JP2019042615A - Mixer and mixing method - Google Patents

Abstract

To provide a mixer and a mixing method in which a change of a density state such as concentration arising when liquid is added, in liquid-liquid mixing, can be quickly solved with appropriate agitation force.SOLUTION: A mixer 1 comprises: a vessel 10 in which first liquid stored therein is mixed with second liquid added through an addition port; a rotary agitation device (20) which agitates the liquid inside the vessel 10; liquid volume measuring instruments (11 and 12) which measure a volume of the liquid stored in the vessel 10; a liquid quality sensor 60 which measures liquid quality in the vessel 10; and a control device 80. The control device 80 controls rotational speed of the rotary agitation device (20) to a rotation speed derived based on a preset relationship between a diffusion distribution on a liquid level and a rotational speed of the rotary agitation device (20) and on measurement using the liquid quality sensor 60, or to a rotation speed derived based on a preset relationship between a liquid level height and a rotational speed of the rotary agitation device (20) and on the liquid level height.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a mixing apparatus and a mixing method for adding and mixing a small volume of liquid to a large volume of liquid.

  In the fields of chemical industry, pharmaceutical industry, food industry, etc., operations for mixing liquids are performed for the purpose of adjusting the concentration. Such a target liquid-liquid mixing is generally performed by adding a small volume and high concentration additive liquid to a large volume main liquid. As a mixing system, there are a semi-batch system in which only the additive liquid is allowed to flow and the main liquid is not discharged, in addition to a continuous system and a batch system. The mixing operation is often performed by mechanical stirring, and a stirring tank equipped with a stirring blade is widely used.

  For example, in the field of biopharmaceutical production, liquids are mixed during fed-batch culture. Fed-batch culture is a culture method that is performed without adding a culture solution or cells while adding a fed solution containing a culture substrate to the culture solution. In fed-batch culture, a fed-batch solution having a relatively high concentration of medium components and the like is added to the culture solution held in the culture tank, and the culture conditions are determined by liquid-liquid mixing of the cultured solution and the fed-batch solution. It is kept.

  Conventionally, in fed-batch culture, a technique for reducing the influence on cells associated with the addition of a high-concentration fed-batch solution has been proposed. For example, Patent Literature 1 discloses a technique for spatially dividing a solution added to a culture solution into a plurality of pieces. In Patent Document 1, as a solution is added by a spray nozzle or added as a plurality of droplets, a local concentration change is quickly eliminated, and the possibility of cell death or damage is reduced. Yes.

JP 2009-089698 A

  Liquid-liquid mixing performed by adding a small volume and a high concentration additive to a large volume of the main liquid is performed in various steps in addition to fed-batch culture as described in Patent Document 1. ing. The additive solution added to the main solution may be affected by the concentration depending on the purpose of mixing, the type of the main solution, etc., even when cells are not present in the main solution. For example, in the manufacture of a biopharmaceutical, a solution may be inactivated by virus in the process of formulating a substance produced by cells such as an antibody drug.

  Virus inactivation treatment by acid treatment is performed by mixing an acidic solution with a solution containing an antibody or the like. And the solution after a process is mixed and neutralized with the alkaline solution. In these operations, addition of an acidic solution or an alkaline solution causes a local concentration change in the solution, so that antibodies, polymers, etc. contained in the solution are denatured or aggregated in a high concentration region, resulting in a product. The quality of the product may deteriorate.

  The influence of the concentration due to the addition of the additive solution can be reduced by appropriately stirring and eliminating the local concentration change quickly. However, as the stirring performance for that purpose, not only the viewpoint of stirring efficiency such as homogenization, stirring power, stirring time, etc. but also consideration of appropriate shearing force and discharge force is required. For example, when cells are present in the main liquid, the cells may be killed or damaged by stirring, and when antibodies, polymers, etc. are present, foaming or denaturation occurs due to stirring. There is a possibility. Therefore, it is desired to perform mixing at an appropriate stirring speed without excessive or insufficient so that excessive stirring force does not act.

  In particular, in recent years, single-use (disposable) containers have been used for cell culture. Single-use containers often have a structure in which a stirring blade is attached to the lower side of the container, and the flow rate tends to be slower toward the liquid surface side. Therefore, there is a demand for means for quickly eliminating a state change such as a concentration caused near the liquid surface by the addition of the additive liquid with an appropriate stirring force.

  Therefore, an object of the present invention is to provide a mixing apparatus and a mixing method capable of quickly eliminating state changes such as concentration when liquid is added during liquid-liquid mixing with an appropriate stirring force.

  In order to solve the above problems, the mixing apparatus according to the present invention has an addition port for adding a liquid at the top, and adds the second liquid from the addition port to the first liquid stored inside. A container to be mixed, a rotary stirrer that is disposed under the container and stirs the liquid in the container, a liquid amount measuring device that measures the amount of liquid stored in the container, and a liquid quality in the container A liquid quality sensor for measuring, and a control device for controlling the rotational speed of the rotary stirrer, wherein the control device determines the rotational speed of the rotary stirrer, the diffusion distribution on the liquid level in the container, and the rotational stirrer. The rotation speed is controlled based on a preset relationship with the rotation speed and the measurement by the liquid quality sensor. Or based on a preset relationship between the height of the liquid level in the container and the rotational speed of the rotary stirrer, and the height of the liquid level in the container determined by measurement by the liquid amount measuring device. To control the rotation speed.

  Further, the mixing method according to the present invention has an addition port for adding a liquid at the top, a container for adding and mixing the second liquid from the addition port to the first liquid stored inside, A rotary stirrer that is disposed in the lower part of the container and stirs the liquid in the container, a liquid amount measuring device that measures the amount of liquid stored in the container, and a liquid quality sensor that measures the liquid quality in the container In the mixing device, the relationship between the diffusion distribution on the liquid level in the container and the rotational speed of the rotary stirrer is set in advance by simulating the flow field and the diffusion field, While mixing the second liquid, the liquid quality in the container is measured, and the rotation speed of the rotary stirrer is set in advance between the diffusion distribution on the liquid level in the container and the rotation speed of the rotary stirrer. Based on the measured relationship and the measurement by the liquid quality sensor. Controlled to a rotational speed derived Te, for mixing while changing the rotational speed based on the measurement by the liquid electrolyte sensor. Alternatively, the relationship between the height of the liquid level in the container and the rotational speed of the rotary stirrer is set in advance by simulating the flow field and the diffusion field, and the second liquid is mixed with the first liquid. While measuring the height of the liquid level in the container, the rotational speed of the rotary stirrer, a preset relationship between the liquid level in the container and the rotational speed of the rotary stirrer, The rotational speed is controlled based on the liquid level in the container determined by the measurement by the liquid amount measuring device, and the rotational speed is changed based on the liquid level in the container. Mix while mixing.

