Method for increasing transitional cell density in seed cultures and achieving high cell density inoculation in production cultures

Abstract

The present application discloses a method for increasing the transfer cell density in a seed culture to achieve a high cell density inoculation in a production culture. The method for increasing the transfer cell density of a culture includes culturing cells in a seed culture by perfusion and subjecting the seed culture to concentration by perfusion.

Description

[Technical Field]

[0001] This application relates to the field of cell culture technology, and in particular to methods for high seeding density culture to enhance expression of target products. [Background technology]

[0002] The production of genetically engineered proteins by mammalian cell culture is a commonly used method for obtaining therapeutic proteins such as monoclonal antibodies. Advances in mammalian cell culture technology have significantly advanced the development and biomanufacturing of therapeutic proteins, such as biopharmaceuticals, including antibodies, fusion proteins, and enzymes. The following is a brief introduction to the production processes commonly used in the biopharmaceutical industry.

[0003] Currently, the domestic and international biopharmaceutical industry mainly uses fed-batch processes to express and produce monoclonal antibodies or fusion proteins by engineered mammalian cell lines.

[0004] Chinese Patent No. 201210057859.7 points out that conventional fed-batch processes (FBs) as a conventional method require process development and optimization to achieve desired production performance. For cGMP (Current Good Manufacturing Practices) production, it often requires bulky cell culture equipment and complex support systems that are repeatedly verified. Traditional fed-batch culture of mammalian cells has several insurmountable drawbacks, such as low cell biomass, low protein expression in a single batch, the inability to eliminate harmful metabolites, and complex process control. Chinese Patent No. 202010592471.1 also notes that during the fed-batch process, medium components are not fully replenished at the site where cell growth is consumed, which weakens the nutrient supply effect of fed-batch culture and therefore affects cell growth efficiency.

[0005] Perfusion is a continuous cell culture mode in which fresh culture medium is continuously supplied to a reactor during the process, and the expression product is continuously recovered along with the discharged cell culture medium. Chinese Patent No. 201210057859.7 points out that the perfusion process can significantly increase cell biomass and protein concentration. However, this process mode consumes a large amount of medium, and the recovery volume requires sufficient storage space and special downstream processing design. Therefore, the perfusion process mode is often used for protein products that have low stability, are prone to changes in quality parameters, and require real-time recovery.

[0006] The process model disclosed in International Application PCT / US2008 / 072612 combines the features of early-stage perfusion and fed-batch culture to achieve higher protein yields. However, this process model fails to enhance seed density accumulation during the growth phase, occupying the production equipment of large-scale reactors for a long time during the cell exponential growth phase, resulting in a longer production period and reduced equipment utilization efficiency.

[0007] WO 2021 / 021973 discloses an intensified fed-batch (IFB) process developed based on the characteristics of the cell culture process described above. In the seed growth stage (N-1), a nutrient-enriched medium, batch feeding, and a perfusion process are applied to rapidly and efficiently accumulate a large number of cells. When the production stage (N) reactor is inoculated, the initial inoculum density increases (10-30×10). 6With perfusion culture (perfusion culture time), higher peak cell densities are achieved during the culture, thereby accumulating higher protein production and improving batch yield and efficiency. However, many challenges remain for the inoculation density and yield to continue reaching higher levels in IFB. If the culture is extended during the growth phase, the cell density cannot effectively reach higher levels due to limited growth capacity; if the perfusion culture time is extended, more perfusion culture medium is required to meet cell consumption. At the same time, the conditions for controlling high seeding density culture, such as dissolved oxygen (DO), pH, pCO2, and foam control, are difficult to maintain stably.

[0008] Chinese Patent No. 201580062059.6 demonstrates the adoption of optimized perfusion culture techniques to obtain ultra-high cell density culture media that can be cryopreserved at high cell densities while maintaining excellent cell viability and quality. The highest cell density reported in that embodiment is 100×10 6 Viable cells / mL are reached. This report indirectly demonstrates that cell culture media with ultra-high cell densities can be successfully prepared using alternating tangential flow membrane filtration (ATF) equipment. The content described in this patent does not involve concentration by perfusion or subsequent high seeding density culture and protein production.

[0009] In order to further increase the cell density at the seeding stage to achieve a higher density inoculum for target protein production, commonly used cell culture processes face the following problems: First, regardless of the process used, the final density of the seed culture will vary depending on differences between cell lines or clones. Many of these cells grow slowly and have difficulty reaching high densities, resulting in inoculation densities that are unfavorable for production. Furthermore, for cells with slower growth rates and lower final densities, more resources are often required to optimize the cultivation process of the seed culture to achieve the expected goals.

[0010] Second, there are many challenges if the inoculum density and IFB yield continue to reach higher levels based on the growth of the current seed culture. If the culture is extended during the growth phase, the cell density may not be able to effectively reach higher levels. If the perfusion time is extended, more perfusion medium is required to meet cell consumption. At the same time, it is difficult to stably maintain conditions for controlling high cell density culture, such as DO, pH, pCO2, and foam control. Increasing the inoculum density without sufficiently increasing the transfer cell density reduces the dilution rate from N-1 to production, affecting the cell culture condition and protein synthesis during production.

