Stirring type bioreactor for cell culture in small-scale test stage

Abstract

The utility model discloses a stirring type bioreactor for cell culture in a lab-scale test stage, which comprises a reaction tank, a stirring component and a ventilation pipeline, the stirring component is mounted on a top cover of the reaction tank and comprises a paddle, and the paddle is mounted on the top cover of the reaction tank. The ventilation pipeline penetrates through and is fixedly mounted on the top cover of the reactor, extends into the reaction tank and is used for introducing gas into the culture solution, the ventilation pipeline comprises a vertical section and a horizontal section, the horizontal section is positioned between the paddle and the bottom surface of the reaction tank, and a ventilation hole is formed in the horizontal section. According to the stirring type bioreactor for cell culture in the small-scale test stage, the horizontal section of the air inlet pipe is arranged between the paddle and the bottom surface of the reaction tank, the ventilation hole is formed in the horizontal section, preferably, the number of the ventilation hole is one, and the ventilation hole is located in the middle of the length direction of the horizontal section, so that the GEV condition which is comparable to or higher than that of a large scale is achieved; risks and negative effects possibly brought by a high GEV level can be recognized in advance in the early research and development stage, and important support is provided for optimization of the cell culture process.

Description

Technical Field

[0001] This utility model relates to the field of cell culture technology, and in particular to a stirred bioreactor for cell culture in the pilot stage. Background Technology

[0002] Antibody drugs occupy an important position in biopharmaceutical manufacturing and are gradually becoming a major direction for development in the biopharmaceutical field. Among them, stirred bioreactors, as the main cell culture system in small-scale, pilot-scale, and large-scale cell culture processes, play a crucial role in the monoclonal antibody production industry. Stirred bioreactors (STRs, for example) use gas distributors for bubbling oxygen supply to ensure that cells can take up sufficient oxygen and nutrients for respiration, growth, metabolism, and other vital activities. Cell damage due to shear forces during cell culture in the reactor is a long-standing problem, generally considered to have three main sources: high energy dissipation in the direct impeller zone; bubble rupture at the liquid surface and headspace interface; and high gas entrance velocity (GEV) at the distributor outlet. For CHO cells, the first two difficulties have been overcome and successfully scaled up for CHO cell culture, mainly because CHO cells can withstand high stirring speeds and shear protectants such as Pluronic F68 can prevent cell damage caused by bubble rupture. However, for the third source, the design of the gas distributor, GEV remains a potential scale-up problem.

[0003] 1. Previous studies have reported little on the effects of high GEV on cell culture, which may have led to insufficient consideration of the negative impact of this factor in the design and operation of bioreactors.

[0004] 2. Effectively reducing high GEVs to prevent cell damage during the transition from small-scale to large-scale production is a challenge. Rapidly addressing the effects of high GEVs in large-scale production is difficult, often requiring the development of new distributors with GEV-reducing capabilities. Furthermore, modifying hardware structures and developing scaled-down models to simulate the effects of high GEVs are ongoing research and exploration.

[0005] Furthermore, based on traditional experience in reactor scale-up, even when strictly adhering to equal P / V scaling-up, the GEV value is difficult to achieve consistently high levels by simply changing process parameters due to inherent differences between large and small-scale reactors. Generally, the GEV value during large-scale bioreactor cultivation is higher than that in small-scale reactors. Therefore, it is urgent to develop a scaled-down model that can reproduce the high GEV effect of large-scale bioreactors in advance. This model would better help us understand and simulate the impact of high GEV on cell culture performance in large-scale bioreactors, maximize the stability of the production process, and have profound significance for the development and characterization of the entire cell culture process. Utility Model Content

[0006] The technical problem to be solved by this invention is to provide a stirred bioreactor for cell culture in the pilot stage, which can simulate GEV conditions comparable to or higher than those in large-scale production. This facilitates early identification of the negative impact of high GEV on cell culture in large-scale bioreactors during the early research and development stage, and helps to understand and predict potential problems in large-scale production.

