A dynamic process control method for high-yield culture of car-nk cells

Abstract

The application belongs to the field of cell therapy process engineering, and discloses a CAR-NK cell high-yield culture method with dynamic process control. The method realizes the dynamic balance of nutrient supply and shear force in the culture system by introducing the strategy of combining culture stage regulation with one-time large-volume liquid replacement in a swing bioreactor. The method realizes online interception and rapid replacement of the culture medium in the high-density cell stage by using the built-in interception membrane of the reactor culture bag, thereby avoiding cell loss and nutrient depletion. Meanwhile, the method effectively maintains the CAR expression level and cell functional activity by cooperating with the optimized culture medium formula and the application of a transfection aid. The method has the advantages of simple operation and high repeatability, is suitable for various swing or closed reactor platforms, has good amplification performance and industrial application value, and can provide an efficient and stable technical scheme for the preclinical preparation and industrial production of CAR-NK cell immunotherapy.

Description

Technical Field

[0001] This invention belongs to the field of cell therapy process engineering, specifically relating to a dynamic process-controlled method for high-yield culture of CAR-NK cells. Background Technology

[0002] Engineered immune cells expressing chimeric antigen receptors (CARs), such as CAR-T cells, have achieved significant efficacy in the treatment of hematologic malignancies. However, their preparation is costly and time-consuming, and they are often accompanied by side effects such as cytokine release syndrome and neurotoxicity. Autologous sources also lead to significant individual variability and limited accessibility. In contrast, NK cells, as innate immune effector cells, are less likely to induce excessive inflammation or neurotoxicity and do not cause graft-versus-host disease, offering a natural safety advantage. CAR-NK cell technology combines the targeted recognition of CARs with the inherent anti-tumor activity of NK cells. It can not only precisely target tumors through CAR-mediated action but also release perforin, granzymes, and IFN-γ, exerting antibody-dependent cell-mediated cytotoxicity (ADCC) effects to broadly eliminate tumor cells and overcome antigen escape. Its allogeneic sources (peripheral blood, umbilical cord blood, or iPSCs) hold promise for enabling readily available, universally applicable drugs, significantly improving treatment standardization and accessibility.

[0003] However, the translation from laboratory research to large-scale clinical and commercial applications faces technical bottlenecks in efficient in vitro expansion. The WAVE bioreactor is the mainstream platform for CAR-NK cell expansion, but NK cells are highly sensitive to shear forces, and CAR-NK cells with high CAR⁺ rates (>70%) grow slowly. Conventional constant-rate, constant-angle culture cannot meet the requirements of low shear and high mass transfer. Under the premise of ensuring cell quality and function, it is difficult to achieve a production scale of hundreds of billions or even trillions, which cannot meet the needs of commercial use. Summary of the Invention

[0004] To address the shortcomings mentioned in the background art, the present invention aims to provide a dynamic process-controlled high-yield CAR-NK cell culture method. By dynamically adjusting the swing speed and angle in stages in a swing bioreactor, combined with a microfiltration membrane for one-time large-volume medium replacement, a balance between nutrient supply and low shear force is achieved in the culture system, significantly improving cell expansion, CAR positivity rate, and functional activity. The process is simple and easy to scale up for industrial production.

