Method for performing a bioprocess on immune or naive cells in a cell culture

Abstract

The present invention relates to a method for performing a bioprocess on cells in a cell culture, wherein the cell culture is present in a cell vessel. The method comprises performing one or more unit operation(s) on the cells in an automated manner and expanding the cells in the cell vessel, wherein expanding is performed prior to and / or after performing the one or more unit operation(s). The invention further relates to a bioreactor module for performing a bioprocess on cells in a cell culture. The invention further relates to a bioprocessing system for performing a bioprocess on cells in a cell culture. The invention further relates to uses of the bioreactor module and the bioprocessing system.

Description

[0001] METHOD FOR PERFORMING A BIOPROCESS ON IMMUNE OR NAIVE

[0002] CELLS IN A CELL CULTURE

[0003] TECHNICAL FIELD OF THE INVENTION

[0004] The present invention relates to a method for performing a bioprocess on cells in a cell culture, wherein the cell culture is present in a cell vessel. The method comprises performing one or more unit operation(s) on the cells in an automated manner and expanding the cells in the cell vessel, wherein expanding is performed prior to and / or after performing the one or more unit operation(s). The invention further relates to a bioreactor module for performing a bioprocess on cells in a cell culture. The invention further relates to a bioprocessing system for performing a bioprocess on cells in a cell culture. The invention further relates to uses of the bioreactor module and the bioprocessing system.

[0005] BACKGROUND OF THE INVENTION

[0006] The term "bioprocess" presently represents a biotechnological process, here a biotechnological process involving the use of immune cell cultures or naive cell cultures. One or more processing steps might be performed on each cell culture. Hence, a bioprocess in this sense might refer to a manufacturing process that involves a sequence of processing steps performed on a cell culture which ultimately will lead to a final product.

[0007] Here, the bioprocess is in the area of cell and gene therapy, for example to manufacture autologous T cells that are modified to express a chimeric antigen receptor (CAR). These cells might be used for the treatment of various types of cancer, including different types of leukemia (blood cancer). Other cell therapies based on naive cells, in particular stem cells and their derivatives, are also of interest.

[0008] It is currently not fully known which factors contribute significantly to the success of automation in cell and gene therapy cell manufacturing. There is a need to devise a bioprocessing system with an extremely high tolerance to errors with the goal of always protecting the cells but at the same time achieving a high grade of automation to reduce manual labor costs and human mistakes. There is also a need to devise a bioprocessing system with a high flexibility and at the same time planning the components and workflows such that the automation is not hindered by complex mechanisms, complex parts or the like. At the same time, with the unknown sources for success of an automated bioprocessing system, there are still many problems to be solved. The wrong type of bioreactor for example may work only for a fraction of the emerging processes, possibly dooming a bioprocessing system or necessitating many individual solutions.

[0009] In bioprocesses in the area of cell and gene therapy with allogenic or autologous production of genetically modified immune cells, compliance of the bioprocess with regulatory demands is also important and the bioprocess is typically highly regulated and needs to be approved by regulatory authorities.

[0010] One specific problem that needs improvement is providing methods and devices with a high flexibility and a low complexity to reduce the number of transfers of the cell culture between cell vessels, perform more unit operations in one cell vessel and thereby speed up the overall process while reducing the likelihood for contamination and errors. Further, high flexibility is also needed as many factors may influence the success of the manufacturing process, with the contribution of each individual factor still being debated. The factors being under investigation include the requirement for even distribution of nutrients, the requirement for agitating the cells, the metabolic state of the cells, the distribution of the cells within the cell vessel and the surface area of the cell culture.

[0011] Some known methods and cell vessels (e.g. Miltenyi Prodigy®) are centrifuge chambers and therefore provide for a high utility but also a high complexity and a high cost for the given flexibility. Also, the order of magnitude of culture volumes handled within those chambers is usually limited. Other known cell vessels (e.g. G-Rex®) are mainly used manually, are specific to certain cell culture volumes and lack necessary features for a high degree of automation.

[0012] It is an object of the present invention to provide a method for performing the different unit operations but also cell expansion in a simplified and efficient manner. It is a further object to provide a cell vessel and surroundings to be used with more flexibility and less complexity. SUMMARY OF THE INVENTION

[0013] The present invention addresses these above-described objects. Specifically, the invention provides a method for performing a bioprocess on cells in a cell culture, wherein the cell culture is present in a cell vessel. The method comprises performing one or more unit operation(s) on the cells in an automated manner. Each such unit operation comprises: i. adding a liquid composition to the cell vessel; ii. incubating the cells in the cell vessel with the added liquid composition; and iii. removing liquid from the cell vessel through at least one filter, and thereby removing at least a part of the added liquid composition.

[0014] The method further comprises expanding the cells in the cell vessel, wherein expanding is performed prior to and / or after performing the one or more unit operation(s).

[0015] The provided method advantageously simplifies performing a bioprocess on cells in a cell culture by applying a cell vessel that allows for performing the one or more unit operation(s) (e.g., activation, transfection, transduction) on the cells in an automated manner. This is realized by removing liquid from the cell vessel through at least one filter, whereby part of an added liquid composition can be removed. The cell vessel may comprise the at least one filter and / or be fluidically connected to at least one filter. The filter achieves a flexible removal of fluid and thus separation from the cells essentially without negatively impacting the sensitive cells, e.g. by fluid transferand / or centrifugation. By using this particularset-up, the unit operation is also performed in an automated manner, e.g. by using an active element such as a pump to add liquid to the vessel, to incubate the cells in a vessel with the added liquid, and to remove liquid from the cell vessel through the at least one filter and thereby remove at least part of the added liquid. At the same time, the cell vessel is suitable for expanding the cells, such that not only a unit operation but also cell expansion can advantageously be performed in the very same cell vessel. This significantly simplifies processes, such as cell therapy manufacturing, compared to conventional approaches in the art, as the laborious manual processes are performed in an automated manner. As a result, handling errors are avoided, improving reproducibility and mitigating contamination risks. Also, by being able to perform multiple unit operations and expansion in the very same cell vessel, the conditions which are applied on the cells can be tightly controlled. Furthermore, transfer steps are avoided, such that overall, the cells are less negatively impacted, and cell loss is reduced. These positive effects are underlined by the Examples disclosed herein. According to a first aspect of the invention, a method for performing a bioprocess on cells in a cell culture is provided, wherein the cell culture is present in a cell vessel, wherein the method comprises performing one or more unit operation(s) on the cells in an automated manner, wherein a unit operation comprises: i. adding a liquid composition to the cell vessel; ii. incubating the cells in the cell vessel with the added liquid composition; and iii. removing liquid from the cell vessel through at least one filter, and thereby removing at least a part of the added liquid composition; and expanding the cells in the cell vessel, wherein expanding is performed prior to and / or after performing the one or more unit operation(s).

[0016] According to a second aspect of the invention, a bioreactor module for performing a bioprocess on cells in a cell culture is provided, wherein the bioreactor module comprises a cell vessel, and at least one filter for removing liquid from the cell vessel through the at least one filter, wherein the bioreactor module is configured for performing in an automated manner one or more unit operations on the cells in the cell vessel, and for expanding the cells in the cell vessel.

[0017] According to a third aspect of the invention, a bioprocessing system for performing a bioprocess on cells in a cell culture is provided, wherein the bioprocessing system comprises the bioreactor module according to the second aspect of the invention.

[0018] According to a fourth aspect of the invention, a use of the bioreactor module according to the second aspect and / or the bioprocessing system according to third aspect for manufacturing a cell therapy product is provided.

[0019] According to a fifth aspect of the invention, a use of the bioreactor module according to the second aspect and / or the bioprocessing system according to third aspect in a method according to the first aspect of the invention is provided. BRIEF DESCRIPTION OF THE FIGURES

[0020] In the following, embodiments of the invention are explained with respect to the drawing. The drawing shows in

[0021] Fig. 1 a bioprocessing system with several bioreactor modules,

[0022] Fig. 2 the bioreactor module with a cell vessel,

[0023] Fig. 3 the cell vessel of the bioreactor module,

[0024] Fig. 4 a view into the cell vessel,

[0025] Fig. 5 a schematic of possible fluid lines of the bioreactor module in a different embodiment,

[0026] Fig. 6 the total cell number (see 1) and daily fold expansion (see 2) of T cells using the bioprocessing system (BRO3) compared to a reference cell culture (BRO2),

[0027] Fig. 7 the viable cell density using the bioprocessing system (BRO3) compared to a reference cell culture (BRO2) during the whole culture (see 1) and the perfusion culture only (see 2),

[0028] Fig. 8 the vessel comprising a binding unit,

[0029] Fig. 9 an example of a preferred embodiment of the method according to the present disclosure, which may be referred to as "sequential embodiment",

[0030] Fig. 10 an example of a preferred embodiment of the method according to the present disclosure, which may be referred to as "parallel embodiment",

[0031] Fig. 11 an example of a preferred embodiment of the method according to the present disclosure, which may be referred to as "transfection short embodiment",

[0032] Fig. 12 an example of a preferred embodiment of the method according to the present disclosure, which may be referred to as "transfection long embodiment",

[0033] Fig. 13 an example of a preferred embodiment of the method according to the present disclosure, which may be referred to as "transfection and transduction embodiment",

[0034] Fig. 14 the total cell number (see 1), total fold expansion (see 2), cell viability (see 3), percentage of activated CD4+ T cells (see 4) and percentage of activated CD8+ T cells (see 5) using T cells from two different donors (BRO1 and BRO3) using the bioprocessing system according to the invention,

[0035] Fig. 15 the percentages of T cell populations over the activation and culturing period in the method according to the invention (BRI and BR2) and a flask culture (flask),

[0036] Fig. 16 the transfection efficiency (see A) and T cell phenotype (see B) of GFP-transfected T cells transfected according to a method of the present disclosure, and Fig. 17 the percentage of T cells expressing a chimeric antigen receptor (CAR) and GFP, respectively, after being transduced with a method according to the invention.

[0037] DETAILED DESCRIPTION

[0038] The following description serves to deepen the understanding of the present disclosure and shall be understood to complement and be read together with the description of exemplary embodiments of the present disclosure as provided in the above section of this description. It is to be understood that this invention is not limited to the particular embodiments, methodologies, protocols and reagents described herein as these may vary within the scope set by the claims. It is also to be understood that terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention which is defined by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

[0039] In the following description, certain elements of the present invention will be described. These elements may be discussed with specific embodiments; however, it should be understood, that they may be combined in any manner and in any number to create additional embodiments. The variously described examples, features and particular embodiments should not be construed to limit the present invention to only the explicitly described embodiments or to the explicitly described combination of features. This description should be understood to disclose and encompass embodiments which combine the explicitly described embodiments with any number of the disclosed and / or preferred elements. Furthermore, any permutations and combinations of all described elements in this application should be considered disclosed by this description unless the context indicates otherwise.

[0040] Method for performing a bioprocess on cells in a cell culture according to a first aspect of the invention

[0041] According to a first aspect of the invention, a method for performing a bioprocess on cells in a cell culture is provided, wherein the cell culture is present in a cell vessel, wherein the method comprises performing one or more unit operation(s) on the cells in an automated manner, wherein a unit operation comprises: i. adding a liquid composition to the cell vessel; ii. incubating the cells in the cell vessel with the added liquid composition; and iii. removing liquid from the cell vessel through at least one filter, and thereby removing at least a part of the added liquid composition; and expanding the cells in the cell vessel, wherein expanding is performed prior to and / or after performing the one or more unit operation(s).

[0042] The provided method advantageously simplifies performing a bioprocess on cells in a cell culture by applying a cell vessel that allows for performing the one or more unit operation(s) (e.g., activation, transfection, transduction) on the cells in an automated manner. This is realized by removing liquid from the cell vessel through at least one filter, whereby part of an added liquid composition can be removed. The cell vessel may comprise the at least one filter and / or be fluidically connected to at least one filter. The filter achieves a flexible removal of fluid and thus separation from the cells essentially without negatively impacting the sensitive cells, e.g. by fluid transferand / or centrifugation. By using this particularset-up, the unit operation is also performed in an automated manner, e.g. by using an active element such as a pump to add liquid to the vessel, to incubate the cells in a vessel with the added liquid, and to remove liquid from the cell vessel through the at least one filter and thereby remove at least part of the added liquid. At the same time, the cell vessel is suitable for expanding the cells, such that not only a unit operation but also cell expansion can advantageously be performed in the very same cell vessel. This significantly simplifies processes, such as cell therapy manufacturing, compared to conventional approaches in the art, as the laborious manual processes are performed in an automated manner. As a result, handling errors are avoided, improving reproducibility and mitigating contamination risks. Also, by being able to perform multiple unit operations and expansion in the very same cell vessel, the conditions which are applied on the cells can be tightly controlled. Furthermore, transfer steps are avoided, such that overall, the cells are less negatively impacted, and cell loss is reduced. These positive effects are underlined by the Examples disclosed herein, showing that higher cell numbers and cell viability can be obtained, indicating more efficient and higher quality cell therapy manufacturing (see Figs. 6 and 7).

[0043] Performing a bioprocess on cells in a cell culture as disclosed herein particularly refers to performing one or more unit operation(s) and typically expanding the cells. However, also further bioprocesses may be performed on the cells, including selecting, washing, harvesting, or formulating the cells, etc., as disclosed herein.

[0044] Performing one or more unit operation(s) typically refers to a unit operation involved in cell therapy manufacturing. Preferred embodiments herein refer to activation and genetic modification. The skilled person is well-aware of unit operations in frame of performing a bioprocess on a cell culture, particularly for cell therapy manufacture. A "unit operation" as disclosed is not dedicated a cell expansion step. This does not mean that cells cannot proliferate or grow throughout the unit operation, but this would rather occur as a side effect throughout the unit operation. Hence, cells may expand throughout the unit operation, but it is not the primary intent of the unit operation to expand cells.

[0045] The method involves performing the unit operation(s) on the cells in "an automated manner". The skilled person is well aware of the term automated manner and conventional means to automate. In the present case, however, by providing the cell vessel and at least one filter which jointly allow for performing the unit operation(s), automation is advantageously achieved. As disclosed herein, further means may be provided to facilitate automation, such as an active element, e.g. a pump, for transferring liquids.

[0046] The term "cell vessel" is well-known in the art and the skilled person understands it. Herein, the term "cell vessel" may be exchangeable used with the term "vessel" or "bag". The cell vessel may be a flexible bag or a rigid vessel or container. The cell vessel preferably comprises a rigid part and a flexible part. Inside the cell vessel, steps of the method, in particular one or more unit operations and expanding are performed.

[0047] The cells are present in a cell culture in the present disclosure. The skilled person is well aware of such terminology. This typically refers to cells being dispersed in a liquid medium and thus form a cell culture. A "liquid medium" according to the present disclosure in particular relates to a composition in which the cells remain to the most extent viable. For instance, the cells may be combined with a cell culture medium for providing an environment in which the cells can proliferate. Also, the cells may be combined with a buffer or storage solution, in which the cells can be stored and / or kept essentially alive. In another embodiment, the cells may be combined with an agent (also referred to as "reagent") in order to manipulate the cells, e.g. an activation agent may be provided to activate the cells and / or a genetic modification agent may be provided to genetically modify the cells. The term "liquid medium" may herein be interchangeable used with the terms "medium", "media", "liquid media". Sometimes, also an activation agent, and / or genetic modification agent, in particular transduction and / ortransfection agent, may be referred to as "liquid medium", such that the term shall encompass such compositions.

[0048] The term "expanding the cells" is also well-known in the art. This typically refers to a process in which the cells multiply or proliferate and therefore increase in number. "Expanding" (which may also be referred to as a "cultivation procedure") of the present disclosure shall not be limited in scope. Indeed, the skilled person is well-aware of various expansion methods or cultivation procedures, which are adequate in order to cultivate cells in a liquid medium. Expanding may include batch cultivation, fed-batch cultivation or perfusion culture of combinations thereof. In addition, expanding may already be achieved if the cells are provided with an adequate environment, in which they may proliferate or at least not become completely apoptotic. In one embodiment, expanding may also include adding a liquid, such as cell culture medium or reagent to the cells present in the vessel according to the present disclosure. According to a preferred embodiment, particularly suitable for manufacture of a cell therapy product, expanding encompasses a preliminary cultivation phase, in which the cells are preliminarily cultivated, preferably wherein cells are predominantly cultivated in a small volume compartment of the vessel (also referred to as "reservoir"); and a subsequent cultivation phase, in which the cells are subsequently cultivated preferably wherein cells are cultivated in the reservoir and a large volume compartment (also referred to as "casing"). Such culture allows for scaling up small cell number, such as typically only obtainable from a patient sample for autologous cell therapy, in the same vessel.

[0049] The vessel is in particular suitable for cultivating cells in a liquid medium in an expansion step. For expansion, the cells are conventionally provided in a liquid medium, which allows for efficient expansion and manipulation of the cells. However, the present disclosure is not limited to cells being exclusively in liquid medium. For instance, the cells may be provided together with a carrier matrix or support matrix for cultivation, wherein these are dispersed in a liquid medium. In addition, the medium may comprise in part a dissolved or undissolved gaseous phase (e.g., culture gas as disclosed herein).

[0050] Unit operation

[0051] The method comprises performing one or more unit operation(s), preferably selected from activation and genetic modification, on the cells in an automated manner. A unit operation herein particularly refers to a unit operation required throughout cell therapy manufacture, e.g. throughout manufacture of therapeutic immune cells, such as CAR T cells or NK cells. Unit operations in frame of a cell therapy manufacturing process are known and may include cell enrichment, cell selection, cell activation, genetic modification of cells, etc. Herein, cell selection is seen as a separate process of the unit operation disclosed herein (see further below disclosure on cell selection). As used herein a unit operation is different from a step of expanding cells (even though cells may also proliferate and thus expand throughout a unit operation).

[0052] According to a preferred embodiment, the unit operation is selected from: activation of the cells, preferably wherein the liquid composition comprises an activation agent; and genetic modification of the cells, in particular transduction and / or transfection, preferably wherein the liquid composition comprises a genetic modification agent, in particular a transduction agent and / or a transfection agent.

[0053] Both of activation of the cells and genetic modification of the cells is usually required in order to manufacture a cell therapy product, in particular cells which have been genetically modified, e.g. to target a specific cell type such as a cancer cell. This particularly refers to manufacture of CAR T cells or other immune cell types which are usually genetically modified for targeting a cell type of interest. In frame of the present disclosure, it is particularly advantageous to perform the necessary unit operations on the very same bioprocessing device using the same cell vessel. For instance, it is particularly advantageous to perform at least one cell activation and at least one genetic modification step of the cells, both of which is preferably performed in the same cell vessel. This is advantageous, as no cell transfer steps are required, reducing risks of contamination and cell damage.

