CN111569055A - Production method and equipment of human influenza vaccine - Google Patents

Production method and equipment of human influenza vaccine Download PDF

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Publication number
CN111569055A
CN111569055A CN202010472879.5A CN202010472879A CN111569055A CN 111569055 A CN111569055 A CN 111569055A CN 202010472879 A CN202010472879 A CN 202010472879A CN 111569055 A CN111569055 A CN 111569055A
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bioreactor
liquid
virus
solution
culture solution
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周蓉
杨刚强
李晓文
侯文礼
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Chengdu Kanghua Biological Products Co Ltd
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Chengdu Kanghua Biological Products Co Ltd
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Abstract

The invention discloses a production method and equipment of human influenza vaccine, and the specific method comprises the steps of utilizing multi-stage bioreactor microcarrier suspension culture to amplify MDCK cells for multiple times, inoculating human influenza virus when the MDCK cells are amplified to required number, finally obtaining virus liquid, and carrying out filtration concentration, inactivation, cracking and purification operations on the virus liquid to obtain the human influenza vaccine. In the multistage bioreactor, after the cell suspension in the previous bioreactor is transferred to the next bioreactor, the next batch of cell suspension is added into the previous bioreactor, so that each bioreactor can produce continuously. The production method and the equipment of the human influenza vaccine can realize the controllable and continuous production of the human influenza vaccine, have short production period and are suitable for large-scale industrial production.

Description

Production method and equipment of human influenza vaccine
Technical Field
The invention relates to the field of biological pharmacy, in particular to a production method and equipment of influenza vaccine for human.
Background
Influenza is an acute viral respiratory infectious disease with high morbidity, wide epidemic and rapid spread caused by influenza viruses, and cannot be completely controlled up to now. Influenza is a disease frequently encountered in daily life of people, and most people get flu at least once in a lifetime. Although most influenza is not very harmful, it is still one of the major killers affecting contemporary human health due to its wide source of infection and numerous transmission routes. The direct cause of influenza development is infection with influenza virus, so the most effective method of preventing viral infection is by injection of influenza vaccine. The wide-range explosion of influenza causes a serious disaster to human beings, and the WHO considers that the main measure for reducing the influenza hazard is to inject influenza vaccine. Therefore, the automation degree of vaccine production is improved, the production period is shortened, the production cost is reduced, and the invention is suitable for preparing influenza vaccines in large-scale industrial production and has very important significance.
At present, the main mode of producing influenza vaccines by domestic enterprises is still to prepare the influenza vaccines by means of traditional chick embryos, the traditional culture mode has many defects, the using amount, the number of operators, the occupied area and the like are disadvantageous, ovalbumin exists in the vaccines prepared from the chick embryos, anaphylactic reaction can be caused, and the quality of vaccine products gradually becomes unable to meet the requirements of people on high-quality vaccines.
The development of modern genetic engineering technology promotes the rapid development of cell culture mode pharmacy, Solvay, Novartis and Med Immune3 enterprises exist in MDCK cell influenza vaccine production enterprises approved to be on the market abroad at present, the scale of the reactor ranges from 50L to 2500L, and research shows that the density in the 10L reactor is 10L6The yield of vaccine prepared from cells/mL of cells corresponds to the yield of 10000 chicken embryos. The production of influenza vaccines by cells in China is still blank at present, so that the application of the cell matrix to the culture of influenza viruses in the production of vaccines has a plurality of advantages, and the quality of the vaccines is greatly improved. Although some companies continue to produce chick embryo derived subunit vaccines, new platforms for the manufacture of influenza vaccines have been developed.
The vaccine produced by MDCK cell has higher homology with human than the vaccine produced in chick embryo, and the cell culture influenza vaccine has been applied in European, North American and other countries for about 10 years, and is proved to be safe and effective.
Microcarrier suspension culture is actually a combination of adherent cell culture technology and suspension culture technology, cells are adsorbed in harmless microcarrier particles in a bioreactor, and the microcarriers are kept in a suspension state under the action of stirring. However, the development of the current domestic cell suspension culture technology is relatively lagged, domestic vaccine production enterprises only use a few bioreactors for suspension culture to produce vaccines, and the scale of the bioreactor has a great gap compared with that of the reactors at abroad.
Disclosure of Invention
Aiming at the problems in the prior art scheme, the invention aims to provide a production method and equipment of human influenza vaccine, which can realize the controllable and continuous production of human influenza vaccine, have short production period and are suitable for large-scale industrial production, and the invention also aims to provide a method for industrially culturing suspension cells in an enlarged manner on a large scale.
In order to achieve the purpose, the technical scheme provided by the invention is as follows:
a production method and equipment of influenza vaccine for human use comprise the following steps:
(1) MDCK cells were expanded on multi-stage bioreactor microcarriers:
digesting well-grown MDCK cells with pancreatin solution, adding cell culture solution to terminate digestion, transferring to a primary bioreactor F microcarrier, and supplementing cell culture solution to make the cell density in the primary bioreactor F be 1-5 × 105Starting stirring, stopping stirring and discharging cell culture liquid from a first-stage bioreactor F after the amplification culture of MDCK cells on microcarriers is finished, washing the microcarriers and cells on the microcarriers by PBS solution, discharging the PBS solution, adding pancreatin solution until the microcarriers are completely submerged, immediately adding cell culture liquid with 2-4 times of the volume of the pancreatin solution after the cells are completely digested and detached from the microcarriers to stop digestion, then conveying cell suspension to more than one secondary bioreactor microcarriers for continuous amplification culture, wherein the operation step on each secondary bioreactor not containing the last stage is that after receiving the cell suspension from the last-stage bioreactor, the cell culture liquid is supplemented to ensure that the density of the MDCK cells is 1-5 × 105Starting amplification culture and stirring after cells/mL, stopping stirring and discharging cell culture solution from the bioreactor when the cell density reaches a culture target, washing the microcarrier and cells on the microcarrier with PBS solution, discharging the PBS solution, adding pancreatin solution until the microcarrier is completely submerged, immediately adding cell culture solution with the volume 2-4 times that of the pancreatin solution to stop digestion after the cells completely disappear from the microcarrier, and then conveying cell suspension to the next bioreactor;
(2) inoculation and propagation of human influenza virus:
after the last stage bioreactor receives the cell suspension from the last stage reactor, the cell culture solution is supplemented to make the MDCK cell density be 1-5 × 105Starting amplification culture and stirring after cells/mL, stopping stirring and discharging cell culture solution from the bioreactor when the cell density reaches the culture target, washing the microcarrier and cells on the microcarrier with PBS solution, discharging the PBS solution, adding human influenza virus into the last stage of biological reverse reactionAdsorbing in a reactor at 33-37 deg.C for 20-40min, and culturing in virus culture solution with the same volume as the cell culture solution;
(3) harvesting virus liquid for multiple times:
(31) in the last-stage bioreactor, stopping stirring to enable the microcarrier to naturally sink after more than 90% of MDCK cells generate pathological changes, collecting supernatant in the bioreactor, wherein the supernatant is virus liquid, and adding virus culture solution to continue culturing after collection is finished;
(32) after culturing for 18-30h, stopping stirring to enable the microcarrier to naturally sink, collecting virus liquid in the bioreactor, and adding virus culture solution to continue culturing after the collection is finished;
(33) repeating the step (32) for multiple times until more than 60% of microcarrier cells fall off; detecting the obtained virus liquid according to the detection procedure of human influenza vaccine, wherein in the detection indexes, the blood coagulation titer of the virus liquid is not less than 1: 160;
(4) and (4) filtering, concentrating, inactivating, cracking and purifying the virus liquid obtained in the step (3) to obtain the influenza vaccine.