[0011] There remains a need in the art to find methods to significantly increase the cell density of seed cultures, achieve high seeding density inoculation and cultivation, significantly increase target protein yield, and significantly reduce production time and perfusion medium consumption, thereby improving cellular protein production with efficient use of equipment and culture medium. Summary of the Invention

[0012] In order to solve the above technical problems, one aspect of the present application is a method for increasing the transitional cell density of a culture, comprising: Culturing cells by perfusion in a seed culture; and subjecting the seed culture to enrichment by perfusion.

[0013] In another aspect, the present application provides a method for high cell density inoculation in production, comprising: a. Obtaining a seed culture, wherein the seed culture is obtained by a method for increasing the transitional cell density of a culture, the method comprising culturing cells in the seed culture by perfusion and subjecting the seed culture to concentration by perfusion; b. inoculating the concentrated seed culture into a production culture medium; c. culturing the cells in the production culture medium to produce the target product.

[0014] [Table 1] [Brief explanation of the drawings]

[0015] The following is a more detailed description of the present application in conjunction with the accompanying drawings. [Figure 1] 1A-1D are schematic diagrams of N-1 perfusion and concentration to achieve high density inoculation in different production process modes according to an embodiment of the present application.

[0016] [Figure 2] 2a and 2b show the cell density (a) and viability (b) curves of perfusion and concentration of N-1 of clone A according to one embodiment of the present application.

[0017] [Figure 3] Figures 3a-d show the UIFB production performance using different N-1 enrichment parameter settings and conventional fed-batch (TFB) performance of clone A in terms of cell growth (a), viability (b), lactate metabolism (c), and product titer (d) according to one embodiment of the present application.

[0018] [Figure 4] 4a and 4b show a comparison of product purity and product quality, including charge variants (a) and N-glycan distribution (b), between two UIFB processes with different perfusion and filtration conditions of clone A and TFB, according to one embodiment of the present application.

[0019] [Figure 5] 5a and b show the cell density (a) and viability (b) curves of the Clone B N-1 perfusion and concentration process according to one embodiment of the present application.

[0020] [Figure 6]6a-d show a comparison of TFB, IFB, IPFB, UIFB, and UI-IPFB production processes using clone B according to one embodiment of the present application in terms of cell growth (a), viability (b), lactate metabolism (c), product titer (d), product purity (e), and charge variants (f).

[0021] [Figure 7] 7a-f show a comparison of TFB, IFB, IPFB and UIFB, UI-IPFB production processes using clone A according to one embodiment of the present application in terms of N-1 cell growth (a), production cell growth (b), viability (c), lactate (d), IVCD and Qp (e), and relative product titer (f).

[0022] [Figure 8] 8a-e show a comparison of 3 L and 250 L UI-IPFB production using clone A according to one embodiment of the present application in terms of N-1 cell growth (a), production cell growth (b), viability (c), lactate (d), and product titer (e).

[0023] [Figure 9] 9a-f show a comparison of UI-IPFB performance of clone C at 2 hours and 24 hours of enrichment according to one embodiment of the present application in terms of N-1 cell growth (a), N-1 viability (b), producer cell growth (c), lactate (d), product titer (e), and product quality results.

[0024] [Figure 10] 10a-g show a comparison between high seed UIPC and UI-CFB and low seed IPC and CFB processes using clone A according to one embodiment of the present application in terms of N-1 cell growth (a), production cell growth (b), viability (c), IVCD (d), product titer (e), Qp (f), and product quality results (g).

[0025] [Figure 11]Figure 11a-f shows the UI-IPFB cell culture performance of clone A at N-1 perfusion and high flow rate concentrations, and N-1 stage VCD (a), production VCD (b), and titer (c), and the cell culture performance of clone C at N-1 stage VCD (d), production VCD (e), and titer (f).

[0026] [Figure 12] 12a and b show the N-1 cell growth profiles of clones A and B with high concentration ratios according to embodiments of the present application in terms of N-1 cell growth (a) and N-1 viability (b). DETAILED DESCRIPTION OF THE INVENTION

