CN103157106A - Method for preparing porcine parvovirus inactivated vaccines - Google Patents
Method for preparing porcine parvovirus inactivated vaccines Download PDFInfo
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- CN103157106A CN103157106A CN2012105781836A CN201210578183A CN103157106A CN 103157106 A CN103157106 A CN 103157106A CN 2012105781836 A CN2012105781836 A CN 2012105781836A CN 201210578183 A CN201210578183 A CN 201210578183A CN 103157106 A CN103157106 A CN 103157106A
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Abstract
The invention relates to a method for preparing porcine parvovirus inactivated vaccines, and belongs to the field of biotechnology. The method for preparing the porcine parvovirus inactivated vaccines includes the following steps: (1) culturing of cells used for vaccine preparation, (2) virus inoculation and culture, (3) virus liquid harvest, (4) virus liquid inactivation, and (5) vaccine preparation. Seed virus used for vaccine preparation and cells are screened, matching degree between the virus and the cells is strengthened, a riptide perfusion type bioreactor culture system is used for improving multiplication titer and harvest yield of the virus, immune effects are improved through the improvement of an emulsification process and a whole production process does not involve other biosafety and public health problems and is suitable for large scale production.
Description
Technical field
The present invention relates to a kind of method of utilizing the torrent filling type bioreactor to prepare porcine parvovirus inactivated vaccines, belong to biological technical field.
Background technology
Porcine parvovirus (Porcineparvo viurs, PPV) be because of due to infected pigs's parvovirus, can cause the breeding difficulty of pig, main manifestations is that farrowing sow is miscarried, stillbirth, fetus and embryo's infection and death, and parent does not show clinical symptoms usually.In swinery all over the world, this virus ubiquity generally is endemicity popular or distribute on most of pig farms, and is difficult to eradicate and causes huge economic loss to pig industry.The infection of PPV there is no effective Therapeutic Method, and therefore, this sick epidemic prevention just seems even more important.
Generally acknowledge that using vaccine is the effective ways of prevention porcine parvovirus, raising sow breeding potential.The PPV vaccine mainly comprises inactivated vaccine, attenuated vaccine, genetic engineering subunit vaccine, genetic engineering live vector vaccine etc. at present, wherein inactivated vaccine has advantages of that safety is good, cost is low, produces antibody time length, does not need cryopreservation, is widely used.
Products in China industry production pig parvoviral is to adopt anchorage-dependent cell mostly at present, as pig testis subculture cells ST, porcine kidney cell PK-15, porcine kidney cell IBRS-2, the traditional spinner culture of many employings or standing cultivation, though this technique follows the several years, but be difficult to break away from tire unstable, the disadvantages such as differences between batches are large, and virus titer is low have greatly limited quality and the output of porcine parvovirus inactivated vaccines.In recent years, along with the development of live vaccine industry, bioreactor was utilized bioreactor to substitute the trend that traditional rolling bottle production technology has become industry development by extensive concern.
Bioreactor can effectively improve cell yield as a kind of new tool, and the stabilization of vaccines differences between batches also are applied to the large-scale production swine parvovirus vaccine.Announced a kind of method that stirring type bioreactor is produced pig parvoviral of using in patent " applying biological reactor suitability for industrialized production swine parvovirus vaccine " (patent No. ZL 201010282134.9).Announced a kind of bioreactor productive culture cell of U.S. NBS company production and then method of producing porcine parvovirus inactivated vaccines used in patent " a kind of preparation method of porcine parvovirus inactivated vaccines " (patent No. ZL 201010277502.0), its volume of culture has reached 40L.But the method for using torrent filling type bioreactor production porcine parvovirus inactivated vaccines has no report.
Summary of the invention
Technical problem to be solved by this invention is that the defective that overcomes prior art provides a kind of method of utilizing the torrent filling type bioreactor to prepare porcine parvovirus inactivated vaccines.
Technical problem of the present invention is realized by following technical scheme.