Further, the density of the microcarrier is 5-40g per liter of culture volume.
Further, the human influenza virus in the step (2) is influenza A and influenza B virus strains provided by WHO.
Further, the preparation method of the cell culture solution in the steps (1) and (2) comprises the following steps: mixing 2-5% of newborn bovine serum, 1% of 3% L-glutamine, 1% of nonessential amino acid and the balance of MEM culture solution, and adjusting pH to 7.4-8.2 with sodium bicarbonate solution; the conditions for the MDCK cell amplification culture in the steps (1) and (2) are set to be that the culture temperature is 36-38 ℃, the dissolved oxygen is 30-50%, and the stirring speed is 40-80 rpm.
Further, the preparation method of the virus culture solution in the steps (2) and (3) comprises the following steps: mixing 3% L-glutamine at volume ratio of 1%, nonessential amino acids at volume ratio of 1%, TPCK trypsin at concentration of 1 μ g/mL, and MEM as the rest, and adjusting pH to 7.4-8.2 with sodium bicarbonate solution; the conditions for virus inoculation and propagation in the steps (2) and (3) are set as follows: the culture temperature is 33-35 ℃, the dissolved oxygen is 30-50%, and the rotation speed is 40-80 rpm.
Furthermore, in the multistage bioreactor, after the cell suspension in the previous stage bioreactor is transferred to the next stage bioreactor, the next batch of cell suspension is added into the previous stage bioreactor.
A production method and equipment of influenza vaccine for human use comprise a primary bioreactor F and more than one secondary bioreactor, a cell culture liquid tank A, a virus culture liquid tank B, a filtering system C, a waste liquid tank D, a liquid receiving tank E, a gas pump J, PBS storage tank K and a plurality of liquid transmission systems, wherein upper liquid inlets of the primary bioreactor F and more than one secondary bioreactor are connected with liquid outlets of a PBS storage tank K, the cell culture liquid tank A and the virus culture liquid tank B through the liquid transmission systems; liquid outlets below the primary bioreactor F and more than one secondary bioreactor are connected with a liquid inlet of the waste liquid tank D through a liquid transmission system; the liquid outlet below each stage of the bioreactor except the final stage is also connected with the liquid inlet on the side wall of the secondary bioreactor through a liquid transmission system, and the liquid outlet below the final stage of the bioreactor is also connected with the liquid inlet above the liquid receiving tank E through a liquid transmission system; a liquid outlet below the liquid collecting tank E is connected with a liquid inlet of the filtering system C through a liquid transmission system; and the liquid transmission system is provided with a control valve and a pressure transmission port, and the pressure transmission port can be connected with the gas pump J through a hose.
Furthermore, the primary bioreactor F and more than one secondary bioreactor are provided with devices for temperature control, sterilization, stirring, cell density monitoring and the like.
Compared with the prior art, the invention has the beneficial effects that: the invention carries out operations such as cell liquid changing, cell digestion and heavy suspension, cell suspension transfer and the like through the multistage bioreactor, realizes the controllable cell amplification, can effectively prevent the cells from being polluted in the proliferation process, improves the product quality, reduces the production cost, and in addition, the multistage bioreactor carries out online culture of different batches of MDCK cells, shortens the vaccine production time, improves the vaccine production efficiency, can quickly produce a large amount of vaccines for patients when flu is popular on a large scale, can reduce the cost for producers and improve the profit space.
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The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention.
In the drawings: figure 1 is a system block diagram of one embodiment of the present invention,
FIG. 2 is a schematic view of the overall structure of a multistage bioreactor.
Reference numerals: A-500L cell culture fluid tank; B-1000L virus culture solution tank; c-a filtration system; d-a waste liquid tank; E-1000L liquid collecting tank; F-7.5L of a primary bioreactor; G-50L secondary bioreactor; H-500L three-stage bioreactor; j-gas pump; a K-PBS storage tank; l-liquid delivery system 1; m-a liquid delivery system 2; an N-control valve; an O-pressure transfer port.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise noted, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available.
The percent in the present invention means mass percent unless otherwise specified; except where otherwise stated, the percent of solution is the grams of solute contained in 100 mL; the percentage between the liquids is the ratio of the volumes at 20 ℃.
A production method and equipment of influenza vaccine for human use comprise the following steps:
(1) MDCK cells were expanded on multi-stage bioreactor microcarriers:
taking well-grown MDCK cells, digesting with pancreatin solution, adding cell culture solution to terminate digestionTransferring to microcarrier of the first-stage bioreactor F, and supplementing cell culture solution to make the cell density in the first-stage bioreactor F be 1-5 × 105Starting stirring, stopping stirring and discharging cell culture liquid from a first-stage bioreactor F after the amplification culture of MDCK cells on microcarriers is finished, washing the microcarriers and cells on the microcarriers by PBS solution, discharging the PBS solution, adding pancreatin solution until the microcarriers are completely submerged, immediately adding cell culture liquid with 2-4 times of the volume of the pancreatin solution after the cells are completely digested and detached from the microcarriers to stop digestion, then conveying cell suspension to more than one secondary bioreactor microcarriers for continuous amplification culture, wherein the operation step on each secondary bioreactor not containing the last stage is that after receiving the cell suspension from the last-stage bioreactor, the cell culture liquid is supplemented to ensure that the density of the MDCK cells is 1-5 × 105Starting amplification culture and stirring after cells/mL, stopping stirring and discharging cell culture solution from the bioreactor when the cell density reaches a culture target, washing the microcarrier and cells on the microcarrier with PBS solution, discharging the PBS solution, adding pancreatin solution until the microcarrier is completely submerged, immediately adding cell culture solution with the volume 2-4 times that of the pancreatin solution to stop digestion after the cells completely disappear from the microcarrier, and then conveying cell suspension to the next bioreactor;
the proliferation of the MDCK cells is limited by the surface area of a matrix, the large-scale production of vaccines is influenced, the problem that the growth area of the MDCK cells is limited can be solved through the microcarrier suspension culture of a multi-stage bioreactor in the MDCK cell culture process, the high-density rapid growth of the cells is realized, and the controllable and sterile large-scale production of the MDCK cells is realized through the step-by-step amplification culture of the multi-stage bioreactor.