[0027] The present application relates to a method for increasing the transitional cell density of a culture. The method for increasing the transitional cell density of a culture includes culturing cells by perfusion in a seed culture. In the present application, the seed culture may be any liquid culture medium known in the art suitable for culturing cells, and may be adjusted depending on the cells being 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 cells" refers to mammalian-derived cell lines selected and domesticated according to production conditions in the pharmaceutical industry to produce biopharmaceuticals. Post-translational modifications of expressed proteins are advantageous for maintaining the biological activity, stability, and antigenicity of the proteins. Many cell lines can be obtained from commercial sources, 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, and the like. 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, such as a fusion protein, an antibody, or an enzyme. As used herein, the term "exogenous protein" refers to a target protein encoded by a gene foreign to a host cell in the field of genetic engineering, whereby a protein of practical value is effectively amplified and expressed in a host cell through DNA recombinant technology.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-associated antigens. In the present application, the culturing step can be any process capable of causing 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 to 10 days, preferably 2 to 5 days. In one embodiment of the present application, the inoculation density of the perfusion culture is 0.3 to 10 x 10. 6 cells / mL, preferably 1-3 x 10 6 In the present application, the conditions for the culturing step may include any parameter suitable for the growth of the cells to be cultured, including, but not limited to, temperature, humidity, DO, pH, pCO2, and foam control. In one embodiment of the present application, the temperature for the culturing step is 35.5 to 37.5°C, preferably 36 to 37°C, and more preferably about 36.5°C. In one embodiment of the present application, the CO2 aeration rate for the culturing step is set to 5 to 10%, preferably 6 to 8%. In one embodiment of the present application, the pH for the culturing step is 6.6 to 7.4, preferably about 6.7 to 7.2. In one embodiment of the present application, the humidity for the culturing step is 70 to 85%, preferably about 80%. In one embodiment of the present application, the DO for the culturing step is 30 to 50%, preferably about 40%. In one embodiment of the present application, the cell density for the culturing step is 10 to 200 x 10 6 cells / mL, preferably 20 to 100 × 10 6 In one embodiment of the present application, the perfusion flow rate is 5 to 20 liters per square meter per hour (LMH). In one embodiment of the present application, the perfusion flow rate is 10 to 16 LMH. In one embodiment of the present application, the perfusion flow rate is about 5, 10, 12.5, 16, or 20 LMH.

[0028] The method for increasing the transitional cell density of a culture further comprises subjecting the seed culture to concentration by perfusion. In one embodiment of the present application, the seed culture is concentrated by filtering the medium using a perfusion device including a filtration device, so that the volume of the discharged waste culture medium is greater than the volume of the added new culture medium. In the present application, a suitable filtration device may 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 comprises 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 comprises a hollow fiber membrane, which may comprise an ultrafiltration membrane or a microfiltration membrane, for example, a 50 kDa ultrafiltration membrane or a 0.2 micron microfiltration membrane, preferably a microfiltration membrane. In one embodiment of the present application, the perfusion concentration step is initiated on the last day of the culturing step or after the end of the culturing step. In one embodiment of the present application, the perfusion concentration step lasts for 1 to 24 hours, preferably 2 to 5 hours. In one embodiment of the present application, the perfusion concentration step lasts for 1 to 24 hours, preferably 2 to 10 hours, and more preferably 3 to 5 hours. In another embodiment of the present application, the perfusion concentration step is initiated 1 to 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 to 72 hours, preferably 24 to 48 hours, and more preferably about 24 hours. In one embodiment of the present application, 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 prior to concentration. In one embodiment of the present application, the filtration ratio in the perfusion concentration step is 40 to 140. In the present application, "filtration ratio" refers to the liquid flow rate entering 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 in the perfusion concentration step is 40 to 400 × 10 6 cells / mL or more, e.g., 100-120 x 10 6 pieces / mL, 180~200×10 6pieces / mL, 300~400×10 6 In one embodiment of the present application, the culture obtained in the perfusion concentration step can be used as a seed culture for subsequent cell culture, can be directly cryopreserved or used, or can be used for other suitable applications known in the art. In one embodiment of the present application, the perfusion flow rate is 5 to 20 liters per square meter hour (LMH). In one embodiment of the present application, the perfusion flow rate is 10 to 16 LMH. In one embodiment of the present application, the perfusion flow rate is about 5, 10, 12.5, 16, or 20 LMH.

[0029] The present application also relates to a method for high cell density inoculation in production, the method comprising obtaining a concentrated seed culture, the seed culture comprising a culture obtained by a method for increasing the transitional cell density of a culture, the method comprising culturing cells in the seed culture by perfusion and subjecting the seed culture to concentration by perfusion. In one embodiment of the present application, the method for increasing the transitional cell density of a culture is as described above.

[0030] The method for high cell density inoculation in production further comprises inoculating the concentrated seed culture into a production culture medium. In one embodiment of the present application, the inoculation dilution ratio in this step is at least 3, preferably 4 to 6. In one embodiment of the present application, the inoculation density in this step is 10 to 100 x 10 6 cells / mL, e.g., 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 x 10 6 In the present application, the production culture medium involved in this step can be any culture medium known in the art that can promote cell growth or target protein expression.

