CN116200260B

CN116200260B - Disposable biological safety bioreactor and monitoring method

Info

Publication number: CN116200260B
Application number: CN202310500667.7A
Authority: CN
Inventors: 钟瑜; 林静雯; 姚慰炜; 陈路
Original assignee: Sichuan University
Current assignee: Sichuan University
Priority date: 2023-05-06
Filing date: 2023-05-06
Publication date: 2023-07-04
Anticipated expiration: 2043-05-06
Also published as: CN116200260A; CN116948817A

Abstract

The invention belongs to the technical field of bioengineering, in particular to a biological engineering methodA disposable bio-safe bioreactor and a monitoring method, wherein the disposable bio-safe bioreactor comprises: a reaction pouch for providing a biological growth or reaction environment; a monitoring unit for monitoring a reaction pouch, the monitoring unit comprising: for monitoring the internal pressure, foam, dissolved oxygen rate and CO of the reaction bag ₂ Monitoring unit of concentration, pH value, liquid turbidity; the reaction bag is correspondingly provided with an air inlet and an air outlet; wherein, the air inlet is provided with a sterile filter device to prevent pollution; the air outlet is provided with a high-efficiency filtering or sterile filtering unit for filtering the aerosol containing pathogenic microorganisms or other possible environmental and biological pollution or influence, so as to prevent the aerosol from being polluted; a leakage sensor for monitoring leakage conditions and a safety sterilization module for handling leakage problems and pollution prevention are provided. The disposable bioreactor provided by the invention is especially suitable for a miniaturized cultivation scene of cells or microorganisms.

Description

Disposable biological safety bioreactor and monitoring method

Technical Field

The invention belongs to the technical field of bioengineering, and particularly relates to a disposable biological safety type bioreactor and a monitoring method.

Background

A disposable bioreactor is a new type of bioreactor, typically made of disposable plastic or stainless steel, which can be used in the following fields in general: such as cell culture, bacterial culture, virus culture, monoclonal antibody and the like. However, the existing bioreactor is generally applied to large-scale cell or microorganism culture (such as commercial application), and is difficult to meet the small-scale experimental requirement. For example, when researchers are in the early stages of vaccine incubation, it is often necessary to first perform multiple batches of small-scale virus cultures in the laboratory. However, the existing biosafety reactor has certain defects in terms of cost, safety and operation convenience for small-scale virus culture.

For example, chinese patent publication No. CN102492607B discloses a disposable bioreactor system and method. Wherein the disposable bioreactor system comprises: a disposable container comprising at least one sample inlet, at least one discharge outlet, at least one collection port, the integrity of the sterile environment being protected using a sterile filter connected to all external open ports; a structure for supporting the disposable container; one or more sensors that sense one or more parameters of the biological material in the container; a heater for heating the contents of the container, the heater having a thermostat; and a mixing system provided with the system such that the biological material contained in the disposable container is mixed. The whole volume of the bioreactor system is larger, a large-capacity bioreactor is adopted, a series of auxiliary supporting equipment is needed, the cost is high, and higher requirements are also put forward on experimental operation space. And, there is a large difference in practical application process due to large-scale microorganism or cell culture and microminiature microorganism or cell culture. Therefore, even if the conventional equipment is reduced in volume at equal ratio, it is difficult to satisfy the laboratory microminiature experiment requirements. Also for example, chinese patent application publication No. CN103945928A discloses a single-use mixing and bioreactor system that would have similar problems when applied to microminiature experiments.

To further adapt to the requirements of microminiature experiments, some designs of small-sized reactors have also been proposed in the prior art, for example, chinese patent application with application publication No. CN102212474a, which discloses a small-sized bioreactor comprising: the cell culture device comprises a cell culture tank body, a temperature control sleeve, a liquid (gas) supplementing device, a cell optical density detecting sleeve and a culture control device, wherein the cell culture tank body is connected with the liquid supplementing device, and the temperature control sleeve, the cell optical density detecting sleeve and the culture control device are matched to provide optimized growth conditions for cell culture. However, for such a small bioreactor, steam sterilization of the whole is required after each reaction, and for batch experiments, the operation is complicated and the application cost is higher. Moreover, when the bioreactor is used for culturing pathogenic microorganisms such as bacteria or viruses, it is difficult to ensure that the inside and outside environment of the reactor are not disturbed (for example, pathogenic bacteria in the reactor may overflow to pollute the experimental environment and even threaten the safety of operators).

Thus, there is a need for a bioreactor that is adaptable to small scale microbial or cell culture scenarios.

Disclosure of Invention

The invention aims to provide a disposable biological safety type bioreactor and a monitoring method, which are used for partially solving or relieving the defects in the prior art and can effectively solve the difficulties of cells or microorganisms in the miniaturized culture process.

In order to solve the technical problems, the invention adopts the following technical scheme: a disposable biosafety bioreactor comprising:

the reaction bag comprises a first space and a second space in sequence in an inner accommodating cavity of the reaction bag; wherein, when the bioreactor is in a working state, the first space is used for containing gas, and the second space is used for containing liquid; wherein, the top area of the first space is also provided with a condensed water guiding area for guiding condensed water to be condensed in the condensed water guiding area and to flow back to the second space through a guiding path provided by the condensed water guiding area;

the working state monitoring module is used for monitoring working state parameters of the bioreactor, and the working state parameters comprise: the liquid has hydraulic pressure, foam generation, dissolved oxygen rate and CO ₂ Concentration, pH, and turbidity; correspondingly, the working state monitoring module comprises: a first pressure monitoring unit for monitoring the hydraulic pressure, a foam monitoring unit for monitoring the foam generation condition, an oxygen dissolution rate monitoring unit for monitoring the oxygen dissolution rate, and a control unit for controlling the CO ₂ A concentration monitoring unit for monitoring the pH value, a pH monitoring unit for monitoring the pH value, and a turbidity monitoring unit for monitoring the turbidity; wherein the monitoring ends of the monitoring units are arranged on the wall surface of the second space;

the first space is also provided with at least one first inlet for introducing first gas and at least one second inlet for introducing first liquid, and the first inlet and the second inlet are both arranged outside the condensed water guiding area; wherein when the bioreactor is in a working state, the opening of the first inlet is higher than the second inlet;

the lower end region of the second space is further provided with at least one first outlet for liquid sampling.

In some embodiments, the top wall surface inside the condensate guiding zone is configured as a slope and the edge of the condensate guiding zone is provided with an annular rail providing a guiding path for condensation, backflow of the condensate.

In some embodiments, further comprising: the stirring device comprises a stirring shaft and a stirring paddle arranged at the tail end of the stirring shaft; the central area of the condensed water guiding area is provided with a mounting hole for mounting the stirring device, the stirring shaft is fixedly mounted on the reaction bag through the mounting hole, the stirring paddle extends into the second space, and the monitoring end of the working state monitoring module is higher than the horizontal height of the stirring paddle; wherein, the stirring shaft is provided with a heating unit at one side of the condensed water guiding area or near the condensed water guiding area, and the heating unit is used for avoiding the formation of condensed water at the outer side of the condensed water guiding area.

In some embodiments, the incline angle of the incline is 1 ° -5 °.

In some embodiments, an annular groove for providing the guiding path is arranged in the annular guide rail, and a condensed water outlet is arranged on the annular groove, and the bottom surface of the annular groove is obliquely arranged, so that the condensed water outlet is the lowest horizontal point of the annular groove, and condensed water in the annular groove is guided to flow out through the condensed water outlet; wherein the condensed water outlet is arranged along a direction away from the first inlet and the second inlet.

In some embodiments, further comprising:

the first liquid leakage monitoring module is correspondingly arranged at the bottom of the reaction bag and used for monitoring whether the reaction bag leaks liquid or not;

a safety sterilization module, the safety sterilization module comprising: a recovery unit connected to the reaction bag and a sterilization unit; the recovery unit includes: recovery bag, and be used for connecting the recovery bag with the recovery pipeline of reaction bag, be provided with the third control valve in the recovery pipeline, disinfection unit includes: a chamber for storing sterilizing substances, and a sterilizing pipeline for connecting the chamber and the reaction bag, wherein a fourth control valve is arranged in the sterilizing pipeline;

The third control valve and the fourth control valve are in communication connection with the working state monitoring module and the first liquid leakage monitoring module.

In some embodiments, the first inlet is provided with a filter device comprising: a virus filtration device, and/or a bacteria filtration device.

In some embodiments, the first space is further provided with a second outlet for the exhaust gas, and the second outlet is provided therein with a drying device for filtering the aerosol, and/or a sterile filtering device for filtering.