(2) Inoculation and propagation of human influenza virus:
after the last stage bioreactor receives the cell suspension from the last stage reactor, the cell culture solution is supplemented to make the MDCK cell density be 1-5 × 105Starting amplification culture and stirring after cells/mL, stopping stirring and discharging cell culture solution from the bioreactor when the cell density reaches the culture target, and washing with PBS solutionAfter the PBS solution is discharged from the microcarrier and the cells on the microcarrier, adding the human influenza virus into a last-stage bioreactor for adsorption, wherein the adsorption temperature is 33-37 ℃, the adsorption time is 20-40min, and after adsorption, adding a virus culture solution with the same volume as the cell culture solution for culture;
(3) harvesting virus liquid for multiple times:
(31) in the last-stage bioreactor, stopping stirring to enable the microcarrier to naturally sink after more than 90% of MDCK cells generate pathological changes, collecting supernatant in the bioreactor, wherein the supernatant is virus liquid, and adding virus culture solution to continue culturing after collection is finished;
(32) after culturing for 18-30h, stopping stirring to enable the microcarrier to naturally sink, collecting virus liquid in the bioreactor, and adding virus culture solution to continue culturing after the collection is finished;
(33) repeating the step (32) for multiple times until more than 60% of microcarrier cells fall off; detecting the obtained virus liquid according to the detection procedure of human influenza vaccine, wherein in the detection index, the blood coagulation titer in the virus liquid is not lower than 1: 160;
(4) and (4) filtering, concentrating, inactivating, cracking and purifying the virus liquid obtained in the step (3) to obtain the influenza vaccine.
The filtering and concentrating operation of the step (4) comprises the following steps: a filter element made of PP (polypropylene) with the diameter of 0.45 mu m and an ultrafiltration part are arranged in the filter system C, and the ultrafiltration part comprises a plurality of 100KD ultrafiltration membranes which are connected in parallel; primarily filtering the virus liquid by a filter element in a filtering system C to realize the primary separation of cell residues, particle impurities and other substances from virus particles, performing ultrafiltration concentration by a 100KD ultrafiltration membrane, adjusting the rotating speed of a sanitary rotor pump of an ultrafiltration part to ensure that the pressure of a liquid inlet of the ultrafiltration part is less than or equal to 2.0bar and the pressure of a backflow port is less than or equal to 1.0bar, repeatedly circulating the filtered virus liquid through the ultrafiltration part to realize the concentration of the virus liquid and the removal of impurities, and harvesting the concentrated virus liquid; the concentrated virus solutions harvested multiple times were pooled.
The inactivation operation of the step (4) comprises the following steps: adding beta-propiolactone with proper concentration into the concentrated virus liquid, uniformly mixing, inactivating the virus in an environment of 2-8 ℃, regularly or continuously mixing the virus liquid at the early stage of the inactivation process, and hydrolyzing at proper temperature after the inactivation is finished;
the cracking operation of the step (4) comprises the following steps: after inactivation is finished, adding a Triton X100 (polyethylene glycol octyl phenyl ether) cracking agent with a proper concentration to perform virus cracking under proper conditions;
the purification operation steps of the step (4) are as follows: and (3) performing column chromatography on the split virus liquid by using a Sepharose 6FF gel as a chromatography medium, taking a PBS solution as a mobile phase, monitoring by ultraviolet rays with the wavelength of 280nm, and collecting corresponding influenza virus peaks, wherein the sample loading amount is not more than 10% of the column volume.
Further, the density of the microcarrier is 5-40g per liter of culture volume.
Further, the influenza virus strains in the step (2) are influenza A and B virus strains provided by WHO (World Health Organization).
Further, the preparation method of the cell culture solution in the steps (1) and (2) comprises the following steps: mixing 2-5% of newborn bovine serum, 1% of 3% L-glutamine and 1% of nonessential amino acid, and the balance MEM culture medium (minimum essential medium), and adjusting pH to 7.4-8.2 with sodium bicarbonate solution; the conditions for the MDCK cell amplification culture in the steps (1) and (2) are set to be that the culture temperature is 36-38 ℃, the dissolved oxygen is 30-50%, and the stirring speed is 40-80 rpm.
Further, the preparation method of the virus culture solution in the steps (2) and (3) comprises the following steps: mixing 3% L-glutamine at volume ratio of 1%, nonessential amino acid at volume ratio of 1%, TPCK (Tosyl phenyl chloromethyl ketone, p-toluene phenylalanine chloromethyl ketone) trypsin at concentration of 1 μ g/mL, and MEM culture solution as the rest, and adjusting pH to 7.4-8.2 with sodium bicarbonate solution; the conditions for virus inoculation and propagation in the steps (2) and (3) are set as follows: the culture temperature is 33-35 ℃, the dissolved oxygen is 30-50%, and the rotation speed is 40-80 rpm.
Furthermore, in the multistage bioreactor, after the cells in the previous stage bioreactor are transferred to the next stage bioreactor, the previous stage cell suspension is added into the next stage bioreactor.
