Method of operating a bioprocessing system and bioprocessing system

Abstract

The present disclosure relates to a method (100) of operating a bioprocessing system, the method comprising: - generating (102) a cell culture mixture in a fermentor (4); - simultaneously with the generating (102) step, directing (104) a flow of the cell culture mixture from the fermentor (4) into the interior of a centrifuge (6) comprising an enlarged surface insert; and - simultaneously with the generating (102) step and the directing (104) step, continuously returning (106) a liquid flow from the interior of the centrifuge (6) to the fermentor (4).

Description

[Technical field]

[0001] The present invention relates to a method for operating a bioprocessing system and to a bioprocessing system.The present invention further relates to a computer program and a computer program product. [Background technology]

[0002] WO 2017 / 025210 discloses a disposable bioprocessing system for continuous processing of microorganisms in the pharmaceutical industry, integrating a single-use bioreactor, a single-use pump and a single-use cross-flow filter, the system providing a perfusion mode processing.

[0003] In contrast to batch operation, which is a method of operation where no fresh medium is added and no spent medium and / or liquid is removed, and fed-batch operation, where the process begins with a small amount of medium and fresh medium is added but no liquid is removed until after the end of the process, perfusion mode operation refers to a method of operation where the medium is replaced sequentially, fresh nutrients are added sequentially, and product, i.e., spent medium, is removed throughout the entire culture period. Perfusion mode operation can typically last 4-8 times longer than batch operation and 2-4 times longer than fed-batch operation.

[0004] Many common methods for producing biological proteins in recombinant mammalian and microbial cells rely on fed-batch culture, in which cells are grown to high cell density and then typically exposed to an induction medium or inducer to trigger protein production. If the cells secrete the desired protein, it is advantageous to switch from fed-batch culture to continuous perfusion culture, which can maintain high cell density and high productivity over a much longer culture period. During continuous perfusion culture, live and productive cells are retained or recycled to the bioreactor, while secreted proteins are continuously recovered from the bioreactor for downstream purification processes.

[0005] Some key advantages of continuous perfusion culture over fed-batch culture are: (1) the secreted protein product is continuously removed from the bioreactor without exposing the product to potential degradation by proteolytic and / or glycolytic enzymes released into the culture medium from dead cells; (2) to achieve high cell densities in the continuous perfusion bioreactor, live and productive cells are retained or recirculated where they continue to produce valuable protein for much longer culture periods within the controlled bioreactor environment rather than being killed and removed from the bioreactor at the end of each fed-batch culture; and (3) unlike the dynamically changing concentrations of nutrients and waste products in fed-batch culture, the perfusion bioreactor environment can be maintained much closer to steady-state conditions by continuously adding fresh nutrient medium and removing waste products along with the recovered protein product, thereby maintaining a more consistent designed product quality.

[0006] Perfusion mode processes typically utilize membrane-type cross-flow filters (CFF) or similar filters such as alternating tangential flow (ATF) filters. The cell culture stream, also referred to herein as the cell culture mixture, passes from the fermentor through the filter. A portion of the cell culture mixture passes through the membrane and is removed from the cells of the cell culture. This portion forms the filtrate, a product that is collected for downstream processing. The remaining portion of the cell culture mixture, the retentate, containing the cells, is conducted back through the membrane to the fermentor.

[0007] The main disadvantage of filter technology is that the permeability of the membrane gradually decreases during use. This aspect is particularly relevant for bioprocessing systems operating in perfusion mode, which may run for weeks or months. Therefore, to ensure the proper functioning of the bioprocessing system over time, the membranes or filters must be replaced regularly or else large membrane areas must be provided, which would otherwise be impractical. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] International Publication No. 2017 / 025210 Brochure [Patent Document 2] EP3666394 Summary of the Invention [Problem to be solved by the invention]

[0009] It would be advantageous to achieve a method for operating a bioprocessing system and / or a bioprocessing system which overcomes or at least mitigates the above-mentioned drawbacks. In particular, it would be desirable to enable a perfusion mode operation of a compact bioprocessing system over long periods of time. In order to solve this problem inter alia, a method for operating a bioprocessing system and / or a bioprocessing system are provided having the features set out in the independent claims. [Means for solving the problem]

[0010] According to one aspect, there is provided a method of operating a bioprocessing system, comprising: - producing a cell culture mixture in a fermenter; - simultaneously with the step of producing the cell culture mixture, directing a flow of the cell culture mixture from the fermenter into the interior of a centrifuge equipped with a surface enlarging insert; - continuously returning a flow of liquid from the interior of the centrifuge back to the fermenter simultaneously with the steps of generating a cell culture mixture and directing a flow of the cell culture mixture. A method is provided that includes:

[0011] According to a further aspect, there is provided a bioprocessing system comprising a fermenter for producing a cell culture mixture therein, a centrifuge with a surface-enlarging insert, a receiving vessel, a conduit system interconnecting the fermenter, the centrifuge and the receiving vessel, a flow control device disposed within the conduit system, and a control system configured to control at least a portion of the centrifuge and the flow control device. The control system comprises: - directing the flow of the cell culture mixture from the fermenter into the interior of the centrifuge, and at the same time - Continuous return flow of liquid from inside the centrifuge back to the fermenter It is structured as follows.

[0012] Each of these methods and bioprocessing systems provides for a continuous return of liquid flow from the interior of the centrifuge with the surface-enlarged insert to the fermenter, thus enabling perfusion mode operation of the bioprocessing system with the centrifuge with the surface-enlarged insert. More specifically, the interior of the centrifuge with the surface-enlarged insert has a volume into which the cells of the cell culture mixture enter, and providing a continuous return from the interior of the centrifuge to the fermenter ensures that stagnant conditions are avoided within this volume, i.e., the interior of the centrifuge with the surface-enlarged insert. A stagnant cell culture mixture or a cell-containing portion of a stagnant cell culture mixture, if returned to the fermenter, would deteriorate and degrade the production of the cell culture mixture in the fermenter. Each of these methods and bioprocessing systems is therefore adapted for perfusion mode operation by providing a continuous return of liquid flow from the interior of the centrifuge with the surface-enlarged insert to the fermenter.

[0013] It has been found that centrifuges equipped with surface-enlarging inserts can be used continuously not only during the recovery of produced biomolecules in the final stage of a bioprocess, but also during several stages of a bioprocess operating in perfusion mode, for example to remove culture medium containing substances of interest and cellular debris without damaging the living cells that may be continuously returned to the fermenter.

[0014] Thus, further advantages of centrifuges with surface-enlarging inserts can be utilized in the context of perfusion mode operation of bioprocessing systems, such as: space efficiency; efficient separation of cells; processing of cells that are separated by continuous transport out of the centrifuge together with the separated heavy phase, which is gentler compared to filter technologies where cells accumulate on the filter surface over shorter or longer periods of time; self-cleaning capabilities; continuous operation over longer periods of time without part replacement, which may be necessary in the context of filter technologies.

[0015] Furthermore, the use of a centrifuge with surface-enlarging inserts provides efficient fractionation or classification of the cell culture mixture when separated in the centrifuge. That is, the maximum particle size that is separated by the centrifuge together with the separated light phase, the so-called critical particle, can be efficiently controlled by controlling the rotation speed of the centrifuge rotor and the flow of the cell culture mixture into the centrifuge. Thus, the critical particle can be set to include intact whole cells together with the heavy phase and exclude them from the light phase.

[0016] Cellular debris and other debris (smaller than whole cells) flow with both the light and heavy phases. Thus, along with the separated light phase, which is not returned to the fermenter, unwanted debris is also removed from the fermenter.

[0017] In comparison with filter technology, the use of a centrifuge with surface-enlarging inserts allows, firstly, the threshold particle to be easily adjusted, and, secondly, and more importantly, the threshold particle to be maintained throughout the entire life of the centrifuge. A drawback of filter technology is that as particles are deposited on the filter membrane, its permeability decreases, so that over time, a larger amount of particles smaller than the intended particles remain in the retentate and are returned to the fermenter. The cell culture mixture is produced in a fermenter of a bioprocessing system. The term "fermenter" as used herein relates to any vessel capable of producing a cell culture mixture, which may alternatively be called a bioreactor or reactor. The fermentor may alternatively be written as a fermenter. The fermenter may be, for example, a so-called Stirred-Tank-Reactor (STR), which integrates an aeration device and an agitation or mixing device for forced exchange of nutrients and gases with the cells in the cell culture mixture. In this specification, the term "bioprocess" is used for the process carried out in the bioprocessing system.

[0018] Fermentation and / or culturing occurs in a fermenter during the step of producing a cell culture mixture therein. The terms "fermentation" and "culturing" as used herein relate to hosting in a fermenter live unicellular organisms, prokaryotes, eukaryotic cells, human cells, microorganisms such as CHO and bacteria or mammalian cells expressing extracellular biomolecules for industrial purposes. The fermentation / culturing produces the cells of the cell culture mixture and the expressed biomolecules. The fermentation occurs throughout the culture period. The culture period can last for several weeks, during most of which a product including the extracellular biomolecules is produced by the perfusion mode operation of the bioprocessing system. The fermentation can be for example to express an extracellular biomolecule, such as an antibody or a protein, from a mammalian cell culture mixture. Thus, the product includes the extracellular biomolecule. For example, the cell culture mixture can include cells, such as CHO (Chinese Hamster Ovary) cells, and a growth medium configured to promote the growth of the cells. CHO is a very popular microbial and mammalian cell line as an expression platform for proteins in the pharmaceutical industry.

[0019] Additionally, within the fermentor, conditions to promote cell growth are provided, such as by providing a growth medium, adding oxygen, and maintaining an appropriate temperature range.

[0020] The growth medium, also called culture medium, is a liquid whose components promote the growth of cells in a cell culture mixture and include water, glucose, salts, and a source of amino acids, among others. Clean air or a gas such as oxygen is added to the growth medium, e.g., the growth medium in a fermenter.

[0021] The term "cell culture mixture" as used herein refers to the unseparated liquid contents, culture broth, in a fermenter or bioreactor consisting of cells, debris, microorganisms, nutrient medium, waste, products, etc. During a first period, the cell culture mixture can be separated into a light phase and a heavy phase, for example in a centrifuge with a surface-enlarging insert. This separation increases the cell density of the heavy phase above that of the incoming cell culture mixture, and as such, is returned as a liquid stream to the aforementioned fermenter and mixed again with the cell culture mixture in the fermenter.

[0022] In this specification, a centrifuge with a surface-enhancing insert may alternatively be referred to as a centrifuge. The centrifuge comprises a rotor defining a separation space. The surface-enhancing insert is disposed in the separation space inside the rotor. The surface-enhancing insert may comprise, for example, an axially arranged separation member or a frusto-conical separation disk disposed in a disk stack. An inlet is provided for directing the cell culture mixture into the separation space, i.e., the interior of the centrifuge. Additionally, an outlet for the separated light phase and an outlet for the separated heavy phase are also provided.

[0023] When separation is performed in a centrifuge, for example during a first period, the rotor is rotated at a speed such that the cell culture mixture is separated into a light phase and a heavy phase.

