Method for operating a bioprocess plant for producing bioproducts

Abstract

The invention relates to a method for operating a biological process plant (1) for producing biological products, in which the biological process plant (1) comprises a source container (2) for cell culture, a harvest container (3) for biological production and a clarification arrangement (4) with a centrifuge (12), in which the source container (2) is operated in a circulating production mode, the circulating production mode comprises the following steps: a) initiating the circulating production mode in the source container (2), b) culturing the cells in the source container (2), thereby obtaining a cell culture fluid (7) comprising the cultured cells, c) discharging a discharge portion (16) of the cell culture fluid (7) from the source container (2), d) combining a restart portion (17) of the cell culture fluid (7) with a fresh culture medium (8) and repeating step b), e) repeating steps c) and d) at least once, and / or f) discharging the cell culture fluid (7) obtained from step d) from the source container (2) in which the cyclic production mode is stopped, obtaining a discharged portion (16), wherein the method further comprises the steps of i) centrifuging the discharge portion (16) via the centrifuge (12), thereby separating the discharge portion (16) into at least a centrifuged discharge portion (19) and a supernatant (14) and, preferably, a biological product, ii) operating the harvest container (3) in a production mode wherein steps i) and ii) are performed at least twice.

Description

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

[0002] The term "bioprocess" currently represents any kind of biotechnological process, in particular biopharmaceutical process. Therefore, the proposed method can be applied to various fields of biotechnology and for different kinds of bioprocesses. An example of a bioprocess is the use of a bioreactor to cultivate microorganisms (such as bacteria or mammalian cells) under given conditions to produce a bioproduct. Typically, the cell culture fluid is transferred from the bioreactor to a downstream process to separate the bioproduct from the cells and purify the bioproduct.

[0003] The term "biological product" generally refers to a product produced by cultured cells. The cultivation of cells can currently be used to produce biopharmaceuticals, particularly proteins, such as human insulin, growth factors, hormones or vaccine proteins, particularly antibodies, antibody derivatives, exosomes, etc. Biological products can also come from non-biopharmaceutical groups, such as enzymes for food processing, laundry detergent enzymes, biodegradable plastics or biofuels. In addition or as an alternative, cells can also be cultured to produce viral vectors, including purified lentiviral vectors, etc., and used for applications in the emerging field of cell and gene therapy.

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

[0005] The term "cell" preferably represents a mammalian cell, including HEK293 cells, CHO cells, etc. Preferably, the cell has been genetically modified (preferably by transfection) to produce a desired bioproduct. Preferably, the bioproduct is secreted from the cell into the culture medium. Alternatively, the bioproduct remains in the cell.

[0006] The discussed method of operating a bioprocess facility for producing a bioproduct can be applied to various fields of biotechnology and to different kinds of bioprocesses. Exemplarily, the method can be applied to the production of monoclonal antibodies using a mammalian CHO suspension cell line.

[0007] When operating a bioprocess facility for the production of biological products, process efficiency is particularly important. In particular, process efficiency is related to cost efficiency and the floor space of the process. The increased demand for biopharmaceuticals (including monoclonal antibodies, vaccines, etc.) has driven high cost efficiency and increased flexibility. Another reason for cost-effective process design is that the material cost associated with the bioprocess of the type is generally high. The reason for this is that sterility during the process must be maintained. Therefore, bioprocesses generally involve the use of disposable materials and / or cost-intensive sterilization procedures, such as heat sterilization, etc.

[0008] Cost efficiency is not only related to material cost, but also to effective process design. Usually, in order to make the container (especially bioreactor) reach its state of producing biological products, it is necessary to carry out seed culture involving multiple culture steps in the increased culture volume. This seed culture requires a lot of time, because in each step of the seed culture, the cells need to grow to a certain cell density to inoculate the next step, and the cell growth rate may be quite low. In addition, and depending on the final production scale, a large number of seed culture bioreactors may be required, and each seed culture bioreactor needs to be operated continuously. Therefore, it is necessary to improve process efficiency.

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

[0010] Document WO 99 / 33955A1 relates to the cultivation of preferably anchorage-dependent cells and discloses a method of operating a bioprocess facility for producing a bioproduct, wherein the bioprocess facility comprises a source container for cell cultivation and a harvest container for bioproduction. It has been recognized that a portion of the cells from the source container may be reused as a restart portion, while another portion of the cells is used for bioproduction. However, there is still a need to improve production efficiency.

[0011] Document WO 2020 / 010080A1 relates to a method for culturing mammalian cells in a source container and strongly increasing the cell concentration by conditioning the culture medium before transferring it to a harvest container. This is used to achieve cell densities as in perfusion culture without using perfusion culture.

[0012] Improving known existing technologies to achieve higher process efficiencies is a challenge.

[0013] The invention is based on the problem of improving known methods such that further optimization with respect to the given challenges is achieved.

[0014] This object is achieved by the features of claim 1 .

[0015] The main realization of the present invention is that operating the source container in a cyclic production mode to produce cells and operating the harvest container in a non-cyclic production mode to produce biological products enables efficient supply of cells to the harvest container and allows separate optimization of the two process steps, including process parameters such as initial viable cell concentration and selected culture medium.

[0016] The source vessel is used to culture cells, and a portion of those cells is transferred to a harvest vessel, where the cells primarily produce the bioproduct. Between these steps, the cells are centrifuged, allowing the bioproduct produced in the source vessel to be harvested as an adjunct, further allowing the viable cell concentration to be increased, allowing for the removal of potentially toxic byproducts, and allowing for the process conditions to be varied between vessels. In particular, there may be different optimal process conditions for the production of cells and bioproducts.

[0017] By using a separate container, cell production in the source container is particularly simple, while the non-cyclic operation of the harvest container enables the process conditions in the harvest container to be adjusted to favor bioproduction without having to keep the cells alive. In addition, by using the first container for repeated cell production, time, labor and cost-intensive seed cultivation can be greatly reduced.

[0018] By including a clarification arrangement with a centrifuge, process conditions between the source container and the harvest container can be adjusted in a particularly simple manner, since the cells can be easily washed before transfer to the harvest container. In this way, dead cells and cell debris can also be at least partially removed from the cell culture fluid, which leads to an increase in the viability of the culture. In addition, washing can remove or at least reduce potential cell growth inhibitory compounds, such as by-products, such as lactate and / or ammonium, from the culture medium. This can enhance subsequent cultivation in the harvest container and / or simplify further purification of the biological product.

[0019] Furthermore, the use of a centrifuge enables easy separation of the biological product and the cells, so that the biological product can also be recovered during transfer from the source container to the harvest container.

[0020] By operating the source container in a cyclic production mode and the harvest container in a non-cyclic production mode, process costs and floor space can be reduced because no large media storage is required and no means for cell retention need to be provided by either container, especially compared to perfusion-based processes. Utilizing two separate containers further provides the option of adjusting the culture time in each container relative to each other, resulting in increased equipment utilization. This further enhances process efficiency. By using the restart portion of the cells to start the next culture in the source container, downtime of the source container is greatly reduced because the source container may be used directly for the next production cycle without having to be cleaned.

