CN102600464A - Avian influenza virus inactivated vaccine and preparation method thereof - Google Patents
Avian influenza virus inactivated vaccine and preparation method thereof Download PDFInfo
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Abstract
The invention discloses an avian influenza virus inactivated vaccine and a preparation method thereof. Adaptive avian influenza viruses are adaptive to screening and domestication of a continuous cell line, and after the viruses are adaptive to cells, the number of the viruses is continuously increased until the viruses are inoculated into a cell culture bottle or a bioreactor to culture. After the obtained culture is inactivated, the obtained culture and mineral oil adjuvant are mixed and emulsified so as to prepare the vaccine. A virus reproduction method for producing an avian vaccine by means of utilizing a large quantity of chicken embryo is omitted, resource consumption is reduced, environmental pollution is decreased, biosafety is guaranteed, cost is lowered, and production efficiency is improved. By the aid of the preparation method, high-potency viruses can be produced to prepare corresponding vaccines, and requirements of fast and high-efficient vaccine production are met.
Description
Technical field
The present invention relates to method for preparing of a kind of vaccine and products thereof, especially relate to a kind of method for preparing of inactivated avian influenza vaccine and the product that obtains of method thus.
Background technology
The production of current inactivated avian influenza vaccine mainly is to adopt the Embryo Gallus domesticus breeding method; This training method is through bird flu virus is inoculated in the chick embryo allantoic liquid; Along with the growth of Embryo Gallus domesticus, viral massive duplication breeding connects the method that malicious chick embryo allantoic liquid obtains a large amount of viruses through results.Though this technology is simple, serious waste of resources, labor intensity is big, the production cycle is long, production efficiency is low, and tail product harmless treatment difficulty is big, has the bio-safety risk.
The suspension culture technology is used widely in international field of biological pharmacy already, and in the past in decades, global large-scale culture technical development is rapid, cultivates from using attached cells such as rolling bottle, CellCube, develops into the bioreactor mass cell and cultivates.Reaction vessel constantly enlarges in the Industrial Application scale; The bioreactor utilization rate had reached more than 70% during present global bio-pharmaceuticals was produced; In the production of veterinary biologics, also be widely used; Be distributed in the production firm of Europe, Africa and 8 countries of South America like Wellcome group, use this skilled industry production foot-and-mouth disease vaccine and veterinary rabies vaccine.
The heavy industrialization culture of animal cells is started late in China's medicine biological technique industry; Lag far behind international most advanced level; Domestic most biological medicine manufacturing enterprise's cell culture scale and the application of bioreactor still rested on starting period, laboratory scale develops comparatively slow.Family surplus domestic human and the live vaccine manufacturing enterprise 100 has only 3～5 households to carry out animal cell culture technology production vaccine, less than 10% with manufacturing enterprise's application response device of vaccine at present.
In addition, still do not adopt the technology of mode large-scale production inactivated avian influenza vaccines such as cell and bioreactor at present.
Summary of the invention
One object of the present invention is to provide a kind of method of utilizing modes such as cell and bioreactor to produce inactivated avian influenza vaccine.This method can be carried out large-scale production, and the safety of goods is good, and is less by the side reaction that vaccination causes.
Another object of the present invention is to provide a kind of inactivated avian influenza vaccine, and this vaccine utilizes cell and bioreactor to produce, and goods are safe, and the side reaction that is caused by vaccination is few.
In order to solve the problems of the technologies described above, the present inventor has carried out research repeatedly, finds that following technical scheme can solve the problems of the technologies described above.
The present invention provides a kind of method for preparing of inactivated avian influenza vaccine, comprises the steps:
1) seedling going down to posterity and cultivating: cell through EDTA-pancreatin cell dispersion liquid had digestive transfer culture, is cultivated in Tissue Culture Flask or bioreactor with cell growth medium, formed cultured cell with cell;
2) virus adapts to the cell domestication: use cell maintenance medium that bird flu virus is diluted to the viral liquid that concentration is 0.1～5wt%; This virus liquid is inoculated into the culture plate that grows up to cell monolayer; Add the TPCK pancreatin, behind incubator absorption 0.5～3h, replenish cell maintenance medium, place incubator to cultivate; Based on identical said method,, continuous passage produces seed culture of viruses thereby making virus adapt to the bird flu that it is good that cell obtains domestication;
3) randomly, set up viral seed lot: get to hang down and set up primordial seed, basic seed and/or seeding for seed culture of viruses;
4) cultivation of virus is used in seedling: with the cultured cell inoculation step 2 of step 1)) domestication bird flu production seed culture of viruses well, interpolation TPCK pancreatin behind absorption 0.5～3h, adds cell maintenance medium continuation cultivation;
5) seedling is with the results of viral liquid: behind Virus culture to 10～50h, sampling detects glucose residual content and the viral liquid blood clotting valency in the culture medium, when two detected values all tend towards stability, gathers in the crops viral liquid;
6) randomly, process the vaccine finished product: the vaccine finished product is processed in viral liquid centrifugalize, deactivation, the emulsifying that will gather in the crops.
