Method for analyzing shear force of bioreactor and method for manufacturing bioreactor

Abstract

The present application relates to a kind of shear force analysis method of bioreactor and bioreactor manufacturing method.Shear force analysis method includes the following steps: calibration step, the operating state of bioreactor is simulated by CFD simulation, and the operating parameters when CFD simulation is calibrated by comparing the actual operating parameters of bioreactor;Simulation step, the shear force data of bioreactor is obtained by CFD simulation simulation;Data processing, shear force data is processed to obtain the curve graph of shear force with the change of calculation domain.The present application is processed by CFD simulation simulation data and draws curve graph, realizes the overall evaluation to the size and distribution of the shear force in reactor, and based on this, the bioreactor with smaller shear force is developed.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of bioreactor, in particular to a shear force analysis method of bioreactor and a bioreactor manufacturing method. BACKGROUND

[0002] In the field of bioreactor, the current method for analyzing the size of the reactor shear force is to use the tip speed or energy dissipation method.

[0003] The impeller tip speed, which calculates the impeller edge linear speed (v = πDN, D is the impeller diameter, and N is the rotation speed), is commonly used in stirred reactors.

[0004] The energy dissipation rate, which is calculated based on the input power (P = 2πN × torque) and the reactor volume (V): ε = P / (ρV). High ε value corresponds to high shear force, which needs to be combined with fluid viscosity (μ) to evaluate the Kolmogorov microscale (λ).

[0005] Both of the above methods are indirect calculations to evaluate the shear force, although they are widely used, but still have some defects. The method of evaluating the shear force by the impeller tip speed is simple and convenient to calculate, but the scope of application is narrow, and it has certain reference significance only on the same reactor or geometrically similar reactors. The tip speed is an indirect index for evaluating the shear force, and cannot directly reflect the size of the shear force. The method of evaluating the shear force by energy dissipation is related to the input power of stirring, which reflects the average energy dissipation, and cannot well evaluate the maximum shear force and the distribution mode of the shear force in the reactor.

[0006] The prior art cannot comprehensively evaluate the size and distribution of the shear force in the reactor, and cannot realize accurate quantitative evaluation of the shear force. In view of the above problems, no effective solution has been proposed. SUMMARY

[0007] Therefore, it is necessary to provide a shear force analysis method of bioreactor and a bioreactor manufacturing method in view of the above technical problems.

[0008] In a first aspect, the present application provides a shear force analysis method of bioreactor, comprising:

[0009] The calibration step calibrates the operating parameters of the CFD simulation by comparing the actual operating parameters of the bioreactor with the operating parameters of the CFD simulation;

[0010] The simulation step obtains the shear force data of the bioreactor by CFD simulation;

[0011] Data processing, processing the shear force data to obtain a curve of shear force varying with the calculation domain;

[0012] The processing of the shear force data comprises: selecting a proper number of bins to process the data by binning;

[0013] The calculation domain is the volume of fluid in the bioreactor or the area of the contact surface between the fluid and the bioreactor during CFD simulation.

[0014] In some embodiments, the processing of the shear force data comprises: generating a probability distribution function (PDF) and a cumulative probability function (CDF) from the CFD variable field, and obtaining the curve of shear force varying with the calculation domain according to the CDF;

[0015] The calculation method of the probability distribution function (PDF) and the cumulative probability function (CDF) is as follows:

[0016] Each grid cell i in the shear force data has a variable value φ i and a cell volume V i / a cell area A i , and the variable value φ i corresponding to the cell volume V i / the cell area A i is denoted as M i , and the total volume of the entire calculation domain is:

[0017] M total =∑ i M i

[0018] According to the target resolution, the data is binned into N intervals:

[0019] [b0,b1),[b1,b2),…,[b N-1 ,b N );

[0020] The calculation domain sum of each bin is:

[0021]

[0022] The discrete PDF can be obtained by normalizing each sum:

[0023]

[0024] The CDF can be obtained by adding each bin of each PDF to all the previous bins:

[0025]

[0026] The curve of shear force varying with the calculation domain is obtained according to the CDF.