  The mixing device and the mixing method according to the present invention can quickly eliminate a state change such as a concentration generated when a liquid is added during liquid-liquid mixing with an appropriate stirring force.

It is a schematic diagram which shows the structure of the mixing apparatus which concerns on embodiment of this invention. It is a flowchart which shows an example of the mixing method of the liquid performed using a mixing apparatus. It is a flowchart which shows the other example of the mixing method of the liquid performed using a mixing apparatus. It is a schematic diagram which shows the structure of the mixing apparatus which concerns on the modification of this invention. It is a schematic diagram which shows the structure of the mixing apparatus which concerns on 1st Example of this invention. It is a schematic diagram which shows the structure of the mixing apparatus which concerns on 2nd Example of this invention.

  Hereinafter, a mixing apparatus and a mixing method according to an embodiment of the present invention will be described with reference to the drawings. In addition, about the structure which is common in each following figure, the same code | symbol is attached | subjected and the overlapping description is abbreviate | omitted.

FIG. 1 is a schematic diagram illustrating a configuration of a mixing apparatus according to an embodiment of the present invention.
As shown in FIG. 1, the mixing apparatus 1 according to the present embodiment includes a mixing container (container) 10, a rotary stirrer having a stirring blade 20, a main liquid inflow line (first liquid inflow line) 110, and an additive liquid An inflow line (second liquid inflow line) 120, a mixed liquid outflow line 130, a main liquid flow rate sensor (liquid amount measuring device) 11, an additive liquid flow rate sensor (liquid amount measuring device) 12, a sterile connector 16, The valve 18, the additive solution container 30, the metering pump 40, the inflow portion 50, the liquid quality sensor 60, the analysis device 70, and the control device 80 are provided.

  The mixing device 1 is a device that mixes liquids by mechanical stirring. In FIG. 1, a small volume of additive liquid (second liquid) is added to a large volume of main liquid (first liquid) introduced and stored in the mixing container 10, and the main liquid and Liquid-liquid mixing with the additive liquid is performed. As the main liquid and additive liquid, various solutions, dispersion liquids, single component liquids and the like are used depending on the purpose of mixing.

  As will be described later, in the mixing apparatus 1, diffusion on the liquid surface in the mixing container 10 is determined by the rotation speed (rotational speed) of the stirring blade 20 (rotary stirrer) being an indicator of the mixing state of the liquid surface in the mixing container 10. It is controlled based on the distribution or the height of the liquid level in the mixing container 10.

  As shown in FIG. 1, the mixing container 10 has an addition port for adding a liquid at the top. In FIG. 1, the mixing container 10 has a cylindrical shape, and two addition ports are provided on the upper surface of the mixing container 10. A main liquid inflow line 110 for introducing the main liquid into the mixing container 10 is connected to one addition port. Further, an additive liquid inflow line 120 for adding the additive liquid to the main liquid in the mixing container 10 is connected to the other addition port (inflow part 50).

  A main liquid flow sensor 11 that measures the flow rate of the main liquid introduced into the mixing container 10 is installed in the main liquid inflow line 110. The amount of main liquid introduced and stored in the mixing container 10 is measured by the main liquid flow sensor 11 when introduced into the mixing container 10.

  Further, a sterile connector 16 is connected to the main liquid inflow line 110 between the mixing container 10 and the main liquid flow rate sensor 11. The aseptic connector 16 enables the pipes to be connected to each other while maintaining the aseptic condition in the mixing container 10. The main liquid inflow line 110 is formed by connecting pipes through the sterile connector 16.

  The additive liquid inflow line 120 connects the mixing container 10 and the additive liquid container 30 that stores the prepared additive liquid. The additive liquid inflow line 120 includes a valve 18 that opens and closes the additive liquid inflow line 120 in the vicinity of the inlet / outlet of the additive liquid container 30. The additive liquid inflow line 120 has an inflow portion 50 at the end on the mixing container 10 side. The inflow part 50 is located above the liquid level of the main liquid stored in the mixing container 10 and is open to face the liquid level.

  In the additive liquid inflow line 120, a metering pump 40 that introduces the additive liquid stored in the additive liquid container 30 into the mixing container 10, and an additive liquid flow rate sensor 12 that measures the flow rate of the additive liquid introduced into the mixing container 10. And are installed. The additive liquid is quantitatively added to the liquid in the mixing container 10 by the metering pump 40. Further, the addition amount of the additive liquid is measured by the additive liquid flow sensor 12 while being introduced into the mixing container 10.

  The aseptic connector 16 is connected to the additive solution inflow line 120 between the mixing container 10 and the additive solution flow rate sensor 12. The aseptic connector 16 enables the pipes to be connected to each other while maintaining the aseptic condition in the mixing container 10. The additive liquid inflow line 120 is formed by connecting pipes through the sterile connector 16.

  As shown in FIG. 1, the mixing container 10 has a discharge port in the lower part for discharging the mixed liquid obtained by mixing. A mixed liquid outflow line 130 for allowing the mixed liquid to flow out of the mixing container 10 is connected to the discharge port. A sterile connector 16 is connected to the mixed liquid outflow line 130. The mixed liquid outflow line 130 is formed by connecting pipes through the sterile connector 16.

  The rotary stirrer having the stirring blade 20 is disposed at the lower part of the mixing container 10. The stirring blade 20 is disposed at the center of the bottom of the mixing container 10, and mixing is performed by mechanical stirring by the rotation of the stirring blade 20. The motor (not shown) for driving the stirring blade 20 is variably controlled by the control device 80 for the rotation speed. The rotation speed of the stirring blade 20 is controlled to a continuous arbitrary value by inverter control, for example.