[0031] The method for high cell density inoculation in production further includes culturing cells in a production culture medium to produce the target product. In the present application, this step can be any process that allows cell growth or expression of the target protein. In one embodiment of the present application, the cell culture in this step continues for 6 to 20 days, preferably 8 to 18 days, and more preferably about 12 to 14 days. In one embodiment of the present application, this step includes shifting 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 decreasing the temperature of the cell culture. In one embodiment of the present application, this step includes maintaining an essentially constant temperature of the cell culture. In one embodiment of the present application, the decrease in temperature of the cell culture is initiated 0 to 5 days after inoculation. In one embodiment of the present application, the decrease in temperature of the cell culture is initiated 0 to 1 day after inoculation. In one embodiment of the present application, the decrease in temperature of the cell culture is initiated within 8 hours after inoculation. In one embodiment of the present application, the decrease in temperature of the cell culture is initiated 1 day after inoculation. In one embodiment of the present application, lowering the temperature of the cell culture comprises culturing the cells at a temperature lower than the temperature of the seed solution. In one embodiment of the present application, the culture temperature is 29 to 37°C, preferably 36 to 37°C, and more preferably about 36.5°C, and the shift temperature of the cell culture in this step is 29 to 35°C, preferably 30 to 34°C, and more preferably about 31 to 33°C. In one embodiment of the present application, lowering the temperature of the cell culture comprises first culturing the cells at an initial temperature and then, after the predetermined conditions are reached, culturing the cells at a shift temperature lower than the initial temperature. In one embodiment of the present application, the initial temperature is 36 to 37°C, preferably 35 to 37°C, and more preferably about 36.5°C. In one embodiment of the present application, the shift temperature is 25 to 35°C, preferably 30 to 32°C, and more preferably about 31°C. In one embodiment of the present application, the predetermined conditions comprise culturing the cells at the initial temperature for a predetermined time, for example, 12 to 48 hours, preferably about 24 hours. In one embodiment of the present application, the predetermined conditions are a predetermined cell density, for example, 16 to 20 × 10 6The cell density in cells / mL is included. In one embodiment of the present application, the process includes fed-batch culture with a feed medium and / or medium exchange and / or perfusion. In one embodiment of the present application, the process includes fed-batch culture with a feed medium. In one embodiment of the present application, the process includes medium exchange by perfusion. In one embodiment of the present application, the process includes continuous perfusion culture with constant exchange of medium by perfusion. In the present application, the culture medium included in the medium exchange by perfusion may be any culture medium known in the art that can promote cell growth or express a target protein. In one embodiment of the present application, the culture medium included in the medium exchange by perfusion is the same as the culture medium included in the seed culture. In one embodiment of the present application, the culture medium included in the medium exchange by perfusion is different from the culture medium included in the seed culture. In one embodiment of the present application, the exchange volume of the culture medium by perfusion may be 0.5 to 6 times, 1.5 to 3 times, or 1.5 to 2 times the initial working volume of the culture medium. In one embodiment of the present application, the medium exchange by perfusion may last for 6 to 48 hours. In one embodiment of the present application, medium exchange by perfusion can be initiated on days 2 to 7 and / or 5 to 12 after inoculation. In one embodiment of the present application, this process comprises intermittent perfusion fed-batch culture. In one embodiment of the present application, this process comprises continuous perfusion production, for example, intensive perfusion culture or concentrated fed-batch culture.

[0032] According to the method of the present application, during and / or after the perfusion seed culture stage, the culture is concentrated and filtered using a hollow fiber membrane perfusion device. By increasing the discharge of cell culture effluent, the final cell density is concentrated to 1.5, 2.0, 3.0, 4.0, or even 5.0 times the original cell density, while maintaining the conditions necessary for cell culture, including temperature, dissolved oxygen, pH, aeration, and medium supply, minimizing the concentration time and eliminating adverse effects on cell health. Transferring the concentrated seed solution to the N-producing bioreactor significantly increases the initial inoculum VCD. A schematic diagram of this process is shown in Figure 1. A description and comparison of some key process features are listed in Table 1.

[0033] [Table 2] The technical solutions of the present application will be described below clearly and completely in conjunction with specific examples. It is clear that the described embodiments are not the entire examples but are part of the present application. Based on the examples of the present application, all other examples obtained by those skilled in the art without creative work belong to the protection scope of the present application. [Example]

[0034] The technical solutions of the present invention will be described in more detail below in conjunction with specific examples. The following examples are merely illustrative and do not constitute any limitations on the technical solutions of the present invention. Specific technical parameters such as materials, processes, conditions, values, or numerical ranges in the following examples are merely illustrative and are not exhaustive or limiting.

[0035] The cells used in the following examples are the CHO-K1 cell line from WuXi Biologics. They are stably transfected to express different types of monoclonal antibodies, such as IgG1 and IgG4. Clone A (WuXia 3.0 cell line 196-N079-05-20) was used in Examples 1, 3, 5, 6, and 7, clone B (WuXia 2.0 cell line 154A-01-010) was used in Examples 2 and 7, and clone C (WuXia 3.0 cell line 196-N079-06-08) was used in Examples 4 and 6.

[0036] The following media used in the examples below are Actipro (Hyclone, Cat. No. SH31037), CD CHO (Gibco, Cat. No. 12490), Cell Boost 7a (HyClone, Cat. No. SH31026), Cell Boost 7b (HyClone, Cat. No. SH31027), WXBM and WXFM (proprietary in-house media from WuXi Biologics). Key equipment information relevant to the experimental examples is shown in Table 2. Further details regarding materials and media preparation are listed in Tables 3 and 4, respectively.