[0021] A method for operating a bioprocess facility for producing a bioproduct is proposed, wherein the bioprocess facility comprises a source vessel for cell culture, a harvest vessel for bioproduction and a clarification arrangement with a centrifuge, wherein the source vessel is operated in a cyclic production mode, wherein the cyclic production mode comprises the following steps in this order:

[0022] a) starting said cyclic production mode in said source container with initial cells and culture medium,

[0023] b) culturing the cells in the source container to obtain a cell culture fluid containing the cultured cells, whereby the source container and the culture medium are configured to provide source culture environmental conditions for culturing the cells,

[0024] c) discharging a discharged portion of the cell culture fluid from the source container,

[0025] d) combining the restarted portion of the cell culture fluid with fresh culture medium and repeating step b),

[0026] e) repeating steps c) and d) at least once, and / or

[0027] f) discharging the cell culture fluid obtained in step d) from the source container that stops the circulation production mode to obtain a discharge portion,

[0028] The method further comprises the following steps:

[0029] i) transferring the effluent portion to the clarification arrangement and centrifuging the effluent portion via the centrifuge, thereby separating the effluent portion into at least a centrifuged effluent portion, a supernatant (and, preferably, a biological product),

[0030] ii) transferring at least a portion of the centrifuged discharge portion from step i) and fresh culture medium into the harvest container and operating the harvest container in a production mode, whereby the harvest container and the culture medium are configured to provide harvest culture environmental conditions to produce the biological product in the harvest container,

[0031] wherein steps i) and ii) are performed at least twice with the discharge portion from the performance of step c) and / or step f).

[0032] The term "cell culture medium" refers to a suspension of solid cells and possible cell debris in a culture medium and describes both the entire culture medium and the respective cells cultured therein.

[0033] According to claim 2, the cultivation in the source container and the cultivation in the harvest container are carried out in parallel for a large amount of time. Thus, the process efficiency is significantly enhanced because the equipment utilization increases as the container is utilized to a large extent.

[0034] Claim 3 relates to the synchronization between the cultivation time in the source container and the production time in the harvest container. Especially when the number of available container slots is limited, the synchronization between the cultivation time and the production time is important to keep the process as efficient as possible. By synchronizing the cultivation time, a large amount of downtime of any one container can be reduced, which improves equipment utilization and thus improves process efficiency.

[0035] By repeating the cyclic production mode without disrupting the synchronization between the source container tank and the harvest container tank (claim 4), the duration of the seed culture is significantly shortened, because for each successive transfer of cells to the harvest container, all steps of the seed culture do not need to be repeated. In addition, each subsequent cyclic production in the source container can be carried out at the same source container tank, which reduces the overall footprint of the process because no additional container tank is required. In addition, this also improves equipment utilization, because not only the container itself, but also the infrastructure at the corresponding container tank is utilized as much as possible.

[0036] According to claim 5, after the production of the biological product, the harvested portion from the harvest container is subjected to a clarification arrangement. The use of a centrifuge in the clarification arrangement is particularly advantageous here, since it allows the discharge from the source container and the harvest container to be carried out similarly in terms of process and in terms of equipment, resulting in easier work steps and also in a higher equipment usage time by using the same centrifuge.

[0037] The remaining culture time and / or the remaining production time can be determined by measuring at least one parameter of the source container and / or the harvest container using a sensor arrangement (claim 6). This allows to adjust some process step times, in particular the duration of step b), and thereby to maintain synchronization between the culture time in the source container and the culture time in the harvest container even when unexpected changes occur.

[0038] Providing source electronic process control to control source culture environmental conditions, as proposed in claim 7, is advantageous because the source container can be controlled to change its culture conditions, cell growth rate, etc., if necessary. For example, if the harvest container is used for production for longer than planned, the source container can be adjusted to reduce the cell growth rate. Thus, the target number of cells is reached later, synchronizing the source container and the harvest container, rather than having to discard part of the cell culture fluid from the source container when its target cell amount is reached prematurely, or running the risk that the cells lose their viability and may no longer be used. In addition, if bioproduction is already occurring in the source container, the amount of biological product in the source container may increase because adjustments to the source container may result in the initiation and / or increase of bioproduction. However, it remains a priority to produce the target amount of cells in the source container and maintain a certain viability of the cells.

[0039] By predicting the time when the termination condition is reached in the harvest container and adjusting the culture conditions in the source container based on this prediction (claim 8), process flexibility is greatly increased because production can continue in the harvest container and cell growth in the source container can be adjusted accordingly. In this way, the production time in the harvest container can be changed without having to repeat the complete seed culture. In particular, the source container may not be optimized for maximum cell growth, but still has some room to increase (and preferably also reduce) cell growth, thereby providing a control reserve and producing more biological products in the source container. Providing a control reserve is a particularly interesting possibility provided by the proposed method. Previously, the harvest container was usually timed to the seed culture, even when the cells may still have high vitality and good production rate, which did not allow for greater deviations in the harvest container operation time. By being able to use cells for a longer time without desynchronizing the seed culture, the cost and waste of each product is reduced.

[0040] Possible options for synchronizing the source container and the harvest container are detailed in claim 9. In order to achieve a predetermined amount of cells in the source container for the subsequent production of the biological product in the harvest container, a synchronization strategy can be implemented.

[0041] By increasing the cell concentration between step b) and step ii), the subsequent production of the biological product in step ii) can be accelerated (claim 10). Increasing the viable cell concentration by centrifugation is particularly advantageous, since not only can the cells be separated from the already produced biological product, but the cells can also be washed and / or transferred to another culture medium.

[0042] By changing at least one process parameter of the source culture environment conditions compared to the harvest culture environment conditions, cell culture and bioproduction can be effectively separated between containers (claim 11). In the harvest container, cell viability does not need to be kept above a certain threshold, because the cells can be discharged at the end of production. The use of a centrifuge further makes it much easier to change the culture environment between cell culture and bioproduct production.

[0043] In a preferred embodiment according to claim 12, for multiple repetitions of steps b) and ii), the source culture environment and / or the harvest culture environment may be at least initially identical, so that the entire method is a repetition of the same parallel culture and production cycle.

[0044] By using batch and / or fed-batch culture conditions (claim 13), the overall setup is simplified, especially compared to perfusion-based systems, because no means are required for keeping the cells in any container during the culture period. This improves the cost efficiency of the entire process and simplifies the operation of the bioprocess facility due to reduced process complexity. In addition, no large storage space is required to continuously supply the required amount of culture medium. In contrast to perfusion culture, the risk of contamination, the floor space and complexity in the form of culture medium waste and storage are reduced.

[0045] In claim 14, the properties of the centrifuge as well as the cell washing step and the cell discharge step are defined. These specifications allow for an optimized preparation of the cells during forward or reverse operation of the centrifuge, as well as optimized conditions for subsequent bioproduction. The defined centrifuge is particularly suitable for the proposed method.

[0046] By transfecting the cells after step c), bioproduction can only be performed after the cells have been discharged from the source container. In this way, no bioproduction occurs in the source container, which is a particularly effective option for separating bioproduction from cell growth (claim 15).

