CN110791431A - Online ultrasonic stripping cell disruption bioreactor and virus harvesting method - Google Patents
Online ultrasonic stripping cell disruption bioreactor and virus harvesting method Download PDFInfo
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Abstract
The invention discloses an online ultrasonic stripping cell disruption bioreactor and a method for harvesting viruses, which comprises a bioreactor, an ultrasonic device and an online cell counting device; the bioreactor is used for culturing cells or/and viruses or/and inoculating the cells and the viruses; the ultrasonic device is used for stripping and ultrasonically crushing cells on a carrier in the bioreactor; the online cell counting device is used for counting cells in the bioreactor so as to determine the virus inoculation time, the virus inoculation amount and the cell stripping and ultrasonic disruption starting time of the cells. The method has the advantages of simple and controllable process, short time consumption, high efficiency, low pollution risk, less virus loss, high virus yield, stable product quality and the like.
Description
Technical Field
The invention relates to the technical field of bioengineering, in particular to an online ultrasonic stripping cell disruption bioreactor and a method for harvesting viruses.
Background
The bioreactor is a device which provides a suitable environment for cell proliferation or biochemical reaction by taking living cells or enzymes as biocatalysts, and is a key device in the biological reaction process. The structure, operation mode and operation condition of the bioreactor are closely related to the quality, yield (conversion rate) and energy consumption of the biochemical products.
With the continuous development of biotechnology, large-scale animal cell bioreactor culture technology is gradually replacing glass bottle static culture and rotary culture technology, because it can provide the best living environment for the cultured object in time, thus the most efficient improvement of product yield and quality, which should be said to be a qualitative leap in animal cell culture history, and also be a major breakthrough of biopharmaceutical technology, it not only simplifies the process, but also can produce high quality product. However, some viral vaccines cannot be produced by using the existing bioreactor, such as rotavirus vaccine, EV71 virus vaccine, CA16 virus vaccine, hepatitis a virus vaccine, varicella virus vaccine, and the like, at present, these vaccines can only be produced by using a cell factory, which usually has a small space and oxygen cannot meet the cell growth requirement, and the growth of cells can be restricted, so that the production efficiency is not high, the operation is complicated, the pollution control difficulty is high, and when preparing the vaccines, particularly for preparing intracellular virus vaccines with strong cell binding property, cultured cells with virus need to be stripped from the cell factory to be collected and crushed, so that free virus particles can be obtained.
At present, the collection of the toxic cells is mainly carried out by the following two methods:
the first freezing method specifically comprises the following steps: the cell factory is put into a refrigerator for freezing, then taken out, thawed and harvested, wherein the freezing time is long, the virus loss is large, a large amount of ultralow-temperature preservation equipment is required to be arranged, and the operability is poor;
the second digestion method specifically comprises the following steps: adding digestive juice containing digestive enzyme into a cell culture vessel, cutting off adhesion molecules between cells and the cell culture vessel, stripping and collecting the cells from the cell culture vessel, and removing introduced exogenous substances through centrifugation to realize the harvest of the cells.
In view of the above, there is a need to develop a safe and efficient online ultrasonic peeling and cell disruption bioreactor and a method for harvesting cells or viruses by online ultrasonic peeling and cell disruption.
Disclosure of Invention
Aiming at the problems in the background art, the invention aims to provide an on-line ultrasonic stripping broken cell bioreactor with simple and controllable process, short time consumption, high efficiency and small pollution risk and a method for harvesting viruses by using the same.
In order to achieve the purpose, the invention adopts the technical scheme that:
an online ultrasonic stripping cell disruption bioreactor comprises a bioreactor, an ultrasonic device and an online cell counting device;
the bioreactor, the bioreactor and the method are used for culturing cells or/and viruses or/and inoculating the cells and the viruses;
the ultrasonic device is used for peeling and ultrasonically breaking cells which are positioned in the bioreactor and attached to the cell carrier;
the on-line cell counting device is used for counting the cells in the bioreactor so as to determine the virus inoculation time, the virus inoculation amount and the cell stripping and ultrasonic disruption starting time of the cells.
Further, the bioreactor comprises a tank body, a tank cover arranged at the top of the tank body, a fixed bed arranged in the tank body and a bioreactor control cabinet arranged beside the tank body;
the tank body is also provided with a DO dissolved oxygen electrode, a PH electrode, a temperature probe, a deep breather pipe, a liquid inlet pipe, a liquid outlet pipe, an alkali adding pipe, a sampling valve and a tank bottom valve; the DO dissolved oxygen electrode, the PH electrode, the temperature probe and the sampling valve are all arranged at the lower part of the outer wall of the tank body, the liquid inlet pipe, the liquid outlet pipe and the alkali adding pipe are all arranged at the upper part of the outer wall of the tank body, the tank bottom valve is arranged at the bottom of the outer wall of the tank body, one end of the deep breather pipe extends out of the outer wall of the upper part of the tank body, and the other end of the deep breather pipe extends into the tank body, penetrates through the fixed bed and extends to the upper part;
the tank cover is also provided with a CIP cleaning ball, a magnetic stirrer and a respirator, one end of the CIP cleaning ball, one end of the magnetic stirrer and one end of the respirator penetrate through the tank cover and extend into the tank body, and the other end of the CIP cleaning ball, the magnetic stirrer and the respirator are exposed outside the tank cover;
a plurality of cell carriers are filled in the fixed bed and used for providing adherent adherence support for cell growth;
the DO dissolved oxygen electrode, the PH electrode, the temperature probe, the sampling valve, the tank bottom valve, the magnetic stirrer and the respirator are all electrically connected with a master controller arranged in the bioreactor main control cabinet, and the master controller is electrically connected with a main control display screen arranged outside the bioreactor main control cabinet.
