Method for improving transfer cell density in seed culture solution and realizing high cell density inoculation in production culture solution

Abstract

Methods of increasing the density of transferred cells in a seed culture solution and enabling high cell density vaccination in a production culture solution are disclosed. The method for improving the cell transfer density of the culture solution comprises the following steps: culturing cells by perfusion in a seed culture solution; and concentrating the seed culture solution by perfusion.

Description

Technical Field

[0001] The present application relates to the field of cell culture technology, and in particular to a method for increasing the expression of a target product through high inoculation density culture. Background Art

[0002] Genetically engineered protein production via mammalian cell culture is a common method for obtaining therapeutic proteins, such as monoclonal antibodies. Advances in mammalian cell culture technology have significantly advanced the development and biomanufacturing of biopharmaceutical products, including antibodies, fusion proteins, and enzymes. The following briefly describes the production processes currently used in the biopharmaceutical industry.

[0003] At present, the domestic and international biopharmaceutical industries use mammalian engineered cell lines to express and produce monoclonal antibodies or fusion proteins, mainly using the fed-batch process.

[0004] CN201210057859.7 points out that the traditional batch fed-batch process (FB) as a conventional method requires process exploration and optimization to obtain the required production performance. In cGMP (Current Good Manufacture Practices) production, repeatedly verified, huge cell culture equipment and complex supporting systems are often required. That is, the traditional batch fed-batch culture of mammalian cells has some shortcomings that are difficult to overcome, such as low cell biomass, low protein expression in a single batch, inability to eliminate harmful metabolites, complex process control, etc. CN202010592471.1 also points out that since the culture medium components are not fully replenished to the area consumed by cell proliferation during the batch fed-batch process, the supplementary effect of nutrients in the batch fed-batch culture is weakened, thereby affecting the cell proliferation effect.

[0005] Perfusion is a continuous cell culture process in which fresh culture medium is continuously injected into the reactor, and the expressed product is continuously harvested along with the discharged cell culture fluid. CN201210057859.7 indicates that this perfusion process can significantly increase cell biomass and protein concentration. However, this process consumes a large amount of culture medium, and the harvested fluid volume also requires sufficient storage space and specialized downstream processing designs. Therefore, the perfusion process is generally used for protein products with poor stability, prone to quality parameter fluctuations, and requiring real-time harvesting.

[0006] The process model disclosed in PCT / US2008 / 072612 combines the advantages of early perfusion and batch fed-batch culture to achieve higher protein yields. However, this process model fails to enhance the accumulation of cell seed density during the expansion phase, resulting in a prolonged period of occupancy of large-scale reactor production facilities during the exponential cell proliferation phase, resulting in a longer production period and lower equipment utilization.

[0007] WO 2021 / 021973 discloses an enhanced fed batch (IFB) process developed based on the characteristics of the above cell culture process. In the seed expansion stage (N-1), a nutrient-enriched culture medium, batch feeding and perfusion process are used to quickly and efficiently accumulate a large number of cells. When inoculated into the production stage (N) reactor, the initial inoculation density is increased (10-30×10 6 cells / ml) and achieve higher peak cell densities during culture, thereby accumulating higher protein yields and improving batch yield and efficiency. However, increasing IFB seeding density and yield to higher levels also faces numerous challenges: prolonged culture during the expansion phase prevents the cell density from effectively reaching higher levels due to limited proliferation potential; prolonged perfusion culture requires a larger amount of perfusion medium to meet cell consumption. Furthermore, maintaining stable culture conditions at high seeding densities, such as dissolved oxygen (DO), pH, pCO2, and foam control, is difficult.

[0008] CN201580062059.6 uses optimized perfusion culture technology to support the production of ultra-high cell density cultures that can be cryopreserved at high cell density while maintaining excellent cell viability and quality. The highest cell density reported in its implementation case reached 100×10 6 viable cells / mL. This report indirectly demonstrates that ultra-high cell density cell culture fluid can be successfully prepared using alternating tangential flow filtration (ATF). The patent does not involve concentration by perfusion, nor does it involve subsequent high-inoculation density culture and protein production.

[0009] In order to further increase the cell density at the seed stage to achieve higher density inoculation for target protein production, common cell culture processes face the following problems:

[0010] First, regardless of the process used, the final density of the seed culture will vary significantly depending on the cell line or clone. Many cell lines grow slowly, making it difficult to achieve high densities, resulting in suboptimal seeding densities. Furthermore, for cells with slower growth rates and lower final densities, more resources are often required to optimize the seed culture process to achieve the desired results.

[0011] Second, based on the current expansion of seed culture medium, further increasing IFB inoculation density and productivity to higher levels faces numerous challenges. If culture is prolonged during the expansion phase, the cell density may not be effectively reached at higher levels. If perfusion time is extended, a larger amount of perfusion medium will be required to meet cell consumption. Simultaneously, it is difficult to maintain stable control of high cell density culture conditions, such as DO, pH, pCO2, and foam control. If the inoculation density is increased without sufficiently increasing the transfer cell density, the dilution ratio from N-1 to production will decrease, affecting the cell culture state and protein synthesis during production.

[0012] There is still a need in this field to find a method for significantly increasing the cell density of the seed culture solution, achieving high inoculation density inoculation and culture, while greatly improving the target protein yield, and significantly shortening the production time and perfusion culture medium consumption, thereby effectively utilizing facilities and culture medium to improve the protein production output of cells. Summary of the Invention

[0013] To solve the above technical problems, one aspect of the present application provides a method for increasing the density of transferred cells in a culture medium, the method comprising:

[0014] Growing the cells by perfusion in a seed culture medium; and

[0015] The seed culture solution is concentrated by perfusion.

