free medium and adapts to cell line.
2. the foundation of serum-free Vero cells seed bank
The CCL-81Vero cells introduced to ATCC, are directly expanded with the recovery of VP-SFM serum free mediums, culture and passage
Increase.Set up the main seed bank of serum-free Vero cells and work seed bank.
3. serum-free viral vaccine strain seed bank is set up
Using VP-SFM serum free mediums, recovery, passage and amplification serum-free Vero cell work seed bank cells connect
Viral vaccine strain is planted, is harvested after culture amplification and frozen, set up the serum-free main seed bank of viral vaccine strain, then with without blood
The clear main seed bank seed culture of viruses inoculation serum-free Vero cell work seed bank cells of viral vaccine strain, harvest after culture amplification and freeze
Deposit, set up serum-free viral vaccine strain work seed bank.
4. culture, amplification of the bioreactor microcarrier serum free medium to Vero cells
Recovery, culture and the passage amplification of 4.1 serum-free Vero cell work seed bank cells
Using VP-SFM serum free mediums, recovery, passage and amplification serum-free Vero cell work seed bank cells are made
It is the basal cell of bioreactor culture.
4.2 microcarriers prepare
Appropriate microcarrier is weighed, is soaked with 0.05mol/L PBS (pH 7.2), change liquid 3~4 times.With PBS in 4 DEG C of immersions
Overnight.After autoclaving, carrier is imported in tank body with fluid filling pump, adds serum free medium balance overnight.
4.3 bioreactor micro-carriers cell cultures
4.3.1 cell imports bioreactor and cell attachment
The cell of results is transferred in cell inoculation bottle, aseptic connection cell inoculation bottle and bioreactor, using just
Pressure is seeded cells into tank；Supplement serum-free medium to working volume；Adjustment biological respinse culture parameters.
4.3.2 bioreactor microcarrier serum-free cell culture
After cell is inoculated with 24 hours, rotating speed of agitator is adjusted.Start perfusion cell amplification culture medium.Seen under daily sampling mirror
Cell growth state is examined, in combination with oxygen consumption and PH situations of change adjustment perfusion flow.When the 6-7 days cell quantities reach
0.9-1.1×107During individual/ml, the preparation of virus inoculation is carried out.
5. the bioreactor microcarrier free serum culture of serum-free viral vaccine strain, amplification and virus liquid are harvested
5.1 bioreactor microcarrier cultured cells virus inoculations, culture and virus harvest
5.1.1 virus inoculation
Bioreactor microcarrier Cultivation of Vero 7 days or so, treats that full ball rate, up to more than 80%, changes liquid on microcarrier,
Sampling carries out cell count, and serum free medium is added by MOI0.001-0.01, is then carried out on carrier with 25-30 revs/min
Cell and viral absorption, about 30-60 minutes, 33-35 DEG C of temperature.After the completion of absorption, rotating speed of agitator is adjusted to 50 revs/min
Clock.
5.2 Virus cultures and results
After cell is completed to the absorption of virus, rotating speed of agitator is carried to 50-60 revs/min, 35 DEG C of temperature.Sampling daily
Microscopic observation cytopathy situation.After 3-5 or so, viral peak is arrived, and harvests virus liquid.
Rotavirus people-cow genome reassortant G1 (D × UK), G2 (DS-1 × UK), G3 (P × UK), G4 (ST-3 × UK),
The culture of the strains such as CDC-9 (G1P8), varicella virus Oka attenuated vaccine strains, hepatitis A virus vaccine strain needs thawing cell
Could releasing virus particle Virus culture, in this viroid, if lesion is slower, cell hold time it is more long, using small
The Virus culture of volume bioreactor can select perfusion；If three days or so after virus inoculation can reach viral peak
Phase, perfusion culture can not be carried out using the larger bioreactor of volume, virus liquid can be harvested when viral peak is arrived and is entered
The treatment of row next step.