Further, ultrasonic device contains supersonic generator, ultrasonic transducer and vibrting spear, supersonic generator and ultrasonic transducer all set up in bioreactor master control cabinet, vibrting spear one end is uncovered and is established at the cover top and be connected with ultrasonic transducer electricity, and the other end passes bioreactor's cover and stretches into in the fixed bed that the internal setting of jar was equipped with, ultrasonic transducer still is connected with supersonic generator electricity, supersonic generator still is connected with bioreactor master control cabinet's master controller electricity.
Furthermore, the online cell counting device comprises an online cell probe, a transmitting module and a current output module, wherein the transmitting module and the current output module are arranged in the bioreactor main control cabinet, one end of the online cell probe is exposed at the top of the tank cover and is electrically connected with the transmitting module, the other end of the online cell probe penetrates through the tank cover of the bioreactor and extends into a fixed bed arranged in the tank body, the transmitting module is also electrically connected with the current output module, and the current output module is also electrically connected with a main controller of the bioreactor main control cabinet.
Further, the magnetic stirrer comprises a magnetic motor, a stirring shaft, an upper stirring paddle, a middle stirring paddle and a lower stirring paddle, wherein one end of the stirring shaft extends out of the tank cover and is connected with the magnetic motor, the other end of the stirring shaft sequentially penetrates through the tank cover and the fixed bed and extends to the upper part of the bottom ventilation disc, and the upper stirring paddle, the middle stirring paddle and the lower stirring paddle are sequentially fixed on the stirring shaft in a penetrating manner from top to bottom and are all positioned in the tank body;
wherein, the upper stirring paddle is positioned above the fixed bed arranged in the tank body; the middle stirring paddle is positioned in the middle of the fixed bed arranged in the tank body, and the lower stirring paddle is positioned below the fixed bed arranged in the tank body and above the bottom vent disc.
Further, the fixed bed comprises a middle cylinder, an upper screen plate arranged at the upper part of the middle cylinder and a lower screen plate arranged at the lower part of the middle cylinder; the annular cavity formed by the upper screen plate, the lower screen plate, the middle cylinder and the tank body of the bioreactor is filled with a cell carrier for adherent growth of cells.
Furthermore, the upper stirring paddle of the magnetic stirrer is positioned above the upper mesh plate of the fixed bed, the middle stirring paddle is positioned in the middle cylinder of the fixed bed, and the lower stirring paddle is positioned below the lower mesh plate of the fixed bed and above the bottom vent disc.
Further, the online ultrasonic stripping cell disruption bioreactor further comprises a tank cover lifting device, wherein the tank cover lifting device is used for lifting a tank cover of the bioreactor, and then the opening and closing of the tank cover and the tank body of the bioreactor are realized.
Further, the online ultrasonic stripping and cell disruption bioreactor also comprises a tank support, and the tank support is used for supporting a tank of the bioreactor.
A method for harvesting viruses by on-line ultrasonic stripping of disrupted cell bioreactors comprising the steps of:
s1, adding a PBS buffer solution into the tank body of the bioreactor to a specified position, and maintaining the pressure for 30 minutes;
s2, when the pressure of the bioreactor is kept for 30 minutes and does not drop, the sealing of the bioreactor is qualified, and at the moment, a sterilization program carried by the bioreactor is started, so that the online sterilization of the bioreactor is automatically completed, and after the sterilization is completed, the temperature is naturally reduced for standby;
s3, draining the PBS buffer solution in the tank body through a tank bottom valve, adding the cell growth solution into the tank body through a liquid inlet pipe, setting corresponding parameters on a main control display screen of a control cabinet of the bioreactor for pre-culture, and obtaining the qualified product when no bacteria grow in the tank body of the bioreactor for 1-2 days;
s4, discharging the cell growth liquid pre-cultured in the tank body through a tank bottom valve, adding new cell growth liquid and cell suspension into the tank body through a liquid inlet pipe, then opening a magnetic stirrer to work, uniformly mixing the newly added liquid through an upper stirring paddle of the magnetic stirrer, simultaneously increasing the dissolution of oxygen, enabling the mixed liquid in the tank body to flow through a middle stirring paddle of the magnetic stirrer, carrying out nutrition exchange on cells, uniformly mixing the liquid at the bottom of the tank body through a lower stirring paddle of the magnetic stirrer, and simultaneously increasing the dissolution of oxygen; meanwhile, the online cell counting device is controlled by the bioreactor control cabinet to start cell counting work, and corresponding cell culture parameters are set on a main control display screen of the bioreactor control cabinet for cell culture;
