CN102327609A - Production method of encephalitis B vaccine - Google Patents
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Abstract
The invention provides a production method of an encephalitis B vaccine, which comprises the following steps: adding Vero seed cells to a 300-liter bioreactor, and carrying out perfusion culture together with a microcarrier; after the perfusion culture is carried out for 5-6 days, depositing the microcarrier and the cells, replacing a culture medium with a main medium, vaccinating encephalitis B viruses, and continuously carrying out the perfusion culture; and collecting a virus culture solution, filtering, inactivating and purifying the virus culture solution. By using the method, the density of the cells for production is improved; the production capacity is improved greatly; the large-scale and high-density culture is achieved; the difference between batches of products and the instability of a culture period are eliminated; the quality of the products is guaranteed to be stable and uniform; the product quality is improved; the difficult problems of the amplification culture process of the bioreactor are solved; the high-density and large-scale culture of mammalian cells by using the bioreactor is achieved.
Description
Technical field
The present invention relates to a kind of production method of Vaccinum Encephalitis B, be specifically related to a kind of method of utilizing bioreactor to produce the inactivation of viruses Vaccinum Encephalitis B.
Background technology
Traditional inactivated virus vaccine; For example the production technology of Vaccinum Encephalitis B adopts rolling bottle, fixed bed and Duo Tai mini-reactor parallel connection technology to come cultured cell and virus more; This technology exists many defectives such as inefficiency of production, unstable product quality, has limited the suitability for industrialized production and the clinical practice of viral vaccine.At present; The cultivation of adopting bioreactor technology to carry out cell and virus has become the focus of viral vaccine research and development in the world; Pasteur Institut uses the 500L bioreactor and has successfully industrially produced Vero cell rabies vaccine and poliomyelitis vaccine, has obtained remarkable economic efficiency and social benefit.Therefore, the technology of utilizing bioreactor to produce Vaccinum Encephalitidis Epidemicae is carried out deep research, and then realize that suitability for industrialized production has broad prospects and positive meaning.
Summary of the invention
Therefore, the purpose of this invention is to provide a kind of method of utilizing bioreactor to produce the inactivation of viruses Vaccinum Encephalitis B, the production capacity of this method significantly improves and product quality stable uniform more.
Be used to realize that the technical scheme of above-mentioned purpose is following:
A kind of production method of Vaccinum Encephalitis B, this production method may further comprise the steps:
(1) the Vero seed cell is added in 300 liters of bioreactors, carry out perfusion with microcarrier and cultivate, wherein culture medium comprises the M199 (GIBCO culture medium handbook is seen in the constituent of M199 dehydrated medium) of 9.55g/L, the NaHCO of 2.2g/L
3, 1.5g/L the hyclone of glutamine and 10% (volume) of glucose, 0.2g/L; Condition of culture comprises: mixing speed is 35～40 rev/mins, and pH is 7.2～7.4, and dissolved oxygen is 40～50% saturations, and temperature is 36.5～37.5 ℃, and the perfusion rate of culture medium is 315 liters/day;
(2) perfusion was cultivated after 5～6 days, sedimentation microcarrier and cell, and to keep liquid replacement culture medium, the inoculation encephalitis b virus is proceeded perfusion and is cultivated, and wherein keeps the M199 culture medium that liquid comprises 9.55g/L, the NaHCO of 2.2g/L
3, the sucrose of 1.0g/L and the human albumin of 0.1g/L; Condition of culture comprises: mixing speed is 35～40 rev/mins, and pH is 7.5～7.6, and dissolved oxygen is 40～50% saturations, and temperature is 33.5～34.5 ℃, and the perfusion rate of keeping liquid is 315 liters/day;
(3) collect virus-culturing fluid, filtration, deactivation and purification.
In aforementioned production method, preferably, encephalitis b virus is 0.01 inoculation according to infection multiplicity (MOI).
In aforementioned production method, the method for preparing of Vero seed cell can may further comprise the steps: with the recovery the Vero cell successively at 175cm
2Cultivate in square vase, 2 liters of rolling bottles, 14 liters of bioreactors and 75 liters of bioreactors.