[0005] The objective of this invention can be achieved through the following technical solutions: A method for high-yield CAR-NK cell culture with dynamic process control includes the following steps: S1. NK cells were obtained by sorting peripheral blood or umbilical cord blood PBMCs with magnetic beads and seeded in T bottles at a density of 1×105 to 10×105 cells / mL in activation medium. K562 feeder cells were added to make the K562 / NK ratio 0.5:1 to 5:1 and then statically cultured. S2. After incubating to D3, mix and count, then add 0.5 to 2 times the volume of activation medium and continue static incubation; S3. After culturing to D4, mix and count, add retrovirus and transfection promoter, mix well and continue static transfection culture to obtain CAR-NK cells; S4. After culturing to D5, mix and count the cells, adjust the cell density to 0.1×105~3×105 cells / mL with amplification medium, and inoculate 15~30 L culture bags. Sort the cells in a WAVE rocking reactor for suspension culture at a speed of 3~10 rpm and an angle of 3°~10°. S5. Samples were taken and counted at D7, 8, and 9 respectively; during D8 and D9, the plants were cultured with the following parameters: rotation speed 3-10 rpm and angle 5°-12° at D8, and rotation speed 3-10 rpm and angle 7°-14° at D9. S6. In the D9 stage, the amplification medium is connected and replaced with a microfiltration membrane at a rate of 0.2 to 2 times the culture volume every 24 hours to achieve online cell retention and large-volume medium replacement; S7. After culturing to day 10, CAR-NK cells were harvested, and the total harvest, expansion fold, CAR positivity rate, and in vitro killing activity were detected.

[0006] More preferably, in step S5, only sampling and counting are performed in stage D7, and then cultivation continues without adjusting the WAVE parameters.

[0007] More preferably, the volume of the replacement culture medium during the 24-hour period is 0.5 to 1.5 times the volume of the original culture system.

[0008] More preferably, the activation culture medium consists of: basal culture medium, supplement, cytokine III and 5%–10% serum substitute; the amplification culture medium consists of: basal culture medium, supplement, cytokine III and 1%–4% serum substitute.

[0009] More preferably, the swing speed of the WAVE swing bioreactor is 3 rpm to 10 rpm, and the swing angle is 3° to 14°.

[0010] More preferably, the microfiltration membrane is a microfiltration membrane with a pore size of 1 μm to 10 μm.

[0011] More preferably, this method is suitable for the industrial-scale production and preclinical preparation of CAR-NK cells.

[0012] The beneficial effects of this invention are: This invention achieves a dynamic balance between nutrient supply and shear force in the culture system by dynamically adjusting the swing rate and angle in stages within a swing-type bioreactor, and by combining this with a large-volume medium exchange via microfiltration membrane during the high-density cell phase. This significantly improves the expansion efficiency and functional performance of CAR-NK cells. Compared to traditional constant-rate, constant-angle culture, the early low-shear environment ensures cell survival, while the gradually increasing swing parameters accelerate mixing and oxygen transfer, resulting in a nearly three-fold increase in cell expansion. Simultaneously, continuous online large-volume medium exchange replenishes nutrients, eliminating metabolic inhibition and playing a crucial role in maintaining CAR expression levels and cell-killing activity. This method is simple to operate, with quantifiable parameters that are easy to replicate and scale up. It is applicable to various swing-type or closed reactor platforms, exhibits good process stability and batch-to-batch consistency, and meets the needs of preclinical preparation and industrial production of CAR-NK cells. Attached Figure Description

[0013] The invention will now be further described with reference to the accompanying drawings.

[0014] Figure 1 The growth curves of cells cultured under the methods of Examples 1-3 and Comparative Examples 1-2 are shown. Figure 2 The cell viability curves are shown for the culture methods of Examples 1-3 and Comparative Examples 1-2. Figure 3 The cell fold growth curves are for Examples 1-3 and Comparative Examples 1-2 under the culture methods. Figure 4 This is a bar chart comparing cell yield under the culture methods of Examples 1-3 and Comparative Examples 1-2; Figure 5 This is a bar chart comparing the CAR positivity rates of cells cultured under the methods of Examples 1-3 and Comparative Examples 1-2; Figure 6 The bar chart shows the comparison of cell killing activity under the culture methods of Examples 1-3 and Comparative Examples 1-2. Detailed Implementation

[0015] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0016] Example 1 10 mL of peripheral blood was collected from healthy donors and peripheral blood mononuclear cells (PBMCs) were separated by centrifugation. NK cells were obtained by magnetic bead sorting and resuspended in activation medium. Irradiated K562 feeder cells were added at a K562 / NK ratio of 5:1. The NK cell density was adjusted to 5 × 10⁵ cells / mL, and the cells were dispensed into multiple T225 culture flasks, 50 mL per flask, and incubated statically at 37 °C with 5% CO₂. On day 3 of culture, the cells were mixed, sampled, and counted. Then, 100 mL of activation medium was added to each flask. On day 4, the cells in the culture flasks were mixed, sampled, and counted. A certain amount of retroviral vector was added to each flask at MOI=10, along with 1.5 mL of transfection promoter (1 mg / mL). The flasks were incubated statically for another 24 h to complete transfection.

