CN111549002A - Method for producing canine parvovirus monoclonal antibody by using high-density culture mode - Google Patents
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Abstract
The invention belongs to the technical field of canine parvovirus monoclonal antibodies, and particularly relates to a method for producing canine parvovirus monoclonal antibodies by using a high-density culture mode; the bioreactor comprises a flow storage bin and a reaction bin; the upper surface of the reaction bin is fixedly connected with a flow storage bin through an inclined plate; the flow storage bin comprises a liquid storage tank and a liquid collection tank; a supporting plate is arranged in the inner wall of the flow storage bin and is designed in an arc shape; a liquid storage cover is sleeved above the liquid storage tank; the outer surface of the liquid storage tank is fixedly connected with the liquid storage tank; first through holes are uniformly distributed in the inner wall of the liquid storage tank; a control valve is fixedly connected in the inner wall of each first through hole; the invention is mainly used for solving the problems that the cell density of the prior art for producing the canine parvovirus monoclonal antibody by culturing hybridoma cells in a bioreactor is low, the titer of the cultured antibody is low, the product needs to be concentrated, and serum needs to be added in the culture, so that the downstream process becomes complicated, and the risk of side reaction is increased.
Description
Technical Field
The invention belongs to the technical field of canine parvovirus monoclonal antibodies, and particularly relates to a method for producing canine parvovirus monoclonal antibodies by using a high-density culture mode.
Background
Canine parvovirus disease (CPVD) is a viral infectious disease caused by infection with Canine Parvovirus (CPV), which has two main clinical manifestations, one is hemorrhagic enteritis type, showing severe vomiting, bloody stool, and a large reduction in white blood cells; one type is non-suppurative myocarditis, and the sick dog dies suddenly. The disease has the advantages of high transmission speed and high incidence rate, the death rate of puppies reaches 70 percent, the death rate of adult dogs is relatively low, but the phenomenon of recessive virus infection generally exists. In 1978, Kelly et al first isolated CPV from dogs with hemorrhagic enteritis and subsequently reported the prevalence of the disease in several countries and regions of the world. In 1982, the first reports of hemorrhagic enteritis of dogs caused by similar CPV in China were reported by Begonist et al; CPV was isolated in 1983 by Xuhankun et al. Therefore, the CPVD can be rapidly generated and spread in dog groups in northeast, northeast and south China and the like, and brings great harm to the healthy development of the dog breeding industry.
At present, the canine parvovirus disease treatment methods mainly comprise symptomatic therapy, fluid infusion therapy, fasting therapy and specific therapy. In recent years, monoclonal antibody technology has also been widely used in various fields of animal infectious disease research. Compared with the hyperimmune serum, the monoclonal antibody has the advantages of strong specificity, uniform texture, high titer, rapid, large-scale and unlimited production and the like. However, most of the monoclonal antibodies produced at present are of murine origin, and have great disadvantages in clinical application. Mainly because the affinity of the murine monoclonal antibody and an Fc segment receptor on the surface of immune cells such as NK (natural killer) and the like is weak, the generated antibody-dependent cell-mediated cytotoxicity (ADCC) is weak, the binding capacity of the murine monoclonal antibody and a complement component is low, the killing capacity of the murine monoclonal antibody to tumor cells is weak, the half-life period of the murine monoclonal antibody in human blood circulation is short, and the time for the murine monoclonal antibody to exert the ADCC function is short; and the mouse monoclonal antibody also has immunogenicity, and is easy to cause host anaphylactic reaction. Therefore, on one hand, the titer of the monoclonal antibody is reduced, and on the other hand, serious consequences are brought to patients, so that the murine monoclonal antibody can be applied to clinic only after being improved. This has prompted continued efforts to explore more desirable antibody production techniques. For the introduction of canine parvovirus monoclonal antibodies, see journal: zhouying, establishment of a method for detecting canine parvovirus antigen and antibody, Jilin agriculture university, 2017(12), however, the existing method for culturing canine parvovirus monoclonal antibody still has certain problems, and specifically comprises the following aspects:
the existing technology for producing canine parvovirus monoclonal antibody by culturing hybridoma cells in a bioreactor has the disadvantages of low cell density, low titer of the cultured antibody, concentration of the product and serum addition during culture, so that the downstream process is complicated and the risk of side reaction is increased.
In the invention, it is found that hybridoma cells can be cultured at high density by adopting a bioreactor fed-batch culture mode to produce high-titer therapeutic canine parvovirus monoclonal antibodies. The therapeutic canine parvovirus monoclonal antibody produced by the method has high titer, reduces the risk of side reaction by serum-free culture, simplifies the downstream process and obviously improves the quality of the therapeutic canine parvovirus monoclonal antibody.
In view of the above, in order to overcome the above technical problems, the present inventors have designed and developed a method for producing a canine parvovirus monoclonal antibody using a high-density culture method, which comprises culturing hybridoma cells secreting an anti-canine parvovirus monoclonal antibody at high density in a bioreactor and harvesting the anti-canine parvovirus monoclonal antibody.
Disclosure of Invention
In order to make up for the defects of the prior art, the invention provides a method for producing a canine parvovirus monoclonal antibody by using a high-density culture mode, which is mainly used for solving the problems that the cell density of the conventional technology for producing the canine parvovirus monoclonal antibody by using hybridoma cells cultured by a bioreactor is low, the titer of the cultured antibody is low, the product needs to be concentrated, serum needs to be added in the culture, the downstream process becomes complicated, and the risk of side reactions is increased.
