Method for establishing shrinking model for simulating uneven mixing of large tank
Through the dual reactor system and specific pH control method, the simulation problem of large tank mixing uneven is solved, and the accurate simulation of cell growth and product quality in small-scale reactors is achieved, which is suitable for prokaryotic and eukaryotic cell culture.
Patent Information
- Application Number
- CN202410031148.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-01-09
- Publication Date
- 2025-07-11
AI Technical Summary
The prior art cannot effectively simulate the uneven mixing of large tanks, resulting in abnormal phenomena such as local pH increase and osmotic pressure increase in cell culture medium in small-scale reactors. The existing shrinking model cannot simulate mixing time and mass transfer at the same time, affecting cell viability and metabolic performance.
Using a dual reactor system, the main reactor and the auxiliary reactor are connected through pipelines to control the flow rate to simulate the mixing time of the large tank, and the mixing unevenness of the large tank is simulated through pH control and stirring speed. The main reactor controls the upper limit of CO2, the auxiliary reactor controls the lower limit of alkali, and the additives are added in the auxiliary reactor to simulate local uneven phenomena.
It has achieved the simulation of uneven mixing of large tanks in small-scale reactors, simulated the cell microenvironment, and the cell growth and product quality are close to that of large tanks, which is suitable for industrial promotion.
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Figure CN120299502A_ABST
Abstract
Description
Technical Field
[0001] The present disclosure relates to the field of cell culture, and particularly to a method for establishing a reduced model simulating uneven mixing in a large-scale tank. Background Art
[0002] In the field of biopharmaceuticals, with the popularization and application of the QbD (Quality by Design) concept, it is crucial to establish a qualified reduced model simulating large-scale cell culture processes. In addition to being used in process characterization studies to determine the impact of parameters on CQAs (Critical Quality Attributes) to support the production of process validation batches, it has many other uses, such as closing process deviations in large-scale tank processes, conducting cell bank stability studies, screening and validating raw materials and excipients, and simulating process evaluations under GMP conditions. Currently, the scale of domestic commercial large-scale bioreactors ranges from pilot scales of 200L and 500L to production scales of 2000L, 6000L, and even 20000L, which are often hundreds or even thousands of times that of small tanks. With the increase in scale, the cell culture medium has a longer mixing time and poorer mass transfer effect, resulting in the additives such as sugar, alkali, and feeding being locally enriched first and then mixed uniformly throughout the whole, which will cause abnormal phenomena such as local pH increase and osmotic pressure increase in the cell liquid that first contacts the additives. Compared with small-scale bioreactors, there are lower cell peaks, more difficult-to-maintain cell viability, and poorer metabolic performance. Therefore, it is necessary to achieve a more consistent large-scale tank simulation in small tanks.
[0003] Currently, the general principle for establishing a reduced model is that process parameters independent of scale need to be consistent with the commercial large-scale tank scale (such as pH, DO (dissolved oxygen), T control); process parameters dependent on scale need to be reduced according to certain scaling principles (such as aeration, agitation). The prior art usually uses the principle of equal VVM (aeration volume per unit volume per unit time) for bottom aeration to set the aeration of the reduced model, and usually uses the principles of equal P / V (agitation power per unit volume), Kla (volume mass transfer coefficient), or agitation tip linear velocity (πND) for agitation to set the agitation speed of the reduced model. However, neither P / V, Kla, nor πND can simulate the mixing time of the large-scale tank. If the agitation is reduced to simulate the mixing time, although it can achieve mass transfer close to that of the large-scale tank, it will affect other momentum transfer and heat transfer, resulting in the mismatch of the reduced model. Therefore, it is urgent to establish a better reduced model simulating uneven mixing in a large-scale tank. Summary of the Invention
[0005] The purpose of the present disclosure is to establish a method for establishing a reduced model simulating uneven mixing in a large-scale tank to realize simulating the process of the large-scale tank with the reduced model.