[0016] In another aspect, the present application provides a method for high cell density inoculation in production, the method comprising:

[0017] a. obtaining a seed culture solution, wherein the seed culture solution is obtained by increasing the transfer cell density of the culture solution, the method for increasing the transfer cell density of the culture solution comprising: culturing cells by perfusion in the seed culture solution; and concentrating the seed culture solution by perfusion;

[0018] b. inoculating the concentrated seed culture into the production medium; and

[0019] c. Culturing the cells in the production medium to produce the desired product.

[0020] abbreviation

[0021]

[0022] BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The present application will be described in more detail below with reference to the accompanying drawings, in which:

[0024] Figure 1 This is a schematic diagram of N-1 perfusion concentration in different production process modes to achieve high-density inoculation according to one embodiment of the present application.

[0025] Figure 2 ab show the cell density (a) and viability (b) curves of clone AN-1 concentrated by perfusion according to one embodiment of the present application.

[0026] Figure 3 ad show the UIFB production performance and traditional fed-batch (TFB) performance of clone A using different N-1 concentration parameter settings according to one embodiment of the present application, with respect to: cell growth (a), viability (b), lactate metabolism (c) and product titer (d).

[0027] Figure 4 ab show the product quality comparison of two UIFB processes and TFB of clone A under different perfusion and filtration conditions according to one embodiment of the present application, including: product purity and charge isomers (a), and N-glycan distribution (b).

[0028] Figure 5 ab show the cell density (a) and viability (b) curves of the perfusion concentration process of clone B N-1 according to one embodiment of the present application.

[0029] Figure 6 af show a comparison of TFB, IFB, IPFB, UIFB and UI-IPFB production processes using clone B in terms of cell growth (a), viability (b), lactate metabolism (c), product titer (d), product purity (e) and charge isomers (f) according to one embodiment of the present application.

[0030] Figure 7 af show a comparison of N-1 cell growth (a), production cell growth (b), viability (c), lactate (d), IVCD and Qp (e) and relative product titer (f) using TFB, IFB, IPFB and UIFB, UI-IPFB production processes of clone A according to one embodiment of the present application.

[0031] Figure 8 ae show a comparison of N-1 cell growth (a), production cell growth (b), viability (c), lactate (d), and product titer (e) between 3L and 250LUI-IPFB production using clone A according to one embodiment of the present application.

[0032] Figure 9af show a comparison of the performance of clone C UI-IPFB between 2 hours and 24 hours of concentration according to one embodiment of the present application for: N-1 cell growth (a), N-1 viability (b), producer cell growth (c), lactate (d), product titer (e) and product quality results (f).

[0033] Figure 10 ag shows a comparison of N-1 cell growth (a), production cell growth (b), viability (c), IVCD (d), product titer (e), Qp (f) and product quality results (g) between the high inoculation UIPC and UI-CFB and low inoculation IPC and CFB processes using clone A according to one embodiment of the present application.

[0034] Figure 11 af show the N-1 perfusion and high-throughput concentration and UI-IPFB cell culture performance of clone A for: N-1 stage VCD (a), production VCD (b) and titer (c); and the cell culture performance of clone C for: N-1 stage VCD (d), production VCD (e) and titer (f).

[0035] Figure 12 ab show N-1 cell growth profiles of clones A and B at high enrichment ratios in terms of N-1 cell growth (a) and N-1 viability (b) according to one embodiment of the present application. DETAILED DESCRIPTION

[0036] The present application relates to a method for increasing the transfer cell density of a culture medium. The method for increasing the transfer cell density of a culture medium comprises culturing cells by perfusion in a seed culture medium. In the present application, the seed culture medium can be any liquid culture medium known in the art suitable for culturing cells and can be adjusted accordingly depending on the cells to be cultured. In one embodiment of the present application, the cell density refers to the viable cell density (VCD). In one embodiment of the present application, the cells express a target product. In one embodiment of the present application, the target product comprises a target protein or polypeptide. In one embodiment of the present application, the cells are genetically engineered to express the target protein or polypeptide. In one embodiment of the present application, the cells comprise mammalian cells. In one embodiment of the present application, the mammalian cells are cells that recombinantly express the target protein or polypeptide, also known as host cells. As used herein, "host cell" refers to a mammalian cell line selected and domesticated according to production conditions for the production of biologics in the pharmaceutical industry. Post-translational modification of the proteins expressed thereby has advantages in maintaining the biological activity, stability, and antigenicity of the proteins. Many cell lines are commercially available, such as the American Type Culture Collection (ATCC). Non-limiting examples of mammalian cells that can be used in the present application include CHO, BHK, HEK293, etc. In a preferred embodiment of the present application, the mammalian cells include Chinese hamster ovary cells (CHO), such as CHO-K1 cells. In one embodiment of the present application, the target protein includes a therapeutic protein. In one embodiment of the present application, the target protein is an exogenous protein, including, for example, a fusion protein, an antibody or an enzyme. As used herein, "exogenous protein" refers to a target protein encoded by a gene that is foreign to the host cell within the scope of genetic engineering, wherein DNA recombination technology is used to effectively amplify and express proteins with practical value in the host cell. In one embodiment of the present application, the target protein is a monoclonal antibody, including but not limited to antibodies targeting tumor-specific antigens, tumor-associated antigens and / or autoimmune-related antigens. In the present application, the culturing step can be any process that can carry out cell proliferation in a culture medium. In one embodiment of the present application, the culturing step includes perfusion culture. In one embodiment of the present application, the duration of the culturing step is 1-10 days, preferably 2-5 days. In one embodiment of the present application, the inoculation density of the perfusion culture is 0.3-10×10 6 cells / mL, preferably 1-3×10 6cells / mL. In the present application, the conditions of the culture step may include any parameters suitable for the proliferation of the cells to be cultured, including but not limited to, for example, temperature, humidity, DO, pH, pCO2, and foam control. In one embodiment of the present application, the temperature of the culture step is 35.5-37.5°C, preferably 36-37°C, and more preferably about 36.5°C. In one embodiment of the present application, the CO2 ventilation ratio of the culture step is set to 5-10%, preferably 6-8%. In one embodiment of the present application, the pH of the culture step is 6.6-7.4, preferably about 6.7-7.2. In one embodiment of the present application, the humidity of the culture step is 70-85%, preferably about 80%. In one embodiment of the present application, the DO of the culture step is 30-50%, preferably about 40%. In one embodiment of the present application, the cell density of the culture step is 10-200×10 6 cells / mL, preferably 20-100×10 6 In one embodiment of the present application, the flux rate of perfusion is 5-20 liters / square meter hour (LMH). In one embodiment of the present application, the flux rate of perfusion is 10-16LMH. In one embodiment of the present application, the flux rate of perfusion is about 5,10,12.5,16 or 20LMH.