In method for preparing of the present invention, preferably, the cultured cell in the step 1) is in 40 generations.
In method for preparing of the present invention, preferably, the cell growth medium in the step 1) is the DMEM culture medium that contains 8～10wt% serum, low blood serum medium of DMEM or the serum-free medium that contains 3～5wt% serum.
In method for preparing of the present invention, preferably, condition of culture is in the step 1): dissolved oxygen 30～60%, pH are 7.0～7.3, temperature is that 36.5～37.5 ℃ and/or reaction vessel rotating speed are 40-50rpm.Preferably, carrying out reaction vessel in the step 1) controls and/or cell suspension cultures automatically.
The addition of the TPCK pancreatin in method for preparing of the present invention, preferably, step 2) is 0.1～3.0 μ g/ml, and promptly adding TPCK pancreatin TPCK pancreas enzyme concentration in system is 0.1～3.0 μ g/ml.
In method for preparing of the present invention, preferably, step 2) condition of culture in is that pH is 7.0～7.2, temperature is 36～37 ℃ and/or CO
2Concentration is 5%.Preferably, carry out virus step 2) and adapt to cell culture.
In method for preparing of the present invention, preferably, the primordial seed in the step 3) is that 2～5 generations, basic seed are that 6～9 generations and/or seeding were no more than for 13 generations.
In method for preparing of the present invention, preferably, condition of culture is that dissolved oxygen 40～60%, pH are 7.0～7.2 in the step 4), temperature is that 36～37 ℃ and/or reaction vessel rotating speed are 50rpm.
In method for preparing of the present invention, preferably, step 2) cell maintenance medium is DMEM culture medium or the serum-free medium that contains 1～2wt% serum in; And/or cell maintenance medium is DMEM culture medium or the serum-free medium that contains 1～2wt% serum in the step 4).
In method for preparing of the present invention, preferably, the addition of TPCK pancreatin is 0.1～3.0 μ g/ml in the step 4), and promptly adding TPCK pancreatin TPCK pancreas enzyme concentration in system is 0.1～3.0 μ g/ml.
In method for preparing of the present invention, preferably, viral liquid glucose residual content is stable at 0.5～1.5g/L and viral liquid blood clotting valency HA>=2 in the step 5)
9
In method for preparing of the present invention, preferably, in step 6), the freezing continuous flow centrifuge of high speed, its rotating speed >=9000rpm are adopted in centrifugalize.Preferably, in step 6), formalin is adopted in deactivation.Preferably, the formalin final concentration is 0.1vt%.In addition, 37 ℃ of deactivations more than 16 hours.
In method for preparing of the present invention; Preferably, in step 6), adopt following method to carry out emulsifying: 2～3 weight portion oil phases are placed emulsion tank; Add 2～1 weight portion waters (being the water of inactivation of viruses liquid preparation) when stirring, fully carry out emulsifying behind the mixing.
In method for preparing of the present invention, preferably, said bioreactor is a torrent formula bioreactor, and its cell culture vector is the polyester fiber carrier.
In method for preparing of the present invention, preferably, said cell is MDCK MDCK, African green monkey kidney cell VERO or CEF DF-1.
In method for preparing of the present invention, preferably, described inactivated avian influenza vaccine is H9 hypotype, F strain inactivated avian influenza vaccine.
In method for preparing of the present invention, preferably, step 2) the TPCK pancreas enzyme concentration is 1.2 μ g/ml or in the step 4).
The present invention also provides a kind of inactivated avian influenza vaccine, and it obtains through method for preparing of the present invention.
Adopt method for preparing of the present invention, can realize the large-scale production of inactivated avian influenza vaccine.
In addition, use the airtight bioreactor high density of simulation body physiological environment, suspended culture cell in enormous quantities to produce bird flu virus, total enclosing, channelization system have reduced the chance of contamination of cells, virus; The raising of automaticity has reduced the factor of manual operation; Through automatic adjusting pH, manufacturing parameters such as dissolved oxygen, temperature and hunting speed realize efficiently expressing of cell and virus.The volumetrical expansion of bioreactor has promoted the homogeneity of end product, is applied in the production of vaccine aspect, can increase the safety of goods, reduces the side reaction that is caused by vaccination, improves the quality and the output of vaccine.
Have, this method overcomes the production bottleneck of avian influenza vaccine again, substitutes the Embryo Gallus domesticus production technology with cell proliferation virus.
The specific embodiment
In the following description, unless stated otherwise, " % " all representes percetage by weight, i.e. " wt% ".
In the present invention, so-called " bird flu " (Bird Flu or Avian Influenza) is a kind of acute infectious disease that is caused by bird flu virus (AIV), and known its can infect the mankind at present.