  The mixing container 10 is made of a synthetic resin and has flexibility that can be folded. The stirring blade 20, the main liquid inflow line 110, the additive liquid inflow line 120, and the mixed liquid outflow line 130 are made of synthetic resin. As the synthetic resin, for example, polycarbonate, polyethylene, polyvinylidene fluoride, or the like, or a material made of a combination thereof is used. By forming these members with inexpensive synthetic resin, it is possible to use them in a single-use (disposable) type.

  The liquid quality sensor 60 is installed below the liquid level on the inner wall of the mixing container 10. The liquid quality sensor 60 measures the state of liquid in the mixing container 10 (liquid quality), that is, the concentration of solute, dispersoid, chemical species, etc., and the state quantity such as temperature. The liquid quality sensor 60 is composed of various sensors that measure the pH, dissolved oxygen concentration, carbon dioxide concentration, organic substance concentration, redox potential, turbidity, temperature, etc. of the liquid according to the purpose of mixing.

  Specific examples of the liquid quality sensor 60 include a pH sensor, a dissolved oxygen sensor, a carbon dioxide gas sensor, an organic carbon sensor, a biosensor, an ion sensor, a redox potential sensor, an absorbance sensor, a turbidity sensor, and a temperature sensor. It can be used according to the purpose of mixing. As the liquid quality sensor 60, one machine may be provided, or a plurality of machines may be provided. Also, a plurality of types that measure different liquid concentrations and state quantities may be used in combination.

  When a plurality of liquid quality sensors 60 are provided in the mixing container 10, each is preferably disposed at two or more locations in the mixing container 10, and disposed at different positions in the height direction of the mixing container 10. Is more preferable. If a plurality of liquid quality sensors 60 are arranged at different positions, the liquid quality (concentration, etc.) of the liquid in the mixing container 10 can be measured at multiple points. Therefore, as will be described later, the mixed state (diffusion distribution) necessary for controlling the rotation speed of the stirring blade 20 can be accurately estimated from the analysis of the flow field and the diffusion field.

  The analysis device 70 analyzes the mixed state of the liquid in the mixing container 10. The analysis device 70 has a function of performing simulation by computational fluid dynamics (CFD) analysis, for example, and simulating the flow field and diffusion field in the mixing container 10. For example, the flow field and the diffusion field in the mixing container 10 in which a predetermined amount of main liquid is stored are obtained by steady analysis using an appropriate coordinate system such as rotational coordinates and stationary coordinates. Then, a two-dimensional or three-dimensional spatial distribution indicating a distribution of flow velocity, concentration, temperature, etc., which is used for determining the rotational speed of the stirring blade 20 by performing unsteady analysis with the flow field and diffusion field by the steady analysis as initial values. A (diffusion distribution) model is obtained as a distribution that varies along the time axis.

  The analysis device 70 can include commercially available thermal fluid analysis software such as “ANSYS FULL” (manufactured by ANSYS) for CFD analysis. CFD analysis can be performed by connecting a transport equation of a chemical species to a Navier-Stokes equation or the like as a basic equation. As the boundary condition, an appropriate condition is added depending on which mixing method is adopted among the continuous method, the batch method, and the semi-batch method. For example, the inflow condition of the additive liquid can be added by measuring in advance the size, shape, distribution, etc. of the droplet of the additive liquid added from the inflow part 50.

  The CFD analysis is performed in addition to the shape and size of the mixing vessel 10, the temperature and pressure of the main liquid and the additive liquid, the viscosity of the solvent, the solute, etc., the thermal conductivity, the heat capacity, the material diffusion coefficient, and the stirring blade 20 The shape, dimensions, installation position, number of steps, etc. may be set. The flow field and diffusion field in the mixing vessel 10 are analyzed in advance for each rotation speed of the stirring blade 20. The numerical information of the spatial distribution (diffusion distribution) model that changes along the time axis such as the flow velocity, concentration, and temperature obtained as a result is stored in the analysis device 70 in advance.

  The analysis device 70 has a function of estimating the mixing state (diffusion distribution) on the liquid surface in the mixing container 10 by using the result of simulating the flow field and diffusion field in the mixing container 10. For example, using a spatial distribution (diffusion distribution) model obtained by analyzing in advance, an estimated value on the liquid level when the liquid state (concentration, etc.) at the measurement position where the liquid quality sensor 60 is installed is a predetermined value. Or the estimated value in the liquid level when the height of the liquid level in the mixing container 10 is a predetermined value is extracted.

  The analysis device 70 has information for controlling the rotational speed of the stirring blade 20 corresponding to the estimated diffusion distribution (estimated value) at the liquid level in the mixing container 10 and the height of the liquid level in the mixing container 10. Stored in advance. For example, the control information in which the diffusion distribution on the liquid level in the mixing vessel 10 and the minimum rotation speed of the stirring blade 20 necessary for mixing the liquid level in the mixing vessel 10 are linked, or the liquid level in the mixing vessel 10 And control information in which the height of the mixing blade 10 and the minimum rotation speed of the stirring blade 20 necessary for mixing the liquid level in the mixing container 10 are linked.

  The analysis device 70 refers to the control information stored in advance, and obtains the mixing state (diffusion distribution) on the liquid surface in the mixing container 10 and the liquid surface in the mixing container 10 which are obtained by measurement at a predetermined time interval. And a function for determining a new control target of the rotational speed of the stirring blade 20 based on the height of the stirring blade 20. A new control target signal of the rotational speed determined by the analysis device 70 is transmitted to the control device 80.

  The control device 80 has a function of controlling the rotation speed of the stirring blade 20 (rotary stirrer), the output of the metering pump 40, and the opening and closing of the valve 18. The output of the metering pump 40 is controlled by proportional control, PI control, PID control or the like based on the measurement by the liquid quality sensor 60. The rotation speed of the stirring blade 20 is controlled to a predetermined basic rotation speed according to the purpose of mixing at the start of mixing. During mixing, the rotational speed determined by the analysis device 70 is changed to a new control target (new target value).

  In the mixing container 10 as shown, it is difficult to directly measure the state (concentration, etc.) of the liquid level in the mixing container 10 by the liquid quality sensor 60 due to the structure of the container. However, by analyzing the flow field and the diffusion field in the mixing container 10 in a simulated manner, the mixing state (diffusion on the liquid surface in the mixing container 10 is determined from the measurement value at the measurement position where the liquid quality sensor 60 is installed. Distribution) can be estimated.