[0037] The method for increasing the transfer cell density of the culture fluid also includes perfusion concentration of the seed culture fluid. In one embodiment of the present application, the seed culture fluid is concentrated by filtering the culture fluid using a perfusion device including a filtration device, and the volume of the waste culture fluid discharged is greater than the volume of the new culture fluid added. In the present application, the applicable filtration device can be any tangential flow filtration device known in the art that is suitable for filtering cells. In one embodiment of the present application, the perfusion concentration step includes the use of a tangential flow filtration device. In one embodiment of the present application, the tangential flow filtration device includes an ATF device and a TFF device. In one embodiment of the present application, the tangential flow filtration device includes a hollow fiber membrane, which may include an ultrafiltration membrane or a microfiltration membrane, for example, a 50kDa ultrafiltration membrane or a 0.2 micron microfiltration membrane, preferably a microfiltration membrane. In one embodiment of the present application, the perfusion concentration step begins on the last day of the culturing step or after the culturing step. In one embodiment of the present application, the perfusion concentration step lasts for 1-24 hours, preferably 2-5 hours. In one embodiment of the present application, the perfusion concentration step lasts for 1-24 hours, preferably 2-10 hours, and more preferably 3-5 hours. In another embodiment of the present application, the perfusion concentration step starts 1-3 days, for example, 1 or 2 days, before the end of the culturing step. In another embodiment of the present application, the perfusion concentration step lasts for 24-72 hours, preferably 24-48 hours, and more preferably about 24 hours. In one embodiment of the present application, the cell density of the perfusion concentration step is at least 1.5 times, preferably at least 2 times, the cell density of the culturing step before concentration. In one embodiment of the present application, the filtration ratio of the perfusion concentration step is 40-140. In the present application, "filtration ratio" refers to the value of the liquid flow rate flowing into the tangential flow filtration device divided by the liquid flow rate passing through the tangential flow filtration device. In one embodiment of the present application, the cell density of the perfusion concentration step is 40-400×10 6 cells / mL or higher, for example, 100-120×10 6 cells / mL, or 180-200×10 6 cells / mL, or 300-400×10 6 In one embodiment of the present application, the culture fluid obtained through the perfusion concentration step can be used as the seed culture fluid of subsequent cell culture, or directly frozen and preserved or used, or suitable other purposes known in the art. In one embodiment of the present application, the flux rate of perfusion is 5-20 liters / square meter hour (LMH). In one embodiment of the present application, the flux rate of perfusion is 10-16LMH. In one embodiment of the present application, the flux rate of perfusion is about 5,10,12.5,16 or 20LMH.

[0038] This application also relates to a method for high-cell-density seeding during production. The method includes obtaining a concentrated seed culture solution, wherein the seed culture solution comprises a culture solution obtained by increasing the transfer cell density of the culture solution. The method for increasing the transfer cell density of the culture solution comprises: culturing cells by perfusion in the seed culture solution; and concentrating the seed culture solution by perfusion. In one embodiment of the present application, the method for increasing the transfer cell density of the culture solution is as described above.

[0039] The method for high cell density inoculation in production further comprises inoculating the concentrated seed culture solution into the production medium. In one embodiment of the present application, the inoculation dilution ratio of this step is at least 3 times, preferably at least 4-6 times. In one embodiment of the present application, the inoculation density of this step is 10-100×10 6 cells / mL, for example, 10-20×10 6 cells / mL, 20-25×10 6 cells / mL, 30-60×10 6 cells / mL, 40-80×10 6 cells / mL or 50-80×10 6 In the present application, the production culture medium included in this step can be any culture medium known in the art that can promote cell proliferation or target protein expression.