" inactivated vaccine " of the present invention be a kind of adopt physical method, chemical method etc. kill causal organism and make be used for premunitive biological product.Usually, inactivated vaccine is earlier to virus or antibacterial culturing, then with its deactivation.The mode of deactivation be known in the art those, for example heat inactivation perhaps uses the formalin solution deactivation.Inactivated vaccine can be made up of whole virus or antibacterial, also can consist of split vaccine by their crack fragment.
In the present invention, the method for preparing of inactivated avian influenza vaccine comprises the steps:
1) seedling going down to posterity and cultivating with cell;
2) virus adapts to the cell domestication;
3) randomly, set up viral seed lot;
4) seedling is with the cultivation of virus;
5) seedling is with the results of viral liquid;
6) randomly, process the vaccine finished product.
In the present invention, step 3) is chosen wantonly, that is to say, method for preparing of the present invention can only comprise step 1), 2), 4), 5), 6).Preferably, the present invention includes step 1)～6).
In the present invention, step 6) can adopt mode known in the art, also can adopt mode described in the invention, thereby also chooses wantonly.Even the omission step 6) also belongs to scope of the present invention.Certainly, preferably, comprise step 6) in the method for preparing of the present invention.
To describe selection and the propagative viruses of vaccine of the present invention, virus kind below in detail with cell.
< kind of vaccine, culture medium, virus and cell >
In the present invention, inactivated avian influenza vaccine comprises H5 hypotype, H9 hypotype etc., is preferably the H9 hypotype, more preferably the inactivated avian influenza vaccine of H9 hypotype, F strain.
In the present invention, culture medium comprises cell growth medium, cell maintenance medium etc.The used cell growth medium of the present invention comprises the DMEM culture medium that contains 8～10wt% serum, low blood serum medium of DMEM and the serum-free medium that contains 3～5wt% serum; Be preferably the DMEM that contains 3～5wt% serum low blood serum medium, more preferably serum-free medium.In the present invention, used cell maintenance medium comprises DMEM culture medium or the serum-free medium that contains 1～2wt% serum.
In the present invention, used bird flu virus is preferably the bird flu Embryo Gallus domesticus and adapts to poison, bird flu duck embryo adaptation poison, and more preferably the bird flu Embryo Gallus domesticus adapts to poison.
In the present invention, propagative viruses is with the not special restriction of the kind of cell, as long as it can make bird flu virus breed.Propagative viruses is preferably MDCK, VERO or DF-1 cell with cell.These cells all are known in the art, for example CN101189326A, CN1894400A disclosed those.For example, African green monkey kidney cell (VERO cell) is from the kidney epithelial cell of cercopithecus aethiops, to isolate and culture.The DF-1 cell is a chick embryo fibroblast, and its manufacturing enterprise comprises that Shanghai visits Lik-Sang thing Science and Technology Ltd. etc.These cells all can be stablized and go down to posterity, and in the regulation generation, do not have exogenous virus, no oncogenicity, teratogenecity, bird flu virus are had sensitivity preferably, on a large scale continuous culture.
To describe method for preparing of the present invention in detail below.
< the preparation process of vaccine >
1, seedling going down to posterity and cultivating with cell
Get well-grown MDCK, VERO or DF-1 cell monolayer; Through EDTA-pancreatin cell dispersion liquid had digestive transfer culture; Cultivate with cell growth medium 37 ℃ of continuation in Tissue Culture Plate/bottle; When forming good monolayer, be used for continuing to go down to posterity, connect poison or being inoculated in Tissue Culture Flask or bioreactor carries out suspension culture.
In the present invention, EDTA-pancreatin cell dispersion liquid is known in the art those, preferably uses the Digestive system of 0.02wt%EDTA+0.25wt% pancreatin, for example uses the product of Shanghai Hu Feng bio tech ltd.
In the present invention, cell growth medium is known in the art those, and for example preamble is said.Here repeat no more.
In the present invention; Preferably; (be preferably 2～8 generations, more preferably 3～6 generations) cell was decided to be germinal cell, 10～20 generations (be preferably 12～18 generations, more preferably 13～16 generations) and was decided to be basic cell, 20～35 generations (be preferably 22～32 generations, more preferably 25～28 generations) and is decided to be working cell 1～10 generation, produced and need be controlled at for 40 generations with interior (for example 36～39 generations) with cell.
In the present invention, cell growth medium is preferably the DMEM culture medium that contains 8～10% serum.The DMEM culture medium is the product that U.S. GIBCO company produces.Serum is preferably Ox blood serum.Ox blood serum is the product that the U.S. flies generation that science international corporation, the production of sea clone's biochemistry Products Co., Ltd.
