CN102327609B - Production method of encephalitis B vaccine - Google Patents
Production method of encephalitis B vaccine Download PDFInfo
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- CN102327609B CN102327609B CN201010264218.XA CN201010264218A CN102327609B CN 102327609 B CN102327609 B CN 102327609B CN 201010264218 A CN201010264218 A CN 201010264218A CN 102327609 B CN102327609 B CN 102327609B
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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 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 technique exists many defects such as inefficiency of production, unstable product quality, has limited suitability for industrialized production and the clinical practice of viral vaccine.At present, employing bioreactor technology carries out cell and viral cultivation has become the focus of viral vaccine research and development in the world, Pasteur Institut's application 500L bioreactor has successfully industrially produced Vero cell rabies vaccine and poliomyelitis vaccine, has obtained significant economic benefit and social benefit.Therefore, the technique of utilizing bioreactor to produce Vaccinum Encephalitidis Epidemicae is carried out to deep research, and then realize that suitability for industrialized production has broad prospects and positive meaning.
Summary of the invention
Therefore, the object of this invention is to provide a kind of method of utilizing bioreactor to produce inactivation of viruses Vaccinum Encephalitis B, the production capacity of the method significantly improves and product quality stable uniform more.
As follows for realizing the technical scheme of above-mentioned purpose:
A production method for Vaccinum Encephalitis B, this production method comprises the following steps:
(1) Vero seed cell is added in 300 liters of bioreactors, carry out perfusion cultivation together with microcarrier, the NaHCO of the M199 that wherein culture medium comprises 9.55g/L (GIBCO culture medium handbook is shown in the constituent of M199 dehydrated medium), 2.2g/L
3, the glucose of 1.5g/L is, the hyclone of the glutamine of 0.2g/L and 10% (volume/volume); Condition of culture comprises: mixing speed is 35～40 revs/min, 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, and sedimentation microcarrier and cell are replaced culture medium with maintenance medium, and inoculation encephalitis b virus is proceeded perfusion and cultivated, the M199 culture medium that wherein maintenance medium 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 revs/min, 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 maintenance medium is 315 liters/day;
(3) collect virus-culturing fluid, filtration, deactivation purification.
In aforementioned production method, preferably, encephalitis b virus is 0.01 inoculation according to infection multiplicity (MOI).
In aforementioned production method, the preparation method of Vero seed cell can comprise the following steps: by recovery Vero cell successively at 175cm
2in square vase, 2 liters of rolling bottles, 14 liters of bioreactors and 75 liters of bioreactors, cultivate.
Preferably, 175cm
2the M199 culture medium that culture medium in square vase is the hyclone that comprises 10% (volume/volume); Condition of culture comprises: temperature is 37 ℃, passes into the CO of 5% (volume/volume)
2.More preferably, 175cm
2after cultural method in square vase is rapid thawing of cell that liquid nitrogen is preserved, by 1 * 10
5/ cm
2inoculum density proceed to 175cm
2in square vase, drip culture medium and at the CO of 37 ℃ and 5% (volume/volume)
2lower cultivation, digests after growing to fine and close monolayer, then with the inoculative proportion of 1: 4 (volume/volume), the cell suspension obtaining through digestion is added in new culture medium, at the CO of 37 ℃ and 5% (volume/volume)
2lower cultivation is after 3～4 days, then is added in new culture medium with 1: 4 (volume/volume) inoculative proportion, at the CO of 37 ℃ and 5% (volume/volume)
2lower cultivation 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, the glucose of 1.5g/L is, the hyclone of the glutamine of 0.2g/L and 10% (volume/volume); 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, the glucose of 1.5g/L is, the hyclone of the glutamine of 0.2g/L and 10% (volume/volume); Condition of culture comprises: mixing speed is 35～40 revs/min, 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 through aperture, being the membrane filtration of 1 μ m and 0.45 μ m successively, then the filter membrane ultrafiltration that is 300KD through molecular cut off.