[0040] The method of performing high cell density inoculation in production also includes performing cell culture in the production medium to produce the target product. In the present application, this step can be any process that can cause cell proliferation or target protein expression. In one embodiment of the present application, the cell culture in this step lasts for 6-20 days, preferably 8-18 days, and more preferably about 12-14 days. In one embodiment of the present application, this step includes switching the temperature of the cell culture or maintaining a constant temperature of the cell culture. In one embodiment of the present application, this step includes lowering the temperature of the cell culture. In one embodiment of the present application, this step includes maintaining the temperature of the cell culture substantially constant. In one embodiment of the present application, lowering the temperature of the cell culture starts on the 0th to 5th day after inoculation. In one embodiment of the present application, lowering the temperature of the cell culture starts on the 0th to 1st day after inoculation. In one embodiment of the present application, lowering the temperature of the cell culture starts within 8 hours after inoculation. In one embodiment of the present application, lowering the temperature of the cell culture starts on the 1st day after inoculation. In one embodiment of the present application, lowering the temperature of the cell culture includes performing cell culture at a temperature lower than the seed solution temperature. In one embodiment of the present application, the temperature of the culture medium is 29-37°C, preferably 36-37°C, more preferably about 36.5°C, and the switching temperature of the cell culture in this step is 29-35°C, preferably 30-34°C, more preferably about 31-33°C. In one embodiment of the present application, lowering the temperature of the cell culture comprises first culturing the cells at the starting temperature, and then culturing the cells at the switching temperature after reaching the predetermined conditions, wherein the switching temperature is lower than the starting temperature. In one embodiment of the present application, the starting temperature is 36-37°C, preferably 35-37°C, more preferably about 36.5°C. In one embodiment of the present application, the switching temperature is 25-35°C, preferably 30-32°C, more preferably about 31°C. In one embodiment of the present application, the predetermined conditions include a predetermined time, for example, culturing cells at the starting temperature for 12-48 hours, preferably about 24 hours. In one embodiment of the present application, the predetermined conditions include a predetermined cell density, such as 16-20×10 6In one embodiment of the present application, the step includes batch culture and / or medium exchange and / or perfusion using a feed medium. In one embodiment of the present application, the step includes batch culture using a feed medium. In one embodiment of the present application, the step includes performing medium exchange by perfusion. In one embodiment of the present application, the step includes continuous perfusion culture with constant medium exchange by perfusion. In the present application, the medium included in the medium exchange by perfusion can be any medium known in the art that can promote cell proliferation or expression of a target protein. In one embodiment of the present application, the medium included in the medium exchange by perfusion is the same as the medium included in the seed culture solution. In one embodiment of the present application, the medium included in the medium exchange by perfusion is different from the medium included in the seed culture solution. In one embodiment of the present application, the amount of medium exchanged by perfusion can be 0.5-6 times, 1.5-3 times, or 1.5-2 times the volume of the starting working volume. In one embodiment of the application, the medium can be changed by perfusion for a sustainable 6-48 hour. In one embodiment of the application, the medium can be changed by perfusion for a sustainable 6-48 hour. In one embodiment of the application, the medium can be changed by perfusion for a sustainable 6-48 hour. In one embodiment of the application, the medium can be changed by perfusion for a sustainable 6-48 hour. In one embodiment of the application, the medium can be changed by perfusion for a sustainable 2-7 day and / or 5-12 day after inoculation. In one embodiment of the application, the step includes intermittent perfusion batch feed culture. In one embodiment of the application, the step includes continuous perfusion production, such as enhanced perfusion culture or concentrated batch feed culture.

[0041] By the method of the present application, during and / or after the perfusion seed culture stage, a perfusion device with a hollow fiber membrane is used to concentrate and filter the culture fluid. By increasing the discharge of cell culture waste liquid, the final cell density is concentrated to 1.5, 2.0, 3.0, 4.0 or even 5.0 times the original cell density; at the same time, the necessary conditions for cell culture, including temperature, dissolved oxygen, pH, ventilation, culture medium supply, etc., are maintained, and the concentration time is minimized to eliminate adverse effects on the cell state. The concentrated seed liquid is transferred to the N production bioreactor, and the initial inoculation VCD is significantly improved. The schematic diagram of the process is shown in FIG. Figure 1 Some main process characteristics are described and compared in Table 1.

[0042] Table 1 Comparison of production cell culture processes and characteristics

[0043]

[0044]

[0045] The following will clearly and completely describe the technical solutions of this application in conjunction with specific embodiments. Obviously, the described implementation methods are part of this application, not all examples. Based on the examples in this application, all other examples obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0046] Example

[0047] The technical solutions of the present invention will be described in more detail below with reference to specific embodiments. The following embodiments are merely illustrative and do not constitute any limitation on the technical solutions of the present invention. The specific materials, steps, conditions, values, or numerical ranges and other technical parameters in the following embodiments are merely illustrative and are not intended to be exhaustive or limiting.

[0048] The cells used in the following examples are CHO-K1 cell lines from WuXi Biologics that have been stably transfected to express different types of monoclonal antibodies (e.g., IgG1 and IgG4). Clone A (WuXia 3.0 platform cell line 196-N079-05-20) was used in Examples 1, 3, 5, 6, and 7, Clone B (WuXia 2.0 platform cell line 154A-01-010) was used in Examples 2 or 7, and Clone C (WuXia 3.0 platform cell line 196-N079-06-08) was used in Examples 4 and 6.

