A method for operating a bioprocessing configuration including a clarification device for removing cell debris from cell broth.

Abstract

The present invention relates to a method for operating a bioprocessing configuration (1) including a clarification device (2) for removing cell debris from a cell broth (10), wherein the clarification device (2) includes a fluidized bed centrifuge (8) having at least one centrifugation chamber (11), and the cell broth (10) is separated into at least a waste fraction (12) and an excrete fraction (13), and the separation of the cell broth (10) into the waste fraction (12) and the excrete fraction (13) is performed by the centrifugation chamber (11) The present invention relates to a method comprising a loading step (14) of loading cells from cell broth (10) into a centrifugation chamber (11) to a volume less than or equal to the maximum cell loading capacity; an overloading step (15) of further loading cell broth (10) into the centrifugation chamber (11) to cause cells to flow out of the centrifugation chamber (11); collecting the cells that flowed out during the overloading step (15) as a waste fraction (12); and collecting cells from the chamber as an exhaust fraction (13).

Description

Technical Field

[0001] The present invention relates to a method of operating a bioprocessing configuration including a clarification device for removing cell debris from a cell broth according to claim 1, and a control system configured to control the bioprocessing configuration to execute the proposed method according to claim 15.

[0002] The term "bioprocess" currently represents any type of biotechnology process that uses cells to produce a product or cultures cells as a product, particularly a biopharmaceutical process. To achieve a highly productive bioprocess, it is important to maintain cell viability. In existing culture scenarios, extended growth and thus high cell viability over a long period are typically achieved by medium exchange in a perfusion process scenario using a membrane device for cell retention. However, these devices tend to clog over time and lose unused medium due to backmixing, and are only capable of retaining all (live and dead) cells. Therefore, cell bleed (discharging a specific amount of cell broth) is sometimes necessary to provide space for dividing cells and remove already dead cells from the process. As a result, the process loses efficiency due to non-specific removal of some still-productive cells.

[0003] Reducing the footprint of a continuous bioprocess and increasing the production of a discontinuous bioprocess product is a challenge.

[0004] The present invention is based on the problem of improving known methods to achieve further optimization with respect to this problem.

[0005] The above object is solved by the features of claim 1.

[0006] The main realization of this invention is that a fluidized bed centrifuge can increase production efficiency in long-term culture scenarios by selectively retaining cells while dead cells are eluted, and thus enriching the bulk of live cells. This can be achieved by overloading the centrifuge chamber. Typically, the centrifuge chamber is loaded to its maximum cell loading capacity, after which a significant increase in the number of cells eluting the chamber is observed. Now it is found that the cells eluting the centrifuge chamber contain significantly more dead cells than the cells remaining in the chamber. The reason for this effect is that dead cells accumulate at the elution boundary, being smaller and therefore being eluted first when the chamber is overloaded. By deliberately utilizing this effect, the overall viability of cells in containers such as bioreactors can be increased. This allows for the disposal of extractions in continuous processes or longer operation in batch processes. Compared to filters, fluidized bed centrifuges have many advantages as versatile devices.

[0007] More specifically, a method has been proposed for operating a bioprocessing configuration including a clarification device for removing cell debris from a cell broth, wherein the clarification device includes a fluidized bed centrifuge having at least one centrifugation chamber, and the cell broth is separated into at least a waste fraction and an exhaust fraction, and the separation of the cell broth into a waste fraction and an exhaust fraction includes a loading step of loading cells of the cell broth into the centrifugation chamber to a volume less than or equal to the maximum cell loading capacity of the centrifugation chamber, an overloading step of further loading the cell broth into the centrifugation chamber, thereby causing cells to flow out of the centrifugation chamber, collecting the cells that flowed out during the overloading step as a waste fraction, and collecting cells from the chamber as an exhaust fraction.