Preferably, 175cm
2Culture medium in the square vase is the M199 culture medium that comprises the hyclone of 10% (volume); Condition of culture comprises: temperature is 37 ℃, feeds the CO of 5% (volume)
2More preferably, 175cm
2Cultural method in the square vase is after the cell that liquid nitrogen is preserved is melted rapidly, by 1 * 10
5/ cm
2Inoculum density change 175cm over to
2In the square vase, drip culture medium and at the CO of 37 ℃ and 5% (volume)
2Following cultivation digests after growing to fine and close monolayer, and the inoculative proportion with 1: 4 (volume) will be added to through the cell suspension that digestion obtains in the new culture medium again, at the CO of 37 ℃ and 5% (volume)
2Under cultivate 3～4 days after, be added in the new culture medium with 1: 4 (volume) inoculative proportion again, at the CO of 37 ℃ and 5% (volume)
2Under cultivated 3～4 days.
Preferably, the culture medium in 2 liters of rolling bottles comprises the M199 culture medium of 9.55g/L, the NaHCO of 1.8g/L
3, 1.5g/L the hyclone of glutamine and 10% (volume) of glucose, 0.2g/L; Condition of culture comprises: temperature is 37 ℃, and rotating speed is 1/8 rev/min.More preferably, the inoculum density of cultivating in 2 liters of rolling bottles is 1.0～2.0 * 10
5/ cm
2, incubation time is 3～4 days.
Preferably, the culture medium in 14 liters and 75 bioreactors comprises the M199 culture medium of 9.55g/L, the NaHCO of 2.2g/L
3, 1.5g/L the hyclone of glutamine and 10% (volume) of glucose, 0.2g/L; Condition of culture comprises: mixing speed is 35～40 rev/mins, and pH is 7.2～7.4, and dissolved oxygen is 40～50% saturations, and temperature is 36.5～37.5 ℃, and the perfusion rate of culture medium is 315 liters/day, and adds microcarrier.
In aforementioned production method, preferably, filter to comprise being the membrane filtration of 1 μ m and 0.45 μ m successively through the aperture, be the filter membrane ultrafiltration of 300KD through molecular cut off again.
In aforementioned production method, preferably, the inactivator of deactivation is a beta-propiolactone.Beta-propiolactone (BPL) uses as inactivator in the production of various human and animal vaccine at present widely.It can be decomposed into nontoxic hydracrylic acid fully in body, this is the product after a kind of body fat mass in human body metabolism, and is nontoxic to human body and animal.BPL is a kind of avirulent liquid, can be hydrolyzed to a kind of nontoxic material at 37 ℃ voluntarily after following 2 hours, also can add sodium sulfite and stop its reaction.BPL is very capable to the deactivation of virus, can also keep simultaneously the good immunogenicity of virus, adds that its hydrolyzate acetone acid is harmless, formally uses so just be selected as the inactivator input of mad dog inactivated vaccine as far back as 1984.
In aforementioned production method, preferably, the method for purification is sieve chromatography and/or ion exchange membrane chromatography.
In aforementioned production method, the consumption of microcarrier is every liter of culture medium 5.0g.Microcarrier in the method for the present invention is preferably the Cytodex 1 of GE company.At present, Cytodex 1 has been widely used in production of vaccine, all adopts Cytodex 1 to carry out extensive adhere-wall culture like rabies vaccine, poliomyelitis vaccine, influenza vaccines etc., and its safety is fully proved.Through the phosphate buffer solution immersion treatment, method for using was with reference to the operation instruction of microcarrier Cytodex 1 in detail before this microcarrier used.
Compare with traditional production process, production method of the present invention has following clear superiority because of successfully adopting bioreactor large-scale culture VERO cell and virus:
1, the cell density that is used to produce improves, and has improved production capacity greatly, has realized extensive, High Density Cultivation.
Because the conditionally complete in the bioreactor is controlled, and adopt low-shearing force to stir and still breather, help guaranteeing that cell growth and virus breeding are in the suitable environment always.Bioreactor culture VERO cell, (density can reach 4 * 10 to the microcarrier of use 5g/L
6Individual/ml), the TCS in the bioreactor of 10L working volume can reach 4 * 10
10, be equivalent to 400 2L rolling bottle cultured cells sums.The cultured cells sum is equivalent to 300 15L rolling bottle cultured cells quantity in the 300L bioreactor.