[0049] The following culture media used in the following examples are ActiPro (Hyclone, Cat No.: SH31037); CDCHO (Gibco, Cat No.: 12490); CellBoost 7a (HyClone, Cat No.: SH31026); CellBoost 7b (HyClone, Cat No.: SH31027); WXBM and WXFM (WuXi Biologics' proprietary in-house culture media). Key equipment information related to the experimental examples is listed in Table 2. Further details regarding material and culture medium preparation are listed in Tables 3 and 4, respectively.

[0050] In addition to the specific methods, equipment, and materials used in the embodiments, based on the understanding of the prior art by those skilled in the art and the description of the present invention, any methods, equipment, and materials in the prior art that are similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention may also be used to implement the present invention.

[0051] Table 2: List of main experimental equipment

[0052]

[0053]

[0054] Table 3: Main material information list

[0055]

[0056] Table 4: List of main culture medium formula information

[0057]

[0058] In order to more clearly illustrate the technical solution of the present invention, further description will be given in conjunction with embodiments.

[0059] Example 1: N-1 perfusion and concentration using UIFB produced from clone A

[0060] To evaluate the impact of ATF perfusion parameters on the N-1 concentration process, clone A was used to investigate different perfusion rates and filtration ratios during the N-1 concentration phase. The concentrated, high-cell-density seed culture was used to inoculate the production bioreactor. Detailed process parameters for the N-1 and production bioreactor are summarized in Tables 5 and 6, respectively.

[0061] For vial thawing, first thaw a vial of frozen cells in a 37.0 ± 1.0°C water bath for 120-150 seconds. Then transfer the cells to a shake flask filled with pre-warmed subculture medium (CD CHO). Subsequently, transfer the shake flask to a shaking incubator with the following settings: 36.5°C incubation temperature; 110 rpm shaker speed, 50 mm orbital diameter; and 6% CO2. After at least 5 minutes, remove a sample from the shake flask for VCD and viability measurement. Based on the results, adjust the inoculation cell density to 0.10-0.20 × 10 6 cells / mL target VCD. After 72±12 hours of culture, when the transfer cell density is 1.5~2.2×10 6 When the cells are within the range of 10 cells / mL, the cells are ready for passage. For the cell expansion before N-1 in the following examples, the cells were thawed and passaged in shake flasks under the same conditions and settings.

[0062] During the N-1 concentration process, the perfusion flow rate can be terminated or maintained, and the harvest flow rate can be increased as described in Table 5. Within 2 to 5 hours of concentration, the cell culture volume is reduced to approximately 50% of the initial culture volume. In this example, the effects of different N-1 concentration conditions were investigated, and the results on cell culture performance and protein production during the UIFB production phase were investigated.

[0063] Table 5N-1 Perfusion Concentration Process Parameters

[0064]

[0065]

[0066] Table 6 TFB and UIFB production bioreactor parameters

[0067]

[0068] result

[0069] like Figure 2 As shown in ab, the different N-1 concentration parameters listed in Table 5 had no significant effect on N-1 VCD and viability. In this study, the peak VCD of N-1 of cell line A reached ∼50×10 6 cells / mL and then concentrated to ∼100×10 cells / mL within 2-3 hours via an ATF controller. 6 cells / mL.

[0070] like Figure 3 As shown in ad, the high seeding density UIFB process significantly increased the peak VCD to ∼30×10 6 cells / mL, compared to ~15×10 6 Due to the higher inoculation cell density, the titer increased from 3.0 g / L to 5.0-6.0 g / L on days 8-10, which was 60%-100% higher than that of the TFB control.

[0071] like Figure 4 As shown in ab, PQA results obtained for the UIFB conditions and the TFB control were comparable in terms of product-related purity and N-glycosylation. All process conditions exhibited comparable levels of aggregation and fragmentation. Regarding charge variants, a slight increase in the main peak and a slight decrease in the acidic peak were observed with the UIFB conditions. In N-glycan analysis, UIFB showed slightly lower levels of Man5 and G0 species than the TFB control. Other glycosylation patterns were not significantly different.

[0072] Example 2: N-1 perfusion and concentration of UI-IPFB produced using clone B

[0073] In this study, the N-1 perfusion and concentration process at different ATF2 filtration ratios was further evaluated using clone B. The ATF flow rate and harvest rate were adjusted according to Table 7 to achieve the target ATF filtration ratio during the N-1 concentration process.

[0074] Table 7N-1 Study on Filtration Ratio in Concentration

[0075]

[0076] After N-1, use 5 kinds of different production culture media from the seed inoculation of representative conditions, and described representative conditions comprises batch seed expansion, perfusion seed expansion and concentrated perfusion seed expansion.Production culture pattern comprises that traditional batch formula flow-addition (TFB), enhanced batch formula flow-addition (IFB), intermittent perfusion batch formula flow-addition (IPFB), super enhanced batch formula flow-addition (UIFB) and super enhanced intermittent perfusion batch formula flow-addition (UI-IPFB) are cultivated.Inoculation density, perfusion scheme and control parameters are listed in table 8.

[0077] Table 8 Production parameters of different culture modes

[0078]

[0079]

[0080] result

[0081] like Figure 5 As shown in ab, with different ATF filtration ratios from 40 to 140, the VCD was finally transferred from N-1 perfusion amplification to 50-60×10 6 cells / mL reached 180-200×10 6 During the concentration process, no hollow fiber membrane clogging or increase in transmembrane pressure was observed at any ATF filtration ratio evaluated. Furthermore, no differences in cell growth and viability were observed under the different N-1 conditions. Therefore, concentrated seeds from N-1 BR#01 and BR#04 were used to inoculate both the UIFB and UI-IPFB production bioreactors.