The invention also provides a monitoring method based on the disposable biological safety type bioreactor, which comprises the following steps: the first pressure monitoring unit is used for monitoring the hydraulic pressure in the reaction bag, and the first liquid leakage monitoring module is correspondingly arranged at the bottom of the reaction bag, and the method comprises the following steps:

s102, introducing a preset amount of gas or liquid into the reaction bag through a first inlet or a second inlet on the reaction bag so as to culture microorganisms or cells;

s104, monitoring the hydraulic pressure of the first pressure monitoring unit and the leakage value of the first leakage monitoring module in real time;

S106, judging the risk state of the bioreactor according to the hydraulic pressure and the leakage value, wherein the risk state comprises the following steps: a safe state, and/or a pending state, and/or a weeping state;

wherein S106 includes:

s61, judging the depressurization risk of the reaction bag according to the hydraulic variation parameters; when the change parameter belongs to a preset first depressurization threshold value, the depressurization risk is first-level, when the change parameter belongs to a preset second depressurization threshold value, the depressurization risk is second-level, and otherwise, the depressurization risk is zero-level;

s62, judging the leakage risk of the reaction bag according to the leakage value, wherein the leakage risk is first-level when the leakage value belongs to a preset first leakage threshold value, is second-level when the leakage value belongs to a preset second leakage threshold value, and is zero-level otherwise;

s63, judging the risk state according to the depressurization risk in S61 and/or the leakage risk in S62; when the depressurization risk is a second level and/or the leakage risk is a second level, the risk state is a leakage state; when the depressurization risk and the weeping risk are zero-order, the risk state is a safety state; otherwise, the risk state is a pending state.

In some embodiments, a condensed water guiding area is further formed at the top area of the first space in the reaction bag, and is used for guiding condensed water to be condensed in the condensed water guiding area and flow back to the second space through a guiding path provided by the condensed water guiding area; correspondingly, the method further comprises the steps of:

s108, when the risk state is detected to be a pending state or a liquid leakage state, respectively sending corresponding early warning signals to a user;

s110, responding to a feedback signal sent by the user, and secondarily monitoring the depressurization risk and/or the leakage risk of the bioreactor, or receiving or correcting the early warning signal.

In some embodiments, the step S110 includes:

when the depressurization risk is first order and the leakage risk is zero order, responding to a first feedback signal sent by the user to heat the condensed water guiding area;

when the pressure reduction risk is monitored to be reduced to zero level in the heating process or after the heating process, the risk state is corrected to be a safe state in response to a first correction signal sent by a user, and otherwise, the risk state is corrected to be a liquid leakage state in response to a second correction signal sent by the user.

The beneficial technical effects are as follows:

the invention provides a biological safety bioreactor suitable for carrying out small-scale batch culture on highly pathogenic microorganisms, which combines the scheme of setting an internal circulation type condensed water guide path and selecting limited key parameter combinations (such as internal hydraulic pressure and external leakage value) for monitoring, and jointly provides a high-sensitivity and low-cost biological reactor suitable for culturing the highly pathogenic microorganisms.

Furthermore, the invention provides a monitoring method capable of realizing manual and automatic mutual coordination aiming at the selected key parameter group, and the monitoring method utilizes limited double-branch data to synchronously monitor and comprehensively evaluate the real working state of the bioreactor so as to reduce the intervention of manual necessity to a certain extent. Meanwhile, the method can also automatically correct or audit the comprehensive evaluation result (such as a risk state) so as to further improve the sensitivity and accuracy of the monitoring method (reduce the emission of error signals). Wherein, through the cooperation with the comdenstion water guide function of inner loop formula, still can reduce the interference of comdenstion water to the pressure variation value, and then help improving pressure sensor's (like first pressure monitoring unit) monitoring accuracy.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. Like elements or portions are generally identified by like reference numerals throughout the several figures. In the drawings, elements or portions thereof are not necessarily drawn to scale. It will be apparent to those of ordinary skill in the art that the drawings in the following description are of some embodiments of the invention and that other drawings may be derived from these drawings without inventive faculty.

FIG. 1 is a schematic view showing a first structure of a disposable bioreactor in an exemplary embodiment of the present invention;

FIG. 2 is a second schematic structural view of a disposable bioreactor in an exemplary embodiment of the present invention;

FIG. 3 is a schematic view showing an internal structure of a disposable bioreactor according to an exemplary embodiment of the present invention;

FIG. 4 is a schematic view of the structure of a condensed water guide area according to an exemplary embodiment of the present invention;

FIG. 5 is a schematic view of the top wall structure in another exemplary embodiment of the present invention;

FIG. 6 is a first schematic structural view of a supporting device according to an exemplary embodiment of the present invention;

FIG. 7 is a second schematic view of a supporting device according to an exemplary embodiment of the present invention;

FIG. 8 is a schematic cross-sectional view of a quick-connect device for a sensor according to an exemplary embodiment of the present invention;

FIG. 9 is a schematic diagram of a sensor quick-connect device according to an exemplary embodiment of the present invention;

FIG. 10 is a flow chart of an automated control method according to an exemplary embodiment of the invention;

FIG. 11 is a flow chart of an automated control method in accordance with another exemplary embodiment of the present invention;

fig. 12 is a block diagram of an automation control system in accordance with an exemplary embodiment of the present invention.

Reference numeral identification summary:

1 is a reaction bag, 11 is a top wall surface, 12 is an annular guide rail, 12-1 is an annular groove, 13 is a first space, and 14 is a second space; 2 is a stirring device, 21 is a stirring paddle, 22 is a stirring shaft, and 23 is a heating unit; 3 is a working state monitoring module; 4 is a first inlet; 5 is a first outlet (also referred to as a sampling port); 6 is a second outlet, 61 is a drying device, and 62 is a filtering device; 7 is a supporting main body, 71 is a leakage monitoring sensor, 72 is a base, 73 is a first side wall, and 74 is a second side wall; 81 is a disposable sensor probe, 82 is a probe terminal, 83 is a power terminal, and 84 is an electrode; l1 is a horizontal plane, and L2 is a guide path.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. It will be apparent that the described embodiments are some, but not all, embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

In this document, suffixes such as "module", "component", or "unit" used to represent elements are used only for facilitating the description of the present invention, and have no particular meaning in themselves. Thus, "module," "component," or "unit" may be used in combination.

The terms "upper," "lower," "inner," "outer," "front," "rear," "one end," "the other end," and the like herein refer to an orientation or positional relationship based on that shown in the drawings, merely for convenience of description and to simplify the description, and do not denote or imply that the devices or elements referred to must have a particular orientation, be constructed and operated in a particular orientation, and thus should not be construed as limiting the invention. Furthermore, the terms "first," "second," and the like, are used for descriptive purposes only and are not to be construed as indicating or implying relative importance.

The terms "mounted," "configured to," "connected," and the like, herein, are to be construed broadly as, for example, "connected," whether fixedly, detachably, or integrally connected, unless otherwise specifically defined and limited; the two components can be mechanically connected, can be directly connected or can be indirectly connected through an intermediate medium, and can be communicated with each other. The specific meaning of the above terms in the present invention will be understood in specific cases by those of ordinary skill in the art.

Herein, "and/or" includes any and all combinations of one or more of the associated listed items.

Herein, "plurality" means two or more, i.e., it includes two, three, four, five, etc.

It should be noted that, in this document, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising one … …" does not exclude the presence of other like elements in a process, method, article, or apparatus that comprises the element.

As used in this specification, the term "about" is typically expressed as +/-5% of the value, more typically +/-4% of the value, more typically +/-3% of the value, more typically +/-2% of the value, even more typically +/-1% of the value, and even more typically +/-0.5% of the value.

In this specification, certain embodiments may be disclosed in a format that is within a certain range. It should be appreciated that such a description of "within a certain range" is merely for convenience and brevity and should not be construed as a inflexible limitation on the disclosed ranges. Accordingly, the description of a range should be considered to have specifically disclosed all possible sub-ranges and individual numerical values within that range. For example, the description of ranges 1-6 should be considered as having specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within such ranges, e.g., 1,2,3,4,5, and 6. The above rule applies regardless of the breadth of the range.

Herein, a "communication connection" may be a direct communication connection between two or more parties, or may be an indirect communication connection implemented by a third party (e.g., a central control module or other central control system).

Example 1

As shown in fig. 1-5, the present invention provides in a first aspect a disposable biosafety bioreactor suitable for batch miniaturized microbial or cell culture in a laboratory.