in the cell culture process, a PH electrode is used for detecting the PH value of liquid in the tank body, a DO dissolved oxygen electrode is used for detecting the dissolved oxygen condition of the liquid in the tank body, and a temperature probe is used for detecting the temperature of the liquid in the tank body;
if the PH electrode detects that the PH value of the liquid in the tank body is lower, adding alkali materials into the tank body from an alkali adding pipe;
if the PH electrode detects that the PH value of the liquid in the tank body is higher, the carbon dioxide is introduced into the tank body from the deep vent pipe;
if the DO dissolved oxygen electrode detects that the dissolved oxygen of the liquid in the tank body is low, oxygen is introduced into the tank body from the deep layer vent pipe;
s5, when the on-line cell counting device counts that the cell number in the tank body meets the process requirements, starting to inoculate the virus, and meanwhile, setting corresponding virus culture parameters on a main control display screen of a bioreactor control cabinet for virus culture;
s6, when the virus culture meets the process requirements, draining the virus culture solution in the tank body through a tank bottom valve, after the virus culture solution is drained, pumping a washing solution into the tank body through a CIP (cleaning in Place) cleaning ball to clean animal-derived protein in the tank body, after the washing is finished, adding the virus culture solution without the animal-derived protein into the tank body through a liquid inlet pipe, and continuing to culture;
s7, when the number of cells in the tank body is measured by the on-line cell counting device to be reduced, the cells are shown to be diseased, when the number of the cells is reduced to a certain number, the animal-derived protein-free virus culture solution added in the step S6 is drained through a tank bottom valve, after the cells are drained completely, a precooled freeze-drying protective agent is added into the tank body through a liquid inlet pipe, meanwhile, ultrasonic parameters are set on a main control display screen of a control cabinet of the bioreactor, a magnetic stirrer is turned on, an ultrasonic device is turned on, the freeze-drying protective agent in the tank body is stirred and circulated while ultrasonic is carried out, and after the ultrasonic effect meets the requirements of a virus suspension harvesting process, stripped cell suspensions or broken cell virus suspensions are collected from the tank bottom valve and are frozen and stored;
wherein, the precooling freeze-drying protective agent added into the tank body needs to submerge an upper net plate of the fixed bed.
Compared with the prior art, the invention has the advantages that: the method has the advantages of simple and controllable process, short time consumption and high efficiency, can effectively reduce pollution risk, can also reduce virus loss, improves the virus yield, and simultaneously greatly ensures the stability of product quality.
Drawings
FIG. 1 is a schematic diagram of the online ultrasonic peeling disrupted cell bioreactor of the present invention;
FIG. 2 is a schematic structural diagram I of the online ultrasonic peeling disrupted cell bioreactor of the present invention;
FIG. 3 is a schematic structural diagram II of the online ultrasonic peeling disrupted cell bioreactor of the present invention;
description of reference numerals:
100. a bioreactor; 101. a tank body; 102. a can lid; 103. a fixed bed; 103a, a middle cylinder; 103b, an upper net plate; 103c, a lower net plate; 104. a bioreactor control cabinet; 104a, a master controller; 104b, a main control display screen; 105. a DO dissolved oxygen electrode; 106. a PH electrode; 107. a temperature probe; 108. a deep layer aeration pipe; 109. a liquid inlet pipe; 110. a liquid outlet pipe; 111. adding an alkali tube; 112. a sampling valve; 113. a tank bottom valve; 114. a bottom vent plate; 115. cleaning balls by CIP; 116. a magnetic stirrer; 116a, a magnetic motor; 116b, a stirring shaft; 116c, an upper stirring paddle; 116d, a middle stirring paddle; 116e, a lower stirring paddle; 117. a respirator;
200. an ultrasonic device; 201. an ultrasonic generator; 202. an ultrasonic transducer; 203. a vibrating rod;
300. an on-line cell counting device; 301. an online cell probe; 302. a transmitting module; 303. a current output module;
400. a cell carrier;
500. an annular cavity;
600. a tank cover lifting device;
700. a tank body bracket.
Detailed Description
In order to make the technical means, the creation features, the achievement purposes and the effects of the invention easy to understand, the following description further explains how the invention is implemented by combining the attached drawings and the detailed implementation modes.
Referring to fig. 1, the present invention provides an online ultrasonic peeling and cell disruption bioreactor, which comprises a bioreactor 100, an ultrasonic device 200 and an online cell counting device 300; wherein:
a bioreactor 100 for culturing cells and/or viruses and/or inoculating cells and viruses;
an ultrasonic device 200 for peeling and ultrasonically breaking the cells which are positioned in the bioreactor 100 and attached on the cell carrier 400;
the on-line cell counting device 300 is used for counting the cells in the bioreactor 100 so as to determine the virus inoculation time, the virus inoculation amount and the cell stripping ultrasonic disruption starting time.