[0082] from Figure 6 ad, the initial seeding density of TFB was 1.0×10 6 cells / mL, and the initial seeding density of IFB and IPFB was 10×10 6 cells / mL, and the initial seeding density of UIFB and UI-IPFB was 35×10 6 During the culture process, the peak cell densities of TFB, IFB, UIFB, and UI-IPFB reached 26, 30, 49, and 49×10 6 cells / mL. For the UIFB process, lactate rapidly accumulated to ~3 g / L on day 8, and cell viability began to gradually decline, falling below 60% on day 10. Similarly, IFB promoted lactate accumulation from day 10, reaching ~3 g / L on day 13, while viability decreased to ~60%. For UI-IPFB, which underwent intermittent perfusion during culture, lactate did not increase, and cell viability remained at ~70% until day 13.

[0083] Results showed that UI-IPFB produced 12.1 g / L of protein on day 13, double the titer of UIFB. Compared to IFB, UIFB's titer reached 6.1 g / L on day 8, while IFB reached 4.6 g / L on day 13. Due to IPFB's superior cell density and viability, the final titer reached 6.3 g / L, comparable to that of UIFB and 1.3-1.4 times that of TFB and IFB.

[0084] from Figure 6 The product-related purity, as shown by SEC and CE profiles, was found to be similar between the different production culture modes. Regarding the charge distribution of iCIEF, the different production modes produced comparable profiles, but intermittent perfusion in IPFB and UI-IPFB showed a higher main peak and a lower acidic peak.

[0085] Example 3: Production of UIFB, IPFB and UI-IPFB using Clone A

[0086] In this study, different culture modes were compared in a 3-L bench-scale bioreactor using clone A. The best performing process from the UI-IPFB process was then further scaled up to a 250-L single-use bioreactor.

[0087] Process parameter settings for N-1 and production stages

[0088] N-1 perfusion concentration in 3L bioreactors and 50L disposable bioreactors followed the procedures shown in Table 9. The production process parameters for 3L and 250L production are listed in Table 10.

[0089] Table 9 N-1 process parameters at 3L and 50L scales

[0090]

[0091] Table 10 Production parameters of different culture modes

[0092]

[0093]

[0094] result

[0095] like Figure 7 As shown, using N-1 enrichment culture, the production inoculum density of UIFB and UI-IPFB doubled to ∼20 × 10 6 cells / mL, while the production seeding densities of TFB, IFB and IPFB were 1.0×10 6 cells / mL and 10×10 6 cells / mL.

[0096] 20×10 6 UI-IPFB cultures seeded with 10 cells / mL showed improved performance, with a peak VCD of ∼35 × 10 6 cells / mL, the highest value among all conditions, and the viability was maintained at more than 60% until day 14. 6 The IFB process with 20×10 6 UIFB inoculated with 10 cells / mL was terminated early because the viability dropped to ~50%. In addition, lactate also peaked on the 8th and 9th days, respectively. There was no significant difference between the Qp of TFB and IFB. Thanks to the common advantages of ultra-high inoculation and IPFB culture mode, the cumulative integral of viable cell density (IVCD) of UI-IPFB increased by 40-70% compared with IFB and UIFB controls. In addition, the lactate peak under IPFB conditions was also effectively suppressed. Compared with IFB and UIFB controls, the Qp of UI-IPFB increased by ~70%. Overall, the final titer reached ~4 times that of TFB and ~2 times that of IFB and UIFB controls.

[0097] like Figure 8 As shown, the UI-IPFB process for cell line A was scaled up from 3L to 250L single-use bioreactors (SUBs), using a 50L SUB for N-1 perfusion and concentration, demonstrating highly comparable in-process trends in cell growth, lactate metabolism, and protein production. This highly comparable performance demonstrates the scalability of both N-1 perfusion concentration and UI-IPFB production for large-scale manufacturing.

[0098] Example 4: N-1 enrichment strategy of UI-IPFB using clone C

[0099] N-1 perfusion and concentration strategy

[0100] As shown in Table 11, for concentration method I, N-1 cultures were concentrated within 2 to 3 hours after perfusion on day 6 by increasing the harvest flow rate to 10 VVD, reducing the culture volume by ∼50%. For method II, N-1 cultures were concentrated starting on day 5 until the end of perfusion on day 6, reducing the culture volume by ∼50% by increasing the ATF harvest flow rate by an additional 0.5 VVD while maintaining the perfusion rate constant. The performance of N-1 cell cultures was evaluated using these two concentration methods.

[0101] For UI-IPFB production, both concentration methods used a similar 4:1 split ratio for inoculation, reaching starting densities of 35 and 45 × 10 6The detailed production parameters of both seeds followed similar UI-IPFB operation, as listed in Table 10.

[0102] Table 11 N-1 process parameters of different concentration methods

[0103]

[0104] result

[0105] like Figure 9 As shown, using two different N-1 enrichment methods, the transfer VCD reached ∼179 and ∼136 × 10 6 To maintain a similar split ratio of 4:1 from N-1 to production, the final seeding densities were ∼37 and ∼50 × 10 6 cells / mL. Higher seeding density increased the peak VCD to more than 70×10 6 cells / mL, compared to a lower seeding density of ~58×10 6 cells / mL. With the exception of VCD, both production conditions had similar viability profiles. At the end of production, the two UI-IPFB conditions using two different inoculation densities produced 18.0 g / L and 15.6 g / L of product, respectively, compared to ~4.0 g / L in TFB (data not shown). PQA results obtained with the N-1 24-hour and 2-hour concentration conditions were comparable in terms of product-related purity, charge variants, and N-glycosylation.