2, production technology of the present invention has been eliminated the unstability of product batches differences and cultivation cycle, guarantees the stable uniform of product quality, has also improved product quality.
The technology that viral vaccine is produced in rolling bottle, fixed bed and the parallel connection of Duo Tai bioreactor all inevitably exists batch differences big, and the problem of poor stability is difficult to guarantee product quality homogeneity.Result of study of the present invention shows; All there is evident difference in the encephalitis b P3 strain of rolling bottle and bioreactor culture at benefit knot antigen and virus titer; The virus titer of bioreactor culture and benefit knot antigen are apparently higher than rolling bottle, and the benefit knot antigen that rolling bottle is cultivated is merely the benefit of bioreactor culture and ties 1/2 of antigen amount.
3, solve the difficult problem of bioreactor amplification culture technology, realized bioreactor high density, large-scale culture mammalian cell.
Utilizing bioreactor high density, large-scale culture mammalian cell always is that the bottleneck of vaccine research and production, especially rolling bottle and fixed bed training method can't realize the technology amplification, is devoted to the research of this respect in the last few years both at home and abroad always.The present invention is through groping to the amplification step by step of 300L bioreactor from rolling bottle, 14L, 75L; Confirm the optimization production condition of macro-organism reaction vessel cultured cell and propagative viruses, really realized utilizing bioreactor high density, large-scale culture mammalian cell.
4, successive perfusion is cultivated homogeneity and the stability that has guaranteed virus stock solution used.
Abroad at present batch culture virus and modes of in batches gathering in the crops of adopting more; Domestic rolling bottle or many small-sized bioreactor viruses of cultivating in parallel of adopting more; The mode that merges the viral liquid of results then, this is difficult to solve, and batch differences is big, the problem of unstable product quality.The mode of continuous perfusion, continuous results is adopted in this research, has both guaranteed the homogeneity of product, and the virus stock solution used of results helps keeping antigenic stable again 4 ℃ of following preservations.
Description of drawings
Below, specify embodiment of the present invention in conjunction with accompanying drawing, wherein:
Fig. 1: the virus titer curve of different incubation times in 300 liters of bioreactors;
Fig. 2: the virus titer curve of different incubation times in 2 liters of rolling bottles.
The specific embodiment
Below in conjunction with the specific embodiment the present invention is further described in detail, the embodiment that provides has been merely and has illustrated the present invention, rather than in order to limit scope of the present invention.
Embodiment 1
1, the recovery of VERO cell seed
1.1VERO the source of cell strain
The primary source of VERO cell is ATCC (being numbered F-12313), has set up master cell bank and working cell storehouse through the amplification of going down to posterity, and has been stored in the liquid nitrogen.Chief cell was 129 generations; Working cell was 133 generations, and frozen density is 4 * 10
6/ ml.
1.2VERO the recovery of seed cell
Get one in the interior work storehouse cell of preserving of liquid nitrogen container, after 39 ℃ of warm water melt rapidly, cell suspension is pressed about 1 * 10
5/ cm
2Inoculum density changes 175cm over to
2In the square vase, (M199 (GIBCO culture medium handbook is seen in the constituent of M199 dehydrated medium) that contains 10% (volume) hyclone is to 60mL, 37 ℃ and 5%CO progressively to drip culture medium
2Cultivate in the incubator.
2, at 175cm
2Cultivate the first order seed cell in the square vase
The cell culture of recovery grew to fine and close monolayer in 3～4 days; Use the digestion of 0.25% (g/ml) trypsinization liquid to disperse; Inoculative proportion with 1: 4 (volume) forwards cell suspension in the new culture bottle to again, adds the culture medium (60ml) of appropriate amount, 37 ℃, 5%CO
2Cultivate in the incubator after 3～4 days that amplification culture is once again with the ratio of 1: 4 (volume).
3, in the 2L rolling bottle, cultivate the secondary seed cell
3.1 culture medium
M199 culture medium 9.55g/L, NaHCO
31.8g/L, glucose 1.5g/L, glutamine 0.2g/L, 10% (volume) hyclone.
3.2 rolling bottle is cultivated
Select 2L rolling bottle and four layers of Rotary Machine, keeping inoculum density is 1.0～2.0 * 10
5/ cm
2, the culture medium of adding 500～600ml, it is 37 ℃ that temperature is set, rotating speed is 1/8 rev/min, cultivates 3～4 days.