The technical scheme adopted by the invention for solving the technical problems is as follows: a method for producing canine parvovirus monoclonal antibody by using a high-density culture mode comprises the following steps:
s1: taking out hybridoma cells from a working cell bank, rapidly transferring the seed cells from liquid nitrogen to a water bath at 37 ℃ for resuscitation by adopting a rapid thawing method, and performing cell expansion culture in a 125ml shake flask by using a serum-free hybridoma cell culture solution; the propagation speed of the hybridoma cells can be improved by culturing the cells by using the serum-free hybridoma cell culture solution, so that the efficiency of culturing the parvovirus monoclonal antibody by the dog is improved;
s2: thoroughly cleaning a bioreactor, installing and debugging the bioreactor after high-temperature and high-pressure sterilization, correcting a pH electrode and a dissolved oxygen electrode, starting four gas supply devices of oxygen, nitrogen and carbon dioxide, and setting culture conditions of the bioreactor: the temperature is 36-37 ℃, the pH value is 6.75-6.95, the dissolved oxygen is 20-50%, the rotating speed of a stirring paddle is 60-100 r/min, and the like; the bioreactor can be in an aseptic state by cleaning and high-temperature sterilization, so that the influence of bacteria and impurities in the bioreactor on the cell propagation speed is prevented, and the bioreactor can be adjusted to improve the most suitable survival and propagation conditions for the cells, so that the cell propagation efficiency can be further improved;
s3 when the seed proliferation of the producing cell reaches the proper total amount, the final cell concentration is 8 × 105～1×106When the cell/ml is within the range, inoculating the cell seeds into a liquid storage tank in the bioreactor at the moment, and starting the bioreactor;
s4: when the cell proliferation enters the middle logarithmic phase for 24 hours, sampling and counting, reserving a sample for titer detection, adding a serum-free fed-batch culture medium according to 4 percent of the total volume, then sampling and counting every 24 hours, reserving a sample for titer detection, adding a serum-free fed-batch culture medium according to 4 percent of the total volume, and harvesting a culture solution until the hemagglutination inhibition titer of the sample reaches more than 1: 2560; serum-free culture medium is fed into the bioreactor within 24 hours, so that the density of cell seeds can be improved, and a canine parvovirus monoclonal antibody product with higher titer is obtained, wherein the hemagglutination inhibition titer of the product can reach more than 1:2560, which is 10-20 times that of the prior art;
s5: collecting the nutrient solution after the culture of the nutrient solution is finished, sterilizing and filtering the collected nutrient solution, aseptically preparing, and quantitatively subpackaging into a sterilization container to obtain the canine parvovirus monoclonal antibody;
wherein the bioreactor comprises a flow storage bin and a reaction bin; the upper surface of the reaction bin is fixedly connected with a flow storage bin through an inclined plate; the flow storage bin comprises a liquid storage tank and a liquid collection tank; a supporting plate is arranged in the inner wall of the flow storage bin and is designed in an arc shape; a liquid storage cover is sleeved above the liquid storage tank, and a pressure valve is arranged on the lower surface of the liquid storage cover; the outer surface of the liquid storage tank is fixedly connected with the liquid storage tank; first through holes are uniformly distributed in the inner wall of the liquid storage tank and are communicated with the liquid storage tank; a control valve is fixedly connected in the inner wall of each first through hole, and the control valve is in a closed arrangement in an initial state; the reaction bin comprises a reaction box, and the reaction box is designed in an I shape; the end surface of the bottom of the reaction tank is fixedly connected with a reaction tank, and the reaction tank is designed in an arc shape; the upper surface of the reaction box is fixedly connected with a motor, and the motor is electrically connected with a controller through a lead; the motor driving shaft is fixedly connected with a round rod, and the round rod penetrates through the reaction box and extends into the reaction tank; the outer surface of one side of the round rod extending into the reaction box is fixedly connected with a first stirring paddle; the outer surface of one side of the round rod extending into the reaction tank is fixedly connected with a second stirring paddle; first rotating blades which are uniformly arranged are rotatably connected around the first stirring paddle and in the inner wall of the reaction box through a rotating shaft; first guide pipes are uniformly arranged in the inner wall of the reaction box, and the first guide pipes are all in elastic design; the other end of each first conduit extends into the liquid collecting tank and is arranged above the supporting plate; an air inlet pipe is arranged in the inner wall of the reaction tank at the central line position of the reaction tank and is used for introducing four gases of air, oxygen, nitrogen and carbon dioxide;
when the method works, the cell density of the cell cultured by the existing technology for producing the canine parvovirus monoclonal antibody by culturing the hybridoma cells in the bioreactor is low, the titer of the cultured antibody is low, the product needs to be concentrated, serum needs to be added in the culture, the downstream process becomes complicated, and the risk of side reaction is increased7The cell/ml is 2-3 times of the density of hybridoma cells cultured by the existing bioreactor, so that a product with higher titer can be obtained, the hemagglutination inhibition titer of the product can reach more than 1:2560, which is 10-20 times of the prior art, the product can be prepared and packaged without concentration, the downstream process is greatly simplified, the risks of exogenous virus and mycoplasma pollution, endotoxin overproof and the like can be greatly reduced by using a serum-free culture medium, side reactions are remarkably reduced, when the bioreactor manufactured by the invention is used, firstly, a liquid storage cover above a liquid storage tank is opened, the serum-free hybridoma cell culture solution and nutrient solution are injected into the liquid storage tank, the serum-free culture medium accounting for four percent of the total volume is injected into the liquid storage tank, then, the liquid storage cover is closed, and at the moment, a controller controls and controls the liquid storage cover toStarting the bioreactor, introducing four gases of air, oxygen, nitrogen and carbon dioxide into the bioreactor through an air inlet pipe, enabling the serum-free hybridoma cell culture solution and the nutrient solution in the liquid storage tank to flow into the reaction box through the first guide pipe under the action of gravity, wherein the first guide pipe corresponds to the first stirring paddle, and the serum-free hybridoma cell culture solution and the nutrient solution flowing into the reaction box through the first guide pipe can be dispersed by the first stirring paddle, so that the cell seeds can be uniformly dispersed in the process, the propagation density of the cell seeds can be improved, and the cell seeds can be prevented from being excessively dense, so that the propagation speed of the cell seeds is reduced, and the serum-free hybridoma cell culture solution and the nutrient solution can be further dispersed when flowing through the first rotating paddle due to the fact that the first rotating paddles are uniformly and fixedly connected in the inner wall of the reaction box, thereby can provide comparatively suitable reproduction environment for the cell seed, when the liquid in the reaction box is too much, can flow into the reaction box, because be equipped with the second stirring rake in the reaction box, thereby can