2, virus adapts to the cell domestication:
With cell maintenance medium H9 subtype avian influenza embryo toxicity virus liquid is diluted; Again viral liquid is inoculated into respectively on 24 well culture plates that cover with cell monolayer, adds the TPCK pancreatin, in 37 ℃ of incubator absorption; Add cell maintenance medium; Put in the CO2 gas incubator and cultivate, gather in the crops in good time, but make virus adapt to cell as 2～4 generations of cytopathy blind passage not occurring according to cytopathy.
Based on above-mentioned same procedure, continuous passage makes virus adapt to cell, measures viral liquid blood clotting valency HA and 50% histiocyte infective dose TCID
50According to testing result, judge the adaptedness of viral pair cell, when viral liquid blood clotting valency, HA>=2
9, every 0.1ml viral level TCID
50>=10
7.00, confirm that virus adapts to cell and tames successfully.
In above-mentioned steps of the present invention, the concentration of above-mentioned viral liquid is preferably 0.1～5wt%, more preferably 1.0～3.0wt%, more preferably 1～1.5wt%.
In above-mentioned steps of the present invention, above-mentioned adsorption time is preferably 0.5～3h, more preferably 0.8～2h, more preferably 1h.
In the present invention, the TPCK pancreatin is called TPCK again and handles pancreatin, and for known in the art, its CAS number is 9002-07-7, can be purchased the company from Sigma-Aldrich.In above-mentioned steps, adding TPCK pancreatin to its concentration is 0.1～3.0 μ g/ml, is preferably 0.5～1.5 μ g/ml, more preferably 1.2 μ g/ml.
In the present invention, cell maintenance medium is preferably the DMEM culture medium that contains 1～2% serum.The used DMEM culture medium of the present invention is the product that U.S. GIBCO company produces.Serum is preferably Ox blood serum.The used Ox blood serum of the present invention is the product that the U.S. flies generation that science international corporation, the production of sea clone's biochemistry Products Co., Ltd.
3, the foundation of the viral seed lot of H9 hypotype (F strain)
Choosing tires stable lowly sets up three grades of seed lots for cell toxicant, and wherein primordial seed is that 2～5 generations (being preferably for 3～4 generations), basic seed are that 13 generations of 6～9 generations (being preferably for 7～8 generations), seeding are with interior (for example 10～12 generations).The stable criterion of wherein tiring is following:
The viral liquid blood clotting valency of primordial seed is 2
2≤HA＜2
5, every 0.1ml viral level 10
2.00≤TCID
50＜10
4.00
The viral liquid blood clotting valency of basis seed is 2
5≤HA≤2
9, every 0.1ml viral level 10
4.00≤TCID
50≤10
7.00
Viral liquid blood clotting valency HA>=2 of seeding
9, every 0.1ml viral level TCID
50>=10
7.00
4, bioreactor culture cell, propagative viruses
(1) pH probe, dissolved oxygen probe and the temperature probe on the calibration bioreactor, and the disinfecting bucket mesohigh sterilization of packing into; Torrent bag and infusion bag inflation pressurize are spent the night.
(2) during pH probe, dissolved oxygen probe and the temperature probe after will sterilizing packed the qualified torrent bag of pressurize into, be installed on the bioreactor, connect torrent bag and infusion bag; Adding pH is 7.2 BS, starts the bioreactor robot control system(RCS), and adjustment automatic control parameter is that pH is 7.0～7.3; Temperature is 36～37 ℃; The reaction vessel rotating speed is 40-50rpm, and BS circulates in system, spends the night.
(3) system automatic control suspends; Discharge BS; Add the DMEM culture medium contain 8～10% serum (perhaps for the DMEM culture medium that contains 3～5% serum, or be serum-free medium), start the bioreactor robot control system(RCS), adjustment automatic control parameter is that pH is 7.0～7.3; Temperature is 36.5～37.5 ℃, and the reaction vessel rotating speed is 40-50rpm.
In above-mentioned steps of the present invention, the DMEM culture medium is known in the art, and it is a kind of culture medium that contains each seed amino acid and glucose.For example, the DMEM culture medium that magnificent bio tech ltd is produced is won in U.S. GIBCO company or Shanghai.
(4) all change the culture medium in the infusion bag over to the torrent bag, the circulation of stopped reaction device culture medium.The Cell sap that digestion is good changes in the infusion bag, and TCS is not less than 2 * 10
9Cells starts the culture medium circulation behind the absorption certain hour, adjustment dissolved oxygen 30～60%, and pH is 7.0～7.3, and temperature is 36.5～37.5 ℃, and the reaction vessel rotating speed is 40～60rpm, the beginning cell culture.
In above-mentioned steps of the present invention (4), adsorption time can be 0.5～3h, is preferably 0.8～2.0h, more preferably 1.0～1.5h.