[0054] On the other hand, the method according to the present disclosure may only have one unit operation, since some unit operations may also be performed prior to the method according to the present disclosure. For instance, the cells may be activated prior to being present in the cell vessel, such that the activated cells only require genetic modification as unit operation in order to perform the bioprocess disclosed herein, especially cell therapy manufacture. Also, the cells may be activated by the method according to the present disclosure and expanded, whereas genetic modification may be performed subsequently, either in the same cell vessel or another cell vessel. While such embodiments are within the scope of the present disclosure, it is clear to the skilled person in the art that performing as many unit operations efficiently as possible in the same vessel is highly advantageous for simplifying the process and avoiding negative effects associated with a cell transfer step into another cell vessel (e.g., contamination risks, cell damage risks).

[0055] Hence, according to a preferred embodiment, the method comprises performing more than one unit operation, preferably, wherein the method comprises at least two unit operations, more preferably wherein the at least two unit operations comprise performing activation and genetic modification of the cells. A specific embodiment of the method comprising performing activation and genetic modification of the cells is illustrated in Figs. 9 to 12.

[0056] According to a further preferred embodiment, the method comprises performing more than one unit operation, preferably, wherein the method comprises at least three unit operations, more preferably wherein the at least three unit operations comprise performing activation and two genetic modifications of the cells, such as activation, transfection and / or transduction. A specific embodiment of the method comprising performing activation and two genetic modifications of the cells is illustrated in Fig. 13.

[0057] According to a preferred embodiment, performing the one or more unit operation(s) is repeated one or more time(s), preferably at least one more time, particularly performing a different unit operation when performing the unit operation one more time.

[0058] Liquid composition

[0059] The method according to the first aspect comprises performing one or more unit operation(s) on the cells in an automated manner, wherein a unit operation comprises i. adding a liquid composition to the cell vessel; ii. incubating the cells in the cell vessel with the added liquid composition; and iii. removing liquid from the cell vessel through at least one filter, and thereby removing at least a part of the added liquid composition.

[0060] A "liquid composition" as disclosed herein refers to a composition that is predominantly in liquid state when being added to the cell vessel. This does not exclude that the composition may be frozen and thus in solid form prior or subsequent to adding the liquid composition in step i. Furthermore, this does not exclude that the liquid composition further comprises matter in solid or gaseous state. For instance, the liquid composition may comprise solid particles, e.g. which may be used in frame of a cell activation step. The liquid composition may further comprise gas bubbles. Nevertheless, as disclosed herein, the liquid composition is predominantly in liquid state when being added to the cell vessel, as this simplifies the addition to the cell vessel (compared to solid or gaseous states) allowing very precise control of the added volume of liquid composition. In addition, the cells of the cell culture are usually present in a liquid medium, such as a cell culture medium or storage / cryo-buffer, such that the addition of the liquid composition allows easy mixing and is adequate for being contacted with the cells. The composition typically comprises a solvent, such as water, in which one or more compounds are dissolved or dispersed. For instance, the composition may comprise water and agents, e.g. for activation and / or genetic modification of the cells. In addition, the liquid composition may comprise further compounds required or supportive for the cell, such as salts, amino acids, and / or nutrients.

[0061] According to a preferred embodiment, the liquid composition added in step i. comprises:

[0062] (i) an activation agent;

[0063] (ii) a genetic modification agent, preferably a transduction agent or a transfection agent, and optionally an enhancer; or (iii) an activation agent and a genetic modification agent, preferably a transduction agent or a transfection agent, and optionally an enhancer.

[0064] An "agent" as disclosed herein particularly refers to a compound that achieves the desired outcome, e.g., activation of the cells or genetic modification of the cells. This becomes immediately clear to the skilled person when reading the term "agent" in frame of the present disclosure. The respective agent as part of the liquid composition may be added to the cell vessel, preferably wherein the cell culture is present in the cell vessel prior to adding the agent. Various agents are known in the field in order to achieve the desired outcome.

[0065] For instance, an activation agent should adequately activate the cells, such as T cells, for ex vivo expansion. T-cell activation usually needs a primary specific signal via the T-cell receptor (Signal 1) and costimulatory signals such as CD28, 4-1BB, or 0X40 (Signal 2). T-cell activation is also required for the transduction of the CAR cDNA via retroviral vectors. An activation agent may be selected from cell-based activation agents, e.g., antigen-presenting cells, such as dendritic cells, which are the endogenous activators of T-cell responses. Another cell-based T-cell activation approach is through artificial antigen-presenting cells. An activation agent may also be selected from beads-based activation agents, Invitrogen CTS Dynabeads CD3 / 28, the Miltenyi MACS GMP ExpAct Treg beads, Miltenyi MACS GMP TransAct CD3 / 28 beads, and the Juno Stage Expamer technology. These reagents have largely simplified the ex vivo T-cell activation procedure. An activation agent may specifically be selected from antibody-coated magnetic beads, from antibody-coated nanobeads, or utilizing the expamer technology (based on the streptamer technology).

[0066] The genetic modification agent as disclosed herein refers to an agent that is capable of genetically modifying at least a fraction of the cells of the cell culture. This may be required in order to genetically modify cel Is for cel I therapy, e.g. CAR T cell therapy. The genetic modification agent is preferably a transduction agent or a transfection agent. The transfection agent may be a lipid nanoparticle for example (e.g. described in WO 2024 / 008967 Al). Non-viral methods, such as reagent-based transfection, reduce the risk of unwanted side effects associated with viral vectors, while also simplifying the manufacturing process. Non-viral, reagent-based transfection is preferably performed using a formulation of GFP-mRNA in liposomes, yielding a high transfection efficiency. After transfection, the cells may be expanded.

[0067] The transduction agent may be a viral vector, in particular a lentiviral or adenoviral vector. Current CAR-T cell therapies largely rely on stable CAR expression upon delivery by viral and nonviral gene transfer systems. There are three major types of stable gene expression vectors used for clinical applications: o-retroviral vectors, lentiviral vectors, and the transposon / transposase system. Messenger RNA transfer-mediated gene expression is another method to introduce CARs into cells while avoiding long-term expression.

[0068] An "enhancer" as disclosed herein is particularly provided in order to improve the unit operation, e.g. activation or genetic modification of the cells. Such enhancers are known in the field and recognized by the skilled person. For instance, sodium butyrate is a well-known compound which can improve transfection.

[0069] According to a preferred embodiment, the method comprises performing more than one unit operation, preferably wherein performing more than one unit operation comprises repeating steps i., to iii., one or more times. Such repeating typically refers to repeating such steps sequentially, i.e. steps i. to iii. a first time and then repeating steps i. to iii. one or more times. This may also be encompassed when referred to performing two or more unit operations. In addition, or alternatively, performing more than one unit operation comprises adding a liquid composition to the cell vessel comprising agents for performing more than one unit operation, preferably the liquid composition comprises an activation agent and a genetic modification agent. In such embodiment, two unit operations are performed simultaneously, e.g. activation and genetic modification. In addition, or alternatively, performing more than one unit operation comprises repeating step i. prior to step ii. and / or prior to step iii. by adding a further liquid composition to the cell vessel, in particular wherein

[0070] (i) the liquid composition in step i. comprises an activation agent, and

[0071] (ii) the further liquid composition comprises a genetic modification agent.

[0072] In addition, or alternatively, performing more than one unit operation comprises repeating step i. prior to step ii. and / or prior to step iii. by adding a further liquid composition to the cell vessel, in particular wherein

[0073] (i) the liquid composition in step i. comprises a genetic modification agent, and

[0074] (ii) the further liquid composition comprises an activation agent.

[0075] In such embodiments, the liquid composition and the further liquid composition are simultaneously present, particularly when incubating these with the cells in step ii. As a result, two (or more) unit operations can occur at the same time without the need for separate incubation and removal steps. An embodiment of such dual unit operation is illustrated in Figs. 10 and 11.

[0076] According to a preferred embodiment, the method comprises adding a further liquid composition to the cell vessel after step i. and prior to step ii., such that the further liquid composition is present in the cell vessel when incubating the cells in step ii. and such that removing in step iii. comprises removing at least part of the further liquid composition through the filter.

[0077] Cells in the cell vessel

[0078] According to the method of the first aspect, the cell culture is present in a cell vessel. In the same cell vessel the unit operation and expansion are performed, which is advantageous, as transfer steps are avoided, which can put the bioprocess at risk, e.g. due to contamination or cell damage. The cell culture is preferably present in the cell vessel prior to performing the one or more unit operations, as then only the liquid composition needs to be handled in an automated manner. On the other hand, it is not excluded that the cells are added throughout a unit operation. For instance, after adding a liquid composition to the cell vessel, the cells of the cell culture may be added to the cell vessel, such that these can be incubated subsequently.

[0079] Hence, according to a preferred embodiment, prior to performing the method according to the present disclosure, the cells of the cell culture are transferred into the cell vessel. According to an alternative or additional embodiment, the cells of the cell culture are added after step i. and prior to step ii. of the one or more unit operation(s), specifically the first unit operation of more than one unit operation is performed. Transferring is preferably performed in an automated manner, e.g. by pumping the cell culture comprising the cells into the cell vessel, e.g. via fluidic lines. This avoids manual handling errors.

[0080] Measuring

[0081] According to a preferred embodiment, the method comprises measuring the cell density one or more time(s), preferably measuring the cell density at least prior to step i. Measuring the cell density can be achieved by methods known in the art. For instance, the cell density may be measured using microscopy-based or capacitance-based measurement approaches. Measuring may be performed prior to step i. either when the cells are present in the cell vessel or outside the cell vessel. Preferably measuring the cell density is performed when the cell culture is present in the cell vessel.

[0082] Measuring of the cell density wherein the cell culture is present in the cell vessel may be performed manually, e.g. by obtaining a sample of the cell culture from the cell vessel and performing a cell measurement on the obtained sample. Preferably, measuring is performed in an automated manner. According to one embodiment, the method comprises measuring the cell density of the cell culture in the cell vessel one or more time(s) in an automated manner, preferably, by providing a suitable sensor or measurement device for measuring the cell density. For instance, the cell vessel may comprise or be connected to a sensor for measuring the cell density. In a preferred alternative, the cell vessel is fluid ical ly connected to a recirculation loop, which comprises or can be coupled to a suitable sensor or measurement device for measuring the cell density. In one example, the suitable sensor or measurement device for measuring the cell density may be based on microscopy.

[0083] Measuring of the cell density may be performed one or more times. It is particularly advantageous to measure the cell density throughout expanding the cells in order to monitor the expansion process. It is also advantageous to measure the cell density prior to and / or subsequent to a unit operation for monitoring and adjusting parameters of the unit operation. For instance, it can be measured whether the unit operation had a positive or negative impact on the cell when measuring the cell density priorto and subsequent to a unit operation. Measuring the cell density priorto the unit operation can further have the advantage that the volume of liquid composition and particularly the amount of agent added to the cell vessel can be adjusted in accordance with the cell density.

[0084] Hence, according to a preferred embodiment, the method comprises on basis of the measured cell density automatically adding a suitable volume of liquid composition to the cell vessel in step i., in particular wherein the liquid composition comprises an activation agent and / or genetic modification agent, such that the volume of the liquid composition is suitable, preferably adjusted, for activation and / or genetic modification of the cells.

[0085] According to a preferred embodiment, the method further comprises one or more of: priorto performing the one or more unit operation(s) on cells in a cell culture in an automated manner, introducing the cell culture into the cell vessel, preferably wherein introducing is performed in an automated manner; and / or removing liquid from and / or adding liquid to the cell vessel priorto step i., preferably wherein the cell density is adjusted.

[0086] Introducing of the cell culture into the cell vessel in an automated manner is advantageous, as handling errors are minimized. Removing liquid from and / or adding liquid to the cell vessel to adjust the cell density prior to step i. and thus prior to performing a unit operation allows for adjusting the cell density in accordance with the requirements for the unit operation. For instance, the cell density may be adjusted for performing a cell activation and / or genetic modification of the cells as unit operation, such that adequate or even optimized conditions for the respective unit operation in terms of cell density are achieved. "Adjusted" herein does not need to mean optimized by suitable for the respective unit operation. Specific embodiments of the method according the first aspect

[0087] According to a preferred embodiment, the method has the following characteristics: optionally, prior to step i., the method comprises measuring the cell density, preferably in an automated manner; the liquid composition in step i. comprises an activation agent, which is at least partially removed in step iii.; the method comprises performing a further unit operation, wherein the liquid composition in step i. comprises a genetic modification agent, preferably a transduction agent; and optionally, wherein subsequent to step iii. another liquid is added, preferably being a cell culture medium.

[0088] One particular process of such embodiment is outlined in Fig. 9. Optionally, between the steps of such embodiment, a washing composition may be added to the cell vessel and thus to the cells and subsequently removed for washing the cells, e.g. after activation and / or after genetic modification. Such washing may be conducted one or more times.

[0089] According to a preferred embodiment, the method has the following characteristics: optionally, prior to step i., the method comprises measuring the cell density, preferably in an automated manner; the liquid composition in step i. comprises o an activation agent, o a genetic modification agent, preferably a transduction agent or a transfection agent, and o optionally an enhancer, which are at least partially removed in step iii.; and optionally, wherein subsequent to step iii. another liquid is added, preferably being a cell culture medium.

[0090] Optionally, between the steps of such embodiment, a washing composition may be added to the cell vessel and thus to the cells and subsequently removed for washing the cells, e.g. after removing step iii. Such washing may be conducted one or more times.

[0091] According to a preferred embodiment, the method has the following characteristics: optionally, priorto step iii., the method comprises measuring the cell density, preferably in an automated manner; the liquid composition in step i. comprises an activation agent, which is at least partially removed in step iii.; the method comprises adding a further liquid composition to the to the cell vessel (4) after step i. and prior to step ii., such that o the further liquid composition is present in the cell vessel (4) when incubating the cells in step ii., and o removing in step iii. comprises removing at least part of the further liquid composition, wherein the further liquid composition comprises o a genetic modification agent, preferably a transduction agent or a transfection agent, and o optionally an enhancer; and optionally, wherein subsequent to the step iii. another liquid is added, preferably being a cell culture medium.

[0092] One particular process of such embodiment is outlined in Figs. 10 and 11. Optionally, between the steps of such embodiment, a washing composition may be added to the cell vessel and thus to the cells and subsequently removed for washing the cells, e.g. after removing step iii. Such washing may be conducted one or more times.

[0093] According to a preferred embodiment, the cells in the cell vessel are expanded after performing the one or more unit operation(s), and wherein the method comprises performing one or more unit further operation(s) after expanding the cells in the cell vessel, preferably wherein such method has the following characteristics: optionally, prior to step i., the method comprises measuring the cell density, preferably in an automated manner; the liquid composition in step i. comprises an activation agent, which is at least partially removed in step iii.; after step iii., expanding the cells in the cell vessel (4); after expanding the cells, adding a further liquid composition to the cell vessel comprising a genetic modification agent, preferably a transduction agent or transfection agent, more preferably a transfection agent, and incubating the cells in the cell vessel (4) with the further liquid composition; and optionally, harvesting the cells.

[0094] One particular process of such embodiment is outlined in Fig. 12. Optionally, between the steps of such embodiment, a washing composition may be added to the cell vessel and thus to the cells and subsequently removed for washing the cells, e.g. after removing step iii. Such washing may be conducted one or more times. According to a preferred embodiment, the method has the following characteristics: optionally, priorto step iii., the method comprises measuring the cell density, preferably in an automated manner; performing a first unit operation, wherein the liquid composition in step i. comprises an activation agent, which is at least partially removed in step iii.; the method comprises performing a second unit operation, wherein the liquid composition in step i. comprises a genetic modification agent, preferably a transfection agent; optionally, wherein subsequent to performing a second unit operation another liquid is added, preferably being a cell culture media; the method comprises performing a third unit operation, wherein the liquid composition in step i. comprises a genetic modification agent, preferably a transduction agent; optionally, wherein subsequent to performing a third unit operation another liquid is added, preferably being a cell culture media; optionally, expanding the cells in the cell vessel; and optionally, harvesting the cells.

[0095] One particular process of such embodiment is outlined in Fig. 13. Optionally, between the steps of such embodiment, a washing composition may be added to the cell vessel and thus to the cells and subsequently removed for washing the cells, e.g. after removing step iii. Such washing may be conducted one or more times.

[0096] Step i.

[0097] The method according to the first aspect of the invention comprises performing one or more unit operation(s) on the cells in an automated manner, wherein a unit operation comprises step i. adding a liquid composition to the cell vessel. The skilled person is aware of different means and system for "adding" of a liquid composition in an automated manner, such that the particular configuration shall not be limited in the present disclosure. For instance, the skilled person is aware of a pump-based system for adding a liquid composition in an automated manner, such as peristaltic pumps.

[0098] Step ii.

[0099] The method according to the first aspect of the invention comprises performing one or more unit operation(s) on the cells in an automated manner, wherein a unit operation comprises step ii. incubating the cells in the cell vessel with the added liquid composition. Incubating facilitates the respective unit operation and typically occurs under the respective required conditions. For instance, incubating may be performed under adequate temperature, humidity and atmosphere for performing the respective unit operation. According to a preferred embodiment, step ii. further comprises agitating the cells in the cell vessel, such as by a rocking motion or wave motion.

[0100] Step Hi.

[0101] The method according to the first aspect of the invention comprises performing one or more unit operation(s) on the cells in an automated manner, wherein a unit operation comprises step iii. removing liquid from the cell vessel through at least one filter, and thereby removing at least a part of the added liquid composition. Such removal step has the advantage that a respective liquid composition can be removed from the cell vessel, such that the contact between the cells and the liquid composition is reduced or mitigated. By optional washing steps, the liquid composition may be essentially completely removed from the cell vessel. At the same time, the cells essentially remain within the cell vessel. The skilled person is aware of different means and system for "removing'' a liquid composition in an automated manner, such that the particular configuration shall not be limited in the present disclosure. For instance, the skilled person is aware of a pump-based system for adding a liquid composition in an automated manner, such as peristaltic pumps.

[0102] For example, the optional washing step may consist of perfusing, preferably wherein an amount of liquid equal to at least lx, 2x, 3x, 5x or lOx the volume of the cell culture is exchanged. It is preferred that the perfusing adds liquid medium, such as fresh cell culture medium, to the cell culture. It is further preferred that the perfusing is carried out for a time period of sufficient length such that the cultured cells are not negatively affected by the act of perfusion. For example, the perfusion speed may be 15 VVD or less, 10 VVD or less, 8 VVD or less, or 4 VVD or less.