  For example, on the spatial distribution (diffusion distribution) model obtained by analyzing in advance, if the state (concentration, etc.) of a certain calculation grid matches the measured value, the state (concentration, etc.) of the other calculation grid is It can be estimated that it is equal to the measured value that should be measured at the position of the calculation grid. Therefore, the mixing state (diffusion distribution) on the liquid surface in the mixing container 10 can be estimated by calculation using the estimated value of the liquid surface state (concentration, etc.).

  The mixing state (diffusion distribution) on the liquid surface in the mixing container 10 can be quantified as, for example, the degree of mixing (Γ). By taking the additive liquid added to the main liquid as a concentration lump having a certain concentration, macro changes due to convective mixing, i.e. changes in the size and movement of the concentration lump, and micro changes due to diffusion mixing, i.e. from concentration lump. The degree of mixing (Γ) can be calculated by CFD analysis separately from the change due to the diffusion of.

The degree of mixing (Γ) can be obtained by performing particle tracing calculation based on a chemical species transport model in CFD analysis. The degree of mixing (Γ) is expressed by the following formula (I) as i: observation point number, V: volume, φ: passive scalar, superscript bar: volume weighted average value. As the degree of mixing (Γ), in the case where the dispersion value in the final complete mixing state can be obtained, a formula for calculating the degree of mixing of Lacey may be used.

  As a new rotation speed (new target value) of the stirring blade 20 determined based on the mixing state (diffusion distribution) on the liquid surface and the height of the liquid surface, for example, the liquid surface has a target degree of mixing (Γ Any rotation speed can be set as long as mixing of the liquid in the mixing container 10 proceeds, such as a minimum rotation speed that becomes) and a rotation speed at which the time variation of the liquid level mixing degree (Γ) becomes a predetermined value or less. it can. Since the calculated value of the degree of mixing (Γ) is “0” in the initial state and “1” in the complete mixing state, the rotation speed can be set by corresponding an intermediate value between 0 and 1. . Usually, the rotation speed is set to be higher as the liquid level is increased by the addition of the additive liquid. However, it is possible to freely determine when to set the rotation speed at which the complete mixing state is achieved in accordance with the purpose of mixing.

  The degree of mixing (Γ) used for setting may be a calculated value based on the entire calculation grid including the entire liquid level, or a calculated value based on a part of the calculation grid including the drop point of the additive liquid. Good. Further, the calculation may be performed from only the uppermost calculation grid corresponding to the liquid level, or may be performed from a plurality of calculation grids including the uppermost layer.

  Next, an example of a liquid mixing method performed using the mixing apparatus 1 will be specifically described.

  In the mixing device 1, the rotation speed of the stirring blade 20 (rotary stirrer) can be controlled using the mixing state (diffusion distribution) on the liquid surface in the mixing container 10 as an index. In this method, the rotational speed of the stirring blade 20 is determined based on the preset relationship between the diffusion distribution on the liquid level in the mixing vessel 10 and the rotational speed of the stirring blade 20 and the measurement by the liquid quality sensor 60. Control to led rotation speed.

FIG. 2 is a flowchart showing an example of a liquid mixing method performed using a mixing apparatus.
FIG. 2 illustrates a flow of obtaining a mixed liquid adjusted to a predetermined pH by mixing the main liquid and the additive liquid in the mixing container 10 including a pH sensor as the liquid quality sensor 60.

  In this method, the mixing apparatus 1 has a relationship between the diffusion distribution on the liquid level in the mixing vessel 10 and the rotational speed of the stirring blade 20 by analyzing the flow field and the diffusion field in the mixing vessel 10 in advance. Is set. For example, on the basis of a spatial distribution (diffusion distribution) model obtained as a result of simulation analysis of the flow field and diffusion field, the diffusion distribution on the liquid surface and the appropriate rotation speed of the stirring blade 20 are linked to each other for control purposes. It is preset as information.

  In this method, first, the amount of main liquid is measured, and a predetermined amount of main liquid is stored in the mixing container 10 (step S10). For example, the main liquid flow rate sensor 11 measures the flow rate of the main liquid introduced into the mixing container 10 while the main liquid is introduced through the main liquid inflow line 110. The amount of the main liquid introduced into the mixing container 10 is matched with the initial amount when the flow field and the diffusion field are simulated.

  Subsequently, the target pH (target value) of the liquid mixture to be obtained by mixing is set (step S11). The target pH is matched with the value in the final state when the flow field and the diffusion field are simulated.

  Subsequently, mixing is started in the mixing container 10 (step S12). Mixing in the mixing container 10 is performed while controlling the addition amount of the additive liquid by adjusting the flow rate or addition time and rest time (duty ratio). The rotation speed of the stirring blade 20 is controlled to a predetermined basic rotation speed at the start of stirring.

  Subsequently, while mixing is performed in the mixing container 10, the pH (measured value) of the liquid in the mixing container 10 is measured (step S13). The pH of the liquid is measured at an appropriate measurement interval by a pH sensor installed on the inner wall of the mixing container 10. A measurement signal of pH (measurement value) at a measurement position by the pH sensor is transmitted to the analysis device 70.

  Subsequently, the analyzing device 70 uses the stirring blade based on the preset relationship between the mixing state (diffusion distribution) at the liquid level in the mixing vessel 10 and the rotation speed of the stirring blade 20 and the measurement by the pH sensor. 20 new rotational speeds (new target values) are determined (step S14). The new rotation speed (new target value) is obtained from the measured pH (measurement value) from, for example, a table (control information) in which the diffusion distribution on the liquid surface and the appropriate rotation speed of the stirring blade 20 are linked. The rotation speed value corresponding to the estimated diffusion distribution is extracted and selected.

  Subsequently, based on the difference between the preset target pH (target value) of the liquid in the mixing container 10 and the measured value by the pH sensor, the amount of addition liquid added from the addition port of the mixing container 10 is determined. Determine (step S15). The addition amount of the addition liquid is set under the adjustment of the flow rate or the addition time and the rest time (duty ratio) so that the local concentration change in the mixing container 10 is reduced.

  Subsequently, the control device 80 measures the rotational speed of the stirring blade 20 with a preset relationship between the mixing state (diffusion distribution) on the liquid surface in the mixing vessel 10 and the rotational speed of the stirring blade 20 and a pH sensor. Then, the rotation speed (new target value) derived based on the above is controlled (step S16). The rotation speed of the stirring blade 20 is normally changed to a rotation speed higher than the basic rotation speed.