(5) every day, timing sampling was measured the glucose residual content; Calculate consumption sugar and measure when reaching 1.4g/L, stop to cultivate, discharge cell culture fluid; The BS that changes 37 ℃ over to cleans cell; Discharge BS, in infusion bag, insert and contain H9 hypotype (F strain) bird flu virus of 1～1.5wt% and the virus of TPCK pancreatin is kept liquid, the absorption certain hour.Adjusting culture parameters then is dissolved oxygen 40～60%, and pH is 7.0～7.2, and temperature is 36～37 ℃, and the reaction vessel rotating speed is 40～60rpm, starts automatic control, carries out Virus culture.
In above-mentioned steps of the present invention (5), the concentration that the virus of TPCK pancreatin is kept TPCK pancreatin in the liquid can be 0.1～3.0 μ g/ml, is preferably 0.5～1.5 μ g/ml, more preferably 1.2 μ g/ml.Because virus is kept the amount of the amount of liquid much larger than bird flu virus, thereby virus is kept, and the TPCK pancreas enzyme concentration is equivalent to the addition of TPCK pancreatin in whole system in the liquid.
In above-mentioned steps of the present invention (5), the reaction vessel rotating speed is preferably 50～60rpm, more preferably 50rpm.
In above-mentioned steps of the present invention (5), the selection of adsorption time is identical with step (4), repeats no more here.
The not special restriction of bioreactor of the present invention is as long as can carry out cell culture, virus breeding.Bioreactor of the present invention is preferably torrent formula bioreactor.For example the general torrent formula bioreactor of producing is pacified in Hangzhou.This bioreactor is fit to the cell of all anchorage dependences growths, need be to the domestication that suspends of corresponding cell, and bioreactor automatization, intelligent degree are high simultaneously, take up an area of for a short time, easy to operate, are convenient to output and amplify fast, and quality balance is stablized.
The production consumptive material of the used bioreactor of the present invention can be disposable disposal type culture bag.The material cell growth does not have any harmful effect, and good dissolved oxygen can be provided, and does not discharge poisonous and harmful substance, and air-tightness is good, the tolerance mechanical tension; Do not need to clean, sterilize energy savings and corresponding human resources.
5, seedling is with the results of viral liquid
After cultivating 10～50h, whenever at a distance from 2～3 hours sampling and measuring glucose residual contents and viral liquid blood clotting valency, viral liquid glucose residual content be stable at 0.5～1.5g/L (be preferably 0.6～1.3g/L, more preferably 0.8～1.2g/L), viral liquid blood clotting valency HA>=2
9Time results venom, subsequent use in-20 ℃ of preservations.
In above-mentioned steps of the present invention, the mensuration of cultivate 10～50h, be preferably 20～40h, more preferably 30h carrying out glucose residual content and viral liquid blood clotting valency afterwards.
6, vaccine product is processed in viral liquid purification, deactivation and emulsifying
After gathering in the crops viral liquid freeze thawing 2 times, centrifugal with the freezing continuous flow centrifuge warp >=9000rpm of high speed, the results separating medium is used the formalin deactivation, 0.1%, 37 ℃ of deactivation of formalin final concentration 16 hours.
3 parts of oil phases are placed emulsion tank, add the water of 1 part of deactivation liquid preparation in the time of stirring, fully pass through pipeline-type emulsifying machine emulsifying 2 times behind the mixing again, process inactivated avian influenza vaccine (H9 hypotype, F strain).Quantitatively packing seals, and labels, and puts 2～8 ℃ of preservations.
In the present invention, the not special restriction of the composition of oil phase is as long as it can be used in vaccine production.Preferably, oil phase of the present invention comprises white oil and first surface activating agent.With respect to 100 weight portion oil phases, the consumption of white oil is the 80-100 weight portion, is preferably the 85-98 weight portion, more preferably the 90-94 weight portion.With respect to 100 weight portion oil phases, the consumption of first surface activating agent is the 0-20 weight portion, is preferably the 2-15 weight portion, more preferably the 6-10 weight portion.
In the present invention, preferably, white oil is the injection white oil.The used first surface activating agent of the present invention is preferably Span (being called Si Ben or span) or Tween (being tween), more preferably Span.The instance of Span includes but not limited to Span-20, Span-40, Span-60, Span-80 or Span-85.The instance of Tween includes but not limited to Tween-20, Tween-40, Tween-60 or Tween-80.
In the present invention, the preparation process of oil phase is known in the art.For example, after white oil and the mixing of first surface activating agent, autoclaving prepares oil phase.
In the present invention, the not special restriction of the composition of water is as long as it can be used in vaccine production.Water of the present invention comprises the AIV antigen liquid of deactivation behind the purification, preferably also comprises the second surface activating agent.With respect to 100 weight portion waters, the consumption of antigen liquid is the 80-100 weight portion, is preferably the 85-98 weight portion, more preferably the 90-96 weight portion.With respect to 100 weight portion waters, the consumption of second surface activating agent is the 0-20 weight portion, is preferably the 2-15 weight portion, more preferably the 4-10 weight portion.