A kind of method for preparing porcine parvovirus inactivated vaccines, it utilizes the torrent filling type bioreactor to be culture systems, described torrent filling type bioreactor comprises the first peristaltic pump, the second peristaltic pump, the first silica gel tube, the second silica gel tube, torrent tank, infusion bag, porose silica gel tube and polyester fiber scraps of paper carrier; Described infusion bag is the cell culture bags that includes polyester fiber scraps of paper carrier, and two infusion bag structures and function are identical, are connected with the torrent tank in an identical manner, realize mass exchange by the external source pump;
The preparation of inactivated vaccine is carried out according to the following steps:
A. the seedling cultivation of cell
The cell suspension mixed liquor that is seed cell and cell growth medium is seeded to the infusion bag (6) of torrent filling type bioreactor, cell is attached on polyester fiber scraps of paper carrier (8), inoculum density is every gram carrier inoculation 0.8～1.2 * 10
7Individual cell, set device parameter: temperature T 1:37.5～38 ℃, T2:43～46 ℃, T3:35.5～37 ℃, pH:7.2～7.8, DO:30%～70%, hunting speed 40～55r/min, air mass flow 100ml/min～1000ml/min, active cell is cultivated program, carries out cell culture, obtains the seedling cell;
B. virus inoculation and cultivation
When the cell odd-numbered day, consumption sugar tended to be steady, showing that cell reaches connects malicious requirement, liquid in emptying reactor, with pig parvoviral kind poison inoculation seedling cell, set device parameter: temperature T 1:35～38 ℃, T2:37～40 ℃, T3:34～36 ℃, pH:7.3～7.8, DO:30%～70%, hunting speed 35 ~ 50r/min, air mass flow 400 ml/min～4000ml/min, start the Virus culture program, the amplification cultivation pig parvoviral;
C. virus liquid is gathered in the crops
After Virus culture 40～42h, the virus liquid in results torrent tanks (5) and infusion bag (6) is gathered in the crops supernatant with after infusion bag (6) multigelation 2～3 times, and after mixing ,-20 ℃ save backup; Detecting virus liquid tires and the blood clotting valency;
D. virus liquid deactivation
Add inactivator, inactivation of viruses in the virus liquid of results;
E. vaccine preparation
The virus liquid of deactivation adds adjuvant, and emulsifying is prepared into porcine parvovirus inactivated vaccines.
The above-mentioned method for preparing porcine parvovirus inactivated vaccines, in step a, seed cell is porcine kidney cell IBRS-2 or pig testis subculture cells ST, be the cell through the screening of limiting dilution assay clone purification, use limiting dilution assay cell to be screened is carried out monoclonal, amplification culture is set up the monoclonal cell bank, by blood clotting and TCID
50The high-adaptability monoclonal cell strain of screening swine parvovirus vaccine strain.
The above-mentioned method for preparing porcine parvovirus inactivated vaccines, in step a, adopt the stage fed-batch mode to replenish cell growth medium in cell cultivation process, when the residual sugar amount during lower than 2g/L, stream adds additional cell growth medium, the stream dosage is not less than 1g/L with culture fluid residual sugar amount and is as the criterion, and when the culture fluid volume reaches effective volume of culture, changes liquid.
The above-mentioned method for preparing porcine parvovirus inactivated vaccines in step a, increases air mass flow day by day, every 24 o'clock increase 100ml/min～1000ml/min.
The above-mentioned method for preparing porcine parvovirus inactivated vaccines, in step b, pig parvoviral kind poison is the good species poison through the screening of plaque method purification.
The above-mentioned method for preparing porcine parvovirus inactivated vaccines, in step b, to plant poison before virus inoculation is mixed in cell maintenance medium, kind of poison is 1%～3% to mix with cell maintenance medium by V/V, the cell maintenance medium that will be mixed with virus liquid during virus inoculation pumps in reactor assembly, need not absorption, directly start Virus culture program amplification cultivation virus.
The above-mentioned method for preparing porcine parvovirus inactivated vaccines in step b, begins Continuous-flow and adds additional n doubly concentrated DMEM liquid and the 200g/L glucose of containing during Virus culture 12h, flow acceleration is not less than 1g/L with culture fluid residual sugar amount and is as the criterion; Described n equals 2 ~ 5.
The above-mentioned method for preparing porcine parvovirus inactivated vaccines, described n doubly concentrated DMEM liquid making method is: after 1 part of DMEM dehydrated medium adds deionized water to be configured to 1 * DMEM liquid according to the operation instructions consumption, add again (n-1) part without the Na-DMEM dehydrated medium, dissolving; Described n equals 2 ~ 5.
The above-mentioned method for preparing porcine parvovirus inactivated vaccines, in step e, add tween 80 to be prepared into water by V/V 6～13% in the virus liquid of deactivation, add Si Ben-80 to be prepared into oil phase by V/V 7～11% in the import white oil, emulsifying after water is mixed in V/V 1:2 ratio with oil phase is prepared into porcine parvovirus inactivated vaccines.
Compared with prior art, the present invention has following beneficial effect:
1. in the present invention, seedling through purification screening, has improved the sensitivity of cell to virus with cell, has guaranteed the state homogeneous of seed cell, and difference between reducing batch is guaranteed the stable of processing parameter and product quality.
2. in the present invention, compare with conventional torrent pouring bioreactor culture system, increased an infusion bag, effectively increased the cell attachment area, cell quantity nearly doubles, and significantly improves virus liquid output.
3. increase day by day air mass flow in cell cultivation process of the present invention, gas circulation in the quickening system is conducive to CO in culture fluid
2Overflow, reduce NaHCO
3Use amount.
4. the pig parvoviral of the present invention's inoculation is through the screening of plaque method purification, and this strain multiplication capacity is strong, and immunogenicity is good, has both increased the titre of semi-finished product venom, has promoted again the immune effect of finished product vaccine.
5. in the present invention, adopt synchronous inocalation method during the pig parvoviral inoculation, need not absorption, simplify the operation, save manpower.
6. the concentrated nutrient solution that uses of the present invention, the several amino acids that can double content for virus breeding provides sufficient nutrition, is conducive to improve viral yield.