[0008] The maximum cell loading capacity of a centrifuge chamber can be experimentally estimated with sufficient accuracy for this purpose. The maximum cell loading capacity is reached when further loading of cell broth into the centrifuge chamber results in a significant increase in the number of cells exiting the chamber through its outlet. The paper "Martin Saballus, Lucy Nisser, Markus Kampmann, Gerhard Greller, A novel clarification approach for intensified monoclonal antibody processes with 100 million cells / mL using a single-use fluidized bed centrifuge, Biochemical Engineering Journal, Volume 167, 2021, 107887, ISSN 1369-703X, https: / / doi.org / 10.1016 / j.bej.2020.107887" provides a formula for calculating the maximum cell broth volume based on the maximum cell loading capacity and shows how significantly the number of cells exiting the centrifuge chamber increases when the maximum cell loading capacity is reached.

[0009] According to claim 2, the valve may be switched as a transition between the loading step and the overloading step. The valve may also be a valve for rerouting the fluid discharge from the centrifugal chamber. Furthermore, according to claim 3, during the loading step, the fluid discharge from the chamber may be collected as a supernatant fraction separate from the waste fraction. It is preferable not to discard the fluid discharge during the loading step. The proposed embodiment allows for the selective designation of cells discharged during the overloading step as waste, which contain significantly more dead cells than other fractions of the cell broth.

[0010] After the overloading step, the cells in the centrifugation chamber may be drained in the reverse direction (Claim 4). In a very preferred embodiment according to Claim 4, a portion of the supernatant fraction is used to drain the cells, thereby not adding fresh culture medium to the cells.

[0011] Furthermore, according to claim 5, the proposed method allows the waste fraction and optionally the supernatant fraction to be returned to the container to increase the cell viability of the container. Both fractions can be returned without diluting the cell broth and / or without washing the cells, or in combination with the addition of fresh culture medium. Claim 6 extends this embodiment to further generate the product in the container after increasing the cell viability. According to claim 7, the increase in viability can be repeated several times. If a continuous or fed-batch process is desired, the volume removed by the waste fraction can be replaced with fresh culture medium.

[0012] Typically, fluidized bed centrifuges are primed with a buffer. The embodiment described in claim 9 includes priming the fluidized bed centrifuge with cell broth, which can be later returned to a container, in order to reduce the culture medium consumption and footprint of the bioprocess.

[0013] The amount of cell broth introduced into the centrifugation chamber during the overloading step, and therefore the amount of cells discharged from the centrifugation chamber, may depend on a measurement of the viability of the fluid discharge from the centrifugation chamber, according to claim 10.

[0014] Alternatively, according to claim 11, the amount of cells added to the centrifugation chamber during the overloading step may be predetermined. The predetermined amount may depend on a measurement of the viability of the cell broth. A good estimate of the number of cells added to the chamber after reaching the maximum cell loading capacity has been found to be near the number of dead cells in the centrifugation chamber. The ratio of live cells to dead cells that drain out of the centrifugation chamber then reaches a good compromise.

[0015] Repeatedly increasing the cell viability of cell broth can be an automated process (Claim 12).

[0016] In one embodiment of claim 13, the fluidized bed centrifuge may operate with the same parameters during the loading step and the overloading step to avoid fluidized bed destabilization, and thus perform separation efficiently. In another embodiment of claim 13, no washing of cells in the centrifugation chamber is performed.

[0017] Claim 14 describes a preferred embodiment of the bioprocess.

[0018] Another equally important teaching by claim 15 relates to a control system configured to control a bioprocessing configuration in order to carry out the proposed method.

[0019] All explanations given regarding the proposed method are fully applicable.

[0020] Embodiments of the present invention will be described below with reference to the drawings. [Brief explanation of the drawing]

[0021] [Figure 1] This shows a bioprocessing configuration configured to repeatedly remove waste fractions from a bioreactor. [Figure 2] This shows the loading, overloading, and emptying of the centrifugal chamber. [Figure 3] This outlines the process of repeatedly removing waste fractions. [Figure 4] a) to c) show the theory and confirmatory measurements. d) shows the results of the first test. [Figure 5] a) shows the results of the second test. b) shows the results of the third test. c) to e) show the results of the fourth test.