The used second surface activating agent of the present invention can be identical or different with the first surface activating agent, is preferably the two difference.The second surface activating agent is preferably Tween.The instance of Tween includes but not limited to Tween-20, Tween-40, Tween-60 or Tween-80.
In the present invention, the preparation of water is known in the art.For example, the AIV antigen liquid of deactivation behind the purification is mixed with the second surface activating agent of sterilization, fully shake, dissolve fully up to the second surface activating agent.
Viral liquid purification process of the present invention simply, fast, is effectively removed foreign protein, and the virus loss is few, and the viral liquid response rate is high.
To further describe the method for inspection of inactivated vaccine below.
< method of inspection of vaccine >
(1) detection method of character
1. outward appearance
Adopt bore hole observation post to get the outward appearance of emulsion.
2. dosage form
Get a cleaning suction pipe, draw the 1ml vaccine and drip in cold water, except that the 1st, observe all the other drops whether diffusing phenomenon take place.
3. stable
Draw the 10ml vaccine and add in the centrifuge tube, in centrifuge,, take out centrifuge tube, measure the pipe end and separate out the volume of aqueous portion with centrifugal 15 minutes of 3000rpm (being r/min).
It is qualified being no more than 0.5ml;
It is defective surpassing 0.5ml.
4. viscosity
With the dynamic viscosities of rotary viscosimeter at 20 ± 0.1 ℃ of mensuration vaccines.
It is qualified being no more than 200cP;
It is defective surpassing 200cP.
(2) steriling test method
Whether test by existing " Chinese veterinary drug allusion quotation " (version in 2010) appendix, detecting has bacteria growing.
(3) safety verification method
With 10 of 10～14 age in days SPF chickens, each muscle or subcutaneous injection vaccine 1.0ml observed 14, observed the part and the systemic adverse reactions that cause because of vaccinate whether to occur.
(4) method for testing efficacy
1. serological method
With 15 of 21～28 age in days SPF chickens, 10 inactivated avian influenza vaccine (H9 hypotype, F strain) 0.2ml that each cervical region is subcutaneous or intramuscular injection the present invention prepares, 5 compare in addition.Inoculation back 21～28 days, every chicken is taken a blood sample respectively, and separation of serum is with bird flu virus H9 hypotype antigen measuring HI antibody.Calculate the geometrical mean (GMT) of HI antibody titer.
The geometrical mean of immune group HI antibody titer should be not less than 1: 64, and matched group HI antibody titer should not be higher than 1: 4, for qualified;
The geometrical mean of immune group HI antibody titer is lower than 1: 64, or matched group HI antibody titer is higher than 1: 4, for defective.
2. immune counteracting toxic substances method
With 15 of 21～28 age in days SPF chickens, 10 inactivated avian influenza vaccine (H9 hypotype, F strain) 0.2ml that each cervical region is subcutaneous or intramuscular injection the present invention prepares, 5 compare in addition.The wing intravenous injection is carried out, every chicken 0.2ml with the AIV F seed culture of viruses that dilutes at 1: 10 in inoculation back 21～28 days.Behind the counteracting toxic substances the 5th day, gather the cloaca swab of every chicken respectively, each sample is through 5 pieces of allantoic cavity inoculation 9～11 age in days SPF Embryo Gallus domesticus, and every embryo 0.2ml is hatched and was observed 5, and the HA that measures Embryo Gallus domesticus liquid by embryo tires.
As long as have the HA of 1 piece of fluid of chick embryo to tire >=1: 16 (micromethod) in 5 pieces of Embryo Gallus domesticus of each sample inoculation, can be judged to virus and separate positive.Virus is separated negative sample, judge again after answering blind passage 1 time.
Measure in the immune group in 10 chickens virus and separate the number of elements of negative chicken, to have at least 9 fowl disease poison to separate negative if having, and then is qualified, otherwise is defective;
Measure in the matched group virus in 5 chickens and separate the number of elements of male chicken, should be all positive.
(5) formaldehyde, antiseptic mercurials determination of residual amount method
Measure by existing " Chinese veterinary drug allusion quotation " (version in 2010) appendix respectively, observe the regulation that whether meets the veterinary biologics general rule.
Embodiment 1
<1>The selection of bioreactor
The general torrent formula bioreactor of Hangzhou peace, the 100L volume of culture.
<2>Seedling is with the selection of cell
(1) select MDCK MDCK (available from China Veterinery Drug Inspection Office) to use cell for seedling.
(2) cell generation
1～10 generation cell be decided to be germinal cell, 10～20 generations and be decided to be basic cell, 20～35 generations and be decided to be working cell, produce and need be controlled in 40 generations with cell.Liquid nitrogen is preserved, and storage life is decided to be 36 months.
(3) cell characteristics
Test by existing " Chinese veterinary drug allusion quotation " (version in 2010) appendix, should not have antibacterial, mycete, mycoplasma and exogenous virus and pollute, responsive to the H9 subtype avian influenza virus.