[0103] Alternatively, the optional washing step may comprise the following steps: removing a first quantity of liquid from the cell vessel, adding a second quantity of a liquid medium to the cell vessel, and rocking the cell vessel for a period of time.

[0104] Therein, the first and second quantities are preferably the same volume, e.g. up to 33% or up to 50% of the volume of the cell culture. The period of time is preferably an amount of time sufficient for thorough mixing of the contents of the cell vessel, such as at least 1 minute, preferably at least 5 minutes or at least 10 minutes, e.g. 30 minutes.

[0105] The cell culture cultured in the cell vessel may benefit from an optional washing step. For example, after T cell activation, washing and thereby removing the activation reagent (e.g., activation beads) may render the cells more susceptible to uptake of a transgene, in turn resulting in an improved transfection ortransduction rate when the washing step has been carried out. The methods of the present invention may thus result in improved automated transfection / transduction.

[0106] Expansion

[0107] The method according to the first aspect comprises expanding the cells in the cell vessel, wherein expanding is performed prior to and / or after performing the one or more unit operation(s). Preferably, the cells undergo at least one unit operations prior to being expanded, in some cases also more than one unit operations. In some cases, the expansion occurs after performing one or more unit operation(s) but one or more further unit operation(s) may be performed after expansion. Different embodiments are illustrated in Figs. 9 to 13.

[0108] According to a preferred embodiment, the step of expanding the cells in the cell vessel involves agitating the cells, such as by a rocking motion or wave motion.

[0109] According to a preferred embodiment, expansion comprises one or more of batch, fed-batch, and / or perfusion culture, preferably, expansion at least comprises perfusion culture.

[0110] According to a preferred embodiment, a volume of the cell culture in the cell vessel increases during the step of expanding the cells, preferably after performing the at least one unit operation on the cells.

[0111] According to a preferred embodiment, the volume of the cell culture in the cell vessel increases by at least 10 %, preferably at least 50 %, more preferably at least 100 %, more preferably at least 200 %, more preferably at least 300 %, more preferably at least 400 %, more preferably at least 500 %, more preferably at least 600 %, more preferably at least 700 %, more preferably at least 800 %, more preferably at least 900 % during the step of expanding the cells.

[0112] According to a preferred embodiment, the volume of the cell culture in the cell vessel at the end of the step of expanding the cells is at least 100 ml, preferably at least 300 ml, more preferably at least 500 ml, more preferably at least 1 1.

[0113] According to a preferred embodiment, the volume of the cell culture in the cell vessel is at least 5 ml prior to step i. Preferably, the volume of the cell culture in the cell vessel at most 250 ml, preferably 200 ml, more preferably 150 ml, more preferably 120 ml, e.g., 100 ml or 80 ml. According to a preferred embodiment, the volume of the cell culture in the cell vessel in is at most 100 ml prior to and / or subsequent to step i.

[0114] According to a preferred embodiment, the unit operation is performed with a volume of cell culture and added fluid in the cell vessel selected from the range of 1 to 200 ml, preferably 5 to 100 ml, particularly, wherein the step of expanding the cells is performed after performing the unit operation(s).

[0115] According to a preferred embodiment, the method comprises removing liquid from the cell vessel and thereby decreasing a volume by at least 10 %, preferably at least 30 %, more preferably at least 50 %, of the cell culture, in particular after performing the unit operation(s) and / or expanding the cells.

[0116] Further optional steps of the method according to the first aspect

[0117] According to a preferred embodiment, the method further comprises a step of washing the cells one or more time(s) after performing the unit operation(s), preferably wherein washing comprises: i. adding a washing liquid composition to the cell vessel; ii. optionally, incubating the cells in the cell vessel with the added washing fluid; and iii. removing fluid from the cell vessel through at least one filter, and thereby removing at least a part of the added washing fluid.

[0118] According to a preferred embodiment, the method further comprises harvesting the cells, preferably after performing the unit operation(s) and expanding the cells. According to a preferred embodiment, harvesting comprises transferring the cell culture from the cell vessel, preferably through a port of the cell vessel, optionally in an automated manner. According to a preferred embodiment, harvesting comprises removing liquid from the cell vessel through the at least one filter for increasing the cell density and afterwards, transferring the concentrated cell culture from the cell vessel, preferably through a port of the cell vessel, optionally in an automated manner.

[0119] According to a preferred embodiment, the method further com prises formulating the cell culture into a cell therapy product. According to a preferred embodiment, formulating is performed after performing the unit operation(s) and expanding the cells. According to a preferred embodiment, formulating comprises: i. removing liquid from the cell vessel through the at least one filter; ii. adding a liquid for formulating the cells culture into a cell therapy product to the cell vessel; and iii. optionally, transferring the cell therapy product from the cell vessel, preferably through a port of the cell vessel, optionally in an automated manner.

[0120] Bioprocessing system

[0121] According to a preferred embodiment, the method comprises using a bioprocessing system comprising a control system and at least one active element, wherein the control system implements control instructions to cause the at least one active element to advance the unit operation.

[0122] According to a preferred embodiment, the control system generates control instructions to cause the active element to add the liquid composition to the cell vessel in step i. and / or remove the liquid from the cell vessel through the at least one filter in step iii.

[0123] According to a preferred embodiment, the at least one active element comprises at least one pump for adding the liquid composition to the cell vessel and / or for removing liquid from the cell vessel through the at least one filter.

[0124] According to a preferred embodiment, the cell vessel comprises two or more compartments, preferably a small volume compartment and a distinct large volume compartment. "Distinct" does not mean that a fraction of the small volume compartment and the large volume compartment overlap or are in contact with each other, however, that the vessel is typically divided in two compartments. Typically, the cells would be initially in one compartment, e.g. when performing one or more unit operation(s) in the small volume compartment, whereas throughout expanding the cells, these are typically in both compartments. Such cell vessel advantageously be used for cultivating cells from small to large scale. Specifically, the small volume compartment defines a volume, in which the cells can be initially cultivated and / or undergo the unit operation(s). Such volume is generally lower than the volume of the large volume compartment, allowing for small-scale culture. At the same time the small volume compartment by being adjacently arranged to the small volume compartment is preferably fluidically connected for fluidical exchange providing access to the typically larger volume of the volume defined by the large volume compartment. Therefore, the cells can be expanded with increasing volume in an adequate environment, i.e., initially in the small volume compartment volume, followed by culture in the volume of the large volume compartment (and small volume compartment volume). Thereby, scale-up is achieved without the necessity to change the vessel (e.g., by transfer to a larger vessel). At the same time, the small volume compartment and large volume compartment provide an adequate culture environment for the respective needs throughout culture. For instance, initially the cells may be present in a small volume compartment, requiring small surface areas in order to minimize evaporation effects. Later in culture, more volume is needed for obtaining a suitably high cell number, such that the large volume compartment provides an adequate extension of the small volume compartment. Such culture mode is particularly advantageous for manufacture of a cell therapy product, such as autologous immune cell therapy (e.g. CAR T-cells), since initially the cell number obtained from a patient sample is comparably low and requires expansion for manufacturing the cell therapy product. Thus, despite a low cell number and also a low culture volume, the cells can be cultured in the very same vessel during the complete expansion process. This not only simplifies the overall process but also makes it less prone to errors and reduces contamination risks. As is demonstrated in the Examples below, also higher daily expansion rates and cell viabilities can be achieved, such that cultivation duration can be shortened and product quality is improved (see e.g., Figs. 6 and 7).

[0125] According to a preferred embodiment, the cell vessel comprises a pre-shaped reservoir forming a small volume compartment. By being "pre-shaped", the reservoir is dimensionally stable such that the reservoir volume is fully developed and does not require provision of a further device or system, e.g. rocker / rocking motion platform, in orderto form the reservoir. "Pre-shaped" does not exclude that the reservoir can be generally deformed, however, it is to be understood that the reservoir has a certain defined shape independent of other (external) devices or system (such as a rocker platform). It is possible that the reservoir returns to its pre-shaped form when a deformation force decreases or disappears. By being pre-shaped, the reservoir can be reproducibly and stably provided enabling a more robust cultivation process. In particular, it can be achieved to provide the same volume (here reservoir volume) for each culture batch. Furthermore, by being pre-shaped, the reservoir is more resistant to deformation and material rupture compared to a reservoir or chamber that forms only when combined with a culture platform or system, such that failure or contamination risks are reduced.

[0126] According to a preferred embodiment, cell vessel comprises a casing forming a large volume compartment, wherein the casing is preferably flexible. A "casing" is well-known to the person skilled in the art. Typically, a casing of a vessel refers to a bag or pouch, which is preferably flexible but may also be rigid or semi-rigid. Preferably, the casing in the present disclosure is at least partially flexible to adapt throughout culture and increasing culture volumes during expansion. The vessel according to the present disclosure may further comprise a small volume compartment, which may be connected or from an integral part of the large volume compartment being the casing. Generally, the casing may be pre-shaped. However, it is also possible that the casing is flexible and particularly obtains its form during the cultivation procedure, for example because of rising pressure or weight inside the vessel.

[0127] The casing may preferably be a single-use bag which are widely used in the field of cell culture, for instance in single-use bioreactors or for storing or conveying cell fluids, such as cell medium, culture buffer or cell culture. Such bags may comprise at least one casing part, which may also be referred to as "wall''. Hence, according to a preferred embodiment, the casing comprises a casing part. Such casing part may be folded and sealed together in order to form a bag-like structure. Typically, a bag may comprise two (large) walls (here casing parts) sealed to one another. Once expanded, they have a limited volume and remain relatively thin, which justifies the fact that they are often called 2D bags (D meaning dimensions). 3D bags are also known and typically comprise two end walls and a side wall that can be folded flat or deployed unfolded, sealed to one another, with the volume able to reach up to 3,000 liters, and even more. Hence, according to a preferred embodiment, the casing comprises two or more casing parts, such as an upper casing part and a lower casing part. Such casing parts may be connected in orderto form a bag-like structure (also referred to as 2D bags). It is also within the scope of the present disclosure that the casing comprises three casing parts, or four casing parts, or five casing parts or more than five casing parts. This may allow forming 3D casings (e.g., 3D bags).

[0128] The casing part may be composed of a multilayer film comprising a contact layer which in contact with the medium that fills the casing, a barrier layer and an outer layer which is in contact with the external environment of the casing, the three layers being connected one to each other with a tie layer. In case the casing is to be filled with a biopharmaceutical product, the contact layer should be made from a material that can be in contact with this biopharmaceutical product without causing degradation of the film and of the biopharmaceutical product. Furthermore, it must be sealable on itself. For that purpose, the material is generally selected from polyolefins, such as polyethylene. The barrier layer provides a barrier to the passage of gases such as oxygen, carbon dioxide and is typically made from ethylene vinyl alcohol (EVOH). The outer layer contributes to the mechanical strength of the casing. For that purpose, it must be sufficiently flexible to withstand high mechanical stress but not be too much stretchable in order to prevent deformation of the casing when it is filled.

[0129] According to a preferred embodiment, the method has following characteristics: during performing the unit operation(s), the cell culture is located in the small volume compartment; and / or during expanding the cells in the cell vessel, preferably after step iii., the volume of the cell culture increases such that the cell culture is preferably located in the small volume compartment and the large volume compartment.

[0130] According to a preferred embodiment, the one or more of the at least two compartments comprise at least one filter and / or is / arefluidically connected to at least one filter. The term "filter" is well-known by the person skilled in the art. A "filter" is typically used to remove particles from a fluid process stream and comprises a porous medium. A filtration process is a process by which particles are removed from a fluid by passing the fluid through a porous medium. Herein, the filter is particularly used in order to retain the cells within the vessel. The remaining filtered cells may be referred to as "retentate". The fluid passing the filter essentially does not comprise the cells. The passing fluid may be referred to as "permeate". The selection of a filter is within the skills of the person of ordinary skill in the art. In particular, the skilled person is well aware of various filters for retaining cells but allowing liquid to pass. Selection of an adequate pore size or molecular cutoff is within the routine of the skilled person. Hence, the present disclosure shall not be limited to a particular filter material or pore size, insofar as the material and pore size should be suitable for cell culture.

[0131] Preferably, the filter is configured to allow liquid to flow through but essentially retain cells. This does not mean that all cells are completely retained but the majority of cells shall be retained. Also, this does not exclude that cell debris or apoptotic cells (which are oftentimes smaller in size than viable cells) pass the filter.

[0132] According to one embodiment, the at least one filter comprises a filter medium selected from a surface filter and a depth filter.

[0133] The term "membrane" is known in the art. Various membrane materials can be used, including polyvinylidene difluoride (PVDF), polyethersulfone (PES), or polysulfone (PS). In one embodiment, the membrane is a PES membrane, optionally having a cutoff of 0.5 to 5 micrometer, such as 1.2 micrometer.

[0134] According to a preferred embodiment, the small volume compartment comprises at least one filter and / or is flu idically connected to at least one filter, and / or the large volume compartment comprises at least one filter and / or is fluid ical ly connected to at least one filter. According to a preferred embodiment, the large volume compartment comprises at least one filter which is a main membrane, preferably the main membrane is not used during performing the unit operation(s), and / or, that the main membrane is used for perfusion during expanding the cells in the cell vessel. According to one embodiment, the at least one filter comprises a main membrane, and / or a reservoir membrane, preferably both of a main membrane and a reservoir membrane. A "main membrane" herein typically refers to a membrane that is arranged at the large volume compartment, which may also be referred to as a "casing". A "reservoir membrane" herein typically refers to a membrane that is arranged at the small volume compartment, which may also be referred to as the "reservoir". The reservoir membrane advantageously allows to perform a filtration process at larger scale, e.g. when the cell culture is present in the casing (and typically also the reservoir). The main membrane can therefore be advantageously used to perform medium to large volume liquid filtrations, e.g. as may be required throughout a perfusion culture.

[0135] According to a preferred embodiment, the small volume compartment comprises at least one filter which is a reservoir membrane, preferably the reservoir membrane is used for performing the unit operation(s), in particular by removing liquid from the cell vessel through the reservoir membrane in step iii.

[0136] According to a preferred embodiment, the at least one filter is configured to allow liquid to flow through but essentially retain the cells; and preferably, is not applied for gas exchange, in particular for gas exchange between the cell culture in contact with the filter and the gas in contact with the other side of the filter.

[0137] According to a preferred embodiment, the at least one filter is a membrane, such as a planar membrane, a hollow fiber membrane and / or floating membrane. A floating membrane may also be referred "pouch membrane", in particular being f lu id ically connected to the outside of the vessel, especially at least to the waste container (which may also be referred to as the "waste container").

[0138] According to a preferred embodiment, the cell vessel comprises at least one filter and / or is fluidically connected to at least one filter, a tube set connected to the cell vessel and presterilized together with the cell vessel.

[0139] According to a preferred embodiment, the tube set comprises: at least one disposable head for a pump; at least one, in particular magnetic, clocking element for a sensor; a recirculation loop; a sealed and weldable connection for a media container; and / or a sealed and weldable connection for a waste container.

[0140] According to a preferred embodiment, the bioprocessing system further comprises a bioreactor module comprising the cell vessel.

[0141] According to a preferred embodiment, the bioprocessing system, preferably the bioreactor module, further comprises a rocker element for imposing a rocking motion onto the cell vessel.

[0142] According to a preferred embodiment, the bioreactor module comprises at least one sensor for sensing a characteristic of the cell culture, preferably wherein the control system during the unit operation receives sensor data from the at least one sensor and derives from the sensor data the control instructions.

[0143] According to a preferred embodiment, the at least one sensor comprises a sensor measuring a characteristic of the cell culture, in particular a o number of cells per volume; o a cell size; o a cell viability; and / or o a pH value of the cell culture; and / or a balance weighing o the cell culture; o an amount of liquid added to the cell culture; and / or o an amount of liquid removed from the cell culture.

[0144] According to a preferred embodiment, control system determines from a measured cell size a point in time at which to start the genetic modification, generates control instructions to control the at least one pump to add the fluid in step i. comprising a genetic modification agent to the cell vessel at the point in time and implements the control instructions.

[0145] According to a preferred embodiment, the bioreactor module further comprises: a media container connected to the cell vessel; a waste container connected to the cell vessel; a bypass line for connecting a container outside the bioreactor module to the cell vessel; a control unit as part of the control system; and / or a heating element for heating the cell vessel.

[0146] According to a preferred embodiment, the bioreactor module further comprises a recirculation loop leading from the cell vessel to the cell vessel.

[0147] According to a preferred embodiment, the recirculation loop has one or more of the following characteristics: it comprises at least one sensor, in particular a sensor for cell counting; it is connected to the media container in particular through a bypass line; it is connected to the waste container, in particular through a recirculation loop membrane, and / or the bypass line.

[0148] According to a preferred embodiment, the bioreactor module comprises a support structure for carrying the cell vessel, and optionally, the media container.

[0149] According to a preferred embodiment, the bioreactor module comprises at least one tube holder holding at least one tube in a defined position relative to a support structure.

[0150] According to a preferred embodiment, the tube holder comprises a tube mover mechanism for extending and / or retracting the tube relative to the tube holder.

[0151] According to a preferred embodiment, the tube holder is configured for interacting with a connection arrangement for aseptically connecting and / or disconnecting containers, preferably wherein the connection arrangement comprises a tube welding unit to weld and / or cut and seal the tube, preferably in an automated manner. The welding unit may employ thermal, ultrasonic, or other suitable welding techniques to ensure a secure and sterile seal. To verify the integrity of the sealed connection, the bioprocessing system may further include a seal testing unit capable of assessing whether the sealing process was successful. This testing may be performed by applying pressure, vacuum, or other diagnostic methods to detect leaks or structural weaknesses in the sealed region.

[0152] According to a preferred embodiment, the bioprocessing system comprises a connection arrangement for aseptically connecting and / or disconnecting containers, preferably, that the connection arrangement comprises a tube welding unit for welding together and / or cutting and closing off tubes. According to a preferred embodiment, the method comprises welding a tube to the bypass line and / or a tube connected to the media container and / or a tube connected to the waste container by the tube welding unit.

[0153] According to a preferred embodiment, the bioprocessing system has one or more of the following characteristics: it comprises a frame; it comprises at least one expansion location at which the expansion operation and preferably the unit operation is performed, in particular supported by the bioprocessing system; it performs a replenishing of the media container; and / or it comprises a transport mechanism which transports the bioreactor module, in particular out of the expansion location and / or into the expansion location.