[0017] On day 5, all transfected cells were mixed, counted, and adjusted to 0.2 × 10⁵ cells / mL with amplification medium. 25 L of cell suspension was placed into a WAVE culture bag and connected to a WAVE rocking reactor. Initial rocking parameters were set: 6 rpm, rocking angle 6°, temperature 37 °C, and suspension culture was started. On day 7, samples were taken for counting, and culture continued to the next stage. On day 8, after sampling and counting, the rocking rate was adjusted to 6 rpm and the angle to 7°, and culture continued for 24 h. On day 9, after sampling and counting, the rocking rate was adjusted to 7 rpm and the angle to 10°. A storage bag containing 25 L of fresh amplification medium was connected, and perfusion was started through the microfiltration membrane of the culture bag at a rate of 25 L of medium replacement every 24 hours, continuing culture at 7 rpm / 10°. Culture was completed on day 10, and cells were harvested. Samples were taken to calculate the total CAR-NK cell harvest, cell expansion fold, CAR positivity rate, and in vitro killing activity.

[0018] Example 2 The culture method for the first 7 days in this embodiment is the same as that in Example 1. On the 8th day, after taking samples and counting, the WAVE swing parameters were adjusted to 6 rpm and 8°, and the culture was continued for 24 h. On the 9th day, after taking samples and counting, the parameters were adjusted to 8 rpm and 12°. A storage bag containing 25 L of fresh amplification medium was connected, and perfusion was started through the microfiltration membrane of the culture bag at a rate of replacing 25 L of medium every 24 hours. The suspension culture was carried out at 8 rpm and 12°. The culture was then carried out until the 10th day, and the cells were harvested. The total harvest of CAR-NK cells, the cell expansion fold, the CAR positivity rate, and the in vitro killing activity were measured.

[0019] Example 3 The culture method for the first 8 days in this embodiment is the same as that in Example 1. After sampling on the 9th day, the rocking parameter is adjusted to 7 rpm / 10°. A storage bag containing 12.5 L of fresh amplification medium is connected, and perfusion is started through the microfiltration membrane of the culture bag at a rate of replacing 12.5 L of medium every 24 hours. The culture is carried out at 7 rpm / 10°. The culture is then carried out until the 10th day, and the cells are harvested. The total amount of CAR-NK cells harvested, the cell expansion fold, the CAR positivity rate, and the in vitro killing activity are measured.

[0020] Comparative Example 1 The culture method for the first four days of this comparative example was the same as that for the first four days of Example 1. On day 5, all transfected cells were mixed, counted, and adjusted to 0.2 × 10⁵ cells / mL with amplification medium. 25 μL of cell suspension was placed into a WAVE culture bag and connected to a WAVE rocking reactor. Initial rocking parameters were set as follows: 2 rpm, rocking angle 8°, and temperature 37 °C for suspension culture. Samples were taken and counted on days 7, 8, and 9, and cultured at 2 rpm / 8°. On day 10, the reactor was stopped, all cell suspension was collected, viable cell counts were performed to calculate the fold increase, and CAR positivity rate and in vitro cytotoxic activity were detected.