6. the clarification of virus liquid and ultrafiltration concentration
In-depth filtration is carried out with 0.45-0.8 μm, to remove cell fragment, then with 300KD-1000KD film bags,
(5mmol/L phosphate, pH7.2,0.9%NaCl), concentrates 30-80 times.
7. inactivate
Per 1000ml be concentrated by ultrafiltration liquid press final concentration 1/4000, plus 10% dilute beta-propiolactone 2.5ml, fully shake up,
Put 4 DEG C 24 hours.8.2 put 37 DEG C hydrolyzes for 2 hours.
Oral Rotavirus vaccine and other attenuated live vaccines do not need inactivation step.
8. gel chromatography
Virus liquid after concentration and assay approval carries out pure through index-20Sepharose 4FF agarose gel column chromatographies
Change.Each applied sample amount 800ml~1500ml.Monitored according to Ultraviolet Detector, corresponding peak shape is intuitively reflected by recorder, in time
Collect, be stoste after purification.
9. stoste is prepared
According to determine protein content and antigenic content prepared, it is degerming, vaccinogen liquid is mixed with dilution, plus
Enter final concentration of 1% human serum albumin, 5% lactose, 5% sucrose, 1.8% dextran, 0.1% sodium glutamate used as stabilizers,
As stoste.Stoste is saved backup under the conditions of should being placed in 2-8 DEG C.
Key point of the invention and effect
1. virus is prepared due to the virus using serum free medium Cultivation of Vero and with Vero cells as host
Property vaccine：1) due to eliminating the security risks that animal derived serum (mainly cow's serum) brings；2) serum pair is eliminated
The suppression of viral growth, beneficial to raising virus titer.
2. the virus with bioreactor microcarrier Cultivation of Vero and with Vero cells as host is viral to prepare
Vaccine：1) technical process is more controllable, and product homogeneity is more preferable；2) due to using bioreactor microcarrier cultured cells and disease
Poison can ensure gas exchanges and the nutritional ingredient supply of cell, can increase institute's cultured cells density, and virus is cultivated in raising
Titre.3) free serum culture is combined with bioreactor microcarrier culture, carries out viral with Vero cells as matrix
The preparation of vaccine, can prepare the serum-free viral vaccine stoste of good homogeneity, high titre, high-quality and high security,
For the viral vaccine of large-scale production serum free medium production Vero cellular matrixs lays the foundation.
3. biological anti-device and free serum culture, increased cell culture density, improve viral antigen titre, cell density
Reach 1.5-2 × 107Cells/ml, make one-ox again with G1 serogroup vaccines strain reach 8.0LogLD50/mL.
4. vaccine finished product need not carry out the calibrating of bovine serum albumin residual.Do not exist object of inoculation in clinical vaccine inoculation
Adverse reaction to cow's serum allergy, improves the security of final products.《Pharmacopeia》Regulation uses cow's serum as culture medium
Auxiliary element, then cow's serum residual quantity must not exceed 50ng/ml, product of the present invention is free of cow's serum, and product code will not set
Cow's serum remains calibrating project.
5. the combination of bioreactor Microcarrier Culture Techniques and free serum culture technology, beneficial to the quality control of product,
Vaccine product homogeneity is more preferable.For the preparation of bioreactor microcarrier free serum culture viral vaccine is laid a good foundation.
The present invention will be described by the following examples
(the bioreactor microcarrier free serum culture Vero cells people-ox reassorted rotavirus G1 serotypes of embodiment one
The preparation of (D × UK) vaccinogen liquid)
Using the CCL-81Vero cells introduced from ATCC, the serum free medium VP- for directly being produced with GIBCO companies
The culture of SFM, obtains serum free medium adapted strain.Free serum culture Vero cells seed bank is set up on this basis and without blood
Clear people-ox reassorted rotavirus G1 serotype (D × UK) strains seed bank (people-ox reassorted rotavirus G1 serum that NIH is provided
Type (D × UK) strain).Using serum free medium VP-SFM to Vero cell work seed banks cell recovery, culture, passage,
Amplification.After cell amplification, using NBS (New Brunswich Scientific) company 14L bioreactors (Celligen
310 Bioreactor System With Cell Lift Impeller) and by GE companies (General Electric
Company) the microcarrier cytodex-1 of production, with 8-20g/L high density Microcarrier Culture Techniques, using serum free medium,
Extensive continuously perfused culture high density Vero cells, can reach 1.5x107The cell density of/milliliter, after about 6-8 days, full ball
Rate is more than 85%, and inoculation serum-free people-ox reassorted rotavirus G1 serotype (D × UK) strain work seed bank seed culture of viruses carries out nothing
Serum free culture system, after about 3 days, during virus amplification to peak, by Virus culture, harvests virus liquid, and harvest liquid virus titer reaches
More than 8.5LD50/ milliliters, the step such as clarified, ultrafiltration concentration prepares serum-free people and uses rotavirus stoste.