  Subsequently, the control device 80 controls the addition amount of the additive liquid added from the addition port of the mixing container 10 to the addition amount determined based on the measurement (step S17). The control device 80 changes the output of the metering pump 40 and performs proportional control, PI control, PID control, and the like.

  Subsequently, it is determined whether or not the difference between the preset target pH (target value) of the liquid mixture and the pH (measured value) measured by the pH sensor is within an allowable value range (step S18). .

  In step S18, if the difference between the target pH (target value) and the measured pH (measured value) is not within the allowable value range (step S18; No), the liquid mixture is not adjusted to the predetermined pH, so Is returned to step S13. Then, mixing of the main liquid and the additive liquid is continued at an appropriate measurement interval. If the process cannot be completed even after a predetermined number of repetitions, an alert is displayed and, depending on the case, the process is automatically stopped.

  On the other hand, when the difference between the target pH (target value) and the measured pH (measured value) is within the allowable range in step S18 (step S18; Yes), the mixed solution is adjusted to a predetermined pH. Therefore, the mixing of the liquid in the mixing container 10 is finished. While the operation of the stirring blade 20 is stopped, the introduction of the additive liquid into the mixing container 10 is stopped, and the valve 18 is closed. The above mixing method can be performed in the same manner when the concentration and state quantity of other components are used as the liquid quality instead of pH.

  According to the liquid mixing method performed using the mixing apparatus 1 described above, the rotational speed of the stirring blade 20 is changed using the mixing state (diffusion distribution) on the liquid surface in the mixing container 10 as an index. For this reason, when an additive liquid is added to the main liquid, a change in state such as concentration near the liquid surface can be quickly eliminated with an appropriate stirring force. In general, the stirring blade 20 is disposed at the lower part of the mixing container 10 and the stirring force tends to be weaker toward the liquid surface side, without directly measuring the liquid quality (concentration, etc.) of the liquid near the liquid surface, Mixing can be performed appropriately using the liquid quality sensor 60 installed at an arbitrary position.

  In addition, according to the liquid mixing method performed using the mixing device 1, it is possible to prevent the concentration lump of the high concentration additive liquid from continuing to contact the main liquid. Therefore, when cells are contained in the main solution, cells are prevented from being killed or damaged by a high concentration of addition solution, and when the main solution contains macromolecules such as proteins. In this case, the substance is prevented from being denatured or aggregated by a high concentration additive solution.

  In addition, according to the liquid mixing method performed using the mixing apparatus 1, it is possible to perform mixing while controlling to the minimum rotational speed necessary for mixing the liquid surface. Therefore, excessive stirring is not performed, and it is possible to prevent the solute, dispersoid, and the like contained in the main liquid and the additive liquid from being altered by the shearing force and impact force of the stirring blade 20. In addition, it is possible to prevent the liquid in the mixing container 10 from foaming and waste of the drive energy of the stirring blade 20.

  Next, another example of the liquid mixing method performed using the mixing apparatus 1 will be specifically described.

  In the mixing apparatus 1, the rotation speed of the stirring blade 20 (rotary stirrer) can be controlled using the height of the liquid level in the mixing container 10 as an index. In this method, the rotational speed of the stirring blade 20 is set to a predetermined relationship between the height of the liquid level in the mixing vessel 10 and the rotational speed of the stirring blade 20, the main liquid flow rate sensor (liquid amount measuring device) 11, And it controls to the rotational speed induced | guided | derived based on the height of the liquid level in the mixing container 10 calculated | required by the measurement by the addition liquid flow sensor (liquid quantity measuring device) 12. FIG.

FIG. 3 is a flowchart showing another example of the liquid mixing method performed using the mixing apparatus.
FIG. 3 illustrates a flow of obtaining a liquid mixture adjusted to a predetermined pH by mixing the main liquid and the additive liquid in the mixing container 10 including a pH sensor as the liquid quality sensor 60.

  In this method, in the mixing apparatus 1, the relationship between the height of the liquid level in the mixing container 10 and the rotation speed of the stirring blade 20 is set in advance by simulating the flow field and diffusion field in the mixing container 10. Is done. For example, on the basis of a spatial distribution (diffusion distribution) model obtained as a result of simulation analysis of the flow field and diffusion field, the height of the liquid level and an appropriate rotation speed of the stirring blade 20 are associated with each other. Is preset.

  In this method, similarly to the method shown in FIG. 2, first, the amount of the main liquid is measured, and a predetermined amount of the main liquid is stored in the mixing container 10 (step S20). And the target pH (target value) of the liquid mixture which is going to be obtained by mixing is set (step S21), and mixing is started in the mixing container 10 (step S22).

  Subsequently, while mixing is performed in the mixing container 10, the height of the liquid level in the mixing container 10 and the pH (measured value) of the liquid in the mixing container 10 are measured (step S23). The height of the liquid level is obtained at appropriate time intervals by measurement using the main liquid flow sensor 11 and measurement using the additive liquid flow sensor 12. For example, measurement signals from the main liquid flow sensor 11 and the additive liquid flow sensor 12 are transmitted to the analysis device 70. The pH of the liquid is measured at appropriate measurement intervals by a pH sensor installed on the inner wall of the mixing container 10. A measurement signal of pH (measurement value) at a measurement position by the pH sensor is transmitted to the analysis device 70.

  Subsequently, the analysis device 70 determines a preset relationship between the height of the liquid level in the mixing container 10 and the rotation speed of the stirring blade 20, measurement by the main liquid flow sensor 11, and measurement by the additive liquid flow sensor 12. The new rotation speed (new target value) of the stirring blade 20 is determined based on the height of the liquid level in the mixing container 10 determined by (Step S24). The new rotation speed (new target value) corresponds to the height of the liquid level measured from, for example, control information such as a table in which the liquid level and the appropriate rotation speed of the stirring blade 20 are linked. Selection is made by extracting the rotational speed value.

  Subsequently, based on the difference between the preset target pH (target value) of the liquid in the mixing container 10 and the measured value by the pH sensor, the amount of addition liquid added from the addition port of the mixing container 10 is determined. Determine (step S25). The addition amount of the addition liquid is set under the adjustment of the flow rate or the addition time and the rest time (duty ratio) so that the local concentration change in the mixing container 10 is reduced.