[0037] In addition to the specific methods, devices, and materials used in the examples, any methods, devices, and materials in the prior art that are similar or equivalent to the methods, devices, and materials described in the examples of the present invention, upon the knowledge of the skilled artisan and upon recording of the present invention, can also be used to practice the present invention.

[0038] [Table 3] [Table 4] [Table 5] In order to explain the technical solutions of the present invention more clearly, they will be further described in conjunction with examples.

[0039] Example 1: N-1 perfusion and concentration for UIFB production using clone A To evaluate the effect of ATF perfusion parameters on the N-1 concentration process, tests with different perfusion rates and filtration ratios during the N-1 concentration phase were conducted using clone A, and the concentrated high-cell-density seed culture was used to inoculate the production bioreactor. The detailed process parameters for the N-1 and production bioreactors are summarized in Tables 5 and 6, respectively.

[0040] For vial thawing, one vial of frozen cells was first thawed in a water bath at 37.0 ± 1.0 °C for 120–150 seconds. The cells were then transferred to a shake flask containing prewarmed passage medium (CD CHO). The shake flask was then transferred to a shaking incubator with the following settings: 36.5 °C culture temperature; 110 rpm shaker speed with a 50 mm orbital diameter; and 6% CO2. After at least 5 minutes, one sample was taken from the shake flask for VCD and viability measurements. Based on the results, the seeding cell density was adjusted to 0.10–0.20 × 10 cells / mL. 6 After 72 ± 12 hours of culture, the cells were ready for passage, at which point the transitional cell density was 1.5–2.2 × 10 cells / mL. 6For pre-N-1 cell expansion in the following examples, cells were thawed and passaged in shake flasks under the same conditions and settings.

[0041] During N-1 enrichment, the perfusion flow could be stopped or maintained, and the harvest flow was increased as specified in Table 5. Within 2-5 hours of enrichment, the cell culture volume decreased to approximately 50% of the initial culture volume. In this example, the effect of different conditions of N-1 enrichment and the resulting cell culture performance and protein production during the UIFB production phase were investigated.

[0042] [Table 6] [Table 7] result As shown in Figure 2a-b, the different N-1 enrichment parameters listed in Table 5 had no obvious effect on N-1 VCD and viability. In this study, the N-1 peak VCD of cell line A was approximately 50 × 10 after 3 days of perfusion. 6 cells / mL, then approximately 100 x 10 cells / mL within 2-3 hours via the ATF controller. 6 Concentrated to cells / mL.

[0043] As shown in Figure 3a-d, the UIFB process with high seeding density is approximately 15 × 10 6 Compared to cells / mL, the peak VCD was approximately 30 x 10 6 With higher inoculation cell densities, titers increased from 3.0 g / L to 5.0–6.0 g / L on days 8–10, 60–100% higher than those of the TFB control.

[0044] As shown in Figure 4a-b, comparable PQA results were obtained between the UIFB condition and the TFB control in terms of product-related purity and N-glycosylation. All process conditions showed comparable levels of aggregation and fragmentation. Regarding charge variants, a slightly higher main peak and a lower acidic peak were observed in the UIFB condition. In N-glycan analysis, the UIFB showed slightly lower levels of Man5 and G0 species than those from the TFB control. Other than that, there were no significant differences in other glycosylation patterns.

[0045] Example 2: N-1 perfusion and concentration for UI-IPFB production using clone B In this study, the N-1 perfusion and concentration process with different ATF2 filtration ratios was further evaluated using clone B. The ATF flow rate and recovery rate were adjusted according to Table 7 to achieve the target ATF filtration ratio during the N-1 concentration process.

[0046] [Table 8] After N-1, five different production cultures were inoculated using seeds from representative conditions, including batch seed growth, perfusion seed growth, and concentrated perfusion seed growth. The production culture modes included conventional fed-batch (TFB), enriched fed-batch (IFB), intermittent perfusion fed-batch (IPFB), ultra-enriched fed-batch culture (UIFB), and ultra-enriched intermittent perfusion fed-batch (UI-IPFB). The seeding density, perfusion schedule, and control parameters are listed in Table 8.

[0047] [Table 9] result As shown in Figure 5a–b, at different ATF filtration ratios of 40–140, the final transitional post-VCD concentration was 50–60 × 10 after day 6 of N-1 perfusion growth. 6 cells / mL to 180-200 x 10 6The concentration reached 1000 cells / mL. Neither hollow fiber membrane fouling nor an increase in transmembrane pressure was observed at any of the ATF filtration ratios evaluated during concentration. Furthermore, no differences in cell growth or viability were identified among 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.

[0048] From Figure 6a-d, the initial seeding density was 1.0 × 10 for TFB. 6 cells / mL, 10 x 10 for IFB and IPFB 6 cells / mL, and 35 × 10 for UIFB and UI-IPFB 6 During the culture, the peak cell densities of TFB, IFB, UIFB, and UI-IPFB were 26, 30, 49, and 49 × 10 cells / mL, respectively. 6 In the UIFB process, lactate rapidly accumulated to approximately 3 g / L on day 8, and viability began to gradually decrease, dropping to less than 60% on day 10. Similarly, IFB promoted lactate accumulation from day 10, reaching approximately 3 g / L on day 13, while viability dropped to approximately 60%. For UI-IPFB with intermittent perfusion during culture, no increase in lactate was observed, and cell viability remained approximately 70% until day 13.