[0007] To solve the above-mentioned technical problems, the present invention provides a stirred bioreactor for small-scale cell culture, comprising:

[0008] A reaction vessel, used to hold culture medium for cell culture;

[0009] A stirring assembly, which is installed on the top cover of the reaction vessel, the stirring assembly including impeller blades;

[0010] A venting pipe, which passes through and is securely installed on the top cover of the reactor and extends into the interior of the reaction vessel for introducing gas into the culture medium, the venting pipe includes a vertical section and a horizontal section, the horizontal section being bent relative to the vertical section, the horizontal section being positioned between the impeller and the bottom surface of the reaction vessel, and the horizontal section having vent holes.

[0011] Preferably, the number of vent holes is one, and it is located at the middle position along the length of the horizontal segment.

[0012] Preferably, the vent hole faces the bottom surface of the reaction vessel.

[0013] Preferably, the diameter of the vent hole is 0.15-0.50 mm.

[0014] Preferably, the diameter of the vent is 0.25 mm.

[0015] Preferably, the stirring assembly further includes a motor, the output end of which is connected to the bottom end of the stirring shaft;

[0016] The blades are installed at the top of the stirring shaft.

[0017] Preferably, the motor is mounted on the top cover of the reaction vessel.

[0018] Preferably, the bioreactor also includes a harvesting pipeline for pumping seed liquid into the reaction vessel and pumping out harvested liquid.

[0019] Preferably, the harvesting pipeline is installed on the top cover of the reaction vessel and extends into the reaction vessel near the bottom.

[0020] Preferably, the bioreactor is fixed by a support.

[0021] This invention provides a stirred bioreactor for cell culture in a pilot-scale stage. The horizontal section of its air inlet pipe is positioned between the impeller and the bottom of the reaction vessel, and a vent is opened in the horizontal section. Preferably, there is one vent, located in the middle of the length of the horizontal section. This achieves GEV conditions comparable to or higher than those at large-scale production, which helps to identify the potential risks and negative impacts of high GEV levels in the early stages of research and development, and provides important support for optimizing the cell culture process. Attached Figure Description

[0022] To more clearly illustrate the technical solution of this utility model, the drawings used in this utility model will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this utility model. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0023] Figure 1 This is a schematic diagram of the structure of a stirred bioreactor used for cell culture in the pilot-scale stage of Embodiment 1 of this utility model;

[0024] Figure 2 This is a schematic diagram of the support frame of the stirred bioreactor used for cell culture in the pilot stage of Embodiment 1 of this utility model;

[0025] Figure 3A Example 2 shows the change in cell density (VCD) over time;

[0026] Figure 3B The graph showing the change in cell viability over time in Example 2;

[0027] Figure 3C Example 2: Glucose variation over time;

[0028] Figure 3D The graph showing the change of lactate over time in Example 2;

[0029] Figure 3E The graph showing the change of ammonium over time in Example 2;

[0030] Figure 3F The graph showing the change in osmolality over time in Example 2;

[0031] Figure 3G Changes in lactate dehydrogenase (LDH) over time in Example 2;

[0032] Figure 3H The change of pCO2 over time in Example 2;

[0033] Figure 3I The change of total O2 over time in Example 2;

[0034] Figure 3J The change of total CO2 over time in Example 2;

[0035] Figure 3K The change of total gas over time in Example 2;

[0036] Figure 3L Changes in total IgG HB over time in Example 2;

[0037] Figure 4 This refers to the GEV levels of cells in different model reactors in Example 2.

[0038] In the diagram, 1-reaction vessel; 11-top cover; 2-stirring assembly; 21-blade; 22-motor; 23-stirring shaft; 3-venting pipe; 31-vertical section; 32-horizontal section; 33-venting hole; 4-support; 41-fixing ring; 42-support leg. Detailed Implementation

[0039] The technical solutions of this utility model will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this utility model. All other embodiments obtained by those skilled in the art based on the embodiments of this utility model without creative effort are within the scope of protection of this utility model.

[0040] Example 1: Stirred Bioreactor for Small-Scale Cell Culture

[0041] refer to Figure 1 This invention illustrates a stirred bioreactor for small-scale cell culture, comprising:

[0042] A reaction vessel, used to hold culture medium for cell culture;

[0043] A stirring assembly, which is installed on the top cover of the reaction vessel, the stirring assembly including impeller blades;

[0044] A venting pipe, which passes through and is securely installed on the top cover of the reactor and extends into the interior of the reaction vessel for introducing gas into the culture medium, the venting pipe includes a vertical section and a horizontal section, the horizontal section being bent relative to the vertical section, the horizontal section being located between the impeller and the bottom surface of the reaction vessel, and the horizontal section having vent holes.