[0027] In some embodiments, the number of bins is 90-110.

[0028] In a second aspect, the present application provides a bioreactor manufacturing method, comprising the following steps:

[0029] Step 1: according to the shear force analysis method of the bioreactor described above, the curve of shear force changing with the calculation domain is calculated and obtained;

[0030] Step 2: according to the curve of shear force changing with the calculation domain, the overall shear force distribution in the internal structure of the bioreactor is compared;

[0031] Step 3: according to the overall shear force distribution, the structure of the bioreactor is adjusted to obtain a bioreactor structure with lower shear force;

[0032] Step 4: according to the obtained bioreactor structure with lower shear force, the corresponding bioreactor is manufactured.

[0033] In some embodiments, the bioreactor structure with lower shear force comprises:

[0034] The ratio of the liquid level height of the maximum working volume to the inner diameter of the tank body is 1.6-2.1;

[0035] The stirring shaft is rotatably arranged in the tank body, and the axis of the stirring shaft is parallel or coincides with the axis of the tank body;

[0036] The upper impeller comprises an upper hub connected with the stirring shaft and three upper blades uniformly arranged along the circumference of the upper hub, and the ratio of the diameter of the upper impeller to the inner diameter of the tank body is 0.4-0.5; the installation angle of the upper blade is 45°-65°;

[0037] The baffle is arranged on the inner wall of the tank body, the extension direction of the baffle is the same as the axial direction of the tank body, and the number of baffles is 3 or 4;

[0038] The lower impeller comprises a lower hub connected with the stirring shaft and four lower blades symmetrically arranged on the lower hub, the ratio of the diameter of the lower impeller to the inner diameter of the tank body is 0.4-0.5, the distance between the lower impeller and the bottom of the tank body is 0.7-0.85 times the diameter of the lower blade, the distance between the lower impeller and the upper impeller is 1-2 times the diameter of the lower blade, and the installation angle of the lower blade is 30°-45°.

[0039] In some embodiments, the size and weight of the lower blade are greater than those of the upper blade; the lower blade is designed as a three-dimensional streamline in an arc shape; and the upper blade has a bending design.

[0040] In some embodiments, the ratio of the liquid level of the maximum working volume of the tank body to the inner diameter of the tank body is 2.1; the ratio of the diameter of the upper impeller to the inner diameter of the tank body is 0.45; the maximum working volume of the tank body is less than or equal to 3000L, and the number of baffles is 3; the maximum working volume of the tank body is greater than 3000L, and the number of baffles is 4.

[0041] In some embodiments, the distance between the lower impeller and the bottom of the tank body is 0.8 times the diameter of the lower blade; the distance between the lower impeller and the upper impeller is 1.58 times the diameter of the lower blade.

[0042] In some embodiments, the installation angle of the upper blade is 58°; the installation angle of the lower blade is 38°.

[0043] In a third aspect, the present application provides a bioreactor prepared by any of the above methods.

[0044] By implementing the above scheme of the present application, the following beneficial effects can be obtained:

[0045] 1. The shear force data of the bioreactor is obtained by CFD simulation, which is subjected to binning processing and plotted into a curve of shear force varying with the calculation domain, so that the size and distribution of the shear force in the reactor can be comprehensively evaluated, and tools can be provided for developing a bioreactor with lower shear force.

[0046] 2. According to the shear force analysis method, the structure of the bioreactor is adjusted to obtain a new bioreactor structure with lower shear force.

[0047] 3. The bioreactor prepared by the new structure realizes the improvement of cell culture density and yield. BRIEF DESCRIPTION OF DRAWINGS

[0048] Figure 1 Fig. 1 is a structural schematic diagram of the bioreactor of the present application;

[0049] Figure 2 Fig. 2 is a curve of shear force varying with the calculation domain obtained by the shear force analysis method of the bioreactor of the present application.