[0021] Comparative Example 2 The culture method for the first 4 days in this comparative example was the same as that for the first 4 days in Example 1. On day 5, all transfected cells were mixed, counted, and adjusted to 0.2 × 10⁵ cells / mL with amplification medium. 25 L of cell suspension was placed into a WAVE culture bag and connected to a WAVE rocking reactor. The cells were cultured in suspension at a constant speed of 2 rpm and an angle of 8°. After daily sampling and counting from day 7 to day 9, a storage bag containing fresh amplification medium was connected, and perfusion culture was started by replacing 2.5 L of medium every 24 hours through the microfiltration membrane of the culture bag. On day 10, the reactor was stopped, all cells were collected, viable cell counts were performed to calculate the fold increase, and the CAR positivity rate and in vitro killing activity were detected.

[0022] Performance testing Cell growth curve After inoculation into the reactor, aseptic samples were taken from the culture bags at various sampling time points. 20 μL of cell culture was pipetted, mixed with 20 μL of AO / PI fluorescent dye, and then 20 μL was added to the wells of a cell counting plate. The plate was then used to count the cells using a fluorescence cell counter, and the viable cell density (cells / mL) and cell viability were recorded. The density and viability curves of cell growth in the reactor culture bags were plotted with culture time (days) on the x-axis and viable cell density and viability on the y-axis, respectively. Reagents and incubation conditions were kept consistent throughout the test, and the experiment was repeated three times, with the average value taken. The results are shown in Tables 1 and 2 below.

[0023] Table 1. Viable cell density at different culture times (×10⁵ cells / mL)

[0024] As shown in Table 1, cells in Examples 1–3 grew rapidly in the culture bags, with viable cell densities reaching 55.0, 47.0, and 40.0 × 10⁵ cells / mL on Day 10, respectively, significantly superior to 15.0 × 10⁵ in Comparative Example 2 and 12.0 × 10⁵ in Comparative Example 1. This invention protects cell survival through early low-speed, small-angle growth, while gradually increasing parameters in the later stages to enhance mixing and oxygen transfer. Simultaneously, perfusion replacement of an equal volume of culture medium on Day 9 replenishes nutrients and eliminates metabolic inhibition, effectively offsetting growth inhibition caused by nutrient depletion and shear damage during the high-density period. This achieves an intermittent "increase-decrease-increase" dynamic balance, greatly improving amplification efficiency and CAR expression stability. In contrast, Comparative Example 1, with its constant speed and angle and no medium change, resulted in a sustained low-level, slow cell growth. Although Comparative Example 2 involved small-volume, multiple medium changes, the peak-to-trough amplitude and final yield were still far inferior to the examples of this invention because the cumulative metabolic inhibition was not addressed.

[0025] Table 2 Cell viability at different culture times

[0026] As shown in Table 2, all groups maintained a high viability of approximately 93% immediately after inoculation into the reactor culture bags on day 5, indicating good activation and initial culture conditions and limited impact of the transfection process on cell viability, resulting in relatively consistent cell status. As the culture time extended to day 9, the cell viability of the comparative examples gradually decreased, with Comparative Example 1 reaching 85.6% and Comparative Example 2 reaching 87.9%, while Examples 1–3 remained relatively stable at 95.3%, 94.1%, and 91.7%, respectively, significantly higher than conventional processes. This indicates that the phased dynamic adjustment strategy of this invention effectively alleviated mixing, oxygen transfer, and shear stress during the high-density phase. By day 10, Example 1 maintained a viability of 92.7%, and Examples 2 and 3 reached 91.7% and 90.6%, respectively, while Comparative Examples 1 and 2 only reached 83.2% and 85.2%. These results fully demonstrate that by using a microfiltration membrane to replace the culture medium and gradually increasing the rocking parameters in the D9 stage, this invention can significantly improve the survival rate and functional stability of cells in the later stages while ensuring high cell expansion efficiency, thus providing a more reliable process guarantee for the industrial-scale production of CAR-NK cells.

[0027] Cell ploidy assay The viable cell concentration (cells / mL) was measured based on the sampling count at each time point, and then the fold change was calculated by dividing the daily concentration by the D5 concentration. The entire measurement was repeated three times and the average value was taken. The results are shown in Table 3.