[0024] The outlet for the separated heavy phase from the centrifuge is suitably provided concentrically or close to the rotor's axis of rotation. In this way, the cells in the separated heavy phase are subjected to lower shear forces and are therefore treated more gently as the heavy phase exits the centrifuge rotor and is returned to the fermenter. Similarly, the inlet for the cell culture mixture of the centrifuge can also be provided concentrically or close to the rotor's axis of rotation.

[0025] The conduit system can include one or more of tubes, hoses, pipes, etc. configured to conduct liquids, such as a cell culture mixture, a separated light phase, and a separated heavy phase, within the bioprocessing system and / or to and / or from external components connected to the bioprocessing system.

[0026] The flow control device can include one or more pumps and valves configured to pump and direct liquids through the bioprocessing system and / or to and / or from external components connected to the bioprocessing system.

[0027] The term "control system" as used herein refers to a programmable logic control (PLC), personal computer (PC), embedded computer (EC), or similar device incorporating a central processing unit (CPU) electronic device with computing power. The control system has various inputs and outputs (I / O) for the centrifuge and flow control devices. The control system incorporates software, algorithms, process recipes in its on-board memory to control the centrifuge and flow control devices. Optionally, the control system can further control aspects of the fermenter to manage and analyze the cultivation or fermentation process according to the process recipe, for example for ongoing process alignment. The control system can communicate with various sensors and actuators to modify process parameters.

[0028] The term "product" is used herein for the medium that is expected to be produced or generated in the bioprocessing system. The light phase that is separated in the centrifuge with the surface-enlarging insert may form the product or may instead contain the product. This product may be subjected to downstream processing to extract a substance of interest, such as an extracellular biomolecule, e.g., an antibody or a protein. That is, this product may be an intermediate product in the larger context of producing a final product.

[0029] The term "cell" as used herein is generally divided into: 1. living unicellular organisms, microorganisms such as fungi, algae, mosses, plankton, yeasts, protozoa, eukaryotes, archaea, microfauna, extremophiles and plant cells, 2. living attached or semi-attached or planktonic cells such as animal cells, insect cells, mammalian cells, human cells, stem cells, 3. prokaryotes and various bacteria such as E. coli, etc. Most of the above may be genetically engineered to provide a specific substance on demand.

[0030] The term "perfusion" mode operation as used herein refers to a method or principle of operation of a fermenter in which the medium is exchanged sequentially, fresh nutrients are added sequentially, and spent medium / product is removed throughout the majority of the culture period. Microbial and mammalian cells maintained in perfusion mode operation typically last 4-8 times longer than in batch operation and 2-4 times longer than in fed-batch operation. Purely by way of example, perfusion mode operation may last for days, a week or weeks, or a month or months.

[0031] Perfusion mode operation supports efficient facility utilization by offering the possibility to use smaller bioreactors / fermenters. Compared to batch or fed-batch operation, perfusion mode operation allows the cells of the cell culture mixture to remain in the exponential growth phase for a longer period of time and reach a higher viable cell density.

[0032] Further, perfusion mode operation involves a continuous return of liquid flow from the centrifuge to the fermenter, and the step of continuously returning liquid flow from inside the centrifuge to the fermenter may be performed for more than 90%, e.g., more than 95%, of the entire culture period.

[0033] The term "single-use" as used herein refers to equipment designed to be used only once, for example for one complete cultivation period of a fermentation process operated in perfusion mode, and to be disposed of after use. This equipment is usually supplied sterile and ready to use, such as single-use parts of centrifuges and single-use fermenters.

[0034] According to an embodiment, the method includes, during a first period of time: - separating the flow of cell culture mixture into a light phase and a heavy phase in a centrifuge, the step of continuously returning the flow of liquid from inside the centrifuge to the fermenter comprising: - during a first period of time: - Returning the heavy phase separated in the centrifuge to the fermenter as a liquid flow to the fermenter. In this manner, the centrifuge with the surface-enlarging insert can be utilized to separate the cell culture mixture from the fermenter into a light phase and a heavy phase during a first period of time. More specifically, during a first period of time, the light phase including the product produced in the bioprocessing system can be separated while the cells of the cell culture mixture are returned to the fermenter together with the heavy phase. Thus, a perfusion mode operation of the bioprocessing system including the centrifuge with the surface-enlarging insert can be provided.

[0035] The light phase separated in the centrifuge is a liquid. The heavy phase separated in the centrifuge is a suspension containing the cells of the cell culture mixture suspended in the same liquid as the liquid forming the light phase. The separated light phase may contain extracellular biomolecules expressed by the cells during fermentation. During the first period, the separated light phase may contain debris. Such debris may have a smaller size than the cells of the cell culture mixture. That is, the cells may be separated together with the heavy phase. Furthermore, some of the particles having a smaller size than the cells of the cell culture mixture may be separated together with the heavy phase.

[0036] During a first period of time, the steps of generating a cell culture mixture in a fermentor and directing a flow of the cell culture mixture from the fermentor into the interior of a centrifuge are performed.

[0037] According to an embodiment, in the step of separating the flow of cell culture mixture into a light phase and a heavy phase, the separated light phase flow may form up to 5% V / V (volume %) of the flow of cell culture mixture, in such a way that a suitable small amount of light phase / product can be removed from the cell culture mixture in a perfusion operation mode of the bioprocessing system.

[0038] According to an embodiment, the method includes, during a first period of time: - conducting the light phase separated in the centrifuge into a receiving vessel; In this way, the light phase / product can be collected in a convenient manner for further processing.

[0039] Based on the amount of separated light phase directed to the receiving vessel, fresh growth medium can be fed to the fermenter, in such a way that the light phase being removed from the fermenter can be replaced with growth medium, which promotes cell growth and the production of required substances by the cells.

[0040] According to an embodiment, prior to the first period of time, the method further comprises: - priming the centrifuge with a liquid other than the cell culture mixture, such as growth medium In this manner, the centrifuge and the bioprocessing system can be prepared for perfusion mode operation. By priming the centrifuge with liquid other than the cell culture mixture, the cell culture mixture from the fermentor can flow uniformly into the interior of the centrifuge at the beginning of the first period.

[0041] According to an embodiment, the step of continuously returning the flow of liquid from the interior of the centrifuge back to the fermenter comprises, during a second period of time: - returning the flow of unseparated cell culture mixture from the interior of the centrifuge to the fermentor as a liquid flow to the fermentor. In this way, different operation of the centrifuge is provided during a first period and a second period, respectively. During the first period, the centrifuge with the surface-enlarging insert can be utilized to separate the cell culture mixture from the fermenter into a light phase and a heavy phase, and during the second period, the cell culture mixture can be returned through the centrifuge to the fermenter. Thus, a perfusion mode operation of the bioprocessing system with the centrifuge with the surface-enlarging insert is provided when the separation capacity of the centrifuge exceeds the separation of the cell culture mixture at the relevant flow rate from the fermenter.

[0042] More specifically, during a first period, the light phase containing the product produced in the bioprocessing system can be separated, while the cells of the cell culture mixture can be returned to the fermenter together with the heavy phase. During a second period, the centrifuge can be inoperative and in a standby state, in the sense that no separation of the light and heavy phases takes place therein. Instead, the cell culture mixture simply returns to the fermenter through the interior of the centrifuge. Thus, during the second period, the cell culture mixture in the centrifuge is continuously exchanged. Thus, it is possible to avoid a deterioration of the cell culture mixture in the centrifuge when the light phase containing the product is not removed from the cell culture mixture during the second period.

[0043] During the second period, no separation may take place in the centrifuge. During the second period, the continued flow of the cell culture mixture through the centrifuge may serve the purpose of preventing stagnation of the cell culture mixture, in particular stagnation of the cell culture mixture within the centrifuge. Such a stagnant cell culture mixture may otherwise degrade the light and / or heavy phases to be separated in a subsequent separation operation.

[0044] Also during a second time period, the steps of producing a cell culture mixture in a fermentor and directing a flow of the cell culture mixture from the fermentor into the interior of a centrifuge are performed.

[0045] After the second period, e.g., for a majority of the entire culture period, a further first period can be initiated, then a further second period can be initiated, and so on. Thus, the method and bioprocessing system can be adapted for intermittent separation of light and heavy phases from a cell culture mixture.

[0046] According to these embodiments, the inventors of the present invention have realized that in order to utilize a centrifuge with surface-enlarged inserts and its advantages in a bioprocessing system operated in perfusion mode, the separation in the centrifuge may have to be performed intermittently. Depending on the size and therefore the capacity of the disc stack centrifuge, the efficient separation provided by the centrifuge, and the size of the fermenter, it is not possible to operate the centrifuge continuously throughout the entire cultivation period. The through flow would be such that the separated light phase would not contain the contents expected from the cell culture mixture produced in the fermenter. Therefore, the inventors of the present invention provide a method and a bioprocessing system that provide conditions for intermittent operation without the risk of degrading the separated light and heavy phases or the cell culture mixture.

[0047] According to an embodiment, during the step of returning the flow of the cell culture mixture during the second time period, the rotor of the centrifuge may be stationary or may rotate at a lower rotational speed than during the step of separating the flow of the cell culture mixture during the first time period, or may rotate in a rotational direction opposite to the rotational direction during the step of separating the flow of the cell culture mixture during the first time period. In this way, it can be ensured that no or substantially no separation occurs in the centrifuge during the second time period. Thus, accumulation of separated cells during the second time period can be avoided.

[0048] According to the embodiment, the flow of the cell culture mixture can be directed into the interior of the centrifuge during the first and second time periods at substantially the same flow rate q. In this way, a uniform flow of the cell culture mixture can be maintained over the first and second time periods. Thus, a correspondingly predetermined flow rate of the culture mixture can at least not be exceeded, thereby ensuring that the cells of the cell culture mixture are not subjected to harmful shear stresses in the bioprocessing system. Furthermore, it can be avoided to reset one or more pumps configured to transport the cell culture mixture and the heavy phase through the bioprocessing system between the first and second time periods.

[0049] According to an embodiment, the method further comprises, during the third period: - stopping the continuous flow of liquid from inside the centrifuge back into the fermenter; - directing a flow of the cell culture mixture from the fermenter to a centrifuge; - separating the cell culture mixture stream into a light phase and a heavy phase in a centrifuge; - conducting the heavy phase separated in the separating step into a receiving vessel; In this way, the cell concentration of the cell culture mixture can be reduced. Thus, for example, if the cell concentration of the cell culture mixture is above a limit value, during the third period, the method and bioprocessing system provide for a reduction in the cell concentration by directing the separated heavy phase to a separate receiving tank instead of returning the separated heavy phase to the fermenter as in the first period.

[0050] According to an embodiment, during the third time period, the rotor of the centrifuge may rotate at a higher rotation speed than during the first time period. In this way, the separated heavy phase during the third time period may have a higher particle concentration than the separated heavy phase during the first time period. Thus, a reduction in the cell concentration of the cell culture mixture may be efficiently performed, and may further include particles smaller than whole cells.

[0051] Following the third period, the first period comprising separating the cell culture mixture and returning the separated heavy phase to the fermentor, or the second period comprising returning the unseparated cell culture mixture to the fermentor, can be repeated.

