Intermittent perfusion fed-batch culture

Abstract

Methods of intermittent perfusion fed-batch culture are provided, including fed-batch processes that include one or more intermittent perfusion stages during mid- to late-phase cultivation to improve productivity and product quality.

Description

[Technical Field]

[0001] The present invention relates to the field of cell culture, and in particular to cell culture methods that improve productivity and product quality in recombinant protein production. [Background technology]

[0002] Over the past decade, various cell culture modes have been developed for the production of recombinant proteins, such as monoclonal antibodies and fusion proteins. Among them, fed-batch culture is the most widely adopted technique for large-scale production due to its ease of operation and scalability. Perfusion culture, another common culture strategy involving continuous medium exchange, often requires specialized manufacturing designs for large-volume medium supply, scalable cell retention systems, and corresponding downstream processing capabilities for continuous harvesting, but also offers clear advantages in both productivity and product quality compared to fed-batch culture.

[0003] A fed-batch process typically lasts approximately 14 days and follows four distinct phases of the growth curve: lag, exponential, stationary, and death. The cell death phase directly reflects the deterioration of cell health, typically caused by the accumulation of toxic metabolites in the cell culture. Increased cell death can be further exacerbated by an environment in which proteases, reductases, glycosylases, and sialidases are released in large quantities into the cell culture, degrading proteins and altering protein glycan structures. Ultimately, cell death leads to reduced production and premature termination of the culture process. In the fed-batch system, cell death can be mitigated or postponed to some extent by providing a bolus of nutrients, but it cannot fundamentally resolve all problems and concerns.

[0004] Media optimization has been widely used to improve cell viability and enhance productivity. Adjusting feeding strategies or supplementing with additives can better maintain cell viability and cell health. Unfortunately, not all media optimization efforts are effective or suitable for all cell lines. Optimizing process parameters, such as temperature shift strategies, is another approach that has proven effective in improving cell performance. However, again, not all of these adjustments have been widely feasible. Another way to maintain cell viability is to employ genetic engineering strategies to inhibit cell death. These include, for example, overexpression of negative regulators of apoptosis (BCL-2, BCL-xL, and MCL-1), knockout of positive regulators of apoptosis (BAK and BAX), and overexpression of HSP27, HSP70, or both to attenuate apoptosis (Matthew N. Henry et al., Biotechnology and Bioengineering. 2020; 117:1187-1203). However, cell line engineering is always subject to a complex process of design, execution, and validation, and in essence, none of the above approaches can fully solve the fundamental problem of toxic by-product accumulation, which is the main root cause of cellular performance decline during the mid- to late-stage of cell culture.

[0005] The perfusion process has become popular primarily because it allows for extended cell culture periods by continuously removing spent medium and replenishing it with fresh medium. However, current strategies involve high capital investments to set up all the necessary facilities, laborious medium preparation, and increased product manufacturing costs. Disposal of large amounts of spent medium is also a concern for manufacturers. Therefore, the application of a fully perfusion process may not be a suitable option for all recombinant cell production processes. Summary of the Invention [Problem to be solved by the invention]

[0006] Therefore, there is a need for improved methods of cell culture, particularly mammalian cell culture, that can more efficiently increase productivity and improve product quality in a wider range of recombinant production processes. [Means for solving the problem]

[0007] Generally, a new cell culture process based on an intermittent perfusion fed-batch (IPFB) mode according to the present invention is provided herein to improve productivity, product quality, and upstream economics in recombinant protein production. This new culture mode is primarily built on the traditional fed-batch mode by introducing one or more perfusion stages at mid- to late-stages.

[0008] According to one aspect, the present invention provides a cell culture method comprising a fed-batch process comprising at least a first perfusion step starting 0-7 days after a temperature shift or 0-5 days after peak VCD (viable cell density).

[0009] Optionally, and preferably, in some cases, the method of the present invention may further comprise one or more additional perfusion steps, each independently beginning 1 to 5 days, e.g., 2, 3, or 4 days, after the end of the previous perfusion step.

[0010] Optionally, and preferably, in some cases, the methods of the present invention include a seed propagation step comprising perfusion culture and / or enhanced fed-batch process culture to provide inoculum for the fed-batch process.