[0022] Figure 1 shows a bioprocessing configuration 1, which here, and also preferably, comprises a clarification device 2 and a bioreactor 3. Further optionally present components of the bioprocessing configuration 1 include a bioreactor control unit 4 and a centrifuge control unit 5 as part of a control system 6. A fluid line connecting the valve configuration 7 and the fluidized bed centrifuge 8 of the clarification device to the bioreactor 3 is also shown.

[0023] The bioreactor 3 can be used to produce a product 9 such as a monoclonal antibody by culturing cells in the bioreactor 3. Here, and also preferably, the production is a continuous process. Generally, the product 9 can be produced by the cells or the cells themselves.

[0024] A method of operating a bioprocessing configuration 1 including a clarification device 2 to remove cell debris, particularly dead cells, from the cell broth 10 is proposed. During continuous production and any other production, dead cells and cell debris accumulate in the bioreactor 3, occupying space from live cells during the processing period, which releases inhibitory substances and reduces cell growth and production efficiency. For continuous processing, it is necessary to remove the inhibitory substances so as to keep the bioreactor 3 operating. Therefore, it is particularly beneficial to remove cell debris, particularly dead cells, in order to reduce the release of these inhibitory substances. In the case of discontinuous processing, removing cell debris can still be advantageous in order to increase the efficiency of the process.

[0025] The clarification device 2 comprises a fluidized bed centrifuge 8 having at least one centrifugal separation chamber 11. As is generally known, the fluidized bed centrifuge 8 functions by creating a balance of forces between the centrifugal force and the fluid flow and suspending the cells inside the centrifugal separation chamber 11.

[0026] Here, and also preferably, the cell broth 10 is contained in a container, here the bioreactor 3. From this, it is proposed to separate the cell broth 10 into at least a waste fraction 12 and an effluent fraction 13.

[0027] Separating cell broth 10 into waste fraction 12 and effluent fraction 13 includes a loading step 14, during which the centrifuge chamber 11 is loaded with the cells of cell broth 10 up to a volume below the maximum cell loading capacity of the centrifuge chamber 11. Regarding the definition of the maximum cell loading capacity, reference is made in the introductory part of the description. In one embodiment, and / or where in doubt, the maximum cell loading capacity can be set as the volume at which the output of the chamber reaches a total number of cells / mL of less than 500,000 cells / mL.

[0028] Separating cell broth 10 into waste fraction 12 and effluent fraction 13 further includes an overloading step 15 of further loading the centrifuge chamber 11 with cell broth 10, thereby causing cells to flow out of the centrifuge chamber 11. FIG. 2 shows the loading step 14 in which cells from cell broth 10 are loaded into the centrifuge chamber 11 while the fluid from cell broth 10 and then the supernatant pass through the centrifuge chamber 11. Specifically, FIG. 2 shows the chamber inlet 16 through which cell broth 10 enters the centrifuge chamber 11, the chamber outlet 17 through which a part of cell broth 10 exits the centrifuge chamber 11, and different loading and operating states of the fluidized bed centrifuge 8. In FIG. 2a), the centrifuge chamber 11 is not fully loaded and the elution boundary 18 is still well before reaching the widest part of the centrifuge chamber 11. FIG. 2b) shows that the centrifuge chamber 11 is further loaded and the elution boundary 18 has approximately reached the widest part of the centrifuge chamber 11. Smaller cells flow out of the centrifuge chamber 11 when replaced by other cells. The smaller the dead cells are, the relatively larger amount of dead cells flows out. FIG. 2c) shows the continuous reverse operation of the centrifuge chamber 11 to let the remaining cells flow out of the centrifuge chamber 11 through the chamber inlet 16. Here, also preferably, the loading step 14 and the overloading step 15 are separate steps involving distinguishable transitions at the physical level and / or control level of the bioprocessing configuration 1.