A production method and equipment of influenza vaccine for human use, comprising a primary bioreactor F and more than one secondary bioreactor, a cell culture liquid tank A, a virus culture liquid tank B, a filtering system C, a waste liquid tank D, a liquid receiving tank E, a gas pump J, PBS storage tank K and a plurality of liquid transmission systems, such as a liquid transmission system 1L and a liquid transmission system 2M in figure 2, wherein upper liquid inlets of the primary bioreactor F and more than one secondary bioreactor are connected with liquid outlets of a PBS storage tank K, the cell culture liquid tank A and the virus culture liquid tank B through the liquid transmission systems; liquid outlets below the primary bioreactor F and more than one secondary bioreactor are connected with a liquid inlet of the waste liquid tank D through a liquid transmission system; the liquid outlet below each stage of the bioreactor except the final stage is also connected with the liquid inlet on the side wall of the secondary bioreactor through a liquid transmission system, and the liquid outlet below the final stage of the bioreactor is also connected with the liquid inlet above the liquid receiving tank E through a liquid transmission system; a liquid outlet below the liquid collecting tank E is connected with a liquid inlet of the filtering system C through a liquid transmission system; the liquid transmission system is provided with a control valve and a pressure transmission port, such as a control valve N and a pressure transmission port O in fig. 2, the pressure transmission port can be connected with a gas pump J through a hose, when no material is transmitted on the liquid transmission system, the pressure transmission port is in a closed state, when the liquid transmission system is required to realize material transmission, the pressure transmission port on the liquid transmission system is connected with the gas pump J through a hose, and the aseptic online transmission of the material is realized by controlling the connection mode of the corresponding control valve and the gas pump J.
Furthermore, the primary bioreactor F and more than one secondary bioreactor are provided with devices for temperature control, sterilization, stirring, cell density monitoring and the like.
The following are specific examples:
EXAMPLE 1 production method and apparatus for influenza A Virus vaccine
The method comprises the following steps:
(1) MDCK cells were expanded on multi-stage bioreactor microcarriers:
taking one or more freezing tubes filled with MDCK cells from a liquid nitrogen tank, quickly dissolving the MDCK cells by using a 38 ℃ water bath, inoculating the dissolved MDCK cells into a T75 square bottle, adding a cell culture solution into a T75 square bottle, culturing in a 37 ℃ environment, and performing bottle-splitting amplification culture according to a ratio of 1:8 after the cells grow into a compact monolayer.
Pre-treating a microcarrier: weighing appropriate dry Cytodex1 type microcarriers, adding PBS solution according to the proportion of Cytodex1 microcarriers to PBS solution =1g to 50mL, soaking and expanding for 3 hours at room temperature with mild stirring, stopping stirring after soaking, naturally settling for 11min, then removing supernatant PBS solution by using a siphon method, then adding PBS solution according to the proportion of Cytodex1 microcarriers to PBS solution =1g to 50mL, gently stirring for 3min, then stopping stirring, naturally settling for 11min, and then removing supernatant PBS solution by using a siphon method. Finally, the PBS solution is added according to the ratio of Cytodex1 microcarrier to PBS solution =1g to 50mL to prepare the Cytodex1 type microcarrier solution. Adding a proper amount of Cytodex1 type microcarrier solution into a bioreactor through a feed inlet of the bioreactor, opening a steam valve, starting a stirring device in the bioreactor at the stirring speed of 40-80rpm, sterilizing the Cytodex1 type microcarrier by high-pressure pure steam on line, and sterilizing for 30min at 121 ℃. And standing after the sterilization is finished, opening a corresponding control valve to connect the bioreactor and a waste liquid tank D after the temperature of the microcarrier in the bioreactor is reduced to below 37 ℃, simultaneously connecting a gas pump J with a pressure transmission port of a corresponding liquid transmission system, pumping supernatant in the bioreactor into the waste liquid tank D, closing the corresponding control valve to block the connection of the bioreactor and the waste liquid tank D, and simultaneously blocking the connection of the gas pump J with the corresponding liquid transmission system. And opening a corresponding control valve to connect the bioreactor with the 500L cell culture solution tank A, simultaneously connecting a gas pump J with a pressure transmission port of a corresponding liquid transmission system, pumping the cell culture solution in the 500L cell culture solution tank A into the bioreactor through the gas pump J, closing the corresponding control valve to block the connection of the gas pump J with the bioreactor and the 500L cell culture solution tank A, and simultaneously blocking the connection of the gas pump J with the corresponding liquid transmission system. Starting a stirring device in the bioreactor, wherein the stirring speed is 60rpm, and the stirring time is 5 min; and (3) closing a stirring device in the bioreactor, naturally settling the microcarrier for 15min, opening a corresponding control valve to connect the bioreactor and the waste liquid tank D, simultaneously connecting a gas pump J with a pressure transmission port of a corresponding liquid transmission system, pumping supernatant in the bioreactor into the waste liquid tank D, closing the corresponding control valve to block the connection between the bioreactor and the waste liquid tank D, and simultaneously blocking the connection between the gas pump J and the corresponding liquid transmission system.
Digesting appropriate amount of well-grown MDCK cells with 0.25% pancreatin solution, observing cell surface digestion condition, pouring out 0.25% pancreatin solution when the cells become honeycomb drawing-wire shape, adding cell culture solution to stop digestion, gently blowing and beating cells with a pipette, counting, collecting cell containing 5 × 108A cell suspension of MDCK cells was added to the 7.5L bioreactor F through the feed inlet of the 7.5L primary bioreactor F. Opening a corresponding control valve to connect the 7.5L primary bioreactor F with the 500L cell culture solution tank A, simultaneously connecting a gas pump J with a pressure transmission port of a corresponding liquid transmission system, pumping the cell culture solution in the 500L cell culture solution tank A into the 7.5L primary bioreactor F through the gas pump J, wherein the volume of the pumped cell culture solution is 4L, closing the corresponding control valve to block the connection of the 7.5L primary bioreactor F with the 500L cell culture solution tank A, and simultaneously blocking the connection of the gas pump J with the corresponding liquid transmission system. The stirring device in the 7.5L bioreactor F was opened and the culture parameters were set as: the culture temperature is 37 ℃, the dissolved oxygen content is 50%, the pH value is 7.4, and the rotating speed is 60 rpm.
During the culture, at 11 am per day: 00 the following operations are carried out: closing a stirring device in a 7.5L primary bioreactor F, naturally settling for 15min, then opening a corresponding control valve to connect the 7.5L primary bioreactor F with a waste liquid tank D, simultaneously connecting a gas pump J with a pressure transmission port of a corresponding liquid transmission system, pumping 1L of cell culture liquid in the 7.5L primary bioreactor F into the waste liquid tank D, closing the corresponding control valve to block the connection between the 7.5L primary bioreactor F and the waste liquid tank D, and simultaneously blocking the connection between the gas pump J and the corresponding liquid transmission system. Opening a corresponding control valve to connect the 7.5L primary bioreactor F with the 500L cell culture solution tank A, simultaneously connecting a gas pump J with a pressure transmission port of a corresponding liquid transmission system, pumping the cell culture solution in the 500L cell culture solution tank A into the 7.5L primary bioreactor F through the gas pump J, wherein the volume of the pumped cell culture solution is 1L, closing the corresponding control valve to block the connection of the 7.5L primary bioreactor F with the 500L cell culture solution tank A, and simultaneously blocking the connection of the gas pump J with the corresponding liquid transmission system.