[0052] According to an embodiment, the method further comprises, during the fourth period: - stopping the step of continuously returning the flow of liquid from inside the centrifuge back to the fermenter if not previously performed; - directing all of the cell culture mixture from the fermenter into a centrifuge; - separating the cell culture mixture into a light phase and a heavy phase in a centrifuge, the light phase forming 50-95% V / V of the cell culture mixture; - conducting the light phase separated in the centrifuge into a receiving vessel; In this way, the fermenter can be emptied at the end of the cultivation period and during a fourth period the light phase / product can be extracted from the cell culture mixture.

[0053] How large a proportion of the light phase forms compared to the cell culture mixture depends on the cell concentration in the relevant cell culture mixture.

[0054] According to an embodiment, following the third and / or fourth period of time, the method further comprises: - diluting the heavy phase in the receiving vessel with a liquid to produce a diluted heavy phase; - transferring the diluted heavy phase from the receiving vessel to a centrifugal device, such as a centrifuge, during a fifth time period; - separating the diluted heavy phase into a light phase and a further heavy phase in a centrifuge during a fifth time period; - conducting the light phase into a receiving vessel; In this manner, the yield from the bioprocess can be increased. The heavy phase separated during one or more of the first, third and fourth time periods can be diluted with liquid. By separating the diluted heavy phase, a light phase containing a desired material that may otherwise be wasted can be extracted.

[0055] According to an embodiment, during the fifth time period, the rotor of the centrifuge can be rotated at a higher rotational speed than during the first time period, such that the separated heavy phase during the fifth time period has a higher particle concentration than the separated heavy phase during the first time period. During the fifth period a larger portion of the lighter phase can separate than during the first period.

[0056] According to an embodiment, following the third, fourth and / or fifth time periods, the method further comprises, during a sixth time period: - flushing the centrifuge with a liquid, such as a growth medium; - operating a centrifuge; - directing at least a portion of the liquid through a light phase outlet of the centrifuge into a receiving vessel; In this manner, the residual product containing the desired material can be flushed out of the centrifuge, thus making such residual product available for downstream processing.

[0057] According to an embodiment, following any one of the fourth, fifth and / or sixth time periods, the method further comprises: - conducting the light phase collected in the receiving vessel to a centrifuge or centrifugal device; - separating the light phase into a light fraction and a heavy fraction in a centrifuge or centrifugal device, the heavy fraction containing residual debris from the cell culture mixture; - transferring the light fraction for further processing, such as extraction of extracellular material from the light fraction; In this manner, the light phase separated during any one of the fourth, fifth and / or sixth time periods may be freed of at least a majority of the debris it may contain, thereby providing the light phase in the form of a separated light fraction and thereby preparing the light phase for downstream processing.

[0058] According to an embodiment, the centrifuge with the surface-enlarging insert can be a modular centrifuge with a base unit and a replaceable separation insert mounted within the base unit. The method includes: - replacing the used replaceable separation insert with an unused replaceable separation insert prior to directing the flow of the cell culture mixture from the fermenter into the interior of a centrifuge equipped with a surface-enlarging insert. In this way, a centrifuge can be provided that is sterile and ready to perform one or several separation operations of the centrifuge in the method of operating a bioprocessing system. Thus, a single-use centrifuge is provided.

[0059] The bioprocessing systems discussed herein, and in particular their control systems, can be configured to perform one or more of the method steps discussed herein, such that the bioprocessing systems can benefit from the same or similar advantages as discussed with respect to the corresponding features and / or steps of this method of operating a bioprocessing system.

[0060] According to a further aspect, there is provided a method of operating a bioprocessing system comprising a fermentor for producing a liquid cell culture mixture therein using a growth medium and a centrifuge comprising an enlarged surface insert, the method comprising: - continuously directing a flow of the cell culture mixture from the fermentor into an interior of a centrifuge during a first period of time; - continuously separating the flow of the cell culture mixture in the centrifuge into a light phase and a heavy phase during a first period of time, the light phase forming up to 5% V / V of the flow of the cell culture mixture; - during a first period, simultaneously with the step of separating, returning the heavy phase to the fermenter; A method is provided that includes:

[0061] According to a further aspect, there is provided the use of a centrifuge with an enlarged surface insert for separating a heavy cell-containing phase from a cell culture mixture during a fermentation process operated in perfusion mode.

[0062] Two aspects of the method of operating a bioprocessing system have been discussed above, one relating to the bioprocessing system and one relating to the use of a centrifuge. Each of these aspects of the two methods, the bioprocessing system and the use of a centrifuge, can utilize features of the various embodiments discussed herein. Thus, the inventive features and advantages discussed in the following detailed description also pertain to all aspects of the invention.

[0063] According to a further aspect, there is provided a computer program comprising instructions which, when executed by a computer, cause the computer to perform a method according to any one of the aspects and / or embodiments discussed herein.

[0064] According to a further aspect, there is provided a computer-readable storage medium comprising instructions that, when executed by a computer, cause the computer to perform a method according to any one of the aspects and / or embodiments discussed herein.

[0065] Further features and advantages of the present invention will become apparent upon consideration of the appended claims and the following detailed description.

[0066] Various aspects and / or embodiments of the present invention, including particular features and advantages of the present invention, will be readily understood from the exemplary embodiments discussed in the following detailed description and accompanying drawings. [Brief description of the drawings]

[0067] [Figure 1] FIG. 1 illustrates an embodiment of a bioprocessing system. [Diagram 2] FIG. 1 illustrates a further embodiment of a bioprocessing system. [Diagram 3] FIG. 1 illustrates an embodiment of a centrifuge utilized within a bioprocessing system. [Figure 4] FIG. 2 illustrates a control system according to an embodiment. [Diagram 5] FIG. 1 illustrates an embodiment of a method of operating a bioprocessing system. [Figure 6] 1 illustrates an embodiment of a computer-readable storage medium. [Figure 7] FIG. 1 shows particle distribution in a cell culture mixture. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0068] Aspects and / or embodiments of the present invention are now more fully described. Like numbers refer to like elements throughout. For brevity and / or clarity, well-known functions or constructions may not be described in detail.

[0069] FIG. 1 illustrates an embodiment of a bioprocessing system 2 .

[0070] The bioprocessing system 2 comprises a fermenter 4 for producing a cell culture mixture therein, a centrifuge 6 with an enlarged surface insert, a receiving vessel 8, a conduit system 10, flow control devices 12, 14, and a control system 16.

[0071] The receiving vessel 8 may be configured to receive the light phase separated in the centrifuge 6. The receiving vessel 8 may be configured for intermediate storage of the separated light phase.

[0072] The centrifuge 6 may be a disc stack centrifuge 6, discussed below with reference to FIG. 3. However, the bioprocessing system 2, in its broadest aspects, is not limited to a particular type of centrifuge 6 with a surface-enhancing insert. Thus, according to some embodiments, the centrifuge 6 may be configured for single-use operation and may include a replaceable separation insert. According to alternative embodiments, the centrifuge 6 may be configured for permanent and / or long-term use. In such embodiments, the centrifuge 6 may be washed between culture periods.

[0073] A conduit system 10 is arranged to interconnect the fermenter 4, the centrifuge 6 and the receiving vessel 8. Flow control devices 12, 14 are arranged in the conduit system 10. The flow control devices may comprise one or more pumps 12 and / or one or more valves 14. The flow control devices 12, 14 are arranged to control the flow of one or more of the cell culture mixture, the heavy phase separated in the centrifuge 6 and the light phase separated in the centrifuge 6. Furthermore, the centrifuge 6 may also have a flow control function in the conduit system 10, for example when the centrifuge rotor is spinning.

[0074] The control system 16 is configured to control at least a portion of the centrifuge 6 and the flow control devices 12,14.

[0075] System 2 is configured for perfusion mode operation utilizing a centrifuge with an enlarged surface area insert.

[0076] Figure 2 shows a further embodiment of a bioprocessing system 2. The bioprocessing system 2 of Figure 2 comprises the same basic features as the system 2 of Figure 1 discussed above. The bioprocessing system 2 according to the embodiment of Figures 1 and / or 2, as well as its various components, may optionally include one or more of the following listed features.

[0077] The bioprocessing system 2 may comprise a receiving vessel 18, and the conduit system 10 may interconnect the centrifuge 6 and the receiving vessel 18. The receiving vessel 18 may be configured to receive the heavy phase separated in the centrifuge 6. The receiving vessel 18 may be configured to store the separated heavy phase, such as for intermediate storage of the separated heavy phase. The configuration of the control system 16 for integrating the receiving vessel 18 into the operation of the bioprocessing system 2 is discussed below.

[0078] The conduit system 10 may comprise a first conduit section 21 extending from the fermenter 4 to the inlet of the centrifuge 6, a second conduit section 22 extending from the heavy phase outlet of the centrifuge 6 to the fermenter 4 and a third conduit section 23 extending from the light phase outlet of the centrifuge 6 to the receiving vessel 8. In this way, the conduit system 10 may interconnect the centrifuge 6, the fermenter 4 and the receiving vessel 8.

[0079] - the flow control device 12 may include one or more of a feed pump 12' disposed in the first conduit section 21, a heavy phase pump 12'' disposed in the second conduit section 22, and a light phase pump 12''' disposed in the third conduit section 23. The feed pump 12' may be utilized to transport the cell culture mixture from the fermenter 4 to the centrifuge 6. According to some embodiments, the feed pump 12' may also be utilized to transport the cell culture mixture through the centrifuge 6 and back to the fermenter 4. The heavy phase pump 12'' may be utilized to transport the separated heavy phase and back to the fermenter 4. According to some embodiments, the heavy phase pump 12'' may be utilized to transport the cell culture mixture from the centrifuge 6 and back to the fermenter 4. According to some embodiments, the heavy phase pump 12'' may be utilized to transport the separated heavy phase to the receiving vessel 18. The light phase pump 12'''' may be utilized to transport the separated light phase to the receiving vessel 8. Additionally, the centrifuge 6 may have a pumping effect on one or more of the cell culture mixture and the separated light phase as such.

[0080] The flow control device 14 may include one or more of a first valve device 14' arranged in the first conduit section 21, a second valve device 14'' arranged in the second conduit section 22, and a third valve device 14''' arranged in the third conduit section 23. In this way, further control of the liquids flowing through the bioprocessing system 2 may be provided. For example, the first valve device 14' may control the flow of the cell culture mixture from the fermenter 4 to the centrifuge 6. The second valve device 14'' may control the flow of the heavy phase and / or the cell culture mixture from the centrifuge 6 back to the fermenter 4 and / or from the centrifuge 6 to the receiving vessel 18. The third valve device 14''' may control the flow of the light phase from the centrifuge 6 to the receiving vessel 8 or to the optional waste vessel 60.

[0081] In the embodiment of FIG. 1, each of the illustrated valve devices 14'-14''' may include a single controllable valve capable of continuously or incrementally controlling the flow of the cell culture mixture, the separated heavy phase, or the separated light phase. In the embodiment of FIG. 2, each of the illustrated valve devices 14'-14''' may include a valve train or multiple inlet / outlet valves capable of continuously or incrementally controlling the flow of the cell culture mixture, the separated heavy phase, or the separated light phase. Alternatively, one or more of the valve devices 14'-14''' of the embodiment of FIG. 1 may be utilized in the embodiment of FIG. 2, depending, for example, on the number and functionality of vessels, tanks, and conduits included in the associated bioprocessing system. The valve devices 14'-14''' may include sanitary valves and / or valve trains, for example sanitary valves and / or valve trains meeting the sanitary requirements of the biotechnology industry.