[0049] As a result, UI-IPFB produced 12.1 g / L of protein on day 13, twice the titer of UIFB. Compared to IFB, the titer of UIFB reached 6.1 g / L on day 8, compared with 4.6 g / L from IFB on day 13. Due to better cell density and viability maintenance in IPFB, the final titer was 6.3 g / L, which was comparable to that of UIFB and 1.3-1.4 times higher than that of TFB and IFB.

[0050] From Figure 6, it was found that the product-related purity as shown in the SEC and CE profiles was all similar among the different production culture modes. For the charge distribution from iCIEF, the different production modes produced comparable profiles, except that those due to intermittent perfusion in IPFB and UI-IPFB showed a higher main peak and a lower acidic peak.

[0051] Example 3: Production of UIFB, IPFB, and UI-IPFB using clone A In this study, a comparison of different cultivation modes was performed in a 3 L bench-scale bioreactor using clone A. The best production performance from the UI-IPFB process was then further scaled up to a 250 L disposable bioreactor.

[0052] Process parameter setting for N-1 and production stages N-1 perfusion and concentration in the 3 L bioreactor and 50 L disposable bioreactor followed the procedures as described in Table 9, and the production process parameters for the 3 L and 250 L production are listed in Table 10.

[0053] [Table 10] [Table 11] result As shown in Figure 7, in the N-1 enrichment culture, the production seeding density of UIFB and UI-IPFB was approximately 20 × 10 6 The production seeding cell densities for TFB, IFB, and IPFB were 1.0 x 10 cells / mL, respectively. 6 cells / mL and 10 x 10 6 cells / mL.

[0054] 20×10 6 UI-IPFB cultures seeded at 100 cells / mL had peak VCDs of approximately 35 × 10 cells / mL, the highest among all conditions. 6showed improved ability to reach 10 × 10 cells / mL, with viability remaining above 60% through day 14. However, 6 IFB process and 20 x 10 cells / mL seeded 6 The UIFB culture, in cells / mL, ultimately ended prematurely due to a decrease in viability to approximately 50%. Furthermore, lactate also spiked on days 8 and 9, respectively. No obvious difference in Qp was observed between TFB and IFB. Due to the combined benefits of the ultra-high seeding and IPFB culture modes, the integral cumulative viable cell density (IVCD) of UI-IPFB increased by 40-70% compared with the IFB and UIFB controls. Furthermore, lactate spikes were also effectively suppressed under IPFB conditions. Qp from UI-IPFB increased by approximately 70% compared with the IFB and UIFB controls. Overall, the final titer reached approximately four times that of TFB and approximately twice that of the IFB and UIFB controls.

[0055] As shown in Figure 8, the UI-IPFB process for cell line A was scaled up from 3 L to 250 L disposable bioreactors (SUBs) using 50 L SUBs for N-1 perfusion and concentration, which showed highly comparable in-process trends in cell growth, lactate metabolism, and protein production. The highly comparable performance demonstrated the process scalability of both N-1 perfusion and concentration and UI-IPFB production in large-scale manufacturing.

[0056] Example 4: N-1 enrichment strategy of UI-IPFB using clone C N-1 perfusion and concentration strategy As listed in Table 11, for concentration approach I, N-1 cultures were concentrated by reducing the culture volume by approximately 50% by increasing the harvest flow rate to 10 VVD within 2–3 hours after N-1 perfusion on day 6. Meanwhile, for approach II, N-1 culture concentration was maintained unchanged from day 5 until the end of perfusion on day 6, while the ATF harvest flow rate was further increased by 0.5 VVD, reducing the culture volume by approximately 50%. N-1 cell culture performance was evaluated with these two concentration approaches.

[0057] For UI-IPFB production, the inocula from the two enrichment approaches followed a similar split ratio of 4:1, with initial densities of 35 and 45 x 10, respectively. 6 The detailed production parameters from both seeds followed similar UI-IPFB operations, as listed in Table 10.

[0058] [Table 12] result As shown in Figure 9, the transition VCDs for the two different N-1 enrichment approaches were approximately 179 and 136 × 10, respectively. 6 To maintain a similar split ratio of 4:1 from N-1 to production stage, the resulting seeding densities were approximately 37 and 50 x 10 cells / mL, respectively. 6 The higher seeding density was approximately 58 x 10 cells / mL from the lower seeding density. 6 cells / mL, compared to a peak VCD of 70 x 10 6 The seeding density was increased to over 100 cells / mL. With the exception of VCD, both production conditions shared similar viability profiles. At the end of production, the two UI-IPFB conditions with two different seeding densities produced 18.0 g / L and 15.6 g / L of product, respectively, compared to approximately 4.0 g / L in TFB (data not shown). Comparable PQA results were obtained between the N-1 24-hour and 2-hour enrichment conditions in terms of product-related purity, charge variants, and N-glycosylation.

