CN103157106B - Method for preparing porcine parvovirus inactivated vaccines - Google Patents
Method for preparing porcine parvovirus inactivated vaccines Download PDFInfo
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- CN103157106B CN103157106B CN201210578183.6A CN201210578183A CN103157106B CN 103157106 B CN103157106 B CN 103157106B CN 201210578183 A CN201210578183 A CN 201210578183A CN 103157106 B CN103157106 B CN 103157106B
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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 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 conventionally.In swinery all over the world, this virus ubiquity, is generally 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.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 by extensive concern, and utilizing bioreactor to substitute traditional rolling bottle production technology has become the trend of industry development.
Bioreactor, as a kind of new tool, can effectively improve cell yield, and stabilization of vaccines differences between batches, are also applied to large-scale production swine parvovirus vaccine.In patent " applying biological reactor suitability for industrialized production swine parvovirus vaccine " (patent No. ZL 201010282134.9), announce a kind of method of applying stirring type bioreactor production pig parvoviral.In patent " a kind of preparation method of porcine parvovirus inactivated vaccines " (patent No. ZL 201010277502.0), announced a kind of bioreactor productive culture cell of NBS company of U.S. production and then method of producing porcine parvovirus inactivated vaccines applied, its volume of culture has reached 40L.But the method that application torrent filling type bioreactor is produced porcine parvovirus inactivated vaccines has no report.
Summary of the invention
Technical problem to be solved by this invention is that the defect that overcomes prior art provides a kind of method of utilizing 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 of preparing porcine parvovirus inactivated vaccines, it utilizes torrent filling type bioreactor for 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 are identical with function, is connected in an identical manner with torrent tank, realizes mass exchange by external source pump;
The preparation of inactivated vaccine is carried out according to the following steps:
A. the cultivation of cell for seedling
The mixed liquor that is seed cell and cell growth medium by cell suspension is seeded to the infusion bag (6) of torrent filling type bioreactor, makes cell be attached to polyester fiber scraps of paper carrier (8) above, and inoculum density is every gram of carrier inoculation 0.8～1.2 × 10
7individual cell, set device parameter: temperature T 1:37.5～38 DEG C, T2:43～46 DEG C, T3:35.5～37 DEG C, 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 seedling cell;
B. virus inoculation and cultivation
In the time that the cell odd-numbered day, consumption sugar tended to be steady, showing that cell reaches connects malicious requirement, liquid in emptying reactor, by pig parvoviral kind poison inoculation seedling cell, set device parameter: temperature T 1:35～38 DEG C, T2:37～40 DEG C, T3:34～36 DEG C, pH:7.3～7.8, DO:30%～70%, hunting speed 35 ~ 50r/min, air mass flow 400 ml/min～4000ml/min, start Virus culture program, amplification cultivation pig parvoviral;
C. virus liquid results
After Virus culture 40～42h, the virus liquid in results torrent tanks (5) and infusion bag (6), gathers in the crops supernatant by after infusion bag (6) multigelation 2～3 times, and after mixing ,-20 DEG C save backup; Detecting virus liquid tires and blood clotting valency;
D. virus liquid deactivation
In the virus liquid of results, add inactivator, inactivation of viruses;
E. vaccine preparation
The virus liquid of deactivation adds adjuvant, and emulsifying is prepared into porcine parvovirus inactivated vaccines.
The above-mentioned method of 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, cell to be screened is carried out monoclonal by application limiting dilution assay, amplification culture is set up monoclonal cell bank, by blood clotting and TCID
50the high-adaptability monoclonal cell strain of screening swine parvovirus vaccine strain.
The above-mentioned method of preparing porcine parvovirus inactivated vaccines, in step a, in cell cultivation process, adopt stage fed-batch mode to supplement cell growth medium, when residual sugar amount is during lower than 2g/L, stream adds supplementary cell growth medium, stream dosage is not less than 1g/L with culture fluid residual sugar amount and is as the criterion, and in the time that culture fluid volume reaches effective volume of culture, changes liquid.
The above-mentioned method of preparing porcine parvovirus inactivated vaccines, in step a, increases air mass flow day by day, increases 100ml/min～1000ml/min at every 24 o'clock.