  Subsequently, the control device 80 sets the rotational speed of the stirring blade 20 to the preset relationship between the height of the liquid level in the mixing container 10 and the rotational speed of the stirring blade 20 and the height of the liquid level in the mixing container 10. Then, the rotation speed (new target value) derived based on the above is controlled (step S26). Note that the height of the liquid level is obtained by the analysis device 70 based on the measurement by the main liquid flow sensor 11 and the measurement by the additive liquid flow sensor 12. The rotation speed of the stirring blade 20 is normally changed to a rotation speed higher than the basic rotation speed.

  Then, the control apparatus 80 controls the addition amount of the addition liquid added from the addition port of the mixing container 10 to the addition amount determined based on measurement (step S27). The control device 80 changes the output of the metering pump 40 and performs proportional control, PI control, PID control, and the like.

  Subsequently, it is determined whether or not a difference between a preset target pH (target value) of the liquid mixture and a pH (measured value) measured by the pH sensor is within an allowable value range (step S28). .

  In step S28, if the difference between the target pH (target value) and the measured pH (measured value) is not within the allowable value range (step S28; No), the mixed liquid is not adjusted to the predetermined pH, so Is returned to step S23. Then, mixing of the main liquid and the additive liquid is continued at an appropriate measurement interval. If the process cannot be completed even after a predetermined number of repetitions, an alert is displayed and, depending on the case, automatically stops.

  On the other hand, in step S28, when the difference between the target pH (target value) and the measured pH (measured value) is within the allowable range (step S18; Yes), the analysis device 70 determines that the mixed solution has a predetermined pH. Therefore, the mixing of the liquid in the mixing container 10 is finished. While the operation of the stirring blade 20 is stopped, the introduction of the additive liquid into the mixing container 10 is stopped, and the valve 18 is closed. The above mixing method can be performed in the same manner when the concentration and state quantity of other components are used as the liquid quality instead of pH.

  According to the liquid mixing method performed using the mixing apparatus 1 described above, the rotational speed of the stirring blade 20 is changed using the height of the liquid level in the mixing container 10 as an index. For this reason, when an additive liquid is added to the main liquid, a change in state such as concentration near the liquid surface can be quickly eliminated with an appropriate stirring force. In general, the stirring blade 20 is disposed at the lower part of the mixing vessel 10 and the stirring force tends to be weaker toward the liquid surface side. However, even in a state where the addition amount of the additive liquid is accumulated and the liquid level is high. Can be mixed properly. Further, similar to the method shown in FIG. 2, it is possible to prevent alteration of the main liquid and solute, foaming, waste of driving energy, and the like.

  Next, the structure of the mixing apparatus according to the modification of the present invention will be described.

FIG. 4 is a schematic diagram showing a configuration of a mixing apparatus according to a modification of the present invention. Fig.4 (a) is a schematic diagram which shows the whole structure of the mixing apparatus which concerns on a modification, FIG.4 (b) is a schematic cross section of the spray type nozzle with which a mixing apparatus is equipped.
As shown to Fig.4 (a), the mixing apparatus (1) based on the said embodiment is mixing of the structure which adds an addition liquid (2nd liquid) to several places from several inflow parts (50a, 50b). It may be an apparatus (mixing apparatus 2 according to a modification).

  In the mixing apparatus 2 according to the modified example, three addition ports are provided on the upper surface of the mixing container 10A. The additive liquid inflow line 120 for adding the additive liquid (second liquid) to the liquid in the mixing container 10A is branched on the mixing container 10A side, and two inflow portions (50a, 50a, 50b). The two inflow portions (50a, 50b) are located at each of the two addition ports discretely arranged on the upper surface of the mixing container 10A, and open so as to face the liquid surface.

  In the mixing operation using the mixing device 2, the additive liquid to be mixed with the main liquid is added simultaneously from each of the two inflow portions (50a, 50b). In obtaining a mixed liquid adjusted to a predetermined concentration or the like, a required addition amount of a required addition amount can be spatially divided into a plurality and added to the main liquid.

  Moreover, as shown in FIG.4 (b), as for the mixing apparatus (1) which concerns on the said embodiment, and the mixing apparatus (2) which concerns on a modification, the addition liquid inflow line 120 is a mixing container (10,10A). You may equip the inflow part (50, 50a, 50b) of the side terminal with the spray type nozzle 50A.

  The spray type nozzle 50A is provided for spraying a liquid in a wide range as a mist or a fine droplet. The spray nozzle 50 </ b> A shown in FIG. 4B has a high-pressure air chamber 51 and a solution chamber 52. For example, when high-pressure air of 0.5 to 10 kPa is supplied to the high-pressure air chamber 51 and the additive solution is supplied to the solution chamber 52, the additive solution is made into a mist-like or fine droplet form, and a wide range of the liquid level of the main liquid. Can be sprayed on.

  According to the mixing device 2 and the spray type nozzle 50A, when a predetermined amount of additive liquid is introduced into the mixing container (10, 10A), the droplets of the additive liquid and the main liquid in the mixing container (10, 10A). The contact area with the liquid is increased. Further, since the droplets of the additive liquid enter the liquid surface in the mixing container (10, 10A) from spatially different positions, convective mixing by stirring proceeds. Therefore, the main liquid and a predetermined amount of the additive liquid can be mixed more rapidly, and the state change such as the concentration caused by the addition of the additive liquid can be eliminated more quickly.

  Next, the configuration of the mixing apparatus according to the embodiment of the present invention will be described.

FIG. 5 is a schematic diagram showing the configuration of the mixing apparatus according to the first embodiment of the present invention.
As shown in FIG. 5, the mixing apparatus (1) according to the above embodiment may constitute an apparatus for feeding and culturing cells (mixing apparatus 3 according to the first example).

  In the mixing apparatus 3 according to the first embodiment, a seed culture solution in which desired cells are seeded in the mixing container 10 is introduced, and a small volume of the culture medium stored in the mixing container 10 is flowed. Additives or alkaline solutions are added. The culture solution is fed-batch cultured under a predetermined culture condition while a fed-batch solution containing a medium component at a high concentration or an alkaline solution for keeping the pH of the culture solution near neutral is added.