In aforementioned production method, preferably, the inactivator of deactivation is beta-propiolactone.Beta-propiolactone (BPL) is now used as inactivator widely in the production of various human and animal vaccine.It can be decomposed into nontoxic hydracrylic acid completely in body, and this is the product after a kind of body fat mass in human body metabolism, nontoxic to human body and animal.BPL is a kind of avirulent liquid, at 37 ℃, after 2 hours, can be hydrolyzed to voluntarily a kind of nontoxic material, also can add sodium sulfite to stop its reaction.BPL is very capable to viral deactivation, can also keep the good immunogenicity of virus simultaneously, adds that its hydrolyzate acetone acid is harmless, therefore be just selected as the inactivator of mad dog inactivated vaccine as far back as 1984, drops into formal use.
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 method of the present invention is preferably the Cytodex 1 of GE company.At present, Cytodex 1 has been widely used in production of vaccine, and as rabies vaccine, poliomyelitis vaccine, influenza vaccines etc. all adopt Cytodex 1 to carry out extensive adhere-wall culture, its safety is fully proved.Before this microcarrier is used, through phosphate buffer solution immersion treatment, using method is with reference to the operation instruction of microcarrier Cytodex 1 in detail.
Compare with traditional production technology, production method of the present invention is because successfully adopting bioreactor large-scale culture VERO cell and virus to have following clear superiority:
1, for the production of cell density improve, greatly improved production capacity, realized extensive, High Density Cultivation.
Because the conditionally complete in bioreactor is controlled, and adopt low-shearing force to stir and still breather, be conducive to guarantee that Growth of Cells and virus breeding are always in suitable environment.Bioreactor culture VERO cell, (density can reach 4 * 10 to the microcarrier of use 5g/L
6individual/ml), the total cellular score in the bioreactor of 10L working volume can reach 4 * 10
10, be equivalent to the total cellular score of 400 2L spinner cultures.In 300L bioreactor, cultured cells sum is equivalent to the cell quantity of 300 15L spinner cultures.
2, production technology of the present invention has been eliminated the unstability of difference and cultivation cycle between product batches, guarantees the stable uniform of product quality, has also improved product quality.
Between rolling bottle, fixed bed and Duo Tai bioreactor technique of producing viral vaccine in parallel all inevitably exists batch, difference is large, and the problem of poor stability is difficult to guarantee the homogeneity of product quality.Result of study of the present invention shows, at benefit knot antigen and virus titer, all there is obvious difference in the encephalitis b P3 strain of rolling bottle and bioreactor culture, the virus titer of bioreactor culture and benefit knot antigen are apparently higher than rolling bottle, and the benefit of spinner culture knot antigen is only that the benefit of bioreactor culture is tied 1/2 of antigen amount.
3, solve a difficult problem for Bioreactor scaleup culture process, realized bioreactor high density, large-scale culture mammalian cell.
Utilizing bioreactor high density, large-scale culture mammalian cell is always that the bottleneck of vaccine research and production, especially rolling bottle and fixed bed training method cannot realize technique amplification, is devoted in the last few years the research of this respect 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, determined the optimization working condition of macro-organism bioreactor culture cell and propagative viruses, really realized and utilized bioreactor high density, large-scale culture mammalian cell.
4, continuous perfusion is cultivated homogeneity and the stability that has guaranteed virus stock solution used.
External many employing batch culture virus and the in batches mode of results at present, domestic rolling bottle or many small-sized biological reactor viruses of cultivating in parallel of adopting more, then the mode that merges results virus liquid, this be difficult to solve batch between the problem of large, the unstable product quality of difference.This research adopts the mode of continuous perfusion, continuous results, has both guaranteed the homogeneity of product, and the virus stock solution used of results is preserved and is conducive to again keep the stable of antigen at 4 ℃.
Accompanying drawing explanation
Below, describe by reference to the accompanying drawings embodiment of the present invention in detail, 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 providing is only in order to illustrate the present invention, rather than in order to limit the scope of the invention.