[0006] The present disclosure provides a method for establishing a reduced model simulating uneven mixing in a large-scale tank, including the following steps:
[0007] (1) Detect the mixing time of the large tank;
[0008] (2) Set the parameters of the scaled - down model according to (1);
[0009] Among them, in (2), the scaled - down model includes a main reactor and an auxiliary reactor. The main reactor and the auxiliary reactor are connected by a pipeline. Control the liquid flow in the pipeline to make the inlet and outlet flow rates of the reactor equal, and set the flow rate as the ratio of the working volume of the auxiliary reactor to the mixing time of the large tank.
[0010] In some embodiments, both the main reactor and the auxiliary reactor in the present disclosure are equipped with pH, T, and DO controls. The pH control of the main reactor is only coupled with CO2 to control the upper pH limit, and the pH control of the auxiliary reactor is only coupled with alkali to control the lower pH limit; the bottom aeration and stirring speed settings of the main reactor and the auxiliary reactor are set after calculation according to the principles of being equal to the large - tank VVM, P / V, or Kla respectively.
[0011] In some embodiments, the mixing time of the large tank in the present disclosure is detected by the following methods: pH electrode method, conductivity method, optical method, temperature difference method, laser - induced fluorescence method, electrical resistance tomography method, liquid crystal temperature recording method, computer tomography method.
[0012] In some embodiments, additives are added to the auxiliary reactor in the present disclosure, and the additives are selected from feeding, amino acids, sugars, alkalis, and acids.
[0013] In some embodiments, the maximum capacities of the main reactor and the auxiliary reactor in the present disclosure are the same. Preferably, the maximum capacity is about 2L - 5L, and more preferably, the maximum capacity is about 3L.
[0014] In some embodiments, the working volume of the main reactor in the present disclosure is larger than the working volume of the auxiliary reactor.
[0015] In some embodiments, the working volume of the main reactor in the present disclosure is about 1.8L, and the working volume of the auxiliary reactor is about 0.8L.
[0016] In some embodiments, the method of the present disclosure is applicable to the cultivation of prokaryotic and eukaryotic cells.
[0017] The present disclosure also provides a scaled - down model for establishing uneven mixing in a large tank, which includes:
[0018] (1) A main reactor and an auxiliary reactor, both of which have an aeration device and a stirring device;
[0019] (2) A pipeline connecting the main reactor and the auxiliary reactor;
[0020] (3) A flow rate controller that controls the flow rate of the liquid in the pipeline.
[0021] In some embodiments, the flow rate controller in the scaled-down model of the present disclosure is a peristaltic pump.
[0022] In some embodiments, the pipeline in the scaled-down model of the present disclosure is a hollow silica gel tube.
[0023] In some embodiments, the number of pipelines connecting two reactors in the scaled-down model of the present disclosure is two.
[0024] In some embodiments, the scaled-down model of the present disclosure is applicable to prokaryotic and eukaryotic cell culture.
[0025] The method for establishing a scaled-down model that simulates the uneven mixing in a large tank of the present disclosure has the following beneficial effects:
[0026] Compared with the general scaled-down model with only one reactor, the scaled-down model established by the present disclosure has two reactors, and only couples the pH control of the main reactor with CO2 to control the upper pH limit, and only couples the pH control of the auxiliary reactor with alkali to control the lower pH limit. This makes the PCO2 of the main reactor closer to that of the large tank, and the local uneven effects caused by alkali supplementation and feeding in the auxiliary reactor can simulate the large tank. Therefore, it can simulate the phenomenon of abnormal increase in local osmotic pressure or pH caused by uneven mixing in the large tank to comprehensively simulate the cell microenvironment in the large tank. The cell growth and product quality after simulation are closer to those in the large tank, and the effect advantages are obvious. In addition, the method for establishing a scaled-down model that simulates the uneven mixing in a large tank of the present disclosure has a simple process, is easy to implement, and is suitable for industrial promotion. BRIEF DESCRIPTION OF THE DRAWINGS
[0027] Figure 1 Legend of the scaled-down model of the present disclosure.
[0028] Figure 2 Cell growth curve graph.
[0029] Figure 3 Product expression level comparison graph.
[0030] Figure 4 Product charge comparison graph. DETAILED DESCRIPTION OF THE EMBODIMENTS
[0031] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.
[0032] Before the present disclosure is described in detail below, it should be understood that the present disclosure is not limited to the specific methodologies, protocols, and reagents described herein, as these may vary. It should also be understood that the terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the scope of the present disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.