<3>Seed culture of viruses is used in seedling
(1) seedling uses seed culture of viruses to be the cell toxicant F strain of H9 subtype avian influenza virus after passage adapts to, and wherein primordial seed is that 2～5 generations, basic seed are that 6～9 generations, seeding were no more than for 13 generations.Below-70 ℃, storage life is 18 months.Below-20 ℃, storage life is 3 months.
(2) pure
Undertaken by existing " Chinese veterinary drug allusion quotation " (version in 2010) appendix, should not have antibacterial, mycete, mycoplasma and exogenous virus and pollute.
(3) imitate inspection seed culture of viruses standard
Seed culture of viruses is H9 hypotype (F strain) bird flu embryo toxicity, and every 0.1ml viral level answers>=10
7.0EID
50, toxic Embryo Gallus domesticus liquid should>=1: 256 to 1% chicken red blood cell agglutination titer, and the seed culture of viruses generation is E2～E8 generation.
<4>The vaccine manufacturing approach
(1) utilizes the bioreactor culture cell
The Cell sap that digestion is good changes in the infusion bag, and TCS is not less than 2 * 10
9Individual cell adsorbs startup culture medium circulation after 1 hour, adjustment dissolved oxygen 30～60%, and pH is 7.0～7.3, and temperature is 36.5～37.5 ℃, and the reaction vessel rotating speed is 40-50rpm, the beginning cell culture.
(2) utilize bioreactor breeding seedling to use venom
Cell culture finishes, and discharges cell culture fluid, and the BS that changes 37 ℃ over to cleans cell, discharges BS, in infusion bag, inserts to contain 1～1.5% H9 hypotype (F strain) bird flu virus and the virus of 1.2 μ g/ml TPCK pancreatin is kept liquid, adsorbs 1 hour.Adjusting culture parameters then is dissolved oxygen 40～60%, and pH is 7.0～7.2, and temperature is 36～37 ℃, and the reaction vessel rotating speed is 50rpm, starts automatic control, carries out Virus culture.
(3) results venom
After 24 hours, whenever at a distance from 4 hours sampling and measuring glucose residual contents and viral liquid blood clotting valency, when viral liquid glucose residual content is stable at 0.5～1.5g/L, viral liquid blood clotting valency HA>=2
9Time results venom, subsequent use in-20 ℃ of preservations, storage life is no more than 6 months.
(4) centrifugalize
After will gathering in the crops viral liquid freeze thawing 2 times, centrifugal with the freezing continuous flow centrifuge warp >=9000rpm of high speed, the results separating medium is used the formalin deactivation, formalin final concentration 0.1vt%, 37 ℃ of deactivations 16 hours obtain the AIV antigen liquid of deactivation behind the purification.
(5) finished product
The preparation of oil phase: get 94 parts of injection white oils, add 6 parts of Si Ben-80 (Span-80) again, after the mixing, autoclaving is subsequent use.
The preparation of water: with 96 parts of the AIV antigen liquids of deactivation behind the purification, add 4 parts of the tween 80s (Tween-80) of sterilization, fully shake, dissolve fully up to tween 80.
The preparation of vaccine finished product: get 3 parts of oil phases in emulsion tank, add 1 part of water in the time of stirring, fully behind the mixing again through pipeline examination mulser emulsifying 2 times; Process inactivated avian influenza vaccine (H9 hypotype, F strain), quantitatively packing; Seal; Label, put 2～8 ℃ of preservations, storage life is no more than 12 months.
The product inspection result of products obtained therefrom is referring to table 1.
Embodiment 2-3
Except selecting African green monkey kidney cell line VERO (available from China Veterinery Drug Inspection Office), CEF respectively for use is the DF-1 (available from China Veterinery Drug Inspection Office); Other steps are all identical with embodiment 1; Obtain inactivated avian influenza vaccine (H9 hypotype, F strain).The product inspection result of products obtained therefrom is referring to table 1.
Table 1, vaccine product inspection result
Can know by table 1, vaccine of the present invention, character is stable, modest viscosity, safety is good.
The efficacy test result of table 2, vaccine finished product
Can know that by table 2 vaccine of the present invention has good immune effect.
The method of the used production inactivated avian influenza vaccine of the present invention; Both can solve Embryo Gallus domesticus self and receive the exogenous virus pollution problems; Avoid the difficulty of a large amount of useless embryo harmless treatments again, practiced thrift available resources, reduce energy resource consumption, effectively ensured bio-safety and public health.