stir serum-free hybridoma cell culture solution once more, can further diffuse the cell seed in this in-process, thereby indirectly improve the density of cell seed, when the gas in the reaction bin expands, gas can flow into the liquid reserve tank through the first pipe, when gas flows into the liquid reserve tank, can press serum-free hybridoma cell culture solution in the liquid reserve tank into the reaction bin completely, open the liquid reserve lid this moment, when the cell seed breeds to the middle stage of logarithm in the reaction tank, after closing the liquid reserve lid, the controller control valve in the first through-hole opens this moment, when the control valve opens, serum-free culture medium in the liquid reserve tank can flow into the liquid reserve tank through the first through-hole, gas in the liquid reserve tank can flow into the liquid reserve tank through the first through hole at this moment, when gas flows into the liquid reserve tank, can be with the liquid reserve tank of impressing at the uniform velocity of the serum-free culture medium in the liquid reserve tank, gas in the reaction storehouse can be when flowing into the liquid reserve tank through first pipe this moment, can be with the serum-free culture medium in the liquid reserve tank through first pipe in the reaction box of impressing, when the serum-free culture medium flows into the reaction box, first stirring rake can carry out primary stirring to the serum-free culture medium, thereby can make no bloodThe clear culture medium is diffused in the serum-free hybridoma cell culture solution, and can provide nutrient substances for cell seeds in the culture solution in the process, thereby improving the propagation speed of cell seeds, when the serum-free culture medium flows into the reaction tank, the second stirring paddle can stir the serum-free culture medium again, thereby further improving the contact degree of the cell seeds and the culture solution and simultaneously improving the hemagglutination inhibition titer of the cell seeds, when the liquid in the liquid collecting box is fed completely, the controller closes the control valve, and adds serum-free culture medium into the liquid collecting box again, and feeds the serum-free culture medium into the reaction bin every hour, so as to circulate, because the pressure valve is arranged at the bottom of the liquid storage cover, the gas pressure in the reactor can be discharged out of the reactor, in the process, the gas pressure in the reactor can be prevented from being too high, and the propagation process of cell seeds can be prevented from being influenced.
Preferably, the upper end of the round rod extends into the liquid storage tank and is fixedly connected with the supporting plate; the supporting plate is rotatably connected with the extrusion box; a circular groove is formed in the inner wall of the circular rod and penetrates through the circular rod; elongated slots are formed in the inner walls of the blades of the first stirring paddles, the elongated slots are communicated with the circular slots, and uniformly-distributed circular holes are formed in the inner walls of the elongated slots; the internal structures of the first stirring paddle and the second stirring paddle are completely the same;
during operation, because the round bar extends to the liquid storage tank, in the process that the motor drives the round bar to rotate, the round bar can drive the supporting plate to rotate, the culture medium in the liquid storage tank can be driven to rotate in the process, the culture medium in the liquid storage tank can be prevented from precipitating in the process, and therefore the diffusion degree of the culture medium is reduced.
Preferably, the upper surface of the supporting plate is fixedly connected with uniformly arranged stirring rods; the outer surface of each stirring rod is fixedly connected with a spiral blade;
the during operation, because the backup pad upper surface links firmly the puddler of evenly arranging, can drive the puddler at backup pad pivoted in-process and rotate, the puddler can stir the culture medium in the liquid reserve tank at this in-process, thereby can further improve the diffusion degree of culture medium, make the nutrient substance in the culture medium distribute evenly, because the puddler surface has linked firmly the spiral leaf, can drive the spiral leaf and remove in the culture medium at the puddler pivoted in-process, the diffusion degree of culture medium can further be improved at this in-process, thereby prevent that the nutrient substance in the culture medium from distributing unevenly.
Preferably, a second conduit is arranged in the inner wall of the reaction box in each first conduit cavity, and the other end of each second conduit extends into a cavity above the liquid collecting box; a one-way valve is arranged at the position of the pipe head of the second guide pipe extending into the liquid collecting tank; the inner wall of the liquid storage tank is made of rubber material; the inner wall of the liquid storage tank is fixedly connected with a sawtooth-shaped memory alloy with the thickness of one to two centimeters; small holes which are uniformly distributed are reserved in the inner wall of each second conduit; a liquid injection valve is fixedly connected in the inner wall above the liquid collecting box;
when the device works, the second guide pipe extends into the cavity above the liquid collecting box, gas in the reaction bin can flow into the liquid collecting box through the second guide pipe, when the gas flows into the liquid collecting box, the liquid collecting box is squeezed due to the fact that the pressure in the liquid collecting box is increased, when the liquid collecting box is squeezed, the inner wall of the liquid collecting box is bent towards the opposite side, the control valve is opened, serum-free culture medium in the liquid collecting box can be quickly pressed into the liquid collecting box after the control valve is opened, after the serum-free culture medium is pressed into the liquid collecting box, the air pressure in the liquid collecting box and the liquid collecting box is gradually balanced, as the inner wall of the liquid collecting box is made of rubber materials and the inner wall is fixedly connected with memory alloy, the bent liquid collecting box can be slowly restored to the initial state, the culture liquid in the reaction tank can be pumped into the liquid collecting box through the first guide pipe in the restoration process of the liquid collecting box, when the culture, can carry out the primary mixing with serum-free medium, thereby can improve cell seed and serum-free medium's the degree of melting mutually, after atmospheric pressure in the liquid reserve tank reaches balance, the serum-free medium in the liquid reserve tank flows into the reaction tank through first pipe this moment, because it has evenly arranged's aperture to open in the second pipe inner wall, gas in the second pipe can pass through the aperture blowout external, when first pipe is when flowing liquid, spun gas can blow the medium in the first pipe, the diffusion degree of medium can be improved at this in-process, the activity of medium can also be improved simultaneously, thereby improve the reproductive efficiency of cell seed.
Preferably, an arc-shaped groove is formed in the inner wall of the reaction tank, and the arc-shaped groove is communicated with the air inlet pipe uniformly; second through holes are uniformly distributed in the inner wall of the arc-shaped groove and are communicated with the reaction tank;
during operation, because intake pipe and arc wall intercommunication, the gas that flows into in the arc wall can flow into the reaction tank through the second through-hole of evenly arranging in, at this in-process, can make air, oxygen, nitrogen gas and the carbon dioxide that lets in fully contact with the culture solution to can provide required material for the cell seed, still can improve the environment of comparatively suitable existence and reproduction for the cell seed simultaneously.