[0106] This study demonstrates that both enrichment methods for N-1 perfusion expansion achieve the same goal of increasing the density of transferred cells. The choice of enrichment strategy can be determined based on the relative cell growth performance of each cell line using the two methods.

[0107] Example 5: High cell density seeding in continuous production using clone A

[0108] In this study, we further evaluated the effect of high seeding densities achieved by N-1 perfusion concentration in continuous culture processes such as IPC and CFB, and compared them with conventional IPC and CFB processes using low seeding densities in 3-day batch expansion.

[0109] Cell culture processes in the production phase

[0110] The N-1 perfusion concentration culture process used in this study follows the same process as described in Example 4. For all conditions in this example, laboratory-scale production was carried out in a 3-liter glass bioreactor connected to an ATF2 hollow fiber. The pore sizes of the ATF hollow fiber filters used in production were 50 kDa and 0.20 μm for CFB and IPC, respectively. The pH value was controlled in the range of 6.7-7.2, and the DO was controlled at 40%. Constant stirring was set at 285 rpm and switched to 320 rpm when the oxygen demand reached the preset standard of 0.2 VVM. During the production process, the starting temperature was set to 36.5 ° C and when the VCD reached 40 to 45 × 10 6 When the VCD exceeds 20×10 6 cells / mL, the perfusion rate of CFB and IPC was 1.0VVD, and when VCD was lower than 20×10 6 When the perfusion rate is 0.3-0.5 VVD, a certain proportion of feed medium is mixed into the perfusion medium to support high-density cell culture and production.

[0111] result

[0112] The results of the high seeding density continuous process are as follows Figure 10 The peak VCD of clone A reached 60×10 after 3 days of perfusion. 6 cells / mL and then concentrated to ∼120×10 6 cells / mL( Figure 10 a) Using concentrated cultures, the production inoculum density reached ~30 × 10 6 cells / mL with a split ratio of 4:1.

[0113] like Figure 10 As shown, UI-CFB and UIPC cultures showed a decrease in cell exponential growth phase and reached ∼60 × 10 6 cells / mL, while 0.5×10 6 The low seeding density of 10 cells / mL resulted in a prolonged culture termination due to the longer cell proliferation phase, although similar peak VCD was achieved on day 9, 6 days later than the UI-CFB and UIPC conditions. Thus, the cumulative IVCD of the UI-CFB and UIPC conditions increased by 15-20% compared to their corresponding CFB and IPC controls. Moreover, the high seeding density obtained from N-1 perfusion concentration also contributed to the higher Qp and ultimate productivity. Compared to the low seeding CFB and IPC, the Qp of UI-CFB and UIPC increased by ∼100% and ∼25%, respectively ( Figure 10f). Under UI-CFB conditions, the cumulative volume productivity reached 2 times that of low-inoculation CFB, and in UIPC production reached 1.5 times that of low-inoculation IPC ( Figure 10 e). Overall, comparable product-related purity was observed for SEC and SE-SDS under the different conditions investigated, but slightly higher levels of aggregation of HMW species were detected in UIPC, CFB, and UI-CFB ( Figure 10 g). Regarding charge variants, lower acidic species were observed in the UI-CFB product, and higher basic modifications were identified under both CFB and UI-CFB conditions. Regarding N-glycan modifications, lower galactosylation (as indicated by %G1F and %G2F) and higher Man5 were observed under UIPC conditions compared to the IPC control, while no significant differences were observed between the CFB and UI-CFB products.

[0114] Example 6: High-throughput studies of N-1 enrichment using clones A and C

[0115] In this study, both clone A and clone C were used for N-1 concentration robustness studies. In terms of culture medium, commercially available and in-house proprietary basal and feed media of the cell lines were used in this study to compare different cell culture processes. In this study, both microfiltration (MF) and ultrafiltration (UF) high throughput ranges were evaluated to demonstrate the robustness of N-1 perfusion concentration of UI-IPFB.

[0116] Process parameters in the N-1 and production stages

[0117] In this study, ATF hollow fiber filters with different pore sizes were used, including a 0.20 μm microfiltration filter (MF) and a 50 kDa ultrafiltration filter (UF). The normal recommended flux rate for ATF perfusion is limited to less than 5 liters / square meter hour (LMH), and the optimal range is 1 to 3 LMH. In this study, high flux rates of 5, 10, 12.5, 16, and 20 LMH were evaluated. In addition, the N-1 perfusion culture used in this study and the subsequent UI-IPFB production followed the same parameters as shown in Tables 9 and 10.

[0118] result

[0119] like Figure 11 As shown, when using clone A ( Figure 11 a) and clone C( Figure 11 d), no significant differences were observed when both microfiltration (MF) and ultrafiltration (UF) were used at high flux.

[0120] For clone A, the N-1 perfusion culture reached 40-45×10 6The culture was then concentrated to 90-100 × 10 cells / mL by both MF and UF. 6 cells / mL, with ATF fluxes ranging from 5 to 16 LMH. No ATF hollow fiber membrane clogging or leakage was observed during the cell culture concentration process. Cultures with the highest fluxes of 16 and 5 LMH were inoculated into the UI-IPFB production bioreactor to verify performance. Comparable cell culture performance and productivity results were achieved, with final productivity reaching 11 to 13 g / L on day 14.