[0047] The term "transfection" refers to the transfer of genetic material to genetically modify cells so that the cells produce the desired biological product. Cell transfection can be performed in different ways, including the following techniques, such as electroporation, treatment with chemicals (such as calcium phosphate), microinjection, etc. Preferably, and as will be explained later, transfection is performed by treatment with chemicals. Alternatively, transfection can also be performed using viruses or viral vectors.

[0048] Below, the embodiment of the present invention is explained with reference to the accompanying drawings.

[0049] Figure 1 , a bioprocess facility carrying out the proposed method of operating a bioprocess facility according to an embodiment of the present invention,

[0050] Figure 2 , steps a)-e) of the proposed method of operating a bioprocess facility,

[0051] Figure 3 , steps e)-iii) of the proposed method of operating a bioprocess facility,

[0052] Figure 4 , an example of simultaneous cultivation in a source container and a harvest container,

[0053] Figure 5 , a source container in a source container tank, a harvest container in a harvest container tank, and a clarification device in an exemplary embodiment of the present application, and

[0054] Figure 6 , proof-of-concept experiment.

[0055] The proposed method of operating a bioprocess facility 1 is Figure 1 Described in. Figure 1 Two cycles of a preferred embodiment of the method are described, whereby Figure 1 The process is schematically shown from left to right over time. A bioprocess facility 1 comprises a source vessel 2 for cell culture, a harvest vessel 3 for bioproduction and at least one clarification device 4 .

[0056] In a preferred embodiment, and as Figure 1 As described, the source container 2 is a bioreactor. It is further preferred that the material of the source container 2 is a disposable material. Alternatively, the source container 2 can be a stainless steel tank bioreactor. Here and preferably, the source container 2 comprises at least one source container inlet port 5 for adding liquid. Each source container inlet port 5 can be connected to a corresponding liquid pipeline for providing liquid.

[0057] The term "liquid" refers to any process liquid handled in the proposed bioprocess facility 1. It includes reagents added to control source culture environmental conditions and / or harvest culture environmental conditions, such as acids or bases to control pH, but also washing solutions such as buffers used. In general, the term liquid may also refer to culture medium and / or feed medium 6 and / or cell culture fluid 7.

[0058] The term "medium" refers to the fact that cells used for a biological process grow in or on a specially designed solid, semisolid or liquid medium, which supplies the nutrients required by the corresponding organism or cells. There are a variety of culture media, but usually contain at least a carbon source, a nitrogen source, water, salts and trace nutrients. Here, preferably a chemically defined culture medium 8 is used, which means that all components of the culture medium and their corresponding concentrations are known. However, it is also possible to use a chemically undefined culture medium, which may contain unknown nutrients and / or unknown amounts of nutrients. A typical example of a chemically undefined culture medium is a culture medium containing FBS (fetal bovine serum).

[0059] As described above, the bioprocess facility 1 further comprises a harvesting vessel 3. In a preferred embodiment and as Figure 1 As described, the harvest container 3 is a bioreactor. It is further preferred that the material of the harvest container 3 is a disposable material. Alternatively, the harvest container 3 can be a stainless steel tank bioreactor. Preferably, the harvest container 3 further comprises at least one harvest container inlet port 9 for adding liquid. Each harvest container inlet port 9 can be connected to a corresponding liquid pipeline for providing liquid.

[0060] In a preferred embodiment and as Figure 1 As described, different harvesting containers 3 are used throughout the method, which will be explained further below.

[0061] like Figure 1 As described, the source container 2 and / or the harvest container 3 preferably comprises at least one source container outlet port 10 and / or at least one harvest container outlet port 11. Preferably, the corresponding outlet ports are used to extract liquid from the source container 2 and / or the harvest container 3. It is further preferred that the cell culture fluid 7 is extracted from the source container 2 and / or the harvest container 3.

[0062] In a preferred embodiment, the source container 2 and / or the harvest container 3 are designed to receive a liquid volume greater than 5 liters, preferably greater than 15 liters, and more preferably greater than 50 liters. Preferably, the source container 2 and the harvest container 3 are designed to receive the same maximum liquid volume. It is further preferred that the source container 2 and the harvest container 3 have the same geometry. Preferably, the layout and number of inlet ports and / or outlet ports are also the same in the source container 2 and the harvest container 3. This makes the operation of both containers simpler.

[0063] However, the source container 2 and the harvest container 3 may also differ in at least one or all of the above aspects. For example, the maximum liquid volume of the harvest container 3 may be lower than the maximum liquid volume of the source container 2.

[0064] As also described above, the bioprocess facility 1 further comprises a clarification arrangement 4. Here and preferably, the clarification arrangement 4 uses centrifugal force to perform a physical process to remove suspended solids, preferably cells, from the liquid phase. In general, the proposed clarification arrangement 4 can be used to separate any solid / liquid components from each other, including but not limited to cells and culture medium. Preferably, the clarification arrangement 4 comprises a centrifuge 12 for clarifying the cell culture liquid 7 by centrifugation. "Centrifugation" is a term for sedimentation of particles in an artificial gravity field by the generated centrifugal force, wherein a significant reduction in separation time is achieved via a large acceleration force.

[0065] In order to carry out the proposed method, it is preferred and also Figure 1 Indicated in FIG. 1 , the source container 2 and the centrifuge 12 can be fluidically coupled. Further preferably, the centrifuge 12 and the harvest container 3 can also be fluidically coupled.

[0066] The term "fluid connection" means establishing a fluid connection that can be used to transfer a fluid (preferably a liquid, more preferably at least a portion of a cell culture fluid 7) from a source container 2 to a centrifuge 12 and / or from a harvest container 3 to a centrifuge 12, and / or to transfer a fluid (preferably a liquid, more preferably centrifuged 12 cell culture fluid 7) from a centrifuge 12 to a harvest container 3 and / or to a waste 13 location, and / or to transfer a supernatant 14 containing a biological product from a centrifuge 12 to a further purification step 29.

[0067] The centrifuge 12 may be operated in forward operation and / or in reverse operation and includes a liquid network of interconnected liquid lines to establish various fluid connections to and from the centrifuge 12 .

[0068] A method for operating a bioprocess facility 1 for producing a bioproduct is presented in detail, wherein the bioprocess facility 1 comprises a source container 2 for cell culture, a harvest container 3 for bioproduction and a clarification arrangement 4 with a centrifuge 12 .

[0069] The source container 2 is operated in a cyclic production mode. The cyclic production mode comprises the following steps in this order:

[0070] a) starting a cyclic production mode in the source container 2 with the initial cells 15 and the culture medium 8,

[0071] b) culturing cells in the source container 2 to obtain a cell culture solution 7 containing the cultured cells, whereby the source container 2 and the culture medium 8 are configured to provide a source culture environment condition for the culture of the cells,

[0072] c) a discharge portion 16 for discharging the cell culture fluid 7 from the source container 2,

[0073] d) combining the restarting portion 17 of the cell culture fluid 7 with the fresh culture medium 8 and repeating step b),

[0074] e) repeating steps c) and d) at least once, and / or

[0075] f) discharging the cell culture fluid 7 obtained in step d) from the source container 2 in which the circulation production mode is stopped, and obtaining a discharge portion 16.