[0059] This study demonstrated that both enrichment approaches in N-1 perfusion growth could serve the same purpose: increasing the transitional cell density. The choice of enrichment strategy could be determined according to the comparative cell growth performance for each cell line from both approaches.

[0060] Example 5: High cell density inoculation for continuous production using clone A This study further evaluated the effect of high seeding densities achieved by N-1 perfusion and concentration in continuous culture processes such as IPC and CFB. Lower seeding densities were inoculated from 3-day batch growth compared to conventional IPC and CFB processes.

[0061] Cell culture process in production The N-1 perfusion and enrichment culture process used in this study followed the same procedure as described in Example 4. Bench-scale production was carried out in a 3-L glass bioreactor connected to an ATF2 hollow fiber filter for all conditions in this example. The pore sizes of the ATF hollow fiber filters used in production were 50 kDa and 0.20 μm for the CFB and IPC, respectively. pH was controlled within the range of 6.7–7.2, and DO was controlled at 40%. Constant agitation was set at 285 rpm and shifted to 320 rpm when oxygen demand reached a preset criterion of 0.2 VVM. During production, the initial temperature was set at 36.5°C, and then the VCD was adjusted to 40–45 × 10 6 When the VCD reached 20 × 10 cells / mL, the temperature was shifted to 33°C. 6 cells / mL is 1.0 VVD, and VCD is 20 × 10 6 When the VVD was less than 0.3-0.5 cells / mL, a specific ratio of feed medium was mixed in the perfusion medium to support high-density cell culture and production.

[0062] result The results of the high seeding density continuous process test are shown in Figure 10. The N-1 peak VCD of clone A was approximately 60 x 10 after 3 days of perfusion. 6 cells / mL, then approximately 120 x 10 6 The enrichment cultures were concentrated to approximately 30 × 10 cells / mL (Figure 10a). For enrichment cultures, the production seeding density was approximately 30 × 10 cells / mL with a split ratio of 4:1. 6 cells / mL was reached.

[0063] As shown in Figure 10, UI-CFB and UIPC cultures showed a shortened exponential cell growth phase, with approximately 60 x 10 6cells / mL, while the peak VCD was reached at 0.5 x 10 6 Although the lower seeding density of cells / mL resulted in a longer culture period due to a longer cell expansion phase, similar peak VCDs were obtained on day 9 after day 6 in the UI-CFB and UIPC conditions. As a result, the cumulative VCDs of the UI-CFB and UIPC conditions were elevated by 15–20% compared to their corresponding CFB and IPC controls. Furthermore, the higher seeding density obtained from N-1 perfusion and concentration also contributed to higher Qp and final productivity. The Qp of the UI-CFB and UIPC increased by approximately 100% and 25%, respectively, compared to the low-seeded CFB and IPC (Figure 10f). The cumulative volumetric productivity of the UI-CFB condition reached 2-fold that of the low-seeded CFB, and UIPC production reached 1.5-fold that of the low-seeded IPC (Figure 10e). Overall, comparable product-related purity from SEC and SE-SDS was observed in the different conditions tested, except for slightly higher levels of aggregation, as seen from HMW species, detected in UIPC, CFB, and UI-CFB (Figure 10g). Regarding charge variants, less acidic species were found in the UI-CFB product, and more basic modifications were identified in both the CFB and UI-CFB conditions. Regarding N-glycan modifications, lower galactosylation and higher Man5, as indicated by %G1F and %G2F, were found in the UIPC condition compared to the IPC control, but no significant differences were found between the CFB and UI-CFB products.

[0064] Example 6: High flow rate study of N-1 enrichment using clones A and C In this study, an N-1 concentration robustness test was performed using both Clone A and Clone C. For culture media, both commercially available and in-house proprietary basal media and feed media for the cell lines were used for comparison of different cell culture processes in this study. High flow rate ranges for both microfiltration (MF) and ultrafiltration (UF) were evaluated to demonstrate the robustness of N-1 perfusion and concentration for UI-IPFB in this study.

[0065] Process parameters for N-1 and production stages ATF hollow fiber filters with different pore sizes, including a 0.20 μm microfiltration filter (MF) and a 50 kDa ultrafiltration filter (UF), were used in the study. The normal recommended flow rate for ATF perfusion is specified to be less than 5 liters per square meter per hour (LMH), with an optimal range of 1 to 3 LMH. In this study, higher flow rates of 5, 10, 12.5, 16, and 20 LMH were evaluated. Otherwise, the N-1 perfusion culture used in this study and the subsequent UI-IPFB production followed the same parameters as those listed in Tables 9 and 10.

[0066] result As shown in Figure 11, no obvious differences were observed when using clones A (Figure 11a) and C (Figure 11d) with both MF and UF at high flow rates.