7. the present invention adopts the prioritization scheme of emulsifying process parameter for groping through screening, can guarantee the optimum emulsification particle diameter of finished product vaccine, is conducive to early release and steady in a long-term release of antigen, promotes the immune effect of vaccine.
Description of drawings
Fig. 1 is that the fluid circulation system of torrent filling type bioreactor forms schematic diagram;
Fig. 2 is process route chart of the present invention;
(wherein, A is emulsifying process of the present invention to the contrast of two kinds of emulsifying process stability of Fig. 3, and centrifugal 20 minutes of 8000r/min separates out without water; B is conventional emulsifying process, centrifugal 20 minutes of 8000r/min, the pipe end water of separating out is 0.2ml approximately);
Fig. 4 is the vaccine HI antibody horizontal curve chart of two kinds of emulsifying process preparations.
In accompanying drawing or word, each label (or symbol) inventory is: 1. the first peristaltic pump (perfusion Pump for giving-out), 2. the second peristaltic pump (perfusion liquid feeding pump), 3. the first silica gel tube, 4. the second silica gel tube, 5. torrent tank, 6. infusion bag, 7. porose silica gel tube, 8. polyester fiber scraps of paper carrier;
T1 is torrent pot liquid temperature, and T2 is the temperature of torrent tank heating film, and T3 is infusion bag external environment temperature, pH is torrent pot liquid pH value, DO is torrent pot liquid dissolved oxygen rate, and hunting speed is the hunting speed of torrent tank, and air mass flow is torrent tank inner air flow amount.
The specific embodiment
Below in conjunction with the specific embodiment, the present invention is described in further detail, wherein each embodiment only be used for explanation the present invention and and non-limiting scope of patent protection of the present invention.
Embodiment 1 limiting dilution assay clone purification screening seedling cell (porcine kidney cell IBRS-2)
Porcine kidney cell IBRS-2 is available from China Veterinery Drug Inspection Office.
The swine parvovirus vaccine strain is pig parvoviral RP2 strain, it screens through plaque method purification, be deposited in Chinese Typical Representative culture collection center on November 14th, 2012, culture title: pig parvoviral RP2 strain (Porcine parvovirus, PPV), preserving number is that CCTCC NO:V201250 preservation address is Wuhan, China university.Described virus is this area common virus, therefore its feature description is omitted.
The formula of cell growth medium is that volumn concentration is 90% DMEM in high glucose liquid, 10% new-born calf serum, and adjusting pH value is 7.4;
Cell maintenance medium is that volumn concentration is 96% DMEM in high glucose liquid, 4% new-born calf serum, and adjusting pH value is 7.4;
A. monoclonal cell strain preparation
A. the preparation of cell suspension
Cultivate porcine kidney cell IBRS-2 in the cell bottle, when cell covers with monolayer into densification, clear-cut, digest with 0.025% pancreatin, make cell suspension.
B. the limiting dilution of cell
Prepared IBRS-2 cell suspension is carried out cell counting, add the DMEM culture fluid diluting cells suspension of serum-free, make cell density be about 1.0 * 10
5Individual/ml; 10 times of gradient dilutions to 10 of IBRS-2 cell suspension after diluting with serum-free DMEM culture fluid
3Individual/ml, then use 10 times of gradient dilutions to 10 of cell growth medium
1Individual/ml, i.e. 1 cell of every 0.1ml.
C. the clone of cell cultivates
The cell suspension that above-mentioned dilution is good adds in 96 porocyte culture plates, the 0.1ml/ hole, and microscopically is observed, and discards the not good cell hole of non-single cell and cell state, is placed in 37 ℃, 5% CO
2Cultivate, every day, observation of cell growth conditions and in time change liquid, treated that cell proliferation is cloning community, chooses the good cell hole of growth conditions, digests with 0.025% pancreatin, makes suspension, repeats above-mentioned cloning process 1 ~ 2 time, until filter out the monoclonal hole of growing.
D. the amplification culture of monoclonal cell strain
Choose monoclonal growth hole, use 0.025% trypsinization, with the calf serum neutralization, resuspended with cell growth medium, be transferred to 24 porocyte culture plates and carry out amplification culture, when cell proliferation is about 80% to degree of converging, use 0.025% trypsinization, add cell growth medium, be inoculated in amplification culture in Tissue Culture Flask, cultivate 6 ~ 10 bottles, carry out the foundation of the frozen and cell bank of monoclonal cell.
B. the screening of pig parvoviral high-adaptability cell strain
A. the digestion bed board of IBRS-2 monoclonal cell strain
The IBRS-2 monoclonal cell strain of covering with monolayer is also counted with 0.025% trypsinization, and every strain monoclonal cell paving 24 porocyte culture plates are established multiple hole, 30000 cells/well.Be placed in 37 ℃, 5% CO
2Lower cultivation 24h.
B. IBRS-2 monoclonal cell strain Pigs Inoculated parvovirus
Discard the cell growth medium in 24 porocyte culture plates, add 0.1M PBS, V:V 0.1% inoculum concentration Pigs Inoculated parvovirus vaccine strain after purge 2 ~ 3 times, is pressed in the 0.8ml/ hole, and the 1.0ml/ hole is placed in 37 ℃, 5% CO
2Cultivate-20 ℃ of 3 freeze thawing repeatedly after 72h, and results virus liquid.