[0050] Figure 3 Fig. 3 is a data graph of CHO cell culture using the hydrofoil type bioreactor and the elephant ear type bioreactor of the preferred embodiments of the present application, respectively.

[0051] Figure 4 Fig. 4 is a comparison graph of cell production curves of CHO cell culture using the hydrofoil type bioreactor and the elephant ear type bioreactor of the preferred embodiments of the present application, respectively.

[0052] Figure 5 Cell viability plot for CHO cell culture using the preferred embodiment of the hydrofoil bioreactor versus the ear bioreactor.

[0053] Figure 6 Data plot for CHO cell culture using the preferred embodiment of the hydrofoil bioreactor versus the hydrofoil bioreactor of a different configuration.

[0054] Figure 7 Cell production plot for CHO cell culture using the preferred embodiment of the hydrofoil bioreactor versus the hydrofoil bioreactor of a different configuration.

[0055] Figure 8 Cell viability plot for CHO cell culture using the preferred embodiment of the hydrofoil bioreactor versus the hydrofoil bioreactor of a different configuration.

[0056] BRIEF DESCRIPTION OF DRAWINGS

[0057] Tank 1, agitator shaft 2, upper impeller 3, upper impeller hub 31, upper impeller blade 32, baffle 4, lower impeller 5, lower impeller hub 51, lower impeller blade 52. DETAILED DESCRIPTION

[0058] The technical solutions in the embodiments of the present application will be described clearly and completely below. Obviously, the described embodiments are only a part of the embodiments of the present application, but not all the embodiments. The following description of at least one exemplary embodiment is actually only illustrative, but not as any limitation on the present application and its application or use. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor are within the scope of protection of the present application.

[0059] In some embodiments of the present application, a shear force analysis method of a bioreactor is provided, comprising:

[0060] The calibration step calibrates the operating parameters of the CFD simulation to the actual operating parameters of the bioreactor by simulating the operating state of the bioreactor by CFD simulation;

[0061] Calibration is a key step of the shear force analysis method of the present application. In order to obtain accurate shear force data, torque and input power calibration and mixing time calibration need to be performed respectively. After these two steps of calibration, the CFD simulation can obtain accurate shear force data.

[0062] The simulation step obtains the shear force data of the bioreactor by CFD simulation.

[0063] Data processing, processing the shear force data to obtain a curve of shear force varying with the calculation domain;

[0064] In some embodiments, the curve of shear force varying with the calculation domain is obtained by binning the original data of the CFD simulation.

[0065] The data amount of the original data of the CFD simulation is too large to be directly processed by drawing software, and the application reduces the requirement for computer hardware resources and improves the processing efficiency by binning the data while ensuring that the data can be used for bioreactor optimization. In the application, the number of bins is preferably between 90 and 110.

[0066] In some embodiments, the processing method of the shear force data comprises:

[0067] The probability distribution function (PDF) and the cumulative probability function (CDF) are generated from the CFD variable field, and the construction of the PDF and the CDF can qualitatively and intuitively analyze and compare the distribution characteristics of the shear force.

[0068] There is a limited area Ω (volume or area, i.e. the calculation domain of the CFD), which is filled with a continuous scalar field φ (a), and a can represent a point in a three-dimensional space or a two-dimensional plane. The PDF p (φ) is constructed based on this. According to the PDF theory, p (φ) is defined by the following equation:

[0069]

[0070] Where p (φ) is the PDF, M Ω is the calculation domain, δ (x) represents the Dirac delta function, and φ (a) represents the continuous scalar field;

[0071] The CDF is obtained by integrating the PDF,

[0072]

[0073] Where C (φ) is the CDF.

[0074] In practical applications, CFD cannot handle continuous fields, but is based on discrete grids of finite volumes and areas, and the generated flow field data is also discrete, so an approximate method is needed to construct the discretized PDF (standardized histogram, the sum of the probabilities of each bin = 1) and CDF.