[0028] Table 3. Cell expansion fold results at different culture times

[0029] As shown in Table 3, the cell doubling rates of Examples 1–3 of this invention during D7–D10 were significantly higher than those of the comparative groups, with Example 1 showing the most outstanding performance: reaching 17.5 times on D7, 50 times on D8, and 275 times on D10, while the comparative group only reached 7.5 to 60 times. Although Examples 2 and 3 were slightly lower than Example 1, they were significantly higher than the comparative groups 2 and 1. This indicates that the innovative process of introducing phased dynamic adjustment of the rocking parameters and a large-volume medium change on D9 promotes cell survival and initial expansion in the early stage through a low-shear environment, and successfully alleviates nutrient depletion and shear stress in high-density culture by improving mixing and oxygen transfer efficiency and timely replenishing the culture medium in the middle and late stages.

[0030] 3. Yield Measurement The total cell yield under different culture processes was calculated by multiplying the viable cell concentration (cells / mL) obtained from sampling and counting at the end of D10 culture by the cell volume of 25L at harvest time for each group. The results are shown in Table 4 below.

[0031] Table 4 Yields of different methods

[0032] As shown in Table 4, Examples 1–3, which employ the dynamic phased parameter adjustment and single large-volume liquid replacement of this invention, achieved a total yield of 13.75 × 10¹ on D10. 0 11.75×10¹ 0 and 10.00×10¹ 0 The number of cells / L is significantly higher than that of Comparative Example 1 (3.00 × 10¹). 0 And 3.75 × 10¹ of Comparative Example 2 0 This invention improves mixing and oxygen transport efficiency by increasing the swaying rate and angle during the D8–D9 stages, and achieves rapid nutrient replenishment and metabolic waste removal during the high-density period by replacing an equal volume of culture medium through microfiltration membrane perfusion. This successfully breaks through the production bottleneck of conventional constant-rate, constant-angle, or fractional feeding processes, enabling cells to proliferate continuously and efficiently in a high-density environment.

[0033] 4. CAR positivity rate determination After washing the harvested CAR-NK cells (D10) with PBS, 1×10⁶ cells were taken and resuspended in PBS containing 3% BSA to 100 µL. Fc-tagged GPC3 protein (5 µg / mL) was added and incubated at 4 °C in the dark for 30 min. The cells were then washed twice with PBS, resuspended in 100 µL of PBS, and anti-Fc antibody-APC (2 µg / mL) was added and incubated at 4 °C in the dark for 30 min. After washing again, the cells were resuspended in 300 µL of PBS. Flow cytometry was used to collect and calculate the percentage of APC-positive cells among the total metabolically viable cells. The results are shown in Table 5 below.

[0034] Table 5 CAR Positive Rate in Each Group

[0035] Table 5 shows that Examples 1–3 all outperformed the two comparative examples in CAR positivity rate, with Example 1 showing the highest CAR expression level of 75%. This result indicates that the present invention, through phased dynamic adjustment of rocking parameters and a single large-volume medium change during cell expansion, not only improved the culture environment and enhanced cell survival and proliferation but also reduced the inhibitory effects of post-transfection cytokines and metabolic waste, thus effectively maintaining CAR expression. In contrast, Comparative Examples 1 and 2, due to a lack of efficient medium changes or only partial feeding, suffered from nutrient and shear stress imbalances, leading to cell damage and decreased CAR expression. Therefore, the process of the present invention achieves high yield while maintaining high phenotypic quality of CAR-NK cells, providing a key technical guarantee for large-scale preclinical preparation.

[0036] 5. Lethality test For the assay of cytotoxic activity, CAR-NK cells were harvested on day 10, washed with PBS, and counted. Effector cells and target cells (HepG2) were prepared at an E:T ratio of 5:1. 1.5 × 10⁴ target cells were seeded in 96-well plates, and 7.5 × 10⁴ CAR-NK cells were added for co-culturing for 24 h. The experiment included a spontaneous release group (effector cells only) and a negative group (target cells only). After co-culture, 100 μL of supernatant was collected, and the secretion of the major cytotoxic factor IFN-γ was detected by ELISA. The results are shown in Table 6 below.