The bioreactor comprises: a reaction bag 1, wherein an inner accommodating chamber of the reaction bag 1 sequentially comprises a first space 13 and a second space 14; wherein, when the bioreactor is in an operating state, the first space 13 is used for containing gas (such as air, oxygen, carbon dioxide, etc.), the second space 14 is used for containing liquid (such as cell culture solution, water, etc.), and a condensed water guiding area is formed at the top area of the first space and used for guiding condensed water to aggregate in the condensed water guiding area and flow back to the second space through a guiding path provided by the condensed water guiding area;

an operating state monitoring module 3 for monitoring operating state parameters of the bioreactor, the operating state parameters comprising: the liquid has hydraulic pressure, foam generation, dissolved oxygen rate and CO ₂ Concentration, pH, and turbidity; correspondingly, the working state monitoring module comprises a monitoring unit: a first pressure monitoring unit for monitoring the hydraulic pressure, a foam monitoring unit for monitoring the foam generation condition, an oxygen dissolution rate monitoring unit for monitoring the oxygen dissolution rate, and a CO monitoring unit ₂ A carbon dioxide monitoring unit for monitoring the concentration, a pH monitoring unit for monitoring the pH value, and a turbidity monitoring unit for monitoring the turbidity; wherein monitoring ends (e.g., monitoring ends of sensors) of the respective monitoring units are disposed on a wall surface of the second space to ensure that respective operation state parameters of the liquid can be accurately monitored;

at least one first inlet 4 for introducing a first gas (e.g., compressed air, carbon dioxide, nitrogen, oxygen, etc.) and at least one second inlet for introducing a first liquid (e.g., cell culture liquid, bacteria to be cultured, water, etc.) are provided at the first space, and both the first inlet and the second inlet are provided outside the condensed water guiding region, so as to avoid blockage and pollution of the first inlet or the second inlet by condensed water in the reaction process; wherein the opening of the first inlet is higher than the second inlet (or, in other embodiments, the position of the second inlet may be unrestricted) when the bioreactor is in an operational state (a placed state as shown in fig. 1) to avoid water or other liquid substances discharged through the second inlet from flowing through the first inlet to clog or contaminate it;

The lower end region of the second space is further provided with at least one first outlet 5 for liquid sampling.

In other embodiments, in combination with specific growth characteristics of the microorganism, some or all of the first inlet or the second inlet may be provided at the wall of the second space 14, so that part of the gas or liquid sample may be directly introduced into the culture liquid in the second space.

In some embodiments, the reaction pouch is a disposable bioreactor pouch, and the disposable bioreactor pouch comprises, in order from the inside to the outside: a contact layer for direct contact with microorganisms or cell cultures or the like, a barrier layer for gas barrier and a structural layer for providing mechanical stability.

Specifically, in some embodiments, the volume of the reaction pouch is preferably set to 1.5L, 2L, 2.5L, or 5L.

In particular, in some embodiments, the bioreactor bag is a disposable bag composed of three layers of plastic material, one layer made of polyethylene terephthalate or LDPE to provide mechanical stability. The intermediate layer is made of PVA or PVC and serves as a gas barrier. Finally, the contact layer with the cell culture is made of PVA or PP.

In order to be able to perform a stable microorganism or cell culture in a small volume of a bioreactor bag, for example, it is ensured that other structures associated with the reactor bag (e.g. the first inlet, the second inlet, etc.) can operate stably. The volume (or capacity) of the second space is typically smaller than the volume of the first space. Preferably, the second space is about 20% -50% of the volume of the interior receiving chamber of the reaction pouch.

For example, in some embodiments, when the bioreactor bag is 5L, the second space is about 2L.

During the experiment it was necessary to ensure that the total amount of liquid added did not exceed the volume of the second space.

For example, in some embodiments, a dividing line is provided in the first space and the second space for determining whether the liquid is excessively added.

It will be appreciated that the expression "first space" and "second space" used in the present invention merely distinguishes the reaction pouch interior receiving chamber from a spatial function perspective. Therefore, the first space and the second space may be spaces that communicate without obstacles in practice, as shown in fig. 1 and 3.

In some embodiments, as shown in fig. 4, the top wall 11 inside the condensate guiding zone is configured as a slope, and the edge of the condensate guiding zone is provided with an annular guide rail 12, the annular guide rail 12 providing a guiding path L2 for the condensate to condense and reflux.

For example, in some embodiments, the top of the first space (or, only the condensate water guiding region) is provided as a slope having a certain inclination angle. Specifically, the top of the first space may be provided in an inverted conical shape.

For example, in some embodiments, the edge of the condensed water guiding area is provided with a circle of circular guide rail, when the air humidity of the first space is high, condensation can be preferentially performed on the inclined surface (i.e. the top wall surface 11) to form water drops, the water drops flow downwards along the inclined surface and collect on the inner side of the guide rail, and when the collected water drops gradually increase, the water drops flow downwards under the action of gravity and flow back into the second space.

In some embodiments, the wall surface of the reaction pouch inside the condensed water guide region is configured as a slope, and the inclination angle α (or gradient) of the slope is preferably set to 1 ° -5 °. Wherein, inclination refers to the included angle between the wall surface of the condensed water guiding area and the horizontal plane L1 when the bioreactor is vertically placed.

Preferably, the incline angle of the incline is 2-3 °.

Further, in some embodiments, as shown in fig. 5, the annular rail is of a design with a gradually decreasing curvature from its first end to its second end (i.e., condensate outlet end). Specifically, the curvature of each point of the guide path of the endless guide rail 12 gradually decreases in the direction from the first end to the second end thereof, so that the second end of the guide path is lower than the first end, that is, the condensate water outlet end is at the lowest position.

In some embodiments, as shown in fig. 3, the bioreactor further comprises: a stirring device 2, wherein the stirring device 2 comprises a stirring shaft 22 and a stirring paddle 21 arranged at the tail end of the stirring shaft; a mounting hole for mounting the stirring device 2 is formed in the central area of the condensed water guiding area, the stirring shaft 22 is fixedly mounted on the reaction bag through the mounting hole, and the stirring paddle 21 extends into the second space;

wherein the stirring shaft 22 is provided with a heating unit 23 (for example, a constant temperature heating plate can be provided) at one side at or near the condensed water guiding region, so as to avoid condensed water from forming at the outer side of the condensed water guiding region; the monitoring end of the working state monitoring module is higher than the horizontal height of the stirring paddle so as to reduce the interference of the stirring paddle on the working stability of the working state monitoring module.

In the embodiment of the invention, in order to avoid that excessive condensed water is generated in the first space due to too low temperature and too high humidity, so that the condensed water may be condensed outside the condensed water guiding area or the condensed water in the condensed water guiding area overflows, an electric heating plate is arranged at a position of the stirring shaft 22, which is close to the condensed water guiding area and far from the liquid level, for example, the electric heating plate can start a heating function when the humidity is too high or the temperature is too low.

It will be appreciated that since the stirring device is typically made of metal, when the stirring device is not in operation for heating, the top of the stirring shaft will typically aggregate to form a volume of condensate water, some of which will flow back along the stirring shaft to the second space, and some of which will flow back along the guiding path under the influence of the condensate water guiding zone.

In some embodiments, the stirring device may be a magnetic stirring device.

In the embodiment of the invention, in order to control the volume and the application cost of the miniaturized bioreactor, an internal circulation type condensate water reflux scheme (without an external drainage pipeline) is provided, so that the blockage or even pollution of condensate water to each inlet or outlet in the reaction process can be avoided, and meanwhile, the internal stability of the bioreactor can be further ensured (the liquid loss is reduced) by the internal circulation type condensate water reflux scheme.

In some embodiments, as shown in fig. 4, an annular groove 12-1 for providing the guiding path L2 is provided in the annular guide rail 12, and a condensed water outlet is provided on the annular groove 12-1, and the bottom surface of the annular groove is inclined, so that the condensed water outlet is the lowest horizontal point of the annular groove, and the condensed water condensed in the annular groove is guided to flow out through the condensed water outlet; wherein the condensed water outlet is arranged along a direction away from the first inlet and the second inlet.

In the embodiment of the invention, by the low-outlet design of the inclined grooves, the condensate water can be further guided and promoted to form in a limited area (namely a condensate water guiding area), and the condensate water is prevented from overflowing due to excessive condensation. Particularly, when the highly pathogenic viruses or bacteria are cultured, concentrated formation and backflow of condensed water can ensure stable performance of experiments, and meanwhile, the possibility that the highly pathogenic viruses or bacteria overflow through a pipeline and pollute the external environment can be reduced.

In some embodiments, the first inlet (i.e. gas inlet) for introducing gas is provided with a first control valve (i.e. gas inlet valve), the second inlet (i.e. liquid inlet) for introducing liquid is provided with a second control valve (i.e. liquid inlet valve), the first space is provided with a second pressure monitoring unit (i.e. gas pressure monitoring unit) for monitoring the gas pressure inside the first space, and the first pressure monitoring unit (i.e. hydraulic pressure monitoring unit) is in communication connection with the second control valve, and the second pressure monitoring unit is in communication connection with the first control valve and the second control valve, respectively.