The cell density in the 7.5L primary bioreactor F is monitored in real time by a cell density electrode monitoring device in the 7.5L primary bioreactor F, and when the concentration of the MDCK cells cultured in suspension reaches 1 × 106After cells/mL, stopping stirring, naturally settling the microcarriers for 15min, opening a corresponding control valve to connect the 7.5L primary bioreactor F with the waste liquid tank D, simultaneously connecting a gas pump J with a pressure transmission port of a corresponding liquid transmission system, pumping cell culture fluid in the 7.5L primary bioreactor F into the waste liquid tank D, closing the corresponding control valve to block the connection between the 7.5L primary bioreactor F and the waste liquid tank D, and simultaneously blocking the connection between the gas pump J and the corresponding liquid transmission system.
(11) Opening a corresponding control valve to communicate the PBS storage tank K with the 7.5L first-stage bioreactor F, simultaneously connecting a gas pump J with a pressure transmission port of a corresponding liquid transmission system, pumping the PBS solution in the PBS storage tank K into the 7.5L first-stage bioreactor F, wherein the transfer volume of the PBS solution is 2L, closing the corresponding control valve to block the connection of the PBS storage tank K with the 7.5L first-stage bioreactor F, and simultaneously blocking the connection of the gas pump J with the corresponding liquid transmission system; opening a stirrer of a 7.5L primary bioreactor F, closing the stirrer after stirring for 1min to enable the microcarrier to naturally settle, opening a corresponding control valve to connect the 7.5L primary bioreactor F and a waste liquid tank D, simultaneously connecting a gas pump J with a pressure transmission port of a corresponding liquid transmission system, pumping PBS solution in the 7.5L primary bioreactor F into the waste liquid tank D, closing the corresponding control valve to block the connection between the 7.5L primary bioreactor F and the waste liquid tank D, and simultaneously blocking the connection between the gas pump J and the corresponding liquid transmission system. Repeating the step (11) once.
Adding a proper amount of 0.25% pancreatin solution into a feed inlet of a 7.5L bioreactor F to submerge a microcarrier, starting a stirring device, stirring at 80rpm, simultaneously opening a corresponding control valve to connect the 7.5L primary bioreactor F with a 500L cell culture solution tank A, simultaneously connecting a gas pump J with a pressure transmission port of a corresponding liquid transmission system, after cells on the microcarrier fall off, pumping cell culture solution in the 500L cell culture solution tank A into the 7.5L primary bioreactor F through the gas pump J, wherein the volume of the pumped cell culture solution is 3 times of the volume of 0.25% pancreatin solution, closing the corresponding control valve to block the connection of the pump J with the 7.5L primary bioreactor F and the 500L cell culture solution tank A, and simultaneously blocking the connection of the gas pump J with the corresponding liquid transmission system.
Opening the corresponding control valve to communicate the 7.5L primary bioreactor F with the 50L secondary bioreactor G, and simultaneously connecting the gas pump J with the pressure transmission port of the corresponding liquid transmission system, pumping the cell suspension in the 7.5L primary bioreactor F into the 50L secondary bioreactor G, wherein the amount of the microcarrier in the 50L secondary bioreactor G is 400G, closing the corresponding control valve to block the communication between the 7.5L primary bioreactor F and the 50L secondary bioreactor G, and simultaneously blocking the connection between the gas pump J and the corresponding liquid transmission system. Opening the corresponding control valve to connect the 50L secondary bioreactor G with the 500L cell culture solution tank A, simultaneously connecting the gas pump J with the pressure transmission port of the corresponding liquid transmission system, pumping the cell culture solution in the 500L cell culture solution tank A into the 50L secondary bioreactor G through the gas pump J, wherein the volume of the pumped cell culture solution is 400L, closing the corresponding control valve to block the connection of the 50L secondary bioreactor G with the 500L cell culture solution tank A, and simultaneously blocking the connection of the gas pump J with the corresponding liquid transmission system. The stirring device in the 50L secondary bioreactor G was opened and the culture parameters were set as: the culture temperature is 37 ℃, the dissolved oxygen content is 50%, the pH value is 7.4, and the rotating speed is 60 rpm.
During the culture, at 11: 00 the following operations are carried out: closing a stirring device in a 50L secondary bioreactor G, naturally settling for 15min, then opening a corresponding control valve to connect the 50L secondary bioreactor G with a waste liquid tank D, simultaneously connecting a gas pump J with a pressure transmission port of a corresponding liquid transmission system, pumping 10L cell culture liquid in the 50L secondary bioreactor G into the waste liquid tank D, closing the corresponding control valve to block the connection between the 50L secondary bioreactor G and the waste liquid tank D, and simultaneously blocking the connection between the gas pump J and the corresponding liquid transmission system. Opening the corresponding control valve to connect the 50L secondary bioreactor G with the 500L cell culture solution tank A, simultaneously connecting the gas pump J with the pressure transmission port of the corresponding liquid transmission system, pumping the cell culture solution in the 500L cell culture solution tank A into the 50L secondary bioreactor G through the gas pump J, wherein the volume of the pumped cell culture solution is 10L, closing the corresponding control valve to block the connection of the 50L secondary bioreactor G with the 500L cell culture solution tank A, and simultaneously blocking the connection of the gas pump J with the corresponding liquid transmission system.
The cell density in the 50L secondary bioreactor G is monitored in real time by a cell density electrode monitoring device in the 50L secondary bioreactor G, and when the concentration of the MDCK cells cultured in suspension reaches 1 × 106Stopping stirring after cells/mL, naturally settling the microcarrier for 15min, opening a corresponding control valve to connect the 50L secondary bioreactor G with the waste liquid tank D, simultaneously connecting a gas pump J with a pressure transmission port of a corresponding liquid transmission system, pumping cell culture liquid in the 50L secondary bioreactor G into the waste liquid tank D, closing the corresponding control valve to block the connection of the 50L secondary bioreactor G with the waste liquid tank D, and simultaneously blocking the connection of the gas pump J with the corresponding liquid transmission system.