[0029] Separating the cell broth 10 into waste fraction 12 and effluent fraction 13 further includes collecting the cells that bled out during the overloading step 15, preferably all the cells that bled out during the overloading step 15, as waste fraction 12. The waste fraction 12 can then be further processed at a downstream level or discarded. Separating the cell broth 10 into waste fraction 12 and effluent fraction 13 further includes collecting the cells, preferably all the cells, from the chamber as effluent fraction 13 (Figure 2c). The effluent fraction 13 and the waste fraction 12 are separate and distinct fractions. It should be understood that the amount of cells bled out during the overloading step 15 is substantially greater than the normal amount of cells bled out during the loading step 14. Here, also preferably, at some point the waste fraction 12, and / or the liquid effluent of the centrifugation chamber 11 added to the waste fraction 12, contains cells per volume of at least 10% of the cells per volume of the cell broth 10, e.g., at least 5 million cells / mL.

[0030] Here, also preferably, the bioprocessing configuration 1 comprises a valve configuration 7. At least one valve of the valve configuration 7 may be switched during the transition from the loading step 14 to the overloading step 15. Preferably, as shown in Figures 1 and 3, the valve is fluidly connected to the chamber outlet 17 of the centrifugal chamber 11 such that switching the valve reroutes the fluid discharge from the centrifugal chamber 11. The fluid discharge from the chamber is routed along different paths between the loading step 14 and the overloading step 15, with the fluid discharge during the overloading step 15 being collected as a waste fraction 12 and the fluid discharge during the loading step 14 being collected separately. Here, also preferably, the fluid discharge during the loading step 14 is collected in a holding container 19 as shown in Figure 1. Here, also preferably, the valve configuration 7 is controlled by a control system 6 so that the separation of the cell broth 10 in particular can be performed automatically without user intervention.

[0031] Fluid discharge during loading step 14 is here, also preferably as supernatant fraction 20, and is preferably collected in a holding container 19 as shown in Figure 1. Preferably, the waste fraction 12 contains at least 10 times, preferably at least 100 times, and more preferably at least 1,000 times, the number of cells per mL of the supernatant fraction 20.

[0032] According to one embodiment, during the loading and overloading steps 15, the cell broth 10 is introduced into the chamber in the forward direction, and the collection step 21 includes separating the cell broth 10 into a waste fraction 12 and an exhaust fraction 13, during which time the cells are discharged from the chamber in the reverse direction opposite to the forward direction and collected as part of the exhaust fraction 13 (Figure 2c).

[0033] Here, also preferably, and as can be seen from the fluid line connections in the figure, during the collection step 21, the supernatant fraction 20, in particular the supernatant from the holding container 19, is pumped in the reverse direction through the chamber, thereby causing the cells to flow out of the chamber. In this way, the fluid-bed centrifuge 8 can be operated and separation can be performed without the need for buffer. All or part of the supernatant fraction 20 may be returned to the container, in particular during the reverse operation. Instead of the supernatant fraction 20, fresh culture medium 22 may be used to supply the cells in the container directly.

[0034] During forward operation, the cell broth 10 or liquid is generally pumped from the chamber inlet 16 to the chamber outlet 17, and during reverse operation, the liquid is pumped in the opposite direction.

[0035] Moving on to the overall process, according to one embodiment, as already described, the cell broth 10 is extracted from the container of the bioprocessing configuration 1, particularly the bioreactor 3, before the loading step 14, and the waste fraction 13 is returned to the container, preferably at least 50%, preferably at least 75%, more preferably at least 90% of the supernatant fraction 20 is returned to the container. Alternatively, the product 9 can be collected from the supernatant fraction 20. Figure 1 shows a separate fluid line for returning the waste fraction 13 or the supernatant fraction or a portion of the supernatant fraction to the bioreactor 3. In another embodiment, a single fluid line can be used to extract the cell broth 10 from the reactor and return the waste fraction 13 to the bioreactor 3. Thus, the valve configuration 7 can be adapted.