[0075] Each grid element i in the shear force data has a variable value φ i and a cell volume V i / a cell area A i , and the variable value φ iCorresponding unit volume V i / Unit area A i Denoted as M i The total volume of the entire calculation domain is:

[0076] M total =∑ i M i

[0077] According to the target resolution, the data is binned into N intervals:

[0078] [b0,b1),[b1,b2),…,[b N-1 ,b N );

[0079] The calculation domain sum of each bin is:

[0080]

[0081] The discrete PDF is obtained by normalizing each sum:

[0082]

[0083] The CDF is obtained by adding each bin of each PDF to all previous bins:

[0084]

[0085] According to the CDF, a curve of shear force change with calculation domain is obtained.

[0086] In the present application, usually multiple graphs are formed, including: volume as the ordinate, shear strain (unit 1 / s) as the abscissa; area as the ordinate, shear strain (unit 1 / s) as the abscissa; area as the ordinate, shear stress (unit Pa) as the abscissa; multiple graphs are compared with each other, and the shear force distribution of the bioreactor is more comprehensively understood.

[0087] In some other embodiments of the present application, a bioreactor manufacturing method is provided, comprising the following steps:

[0088] Step 1: according to the shear force analysis method of the bioreactor described above, a curve of shear force change with calculation domain is obtained by calculation;

[0089] Step 2: according to the curve of shear force change with calculation domain, the overall shear force distribution in the interior of bioreactors with different structures is compared;

[0090] Step 3: according to the overall shear force distribution, the structure of the bioreactor is adjusted to obtain a bioreactor structure with lower shear force;

[0091] Step 4: According to the obtained low shear bioreactor structure, the corresponding bioreactor is made.

[0092] According to the curve of the shear force varying with the calculation domain, the magnitude of the shear force at each position of different reactors can be easily compared, and the structure optimization of the ultra-low shear reactor is conveniently carried out. By adjusting the mechanical parameters such as the form of the reactor paddle, the installation position, the paddle diameter ratio, and the height diameter ratio, the magnitude of the shear force under the same input power is compared, and the bioreactor with lower shear force (hydrofoil type bioreactor) is developed.

[0093] As shown in Figure 1 , the low shear bioreactor (hydrofoil type bioreactor) structure of the present application comprises:

[0094] a tank body 1, a stirring shaft 2, an upper impeller 3, a baffle 4, and a lower impeller 5.

[0095] The ratio of the liquid level height of the maximum working volume of the tank body 1 to the inner diameter of the tank body is 1.6-2.1, and preferably 2.1 is adopted in the present embodiment.

[0096] The stirring shaft 2 is rotatably arranged in the tank body 1, and the axis of the stirring shaft 2 is parallel or coincides with the axis of the tank body 1.

[0097] The upper impeller 3 comprises an upper hub 31 connected with the stirring shaft 2 and three upper blades 32 uniformly arranged along the circumference of the upper hub 31, and the ratio of the diameter of the upper impeller 3 to the inner diameter of the tank body is 0.4-0.5, and preferably 0.45 is adopted in the present embodiment. The installation angle of the upper blade 32 is 45°-65°, and preferably 58° is adopted in the present embodiment.

[0098] The baffle 4 is arranged on the inner wall of the tank body 1, and the extension direction of the baffle 4 is the same as the axial direction of the tank body 1, which is vertically along the up-down direction in Figure 1 the present embodiment, and the number of the baffles 4 is 3 or 4. When the maximum working volume of the tank body 1 is less than or equal to 3000L, the number of the baffles 4 is 3; and when the maximum working volume of the tank body 1 is greater than 3000L, the number of the baffles 4 is 4.

[0099] The lower impeller 5 comprises a lower hub 51 connected with the stirring shaft 2 and four lower blades 52 symmetrically arranged on the lower hub 51. The ratio of the diameter of the lower impeller 5 to the inner diameter of the tank body is 0.4-0.5, preferably 0.45 in the embodiment. The distance between the lower impeller 5 and the bottom of the tank body 1 is 0.7-0.85 times the diameter of the lower blade 52, preferably 0.8 times in the embodiment. The distance between the lower impeller 5 and the upper impeller 3 is 1-2 times the diameter of the lower blade 52, preferably 1.58 times in the embodiment. The installation angle of the lower blade 52 is 30°-45°, preferably 38° in the embodiment.