(12) Opening a corresponding control valve to communicate the PBS storage tank K with the 50L secondary bioreactor G, simultaneously connecting a gas pump J with a pressure transmission port of a corresponding liquid transmission system, pumping the PBS solution in the PBS storage tank K into the 50L secondary bioreactor G, wherein the transfer volume of the PBS solution is 20L, closing the corresponding control valve to block the connection of the PBS storage tank K with the 50L secondary bioreactor G, and simultaneously blocking the connection of the gas pump J with the corresponding liquid transmission system; opening a stirrer of a 50L secondary bioreactor G, closing the stirrer after stirring for 1min to naturally settle microcarriers, opening a corresponding control valve to connect the 50L secondary bioreactor G with a waste liquid tank D, simultaneously connecting a gas pump J with a pressure transmission port of a corresponding liquid transmission system, pumping PBS solution in the 50L secondary bioreactor G into the waste liquid tank D, closing the corresponding control valve to block the connection of the 50L secondary bioreactor G with the waste liquid tank D, and simultaneously blocking the connection of the gas pump J with the corresponding liquid transmission system. Repeating the step (12) once.
Adding a proper amount of 0.25% pancreatin solution into a feed inlet of a 50L secondary bioreactor G to submerge the microcarrier, starting a stirring device, stirring at 80rpm, simultaneously opening a corresponding control valve to connect the 50L secondary bioreactor G with a 500L cell culture solution tank A, simultaneously connecting a gas pump J with a pressure transmission port of a corresponding liquid transmission system, after cells on the microcarrier fall off, pumping cell culture solution in the 500L cell culture solution tank A into the 50L secondary bioreactor G, wherein the volume of the pumped cell culture solution is 3 times of the volume of 0.25% pancreatin solution, closing the corresponding control valve to block the connection of the 50L secondary bioreactor G and the 500L cell culture solution tank A, and simultaneously blocking the connection of the gas pump J with the corresponding liquid transmission system.
Opening the corresponding control valve to communicate the 50L secondary bioreactor G with the 500L tertiary bioreactor H, and simultaneously connecting the gas pump J with the pressure transmission port of the corresponding liquid transmission system, pumping the cell suspension in the 50L secondary bioreactor G into the 500L tertiary bioreactor H, wherein the amount of the microcarrier in the 500L tertiary bioreactor H is 4kg, closing the corresponding control valve to block the communication between the 50L secondary bioreactor G and the 500L tertiary bioreactor H, and simultaneously blocking the connection between the gas pump J and the corresponding liquid transmission system. Opening the corresponding control valve to connect the 500L three-stage bioreactor H and the 500L cell culture solution tank A, simultaneously connecting the gas pump J with the pressure transmission port of the corresponding liquid transmission system, pumping the cell culture solution in the 500L cell culture solution tank A into the 500L three-stage bioreactor H through the gas pump J, wherein the volume of the pumped cell culture solution is 400L, closing the corresponding control valve to block the connection of the 500L three-stage bioreactor H and the 500L cell culture solution tank A, and simultaneously blocking the connection of the gas pump J with the corresponding liquid transmission system. The stirring device in the 500L tertiary bioreactor H was turned on, and the culture parameters were set as: the culture temperature is 37 ℃, the dissolved oxygen content is 50%, the pH value is 7.4, and the rotating speed is 60 rpm.
During the culture, at 11: 00 the following operations are carried out: closing a stirring device in a 500L three-stage bioreactor H, naturally settling for 15min, then opening a corresponding control valve to connect the 500L three-stage bioreactor H and a waste liquid tank D, simultaneously connecting a gas pump J with a pressure transmission port of a corresponding liquid transmission system, pumping 100L cell culture solution in the 500L three-stage bioreactor H into the waste liquid tank D, closing the corresponding control valve to block the connection between the 500L three-stage bioreactor H and the waste liquid tank D, and simultaneously blocking the connection between the gas pump J and the corresponding liquid transmission system. Opening the corresponding control valve to connect the 500L three-stage bioreactor H and the 500L cell culture solution tank A, simultaneously connecting the gas pump J with the pressure transmission port of the corresponding liquid transmission system, pumping the cell culture solution in the 500L cell culture solution tank A into the 500L three-stage bioreactor H through the gas pump J, wherein the volume of the pumped cell culture solution is 100L, closing the corresponding control valve to block the connection of the 500L three-stage bioreactor H and the 500L cell culture solution tank A, and simultaneously blocking the connection of the gas pump J with the corresponding liquid transmission system.
(2) Inoculation and culture of influenza a virus:
the cell density in the 500L three-stage bioreactor H is monitored in real time by a cell density electrode monitoring device in the 500L three-stage bioreactor H, and when the concentration of MDCK cells cultured in suspension reaches 1 × 106Stopping stirring after cells/mL, naturally settling the microcarrier for 15min, opening a corresponding control valve to connect the 500L tertiary bioreactor H and the waste liquid tank D, simultaneously connecting a gas pump J with a pressure transmission port of a corresponding liquid transmission system, pumping cell culture liquid in the 500L tertiary bioreactor H into the waste liquid tank D, closing the corresponding control valve to block the connection between the 500L tertiary bioreactor H and the waste liquid tank D, and simultaneously blocking the connection between the gas pump J and the corresponding liquid transmission system.
(21) Opening a corresponding control valve to communicate the PBS storage tank K with the 500L three-stage bioreactor H, simultaneously connecting a gas pump J with a pressure transmission port of a corresponding liquid transmission system, pumping the PBS solution in the PBS storage tank K into the 500L three-stage bioreactor H, wherein the transfer volume of the PBS solution is 200L, closing the corresponding control valve to block the connection of the PBS storage tank K with the 500L three-stage bioreactor H, and simultaneously blocking the connection of the gas pump J with the corresponding liquid transmission system; opening a stirrer of the 500L three-stage bioreactor H, closing the stirrer after stirring at 40rpm for 1min to naturally settle the microcarrier, opening a corresponding control valve to connect the 500L three-stage bioreactor H and the waste liquid tank D, simultaneously connecting a gas pump J with a pressure transmission port of a corresponding liquid transmission system, pumping the PBS solution in the 500L three-stage bioreactor H into the waste liquid tank D, closing the corresponding control valve to block the connection between the 500L three-stage bioreactor H and the waste liquid tank D, and simultaneously blocking the connection between the gas pump J and the corresponding liquid transmission system. Repeating the step (21) once.