The following describes part of the working procedures of the air pressure monitoring unit, the hydraulic pressure monitoring unit, the air inlet valve and the liquid inlet valve:

When the hydraulic pressure monitoring unit monitors that the hydraulic pressure exceeds a preset first pressure threshold value, a first closing signal which indicates stopping liquid feeding is sent to the liquid inlet valve, and the liquid inlet valve responds to the first closing signal to keep the liquid inlet closed;

and when the air pressure monitoring unit monitors that the air pressure exceeds a preset second pressure threshold value, sending a second closing signal for indicating stopping sample injection to an air inlet valve and a liquid inlet valve, and keeping the air inlet valve and the liquid inlet valve closed based on the second closing signal.

In some embodiments, when the hydraulic pressure or the air pressure is detected to exceed the preset pressure threshold value, an early warning signal can be directly sent to the user, and the user intervenes to judge whether the inlets need to be closed or opened.

In order to ensure the safety and reliability of a miniaturized experiment (for example, when the capacity of the reaction bag is only 0.5L, 1L or 2L), the embodiment of the invention can avoid the experiment failure and even the reaction bag rupture caused by excessive liquid filling or air filling by synchronously monitoring the air pressure and the hydraulic pressure.

Further, in some embodiments, further comprising:

and the first liquid leakage monitoring module is arranged at the bottom of the reaction bag and used for monitoring whether the reaction bag leaks liquid or not (for example, the first liquid leakage monitoring module can be a liquid leakage sensor).

Further, in some embodiments, further comprising:

a safety sterilization module, the safety sterilization module comprising: a recovery unit connected to the reaction bag and a sterilization unit; the recovery unit includes: a recovery bag and a recovery pipeline for connecting the recovery bag and the reaction bag, wherein a third control valve (namely a recovery valve) is arranged in the recovery pipeline; the sterilizing unit includes: a chamber for storing sterilizing substances, and a sterilizing duct for connecting the chamber and the reaction bag, wherein a fourth control valve (i.e., a sterilizing valve) is arranged in the sterilizing duct;

and the third control valve and the fourth control valve are in communication connection with the pressure monitoring sensor and the first liquid leakage monitoring module.

In the embodiment of the invention, in order to control the dosage of the sterilizing substances and reduce adverse effects on the surrounding environment of the reaction bag (such as a supporting device for supporting the reaction bag or a biosafety cabinet where the reaction bag is located) in the sterilizing process, a mode of firstly recycling and then sterilizing is adopted.

The following describes a preferred sterilization procedure for the safety sterilization module:

step 21: the safety sterilization module (for example, a recovery valve and a sterilization valve) receives an opening signal (for example, when the leakage of the reaction bag is detected, such as the leakage state of the reaction bag is detected, or a sterilization instruction input by a user) is detected;

Step 22: the recovery valve is opened and the liquid in the second space is pumped to the recovery bag;

after the liquid is completely recovered, or after a certain preset recovery period (e.g., 10 s), step 23 is performed: closing the recovery valve and opening the sterilizing valve, wherein the sterilizing valve is used for introducing sterilizing substances in the cavity into the reaction bag.

In some embodiments, a sterilizing substance for sterilizing the liquid fluid is pre-stored within the recovery bag.

In some embodiments, the sterilizing substance is a second liquid or a second gas, such as hydrogen peroxide gas, for sterilization.

In some embodiments, the recovery bag may be of the same or similar multi-layer design as the disposable bioreactor bag.

In some embodiments, after the entire bioreactor is finished, the safety sterilization module may sterilize the bioreactor in the same or similar manner as steps 21-23.

In the embodiment of the invention, in order to avoid the liquid (containing high-harm microorganisms) in the reaction bag from leaking out under the impact of the liquid in the reaction bag during the process of filling the sterilizing substance, and even aggravate the damage degree of the reaction bag. Before filling the sterilizing substance, the liquid is recovered, and then sterilizing gas or liquid is introduced to perform centralized sterilization on the reaction bag.

In some embodiments, the sterilizing substance may also be introduced into the reaction pouch through the first outlet.

In some embodiments, further comprising: a secure sampling device, the secure sampling device comprising: the sampling tube is connected with the first outlet 5, a fifth control valve is arranged in the sampling tube, a plurality of sampling ports are axially arranged on the sampling tube, the sampling ends of the sampling ports are connected with disposable sampling bags in a sealing mode, and the opening ends of the disposable sampling bags are made of thermoplastic plastics.

After the sampling bag is sampled, the open end of the sampling bag can be subjected to heat treatment through the clamping type heater, so that the sampling bag is automatically sealed under the heat treatment effect and can be separated from the sampling end of the sampling port to be taken out by a user.

In some embodiments, the reaction pouch has a 4-way gas inlet connected thereto. Of course, the number/position of the gas inlets can be adjusted according to the actual requirements.

In some embodiments, the control valve in the gas inlet is a non-return valve.

In some embodiments, the wall surface of the first space is further provided with a second outlet 6 (i.e. an air outlet), the air outlet is connected with an air outlet pipeline, and a filtering device (such as an H14 HEPA filter) is further connected inside the air outlet pipeline, so as to be used for filtering aerosol inside the reaction bag, so as to avoid polluting the external environment. When the reaction bag is used in a biological reaction cabinet, the air outlet pipeline can be communicated with an air outlet pipeline of the biological reaction cabinet.

In some embodiments, the air outlet is provided with a drying device 61. For example, a desiccant is provided inside the duct of the air outlet for filtering contaminants in the air.

Alternatively, in other embodiments, the desiccant may be omitted when an anhydrous disinfectant is employed within the filter assembly 62.

Further, in some embodiments, by monitoring key parameters of the liquid inside the reaction bag, such as pressure (which may be used for reaction liquid content), pH, oxygen dissolution rate, CO ₂ And judging whether the cell culture solution, water, acid solution or alkali solution and the like need to be supplemented into the reactor or not according to the concentration, turbidity and the like so as to complete automatic fluid replacement in the culture process.

Further, in some embodiments, any one of the control valves may employ a one-way valve.

Further, in some embodiments, the amount or rate of addition of liquid may be controlled by peristaltic pumps at the control valve used to control the liquid.

Further, in some embodiments, a sterilization unit, e.g., a 0.22 micron sterilization filter device, is provided at the inlet of the reaction bag for venting (e.g., oxygen, etc.).

It will be appreciated that the disposable bioreactor of the embodiments of the present invention may also be used to achieve continuous culture of microorganisms or cells. For example, in the case of virus culture, when the amount of virus in the disposable bioreactor reaches a certain amount, a part of virus sample in the reactor can be taken out first, and then new culture cell liquid is added into the reactor so that the remaining virus can continue to grow in the reactor, thereby realizing continuous culture of the virus.

In some embodiments, as shown in fig. 8-9, the sensor employs a bio-safe sensor quick release device to achieve tightness, wherein the sensor quick release device comprises: a disposable sensor probe 81, a probe terminal 82, a power terminal 83, an electrode 84. The probe terminal 82 is provided with positive and negative electrode terminals which are connected with the power supply terminal 83 through the electrode 84 so as to transmit current signals (specifically, sensing signals such as hydraulic values, dissolved oxygen rates and other values) to a central control system (such as a central control module) for monitoring sensor parameters in real time. In use, probe terminal 82 is connected to power terminal 83 for transmitting electrical signals.

In some embodiments, the supporting device is provided with an interface for installing the power terminal 83, the interface of the supporting device is made of corrosion-resistant sealing materials, the power terminal 83 can be inserted into the interface in a quick-insertion or threaded connection mode, and the sealing of the junction of the power terminal 83 and the supporting device is ensured, so that no aerosol leaks.

In some embodiments, sampling detection is performed directly in the biosafety cabinet/isolator by controlling the sampling check valve for less than or equal to 20L of disposable bioreactors placed in the biosafety cabinet/isolator.

In some embodiments, greater than 20L of reactor, the sampling tubing is directly connected to the biosafety cabinet/isolator for sampling detection by controlling the sampling check valve.

Alternatively, in some embodiments, the bioreactor further comprises: the central control module is respectively in communication connection with one or more control valves, one or more monitoring units or monitoring modules; in order to be able to monitor and maintain the reaction safety in real time and accurately, the central control module comprises a risk monitoring unit.