[0033] Certain embodiments disclosed herein include numerical ranges, and certain aspects of the present disclosure may be described in terms of ranges. Unless otherwise stated, it should be understood that a numerical range or a description in terms of a range is merely for the purpose of brevity and convenience and should not be considered a strict limitation on the scope of the present disclosure. Thus, a description in terms of a range should be considered to specifically disclose all possible sub-ranges and all possible specific numerical points within that range, as if these sub-ranges and numerical points were explicitly written herein. For example, a description of a range from 1 to 6 should be considered to specifically disclose sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., and the specific numerical points within these ranges, such as 1, 2, 3, 4, 5, 6. This principle applies equally regardless of the width of the stated numerical values. When a range is described, the range includes the endpoints of the range.
[0034] It must be noted that the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from "about" a particular value, and / or to "about" another particular value. When such a range is expressed, another embodiment includes from that particular value and / or to that other particular value. Similarly, when a value is expressed as an approximation by use of the antecedent "about", it will be understood that the particular value forms another embodiment. For example, a pH of about 7.2 may be a pH of 7.2. At the same time, the term "about" includes variations of ±20%, or in some cases ±10%, or in some cases ±5%, or in some cases ±1%, or in some cases ±0.1% of the particular value.
[0035] pH electrode method: Use a culture medium or replace it with PBS to run a large-scale bioreactor. Conduct aeration and stirring according to the process flow. There are multiple pH electrodes distributed at the bottom and top of the tank body, which can monitor the pH inside the large tank in real time. Control it according to the conventional culture process. Add acid solution (such as dilute hydrochloric acid solution) or alkali solution (such as 1M NaOH solution) from the feeding port. Record the initial pH as pH1. Observe the time from the addition point until the pH values of all pH electrodes no longer change, and record the pH at this time as pH2. The pH monitoring value during the process is pH t , when (pH tWhen the absolute values of (pH1 - pH) / (pH2 - pH1) are 0.95 and 1.05 respectively, the average value of the time used for the pH monitoring points in the two processes at this time is the mixing time of the large-scale bioreactor under these process conditions.
[0036] Unless otherwise required by the context, in this disclosure, the terms "large tank", "large-scale bioreactor", "large bioreactor", "large cell reactor" can be used interchangeably, and refer to a bioreactor for large-scale cell culture with a minimum capacity of 200 L. The terms "small tank", "small-scale bioreactor", "small bioreactor", "small cell reactor" can be used interchangeably, and refer to a bioreactor for small-scale cell culture with a maximum capacity of 20 L.
[0037] The technical solutions of the present invention will be further described below in conjunction with the embodiments. However, it should be understood that the embodiments are only used for more detailed and specific explanations, and should not be construed as limiting the present invention in any form. The present disclosure will be specifically described below through embodiments. It is necessary to point out here that the following embodiments are only used for further explanation of the present disclosure and cannot be understood as limiting the protection scope of the present disclosure. Some non-essential improvements and adjustments made by those skilled in the art according to the above-mentioned inventive content still fall within the protection scope of the present disclosure. For those conditions not specified in the embodiments, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. For the reagents or instruments not specified by the manufacturer, they are all conventional products that can be obtained through commercial purchase.
[0038] Example 1: Establishing a scaled-down model to simulate the effect evaluation of uneven mixing in a large tank
[0039] Three batches of 6000 L large tank cultures were used as the control group. Experimental group 1 was simulated using a general scale-down model, and experimental group 2 was simulated using the scale-down model of the present disclosure.
[0040] Experimental group 1: Before the start of the experiment, 1.8 L of cell culture solution was taken out from the large tank in parallel and pumped into a 3 L bioreactor. The pH control (7.05 ± 0.2), temperature control (35 °C), and DO control (50%) were turned on. The aeration was converted according to VVM (the bottom aeration volume was 18 ml / min), and the stirring was converted according to the principle of equal P / V (the 1.8 L working volume was equipped with double stirring paddles, and the stirring speed was 300 rpm). The culture was carried out according to the culture process (the feeding strategy in this experiment was to feed every day from D3 to D15, and a total of 20 - 30% of the initial working volume was fed. The sugar was supplemented to maintain the sugar content in the culture solution at 1 - 8 g / L). During the culture period, aseptic samples were taken and detected once a day or every other day, and the culture ended on the 16th day.