Influenza A virus strain is added into a feed inlet of a 500L three-stage bioreactor H according to the proportion of 0.01MOI, a stirring device in the 500L three-stage bioreactor H is started, the stirring speed is 40rpm, and the influenza A virus is adsorbed for 30min at the temperature of 37 ℃. After adsorption is completed, opening a corresponding control valve to communicate a 500L three-stage bioreactor H with a 1000L virus culture solution tank B, simultaneously connecting a gas pump J with a pressure transmission port of a corresponding liquid transmission system, pumping the virus culture solution in the 1000L virus culture solution tank B into the 500L three-stage bioreactor H, wherein the volume of the pumped virus culture solution is 400L, closing the corresponding control valve to block the communication between the 500L three-stage bioreactor H and the 1000L virus culture solution tank B, and simultaneously blocking the connection between the gas pump J and the corresponding liquid transmission system. Setting culture parameters as follows: the culture temperature is 35 ℃, the dissolved oxygen content is 40%, the pH value is 7.5, and the rotating speed is 40 rpm.
(3) Harvesting virus culture solution:
(31) culturing influenza A virus on a microcarrier of a 500L three-stage bioreactor H for 48 hours, observing the pathological change condition of MDCK cells through an inverted microscope, stopping stirring when the pathological change of the MDCK cells reaches 90%, naturally settling for 15min, opening a corresponding control valve to enable the 500L three-stage bioreactor H to be communicated with a 1000L liquid receiving tank E, simultaneously connecting a gas pump J with a pressure transmission port of a corresponding liquid transmission system, pumping virus culture liquid in the 500L three-stage bioreactor H into the 1000L liquid receiving tank E to obtain virus liquid, closing the corresponding control valve to block the communication between the 500L three-stage bioreactor H and the 1000L liquid receiving tank E, simultaneously blocking the connection between the gas pump J and the corresponding liquid transmission system, and detecting the obtained virus liquid according to a human influenza vaccine detection procedure, wherein the blood coagulation titer is not lower than 1: 160;
(32) opening corresponding control valve to communicate 500L three-stage bioreactor H and 1000L virus culture solution tank B, simultaneously connecting gas pump J with pressure transmission port of corresponding liquid transmission system, pumping the virus culture solution in 1000L virus culture solution tank B into 500L three-stage bioreactor H, the volume of pumped virus culture solution is 400L, closing corresponding control valve to block communication of pump J with 500L three-stage bioreactor H and 1000L virus culture solution tank B, simultaneously blocking connection of gas pump J and corresponding liquid transmission system, setting culture parameters as: the culture temperature is 35 ℃, the dissolved oxygen content is 40%, the pH value is 7.5, and the rotating speed is 40 rpm. Culturing for 18 hours, stopping the stirring of the bioreactor, naturally settling for 15min, opening a corresponding control valve to enable the 500L tertiary bioreactor H to be communicated with the 1000L liquid receiving tank E, simultaneously connecting a gas pump J with a pressure transmission port of a corresponding liquid transmission system, pumping virus culture liquid in the 500L tertiary bioreactor H into the 1000L liquid receiving tank E, closing the corresponding control valve to block the communication between the 500L tertiary bioreactor H and the 1000L liquid receiving tank E, simultaneously blocking the connection between the gas pump J and the corresponding liquid transmission system, and detecting the obtained virus liquid according to a human influenza vaccine detection protocol, wherein the blood coagulation titer is not lower than 1: 160. Repeating the step (32) 3 times.
(4) Filtering and concentrating virus liquid:
opening the corresponding control valve to communicate the 1000L liquid receiving tank E with the ultrafiltration system C, simultaneously connecting the gas pump J with the pressure transmission port of the corresponding liquid transmission system, pumping the virus liquid in the 1000L liquid receiving tank E into the filtration system C, closing the corresponding control valve to block the communication between the 1000L liquid receiving tank E and the ultrafiltration system C, and simultaneously blocking the connection between the gas pump J and the corresponding liquid transmission system. The virus liquid is primarily filtered by a filter element in a filtering system C, so that the primary separation of cell residues, particle impurities and other substances from virus particles is realized, the virus liquid is ultrafiltered and concentrated by a 100KD ultrafiltration membrane of an ultrafiltration part of the filtering system C, the rotating speed of a sanitary rotor pump in the ultrafiltration part is adjusted to ensure that the pressure of a liquid inlet of the ultrafiltration part is less than or equal to 2.0bar and the pressure of a backflow port is less than or equal to 1.0bar, the filtered virus liquid is repeatedly circulated through a plurality of 100KD ultrafiltration membranes connected in parallel in the ultrafiltration part of the filtering system C, so that the concentration of the virus liquid and the removal of impurities are realized, the ultrafiltration system is closed after the virus liquid is concentrated to 1/50 of an original set, the concentrated virus liquid is harvested, and the concentrated virus liquid harvested for.
(5) Virus liquid inactivation and cracking:
adding beta-propiolactone into the concentrated virus liquid according to the concentration proportion of 1/4000, uniformly mixing, putting the mixture in an environment of 4 ℃ for 24h for virus inactivation, using an oscillator to vibrate the uniformly mixed liquid at intervals or continuously in the inactivation process to ensure thorough inactivation, putting the mixture at a proper temperature for hydrolysis after the inactivation is finished, immediately sampling each virus inactivation container after the inactivation is finished, respectively carrying out virus inactivation verification tests by adopting 9-11 day-old chick embryos, and measuring the content of bacterial endotoxin. After inactivation, the virus is lysed by adding a suitable concentration of Triton X100 lysis agent under suitable conditions.
(6) And (3) virus liquid purification:
and (3) performing column chromatography on the virus liquid after cracking, wherein a chromatography medium is Sepharose 6FF gel, the loading amount is 9% of the column volume, a PBS (phosphate buffer solution) solution with the pH of 7.2 is used as a mobile phase, and the corresponding influenza virus peak is collected by ultraviolet monitoring at the wavelength of 280nm, so that the influenza A vaccine is obtained. Purification parameters: the sample loading flow rate is 1L/min, the sample loading volume is 10L, the sample elution speed is 1L/min, the detection wavelength is 280nm, and the eluent is 0.01mol/L PBS solution with pH of 7.2.