[0067] For clone A, the N-1 perfusion culture reached 40–45 × 10 cells on day 5. 6 Once the culture reached 90-100 x 10 cells / mL, the culture was then purified by both MF and UF at an ATF flow rate range of 5-16 LMH. 6 The cells were concentrated to 1000 cells / mL. There was no evidence of blockage or leakage of the ATF hollow fiber membrane during cell culture concentration. The cultures with the highest flow rates of 16 LMH and 5 LMH were then inoculated into a UI-IPFB production bioreactor to demonstrate their performance. Equivalent cell culture performance and productivity results were achieved. Final productivity reached 11-13 g / L on day 14.

[0068] For clone C, the VCD of the N-1 culture was 60-70 x 10 on day 6. 6 The culture was then cultured at 120-140 x 10 cells / mL by MF using both low and high flow rates of 5 and 20 LMH, respectively. 6 The concentrated seed was then added to a 30-35 x 10 cells / mL medium. 6 The UI-IPFB product was seeded with an initial VCD of cells / mL. The peak VCD was approximately 50 × 10 cells / mL under high-flow binding conditions. 6cells / mL, similar to the control seeded from the normal flow rate condition. Ultimately, similar titers were obtained from both conditions.

[0069] Example 7: N-1 perfusion and concentration using clones A and B In this study, N-1 cultures with high concentration ratios were evaluated using clone A and clone B. Regarding culture media, both commercially available and in-house proprietary basal media as well as feed media were used for N-1 perfusion in this study.

[0070] result As shown in Figure 12, concentrations as high as 6.8-fold were achieved after N-1 perfusion, with 400 × 10 6 The final cell density was 400 × 10 cells / mL. 6 It was shown that the concentration can be increased to more than 2, 3, 4, 5, 6, 7 or more cells / mL.

[0071] In summary, the present invention has the following advantages over the prior art: 1. The present invention significantly increases the transitional cell density in N-1 seed cultures or significantly reduces the time required to reach the same cell density in cell growth. 2. Cultivating UIFB with a much higher seeding density than IFB is demonstrated by using seed from cell culture concentrates during or after N-1 perfusion growth. This invention increases the initial seeding VCD, total cell biomass, and improves protein production in fed-batch production cultures. 3. UI-IPFB, which has a much higher seeding density compared to IFB or IPFB production processes, is achieved by cell culture concentration during or after N-1 perfusion growth. The UI-IPFB culture mode solves the low cell viability and low productivity that often occur in IFB or UIFB processes by using limited cycles of intermittent perfusion in production to remove the accumulation of toxic by-products and replenish the cell culture with fresh medium, thereby improving cell culture performance. UI-IPFB has been demonstrated to significantly improve protein production with similar or better product quality. 4. For multiple cell lines tested, UI-IPFB was shown to achieve significant productivity improvements compared to TFB, IFB, or UIFB. In terms of scalability, UI-IPFB has proven to be scalable and possesses both the operational simplicity of fed-batch culture and the excellent cell health of the perfusion process.

[0072] 5. UI-IPFB provided a more competitive production COG due to a significant increase in titer without the need for facility and equipment changes.

[0073] 6. The high seeding densities achieved by N-1 perfusion and concentration in continuous UIPC and UI-CFB processes also provide significantly improved volumetric and cell-specific productivity, along with a shortened production phase, compared to conventional low-seeded IPC and CFB processes.

[0074] The above are only specific examples of the present application and do not constitute any limitation on the scope of protection of the present application. Those skilled in the art may make various changes or modifications based on the above description. It is not necessary or possible to provide a complete list and description of all implementation methods here. Any similar technical solutions formed by equivalent transformation or equivalent substitution fall within the scope of protection of the claims in this application.

Claims

1. 1. A method for increasing the transitional cell density of a culture, comprising: Culturing cells by perfusion in a seed culture; and subjecting said seed culture to concentration by perfusion.

2. 10. The method of 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 step of culturing includes perfusion culturing, and optionally the duration of the step of culturing is 1 to 10 days, preferably 2 to 5 days, and / or optionally the inoculation density of said perfusion culture is 0.3 to 10 x 10 ６ The method of claim 1, wherein the concentration is greater than or equal to 1000 cells / mL.

4. 2. The method of claim 1, wherein the perfusion concentration step is initiated on the last day of the culturing step or after the end of the culturing step, or 1 to 3 days before the end of the culturing step, optionally the perfusion concentration step lasts for 1 to 24 hours, preferably 2 to 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 step of concentrating by perfusion comprises the use of 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 of the step of concentrating by perfusion is between 40 and 400 x 10 ６ The method of claim 1, wherein the concentration is greater than or equal to 1000 cells / mL.

6. 1. A method for high cell density inoculation in production comprising: a. Obtaining an enriched seed culture, wherein the seed culture is obtained by the method of any one of claims 1 to 5; b. inoculating a production culture medium with the concentrated seed culture; c) culturing cells in said production culture medium to produce the target product.

7. The method according to claim 6, wherein the inoculum dilution ratio in step b is at least 3, preferably 4 to 6.

8. 7. The method of claim 6, wherein step c comprises shifting the temperature of the cell culture or maintaining a constant temperature of the cell culture.

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

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