1, the recovery of VERO cell seed
The source of 1.1VERO cell strain
The primary source of VERO cell is ATCC (being numbered F-12313), has set up master cell bank and working cell storehouse, and be stored in liquid nitrogen through the amplification of going down to posterity.Chief cell was 129 generations; Working cell was 133 generations, and frozen density is 4 * 10
6/ ml.
The recovery of 1.2VERO seed cell
Get one, the interior work storehouse cell of preserving of liquid nitrogen container, after 39 ℃ of warm water melt rapidly, cell suspension is pressed to approximately 1 * 10
5/ cm
2inoculum density proceeds to 175cm
2in square vase, progressively drip culture medium and (contain the M199 (GIBCO culture medium handbook is shown in the constituent of M199 dehydrated medium) of 10% (volume/volume) hyclone to 60mL, 37 ℃ and 5%CO
2in incubator, cultivate.
2, at 175cm
2in square vase, cultivate first order seed cell
The cell culture of recovery grows to fine and close monolayer for 3～4 days, use the digestion of 0.25% (g/ml) trypsinization liquid to disperse, inoculative proportion with 1: 4 (volume/volume) forwards cell suspension in new culture bottle to again, adds the culture medium (60ml) of appropriate amount, 37 ℃, 5%CO
2in incubator, cultivate after 3～4 days that amplification culture is once again with the ratio of 1: 4 (volume/volume).
3, in 2L rolling bottle, cultivate 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/volume) hyclone.
3.2 spinner culture
Select 2L rolling bottle and four layers of Rotary Machine, keeping inoculum density is 1.0～2.0 * 10
5/ cm
2, adding the culture medium of 500～600ml, set temperature is 37 ℃, rotating speed is 1/8 rev/min, cultivates 3～4 days.
4, in 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/volume) hyclone.
The selection of 4.2 reactors
Select NBS or Sartorius mammaliancellculture tank, tank volume is 14L, working volume 10L.
4.3 microcarriers are selected
Cytodex 1 microcarrier (GE company), consumption is 5.0g/L.
Tank inoculation on 4.4
By cultivating the VERO cell of 3～4 days in rolling bottle after digestion, be transferred in blue lid bottle, piping and druming is uniformly dispersed, and accesses in the lump in 14L bioreactor, adds culture medium to 10L.
4.5 parameters are controlled
Speed of agitator: 35～40RPM; PH:7.2～7.4; Dissolved oxygen content (DO): 40-50% saturation; Temperature: 36.5～37.5 ℃.
4.6 perfusions are controlled
Inoculate and open perfusion after 12 hours, perfusion flow is working volumes every days 1.5, cultivates 5～6 days.
5, in 75L bioreactor, cultivate level Four seed cell
The same 4.1. of 5.1 culture medium
5.2 reactors are selected
Select NBS or Sartorius mammaliancellculture tank, tank volume is 75L, working volume 50L.
5.3 microcarrier usings method and consumption are with 4.3
5.4 turn tank inoculation
0.25% (g/ml) trypsinization liquid for cell (containing the EDTA of 0.01% (g/ml)) tank of cultivating in 14L bioreactor 5～6 days is digested outward, stirring vibrating dispersion is even, with after pancreatin inhibitor (Gibco) cessation reaction, by pipeline, cell is proceeded to 75L bioreactor in the lump together with microcarrier, be settled to 50L.
5.5 parameters are controlled
Speed of agitator: 35～40RPM; PH:7.2～7.4; Dissolved oxygen content (DO): 40～50% saturations; Temperature: 36.5～37.5 ℃.
5.6 perfusions are controlled
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 300L bioreactor
6.1 culture medium and maintenance medium formula
The same 4.1. of culture medium
Maintenance medium: M199 culture medium 9.55g/L, NaHCO
32.2g/L, sucrose 1.0g/L, human albumin 0.1g/L.
6.2 reactors are selected
Selection is than Europe or Sartorius mammaliancellculture tank, and tank volume is 300L, working volume 210L.