[0082] Similarly, the conduits and pumps and centrifuges discussed herein may meet the same sanitary requirements.

[0083] - The feed pump 12' can be a centrifugal pump and the heavy phase pump 12'' and the light phase pump 12''' can each be a peristaltic pump. In this way, the centrifugal pump can provide gentle handling of the cell culture mixture, especially when operated at low rotational speeds. Thus, the cell culture mixture can be subjected to low shear forces that do not damage the cells of the cell culture mixture. The peristaltic pump can also function as a flow meter, thus providing flow data of the separated light and heavy phases.

[0084] - the conduit system 10 can include a fourth conduit section 24, which extends to the first conduit section 21. The fourth conduit section 24 can be configured to supply a liquid other than the cell culture mixture to the inlet of the centrifuge 6. In this way, the bioprocessing system 2 can be configured to supply the centrifuge 6 with a liquid different from the cell culture mixture, for example during a start-up procedure, so-called priming, of the bioprocessing system 2. This liquid can for example be a priming liquid, such as a growth medium, which is supplied to the centrifuge 6 before separation of the cell culture mixture in the centrifuge 6 begins. Such a liquid can be stored in a separate container 30.

[0085] During such priming of the bioprocessing system, the priming fluid leaving the light phase outlet of the centrifuge 6 can be directed to a waste container 60.

[0086] The conduit system 10 may include a fifth conduit section 25 extending from the second conduit section 22 to the receiving vessel 18. In this way, the separated heavy phase can be transported from the heavy phase outlet of the centrifuge 6 through the second and fifth conduit sections 22, 25 to the receiving vessel 18.

[0087] - the centrifuge 6 can be arranged above the fermenter 4. In this way a compact bioprocessing system 2 can be provided. In the embodiment of Fig. 1 the centrifuge 6 is shown arranged above the fermenter 4. In the embodiment of Fig. 2 the centrifuge 6 can also be arranged above the fermenter 4. Conversely the centrifuge 6 in the embodiment of Fig. 2 can also be arranged remotely from the fermenter 4.

[0088] The control system 16 may be configured to control the fermenter 4, e.g. a pump (not shown) of the fermenter 4, a valve (not shown) of the fermenter 4, an air inlet 32 ​​to the fermenter 4 and / or a stirring member 34 arranged in the fermenter 4. In this way, a compact control system 16 for the bioprocessing system 2 may be provided.

[0089] Alternatively or additionally, the control system 16 may be configured to control the centrifuge 6 and the flow of liquid to and from the centrifuge 6. The fermenter 4 and the cultivation process therein may be controlled by a separate control system. In such an embodiment, the control system 16 of the centrifuge 6 and the separate control system of the fermenter 4 may communicate with each other.

[0090] FIG. 3 illustrates an embodiment of a centrifuge 6 utilized in the bioprocessing system 2 discussed herein with particular reference to FIGS.

[0091] The centrifuge 6 is a modular centrifuge 6. The modular centrifuge 6 comprises a base unit 40 and an exchangeable separation insert 42. In FIG. 3, a first exchangeable separation insert 42' is mounted on the base unit 40 and a second exchangeable separation insert 42'' is shown next to the centrifuge 6.

[0092] In this way, the centrifuge 6 is configured for single use. That is, after use, the first replaceable separation insert 42' in the base unit 40 is replaced with the second replaceable separation insert 42''. The first replaceable separation insert 42' may then be discarded. For example, the first replaceable separation insert 42' may be replaced after one culture period.

[0093] During use of the modular centrifuge 6, the liquid feed mixture, the heavy phase, and the light phase come into contact only with the associated first or second replaceable separation insert 42', 42". Of course, the conduits in the form of tubes 44 configured to guide the liquid feed mixture to and from the replaceable separation insert 42', 42" also come into contact with the liquid feed mixture and the heavy and light phases. The base unit 40 does not come into contact with either the liquid feed mixture or the heavy and light phases.

[0094] The tubes 44 connected to the exchangeable separation inserts 42', 42'' may form part of the exchangeable separation inserts 42', 42''. The tubes 44 form part of the conduit system 10 (see Figures 1 and 2) and thus lead to the inlet of the centrifuge 6 for the cell culture mixture and from the respective outlets of the centrifuge 6 for the separated light and heavy phases.

[0095] The base unit 40 comprises components for supporting and rotating the interchangeable separation inserts 42′, 42″. That is, the base unit 40 comprises, among other things, a stationary frame 46, a rotating member 47, and a drive unit for rotating the rotating member (not shown).

[0096] The stationary frame 46 is stationary during use of the modular centrifuge. However, such a base unit 40 may be made mobile, for example for installation at different locations in a user's production facility. For this purpose, the stationary frame 46 may be provided with wheels 48.

[0097] The replaceable separation insert 42', 42'' is configured as part of a rotor casing 43 or the like and is disposed inside an internal space of a rotating member 47 of the base unit 40. Together, the rotor casing 43 of the replaceable separation insert 42', 42'' and the rotating member 47 of the base unit 40 form a rotor 49 of the centrifuge 6. The replaceable separation insert 42', 42'' may also include one or more stationary parts 45, 45', such as a part where the tube 44 is connected to the replaceable separation insert 42', 42''.

[0098] The tubes 44 may be arranged differently than in the illustrated embodiment. Any configuration of one, two or three tubes 44 may be provided in the stationary portion of the replaceable separation insert 42', 42''. In the latter case, one stationary portion may include three tubes, and the replaceable separation insert may include only one stationary portion at its upper or lower end.

[0099] The rotor 49 of the centrifuge 6 is configured to rotate about a rotation axis 51. The rotor casing 43 of the replaceable separation insert 42', 42'' defines a separation space therein. The replaceable separation insert 42', 42'' comprises a surface-enlarging insert, such as, for example, a stack of frusto-conical separation discs 53, disposed in the separation space. That is, the centrifuge 6 is a disc-stack centrifuge 6.

[0100] The separation space forms part of the interior of the centrifuge 6 into which and from which the liquid is led.

[0101] In a known manner, during the separation operation of the centrifuge 6, the rotor 49 is rotated about the axis of rotation. The feed mixture to be separated, in this case a cell culture mixture to be separated, is fed through an inlet of the centrifuge 6 into the interior and into the separation space of the centrifuge 6. Due to the density difference between the cells and the liquid in which they are suspended, the cell culture mixture is separated into a light liquid phase and a heavy liquid phase. This separation is facilitated by the gaps between the separating disks of a stack arranged in the separation space. The separated light phase is substantially free of the cells of the cell culture mixture and may contain debris of a size smaller than the cells of the cell culture mixture. The separated heavy phase contains particles, such as cells of the cell culture mixture and any debris smaller than the cells. The heavy phase forms a cell containing suspension, which contains a concentrated mixture of the light phase and the cells.

[0102] For a more detailed description of an exemplary modular centrifuge that may be utilized as the centrifuge 6 of the present invention, reference is made to EP 3 666 394, the disclosure of which is incorporated herein by reference.

[0103] Figure 4 illustrates a control system 16 according to an embodiment, which may be utilized in connection with different aspects and / or embodiments of the present invention. The control system 16 is also illustrated in Figures 1 and 2. The control system 16 comprises at least one control unit 52, which may take the form of substantially any suitable type of processor circuit or microcomputer, such as a circuit for digital signal processing (digital signal processor, DSP), a central processing unit (CPU), a processing unit, processing circuit, a processor, an application specific integrated circuit (ASIC), a microprocessor, or other processing logic capable of interpreting and executing instructions.

[0104] The control system 16 comprises a memory unit 54. The control unit 52 is connected to the memory unit 54, which provides the control unit 52 with, for example, stored program codes, data tables and / or other stored data that the control unit 52 needs to perform calculations and thus be able to control the centrifuge 6 and the control devices 12, 14. The control unit 52 is also adapted to store partial or final results of the calculations in the memory unit 54. The memory unit 54 may comprise a physical device utilized to store data or programs, i.e. sequences of instructions, either temporarily or permanently. According to some embodiments, the memory unit 54 may comprise an integrated circuit including silicon-based transistors. The memory unit 54 may, in different embodiments, comprise, for example, a memory card, a flash memory, a USB memory, a hard disk or another similar volatile or non-volatile storage unit for storing data, such as, for example, a ROM (read only memory), a PROM (programmable read only memory), an EPROM (erasable PROM), an EEPROM (electrically erasable PROM).

[0105] The control system 16 may further comprise one or more sensors 56 (only one is shown in FIG. 4). The control unit 52 receives measurement data, such as, for example, the centrifuge rotation speed, from the one or more sensors 56. The control system 16 sends control data to the centrifuge 6 and the control devices 12, 14, for example to control the centrifuge rotation speed, pump speed, valve device settings.

[0106] Thus, the control unit 52 is configured to receive input signals from the sensor 56 and send output signals to the centrifuge 6 and the control devices 12, 14. These signals may include waveforms, pulses or other attributes that can be detected and sent as information by the control unit 52 and converted directly or indirectly into signals that can be processed by the control unit 52. Each of the connections to the sensor, the centrifuge 6 and the control devices 12, 14 may take one or more of the following forms: a cable, a data bus, e.g., a CAN (Controller Area Network) bus, a MOST (Media Oriented System Transport) bus or other bus configuration, or a wireless connection. In the illustrated embodiment, only one control unit 52 and memory 54 are shown, but the control system 16 may alternatively comprise multiple control units and / or memories.

[0107] Control system 16 and its operation are discussed below, with particular reference to Figures 1-4. Moreover, control system 16 may be configured to carry out embodiments of method 100 discussed herein, with particular reference to Figure 5. Thus, advantages discussed in relation to method 100 also apply to control system 16, and vice versa.

[0108] The control system 16 includes: - directing the flow of the cell culture mixture from the fermenter 4 into the interior of the centrifuge 6, and at the same time - Continuously returning the liquid flow from inside the centrifuge 6 back to the fermenter 4 In this way, a perfusion mode operation of the bioprocessing system 2 comprising the centrifuge 6 with the surface enlarged insert is possible. More specifically, in this way, stagnant liquid in the centrifuge 6 is avoided, which would otherwise be returned to the fermenter 4 and would degrade the cell culture mixture in the fermenter 4 during the cultivation period.

[0109] The liquid may be a heavy phase that has been separated from the cell culture mixture, i.e. a heavy phase that contains a more concentrated cell culture mixture than that directed inside the centrifuge 6, may be returned to the fermenter 4. According to some embodiments, intermittently, the liquid may be the cell culture mixture as directed inside the centrifuge 6, i.e. flowing through the centrifuge 6 without being separated.

[0110] The flow of the cell culture mixture may be directed from the fermentor 4 to the centrifuge 6 through a first conduit section 21 and further through a feed pump 12' and / or a first valve device 14', if present in the system 2.