The above-mentioned method of 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 of preparing porcine parvovirus inactivated vaccines, in step b, before virus inoculation, kind of a poison is mixed in cell maintenance medium, kind of poison is 1%～3% to mix with cell maintenance medium by V/V, when virus inoculation, the cell maintenance medium that is mixed with virus liquid is pumped in reactor assembly, without absorption, directly start Virus culture program amplification cultivation virus.
The above-mentioned method of preparing porcine parvovirus inactivated vaccines, in step b, starts Continuous-flow and adds doubly concentrated DMEM liquid and 200g/L glucose of the supplementary n of containing when 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 of preparing porcine parvovirus inactivated vaccines, described n doubly concentrated DMEM liquid making method is: 1 part of DMEM dehydrated medium adds deionized water to be configured to after 1 × DMEM liquid according to operation instructions consumption, add again (n-1) part without Na-DMEM dehydrated medium, dissolve; Described n equals 2 ~ 5.
The above-mentioned method of preparing porcine parvovirus inactivated vaccines, in step e, in the virus liquid of deactivation, add tween 80 to be prepared into water by V/V 6～13%, in import white oil, add Si Ben-80 to be prepared into oil phase by V/V 7～11%, 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, cell, through purification screening, has improved cell to viral sensitivity for seedling, has ensured the state homogeneous of seed cell, and difference between reducing batch, guarantees the stable of processing parameter and product quality.
2. in the present invention, compared with conventional torrent pouring bioreactor culture system, increased an infusion bag, effectively increased cell attachment area, cell quantity nearly doubles, and significantly improves virus liquid output.
3. in cell cultivation process of the present invention, increase day by day air mass flow, gas circulation in 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, when pig parvoviral inoculation, adopt synchronous inocalation method, without absorption, simplify the operation, save manpower.
6. the concentrated nutrient solution that the present invention uses, the several amino acids that can double content, for virus breeding provides sufficient nutrition, is conducive to improve viral yield.
7. it is the prioritization scheme of groping through screening that the present invention adopts emulsifying process parameter, can ensure 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.
Brief description of the drawings
Fig. 1 is the fluid circulation system composition schematic diagram of torrent filling type bioreactor;
Fig. 2 is process route chart of the present invention;
(wherein, A is emulsifying process of the present invention to two kinds of emulsifying process stability contrasts 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 about 0.2ml of water that separate out at the pipe end);
Fig. 4 is vaccine HI antibody horizontal curve chart prepared by two kinds of emulsifying process.
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.
Detailed description of the invention
Below in conjunction with detailed description of the invention, the present invention is described in further detail, wherein each embodiment is only for illustrating the present invention and non-limiting scope of patent protection of the present invention.
Embodiment 1 limiting dilution assay clone purification screening cell (porcine kidney cell IBRS-2) for seedling
Porcine kidney cell IBRS-2 is purchased from China Veterinery Drug Inspection Office.
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
In cell bottle, cultivate porcine kidney cell IBRS-2, in the time that cell covers with as fine and close monolayer, clear-cut, digest with 0.025% pancreatin, make cell suspension.
B. the limiting dilution of cell
Prepared IBRS-2 cell suspension is carried out to cell counting, add the DMEM culture fluid diluting cells suspension of serum-free, make cell density be about 1.0 × 10
5individual/ml; With serum-free DMEM culture fluid by dilution after 10 times of gradient dilutions to 10 of IBRS-2 cell suspension
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 good cell suspension of above-mentioned dilution is added in 96 porocyte culture plates, 0.1ml/ hole, micro-Microscopic observation, discards the not good cell hole of non-single cell and cell state, is placed in 37 DEG C, 5% CO
2cultivate, every day, observation of cell growth conditions change in time liquid, treated that cell proliferation is cloning community, chose the cell hole that growth conditions is good, digested with 0.025% pancreatin, made suspension, repeated 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 calf serum neutralization, resuspended with cell growth medium, be transferred to 24 porocyte culture plates and carry out amplification culture, in the time that 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
To cover with IBRS-2 monoclonal cell strain 0.025% trypsinization the counting of monolayer, every strain monoclonal cell spreads 24 porocyte culture plates, establishes multiple hole, 30000 cells/well.Be placed in 37 DEG C, 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, 0.8ml/ hole, after purge 2 ~ 3 times, by V:V 0.1% inoculum concentration Pigs Inoculated parvovirus vaccine strain, 1.0ml/ hole, is placed in 37 DEG C, 5% CO
2cultivate ,-20 DEG C of 3 freeze thawing repeatedly after 72h, and gather in the crops 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, in last multiple hole, discard 25 μ l liquid;
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 swine parvovirus vaccine strain
50detect
By 10 times of gradient doubling dilutions of serum-free DMEM for the virus liquid of results, dilution factor is 10 according to a conventional method
-1～10
-10, the virus liquid having diluted is joining in 96 porocyte culture plates of correspondence successively, and each dilution factor is inoculated a tandem, totally 8 multiple holes, 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 DEG C, 5% CO
2cultivate 5d, observation of cell pathological changes after 5d, result is calculated according to 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 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.