Preferably, a rubber layer is fixedly connected to the inner surface of the reaction tank above each second through hole, and the rubber layer is wrapped on the second through holes; micropores which are uniformly distributed are formed in the inner wall of each rubber layer;
the during operation, because the second through-hole outside all links firmly the rubber layer to the gaseous micropore that can evenly arrange of second through-hole outflow can further refine gas at this in-process in flowing into the reaction tank, can make gaseous better melt with the culture solution, thereby can be more comprehensive for the cell seed provide required material and provide suitable living environment, can also prevent simultaneously that the culture solution in the reaction tank from flowing into in the arc wall.
The invention has the following beneficial effects:
1. the invention can culture the cell seeds at high cell density by feeding serum-free culture medium, and the cell density can reach 2-3 × 107The cell/ml is 2-3 times of the density of hybridoma cells cultured by the existing bioreactor, so that a product with higher titer can be obtained, the hemagglutination inhibition titer of the product can reach more than 1:2560 and is 10-20 times of that of the prior art, the product can be prepared and packaged without concentration, the downstream process is greatly simplified, the risks of exogenous virus and mycoplasma pollution, excessive endotoxin and the like can be greatly reduced by using a serum-free culture medium, and the side reaction is obviously reduced.
2. According to the invention, the supporting plate is arranged, the round rod is fixedly connected with the supporting plate, the round rod can drive the supporting plate to rotate in the rotating process of the round rod, the culture medium in the liquid storage tank can be driven to rotate in the process, the culture medium in the liquid storage tank can be prevented from precipitating in the process, and the diffusion degree of the culture medium is reduced.
Drawings
The invention will be further explained with reference to the drawings.
FIG. 1 is a flow chart of a method of the present invention;
FIG. 2 is a main body view of the bioreactor of the present invention;
FIG. 3 is a sectional view of a bioreactor of the present invention;
FIG. 4 is an enlarged view of a portion of FIG. 3 at A;
FIG. 5 is an enlarged view of a portion of FIG. 3 at B;
FIG. 6 is an enlarged view of a portion of FIG. 3 at C;
in the figure: the device comprises a flow storage bin 1, an inclined plate 11, a liquid storage tank 12, a supporting plate 13, a stirring rod 14, a spiral blade 15, a liquid storage cover 16, a first through hole 17, a control valve 18, a liquid collection tank 19, an air valve 191, a reaction bin 2, a reaction tank 21, a motor 22, a round rod 23, a first stirring paddle 24, a second stirring paddle 25, a first rotating blade 26, a round groove 27, a long groove 28, a round hole 29, a first conduit 291, a second conduit 292, a small hole 293, a reaction tank 294, an air inlet pipe 295, an arc-shaped groove 296, a second through hole 297, a rubber layer 298, micropores 299, a pressure valve 211 and a memory alloy 221.
Detailed Description
The method for producing a canine parvovirus monoclonal antibody by a high-density culture method according to an embodiment of the present invention will be described below with reference to FIGS. 1 to 6.
As shown in FIGS. 1 to 6, the present invention provides a method for producing canine parvovirus monoclonal antibody by high density culture, which comprises the following steps:
s1: taking out hybridoma cells from a working cell bank, rapidly transferring the seed cells from liquid nitrogen to a water bath at 37 ℃ for resuscitation by adopting a rapid thawing method, and performing cell expansion culture in a 125ml shake flask by using a serum-free hybridoma cell culture solution; the propagation speed of the hybridoma cells can be improved by culturing the cells by using the serum-free hybridoma cell culture solution, so that the efficiency of culturing the parvovirus monoclonal antibody by the dog is improved;
s2: thoroughly cleaning a bioreactor, installing and debugging the bioreactor after high-temperature and high-pressure sterilization, correcting a pH electrode and a dissolved oxygen electrode, starting four gas supply devices of oxygen, nitrogen and carbon dioxide, and setting culture conditions of the bioreactor: the temperature is 36-37 ℃, the pH value is 6.75-6.95, the dissolved oxygen is 20-50%, the rotating speed of a stirring paddle is 60-100 r/min, and the like; the bioreactor can be in an aseptic state by cleaning and high-temperature sterilization, so that the influence of bacteria and impurities in the bioreactor on the cell propagation speed is prevented, and the bioreactor can be adjusted to improve the most suitable survival and propagation conditions for the cells, so that the cell propagation efficiency can be further improved;
s3 when the seed proliferation of the producing cell reaches the proper total amount, the final cell concentration is 8 × 105～1×106When the cell/ml is within the range, inoculating the cell seeds into the liquid storage tank 12 in the bioreactor at the moment, and starting the bioreactor;
s4: when the cell proliferation enters the middle logarithmic phase for 24 hours, sampling and counting, reserving a sample for titer detection, adding a serum-free fed-batch culture medium according to 4 percent of the total volume, then sampling and counting every 24 hours, reserving a sample for titer detection, adding a serum-free fed-batch culture medium according to 4 percent of the total volume, and harvesting a culture solution until the hemagglutination inhibition titer of the sample reaches more than 1: 2560; serum-free culture medium is fed into the bioreactor within 24 hours, so that the density of cell seeds can be improved, and a canine parvovirus monoclonal antibody product with higher titer is obtained, wherein the hemagglutination inhibition titer of the product can reach more than 1:2560, which is 10-20 times that of the prior art;
s5: collecting the nutrient solution after the culture of the nutrient solution is finished, sterilizing and filtering the collected nutrient solution, aseptically preparing, and quantitatively subpackaging into a sterilization container to obtain the canine parvovirus monoclonal antibody;