[0036] Generally, product 9 can be produced by carrying out a batch, fed-batch, or continuous process in a container, and product 9 is produced by or is cells. Here, also preferably, the production of product 9 continues in the container after the waste fraction 13 is returned to the container.

[0037] The separation of the cell broth 10 from the container into waste fraction 12 and discharge fraction 13 is repeated using the fraction of cell broth 10 from the container at intervals, particularly intervals of up to 8 hours, which may enhance the vitality of the cells in the container. Preferably, the separation is repeated at least another such interval, preferably at least two other such intervals. After each interval, the elements of the disposable configuration of the fluidized bed centrifuge 8 can be changed.

[0038] For example, at least 20%, preferably at least 30%, and / or up to 100% of the volume of the bioreactor 3 can be continuously separated into waste fraction 12 and discharge fraction 13 by the fluidized bed centrifuge 8 during the interval. This may take, for example, one hour. After an interval of, for example, six hours, the process can be repeated to activate, for example, one-third of the volume every six hours. Figure 3 schematically shows the repetition of the process. At the top of Figure 3, the bioreactor 3 is operated until viability reaches a threshold or until the end of the interval. At least some of the cell broth 10 is then subjected to separation into waste fraction 12 and discharge fraction 13 and added back to the bioreactor 3 in the third column of Figure 3. This process is repeated until harvesting at the bottom of Figure 3.

[0039] According to one embodiment, it is proposed that fresh culture medium 22 is added to the container periodically, preferably in multiple repeated steps and / or at least one interval steps, after multiple repetitions of separation.

[0040] Another aspect of the proposed method is that the separation of the cell broth 10 into a waste fraction 12 and an effluent fraction 13 may include a priming step before the loading step 14, during which the centrifugation chamber 11 is primed. It is generally known that the chamber of a fluidized bed centrifuge 8 is primed with a buffer before loading. However, here, also preferably, the chamber is primed with the cell broth 10. In this embodiment, a buffer is not required for priming. The fluid effluent from the centrifugation chamber 11 during the priming step may be returned directly or indirectly to the container. Figures 1 and 3 show the respective valves that can be actuated accordingly. Here, also preferably, the valves of the valve configuration 7 are switched during the transition from the priming step to the loading step 14. Figure 1 also shows that, since a buffer is not required here, the two inlets of the fluidized bed centrifuge 8 may be connected to the container.

[0041] According to one embodiment, the bioprocessing configuration 1 is proposed to include a biomass sensor 23, particularly a capacitive biomass sensor 23, for measuring the cell viability of cells in the fluid discharge of the centrifugal chamber 11. Preferably, the overloading step 15 is stopped based on a signal from the biomass sensor 23. The overloading step 15 may be stopped if the biomass sensor 23 detects a rapid increase in cell viability in the fluid discharge or detects that the viability has reached a threshold.

[0042] It is further possible to include another biomass sensor 23 inside the container or between the container and the fluidized bed centrifuge 8. The overloading step 15 can then be stopped based on signals from both biomass sensors 23.

[0043] In an alternative embodiment, it is proposed that during the overloading step 15, the maximum cell loading capacity of the chamber is exceeded by a predetermined amount of cells added to the chamber. Preferably, the predetermined amount depends on a measure of cell viability in the cell broth 10. More preferably, the predetermined amount is 80% to 120%, preferably 90% to 110%, of the number of dead cells expected to be in the centrifugation chamber 11, based on the viability measurement. Independently, the number of separation steps per interval may depend on the viability measurement.

[0044] As shown in the figure, the bioprocessing configuration 1 may include a bioreactor control unit 4 for controlling the production in the container and / or a centrifuge, preferably a valve configuration 7, for controlling the centrifuge. Both control units may be part of the control system 6 as described.