[0100] Further, in the embodiment, the size and weight of the lower blade 52 are greater than those of the upper blade 32. In the embodiment, two different impellers are used for the upper and lower layers. The upper layer uses a smaller and lighter impeller to reduce the influence of the vibration of the stirring shaft 2 on the shear. The lower impeller 5 is larger and heavier to increase the stability.

[0101] Further, in the embodiment, the lower blade 52 is designed in a three-dimensional streamline shape with an arc. The size of the arc is designed according to the used rotating speed and the corresponding CFD simulated flow field flow rate.

[0102] The upper blade 32 has a bending design and is designed according to the used rotating speed and the corresponding CFD simulated flow field flow rate (satisfying that the discharge flow rate of the impeller is less than 0.3 m / s at the used rotating speed, and the maximum CFD simulated wall surface shear force of the blade tip is not more than 10 Pa. 3

[0103] Figure 2 A curve diagram of the shear force obtained by the shear force analysis method of the bioreactor according to the application varies with the calculation domain. As shown in Figure 2 , the abscissa is the shear force size, and the ordinate is the area percentage, which can comprehensively describe the shear force distribution of the whole reactor, including the distribution of the wall surface shear force (the ordinate is expressed by the area percentage). When the ordinate is the volume percentage, the shear force distribution in the volume space can be described.

[0104] According to Figure 2 , it can be known that the shear force generated by the bioreactor (hydrofoil type bioreactor) of the embodiment is significantly lower than that of the ear type bioreactor.

[0105] The same cell strain and culture medium are used for CHO cell culture in the ear type bioreactor and the bioreactor (hydrofoil type bioreactor) of the embodiment, respectively, and the experimental data are obtained as shown in Figures 3-5 .

[0106] ​According to the experimental data, the cell density of CHO cells cultured in the bioreactor (hydrofoil type bioreactor) of the present embodiment is significantly increased by at least 20% and the maximum cell density is increased by 30-40% compared with the cell density of CHO cells cultured in the bioreactor (ear type bioreactor). The product yield (yield = cell density x viability x culture volume) is also increased by more than 20%.

[0107] The same cell strain and culture medium were used to culture CHO cells in a bioreactor (non-hydrofoil type bioreactor) and the bioreactor (hydrofoil type bioreactor) of the present embodiment, respectively, to obtain the experimental data as shown in Table 2. Figures 6-8

[0108] According to the experimental data, the cell density of CHO cells cultured in the bioreactor (hydrofoil type bioreactor) of the present embodiment is significantly increased by at least 20% and the maximum cell density is increased by 30-40% compared with the cell density of CHO cells cultured in the bioreactor (ear type bioreactor). The product yield (yield = cell density x viability x culture volume) is also increased by more than 20%.

[0109] So far, the embodiments of the present application have been described in detail. In order to avoid obscuring the concept of the present application, some details known in the art are not described. Those skilled in the art can fully understand how to implement the technical solutions of the present application according to the above description.

[0110] Although some specific embodiments of the present application have been described in detail through examples, those skilled in the art should understand that the above examples are only for illustration and are not intended to limit the scope of the present application. Those skilled in the art should understand that the above embodiments can be modified or some technical features can be replaced equivalently without departing from the scope and spirit of the present application. The scope of the present application is defined by the appended claims.​