wherein the bioreactor comprises a flow storage bin 1 and a reaction bin 2; the upper surface of the reaction bin 2 is fixedly connected with a flow storage bin 1 through an inclined plate 11; the flow storage bin 1 comprises a liquid storage tank 12 and a liquid collection tank 19; a supporting plate 13 is arranged in the inner wall of the flow storage bin 1, and the supporting plate 13 is in an arc-shaped design; a liquid storage cover 16 is sleeved above the liquid storage tank 12, and a pressure valve 211 is arranged on the lower surface of the liquid storage cover 16; the outer surface of the liquid storage tank 12 is fixedly connected with a liquid storage tank 19; first through holes 17 are uniformly arranged in the inner wall of the liquid storage tank 12, and the first through holes 17 are communicated with a liquid storage tank 19; a control valve 18 is fixedly connected to the inner wall of each first through hole 17, and the control valve 18 is closed in an initial state; the reaction bin 2 comprises a reaction box 21, and the reaction box 21 is designed in an I shape; the bottom end face of the reaction box 21 is fixedly connected with a reaction tank 294, and the reaction tank 294 is designed in a circular arc shape; the upper surface of the reaction box 21 is fixedly connected with a motor 22, and the motor 22 is electrically connected with a controller through a lead; a round rod 23 is fixedly connected to a driving shaft of the motor 22, and the round rod 23 penetrates through the reaction box 21 and extends into the reaction tank 294; the outer surface of one side of the round rod 23 extending into the reaction box 21 is fixedly connected with a first stirring paddle 24; the outer surface of one side of the round rod 23 extending into the reaction tank 294 is fixedly connected with a second stirring paddle 25; first rotating blades 26 which are uniformly arranged are rotatably connected around the first stirring paddle 24 and in the inner wall of the reaction box 21 through a rotating shaft; the inner wall of the reaction box 21 is provided with first guide pipes 291 which are uniformly arranged, and the first guide pipes 291 are all designed to be elastic; the other end of each of said first conduits 291 extends into the header tank 19 and is disposed above the support plate 13; an air inlet pipe 295 is arranged in the inner wall of the reaction tank 294 at the central line position of the reaction tank 294, and the air inlet pipe 295 is used for introducing four gases of air, oxygen, nitrogen and carbon dioxide;
when the method works, the cell density of the cell cultured by the existing technology for producing the canine parvovirus monoclonal antibody by culturing the hybridoma cells in the bioreactor is low, the titer of the cultured antibody is low, the product needs to be concentrated, serum needs to be added in the culture, the downstream process becomes complicated, and the risk of side reaction is increased7The cell/ml is 2-3 times of the density of hybridoma cells cultured by the existing bioreactor, so that a product with higher titer can be obtained, the hemagglutination inhibition titer of the product can reach more than 1:2560 and is 10-20 times of the prior art, the product can be prepared and packaged without concentration, the downstream process is greatly simplified, the risks of exogenous virus and mycoplasma pollution, excessive endotoxin and the like can be greatly reduced by using a serum-free culture medium, side reactions are remarkably reduced, when the bioreactor manufactured by the invention is used, firstly, a liquid storage cover 16 above a liquid storage tank 12 is opened, serum-free hybridoma cell culture liquid and nutrient solution are injected into a liquid storage tank 12, simultaneously, four percent of the serum-free culture medium is injected into a liquid collection tank, then, the liquid storage cover 16 is closed, at the moment, a controller controls to start the bioreactor, and oxygen is introduced into the bioreactor through an air inlet pipe 295, The serum-free hybridoma cell culture solution and the nutrient solution flowing into the reaction box 21 through the first conduit 291 can be dispersed by the first stirring paddle 24 because the first conduit 291 corresponds to the first stirring paddle 24, and the cell seeds can be uniformly dispersed in the process, so that the propagation of the cell seeds can be improvedThe breeding density is high, meanwhile, the cell seeds are prevented from being too dense, the propagation speed of the cell seeds is reduced, the first rotating blades 26 which are uniformly distributed are fixedly connected in the inner wall of the reaction box 21, when the serum-free hybridoma cell culture solution and the nutrient solution flow through the first rotating blades 26, the serum-free hybridoma cell culture solution can be further diffused, a relatively suitable propagation environment can be provided for the cell seeds, when the liquid in the reaction box 21 is too much, the liquid can flow into the reaction tank 294, the second stirring paddle 25 is arranged in the reaction tank 294, the serum-free hybridoma cell culture solution can be stirred again, the cell seeds can be further diffused in the process, the density of the cell seeds is indirectly improved, when the gas in the reaction bin 2 expands, the gas can flow into the liquid storage box 12 through the first guide pipe 291, when the gas flows into the liquid storage box 12, the serum-free hybridoma cell culture fluid in the liquid storage tank 12 can be completely pressed into the reaction chamber 2, when cell seeds propagate to the middle logarithmic phase in the reaction tank 294, the controller controls the control valve 18 in the first through hole 17 to be opened at the moment, the serum-free culture medium in the liquid collection tank 19 flows into the liquid storage tank 12 through the first through hole 17 after the control valve 18 is opened, the gas in the liquid storage tank 12 flows into the liquid collection tank 19 through the first through hole 17 at the moment, the serum-free culture medium in the liquid collection tank 19 can be pressed into the liquid storage tank 12 at a constant speed when the gas flows into the liquid collection tank 19, the serum-free culture medium in the liquid storage tank 12 can be pressed into the reaction tank 21 through the first conduit 291 when the gas in the reaction chamber 2 flows into the liquid storage tank 12 through the first conduit 291, and the first stirring paddle 24 can perform primary stirring on the serum-free culture medium when the serum-free culture medium flows into the reaction tank 21, so that the serum-free culture medium can be diffused in the serum-free hybridoma cell culture solution, nutrient substances can be provided for cell seeds in the culture solution in the process, the propagation speed of the cell seeds can be improved, when the serum-free culture medium flows into the reaction tank 294, the second stirring paddle 25 can stir the serum-free culture medium again, the contact degree of the cell seeds and the culture solution can be further improved, the hemagglutination inhibition titer of the cell seeds can be improved, and after the liquid flow in the liquid collecting tank 19 is finished, the controller is closed at the momentThe valve 18 is controlled, the serum-free culture medium is added into the liquid collecting box 19 again, the serum-free culture medium flows into the reaction bin 2 every 24 hours, and the circulation is performed, and the pressure valve 211 is arranged at the bottom of the liquid collecting cover 16, so that the gas pressure in the reactor can be discharged out of the reactor, and the phenomenon that the gas pressure in the reactor is too high to influence the propagation process of cell seeds can be prevented in the process.