Claims (18)
1. the method for preparing of an inactivated avian influenza vaccine comprises the steps:
1) seedling going down to posterity and cultivating with cell:
Cell through EDTA-pancreatin cell dispersion liquid had digestive transfer culture, is cultivated in Tissue Culture Flask or bioreactor with cell growth medium, formed cultured cell;
2) virus adapts to the cell domestication:
Use cell maintenance medium that bird flu virus is diluted to the viral liquid that concentration is 0.1～5wt%, this virus liquid is inoculated into the culture plate that grows up to cell monolayer, add the TPCK pancreatin, behind incubator absorption 0.5～3h, replenish cell maintenance medium, place incubator to cultivate;
Based on identical said method,, continuous passage produces seed culture of viruses thereby making virus adapt to the bird flu that it is good that cell obtains domestication;
3) randomly, set up viral seed lot:
To hang down for seed culture of viruses as primordial seed, basic seed and/or seeding;
4) cultivation of virus is used in seedling:
Cultured cell inoculation step 2 with step 1)) bird flu that domestication is good is produced seed culture of viruses, adds the TPCK pancreatin, behind absorption 0.5～3h, adds cell maintenance medium and continues to cultivate;
5) seedling is with the results of viral liquid:
Behind Virus culture to 10～50h, sampling detects glucose residual content and the viral liquid blood clotting valency in the culture medium, when two detected values all tend towards stability, gathers in the crops viral liquid;
6) randomly, process the vaccine finished product:
The vaccine finished product is processed in viral liquid centrifugalize, deactivation, the emulsifying of results.
2. method for preparing according to claim 1 is characterized in that: the cultured cell in the step 1) is in 40 generations.
3. method for preparing according to claim 1 and 2 is characterized in that: the cell growth medium in the step 1) is the DMEM culture medium that contains 8～10wt% serum, DMEM culture medium or the serum-free medium that contains 3～5wt% serum.
4. method for preparing according to claim 1 and 2; It is characterized in that: condition of culture is in the step 1): dissolved oxygen 30～60%, pH are 7.0～7.3, and temperature is 36.5～37.5 ℃; The reaction vessel rotating speed is 40-50rpm, carries out that reaction vessel is controlled automatically, cell suspension cultures.
5. method for preparing according to claim 1 is characterized in that: step 2) in, adding TPCK pancreatin TPCK pancreas enzyme concentration in system is 0.1～3.0 μ g/ml.
6. method for preparing according to claim 1 and 2 is characterized in that: step 2) in condition of culture be: pH is 7.0～7.2, and temperature is 36～37 ℃, CO
2Concentration is 5%, carries out virus and adapts to cell culture.
7. method for preparing according to claim 1 and 2 is characterized in that: the primordial seed in the step 3) is that 2～5 generations, basic seed are that 6～9 generations and seeding were no more than for 13 generations.
8. method for preparing according to claim 1 and 2 is characterized in that: condition of culture is a dissolved oxygen 40～60% in the step 4), and pH is 7.0～7.2, and temperature is 36～37 ℃, and the reaction vessel rotating speed is 50rpm.
9. method for preparing according to claim 1 and 2 is characterized in that:
Step 2) cell maintenance medium is DMEM culture medium or the serum-free medium that contains 1～2wt% serum in; And/or
Cell maintenance medium is DMEM culture medium or the serum-free medium that contains 1～2wt% serum in the step 4).
10. method for preparing according to claim 1 is characterized in that: in the step 4), adding TPCK pancreatin TPCK pancreas enzyme concentration in system is 0.1～3.0 μ g/ml.
11. method for preparing according to claim 1 and 2 is characterized in that: viral liquid glucose residual content is stable at 0.5～1.5g/L in the step 5), viral liquid blood clotting valency HA>=2
9
12. method for preparing according to claim 1 and 2 is characterized in that: in step 6), the freezing continuous flow centrifuge of high speed, its rotating speed >=9000rpm are adopted in centrifugalize; Formalin is adopted in deactivation, and its concentration is 0.1vt%, and 37 ℃ of deactivations are more than 16 hours.
13. method for preparing according to claim 1 and 2; It is characterized in that: in step 6); Adopt following method to carry out emulsifying: the oil phase of 2～3 weight portions is placed emulsion tank, and the water of virus preparation after adding 2～1 weight portion deactivations fully carries out emulsifying behind the mixing in the time of stirring.
14. method for preparing according to claim 1 and 2 is characterized in that: said bioreactor is a torrent formula bioreactor, and its cell culture vector is the polyester fiber carrier.
15. method for preparing according to claim 1 and 2 is characterized in that: said cell is MDCK MDCK, African green monkey kidney cell VERO or CEF DF-1.
16. method for preparing according to claim 1 and 2, described inactivated avian influenza vaccine are H9 hypotype, F strain inactivated avian influenza vaccine.
17., it is characterized in that: step 2 according to claim 5 or 10 described method for preparinies) or step 4) in the TPCK pancreas enzyme concentration be 1.2 μ g/ml.
18. an inactivated avian influenza vaccine is characterized in that, it obtains through each described method for preparing of claim 1～17.
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