[0111] The liquid flow passes from the centrifuge 6 through a second conduit section 22 and a heavy phase pump 12'', and may optionally be directed / returned through a second valve device 14'', if one is present in the system 2.

[0112] According to an embodiment, during a first period of time, the control system 16 - activating the centrifuge 6 in order to separate the flow of the cell culture mixture into a light phase and a heavy phase; - from inside the centrifuge 6, continuously returning the heavy phase separated in the centrifuge 6 as a liquid flow to the fermenter 4 In this manner, perfusion mode operation of bioprocessing system 2 may be provided by returning cells of a cell culture mixture having a heavy phase to fermentor 4, where the returned cells can continue to produce the desired product of the associated bioprocess.

[0113] According to an embodiment, during a first period of time, the control system 16 - directing the light phase separated in the centrifuge 6 into a receiving vessel 8 In this way, the light phase may be collected in a receiving vessel 8, for example for further downstream processing of the light phase or for expectorating therefrom desired substances of a bioprocess.

[0114] The light phase conducted to the receiving vessel 8 may be exchanged with fresh growth medium, for example through the growth medium conduit 33 to the fermenter 4. By way of example, the turnover rate of the volume of the fermenter 4 may be 1 to 2 times per 24 hours, i.e. the light phase separated from the cell culture mixture conducted to the receiving vessel 8 may be equal to the volume of the fermenter 4 or twice the volume of the fermenter 4 per 24 hours.

[0115] Once filled, the receiving vessel 8 may be replaced with an empty receiving vessel. This may be done once or multiple times over the cultivation period. The contents of the filled receiving vessel 8, i.e. the light phase therein, may be subjected to downstream processing. An alternative, or a complement, to replacing the receiving vessel 8 may be to intermittently empty the receiving vessel 8 and transfer the light phase for downstream processing.

[0116] According to an embodiment, during the second time period, the control system 16 - continuously returning the unseparated cell culture mixture from inside the centrifuge 6 as a liquid flow to the fermenter 4 In this way, a continuous flow of unseparated cell culture mixture can be maintained through the centrifuge 6. More specifically, no separation of the cell culture mixture takes place in the centrifuge 6 during the second period. This makes it possible to prevent stagnant liquid in the centrifuge 6 during periods when separation of the cell culture mixture is not required in the bioprocessing system 2. Thus, the conditions for perfusion mode operation of the bioprocessing system 2 can be achieved even when, for example, the bioprocess in the system 2 does not provide for continuous extraction of light phases / products. This is believed to be due to the high separation capacity of the centrifuge 6 compared to the production rate in the fermenter 4 of the cell culture mixture capable of extracting the desired substances.

[0117] According to an embodiment, the control system 16 may be configured to keep the rotor of the centrifuge 6 stationary during the second time period, or to rotate the rotor at a lower rotational speed than during the first time period, or to rotate the rotor in a rotational direction opposite to that during the first time period. In this way, no separation of the cell culture mixture takes place in the rotor of the centrifuge 6 during the second time period.

[0118] When the rotor rotates during the second period, its rotation speed is substantially lower than its rotation speed during the first period. This is true for each of the directions of rotation of the rotor. For example, the rotation speed during the second period may be within a range of 1-10% of the rotation speed during the first period, or within a range of 0.1-20%. By maintaining such rotation of the rotor, it can be ensured that no region with a stagnant cell culture mixture is formed in the separation space during the second period.

[0119] According to an embodiment, the control system 16 may be configured to alternate between the first and second time periods. In this way, the perfusion mode operation of the bioprocessing system 2 can be maintained for longer periods during which the light phase / product is intermittently extracted from the cell culture mixture in the fermenter 4. That is, for each first period, the light phase / product is separated in the centrifuge 6, and for each second period, the extraction of the light phase / product is suspended while the unseparated cell culture mixture is returned from the centrifuge 6 to the fermenter.

[0120] According to an embodiment, the bioprocessing system 2 includes a receiving vessel 18, and the control system 16 controls, during a third period of time, - stopping the continuous return flow of liquid from inside the centrifuge 6 to the fermenter 4, - directing the flow of cell culture mixture from the fermenter 4 to a centrifuge 6, - activating the centrifuge 6 in order to separate the flow of cell culture mixture into a light layer and a heavy phase inside the centrifuge 6; - directing the separated heavy phase in the centrifuge 6 into a receiving vessel 18 In this way, the concentration of cells in the cell culture medium in the fermenter 4 may be reduced. More specifically, over the culture period, the cell content of the cell culture mixture may increase to a level that adversely affects the cell culture mixture and / or the production of the sought substance. In such a situation, the cell concentration of the cell culture mixture in the fermenter 4 may be reduced by diverting the separated heavy phase and the cells contained therein from the fermenter 4 during the third period.

[0121] Compared to the volume of the separated light phase led to the receiving vessel 8, the separated heavy phase led to the receiving vessel 18 can be small, i.e., the separated heavy phase led to the receiving vessel 18 contributes only to a small part of the turnover of the volume of the fermenter 4.

[0122] The third time period may be followed by the second time period or the first time period and the measures performed in association with the time periods. The third time period and the measures performed in association therewith may be repeated intermittently over the culture period to maintain the cell concentration of the cell culture mixture within a desired range.

[0123] According to an embodiment, during the third time period, the control system 16 - directing the light phase separated in the centrifuge 6 into a receiving vessel 8 In this way, also during the third period, the separated light phase may be collected in the receiving vessel 8 while the separated heavy phase is diverted to the receiving vessel 18. That is, also during the third period, the light phase / product may contain the desired substance.

[0124] According to an embodiment, the centrifuge 6 comprises a rotor (as described above), and the control system 16 may be configured to rotate the rotor at a higher rotation speed during the third period than during the first period. In this way, a more concentrated heavy phase can be separated during the third period than during the first period. Thus, less light phase is contained in the suspension forming the separated heavy phase during the third period than during the first period, and therefore less light phase can be sent to the receiving vessel 18 and wasted from the bioprocess. As a result, more light phase can be separated during the third period than during the first period.

[0125] According to an embodiment, during the fourth time period, the control system 16 - stopping the continuous return flow of liquid from inside the centrifuge 6 to the fermenter 4 if this has not been done before, - directing the entire cell culture mixture from the fermenter 4 into a centrifuge 6; - activating the centrifuge 6 in order to separate the cell culture mixture into a light phase and a heavy phase; - directing the light phase separated in the centrifuge 6 into a receiving vessel 8 In this way, the fermentor 4 may be emptied at the end of the relevant cultivation period and a final amount of light phase / product may be extracted from the cell culture mixture in the fermentor 4.

[0126] According to an embodiment, during the fourth time period, the control system 16 - directing the heavy phase separated in the centrifuge 6 into a receiving vessel 18 In this way, the heavy phase separated during the fourth period may be collected in the receiving vessel 18 and not sent back to the fermenter 4, which is thereby emptied during the fourth period.

[0127] According to an embodiment, the bioprocessing system 2 may optionally include a centrifuge device 6', and optionally the conduit system 10 may interconnect the receiving vessel 8 and the centrifuge device 6'. The control system 16 may be configured to: - directing the light phase collected in the receiving vessel 8 to a centrifuge 6 or a centrifugal device 6', - activating the centrifuge 6 or centrifugal device 6' in order to separate the light phase into a light fraction and a heavy fraction, - transferring the light fraction from the centrifuge 6 or centrifugal device 6' In this way, the light phase collected in the receiving vessel 8 may be removed from any debris it may contain, thus preparing the light phase for downstream processing by presenting it in the form of a separated light fraction.

[0128] The transfer of the light fraction may be to a further receiving vessel 50 or directly to downstream processing equipment for further processing of the light fraction, such as extraction of desired substances from the light fraction.

[0129] Centrifugal device 6' is shown diagrammatically in FIG. 2 and forms an optional different centrifuge device 6' other than the centrifuge 6 utilized to separate the light and heavy phases during the first period. Thus, either the same centrifuge 6 or a different centrifuge device 6' may be utilized to separate the light and heavy fractions. Such a different centrifuge device 6' may be the same or a different centrifuge type as centrifuge 6. It should be noted that centrifuge device 6' may be configured for a single use.

[0130] The pipeline system 10 may comprise a sixth conduit section 26 for conducting the light phase from the receiving vessel 8 to the centrifuge 6 or to a centrifuge device 6'.

[0131] If the same centrifuge 6 as in the first period is utilized to separate the light and heavy fractions, the conduit system 10 may comprise a seventh conduit section 27 connected to the third conduit section 23 and to the further receiving vessel 50. The separated light fraction may thus be transferred through the seventh conduit section 27 to the further receiving vessel 50. In such an embodiment, the separated heavy fraction may be directed to a receiving vessel 18 or a different waste receptacle connected to the heavy phase outlet of the centrifuge 6.

[0132] To ensure that the separated light fraction has a low particle content, the centrifugal device 6' may have a higher separation capacity than the centrifuge 6.

[0133] The advantage of utilizing a centrifugal device 6' is that it can be operated in parallel with the centrifuge 6. Thus, the receiving vessel 8 may be emptied or release some of its contents during the incubation period.

[0134] According to an embodiment, the control system 16 may be configured to provide a flow of the cell culture mixture at substantially the same flow rate q to the interior of the centrifuge 6 during the first and second time periods. In this way, there is substantially no difference in the flow rate q between the first and second time periods, which can promote gentle handling of the cell culture mixture and the separated heavy phase during each of the first and second time periods. In particular, the flow rate q can be selected to provide low shear stress to the cell culture mixture and the separated heavy phase within the conduit system 10.

[0135] The flow rate q may vary within ±10% between the first and second time periods.

[0136] As discussed above, according to an embodiment, the centrifuge 6 with the surface-enlarging insert may be a modular centrifuge with a base unit 40 and interchangeable separation inserts 42', 42'' mounted to the base unit 40.

[0137] According to an embodiment in which the conduit system 10 includes a fourth conduit section 24 configured to supply a liquid other than the cell culture mixture to the inlet of the centrifuge 6, the control system 16 may, prior to the first time period, so that liquid other than the cell culture mixture is transported into the centrifuge 6 through the fourth and first conduit sections 24, 21 and out of the centrifuge 6 through the heavy phase outlet and the light phase outlet and is diverted from the second and third conduit sections 22, 23 before reaching the fermenter 4 and the receiving vessel 8, - activating the feed pump 12', the heavy phase pump 12'', and the light phase pump 12'''; - providing a liquid passage from the fourth conduit section 24 to the first conduit section 21 and to the centrifuge 6 using a first valve device 14'; - utilizing a second valve device 14'' to provide a bypass passage from the second conduit section 22; - Utilizing a third valve arrangement 14''' to provide a bypass passage from the third conduit section 23 In this manner, the control system may be configured to supply the centrifuge 6 with a liquid different from the cell culture mixture prior to the first time period. That is, prior to separation of the cell culture mixture in the centrifuge 6, a priming liquid, such as a growth medium, may be supplied to the centrifuge 6 to prime the centrifuge 6 with a liquid of lower cost / value than the cell culture mixture. The priming liquid is supplied in a separate container 30 and conducted to the centrifuge 6 through the fourth conduit section 24.