C. the blood clotting titer determination of swine parvovirus vaccine strain is cultivated in the strain of IBRS-2 monoclonal cell
In 96 hole blood-coagulation-boards, add normal saline 25 μ l/ holes from the 1st hole, hole to 12, get and obtain virus liquid 25 μ l, from the 1st hole, take turns doing 2 times of gradient doubling dilutions, to last multiple hole, last multiple discards 25 μ l liquid in the hole;
Every hole adds 1% Cavia porcellus erythrocyte 25 μ l, and establishes the erythrocyte control wells that does not add virus liquid, after vibration, puts 30～40min under room temperature, observed result.Method is with reference to " People's Republic of China's regulations " in 2000 version.
D. the TCID of IBRS-2 monoclonal cell strain is infected in the swine parvovirus vaccine strain
50Detect
With virus liquid 10 times of gradient doubling dilutions of serum-free DMEM of results, dilution factor is 10 according to a conventional method
-1～10
-10, dilution virus liquid well is joining in 96 porocyte culture plates of correspondence successively, each dilution factor inoculation one tandem, answer holes for totally 8, every hole 100 μ l virus liquids, wherein last two tandems of 96 porocyte plates are in contrast, do not add virus liquid, only add serum-free DMEM, every hole 100 μ l.Then adding cell number to every hole is 2 * 10
5The cell suspension 100 μ l of individual/ml.Be placed in 37 ℃, 5% CO
2Cultivate 5d, observation of cell pathological changes after 5d, result is calculated according to the Reed-Muench method.
C. result
A. the screening of the cultivation of IBRS-2 monoclonal cell strain and the strain of swine parvovirus vaccine plant height adaptability IBRS-2 monoclonal cell.
Obtain IBRS-2 monoclonal cell strain 58 strains by the limiting dilution assay amplification culture.Liquid nitrogen freezing is preserved, frozen 5 of every strain monoclonal cell, and every 1.5ml, density is about 5 * 10
6Individual/ml.
Detect by the blood clotting titre after swine parvovirus vaccine strain infection IBRS-2 monoclonal cell strain 72h, filter out 2 strain IBRS-2 monoclonal cells: the 16th strain and the 34th strain, the blood clotting titre is the highest in 58 strains, is respectively 1: 2048 and 1: 4096.Thus, screening obtains the 16th strain and the 34th strain is the strain of swine parvovirus vaccine plant height adaptability IBRS-2 monoclonal cell, and label is I16 and I34 respectively.
B. the TCID of IBRS-2 monoclonal cell strain is infected in the swine parvovirus vaccine strain
50Titre
Pass through TCID
50Method detects the TCID that I16 and the strain of I34 monoclonal cell are infected in the swine parvovirus vaccine strain
50Result is respectively 10
7.5TCID
50/ ml and 10
7.7TCID
50/ ml.TCID with the common IBRS-2 cell of swine parvovirus vaccine strain infection
50(10
6.5~ 10
7.0TCID
50/ ml) compare, improve approximately 10 times.This shows the TCID of screening obtains IBRS-2 monoclonal cell strain infected pigs parvovirus vaccine strain
50Titre is significantly improved.
I34 porcine kidney cell IBRS-2 is deposited in Chinese Typical Representative culture collection center on November 14th, 2012, and preserving number is CCTCC NO:C2012178, and the preservation address is Wuhan, China university; Described cell is more common, and its feature description is omitted.
The purification that should use the same method filters out a strain pig testis subculture cells ST, is deposited in Chinese Typical Representative culture collection center on November 14th, 2012, and preserving number is CCTCC NO:C2012180, and the preservation address is Wuhan, China university; Described cell is more common, and its feature description is omitted.
Embodiment 2 AP20C type torrent filling type bioreactors prepare porcine parvovirus inactivated vaccines
AP20C type torrent filling type bioreactor used in the present embodiment, infusion bag 6 volumes are that the volume of two infusion bags of 8L(is 4L), torrent tank 5 volumes are 13L, and infusion bag 6 contains 300g polyester fiber scraps of paper carrier 8(two infusion bags and respectively contains the 150g carrier); Theoretical effectively volume of culture is 21L.
Cell is with the porcine kidney cell IBRS-2 of culture presevation in embodiment 1;
Plant poison with pig parvoviral RP2 strain in embodiment 1;
The formula of cell growth medium is volumn concentration 92% DMEM in high glucose liquid, 8% new-born calf serum, and adjusting pH value is 7.4;
Cell maintenance medium is volumn concentration 98% DMEM in high glucose liquid, 2% new-born calf serum, and adjusting pH value is 7.5;
Without the Na-DMEM dehydrated medium for not contain NaCl and phenol red DMEM in high glucose culture medium, by Invitrogen Corporation production and sales.