[0041] Experimental group 2: A scale-down model simulating the uneven mixing in the large tank was established by the method of the present disclosure, specifically as follows:
[0042] 1) Detection of the mixing time in the large tank: Simulate the actual production process of 6000L. A total of 6 calibrated pH electrodes are distributed at different positions at the bottom and top of a 6000L large-scale bioreactor. Add 4750 kg of culture medium solution, turn on the temperature control at 35°C, turn on the stirring at 30 rpm, ventilate at the bottom at 48 slpm, and the tank pressure is 0.2 bar. After the pH electrodes are stable, record the pH reading as pH1 = 7.9 (all 6 pH electrodes are between 7.88 and 7.95, and are uniformly compensated and corrected to 7.90). Add 10 L of 1 mol / L NaOH solution from the feeding port, and monitor the pH (pHt) during the process. Record the change of pH with time by online data or video. When all pH electrodes reach a stable state, record the pH value at this time as 9.36. According to the formula 0.95 = |(pH t1 - 7.9) / (9.36 - 7.9)|, the pH t1 is 9.29. According to the formula 1.05 = |(pH t2 - 7.9) / (9.36 - 7.9)|, the pH t2 is 9.43. According to the recorded pH change curve with time, find the corresponding recorded average times for 9.29 and 9.43, which are 23.3 s and 26.9 s respectively. Calculate the average value to obtain the mixing time of the 6000L large-scale bioreactor under the process parameter conditions of this project as 25.1 s.
[0043] 2) Set the parameters of the scaled-down model: Use 2 bioreactors with a capacity of 3L to culture cells. The working volumes are 1.8L and 0.8L respectively. Each has two pipelines leading to below the liquid level, and the flow rates of the pipelines are controlled by two peristaltic pumps (see the example diagram in Figure 1) Before the experiment, 2.6 L of cell culture medium was taken from the large tank running in parallel, and 1.8 L and 0.8 L were respectively injected into two 3 L bioreactors. pH control was started (7.05 ± 0.2, where the pH of the 1.8 L working volume tank was controlled only by CO2 for the upper limit, and the pH of the 0.8 L working volume tank was controlled only by coupling with an alkali pump for the lower limit), temperature control (35 °C), DO control (50%), aeration was converted according to VVM (the bottom aeration volume of the 1.8 L working volume was 18 ml / min, and the bottom aeration volume of the 0.8 L working volume was 8 ml / min), and stirring was converted according to the principle of equal P / V (the 1.8 L working volume had a double stirrer paddle with a stirring speed of 300 rpm; the 0.8 L working volume had a single stirrer paddle with a stirring speed of 280 rpm). After the pH and T were stable, the peristaltic pump was started to make the inflow and outflow equal, which was 0.8 L / 25.1 s. Cultivation was carried out according to the cultivation process (the feeding strategy was to feed every day from D3 to D15, and a total of 20 - 30% of the initial working volume was fed. Sugar was supplemented to maintain the sugar content in the culture medium at 1 - 8 g / L), and the additives (feed, sugar, alkali) during the cultivation process were added to the 0.8 L working volume reactor. Samples were aseptically taken and detected from the main reactor once a day or every other day during the cultivation to simulate the uneven mixing in the large tank, and the cultivation ended on the 16th day.
[0044] Detection equipment: A Count star cell counter was used for cell number and viability detection, an osmometer was used for osmotic pressure detection, a Cedex biochemical analyzer was used for sugar concentration detection, high-performance liquid chromatography was used for expression level detection, and capillary isoelectric focusing electrophoresis was used for charge distribution detection in the product quality.
[0045] By comparing the simulation of the cell growth curve in the large tank by the reduced model of the present disclosure and the general reduced model through the above scheme, the results are shown in Figure 2 , It can be seen from the results that the cell growth curve of the reduced model of the present disclosure is within the range of the cell growth curves of 3 batches of large tanks, while the cell growth peak value in the general reduced model is significantly higher than the cell growth peak values in the 3 batches of large tanks.