Specifically, the risk monitoring unit is configured to perform the following procedure:

step 11, acquiring a monitoring value (such as an electric signal of a liquid leakage sensor) of the first liquid leakage monitoring module in real time;

step 12, the risk monitoring unit judges the leakage risk according to the leakage value;

in order to accurately monitor the leakage state, the risk of misjudgment is reduced, and the leakage risk is classified into 0 level, 1 level and 2 level according to the magnitude of the leakage value;

then, respectively carrying out safety early warning or safety disinfection and other treatments on the bioreactor according to the specific leakage risk degree;

step 13: when the leakage risk is 1, the risk monitoring unit judges whether the hydraulic pressure is abnormal or not;

If yes, corresponding disinfection signals are generated to the recovery valve and the disinfection valve;

if not, continuing to monitor the leakage risk and the hydraulic pressure, and if the monitored hydraulic pressure monitoring value increases or the hydraulic pressure is abnormal, generating corresponding disinfection signals to the recovery valve and the disinfection valve;

step 14: when the leakage risk is 2, corresponding starting signals are directly generated to the recovery valve and the disinfection valve.

In some embodiments, when the risk of leakage is detected, an early warning signal can also be generated to the staff, and the staff can manually judge whether to continue the experiment or not.

In the embodiment of the invention, two limited parameters of external liquid leakage and internal hydraulic pressure are selected for monitoring, so that a monitoring method with high sensitivity and relatively low cost (namely, a double-mechanism risk assessment method) is provided.

In fact, current bacterial or viral cultures that are infectious, highly pathogenic often need to be performed in high-level biosafety laboratories (e.g., P3 laboratories). The embodiment of the invention integrates various key state monitoring, double-mechanism risk assessment and safety protection functions in a single small-sized bioreactor, so that when the small-sized bioreactor is used for culturing high-harm microorganisms, the internal environment stability of the reactor can be ensured through the monitoring of key parameters and the design of internal circulation type condensate water reflux, and meanwhile, autonomous risk monitoring and sterilization can be finished according to low-cost double-mechanism monitoring.

Therefore, the small bioreactor can be directly applied to culturing cells or microorganisms in a biosafety cabinet (BSC), so that the cost of manpower and material resources for culturing high-hazard microorganisms can be reduced to a certain extent.

Example two

The invention also provides a monitoring method which can be applied to the bioreactor in any embodiment of the invention to realize microorganism culture.

Preferably, in some embodiments, as shown in fig. 10, the method comprises the steps of:

s106, judging the risk state of the bioreactor according to the hydraulic pressure and the leakage value, wherein the risk state comprises the following steps: a safe state, and/or a pending state, and/or a weeping state; wherein S106 includes:

s61, judging the depressurization risk of the reaction bag according to the change parameter (such as the pressure reduction value or the pressure reduction speed) of the hydraulic pressure; when the change parameter belongs to a preset first depressurization threshold value, the depressurization risk is first-level, when the change parameter belongs to a preset second depressurization threshold value, the depressurization risk is second-level, and otherwise, the depressurization risk is zero-level;

S62, judging the leakage risk of the reaction bag according to the leakage value (for example, in some embodiments, the leakage value is an electric signal value of a leakage sensor), wherein the leakage risk is first-level when the leakage value belongs to a preset first leakage threshold value, is second-level when the leakage value belongs to a preset second leakage threshold value, and otherwise, the leakage risk is zero-level;

s63, judging the risk state according to the depressurization risk in S61 and/or the leakage risk in S62; wherein, the liquid crystal display device comprises a liquid crystal display device,

when the depressurization risk is a second level and/or the leakage risk is a second level, the risk state is a leakage state; when the depressurization risk and the weeping risk are zero-order, the risk state is a safety state; otherwise, the risk state is a pending state.

In some embodiments, a top region of the first space is further formed with a condensed water guiding region for guiding condensed water to be condensed in the condensed water guiding region and to flow back into the second space through a guiding path provided by the condensed water guiding region, the method further comprising the steps of:

In some embodiments, the step S110 includes:

Specifically, in some embodiments, when the risk status is monitored to be pending, an early warning signal needs to be sent to the user to remind the user to timely check the working status of the bioreactor (for example, whether there is a leakage tendency or a slight leakage phenomenon, or whether there is a problem of excessive condensation water aggregation, etc.).

For example, in some embodiments, when the risk state is detected to be a pending state, the user receives a corresponding early warning signal, at which time the user may choose to heat the condensed water guiding region (preferably, may heat the condensed water guiding region by a heating unit disposed on the stirring shaft) so as to limit or reduce formation of condensed water, thereby judging whether the current bioreactor has a problem of excessive condensation of condensed water, if so, the risk state may be corrected to a safe state by the user when the reduced-pressure risk returns to a normal state (such as zero level).

It will be appreciated that especially for bioreactors having a volume of only a few liters (e.g. 1L), the total amount of liquid inside the reactor is relatively limited and thus the hydraulic pressure is also relatively susceptible to multiple factors such as stirring speed, stirring force, leakage of liquid, condensation of condensed water, etc. At this time, it is difficult to find a problem at the first time if the monitoring difference value (corresponding to the second step-down threshold value) of the sensor is set too high, but if the monitoring difference value of the sensor is set too low, normal operation of the false alarm interference test may frequently occur. In the embodiment of the invention, the formation or backflow of the condensed water is controlled or influenced by utilizing the internal circulation condensed water guide area, so that the sensor can not interfere with the normal operation of the test due to excessive false alarm even if the sensor is in the condition of low monitoring difference (namely high sensitivity). In other words, the invention can adjust the accuracy of the leakage monitoring to a certain extent.

For example, in some embodiments, the monitored difference may be set to about 5% or even lower, specifically when the hydraulic pressure difference varies by 5% or more, then the bioreactor is considered to currently have a higher risk of weeping.

Specifically, in some embodiments, the risk state may be converted to the safe state when it is monitored that the hydraulic pressure is restored to the desired level (the desired level may be determined according to the actual amount of liquid added) within the preset second time, and the risk of leakage is unchanged.

Or in some embodiments, when it is monitored that the hydraulic variation parameter does not change significantly (for example, still falls within the range of the first depressurization threshold value) within the preset third time, a corresponding early warning signal is sent to the user so as to prompt the user that there is a risk of excessive condensed water.

It will be appreciated that the various thresholds in embodiments of the present invention may be adaptively set by the user in connection with the actual situation (e.g., volume of the reactor, amount of liquid added, etc.).

According to the embodiment of the invention, the limited parameters are monitored in real time, so that a low-cost and high-precision monitoring mode is provided for the working state (particularly the risk state) of the bioreactor, on one hand, the manual monitoring pressure of a worker can be reduced to a certain extent, and on the other hand, the interference caused by transitional monitoring (caused by the error signal suspension test and the waste of manpower and material resources) can be avoided.

In some embodiments, prior to S102, further comprising the steps of: s100, testing the bioreactor in advance; wherein, the S100 includes:

introducing gas into the reaction bag through the first inlet, and keeping the other inlets and outlets of the reaction bag closed at the same time;

ending ventilation and keeping the corresponding first inlet closed;

collecting a first air pressure change value of the reaction bag in a first time through the first pressure monitoring unit, and judging whether the bioreactor passes a pre-test (namely whether the sealing performance is good) or not according to the first air pressure change value; wherein when the first air pressure variation value belongs to a preset safety threshold value (specifically, when the air pressure variation is small or the variation value is 0), the bioreactor passes a pre-test.

Example III

As shown in fig. 6 and 7, the present invention also provides a supporting device for supporting the above bioreactor in order to further improve the safety of the operation during the cultivation of highly pathogenic microorganisms. As shown in fig. 6, it includes:

a temperature-controllable support body 7 for supporting the bioreactor; the bioreactor comprises: a reaction pouch, the support body 7 comprising: a sidewall structure formed by a first sidewall 73 and a second sidewall 74 together, a base 72 formed below the sidewall structure, and a top cover for sealing above the sidewall structure, wherein the sidewall structure and the base 72 support the reaction bag; the top cover is provided with at least one third inlet for leading out at least one pipeline connected with a first inlet and a second inlet in the reaction bag, and the first inlet and the second inlet are respectively used for adding gas and liquid into the reaction bag;

And a heating module for heating the reaction pouch, the heating module comprising: the heating cavity is arranged on the first side wall and/or the second side wall, is used for containing a heat conducting medium, and is also provided with a medium inlet and a medium outlet for the heat conducting medium to enter and exit; a temperature monitoring unit (e.g., a temperature sensor) for monitoring the temperature of the heat-conducting medium, and a heating unit communicatively connected to the temperature monitoring unit and for heating the heat-conducting medium;

and a first leakage monitoring module (such as a leakage monitoring sensor 71) disposed in a central region of the bottom, the first leakage monitoring module being configured to monitor whether leakage occurs in the reaction bag and/or the heating cavity.