[0154] Cells

[0155] According to a preferred embodiment, the cells are mammalian cells, preferably immune or naive cells. According to one embodiment, the cells are immune cells, preferably T cells. The term "immune cells" is well known in the art and the skilled person is well-aware and understands the term "immune cells". Immune cells generally refer to types of white blood cells. Any type of white blood cells may be used here. Combinations of different types are also conceivable. According to a preferred embodiment, however, the term "immune cells" herein refers to a single type of cells, e.g. T-cells or subtypes thereof, or NK-cells or subtypes thereof. Hence, the term "immune cells" includes a variety of cells, for example, but not limited to dendritic cells, T lymphocytes, also referred to as T cells, B lymphocytes, natural killer cells, macrophages or the like. Immune cells may also include subtypes of immune cells, for example tumor-infiltrating lymphocytes or different types of T cells. Subtypes of a certain type of immune cells may be classified based on the type of antigen present at the cell surface. Hence, the term immune cells may for example refer to T cells comprising the surface antigen CD4 ("CD4+ T cells").

[0156] Preferably, the T cells are genetically modified in one of the unit operations to express a chimeric antigen receptor (CAR). Consequently, the term "CAR-T cells" describes T cells that have been genetically modified to express a CAR. Hence, preferably, the genetically modified T cells are genetically modified CAR-T cells.

[0157] According to a preferred embodiment, the cell culture comprises the cells and a liquid, in which the cells are present, preferably the liquid being a cell culture medium. According to one embodiment, the cells are dedicated for cell and / or gene therapy, preferably dedicated for cell therapy.

[0158] Cell selection

[0159] According to a preferred embodiment, the method comprises a cell selection, preferably prior to performing the unit operation(s). Cell selection is advantageous in order to select specific cells within a population of cells, which then undergo the one or more unit operation(s). Particularly, in manufacture of cell therapies, cell selection is performed. The present disclosure hence allows performing cell selection within the method according to the first aspect or outside of the method, e.g. by providing cells in a cell culture which have already undergone a cell selection.

[0160] According to a preferred embodiment, the cell selection comprises: positive cell selection, wherein a target fraction of the cells directly or indirectly binds to a binding matrix of a binding unit; or negative cell selection, wherein a target fraction of the cells does not directly or indirectly bind to a binding matrix of a binding unit.

[0161] According to a preferred embodiment, cell selection comprises: adding a mixture of cells to the cell vessel, contacting the mixture of cells with a binding unit, wherein at least a fraction of the cells directly or indirectly binds to a binding matrix of the binding unit, and removing liquid from the cell vessel, thereby removing at least part of the cells, which do not directly or indirectly bind to the binding matrix.

[0162] According to a preferred embodiment, the binding matrix is configured for allowing direct or indirect binding of a ligand expressed by at least a fraction of the cells.

[0163] According to a preferred embodiment, indirect binding comprises adding at least one binding reagent to the cell vessel, such that the mixture of cells is contacted with the binding reagent, and it binds to at least a fraction of the cells, and wherein the binding reagent is configured for binding to the binding matrix.

[0164] According to a preferred embodiment, the cell vessel comprises a binding unit; and / or is fluidically connected to at least one a binding unit, wherein the binding unit comprises a binding matrix configured for allowing direct or indirect binding of a ligand expressed by at least a fraction of the cells. Such binding unit is particularly suitable for performing cell selection. In particular, by providing a binding unit, at least a fraction of the cells in the liquid medium may be sorted by expressing a ligand that binds to the binding matrix. Cells that do not express such ligand would not bind to the binding matrix. Such binding may be referred to as "direct" binding of the matrix to the ligand expressed in at least a fraction of the cells. Alternatively, one or more binding reagent(s) may be provided, which allows for "indirect" binding of the binding matrix to the ligand expressed in at least a fraction of the cells. In such case, the one or more binding reagent(s) usually bind to the ligand expressed by the cells, e.g. an antibody or antibody fragment capable of binding to the ligand expressed by the cells. The binding reagent then usually further comprises a moiety for binding to the binding matrix, e.g. biotin, avidin, streptavidin, such that the cells that express the ligand indirectly (i.e., via the binding reagent) bind to the binding matrix. When providing more than one binding reagent, a first binding reagent may bind to the ligand expressed by the cells, whereas the second binding reagent binds to the binding matrix and both, the first and second binding reagent bind to each other. Further configurations or chemistries may be applied, which achieve the desired outcome, i.e. direct or indirect binding of the cells expressing the ligand to the binding matrix. The skilled person is well-aware of suitable binding matrices and chemistries for allowing binding of a ligand expressed by at least a fraction of the cells.

[0165] The binding unit may be fluidically connected to the vessel, wherein such fluidic connection may be provided in form of tubes. There may be more than one fluidic connection, such as preferably two fluidic connections, in particular at different sides or positions of the binding unit. For instance, there may be one fluidic connection from the vessel to the entrance of the binding unit and one fluidic connection from the exit of the binding unit to the vessel. Further fluidic connections may be present, which allow also fluid to be transferred through the binding unit into another container, e.g. waste container or storage container. The fluidic connection(s) between the vessel and the binding unit may be located at the casing and / or the reservoir of the vessel. Preferably, there is one fluidic connection between the reservoir and the binding unit and another fluidic connection between the casing and the binding unit. Other configurations are also possible, for instance, there may be two fluidic connections between the casing and the binding unit. Or, alternatively, two fluidic connections between the reservoir a nd the binding unit.

[0166] After binding of the at least fraction of the cells, liquid medium present in the vessel can be removed, e.g. through the binding matrix or a fluidic line connected to vessel, and with the liquid the unbound cells (which then do not express the ligand). As a result, only cells expressing the ligand would remain in the vessel, i.e., a positive cell selection is performed. In orderto release the cells from the binding matrix, these may be eluted as commonly known in the art (e.g. by providing a release agent, such as salt, biotin, avidin or streptavidin). Eluted cells would then be released into the liquid medium present in the vessel. Preferably, the cells can be recirculated into the vessel, e.g. by pressing liquid through the binding unit which elutes the cells and then into a fluidic line connected to the vessel, such that the released cells recirculate into the cells. Afterwards, the binding unit may be closed or becomes inactive. The binding unit may also still be left "open", so in principle being accessible to the cells. However, no force or pressure is preferably applied towards the binding unit, such that the cells are not actively moved into the binding unit. The binding unit may also be closed, e.g., by closing a valve or a cover that closes the binding unit from the vessel.

[0167] Also, a negative cell selection step may be performed. For instance, after binding of the at least fraction of the cells, liquid medium present in the vessel can be removed, e.g. through the binding matrix or a fluidic line connected to vessel, and with the liquid the unbound cells (which then do not express the ligand). These unbound cells can then be collected, e.g. in a container. In order to release the cells from the binding matrix, these may be eluted as commonly known in the art. Eluted cells would then be released into the liquid medium present in the vessel and can then be removed through a waste port. Preferably, the cells can be released from the binding unit, e.g. by pressing liquid through the binding unit which elutes the cells and then into a fluidic line connected to a waste container, such that the released cells are removed from the vessel. Afterwards, the collected cells may be recirculated back to the vessel.

[0168] According to some embodiments of the present disclosure more than one cell selection step is performed, such as at least two cell selection steps. These may comprise positive and / or negative cell selection, as disclosed herein.

[0169] According to a preferred embodiment, the binding matrix is provided as a resin or monolithic material. Various materials are known in the art for binding matrices. Particularly preferred may be a binding matrix which comprises resin beads. A binding matrix may be covered or enclosed by one or more membranes, which separates the cells from the binding matrix, as long as no force is applied that would drag the cells in contact with the binding matrix.

[0170] According to a preferred embodiment, the binding unit is located within or adjacent to a small volume compartment of the cell vessel, preferably wherein the binding unit is arranged adjacent to a filter of the small volume compartment. By providing the binding unit located within or adjacent to the small volume compartment, small volumes of cell culture or cell suspension can be handled. Specifically, the cells may be provided as a mixture of cells, which need to undergo a cell selection step utilizing the binding matrix. Since the cells are in such stage oftentimes present in low volume cell cultures, it is advantageous to provide the binding unit in the small volume compartment of the vessel, which can be advantageously used to handle and culture low volume cell cultures. In addition, the small volume compartment is preferably pre-shaped, such that it is mechanically more stable than a reservoir that only shapes upon interaction with other devices throughout culture. Thus, the binding unit has then a fixed position, allowing for precise cell selection.

[0171] In the embodiments, wherein the binding unit is located within or adjacent to the small volume compartment of the vessel, the binding unit may be provided as a column, wherein the column is predominantly present internally, i.e., within the vessel, particularly within the small volume compartment, such that the column reaches into its volume. Such "internal column" configuration advantageously allows for the cells present in the cell vessel and small volume compartment, respectively, to be in direct connect with the column, such that no additional fluid transfer step is required. In such configuration, it may be advantageous to provide the binding matrix within the column covered by a membrane, such that the cells are not always in contact with the binding matrix in order to control the cell selection. As a result, the cells only interact with the binding matrix, once a force is put on the cells that drags them through the membrane into the column and thus in contact with the binding matrix. As a result, the cell selection is only occurring when such force is applied. According to one embodiment, an active element, e.g. a pump, is provided that manipulates the fluid in the vessel such that the fluid is dragged into the column. For instance, a pump may draw the fluid and with it the cells present in the fluid within the vessel into the column, such that the cells come in contact with the binding matrix, such that at least a fraction of the cells bind to the binding matrix. According to one embodiment, the binding unit further comprises a valve, which is configured for closing the fluid lines exiting the binding unit. This advantageously closes the binding matrix, such that cells are not dragged actively into the binding unit and thus in contact with the binding matrix.

[0172] According to a preferred embodiment, the binding unit is provided as a column, preferably wherein the opening of the column is arranged within an opening of a small volume compartment of the cell vessel, more preferably an opening at the bottom of the small volume compartment, and / or the column is fl uidica I ly connected to the cell vessel, preferably a reservoir, wherein fluidic connections between the cell vessel and the column are provided, in particular wherein the fluidic connections are provided by one or more tubes, optionally further comprising a valve. This has the advantage that the column can be used as known in the art for cel I selection columns, however, as implementation of the cell vessel, such that cell selection can be performed with the same vessel, in which cell culture can take place, as well as the at least one unit operation, e.g., cell activation, cell transduction, cell transfection, but also media exchange, and / or volume reduction. Hence, relevant steps for manufacture of a cell therapy product can be performed within the same vessel, significantly simplifying the process. Additionally or alternatively, the binding unit is provided as a column which is fl uidica I ly connected to the vessel, preferably the reservoir, wherein fluidic connections between the vessel and the column are provided, in particular wherein the fluidic connections are provided by one or more tubes, optionally further comprising a valve. Fl uidica I ly connecting the column and the reservoir has the advantage that the binding unit is separate from the vessel allowing for separate control. At the same time both are usually in close proximity, such that for instance a temperature control would effectively heat the vessel and the column, which is advantageous for maintaining high cell viability. Optionally, the fluidic connection, such as one or more tubes further comprise a valve. This has the advantage that the fluidic connection between the binding unit (in particular column) and the vessel (in particular reservoir) can be closed, which can be done subsequent to the cell selection.

[0173] Providing the binding unit as a column as disclosed herein the term "column'' shall not be limited in any specific geometry, as is known in the art. While typically columns may be provided in a pillar or column shape, also other shapes are known in the field. A column herein may also refer to a bag-like shape, wherein two or more walls are connected forming a casing. As typically casings who have a bag-like shape are flexible, such column configuration may also be referred to as "flexible column".

[0174] According to a preferred embodiment, the binding unit comprises a flexible column, comprising a casing, an entrance port and an exit port, wherein the casing comprises the binding matrix. The flexible column may further comprise two membranes, which confine the casing volume in which the binding matrix is present, e.g. one membrane at the entrance and one membrane at the exit of the flexible column. The flexible column may be fluid ical ly connected to the vessel, wherein preferably such connection comprises valve to controlling the fluid flow between the vessel and the flexible column. According to a preferred embodiment, the binding unit is provided as a floating unit configured for float within the cell vessel. When providing the binding unit as a floating unit, this may be provided in form of a pouch, which can float within a fluid of the vessel. Such floating unit is typically connected via fluidic line(s) going through the vessel to the outside. Hence, the floating unit can float but within a certain range limited to the length of the fluidic line(s). A floating unit has the advantage that it is present within the vessel, such that contact between the floating unit and the cells within the vessel is enabled. In order to achieve interaction between the cells and the binding matrix of the binding unit, the cells may be forced or pushed into the floating binding unit by overpressure within the vessel and / or under pressure within the binding unit, e.g. created by the fluidic line(s). Bound cells can then, as explained above, be retained within the binding matrix, whereas unbound cells exit the binding unit through the fluidic line(s).

[0175] According to a preferred embodiment, the binding unit is by an external loop fluidically connected to the cell vessel and / or a waste port, preferably both. A specific configuration of such embodiment can be seen in Fig. 8. By providing an external loop it is possible to recirculate the cells though the binding unit into the vessel, particularly the small volume compartment, and thus the vessel again. This has the advantage that cells can undergo multiple rounds of binding to achieve binding of as many cells as possible expressing the ligand that bindstothe binding matrix. Furthermore, this has the advantage that cells that bind to the binding matrix and are subsequently eluted, can exit the binding unit and be recirculated into the cell vessel, in particular into the small volume compartment. Furthermore, by connecting the binding unit to a waste port, it is possible to transfer the unbound cells into a waste container.

[0176] According to a preferred embodiment, the external loop comprises at least one valve, preferably one valve for controlling the fluid flow between the binding unit and the waste port and another valve controlled the fluid flow between the cell vessel and the waste port, preferably wherein both valves are for controlling the fluid flow between the binding unit and the cell vessel via the external loop. A specific configuration of such embodiment can be seen in Fig. 8. By providing the valves, the fluid flow can be controlled, allowing for fluid flow into a waste container and / or recirculation into the vessel, particularly into the small volume compartment. Bioreactor module for performing a bioprocess on cells in a cell culture according to a second aspect of the invention

[0177] According to a second aspect of the invention, a bioreactor module for performing a bioprocess on cells in a cell culture is provided, wherein the bioreactor module comprises a cell vessel, and at least one filter for removing liquid from the cell vessel through the at least one filter, wherein the bioreactor module is configured for performing in an automated manner one or more unit operations on the cells in the cell vessel, and for expanding the cells in the cell vessel.

[0178] The bioreactor module according to the second aspect advantageously simplifies performing a bioprocess on cells in a cell culture by applying a cell vessel that allows for performing the one or more unit operation(s) (e.g., activation, transfection, transduction) on the cells in an automated manner. At the same time, the cell vessel of the bioreactor module is suitable for expanding the cells, such that not only a unit operation but also cell expansion can advantageously be performed in the very same cell vessel. This significantly simplifies processes, such as cell therapy manufacturing, compared to conventional approaches in the art, as the laborious manual processes are performed in an automated manner. As a result, handling errors are avoided, improving reproducibility and mitigating contamination risks. Also, by being able to perform multiple unit operations and expansion in the very same cell vessel, the conditions which are applied on the cells can be tightly controlled. Furthermore, transfer steps are avoided, such that overall, the cells are less negatively impacted, and cell loss is reduced. These positive effects are highlighted by the Examples disclosed herein, showing that higher cell numbers and cell viability can be obtained, indicating more efficient and higher quality cell therapy manufacturing (see Figs. 6 and 7).

[0179] The individual features and preferred embodiments of the bioreactor module according to the second aspect correspond to the individual features and embodiments of the method according to the first aspect insofar as these directly or indirectly relate to the bioreactor module. Therefore, it is referred to the above disclosure which shall equally be applicable to the bioreactor module according to the second aspect. Further features will now be described in detail.

[0180] According to a preferred embodiment, the bioreactor module further comprises one or both of: at least one active element for influencing the cell culture in the bioreactor module, preferably for adding a fluid to and / or removing a fluid from the cell vessel, preferably wherein the fluid is a liquid; and at least one sensor for sensing a characteristic of the cell culture.

[0181] According to a preferred embodiment, the at least one active element comprises at least one pump for adding a fluid to the cell vessel and / or for removing fluid from the cell vessel through the at least one filter.

[0182] According to a preferred embodiment, the cell vessel comprises one or more of the following: two or more compartments, preferably a small volume compartment and a distinct large volume compartment; a pre-shaped reservoir forming a small volume compartment; a casing forming the large volume compartment, preferably wherein the casing is flexible; a main membrane; and / or the control unit and / or an interface to the control system, wherein the control unit is adapted to.

[0183] According to a preferred embodiment, the bioreactor module has the following characteristics: during performing the unit operation(s), the cell culture is located in the small volume compartment; and / or during expanding the cells in the cell vessel, the volume of the cell culture increases such that it is preferably located in the small volume compartment and the large volume compartment.

[0184] According to a preferred embodiment, the cell vessel comprises at least two compartments, wherein at least one of the compartments comprises the at least one filter and / or is / are fluidically connected to the at least one filter.

[0185] According to a preferred embodiment, the small volume compartment comprises the at least one filter and / or is fluidically connected to the at least one filter, and / or the large volume compartment comprises the at least one filter and / or is fluidically connected to the at least one filter.

[0186] According to a preferred embodiment, the large volume compartment comprises the at least one filter which is a main membrane, preferably the main membrane is not used during performing the unit operation(s), and / or, that the main membrane is used for perfusion during expanding the cells in the cell vessel.

[0187] According to a preferred embodiment, the small volume compartment comprises at least one filter which is a reservoir membrane, preferably the reservoir membrane is used for performing the unit operation(s), in particular by removing fluid from the cell vessel through the reservoir membrane.

[0188] According to a preferred embodiment, the bioreactor module comprises a control unit and / or an interface to the control system, wherein the control unit is adapted to, after a cell culture was introduced into the cell vessel, subject the cells to at least one unit operation and an, in particular subsequent, expansion operation inside the cell vessel, and, during the unit operation and / or the expansion operation, implement control instructions to cause the at least one active element to influence the cell culture and thereby advance the unit operation and / or the expansion operation, and / or, wherein the interface to the control system is adapted to allow the control system to implement control instructions to cause the at least one active element to influence the cell culture and thereby advance the unit operation and / or the expansion operation, preferably, wherein the control unit is adapted to receive sensor data from the at least one sensor and derive from the sensor data control instructions and / or, wherein the interface to the control system is adapted to allow the control system to receive sensor data from the at least one sensor.

[0189] According to a preferred embodiment, the cell vessel comprises a tube set connected to the cell vessel, preferably wherein the tube set is pre-sterilized together with the cell vessel.