[0045] The bioreactor control unit 4 and the centrifuge control unit 5 may be part of a single control unit or communicated together to perform control for automatically recovering the cell broth 10 from the container, separating the cell broth 10, and repeatedly returning the discharge fraction 13 and optionally the supernatant fraction 20 to the container. The bioreactor control unit 4 and / or the centrifuge control unit 5 and / or the control system 6 may have one or more interfaces for controlling one or more pumps and / or one or more valves of the valve configuration 7 and may have at least one processor configured to perform specified operations.

[0046] According to one embodiment, the rotational speed of the centrifugal chamber 11 and the flow rate at the input of the centrifugal chamber 11 are proposed to remain substantially unchanged or not changed at all between the loading step 14 and the overloading step 15. Here, preferably, no washing step is performed during the separation of the cell broth 10.

[0047] Here, the term "substantially unchanged" means that the fluidized bed is not disturbed in a way that would destabilize it.

[0048] According to one embodiment, it is proposed that the container has a volume of at least 1,000 L, preferably at least 1,500 L, more preferably at least 2,000 L, and / or that the cell broth 10 contains more than 25 million cells / mL, preferably more than 40 million cells / mL, and preferably the cells are CHO cells.

[0049] Another equally important teaching relates to a control system 6 configured to control the bioprocessing configuration 1 in order to carry out the proposed method.

[0050] All explanations given regarding the proposed method are fully applicable.

[0051] In the proof-of-concept experiment, a clarification device in a fluidized bed centrifuge was implemented as part of a bioprocessing configuration for an eluate-based cell sorting approach. Figure 4 shows the theoretical and confirmatory measurements in a) to c). Figure 4d) shows the results of the first test, Figure 5a) shows the second test, Figure 5b) shows the third test, and Figures 5c) to e) show the results of the fourth test.

[0052] In the first trial, we investigated and optimized cell clarification equipment using a small fluidized bed centrifuge with a 15 mL scale multi-parallel bioreactor system, employing a Chinese hamster ovary (CHO) cell line established for the production of mAb reference products designated by the National Institute of Standards and Technology (NIST).

[0053] In the second trial, a fluidized bed centrifuge clarification system for cell sorting was tested in an upscale proof-of-concept study using 2 L of CHO-supplied batch broth for industrially relevant mAb production.

[0054] To evaluate the experimental results, cell debris (non-living cells) was stained using the trypan blue method. The cell capture position in the fluidized bed centrifuge was calculated according to Kelly et al. 2016 ("William Kelly, Jonathan Rubin, Jennifer Scully, Hari Kamaraju, Piotr Wnukowski, Ravinder Bhatia, Understanding and modeling retention of mammalian cells in fluidized bed centrifuges, Biotechnol Progress, Volume 32, 2016, 1520-1530, https: / / doi.org / 10.1002 / btpr.2365"), taking into account the chamber dimensions and rotation speed, fluid flow rate and viscosity, cell and fluid density, and cell size.

[0055] The smallest cells (living cells) and cell debris (non-living cells) captured in the chamber are found near the elution boundary, but most of the small cell debris passes through the elution boundary and flows out of the chamber (Figure 4a) for the following reasons.

[0056] From the inlet to the outlet of the chamber, the fluid velocity decreases sharply due to the dimensions of the pear-shaped chamber, but the unobstructed settling velocity in the opposite direction decreases only slightly due to the decrease in g-force near the center of the rotor (Figure 4b). The cells float where the fluid velocity equals the settling velocity.

[0057] Since we can assume that all physical properties of the culture medium and cells are constant except for cell size, larger cells are abundant at the front of the chamber, and smaller cells are abundant at the wider chamber outlet. Therefore, the most likely explanation for the abundance of cell debris (non-living cells) at the chamber outlet is that they have collapsed and are thus smaller than living cells.

[0058] As shown in Figure 4c), the composition of the cells in the chamber was experimentally confirmed.