[0045] Specifically, there is one vent hole, which is located at the middle of the length of the horizontal section, and preferably the vent hole faces the bottom of the reaction vessel.

[0046] In this embodiment, the reaction vessel used in the pilot-scale stage has a volume of 3L. During cell culture, the stirring component stirs the culture medium, and at the same time, the venting pipe is used to supply oxygen to the culture medium. The gas enters the culture medium in the reaction vessel through the vent hole in the horizontal section of the venting pipe. In this embodiment, by opening the vent hole in the horizontal section and placing the vent hole at the middle position along the length of the horizontal section, the GEV effect comparable to or higher than that on a large scale is simulated in the small-scale bioreactor in the pilot-scale stage. This helps to identify the risks and negative impacts that high GEV levels may bring in the early research and development stage, and provides important support for the optimization of the cell culture process.

[0047] In one specific embodiment of this example, the diameter of the vent hole is 0.15-0.50 mm, preferably 0.25 mm.

[0048] In one specific embodiment of this invention, the stirring assembly further includes a motor, the output end of which is connected to the bottom end of the stirring shaft; the impeller is mounted at the top end of the stirring shaft. Understandably, the output end of the motor drives the stirring shaft to rotate, thereby driving the impeller to rotate, thus achieving stirring of the culture medium. Preferably, the motor is mounted on the top cover of the reaction vessel.

[0049] In one specific embodiment of this invention, the bioreactor further includes a harvesting pipeline for pumping seed liquid into the reaction tank and pumping out harvested liquid. Preferably, the harvesting pipeline is installed on the top cover of the reaction tank and extends into the reaction tank near the bottom.

[0050] In one specific embodiment of this example, the bioreactor is fixed by a support. Understandably, the support is used to fix the reaction vessel to maintain stability during cell culture; the structure of the support is, for example, as shown below. Figure 2As shown, the reactor includes a fixing ring and legs. The outer wall of the reactor is connected to the fixing ring circumferentially. There are, for example, two fixing rings, located near the top and bottom of the reactor, respectively. There are, for example, at least three legs, distributed circumferentially along the fixing ring. If you do not wish to be constrained by this, any support used in the prior art to stabilize a bioreactor can be applied to this embodiment.

[0051] Example 2: High GEV Simulation and Validation of a Stirred Bioreactor for Cell Culture in a Small-Scale Pilot-Scale Phase

[0052] Reactors: 3L reactor used in pilot-scale tests in the prior art (3L control group), 3L reactor of Embodiment 1 of this utility model (3L high GEV model), 200L reactor for large-scale production (200L STR);

[0053] Cells to be cultured: CHO cell line (WXCHO-K1)

[0054] Cultivation process: 14 days, see Table 1 for details:

[0055] Table 1

[0056] ,

[0057] In this embodiment, the total gas flow rate was monitored daily during the culture process. The GEV results for the three reactors were calculated using the formula (GEV (m / s) = Qs / A) and are listed in Table 2. The GEV levels of cells in different reactor models are plotted as follows. Figure 4 As shown:

[0058] Table 2

[0059] ,

[0060] Table 1 and Figure 4 It can be seen that the GEV level of the 3L reactor used in the pilot test in the prior art is relatively low, ranging from 0.02 m / s to 0.79 m / s; in the large-scale 200 L STR, the GEV level is controlled to vary from 0.41 m / s to 3.78 m / s; while the reactor of one embodiment of the present invention successfully simulated a higher GEV level, fluctuating between 0.00 m / s and 36.01 m / s.