Specific embodiment is as follows
Experiment material
Cell and seed culture of viruses：Vero cells are the CCL-81 from ATCC；Seed culture of viruses is provided for NIH
People-ox reassorted rotavirus G1 serotype (D × UK) vaccine strain.
Main agents and consumptive material：VP-SFM serum free mediums and pancreatin are purchased from GIBCO companies；T75, T175, T225 are thin
Born of the same parents' blake bottle is CORNING Products；Microcarrier cytodex-1, cytodex-3, by GE companies (General Electric
Company) produce.
Activated viral agent：Serum-free medium containing finite concentration (10~40 μ g/mL) trypsase.
Viral maintaining liquid：The nutrient solution of the serum-free containing finite concentration (1~4 μ g/mL) trypsase.
Key instrument equipment：Fluorescence inverted microscope is purchased from Chongqing Photoelectric Co., Ltd., model IBE2003；It is inverted life
Thing microscope, model XDS-1B is produced by Shanghai Cai Kang optical instrument factories；Electric-heated thermostatic water bath is purchased from the grand limited public affairs of upper Nereid
Department, model DK-8D；CO2gas incubator, model MCO-20AIC, Sanyo's production；14L bioreactors, model
The Bioreactor System With Cell Lift Impeller of Celligen 310 are NBS (New Brunswich
Scientific) company's production.
2. the foundation of serum-free Vero cells seed bank
Recovery, passage amplification and the foundation of the main seed bank of serum-free Vero cells of 2.1 cells
2.1.1 cell recovery
The CCL-81Vero cells introduced from ATCC are taken out from liquid nitrogen container, 38-40 DEG C is immediately placed in after verification label
(cell must be completely dissolved in 1~2 minute) instant in warm water；Low speed (1000rpm) takes out after being centrifuged about 3-5 minutes.Nothing
Under the conditions of bacterium, the supernatant in cryopreservation tube is discarded, appropriate serum free medium VP-SFM culture mediums are added with suction pipe, gently blown and beaten
After cell precipitation, sucking-off is put in sterilized glass square vase, supplies serum free medium VP-SFM culture mediums, shakes up lid plug, and
Carry out mark.Glass square vase is placed in 37 DEG C of thermostatic chamber quiescent culture 48-96h.
2.1.2 passage amplification and the foundation of the main seed bank of serum-free Vero cells
Microscopy adapts to the Vero cells of serum free medium, is operated under aseptic condition, and the old nutrient solution in square vase is discarded,
Appropriate 0.125% trypsin solution (containing 0.04%EDTA-2Na) is added, makes its infiltrating cells face and bottle wall.Treat that cell face is in hair
During vitreousness, digestive juice is discarded；Remaining digestive juice continuation is acted on, when cell face is loose, appropriate VP-SFM is added without blood
Clear culture medium makes cell be uniformly dispersed, and a point kind (1 is carried out according to cell concentration:2~4), and VP-SFM serum free mediums are supplied, put 37
DEG C thermostatic chamber quiescent culture 48-96h.