[0138] The light phase outlet may divert liquid other than the cell culture mixture to a waste container 60. The heavy phase outlet may divert liquid other than the cell culture mixture to a receiving vessel 18.

[0139] According to an embodiment, during a first period of time, the control system 16 - activating the feed pump 12', the heavy phase pump 12'', and the light phase pump 12'''; - providing a liquid passage from the fermenter 4 to the first conduit section 21 and the centrifuge 6 using a first valve device 14'; - providing a heavy-duty passage through the second conduit section 22 to the fermenter 4 using a second valve device 14''; - Utilizing a third valve device 14''' to provide a light passageway through the third conduit section 23 to the receiving vessel 8. In this manner, the control system 16 may be configured to transfer the cell culture mixture from the fermentor 4 to the centrifuge 6. The separated light and heavy phases therein may also be transferred in this manner to the receiving vessel 8 and the fermentor 4, respectively.

[0140] According to an embodiment, during the second time period, the control system 16 - activating the feed pump 12' and the heavy phase pump 12'', - maintaining the first and second valve devices 14', 14'' open, - maintaining the third valve device 14''' in a closed state, for example to provide a cell culture mixture passage from the fermenter 4 through the first conduit section 21 to the centrifuge 6 and from there through the second conduit section 22 back to the fermenter 4; In this manner, the control system 16 may be configured to prevent stagnation of the cell culture mixture within the centrifuge 6 when no separation is occurring therein, i.e., the cell culture mixture is not degraded during the second period of time when no separation of the cell culture mixture is occurring within the centrifuge 6.

[0141] According to an embodiment in which the conduit system 10 comprises a fifth conduit section 25 and a receiving vessel 18, the control system 16 controls, during the third time period: The cell culture mixture is transferred from the fermenter 4 through the first conduit section 21 to the centrifuge 6 and separated in the centrifuge 6 into a light phase, which is led through the third conduit section 23 to the receiving vessel 8, and a heavy phase, which is led through the first and fifth conduit sections 21, 25 to the receiving vessel 18, - activating the feed pump 12' and the heavy phase pump 12'', - Activate centrifuge 6; - Utilizing the second valve device 14'' and the fifth conduit section 25 to provide a heavy phase passage from the heavy phase outlet to the receiving vessel 18. In this manner, the control system 16 may be configured to divert the separated heavy phase from the fermentor 4. Instead of being transferred to the fermentor 4, the separated heavy phase may be transferred from the centrifuge 6 through the second and fifth conduit sections 22, 25 to the receiving vessel 18.

[0142] Figure 5 shows an embodiment of a method 100 for operating a bioprocessing system. The bioprocessing system may be a system 2 according to any one of the embodiments discussed in relation to Figures 1-3 and comprises a control system 16 as discussed in relation to Figures 1-4. In the following, reference will also be made to Figures 1-4. The features and advantages discussed in relation to the method 100 also apply to the control system 16 and vice versa.

[0143] A method 100 for operating a bioprocessing system includes: - 102 generating a cell culture mixture in a fermenter 4; - simultaneously with the step of generating a cell culture mixture 102, a step of directing 104 a flow of the cell culture mixture from the fermenter 4 into the interior of a centrifuge 6 equipped with an insert for enlarging the surface; - generating 102 a cell culture mixture and directing 104 a flow of the cell culture mixture, simultaneously with continuously returning 106 a flow of liquid from the interior of the centrifuge 6 to the fermenter 4; Includes.

[0144] Thus, perfusion mode operation of a bioprocessing system 2 comprising a centrifuge 6 with a surface-enlarging insert is made possible by continuous return flow of liquid from the interior of the centrifuge 6 to the fermenter 4 .

[0145] The step of producing a cell culture mixture 102 involves culturing the cells of interest in a fermentor 4 by known methods, utilizing, inter alia, a growth medium, oxygen, and favorable temperature conditions.

[0146] Therefore, during the 102 steps of generation, either continuously or intermittently, - feeding a growth medium into the fermenter 4 through a growth medium conduit 33; - feeding oxygen into the fermenter 4 through the oxygen or air inlet 32; - stirring the cell culture mixture in the fermenter 4 with a stirring member 34; - controlling the temperature inside the fermenter 4 by means of a cooling and / or heating element 35; may be provided.

[0147] It should be noted that the step of continuously returning 106 the flow of liquid from inside the centrifuge 6 to the fermentor 4 may be performed for more than 90%, such as more than 95% of the duration of the entire culture period.

[0148] According to an embodiment, the method 100 includes, during a first period of time: - separating the cell culture mixture flow into a light phase and a heavy phase in a centrifuge 107, wherein the step of continuously returning the liquid flow from inside the centrifuge 6 to the fermenter 4 106 includes, during a first period of time: - 108 step of returning the heavy phase separated in the centrifuge 6 to the fermenter as a liquid flow to the fermenter 4 may include:

[0149] That is, during a first period of time, the cell culture mixture from fermentor 4 is allowed to separate into a light phase and a heavy phase, and the cell-containing heavy phase of the cell culture mixture is returned to fermentor 4.

[0150] According to an embodiment, in step 107 of separating the cell culture mixture flow into a light phase and a heavy phase, the separated light phase flow may form up to 5% V / V (volume %) of the cell culture mixture flow.

[0151] That is, a suitably small amount of light phase / product may be extracted from the cell culture mixture during separation of the cell culture mixture. The amount of light phase may be a function of, among other things, the fermentor 4 volume, the flow of the cell culture mixture from the fermentor 4, and the cell density of the cell culture mixture.

[0152] According to an embodiment, the method includes, during a first period of time: 112 step of directing the light phase separated in the centrifuge 6 into a receiving vessel 8; may include:

[0153] That is, the light phase / product may be collected for subsequent processing.

[0154] According to an embodiment, during the first period, the heavy phase comprises growth medium and whole cells, and the light phase comprises growth medium and debris from the cell culture mixture having a particle size smaller than the whole cells, and may be substantially free of whole cells.

[0155] The growth medium may be consumed by the cells at various stages. However, during the cultivation period, fresh growth medium is fed to the fermenter 4 to drive cell growth. Similarly, during, for example, a first period, a light phase comprising the growth medium is extracted from the fermenter 4 to the receiving vessel 8. As mentioned above, the light phase contains the desired substances that are the desired end products when the light phase is subjected to downstream processing in the bioprocessing system 2. Thus, fresh growth medium also replaces the light phase that was led to the receiving vessel 8.

[0156] According to an embodiment, during a first period, the flow of the cell culture mixture is conducted at a flow rate q, in which case the centrifuge 6 may be operated at an area equivalent Ae such that whole cells having a diameter d are separated from the cell culture mixture into a heavy phase at a flow rate q. In this way, the cells in the cell culture mixture may be separated together with the heavy phase as they pass through the centrifuge 6 and returned to the fermenter 4. Smaller particles such as debris, e.g., debris from cell lysis, may follow the light phase as well as the heavy phase.

[0157] Thus, the threshold particle d may be selected to be smaller than the diameter d of a whole cell of the cell culture mixture during the first period. As an example, the diameter d of the whole cell may be in the range of 10 to 30 μm.

[0158] More specifically, the area equivalent Ae is a measure of the separation capacity of the centrifuge 6 equipped with the surface-enhancing insert. The higher the area equivalent, the higher the separation performance of the centrifuge 6.

[0159] Here, the area equivalent Ae, exemplified in the context of a centrifuge with a stack of separating discs, is calculated according to the following formula:

[0160]

number

[0161] where g = gravitational constant, N = number of truncated conical separating disks of the separating insert, α = half the apex angle of the truncated conical separating disk, i.e. the angle between the axis of rotation and the inner surface of one of the truncated conical separating disks, ω = rotation speed of the rotor of the centrifuge 6, r2 = outer radius of the truncated conical separating disk, r1 = inner radius of the truncated conical separating disk.

[0162] Since Ae has a rotational speed component ω, Ae can be manipulated by adjusting the rotational speed of the rotor of the centrifuge 6.

[0163] The limiting particle by Ambler in 1959 and later Axelsson / Madsen describes the diameter d of the largest particle that is separated with the light phase in a suspension, i.e. in this case a cell culture mixture.

[0164] The following equations and relationships are correct:

[0165] The flow rate q of the suspension passing through the centrifuge is given by: q = v g Ae (2) where v g is the Stokes settling velocity and Ae is the area equivalent given by equation (1) above.

[0166] From Stokes' law we get:

[0167]

number

[0168] where ρ is the density difference between the particle of interest and its surrounding medium, d is the particle size, and μ is the viscosity of the suspension.

[0169] The critical particle d can be calculated from equations (1), (2), and (3).

[0170] The limiting particle size d is therefore a function of the flow rate q as well as the rotational speed ω of the rotor of the centrifuge 6 .

[0171] FIG. 7 shows an example of particle size distribution of a cell culture mixture in a fermenter 4. In this example, the size of the whole cells is about 11 μm, as shown by the large peak of the curve. The smaller peak of the curve shows the main size of the debris contained in the cell culture mixture. However, debris in the range of <0.1-10 μm, such as debris from cell lysis, is also contained in the cell culture mixture. In this example, the preferred size of the limiting particle d is 10 μm, as shown by the dashed line.

[0172] That is, whole cells are separated with the heavy phase. Smaller sized debris is contained in both the heavy and light phases. Thus, a portion of the debris is removed from fermenter 4 with the separated light phase and thereby removed from the fermentation process therein.

[0173] In the cell culture mixture, as in other Newtonian fluids, viscous stresses in the form of shear stresses result from its flow. The maximum shear stress that the cell culture mixture and / or the separated heavy phase may experience as it flows through the conduit system 10 may be one that does not cause damage to the cells contained therein. The flow rate within the conduit system 10 may be limited depending on the shear stress that the cells may experience therein. For example, the flow rate may be limited to a flow rate at which at least 95% of the cells survive passing through the conduit system.

[0174] In the centrifuge 6, the cell culture mixture and the separated heavy phase are subjected to the highest shear stresses at the cell culture mixture inlet and heavy phase outlet, where the cell culture mixture and the separated heavy phase are accelerated and decelerated. The inlets and outlets concentric with the rotation axis of the rotor of the centrifuge 6 provide the most gentle acceleration and deceleration.

[0175] Returning to FIG. 5 and FIGS. 1-4, according to an embodiment, prior to the first time period, the method 100 includes: - priming 114 the centrifuge 6 with a liquid other than the cell culture mixture, such as a growth medium. In this way, priming 114 of the centrifuge 6 with a liquid other than the cell culture medium can ensure a uniform flow of the cell culture mixture into the interior of the centrifuge 6 at the start of the first time period.

[0176] In the priming 114 step, the interior of the centrifuge 6, such as the separation space of the centrifuge 6, may be filled with a liquid other than the cell culture mixture.

[0177] The priming 114 step may also include priming parts or the entire conduit system 10, such as the conduit sections leading from and / or to the fermenter 4. Thus, the cell culture mixture can be gently introduced into such conduit sections leading to the interior of the centrifuge 6, and the separated heavy phase can be gently introduced into the conduit sections leading from the centrifuge 6 back to the fermenter 4.