[0061] During the culture period, samples were taken daily for pH and pCO2 testing using a blood gas analyzer (BGA), VCD and viability testing using a Vi-CELL analyzer, and biochemical parameters such as glucose, lactate, ammonium, LDH, and IgG HB measured using a Cedex biochemical analyzer. The osmotic pressure of the test samples was also measured using an osmometer. The test results are as follows: Figure 3A-3L As shown, where Figure 3A This is a graph showing the change in cell density (VCD) over time. Figure 3B This is a graph showing the change in cell viability over time. Figure 3C This is a graph showing the change in glucose over time. Figure 3D This is a graph showing the change in lactate over time. Figure 3E This is a graph showing the change in ammonium concentration over time. Figure 3F This is a graph showing the change in osmotic pressure over time. Figure 3G It is the change of lactate dehydrogenase (LDH) over time. Figure 3H It is the change of pCO2 over time. Figure 3I It is the change of total O2 over time. Figure 3J It represents the change in total CO2 over time. Figure 3K It represents the change in total gas volume over time; Figure 3L This represents the change in total IgG HB over time. Finally, on the day of receipt, a supernatant sample (3000g, 10min; filtered) was collected for Product Quality Assurance (PQA) testing, and the results are shown in Table 3.

[0062] Table 3

[0063] ,

[0064] Among them, SDS-Caliper NR: Sodium Dodecyl Sulfate Caliper Non-reduced microfluidic chip capillary electrophoresis;

[0065] iCIEF: Imaged Capillary Isoelectric Focusing.

[0066] SEC: Size-exclusion Chromatography.

[0067] UPLC: Ultra-performance liquid chromatography.

[0068] Figure 3 and Figure 4The results show that higher GEV levels during culture have a greater impact on cell culture performance. In the high-GEV model's 3 L reactor, decreased cell viability and viability were observed in the later stages of culture, attributed to the high injection velocity at the gas outlet of the novel gas distributor, leading to shear forces that caused cell damage. Simultaneously, compared to the control group, higher concentrations of lactate dehydrogenase (LDH) were detected in the high-GEV model, and the LDH concentration increased with culture time, further confirming that the cells suffered some degree of damage. From Day 8, antibody expression in the three different models was monitored using Cedex titer assays until harvest day. Although VCD and viability were affected in the high-GEV model in the later stages of culture, titer expression levels remained largely similar. Finally, the harvest day samples from the three different models were purified using a one-step purification process for product quality analysis. The results showed that the purity of the SEC Monomer and SDS-Caplier in all three models was above 94%; the main peak of iCIEF was above 55.0% in all models; and the levels of N-Glycan and Man5 were good, both below 10.0%. Therefore, high GEV levels did not negatively affect cell quality properties and were comparable to the control group. This experiment confirms that the high GEV effect impairs cell performance to some extent, which is particularly noteworthy when scaled up to large-scale bioreactors.

[0069] The above description is only a preferred embodiment of the present utility model and is not intended to limit the scope of protection of the present utility model. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present utility model should be included within the scope of protection of the present utility model.

Claims

1. A stirred bioreactor for small-scale cell culture, characterized in that, include: A reaction vessel, used to hold culture medium for cell culture; A stirring assembly, which is installed on the top cover of the reaction vessel, the stirring assembly including impeller blades; A venting pipe, which passes through and is securely installed on the top cover of the reactor and extends into the interior of the reaction vessel for introducing gas into the culture medium, the venting pipe includes a vertical section and a horizontal section, the horizontal section being bent relative to the vertical section, the horizontal section being located between the impeller and the bottom surface of the reaction vessel, and the horizontal section having vent holes.

2. The bioreactor as described in claim 1, characterized in that, The number of vent holes is one, and it is located at the middle position along the length of the horizontal segment.

3. The bioreactor as described in claim 2, characterized in that, The vent faces the bottom of the reaction vessel.

4. The bioreactor as described in claim 3, characterized in that, The diameter of the vent is 0.15-0.50 mm.

5. The bioreactor as described in claim 4, characterized in that, The vent has a diameter of 0.25 mm.

6. The bioreactor as described in claim 1, characterized in that, The stirring assembly also includes a motor, the output end of which is connected to the bottom end of the stirring shaft; The blades are installed at the top of the stirring shaft.

7. The bioreactor as described in claim 6, characterized in that, The motor is mounted on the top cover of the reaction vessel.

8. The bioreactor as described in claim 1, characterized in that, The bioreactor also includes a harvesting pipeline for pumping seed liquid into the reaction vessel and pumping out harvested liquid.

9. The bioreactor as described in claim 8, characterized in that, The harvesting pipeline is installed on the top cover of the reaction vessel and extends into the reaction vessel near the bottom.

10. The bioreactor as described in claim 1, characterized in that, The bioreactor is fixed by a support frame.

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