[0059] In the first test, a significant difference in the elution of live cells and cell debris (non-live cells) may be shown to enable an effective clarification device for cell sorting. A small fluidized bed centrifuge (Ksep50; 2,000 × g) was used. CHO cell broth (total 13.4 × 10⁶ cells / mL; 76.5% viability; live cell diameter 20.2 μm) was added to the chamber (50 mL / min). Fractions of the supernatant during loading and overloading were collected and further investigated. The breakthrough of live cells and cell debris (non-live cells) in the supernatant against loading is depicted in Figure 4d).

[0060] In the second trial, it was successfully demonstrated that the clarification apparatus for cell sorting increased cell viability by more than 30%. Furthermore, the majority of whole viable cells were recovered from the sorted feed (cell broth) of the sorted viable cell fraction. This proof-of-concept trial of sorting bulk CHO cells in a clarification apparatus applying the developed overloading recipe for a fluidized bed centrifuge was successfully performed by sorting a bulk low-viable CHO cell broth feed (2.0 L; total 33.4 × 10⁶ cells / mL; 50.7% viability; viable cell diameter 19.8 μm) by overloading a technology-scale fluidized bed centrifuge (KSep400; 1,000 g) to approximately 100% overloading, which produced a waste fraction containing the majority of dead cells and an effluent fraction containing the majority of viable cells (Figure 5a).

[0061] Furthermore, it was found that T cells could be sorted in a similar manner to CHO cells (the difference was not very significant because, as is typical with T cells, the sorting feed (cell broth) already had a high viability). Using a small fluidized bed centrifuge (Ksep50), the supernatant fraction was collected during (excess) loading and washing to investigate a clarification device for sorting relatively small (6-9 μm) and shear-sensitive T cells (Figure 5b).

[0062] Another proof-of-concept study of bulk T cell sorting applying the developed fluidized bed centrifuge overloading recipe showed that the clarification device for cell sorting could increase T cell viability from 94.5% to 96.3% (Figures 5c)-e). Interestingly, in this context, it was also found that even long processing times (30 minutes) did not adversely affect the T cells.

Claims

1. A method for operating a bioprocessing configuration (1) including a clarification device (2) for removing cellular debris from a cell broth (10), wherein the clarification device (2) includes a fluidized bed centrifuge (8) having at least one centrifugal chamber (11), and the cell broth (10) is separated into at least a waste fraction (12) and an excrete fraction (13), and the separation of the cell broth (10) into the waste fraction (12) and the excrete fraction (13) is A loading step (14) involves loading cells from the cell broth (10) into the centrifugal chamber (11) to a volume less than or equal to the maximum cell loading capacity of the centrifugal chamber (11). An overloading step (15) is performed by further loading cell broth (10) into the centrifugal chamber (11) thereby causing cells to flow out of the centrifugal chamber (11). The cells that leaked out during the overloading step (15) are collected as the waste fraction (12), and A method comprising collecting cells from the chamber as the waste fraction (13).

2. The method according to claim 1, wherein the bioprocessing configuration (1) comprises a valve configuration (7), and at least one valve of the valve configuration (7) is switched during the transition from the loading step (14) to the overloading step (15), preferably the valve is fluidly connected to the chamber outlet (17) of the centrifugal chamber (11) so as to reroute the fluid discharge from the centrifugal chamber (11) by switching the valve, and the fluid discharge from the chamber is routed differently between the loading step (14) and the overloading step (15), such that the fluid discharge during the overloading step (15) is collected as the waste fraction (12) and the fluid discharge during the loading step (14) is collected separately.

3. The method according to claim 2, wherein the fluid discharge during the loading step (14) is preferably collected as a supernatant fraction (20) in a holding container (19), and preferably the waste fraction (12) contains at least 10 times, preferably at least 100 times, and more preferably at least 1,000 times more cells / mL than the supernatant fraction (20).