A, preparation:
The checking system air-tightness, calibration electrodes, PBS processes scraps of paper carrier 8, connects infusion bag 6 and torrent tank 5, soaks scraps of paper carrier 8, balance sysmte with cell growth medium.
B, preparation work, concrete steps are as follows:
A. seedling is cultivated with early stage with the recovery of cell
Take out frozen IBRS-2 cell recovery from liquid nitrogen container after, with cell growth medium in 37 ℃ of cultivations, grow up to after good monolayer digestion go in the 10L rolling bottle cultivate, standby.
B. seedling inoculation, the cultivation of cell in bioreactor
Get 8 rolling bottles that cover with cell monolayer, use 0.025% trypsin digestion cell, preparation cell suspension 10L(contains cell and cell growth medium), total cellular score approximately 3.3 * 10
9Individual.
Two step inocalation methods, the first step: respectively squeeze into cell suspension 4.0L, standing adsorption 1h in two infusion bags from the bottom are adopted in the cell inoculation; Second step: the liquid in two infusion bags is respectively got 1L, respectively squeeze into 1L remaining cell suspension, standing adsorption 0.5h from two infusion bag top deep layer pipes; Simultaneously in the preheating of the interior additional 5L cell growth medium of torrent tank 5; The cell inoculum density is: every gram carrier inoculation 1.1 * 10
7Individual cell.
After adsorption process is completed, device parameter is set as follows: T1:37.5 ℃, T2:44 ℃, T3:36.5 ℃, PH:7.3, DO:40～70%, hunting speed 45r/min, circulation rate 400ml/min, air mass flow 100ml/min.Active cell is cultivated program, carries out cell culture.Day by day increase air mass flow, every 24 o'clock increase 100ml/min.Cell growth medium circumfusion speed increases gradually with the increase of incubation time, makes silica gel tube 3, the interior liquid color of silica gel tube 4 keep basically identical.
Take a sample every day and survey the residual sugar amount 2 times, calculate odd-numbered day consumption sugar amount, cell culture 48h culture fluid residual sugar amount begins stream and adds additional cell growth medium lower than 2g/L, and flow acceleration is 11ml/min;
During cell culture 60h, pump culture fluid 13L, fill into cell growth medium 4L after stream add additional cell growth medium, flow acceleration is 11ml/min.
During cell culture 72h, the culture fluid volume is all got culture fluid near effective volume 21L, changes to the fresh cell growth medium of 20L and continues to cultivate.
C. pig parvoviral inoculation seedling cell, continue to cultivate
After cell inoculation consumption sugar amount reaches 3.42g/L/24h (96h) odd-numbered day on the 4th, and tends to be steady, and determines that cell density has reached to connect malicious requirement.Liquid in emptying reactor will contain cell maintenance medium 14L and virus liquid 420ml(blood clotting valency is 1:4096, and tiring is 10
7.7TCID
50/ ml) the malicious suspension of kind is squeezed in system, device parameter is set as follows: T1:37 ℃, and T2:39 ℃, T3:36 ℃, pH:7.5, DO:40～70%, hunting speed 45r/min, circulation rate 500ml/min, air mass flow 400 ml/min start the Virus culture program and begin to cultivate.
Beginning stream after virus inoculation 12h adds and replenishes 200g/L glucose and 3 times of concentrated DMEM liquid (after 1 part of DMEM dehydrated medium adds deionized water to be configured to 1 * DMEM liquid according to the operation instructions consumption, add again 2 parts without the Na-DMEM dehydrated medium, dissolving forms), 3 times of concentrated DMEM acceleration of liquid are 3.14ml/min, and 200g/L glucose feeding speed is 0.1ml/min.
D. gather in the crops virus liquid, inactivation of viruses
After Virus culture 41h, the virus liquid in results torrent tank 5 and infusion bag 6 is gathered in the crops supernatant after infusion bag 6 freeze thawing 3 times, with whole virus liquid mixings of results, to get altogether the 20.9L virus liquid, and is standby.
The virus liquid of results is measured and is tired and the blood clotting valency, and every 1ml contains virus 10
8.3TCID
50, be 1:4096 to the guinea-pig red blood cell agglutination titer.
Add 21mL divinyl imines inactivator in the 20.9L virus liquid of results, 37 ℃ of deactivation 24h stop.After deactivation is completed, the deactivation check is carried out in sampling, is placed in 4 ℃ and saves backup.
E. vaccine preparation
Add the 1.8L tween 80 to be prepared into water in the virus liquid of deactivation, in 43L import white oil, by adding 4L Si Ben-80 to be brewed into oil phase, emulsifying after water is mixed in V/V 1:2 ratio with oil phase is prepared into pig small virus inactivated vaccine.
F. safety verification
Piglet safety check: with 2 of 4～6 healthy susceptible piglets in age in week (serum HI antibody titer should not higher than 1:8), each musculi colli vaccinate 4ml, Continuous Observation 21 days, after immunity, piglet spirit, appetite are without significant change, without abnormal clinical response, the piglet safety check is qualified.