  In the mixing device 3, a fed solution containing a high concentration medium component or the like is prepared in a fed solution container 30 a connected to the added solution inflow line 120. An alkaline solution for adjusting the pH of the culture solution is prepared in an alkaline solution container 30b connected to the additive solution inflow line 120. On the wall surface of the mixing container 10, a pH sensor 60a, a dissolved oxygen sensor 60b, a temperature sensor 60c, and a medium sensor 60d are provided as liquid quality sensors. In addition, an air diffuser 90 is installed inside the mixing container 10, and a mixed gas of air and carbon dioxide or the like is supplied from the air diffuser 100 to the air diffuser 90.

  The feed solution and the alkaline solution are controlled using the mixing state (diffusion distribution) at the liquid level in the mixing container 10 as an index (see FIG. 2), or controlled using the height of the liquid level in the mixing container 10 as an index. According to (see FIG. 3), it is added to the culture solution stored in the mixing container 10. For the fed-batch solution, the medium sensor 60d includes, for example, an organic carbon sensor, biosensor, ion sensor, oxidation-reduction potential sensor, and the like. The mixed state (diffusion distribution) in can be estimated and controlled. Note that the minimum rotation speed of the stirring blade 20 may be determined based on CFD analysis in consideration of interphase mass transfer of the gas supplied from the diffuser 100.

  Examples of the measurement target by the medium sensor 60d include medium components such as glutamine and glucose, components that inhibit the production of substances by cells as the concentration increases, substances that kill or damage cells, and cell metabolism Inhibiting substances, substances that alter the components in the culture solution, substances that generate by-products, and the like. The medium sensor 60d may measure the quality (concentration, etc.) of other additive liquids instead of the illustrated fed-batch liquid.

  The mixing device 3 can be used by being incorporated in a separation and purification system for separating and purifying a biological material. For example, for the purpose of producing an antibody drug, it can be used in a separation and purification system for separating and purifying an antibody from a culture solution in which antibody-producing cells are cultured. The culture solution in which the substance-producing cells are cultured flows out to the purification column through the mixed solution outflow line 130 to collect the purified substance.

  In this way, when a mixing device for feeding cells is configured, when a fed-batch solution or an alkaline solution is added to the culture solution, extreme changes in the concentration of medium components and pH changes that occur near the liquid level Can be quickly eliminated with a sufficient stirring force. In general, it is known that when a fed-batch solution containing a medium component at a high concentration is added, a region with a high nutrient concentration is locally formed, and the metabolic rate of the cells changes in that region. However, since such a region is eliminated by an appropriate stirring force, cells cultured in the mixing container 10 can be uniformly grown under an appropriate nutrient concentration to produce a substance with high efficiency. it can. In addition, it is possible to prevent cells and the like from aggregating due to the alkalinity of the culture solution and from modifying the three-dimensional structure and chemical structure of the target substance.

FIG. 6 is a schematic diagram showing the configuration of the mixing apparatus according to the second embodiment of the present invention.
As shown in FIG. 6, the mixing apparatus (1) according to the above-described embodiment is an apparatus for inactivating a virus-containing liquid (virus-containing liquid) (mixing apparatus according to the second example). 4) may be configured.

  In the mixing apparatus 4 according to the second embodiment, the virus-containing liquid is introduced into the mixing container 10, and a small-volume acidic solution is added to the large-capacity virus-containing liquid stored in the mixing container 10. The virus-containing solution is acidified to about pH 3 by adding a high-concentration acidic solution, and the contaminated virus is inactivated. Then, the inactivated liquid is neutralized by adding an alkaline solution, and then recovered as a processed liquid (virus inactivating liquid).

  In the mixing device 4, an acidic solution for inactivating viruses is prepared in an acidic solution container 30 c connected to the additive solution inflow line 120. Further, an alkaline solution for neutralizing the acidified virus-containing solution is prepared in an alkaline solution container 30 d connected to the additive solution inflow line 120. A pH sensor is provided as a liquid quality sensor 60 on the wall surface of the mixing container 10.

  The acidic solution and the alkaline solution are controlled using the mixing state (diffusion distribution) at the liquid level in the mixing container 10 as an index (see FIG. 2), or controlled using the liquid level in the mixing container 10 as an index ( According to FIG. 3), it is added to the virus-containing liquid stored in the mixing container 10, and the pH of the liquid in the mixing container 10 is adjusted with stirring.

  The mixing device 4 can be used by being incorporated in a separation and purification system for separating and purifying a biological material. For example, it can be used in a separation and purification system for the purpose of producing an antibody drug, in which an antibody is separated and purified from a culture solution in which antibody-producing cells are cultured. In such a process of separation and purification, if the virus is alive in the culture solution or in the cells, the virus-containing solution may be mixed with the product to impair the safety of the antibody drug. The virus is inactivated by holding for a period of time.

  In the separation and purification system, for example, the main liquid inflow line 110 is connected to a purification column that performs purification by affinity chromatography, cation exchange chromatography, anion exchange chromatography, or the like, and the culture solution in which the substance-producing cells are cultured is connected to the purification column. After purifying through, it can be introduced into the mixing vessel 10 and subjected to virus inactivation treatment. Then, the treated liquid (virus inactivating liquid) is caused to flow out through the mixed liquid outflow line 130 to another purification column or a product recovery container.

  In this way, when a mixing device for virus inactivation treatment is configured, when an acidic solution or alkaline solution is added to the virus-containing liquid, extreme pH changes that occur near the liquid surface can be quickly resolved with an appropriate stirring force. can do. For this reason, it is possible to prevent the antibody or the like contained in the virus-containing liquid from aggregating due to the acidification or alkalinization of the solution or from the modification of the three-dimensional structure or chemical structure. Moreover, since an acidic solution or an alkaline solution can be quickly mixed and processed in a short time, it is possible to avoid deterioration of antibodies and the like during the processing. That is, a biological substance or the like can be separated and purified with high productivity and recovered.

  As mentioned above, although embodiment and the modification of this invention were demonstrated, this invention is not limited to the said form, A various change is possible in the range which does not deviate from the meaning of this invention. For example, a part of a configuration of an embodiment or a modification is replaced with a configuration of another embodiment or a modification, or a part of a configuration of a certain embodiment or a modification is added to another embodiment or a modification. It is also possible to omit a part of the configuration of a certain embodiment or modification.