The blood clotting titre infecting after IBRS-2 monoclonal cell strain 72h by swine parvovirus vaccine strain detects, and filters out 2 strain IBRS-2 monoclonal cells: the 16th strain and the 34th strain, 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 swine parvovirus vaccine strain
50titre
Pass through TCID
50method detects the TCID of swine parvovirus vaccine strain infection I16 and the strain of I34 monoclonal cell
50result is respectively 10
7.5tCID
50/ ml and 10
7.7tCID
50/ ml.Infect the TCID of common IBRS-2 cell with swine parvovirus vaccine strain
50(10
6.5~ 10
7.0tCID
50/ ml) compare, improve approximately 10 times.The TCID of the IBRS-2 monoclonal cell strain infected pigs parvovirus vaccine strain that as can be seen here, screening obtains
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 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 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 are prepared 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 respectively containing 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;
Be not contain NaCl and phenol red DMEM in high glucose culture medium without Na-DMEM dehydrated medium, by Invitrogen Corporation production and sales.
A, preparation:
Checking system air-tightness, calibration electrodes, PBS processes scraps of paper carrier 8, connects infusion bag 6 and torrent tank 5, by cell growth medium immersion scraps of paper carrier 8, balance sysmte.
B, preparation work, concrete steps are as follows:
A. seedling is cultivated with early stage with the recovery of cell
From liquid nitrogen container, take out after frozen IBRS-2 cell recovery, with cell growth medium in 37 DEG C of cultivations, grow up to after good monolayer digestion go in 10L rolling bottle cultivate, for subsequent use.
B. seedling inoculation, cultivation in bioreactor with cell
Get 8 rolling bottles that cover with cell monolayer, use 0.025% trypsin digestion cell, prepare cell suspension 10L(containing cell and cell growth medium), total cellular score approximately 3.3 × 10
9individual.
Cell inoculation adopts two step inocalation methods, the first step: respectively squeeze into cell suspension 4.0L, standing adsorption 1h in two infusion bags from bottom; Second step: the liquid in two infusion bags is respectively got to 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 supplementary 5L cell growth medium of torrent tank 5; Cell inoculum density is: every gram of carrier inoculation 1.1 × 10
7individual cell.
After adsorption process completes, device parameter is set as follows: T1:37.5 DEG C, T2:44 DEG C, T3:36.5 DEG C, 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, increase 100ml/min at every 24 o'clock.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.
Sample 2 every day and survey residual sugar amount, calculate odd-numbered day consumption sugar amount, cell culture 48h culture fluid residual sugar amount, lower than 2g/L, starts stream and adds supplementary cell growth medium, and flow acceleration is 11ml/min;
When cell culture 60h, pump culture fluid 13L, fill into cell growth medium 4L after stream add supplementary cell growth medium, flow acceleration is 11ml/min.
When cell culture 72h, culture fluid volume approaches effective volume 21L, and culture fluid is all got, and changes to the fresh cell growth medium of 20L and continues to cultivate.
C. pig parvoviral inoculation seedling cell, continues 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 be 1:4096 containing cell maintenance medium 14L and virus liquid 420ml(blood clotting valency, and tiring is 10
7.7tCID
50/ ml) the malicious suspension of kind squeeze in system, device parameter is set as follows: T1:37 DEG C, T2:39 DEG C, T3:36 DEG C, pH:7.5, DO:40～70%, hunting speed 45r/min, circulation rate 500ml/min, air mass flow 400 ml/min, start Virus culture program and start to cultivate.