[0076] Figure 1 Shows most of the entire method, and Figure 2 The steps a) to e) are shown as being named separately. Steps a) to f) involve the source container 2, and the harvest container 3 will be explained further below. The source container 2 is operated in a cyclic production mode to culture cells. In step a), the initial cells 15 are added to the Figure 2 In source vessel 2, depicted on the upper left, cell culture is initiated. Figure 2It is shown how to combine the initial cells 15 and the culture medium 8 in the source container 2 in a conventional manner to culture the initial cells 15 and increase the absolute number of cells in the source container 2. The initial cells 15 can be the result of seed culture, and the culture in the source container 2 can be the last step of seed culture. Preferably, the initial concentration of viable cells in the source container 2 is adjusted to a predetermined value. Preferably, this adjustment is performed by adding the culture medium 8 until a predetermined cell concentration is reached.

[0077] Step b) comprises culture. The source container 2 and the culture medium 8 provide the source culture environment conditions (particularly the predetermined source culture environment conditions) together. The culture environment conditions include all relevant parameters of cell culture and biological product production, such as temperature, stirring speed, pH value, nutrient concentration, etc. The culture environment conditions partially change over time. Starting from the initial culture environment conditions, cells consume nutrients and secrete byproducts. According to the culture environment conditions and cells, during the culture in the source container 2, biological products can also be produced to a certain extent. This will be further explained below. Here and preferably, the initial source culture environment conditions and / or the initial harvest culture environment conditions to be explained are predetermined, meaning that there is a plan for the initial source and / or harvest culture environment conditions. Preferably, for the source and / or harvest container 3, the later culture environment conditions and therefore all culture environment conditions are predetermined. However, this does not necessarily mean that each parameter is perfectly controlled. Preferably, the source culture environment conditions are adjusted to provide conditions that lead to optimized cell growth and / or the high overall vitality of the cell.

[0078] In order to maintain optimal cell growth conditions and keep the viability of the cells above a predetermined value, it may be necessary to feed the feed medium 6 during the cultivation of the cells in step b), because certain medium components (e.g., carbon sources, such as glucose) may be depleted from the medium 8 during the cultivation because they are completely consumed by the cells. However, depending on the cultivation behavior and the cultivation time, the feed medium 8 is only optional and may not be required in any case.

[0079] It should be noted here that the feed medium 6 can be the same as the initial culture medium 8 or, in a preferred embodiment, the feed medium 6 differs from the culture medium 8 in at least one type and / or at least one concentration of components.

[0080] Preferably, at least one of the source container inlet ports 5 is a feed port with a feed line 18 for controlled feeding of the culture medium 8 according to a predetermined feeding scheme during the cultivation. Such a feeding scheme can be designed as a pulse or continuous or mixed feeding scheme, wherein preferably the feeding scheme is individually controllable and adjustable. It should be noted that the feeding of the culture medium 8 results in an increase in the volume of the culture liquid in the source container 2, which depends on the feeding scheme and the feed medium 6.

[0081] Here and preferably, after the cultivation of the cells in step b) reaches a predetermined termination condition (preferably a target absolute number of cells or a target cell concentration), step b) is terminated and step c is initiated. Here and preferably, step b) focuses on providing a rapid increase in the number of cells while keeping the cells alive, rather than on the production of a biological product. A biological product may or may not be produced in step b). If any, the biological product produced in step b) may have been secreted into the cell culture fluid 7, or may be located intracellularly. In the former case, the biological product may be harvested, as will be explained later, in the latter case, the biological product is preferably not harvested until after the cells have been used to produce a biological product in the harvesting container 3 (as will be explained).

[0082] In step c), the outflow portion 16 of the cell culture fluid 7 is removed from the source container 2. Figure 1 2 is shown moving downward from the source container 2. Preferably, and as will be described in further detail below, the cell culture fluid 7 is not completely drained from the source container 2, but rather a restart portion 17 is retained in the source container 2. In step d), this restart portion 17 is combined with fresh culture medium 8 to culture the cells again in a repetition of step b).

[0083] At least one repetition of this step b) allows the culture in the source container 2 to be cyclic. Instead of using all of the cell culture fluid 7 to produce the biological product, the start of the next seed culture is skipped and the restart section 17 is used to culture the cells again in the same source container 2. Here and preferably, the restart section 17 stays in the source container 2 and is not temporarily removed from the source container 2 between repetitions of step b). In this way, the source container 2 can be used for more than one cycle without changing or cleaning the source container 2. This is accomplished by Figure 1 The dashed line in FIG. 2 is used for source container 2. Figure 1 When the top of the Figure 2 A portion of step d) is depicted, showing a repetition of step b).

[0084] The repetition of step b), preferably each repetition of step b), may be identical to the first execution of step b) in some or all aspects, in particular with respect to the source environmental conditions or some source environmental conditions. All aspects described herein with respect to step b) may be identical for at least one, preferably all executions of step b), alone or in any combination.

[0085] When starting step c) or ending step b), the restarting part 17 and the discharge part 16 preferably amount to at least 95%, preferably at least 99%, more preferably 100% of the cell culture fluid 7 in the source container 2.

[0086] Preferably, at the beginning of step c) or at the end of step b), the discharge portion 16 is at least 50%, preferably at least 70%, more preferably at least 90% of the volume of the cell culture fluid 7. Preferably, the cell culture fluid 7 is discharged from the source container 2 using the source container outlet port 10. As will be explained in detail below, the cell culture fluid 7 is preferably transferred to a centrifuge 12.

[0087] In step e), steps c) and d) can be repeated at least once, preferably at least twice, and more preferably at least three times. For each repetition, the discharge portion 16 is transferred to the harvest container 3, as will be explained. Thus, the source container 2 cyclically produces the cell culture fluid 7 for the production of the biological product, without requiring seed culture each time.

[0088] exist Figure 3 Here and preferably, the discharge portion 16 (which is preferably at least 95%, preferably at least 99%, more preferably 100% of the cell culture fluid 7 in the source container 2) is transferred again to the harvest container 3, as will be explained. Figure 2 Steps a) to e) are shown. Figure 2 The values ​​in each of the three rows are read from left to right. VCC is the viable cell concentration in source container 2, which increases and decreases with the different method steps. The volume in source container 2 may change during a step (as shown) or may not change, depending on the details of the process.

[0089] Whether coming from step d) or step f), the discharge portion 16 is not transferred directly to the harvest container 3 but is sent to the clarification installation 4 .

[0090] Now go to Figure 1 The first row below and Figure 3 The method further comprises the following steps:

[0091] i) transferring the effluent fraction 16 resulting from step c) or f) to a clarification device 4 and centrifuging the effluent fraction 16 via a centrifuge 12, thereby separating the effluent fraction 16 into at least a centrifuged effluent fraction 19 and a supernatant 14 (and, preferably, a biological product),

[0092] ii) transferring at least a portion of the centrifuged discharge portion 19 from step i) and fresh culture medium 8 into a harvest container 3, and operating the harvest container 3 in a production mode (here and preferably, a non-circulating production mode), whereby the harvest container 3 and the culture medium 8 are configured to provide a harvest culture environmental condition for producing the biological product in the harvest container 3,

[0093] Thereby steps i) and ii) are performed at least twice with the discharge portion 16 resulting from the performance of step c) and / or step f).