[0190] According to a preferred embodiment, the tube set comprises at least one disposable pump head; at least one, in particular magnetic, docking element for a sensor; a recirculation loop; a sealed and weldable connection for a media container; and / or a sealed and weldable connection for a waste container. Bioprocessing system for performing a bioprocess on cells in a cell culture according to a third aspect of the invention

[0191] According to a third aspect of the invention, a bioprocessing system for performing a bioprocess on cells in a cell culture is provided, wherein the bioprocessing system comprises the bioreactor module according to the second aspect of the invention.

[0192] According to a preferred embodiment, the bioprocessing system comprises a control system which is adapted to, during the unit operation(s) and / or the expansion step, implement control instructions to cause the at least one active element to influence the cell culture and thereby advance the unit operation and / or the expansion operation.

[0193] The bioprocessing system according to the third aspect advantageously simplifies performing a bioprocess on cells in a cell culture by applying the bioreactor module according to the second aspect. The positive effects are also highlighted by the Examples disclosed herein, showing that higher cell numbers and cell viability can be obtained, indicating more efficient and higher quality cell therapy manufacturing (see Figs. 6 and 7).

[0194] The individual features and preferred embodiments of the bioprocessing system according to the third aspect correspond to the individual features and embodiments of the method according to the first aspect and bioreactor module according to the second aspect insofar as these directly or indirectly relate to the bioprocessing system. Therefore, it is referred to the above disclosure which shall equally be applicable to the bioprocessing system according to the third aspect.

[0195] Uses according to a fourth and fifth aspect of the invention

[0196] According to a fourth aspect of the invention, a use of the bioreactor module according to the second aspect and / or the bioprocessing system according to third aspect for manufacturing a cell therapy product is provided, preferably wherein manufacturing comprises at least one of the following: optionally, selecting cells one or more times, in particular by binding at least a fraction of the cells expressing a ligand, preferably selecting a subpopulation of T cells; activating cells, preferably activating T cells; transducing cells, preferably transducing T cells; transfecting cells, preferably transfecting T cells; and / or expanding cells, preferably T cells, more preferably genetically modified T cells. According to a fifth aspect of the invention, a use of the bioreactor module according to the second aspect and / or the bioprocessing system according to third aspect in a method according to the first aspect of the invention is provided.

[0197] The uses according to the fourth and fifth aspect advantageously simplify performing a bioprocess on cells in a cell culture for manufacturing a cell therapy product. The individual features and preferred embodiments of the uses correspond to the individual features and embodiments of the other aspects of the invention insofar as applicable. Therefore, it is referred to the above disclosure which shall equally be applicable to the uses.

[0198] Further embodiments of the present disclosure

[0199] The following embodiments provide further advantages of the present disclosure:

[0200] 1. A method for performing a bioprocess on cells in a cell culture, wherein the cell culture is present in a cell vessel (4), wherein the method comprises performing one or more unit operation(s) on the cells in an automated manner, wherein a unit operation comprises: i. adding a liquid composition to the cell vessel (4); ii. incubating the cells in the cell vessel (4) with the added liquid composition; and iii. removing liquid from the cell vessel (4) through at least one filter, and thereby removing at least a part of the added liquid composition; and expanding the cells in the cell vessel (4), wherein expanding is performed prior to and / or after performing the one or more unit operation(s).

[0201] 2. The method according to embodiment 1, wherein the unit operation is selected from: activation of the cells, preferably wherein the liquid composition comprises an activation agent; and genetic modification of the cells, in particular transduction and / or transfection, preferably wherein the liquid composition comprises a genetic modification agent, in particular a transduction agent and / or a transfection agent.

[0202] 3. The method according to embodiment 1 or 2, wherein the method comprises performing more than one unit operation, preferably, wherein the method comprises at least two unit operations, more preferably wherein the at least two unit operations comprise performing activation and genetic modification of the cells. The method according to one or more of embodiments 1 to 3, wherein the method comprises performing more than one unit operation, preferably, wherein the method comprises at least three unit operations, more preferably wherein the at least three unit operations comprise performing activation and two genetic modifications of the cells, such as activation, transfection and / or transduction. The method according to one or more of embodiments 1 to 4, wherein the liquid composition added in step i. comprises:

[0203] (i) an activation agent;

[0204] (ii) a genetic modification agent, preferably a transduction agent or a transfection agent, and optionally an enhancer; or

[0205] (iii) an activation agent and a genetic modification agent, preferably a transduction agent or a transfection agent, and optionally an enhancer. The method according to one or more of embodiments 1 to 5, wherein the method comprises performing more than one unit operation, preferably wherein performing more than one unit operation comprises: repeating steps i., to iii., one or more times; adding a liquid composition to the cell vessel (4) comprising agents for performing more than one unit operation, preferably the liquid composition comprises an activation agent and a genetic modification agent; repeating step i. prior to step ii. and / or prior to step iii. by adding a further liquid composition to the cell vessel (4), in particular wherein

[0206] (i) the liquid composition in step i. comprises an activation agent, and

[0207] (ii) the further liquid composition comprises a genetic modification agent; and / or repeating step i. prior to step ii. and / or prior to step iii. by adding a further liquid composition to the cell vessel (4), in particular wherein

[0208] (i) the liquid composition in step i. comprises a genetic modification agent, and

[0209] (ii) the further liquid composition comprises an activation agent. The method according to one of more of embodiments 1 to 6, wherein the method comprises measuring the cell density one or more time(s), preferably measuring the cell density at least prior to step i. The method according to embodiment 7, wherein the method comprises on basis of the measured cell density automatically adding a suitable volume of liquid composition to the cell vessel (4) in step i., in particular wherein the liquid composition comprises an activation agent and / or genetic modification agent, such that the volume of the liquid composition is suitable, preferably adjusted, for activation and / or genetic modification of the cells. The method according to one of more of embodiments 1 to 8, wherein the method further comprises one or more of: prior to performing the one or more unit operation(s) on cells in a cell culture in an automated manner, introducing the cell culture into the cell vessel (4), preferably wherein introducing is performed in an automated manner; and / or removing liquid from and / or adding liquid to the cell vessel (4) prior to step i., preferably wherein the cell density is adjusted. The method according to one of more of embodiments 1 to 9, wherein performing the one or more unit operation(s) is repeated one or more time(s), preferably at least one more time, particularly performing a different unit operation when performing the unit operation one more time. The method according to one of more of embodiments 1 to 10, wherein the method comprises adding a further liquid composition to the cell vessel (4) after step i. and priorto step ii., such that the further liquid composition is present in the cell vessel (4) when incubating the cells in step ii. and such that removing in step iii. comprises removing at least part of the further liquid composition through the filter. The method according to one or more of embodiments 1 to 11, wherein the method has the following characteristics: optionally, prior to step i., the method comprises measuring the cell density, preferably in an automated manner; the liquid composition in step i. comprises an activation agent, which is at least partially removed in step iii.; the method comprises performing a further unit operation, wherein the liquid composition in step i. comprises a genetic modification agent, preferably a transduction agent; and optionally, wherein subsequent to step iii. another liquid is added, preferably being a cell culture medium. The method according to one or more of embodiments 1 to 11, wherein the method has the following characteristics: optionally, prior to step i., the method comprises measuring the cell density, preferably in an automated manner; the liquid composition in step i. comprises o an activation agent, o a genetic modification agent, preferably a transduction agent or a transfection agent, and o optionally an enhancer, which are at least partially removed in step iii.; and optionally, wherein subsequent to step iii. another liquid is added, preferably being a cell culture medium.

[0210] 14. The method according to one or more of embodiments 1 to 11, wherein the method has the following characteristics: optionally, prior to step iii., the method comprises measuring the cell density, preferably in an automated manner; the liquid composition in step i. comprises an activation agent, which is at least partially removed in step iii.; the method comprises adding a further liquid composition to the to the cell vessel (4) after step i. and prior to step ii., such that o the further liquid composition is present in the cell vessel (4) when incubating the cells in step ii., and o removing in step iii. comprises removing at least part of the further liquid composition, wherein the further liquid composition comprises o a genetic modification agent, preferably a transduction agent or a transfection agent, and o optionally an enhancer; and optionally, wherein subsequent to the step iii. another liquid is added, preferably being a cell culture medium.

[0211] 15. The method according to one or more of embodiments 1 to 14, wherein the step of expanding the cells in the cell vessel (4) involves agitating the cells, such as by a rocking motion or wave motion.

[0212] 16. The method according to embodiment 15, wherein expansion comprises one or more of batch, fed-batch, and / or perfusion culture, preferably, expansion at least comprises perfusion culture.

[0213] 17. The method according to one or more of embodiments 1 to 16, wherein the cells in the cell vessel (4) are expanded after performing the one or more unit operation(s), and wherein the method comprises performing one or more unit further operation(s) after expanding the cells in the cell vessel (4), preferably wherein such method has the following characteristics: optionally, prior to step i., the method comprises measuring the cell density, preferably in an automated manner; the liquid composition in step i. comprises an activation agent, which is at least partially removed in step iii.; after step iii., expanding the cells in the cell vessel (4); after expanding the cells, adding a further liquid composition to the cell vessel comprising a genetic modification agent, preferably a transduction agent or transfection agent, more preferably a transfection agent, and incubating the cells in the cell vessel (4) with the further liquid composition; and optionally, harvesting the cells.

[0214] 18. The method according to one or more of embodiments 1 to 16, wherein the method has the following characteristics: optionally, prior to step iii., the method comprises measuring the cell density, preferably in an automated manner; performing a first unit operation, wherein the liquid composition in step i. comprises an activation agent, which is at least partially removed in step iii.; the method comprises performing a second unit operation, wherein the liquid composition in step i. comprises a genetic modification agent, preferably a transfection agent; optionally, wherein subsequent to performing a second unit operation another liquid is added, preferably being a cell culture media; the method comprises performing a third unit operation, wherein the liquid composition in step i. comprises a genetic modification agent, preferably a transduction agent; optionally, wherein subsequent to performing a third unit operation another liquid is added, preferably being a cell culture media; optionally, expanding the cells in the cell vessel (4); and optionally, harvesting the cells.

[0215] 19. The method according to one or more of embodiments 1 to 18, wherein step ii. further comprises agitating the cells in the cell vessel (4), such as by a rocking motion or wave motion.

[0216] 19a. The method according to one or more of embodiments 1 to 19, wherein step iii. further comprises one or more washing steps to essentially completely remove the liquid composition from the cell vessel.

[0217] 19b. The method according to embodiment 19a, wherein the washing step consists of perfusing, preferably wherein an amount of liquid equal to at least lx, 2x, 3x, 5x or lOx the volume of the cell culture is exchanged, further preferably wherein the perfusion speed is 15 VVD or less, 10 VVD or less, 8 VVD or less, or 4 VVD or less. c. The method according to embodiment 19a, wherein the washing step comprises the following steps: removing a first quantity of liquid from the cell vessel, adding a second quantity of a liquid medium to the cell vessel, and rocking the cell vessel for a period of time. d. The method according to embodiment 19c, wherein the first and second quantities are the same volume, e.g. up to 33% or up to 50% of the volume of the cell culture, preferably wherein the period of time is at least 1 minute, preferably at least 5 minutes or at least 10 minutes, e.g. 30 minutes. . The method according to one or more of embodiments 1 to 19d, wherein a volume of the cell culture in the cell vessel (4) increases during the step of expanding the cells, preferably after performing the at least one unit operation on the cells. . The method according to embodiment 20, wherein the volume of the cell culture in the cell vessel (4) increases by at least 10 %, preferably at least 50 %, more preferably at least 100 %, more preferably at least 200 %, more preferably at least 300 %, more preferably at least 400 %, more preferably at least 500 %, more preferably at least 600 %, more preferably at least 700 %, more preferably at least 800 %, more preferably at least 900 % during the step of expanding the cells. . The method according to embodiment 20 or 21, wherein the volume of the cell culture in the cell vessel (4) at the end of the step of expanding the cells is at least 100 ml, preferably at least 300 ml, more preferably at least 500 ml, more preferably at least 1 1. . The method according to one or more of embodiments 1 to 22, wherein the volume of the cell culture in the cell vessel (4) is at least 5 ml prior to step i. . The method according to one or more of embodiments 1 to 23, wherein the volume of the cell culture in the cell vessel (4) in is at most 100 ml prior to and / or subsequent to step i.. The method according to one or more of embodiments 1 to 24, wherein the unit operation is performed with a volume of cell culture and added fluid in the cell vessel (4) selected from the range of 1 to 200 ml, preferably 5 to 100 ml, particularly, wherein the step of expanding the cells is performed after performing the unit operation(s). . The method according to one or more of embodiments 1 to 25, wherein the method comprises removing liquid from the cell vessel (4) and thereby decreasing a volume by at least 10 %, preferably at least 30 %, more preferably at least 50 %, of the cell culture, in particular after performing the unit operation(s) and / or expanding the cells. . The method according to one or more of embodiments 1 to 25, wherein the method further comprises a step of washing the cells one or more time(s) after performing the unit operation(s), preferably wherein washing comprises: - i. adding a washing liquid composition to the cell vessel (4); ii. optionally, incubating the cells in the cell vessel (4) with the added washing fluid; and iii. removing fluid from the cell vessel (4) through at least one filter, and thereby removing at least a part of the added washing fluid.

[0218] 28. The method according to one or more of embodiments 1 to 27, wherein the method further comprises harvesting the cells, preferably after performing the unit operation(s) and expanding the cells.

[0219] 29. The method according to embodiment 28, wherein harvesting comprises transferring the cell culture from the cell vessel (4), preferably through a port of the cell vessel, optionally in an automated manner.

[0220] 30. The method according to embodiment 28 or 29, wherein harvesting comprises removing liquid from the cell vessel (4) through the at least one filter for increasing the cell density and afterwards, transferring the concentrated cell culture from the cell vessel (4), preferably through a port of the cell vessel, optionally in an automated manner.

[0221] 31. The method according to one or more of embodiments 1 to 30, wherein the method further comprises formulating the cell culture into a cell therapy product.

[0222] 32. The method according to embodiment 31, wherein formulating is performed after performing the unit operation(s) and expanding the cells.

[0223] 33. The method according to embodiment 31 or 32, wherein formulating comprises: i. removing liquid from the cell vessel (4) through the at least one filter; ii. adding a liquid for formulating the cells culture into a cell therapy product to the cell vessel (4); and iii. optionally, transferring the cell therapy product from the cell vessel (4), preferably through a port of the cell vessel, optionally in an automated manner.

[0224] 34. The method according to one of more of embodiments 1 to 33, wherein the method comprises using a bioprocessing system (1) comprising a control system (2) and at least one active element (5), wherein the control system (2) implements control instructions to cause the at least one active element (5) to advance the unit operation.

[0225] 35. The method according to embodiment 34, wherein the control system (2) generates control instructions to cause the active element (5) to add the liquid composition to the cell vessel (4) in step i. and / or remove the liquid from the cell vessel (4) through the at least one filter in step iii.

[0226] 36. The method according to embodiment 34 or 35, wherein the at least one active element (5) comprises at least one pump (8) for adding the liquid composition to the cell vessel (4) and / or for removing liquid from the cell vessel (4) through the at least one filter. The method according to one or more of embodiments 1 to 36, wherein the cell vessel (4) comprises two or more compartments, preferably a small volume compartment (13) and a distinct large volume compartment (14). The method according to one or more of embodiments 1 to 37, wherein the cell vessel (4) comprises a pre-shaped reservoir (15) forming a small volume compartment (13). The method according to one or more of embodiments 1 to 38, wherein cell vessel (4) comprises a casing (16) forming a large volume compartment (14), wherein the casing is preferably flexible. The method according to one or more of embodiments 37 to 39, wherein the method has following characteristics: during performing the unit operation(s), the cell culture is located in the small volume compartment (13); and / or during expanding the cells in the cell vessel (4), preferably after step iii., the volume of the cell culture increases such that the cell culture is preferably located in the small volume compartment (13) and the large volume compartment (14). The method according to one or more of embodiments 37 to 40, wherein the one or more of the at least two compartments comprise at least one filter and / or is / are fluidically connected to at least one filter. The method according to one or more of embodiments 37 to 41, wherein the small volume compartment (13) comprises at least one filter and / or is fluidically connected to at least one filter, and / or the large volume compartment (14) comprises at least one filter and / or is fluidically connected to at least one filter. The method according to embodiment 42, wherein the large volume compartment (14) comprises at least one filter which is a main membrane (7), preferably the main membrane (7) is not used during performing the unit operation(s), and / or, that the main membrane (7) is used for perfusion during expanding the cells in the cell vessel (4). The method according to embodiment 42 or 43, wherein the small volume compartment (13) comprises at least one filter which is a reservoir membrane (17), preferably the reservoir membrane (17) is used for performing the unit operation(s), in particular by removing liquid from the cell vessel (4) through the reservoir membrane in step iii. The method according to one or more of embodiments 1 to 44, wherein the at least one filter is configured to allow liquid to flow through but essentially retain the cells; and preferably, is not applied for gas exchange, in particularfor gas exchange between the cell culture in contact with the filter and the gas in contact with the other side of the filter. The according to one or more of embodiments 1 to 45, wherein the at least one filter is a membrane, such as a planar membrane, a hollow fiber membrane and / or floating membrane. The according to one or more of embodiments 1 to 46, wherein the cell vessel (4) comprises at least one filter and / or is fluidically connected to at least one filter, a tube set connected to the cell vessel (4) and presterilized together with the cell vessel (4). The method according to embodiment 47, wherein the tube set comprises: at least one disposable head for a pump (8); at least one, in particular magnetic, docking element for a sensor; a recirculation loop (24); a sealed and weldable connection for a media container (10); and / or a sealed and weldable connection for a waste container (18). The method according to one or more of embodiments 34 to 48, wherein the bioprocessing system further comprises a bioreactor module (3) comprising the cell vessel (4). The method according to one or more of embodiments 34 to 49, wherein the bioprocessing system (1), preferably the bioreactor module (3), further comprises a rocker element (9) for imposing a rocking motion onto the cell vessel (4). The method according to embodiment 49 or 50, wherein the bioreactor module (3) comprises at least one sensor (6) for sensing a characteristic of the cell culture, preferably wherein the control system (2) during the unit operation receives sensor data from the at least one sensor (6) and derives from the sensor data the control instructions. The method according to embodiment 51, wherein the at least one sensor (6) comprises a sensor measuring a characteristic of the cell culture, in particular a o number of cells per volume; o a cell size; o a cell viability; and / or o a pH value of the cell culture; and / or a balance weighing o the cell culture; o an amount of liquid added to the cell culture; and / or o an amount of liquid removed from the cell culture.

[0227] 53. The method according to embodiment 51 or 52, wherein control system (2) determines from a measured cell size a point in time at which to start the genetic modification, generates control instructions to control the at least one pump (8) to add the fluid in step i. comprising a genetic modification agent to the cell vessel (4) at the point in time and implements the control instructions.

[0228] 54. The method according to one or more of embodiments 49 to 53, wherein the bioreactor module (3) further comprises: a media container (10) connected to the cell vessel (4); a waste container (18) connected to the cell vessel (4); a bypass line (22) for connecting a container outside the bioreactor module (3) to the cell vessel (4); a control unit as part of the control system (2); and / or a heating element for heating the cell vessel (4).

[0229] 55. The method according to one or more of embodiments 49 to 54, wherein the bioreactor module (3) further comprises a recirculation loop (24) leading from the cell vessel (4) to the cell vessel (4).

[0230] 56. The method according to embodiment 55, wherein the recirculation loop (24) has one or more of the following characteristics: it comprises at least one sensor (6), in particular a sensor for cell counting; it is connected to the media container (10) in particular through a bypass line (22); it is connected to the waste container (18), in particular through a recirculation loop membrane (12), and / or the bypass line (22).

[0231] 57. The method according to one or more of embodiments 49 to 56, wherein the bioreactor module (3) comprises a support structure (19) for carrying the cell vessel (4), and optionally, the media container (10)

[0232] 58. The method according to one or more of embodiments 49 to 57, wherein the bioreactor module (3) comprises at least one tube holder (38) holding at least one tube in a defined position relative to a support structure (19).

[0233] 59. The method according to embodiment 58, wherein the tube holder (38) comprises a tube mover mechanism for extending and / or retracting the tube relative to the tube holder (38).

[0234] 60. The method according to embodiment 58 or 59, wherein the tube holder is configured for interacting with a connection arrangement (36) for aseptically connecting and / or disconnecting containers, preferably wherein the connection arrangement (36) comprises a tube welding unit (37) to weld and / or cut and seal the tube. 61. The method according to one or more of embodiments 34 to 60, wherein the bioprocessing system (1) comprises a connection arrangement (36) for aseptically connecting and / or disconnecting containers, preferably, that the connection arrangement (36) comprises a tube welding unit (37) for welding together and / or cutting and closing off tubes.

[0235] 62. The method according to embodiment 61, wherein the method comprises welding a tube to the bypass line (22) and / or a tube connected to the media container (10) and / or a tube connected to the waste container (18) by the tube welding unit (37).

[0236] 63. The method according to one or more of embodiments 34 to 62, wherein the bioprocessing system (1) has one or more of the following characteristics: it comprises a frame (32); it comprises at least one expansion location (33) at which the expansion operation and preferably the unit operation is performed, in particular supported by the bioprocessing system (1); it performs a replenishing of the media container (10); and / or it comprises a transport mechanism (34) which transports the bioreactor module (3), in particular out of the expansion location (33) and / or into the expansion location (33).

[0237] 64. The method according to one or more of embodiments 1 to 63, wherein the cells are mammalian cells, preferably immune or naive cells.

[0238] 65. The method according to one or more of embodiments 1 to 64, wherein the cell culture comprises the cells and a liquid, in which the cells are present, preferably the liquid being a cell culture medium.

[0239] 66. The method according to one or more of embodiments 1 to 65, wherein the method comprises a cell selection, preferably prior to performing the unit operation(s).

[0240] 67. The method according to embodiment 66, wherein the cell selection comprises: positive cell selection, wherein a target fraction of the cells directly or indirectly binds to a binding matrix of a binding unit; or negative cell selection, wherein a target fraction of the cells does not directly or indirectly bind to a binding matrix of a binding unit.

[0241] 68. The method according to embodiment 66 or 67, wherein cell selection comprises: adding a mixture of cells to the cell vessel (4), contacting the mixture of cells with a binding unit, wherein at least a fraction of the cells directly or indirectly binds to a binding matrix of the binding unit, and removing liquid from the cell vessel (4), thereby removing at least part of the cells, which do not directly or indirectly bind to the binding matrix. 69. The method according to embodiment 68, wherein the binding matrix is configured for allowing direct or indirect binding of a ligand expressed by at least a fraction of the cells.

[0242] 70. The method according to embodiment 68 or 69, wherein indirect binding comprises adding at least one binding reagent to the cell vessel (4), such that the mixture of cells is contacted with the binding reagent, and it binds to at least a fraction of the cells, and wherein the binding reagent is configured for binding to the binding matrix.

[0243] 71. The method according to one or more of embodiments 66 to 70, wherein the cell vessel (4) comprises a binding unit; and / or is fluidically connected to at least one a binding unit, wherein the binding unit comprises a binding matrix configured for allowing direct or indirect binding of a ligand expressed by at least a fraction of the cells.

[0244] 72. The method according to embodiment 71, wherein the binding matrix is provided as a resin or monolithic material.

[0245] 73. The method according to embodiment 71 or 72, wherein the binding unit is located within or adjacent to a small volume compartment (13) of the cell vessel, preferably wherein the binding unit is arranged adjacent to a filter of the small volume compartment (13).

[0246] 7 . The method according to one or more of embodiments 71 to 73, wherein the binding unit is provided as a column, preferably wherein the opening of the column is arranged within an opening of a small volume compartment (13) of the cell vessel, more preferably an opening at the bottom of the small volume compartment (13), and / or the column is fluidically connected to the cell vessel (4), preferably a reservoir, wherein fluidic connections between the cell vessel (4) and the column are provided, in particular wherein the fluidic connections are provided by one or more tubes, optionally further comprising a valve.

[0247] 75. The method according to one or more of embodiments 71 to 74, wherein the binding unit is provided as a floating unit configured for float within the cell vessel (4).

[0248] 76. The method according to one or more of embodiments 71 to 75, wherein the binding unit is by an external loop fluidically connected to the cell vessel (4) and / or a waste port, preferably both.

[0249] 77. The method according to embodiment 76, wherein the external loop comprises at least one valve, preferably one valve for controlling the fluid flow between the binding unit and the waste port and another valve controlled the fluid flow between the cell vessel (4) and the waste port, preferably wherein both valves are for controlling the fluid flow between the binding unit and the cell vessel (4) via the external loop. 78. A bioreactor module for performing a bioprocess on cells in a cell culture, wherein the bioreactor module (3) comprises a cell vessel (4), and at least one filter for removing liquid from the cell vessel (4) through the at least one filter, wherein the bioreactor module is configured for performing in an automated manner one or more unit operations on the cells in the cell vessel (4), and for expanding the cells in the cell vessel (4).

[0250] 79. The bioreactor module according to embodiment 78, wherein the bioreactor module further comprises one or both of: at least one active element (5) for influencing the cell culture in the bioreactor module (3), preferably for adding a fluid to and / or removing a fluid from the cell vessel (4), preferably wherein the fluid is a liquid; and at least one sensor (6) for sensing a characteristic of the cell culture.

[0251] 80. The bioreactor module according to embodiment 79, wherein the at least one active element (5) comprises at least one pump (8) for adding a fluid to the cell vessel (4) and / or for removing fluid from the cell vessel (4) through the at least one filter.

[0252] 81. The bioreactor module according to one or more of embodiments 78 to 80, wherein the cell vessel (4) comprises one or more of the following: two or more compartments, preferably a small volume compartment (13) and a distinct large volume compartment (14); a pre-shaped reservoir (15) forming a small volume compartment (13); a casing (16) forming the large volume compartment (14), preferably wherein the casing is flexible; a main membrane (7); and / or the control unit and / or an interface to the control system (2), wherein the control unit is adapted to.

[0253] 82. The bioreactor module according to embodiment 81, wherein the bioreactor module (3) has the following characteristics: during performing the unit operation(s), the cell culture is located in the small volume compartment (13); and / or during a expanding the cells in the cell vessel (4), the volume of the cell culture increases such that it is preferably located in the small volume compartment (13) and the large volume compartment (14).

[0254] 83. The bioreactor module according to one or more of embodiments 78 to 82, wherein the cell vessel (4) comprises at least two compartments, wherein at least one of the compartments comprises the at least one filter and / or is / are fluidically connected to the at least one filter. The bioreactor module according to one or more of embodiments 81 to 83, wherein the small volume compartment (13) comprises the at least one filter and / or is fluidically connected to the at least one filter, and / or the large volume compartment (14) comprises the at least one filter and / or is fluidically connected to the at least one filter. The bioreactor module according to embodiment 84, wherein the large volume compartment (14) comprises the at least one filter which is a main membrane (7), preferably the main membrane (7) is not used during performing the unit operation(s), and / or, that the main membrane (7) is used for perfusion during expanding the cells in the cell vessel (4). The bioreactor module according to embodiment 84 or 85, wherein the small volume compartment (13) comprises at least one filter which is a reservoir membrane (17), preferably the reservoir membrane (17) is used for performing the unit operation(s), in particular by removing fluid from the cell vessel (4) through the reservoir membrane. The bioreactor module according to one or more of embodiments 78 to 86, wherein the bioreactor module (3) comprises a control unit and / or an interface to the control system (2), wherein the control unit is adapted to, after a cell culture was introduced into the cell vessel (4), subject the cells to at least one unit operation and an, in particular subsequent, expansion operation inside the cell vessel (4), and, during the unit operation and / or the expansion operation, implement control instructions to cause the at least one active element (5) to influence the cell culture and thereby advance the unit operation and / or the expansion operation, and / or, wherein the interface to the control system (2) is adapted to allow the control system (2) to implement control instructions to cause the at least one active element (5) to influence the cell culture and thereby advance the unit operation and / or the expansion operation, preferably, wherein the control unit is adapted to receive sensor data from the at least one sensor (6) and derive from the sensor data control instructions and / or, wherein the interface to the control system (2) is adapted to allow the control system (2) to receive sensor data from the at least one sensor (6). The bioreactor module according to one or more of embodiments 78 to 87, wherein the cell vessel (4) comprises a tube set connected to the cell vessel (4), preferably wherein the tube set is pre-sterilized together with the cell vessel (4). The bioreactor module according to embodiment 88, wherein the tube set comprises at least one disposable pump (8) head; at least one, in particular magnetic, docking element for a sensor; a recirculation loop (24); a sealed and weldable connection for a media container (10); and / or a sealed and weldable connection for a waste container (18).

[0255] 90. A bioprocessing system for performing a bioprocess on cells in a cell culture, wherein the bioprocessing system (1) comprises the bioreactor module (3) according to one or more of embodiments 78 to 89.

[0256] 91. The bioprocessing system according to embodiment 90, wherein the bioprocessing system (1) comprises a control system (2) which is adapted to, during the unit operation(s) and / or the expansion step, implement control instructions to cause the at least one active element (5) to influence the cell culture and thereby advance the unit operation and / or the expansion operation.

[0257] 92. Use of the bioreactor module (3) according to one or more of embodiments 78 to 89 and / or the bioprocessing system according to embodiment 90 or 91 for manufacturing a cell therapy product, preferably wherein manufacturing comprises at least one of the following: optionally, selecting cells one or more times, in particular by binding at least a fraction of the cells expressing a ligand, preferably selecting a subpopulation of T cells; activating cells, preferably activating T cells; transducing cells, preferably transducing T cells; transfecting cells, preferably transfecting T cells; and / or expanding cells, preferably T cells, more preferably genetically modified T cells.

[0258] 93. Use of the bioreactor module (3) according to one or more of embodiments 78 to 89 and / or the bioprocessing system according to embodiment 90 or 91 in a method according to one or more of embodiments 1 to 77.

[0259] Further aspects and features of the present disclosure

[0260] The cell vessel may comprise a membrane that can be used to perform at least an expansion operation and another, in particular preceding, unit operation supported by a control system of a bioprocessing system. To allow for this automation, an active element may be advantageously included as a part of a bioreactor module comprising the cell vessel. The active element allows actively advancing the unit operation or expansion operation. The active advancement of the bioprocess is more than just a control of the ambient temperature which merely provides necessary conditions for the cell culture but does not actively advance the bioprocess. For example, changing parameters of a rocking motion or conceivably also a temperature based on a cell density measurement actively advances the bioprocess. The number of transfers of the cells can be reduced by combining multiple unit operations in a cell vessel. That simplifies the process and also reduces for example damage to cells, or cell excursions into environments that are not conducive to growth during the transfer.

[0261] The bioprocessing system may use sensor feedback to advance the bioprocess.

[0262] Preferably the unit operation is an activation or a genetic modification. In a preferred embodiment, activation and transduction and / or transfection are performed in the cell vessel, reducing the number of necessary vessels for the cell culture throughout the bioprocess. This also simplifies the overall control system.

[0263] Another embodiment relates to preferred active elements which include a pump or a rocker element. The pump may be controlled by the control system to automatically add an agent into the cell vessel for activation and / or genetic modification, thereby advancing the unit operation. Also preferred sensors are disclosed herein.

[0264] A preferred embodiment relates to a determination of a cell size to determine the right point in time to proceed from activation to genetic modification. The cell size may be indicative of the activation state.

[0265] The bioreactor module preferably allows increasing and / or decreasing the volume of the cell culture, preferably by large factors. The main membrane in general also enables an adjustment of the cell concentration, which makes it possible to adjust the cell concentration prior to a unit operation.

[0266] A preferred way to enable these large volume changes of the cell culture is described herein. According to this embodiment the cell vessel comprises two compartments, which may be connected and adjacent to each other, for different culture volumes. In particular, a small volume compartment can be designed to allow for handling small culture volumes, down to 5 ml for example, in a confined space while a large volume compartment handles culture volumes above for example 100 ml. The small volume compartment may be at the bottom of the large volume compartment such that it is also filled when the large volume compartment is used, but having around 100 ml inside the small volume compartment while using the large volume compartment has shown to be acceptable. The main membrane may be part of the large volume compartment. A preferred way of providing the small volume compartment is a pre-shaped reservoir. Additionally or alternatively, a second membrane may be provided in the small volume compartment. It should be noted that the terms "main" and "reservoir" membrane are only descriptive for the preferred embodiments. The main membrane may be located in the reservoir, and everything described for the reservoir may apply to the main membrane instead. It may therefore be the case that only one membrane is present which is used as described for the reservoir membrane herein or that the main membrane is used as described for the reservoir membrane herein and another membrane is present outside the reservoir or even in-side the reservoir.

[0267] One of the membranes may be used to remove from the cell vessel an activation and / or genetic modification agent, in particular controlled by the control system.

[0268] A further embodiment relates to the bioreactor module which may comprise a media container, a waste container, a bypass line, a control unit and / or a heating element.

[0269] Another preferred embodiment provides a recirculation loop leading from the cell vessel to the cell vessel. The recirculation loop may be used for a sensor and / or for adding to or removing liquid from the cell vessel.

[0270] A further embodiment described the bioprocessing system further relating to the integration of the bioreactor module into the broader bioprocessing system and to providing the bioreactor module with support functions.

[0271] A very preferred feature of the bioprocessing system is a connection arrangement for automatically making aseptical connections, in particular by tube welding via a tube welding unit.

[0272] For enabling the tube welding to be made with less complexity, the bioreactor module preferably comprises at least one tube holder holding a tube in a defined position and allowing the tube welding unit to interact with the tube holder.

[0273] A preferred tube set is further described herein.

[0274] In another teaching it is conceivable to leave out the membrane and in particular add a membrane in a recirculation loop connected to the cell vessel. The membrane in the recirculation loop may be used for removing an agent and / or adding a medium, in particular an agent, and / or for perfusion.

[0275] Another teaching, which is of equal importance, relates to a bioreactor module, wherein the bioreactor module comprises a cell vessel for containing the cell culture, at least one active element for influencing the cell culture in the bioreactor module and preferably at least one sensor for sensing a characteristic of the cell culture, wherein the cell vessel comprises a main membrane, wherein the bioreactor module comprises the control unit and / or an interface to the control system, wherein the control unit is adapted to, after a cell culture was introduced into the cell vessel, subject the cells to at least one unit operation and an, in particular subsequent, expansion operation inside the cell vessel, and, during the unit operation and / or the expansion operation, implement control instructions to cause the at least one active element to influence the cell culture and thereby advance the unit operation and / or the expansion operation, and / or, wherein the interface to the control system is adapted to allow the control system to implement control instructions to cause the at least one active element to influence the cell culture and thereby advance the unit operation and / or the expansion operation, preferably, wherein the control unit is adapted to receive sensor data from the at least one sensor and derive from the sensor data control instructions and / or, wherein the interface to the control system is adapted to allow the control system to receive sensor data from the at least one sensor.

[0276] All explanations given with regard to the proposed method are fully applicable to the bioreactor module.

[0277] The bioreactor module may be equipped with the tube set disclosed herein.

[0278] Another teaching, which is of equal importance, relates to a bioprocessing system wherein the bioprocessing system comprises a control unit and a bioreactor module which comprises a cell vessel for containing the cell culture, at least one active element for influencing the cell culture in the bioreactor module and preferably at least one sensor for sensing a characteristic of the cell culture, wherein the cell vessel comprises a main membrane, wherein the bioprocessing system is adapted to, after a cell culture was introduced into the cell vessel, subject the cells to at least one unit operation and an, in particular subsequent, expansion operation inside the cell vessel, wherein the control system is adapted to, during the unit operation and / or the expansion operation, implement control instructions to cause the at least one active element to influence the cell culture and thereby advance the unit operation and / or the expansion operation. All explanations given with regard to the proposed method and the proposed bioreactor module are fully applicable to the bioprocessing system.

[0279] Description of Figures

[0280] Fig. 1 shows a bioprocessing system 1 adapted to perform the proposed method. The bioprocessing system 1 serves to perform several parallel bioprocesses on immune or naive cells. The cells are preferably dedicated for cell and / or gene therapy.

[0281] The term "immune cells" may refer to different types of white blood cells. Hence, the term "immune cells" includes a variety of cells, for example, but not limited to dendritic cells, T lymphocytes, also referred to as T cells, B lymphocytes, natural killer cells, macrophages or the like. Immune cells may also include subtypes of immune cells, for example tumor-infiltrating lymphocytes or different types of T cells. Subtypes of a certain type of immune cells may be classified based on the type of antigen present at the cell surface. Hence, the term immune cells may for example refer to T cells comprising the surface antigen CD4 ("CD4+ T cells").

[0282] Preferably, the cells are genetically modified T cells or are genetically modified to become genetically modified T cells. Preferably, the T cells are genetically modified to express a chimeric antigen receptor (CAR). Consequently, the term "CAR-T cells" describes T cells that have been genetically modified to express a CAR. Hence, preferably, the genetically modified T cells are genetically modified CAR-T cells.

[0283] Disclosed herein is also a method for performing a bioprocess on immune or naive cells in a cell culture using a bioprocessing system 1. The bioprocessing system 1 comprises a control system 2 and a bioreactor module 3. Fig. 1 shows the bioprocessing system 1 which, as is preferably the case, comprises multiple bioreactor modules 3.

[0284] Fig. 2 shows an embodiment of the bioreactor module 3, which comprises a cell vessel 4 for containing the cell culture, at least one active element 5 for influencing the cell culture in the bioreactor module 3 and preferably at least one sensor 6 for sensing a characteristic of the cell culture.

[0285] Fig. 3 shows the cell vessel 4 in a view from above and Fig. 4 shows a view inside the cell vessel 4 with the top part removed. As can be seen in Fig. 4, the cell vessel 4 comprises a main membrane 7, here and preferably even two membranes as will be explained later. For performing a part of the bioprocess, the cell culture is introduced into the cell vessel 4. The cell culture may be introduced into the cell vessel 4 automatically by the bioprocessing system 1 or manually by a user or semi-automatically supported by the bioprocessing system 1.

[0286] After the cell culture was introduced into the cell vessel 4, the cells are subjected to at least one unit operation and an, in particular subsequent, expansion operation inside the cell vessel 4. The bioprocessing system 1 preferably performs the unit operation and the expansion operation and preferably a transition from the unit operation to the expansion operation automatically. The term "automatically" means that no user intervention is needed at the level of the components interacting with the cell culture, other liquids used forthe cell culture, tubes and the like but does not exclude a user having to allow a continuation of the bioprocess through a user interface for example.

[0287] During the unit operation and / or the expansion operation the control system 2 preferably receives sensor data from the at least one sensor 6, for example by measuring analogue sensor signals or reading digital sensor signals and preferably derives from the sensor data control instructions and implements control instructions, in particularthe derived control instructions, to cause the at least one active element 5 to influence the cell culture, in particular inside the cell vessel 4, and thereby advance the unit operation and / or the expansion operation. The control instructions preferably depend on the sensor signals and may depend on other factors like time. "Advancing the unit" operation means that an action of the active element 5 is causal for a next step in the bioprocess to be performed, for example, a rocking motion may be started or a liquid may be pumped. Just keeping a steady state, for example a steady temperature, is not seen as advancing the bioprocess. The expansion can be done partially orfully in batch and / orfed-batch and / or perfusion or any combination thereof. For instance, expansion may encompass an initial batch culture followed by fed-batch and / or perfusion culture.

[0288] According to one embodiment it is proposed that the bioreactor module 3 comprises at least one sensor 6 for sensing a characteristic of the cell culture, wherein the control system 2 during the unit operation and / or the expansion operation receives sensor data from the at least one sensor 6 and derives from the sensor data the control instructions.

[0289] It is preferably the case that the at least one unit operation comprises activation, in particular performed with an activation agent, and / or genetic modification, in particular transduction and / or transfection, in particular performed with a genetic modification agent, in particular a transduction agent and / or a transfection agent. The respective agent is added to the cell culture, preferably inside the cell vessel 4. The transfection agent may be a lipid nanoparticle for example (e.g. described in WO 2024 / 008967 Al). The transduction agent may be a viral vector, in particular a lentiviral or adenoviral vector. It is preferably the case that the activation and a subsequent genetic modification are performed inside the cell vessel 4 and preferably at different cell culture volumes. It is also conceivable to perform selection inside the cell vessel 4, in particular if the selection is performed via an agent and / or the membrane instead of magnetically. Enrichment may also be performed in the cell vessel 4. It is preferably the case that the cell culture does not leave the cell vessel 4 between the start of the unit operation and the end of the expansion operation. This provides several advantages, for example, fewer pumping operations, with liquid transfer itself potentially being error-prone, and no relocation of the cells to a different environment which may cause shear, are necessary.

[0290] The at least one unit operation, in particular activation and genetic modification, and the expansion may be performed fully automatically by the bioprocessing system 1 in the cell vessel 4. Other possible unit operations in particular performable afterthe expansion are concentration and / or media exchange, in particular for subsequent freezing.

[0291] Looking towards the active element 5 and for example shown in Fig. 3, the at least one active element 5 may comprise at least one pump 8 for adding a liquid to the cell culture and / or at least one pump 8 for removing liquid from the cell vessel 4, preferably a pump 8 adapted to do both, and / or a rocker element 9 shown in Fig. 2 for imposing a rocking motion onto the cell vessel 4. The bioreactor module 3 preferably comprises the rocker element 9 and / or the pump 8 or pumps 8 (not shown in Fig. 2).

[0292] Preferably, the control system 2 controls the at least one pump 8 to add the activation agent to the cell culture while the cell culture is located in the cell vessel 4 and / or to add the genetic modification agent and / or culture medium to the cell culture while the cell culture is located in the cell vessel 4. It may be the case that a media container 10 comprising the respective agent is connected to the cell vessel 4 prior to the unit operation and the control system 2 preferably controls the addition of the agent without human intervention. The media container 10 may be connected automatically by the bioprocessing system 1 as disclosed herein.

[0293] The at least one sensor 6 may comprise a sensor measuring a characteristic of the cell culture, in particular a number of cells per volume and / or a cell size and / or cell size distribution and / or a cell viability and / or a pH value of the cell culture and / or a concentration of a metabolite, such as glucose, in the cell culture, and / or a concentration of a cell metabolism by-product, such as lactate, in the cell culture. Additionally or alternatively, the at least one sensor 6 may comprise a balance weighing the cell culture and / or an amount of liquid added to the cell culture and / or an amount of liquid removed from the cell culture. The control system 2 may determine a parameter of a next step in the unit operation or expansion operation based on the cell characteristic, in particular the number of cells per volume and / or the cell size and / or the cell size distribution and / or the cell viability and / or the pH value of the cell culture and / or the concentration of a metabolite, such as glucose, in the cell culture, and / or the concentration of a cell metabolism byproduct, such as lactate, in the cell culture and / or an elapsed time. A next step can also be the end of the unit operation and a transition to the expansion. The cell size may comprise a cell size distribution and / or a mean cell size and / or a median cell size.

[0294] It may in particular be the case that the control system 2 determines from a measured cell size a point in time at which to start the genetic modification, generates control instructions to control the at least one pump 8 to add the genetic modification agent to the cell vessel 4 at the point in time and implements the control instructions. The control system 2 may decide on the point in time based on the cell size reaching a predetermined value and may implement the genetic modification autonomously. It is possible to add several agents together to combine steps. The cell size may be indicative of a state of the activation.

[0295] More generally, the control system 2 may determine from a measured characteristic of the cell culture a point in time at which to start activation and / or transduction and / or transfection and / or expansion, generate control instructions to control the at least one pump 8 to add the activation agent and / or transduction agent and / or transfection agent and / or culture medium to the cell vessel 4 at the point in time and implement the control instructions. The point in time may also depend on a pre-defined minimum elapsed time.

[0296] Further, the control system 2 may determine from a measured cell characteristic of the cell culture an amount of activation agent and / or genetic modification agent, in particular transduction agent and / or transfection agent, and / or culture medium, generate control instructions to control the at least one pump 8 to add the determined amount of activation agent and / or transduction agent and / or transfection agent and / or culture medium to the cell vessel 4 and implement the control instructions.

[0297] It is preferably the case that a volume of the cell culture is actively changed by more than just an addition of the agent. Preferably, a volume of the cell culture is increased between the start of the unit operation and the end of the expansion operation by at least 10 %, preferably at least 50 %, more preferably at least 100 %, more preferably at least 200 %, more preferably at least 300 %, more preferably at least 400 %, more preferably at least 500 %, more preferably at least 600 %, more preferably at least 700 %, more preferably at least 800 %, more preferably at least 900 %. Additionally or alternatively, the volume of the cell culture inside the cell vessel 4 may be at most 100 ml, preferably at most 80 ml, more preferably at most 50 ml, more preferably at most 25 ml, more preferably at most 15 ml, and / or at least 5 ml, at the start of the unit operation and / or at least 100 ml, preferably at least 300 ml, more preferably at least 500 ml, more preferably at least 1 I, at the end of the expansion operation. It is also possible that the bioprocessing system 1 performs a volume decrease of at least 10 %, preferably at least 30 %, more preferably at least 50 %, on the cell culture between the start of the unit operation and the end of the expansion. The volume decrease may be performed through the main membrane 7, through a sampling port (e.g. one of the supply ports 11 described below) or through the recirculation loop membrane 12 described below.

[0298] It is in particular possible that the activation is performed with one of the named volumes below 100 ml. The volume may be increased by one of the named percentages for the genetic modification by adding a feed medium for example and in particular automatically.

[0299] In particular in combination with the rocking motion it is possible to have a wide range of viable cell counts, for example, the viable cell count of the cell culture between the start of the unit operation and the end of the expansion may vary between at least 10*10A6 viable cells per ml and 20*10A6 viable cells per ml, preferably between 5*10A6 viable cells per ml and 25*10A6 viable cells per ml, more preferably between l*10A6 viable cells per ml and 30*10A6 viable cells per ml.

[0300] Fig. 4 shows how in the preferred embodiment the different culture volumes are handled. It is preferably the case that the cell vessel 4 comprises a small volume compartment 13 and a distinct large volume compartment 14. The term "distinct" means that the volume compartments are discernible and functionally different. A boundary between the compartments may not be placed arbitrarily but must be accompanied by a functional and visual boundary.

[0301] During the unit operation, in particular during the whole unit operation, the volume of the cell culture preferably fits into the small volume compartment 13 and is located in the small volume compartment 13 during a period without rocking motion or other mixing. During rocking motion, the liquid may slosh out of the small volume compartment 13. However, the large volume compartment 14 is here and preferably arranged such that the liquid gravitates back into the small volume compartment 13 during a period without rocking motion. During the expansion operation, at least at some point, the volume of the cell culture is preferably too large to fit in the small volume compartment 13 and therefore preferably located in the small volume compartment 13 and the large volume compartment 14, in particular during a period without rocking motion or other mixing. Accordingly, it is preferred that the expansion operation is performed in perfusion mode with the cells being retained within the cell vessel 4 by the main membrane 7.

[0302] As can be seen, preferably, the large volume compartment 14 comprises the main membrane 7. It follows that preferably, the main membrane 7 is not used during the unit operation. The main membrane 7 may be used for perfusion during the expansion operation.

[0303] Fig. 4 also shows that preferably the cell vessel 4 comprises a pre-shaped reservoir 15 forming the small volume compartment 13 and a, in particular flexible, casing 16 forming the large volume compartment 14. In addition thereto or independently, the small volume compartment 13 may comprise a reservoir membrane 17, which is used during the unit operation.

[0304] Both the term "main" and the term "reservoir" in front of membrane serve to distinguish the membranes by their preferred use cases but are not limiting. The main membrane 7 may be located at the lowest point of the cell vessel 4 whether it has a reservoir 15 or not, for example.

[0305] For both membranes several use cases are conceivable, for example selection as already explained. Also, the reservoir membrane 17 may be used for perfusion during the unit operation and / or during the expansion operation. The membranes have a pore size that holds the cells in and is adapted to the respective function. The membrane or membranes may also be functionalized.

[0306] In a preferred use case, the activation agent and / or the genetic modification agent is removed from the cell vessel 4 through one of the membranes, in particular the reservoir membrane 17. For that, preferably, the control system 2 generates control data to cause the pump 8 to remove from the cell vessel 4 the activation agent and / or genetic modification agent. A feed medium or other neutral medium may be added by one pump 8 and a permeate may be drawn through the membrane by the same or another pump 8. The addition of the medium and the removal of the permeate may be performed consecutively or concurrently. This may be repeated two or three times for example to reduce the concentration of the respective agent in the cell vessel 4. It is also imaginable to remove culture medium, in particular cell-free culture medium, from the cell vessel 4 by letting the cells settle to the bottom of the cell vessel 4 and withdrawing medium from above the settled cells by a respective port.

[0307] As shown in Fig. 2, preferably the bioreactor module 3 comprises a media container 10 and / or a waste container 18 connected to the cell vessel 4. The bioreactor module 3 may comprise a support structure 19 and the support structure 19 then carries the cell vessel 4 and the media container 10. It is therefore the case that the bioreactor module 3 can be handled as a unit together with the cell vessel 4 and the media container 10 due to the support structure 19. If handled manually the bioreactor module 3 could be lifted by its support structure 19 which then carries the cell vessel 4 and the media container 10. The bioreactor module 3 may provide temperature control for the media container 10, in particular for cooling an activation and / or genetic modification agent.

[0308] It may however be the case that the media container 10 does not contain sufficient medium and / or not all needed media for the steps of the bioprocess performed inside the cell vessel 4, in particular for the expansion operation. To overcome this shortage, a liquid medium inside the media container 10 is replenished, in particular automatically by the bioprocessing system 1.

[0309] One way to achieve the replenishing may be swapping out the media container 10 for another media container 10. According to one embodiment, it is proposed that the bioreactor module 3 comprises a life support container 20 that comprises the media container 10 and the waste container 18. Preferably, the life support container 20 containing the media container 10 and / or the waste container 18 is swapped out for a second life support container 20 containing a second media container 10 and / or a second waste container 18, in particular automatically in a swapping routine by the bioprocessing system 1.

[0310] In a different embodiment or conceivably even in addition to the swapping it may be the case that in a refill routine the bioprocessing system 1 automatically connects a refill container 21 to the media container 10 and automatically refills the media container 10 and preferably automatically disconnects the refill container 21 from the media container 10.

[0311] Further, it may be the case that the bioreactor module 3 comprises a bypass line 22 for connecting a container outside the bioreactor module 3 to the cell vessel 4, and / or that the bioreactor module 3 comprises a control unit as part of the control system 2, and / or that the bioreactor module 3 comprises a heating element for heating the cell vessel 4. The bypass line 22 bypasses the media container 10 and the waste container 18.

[0312] It may also be the case that the life support container 20 and / or the media container 10 and / or the waste container 18 is weighed by a balance being one of the at least one sensor 6, in particular to determine the amount of liquid added or removed to or from the cell vessel 4.

[0313] According to one embodiment, it is proposed that the bioreactor module 3 comprises a lid 23 for providing a controlled environment for the cell vessel 4, and / or, that the bioreactor module 3 comprises a heating unit for the cell vessel 4.

[0314] Fig. 3 shows in the top right corner that the bioreactor module 3 may comprise a recirculation loop 24 leading from the cell vessel 4 to the cell vessel 4. The recirculation loop 24 may connect supply ports 11 of the cell vessel 4 which are also connected to the media container 10 and / or the waste container 18. Preferably, the recirculation loop 24 comprises at least one branch 25. In Fig. 3 the schematic recirculation loop 24 comprises the same tubes as shown at the cell vessel 4 in the same order. For example, the shown tube leading away from the supply port 11 may comprise a branch 25, preferably towards a pump 8 and / or a sensor and from there return to another branch 25 of a tube connected to the other supply port 11 for example (only in Fig. 5 the recirculation loop 24 returns to a supply port 11). The recirculation loop 24 in a preferred and shown embodiment is a two-way recirculation loop 24. Then the recirculation loop 24 goes from one port, in particularthrough a branch 25, to a loop destination 26, e.g. a sensor. From there, the recirculation loop 24 goes on to an air source, in particular a further gas port 27 of the cell vessel 4. A sample drawn through the recirculation loop 24 is however not routed through the whole recirculation loop 24 but instead to the loop destination 26 and back. The part of the recirculation loop 24 not used for the liquid is then used for air to push the liquid back and allow for the liquid to flow forward. The cell vessel 4 may further comprise a gas inlet port 28, a gas outlet port 29 and / or a waste port 30, as shown.

[0315] Here and preferably, the recirculation loop 24 comprises one of the at least one sensor 6, in particular a sensor for cell counting, in particular at the loop destination 26. Fig. 5 shows an embodiment of fluid lines that may be connected to the cell vessel 4 also including a recirculation loop 24. It can be seen there that, here and preferably, the recirculation loop 24 is connected to the media container 10 and / or the waste container 18, in particular through a recirculation loop membrane 12, and / or the bypass line 22. An additional connection of the media container 10 and the waste container 18 to the cell vessel 4 may be present, too. In Fig. 5, an agent source 31 is connected to the bypass line 22. The fluid lines in Fig. 5 do not correspond completely to the fluid lines in Figs. 2 to 4.

[0316] The recirculation loop 24 may generally allow for non-destructive, preferably non-contact, sensing of characteristics of the cell culture, in particular the cells. It is also conceivable to use destructive sampling or a probe in or attached to the reservoir 15. Examples of suitable probes may include capacitance-based sensors, microscopic devices or the like.

[0317] During the expansion step, a culture medium from the media container 10 may be added to the cell vessel 4 and / or a liquid removed from the cell vessel 4 may be introduced into the waste container 18.

[0318] Adjusting the volume during the unit operation and / or the expansion operation may be performed by retrieving a desired cell characteristic from a recipe, measuring a cell characteristic by the at least one sensor 6, determining a volume adjustment necessary by determining a current volume in the cell vessel 4, e.g., by the balance, by a flow sensor, or by a calculation, optionally increasing the volume in the cell vessel 4 by an addition of a medium, in particular from the media container 10, preferably monitored by the balance. Alternatively, the media container 10 may be prefilled with a predefined amount. A cell number may be adjusted prior to an agent addition based on the characteristic of the cell culture by introducing media to or removing media from the cell vessel 4.

[0319] Turning to the bioprocessing system 1 in Fig. 1, it is preferred that the bioprocessing system 1 comprises a frame 32. The frame 32 may comprise a user interface and may generally carry up to all components of the bioprocessing system 1.

[0320] The bioprocessing system 1 here and preferably comprises at least one expansion location 33 at which the expansion operation and preferably the unit operation is performed, in particular supported by the bioprocessing system 1. The bioprocessing system 1 here and preferably performs a replenishing of the media container 10.

[0321] It is further preferably the case that the bioprocessing system 1 comprises a transport mechanism 34, for example a movable tray mounted on rails (located outside the visible area on the right side in Fig. 1) or a robotic arm, for automatically moving the bioreactor modules 3. The transport mechanism 34 serves for automatically transporting the bioreactor modules 3 including the cell vessel 4 and the media container 10 and automatically transports several of the bioreactor modules 3. Here, the transport mechanism 34 is an elevator and / or loads bioreactor modules 3 from an expansion location 33 and / or into an expansion location 33. Several such expansion locations 33 may be placed in a column as shown. The transport mechanism 34 transports the cell vessel 4 and the media container 10 at the same time and together as both are connected fluidically.