6.3 microcarrier consumptions are with 4.3
6.4 turn tank
0.25% (g/ml) trypsinization liquid for cell (containing the EDTA of 0.01% (g/ml)) tank of cultivating in 75L bioreactor 5～6 days is digested outward, stirring vibrating dispersion is even, with after pancreatin inhibitor (Gibco) cessation reaction, by pipeline, cell is proceeded to 300L bioreactor in the lump together with microcarrier, be settled to 210L.
6.5 culture parameters are with 4.5.
6.6 perfusions are controlled
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 and control parameter, sedimentation microcarrier and cell, take out supernatant culture medium, is replaced by maintenance medium, after parameter stability, according to MOI, is 0.01 virus inoculation.
7.2 parameters are controlled
Speed of agitator: 35～40RPM; PH:7.5～7.6; DO:40～50% saturation; Temperature: 33.5～34.5 ℃.
7.3 gather in the crops virus-culturing fluid in the mode of continuous perfusion
Connect poison and start perfusion after 12 hours, perfusion flow is working volumes every days 1.5, from connecing malicious 48 hours, starts to gather in the crops virus liquid, gathers in the crops continuously 8 days.
8, the clarification filtration of virus harvest liquid and ultrafiltration and concentration
8.1 clarification filtration
Selective membrane area is the 1 μ m of 1 square metre (20 inches) and the series connection of the polyether sulfone filter element of 0.5 μ m, first 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 aseptic phosphoric acid of pH7.2-8.0) buffer to rinse the good ultrafiltration system of balance.
8.2.2 equipment is selected
Use 0.5 square metre of PELLICON 2 ultrafiltration system of Millipore company, 2 PELLICON, 2 ultrafilter membrane bags are installed, the filter membrane that molecular cut off is 300KD.
8.2.3 operating parameter
Setting inlet pressure is 10～20psi, and refluxing opening pressure is 5～10psi, completes the ultrafiltration of 80～120L virus harvest liquid in 2～4 hours; 20～40 times of sample concentration, obtain 2～6L concentrated solution.
9, inactivation of virus:
The preparation of 9.1 deactivation reagent
First beta-propiolactone is diluted to the mother solution of 40 times with water for injection, through 0.22 μ m membrane filtration degerming.
9.2 deactivation operations
It is to place deactivation at 1/4000,2～8 ℃ to final concentration that prediluted beta-propiolactone mother solution is added in concentrated solution, after 24 hours, is hydrolyzed 2 hours at 37 ℃.
10, sieve chromatography
The selection of 10.1 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
First the buffer balance chromatographic column that uses 2 times of column volumes, flow velocity is 20～50cm/h, the sample volume of loading is 8%～12% column volume, results first peak (UV-detector of wavelength 280nm).
11, Ion exchange membrane chromatography:
The selection of 11.1 buffer
Buffer A: pH 7.5～8.5 phosphate buffers, buffer B: pH 7.5～8.0 phosphate buffers add 500mMol/L sodium chloride.
The selection of 11.2 ion exchange membranees
The Sartobind Q ion-exchange chromatography film of Sartorius company.
11.3 chromatography operation
First use the buffer A balance chromatographic film of 10 times of volumes, the antigen samples of loading after sieve chromatography purification, by buffer A (volume ratio: 100%-0%) and buffer B (volume ratio: linear gradient elution 0%-100%) forming, the eluent of collecting between 70mM to 250mM is stock solution after purification.
12, aluminium adjuvant original position adsorption antigen
12.1 reagent
1mol/L NaOH solution, the phosphate buffered solution of 1mol/L pH8.5, the AlCl of 0.2mol/L
3solution, the NaCl solution of 1mol/L, filtration sterilization.
12.2 adsorption methods
By after the antigen filtration sterilization after purification, adding 1mol/L pH8.5 phosphate buffered solution to make its phosphate concn is 0.05mol/L, then stirs and adds the NaOH solution of 1mol/L and the AlCl of 0.2mol/L
3solution, in course of reaction, keeping the pH of system is 7.0 left and right, the Al in reaction system (OH)
3when being 2.0～2.5mg/ml, amount stops.