4. The method according to any one of claims 1 to 3, wherein, during the loading and overloading step (15), the cell broth (10) is introduced into the chamber in the forward direction, and the collection step (21) is to separate the cell broth (10) into the waste fraction (12) and the waste fraction (13), during which the cells flow out of the chamber in the reverse direction opposite to the forward direction and are collected as part of the waste fraction (13), preferably, during the collection step (21), the supernatant fraction (20), in particular the supernatant from the holding container (19), is pumped through the chamber in the reverse direction, thereby causing the cells to flow out of the chamber.

5. The method according to any one of claims 1 to 4, characterized in that the cell broth (10) is extracted from the container of the bioprocessing configuration (1), particularly the bioreactor (3), before the loading step (14), the waste fraction (13) is returned to the container, preferably at least 50%, preferably at least 75%, more preferably at least 90% of the supernatant fraction (20) is returned to the container, or the product (9) is collected from the supernatant fraction (20).

6. The method according to claim 5, characterized in that a batch, fed-batch, or continuous process is carried out in the container to produce product (9), the product (9) is produced by the cells, or the product (9) is the cells, and the production of product (9) continues in the container after the waste fraction (13) is returned to the container.

7. The method according to claim 6, wherein the separation of the cell broth (10) from the container into the waste fraction (12) and the waste fraction (13) is repeated with respect to the fraction of the cell broth (10) from the container at a certain interval, particularly at an interval of up to 8 hours, thereby increasing the vitality of the cells in the container, preferably the separation is repeated at at least another such interval, preferably at least two other such intervals.

8. The method according to claim 7, characterized in that fresh culture medium (22) is added to the container periodically, preferably in multiple repeating steps and / or at least one interval step, after multiple repetitions of the separation.

9. The method according to any one of claims 1 to 8, wherein the separation of the cell broth (10) into the waste fraction (12) and the waste fraction (13) includes a priming step prior to the loading step (14), during which the centrifugation chamber (11) is primed, preferably the chamber is primed with the cell broth (10).

10. The method according to any one of claims 1 to 9, wherein the bioprocessing configuration (1) comprises a biomass sensor (23), particularly a capacitive biomass sensor (23), for measuring the cell viability of the cells in the fluid discharge from the centrifugal chamber (11), and preferably the overloading step (15) is stopped based on the signal from the biomass sensor (23).

11. The method according to any one of claims 1 to 10, characterized in that during the overloading step (15), the maximum cell loading capacity of the chamber is exceeded by a predetermined amount of cells added to the chamber, preferably the predetermined amount being based on a measurement of the viability of the cells in the cell broth (10), and more preferably the predetermined amount being 80% to 120%, preferably 90% to 110%, of the number of dead cells expected to be in the centrifugal chamber (11) based on the measurement of viability.

12. The method according to any one of claims 1 to 11, wherein the bioprocessing configuration (1) comprises a bioreactor control unit (4) and / or a centrifuge and preferably a valve configuration (7) for controlling the generation in the container, and preferably the bioreactor control unit (4) and the centrifuge control unit (5) are part of a single control unit or are communicably coupled to control the automatic recovery of the cell broth (10) from the container, the separation of the cell broth (10), and the repeated return of the waste fraction (13) and optionally the supernatant fraction (20) to the container.

13. The method according to any one of claims 1 to 12, characterized in that the rotational speed of the centrifugal chamber (11) and the flow velocity at the input of the centrifugal chamber (11) do not change substantially or not at all between the loading step (14) and the overloading step (15), and / or a washing step is not performed during the separation of the cell broth (10).

14. The method according to any one of claims 1 to 13, characterized in that the container has a volume of at least 1,000 L, preferably at least 1,500 L, more preferably at least 2,000 L, and / or the cell broth (10) contains more than 25,000,000 cells / mL, preferably more than 40,000,000 cells / mL, and preferably the cells are CHO cells.

15. A control system configured to control a bioprocessing configuration (1) to perform the method according to any one of claims 1 to 14.

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