4, in the 14L bioreactor, cultivate three grades of seed cells
4.1 culture medium
M199 culture medium 9.55g/L, NaHCO
32.2g/L, glucose 1.5g/L, glutamine 0.2g/L, 10% (volume) hyclone.
4.2 the selection of reaction vessel
Select NBS or Sartorius mammalian cell culture tank, tank volume is 14L, working volume 10L.
4.3 microcarrier is selected
Cytodex 1 microcarrier (GE company), consumption is 5.0g/L.
4.4 last jar of inoculation
With cultivating 3～4 days VERO cell in the rolling bottle after digestion, be transferred in the blue lid bottle, piping and druming is uniformly dispersed, and inserts in the lump in the 14L bioreactor, adds culture medium to 10L.
4.5 parameter control
Speed of agitator: 35～40RPM; PH:7.2～7.4; Dissolved oxygen content (DO): 40-50% saturation; Temperature: 36.5～37.5 ℃.
4.6 perfusion control
Inoculate and open perfusion after 12 hours, perfusion flow is working volumes every days 1.5, cultivates 5～6 days.
5, in the 75L bioreactor, cultivate the level Four seed cell
5.1 the same 4.1. of culture medium
5.2 reaction vessel is selected
Select NBS or Sartorius mammalian cell culture tank, tank volume is 75L, working volume 50L.
5.3 microcarrier method for using and consumption are with 4.3
5.4 change a jar inoculation
The cell of cultivating in the 14L bioreactor 5～6 days is digested with 0.25% (g/ml) trypsinization liquid (EDTA that contains 0.01% (g/ml)) jar is outer; The stirring vibrating dispersion is even; After pancreatin inhibitor (Gibco) cessation reaction; Change cell over to the 75L bioreactor in the lump together with microcarrier through pipeline, be settled to 50L.
5.5 parameter control
Speed of agitator: 35～40RPM; PH:7.2～7.4; Dissolved oxygen content (DO): 40～50% saturations; Temperature: 36.5～37.5 ℃.
5.6 perfusion control
Inoculate and open perfusion after 12 hours, perfusion flow is working volumes every days 1.5, cultivates 5～6 days.
6, large-scale culture VERO cell and virus in the 300L bioreactor
6.1 culture medium with keep formula of liquid
The same 4.1. of culture medium
Keep liquid: M199 culture medium 9.55g/L, NaHCO
32.2g/L, sucrose 1.0g/L, human albumin 0.1g/L.
6.2 reaction vessel is selected
Selection is than Europe or Sartorius mammalian cell culture tank, and tank volume is 300L, working volume 210L.
6.3 the microcarrier consumption is with 4.3
6.4 change jar
The cell of cultivating in the 75L bioreactor 5～6 days is digested with 0.25% (g/ml) trypsinization liquid (EDTA that contains 0.01% (g/ml)) jar is outer; The stirring vibrating dispersion is even; After pancreatin inhibitor (Gibco) cessation reaction; Change cell over to the 300L bioreactor in the lump together with microcarrier through pipeline, be settled to 210L.
6.5 culture parameters is with 4.5.
6.6 perfusion control
Inoculate and open perfusion after 24 hours, perfusion flow is working volumes every days 1.5, cultivates 5～6 days.
7, inoculation encephalitis b P3 strain virus is cultivated
7.1 connect malicious method
Suspend the control parameter, sedimentation microcarrier and cell are taken out the supernatant culture medium, are replaced by and keep liquid, treat behind the parameter stability according to MOI to be 0.01 virus inoculation.
7.2 parameter control
Speed of agitator: 35～40RPM; PH:7.5～7.6; DO:40～50% saturation; Temperature: 33.5～34.5 ℃.
7.3 the mode with continuous perfusion is gathered in the crops virus-culturing fluid
Connect poison and begin perfusion after 12 hours, perfusion flow is working volumes every days 1.5, begins to gather in the crops viral liquid in 48 hours from connecing poison, gathers in the crops continuously 8 days.
8, the clarification filtration and the ultrafiltration and concentration of virus results liquid
8.1 clarification filtration
The selective membrane area is the polyether sulfone filter element series connection of the 1 μ m and the 0.5 μ m of 1 square metre (20 inches), and elder generation is through 1 μ m filter membrane, again through 0.45 μ m membrane filtration.