12.3 dilutions
By NaCl solution and the water for injection dilution benefit of 1mol/L, to connecing antigen amount, be 1: 2～1: 4, Al (OH)
3content is 0.5～0.75mg/ml, and NaCl concentration is 0.14～0.16mol/L.
12.4 add additive: 1% (g/ml) glycine and 5% (g/ml) sucrose.
12.5 semi-finished product detect: sterility test.
Embodiment 2
1. the product that prepared by production method of the present invention batch between stability study
040801,040802 and 040,901 3 batch of pilot scale is cultivated to the testing result demonstration of stock solution, the virus titer of three batch sample harvest liquids, benefit knot antigen, protein residue, sterility test and effect are all without significant difference (in Table 1).
The verification result of harvest liquid is cultivated in three batches of pilot scales of table 1
2. in rolling bottle, cultivate the research of VERO cell and breeding encephalitis b P3 strain
VERO cell accesses 2L square vase after square vase goes down to posterity, and carries out amplification culture subsequently in the ratio of 1: 4, cultivates three batches, 16 bottles every batch.Cell culture condition is: the formula of culture medium and maintenance medium is with above-mentioned 6.1, and temperature is 37 ℃, and rotating speed is 1/8 rev/min.After 72 hours, cell covers with and forms homogeneous monolayer, and sucking-off growth-promoting media adds maintenance medium, by 10
-3(volume/volume) concentration access encephalitis b virus, continues to cultivate, and set temperature is 35 ℃, and rotating speed is 1/8 rev/min, every 24 hours results once, gathers in the crops altogether 4 times later, finally merges culture fluid.
The verification result of the harvest liquid of three batches of spinner cultures of table 2
3 virus multiplication CURVE STUDY
In propagative viruses process, bioreactor, since 48 hours, every sampling in 24 hours once, detects virus titer and antigen, and after the each results of spinner culture, sampling detects virus titer and antigen.Result shows: in bioreactor propagative viruses process, virus titer and antigen all keep relative stability, higher than titre and the antigen (seeing Fig. 1 and Fig. 2) of spinner culture.
The viral benefit knot antigen amount of breeding in 4 rolling bottles and bioreactor differs greatly
Mending conjugated antigen testing result shows, the virus of breeding in rolling bottle and bioreactor is mended knot antigen and is 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 that the antigen of isodose needs, rolling bottle is the twice of bioreactor.
Claims (45)
1. a production method for Vaccinum Encephalitis B, this production method comprises the following steps:
(1) Vero seed cell is added in 300 liters of bioreactors, carry out perfusion cultivation together with microcarrier, the M199 culture medium that wherein culture medium comprises 9.55g/L, the NaHCO of 2.2g/L
3, the glucose of 1.5g/L is, the hyclone of the glutamine of 0.2g/L and 10% volume/volume; Condition of culture comprises: mixing speed is 35～40 revs/min, 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, and sedimentation microcarrier and cell are replaced culture medium with maintenance medium, according to infection multiplicity, was that 0.01 inoculation encephalitis b virus is proceeded perfusion cultivation, the M199 culture medium that wherein maintenance medium 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 revs/min, and pH is 7.5～7.6, and dissolved oxygen is 40-50% saturation, and temperature is 33.5-34.5 ℃, and the perfusion rate of maintenance medium is 315 liters/day;
(3) collect virus-culturing fluid, filtration, deactivation purification.
2. production method according to claim 1, is characterized in that, the preparation method of described Vero seed cell comprises the following steps: by recovery Vero cell successively at 175cm
2in square vase, 2 liters of rolling bottles, 14 liters of bioreactors and 75 liters of bioreactors, cultivate.
3. production method according to claim 2, is characterized in that, described 175cm
2culture medium in square vase is the M199 culture medium of the hyclone that comprises 10% volume/volume; Condition of culture comprises: temperature is 37 ℃, passes into the CO of 5% volume/volume
2.