Daily observation of cell form and cytoactive, pass on, by 4-5 free serum culture and passage for three days or so again
After amplification, the final Vero cell lines for obtaining VP-SFM free serum cultures.Suspension cell, is dispensed with 1mL cells pipe, is protected in liquid nitrogen
Deposit, meanwhile, sampling carries out aseptic, mycoplasma etc.《Pharmacopeia》The relevant item calibrating of regulation.After all calibrating projects are qualified, complete
The foundation of the main seed bank of serum-free Vero cells.
The foundation of 2.2 serum-free Vero cell work seed banks
2.2.1 cell recovery
1 or several cells are taken out from the serum-free main seed bank of Vero cells, 38-40 DEG C is immediately placed in after verification label
Warm water in instant (cell must be completely dissolved in 1~2 minute)；Low speed (1000rpm) takes out after being centrifuged about 3-5 minutes.
Under aseptic condition, the supernatant in cryopreservation tube is discarded, after adding appropriate serum free medium gently to blow and beat cell precipitation with suction pipe,
Sucking-off is put in sterilized glass square vase, supplies serum free medium, shakes up lid plug, and carry out mark.Glass square vase is placed in
37 DEG C of thermostatic chamber quiescent culture 48-96h.In strict accordance with sterile working, appropriate VP-SFM culture mediums are taken, regulation pH value is 7.0
~7.4, it is standby.
2.2.2 cell culture, passage amplification and the foundation of serum-free Vero cell work seed banks
Microscopy cell, is operated under aseptic condition, and the old nutrient solution in glass square vase is discarded, and adds appropriate 0.125%
Trypsin solution (contains 0.04%EDTA-2Na), makes its infiltrating cells face and bottle wall.When cell face is in frosted glass state, discards and disappear
Change liquid；Remaining digestive juice continuation is acted on, when cell face is loose, adds appropriate serum free medium cell is uniformly dispersed,
A point kind (1 is carried out according to cell concentration:2~4), and serum free medium is supplied, put 37 DEG C of thermostatic chamber quiescent culture 48-96h.Will be long extremely
The cell of fine and close individual layer digests according to the above method, adds appropriate serum free medium, suspension cell, by passage, amplification, when thin
Cell is collected after born of the same parents' quantity to certain scale, while sampling carries out the calibrating of the relevant items such as mycoplasma, is dispensed with 1mL cells pipe,
Preserved in liquid nitrogen, set up serum-free Vero cell work seed banks.Cell growth state is shown in Fig. 2, and verification of mycoplasma result is shown in figure
3。
3. the foundation of people-ox reassorted rotavirus G1 serotype (D × UK) strain seed bank
The foundation of the main seed bank of 3.1 seeds culture of viruses
3.1.1 the activation of virus
The activator of the g/mL trypsase of μ containing 10-40 is added in virus liquid, 37 DEG C of water-baths act on 60min.
3.1.2 viral inoculation and absorption
The Vero cells in good condition for covering with individual layer are taken, the virus liquid strain after activation is inoculated with Vero with MOI 0.005
Cell.It is placed in 37 DEG C of thermostatic chamber absorption 60min.Serum-free virus maintaining liquid liquid is added to 30mL, is gently mixed.
3.1.3 viral culture with harvest
Cell bottle is continued to be placed in 37 DEG C of thermostatic chamber cultures, in 3 days or so after virus inoculation, viral peak period harvested virus
And sample, cell bottle and the sampling seal of tube are placed in -20 DEG C, and continuous freeze thawing three times is sampled and pressed《Pharmacopeia》The relevant item of regulation
Calibrating, remaining virus liquid is dispensed with seed culture of viruses bottle, posts label, is placed in -80 DEG C and is frozen, and is completed rotavirus people-cow genome and is matched somebody with somebody again
The preparation of vaccine strain (D × UK, G1) main seed bank.
The foundation of 3.2 seeds culture of viruses work seed bank
3.2.1 the activation of virus
The activator of the g/mL trypsase of μ containing 10-40 is added in virus liquid, 37 DEG C of water-baths act on 60min.