The foregoing is a detailed description of the invention, which is described in more detail and specific, but is not to be construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the spirit of the invention, which falls within the scope of the invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims (8)

1. A production method and equipment of influenza vaccine for human use are characterized in that: the method comprises the following steps:
(1) MDCK cells were expanded on multi-stage bioreactor microcarriers:
taking well-grown MDCK cells, digesting with pancreatin solution, adding cell culture solution to terminate digestion, transferring to the first stageSupplementing cell culture solution on the microcarrier of the bioreactor (F) to ensure that the cell density in the primary bioreactor (F) is 1-5 × 105Starting stirring, after the MDCK cells are amplified and cultured on the microcarriers of the primary bioreactor (F), stopping stirring, discharging cell culture solution from the primary bioreactor, washing the microcarriers and the cells on the microcarriers by PBS solution, discharging PBS solution, adding pancreatin solution until the microcarriers are completely submerged, immediately adding cell culture solution with 2-4 times of the volume of the pancreatin solution to stop digestion after the cells are completely digested and detached from the microcarriers, then conveying cell suspension to more than one secondary bioreactor microcarriers for continuous amplification and culture, wherein the operation step on each secondary bioreactor not containing the final stage is that after receiving the cell suspension from the previous primary bioreactor, the cell culture solution is supplemented to ensure that the MDCK cell density is 1-5 × 105Starting amplification culture and stirring after cells/mL, stopping stirring and discharging cell culture solution from the bioreactor when the cell density reaches a culture target, washing the microcarrier and cells on the microcarrier with PBS solution, discharging the PBS solution, adding pancreatin solution until the microcarrier is completely submerged, immediately adding cell culture solution with the volume 2-4 times that of the pancreatin solution to stop digestion after the cells completely disappear from the microcarrier, and then conveying cell suspension to the next bioreactor;
(2) inoculation and propagation of human influenza virus:
after the last stage bioreactor receives the cell suspension from the last stage reactor, the cell culture solution is supplemented to make the MDCK cell density be 1-5 × 105Starting amplification culture and stirring after cells/mL, stopping stirring and discharging cell culture solution from the bioreactor when the cell density reaches a culture target, washing the microcarrier and cells on the microcarrier with PBS solution, adding human influenza virus into the last bioreactor for adsorption after discharging the PBS solution, wherein the adsorption temperature is 33-37 ℃, the adsorption time is 20-40min, and adding virus culture solution with the same volume as the cell culture solution for culture after adsorption is finished;
(3) harvesting virus liquid for multiple times:
(31) in the last-stage bioreactor, stopping stirring to enable the microcarrier to naturally sink after more than 90% of MDCK cells generate pathological changes, collecting supernatant in the bioreactor, wherein the supernatant is virus liquid, and adding virus culture solution to continue culturing after collection is finished;
(32) after culturing for 18-30h, stopping stirring to enable the microcarrier to naturally sink, collecting virus liquid in the bioreactor, and adding virus culture solution to continue culturing after the collection is finished;
(33) repeating the step (32) for multiple times until more than 60% of microcarrier cells fall off; detecting the obtained virus liquid according to the detection procedure of human influenza vaccine, wherein in the detection indexes, the blood coagulation titer of the virus liquid is not less than 1: 160;
(4) and (4) filtering, concentrating, inactivating, cracking and purifying the virus liquid obtained in the step (3) to obtain the influenza vaccine.
2. The method and apparatus for producing influenza vaccine for human use according to claim 1, wherein: the density of the microcarrier is 5-40g per liter of culture volume.
3. The method and apparatus for producing influenza vaccine for human use according to claim 1, wherein: the human influenza virus in the step (2) is influenza A and influenza B virus strains provided by WHO.
4. The method and apparatus for producing influenza vaccine for human use according to claim 1, wherein: the preparation method of the cell culture solution in the steps (1) and (2) comprises the following steps: mixing 2-5% of newborn bovine serum, 1% of 3% L-glutamine, 1% of nonessential amino acid and the balance of MEM culture solution, and adjusting pH to 7.4-8.2 with sodium bicarbonate solution; the conditions for the MDCK cell amplification culture in the steps (1) and (2) are set to be that the culture temperature is 36-38 ℃, the dissolved oxygen is 30-50%, and the stirring speed is 40-80 rpm.
5. The method and apparatus for producing influenza vaccine for human use according to claim 1, wherein: the preparation method of the virus culture solution in the steps (2) and (3) comprises the following steps: mixing 3% L-glutamine at volume ratio of 1%, nonessential amino acids at volume ratio of 1%, TPCK trypsin at concentration of 1 μ g/mL, and MEM as the rest, and adjusting pH to 7.4-8.2 with sodium bicarbonate solution; the conditions for virus inoculation and propagation in the steps (2) and (3) are set as follows: the culture temperature is 33-35 ℃, the dissolved oxygen is 30-50%, and the rotation speed is 40-80 rpm.
6. The method and apparatus for producing influenza vaccine for human use according to claim 1, wherein: in the multistage bioreactor, after the cells in the previous bioreactor are transferred to the next bioreactor, the next batch of cell suspension is added into the previous bioreactor.
7. A production method and equipment of influenza vaccine for human use are characterized in that: comprises a primary bioreactor (F), more than one secondary bioreactor, a cell culture solution tank (A), a virus culture solution tank (B), a filtering system (C), a waste solution tank (D), a liquid receiving tank (E), a gas pump (J), a PBS storage tank (K) and a plurality of liquid transmission systems; liquid inlets above the primary bioreactor (F) and more than one secondary bioreactor are connected with liquid outlets of the PBS storage tank (K), the cell culture liquid tank (A) and the virus culture liquid tank (B) through a liquid transmission system; the lower liquid outlets of the primary bioreactor (F) and more than one secondary bioreactor are connected with the liquid inlet of the waste liquid tank (D) through a liquid transmission system; the liquid outlet below each stage of the bioreactor except the final stage is also connected with the liquid inlet on the side wall of the secondary bioreactor through a liquid transmission system, and the liquid outlet below the final stage of the bioreactor is also connected with the liquid inlet above the liquid receiving tank (E) through a liquid transmission system; a liquid outlet below the liquid collecting tank (E) is connected with a liquid inlet of the filtering system (C) through a liquid transmission system; the liquid transmission system is provided with a control valve and a pressure transmission port, and the pressure transmission port can be connected with a gas pump (J) through a hose.
8. The method and apparatus for producing influenza vaccine for human use according to claim 7, wherein: the primary bioreactor (F) and more than one secondary bioreactor are provided with devices for controlling temperature, sterilizing, stirring and monitoring cell density.
CN202010472879.5A 2020-05-29 2020-05-29 Production method and equipment of human influenza vaccine Pending CN111569055A (en)

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