4. production method according to claim 2, is characterized in that, described 175cm
2after cultural method in square vase is rapid thawing of cell that liquid nitrogen is preserved, by 1 * 10
5/ cm
2inoculum density proceed to 175cm
2in square vase, drip culture medium and at the CO of 37 ℃ and 5% volume/volume
2lower cultivation, digests after growing to fine and close monolayer, then is added in new culture medium with the cell suspension that the inoculative proportion of 1:4 volume/volume will obtain through digestion, at the CO of 37 ℃ and 5% volume/volume
2lower cultivation is after 3～4 days, then is added in new culture medium with 1:4 volume/volume inoculative proportion, at the CO of 37 ℃ and 5% volume/volume
2lower cultivation 3～4 days.
5. production method according to claim 3, is characterized in that, described 175cm
2after cultural method in square vase is rapid thawing of cell that liquid nitrogen is preserved, by 1 * 10
5/ cm
2inoculum density proceed to 175cm
2in square vase, drip culture medium and at the CO of 37 ℃ and 5% volume/volume
2lower cultivation, digests after growing to fine and close monolayer, then is added in new culture medium with the cell suspension that the inoculative proportion of 1:4 volume/volume will obtain through digestion, at the CO of 37 ℃ and 5% volume/volume
2lower cultivation is after 3～4 days, then is added in new culture medium with 1:4 volume/volume inoculative proportion, at the CO of 37 ℃ and 5% volume/volume
2lower cultivation 3～4 days.
6. production method according to claim 2, is characterized in that, the culture medium in described 2 liters of rolling bottles comprises the M199 culture medium of 9.55g/L, the NaHCO of 1.8g/L
3, the glucose of 1.5g/L is, the hyclone of the glutamine of 0.2g/L and 10% volume/volume; Condition of culture comprises: temperature is 37 ℃, and rotating speed is 1/8 rev/min.
7. production method according to claim 3, is characterized in that, the culture medium in described 2 liters of rolling bottles comprises the M199 culture medium of 9.55g/L, the NaHCO of 1.8g/L
3, the glucose of 1.5g/L is, the hyclone of the glutamine of 0.2g/L and 10% volume/volume; Condition of culture comprises: temperature is 37 ℃, and rotating speed is 1/8 rev/min.
8. production method according to claim 4, is characterized in that, the culture medium in described 2 liters of rolling bottles comprises the M199 culture medium of 9.55g/L, the NaHCO of 1.8g/L
3, the glucose of 1.5g/L is, the hyclone of the glutamine of 0.2g/L and 10% volume/volume; Condition of culture comprises: temperature is 37 ℃, and rotating speed is 1/8 rev/min.
9. production method according to claim 5, is characterized in that, the culture medium in described 2 liters of rolling bottles comprises the M199 culture medium of 9.55g/L, the NaHCO of 1.8g/L
3, the glucose of 1.5g/L is, the hyclone of the glutamine of 0.2g/L and 10% volume/volume; Condition of culture comprises: temperature is 37 ℃, and rotating speed is 1/8 rev/min.
10. production method according to claim 2, is characterized in that, the inoculum density of cultivating in described 2 liters of rolling bottles is 1.0～2.0 * 10
5/ cm
2, incubation time is 3～4 days.
11. according to the production method described in any one in claim 2 to 10, it is characterized in that, the culture medium in described 14 liters and 75 bioreactors comprises the M199 culture medium of 9.55g/L, the NaHCO of 2.2g/L
3, the glucose of 1.5g/L is, the hyclone of the glutamine of 0.2g/L and 10% volume/volume; Condition of culture comprises: mixing speed is 35～40 revs/min, 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.
12. according to the production method described in any one in claim 1 to 10, it is characterized in that, described filtration comprises through aperture, being the membrane filtration of 1 μ m and 0.45 μ m successively, then the filter membrane ultrafiltration that is 300KD through molecular cut off.