Referring to fig. 2, in the present invention, a bioreactor 100 comprises a tank 101, a tank cover 102 disposed on the top of the tank 101, a fixed bed 103 disposed in the tank 101, and a bioreactor control cabinet 104 disposed beside the tank 101;
a DO dissolved oxygen electrode 105, a PH electrode 106, a temperature probe 107, a deep aeration pipe 108, a liquid inlet pipe 109, a liquid outlet pipe 110, an alkali adding pipe 111, a sampling valve 112 and a tank bottom valve 113 are also arranged on the tank body 101; the tank cover 103 is also provided with a CIP cleaning ball 115, a magnetic stirrer 116 and a respirator 117, the fixed bed 103 is filled with a plurality of cell carriers 400, and the cell carriers 400 are used for providing adherent attachment support for cell growth;
specifically, the DO dissolved oxygen electrode 105, the PH electrode 106, the temperature probe 107 and the sampling valve 112 are all arranged at the lower part of the outer wall of the tank body 101, the liquid inlet pipe 109, the liquid outlet pipe 110 and the alkali adding pipe 111 are all arranged at the upper part of the outer wall of the tank body 101, the tank bottom valve 113 is arranged at the bottom of the outer wall of the tank body 101, one end of the deep aeration pipe 108 extends out of the outer wall of the upper part of the tank body 101, and the other end of the deep aeration pipe extends into the tank body 101, penetrates through the fixed bed 103 and extends to the upper part of the; one end of the CIP cleaning ball 115, one end of the magnetic stirrer 116 and one end of the breather 117 penetrate through the tank cover 103 and extend into the tank body 101, and the other end of the CIP cleaning ball 115, the magnetic stirrer 116 and the breather 117 are exposed outside the tank cover 103; the DO dissolved oxygen electrode 105, the PH electrode 106, the temperature probe 107, the sampling valve 112, the tank bottom valve 113, the magnetic stirrer 116 and the respirator 117 are all electrically connected with a master controller 104a arranged in the bioreactor main control cabinet 104, and the master controller 104a is electrically connected with a main control display screen 104b arranged outside the bioreactor main control cabinet 104;
wherein, the sampling valve 112 and the tank bottom valve 113 are preferably in an electrically controlled valve structure; the breather 117 is used for ensuring that the interior of the tank body 101 is in a normal pressure state, and simultaneously, the air is sterilized and filtered, so that the interior of the tank body 101 is ensured not to be polluted.
Referring to fig. 1 and 2, in the present invention, the ultrasonic apparatus 200 includes an ultrasonic generator 201, an ultrasonic transducer 202 and a vibrating rod 203, the ultrasonic generator 201 and the ultrasonic transducer 202 are both disposed in the bioreactor main control cabinet 104, one end of the vibrating rod 203 is exposed at the top of the tank cover 102 and electrically connected to the ultrasonic transducer 202, the other end of the vibrating rod 203 passes through the tank cover 102 of the bioreactor 100 and extends into the fixed bed 103 disposed in the tank body 101 and is submerged into the cell carriers 400 filled in the fixed bed 103, the ultrasonic transducer 202 is further electrically connected to the ultrasonic generator 201, and the ultrasonic generator 201 is further electrically connected to the main controller 104a of the bioreactor main control cabinet 104.
Referring to fig. 1 and 2, in the present invention, the online cell counting apparatus 300 includes an online cell probe 301, a transmitting module 302 and a current output module 303, the transmitting module 302 and the current output module 303 are all disposed in the bioreactor main control cabinet 104, one end of the online cell probe 301 is exposed at the top of the tank cover 102 and electrically connected to the transmitting module 302, the other end of the online cell probe passes through the tank cover 102 of the bioreactor 100 and extends into the fixed bed 103 disposed in the tank 101 and is immersed in the cell carriers 400 filled in the fixed bed 103, the transmitting module 302 is also electrically connected to the current output module 303, and the current output module 303 is also electrically connected to the main controller 104a of the bioreactor main control cabinet 104.
Referring to fig. 2 and 3, in the present invention, the magnetic stirrer 116 includes a magnetic motor 116a (e.g., a servo motor), a stirring shaft 116b, and upper, middle, and lower stirring paddles 116c, 116d, and 116e, one end of the stirring shaft 116b extends out of the tank cover 102 and is connected to the magnetic motor 116a, the other end of the stirring shaft passes through the tank cover 102 and the fixed bed 103 in sequence and extends to the upper side of the bottom vent plate 114, and the upper, middle, and lower stirring paddles 116c, 116d, and 116e are sequentially fixed on the stirring shaft 116b from top to bottom and are all located in the tank 101;
wherein, the upper stirring paddle 116c is positioned above the fixed bed 103 arranged in the tank body 101; the middle stirring paddle 116d is located in the middle of the fixed bed 103 in the tank 101, and the lower stirring paddle 116e is located below the fixed bed 103 in the tank 101 and above the bottom vent plate 114.
Referring to FIG. 2, in the present invention, the fixed bed 103 comprises a middle cylinder 103a, an upper screen plate 103b disposed at an upper portion of the middle cylinder 103a, and a lower screen plate 103c disposed at a lower portion of the middle cylinder 103 a; an annular cavity 500 surrounded by the upper mesh plate 103b, the lower mesh plate 103c, the middle cylinder 103a of the fixed bed 103 and the tank 101 of the bioreactor 100 is filled with a cell carrier 400 growing adherently with cells.
Specifically, in the present invention, the upper stirring blade 116c of the magnetic stirrer 116 is positioned above the upper mesh plate 103b of the fixed bed 103, the middle stirring blade 116d is positioned in the middle cylinder 103a of the fixed bed 103, and the lower stirring blade 116e is positioned below the lower mesh plate 103c of the fixed bed 103 and above the bottom vent plate 114.
In order to facilitate the opening and closing of the bioreactor tank cover, a tank cover lifting device 600 for lifting the tank cover 102 of the bioreactor 100 can be arranged according to actual needs to realize the opening and closing of the tank cover 102 and the tank body 101 of the bioreactor 100; the can lid lifting apparatus 600 is conventional and comprises, for example, a lift cylinder, lift arms, and support frame.
In order to facilitate the maintenance of the bioreactor 100 and the discharge of liquid and the collection of virus (or cells), a tank bracket 700 can be arranged according to actual needs to support the tank 101 of the bioreactor 100; the tank support 700 is a conventional one, and is formed by splicing a plurality of square pipes.
In the present invention, the bioreactor 100 can set different culture parameters of cells and/or viruses according to the process requirements through the main control display screen 104b, so as to achieve the goal of quantitative automatic control of the culture growth of the cells and/or viruses.
When inoculating cells, the stirring shaft 116b of the magnetic stirrer 116 can rotate clockwise and counterclockwise under the driving of the magnetic motor 116a, and when the stirring shaft 116b rotates clockwise, the stirring shaft 116b can drive the three stirring paddles arranged thereon to rotate clockwise, so that the cells can pass through the fixed bed 103 from top to bottom; when the stirring shaft 116b rotates anticlockwise, the three stirring paddles arranged on the stirring shaft can be driven to rotate anticlockwise, so that cells can pass through the fixed bed 103 from bottom to top, the purpose of vertically inoculating cells (or viruses) on the fixed bed 103 can be achieved, the cells are uniformly inoculated, and the defect of nonuniform cell inoculation caused by that all the fixed bed bioreactors can only perform unidirectional cell inoculation at present is overcome.
In the invention, the three stirring paddles are arranged on the stirring shaft 116b, which is more beneficial to dissolving oxygen and uniformly mixing liquid, and the animal-derived heterologous protein in the bioreactor can be conveniently cleaned through the arranged CIP cleaning ball; the cell counting device 300 can count cells on line in real time, so that the virus inoculation time, the virus inoculation amount and the starting ultrasonic time are determined; the ultrasonic device 200 can realize the stripping and ultrasonic breaking of the cells attached to the cell carrier 400, thereby facilitating the inoculation of the virus.
Compared with the prior art, when the online ultrasonic stripping and cell disruption bioreactor provided by the invention is used for stripping and disrupting cells and collecting cells or viruses, the online ultrasonic stripping and cell disruption bioreactor has the following advantages: the method has the advantages of simple and controllable process, short time consumption, high efficiency, small pollution risk, less virus loss and high virus yield, and can also greatly ensure the stability of product quality.
Aiming at the online ultrasonic stripping and cell disruption bioreactor, the invention also provides a method for harvesting viruses by the online ultrasonic stripping and cell disruption bioreactor, which specifically comprises the following steps:
s1, adding a PBS buffer solution into the tank body 101 of the bioreactor 100 to a specified position, and maintaining the pressure for 30 minutes;
s2, when the pressure of the bioreactor 100 is kept for 30 minutes without dropping the pressure, the sealing of the bioreactor 100 is qualified, and at the moment, a sterilization program carried by the bioreactor 100 is started, so that the online sterilization of the bioreactor 100 is automatically completed, and after the sterilization is completed, the temperature is naturally reduced for standby;
s3, draining the PBS buffer solution in the tank body 101 through the tank bottom valve 113, adding the cell growth solution into the tank body 101 through the liquid inlet pipe 109, setting corresponding parameters on the main control display screen 104b of the bioreactor control cabinet 104 for pre-culture, and obtaining the qualified product when no bacteria grow in the tank body 101 of the bioreactor 100 for 1-2 days;
s4, discharging the cell growth liquid pre-cultured in the tank body 101 through the tank bottom valve 113, adding new cell growth liquid and cell suspension into the tank body 101 through the liquid inlet pipe 109, then opening the magnetic stirrer 116 to work, uniformly mixing the newly added liquid through the upper stirring paddle 116c of the magnetic stirrer 116, increasing the dissolution of oxygen, enabling the mixed liquid in the tank body 101 to flow through the middle stirring paddle 116d of the magnetic stirrer 116, performing nutrition exchange on cells, uniformly mixing the liquid at the tank bottom through the lower stirring paddle 116e of the magnetic stirrer 116, and increasing the dissolution of oxygen; meanwhile, the on-line cell counting device 300 is controlled by the bioreactor control cabinet 104 to start cell counting work, and corresponding cell culture parameters are set on the main control display screen 104b of the bioreactor control cabinet 104 for cell culture;
in the cell culture process, the PH electrode 106 is used for detecting the PH value of liquid in the tank body 101, the DO dissolved oxygen electrode 105 is used for detecting the dissolved oxygen amount of the liquid in the tank body 101, and the temperature probe 107 is used for detecting the temperature of the liquid in the tank body 101;
if the PH electrode 106 detects that the PH value of the liquid in the tank body 101 is low, adding an alkali material into the tank body 101 through an alkali adding pipe 111;
if the PH electrode 106 detects that the PH value of the liquid in the tank 101 is higher, carbon dioxide is introduced into the tank 101 through the deep vent pipe 108;
if the DO dissolved oxygen electrode 105 detects that the dissolved oxygen in the liquid in the tank 101 is low, oxygen is introduced into the tank 101 through the deep vent pipe 108;
s5, when the on-line cell counting device 300 counts that the cell number in the tank 101 meets the process requirements, starting to inoculate the virus, and meanwhile, setting corresponding virus culture parameters on the main control display screen 104b of the bioreactor control cabinet 104 for virus culture;
s6, when the virus culture meets the process requirements, draining the virus culture solution in the tank body 101 through the tank bottom valve 113, after the virus culture solution is drained completely, pumping a washing solution into the tank body 101 through the CIP cleaning ball 115 to clean the animal-derived protein in the tank body 101, after the washing is finished, adding the virus culture solution without the animal-derived protein into the tank body 101 through the liquid inlet pipe 109, and continuing to culture;
s7, when the number of cells in the tank body 101 starts to decrease through the on-line cell counting device 300, the cells start to be diseased, when the number of cells decreases to a certain number, the animal-derived protein-free virus culture solution added in the step S6 is discharged through the tank bottom valve 113, after the cells are completely discharged, a precooled freeze-drying protective agent is added into the tank body 101 through the liquid inlet pipe 109, meanwhile, corresponding ultrasonic parameters are set on the main control display screen 104b of the bioreactor control cabinet 104, the magnetic stirrer 116 is turned on, the ultrasonic device 200 is started, the freeze-drying protective agent in the tank body 101 is subjected to stirring circulation and ultrasonic treatment, and after the ultrasonic effect meets the requirements of a virus suspension harvesting process, the peeled cell suspension or the crushed cell suspension is collected from the tank bottom valve 113 and is frozen and stored;
wherein, the precooling freeze-drying protective agent added into the tank body 101 is required to submerge the upper net plate 103b of the fixed bed 103.
According to the method for harvesting viruses by the online ultrasonic peeling and cell disruption bioreactor, the online ultrasonic peeling and cell disruption bioreactor provided by the invention can also be used for harvesting cells, the four steps are the same as the steps for harvesting viruses, and the difference is that in step S5, in step S5, when the cell number in the tank body 101 is measured by the online cell counting device 300 to meet the process requirements, liquid in the tank body 101 is drained through the tank bottom valve 113, after the liquid is drained, precooled freeze-drying protective agent is added into the tank body 101 through the liquid inlet pipe 109, the precooled freeze-drying protective agent added into the tank body 101 is required to submerge the upper net plate 103b of the fixed bed 103, corresponding ultrasonic parameters are set on the main control screen 104b of the bioreactor control cabinet 104, the magnetic stirrer 116 is turned on, the ultrasonic device 200 is turned on, and the freeze-drying protective agent in the tank body 101 is subjected to ultrasonic treatment while stirring circulation, and after the ultrasonic effect meets the requirements of the cell suspension harvesting process, collecting the stripped cell suspension from the tank bottom valve 113, and freezing and storing to obtain the required cells.
Finally, the above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all equivalent structures or equivalent processes performed by the present invention or directly or indirectly applied to other related technical fields using the contents of the present specification and the attached drawings are included in the scope of the present invention.
Claims (10)
1. An online ultrasonic stripping cell disruption bioreactor is characterized in that: comprises a bioreactor (100), an ultrasonic device (200) and an online cell counting device (300);
the bioreactor (100) is used for culturing cells or/and viruses or/and inoculating the cells and the viruses;
the ultrasonic device (200) is used for peeling and ultrasonically breaking cells which are positioned in the bioreactor (100) and attached to the cell carrier (400);
the on-line cell counting device (300) is used for counting the cells in the bioreactor (100) so as to determine the virus inoculation time of the cells, the virus inoculation amount and the cell stripping and ultrasonic disruption starting time.
2. The line ultrasonic peel disrupted cell bioreactor of claim 1, wherein: the bioreactor (100) comprises a tank body (101), a tank cover (102) arranged at the top of the tank body (101), a fixed bed (103) arranged in the tank body (101) and a bioreactor control cabinet (104) arranged beside the tank body (101);
a DO dissolved oxygen electrode (105), a PH electrode (106), a temperature probe (107), a deep-layer vent pipe (108), a liquid inlet pipe (109), a liquid outlet pipe (110), an alkali adding pipe (111), a sampling valve (112) and a tank bottom valve (113) are also arranged on the tank body (101); the DO dissolved oxygen electrode (105), the PH electrode (106), the temperature probe (107) and the sampling valve (112) are all arranged at the lower part of the outer wall of the tank body (101), the liquid inlet pipe (109), the liquid outlet pipe (110) and the alkali adding pipe (111) are all arranged at the upper part of the outer wall of the tank body (101), the tank bottom valve (113) is arranged at the bottom of the outer wall of the tank body (101), one end of the deep breather pipe (108) stretches out of the outer wall of the upper part of the tank body (101), and the other end of the deep breather pipe stretches into the tank body (101), penetrates through the fixed bed (103), extends to the upper part of the inner bottom wall of the tank body (101), and;
the tank cover (103) is also provided with a CIP cleaning ball (115), a magnetic stirrer (116) and a respirator (117), one end of the CIP cleaning ball (115), one end of the magnetic stirrer (116) and one end of the respirator (117) penetrate through the tank cover (103) and extend into the tank body (101), and the other end of the CIP cleaning ball (115), one end of the magnetic stirrer (116) and one end of the respirator (117) are exposed outside the tank cover (103);
a plurality of cell carriers (400) are filled in the fixed bed (103), and the cell carriers (400) are used for providing adherent attachment support for cell growth;
the DO dissolved oxygen electrode (105), the PH electrode (106), the temperature probe (107), the sampling valve (112), the tank bottom valve (113), the magnetic stirrer (116) and the respirator (117) are electrically connected with a master controller (104a) arranged in the bioreactor main control cabinet (104), and the master controller (104a) is electrically connected with a main control display screen (104b) arranged outside the bioreactor main control cabinet (104).
3. The wire ultrasonic stripping disrupted cell bioreactor of claim 2, wherein: ultrasonic device (200) contain supersonic generator (201), ultrasonic transducer (202) and vibrting spear (203), supersonic generator (201) and ultrasonic transducer (202) all set up in bioreactor master control cabinet (104), vibrting spear (203) one end is exposed and is established lid (102) top and be connected with ultrasonic transducer (202) electricity, and the other end passes lid (102) of bioreactor (100) and stretches into in fixed bed (103) that are equipped with in jar body (101), ultrasonic transducer (202) still are connected with supersonic generator (201) electricity, supersonic generator (201) still are connected with master controller (104a) electricity of bioreactor master control cabinet (104).
4. The wire ultrasonic stripping disrupted cell bioreactor of claim 2, wherein: the online cell counting device (300) comprises an online cell probe (301), a transmission module (302) and a current output module (303), wherein the transmission module (302) and the current output module (303) are arranged in a bioreactor main control cabinet (104), one end of the online cell probe (301) is exposed at the top of a tank cover (102) and is electrically connected with the transmission module (302), the other end of the online cell probe penetrates through the tank cover (102) of the bioreactor (100) and extends into a fixed bed (103) arranged in the tank body (101), the transmission module (302) is further electrically connected with the current output module (303), and the current output module (303) is further electrically connected with a main controller (104a) of the bioreactor main control cabinet (104).
5. The wire ultrasonic stripping disrupted cell bioreactor of claim 2, wherein: the magnetic stirrer (116) comprises a magnetic motor (116a), a stirring shaft (116b), an upper stirring paddle, a middle stirring paddle and a lower stirring paddle (116c, 116d and 116e), one end of the stirring shaft (116b) extends out of the tank cover (102) and is connected with the magnetic motor (116a), the other end of the stirring shaft sequentially penetrates through the tank cover (102) and the fixed bed (103) and extends to the upper part of the bottom ventilating disc (114), and the upper stirring paddle, the middle stirring paddle and the lower stirring paddle (116c, 116d and 116e) are sequentially fixed on the stirring shaft (116b) in a penetrating mode from top to bottom and are all located in the tank body (101);
wherein, the upper stirring paddle (116c) is positioned above the fixed bed (103) arranged in the tank body (101); the middle stirring paddle (116d) is positioned in the middle of the fixed bed (103) arranged in the tank body (101), and the lower stirring paddle (116e) is positioned below the fixed bed (103) arranged in the tank body (101) and above the bottom vent disc (114).
6. The wire ultrasonic stripping disrupted cell bioreactor of claim 5, wherein: the fixed bed (103) comprises a middle cylinder (103a), an upper screen plate (103b) arranged at the upper part of the middle cylinder (103a) and a lower screen plate (103c) arranged at the lower part of the middle cylinder (103 a); an annular cavity (500) enclosed by the upper mesh plate (103b), the lower mesh plate (103c) and the middle cylinder (103a) of the fixed bed (103) and the tank body (101) of the bioreactor (100) is filled with a cell carrier (400) growing by cell adherence.
7. The wire ultrasonic stripping disrupted cell bioreactor of claim 6, wherein: the upper stirring paddle (116c) of the magnetic stirrer (116) is positioned above the upper mesh plate (103b) of the fixed bed (103), the middle stirring paddle (116d) is positioned in the middle cylinder (103a) of the fixed bed (103), and the lower stirring paddle (116e) is positioned below the lower mesh plate (103c) of the fixed bed (103) and above the bottom vent disc (114).
8. The wire ultrasonic stripping disrupted cell bioreactor according to any one of claims 2 to 7, wherein: the device is characterized by further comprising a tank cover lifting device (600), wherein the tank cover lifting device (600) is used for lifting a tank cover (102) of the bioreactor (100), and then the tank cover (102) of the bioreactor (100) is opened and closed with the tank body (101).
9. The wire ultrasonic stripping disrupted cell bioreactor according to any one of claims 2 to 7, wherein: also comprises a tank bracket (700), wherein the tank bracket (700) is used for supporting the tank (101) of the bioreactor (100).
10. A method of harvesting virus by the line ultrasonication cell disruption bioreactor of claim 7, wherein: comprises the following steps:
s1, adding PBS buffer solution into the tank body (101) of the bioreactor (100) to a specified position, and maintaining the pressure for 30 minutes;
s2, when the pressure of the bioreactor (100) is not dropped for 30 minutes, the bioreactor (100) is sealed to be qualified, and the sterilization program carried by the bioreactor (100) is opened at the moment, so that the online sterilization of the bioreactor (100) is automatically completed, and after the sterilization is completed, the temperature is naturally reduced for standby;
s3, draining the PBS buffer solution in the tank body (101) through a tank bottom valve (113), adding a cell growth solution into the tank body (101) through a liquid inlet pipe (109), setting corresponding parameters on a main control display screen (104b) of a bioreactor control cabinet (104) for pre-culture, and obtaining the qualified product when no bacteria grow in the tank body (101) of the bioreactor (100) for 1-2 days;
s4, discharging the cell growth liquid pre-cultured in the tank body (101) through a tank bottom valve (113), adding new cell growth liquid and cell suspension into the tank body (101) through a liquid inlet pipe (109), then opening a magnetic stirrer (116) to work, uniformly mixing the newly added liquid through an upper stirring paddle (116c) of the magnetic stirrer (116), simultaneously increasing the dissolution of oxygen, enabling the mixed liquid in the tank body (101) to flow through a middle stirring paddle (116d) of the magnetic stirrer (116), performing nutrition exchange on cells, uniformly mixing the liquid at the tank bottom through a lower stirring paddle (116e) of the magnetic stirrer (116), and simultaneously increasing the dissolution of oxygen; meanwhile, the online cell counting device (300) is controlled by the bioreactor control cabinet (104) to start cell counting work, and corresponding cell culture parameters are set on a main control display screen (104b) of the bioreactor control cabinet (104) for cell culture;
in the cell culture process, a PH electrode (106) is used for detecting the PH value of liquid in a tank body (101), a DO dissolved oxygen electrode (105) is used for detecting the dissolved oxygen amount of the liquid in the tank body (101), and a temperature probe (107) is used for detecting the temperature of the liquid in the tank body (101);
if the PH electrode (106) detects that the PH value of the liquid in the tank body (101) is low, adding an alkali material into the tank body (101) through an alkali adding pipe (111);
if the PH electrode (106) detects that the PH value of the liquid in the tank body (101) is higher, the carbon dioxide is introduced into the tank body (101) through the deep vent pipe (108);
if the DO dissolved oxygen electrode (105) detects that the dissolved oxygen of the liquid in the tank body (101) is low, oxygen is introduced into the tank body (101) through the deep-layer vent pipe (108);
s5, when the cell number in the tank body (101) reaches the process requirement, inoculating the virus is started when the online cell counting device (300) counts, and meanwhile, corresponding virus culture parameters are set on a main control display screen (104b) of the bioreactor control cabinet (104) for virus culture;
s6, when the virus culture meets the process requirements, draining the virus culture solution in the tank body (101) through a tank bottom valve (113), after the virus culture solution is drained completely, pumping a washing solution into the tank body (101) through a CIP (cleaning in place) cleaning ball (115), cleaning animal-derived proteins in the tank body (101), after cleaning, adding the virus culture solution without the animal-derived proteins into the tank body (101) through a liquid inlet pipe (109), and continuing to culture;
s7, when the online cell counting device (300) counts that the number of cells in the tank (101) begins to decrease, it indicates that the cells have started to be diseased, and when the number of cells has decreased to a certain number, the animal-derived protein-free virus culture solution added in step S6 is drained through the tank bottom valve (113), and after draining, a precooled freeze-drying protective agent is added into the tank body (101) through the liquid inlet pipe (109), and simultaneously, setting corresponding ultrasonic parameters on a main control display screen (104b) of a bioreactor control cabinet (104), turning on a magnetic stirrer (116), turning on an ultrasonic device (200), stirring and circulating the freeze-drying protective agent in the tank body (101) and carrying out ultrasonic treatment at the same time, after the ultrasonic effect meets the requirements of the virus suspension harvesting process, collecting the stripped cell suspension or the broken cell virus suspension from a tank bottom valve (113), and freezing and storing;
wherein, the precooling freeze-drying protective agent added into the tank body (101) is required to submerge the upper net plate (103b) of the fixed bed (103).
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| CN114181287A (en) * | 2021-12-10 | 2022-03-15 | 商丘安华生物疫苗有限公司 | Preparation method of DNA virus vaccine |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114181287A (en) * | 2021-12-10 | 2022-03-15 | 商丘安华生物疫苗有限公司 | Preparation method of DNA virus vaccine |
| CN114181287B (en) * | 2021-12-10 | 2023-09-19 | 商丘美兰生物工程有限公司 | Preparation method of DNA virus vaccine |
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