[0094] Step i) involves centrifuging the discharge portion 16 via a centrifuge 12, preferably always the same centrifuge 12. This centrifugation can be used to achieve several positive effects.

[0095] The effluent portion 16 can be transferred to the centrifuge 12 via a multi-purpose or temporary liquid line. Multi-purpose means that the liquid line is used for at least two effluent portions 16. A pump arrangement can be used to transfer the effluent portions 16. The effluent portions 16 are then centrifuged 12. This centrifugation can increase the cell concentration so that a higher initial cell concentration can be used in step ii). The cells can be washed, cell debris and / or accumulated by-products can be removed, etc. Preferably, the biological product is separated from the effluent portion 16, especially if the biological product is secreted into the cell culture fluid 7. The biological product can be separated directly by the centrifuge during centrifugation, producing the centrifuged effluent portion 16, or can be contained in the supernatant 14 and separated in a later or earlier step. Centrifugation particularly allows the culture environment conditions between the source container 2 and the harvest container 3 to be changed when the supernatant 14 is removed, and can be replaced by a fresh culture medium 8 with different properties. Removing accumulated by-products further improves the efficiency of subsequent bioproduction.

[0096] At least a portion of the centrifuged effluent 19 (preferably at least 95%, more preferably at least 99%, more preferably 100% of the centrifuged effluent 19) is preferably combined with fresh culture medium 8 in step i) and then used in step ii) for the production of a biological product. Preferably, the cells are combined with fresh culture medium 8 during the centrifugation process and then the centrifuged effluent is transferred to the harvesting container 3. The volume of fresh culture medium 8 may be less than 90%, preferably less than 70%, more preferably less than 50% of the volume of the effluent 16. Here, the culture medium 8 of the centrifuged effluent 19 preferably differs from the culture medium used in step a) and / or the feed medium used in step b) in at least one type and / or amount of nutrients.

[0097] Preferably, the harvest culture environmental conditions in the harvest container 3 are significantly different from the source culture environmental conditions. The former can be optimized for biological product production and / or the latter for cell growth. Exemplarily, the feed medium 6 can be supplied via the harvest container inlet port 9. The feed medium 6 and / or the feeding scheme can be the same feed medium 6 and / or the feeding scheme that can be used in step b) in the source container 2. However, it is preferred that the feed medium 6 and / or the feeding scheme is different from the feed medium 6 and / or the feeding scheme used in step b) in the source container 2 in at least one parameter. An example of such a parameter can be the nutrient composition of the feed medium 6.

[0098] like Figure 1 As described, steps i) and ii) are also repeated, preferably however with a different (especially disposable) harvest container 3 or in a harvest container 3 which is cleaned between executions. The source container 2 and the harvest container 3 are not identical containers.

[0099] Here and preferably, after each execution of step ii), the biological product is extracted from the cell culture fluid 7 produced in the harvest container 3. Since the cells from the harvest container 3 are preferably no longer used, the harvest culture environmental conditions can cause the cells to die during the biological production, reducing the cell viability to a lower value compared to the culture in the source container 2.

[0100] The duration of step b) and / or step ii) may be at least one day, preferably at least two days.

[0101] Here and preferably, the source container 2 and the harvest container 3 are not directly connected in the process. Here and preferably, no cells are transferred from the harvest container 3 to the source container 2.

[0102] Again looking at the centrifuge 12, the centrifuge 12 can be operated in a forward operation for cell separation and / or cell washing. "Forward operation" refers to one of the two possible fluid flow directions of the centrifuge 12, and describes the operation that causes the separation of liquid and solid particles (e.g., culture medium and cells). The liquid obtained in this way is the supernatant 14. This separation allows the separated cells to be washed with a wash buffer (preferably PBS buffer) or a culture medium (preferably culture medium 8) on the one hand, and / or allows the supernatant 14 to be obtained on the other hand.

[0103] Alternatively, the centrifuge 12 can be operated in reverse operation. "Reverse operation" refers to the second of the two possible fluid flow directions of the centrifuge 12, and describes an operation that results in the discharge of separated solid particles (preferably cells). The product obtained in the reverse operation can be the centrifuged discharge portion 19.

[0104] According to one embodiment, it is proposed that for at least one, preferably at least two, more preferably each execution of steps b) and ii), the cultivation of the cells in step b) and the biological production in step ii) are at least partially performed in parallel, preferably, the execution of steps b) and ii) runs in parallel for at least 50%, preferably at least 75%, more preferably at least 90% of the time used for the cultivation in step b) and / or the time used for the biological production in step ii). The time is the time from the initial filling of the corresponding container or the partial refilling with fresh culture medium 8 until the removal of the discharge portion 16 or the removal of at least the main part of the cell culture liquid 7 of the harvesting container 3 for harvesting the biological product. If the biological product is harvested from the harvesting container 3 and / or the discharge portion 16 is discharged in more than one portion, the last portion shall prevail.

[0105] like Figure 4 After performing step b) for example for the first time (n=1) as part of the proposed method, the restarted portion 17 of cells can be combined with fresh culture medium 8 (step d)) and step b can be repeated at least a second time (n+1). However, step b) is preferably repeated until the cyclic production mode is stopped by performing step f) of the proposed method.

[0106] Once the discharge portion 16 of step b) has been subjected to steps c) and i) for the first time (n), the first execution (n) of step ii) can be carried out in the harvest container 3 .

[0107] By performing the second execution (n+1) of step b) in parallel with the first execution (n) of step ii), the two vessels are operated in parallel. By operating the cultures in the vessels in parallel for a predetermined amount of time, the utilization of the vessels and associated equipment is maximized.

[0108] As will be described further below, different strategies can be applied to synchronize the culture time in the two vessels.

[0109] See also Figure 5 By providing slots for the containers, planning the movement of the culture medium and the process times, etc., the bioproduction can be optimized in terms of the overall process planning. Thus, it can be the case that a single source container slot 20 is paired with a single harvest container slot 21, step b) is performed for a culture time (particularly a predetermined culture time), step ii) is performed for a production time (particularly a predetermined production time), the culture time and the production time and thus the source container slot 20 and the harvest container slot 21 are synchronized, so that for at least two consecutive executions of step ii), preferably all executions, the same harvest container slot 21 is used.

[0110] A tank contains exactly one source container or harvest container 3 and preferably at least one or exactly one electronic process control for the container and a defined position.

[0111] Here and preferably, after at least one repetition of steps b), c) and i), preferably after each repetition, the harvest container slot 21 is ready to receive the centrifuged discharge portion 19. When the term "repetition" is used, any execution except the first execution is meant. Thus, the method is synchronized so that not only the harvest container 3 is completed, but also the necessary steps for preparing the harvest container slot 21, such as connecting a new container. Preferably, when the first part of the centrifuged discharge portion 19 is ready to be discharged from the centrifuge 12, the harvest container slot 21 is already ready, or at least it is prepared so quickly that the relevant part of the cells does not die.