The neonatal rat safety check: with 5 of 3～5 age in days neonatal rats, each subcutaneous injection vaccine 0.1ml observed 10, and after immunity, the neonatal rat mental status is good, and without abnormal response, the neonatal rat safety check is qualified.Above evidence vaccine safety is up to the standards.
G. vaccine potency check
Select 8 of 4 monthly age PPV negative antibody first farrowing sows (serum HI antibody titer should not higher than 1: 8), establish simultaneously 4 of contrasts, every incidence intramuscular injection vaccine 2ml, after 28 days, blood sampling, separation of serum is measured HI antibody.Contrast pig antibody is all negative, and immune swine HI tires and is respectively 1:4096,1: 4096,1:4096,1:2048.
Compare with rolling bottle technique: every bottle of results volume 1L of spinner culture technique, virus titer is generally 10
7.0TCID
50/ ml left and right; This viral total output of experiment and 400 10L rolling bottle output maintain an equal level.
In the present embodiment, torrent filling type bioreactor used comprises two kinds, AP20C type and AP200 type.
AP20C type torrent filling type bioreactor is with embodiment 2.
AP200 type infusion bag 6 volumes are that the volume of two infusion bags of 80L(is 40L), torrent tank 5 volumes are 130L, infusion bag 6 contains 3000g polyester fiber scraps of paper carrier 8(two infusion bags and respectively contains the 1500g carrier); Theoretical effectively volume of culture is 210L.
Seedling cell, kind poison are all with embodiment 2.
A, preparation:
Select two kinds of torrent filling type bioreactors of AP20C type and AP200 type, do the front preparation of cultured cell.
B, preparation work, concrete steps are as follows:
A. the seedling cultivation of cell
According to embodiment 2 methods, cultivate seedling cell 96h in AP20C type bioreactor, measure to cell consumption sugar and increase to 3.3g/L/24h and when tending to be steady, with infusion bag 6 inner cells of 0.025% trypsinization, make cell suspension 100L, include porcine kidney cell IBRS-2 approximately 2.9 * 10
10Individual.The cell suspension that makes is inoculated in AP200 type bioreactor infusion bag 6 in two steps: the first step: respectively squeeze into cell suspension 40L, standing adsorption 1h in two infusion bags from the bottom; Second step: the liquid in two infusion bags is respectively got 10L, respectively squeeze into 10L remaining cell suspension, standing adsorption 0.5h from two infusion bag top deep layer pipes; Simultaneously in the preheating of the interior additional 50L cell growth medium of torrent tank 5; The cell inoculum density is: every gram carrier inoculation 0.97 * 10
7Individual cell.
After adsorption process is completed, device parameter is set as follows: T1:37.5 ℃, T2:43 ℃, T3:36.5 ℃, PH:7.3, DO:50～70%, hunting speed 43r/min, circulation rate 4000ml/min, air mass flow 1000ml/min.Active cell is cultivated program, carries out cell culture.Day by day increase air mass flow, every 24 o'clock increase 1000ml/min.Cell growth medium circumfusion speed increases gradually with the increase of incubation time, makes silica gel tube 3, the interior liquid color of silica gel tube 4 keep basically identical.
Take a sample every day and survey the residual sugar amount 2 times, calculate odd-numbered day consumption sugar amount, cell culture 48h culture fluid residual sugar amount begins stream and adds additional cell growth medium lower than 2g/L, and flow acceleration is 105ml/min;
During cell culture 60h, pump culture fluid 130L, fill into cell growth medium 30L after stream add additional cell growth medium, flow acceleration changes 115ml/min into.
During cell culture 72h, the culture fluid volume is all got culture fluid near effective volume 210L, changes to the fresh cell growth medium of 200L and continues to cultivate.
B. pig parvoviral inoculation seedling cell, continue to cultivate
After cell inoculation consumption sugar amount reaches 3.4g/L/24h (96h) odd-numbered day on the 4th, and tends to be steady, and determines that cell density has reached to connect malicious requirement.Liquid in emptying reactor will contain cell maintenance medium 140L and virus liquid 4.2L(blood clotting valency is 1:4096, and tiring is 10
7.7TCID
50/ ml) the malicious suspension of kind is squeezed in system, device parameter is set as follows: T1:37 ℃, and T2:39 ℃, T3:36 ℃, pH:7.5, DO:50～70%, hunting speed 45r/min, circulation rate 5000ml/min, air mass flow 4000 ml/min start the Virus culture program and begin to cultivate.
Begin stream after virus inoculation 12h and add additional 200g/L glucose and 3 times of concentrated DMEM liquid, flow acceleration is 32ml/min, and glucose feeding speed is 1ml/min.
C. gather in the crops virus liquid, inactivation of viruses
After Virus culture 40h, the virus liquid in results torrent tank 5 and infusion bag 6 is gathered in the crops supernatant after infusion bag 6 freeze thawing 3 times, with whole virus liquid mixings of results, to get altogether the 208L virus liquid, and is standby.
The virus liquid of results is measured and is tired and the blood clotting valency, and every 1ml contains virus 10
8.2TCID
50, be 1:2048 to the guinea-pig red blood cell agglutination titer.