8.2 ultrafiltration and concentration
8.2.1 buffer is selected
Select the good ultrafiltration system of (the aseptic phosphoric acid of pH7.2-8.0) buffer flushing balance.
8.2.2 choice of equipment
Use 0.5 square metre of PELLICON 2 ultrafiltration system of Millipore company, 2 PELLICON 2 ultrafilter membrane bags are installed, molecular cut off is the filter membrane of 300KD.
8.2.3 operating parameter
Setting inlet pressure is 10～20psi, and refluxing opening pressure is 5～10psi, accomplishes the ultrafiltration of 80～120L virus results liquid in 2～4 hours; 20～40 times of sample concentration obtain 2～6L concentrated solution.
9, inactivation of virus:
9.1 the preparation of deactivation reagent
Earlier beta-propiolactone is diluted to 40 times mother solution with water for injection, through 0.22 μ m membrane filtration degerming.
9.2 deactivation operation
With prediluted beta-propiolactone mother solution add in the concentrated solution to final concentration be 1/4000,2～8 ℃ of held deactivations, after 24 hours 37 ℃ of following hydrolysis 2 hours.
10, sieve chromatography
10.1 the selection of buffer
The phosphate buffer of pH 7.5～8.5.
10.2 filler is selected
The Sephacryl S-300 chromatographic stuffing of GE company, dress post height is 20～60cm.
10.3 chromatography operation
At first use the buffer balance chromatographic column of 2 times of column volumes, flow velocity is 20～50cm/h, and the sample volume of last appearance is 8%～12% column volume, results first peak (UV-detector of wavelength 280nm).
11, ion exchange membrane chromatography:
11.1 the selection of buffer
Buffer A: pH 7.5～8.5 phosphate buffers, buffer B: pH 7.5～8.0 phosphate buffers add 500mMol/L sodium chloride.
11.2 the selection of ion exchange membrane
The Sartobind Q ion-exchange chromatography film of Sartorius company.
11.3 chromatography operation
Elder generation is with the buffer A balance chromatographic film of 10 times of volumes; The antigen samples of last appearance behind the sieve chromatography purification; With buffer A (volume ratio: 100%-0%) and buffer B (volume ratio: the linear gradient elution that 0%-100%) forms, the eluent of collecting between the 70mM to 250mM is a stock solution behind the purification.
12, aluminium adjuvant original position adsorption antigen
12.1 reagent
1mol/L NaOH solution, the PBS of 1mol/L pH8.5, the AlCl of 0.2mol/L
3Solution, the NaCl solution of 1mol/L, filtration sterilization.
12.2 adsorption method
After the antigen filtration sterilization behind the purification, it is 0.05mol/L that adding 1mol/L pH8.5 PBS makes its phosphate concn, stirs the NaOH solution of adding 1mol/L and the AlCl of 0.2mol/L then
3Solution, keeping the pH of system in the course of reaction is about 7.0, the Al in the reaction system (OH)
3When being 2.0～2.5mg/ml, amount ends.
12.3 dilution
Using NaCl solution and the water for injection dilution benefit of 1mol/L is 1: 2～1: 4 to connecing the antigen amount, Al (OH)
3Content is 0.5～0.75mg/ml, and NaCl concentration is 0.14～0.16mol/L.
12.4 adding additive: 1% (g/ml) glycine and 5% (g/ml) sucrose.
12.5 semi-finished product detect: sterility test.
Embodiment 2
1. the product of production method of the present invention preparation batch between stability study
The testing result of 040801, the 040802 and 040,901 3 batch of pilot scale being cultivated stock solution shows, the virus titer of three lot sample article results liquid, mend tie that antigen, albumen are residual, sterility test and effectiveness all do not have significant difference (seeing table 1).