[0011] According to some embodiments, the cells are host cells engineered to recombinantly express a product of interest, and the method further comprises harvesting the expressed product. The cells are mammalian cells, such as CHO cells, and the product of interest can be a polypeptide, such as a monoclonal antibody. Thus, also provided are methods of producing a product of interest, comprising culturing cells that express the product of interest according to the methods of the invention and harvesting the expressed product of interest.

[0012] The IPFB culture process of the present invention allows for extended culture periods while maintaining cell health, leading to both improved productivity and product quality, while also enabling reduced medium consumption and better cost control compared to traditional perfusion culture. Furthermore, the IPFB mode of the present invention is conveniently adapted to any fed-batch culture process to improve productivity and product quality.

[0013] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. [Brief explanation of the drawings]

[0014] [Figure 1] FIG. 1 shows a schematic diagram of an exemplary setup of an IPFB process according to the present invention.

[0015] [Figure 2] FIG. 2 illustrates an exemplary timeline of the IPFB process according to the present invention.

[0016] [Figure 3] FIG. 3 shows the cell growth profiles of cultures in spin tubes with and without medium exchange, according to the Examples below.

[0017] [Figure 4] Figure 4 shows the cell viability profiles of cultures in spin tubes with and without medium exchange and control cultures.

[0018] [Figure 5] FIG. 5 shows the lactate profiles of the cultures in spin tubes with and without medium exchange and the control.

[0019] [Figure 6]FIG. 6 shows the productivity profiles of the cultures in spin tubes with and without medium exchange.

[0020] [Figure 7] FIG. 7 shows the cell growth profiles of IPFB cultures and control fed-batch cultures in 3 L bioreactors according to the Examples below.

[0021] [Figure 8] FIG. 8 shows the cell viability profiles of IPFB cultures and control fed-batch cultures in 3 L bioreactors according to the Examples below.

[0022] [Figure 9] FIG. 9 shows the lactate profiles of IPFB cultures and control fed-batch cultures in 3 L bioreactors according to the Examples below.

[0023] [Figure 10] FIG. 10 shows the productivity profiles of IPFB cultures and control fed-batch cultures in 3 L bioreactors according to the Examples below.

[0024] [Figure 11] FIG. 11 shows product purity over size variation of IPFB cultures and control fed-batch cultures in 3 L bioreactors according to the examples below.

[0025] [Figure 12] FIG. 12 shows product purity in terms of charge change for IPFB cultures and control fed-batch cultures in 3 L bioreactors according to the examples below.

[0026] [Figure 13] FIG. 13 shows the cell growth profiles of IPFB cultures and control fed-batch cultures in 3 L bioreactors according to the example study on intermittent perfusion rates described below.

[0027] [Figure 14]FIG. 14 shows the cell viability profiles of IPFB cultures and control fed-batch cultures in 3 L bioreactors according to the Examples below.

[0028] [Figure 15] FIG. 15 shows the lactate profiles of IPFB cultures and control fed-batch cultures in 3 L bioreactors according to the Examples below.

[0029] [Figure 16] FIG. 16 shows the productivity profiles of IPFB cultures and control fed-batch cultures in 3 L bioreactors according to the Examples below.

[0030] [Figure 17] FIG. 17 shows the cell growth profiles of IPFB cultures and control fed-batch cultures in 3 L bioreactors according to the following example studies on various intermittent perfusion regimes.

[0031] [Figure 18] FIG. 18 shows the cell viability profiles of IPFB cultures and control fed-batch cultures in 3 L bioreactors according to the Examples below.

[0032] [Figure 19] FIG. 19 shows the lactate profiles of IPFB cultures and control fed-batch cultures in 3 L bioreactors according to the Examples below.

[0033] [Figure 20] FIG. 20 shows the productivity profiles of IPFB cultures and control fed-batch cultures in 3 L bioreactors according to the Examples below. DETAILED DESCRIPTION OF THE INVENTION

[0034] nomenclature As used herein, the singular forms preceded by "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Similarly, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein.

[0035] As used herein, the terms "about," "around," or "approximately," when preceding a numerical value, refer to a range defined by 1%, 2%, 3%, 4%, 5%, 10% or more approximations of the specified value.

[0036] In the present disclosure, one or more features of one embodiment may be combined with any one or more features of another embodiment without departing from the spirit and concept of the invention.

[0037] In this disclosure, all ranges, including ranges defined as being between two specified end values, are inclusive of the specified end values unless otherwise specified. For example, a range of 1 to 10 means a range of 1 to 10.