[0121] For clone C, the VCD of N-1 culture on day 6 was 60-70 × 10 6 The culture was then concentrated to 120-140×10 cells / mL by MF. 6 cells / mL, low-throughput and high-throughput were 5 and 20 LMH, respectively. Afterwards, the concentrated seeds were inoculated into UI-IPFB production, with an initial VCD of 30-35×10 6 cells / mL. Under high-throughput connection conditions, the peak VCD reached ∼50×10 6 cells / mL, similar to the control seeded from normal flux concentration conditions. Ultimately, similar titers were obtained from both conditions.

[0122] Example 7: N-1 perfusion and concentration using clone A and clone B

[0123] In this study, highly enriched N-1 cultures were evaluated using clones A and B. Commercially available and proprietary basal and feed media were used for N-1 perfusion.

[0124] result

[0125] like Figure 12 As shown in Figure 2, the concentration after N-1 perfusion was as high as 6.8 times, and the final cell density was ≥400×10 6 The results showed that the concentration cell density in N-1 perfusion concentration can be increased to 400×10 6 cells / mL or more, and the concentration factor can be 2, 3, 4, 5, 6, 7 times or more.

[0126] In summary, the present invention has the following advantages compared with the prior art:

[0127] 1. The present invention significantly increases the density of transferred cells in N-1 seed culture medium, or significantly reduces the time required to reach the same cell density in cell expansion;

[0128] 2. By using seeds from cell culture concentrate during or after the N-1 perfusion expansion process, we demonstrated that UIFB can be seeded at much higher densities than IFB. This method increases the starting inoculum VCD, total cell biomass, and protein yield in fed-batch production cultures.

[0129] 3. By using cell culture concentrate during or after the N-1 perfusion expansion process, UI-IPFB achieves a seeding density far higher than that achieved with IFB or IPFB production processes. The UI-IPFB culture model uses limited intermittent perfusion cycles to eliminate the accumulation of toxic byproducts and replenish the cell culture with fresh medium, thereby improving cell culture performance and addressing the potential for decreased cell viability and low productivity often encountered in IFB or UIFB processes.

[0130] Demonstrated that UI-IPFB significantly increased protein yield with similar or better product quality;

[0131] 4. UI-IPFB demonstrated significant productivity improvements compared to TFB, IFB, or UIFB for multiple cell lines studied. In terms of scalability, UI-IPFB has been demonstrated to be scalable and combines the operational simplicity of batch culture with the superior cell performance of perfusion processing.

[0132] 5. Since the titer can be greatly increased without modifying facilities and equipment, UI-IPFB provides more competitive COG production.

[0133] 6. Compared with conventional low-seeding IPC and CFB processes, the high seeding density achieved by N-1 perfusion concentration in continuous UIPC and UI-CFB processes also significantly improves the unit volume productivity and unit cell productivity, with a shortened production period.

[0134] The above are only specific application examples of the present application and do not constitute any limitation on the scope of protection of the present application. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list and describe all implementation methods here. Any similar technical solutions formed by equivalent transformation or equivalent replacement fall within the scope of protection of the present application.

Claims

1. A method for increasing the transfer cell density of a culture medium, the method comprising: Cells were cultured by perfusion in seed culture medium; and The seed culture solution is concentrated by perfusion.

2. The method according to claim 1, wherein the cell expresses a target product, preferably a recombinant protein or polypeptide; and / or optionally, the cell comprises a mammalian cell.

3. The method according to claim 1, wherein the culturing step comprises perfusion culture; optionally, the duration of the culturing step is 1-10 days, preferably 2-5 days; and / or optionally, the seeding density of the perfusion culture is 0.3-10×10 6 cells / mL or higher.

4. The method according to claim 1, wherein the perfusion concentration step begins on the last day of the culturing step or after the end of the culturing step, or begins 1-3 days before the end of the culturing step; optionally, the perfusion concentration step lasts for 1-24 hours, preferably 2-5 hours; and / or optionally, the cell density in the perfusion concentration step is at least 1.5 times, preferably at least 2 times, the cell density in the culturing step before concentration.

5. The method according to claim 1, wherein the perfusion concentration step comprises using a tangential flow filtration device; optionally, the tangential flow filtration device comprises an ATF device and a TFF device; optionally, the tangential flow filtration device comprises a hollow fiber membrane; optionally, the hollow fiber membrane comprises an ultrafiltration membrane or a microfiltration membrane; and / or optionally, the cell density in the perfusion concentration step is 40-400×10 6 cells / mL or higher.

6. A method for high cell density inoculation in production, the method comprising: a. Obtaining a concentrated seed culture solution, wherein the seed culture solution is obtained by the method according to any one of claims 1-5; b. inoculating the concentrated seed culture into the production medium; and c. Culturing the cells in the production medium to produce the target product.

7. The method according to claim 6, wherein the inoculation dilution factor of step b is at least 3 times, preferably at least 4-6 times.

8. The method according to claim 6, wherein step c comprises switching the temperature of the cell culture or maintaining the cell culture temperature constant.

9. The method according to claim 6, wherein step c comprises a batch culture with a feed medium and / or medium exchange and / or perfusion, and / or comprises a continuous perfusion culture with constant medium exchange by perfusion.

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