Claims

1. A method of shear analysis of a bioreactor, characterized by, The method comprises the following steps: a calibration step, in which the running state of the bioreactor is simulated by CFD simulation, and the actual running parameters of the bioreactor are compared with the running parameters in the CFD simulation to calibrate the running parameters; a simulation step, in which shear force data of the bioreactor are obtained by CFD simulation; data processing, in which the shear force data are processed to obtain a curve of the shear force varying with a calculation domain; the processing of the shear force data comprises: selecting a suitable number of bins to process the data; the calculation domain is the volume of the fluid in the bioreactor during the CFD simulation, or the area of the contact surface between the fluid and the bioreactor; the processing of the shear force data comprises: generating a probability distribution function (PDF) and a cumulative probability function (CDF) from the CFD variable field, and obtaining the curve of the shear force varying with the calculation domain according to the CDF; the calculation method of the PDF and the CDF is as follows: The variable value φ in each grid cell i in the shear force data i with the cell volume V i / the cell area A i The variable value φ i with the corresponding cell volume V i / the cell area A i is denoted M i The total mass of the entire calculation domain is then: M total =∑ i M i the data are divided into N intervals according to a target resolution: [b0,b1),[b1,b2),...,[b N-1 ,b N ) the calculation domain of each bin is as follows: the discrete PDF is obtained by standardizing each sum: the CDF is obtained by adding each bin of each PDF to all the previous bins: the curve of the shear force varying with the calculation domain is obtained according to the CDF.

2. The shear force analysis method of the bioreactor according to claim 1, characterized in that: the number of bins is 90-110.

3. A method for manufacturing a bioreactor, characterized in that, The method comprises the following steps: Step 1: obtaining the curve of the shear force varying with the calculation domain according to the shear force analysis method of the bioreactor according to any one of claims 1-2; Step 2: comparing the overall shear force distribution in the internal of bioreactors with different structures according to the curve of the shear force varying with the calculation domain; Step 3: adjusting the structure of the bioreactor according to the overall shear force distribution to obtain a bioreactor structure with lower shear force; Step 4: manufacturing a corresponding bioreactor according to the bioreactor structure with lower shear force.

4. The bioreactor manufacturing method according to claim 3, characterized in that: the bioreactor structure with lower shear force comprises: a tank body, the ratio of the liquid level height of the maximum working volume to the inner diameter of the tank body is 1.6-2.1; a stirring shaft, which is rotatably arranged in the tank body, and the axis of the stirring shaft is parallel to or coincides with the axis of the tank body; an upper impeller, which comprises an upper hub connected with the stirring shaft and three upper blades uniformly arranged along the circumference of the upper hub, the ratio of the diameter of the upper impeller to the inner diameter of the tank body is 0.4-0.5, and the installation angle of the upper blade is 45°-65°; a baffle, which is arranged on the inner wall of the tank body, the extension direction of the baffle is the same as the axial direction of the tank body, and the number of the baffles is 3 or 4. The lower impeller comprises a lower hub connected with the stirring shaft and four symmetrical lower blades arranged on the lower hub, the ratio of the diameter of the lower impeller to the inner diameter of the tank body is 0.4-0.5, the distance between the lower impeller and the bottom of the tank body is 0.7-0.85 times the diameter of the lower blade, the distance between the lower impeller and the upper impeller is 1-2 times the diameter of the lower blade, and the installation angle of the lower blade is 30°-45°.

5. The bioreactor manufacturing method of claim 4, wherein, the size and weight of the lower blade are greater than those of the upper blade; the lower blade is designed in a three-dimensional streamline shape; the upper blade is designed with a bending shape.

6. The bioreactor manufacturing method of claim 4, wherein, the ratio of the liquid level of the maximum working volume of the tank body to the inner diameter of the tank body is 2.1; the ratio of the diameter of the upper impeller to the inner diameter of the tank body is 0.45; when the maximum working volume of the tank body is less than or equal to 3000L, the number of baffles is 3; when the maximum working volume of the tank body is greater than 3000L, the number of baffles is 4.

7. The bioreactor manufacturing method of claim 4, wherein, the distance between the lower impeller and the bottom of the tank body is 0.8 times the diameter of the lower blade; the distance between the lower impeller and the upper impeller is 1.58 times the diameter of the lower blade.

8. The bioreactor manufacturing method of claim 4, wherein, the installation angle of the upper blade is 58°; and / or the installation angle of the lower blade is 38°.

9. A bioreactor prepared by any one of the methods of claims 3-8.

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