[0037] Table 6 Killing Activity

[0038] As shown in Table 6, all groups exhibited high cytotoxic activity at the same E:T ratio. However, the dynamic staged regulation and single large-volume medium replacement strategy used in Examples 1–3 enabled CAR-NK cells to maintain extremely high functionality after high-density culture, reaching over 35,000 pg / ml in Examples 1–3, significantly better than the less than 30,000 pg / ml in the control group. This difference indicates that by optimizing nutrient supply and reducing shear damage, this invention not only improves cell expansion efficiency and survival rate but also effectively enhances the in vitro effector function of CAR-NK cells.

[0039] In the description of this specification, references to terms such as "an embodiment," "example," "specific example," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0040] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the claimed invention.

Claims

1. A method for high-yield culture of CAR-NK cells with dynamic process control, characterized in that, Includes the following steps: S1. NK cells were obtained by sorting peripheral blood or umbilical cord blood PBMCs with magnetic beads and seeded in T bottles at a density of 1×105 to 10×105 cells / mL in activation medium. K562 feeder cells were added to make the K562 / NK ratio 0.5:1 to 5:1 and then statically cultured. S2. After incubating to D3, mix and count, then add 0.5 to 2 times the volume of activation medium and continue static incubation; S3. After culturing to D4, mix and count, add retrovirus and transfection promoter, mix well and continue static transfection culture to obtain CAR-NK cells; S4. After culturing to D5, mix and count the cells, adjust the cell density to 0.1×105~3×105 cells / mL with amplification medium, and inoculate 15~30 L culture bags. Sort the cells in a WAVE rocking reactor for suspension culture at a speed of 3~10 rpm and an angle of 3°~10°. S5. Samples were taken and counted at D7, 8, and 9 respectively; during D8 and D9, the plants were cultured with the following parameters: rotation speed 3-10 rpm and angle 5°-12° at D8, and rotation speed 3-10 rpm and angle 7°-14° at D9. S6. In the D9 stage, the amplification medium is connected and replaced with a microfiltration membrane at a rate of 0.2 to 2 times the culture volume every 24 hours to achieve online cell retention and large-volume medium replacement; S7. After culturing to day 10, CAR-NK cells were harvested, and the total harvest, expansion fold, CAR positivity rate, and in vitro killing activity were detected.

2. The method for high-yield CAR-NK cell culture with dynamic process control according to claim 1, characterized in that, In step S5, only sampling and counting are performed in stage D7, and then culture continues without adjusting the WAVE parameters.

3. The method for high-yield CAR-NK cell culture with dynamic process control according to claim 1, characterized in that, The volume of the replacement culture medium during the 24-hour period is 0.5 to 1.5 times the volume of the original culture system.

4. The method for high-yield CAR-NK cell culture with dynamic process control according to claim 1, characterized in that, The activation culture medium consists of: basal culture medium, supplement, cytokine III and 5%–10% serum substitute; the amplification culture medium consists of: basal culture medium, supplement, cytokine III and 1%–4% serum substitute.

5. The method for high-yield CAR-NK cell culture with dynamic process control according to claim 1, characterized in that, The swing speed of the WAVE swing bioreactor is 3 rpm to 10 rpm, and the swing angle is 3° to 14°.

6. The method for high-yield CAR-NK cell culture with dynamic process control according to claim 1, characterized in that, The culture bag microfiltration membrane is a microfiltration membrane with a pore size of 1 μm to 10 μm.

7. The method for high-yield CAR-NK cell culture with dynamic process control according to claim 1, characterized in that, This method is applicable to the industrial-scale production and preclinical preparation of CAR-NK cells.

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