[0038] In this disclosure, when a period or duration or interval length is expressed in days and a time point is expressed in days, it means that the time or timing is counted or identified in days, where the numbers do not necessarily mean an exact multiple of 24 hours.

[0039] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this invention pertains. All publications and patents specifically mentioned herein are incorporated by reference for all purposes. All references cited herein should be construed as indicative of the level of skill in the art, and should not be construed as an admission that the present invention is not entitled to antedate such disclosure by prior invention.

[0040] Embodiments of the present invention The present invention builds on the fed-batch process by implementing one or more intermittent perfusions during the mid- to late-stage culture. Figure 1 shows a schematic diagram of an example of a process setup according to the present invention. The culture vessel (e.g., bioreactor) is equipped with a retention system, and retained cells and, optionally, products in the medium are returned to the culture vessel, while permeate containing waste and, optionally, products is removed and collected as needed. Meanwhile, a perfusion device connected to the culture vessel replenishes fresh medium to maintain a constant culture volume.

[0041] In a traditional fed-batch process, nutrients are supplied in discontinuous boluses without any removal of spent medium. In fed-batch culture, a predetermined cell density is typically inoculated and maintained by bolus feeding of nutrients. It should be noted that, because spent medium replacement is typically not performed in fed-batch processes, metabolic products tend to accumulate as the cell culture progresses, which can have toxic effects on cell growth or target protein production. In particular, the use of high cell densities can exacerbate toxicity through mechanisms of cell apoptosis.

[0042] In contrast, typical perfusion processes involve continuous medium exchange by pumping in fresh medium at equal flow rates and pumping out spent medium while maintaining a large number of viable cells. Removal of spent medium while cells remain cultured can be achieved through cell retention measures. However, a complete perfusion process is expensive. The high costs of medium supply, equipment, maintenance, and spent medium disposal are major concerns, limiting its scope of application.

[0043] The intermittent perfusion fed-batch (IPFB) method of the present invention combines the advantages of both methods, including the operational simplicity of fed-batch culture and superior cell sustainability in the perfusion process. Specifically, one or more intermittent medium changes are introduced using a cell retention device during the mid- to late-stage of the fed-batch process. This approach can reduce the effects of toxicity and further extend the cell culture period with healthier cell conditions and thereby improved production capacity. By limiting the number of medium changes using a cell retention device during the mid- to late-stage of fed-batch culture, both cell viability and viable cell density (VCD) can be better maintained throughout the entire culture period, preferably over an extended culture and production period, resulting in improved productivity and more desirable product quality.

[0044] As used herein, the term "intermittent perfusion (IP)" refers to one or more phases (i.e., periods) of perfusion that are introduced and therefore "intermittent" compared to a full typical fed-batch process and in contrast to a full perfusion process. Because perfusion is primarily a medium exchange, and cells and, optionally, product are maintained within the bioreactor, in this disclosure, "intermittent perfusion" and "medium exchange" can be used interchangeably in the context of IPFB culture.

[0045] As used herein, in the context of fed-batch or IPFB culture, the term "mid-to-late phase" refers to the period after the temperature shift or peak VCD.

[0046] In particular, the present invention provides cell culture methods comprising a fed-batch process including at least a first perfusion stage beginning (a) 0-7 days (e.g., 1, 2, 3, 4, 5, 6 days) after a temperature shift or (b) 0-5 days (e.g., 1, 2, 3, 4 days) after the peak VCD. Thus, the start of the first perfusion stage is typically (but not exclusively) determined according to item (a) if a temperature shift is present, or according to item (b) if a temperature shift is not present.

[0047] According to some embodiments, the methods of the present invention can include a temperature shift, depending on the cell line or clone. The temperature shift can be a decrease below 37°C, e.g., to 33°C or below, e.g., 31°C, 30°C or below. According to some embodiments, the temperature shift occurs when the VCD increases to at least about 50% of the peak VCD, at least about 70% of the peak VCD, at least about 80% of the peak VCD, or at least about 90% of the peak VCD, e.g., about 50%-95%, about 50%-75%, about 70%-90%, or about 85%-95% of the peak. According to some embodiments, the temperature shift occurs on a day between days 0 and 5 of the fed-batch process (i.e., IPFB process), e.g., day 1, day 2, day 3, or day 4, counting the day of inoculation as day 0. For high-density inoculation, an earlier temperature shift is preferred. In the present disclosure, days are counted along the fed-batch process (i.e., IPFB process) starting from the day of inoculation as day 0.

[0048] As a criterion for determining the time of a temperature shift or the start of the first perfusion phase, "peak VCD" may refer to a predetermined peak VCD, i.e., a peak determined in a preliminary matching experiment without a temperature shift. Preliminary matching experiments are typically performed to obtain useful parameters that indicate the profile of an actual run. As is well understood, such a predetermined peak VCD may be identical to or very close to the peak of an actual run.

[0049] According to some embodiments, the first perfusion stage begins on day 2 or later of the fed-batch culture process, such as day 3, 4, 5, 6, 7, 8, 9, 10, or later. For example, the first perfusion stage may begin between days 2 and 7, or between days 4 and 8.

[0050] According to some embodiments, the methods of the present invention include multiple perfusion stages. That is, the methods of the present invention can optionally include one or more additional perfusion stages in addition to the first perfusion stage described above to further improve process performance and product quality. Each of the one or more additional perfusion stages can independently begin 1 to 5 days after the end of the previous perfusion stage, where the adjective "previous" refers to the stage (n-1) immediately preceding the current or target stage (n). Thus, for example, the methods of the present invention can further include at least a second perfusion stage beginning 1 to 5 days after the end of the first perfusion stage, and optionally at least a third perfusion stage beginning 1 to 5 days after the end of the second perfusion stage. According to some embodiments, each of the one or more additional perfusion stages can independently begin 2, 3, or 4 days after the end of the previous perfusion stage. Figure 2 shows an exemplary timeline of an IPFB cultivation process according to the present invention.

[0051] According to some embodiments, one or more additional perfusion steps, such as a second perfusion step, may begin on day 3 or later, e.g., day 5 or later, day 6 or later, day 7 or later, day 8 or later, day 9 or later, day 10 or later, day 11 or later, and / or day 12 or later. According to some embodiments, one or more additional perfusion steps, such as a second perfusion step, may begin between days 5 and 12, or between days 8 and 12, of the process. According to some embodiments, the process of the invention comprises a third perfusion step, which may begin on day 8 or later, e.g., day 9 or later, day 10 or later, day 11 or later, day 12 or later, or in some cases between days 8 and 12.

[0052] Further implementation of intermittent perfusion may promote improvements in productivity and product quality. In particular, even in the IPFB process in which intermittent perfusion is performed multiple times according to the present invention, the amount of medium required for the process can be significantly reduced compared to a complete typical perfusion process.

[0053] According to the present invention, IPFB applies up to 100% medium exchange one or more times during the culture period. According to some embodiments, medium exchange, i.e., each run of intermittent perfusion, is independently performed over a period of 72 hours or less at a perfusion rate of about 0.5-6 VVT (vessel volume per hour). This corresponds to an exchange rate of 39%-100% according to the empirical formula: Exchange rate = (1-1 / em) × 100%, where "m" refers to the perfusion rate at VVT. According to some embodiments, each run of intermittent perfusion is independently performed at a perfusion rate of about 1-3 VVT. According to some embodiments, each run of intermittent perfusion is independently performed over a period of about 6-72 hours, for example, about 12-48 hours or about 24-36 hours.

[0054] Intermittent perfusion involves replacing cell-free spent medium containing cellular waste and toxic metabolites with fresh medium, and cells are retained by a cell retention device connected to the bioreactor via an external loop. Various cell retention techniques and corresponding devices known to be useful in perfusion cultures and systems can be used. According to some embodiments, the cell retention has a cutoff that can retain cells and the product of interest, such as a target polypeptide or monoclonal antibody, within the culture vessel. For example, the cutoff may be 50 KD or less. According to some embodiments, hollow fiber tangential flow filtration, such as standard tangential flow filtration (TFF) or alternating tangential flow filtration (ATF), is used. Hollow fiber tangential flow filtration, particularly ATF, is preferred for mammalian cells. According to some embodiments, the hollow fiber tangential flow filtration has a pore size (e.g., nominal pore size) of 50 KD or less, meaning that a filter (e.g., a cartridge or column) with this pore size is used. According to some embodiments, intermittent perfusion includes ATF as a cell retention technique and device.

[0055] In the present invention, a fed-batch process typically involves culturing a culture medium at a predetermined seeding density, e.g., about 0.3×10 6 ~10×10 6cells / mL, approximately 0.3×10 6 ~50×10 6 Start with seed inoculation at a density of approximately 5 ± 1.0 × 10 cells / mL, or possibly even higher. 6 cells / mL, approximately 10±2.0×10 6 cells / mL, approximately 20±2.0×10 6 cells / mL, approximately 30±2.0×10 6 cells / mL, approximately 40±2.0×10 6 cells / mL, or approximately 50 ± 2.0 × 10 6 According to some embodiments, the cell density may be about 5±1.0×10 cells / mL. 6 ~10±2.0×10 6 A high seeding density of cells / mL is used. The high seeding density is provided by a high seed culture density, which can be achieved by applying an intensive seed propagation method such as perfusion culture or intensive fed-batch culture. Thus, according to some embodiments, the method of the present invention further comprises a seed propagation step comprising perfusion culture and / or intensive fed-batch culture, which inoculates the fed-batch process.

[0056] During cultivation, the feed and carbon source, such as glucose, are periodically replenished, as in a typical fed-batch process. According to some embodiments, the feed is added once every two days, which may begin on day 0. According to some embodiments, the amount of feed is independently 2%-4% of the culture volume. According to some embodiments, the glucose level is maintained at about 3-10 g / L.

[0057] Generally, the methods of the present invention can be used to improve the yield of biomass and / or a desired product. According to some embodiments, the cultured cells are animal cells, such as mammalian cells, in particular CHO cells.

[0058] According to some embodiments, host cells transformed to recombinantly express a product of interest are cultured, and the method further comprises the step of harvesting the expressed product. According to some embodiments, the product of interest may be a peptide, polypeptide, or protein, such as an immunoglobulin or monoclonal antibody or fragment thereof. Thus, there is also provided a method of producing a product of interest, comprising culturing cells expressing the product of interest according to the methods of the invention and harvesting the expressed product of interest.

[0059] As used herein, the term "monoclonal," when used to describe an antibody, refers to uniformity of structure and activity, not the method of production. Monoclonal antibodies can be monovalent or multivalent (e.g., bivalent), and / or monospecific or multispecific (e.g., bispecific). Antibody fragments include (Fab) fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, Fc fragments, variable heavy chain (VH) regions, single-chain antibody fragments (including single-chain variable fragments (scFv)), and single-domain antibody (e.g., sdAb, sdFv, nanobody) fragments.

[0060] The present invention enhances mammalian cell culture by addressing specific challenges and bottlenecks common to fed-batch culture, including low culture viability, reduced VCD, atypical lactate accumulation, and low productivity. As demonstrated in the following examples, IPFB-based cultures according to the present invention can reduce final lactate accumulation, improve VCD and viability persistence, increase productivity, and provide more desirable product quality. As demonstrated in the examples, productivity is improved by at least 30%, and preferably by 150% or more. From the perspective of scalability and operability in large-scale biologics manufacturing, the IPFB of the present invention significantly reduces the complexity and equipment requirements for large-volume medium preparation, storage, and waste disposal typically associated with perfusion culture. Furthermore, the healthier cell state at harvest achieved by IPFB is beneficial for downstream impurity removal and potentially reduces safety concerns. [Example]

[0061] The following examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention.

[0062] Materials and Methods In the following examples, CHO-K1 cell lines recombinantly expressing monoclonal antibodies of the subclass IgG1 were cultured. Unless otherwise specified, the basal culture medium was Actipro medium (Hyclone, Catalog No. SH31037) containing 1% HT Supplement (Gibco, Catalog No. 11067), and the feed medium was CB7a / CB7b (Hyclone, Catalog Nos. SH31026 and SH31027).

[0063] Cell density and viability were monitored by trypan blue exclusion using a Vi-Cell Analyzer equipped with Vi-Cell XR software version 2.04. Lactate accumulation and product mAb titer were monitored using a CedexBio HT Analyzer.

[0064] Example 1: Study of the effect of medium changes on different days First, to investigate the effect of medium exchange on different days, we conducted the experiments summarized in Table 1. Study 1 ("ST No. 1") without medium exchange was employed as a control.

[0065] [Table 1]

[0066] Cells were inoculated into 50 ml spin tubes containing 15 ml of basal culture medium at the indicated seeding density. Feeds were added according to the days and ratios listed in Table 1. Medium exchange was performed by centrifugation at 250 g for 5 minutes, removing the supernatant, and adding an equal volume of fresh medium instead. Ignoring the volume of the cell pellet, such an exchange corresponds to a nearly 100% medium exchange, which corresponds to a perfusion rate of approximately 3 VVT, which will be used in further bioreactor studies.

[0067] result As shown in Figures 3 and 4, the peak VCD largely overlapped among the various processes. However, it is noteworthy that processes that included medium exchange improved the persistence of VCD and cell viability along the later stages, especially after day 10. Meanwhile, medium exchange also reduced the accumulation of residual lactate (Figure 5). The culture in the control group (ST No. 1) was terminated early due to low viability. In contrast, the culture period was extended in studies involving one or more medium exchanges.

[0068] Regarding the final titer of the mAb product, the control ended up with a titer of 2.451 g / L, while all processes that included one or more media changes significantly increased the titer (Figure 6). As shown in the figure, single media changes on days 4, 6, and 8 resulted in similar increases in titer, while a single media change on day 10 resulted in a slightly smaller increase. Furthermore, as shown in the figure, increasing the number of changes further increased the production titer, with ST No. 8 ("D4, D8, D12-3VVT") achieving the highest titer of 6.235 g / L. Overall, incorporating media changes increased product titer by 45% to 154%.

[0069] Example 2: IPFB cultivation in a 3L bioreactor In this study, an IPFB culture system was set up, including an alternating tangential flow filtration (ATF) system as a cell retention device, as shown schematically in Figure 1. The experimental results are summarized in Table 2.

[0070] [Table 2]

[0071] The inoculation density for the 3L bioreactor was 11 x 10 6cells / mL, which was achieved by perfusion culture at the seed stage. In culture "D5-3VVT," intermittent perfusion was initiated on day 5 and performed at a perfusion rate of 3VVT for 24 hours. In culture "D5, D9-3VVT," in addition to the first intermittent perfusion on day 5, a second intermittent perfusion was initiated on day 9, both of which were performed at 3VVT for 24 hours.

[0072] result As shown in Figures 7 and 8, the peak VCD largely overlapped among the various processes, but it was notable that processes with intermittent perfusion improved the persistence of VCD and cell viability along the late stage. Regarding lactate levels, as shown in Figure 9, the control culture began to re-elevate at the late stage as early as day 7. In contrast, the cultures with one intermittent perfusion (D 5-3 VVT) and two intermittent perfusions (D 5, D 9-3 VVT) delayed the re-elevation by 4 and 6 days, respectively.

[0073] Product titers plotted over time are shown in Figure 10. In the control, the titer increased slowly, plateauing after day 12 and ultimately reaching a value of 4.866 g / L; in contrast, in the two IPFB cultures, the titer continued to increase at a steeper slope over the long term until the end of the culture, reaching final values of 7.590 g / L and 7.634 g / L, respectively, representing over 55% and 67% increases in productivity compared to the control. Furthermore, as can be seen, the cultures with two intermittent perfusion runs achieved even higher titers than the cultures with a single run.

[0074] The product quality of the harvested samples produced in the 3L bioreactor after Protein A column purification was examined. As shown in Figures 11 and 12, intermittent perfusion culture improved product purity in terms of both size and charge variation. Furthermore, the results of N-glycan analysis are shown in Table 33. As can be seen, the N-glycan profiles were comparable among the three culture modes tested. These results demonstrated that IPFB not only improves productivity but also facilitates the adjustment of product quality in recombinant production.

[0075] [Table 3]

[0076] Example 3: Effect of intermittent perfusion rate In this study, the effect of perfusion rate was further investigated. Two IPFB cultures in a 3 L bioreactor using different perfusion rates were evaluated compared to a control culture. The results of the experiment are summarized in Table 4.

[0077] [Table 4]

[0078] result As shown in Figures 13 and 14, IPFB cultures with intermittent perfusion at 1VVT and 2VVT exhibited improved VCD and viability persistence throughout the production culture compared to the control, despite the peak VCD largely overlapping between the different perfusion regimes. The two IPFB cultures using lower perfusion rates exhibited a similar delayed lactate spike in the later stages (Figure 15), consistent with what was observed in the example above. Late lactate spikes may indicate mitochondrial dysfunction. Increasing the medium exchange rate, as reflected in the delayed late lactate spike, may support healthier cellular metabolism.

[0079] Due to the healthy cell condition, the product titer in IPFB cultures continued to increase rapidly until the end of the culture, with the final protein production reaching levels 30-40% higher than that of the control (7.220g / L-7.904g / L vs. 5.536g / L, Figure 16). This study suggested that IPFB offers a competitive advantage over conventional fed-batch cultures, even at low perfusion rates. Meanwhile, the IPFB process can be advantageously adapted to manage product costs and meet actual demands at manufacturing facilities.

[0080] Example 4: Study of various intermittent perfusion modalities In this study, more diverse IP modalities were evaluated, including different perfusion rates, durations, and / or intervals. The experiments are summarized in Table 5. Among them, the "Ds5~8-6VVT" culture included one run of intermittent perfusion (IP) starting on day 5 and running for 72 hours at 6VVT until day 8; "D4, D6, D9-0.5VVT" included three IP runs on days 4, 6, and 9, respectively, all running for 6 hours at 0.5VVT, with the second and third runs starting 2 and 3 days after the end of the previous run, respectively; and "D4, D5, D10-1.0VVT" included three IP runs on days 4, 5, and 10, respectively, all running for 6 hours at 1.0VVT, with the second and third runs starting 1 and 5 days after the end of the previous run, respectively. The "control" culture did not include IP.

[0081] [Table 5]

[0082] result As shown in Figures 17 and 18, all experimental groups showed improved VCD and viability persistence throughout the production culture compared to the control group. Figure 19 shows that all experimental groups showed a delayed lactate spike at the later stage, which is consistent with what was observed in the previous example.

[0083] Due to the healthy cells, the product titers in the IPFB cultures continued to increase rapidly until the end of the culture, with final titers of 6.214g / L, 6.340g / L, and 6.636g / L, respectively, indicating protein production levels 36-45% higher than that of the control group (4.583g / L) (Figure 20). This study suggests that the IPFB culture according to the present invention can flexibly accommodate expanded adjustments according to actual demand, significantly benefiting cell growth performance and productivity.

Claims

1. 1. A cell culture method comprising a fed-batch process and one or more perfusion steps, wherein a temperature shift is performed to lower the temperature before a first perfusion step, the first perfusion step being initiated 0-7 days after the temperature shift, and the temperature shift is performed when the viable cell density (VCD) has increased to at least 50% of peak VCD.

2. 2. The method of claim 1, wherein the temperature shift occurs when the VCD increases to 50-70%, 70-90%, or 85-95% of the peak VCD.

3. 10. The method of claim 1, wherein the temperature change shift occurs on a day between day 0 and day 5 of the fed-batch process.

4. 10. The method of claim 1, wherein the first perfusion stage begins on or after day 2, or between days 2 and 7 of the fed-batch process.

5. 10. The method of claim 1, further comprising one or more additional perfusion stages, each independently beginning 1 to 5 days after the end of the previous perfusion stage.

6. 6. The method of claim 5, wherein the one or more additional perfusion steps begin on day 5 of the fed-batch process or later, for example between days 5 and 12.

7. 10. The method of claim 1, wherein the perfusion comprises a retention device such as an alternating tangential flow filtration (ATF) system or a tangential flow filtration (TFF) system.

8. The method of claim 7 , wherein the holding device holds cells in a culture vessel.

9. 10. The method of claim 1, wherein the first perfusion stage and the one or more additional perfusion stages are independently performed at a perfusion rate of 0.5 to 6 VVT.

10. 10. The method of claim 1, wherein the first perfusion step and the one or more additional perfusion steps are carried out independently for 6 to 72 hours.

11. 11. The method of any one of claims 1 to 10, further comprising a seed propagation step comprising a perfusion culture and / or a concentrated fed-batch culture for inoculating the fed-batch process.

12. The fed-batch process ６ ~50 x 10 ６ 2. The method of claim 1, starting with inoculation at a seed density of 100 cells / ml.

13. The method of claim 1 , wherein the cell is a mammalian cell.

14. 10. The method of claim 1, wherein the cell is a host cell transformed to recombinantly express a product of interest, and the method further comprises harvesting the expressed product.

15. 15. The method of claim 14, wherein the product of interest is a polypeptide, such as a monoclonal antibody.

16. A method for producing a target product, comprising culturing cells that express the target product according to the method of any one of claims 1 to 10, and harvesting the expressed target product.

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