After virus inoculation 12h, starting stream adds and supplements 200g/L glucose and 3 times of concentrated DMEM liquid (1 part of DMEM dehydrated medium adds deionized water to be configured to after 1 × DMEM liquid according to operation instructions consumption, add again 2 parts without 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, gathers in the crops supernatant after infusion bag 6 freeze thawing 3 times, and whole virus liquids of results are mixed, and to obtain altogether 20.9L virus liquid, for subsequent use.
The virus liquid of results is measured and is tired and blood clotting valency, and every 1ml is containing virus 10
8.3tCID
50, be 1:4096 to guinea-pig red blood cell agglutination titer.
In the 20.9L virus liquid of results, add 21mL divinyl imines inactivator, 37 DEG C of deactivation 24h stop.After deactivation completes, deactivation inspection is carried out in sampling, is placed in 4 DEG C and saves backup.
E. vaccine preparation
In the virus liquid of deactivation, add 1.8L tween 80 to be prepared into water, 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 healthy susceptible piglets in 4～6 week age (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, piglet safety check is qualified.
Neonatal rat safety check: with 5 of 3～5 age in days neonatal rats, each subcutaneous injection vaccine 0.1ml, observes 10, after immunity, the neonatal rat mental status is good, and without abnormal response, neonatal rat safety check is qualified.Above evidence vaccine safety is up to the standards.
G. vaccine potency inspection
Select 8 of 4 monthly age PPV negative antibody first farrowing sows (serum HI antibody titer should not higher than 1: 8), establish 4 of contrasts simultaneously, every incidence intramuscular injection vaccine 2ml, after 28 days, blood sampling, separation of serum, measures HI antibody.Contrast pig antibody is all negative, and immune swine HI tires and is respectively 1:4096,1: 4096,1:4096,1:2048.
With the comparison of rolling bottle technique: every bottle of spinner culture technique results volume 1L, virus titer is generally 10
7.0tCID
50/ ml left and right; This tests viral total output and 400 10L rolling bottle output maintain an equal level.
Embodiment 3 AP200 type torrent filling type bioreactors are prepared porcine parvovirus inactivated vaccines
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 respectively containing 1500g carrier); Theoretical effectively volume of culture is 210L.
For 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 cultivation of cell for seedling
According to embodiment 2 methods, in AP20C type bioreactor, cultivate seedling cell 96h, when increasing to 3.3g/L/24h and tend to be steady to cell consumption sugar amount, 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 making is inoculated in AP200 type bioreactor infusion bag 6 in two steps: the first step: in two infusion bags, respectively squeeze into cell suspension 40L, standing adsorption 1h from bottom; Second step: the liquid in two infusion bags is respectively got to 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 supplementary 50L cell growth medium of torrent tank 5; Cell inoculum density is: every gram of carrier inoculation 0.97 × 10
7individual cell.
After adsorption process completes, device parameter is set as follows: T1:37.5 DEG C, T2:43 DEG C, T3:36.5 DEG C, 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, increase 1000ml/min at every 24 o'clock.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.
Sample 2 every day and survey residual sugar amount, calculate odd-numbered day consumption sugar amount, cell culture 48h culture fluid residual sugar amount, lower than 2g/L, starts stream and adds supplementary cell growth medium, and flow acceleration is 105ml/min;
When cell culture 60h, pump culture fluid 130L, fill into cell growth medium 30L after stream add supplementary cell growth medium, flow acceleration changes 115ml/min into.
When cell culture 72h, culture fluid volume approaches effective volume 210L, and culture fluid is all got, and changes to the fresh cell growth medium of 200L and continues to cultivate.
B. pig parvoviral inoculation seedling cell, continues 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 be 1:4096 containing cell maintenance medium 140L and virus liquid 4.2L(blood clotting valency, and tiring is 10
7.7tCID
50/ ml) the malicious suspension of kind squeeze in system, device parameter is set as follows: T1:37 DEG C, T2:39 DEG C, T3:36 DEG C, pH:7.5, DO:50～70%, hunting speed 45r/min, circulation rate 5000ml/min, air mass flow 4000 ml/min, start Virus culture program and start to cultivate.