As an embodiment of the present invention, the upper end of the round rod 23 extends into the liquid storage tank 12 and is fixedly connected with the supporting plate 13; the supporting plate 13 is rotatably connected with the extrusion box; a circular groove 27 is formed in the inner wall of the circular rod 23, and the circular groove 27 penetrates through the circular rod 23; elongated slots 28 are formed in the inner walls of the blades of the first stirring paddles 24, the elongated slots 28 are communicated with the circular slots 27, and circular holes 29 are uniformly formed in the inner walls of the elongated slots 28; the internal structures of the first stirring paddle 24 and the second stirring paddle 25 are completely the same;
during operation, because the round rod 23 extends into the liquid storage tank 12, in the process that the motor 22 drives the round rod 23 to rotate, the round rod 23 can drive the support plate 13 to rotate, the culture medium in the liquid storage tank 12 can be driven to rotate in the process, the culture medium in the liquid storage tank 12 can be prevented from precipitating in the process, and therefore the diffusion degree of the culture medium is reduced.
As an embodiment of the invention, the upper surface of the supporting plate 13 is fixedly connected with evenly arranged stirring rods 14; the outer surface of each stirring rod 14 is fixedly connected with a spiral blade 15;
the during operation, because backup pad 13 upper surface links firmly the puddler 14 of evenly arranging, can drive puddler 14 at backup pad 13 pivoted in-process and rotate, puddler 14 can stir the culture medium in liquid reserve tank 12 at this in-process, thereby can further improve the diffusion degree of culture medium, make the nutrient substance in the culture medium distribute evenly, because puddler 14 surface has linked firmly spiral leaf 15, can drive spiral leaf 15 and remove in the culture medium at puddler 14 pivoted in-process, can further improve the diffusion degree of culture medium at this in-process, thereby prevent that the nutrient substance in the culture medium from distributing unevenly.
As an embodiment of the present invention, a second conduit 292 is installed in the inner wall of the reaction box 21 in the cavity of each first conduit 291, and the other end of the second conduit 292 extends into the cavity above the liquid collecting box 19; a check valve is arranged at the position of the pipe head of the second conduit 292 extending into the liquid collecting tank 19; the inner wall of the liquid storage tank 12 is made of rubber material; the inner wall of the liquid storage tank 12 is fixedly connected with a sawtooth-shaped memory alloy 221 with the thickness of one to two centimeters; small holes 293 which are uniformly distributed are reserved in the inner wall of each second conduit 292; a liquid injection valve 191 is fixedly connected in the inner wall above the liquid collecting box 19;
when the device works, the second guide pipe 292 extends into a cavity above the liquid collecting tank 19, gas in the reaction bin 2 can flow into the liquid collecting tank 19 through the second guide pipe 292, when the gas flows into the liquid collecting tank 19, the pressure in the liquid collecting tank 19 is increased, so that the liquid collecting tank 12 is squeezed, when the liquid collecting tank 12 is squeezed, the inner wall of the liquid collecting tank 12 is bent towards the opposite side, the control valve 18 is opened at the moment, serum-free culture medium in the liquid collecting tank 19 is quickly pressed into the liquid collecting tank 12 after the control valve 18 is opened, after the serum-free culture medium is pressed into the liquid collecting tank 12, the air pressure in the liquid collecting tank 12 and the liquid collecting tank 19 is gradually balanced, because the inner wall of the liquid collecting tank 12 is made of rubber material and the memory alloy 221 is fixedly connected in the inner wall, so that the bent liquid collecting tank 12 can be slowly restored to the initial state, and the culture liquid in the reaction tank can be pumped into the liquid collecting tank 12 through the first guide pipe 291 in the, when the culture solution flows into the liquid storage tank 12, the culture solution can be preliminarily mixed with a serum-free culture medium, so that the degree of fusion of cell seeds and the serum-free culture medium can be improved, after the air pressure in the liquid storage tank 12 is balanced, the serum-free culture medium in the liquid storage tank 12 flows into the reaction tank through the first conduit at the moment, the small holes 293 which are uniformly distributed are formed in the inner wall of the second conduit 292, gas in the second conduit 292 can be sprayed out through the small holes 293, when the first conduit 291 flows, the sprayed gas can blow the culture medium in the first conduit 291, the diffusion degree of the culture medium can be improved in the process, meanwhile, the activity of the culture medium can also be improved, and the propagation efficiency of the cell seeds is improved.
As an embodiment of the present invention, an arc-shaped groove 296 is formed in an inner wall of the reaction tank 294, and the arc-shaped groove 296 is communicated with the air inlet pipe 295 uniformly; second through holes 297 which are uniformly distributed are formed in the inner wall of the arc-shaped groove 296, and the second through holes 297 are communicated with the reaction tank 294;
during operation, because intake pipe 295 and arc groove 296 intercommunication, the gas that flows into in the arc groove 296 can flow into reaction tank 294 through the second through-hole 297 of evenly arranging in, at this in-process, can make the air, oxygen, nitrogen gas and the carbon dioxide that lets in fully contact with the culture solution to can provide required material for the cell seed, still can improve comparatively suitable living and environment of propagating for the cell seed simultaneously.
As an embodiment of the present invention, a rubber layer 298 is fixedly attached to the inner surface of the reaction cell 294 above each second through hole 297, and the rubber layer 298 is designed to wrap the second through hole 297; uniformly arranged micropores 299 are formed in the inner wall of each rubber layer 298;
during operation, because second through-hole 297 outside all links firmly rubber layer 298 to the gas that second through-hole 297 flows out can flow into reaction tank 294 through micropore 299 of evenly arranging, can further refine gas at this in-process, can make gas better melt with the culture solution, thereby can be more comprehensive provide required material and provide suitable living environment for the cell seed, can also prevent that the culture solution in the reaction tank 294 from flowing into in the arc 296 simultaneously.