[0046] Furthermore, in terms of product expression, through Figure 2 It can be seen that the product expression level of the reduced model of the present disclosure (1845 mg / ml) is also within the range of the product expression levels of 3 batches of large tanks (1810 - 1990 mg / ml), while the product expression level of the general reduced model (2276 mg / ml) is significantly higher than the product expression level of the large tank, and the simulation results have a large deviation.
[0047] Finally, the key index of product quality - charge in each group was detected by ICIEF, and the results are shown in Figure 3, the main charge peak of the general scaled-down model (67.1%) is significantly lower than the main charge peak range of the large tank product (80.10% - 82.30%), while the main charge peak of the product in the scaled-down model of the present disclosure (81.4%) is within the main charge peak range of the large tank product, indicating that the product quality of the scaled-down model of the present disclosure is very close to that of the product in the large tank, and the effect is significantly better than that of the general scaled-down model.
[0048] The embodiments of the present disclosure described above are merely exemplary, and any person skilled in the art can recognize or determine countless equivalents of specific compounds, materials, and operations without performing tests beyond the routine. All such equivalents are within the scope of the present disclosure and are encompassed by the claims.
Claims
1. A method for establishing a scaled - down model simulating uneven mixing in a large tank, comprising the following steps: (1) Detect the mixing time of the large tank; (2) Set the parameters of the scaled - down model according to (1); Among them, In (2), the scaled - down model includes a main reactor and an auxiliary reactor, which are connected by a pipeline. Control the liquid flow in the pipeline to make the inlet and outlet flow rates of the reactor equal, and set the flow rate as the ratio of the working volume of the auxiliary reactor to the mixing time of the large tank.
2. The method according to claim 1, wherein both the main reactor and the auxiliary reactor are equipped with pH, T, and DO controls. The pH control of the main reactor is only coupled with CO2 to control the pH upper limit, and the pH control of the auxiliary reactor is only coupled with alkali to control the pH lower limit; the bottom aeration and stirring speed settings of the main reactor and the auxiliary reactor are calculated and set according to the principles of being equal to the VVM, P / V, or Kla of the large tank respectively.
3. The method according to claim 1, wherein the mixing time of the large tank is detected by the following methods: pH electrode method, conductivity method, optical method, temperature difference method, laser - induced fluorescence method, electrical resistance tomography method, liquid crystal temperature recording method, computer tomography method.
4. The method according to claim 1, wherein additives are added to the auxiliary reactor, and the additives are selected from feeding materials, amino acids, sugars, alkalis, and acids.
5. The method according to claim 1, wherein the maximum capacities of the main reactor and the auxiliary reactor are the same. Preferably, the maximum capacity is about 2L - 5L, more preferably, the maximum capacity is about 3L. Preferably, the working volume of the main reactor is greater than the working volume of the auxiliary reactor. More preferably, the working volume of the main reactor is about 1.8L, and the working volume of the auxiliary reactor is about 0.8L.
6. The method according to any one of claims 1 - 5, wherein the method is applicable to the cultivation of prokaryotic and eukaryotic cells.
7. A scaled - down model for simulating uneven mixing in a large tank, comprising: (1) A main reactor and an auxiliary reactor, which are equipped with an aeration device and a stirring device; (2) A pipeline connecting the main reactor and the auxiliary reactor; (3) A flow rate controller, which controls the liquid flow rate in the pipeline.
8. The scaled - down model according to claim 7, wherein the maximum capacities of the main reactor and the auxiliary reactor are the same. Preferably, the maximum capacity is about 2L - 5L, more preferably, the maximum capacity is about 3L. Preferably, the working volume of the main reactor is greater than the working volume of the auxiliary reactor. More preferably, the working volume of the main reactor is about 1.8L, and the working volume of the auxiliary reactor is about 0.8L.
9. The scaled - down model according to claim 7, wherein the flow rate controller is a peristaltic pump, and the pipeline is a hollow silica gel tube. Preferably, the number of pipelines is two.
10. The scaled - down model according to any one of claims 7 - 9, wherein the scaled - down model is applicable to the cultivation of prokaryotic and eukaryotic cells.