[0112] The term "pairing" currently refers to transferring cells cultured in source container 2 of source container tank 20 to harvest container 3 located in harvest container tank 21. The pairing of source container tank 20 and harvest container tank 21 is preferably maintained for at least 2 executions of steps b) and ii), and / or preferably maintained for at least 3 weeks, more preferably at least 5 weeks.

[0113] like Figure 5 As depicted, source container slot 20 includes a physical space for receiving source container 2. Preferably, source container slot 20 includes a physical location for mounting source container 2. Figure 5 As described, the source container 2 can be transferred to the space provided by the source container slot 20 using a transfer device. It is further preferred that the source container slot 20 contains a source electronic process control 22, and the source electronic process control 22 is integrated into a source electronic process control unit 23. Preferably, the source electronic process control unit 23 is located next to the source container 2.

[0114] It is further preferred that the source electronic process control unit 23 is connected to at least one sensor placed inside or at the source container 2. Figure 5 As described, the sensor may be a pH probe or a temperature probe or any other sensor suitable for monitoring and preferably also controlling at least one process parameter.

[0115] Preferably, the culture in the source container tank 20 is carried out for a predetermined culture time. After the predetermined culture time, the discharge portion 16 of the cell culture fluid 7 is discharged from the source container 2 and transferred to the centrifuge 12.

[0116] like Figure 5As further described, the source container 2 is connected to a clarification device 4 comprising a centrifuge 12. Here, it should be noted that, in order to maintain clarity, the complex network of fluid lines that conduct the liquid to and from the centrifuge chamber 24 is not described. Preferably, the centrifuge 12 is designed as a fluidized bed centrifuge 12 comprising at least one centrifuge chamber 24. In a preferred embodiment and as Figure 5 As depicted, the centrifuge 12 includes four centrifuge chambers 24 .

[0117] It should be noted here that the connection between the source container 2 and the centrifuge 12 and / or between the harvest container 3 and the centrifuge 12 is preferably maintained only until the corresponding transfer step has been performed. In this way, when no transfer to and / or transfer from the centrifuge 12 and no centrifugation is required, the centrifuge 12 can be used elsewhere.

[0118] like Figure 5 As described, the harvest container slot 21 includes a physical space for receiving the harvest container 3. Preferably, the harvest container slot 21 includes a physical location for mounting the harvest container 3. Figure 5 As described, the harvest container 3 can be transported to the space provided by the harvest container tank 21 using a transport device. It is further preferred that the harvest container tank 21 contains a harvest electronic process control 25, and the harvest electronic process control 25 is integrated into a harvest electronic process control unit 26. Preferably, the harvest electronic process control unit 26 is located next to the harvest container 3.

[0119] It is further preferred that the harvesting electronic process control unit 26 is connected to at least one sensor placed inside or at the harvesting container 3. Figure 5 As described, the sensor may be a pH probe or a temperature probe or any other sensor suitable for monitoring and preferably also controlling at least one process parameter.

[0120] As from Figure 5 It is clear from the above that, preferably, the source container 2 and the harvest container 3 are not directly connected. In addition, and also as Figure 5 As described, once the cell culture fluid 7 has been transferred from the source container 2 to the centrifuge 12, the cell culture fluid 7 is not transferred back to the source container 2. Typically it would be possible to send the whole cell culture fluid 7 from the source container 2 to the centrifuge 12 and transfer the restart portion 17 from the centrifuge directly or indirectly (e.g. via a harvest container 3) back to the same and not cleaned, sterilized or similar source container 2. However, here and preferably, the restart portion 17 stays in the source container 2.

[0121] In a preferred embodiment, steps a) to f) are synchronized in time with the repeated seed culture, so that after step f), step a) is repeated at the same source container tank 20 without destroying the synchronization between the source container tank 20 and the harvest container tank 21. After multiple repetitions of step b), the cells may become genetically unstable. Preferably, without changing the repetitions in the harvest container 3, they are replaced with new initial cells 15 from the seed culture. In this way, production can theoretically be maintained forever.

[0122] According to one embodiment, it is proposed that the method further comprises the following steps

[0123] iii) transferring the harvested portion 27 from the harvesting container 3 to a clarification device 4 and centrifuging the harvested portion 27 via a centrifuge 12, thereby separating the biological product when located in the supernatant 14 from the harvested portion 27. Here and preferably, step iii) is performed after each step ii). Preferably, the harvested portion 27 is the whole cell culture fluid 7 from the harvesting container 3. Likewise, when the biological product is located in the supernatant 14, it can be forwarded to a further purification step 29, while the harvested portion 28 is preferably directed to a waste 13 location. Alternatively, when the biological product is present intracellularly, the harvested portion 28 can be directed to a further purification step and the supernatant can be directed to a waste location.

[0124] In a particularly preferred embodiment, the same clarification setup 4 is used in step i) and step iii).

[0125] According to one embodiment, it is proposed that the bioprocess facility 1 comprises a sensor arrangement, which comprises at least one sensor placed in or at the source container 2 and / or at least one sensor placed in or at the harvest container 3, preferably, at least one sensor is configured to measure at least one process parameter of the following group of the source container 2 and / or the harvest container 3: oxygen concentration, carbon dioxide concentration, pH, temperature, conductivity, pressure, viable cell concentration, viable cell production rate, product concentration, production capacity, stirring speed and / or culture medium, including nutrient concentration, such as carbon source concentration, nitrogen source concentration, amino acid concentration, growth factor concentration, etc. As described, the sensor can be connected to a corresponding electronic process control unit and used to control the corresponding culture environment conditions in the source container 2 and / or the harvest container 3.

[0126] The term “process parameter” is to be understood in a broad sense and comprises at least one process parameter which can be monitored and preferably also controlled within the source container 2 and / or the harvest container 3 .

[0127] It should be noted that additional process parameters can be derived from monitoring more than one process parameter. For example, the change in bioproduct concentration over time (also referred to as production capacity) can be derived by correlating bioproduct concentration with culture time. In addition, cell growth rate can be determined by evaluating the change in viable cell concentration over culture time.

[0128] As already mentioned, it may be the case that the bioprocess facility 1 (in particular the source container tank 20) ​​comprises a source electronic process control 22 for controlling the source culture environment conditions, preferably the source electronic process control 22 being connected to at least one sensor placed in or at the source container 2.

[0129] Furthermore, it may be the case that step ii) is terminated after reaching a predetermined termination condition, preferably during at least one execution (especially each execution) of step ii) at least one sensor measurement associated with the termination condition is repeatedly (especially cyclically or continuously) measured by a sensor placed in or at the harvest container 3, more preferably using the sensor measurement, especially by the harvest electronic process control 25, to predict when the termination condition will be reached, especially by the source electronic process control 22 to control the source culture environment conditions in step c) to synchronize step c) with step ii). Applying the above mentioned sensor or measured process parameters.