In some embodiments, for small-sized bioreactor bags, the circumferential wall of the small-sized bioreactor bag is heated uniformly with water as a heat conducting medium.

In some embodiments, when the temperature inside the reactor is monitored to be lower than the preset culture temperature by the temperature sensor, a proper high-temperature heat conducting medium can be introduced into the heating cavity to heat the reactor. Alternatively, the heat-conducting medium may be directly subjected to the heat treatment by a heating unit (for example, an electric heating pipe or the like may be provided to be inserted into water).

In some embodiments, the bioreactor comprises: the working state monitoring module is used for monitoring working state parameters of the bioreactor, and the working state parameters comprise: liquid pressure, foam generation, dissolved oxygen rate, CO ₂ Concentration, pH, and turbidity; correspondingly, the supporting device further comprises: the central control module is used for being in communication connection with the working state monitoring module, the first liquid leakage monitoring module and the heating module, and controlling the running states of the bioreactor and the supporting device according to the monitoring results of the communication of the working state monitoring module, the first liquid leakage monitoring module and the heating module.

In some embodiments, the bioreactor further comprises: a safety disinfection module in communication with the central control module;

the safety sterilization module includes: a recovery unit and a sterilization unit connected with the reaction bag, the recovery unit comprising: recovery bag, and be used for connecting the recovery bag with the recovery pipeline of reaction bag, be provided with the third control valve in the recovery pipeline, disinfection unit includes: a chamber for storing a sterilizing substance (e.g., hydrogen peroxide sterilizing liquid), a first sterilizing duct for introducing the sterilizing substance into the inside of the reaction bag, and a second sterilizing duct for introducing the sterilizing substance between the support body and the reaction bag, the first and second sterilizing ducts each being provided therein with a fourth control valve, wherein the chamber may be provided on the second side wall.

Further, in some embodiments, the safety sterilization module may also sterilize various lines in the reaction pouch or support device.

Particularly, when high-pathogenicity microorganism culture such as viruses and bacteria is carried out, the supporting device in the embodiment of the invention can detect the working state of the disposable bioreactor in real time, and when a leakage or gas leakage signal is detected, the safety disinfection module is started in time to complete safety disinfection.

In some embodiments, a visual window is further formed on the second side wall, so as to be used for observing the reaction condition inside the reaction bag.

In some embodiments, a second leakage monitoring module is disposed on the second sidewall, and a monitoring end of the second leakage monitoring module contacts a wall surface of the reaction bag when the reaction bag is mounted inside the support body.

Therefore, when the reaction bag is slightly damaged (such as condensed water overflows from the mounting hole of the stirring device or trace leakage exists in the area above the reaction bag), the leakage monitoring sensor arranged on the second side wall can carry out supplementary monitoring on the stability of the reaction bag, so that experimental risks are further reduced.

In some embodiments, the fluid leakage monitoring module includes one or more of the following sensors: a point-type water leakage sensor, a bracket probe type water leakage sensor, a non-positioning type water leakage detector and a positioning type water leakage detector.

In some embodiments, the top cover and the sidewall structure are threadably engaged.

In some embodiments, the top cover and the sidewall structure are cooperatively connected by a snap-in structure, and the area where the top cover and the sidewall structure contact is provided with a corrosion resistant material.

In some embodiments, two leak-proof sterilization interfaces (connected to the sterilization unit) are provided on the side walls of the support device, through which the chamber is sterilized in a cyclic manner, if a leak occurs. Wherein the interface can be flexibly arranged on the side wall or the base of the supporting device.

For example, in some embodiments, two leak-proof interfaces are one inlet and the other outlet. Specifically, during the sterilization process, a certain amount of sterilizing substance may be output to the inside of the supporting device through the inlet (e.g., liquid sterilizing liquid/sterilizing gas may be introduced or sprayed through the interface) and sterilized for a certain period of time, and then discharged through the outlet.

Alternatively, in other embodiments, the incoming sanitizing substance can be recycled. Specifically, the disinfectant is introduced into the inlet, the disinfectant is recovered by a peristaltic pump at the outlet, and the recovered disinfectant is introduced into the supporting device again (e.g. the disinfectant is added into the supporting device again through the inlet).

In some embodiments, after disinfection is completed, the interior of the support device may also be cleaned (e.g., by washing the interior with water) through the inlet, the interface, to extend the service life of the support device.

It will be appreciated that the staff may choose different sterilization modes according to different application requirements (e.g. sterilization of new products or sterilization after the reaction is completed).

Example IV

The invention also provides a monitoring method for the disposable biological safety type bioreactor supporting device, which comprises the following steps:

s200, any one of the supporting devices and the reaction bag provided by the invention are provided; wherein, the reaction pouch comprises: the reaction bag is arranged in the supporting main body of the supporting device, the wall surface of the reaction bag is abutted against or adjacent to at least one side wall of the supporting device, so that the heating efficiency of the heating module for heating the reaction bag is improved, and the bottom of the reaction bag is contacted with the first liquid leakage monitoring module to promote the first liquid leakage monitoring module to monitor the liquid leakage of the bottom of the reaction bag;

S202, introducing a preset amount of gas or liquid into the reaction bag through a first inlet or a second inlet on the reaction bag so as to culture microorganisms or cells;

s204, monitoring the hydraulic pressure of the first pressure monitoring unit and the leakage value of the first leakage monitoring module in real time through a central control module of the supporting device;

s206, judging the risk states of the supporting device and the bioreactor according to the hydraulic pressure and the leakage numerical value.

In some embodiments, the risk status includes: a safe state, and/or a weeping state, and/or a pending state; wherein S206 includes:

judging the depressurization risk of the reaction bag according to the hydraulic variation parameters; when the change parameter belongs to a preset first depressurization threshold value, the depressurization risk is first-level, when the change parameter belongs to a preset second depressurization threshold value, the depressurization risk is second-level, and otherwise, the depressurization risk is zero-level;

judging the leakage risk of the reaction bag according to the leakage value, wherein the leakage risk is first-level when the leakage value belongs to a preset first leakage threshold value, second-level when the leakage value belongs to a preset second leakage threshold value, and otherwise, the leakage risk is zero-level;

Judging the risk state according to the depressurization risk and/or the leakage risk; wherein, the liquid crystal display device comprises a liquid crystal display device,

when the depressurization risk is a second level and/or the leakage risk is a second level, the risk state is a leakage state; when the depressurization risk and the weeping risk are zero-order, the risk state is a safety state; when the depressurization risk is first-order and the leakage risk is zero-order, or when the depressurization risk is zero-order or the leakage risk is first-order, the risk state is a pending state.

It will be appreciated that the monitoring method in this embodiment may include the same steps as in either the first or second embodiment. For example, the method further comprises: the user is optionally given an early warning according to the result of the risk status, or the support device and the reactor are sterilized, etc., which will not be described here again.

In some embodiments, the second side wall of the supporting device is further provided with an air pressure monitoring module for monitoring the air pressure inside the supporting body, and accordingly, before S202, the method further includes the steps of: s208, pre-testing the bioreactor and the supporting device;

wherein, the step S208 includes the steps of:

introducing gas into the reaction bag through the first inlet, and monitoring a second pressure change value in the support main body through the pressure monitoring module; simultaneously keeping the reaction bag, the rest of inlets and outlets of the supporting device closed;

Ending ventilation and keeping the corresponding first inlet closed;

collecting a first air pressure change value of the reaction bag in a preset first time through the first pressure monitoring unit, and monitoring a third air pressure change value of the supporting main body in the first time through the air pressure monitoring module;

judging whether the bioreactor passes a pre-test according to the first air pressure change value, and judging whether the supporting device passes the pre-test according to the second air pressure change value and the third air pressure change value;

when the first air pressure change value belongs to a preset corresponding safety threshold value (namely, the air pressure change is 0 or very tiny), the bioreactor passes a pre-test; when the second air pressure variation value is consistent with the amount of the introduced air (it can be understood that the more the amount of the introduced air is, the larger the air pressure variation value is), and the third air pressure variation value belongs to a preset corresponding safety threshold value, the supporting device passes the pre-test.

For example, in some embodiments, the pouch is considered to be well-qualified when the air pressure does not change significantly after venting.

For example, in some embodiments, when the air pressure within the support body increases significantly during ventilation and remains steady for the first time the test is performed, the support device is considered to be well sealed.

Further, in some embodiments, the support device may also achieve high temperature in situ sterilization.

Further, in some embodiments, pulleys are also provided on the base to facilitate transport of the support device.

Further, in some embodiments, the top of the support device is also provided with mounting holes for mounting the stirring device 2.