After virus inoculation 12h, start stream and add supplementary 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, gathers in the crops supernatant after infusion bag 6 freeze thawing 3 times, and whole virus liquids of results are mixed, and to obtain altogether 208L virus liquid, for subsequent use.
The virus liquid of results is measured and is tired and blood clotting valency, and every 1ml is containing virus 10
8.2tCID
50, be 1:2048 to guinea-pig red blood cell agglutination titer.
In the virus liquid of results, add inactivator divinyl imines 208mL, 37 DEG C of deactivation 24h stop.After deactivation completes, deactivation inspection is carried out in sampling, is placed in 4 DEG C and saves backup.
D. vaccine preparation
In the 208L virus liquid of deactivation, add 17L tween 80 to be prepared into water, 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 healthy susceptible piglets in 4～6 week age (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, piglet safety check is qualified.
Neonatal rat safety check: with 5 of 3～5 age in days neonatal rats, each subcutaneous injection vaccine 0.1ml, observes 10, after immunity, the neonatal rat mental status is good, and without abnormal response, neonatal rat safety check is qualified.Above evidence vaccine safety is up to the standards.
F. vaccine potency inspection
Select 8 of 4 monthly age PPV negative antibody first farrowing sows (serum HI antibody titer should not higher than 1: 8), establish 4 of contrasts simultaneously, every incidence intramuscular injection vaccine 2ml, after 28 days, blood sampling, separation of serum, measures HI antibody.Contrast pig antibody is all negative, and immune swine HI tires and is respectively 1:4096,1: 4096,1:2048,1:2048.
With the comparison of rolling bottle technique: every bottle of spinner culture technique results volume 1L, virus titer is generally 10
7.0tCID
50/ ml left and right; This tests viral total output and 3000 10L rolling bottle output maintain an equal level.
Embodiment 4 emulsifying process of the present invention and conventional emulsifying process comparison
Certain a collection of pig parvoviral liquid semi-finished product, it is tired is 10
8.0tCID
50/ ml is 1:4096 to guinea-pig red blood cell agglutination titer.
A. emulsifying process of the present invention
The preparation of a water joins 3.6L tween in 36.4L pig parvoviral deactivation venom, stirs, and is water;
The preparation of b oil phase joins 7L span-80 in 74L import white oil, rising temperature to 126 DEG C, and after heating 15min, after temperature is down to room temperature, autoclaving (121 DEG C, 20min) is for subsequent use;
The preparation of c vaccine is added to 80L oil phase in emulsion tank, slowly adds 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 4 of contrasts simultaneously, every incidence intramuscular injection vaccine 2ml, after 7d, 15d, 30d, 60d, 90d, 120d, 150d, 180d, 210d after immunity, blood sampling, separation of serum, measures HI antibody.
B. conventional emulsifying process
The preparation of a water joins 1.8L Tween 80 in 43.2L pig parvoviral deactivation venom (32 DEG C), stirs, and is water;
The preparation of b oil phase joins 1080g stearic acid aluminum in 85.5L import white oil, is heated to 90 DEG C of left and right, after aluminium stearate all dissolves, adds class of 4.5L department 80; Rising temperature to 126 DEG C, after heating 2h, after temperature is down to room temperature, autoclaving (121 DEG C, 30min) is for subsequent use;
The preparation of c vaccine is added to 90L oil phase in emulsion tank, slowly adds 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 about 0.2ml of water that separate out at the pipe end;
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 4 of contrasts simultaneously, every incidence intramuscular injection vaccine 2ml, after 7d, 15d, 30d, 60d, 90d, 120d, 150d, 180d, 210d after immunity, blood sampling, separation of serum, measures HI antibody.
C conclusion
A Detection of Stability: the vaccine stability better (Fig. 3 is shown in two kinds of emulsifying process stability contrasts) that vaccine prepared by emulsifying process of the present invention is prepared than conventional emulsifying process.
B effect detects: the antibody horizontal that the vaccine that vaccine prepared by emulsifying process of the present invention is prepared than conventional emulsifying process produces is high, and the persistent period is long, immune effect better (effect testing result is shown in Fig. 4).