The verification result of results liquid is cultivated in three batches of pilot scales of table 1
2. cultivate the research of VERO cell and breeding encephalitis b P3 strain in the rolling bottle
The VERO cell inserts the 2L square vase after square vase goes down to posterity, carry out amplification culture in 1: 4 ratio subsequently, cultivates three batches, 16 bottles every batch.Cell culture condition is: culture medium and the prescription of keeping liquid are with above-mentioned 6.1, and temperature is 37 ℃, and rotating speed is 1/8 rev/min.Cell covers with and forms the homogeneous monolayer after 72 hours, and the sucking-off growth-promoting media adds keeps liquid, by 10
-3(volume) concentration inserts encephalitis b virus, continues to cultivate, and it is 35 ℃ that temperature is set, and rotating speed is 1/8 rev/min, whenever at a distance from 24 hours results once, gathers in the crops altogether 4 times later on, merges culture fluid at last.
Table 2 three is write instructions and transfer the verification result of the results liquid of flask culture
3 virus multiplication CURVE STUDY
In the propagative viruses process, bioreactor whenever at a distance from sampling in 24 hours once, detected virus titer and antigen since 48 hours, and rolling bottle is cultivated the back sampling of each results and detected virus titer and antigen.The result shows: in bioreactor propagative viruses process, virus titer and antigen all keep relative stability, and are higher than titre and antigen (seeing Fig. 1 and Fig. 2) that rolling bottle is cultivated.
The benefit knot antigen amount of the virus of breeding in 4 rolling bottles and the bioreactor differs greatly
Mending the conjugated antigen testing result shows; The virus of breeding in rolling bottle and the bioreactor is mended knot antigen and was respectively 1: 2～1: 4 and 1: 4～1: 8; It is 1: 2 and 1: 4 that amalgamation liquid is mended knot antigen, and with regard to the virus stock solution used of the antigen needs of isodose, rolling bottle is the twice of bioreactor.
Claims (12)
1. the production method of a Vaccinum Encephalitis B, this production method may further comprise the steps:
(1) the Vero seed cell is added in 300 liters of bioreactors, carry out perfusion with microcarrier and cultivate, wherein culture medium comprises the M199 culture medium of 9.55g/L, the NaHCO of 2.2g/L
3, 1.5g/L the hyclone of glutamine and 10% (volume) of glucose, 0.2g/L; Condition of culture comprises: mixing speed is 35～40 rev/mins, and pH is 7.2～7.4, and dissolved oxygen is 40～50% saturations, and temperature is 36.5～37.5 ℃, and the perfusion rate of culture medium is 315 liters/day;
(2) perfusion was cultivated after 5～6 days, sedimentation microcarrier and cell, and to keep liquid replacement culture medium, the inoculation encephalitis b virus is proceeded perfusion and is cultivated, and wherein keeps the M199 culture medium that liquid comprises 9.55g/L, the NaHCO of 2.2g/L
3, the sucrose of 1.0g/L and the human albumin of 0.1g/L; Condition of culture comprises: mixing speed is 35～40 rev/mins, and pH is 7.5～7.6, and dissolved oxygen is the 40-50% saturation, and temperature is 33.5-34.5 ℃, and the perfusion rate of keeping liquid is 315 liters/day;
(3) collect virus-culturing fluid, filtration, deactivation and purification.
2. production method according to claim 1 is characterized in that, said encephalitis b virus is 0.01 inoculation according to infection multiplicity.
3. production method according to claim 1 and 2 is characterized in that, the method for preparing of said Vero seed cell may further comprise the steps: with the recovery the Vero cell successively at 175cm
2Cultivate in square vase, 2 liters of rolling bottles, 14 liters of bioreactors and 75 liters of bioreactors.
4. production method according to claim 3 is characterized in that, said 175cm
2Culture medium in the square vase is the M199 culture medium that comprises the hyclone of 10% (volume); Condition of culture comprises: temperature is 37 ℃, feeds the CO of 5% (volume)
2
5. according to claim 3 or 4 described production methods, it is characterized in that said 175cm
2Cultural method in the square vase is after the cell that liquid nitrogen is preserved is melted rapidly, by 1 * 10
5/ cm
2Inoculum density change 175cm over to
2In the square vase, drip culture medium and at the CO of 37 ℃ and 5% (volume)
2Following cultivation digests after growing to fine and close monolayer, and the inoculative proportion with 1: 4 (volume) will be added to through the cell suspension that digestion obtains in the new culture medium again, at the CO of 37 ℃ and 5% (volume)
2Under cultivate 3～4 days after, be added in the new culture medium with 1: 4 (volume) inoculative proportion again, at the CO of 37 ℃ and 5% (volume)
2Under cultivated 3～4 days.