[0178] According to an embodiment, the step of continuously returning 106 the flow of liquid from the interior of the centrifuge 6 to the fermenter 4 includes, during a second period of time: - 110 step of returning the flow of cell culture mixture from inside the centrifuge 6 to the fermenter 4 as a fluid flow to the fermenter 4. may include:

[0179] During the second period, no or substantially no separation takes place in the centrifuge 6. The cell culture mixture simply flows through the centrifuge 6.

[0180] In this way, different operation of the centrifuge 6 may be provided during the first and second time periods. During both time periods, liquid is returned from the centrifuge 6 to the fermenter 4. Thus, stagnation of the cell culture mixture in the centrifuge 6 can be avoided during perfusion mode operation of the bioprocessing system 2, regardless of whether separation takes place in the centrifuge 6 or not.

[0181] During the second time period, the flow direction of the cell culture mixture within the bioprocessing system 2 can be a first flow direction from the fermentor 4 through the first conduit section 21 to the centrifuge 6 and back to the fermentor 4 through the second conduit section 22. The feed pump 12' and the heavy phase pump 12'' can pump the cell culture mixture in the first flow direction. Alternatively, the heavy phase pump 12'' can be stationary and simply open for flow therethrough, with only the feed pump 12' pumping the cell culture mixture in the first flow direction.

[0182] Alternatively, the flow direction during the second period may be a second flow direction opposite to the first flow direction, i.e. from the fermenter 4 through the second conduit section 22 to the centrifuge 6 and then back to the fermenter 4 through the first conduit section 21. The second flow direction is available if the feed pump 12' and / or the heavy phase pump 12'' are reversible.

[0183] According to an embodiment, during the step of returning the flow of cell culture mixture during the second time period 110, the rotor of the centrifuge may be stationary or may rotate at a lower rotational speed than during the step of separating the flow of cell culture mixture during the first time period 107, or may rotate in a rotational direction opposite to that during the step of separating the flow of cell culture mixture during the first time period 107. In this way, it can be ensured that no or substantially no separation takes place in the centrifuge during the second time period. Thus, accumulation of separated cells can be avoided.

[0184] Naturally, in the case of rotation of the rotor in a direction opposite to that during the first period, the rotation speed is lower than the rotation speed during the first period in order to prevent or at least substantially reduce separation of cells in the separation space of the centrifuge.

[0185] When rotating during the second period, the rotation speed of the rotor of the centrifuge 6 may be within the range of 0.1 to 10% of the rotation speed of the rotor during the first period.

[0186] The advantage of rotating the rotor of the centrifuge 6 in a direction of rotation opposite to that during the first period is to loosen or release the separated cells at the periphery of the separation space of the centrifuge 6. Thus, the cells can be transported back to the fermenter 4 together with the cell culture mixture flowing through the interior of the centrifuge 6 during the second period.

[0187] Similarly, an advantage of applying the reverse flow direction, i.e. the second flow direction described above, is to avoid stagnation areas inside the centrifuge 6 during the second period, which, depending on the structure of the centrifuge 6, may form when the rotor is stationary or rotating at a substantially reduced rotational speed.

[0188] According to an embodiment, during the first and second time periods the flow of the cell culture mixture may be directed into the interior of the centrifuge at substantially the same flow rate q.

[0189] For example, the flow rate q may differ by no more than ±10% between the first and second time periods.

[0190] According to an embodiment, the method 100 includes, during a third period: - a step 115 of stopping the step 106 of continuously returning the flow of liquid from inside the centrifuge back to the fermenter; - directing the flow of the cell culture mixture from the fermenter 4 to a centrifuge 6; - separating the flow of cell culture mixture in a centrifuge 6 into a light phase and a heavy phase 118; - a step 120 of conducting the heavy phase separated in the separating step 118 to a receiving tank 18; In this way, the cell concentration of the cell culture broth can be reduced by directing the heavy phase separated in the separation 118 step to the receiving tank 18 instead of returning it to the fermenter 4.

[0191] According to an embodiment, the method 100 includes, during a third period: - Step 122: Lead the light phase separated in step 118 to a receiving vessel In this manner, the light phase that is separated during the third period can be collected for downstream processing.

[0192] The receiving vessel may be receiving vessel 8 as discussed above and shown in FIG. 2, or the receiving vessel may be a different receiving vessel for the light phase resolved during the separating 118 step.

[0193] According to an embodiment, during the third time period, the rotor of the centrifuge can rotate at a higher rotation speed than during the first time period, in this way, the cell concentration and the content of smaller particles of the cell culture mixture can be efficiently reduced, and more light phases can be separated in the separating step 118 during the third time period than in the separating step 107 during the first time period.

[0194] According to an embodiment, the method 100 includes, during a fourth time period: - stopping step 106 of continuously returning the flow of liquid from inside the centrifuge back to the fermenter, if not done previously, at step 115; - 124 step of directing the entire cell culture mixture from the fermenter 4 to a centrifuge 6; - separating the cell culture mixture into a light phase and a heavy phase in a centrifuge 6, the light phase forming 50-95% V / V of the cell culture mixture; - 130 steps to guide the light phase separated in the centrifuge into a receiving vessel; In this manner, the fermentor may be emptied at the end of the culture period and the light phase / product may be extracted or harvested from the cell culture mixture during a fourth period.

[0195] The receiving vessel may be receiving vessel 8, as discussed above and shown in FIG. 2, or the receiving vessel may be a different receiving vessel for the light phase separated during the separating 126 step.

[0196] According to an embodiment, the method 100 includes, during a fourth time period: - 128 steps of directing the heavy phase separated in the centrifuge 6 to a receiving vessel In this way, the heavy phase separated during the separating 126 step may be diverted to a receiving tank instead of being returned to the fermenter 4. In this way, the fermenter 4 may be emptied during the fourth period.

[0197] The receiving vessel may be receiving vessel 18, as discussed above and shown in FIG. 2, or the receiving vessel may be a different receiving vessel for the heavy phase separated during the separating 126 step.

[0198] According to an embodiment, following the third and / or fourth time period, the method 100 further comprises: - diluting 132 the heavy phase in the receiving vessel 18 with liquid to produce a diluted heavy phase; - during a fifth period 134, transferring the diluted heavy phase from the receiving vessel 18 to a centrifugal device, such as a centrifuge 6; - during a fifth period, separating the diluted heavy phase in a centrifuge into a light phase and a further heavy phase; - 138 steps to direct the light phase to the receiving vessel; In this manner, the heavy phase separated during one or more of the first, third, and fourth time periods may be diluted with a liquid, such as, for example, a growth medium. By separating the diluted heavy phase, an additional light phase containing the desired material may be produced to increase the yield from the bioprocess.

[0199] The receiving vessel may be receiving vessel 8 as discussed above and shown in FIG. 2, or the receiving vessel may be a different receiving vessel for the light phase separated during the separating 136 step.

[0200] According to an embodiment, during the fifth period, the rotor of the centrifuge 6 may be rotated at a higher rotational speed than during the first period. In this way, the heavy phase separated during the fifth period may have a higher concentration of particles than the heavy phase separated during the first period. Thus, a large part of the available light phase may be withdrawn from the diluted heavy phase during the fifth period.

[0201] According to an embodiment, following the third, fourth and / or fifth time periods, the method 100 continues, during a sixth time period: - a step of flushing the centrifuge 6 with a liquid, such as a growth medium, 140; - 142 step of operating the centrifuge 6; - conducting 144 at least a portion of the liquid through the light phase outlet of the centrifuge 6 into a receiving vessel 8; In this manner, the remaining product containing the desired material is flushed from the centrifuge and is thus available for utilization.

[0202] As the centrifuge 6 operates, remaining particles, such as cells, remain within the centrifuge 6 and the portion of the liquid that is directed through the light phase outlet is free of particles, or at least substantially free of particles.

[0203] According to an embodiment, during the step of operating 142 the centrifuge 6, the rotor of the centrifuge 6 may be rotated at a higher rotational speed than during the first period of time. In this way, it can be ensured that the liquid conducted through the light phase outlet during the sixth period of time contains a minimum concentration of particles.

[0204] According to an embodiment, following any one of the first through sixth time periods, the method 100 includes: - 146 a step of directing the light phase collected in the receiving vessel 8 to a centrifuge 6 or a centrifugal device 6'; - separating the light phase into a light fraction and a heavy fraction in a centrifuge 6 or centrifuge device 6' 148, the heavy fraction containing residual debris from the cell culture mixture; - 150 steps to transfer the light fraction for further processing, such as extraction of extracellular material from the light fraction; In this way, the light phase collected in the receiving vessel 8 may be freed from at least the majority of the debris it may contain, thus making it possible to prepare the light phase for downstream processing by presenting it in the form of a separated light fraction.

[0205] Reference is further made to the discussion above relating to corresponding features of the control system 16.

[0206] According to an embodiment, during step 148 of separating the light phase into a light fraction and a heavy fraction in the centrifugal device, the rotor of the centrifuge 6 may rotate at a higher rotational speed than during one or more of the first to sixth periods. In this way, it can be ensured that the light fraction may contain a minimum concentration of particles, i.e. debris with a size substantially smaller than the diameter of a whole cell, is separated from the light fraction during the sixth period.

[0207] According to an embodiment, the centrifuge with the surface-enlarging insert may be a modular centrifuge with a base unit 40 and replaceable separation inserts 42', 42'' mounted within the base unit 40. The method 100 includes: - before the step 104 of directing the flow of the cell culture mixture from the fermenter 4 into the interior of a centrifuge 6 equipped with an enlarged surface insert, a step 152 of replacing the used replaceable separation insert 42' with an unused replaceable separation insert 42''; may include:

[0208] Thus, the centrifuge 6 is ready to perform its separation mission in the method 100 for operating a bioprocessing system 2 .

[0209] As noted above, the first and second periods may be interleaved.

[0210] Additionally or alternatively, the first and third periods may be interleaved.

[0211] A further alternative is to alternate the first, second, and third periods.

[0212] Various embodiments of method 100 are envisaged for the first through sixth periods and their associated steps depending on the requirements of the bioprocess, the capacity of the associated centrifuge 6, the yield of the bioprocess, the cost of operating the bioprocess, etc.

[0213] One or more of the following may be true: a second time period follows the first time period; a first time period follows the second time period; a third time period follows the second or first time period; a first time period follows the third time period; a fourth time period follows one of the first, second, or third time periods; a fifth time period follows the third or fourth time period; a sixth time period follows one of the third, fourth, or fifth time periods.

[0214] Also, the second period and associated method steps may be omitted. Thus, the third or fourth period may directly follow the first period. Alternatively, the third period and associated method steps may be omitted. Thus, the fourth period may directly follow the first or second period. Furthermore, each of the fifth and sixth periods and associated steps is optional.