Claims (7)
1. prepare the method for porcine parvovirus inactivated vaccines for one kind, it is characterized in that, it utilizes torrent filling type bioreactor for 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 are identical with function, are connected in an identical manner with torrent tank (5), realize mass exchange by external source pump;
The preparation of inactivated vaccine is carried out according to the following steps:
A. the cultivation of cell for seedling
The mixed liquor that is seed cell and cell growth medium by cell suspension is seeded to the infusion bag (6) of torrent filling type bioreactor, makes cell be attached to polyester fiber scraps of paper carrier (8) above, and inoculum density is every gram of carrier inoculation 0.8～1.2 × 10
7individual cell, set device parameter: temperature T 1:37.5～38 DEG C, T2:43～46 DEG C, T3:35.5～37 DEG C, 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 seedling cell;
B. virus inoculation and cultivation
In the time that the cell odd-numbered day, consumption sugar tended to be steady, showing that cell reaches connects malicious requirement, liquid in emptying reactor, by pig parvoviral kind poison inoculation seedling cell, set device parameter: temperature T 1:35～38 DEG C, T2:37～40 DEG C, T3:34～36 DEG C, pH:7.3～7.8, DO:30%～70%, hunting speed 35 ~ 50r/min, air mass flow 400 ml/min～4000ml/min, start Virus culture program, amplification cultivation pig parvoviral;
C. virus liquid results
After Virus culture 40～42h, the virus liquid in results torrent tanks (5) and infusion bag (6), gathers in the crops supernatant by after infusion bag (6) multigelation 2～3 times, and after mixing ,-20 DEG C save backup; Detecting virus liquid tires and blood clotting valency;
D. virus liquid deactivation
In the virus liquid of results, add inactivator, inactivation of viruses;
E. vaccine preparation
The virus liquid of deactivation adds adjuvant, and emulsifying is prepared into porcine parvovirus inactivated vaccines;
In described step a, seed cell is porcine kidney cell IBRS-2 or pig testis subculture cells ST, is the cell through the screening of limiting dilution assay clone purification, and cell to be screened is carried out monoclonal by application limiting dilution assay, amplification culture is set up monoclonal cell bank, by blood clotting and TCID
50the high-adaptability monoclonal cell strain of screening swine parvovirus vaccine strain;
In described step a, in cell cultivation process, adopt stage fed-batch mode to supplement cell growth medium, when residual sugar amount is during lower than 2g/L, stream adds supplementary cell growth medium, stream dosage is not less than 1g/L with culture fluid residual sugar amount and is as the criterion, and in the time that culture fluid volume reaches effective volume of culture, changes liquid.
2. the method for preparing porcine parvovirus inactivated vaccines according to claim 1, is characterized in that, in step a, increases day by day air mass flow, increases 100ml/min～1000ml/min at every 24 o'clock.
3. the method for preparing porcine parvovirus inactivated vaccines according to claim 2, is characterized in that, in step b, pig parvoviral kind poison is the good species poison through the screening of plaque method purification.
4. the method for preparing porcine parvovirus inactivated vaccines according to claim 3, it is characterized in that, in step b, before virus inoculation, kind of a poison is mixed in cell maintenance medium, kind of poison is 1%～3% to mix with cell maintenance medium by V/V, when virus inoculation, the cell maintenance medium that is mixed with virus liquid is pumped in reactor assembly, without absorption, directly start Virus culture program amplification cultivation virus.
5. the method for preparing porcine parvovirus inactivated vaccines according to claim 4, it is characterized in that, in step b, start Continuous-flow and add doubly concentrated DMEM liquid and 200g/L glucose of the supplementary n of containing when 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.
6. the method for preparing porcine parvovirus inactivated vaccines according to claim 5, it is characterized in that, described n doubly concentrated DMEM liquid making method is: 1 part of DMEM dehydrated medium adds deionized water to be configured to after 1 × DMEM liquid according to operation instructions consumption, add again (n-1) part without Na-DMEM dehydrated medium, dissolve; Described n equals 2 ~ 5.
7. the method for preparing porcine parvovirus inactivated vaccines according to claim 6, it is characterized in that, in step e, in the virus liquid of deactivation, add tween 80 to be prepared into water by V/V 6～13%, in import white oil, add Si Ben-80 to be prepared into oil phase by V/V 7～11%, 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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