In some embodiments, different configurations of agitation devices may be selected according to different culture objects. For example, when the culture object is a cell, the stirring paddle of the stirring device may be in a blade shape. For another example, when the object of culture is bacteria, the stirring paddle of the stirrer may be in the shape of a stick.

In some embodiments, various monitoring units in the reaction bag can be disposable electrode detection sensors, and the sensors are hermetically connected with the reaction bag so as to prevent leakage. The whole bioreactor is only required to be simply installed and disposable.

The disposable bioreactor provided by the invention can be used for culturing various microorganisms and cells, for example, the disposable bioreactor can be applied to vaccine production.

It should be noted that the present invention preferably provides a miniaturized, highly pathogenic microbial culture with a miniaturized integrated bioreactor and associated support means. Also, such a compact integrated bioreactor may be scaled up according to the actual needs of the user (e.g., from pilot plant to pilot plant), for example, to a bioreactor with a chamber volume of about 100L-200L.

The invention can be widely applied to various microorganisms or cells. For example, the method can be applied to culture of highly pathogenic bacteria and viruses, microorganisms or cells which can generate different degrees of pollution or pathogenic effects on the environment or human beings, and can be applied to culture of various animal and plant cells.

Example five

The invention also provides a semi-automatic control method suitable for the bioreactor and the supporting device, so as to reduce the intervention operation (such as ventilation or disinfection operation and the like) of a user in the process of cultivating the highly pathogenic virus to a certain extent.

FIG. 11 illustrates an exemplary semi-automated control method of the present invention, comprising:

s301 provides a biological reaction system for microbial growth, the biological reaction system comprising: a bioreactor, and a support device for supporting the bioreactor, wherein the bioreactor comprises: the reaction bag comprises a first space and a second space which are respectively used for containing gas and liquid, a first inlet and a second inlet which are respectively used for introducing the gas and the liquid are also arranged in the reaction bag, and a working state monitoring module which is used for monitoring working state parameters of the bioreactor is also arranged on the reaction bag;

S302, responding to a corresponding control signal sent by a user or the working state monitoring module, and introducing gas and/or liquid into the reaction bag through the first inlet and/or the second inlet so as to perform biological culture (microorganism culture or cell culture);

s303, monitoring the pressure of the liquid in the second space to acquire hydraulic pressure, and monitoring the bottom of the reaction bag to acquire a leakage value;

s304, judging the risk state of the biological reaction system through the hydraulic pressure and the leakage value, wherein the risk state comprises the following steps: a safe state, and/or a pending state, and/or a weeping state; wherein S304 includes:

determining the depressurization risk of the reaction bag according to the hydraulic variation parameters; wherein, the step-down risk is divided into respectively according to the numerical value of the change parameter: zero order, first order, and second order;

determining the leakage risk of the reaction bag according to the leakage value; wherein, the leakage risk is divided into according to the leakage value size respectively: zero order, first order, and second order;

judging the risk state according to the depressurization risk and the leakage risk; when the depressurization risk is a second level and/or the leakage risk is a second level, the risk state is a leakage state; when the depressurization risk and the weeping risk are zero-order, the risk state is a safety state; when the depressurization risk is first-order and the leakage risk is zero-order, or when the depressurization risk is zero-order and the leakage risk is first-order, the risk state is a pending state.

In some embodiments, the method further comprises:

s305, when the risk state is detected to be in a pending state or a liquid leakage state, respectively sending corresponding early warning signals to a user;

s306, responding to the feedback signal sent by the user, and secondarily monitoring the depressurization risk and/or the leakage risk of the biological reaction system so as to receive or correct the early warning signal.

Preferably, in some embodiments, the feedback signal may be manually issued by a worker.

Alternatively, in other embodiments, when the feedback signal sent by the worker is not monitored or received within a set time (e.g., within 5S or 15S), the feedback signal preset by the worker may be sent by a computer (which may be communicatively connected to the bioreactor or the support device) correspondingly according to the current risk status.

In some embodiments, a condensate water guiding area is further formed in a top area of the first space, and the condensate water guiding area is used for guiding condensate water to be condensed in the guiding area, and flows back into the second space through a guiding path provided by the condensate water guiding area, and accordingly, the feedback signal includes: a first feedback signal for heating the condensed water guide area, and a second feedback signal for receiving the early warning signal, S306 includes the steps of:

When the depressurization risk is first-order and the leakage risk is zero-order, a heating unit arranged in the condensed water guiding area responds to the first feedback signal to heat the condensed water guiding area;

Preferably, in the embodiment of the present invention, the correction of the risk status needs to be manually operated by a staff member to avoid erroneous judgment of the bioreactor.

Of course, in other embodiments, the risk status may be corrected and evaluated in an automated manner.

In some embodiments, S306 further comprises the steps of:

and when the step-down risk is monitored to be zero level or the leakage risk is monitored to be one level, continuing to monitor the leakage value, and when the leakage value is monitored to be constant in a preset fourth time, correcting the risk state to be a safe state in response to the first correction signal, otherwise, correcting the risk state to be a leakage state in response to the second correction signal.

For example, in some embodiments, when the leak value does not change significantly over a period of time (e.g., 5s or 10 s), the leak from the bioreactor may be excluded by the user.

In some embodiments, the bioreactor system further comprises: the stirring device comprises a stirring shaft and a stirring paddle arranged at the tail end of the stirring shaft; the central area of comdenstion water guide district has seted up and is used for installing agitating unit's mounting hole, just the (mixing) shaft passes through mounting hole fixed mounting is in on the reaction bag, the (mixing) shaft is located or is close to one side of comdenstion water guide district is provided with heating element for avoid or restrict the formation of comdenstion water.

In some embodiments, the bioreactor system further comprises: a safety sterilization module for sterilizing the bioreactor and the support device, the safety sterilization module comprising: a recovery unit connected to the reaction bag through a recovery pipe, and a chamber for storing a sterilizing substance, the chamber being respectively connected to the reaction bag and the supporting means through a sterilizing pipe; the method further comprises the steps of:

S307, when the risk state is detected to be a liquid leakage state, a corresponding disinfection signal is sent to the safety disinfection module;

s308, the safety disinfection module responds to the disinfection signal to recover the liquid in the reaction bag to the recovery unit, and the recovery pipeline is closed after recovery is finished;

s309, after the recovery pipeline is closed, the safety disinfection module responds to the disinfection signals to introduce the disinfection substances into the reaction bag and the supporting device.

In some embodiments, the method further comprises the step of, prior to 301:

s300, testing the stability of the biological reaction system in advance; wherein, the S300 includes:

introducing gas into the reaction bag through the first inlet, and synchronously collecting a second air pressure change value in the support device; simultaneously keeping the reaction bag, the rest of inlets and outlets of the supporting device closed;

ending ventilation and keeping the corresponding first inlet closed;

collecting a first air pressure change value of the reaction bag in a preset first time and a third air pressure change value of the supporting device in the first time;

judging whether the stability of the biological reaction system is qualified or not according to the first, second and third air pressure change values;

When the first air pressure change value belongs to a corresponding preset safety threshold value, and the second air pressure change value and the third air pressure change value are consistent with the amount of the introduced air, the biological reaction system passes the pre-test.

Aiming at small-scale culture of highly pathogenic microorganisms (such as influenza virus and the like), the invention selects limited monitoring factors (namely internal hydraulic pressure and external leakage value) to perform core monitoring on the working state of the bioreactor in the culture process, and provides a low-cost semi-automatic control method capable of reducing manual intervention.

The method can be matched with the condensed water guiding area to further reduce the interference of other factors on risk monitoring so as to reduce the misjudgment condition, thereby reducing the research and development cost to a certain extent (particularly, the method can avoid stopping the test due to misjudgment and excessively consume manpower and material resources).