Add inactivator divinyl imines 208mL in the virus liquid of results, 37 ℃ of deactivation 24h stop.After deactivation is completed, the deactivation check is carried out in sampling, is placed in 4 ℃ and saves backup.
D. vaccine preparation
Add the 17L tween 80 to be prepared into water in the 208L virus liquid of deactivation, in 425L import white oil, by adding 39L Si Ben-80 to be brewed into oil phase, emulsifying after water is mixed in V/V 1:2 ratio with oil phase is prepared into pig small virus inactivated vaccine.
E. safety verification
Piglet safety check: with 2 of 4～6 healthy susceptible piglets in age in week (serum HI antibody titer should not higher than 1: 8), each musculi colli vaccinate 4ml, Continuous Observation 21 days, after immunity, piglet spirit, appetite are without significant change, without abnormal clinical response, the piglet safety check is qualified.
The neonatal rat safety check: with 5 of 3～5 age in days neonatal rats, each subcutaneous injection vaccine 0.1ml observed 10, and after immunity, the neonatal rat mental status is good, and without abnormal response, the neonatal rat safety check is qualified.Above evidence vaccine safety is up to the standards.
F. vaccine potency check
Select 8 of 4 monthly age PPV negative antibody first farrowing sows (serum HI antibody titer should not higher than 1: 8), establish simultaneously 4 of contrasts, every incidence intramuscular injection vaccine 2ml, after 28 days, blood sampling, separation of serum is measured HI antibody.Contrast pig antibody is all negative, and immune swine HI tires and is respectively 1:4096,1: 4096,1:2048,1:2048.
Compare with rolling bottle technique: every bottle of results volume 1L of spinner culture technique, virus titer is generally 10
7.0TCID
50/ ml left and right; This viral total output of experiment and 3000 10L rolling bottle output maintain an equal level.
Embodiment 4 emulsifying process of the present invention and conventional emulsifying process are relatively
Certain a collection of pig parvoviral liquid semi-finished product, it is tired is 10
8.0TCID
50/ ml is 1:4096 to the guinea-pig red blood cell agglutination titer.
A. emulsifying process of the present invention
The preparation of a water joins the 3.6L tween in 36.4L pig parvoviral deactivation venom, stirs, and is water;
B oil phase preparation joins the 7L span-80 in 74L import white oil, rising temperature to 126 ℃, and after heating 15min, after temperature is down to room temperature, autoclaving (121 ℃, 20min) standby;
The preparation of c vaccine is added to the 80L oil phase in emulsion tank, slowly adds the 40L water, water and oil phase V/V 1:2, and emulsifying is prepared into porcine parvovirus inactivated vaccines;
Centrifugal 15 minutes of d Detection of Stability 3000r/min, centrifugal 20 minutes of 8000r/min all separates out without water;
E effect detects selects 8 of 4 monthly age PPV negative antibody first farrowing sows (serum HI antibody titer should not higher than 1:8), establish simultaneously 4 of contrasts, every incidence intramuscular injection vaccine 2ml, after 7d, 15d, 30d, 60d, 90d, 120d, 150d, 180d, 210d after immunity, blood sampling, separation of serum is measured HI antibody.
B. conventional emulsifying process
The preparation of a water joins the 1.8L Tween 80 in 43.2L pig parvoviral deactivation venom (32 ℃), stirs, and is water;
B oil phase preparation joins 1080g stearic acid aluminum in 85.5L import white oil, is heated to 90 ℃ of left and right, until aluminium stearate all after dissolving, adds 4.5L department classes 80; Rising temperature to 126 ℃, after heating 2h, after temperature is down to room temperature, autoclaving (121 ℃, 30min) standby;
The preparation of c vaccine is added to the 90L oil phase in emulsion tank, slowly adds the 45L water, water and oil phase V/V 1:2, and emulsifying is prepared into porcine parvovirus inactivated vaccines;
Centrifugal 15 minutes of d Detection of Stability 3000r/min separates out without water; Centrifugal 20 minutes of 8000r/min, the pipe end water of separating out is 0.2ml approximately;
E effect detects selects 8 of 4 monthly age PPV negative antibody first farrowing sows (serum HI antibody titer should not higher than 1: 8), establish simultaneously 4 of contrasts, every incidence intramuscular injection vaccine 2ml, after 7d, 15d, 30d, 60d, 90d, 120d, 150d, 180d, 210d after immunity, blood sampling, separation of serum is measured HI antibody.
The C conclusion
The a Detection of Stability: the vaccine of emulsifying process preparation of the present invention is than the vaccine stability of conventional emulsifying process preparation better (Fig. 3 is seen in the contrast of two kinds of emulsifying process stability).
B effect detects: the vaccine of emulsifying process preparation of the present invention is higher than the antibody horizontal of the vaccine generation of conventional emulsifying process preparation, and longer duration, immune effect better (the effect testing result is seen Fig. 4).