[0130] Now it is very interesting that in some cases the harvest container 3 can be used for a longer or shorter time than planned and this can be noticed or deliberately decided only during the corresponding step ii). Instead of having to interrupt step ii) due to the imminent end of step b), step b can be planned from the beginning with the use of a control reserve). Here and preferably, step b can be slowed down or accelerated). This results in a duration of step b) that is longer than the theoretically optimal time, but this variation can be used in particular to keep the viability of the cells in the source container 2 above a predetermined threshold and / or to produce more biological product already during step b) and to obtain a control reserve to accelerate step b). In addition to or as an alternative, step b) can be slowed down by changing the source culture environmental conditions in order to produce more biological product and / or to have less cell growth and thereby, for example, to produce less toxic byproducts, which may reduce the overall culture viability.

[0131] Generally, a synchronization strategy may be implemented, particularly applied to the source container 2, to keep the source container and the harvest container 3 and / or their tanks synchronized. Such synchronization is particularly important because cells may not remain viable for an extended period of time in the source container 2 without adjusting the source culture environment conditions and / or at least one process parameter in the source container 2. The goal is to provide a predetermined amount of cells in the source container 2 when the harvest container tank 21 is ready to perform step ii). To this end, the culture time in the source container 2 is synchronized with the culture time in the harvest container 3.

[0132] exist Figure 4 Such a synchronization strategy is described in b. For example, the temperature within the source container 2 can be reduced to extend the culture time in the source container 2. In addition or alternatively, a feed medium can be supplied to the cultured cells, or the feed medium can continue to be supplied to extend the culture time in the source container 2.

[0133] The corresponding process parameters can be predicted based on historical data. In case the prediction is not good enough, a gradual change of the process parameters can be made. Another synchronization strategy can include a change of dividing the cell culture fluid 7 from the source container 2 into a discharge part 16 and a restart part 17. If more cell culture fluid 7 or a higher viable cell concentration has been achieved in the extended step b), the restart part 17 can be reduced absolutely or relatively to allow another longer step b) and / or step ii).

[0134] The predetermined termination condition of the harvesting container 3 can be that at least one process value reaches a certain value, in particular, and Figure 3 As indicated, the viability (Viab) falls below a predetermined threshold, and / or a certain culture time is reached, and / or a certain filling volume is reached, and / or a certain biological product concentration is reached, and / or the ratio between product and by-product concentration falls below a predetermined threshold, and / or a certain amount of by-product is reached, and / or the viable cell concentration reaches a certain value, and / or a certain process value is maintained for a certain amount of time.

[0135] To estimate the time when the culture in harvest vessel 3 will reach the predetermined termination condition, various process parameters with an appropriate process model can be used, such as cell viability, cell specific productivity, product concentration, impurity levels (e.g. host cell impurity concentration), or any CQA of the product.

[0136] According to one embodiment, it is proposed that if a deviation between the predicted time of reaching the termination condition and the planned end point of the production time is detected, in particular by the source electronic process control 22 or the harvest electronic process control 25, a synchronization strategy is executed, preferably comprising adjusting the source culture environment conditions and / or the harvest culture environment conditions to reduce the resulting deviation between the source container 2 and the harvest container 3, in particular to reach a predetermined absolute cell amount in the source container 2 before starting step c). This may result in step c) being started earlier or later.

[0137] Another synchronization strategy may include changing the composition of the feed medium 6 and / or the time point of adding the feed medium 6 and / or changing the target fill volume in the source container 2 .

[0138] According to one embodiment, it is proposed that the initial viable cell concentration in step ii) is higher than the final viable cell concentration in step b), preferably, the cell concentration is increased by centrifugation in step i). This is also particularly advantageous in the case where the source container 2 has a larger volume than the harvest container 3 (e.g. at least 10% larger).

[0139] It may also be the case that at least one process parameter of the source culture environment conditions (particularly initial source culture environment conditions) in at least one execution (particularly each execution) of step b) and the harvest culture environment conditions (particularly initial harvest culture environment conditions) in at least one execution (particularly each execution) of step ii) is set differently, preferably, at least one process parameter is optimized with respect to the production of biological products in the source culture environment conditions for culturing cells, and / or at least one process parameter is optimized with respect to the cultivation of cells in the harvest culture environment conditions for producing biological products. Different here means substantially different, not different within or close to tolerance.

[0140] According to one embodiment, it is proposed that for at least one repetition (especially each repetition) of step b) and / or step ii), the initial source culture environment (especially the source culture environment) and / or the initial harvest culture environment (especially the harvest culture environment) are substantially the same.

[0141] According to one embodiment, it is proposed to carry out the cultivation of the cells in step b) in cyclic production mode under batch culture conditions or fed-batch culture conditions and / or to carry out step ii) in production mode under fed-batch culture conditions.

[0142] This may be the case at least once, preferably at least twice, more preferably each time the respective step is performed.

[0143] According to one embodiment and as already mentioned, it is proposed that the centrifuge 12 is a fluidized bed centrifuge 12, preferably, the centrifuge 12 is operated in forward operation for cell separation and / or cell washing, and in reverse operation for cell discharge, more preferably, the centrifuge 12 is operated in reverse operation for transferring the centrifuged discharge portion 19 to the harvest container 3 between steps i) and ii).

[0144] According to one embodiment, it is proposed to subject the effluent portion 16 to a transfection step after step c), or to produce a biological product from the cells in the source container 2 .

[0145] Here, the use of a centrifuge 12 (preferably a fluidized bed centrifuge) as a clarification device 4 provides a simple method for performing the transfection step. For the definition of transfection, reference is made to the definition given above. For example, by being able to supply washing solution and / or fresh culture medium during the centrifugation in step i), treatment with calcium phosphate for transfection can be easily performed when calcium phosphate and the DNA to be transferred into the cells can be supplied during the centrifugation. Transfection can also be performed outside the source container 2 before or after centrifugation.

[0146] The results of the proof-of-concept experiments are shown in Figure 6 Here, two 5 L glass Univessels were used as source vessel 2 and harvest vessel 3, respectively. Cells were cultured in source vessel 2 for six days until they reached approximately 25 × 10 6 Viable cell concentration (VCC) of cells / mL ( Figure 6 a), solid circle). At this time, if Figure 6 As indicated by the vertical dashed line in a, the discharged portion 16 of the cell culture fluid 7 is discharged from the source container 2 to the harvest container 3, and the cells are cultured in the harvest container 3 for another six days ( Figure 6 a), solid circles). At the same time, the restart portion 17 of the cell culture fluid 7 was combined with the fresh culture medium 8 in the source container 2 so that the initial viable cell concentration (VCC) was 0.3×10 6 cells / mL( Figure 6 a), shaded triangle). Figure 6 This process was repeated several times (diamonds and shaded circles) as indicated in a). The vertical dashed lines indicate the changes in the container.

[0147] The results demonstrate that operating source container 2 in cyclic production mode enables cyclic production of living cells, and that cell growth and viability are not negatively affected by the cyclic production mode.