Example six

Based on the above-mentioned semi-automatic control method, as shown in fig. 12, the present invention also correspondingly provides a semi-automatic system, which includes:

a biological reaction system 10, the biological reaction system comprising: a bioreactor, and a support device for supporting the bioreactor, wherein the bioreactor comprises: the reaction bag comprises a first space and a second space which are respectively used for containing gas and liquid, and a first inlet and a second inlet which are respectively used for introducing gas and liquid are also arranged in the reaction bag, and a working state monitoring module which is used for monitoring working state parameters of the bioreactor is also arranged in the reaction bag;

A sampling module 20 configured to introduce gas and/or liquid into the reaction bag through the first inlet and/or the second inlet for biological culture in response to a corresponding control signal issued by a user or the working state monitoring module;

the leakage monitoring module 30 is configured to monitor the pressure of the liquid in the second space to acquire the hydraulic pressure, and monitor the bottom of the reaction bag to acquire a leakage value;

a risk analysis module 40 configured to determine a risk status of the biological reaction system from the hydraulic pressure and the leakage value, the risk status comprising: a safe state, and/or a pending state, and/or a weeping state; wherein, the risk analysis module includes:

a hydraulic pressure analysis unit 40-1 configured to determine a depressurization risk of the reaction bag according to a variation parameter of the hydraulic pressure; wherein, the step-down risk is divided into respectively according to the numerical value of the change parameter: zero order, first order, and second order;

a leakage analysis unit 40-2 configured to determine a leakage risk of the reaction bag from the leakage value; wherein, the leakage risk is divided into according to the leakage value size respectively: zero order, first order, and second order;

A risk analysis unit 40-3 configured to determine the risk status from the depressurization risk and the weeping risk; when the depressurization risk is a second level and/or the leakage risk is a second level, the risk state is a leakage state; when the depressurization risk and the weeping risk are zero-order, the risk state is a safety state; when the depressurization risk is first-order and the leakage risk is zero-order, or when the depressurization risk is zero-order and the leakage risk is first-order, the risk state is a pending state.

In some embodiments, the sample addition module can be used for adding culture nodes by a user according to culture states such as turbidity and the like in a self-defined mode, so as to judge when culture is required to be stopped, when liquid addition is required to be automatically carried out, and then pH is automatically adjusted, different gases or oxygen are introduced.

In some embodiments, further comprising:

the early warning sending module 50 is configured to send corresponding early warning signals to users respectively when the risk state is detected to be a pending state or a liquid leakage state;

the early warning correction module 60 is configured to respond to the feedback signal sent by the user to perform secondary monitoring on the depressurization risk and/or the leakage risk of the biological reaction system so as to receive or correct the early warning signal.

In some embodiments, a condensate water guiding area is further formed in a top area of the first space, and the condensate water guiding area is used for guiding condensate water to be condensed in the guiding area, and flows back into the second space through a guiding path provided by the condensate water guiding area, and accordingly, the feedback signal includes: a first feedback signal for heating the condensed water guide area, and a second feedback signal for receiving the early warning signal, the early warning correction module 60 includes:

a secondary monitoring unit 60-1 configured to heat the condensed water guiding region in response to the first feedback signal when the reduced pressure risk is at a level one and the leakage risk is at a level zero;

and the correction unit 60-2 is used for correcting the risk state to be a safe state in response to a first correction signal sent by a user when the pressure reduction risk is monitored to be reduced to zero level during or after heating, and otherwise, correcting the risk state to be a liquid leakage state in response to a second correction signal sent by the user.

It should be noted that, the user in the present invention may be a computer connected to the semi-automatic control system, or may be a test operator.

It should be noted that the present invention can be applied to culture of highly pathogenic microorganisms (such as bacteria and viruses), but also to other microorganisms that may cause pollution or influence to the environment to different extents, or other microorganisms that may cause pathogenic effects or influence to human beings or animals and plants to different extents. Of course, the invention can also be applied to the cultivation of various biological cells, such as animal, plant cells or unicellular organisms.

From the above description of the embodiments, it will be clear to those skilled in the art that the above-described embodiment method may be implemented by means of software plus a necessary general hardware platform, but of course may also be implemented by means of hardware, but in many cases the former is a preferred embodiment. Based on such understanding, the technical solution of the present invention may be embodied essentially or in a part contributing to the prior art in the form of a software product stored in a storage medium (e.g. ROM/RAM, magnetic disk, optical disk) comprising several instructions for causing a computer terminal (which may be a mobile phone, a computer, a server, or a network device, etc.) to perform the method according to the embodiments of the present invention.

The embodiments of the present invention have been described above with reference to the accompanying drawings, but the present invention is not limited to the above-described embodiments, which are merely illustrative and not restrictive, and many forms may be made by those having ordinary skill in the art without departing from the spirit of the present invention and the scope of the claims, which are to be protected by the present invention.

Claims

1. A disposable biosafety bioreactor, comprising:

the reaction device comprises a reaction bag (1), wherein an inner accommodating cavity of the reaction bag (1) sequentially comprises a first space (13) and a second space (14); wherein, when the bioreactor is in a working state, the first space is used for containing gas, and the second space is used for containing liquid; wherein, the top area of the first space is also provided with a condensed water guiding area for guiding condensed water to be condensed in the condensed water guiding area and to flow back to the second space through a guiding path provided by the condensed water guiding area; a top wall surface (11) inside the condensed water guiding region is configured as an inclined surface, and an edge of the condensed water guiding region is provided with a ring-shaped guide rail (12) for providing a guiding path for condensed water to aggregate and reflux;

An operating state monitoring module (3) for monitoring operating state parameters of the bioreactor, the operating state parameters comprising: the liquid has hydraulic pressure, foam generation, dissolved oxygen rate and CO ₂ Concentration, pH, and turbidity; correspondingly, the working state monitoring module comprises: a first pressure monitoring unit for monitoring the hydraulic pressure, a foam monitoring unit for monitoring the foam generation condition, an oxygen dissolution rate monitoring unit for monitoring the oxygen dissolution rate, and a control unit for controlling the CO ₂ A concentration monitoring unit for monitoring the pH value, a pH monitoring unit for monitoring the pH value, and a turbidity monitoring unit for monitoring the turbidity; wherein the monitoring ends of the monitoring units are arranged on the wall surface of the second space;

the first space is also provided with at least one first inlet (4) for introducing first gas and at least one second inlet for introducing first liquid, and the first inlet and the second inlet are both arranged outside the condensed water guide area; wherein when the bioreactor is in a working state, the opening of the first inlet is higher than the second inlet; the lower end region of the second space is also provided with at least one first outlet (5) for liquid sampling;

The bioreactor further comprises: the first liquid leakage monitoring module is correspondingly arranged at the bottom of the reaction bag and used for monitoring whether the reaction bag leaks liquid or not; a safety sterilization module, the safety sterilization module comprising: a recovery unit connected to the reaction bag and a sterilization unit; the recovery unit includes: recovery bag, and be used for connecting the recovery bag with the recovery pipeline of reaction bag, be provided with the third control valve in the recovery pipeline, disinfection unit includes: a chamber for storing sterilizing substances, and a sterilizing pipeline for connecting the chamber and the reaction bag, wherein a fourth control valve is arranged in the sterilizing pipeline; and the third control valve and the fourth control valve are both in communication connection with the working state monitoring module and the first liquid leakage monitoring module.

2. The disposable biosafety type bioreactor as recited in claim 1, further comprising: a stirring device (2) comprising a stirring shaft (22) and a stirring paddle (21) arranged at the tail end of the stirring shaft; the central area of the condensed water guiding area is provided with a mounting hole for mounting the stirring device, the stirring shaft is fixedly mounted on the reaction bag through the mounting hole, the stirring paddle extends into the second space, and the monitoring end of the working state monitoring module is higher than the horizontal height of the stirring paddle; wherein the stirring shaft is provided with a heating unit (23) at one side of the stirring shaft, which is positioned at or near the condensed water guiding region, and is used for avoiding condensed water from forming at the outer side of the condensed water guiding region;

And/or the inclined plane has an inclination angle of 1-5 degrees.

3. A disposable biosafety type bioreactor according to claim 2, characterized in that an annular groove (12-1) for providing the guide path is provided in the annular guide rail (12), and a condensed water outlet is provided on the annular groove, and the bottom surface of the annular groove is inclined, so that the condensed water outlet is the lowest horizontal point of the annular groove, and condensed water in the annular groove is guided to flow out through the condensed water outlet; wherein the condensed water outlet is arranged along a direction away from the first inlet and the second inlet.

4. A disposable biosafety type bioreactor according to claim 1, wherein said first inlet is provided with a filter device comprising: a virus filtration device, and/or a bacteria filtration device.

5. A disposable biosafety bioreactor according to claim 1, characterized in that the first space is further provided with a second outlet for the exhaust gas, and that the second outlet is provided with drying means for filtering aerosols and/or sterile filtering means for filtering.

6. A method of monitoring a disposable biosafety bioreactor according to any one of claims 1-5, characterized in that the bioreactor comprises: the first pressure monitoring unit is used for monitoring the hydraulic pressure in the reaction bag, and the first liquid leakage monitoring module is correspondingly arranged at the bottom of the reaction bag, and the method comprises the following steps:

wherein S106 includes:

7. The monitoring method according to claim 6, wherein a condensed water guiding area is further formed at a top area of the first space in the reaction bag, for guiding condensed water to be condensed in the condensed water guiding area and to flow back into the second space through a guiding path provided by the condensed water guiding area; correspondingly, the method further comprises the steps of:

8. The method of claim 7, wherein the step S110 includes: