Method of operating a bioprocessing facility for producing a biological product

Abstract

The present invention relates to a method of operating a bioprocessing facility (1) for producing a biological product, the bioprocessing facility (1) comprising a source receptacle (2) for cell culture, a harvest receptacle (3) for biomanufacturing, and a clarification device (4) using a centrifuge (12), the source receptacle (2) comprising: a) initiating a cyclic production mode within the source receptacle (2); b) culturing cells in a source receptacle (2), thereby obtaining a cell broth (7) containing the cultured cells; c) discharging the discharged fraction (16) of the cell broth (7) from the source receptacle (2); d) combining the reinitiation portion (17) of the cell broth (7) with fresh culture medium (8) and repeating step b); e) repeating steps c) and d) at least once, and / or f) Discharging the cell broth (7) obtained from step d) from the source receptacle (2) to stop the cyclical production mode and obtain a discharge fraction (16). Operates in a cyclical production mode, including The method further comprises: i) centrifuging the effluent fraction (16) through a centrifuge (12), thereby separating the effluent fraction (16) into at least a centrifuged effluent fraction (19), a supernatant (14), and preferably a bioproduct; ii) operating the harvesting receptacle (3) in a production mode; Steps i) and ii) are carried out at least twice.

Description

[Technical Field]

[0001] The present invention relates to a method for operating a bioprocessing facility for producing a bioproduct according to claim 1.

[0002] The term "bioprocess" currently refers to any type of biotechnological process, especially biopharmaceutical processes. Therefore, the proposed method can be applied in various fields of biotechnology and for different types of bioprocesses. One example of a bioprocess is the use of a bioreactor to cultivate microorganisms, such as bacteria or mammalian cells, under given conditions to produce a biological product. Typically, the cell broth is transferred from the bioreactor to downstream processes to separate the biological product from the cells and purify the biological product.

[0003] The term "bioproduct" generally refers to a product produced by cultured cells. Cell cultures can currently be used to produce biopharmaceuticals, particularly proteins such as human insulin, growth factors, hormones, or vaccine proteins, in particular antibodies, antibody derivatives, exosomes, etc. Bioproducts may also be from the group of non-biopharmaceuticals, such as enzymes for food processing, laundry detergent enzymes, biodegradable plastics, or biofuels. Additionally or alternatively, cells may also be cultured to produce viral vectors, including lentiviral vectors, which are purified and used for applications in the emerging field of cell and gene therapy.

[0004] The focus of the present invention is on biopharmaceuticals, more specifically antibodies, and viral vectors that are secreted by cells into the culture medium.

[0005] The term "cell" preferably refers to mammalian cells, including HEK293 cells, CHO cells, etc. The cells are preferably genetically modified, preferably by transfection, to produce a desired bioproduct. Preferably, the bioproduct is secreted from the cell into the culture medium. Alternatively, the bioproduct remains intracellular.

[0006] The present method for operating a bioprocess facility to produce a biological product can be applied to various fields of biotechnology and different types of bioprocesses, for example, the present method can be applied to the production of monoclonal antibodies using a mammalian CHO suspension cell line.

[0007] Process efficiency is particularly important when operating bioprocessing facilities for the production of biological products. Process efficiency, in particular, relates to the cost-effectiveness and footprint of the process. High cost efficiency and increased flexibility have been driven by the growing demand for biopharmaceuticals, including monoclonal antibodies, vaccines, and the like. Another reason for cost-effective process design is the typically high material costs associated with the types of bioprocesses mentioned above. This is because sterility must be maintained throughout the process. As a result, bioprocesses typically involve the use of disposable materials and / or cost-intensive sterilization procedures, such as heat sterilization.

[0008] Cost efficiency is not only related to material costs, but also to efficient process design. Typically, to prepare a receptacle, particularly a bioreactor, for bioproduct production, a seed train, including multiple culture steps of increasing culture volume, must be run. This seed train culture requires cells to grow to a specific cell density at each step of the seed train in order to inoculate the next step, and since the cell growth rate may be relatively low, it takes a considerable amount of time. Furthermore, depending on the final production scale, a significant number of seed train bioreactors may be required, each of which must be run sequentially. As a result, process efficiency must be improved.

[0009] Furthermore, it is desirable to have as high equipment utilization as possible, which means that the receptacles involved in the process are highly utilized, and the process design in terms of the equipment required should be as simple as possible to keep complexity as low as possible.

[0010] WO 99 / 33955 discloses a method of operating a bioprocessing facility for the production of a biological product, preferably relating to the cultivation of anchorage-dependent cells, the bioprocessing facility comprising a source receptacle for cell culture and a harvest receptacle for biomanufacturing. It is understood that a portion of the cells from the source receptacle can be reused as a restart fraction, and another fraction of the cells is used for biomanufacturing. However, there is still a need to improve production efficiency.

[0011] WO 2020 / 010080 relates to a method for culturing mammalian cells in a source receptacle and strongly increasing the cell concentration by conditioning the culture medium before transferring it to a harvest receptacle, which is used to achieve cell densities similar to those of perfusion culture without the use of perfusion culture.

[0012] The challenge is to improve the known prior art to achieve higher process efficiency.

[0013] The present invention is based on the problem of improving known methods to reach further optimization with respect to this task.

[0014] The above object is solved by the features of claim 1.

[0015] A key realization of the present invention is that operating a source receptacle in a cyclic production mode to manufacture cells and a harvest receptacle in acyclic production to manufacture a bioproduct allows for efficient supply of cells to the harvest receptacle and allows for separate optimization of both process steps, including process parameters such as initial viable cell concentration and the selected culture medium.

[0016] The source receptacle is used to culture cells, and a portion of those cells is transferred to a harvest receptacle, where the cells primarily produce the bioproduct. Between these steps, the cells are centrifuged, allowing for concomitant harvesting of the bioproduct produced in the source receptacle, further increasing the viable cell concentration, removing potentially toxic by-products, and allowing for varying process conditions between receptacles. Notably, there may be different optimal process conditions for the cells and the production of the bioproduct.

[0017] The use of separate receptacles makes cell production in the source receptacle particularly simple, and the acyclic operation of the harvest receptacle allows for adjustment of process conditions in the harvest receptacle without the need to keep cells viable, facilitating biomanufacturing. Furthermore, the use of a first receptacle for repeated cell production can significantly reduce the time, labor, and cost of seed train culture.

[0018] By including a centrifuge-based clarification device, cells can be easily washed before being transferred to the harvest receptacle, allowing for particularly simple adjustment of process conditions between the source and the harvest receptacle. In this way, dead cells and cell debris can also be at least partially removed from the cell broth, resulting in increased viability of the culture. Furthermore, washing allows for the removal or at least reduction of by-products, such as compounds that potentially inhibit cell growth, such as lactate and / or ammonium, from the culture medium. This can enhance subsequent cultivation in the harvest receptacle and / or simplify further purification of the bioproduct.

[0019] Additionally, the use of a centrifuge allows for easy separation of the biological product and cells so that the biological product can be harvested even during transfer from the source receptacle to the harvest receptacle.

[0020] Operating the source receptacle in a cyclic production mode and the harvest receptacle in an acyclic production mode reduces process costs and footprint, particularly compared to perfusion-based processes, because no large medium storage is required and no cell retention means need to be provided by either receptacle. The use of two separate receptacles also provides the option to match the culture times in each receptacle to each other, increasing equipment utilization. This further improves process efficiency. By using the restarting fraction of cells to start the next culture in the source receptacle, the source receptacle can be used directly for the next production cycle and does not need to be cleaned, significantly reducing source receptacle downtime.

[0021] A method is provided for operating a bioprocessing facility for producing a biological product, the bioprocessing facility including a source receptacle for cell culture, a harvest receptacle for biomanufacturing, and a centrifuge-based clarification device, the source receptacle operating in a cyclic production mode, the cyclic production mode comprising, in this order: a) initiating a cyclical production mode in a source receptacle with initial cells and culture medium; b) culturing cells in a source receptacle, thereby obtaining a cell broth containing the cultured cells, wherein the source receptacle and the culture medium are configured to provide source culture environment conditions for the culturing of the cells; c) discharging the discharged fraction of cell broth from the source receptacle; d) combining the reinitiation portion of the cell broth with fresh culture medium and repeating step b); e) repeating steps c) and d) at least once, and / or f) Discharging the cell broth obtained from step d) from the source receptacle to stop the cyclic production mode and obtain a discharge fraction. the method further comprising: i) transferring the effluent fraction to a clarification device and centrifuging the effluent fraction through a centrifuge, thereby separating the effluent fraction into at least a centrifuged effluent fraction, a supernatant, and preferably a bioproduct; ii) transferring at least a portion of the centrifuged discharge fraction from step i) and fresh culture medium to a harvest receptacle and operating the harvest receptacle in a production mode, wherein the harvest receptacle and culture medium are configured to provide harvest culture environmental conditions, and producing a biological product in the harvest receptacle; wherein steps i) and ii) are carried out at least twice using the effluent fraction from the execution of step c) and / or step f).

[0022] The term "cell broth" means a suspension of solid cells and possibly cell debris in a culture medium, and refers to the entire culture medium and the individual cells cultured in the culture medium.

[0023] According to claim 2, the cultivation in the source receptacle and the cultivation in the harvest receptacle are carried out in parallel for a significant period of time. In this way, process efficiency is significantly improved as equipment utilization is increased due to greater utilization of the receptacles.

[0024] Claim 3 is directed to synchronizing incubation time in the source receptacle with production time in the harvest receptacle. Synchronizing incubation time with production time is important to keep the process as efficient as possible, especially when the number of available receptacle slots is limited. Synchronizing incubation time can reduce extensive downtime for any one receptacle, thereby improving equipment utilization and, therefore, process efficiency.

[0025] By repeating the cyclic production mode without breaking the synchronization between the source receptacle slot and the harvest receptacle slot (claim 4), the duration of the seed train is significantly reduced, since all steps of the seed train do not need to be repeated for each successive transfer of cells to the harvest receptacle. Furthermore, each subsequent cyclic production in the source receptacle may be performed in the same source receptacle slot, thereby reducing the overall footprint of the process because no additional receptacle slots are required. This also increases equipment utilization, since not only the receptacle itself but also the infrastructure at each receptacle slot is utilized as much as possible.

[0026] According to claim 5, the harvest fraction from the harvest receptacle is subjected to a clarification device after producing the bioproduct. Here, the use of a centrifuge in the clarification device is particularly advantageous, since it allows the discharge from the source and the harvest receptacle to be carried out similarly for each process and each device, resulting in easier work steps and also higher device usage time by using the same centrifuge.

[0027] By using a sensor device to measure at least one parameter of the source receptacle and / or the harvest receptacle (claim 6), it becomes possible to determine the remaining incubation time and / or the remaining production time, which makes it possible to adapt the times of some process steps, in particular the duration of step b), thereby maintaining synchronization of the incubation times in the source receptacle and the harvest receptacle even in the event of unexpected changes.

[0028] Providing a source electronic process control for controlling source culture environmental conditions, as proposed in claim 7, is advantageous because it allows the source receptacle to be controlled to change its culture conditions, cell growth rate, etc. as needed. For example, if the harvest receptacle is used for production for longer than planned, the source receptacle can be adapted to slow down the cell growth rate. This synchronizes the source and harvest receptacles, so that the target number of cells is reached later and instead of having to discard part of the cell broth from the source receptacle if the cells reach their target cell mass prematurely, or risking that the cells lose their viability and are no longer used. Furthermore, if bioproduction is already occurring in the source receptacle, adapting the source receptacle can result in the initiation and / or ramp-up of bioproduction, so that the amount of bioproduct in the source receptacle can be increased significantly. Nevertheless, producing the target amount of cells and maintaining a constant cell viability remains a priority within the source receptacle.

[0029] By predicting the time when the end conditions will be reached in the harvest receptacle and adjusting the culture conditions in the source receptacle based on this prediction (claim 8), production can continue in the harvest receptacle, and cell growth in the source receptacle can be adjusted accordingly, greatly increasing process flexibility. In this way, the production time in the harvest receptacle can be changed without having to repeat the complete seed train. In particular, the source receptacle may not be optimized for maximum cell growth; instead, there is still room to increase, and preferably also decrease, cell growth, thereby providing a control reserve and producing more bioproduct in the source receptacle. Providing a control reserve is a particularly interesting possibility offered by the proposed method. Until now, the harvest receptacle has typically been timed to the seed train, and does not tolerate larger deviations in the harvest receptacle's run time, even if the cells may still have high viability and good production rates. Being able to use cells for longer periods without desynchronizing the seed train reduces waste and cost per product.

[0030] Claim 9 details possible options for synchronizing the source receptacle and the harvest receptacle: A synchronization strategy can be implemented to reach a predetermined cell mass in the source receptacle required for the subsequent production of a biological product in the harvest receptacle.

[0031] By increasing the cell concentration between steps b) and ii), the subsequent production of the biological product in step ii) can be accelerated (claim 10). Increasing the viable cell concentration by centrifugation is particularly advantageous because not only can the cells be separated from the already produced biological product, but the cells can also be washed and / or transferred to another culture medium.

[0032] By changing at least one process parameter of the source culture environment conditions compared to the harvest culture environment conditions, cell culture and bioproduction can be efficiently divided between receptacles (claim 11). In the harvest receptacle, cell viability does not need to be maintained above a certain threshold because cells can be discharged at the end of production. The use of a centrifuge makes it even easier to change the culture environment between the cultivation of cells and the production of bioproducts.

[0033] In a preferred embodiment as claimed in claim 12, the source culture environment and / or the harvest culture environment may, at least initially, be the same for multiple repetitions of steps b) and ii), making the entire method a repetition of the same parallel culture and production cycle.

[0034] By using batch and / or fed-batch culture conditions (claim 13), the overall setup is simplified, especially compared to perfusion-based systems, since no means of retaining cells in any receptacle is required during cultivation. This improves the cost efficiency of the overall process and simplifies the operation of the bioprocessing facility by reducing process complexity. Furthermore, no large storage space is required to continuously supply the required amount of culture medium. In contrast to perfusion culture, contamination risks, footprints in the form of culture medium storage and disposal, and complexity are reduced.

[0035] Claim 14 specifies the characteristics of the centrifuge and the cell washing and cell discharge steps. These specifications allow for optimized preparation of cells during forward or reverse operation of the centrifuge, as well as optimized conditions for subsequent biomanufacturing. The specified centrifuge is particularly well suited for the proposed method.

[0036] By transfecting the cells after step c), biomanufacturing can only be possible after the cells have been expelled from the source receptacle. In this way, no biomanufacturing occurs within the source receptacle, which is a particularly efficient option for separating biomanufacturing from cell growth (claim 15).

[0037] The term "transfection" refers to the transfer of genetic material to genetically modify cells so that they produce a desired bioproduct. Cell transfection can be performed in a variety of ways, including techniques such as electroporation, treatment with chemicals such as calcium phosphate, and microinjection. Preferably, and as described below, transfection is performed by chemical treatment. Alternatively, transfection may be performed using a virus or viral vector.

[0038] In the following, embodiments of the present invention will be described with reference to the drawings, in which: [Brief explanation of the drawings]

[0039] [Figure 1] FIG. 1 shows a bioprocessing facility for carrying out the proposed method for operating a bioprocessing facility according to one embodiment of the present invention. [Figure 2] FIG. 1 shows steps a) to e) of the proposed method for operating a bioprocessing facility. [Figure 3] FIG. 1 shows steps e) to iii) of the proposed method for operating a bioprocessing facility. [Figure 4] FIG. 10 shows an example for synchronizing the cultures in the source and harvest receptacles. [Figure 5] 1 illustrates a source receptacle in a source receptacle slot, a harvest receptacle in a harvest receptacle slot, and a clarification device in an exemplary embodiment of the present application. [Figure 6]FIG. 1 illustrates a proof-of-concept experiment.

[0040] The proposed method for operating a bioprocessing facility 1 is shown in Figure 1. Figure 1 shows two cycles of a preferred embodiment of the method, which shows the method diagrammatically from left to right over time in Figure 1. The bioprocessing facility 1 comprises a source receptacle 2 for cell culture, a harvest receptacle 3 for biomanufacturing, and at least one clarification device 4.

[0041] In a preferred embodiment, and as shown in Figure 1, source receptacle 2 is a bioreactor. It is further preferred that the material of source receptacle 2 is a disposable material. Alternatively, source receptacle 2 may be a stainless steel tank bioreactor. Source receptacle 2 here and preferably includes at least one source receptacle inlet port 5 for adding liquid. Each source receptacle inlet port 5 may be connected to a respective liquid line that supplies liquid.

[0042] The term "liquid" refers to any process liquid handled in the proposed bioprocess equipment 1. This includes reagents added to control the source and / or harvest culture environmental conditions, e.g., acids or bases to control pH values, but also includes wash liquids such as buffers used. In general, the term liquid can also refer to the culture medium and / or feed medium 6 and / or cell broth 7.

[0043] The term "culture medium" refers to the fact that cells used in bioprocesses are grown in or on specially designed solid, semi-solid, or liquid culture media that supply the nutrients required by each organism or cell. Various media exist, but typically contain at least a carbon source, a nitrogen source, water, salts, and trace nutrients. Chemically defined culture media are preferably used, meaning that all components of the medium and their respective concentrations are known. However, it is also entirely possible to use chemically undefined media that may contain unknown nutrients and / or unknown amounts of nutrients. A typical example of a chemically undefined medium is a medium containing FBS (fetal bovine serum).

[0044] As described above, the bioprocessing equipment 1 further comprises a harvest receptacle 3. In a preferred embodiment, and as shown in FIG. 1, the harvest receptacle 3 is a bioreactor. It is further preferred that the material of the harvest receptacle 3 is a disposable material. Alternatively, the harvest receptacle 3 may be a stainless steel tank bioreactor. Preferably, the harvest receptacle 3 further comprises at least one harvest receptacle inlet port 9 for adding liquid. Each harvest receptacle inlet port 9 may be connected to a respective liquid line that supplies liquid.

[0045] In a preferred embodiment, and as shown in Figure 1, different harvesting receptacles 3 are used throughout the method, as further described below.

[0046] 1, source receptacle 2 and / or harvest receptacle 3 preferably include at least one source receptacle outlet port 10 and / or at least one harvest receptacle outlet port 11. Preferably, the respective outlet ports are used to remove liquid from source receptacle 2 and / or harvest receptacle 3. It is further preferred that cell broth 7 be removed from source receptacle 2 and / or harvest receptacle 3.

[0047] In a preferred embodiment, source receptacle 2 and / or harvest receptacle 3 are designed to accept a liquid volume of more than 5 liters, preferably more than 15 liters, and more preferably more than 50 liters. Preferably, source receptacle 2 and harvest receptacle 3 are designed to accept the same maximum liquid volume. It is further preferred that source receptacle 2 and harvest receptacle 3 have the same shape. Preferably, the layout and number of inlet and / or outlet ports are also identical in source receptacle 2 and harvest receptacle 3. This makes operation of both receptacles easier.

[0048] However, it is entirely possible for source receptacle 2 and harvest receptacle 3 to differ in at least one or all of the aspects described above. For example, the maximum liquid volume of harvest receptacle 3 may be less than the maximum liquid volume of source receptacle 2.

[0049] As also mentioned above, the bioprocessing facility 1 further comprises a clarification device 4. Here, and preferably, this clarification device 4 performs a physical process using centrifugal force to remove suspended solids, preferably cells, from the liquid phase. In general, the proposed clarification device 4 can be used to separate any solid / liquid components from each other, including, but not limited to, cells and culture medium. Preferably, the clarification device 4 comprises a centrifuge 12 for clarification of the cell broth 7 by centrifugation. "Centrifugation" is a term for the sedimentation of particles in a gravitational field artificially generated by centrifugal force, whereby significant acceleration forces result in significant reductions in separation time.

[0050] To carry out the proposed method, preferably, the source receptacle 2 and the centrifuge 12 can be in fluid coupling, as also shown in Figure 1. It is even more preferred that the centrifuge 12 and the harvest receptacle 3 are also in fluid coupling.

[0051] The term "fluid coupling" means that a fluid connection is established that can be used to transfer a fluid, preferably a liquid, more preferably at least a portion of the cell broth 7 from the source receptacle 2 to the centrifuge 12 and / or from the harvest receptacle 3 to the centrifuge 12, and / or to transfer a fluid, preferably a liquid, more preferably the centrifuged 12 cell broth 7 from the centrifuge 12 to the harvest receptacle 3 and / or to a waste 13 location, and / or to transfer the supernatant 14 containing the bioproduct from the centrifuge 12 to a further purification step 29.

[0052] The centrifuge 12 can operate in forward and / or reverse operation and includes a fluid network of interconnected fluid lines for establishing different fluid connections with the centrifuge 12 .

[0053] In particular, a method is proposed for operating a bioprocessing facility 1 for producing a biological product, the bioprocessing facility 1 comprising a source receptacle 2 for cell culture, a harvest receptacle 3 for biomanufacturing, and a clarification device 4 using a centrifuge 12.

[0054] The source receptacle 2 is proposed to operate in a cyclic production mode, which comprises, in this order: a) initiating a cyclical production mode in a source receptacle 2 with initial cells 15 and culture medium 8; b) culturing cells in a source receptacle 2, thereby obtaining a cell broth 7 containing the cultured cells, wherein the source receptacle 2 and the culture medium 8 are configured to provide source culture environment conditions for the culturing of the cells; c) discharging the discharged fraction 16 of the cell broth 7 from the source receptacle 2; d) combining the reinitiation portion 17 of the cell broth 7 with fresh culture medium 8 and repeating step b); e) repeating steps c) and d) at least once, and / or f) Discharging the cell broth 7 obtained from step d) from the source receptacle 2 to stop the cyclic production mode and obtain a discharge fraction 16. Includes.

[0055] FIG. 1 shows the bulk of the overall method, and FIG. 2 shows the steps labeled a) through e). Steps a) through f) relate to source receptacle 2, while harvest receptacle 3 is further described below. Source receptacle 2 is run in a cyclical production mode to cultivate cells. In step a), cell cultivation begins by adding initial cells 15 to source receptacle 2, as shown in the upper left of FIG. 2. FIG. 2 shows how initial cells 15 and culture medium 8 are combined in source receptacle 2 in a conventional manner to cultivate the initial cells 15 and increase the absolute number of cells in source receptacle 2. The initial cells 15 may be the result of a seed train, in which cultivation in source receptacle 2 may be the final step. Preferably, the initial concentration of viable cells in source receptacle 2 is adjusted to a predetermined value. Preferably, this adjustment is performed by adding culture medium 8 until the predetermined cell concentration is reached.

[0056] Step b) involves culturing. The source receptacle 2 and the culture medium 8 together provide specifically predefined source culture environment conditions. The culture environment conditions include all relevant parameters for culturing cells and producing a bioproduct, such as temperature, agitation speed, pH value, and nutrient concentration. The culture environment conditions change, in part, over time. Starting from the initial culture environment conditions, the cells consume nutrients and secrete by-products. Depending on the culture environment conditions and the cells, a bioproduct may also be produced to some extent during culturing in the source receptacle 2, as will be further described below. Here, and preferably, the initial source culture environment conditions and / or the initial harvest culture environment conditions not yet described are predefined, meaning that there is a plan for the initial source and / or harvest culture environment conditions. Preferably, for the source and / or harvest receptacle 3, the subsequent culture environment conditions, and therefore all culture environment conditions, are predefined. However, this does not necessarily mean that all parameters are fully controlled. Preferably, the source culture environment conditions are adjusted to provide conditions that result in optimized cell growth and / or high overall viability of the cells.

[0057] Since certain medium components (e.g., carbon sources such as glucose) may be depleted from the culture medium 8 during cultivation as they are completely consumed by the cells, it may be necessary to supply a feeding medium 6 during the cultivation of the cells in step b) in order to maintain optimal cell growth conditions and keep the cell viability above a predetermined value. However, depending on the culture behavior and the cultivation time, the supply of the culture medium 8 is optional and may not be required in any case.

[0058] It should be noted that the feed medium 6 may be the same as the initial culture medium 8. Alternatively, in a preferred embodiment, the feed medium 6 differs from the culture medium 8 in at least one type and / or at least one concentration of a component.

[0059] Preferably, at least one of the source receptacle inlet ports 5 is a feed port having a feed line 18 for controlled supply of culture medium 8 during cultivation according to a predetermined feed profile. Such a feed profile can be designed as a pulsed, continuous, or mixed feed profile, and preferably, the feed profiles are individually controllable and adjustable. Note that the supply of culture medium 8 results in an increase in the culture broth volume in the source receptacle 2 depending on the feed profile and the feed medium 6.

[0060] Here, and preferably, after the culturing of cells in step b) reaches a predetermined end condition, preferably a target absolute cell number or a target cell concentration, step b) is terminated and step c) is initiated. Here, and preferably, step b) focuses on providing a rapid increase in cell number while keeping the cells viable, instead of producing a biological product. The biological product may or may not be produced in step b). If present, the biological product produced in step b) may be secreted into the cell broth 7 or may be located inside the cells. In the former case, the biological product may be harvested as described below; in the latter case, the biological product is preferably not harvested until after the cells have been used to produce a biological product in the harvest receptacle 3, as described below.

[0061] In step c), a drain fraction 16 of cell broth 7 is removed from source receptacle 2. This is shown in FIG. 1 as moving downward from source receptacle 2. Preferably, and as described in further detail below, cell broth 7 is not completely drained from source receptacle 2, and a restart fraction 17 is retained within source receptacle 2. This restart fraction 17 is combined with fresh culture medium 8 in step d) to again culture the cells in a repeat of step b).

[0062] This allows at least one repetition of step b) to cycle the culture in source receptacle 2. Instead of using all of the cell broth 7 to produce a biological product, the start of the next seed train is skipped, and the restarting fraction 17 is used to culture the cells in the same source receptacle 2 again. Here, and preferably, the restarting fraction 17 remains in source receptacle 2 and is not temporarily removed from source receptacle 2 between repetitions of step b). In this way, source receptacle 2 can be used for two or more cycles without replacing or cleaning source receptacle 2. This is indicated by the dashed line used for source receptacle 2 in FIG. 1. The repetition of step b) is indicated by a shift to the right at the top of FIG. 1 and / or as part of step d) in FIG. 2.

[0063] A repetition of step b), preferably each repetition of step b), may in some or all embodiments be identical to the first run of step b), particularly with respect to the source environmental conditions or some of the source environmental conditions. All embodiments described herein with respect to step b) may be identical, alone or in any combination, for at least one, and preferably all, runs of step b).

[0064] The restart fraction 17 and the discharge fraction 16 preferably sum to at least 95%, preferably at least 99%, more preferably 100% of the cell broth 7 in the source receptacle 2 at the start of step c) or the end of step b).

[0065] Preferably, the discharge fraction 16 is at least 50%, preferably at least 70%, more preferably at least 90% of the volume of the cell broth 7 at the start of step c) or at the end of step b). Preferably, the cell broth 7 is discharged from the source receptacle 2 using the source receptacle outlet port 10. The cell broth 7 is preferably transferred to a centrifuge 12, as described in more detail below.

[0066] In step e), steps c) and d) may be repeated at least once, preferably at least twice, and more preferably at least three times. After each repetition, the drain fraction 16 is transferred to a harvest receptacle 3, as described below. Thus, the source receptacle 2 periodically produces cell broth 7 for bioproduction without the need for a seed train each time.

[0067] In step f) shown in Figure 3, cyclic production stops. The drain fraction 16, which is preferably at least 95%, preferably at least 99%, and more preferably 100%, of the cell broth 7 in the source receptacle 2, is now, and preferably, transferred again to the harvest receptacle 3 as described. Figure 2 shows steps a) to e). Figure 2 is to be read from left to right in each of the three rows. VCC is the viable cell concentration in the source receptacle 2, which increases or decreases in different method steps. The volume in the source receptacle 2 may or may not change during the steps (as shown) depending on the details of the process.

[0068] The effluent fraction 16, whether from step d) or step f), is not transferred directly to the harvest receptacle 3, but is sent to the clarification device 4.

[0069] Turning now to the harvesting receptacle 3 side and the transfer to the harvesting receptacle 3 shown below the first line of FIG. 1 and in FIG. 3, the proposed method further comprises: i) transferring the effluent fraction 16 obtained from either step c) or f) to a clarification device 4 and centrifuging the effluent fraction 16 via a centrifuge 12, thereby separating the effluent fraction 16 into at least a centrifuged effluent fraction 19, a supernatant 14, and preferably a bioproduct; ii) transferring at least a portion of the centrifuged discharge fraction 19 from step i) and fresh culture medium 8 to a harvest receptacle 3 and operating the harvest receptacle 3 in a production mode, here and preferably in an acyclic production mode, whereby the harvest receptacle 3 and the culture medium 8 are configured to provide harvest culture environmental conditions and produce a biological product within the harvest receptacle 3; wherein steps i) and ii) are carried out at least twice using the discharge fraction 16 from the execution of step c) and / or step f).

[0070] Step i) concerns the centrifugation of the discharge fraction 16 through a centrifuge 12, preferably always the same centrifuge 12. This centrifugation can be used to achieve several positive effects.

[0071] The effluent fraction 16 can be transferred to the centrifuge 12 via a multi-use or temporary liquid line. By multi-use, we mean that the liquid line is used for at least two effluent fractions 16. A pumping device may be used to transfer the effluent fractions 16. The effluent fractions 16 are then centrifuged (12). This centrifugation may increase the cell concentration so that a higher initial cell concentration can be used in step ii). The cells may be washed to remove cellular debris and / or accumulated by-products. Preferably, the bioproduct is separated from the effluent fraction 16, especially if the bioproduct is secreted into the cell broth 7. The bioproduct may be separated directly by the centrifuge during centrifugation to produce the centrifuged effluent fraction 16, or it may be contained in the supernatant 14 and separated in a later or previous step. Centrifugation, in particular, allows for changing the culture environment conditions between the source receptacle 2 and the harvest receptacle 3, since the supernatant 14 can be removed and replaced with fresh culture medium 8 having different properties. Removal of accumulated by-products further improves the efficiency of subsequent biomanufacturing.

[0072] At least a portion of the centrifuged discharge fraction 19, preferably at least 95%, more preferably at least 99%, more preferably 100%, of the centrifuged discharge fraction 19, is preferably combined with fresh culture medium 8 in step i) and then used in step ii) to produce a bioproduct. Preferably, the cells are combined with fresh culture medium 8 during the centrifugation process, and this centrifuged discharge fraction is then transferred to a harvest receptacle 3. The fresh culture medium 8 may have a volume less than 90% of the volume of the discharge fraction 16, preferably less than 70%, more preferably less than 50% of the volume of the discharge fraction 16. Here, it is preferred that the culture medium 8 of the centrifuged discharge fraction 19 differs in at least one type and / or amount of nutrient from the culture medium used in step a) and / or the feed medium used in step b).

[0073] Preferably, the harvest culture environmental conditions in the harvest receptacle 3 are substantially different from the source culture environmental conditions. The former may be optimized for biomanufacturing and / or the latter may be optimized for cell growth. For example, a feed medium 6 may be supplied through the harvest receptacle inlet port 9. The feed medium 6 and / or feeding profile may be the same feed medium 6 and / or feeding profile as that used in step b) in the source receptacle 2. However, it is preferred that the feed medium 6 and / or feeding profile differ in at least one parameter from the feed medium 6 and / or feeding profile used in step b) in the source receptacle 2. One example of such a parameter may be the nutrient composition of the feed medium 6.

[0074] As shown in Figure 1, steps i) and ii) are also repeated, but preferably using a different, particularly disposable, harvest receptacle 3 or in a harvest receptacle 3 that is cleaned between runs. The source receptacle 2 and the harvest receptacle 3 are not the same receptacle.

[0075] Here, and preferably after each execution of step ii), a biological product is extracted from the cell broth 7 produced in harvest receptacle 3. Preferably, since there is no further use of the cells from harvest receptacle 3, the harvest culture environmental conditions may be such that the cells die during biomanufacturing, reducing cell viability to a lower value compared to the culture in source receptacle 2.

[0076] The duration of step b) and / or step ii) may be at least 1 day, preferably at least 2 days.

[0077] Here, and preferably, the source receptacle 2 and the harvest receptacle 3 are not directly connected per process. Here, and preferably, the cells are not transferred from the harvest receptacle 3 to the source receptacle 2.

[0078] Turning again to the centrifuge 12, the centrifuge 12 can be operated in forward operation for cell separation and / or cell washing. "Forward operation" refers to one of the two possible fluid flow directions of the centrifuge 12 and describes an operation that leads to the separation of liquid and solid particles, such as culture medium and cells. The liquid thus obtained is the supernatant 14. This separation makes it possible, on the one hand, to wash the separated cells with a washing buffer, preferably a PBS buffer, or a medium, preferably a culture medium 8, and / or, on the other hand, to obtain the supernatant 14.

[0079] Alternatively, the centrifuge 12 can be operated in reverse operation. "Reverse operation" refers to the second of two possible fluid flow directions of the centrifuge 12 and describes operation that results in the discharge of separated solid particles, preferably cells. The resulting product of reverse operation can be a centrifuged discharge fraction 19.

[0080] According to one embodiment, it is proposed that for at least one, preferably at least two, more preferably each run of steps b) and ii), the culturing of cells in step b) and the bioproduction in step ii) are carried out at least partially in parallel, preferably the runs of steps b) and ii) are carried out in parallel for at least 50%, preferably at least 75%, more preferably at least 90% of the time used for culturing in step b) and / or bioproduction in step ii) and / or the time used for carrying out steps b) and / or ii). The times mentioned are the time from the initial filling of the respective receptacle, or partially, from refilling with fresh culture medium 8, to removing the discharge fraction 16 or removing at least a necessary portion of the cell broth 7 from the harvest receptacle 3 for harvesting the bioproduct. If the bioproduct is harvested from the harvest receptacle 3 and / or the discharge fraction 16 is discharged in several fractions, the last fraction is of interest.

[0081] As shown in Figure 4a, as part of the proposed method, for example, after step b) has been performed for the first time (n=1), a restarting portion 17 of cells may be combined with fresh culture medium 8 (step d)), and step b) may be repeated at least once more (n+1). However, it is preferred that step b) be repeated until the cyclical production mode is stopped by performing step f) of the proposed method.

[0082] As soon as steps c) and i) have been carried out for the first time (n) on the discharge fraction 16 of step b), the first execution (n) of step ii) can take place in the harvesting receptacle 3 .

[0083] Both receptacles are run in parallel by running a second run (n+1) of step b) in parallel with the first run (n) of step ii). By running the cultures in the receptacles in parallel for a predetermined amount of time, utilization of the receptacles and associated equipment is maximized.

[0084] As will be further explained below, different strategies can be applied to synchronize the culture time in both receptacles.

[0085] 5, biomanufacturing can be optimized with respect to a general process plan by providing slots for receptacles, planning media movement and process times, etc. Thus, it may be the case that a single source receptacle slot 20 is paired with a single harvest receptacle slot 21, step b) is performed for a specifically predetermined incubation time, and step ii) is performed for a specifically predetermined production time, with the incubation time and production time, and therefore the source receptacle slot 20 and harvest receptacle slot 21, being synchronized such that the same harvest receptacle slot 21 is used for at least two consecutive runs of step ii), preferably all runs.

[0086] One slot comprises exactly one source or harvest receptacle 3, preferably a receptacle and at least one or exactly one electronic process control device for a given position.

[0087] Here, and preferably after at least one repetition of steps b), c), and i), and preferably after each repetition, harvest receptacle slot 21 is already prepared to receive centrifuged discharge fraction 19. When the term "repetition" is used, it refers to any run except the first run. Thus, the method is synchronized so that not only is harvest receptacle 3 finished, but also the steps necessary to prepare harvest receptacle slot 21, such as connecting a new receptacle, are performed. Preferably, harvest receptacle slot 21 is already prepared when the first fraction of centrifuged discharge fraction 19 is ready to be discharged from centrifuge 12, or at least is prepared immediately to prevent the death of a relevant portion of the cells.

[0088] The term "pairing" here means that cells cultured in source receptacle 2 in source receptacle slot 20 are transferred to harvest receptacle 3 located in harvest receptacle slot 21. The pairing of source receptacle slot 20 with harvest receptacle slot 21 is preferably maintained for at least two runs of steps b) and ii) and / or preferably for at least 3 weeks, more preferably at least 5 weeks.

[0089] As shown in Fig. 5, the source receptacle slot 20 provides a physical space for receiving the source receptacle 2. Preferably, the source receptacle slot 20 provides a physical location for installing the source receptacle 2. As shown in Fig. 5, a transport means can be used to transport the source receptacle 2 into the space provided by the source receptacle slot 20. It is further preferred that the source receptacle slot 20 includes a source electronic process controller 22, and that the source electronic process controller 22 is integrated into a source electronic process control unit 23. Preferably, the source electronic process control unit 23 is located adjacent to the source receptacle 2.

[0090] It is further preferred that the source electronic process control unit 23 is connected to at least one sensor located within or at the source receptacle 2. As shown in Figure 5, this sensor may be a pH probe or a temperature probe or any other sensor suitable for monitoring, and preferably also controlling, at least one process parameter.

[0091] Preferably, the incubation in the source receptacle slot 20 is carried out for a predetermined incubation time, after which the discharge fraction 16 of the cell broth 7 is discharged from the source receptacle 2 and transferred to the centrifuge 12.

[0092] As further shown in Figure 5, the source receptacle 2 is connected to a clarification device 4 comprising a centrifuge 12. Note that the complex network of fluid lines conducting liquid to and from the centrifuge chambers 24 is not shown to maintain clarity. Preferably, the centrifuge 12 is designed as a fluidized bed centrifuge 12 comprising at least one centrifuge chamber 24. In a preferred embodiment, the centrifuge 12 comprises four centrifuge chambers 24, as shown in Figure 5.

[0093] It is noted that the connection between the source receptacle 2 and the centrifuge 12 and / or the harvest receptacle 3 and the centrifuge 12 is preferably maintained only until the respective transfer steps are performed. In this way, no transfer to and / or from the centrifuge 12 is required and the centrifuge 12 may be utilized elsewhere when centrifugation is not required.

[0094] As shown in Figure 5, the harvesting receptacle slot 21 provides a physical space for receiving the harvesting receptacle 3. Preferably, the harvesting receptacle slot 21 provides a physical location for installing the harvesting receptacle 3. As shown in Figure 5, a transport means can be used to transport the harvesting receptacle 3 into the space provided by the harvesting receptacle slot 21. It is further preferred that the harvesting receptacle slot 21 includes a harvesting electronic process controller 25, and that the harvesting electronic process controller 25 is integrated into a harvesting electronic process control unit 26. Preferably, the harvesting electronic process control unit 26 is located adjacent to the harvesting receptacle 3.

[0095] It is further preferred that the harvest electronic process control unit 26 is connected to at least one sensor located within or at the harvest receptacle 3. As shown in Figure 5, this sensor may be a pH probe or a temperature probe or any other sensor suitable for monitoring, and preferably also controlling, at least one process parameter.

[0096] As is apparent from Figure 5, source receptacle 2 and harvest receptacle 3 are preferably not directly connected. Furthermore, as also shown in Figure 5, once cell broth 7 is transferred from source receptacle 2 to centrifuge 12, cell broth 7 is not returned to source receptacle 2. Generally, it would be possible to feed the entire cell broth 7 from source receptacle 2 to centrifuge 12 and return the restart fraction 17 from the centrifuge directly or indirectly, for example via harvest receptacle 3, to the same source receptacle 2, which has not been washed, sterilized, etc. However, here and preferably, restart fraction 17 remains inside source receptacle 2.

[0097] In a preferred embodiment, steps a) to f) are synchronized in time with the repeating seed train, and step a) is repeated in the same source receptacle slot 20 after step f) without breaking the synchrony between the source receptacle slot 20 and the harvest receptacle slot 21. After several repetitions of step b), the cells may become genetically unstable. Preferably, they are replaced with new initial cells 15 from the seed train without changing the repetition in the harvest receptacle 3. In that way, production could theoretically be maintained indefinitely.

[0098] According to one embodiment, the method further comprises: iii) transferring the harvest fraction 27 from the harvest receptacle 3 to the clarification device 4 and centrifuging the harvest fraction 27 via the centrifuge 12, thereby separating the bioproduct from the harvest fraction 27, if present in the supernatant 14; Here, and preferably, step iii) is performed after each step ii). Preferably, harvest fraction 27 is the complete cell broth 7 from harvest receptacle 3. Again, if the bioproduct is in supernatant 14, it may be sent to further purification steps 29, while harvest fraction 28 is preferably directed to waste 13. Alternatively, if the bioproduct is intracellular, harvest fraction 28 may be directed to further purification steps and the supernatant may be directed to waste.

[0099] In a particularly preferred embodiment, the same clarification device 4 is used in steps i) and iii).

[0100] According to one embodiment, it is proposed that the bioprocess installation 1 comprises a sensor device, the sensor device comprising at least one sensor arranged in or at the source receptacle 2 and / or at least one sensor arranged in or at the harvest receptacle 3, preferably the at least one sensor being configured to measure at least one process parameter from the group of oxygen concentration, carbon dioxide concentration, pH, temperature, conductivity, pressure, viable cell concentration, viable cell production rate, product concentration, productivity, agitation speed and / or culture medium, including concentrations of nutrients such as carbon source concentration, nitrogen source concentration, amino acid concentration, growth factor concentration for the source receptacle 2 and / or the harvest receptacle 3. As mentioned above, the sensors may be connected to respective electronic process control units and used to control the respective culture environmental conditions in the source receptacle 2 and / or the harvest receptacle 3.

[0101] The term "process parameter" should be understood broadly and includes at least one process parameter that can be monitored and preferably also controlled within the source receptacle 2 and / or the harvest receptacle 3.

[0102] It should be noted that additional process parameters may be derived from monitoring two or more process parameters. For example, the change in bioproduct concentration over time, which may also be referred to as productivity, may be derived by relating the bioproduct concentration to the culture time. Furthermore, the cell growth rate may be determined from assessing the change in viable cell concentration over the culture time.

[0103] As already mentioned, the bioprocessing equipment 1, in particular the source receptacle slot 20, may be equipped with a source electronic process controller 22 for controlling the source culture environmental conditions, and preferably the source electronic process controller 22 may be connected to at least one sensor located in or at the source receptacle 2.

[0104] Furthermore, step ii) may terminate after a predetermined end condition is reached, and preferably at least one sensor measurement related to the end condition is repeatedly, in particular periodically or continuously, measured by a sensor located in or at the harvest receptacle 3 at least once, in particular during each run of step ii), and more preferably the sensor measurement is used, in particular by the harvest electronic process controller 25, to predict the time when the end condition will be reached, and the source culture environment conditions of step c) may be controlled, in particular by the source electronic process controller 22, to synchronize step c) with step ii). The above sensor or measured process parameters are applied.

[0105] What is interesting here is that, in some cases, the harvest receptacle 3 may be used longer or shorter than planned, which may only be noticed or intentionally decided during the respective step ii). Instead of having to interrupt step ii) because step b) is nearing completion, step b) may be planned with a control reserve from the beginning. Here, and preferably, step b) can be slowed down or accelerated. While this would result in a longer duration of step b) than the theoretical best, this change may be particularly used to maintain the viability of the cells in the source receptacle 2 above a predetermined threshold and / or to produce more bioproducts already during step b), thereby gaining a control reserve for accelerating step b). Additionally or alternatively, step b) may be slowed down by modifying the source culture environment conditions to produce more bioproducts and / or have less cell growth, thereby, for example, producing less toxic by-products, which may reduce the overall culture viability.

[0106] In general, a synchronization strategy can be implemented, and applied particularly to the source receptacle 2, to maintain synchronization of the source and harvest receptacles 3 and / or their slots. Such synchronization is particularly important because cells cannot be maintained viably in the source receptacle 2 for extended periods of time without adjusting the source culture environmental conditions and / or at least one process parameter in the source receptacle 2. The objective is to provide a predetermined amount of cells in the source receptacle 2 when the harvest receptacle slot 21 is prepared to perform step ii). For this purpose, the culture time in the source receptacle 2 is synchronized with the culture time in the harvest receptacle 3.

[0107] Such a synchronization strategy is shown in Figure 4b. For example, the temperature in source receptacle 2 may be reduced to extend the incubation time in source receptacle 2. Additionally or alternatively, feed medium may be supplied to the cultured cells, or the supply of feed medium may be continued to extend the incubation time in source receptacle 2.

[0108] Each process parameter can be predicted based on historical data. If the prediction is not sufficient, incremental changes to the process parameters may be made. Another synchronization strategy may involve changing the partitioning of cell broth 7 from source receptacle 2 to drain fraction 16 and restart fraction 17. If more cell broth 7 or a higher viable cell concentration is reached in the longer step b), the restart fraction 17 can be reduced, either absolutely or relatively, to allow for another longer step b) and / or step ii).

[0109] The predetermined termination condition for the harvest receptacle 3 may be that at least one process value reaches a certain value, in particular, and as shown in FIG. 3, that the viability (viab) falls below a predetermined threshold, and / or that a certain incubation time is reached, and / or that a certain fill volume is reached, and / or that a certain bioproduct concentration is reached, and / or that the ratio of product and by-product concentrations falls below a predetermined threshold, and / or that a certain amount of by-product is reached, and / or that the viable cell concentration reaches a certain value, and / or that a certain process value has been maintained for a certain time.

[0110] To estimate the time for the culture in harvest receptacle 3 to reach a predetermined end condition, various process parameters with appropriate process models can be used, such as cell viability, cell-specific productivity, product concentration, impurity levels (e.g., host cell impurity concentrations) or any CQA of the product.

[0111] According to one embodiment, it is proposed that when a deviation between the predicted time of reaching the end condition and the planned end of the production time is detected, in particular by the source electronic process controller 22 or the harvest electronic process controller 25, a synchronization strategy is executed, preferably comprising adapting the source and / or harvest culture environmental conditions to reduce the resulting deviation between the source receptacle 2 and the harvest receptacle 3, in particular so that a predetermined absolute cell number is reached in the source receptacle 2 before starting step c), thereby allowing step c) to be started earlier or later.

[0112] Alternative synchronization strategies may include changing the composition of the feed medium 6 and / or the time point at which the feed medium 6 is added, and / or changing the target fill volume in the source receptacle 2.

[0113] According to one embodiment, it is proposed that the initial viable cell concentration in step ii) is higher than the final viable cell concentration in step b), preferably the cell concentration being increased by centrifugation in step i), which may also be particularly advantageous if the source receptacle 2 has a larger volume than the harvest receptacle 3, for example at least 10% larger.

[0114] At least one process parameter of the source culture environmental conditions, particularly the initial source culture environmental conditions, in at least one run, particularly each run, of step b) and at least one process parameter of the harvest culture environmental conditions, particularly the initial harvest culture environmental conditions, in at least one run, particularly each run, of step ii) may be set to be different, preferably such that at least one process parameter in the source culture environmental conditions is optimized for culturing cells rather than for producing a biological product and / or such that at least one process parameter in the harvest culture environmental conditions is optimized for producing a biological product rather than for culturing cells. Different here means substantially different, not different within a tolerance or approximate tolerance.

[0115] According to one embodiment, it is proposed that the initial source culture environment, in particular the source culture environment and / or the initial harvest culture environment, in particular the harvest culture environment, are essentially identical for at least one, in particular each repetition of step b) and / or step ii), respectively.

[0116] According to one embodiment, it is proposed that the cultivation of the cells in step b) of the cyclic production mode is carried out under batch or fed-batch conditions and / or that the production mode of step ii) is carried out under fed-batch conditions.

[0117] This may occur at least once, preferably at least twice, and more preferably each time each step is performed.

[0118] According to one embodiment, as already mentioned, it is proposed that the centrifuge 12 is a fluidized bed centrifuge 12, preferably that the centrifuge 12 operates in a forward direction for cell separation and / or cell washing and in a reverse direction for cell discharge, more preferably that the centrifuge 12 operates in a reverse direction for transferring the centrifuged discharge fraction 19 between steps i) and ii) to a harvest receptacle 3.

[0119] According to one embodiment, it is proposed that a transfection step is carried out on the output fraction 16 after step c) or that a biological product is produced by the cells in the source receptacle 2 .

[0120] Here, the use of a centrifuge 12, preferably a fluidized bed centrifuge, as the clarification device 4 provides a simple means for carrying out the transfection step. For the definition of transfection, see the definition above. For example, by being able to supply wash solution and / or fresh culture medium during the centrifugation of step i), treatment with calcium phosphate for transfection can be easily carried out, since the DNA and calcium phosphate to be transferred to the cells can be supplied during centrifugation. Transfection can also be carried out outside the source receptacle 2, before or after centrifugation.

[0121] The results of a proof-of-concept experiment are shown in Figure 6. Here, two 5 L glass monoliths were used as source receptacle 2 and harvest receptacle 3, respectively. Cells were cultured in source receptacle 2 for 6 days until a viable cell concentration (VCC) of approximately 25 x 10^6 cells / milliliter was reached (Figure 6a, full circle). At this point, as indicated by the dashed vertical line in Figure 6a, the discharged fraction 16 of cell broth 7 was drained from source receptacle 2 to harvest receptacle 3, and the cells were cultured in harvest receptacle 3 for an additional 6 days (Figure 6a, full circle). Simultaneously, the reinitiation fraction 17 of cell broth 7 was combined with fresh culture medium 8 in source receptacle 2 to achieve an initial viable cell concentration (VCC) of 0.3 x 10^6 cells / mL (Figure 6a, shaded triangle). This process was repeated several times (diamonds and shaded circles), as shown in Figure 6a. The dashed vertical lines indicate the change in receptacle.

[0122] The results demonstrate that operating source receptacle 2 in a cyclic production mode allows for cyclic production of live cells, and that cell growth and viability are not adversely affected by the cyclic production mode.