[0148] like Figure 6 As further indicated in the results, it was demonstrated that the non-cyclic operation of the harvest vessel 3 enabled the process conditions in the harvest vessel 3 to be adjusted to favor bioproduction ( Figure 6b) without having to keep cells alive ( Figure 6 a). Although cell viability decreases during operation of the harvest vessel 3 in production mode ( Figure 6 a), but the concentration of the biological product (in this case, the antibody) increases disproportionately. In summary, the proposed method not only reveals similar performance compared to previous processes, but also shows continuous properties that further enhance the process efficiency.

[0149] Reference Numbers

[0150] 1 Bioprocess facilities

[0151] 2 Source Container

[0152] 3 Harvest Container

[0153] 4 Clarify settings

[0154] 5 Source container entry port

[0155] 6 Feed medium

[0156] 7 Cell culture medium

[0157] 8 Culture medium

[0158] 9 Harvest container inlet port

[0159] 10 Source container outlet port

[0160] 11 Harvest container outlet port

[0161] 12 Centrifuge

[0162] 13 Waste

[0163] 14 Supernatant

[0164] 15 Initial cells

[0165] 16 Discharge section

[0166] 17 Restart Section

[0167] 18 Feed line

[0168] 19 Centrifugal discharge

[0169] 20 source container slots

[0170] 21 Harvest Container Slots

[0171] 22 Source Electronic Process Control

[0172] 23 Source Electronic process control unit 24 Centrifuge chamber 25 Harvest Electronic process control

[0173] 26 Harvest electronic process control unit 27 Harvest section

[0174] 28 Centrifuge the harvested fraction

[0175] 29 Purification Steps

Claims

1. A method for operating a bioprocess facility (1) for producing a bioproduct, wherein the bioprocess facility (1) comprises a source container (2) for cell culture, a harvest container (3) for bioproduction and a clarification device (4) with a centrifuge (12), wherein the source container (2) is operated in a cyclic production mode, The cyclic production mode comprises the following steps in this order: a) starting the cyclic production mode in the source container (2) with the initial cells (15) and the culture medium (8), b) culturing the cells in the source container (2) to obtain a cell culture solution (7) containing the cultured cells, whereby the source container (2) and the culture medium (8) are configured to provide source culture environmental conditions for the cultivation of the cells, c) discharging a discharge portion (16) of the cell culture fluid (7) from the source container (2), d) combining the restarted portion (17) of the cell culture fluid (7) with fresh culture medium (8) and repeating step b), e) repeating steps c) and d) at least once, and / or f) discharging the cell culture fluid (7) obtained in step d) from the source container (2) that stops the circulation production mode, and obtaining a discharge portion (16), The method further comprises the following steps: i) transferring the effluent portion (16) to the clarification device (4) and centrifuging the effluent portion (16) via the centrifuge (12), thereby separating the effluent portion (16) into at least a centrifuged effluent portion (19) and a supernatant (14) and, preferably, a biological product, ii) transferring at least a portion of the centrifuged discharge portion (19) from step i) and fresh culture medium (8) into the harvest container (3), and operating the harvest container (3) in a production mode, whereby the harvest container (3) and the culture medium (8) are configured to provide harvest culture environmental conditions to produce the biological product in the harvest container (3), Steps i) and ii) are performed at least twice with the discharge portion (16) coming from the performance of step c) and / or step f).

2. The method according to claim 1, characterized in that For at least one, preferably at least twice, and more preferably each time, execution of steps b) and ii), the culturing of the cells in step b) and the biological production in step ii) are performed at least partially in parallel, preferably, the execution of steps b) and ii) runs in parallel for at least 50%, preferably at least 75%, and more preferably at least 90% of the time used for the culturing in step b) and / or the time used for the biological production in step ii), and / or the time used for executing steps b) and / or step ii).

3. The method according to claim 1 or 2, characterized in that: A single source container slot (20) is paired with a single harvest container slot (21), step b) is performed for a culture time, in particular a predetermined culture time, and step ii) is performed for a production time, in particular a predetermined production time, the culture time being synchronized with the production time and therefore with the source container slot (20) and the harvest container slot (21), so that the same harvest container slot (21) is used for at least two consecutive executions of step ii), preferably all executions.

4. The method according to claim 3, characterized in that Steps a) to f) are synchronized with repeated seed cultivation, so that after step f), step a) is repeated at the same source tank (20) without destroying the synchronization between the source tank (20) and the harvest tank (21).

5. The method according to any one of the preceding claims, characterized in that The method further comprises the following steps iii) transferring the harvested portion (27) from the harvest container (3) to the clarification device (4) and centrifuging the harvested portion (27) via the centrifuge (12) to separate the biological product from the harvested portion (27), Preferably, step iii) is performed after each step ii).

6. The method according to any one of the preceding claims, characterized in that The bioprocess facility (1) comprises a sensor arrangement, which comprises at least one sensor placed in or at the source container (2) and / or at least one sensor placed in or at the harvest container (3), preferably, at least one sensor is configured to measure at least one process parameter of the source container (2) and / or the harvest container (3) from the following group: carbon source concentration, nitrogen source concentration, amino acid concentration, growth factor concentration, oxygen concentration, carbon dioxide concentration, pH, temperature, conductivity, pressure, biomass concentration, biomass production rate, product concentration, production capacity, oxygen uptake rate and / or stirring speed.

7. The method according to any one of the preceding claims, characterized in that The bioprocess facility (1), in particular the source container tank (20), comprises a source electronic process control (22) for controlling the source culture environment conditions, and preferably the source electronic process control (22) is connected to the at least one sensor placed in or at the source container (2).

8. The method according to any one of the preceding claims, characterized in that Step ii) is terminated after a predetermined termination condition is reached. Preferably, during at least one execution of step ii), in particular during each execution, at least one sensor measurement related to the termination condition is repeatedly, in particular cyclically or continuously, measured by the sensor placed in or at the harvest container (3). More preferably, the sensor measurement is used to predict the time when the termination condition is reached, in particular by the harvest electronic process control (25). The source culture environment conditions in step b) are controlled, in particular by the source electronic process control (22), so that step b) is synchronized with step ii).

9. The method according to claims 3 and 8, characterized in that If a deviation between the predicted time for reaching the termination condition and the planned end point of the production time is detected, in particular by the source electronic process control (22) or the harvest electronic process control (25), a synchronization strategy is executed, preferably, the synchronization strategy comprises adjusting the source culture environment conditions and / or the harvest culture environment conditions to reduce the resulting deviation between the source container (2) and the harvest container (3), in particular to achieve a predetermined absolute cell amount in the source container (2) before starting step c).

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

11. The method according to any one of the preceding claims, characterized in that In at least one execution of step b), in particular, the source culture environment conditions in each execution, in particular, at least one process parameter of the initial source culture environment conditions and in at least one execution of step ii), in particular, the harvest culture environment conditions in each execution, in particular, at least one process parameter of the initial harvest culture environment conditions are set differently. Preferably, the at least one process parameter is optimized with respect to the production of the biological product in the source culture environment conditions for culturing cells, and / or the at least one process parameter is optimized with respect to the cultivation of cells in the harvest culture environment conditions for producing the biological product.

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

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

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

15. The method according to any one of the preceding claims, characterized in that After step c), the discharge portion (16) is subjected to a transfection step, or the biological product is produced from the cells in the source container (2).

Images