Claims (9)
1. method for preparing porcine parvovirus inactivated vaccines, it is characterized in that, it utilizes the torrent filling type bioreactor to be culture systems, described torrent filling type bioreactor comprises the first peristaltic pump (1), the second peristaltic pump (2), the first silica gel tube (3), the second silica gel tube (4), torrent tank (5), infusion bag (6), porose silica gel tube (7) and polyester fiber scraps of paper carrier (8); Described infusion bag (6) is for including the cell culture bags of polyester fiber scraps of paper carrier, and two infusion bag structures and function are identical, are connected with torrent tank (5) in an identical manner, realize mass exchange by the external source pump;
The preparation of inactivated vaccine is carried out according to the following steps:
A. the seedling cultivation of cell
The cell suspension mixed liquor that is seed cell and cell growth medium is seeded to the infusion bag (6) of torrent filling type bioreactor, cell is attached on polyester fiber scraps of paper carrier (8), inoculum density is every gram carrier inoculation 0.8～1.2 * 10
7Individual cell, set device parameter: temperature T 1:37.5～38 ℃, T2:43～46 ℃, T3:35.5～37 ℃, pH:7.2～7.8, DO:30%～70%, hunting speed 40～55r/min, air mass flow 100ml/min～1000ml/min, active cell is cultivated program, carries out cell culture, obtains the seedling cell;
B. virus inoculation and cultivation
When the cell odd-numbered day, consumption sugar tended to be steady, showing that cell reaches connects malicious requirement, liquid in emptying reactor, with pig parvoviral kind poison inoculation seedling cell, set device parameter: temperature T 1:35～38 ℃, T2:37～40 ℃, T3:34～36 ℃, pH:7.3～7.8, DO:30%～70%, hunting speed 35 ~ 50r/min, air mass flow 400 ml/min～4000ml/min, start the Virus culture program, the amplification cultivation pig parvoviral;
C. virus liquid is gathered in the crops
After Virus culture 40～42h, the virus liquid in results torrent tanks (5) and infusion bag (6) is gathered in the crops supernatant with after infusion bag (6) multigelation 2～3 times, and after mixing ,-20 ℃ save backup; Detecting virus liquid tires and the blood clotting valency;
D. virus liquid deactivation
Add inactivator, inactivation of viruses in the virus liquid of results;
E. vaccine preparation
The virus liquid of deactivation adds adjuvant, and emulsifying is prepared into porcine parvovirus inactivated vaccines.
2. the method for preparing porcine parvovirus inactivated vaccines according to claim 1, it is characterized in that, in step a, seed cell is porcine kidney cell IBRS-2 or pig testis subculture cells ST, be the cell through the screening of limiting dilution assay clone purification, use limiting dilution assay cell to be screened is carried out monoclonal, amplification culture is set up the monoclonal cell bank, by blood clotting and TCID
50The high-adaptability monoclonal cell strain of screening swine parvovirus vaccine strain.
3. the method for preparing porcine parvovirus inactivated vaccines according to claim 2, it is characterized in that, in step a, adopt the stage fed-batch mode to replenish cell growth medium in cell cultivation process, during lower than 2g/L, stream adds additional cell growth medium when the residual sugar amount, and the stream dosage is not less than 1g/L with culture fluid residual sugar amount and is as the criterion, when the culture fluid volume reaches effective volume of culture, change liquid.
4. the method for preparing porcine parvovirus inactivated vaccines according to claim 3, is characterized in that, in step a, increases day by day air mass flow, every 24 o'clock increase 100ml/min～1000ml/min.
5. the method for preparing porcine parvovirus inactivated vaccines according to claim 4, is characterized in that, in step b, pig parvoviral kind poison is the good species poison through the screening of plaque method purification.
6. the method for preparing porcine parvovirus inactivated vaccines according to claim 5, it is characterized in that, in step b, to plant poison before virus inoculation is mixed in cell maintenance medium, kind of poison is 1%～3% to mix with cell maintenance medium by V/V, the cell maintenance medium that will be mixed with virus liquid during virus inoculation pumps in reactor assembly, need not absorption, directly starts Virus culture program amplification cultivation virus.
7. the method for preparing porcine parvovirus inactivated vaccines according to claim 6, it is characterized in that, in step b, begin Continuous-flow during Virus culture 12h and add additional n doubly concentrated DMEM liquid and the 200g/L glucose of containing, flow acceleration is not less than 1g/L with culture fluid residual sugar amount and is as the criterion; Described n equals 2 ~ 5.
8. the method for preparing porcine parvovirus inactivated vaccines according to claim 7, it is characterized in that, described n doubly concentrated DMEM liquid making method is: after 1 part of DMEM dehydrated medium adds deionized water to be configured to 1 * DMEM liquid according to the operation instructions consumption, add again (n-1) part without the Na-DMEM dehydrated medium, dissolving; Described n equals 2 ~ 5.
9. the method for preparing porcine parvovirus inactivated vaccines according to claim 8, it is characterized in that, in step e, add tween 80 to be prepared into water by V/V 6～13% in the virus liquid of deactivation, add Si Ben-80 to be prepared into oil phase by V/V 7～11% in the import white oil, emulsifying after water is mixed in V/V 1:2 ratio with oil phase is prepared into porcine parvovirus inactivated vaccines.
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