3.2.2 viral inoculation and absorption
The Vero cells in good condition for covering with individual layer are taken, the virus liquid strain after activation is inoculated with Vero with MOI 0.005
Cell.It is placed in 37 DEG C of thermostatic chamber absorption 60min.Add containing trypsase (2-4 μ g/mL) serum-free virus maintaining liquid liquid extremely
30mL, gently mixes.
3.2.3 viral culture with harvest
Cell bottle is continued to be placed in 37 DEG C of thermostatic chamber cultures, in 3 days or so after virus inoculation, viral peak period harvested virus
And sample, cell bottle and the sampling seal of tube are placed in -20 DEG C, and continuous freeze thawing three times is sampled and pressed《Pharmacopeia》The relevant item of regulation
Calibrating, remaining virus liquid is dispensed with seed culture of viruses bottle, posts label, is placed in -80 DEG C and is frozen, and is completed rotavirus people-cow genome and is matched somebody with somebody again
The preparation of vaccine strain (D × UK, G1) work seed bank.Vero is thin for serum-free rotavirus work seed bank virus infection serum-free
Born of the same parents' work seed bank cell situation is shown in Fig. 4.
4. culture, amplification of the bioreactor microcarrier serum free medium to Vero cells
Recovery, culture and the passage amplification of 4.1 serum-free Vero cell work seed bank cells
Use VP-SFM serum free mediums, recovery, passage and amplification serum-free Vero cell work seed bank cells
4.2 microcarriers prepare
According to bioreactor working volume, calculated by carrier input amount 8-18g/L, weigh appropriate microcarrier, used
0.05mol/L PBS (pH 7.2) soak, and change liquid 3~4 times.With PBS in 4 DEG C of soaked overnights.After autoclaving, carrier with plus
Liquid pump is imported in tank body, adds serum free medium, 50 revs/min of rotating speed, 37 DEG C of temperature, PH7.2-7.6, DO20-60, balance
Overnight.
4.3 bioreactor micro-carriers cell cultures
4.3.1 cell imports bioreactor and cell attachment：
The cell of results is transferred in cell inoculation bottle, the volume of cell suspension is controlled, sampling carries out cell count, protects
Demonstrate,prove postvaccinal cell density and be not less than 1.0-1.5 × 106Individual/ml.Aseptic connection cell inoculation bottle and biological reactor, use
Malleation is seeded cells into tank.Supplement serum-free medium to working volume.Adjustment biological respinse culture parameters：Temperature is 37
DEG C, DO20-60, throughput is 0.5LPM, 25-35 revs/min of rotating speed of agitator, 37 DEG C of temperature, PH7.2-7.6.
4.3.2 bioreactor microcarrier serum-free cell culture
After cell is inoculated with 24 hours, adjustment rotating speed of agitator is to 50-60 revs/min.Start perfusion cell amplification culture medium.
Be within the first to three day 0.5-1 tank volume/perfusion flow, be within second day 2.5 tanks the 3rd day to the 7th day for 1-3 tank volume/
Perfusion flow.Observation of cell growth conditions under daily sampling mirror, in combination with oxygen consumption and PH situations of change adjustment perfusion flow.
When the 6-7 days cell quantities reach 0.9-1.1 × 107During individual/ml, the preparation of virus inoculation is carried out.Biological anti-master answers the micro- load of device
Body free serum culture Vero cell attachments and growing state are shown in Fig. 5.
5. the bioreactor carrier free serum culture of serum-free viral vaccine strain, amplification and virus liquid are harvested
5.1 activated viral
The activator of the g/mL trypsase of μ containing 10-40 is added in virus liquid, 37 DEG C of water-baths act on 60min.
5.2 bioreactor microcarrier cultured cells virus inoculations
5.1.1 virus inoculation
Bioreactor microcarrier Cultivation of Vero 7 days or so, treats that full ball rate, up to more than 80%, changes liquid on microcarrier,
Sampling carries out cell count, and virus is added by MOI0.001-0.01, and supplements trypsase to 2-4 μ g/mL, then with 25-30
Rev/min carry out the absorption of cell and virus on carrier, about 30-60 minutes, 33-35 DEG C of temperature.After the completion of absorption, will stir
Oar rotating speed is adjusted to 50 revs/min.