6. according to each described production method in the claim 3 to 5, it is characterized in that the culture medium in said 2 liters of rolling bottles comprises the M199 culture medium of 9.55g/L, the NaHCO of 1.8g/L
3, 1.5g/L the hyclone of glutamine and 10% (volume) of glucose, 0.2g/L; Condition of culture comprises: temperature is 37 ℃, and rotating speed is 1/8 rev/min.
7. according to each described production method in the claim 3 to 6, it is characterized in that the inoculum density of cultivating in said 2 liters of rolling bottles is 1.0～2.0 * 10
5/ cm
2, incubation time is 3～4 days.
8. according to each described production method in the claim 3 to 7, it is characterized in that the culture medium in said 14 liters and 75 bioreactors comprises the M199 culture medium of 9.55g/L, the NaHCO of 2.2g/L
3, 1.5g/L the hyclone of glutamine and 10% (volume) of glucose, 0.2g/L; Condition of culture comprises: mixing speed is 35～40 rev/mins, and pH is 7.2～7.4, and dissolved oxygen is 40～50% saturations, and temperature is 36.5～37.5 ℃, and the perfusion rate of culture medium is 315 liters/day, and adds microcarrier.
9. according to each described production method in the claim 1 to 8, it is characterized in that said filtration comprises being the membrane filtration of 1 μ m and 0.45 μ m successively through the aperture, is the filter membrane ultrafiltration of 300KD through molecular cut off again.
10. according to each described production method in the claim 1 to 9, it is characterized in that the inactivator of said deactivation is a beta-propiolactone.
11., it is characterized in that the method for said purification is sieve chromatography and/or ion exchange membrane chromatography according to each described production method in the claim 1 to 10.
12., it is characterized in that the consumption of said microcarrier is every liter of culture medium 5.0g according to each described production method in the claim 1 to 11.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105548536A (en) * | 2015-12-08 | 2016-05-04 | 天津瑞普生物技术股份有限公司 | Method for preparing positive serum of antibody against porcine Japanese encephalitis virus |
| CN106754762A (en) * | 2016-11-22 | 2017-05-31 | 中牧实业股份有限公司 | A kind of antigen of encephalitis B live vaccine and preparation method and application |
| CN111569055A (en) * | 2020-05-29 | 2020-08-25 | 成都康华生物制品股份有限公司 | Production method and equipment of human influenza vaccine |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101028514A (en) * | 2006-06-30 | 2007-09-05 | 辽宁成大生物股份有限公司 | Method for producing vaccine for man by biological reactor |
| CN101062411A (en) * | 2006-04-24 | 2007-10-31 | 江苏延申生物科技股份有限公司 | Method for large-scale production of influenza vaccine by using bioreactor |
| CN101732708A (en) * | 2009-11-10 | 2010-06-16 | 浙江天元生物药业股份有限公司 | Preparation method of vaccine for epidemic encephalitis B |
-
2010
- 2010-08-27 CN CN201010264218.XA patent/CN102327609B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101062411A (en) * | 2006-04-24 | 2007-10-31 | 江苏延申生物科技股份有限公司 | Method for large-scale production of influenza vaccine by using bioreactor |
| CN101028514A (en) * | 2006-06-30 | 2007-09-05 | 辽宁成大生物股份有限公司 | Method for producing vaccine for man by biological reactor |
| CN101732708A (en) * | 2009-11-10 | 2010-06-16 | 浙江天元生物药业股份有限公司 | Preparation method of vaccine for epidemic encephalitis B |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105548536A (en) * | 2015-12-08 | 2016-05-04 | 天津瑞普生物技术股份有限公司 | Method for preparing positive serum of antibody against porcine Japanese encephalitis virus |
| CN105548536B (en) * | 2015-12-08 | 2018-10-16 | 天津瑞普生物技术股份有限公司 | A kind of preparation method of Latex agglutination test Positive Sera |
| CN106754762A (en) * | 2016-11-22 | 2017-05-31 | 中牧实业股份有限公司 | A kind of antigen of encephalitis B live vaccine and preparation method and application |
| CN111569055A (en) * | 2020-05-29 | 2020-08-25 | 成都康华生物制品股份有限公司 | Production method and equipment of human influenza vaccine |
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