[0215] According to a further aspect, a method of operating a bioprocessing system, the bioprocessing system comprising a fermentor for producing a liquid cell culture mixture therein using a growth medium, and a centrifuge comprising an enlarged surface insert, the method comprising: - continuously directing a flow of the cell culture mixture from the fermentor into an interior of a centrifuge during a first period of time; - continuously separating the flow of the cell culture mixture in the centrifuge into a light phase and a heavy phase during a first period of time, the light phase forming up to 5% V / V of the flow of the cell culture mixture; - returning the heavy phase to the fermenter during a first period of time simultaneously with the separating step. A method is provided, comprising:

[0216] According to a further aspect, there is provided a use of a centrifuge comprising a surface-enlarging insert for separating cells containing a heavy phase from a cell culture mixture during a fermentation process operated in perfusion mode.

[0217] More specifically, this use may relate to the use of a disc stack centrifuge for separating cells comprising a heavy phase and cell debris comprising a light phase from a cell culture mixture during a fermentation process operated in perfusion mode.

[0218] The centrifuge can thus be used before and at the end of the fermentation step of a fermentation process, which may be for producing biomolecules.

[0219] According to a further aspect, there is provided a computer program comprising instructions which, when the program is executed by a computer, cause the computer to perform the method 100 according to any one of the aspects and / or embodiments discussed herein.

[0220] Those skilled in the art will understand that the method 100 for operating a bioprocessing system may be realized by programmed instructions. These programmed instructions are typically constituted by a computer program, which when executed in the computer or computing unit 52 ensures that the computer or computing unit 52 performs the desired control, such as one or more of the method steps 102-152 according to the present invention. The computer program is typically part of a computer readable storage medium, including suitable digital storage media, on which the computer program is stored.

[0221] FIG. 6 illustrates an embodiment of a computer-readable storage medium 99 comprising instructions that, when executed by a computer or computing unit 52, cause the computer or computing unit 52 to perform one or more of the steps of a method 100 according to any one of the aspects and / or embodiments discussed herein.

[0222] The computer readable storage medium 99 may be provided in the form of a data carrier, which, for example, carries computer program code for executing at least some of the steps 102 to 152 according to some embodiments when loaded into one or more computing units 52. The data carrier may be, for example, a ROM (Read Only Memory), a PROM (Programmable Read Only Memory), an EPROM (Erasable PROM), a flash memory, an EEPROM (Electrically Erasable PROM), a hard disk, a CD ROM disk, a memory stick, an optical storage device, a magnetic storage device, or any other suitable medium such as a disk or tape capable of holding machine-readable data in a non-transitory manner. The computer readable storage medium may further be provided as computer program code on a server and downloaded to the computing unit 52 remotely, for example, via an Internet or intranet connection or via other wired or wireless communication systems.

[0223] The computer readable storage medium 99 shown in FIG. 6 is a non-limiting example in the form of a USB memory stick.

[0224] It should be understood that the foregoing is a representation of various exemplary embodiments, and that the present invention is defined solely by the appended claims. Those skilled in the art will recognize that the exemplary embodiments may be modified, and that different features of the exemplary embodiments may be combined to create embodiments other than those described herein, without departing from the scope of the present invention as defined by the appended claims. [Explanation of symbols]

[0225] 2. Bioprocessing System 4 Fermentation tank 6. Centrifuge 6' Centrifuge 8 Receiving Container 10. Pipe System 12' Feed Pump 12'' Heavy Phase Pump 12'' Light Phase Pump 14' First valve device 14'' Second Valve Unit 14'' Third valve gear 16 Control System 18 Receiving tank 21 First Pipe Section 22 Second Pipe Section 23 Third Pipe Section 24 Fourth Pipe Section 25 Fifth Pipeline Section 26 6th Pipeline Section 27 Seventh Pipeline 30 Separate containers 32 Air inlet 33 Growth medium conduit 34 Stirring member 35 Cooling and / or heating elements 40 Base Unit 42 Interchangeable Separation Inserts 42' replaceable separation insert 42'' Replaceable Separation Insert 43 Rotor casing 44 Tube 45 Stationary part 45' Stationary part 46 Still Frames 47 Rotating member 48 Wheels 49 Rotor 51 Rotation axis 52 Control Unit 53 Cone-shaped separating disk 54 Memory Unit 56 Sensors 60 Waste liquid container 99 Computer-readable storage medium

Claims

1. A method (100) of operating a bioprocessing system (2) adapted for perfusion mode operation, comprising: - Producing (102) a cell culture mixture in a fermenter (4), - simultaneously with said step of producing (102) said cell culture mixture, directing (104) a flow of the cell culture mixture from said fermenter (4) into the interior of a centrifuge (6), said separator comprising a rotor defining a separation space in which a surface-enhancing insert is arranged; - simultaneously with said step of producing (102) said cell culture mixture and said step of directing (104) said flow of cell culture mixture, continuously returning (106) a flow of liquid from said interior of said centrifuge (6) to said fermenter (4); Including, The method includes, during a first period of time: - separating (107) said flow of cell culture mixture into a light phase and a heavy phase in said centrifuge (6), said step of continuously returning (106) a flow of liquid from said interior of said centrifuge (6) to said fermenter (4) comprising, during said first period of time: - returning (108) the heavy phase separated in the centrifuge (6) to the fermenter (4) as the flow of liquid to the fermenter (4). Including, The method (100), wherein during the first period, the heavy phase comprises growth medium and whole cells, and the light phase comprises the growth medium and debris from the cell culture mixture that is smaller in particle size than whole cells, and does not include whole cells.

2. 2. The method (100) of claim 1, wherein in the step of separating (107) the flow of cell culture mixture into a light phase and a heavy phase, the separated light phase flow forms up to 5% V / V of the flow of cell culture mixture.

3. During the first period of time, - directing (112) the light phase separated in the centrifuge (6) into a receiving vessel (8); The method of claim 1 , comprising:

4. Prior to the first period of time, the method further comprises: - priming (114) said centrifuge (6) with a liquid other than the cell culture mixture The method (100) of any one of claims 1 to 3, comprising:

5. The step of continuously returning (106) a flow of liquid from the interior of the centrifuge (6) to the fermenter (4) comprises, during a second period of time: - returning (110) said flow of unseparated cell culture mixture from said interior of said centrifuge (6) to said fermenter (4) as said flow of liquid to said fermenter (4). A method (100) for operating a bioprocessing system (2) according to any one of claims 1 to 4, comprising:

6. 6. The method (100) according to claim 5, wherein during the step of returning (110) the flow of cell culture mixture during the second time period, the rotor of the centrifuge (6) is stationary or rotates at a lower rotational speed than during the step of separating (107) the flow of cell culture mixture during the first time period or rotates in a rotational direction opposite to the rotational direction during the step of separating (107) the flow of cell culture mixture during the first time period.

7. The method (100) of claim 5 or 6, wherein the first time period and the second time period are alternately repeated.

8. 8. The method (100) according to any one of claims 5 to 7, wherein during the first and second time periods the flow of the cell culture mixture is directed into the interior of the centrifuge (6) with the same flow rate q.

9. During the third period, - stopping (115) said step of continuously returning (106) the flow of liquid from said interior of said centrifuge (6) to said fermenter (4); - directing (116) the flow of said cell culture mixture from said fermenter (4) into said centrifuge (6); - separating (118) said flow of cell culture mixture into a light phase and a heavy phase in said centrifuge (6); - conducting (120) the heavy phase separated in the step of separating (118) into a receiving tank (18); The method (100) of any one of claims 5 to 8, comprising:

10. During the third period, - conducting (122) the light phase separated in the separating (118) step into a receiving vessel (8); 10. The method of claim 9, comprising:

11. 11. The method (100) according to claim 9 or 10, wherein during the third period the rotor of the centrifuge (6) rotates at a higher rotational speed than during the first period.

12. During the fourth period, - stopping (115) said step of continuously returning (106) the flow of liquid from said interior of said centrifuge (6) to said fermenter (4); - directing (124) all of said cell culture mixture from said fermenter (4) into said centrifuge (6); - separating (126) the cell culture mixture into a light phase and a heavy phase in the centrifuge (6), the light phase forming 50-95% V / V of the cell culture mixture; - conducting (130) the light phase separated in the centrifuge (6) into a receiving vessel (8); The method (100) of any one of claims 9 to 11, comprising:

13. During the fourth period, - conducting (128) the heavy phase separated in the centrifuge (6) into a receiving tank (18); 13. The method (100) of claim 12, comprising:

14. Following the third and / or fourth time period, the method (100) further comprises: - diluting (132) said heavy phase in said receiving tank (18) with a liquid to produce a diluted heavy phase; - during a fifth period of time, transferring (134) said diluted heavy phase from said receiving tank (18) to a centrifuge; - separating (136) said diluted heavy phase in said centrifuge during said fifth period into a light phase and a further heavy phase; - conducting (138) said light phase into said receiving vessel (8); 14. The method (100) of claim 12 or 13, comprising:

15. 15. The method (100) of claim 14, wherein during the fifth period of time, the rotor of the centrifuge (6) rotates at a higher rotational speed than during the first period of time.

16. Following the third, fourth and / or fifth time periods, the method (100) comprises, during a sixth time period: - flushing (140) said centrifuge (6) with a growth medium; - operating (142) said centrifuge (6); - directing (144) at least a portion of said liquid through a light phase outlet of said centrifuge (6) into said receiving vessel (8); 16. The method (100) of claim 14 or 15, comprising:

17. 17. The method (100) of claim 16, wherein during the step of operating (142) the centrifuge (6), the rotor of the centrifuge (6) rotates at a higher rotational speed than during the first period of time.

18. Following any one of the first through sixth time periods, the method (100) further comprises: - conducting (146) the light phase collected in the receiving vessel (8) into the centrifuge (6) or centrifugal device (6'); - separating (148) the light phase in the centrifuge (6) or in the centrifuge device (6') into a light fraction and a heavy fraction, the heavy fraction comprising residual debris from the cell culture mixture; - transferring (150) said light fraction for the extraction of extracellular material from said light fraction; 18. The method (100) of claim 16 or 17, comprising:

19. 20. The method (100) according to claim 18, wherein during the step of separating the light phase into a light fraction and a heavy fraction in the centrifuge (148), the rotor of the centrifuge (6) rotates at a higher rotational speed than during one or more of the first to sixth time periods.

20. The centrifuge (6) with a surface-enlarging insert is a modular centrifuge (6) with a base unit and a replaceable separation insert mounted in the base unit, and the method (100) comprises: - replacing a used replaceable separation insert with an unused replaceable separation insert prior to said step of directing (104) the flow of the cell culture mixture from said fermenter (4). The method (100) of any one of claims 1 to 19, comprising:

21. A bioprocessing system (2) configured to operate in a perfusion mode of operation, comprising a fermenter (4) for producing a cell culture mixture therein, a centrifuge (6) with a rotor defining a separation space in which a surface-enlarging insert is disposed, a receiving vessel (8), a conduit system interconnecting said fermenter (4), said centrifuge (6) and said receiving vessel (8), flow control devices disposed in said conduit system, and a control system configured to control at least a portion of said centrifuge (6) and said flow control devices, said control system being configured to: - directing a flow of cell culture mixture from said fermenter (4) into said centrifuge (6), while - Continuously returning the flow of liquid from the interior of the centrifuge (6) to the fermenter (4). It is structured as follows: A bioprocessing system (2), wherein the control system is further configured to perform one or more of the steps of the method according to any one of claims 1 to 20.

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