The specific working process is as follows:
when the bioreactor manufactured by the invention is used, firstly, the liquid storage cover 16 above the liquid storage box 12 is opened, the serum-free hybridoma cell culture solution and the nutrient solution are injected into the liquid storage box 12, simultaneously, the serum-free culture medium accounting for four percent of the total volume is injected into the liquid collection tank, then, the liquid storage cover 16 is closed, at the moment, the controller controls the bioreactor to be started, air, oxygen, nitrogen and carbon dioxide are introduced into the bioreactor through the air inlet pipe 295, at the moment, the serum-free hybridoma cell culture solution and the nutrient solution in the liquid storage tank flow into the reaction box 21 through the first conduit 291 under the action of gravity, as the first conduits 291 correspond to the first stirring paddles 24, the serum-free hybridoma cell culture solution and the nutrient solution flowing into the reaction box 21 through the first conduit 291 can be dispersed by the first stirring paddles 24, and as the first rotating blades 26 which are uniformly arranged are fixedly connected in the inner wall of the reaction box 21, when the serum-free hybridoma cell culture solution and the nutrient solution flow through the first rotating blade 26, the serum-free hybridoma cell culture solution can be further diffused, when the liquid in the reaction tank 21 is excessive, the serum-free hybridoma cell culture solution can flow into the reaction tank 294, and the second stirring paddle 25 is arranged in the reaction tank 294, so that the serum-free hybridoma cell culture solution can be stirred again, the cell seeds can be further diffused in the process, so that the density of the cell seeds is indirectly improved, when the gas in the reaction chamber 2 expands, the gas can flow into the liquid storage tank 12 through the first conduit 291, when the gas flows into the liquid storage tank 12, the serum-free hybridoma cell culture solution in the liquid storage tank 12 can be completely pressed into the reaction chamber 2, at the moment, the liquid storage cover 16 is opened, when the cell seeds propagate to the middle logarithmic phase in the reaction tank 294, the liquid storage cover 16 is closed, at the moment, the controller controls the control valve 18 in the first through hole 17 to be opened, after the control valve 18 is opened, the serum-free culture medium in the liquid collecting tank 19 flows into the liquid collecting tank 12 through the first through hole 17, at this time, the gas in the liquid collecting tank 12 flows into the liquid collecting tank 19 through the first through hole 17, when the gas flows into the liquid collecting tank 19, the serum-free culture medium in the liquid collecting tank 19 can be pressed into the liquid collecting tank 12 at a constant speed, at this time, the serum-free culture medium in the liquid collecting tank 12 can be pressed into the reaction tank 21 through the first conduit 291 when the gas in the reaction chamber 2 flows into the liquid collecting tank 12 through the first conduit 291, when the serum-free culture medium flows into the reaction tank 21, the first stirring paddle 24 can primarily stir the serum-free culture medium, when the serum-free culture medium flows into the reaction tank 294, the second stirring paddle 25 can stir the serum-free culture medium again, so that the contact degree between the cell seeds and the culture medium can be further improved, after the liquid in the liquid collecting tank 19 is fed, the controller closes the control valve 18, and adds serum-free culture medium into the liquid collecting tank 19 again, and feeds the serum-free culture medium into the reaction chamber 2 every 24 hours, so as to circulate, because the second conduit 292 extends into the cavity above the liquid collecting tank 19, the gas in the reaction chamber 2 can flow into the liquid collecting tank 19 through the second conduit 292, when the gas flows into the liquid collecting tank 19, the pressure in the liquid collecting tank 19 is increased, so as to press the liquid storage tank 12, when the liquid storage tank 12 is pressed, the inner wall of the liquid storage tank 12 is bent to the opposite side, at this time, the control valve 18 is opened, when the control valve 18 is opened, the serum-free culture medium in the liquid collecting tank 19 is quickly pressed into the liquid storage tank 12, after the serum-free culture medium is pressed into the liquid storage tank 12, the air pressure in the liquid storage tank 12 and the liquid collecting tank 19 is gradually balanced, because the inner wall of the liquid storage tank 12 is made of rubber material and the, thereby can make crooked liquid reserve tank 12 slow recovery to initial condition, can be through in the culture solution suction liquid reserve tank 12 of first pipe 291 in reaction tank 294, when the culture solution flows into liquid reserve tank 12, can carry out preliminary mixing with serum-free culture medium, thereby can improve cell seed and serum-free culture medium's the degree of melting mutually, after the atmospheric pressure in liquid reserve tank 12 reached balanced, serum-free culture medium in the liquid reserve tank 12 flowed into the reaction tank through first pipe this moment.