  For example, the mixing container (10, 10A) is configured to add an additive liquid through a single additive liquid inflow line 120, but a plurality of types of additive liquids can be supplied from a plurality of addition ports through a plurality of lines. You may be set as the structure to add. Moreover, although the said mixing container (10,10A) is made from the synthetic resin which has the flexibility which can be folded, it may be made from metals, such as stainless steel, and the rigidity of the grade which does not deform | transform easily. It may be made of a synthetic resin. The mixing container (10, 10A) may have another shape such as a polygonal shape. Further, the stirring blade 20 may have an appropriate shape such as a turbine blade, a paddle blade, a propeller blade, or a lattice blade, and the number of blade stages, the blade diameter, the blade height, and the like are set to appropriate conditions according to the container. be able to.

  In FIGS. 2 and 3, the measurement by the sensor (see step S13 and step S23) is performed during mixing, but may be performed before the start of mixing. That is, the stirring may be performed at a controlled rotational speed each time a predetermined addition amount of the addition liquid is added. Further, the rotational speed is determined (see Steps S14 and S24) and the addition amount of the additive solution (see Steps S15 and S25), the rotational speed is controlled (see Steps S16 and S26), and the additive solution is added. The control (see step S17 and step S27) may be switched in order.

1,2,3,4 Mixing device 10,10A Mixing container (container)
11 Main liquid flow rate sensor
12 Additive flow sensor (liquid measuring device)
16 Aseptic connector 18 Valve 20 Stirring blade (rotary stirrer)
30 Additive liquid container 40 Metering pump 50 Inflow part 60 Liquid quality sensor 70 Analysis device 80 Control device 110 Main liquid inflow line (first liquid inflow line)
120 Additive liquid inflow line (second liquid inflow line)
130 Mixture outflow line

Claims (12)

A container having an addition port for adding a liquid at the top, and adding and mixing the second liquid from the addition port to the first liquid stored inside;
A rotary stirrer disposed at a lower portion of the container and stirring the liquid in the container;
A liquid amount measuring device for measuring the amount of liquid stored in the container;
A liquid quality sensor for measuring the liquid quality in the container;
A control device for controlling the rotational speed of the rotary stirrer,
The control device derives the rotation speed of the rotary stirrer based on a preset relationship between the diffusion distribution on the liquid level in the container and the rotation speed of the rotary stirrer and measurement by the liquid quality sensor. Mixing device to control to the specified rotational speed. A container having an addition port for adding a liquid at the top, and adding and mixing the second liquid from the addition port to the first liquid stored inside;
A rotary stirrer disposed at a lower portion of the container and stirring the liquid in the container;
A liquid amount measuring device for measuring the amount of liquid stored in the container;
A liquid quality sensor for measuring the liquid quality in the container;
A control device for controlling the rotational speed of the rotary stirrer,
The control device is configured to determine the rotation speed of the rotary stirrer in the container determined by a preset relationship between the liquid level in the container and the rotation speed of the rotary stirrer and the measurement by the liquid amount measuring device. The mixing device that controls the rotational speed derived based on the height of the liquid level.   The mixing according to claim 1 or 2, wherein the addition amount of the second liquid is controlled based on a difference between a target value set in advance for the liquid quality in the container and a measured value by the liquid quality sensor. apparatus.   The mixing apparatus according to claim 1, wherein the liquid quality sensor measures pH, dissolved oxygen concentration, carbon dioxide concentration, organic substance concentration, oxidation-reduction potential, turbidity, or temperature. A plurality of liquid quality sensors;
The mixing device according to claim 1 or 2, wherein each of the plurality of liquid quality sensors is disposed at two or more locations in the container.   The mixing apparatus according to claim 1 or 2, wherein the container, the stirring blade of the rotary stirrer, and the pipe connected to the container are made of synthetic resin.   The mixing apparatus according to claim 1 or 2, wherein the container, the stirring blade of the rotary stirrer, and the pipe connected to the container are made of polycarbonate, polyethylene, polyvinylidene fluoride, or a combination thereof.   The mixing apparatus according to claim 1, wherein the pipe connected to the container includes a sterile connector.   The mixing apparatus according to claim 1, wherein the pipe connected to the addition port includes a spray nozzle at an end on the container side. The container has a plurality of the addition ports;
The mixing apparatus according to claim 1 or 2, wherein a branched pipe for adding the second liquid is connected to each of the plurality of addition ports. A container having an addition port for adding a liquid at the top, and adding and mixing the second liquid from the addition port to the first liquid stored inside;
A rotary stirrer disposed at a lower portion of the container and stirring the liquid in the container;
A liquid amount measuring device for measuring the amount of liquid stored in the container;
In a mixing device comprising a liquid quality sensor for measuring the liquid quality in the container,
Preliminarily set the relationship between the diffusion distribution on the liquid level in the vessel and the rotational speed of the rotary stirrer by analyzing the flow field and the diffusion field in a simulated manner,
While mixing the second liquid with the first liquid, measure the liquid quality in the container,
The rotational speed of the rotary stirrer is set to a rotational speed derived based on a preset relationship between the diffusion distribution on the liquid level in the container and the rotational speed of the rotary stirrer, and measurement by the liquid quality sensor. Control
A mixing method for performing mixing while changing the rotation speed based on measurement by the liquid quality sensor. A container having an addition port for adding a liquid at the top, and adding and mixing the second liquid from the addition port to the first liquid stored inside;
A rotary stirrer disposed at a lower portion of the container and stirring the liquid in the container;
A liquid amount measuring device for measuring the amount of liquid stored in the container;
In a mixing device comprising a liquid quality sensor for measuring the liquid quality in the container,
Preliminarily set the relationship between the height of the liquid level in the vessel and the rotational speed of the rotary stirrer by analyzing the flow field and diffusion field in a simulated manner,
While mixing the second liquid with the first liquid, measure the height of the liquid level in the container,
The rotational speed of the rotary stirrer is determined by a preset relationship between the height of the liquid level in the container and the rotational speed of the rotary stirrer, and the level of the liquid level in the container obtained by measurement by the liquid amount measuring device. And control to the rotation speed led based on
A mixing method in which mixing is performed while changing the rotation speed based on the height of the liquid level in the container.

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