5.1.2 Virus culture and results
After cell is completed to the absorption of virus, rotating speed of agitator is carried to 50-60 revs/min, 35 DEG C of temperature.Sampling daily
Microscopic observation cytopathy situation.After three days or so, viral peak is arrived, 3-7 days, according to carrier cell virus change situation,
Harvest standby after virus liquid, continuous freeze thawing 3 times.
In the present embodiment, Virus culture is carried out using thawing, (such as rotavirus people-cow genome reassortant G1 (D ×
UK), G2 (DS-1 × UK), G3 (P × UK), G4 (ST-3 × UK) and CDC-9 (G1P8) etc. and rotavirus people source attenuated live epidemic disease
Seedling strain grade, varicella virus Oka attenuated vaccine strains, hepatitis A virus vaccine strain, etc. be unable to perfusion culture).Trained in bioreactor
After supporting 3-7 days, situation is become according to cell virus, morphologically normal cell nearly 10% or so on carrier harvests virus liquid.It is biological anti-
The culture of device microcarrier is answered without situation is shown in Fig. 6 before blood culture Vero cell rotavirus virus inoculations and after virus inoculation 96 hours.
6. the clarification of virus liquid and ultrafiltration concentration
In-depth filtration is carried out with 0.45-0.8 μm of filter membrane, to remove cell fragment, then with 300KD-1000KD film bags,
(5mmol/L phosphate, pH7.2,0.9%NaCl), concentrates 30-80 times.
Vaccine formulation
The antigenic content of each milliliter of Rotavirus Vaccine stoste should be 7.5-8.0lgCCID50/mL and separately enter 7.2% (W/
V) and 12% (V/V) 10 sucrose.
Virus stock solution used titre verification result
Using the combined enzyme-linked immunizations of CCID50, individual layer, state are covered with by being seeded to after measuring samples dilution finite concentration
Good MA104 cells, after continuing to cultivate a period of time, virus infection titer are detected with rotavirus detection kit.
Four kinds of virus titer testing results of culture 3 consecutive batches of form
Brief summary
Because rotavirus infection cell does not have stronger suction to virion to extracellular releasing virus and cell fragment
Attached property, it is possible to use less bioreactor carries out continuously perfused culture, it is also possible to carried out using larger bioreactor
Non- perfusion culture., it is necessary to carry out the freeze thawing of three times to harvest liquid after virus liquid is harvested, so that virus is discharged into supernatant, so
Collect vial supernatant again afterwards.Rotavirus belongs to intestinal mucosa virus, and natural immunity approach is Intestinal Mucosal Immunity, people-ox
It is again people source with having a genetic fragment in vaccine strain, remaining is Niu Yuan, and ox source rotavirus is to people's no pathogenicity.Vaccine
It is live oral vaccine, therefore protective agent (multitudinous sugar etc.) is directly added into without other purification steps and inactivation step after ultrafiltration concentration,
Vaccine is after packing.
From titre verification result can be seen that continuous three batches of bioreactors microcarrier free serum culture Vero cells people-
Ox reassorted rotavirus G1 serotype (D × UK) vaccinogen liquids average titer is 8.33lgCCID50/mL, and three batches have seroreaction
The average titer of device culture stoste is 7.83lgCCID50/mL, and three batches of average titers for having the flask culture stoste of serum T 225 are
6.25lgCCID50/mL, three batches of average titers for having serum 3L spinner culture stostes are 6.58, show that free serum culture is conducive to
Infection of the virus to cell, is one of factor that titre is improved, meanwhile, reactor microcarrier culture form is that cell is made
Virus culture provides condition and the sufficient nutrition supply that gas is well exchanged, and is the second factor that titre is improved.For
There is a sensitized vaccine product, Pharmacopoeia of People's Republic of China (three, version in 2015) regulation ox is residual to must not exceed 50ng/ml, and nothing
The calibrating of cow's serum residual protein is not set in sensitized vaccine product calibrating project, calibrating cost is not only reduced, is eliminated yet and is faced
When bed is using product, the individual allergic reaction to cow's serum improves the security of vaccine product.
Embodiment two
On the basis of embodiment one, using the bioreactor compared with low capacity, the bioreactor of 14L is such as less than, can
To carry out perfusion culture.Cell is held time more long, and perfusion can be selected using the Virus culture of small size bioreactor, disease
Malicious peak can harvest virus liquid and be for further processing when arriving.
Embodiments of the present invention and embodiment are illustrated above, various conditions, reagent during preparation and
Parameter is not unique, and vaccine strain and serum free medium etc. are changed also dependent on needs, those skilled in the art
Member the parameter in implementation method and embodiment, vaccine strain and serum free medium can be adjusted according to its knowledge and
Change, this adjustment and change are belonged within the scope of the present invention.
Claims (10)
1. a kind of method that serum-free Vero cells prepare Rotavirus Vaccine stoste, it is characterised in that comprise the steps of,
Step 1. serum free medium Cultivation of Vero, obtains serum free medium and adapts to cell line；
Step 2. adapts to cell line using the serum free medium for obtaining, and sets up serum-free Vero cell seed banks；
Step 3. adapts to cell line using the serum free medium for obtaining, and sets up serum-free rotavirus strain work seed
Storehouse；
Step 4. is recovered using serum free medium, cultivated, passed on, amplification Vero cell work seed bank cells, used as biology
The basal cell of bioreactor culture, after cell amplification, using bioreactor and microcarrier, using serum free medium, continuously
Perfusion culture high density Vero cells；
After step 5. inoculation rotavirus strain work seed bank seed culture of viruses, bioreactor microcarrier free serum culture is carried out,
During virus amplification to peak, results virus liquid, harvest liquid virus titer, clarified, ultrafiltration concentration, inactivation and purification process are obtained
Rotavirus stoste is used to serum-free people.
2. the method that a kind of serum-free Vero cells according to claim 1 prepare rotavirus stoste, it is characterised in that
In the step 2, cell line is directly adapted to serum free medium recovery, culture and the passage amplification serum free medium,
Set up the main seed bank of serum-free Vero cells and work seed bank.
3. the method that a kind of serum-free Vero cells according to claim 2 prepare rotavirus stoste, it is characterised in that
In the step 3, using serum free medium, recover, pass on and amplification serum-free Vero cell work seed bank cells,
Virus inoculation vaccine strain, harvests after culture amplification and freezes, and sets up the serum-free main seed bank of viral vaccine strain, then with nothing
The main seed bank seed culture of viruses inoculation serum-free Vero cell work seed bank cells of serum sickness toxicity vaccine strain, harvest after culture amplification
Freeze, set up serum-free viral vaccine strain work seed bank.
4. the method that a kind of serum-free Vero cells according to claim 3 prepare rotavirus stoste, it is characterised in that
In the step 4, the serum-free Vero cell work seed banks using serum free medium, are recovered, pass on and expanded
Cell, as the basal cell of bioreactor culture, weighs appropriate microcarrier, adds serum free medium balance overnight, will
The cell of results is transferred in cell inoculation bottle, aseptic connection cell inoculation bottle and bioreactor, is connect cell using malleation
Plant in tank, supplement serum-free medium to working volume, adjust bioreactor culture parameter, cell inoculation certain hour
Afterwards, rotating speed of agitator is adjusted, starts perfusion cell amplification culture medium, when cell quantity reaches ormal weight, carry out virus inoculation
Preparation.
5. the method that a kind of serum-free Vero cells according to claim 4 prepare rotavirus stoste, it is characterised in that
In the step 5, full ball rate changes liquid up to more than 80% on microcarrier, and sampling carries out cell count, by MOI0.001-
0.01 adds serum free medium, cell and viral absorption on carrier is carried out, after completing cell to the absorption of virus, in disease
When malicious peak is arrived, virus liquid is harvested.
6. the method that a kind of serum-free Vero cells according to claim 3 prepare rotavirus stoste, it is characterised in that
In the step 3, the activator of the g/mL trypsase of μ containing 10-40 is added in virus liquid, 37 DEG C of water-baths act on 60min,
The Vero cells in good condition for covering with individual layer are taken, the virus liquid strain after activation is inoculated with Vero cells with MOI 0.005, be placed in
37 DEG C of thermostatic chambers adsorb 60min, add serum-free virus maintaining liquid liquid to 30mL, gently mix, and cell bottle is continued to be placed in 37
DEG C thermostatic chamber culture, in after the virus inoculation stipulated time, viral peak period harvests virus and simultaneously samples, cell bottle and the sampling seal of tube
- 20 DEG C are placed in, continuous freeze thawing three times is placed in -80 DEG C and freezes.
7. the method that a kind of serum-free Vero cells according to claim 4 prepare rotavirus stoste, it is characterised in that
In the step 4, using serum free medium, recover, pass on and amplification serum-free Vero cell work seed bank cells,
According to bioreactor working volume, calculated by carrier input amount 8-18g/L, weigh appropriate microcarrier, use 0.05mol/L PBS
Immersion, changes liquid 3~4 times, and with PBS in 4 DEG C of soaked overnights, after autoclaving, carrier is imported in tank body with fluid filling pump, adds nothing
Blood serum medium, 50 revs/min of rotating speed, 37 DEG C of temperature, PH7.2-7.6, DO20-60, balance overnight, the cell of results is transferred to
To in cell inoculation bottle, the volume of cell suspension is controlled, sampling carries out cell count, it is ensured that postvaccinal cell density is not less than
1.0-1.5×106Individual/ml, aseptic connection cell inoculation bottle and biological reactor, are seeded cells into tank using malleation, are mended
Fill serum-free medium to working volume, whole biological respinse culture parameters：Temperature is 37 DEG C, DO20-60, and throughput is
0.5LPM, 25-35 revs/min of rotating speed of agitator, 37 DEG C of temperature, PH7.2-7.6 after cell is inoculated with 24 hours, adjusts agitating paddle
Rotating speed starts perfusion cell amplification culture medium to 50-60 revs/min, is within the first to three day 0.5-1 tank volume/perfusion flow, the
It is within two days that 2.5 tanks the 3rd day to the 7th day are 1-3 tank volume/perfusion flow, observation of cell growth conditions under daily sampling mirror,
In combination with oxygen consumption and PH situations of change adjustment perfusion flow, when the 6-7 days cell quantities reach 0.9-1.1 × 107Individual/
During ml, the preparation of virus inoculation is carried out.
8. the method that a kind of serum-free Vero cells according to claim 5 prepare rotavirus stoste, it is characterised in that
In the step 5, the activator of the g/mL trypsase of μ containing 10-40 is added in virus liquid, 37 DEG C of water-baths act on 60min,
Bioreactor microcarrier Cultivation of Vero 7 days or so, treats that full ball rate, up to more than 80%, changes liquid on microcarrier, and sampling is carried out
Cell count, virus is added by MOI0.001-0.01, and supplements trypsase to 2-4 μ g/mL, then with 25-30 revs/min
Carry out cell and viral absorption, about 30-60 minutes, 33-35 DEG C of temperature, after the completion of absorption, by rotating speed of agitator on carrier
50 revs/min are adjusted to, after completing cell to the absorption of virus, rotating speed of agitator is carried to 50-60 revs/min, and 35 DEG C of temperature is seen
Cytopathy situation is examined, after three days or so, viral peak is arrived, 3-7 days, results virus liquid is standby after continuous freeze thawing 3 times.
9. the method that a kind of serum-free Vero cells according to any one of claim 1 to 8 prepare rotavirus stoste,
Characterized in that,
In the step 5, according to the capacity of bioreactor, for the capacity of the bioreactor less than 14L, perfusion training is carried out
Support.
10. serum-free Rotavirus Vaccine product, it is characterised in that the serum-free rotavirus product is by claim 1 to 9
Any one of be obtained.
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