The foregoing illustrates and describes the principles, general features, and advantages of the present invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (6)
1. A method for producing canine parvovirus monoclonal antibody by using a high-density culture mode is characterized by comprising the following steps:
s1: taking out hybridoma cells from a working cell bank, rapidly transferring the seed cells from liquid nitrogen to a water bath at 37 ℃ for resuscitation by adopting a rapid thawing method, and performing cell expansion culture in a 125ml shake flask by using a serum-free hybridoma cell culture solution;
s2: thoroughly cleaning a bioreactor, installing and debugging the bioreactor after high-temperature and high-pressure sterilization, correcting a pH electrode and a dissolved oxygen electrode, starting four gas supply devices of oxygen, nitrogen and carbon dioxide, and setting culture conditions of the bioreactor: the temperature is 36-37 ℃, the pH value is 6.75-6.95, the dissolved oxygen is 20-50%, the rotating speed of a stirring paddle is 60-100 r/min, and the like;
s3 when the seed proliferation of the producing cell reaches the proper total amount, the final cell concentration is 8 × 105～1×106When the cell/ml is within the range, inoculating the cell seeds into a liquid storage box (12) in the bioreactor at the moment, and starting the bioreactor;
s4: when the cell proliferation enters the middle logarithmic phase for 24 hours, sampling and counting, reserving a sample for titer detection, adding a serum-free fed-batch culture medium according to 4 percent of the total volume, then sampling and counting every 24 hours, reserving a sample for titer detection, adding a serum-free fed-batch culture medium according to 4 percent of the total volume, and harvesting a culture solution until the hemagglutination inhibition titer of the sample reaches more than 1: 2560;
s5: collecting the nutrient solution after the culture of the nutrient solution is finished, sterilizing and filtering the collected nutrient solution, aseptically preparing, and quantitatively subpackaging into a sterilization container to obtain the canine parvovirus monoclonal antibody;
wherein the bioreactor comprises a flow storage bin (1) and a reaction bin (2); the upper surface of the reaction bin (2) is fixedly connected with a flow storage bin (1) through an inclined plate (11); the flow storage bin (1) comprises a liquid storage tank (12) and a liquid collection tank (19); a supporting plate (13) is arranged in the inner wall of the flow storage bin (1), and the supporting plate (13) is designed in an arc shape; a liquid storage cover (16) is sleeved above the liquid storage tank (12), and a pressure valve (211) is arranged on the lower surface of the liquid storage cover (16); the outer surface of the liquid storage tank (12) is fixedly connected with a liquid storage tank (19); first through holes (17) are uniformly distributed in the inner wall of the liquid storage tank (12), and the first through holes (17) are communicated with the liquid storage tank (19); a control valve (18) is fixedly connected to the inner wall of each first through hole (17), and the control valve (18) is arranged in a closed mode in an initial state; the reaction bin (2) comprises a reaction box (21), and the reaction box (21) is designed in an I shape; the bottom end face of the reaction box (21) is fixedly connected with a reaction tank (294), and the reaction tank (294) is designed in an arc shape; the upper surface of the reaction box (21) is fixedly connected with a motor (22), and the motor (22) is electrically connected with a controller through a lead; a round rod (23) is fixedly connected to a driving shaft of the motor (22), and the round rod (23) penetrates through the reaction box (21) and extends into the reaction tank (294); the outer surface of one side of the round rod (23) extending into the reaction box (21) is fixedly connected with a first stirring paddle (24); the outer surface of one side of the round rod (23) extending into the reaction tank (294) is fixedly connected with a second stirring paddle (25); first rotating blades (26) which are uniformly arranged are rotatably connected around the first stirring paddle (24) and in the inner wall of the reaction box (21) through a rotating shaft; first guide pipes (291) are uniformly arranged in the inner wall of the reaction box (21), and the first guide pipes (291) are all in an elastic design; the other end of each first conduit (291) extends into the header tank (19) and is arranged above the supporting plate (13); an air inlet pipe (295) is arranged in the inner wall of the reaction tank (294) at the central line position of the reaction tank (294), and the air inlet pipe (295) is used for introducing four gases of air, oxygen, nitrogen and carbon dioxide.
2. The method for producing canine parvovirus monoclonal antibody by high-density culture according to claim 1, wherein: the upper end of the round rod (23) extends into the liquid storage tank (12) and is fixedly connected with the supporting plate (13); the supporting plate (13) is rotatably connected with the extrusion box; a circular groove (27) is formed in the inner wall of the circular rod (23), and the circular groove (27) penetrates through the circular rod (23); elongated slots (28) are formed in the inner walls of the blades of the first stirring paddles (24), the elongated slots (28) are communicated with the circular slots (27), and circular holes (29) are uniformly distributed in the inner walls of the elongated slots (28); the internal structures of the first stirring paddle (24) and the second stirring paddle (25) are completely the same.
3. The method for producing canine parvovirus monoclonal antibody by high-density culture according to claim 2, wherein: the upper surface of the supporting plate (13) is fixedly connected with uniformly arranged stirring rods (14); the outer surface of each stirring rod (14) is fixedly connected with a spiral blade (15).
4. The method for producing canine parvovirus monoclonal antibody by high-density culture according to claim 1, wherein: a second guide pipe (292) is arranged in the inner wall of the reaction box (21) in each first guide pipe (291) cavity, and the other end of each second guide pipe (292) extends into the cavity above the liquid collecting box (19); the pipe head of the second conduit (292) extending into the liquid collecting tank (19) is provided with a one-way valve; the inner wall of the liquid storage tank 12 is made of rubber material; the inner wall of the liquid storage tank (12) is fixedly connected with a sawtooth-shaped memory alloy (221) with the thickness of one to two centimeters; small holes (293) which are uniformly distributed are trapped in the inner wall of each second conduit (292); a liquid injection valve (191) is fixedly connected in the inner wall above the liquid collecting box (19).
5. The method for producing canine parvovirus monoclonal antibody by high-density culture according to claim 1, wherein: an arc-shaped groove (296) is formed in the inner wall of the reaction tank (294), and the arc-shaped groove (296) is communicated with an air inlet pipe (295); second through holes (297) are uniformly arranged and are formed in the inner wall of the arc-shaped groove (296), and the second through holes (297) are communicated with the reaction tank (294).
6. The method for producing canine parvovirus monoclonal antibody by high-density culture according to claim 5, wherein: a rubber layer (298) is fixedly connected to the inner surface of the reaction tank (294) above each second through hole (297), and the rubber layer (298) is wrapped on the second through holes (297); uniformly arranged micropores (299) are formed in the inner wall of each rubber layer (298).
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| CN112852749A (en) * | 2021-03-27 | 2021-05-28 | 哈尔滨元亨生物药业有限公司 | Hybridoma cell strain C68 capable of efficiently secreting canine parvovirus monoclonal antibody and production method thereof by using bioreactor |
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| CN111979125A (en) * | 2020-08-28 | 2020-11-24 | 安徽惠恩生物科技股份有限公司 | Serum-free cell culture medium |
| CN112852749A (en) * | 2021-03-27 | 2021-05-28 | 哈尔滨元亨生物药业有限公司 | Hybridoma cell strain C68 capable of efficiently secreting canine parvovirus monoclonal antibody and production method thereof by using bioreactor |
| CN112877297A (en) * | 2021-03-27 | 2021-06-01 | 哈尔滨元亨生物药业有限公司 | Method for preparing cat distemper virus monoclonal antibody by using bioreactor |
| CN114561270A (en) * | 2022-03-21 | 2022-05-31 | 广州齐志生物工程设备有限公司 | High-pathogenicity virus culture containerization system |
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