13. production methods according to claim 11, is characterized in that, described filtration comprises through aperture, being the membrane filtration of 1 μ m and 0.45 μ m successively, then the filter membrane ultrafiltration that is 300KD through molecular cut off.
14. according to the production method described in any one in claim 1 to 10, it is characterized in that, the inactivator of described deactivation is beta-propiolactone.
15. production methods according to claim 11, is characterized in that, the inactivator of described deactivation is beta-propiolactone.
16. production methods according to claim 12, is characterized in that, the inactivator of described deactivation is beta-propiolactone.
17. production methods according to claim 13, is characterized in that, the inactivator of described deactivation is beta-propiolactone.
18. production methods according to claim 14, is characterized in that, the inactivator of described deactivation is beta-propiolactone.
19. according to the production method described in any one in claim 1 to 10, it is characterized in that, the method for described purification is sieve chromatography and/or Ion exchange membrane chromatography.
20. production methods according to claim 11, is characterized in that, the method for described purification is sieve chromatography and/or Ion exchange membrane chromatography.
21. production methods according to claim 12, is characterized in that, the method for described purification is sieve chromatography and/or Ion exchange membrane chromatography.
22. production methods according to claim 13, is characterized in that, the method for described purification is sieve chromatography and/or Ion exchange membrane chromatography.
23. production methods according to claim 14, is characterized in that, the method for described purification is sieve chromatography and/or Ion exchange membrane chromatography.
24. production methods according to claim 15, is characterized in that, the method for described purification is sieve chromatography and/or Ion exchange membrane chromatography.
25. production methods according to claim 16, is characterized in that, the method for described purification is sieve chromatography and/or Ion exchange membrane chromatography.
26. production methods according to claim 17, is characterized in that, the method for described purification is sieve chromatography and/or Ion exchange membrane chromatography.
27. production methods according to claim 18, is characterized in that, the method for described purification is sieve chromatography and/or Ion exchange membrane chromatography.
28. according to the production method described in any one in claim 1 to 10, it is characterized in that, the consumption of described microcarrier is every liter of culture medium 5.0g.
29. production methods according to claim 11, is characterized in that, the consumption of described microcarrier is every liter of culture medium 5.0g.
30. production methods according to claim 12, is characterized in that, the consumption of described microcarrier is every liter of culture medium 5.0g.
31. production methods according to claim 13, is characterized in that, the consumption of described microcarrier is every liter of culture medium 5.0g.
32. production methods according to claim 14, is characterized in that, the consumption of described microcarrier is every liter of culture medium 5.0g.
33. production methods according to claim 15, is characterized in that, the consumption of described microcarrier is every liter of culture medium 5.0g.
34. production methods according to claim 16, is characterized in that, the consumption of described microcarrier is every liter of culture medium 5.0g.
35. production methods according to claim 17, is characterized in that, the consumption of described microcarrier is every liter of culture medium 5.0g.
36. production methods according to claim 18, is characterized in that, the consumption of described microcarrier is every liter of culture medium 5.0g.
37. production methods according to claim 19, is characterized in that, the consumption of described microcarrier is every liter of culture medium 5.0g.
38. production methods according to claim 20, is characterized in that, the consumption of described microcarrier is every liter of culture medium 5.0g.
39. production methods according to claim 21, is characterized in that, the consumption of described microcarrier is every liter of culture medium 5.0g.
40. production methods according to claim 22, is characterized in that, the consumption of described microcarrier is every liter of culture medium 5.0g.
41. production methods according to claim 23, is characterized in that, the consumption of described microcarrier is every liter of culture medium 5.0g.
42. production methods according to claim 24, is characterized in that, the consumption of described microcarrier is every liter of culture medium 5.0g.
43. production methods according to claim 25, is characterized in that, the consumption of described microcarrier is every liter of culture medium 5.0g.
44. production methods according to claim 26, is characterized in that, the consumption of described microcarrier is every liter of culture medium 5.0g.
45. production methods according to claim 27, is characterized in that, the consumption of described microcarrier is every liter of culture medium 5.0g.
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| 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 |
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