[0123] As further shown in Figure 6, the results demonstrate that the aperiodic operation of the harvest receptacle 3 allows for adjusting process conditions within the harvest receptacle 3 to facilitate biomanufacturing (Figure 6b) without the need to keep cells viable (Figure 6a). While cell viability declines during operation of the harvest receptacle 3 in production mode (Figure 6a), the concentration of the bioproduct, in this case, the antibody, increases disproportionately. Overall, the proposed method not only reveals similar performance compared to previous processes, but also exhibits continuous characteristics that further enhance process efficiency. [Explanation of symbols]

[0124] 1. Bioprocessing equipment 2 Source Receptacle 3 Harvesting Receptacle 4. Clarification equipment 5 Source Receptacle Inlet Port 6. Feeding medium 7 Cell Broth 8. Culture Medium 9 Harvesting Receptacle Inlet Port 10 Source Receptacle Outlet Port 11 Harvesting receptacle outlet port 12 Centrifuge 13 Waste 14 Supernatant 15 Early cells 16 Excretion fraction 17 restart videos 18 Supply Line 19 Centrifuged effluent fraction 20 Source Receptacle Slots 21 harvesting receptacle slots 22-Source Electronic Process Control Device 23 Source Electronic Process Control Unit 24 Centrifuge chamber 25 Harvest Electronic Process Control Device 26 Harvest Electronic Process Control Unit 27 Harvested Fractions 28 Centrifuged Harvested Fractions 29 Purification process

Claims

1. A method for operating a bioprocessing facility (1) for producing a biological product, the bioprocessing facility (1) comprising a source receptacle (2) for cell culture, a harvest receptacle (3) for biomanufacturing, and a clarification device (4) using a centrifuge (12), said source receptacle (2) operates in a cyclic production mode; The cyclic production mode includes, in this order: a) initiating said cyclical production mode in said source receptacle (2) with starter cells (15) and culture medium (8); b) culturing the cells in the source receptacle (2), thereby obtaining a cell broth (7) containing the cultured cells, wherein the source receptacle (2) and the culture medium (8) are configured to provide source culture environment conditions for the culturing of the cells; c) discharging a discharge fraction (16) of said cell broth (7) from said source receptacle (2); d) combining the restart fraction (17) of the cell broth (7) with fresh culture medium (8) and repeating step b); e) repeating steps c) and d) at least once, and / or f) Discharging the cell broth (7) obtained from step d) from the source receptacle (2) to stop the cyclic production mode and obtain a discharge fraction (16). wherein the method further comprises: i) transferring the effluent fraction (16) to the clarification device (4) and centrifuging the effluent fraction (16) through the centrifuge (12), thereby separating the effluent fraction (16) into at least a centrifuged effluent fraction (19), a supernatant (14) and preferably a bioproduct; ii) transferring at least a portion of the centrifuged effluent fraction (19) from step i) and fresh culture medium (8) to the harvest receptacle (3) and operating the harvest receptacle (3) in a production mode, whereby the harvest receptacle (3) and the culture medium (8) provide harvest culture environmental conditions and are configured to produce the biological product within the harvest receptacle (3); wherein steps i) and ii) are carried out at least twice using the effluent fraction (16) from the execution of step c) and / or step f).

2. 2. The method of claim 1, characterized in that for at least one, preferably at least two, more preferably each run of steps b) and ii), the culturing of cells in step b) and the biomanufacturing in step ii) are carried out at least partly in parallel, preferably the running of steps b) and ii) is carried out in parallel for at least 50%, preferably at least 75%, more preferably at least 90% of the time used for culturing in step b) and / or the time used for biomanufacturing in step ii) and / or the time used for running step b) and / or step ii).

3. 3. The method according to claim 1 or 2, characterized in that a single source receptacle slot (20) is paired with a single harvesting receptacle slot (21), step b) is carried out in particular for a predetermined incubation time, step ii) is carried out in particular for a predetermined production time, and said incubation time and said production time, and therefore said source receptacle slot (20) and said harvesting receptacle slot (21), are synchronized so that the same harvesting receptacle slot (21) is used for at least two consecutive runs of step ii), preferably for all runs.

4. 4. The method of claim 3, wherein steps a) to f) are synchronized with the repeated seed train, and step a) is repeated in the same source receptacle slot (20) after step f) without destroying the synchronism between the source receptacle slot (20) and the harvest receptacle slot (21).

5. The method further comprises: iii) transferring the harvest fraction (27) from the harvest receptacle (3) to the clarification device (4) and centrifuging the harvest fraction (27) through the centrifuge (12), thereby separating the bioproduct from the harvest fraction (27); containing, Preferably, step iii) is carried out after each step ii).

5. The method according to claim 1, wherein the first and second electrodes are connected to each other.

6. 6. The method according to any one of claims 1 to 5, characterized in that the bioprocess installation (1) comprises a sensor device, the sensor device comprising at least one sensor arranged in or at the source receptacle (2) and / or at least one sensor arranged in or at the harvest receptacle (3), preferably at least one sensor configured to measure at least one process parameter from the group of carbon source concentration, nitrogen source concentration, amino acid concentration, growth factor concentration, oxygen concentration, carbon dioxide concentration, pH, temperature, conductivity, pressure, biomass concentration, biomass production rate, product concentration, productivity, oxygen uptake rate and / or agitation speed for the source receptacle (2) and / or the harvest receptacle (3).

7. 7. The method according to any one of claims 1 to 6, characterized in that the bioprocessing equipment (1), in particular the source receptacle slot (20), comprises a source electronic process controller (22) for controlling the source culture environmental conditions, preferably the source electronic process controller (22) being connected to the at least one sensor arranged in or at the source receptacle (2).

8. 8. The method according to claim 1, wherein step ii) is terminated after a predetermined end condition is reached, preferably wherein at least one sensor measurement value related to said end condition is repeatedly, in particular periodically or continuously, measured by said sensor arranged in or at said harvesting receptacle (3) at least once, in particular during each execution of step ii), more preferably wherein said sensor measurement value is used, in particular by a harvesting electronic process control device (25), to predict the time when said end condition will be reached, and wherein the source culture environment conditions of step b) are controlled, in particular by the source electronic process control device (22), in order to synchronize step b) with step ii).

9. 9. The method according to claims 3 and 8, characterized in that, when a deviation between the predicted time of reaching the end condition and the planned end of the production time is detected, in particular by the source electronic process control device (22) or the harvest electronic process control device (25), a synchronization strategy is executed, preferably comprising adapting the source and / or harvest culture environmental conditions to reduce the resulting deviation between the source receptacle (2) and the harvest receptacle (3), in particular so as to reach a predetermined absolute cell number in the source receptacle (2) before starting step c).

10. 10. The method according to any one of claims 1 to 9, characterized in that the initial viable cell concentration in step ii) is higher than the final viable cell concentration in step b), preferably said cell concentration being increased by the centrifugation in step i).

11. 11. The method according to claim 1, wherein at least one process parameter of the source culture environmental conditions, in particular the initial source culture environmental conditions, in at least one run, in step b) and at least one process parameter of the harvest culture environmental conditions, in particular the initial harvest culture environmental conditions, in at least one run, in step ii) are set to be different, preferably wherein in the source culture environmental conditions, the at least one process parameter is optimized for culturing cells rather than for producing a biological product and / or wherein in the harvest culture environmental conditions, the at least one process parameter is optimized for producing a biological product rather than for culturing cells.

12. 12. The method according to any one of claims 1 to 11, characterized in that the initial source culture environment, in particular the source culture environment and / or the initial harvest culture environment, in particular the harvest culture environment, are essentially identical for at least one, in particular each repetition of step b) and / or step ii), respectively.

13. 13. The method according to any one of claims 1 to 12, characterized in that the culturing of the cells in step b) of the cyclic production mode is carried out under batch or fed-batch conditions and / or the production mode of step ii) is carried out under fed-batch conditions.

14. 14. The method according to any one of claims 1 to 13, characterized in that the centrifuge (12) is a fluidized bed centrifuge (12), preferably operating in a forward direction for cell separation and / or cell washing and in a reverse direction for cell discharge, more preferably operating in a reverse direction for transferring the centrifuged discharge fraction (19) to the harvest receptacle (3) between steps i) and ii).

15. 15. The method according to any one of claims 1 to 14, characterized in that a transfection step is carried out on the output fraction (16) after step c) or the biological product is produced by the cells in the source receptacle (2).

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