[0322] The transport mechanism 34 may comprise a transport element that is adapted to hold a bioreactor module 3 and movable in at least one linear direction. The transport element may be movable in exactly one linear direction, in particular vertically, as explained with regard to the elevator embodiment above. A control strategy regarding the control of the cell vessel 4, for example rocking or media supply, of the bioreactor module 3, in particular executed by a control unit comprised by the bioreactor module 3, may depend on a location of the bioreactor module 3. Location information may be provided by the bioprocessing system 1. The bioprocessing system 1 may comprise a dedicated unit operation location 35 for the unit operation or, preferably, for a separate unit operation like selection or enrichment.

[0323] When the interaction between the bioprocessing system 1 and the bioreactor is described the term bioprocessing system 1 relates to the components of the bioprocessing system 1 other than the bioreactor module 3.

[0324] Another relevant aspect of the bioprocessing system 1 is that it preferably comprises a connection arrangement 36 for aseptically connecting and / or disconnecting containers. The connection arrangement 36 may comprise a tube welding unit 37 for welding together and / or cutting and closing off tubes. A preferred connection arrangement 36 is described in EP 4 342 975 Al. The tube welding unit 37 may weld a tube to the bypass line 22 and / or a tube connected to the media container 10 and / or a tube connected to the waste container 18. The tube welding unit 37 may be used for swapping out the media container 10 and / or waste container 18 and / or for refilling the media container 10 and / or waste container 18.

[0325] The tube welding unit 37 here and preferably automatically grabs the tubes. It is preferably the case that the bioreactor module 3, in particular the life support container 20, comprises at least one tube holder 38 holding at least one tube in a defined position relative to the support structure 19 and / or the life support container 20 and that the tube welding unit 37 interacts with the tube holder 38 to weld and / or cut and seal the tube. Fig. 1 shows a close-up onto one embodiment of such tube holders 38. Preferably, the tube holder 38 comprises a tube mover mechanism for extending and / or retracting the tube relative to the tube holder 38. Anyhow, the tube holders 38 enable a precise handling of the tubes without making it necessary for the tube welding unit 37 to somehow locate the tube itself. It is sufficient to locate the tube holder 38 which has a better-defined position and orientation. It is further preferred that the tube welding unit 37 performs an integrity test on the newly established weld to determine that the weld has been correctly established. In case the weld fails the integrity test, it is further preferred that the tube welding unit 37 may cut the tubes and establish a new weld.

[0326] It is preferred that the tubes are cut by a blade. For this, the bioprocessing system 1 may provide a blade supply, particularly a blade box, as well as a blade removal, in particular a waste bin, for supply and discard of the blades. The blade supply and the blade removal may be provided as a combined unit within the bioprocessing system 1 (not shown in detail). The tube welding unit 37 may automatically move to the blade supply / removal unit for blade supply and / or removal.

[0327] It is preferably the case that the bioreactor module 3 comprises a drive unit for moving the tube holder38, in particular linearly along a rail of the bioreactor module 3. The rails can be seen in Fig. 1, too.

[0328] It is preferably also the case that each of the bioreactor modules 3 of the group comprises a receiving interface with a connection for receiving electrical energy and / or a connection for receiving wire-bound signals and / or a connection for receiving gas and / or a connection for an optical sensor, and, that the receiving interface is connectable to a providing interface of the frame 32.

[0329] Preferably, the frame 32 comprises providing interfaces providing electrical energy and / or wirebound signals and / or gas and / or an optical sensor connection at different locations, in particular at the expansion location 33. The bioreactor module 3 may be self-sufficient to some extent and may comprise a battery. With the media container 10 and preferably waste container 18, the bioreactor module 3 may be self-sufficient for at least 12 hours, preferably at least 24 hours. That protects the cells even during a power outage or the like.

[0330] Different providing interfaces may provide a different subset of connections for the receiving interface. For example, gas may be provided only at the expansion locations 33 while energy may be provided by each providing interface.

[0331] Looking at Figs. 3 and 5 it may also be the case that the cell vessel 4 comprises a tube set connected to the cell vessel 4 and pre-sterilized together with the cell vessel 4, that the tube set comprises at least one disposable head for a pump 8 and / or at least one, in particular magnetic, docking element for a sensor, and / or, the recirculation loop 24, and / or, a sealed and weldable connection for a media container 10 and / or a sealed and weldable connection for a waste container 18. Drives for the heads of pumps 8 may be included in the bioreactor module 3.

[0332] Another teaching which is of equal importance relates to a bioreactor module 3 for use in the proposed method, wherein the bioreactor module 3 comprises a cell vessel 4 for containing the cell culture, at least one active element 5 for influencing the cell culture in the bioreactor module 3 and preferably at least one sensor 6 for sensing a characteristic of the cell culture, wherein the cell vessel 4 comprises a main membrane 7, wherein the bioreactor module 3 comprises the control unit and / or an interface to the control system 2, wherein the control unit is adapted to, after a cell culture was introduced into the cell vessel 4, subject the cells to at least one unit operation and an, in particular subsequent, expansion operation inside the cell vessel 4, and, during the unit operation and / or the expansion operation, preferably receive sensor data from the at least one sensor 6 and preferably derive from the sensor data control instructions and implement control instructions, in particular the control instructions, to cause the at least one active element 5 to influence the cell culture and thereby advance the unit operation and / or the expansion operation, and / or, wherein the interface to the control system 2 is adapted to allow the control system 2 to implement control instructions to cause the at least one active element 5 to influence the cell culture and thereby advance the unit operation and / or the expansion operation. Preferably, the interface is adapted to allow the control system 2 to receive sensor data from the at least one sensor 6.

[0333] The bioreactor module 3 can conceivably also be used to produce agents required in the manufacturing process of cell and gene therapies. In particular, it is well possible to use the bioreactor module 3 to produce viral vectors needed for the genetic modification step with the present system as well. In this case, the cells used for vector production, e.g., H EK293 cells, may be transiently transfected as a unit operation which is followed by an expansion operation, or an expansion operation may be performed in the H EK293 cells which is then followed by transfection for genetic modification.

[0334] According to one embodiment, it is proposed that the cell vessel 4 comprises a tube set connected to the cell vessel 4 and pre-sterilized together with the cell vessel 4, that the tube set comprises at least one disposable pump 8 head, and / or at least one, in particular magnetic, docking element for a sensor, and / or the recirculation loop 24, and / or a sealed and weldable connection for a media container 10, and / or a sealed and weldable connection for a waste container 18.

[0335] Another teaching which is of equal importance relates to a bioprocessing system 1 for use in the proposed method, wherein the bioprocessing system 1 comprises a control unit and a bioreactor module 3 which comprises a cell vessel 4 for containing the cell culture, at least one active element 5 for influencing the cell culture in the bioreactor module 3 and preferably at least one sensor 6 for sensing a characteristic of the cell culture, wherein the cell vessel 4 comprises a main membrane 7, wherein the bioprocessing system 1 is adapted to, after a cell culture was introduced into the cell vessel 4, subject the cells to at least one unit operation and an, in particular subsequent, expansion operation inside the cell vessel 4, wherein the control system 2 is adapted to, during the unit operation and / or the expansion operation, implement control instructions to cause the at least one active element 5 to influence the cell culture and thereby advance the unit operation and / or the expansion operation.

[0336] A further embodiment of the cell vessel 4 is illustrated in Fig. 8. As shown therein, the cell vessel 4 comprises a casing 16 and reservoir 15 and can further comprise a binding unit 101. The binding unit as shown is located at the bottom of the reservoir adjacent to the reservoir membrane 17. The binding unit typically comprises a binding matrix (see grey section in 101) which is configured for binding directly or indirectly to the cells for achieving a positive or negative cell selection step. The binding unit 101 may be provided in form of a column as shown in Fig. 8. Alternatively, the binding unit 101 may be provided as a floating unit (not shown), wherein the binding unit 101 may float within the fluid present in the vessel 1. The binding unit 101 when provided as a column may be directly provided in or adjacent to the reservoir (see Fig. 8). Alternatively, the binding unit 101 may be fluid ical ly connected to the vessel 4, preferably to the reservoir 15, such as via a tubing (not shown). The binding unit 101 as shown in Fig. 8 is located adjacent to the reservoir membrane 17. An opening in the reservoir 15 bottom may be provided such that liquid medium can enter the binding unit 101.

[0337] The binding unit 101 as shown in Fig. 8 may comprise an external loop fluidically connected to the reservoir 15 and / or a waste port, preferably both. The external loop may be provided by tubes, which allow for transport of the fluid exiting or entering the binding unit. Such tubes preferably comprise a valve as shown in Fig. 8. The external loop comprises at least one valve, preferably one valve for controlling the fluid flow between the binding unit 101 and the waste port and another valve controlling the fluid flow between the reservoir 15 and the waste port, preferably wherein both valves are for controlling the fluid flow between the binding unit 101 and the reservoir 15 via the external loop. The waste port is preferably connected to a pump allowing fluid transfer operations. Alternatively, the fluid transfer may be achieved by pressurizing the cell vessel 4 such that the fluid is pushed through the binding matrix 101. It is also possible that an additional entrance is provided between the reservoir 5 and the binding matrix 101, e.g., when providing the binding matrix 101 fluidically connected to the reservoir 15, e.g. by a tube, such that a further tube is connected that can provide over or under pressure to binding matrix 101 for performing fluid transfer operations.

[0338] Fig. 9 illustrates a preferred embodiment of the method according to the present disclosure, which may be referred to as "sequential embodiment". As disclosed herein, it shows an embodiment wherein the activation step occurs, followed by genetic modification, here transduction. Shown is furtherthe system setup as well as the steps to be executed, preferably in an automated manner.

[0339] Fig. 10 illustrates a preferred embodiment of the method according to the present disclosure, which may be referred to as "parallel embodiment". As disclosed herein, it shows an embodiment wherein the activation step essentially in parallel to the genetic modification, here transduction. Shown is furtherthe system setup as well as the steps to be executed, preferably in an automated manner.

[0340] Fig. 11 illustrates a preferred embodiment of the method according to the present disclosure, which may be referred to as "transfection short embodiment". As disclosed herein, it shows an embodiment wherein the activation step essentially in parallel to the genetic modification, here transfection. Shown is further the system setup as well as the steps to be executed, preferably in an automated manner.

[0341] Fig. 12 illustrates a preferred embodiment of the method according to the present disclosure, which may be referred to as "transfection long embodiment". As disclosed herein, it shows an embodiment wherein the activation step occurs, followed by expansion, followed genetic modification, here transfection. Shown is further the system setup as well as the steps to be executed, preferably in an automated manner.

[0342] Fig. 13 illustrates a preferred embodiment of the method according to the present disclosure, which may be referred to as "transfection and transduction embodiment". As disclosed herein, it shows an embodiment wherein the activation step occurs, followed by genetic modification, here transfection. Afterwards, the cells undergo transduction and are then expanded. Shown is further the system setup as well as the steps to be executed, preferably in an automated manner. Items according to the present disclosure

[0343] The following items provide further advantageous embodiments of the present disclosure:

[0344] 1. Method for performing a bioprocess on immune or naive cells in a cell culture using a bioprocessing system (1), wherein the bioprocessing system (1) comprises a control system (2) and a bioreactor module (3) which comprises a cell vessel (4) for containing the cell culture, at least one active element (5) for influencing the cell culture in the bioreactor module (3), wherein the cell vessel (4) comprises a main membrane (7), wherein the cell culture is introduced into the cell vessel (4), wherein after the cell culture was introduced into the cell vessel (4), the cells are subjected to at least one unit operation and an, in particular subsequent, expansion operation inside the cell vessel (4), wherein the control system (2) during the unit operation and / or the expansion operation implements control instructions to cause the at least one active element (5) to influence the cell culture and thereby advance the unit operation and / or the expansion operation.

[0345] 2. Method according to item 1, characterized in that the bioreactor module (3) comprises at least one sensor (6) for sensing a characteristic of the cell culture, wherein the control system (2) during the unit operation and / or the expansion operation receives sensor data from the at least one sensor (6) and derives from the sensor data the control instructions.

[0346] 3. Method according to item 1 or 2, characterized in that the at least one unit operation comprise activation, in particular performed with an activation agent, and / or genetic modification, in particular transduction and / or transfection, in particular performed with a genetic modification agent, in particular a transduction agent and / or a transfection agent.

[0347] 4. Method according to one of the preceding items, characterized in that the at least one active element (5) comprises at least one pump (8) for adding a liquid to the cell culture and / or at least one pump (8) for removing liquid from the cell culture and / or a rocker element (9) for imposing a rocking motion onto the cell vessel (4), preferably, that the control system (2) controls the at least one pump (8) to add the activation agent to the cell culture while the cell culture is located in the cell vessel (4) and / or to add the genetic modification agent to the cell culture while the cell culture is located in the cell vessel (4). 5. Method according to one of the preceding items, characterized in that the at least one sensor (6) comprises a sensor measuring a characteristic of the cell culture, in particular a number of cells per volume and / or a cell size and / or a cell viability and / or a pH value of the cell culture, and / or, that the at least one sensor (6) comprises a balance weighing the cell culture and / or an amount of liquid added to the cell culture and / or an amount of liquid removed from the cell culture.

[0348] 6. Method according to item 4 and 5, characterized in that the control system (2) determines from a measured cell size a point in time at which to start the genetic modification, generates control instructions to control the at least one pump (8) to add the genetic modification agent to the cell vessel (4) at the point in time and implements the control instructions.

[0349] 7. Method according to one of the preceding items, characterized in that a volume of the cell culture is increased between the start of the unit operation and the end of the expansion operation by at least 10 %, preferably at least 50 %, more preferably at least 100 %, more preferably at least 200 %, more preferably at least 300 %, more preferably at least 400 %, more preferably at least 500 %, more preferably at least 600 %, more preferably at least 700 %, more preferably at least 800 %, more preferably at least 900 %, and / or, that the volume of the cell culture inside the cell vessel (4) is at most 100 ml, preferably at most 80 ml, more preferably at most 50 ml, more preferably at most 25 ml, more preferably at most 15 ml, and / or at least 5 ml, at the start of the unit operation and / or at least 100 ml, preferably at least 300 ml, more preferably at least 500 ml, more preferably at least 1 I, at the end of the expansion operation, and / or, that the bioprocessing system (1) performs a volume decrease of at least 10 %, preferably at least 30 %, more preferably at least 50 %, on the cell culture between the start of the unit operation and the end of the expansion operation.

[0350] 8. Method according to one of the preceding items, characterized in that the cell vessel (4) comprises a small volume compartment (13) and a distinct large volume compartment (14), preferably, that during the unit operation the volume of the cell culture fits into the small volume compartment (13) and is located in the small volume compartment (13) during a period without rocking motion or other mixing, and, that during the expansion operation the volume of the cell culture is too large to fit in the small volume compartment (13) and preferably located in the small volume compartment (13) and the large volume compartment (14), in particular during a period without rocking motion or other mixing. 9. Method according to item 8, characterized in that the large volume compartment (14) comprises the main membrane (7), preferably, that the main membrane (7) is not used during the unit operation, and / or, that the main membrane (7) is used for perfusion during the expansion operation.

[0351] 10. Method according to one of the preceding items, characterized in that the cell vessel (4) comprises a pre-shaped reservoir (15) forming the small volume compartment (13) and a, in particular flexible, casing (16) forming the large volume compartment (14), and / or, that the small volume compartment (13) comprises a reservoir membrane (17), preferably, that the reservoir membrane (17) is used during the unit operation.

[0352] 11. Method according to one of the preceding items, characterized in that the activation agent and / or the genetic modification agent is removed from the cell vessel (4) through one of the membranes, in particular the reservoir membrane (17), preferably, that the control system (2) generates control data to cause the pump (8) to remove from the cell vessel (4) the activation agent and / or the genetic modification agent.

[0353] 12. Method according to one of the preceding items, characterized in that the bioreactor module

[0354] (3) comprises a media container (10) and / or a waste container (18) connected to the cell vessel

[0355] (4), and / or, that the bioreactor module (3) comprises a bypass line (22) for connecting a container outside the bioreactor module (3) to the cell vessel (4), and / or, that the bioreactor module (3) comprises a control unit as part of the control system (2), and / or, that the bioreactor module (3) comprises a heating element for heating the cell vessel (4).

[0356] 13. Method according to one of the preceding items, characterized in that the bioreactor module (3) comprises a recirculation loop (24) leading from the cell vessel (4) to the cell vessel (4), preferably, that the recirculation loop (24) comprises one of the at least one sensor (6), in particular a sensor for cell counting, and / or, that the recirculation loop (24) is connected to the media container (10) and / or the waste container (18), in particular through a recirculation loop membrane (12), and / or the bypass line (22).

[0357] 14. Method according to one of the preceding items, characterized in that the bioprocessing system (1) comprises a frame (32), and / or, that the bioprocessing system (1) comprises at least one expansion location (33) at which the expansion operation and preferably the unit operation is performed, in particular supported by the bioprocessing system (1), and / or, that the bioprocessing system (1) performs a replenishing of the media container (10), and / or, that the bioprocessing system (1) comprises a transport mechanism (34) which transports the bioreactor module (3), in particular out of the expansion location (33) and / or into the expansion location (33).

[0358] 15. Method according to one of the preceding items, characterized in that the bioprocessing system (1) comprises a connection arrangement (36) for aseptically connecting and / or disconnecting containers, preferably, that the connection arrangement (36) comprises a tube welding unit (37) for welding together and / or cutting and closing off tubes, more preferably, that the tube welding unit (37) welds a tube to the bypass line (22) and / or a tube connected to the media container (10) and / or a tube connected to the waste container (18).

[0359] 16. Method according to item 15, characterized in that the bioreactor module (3) comprises at least one tube holder (38) holding at least one tube in a defined position relative to the support structure (19) and that the tube welding unit (37) interacts with the tube holder (38) to weld and / or cut and seal the tube, preferably, that the tube holder (38) comprises a tube mover mechanism for extending and / or retracting the tube relative to the tube holder (38).

[0360] 17. Method according to one of the preceding items, characterized in that the cell vessel (4) comprises a tube set connected to the cell vessel (4) and presterilized together with the cell vessel (4), that the tube set comprises at least one disposable head for a pump (8) and / or at least one, in particular magnetic, docking element for a sensor, and / or, the recirculation loop (24), and / or, a sealed and weldable connection for a media container (10) and / or a sealed and weldable connection for a waste container (18).

[0361] 18. Bioreactor module in particular for use in the method according to one of the preceding items, wherein the bioreactor module (3) comprises a cell vessel (4) for containing the cell culture, at least one active element (5) for influencing the cell culture in the bioreactor module (3) and preferably at least one sensor (6) for sensing a characteristic of the cell culture, wherein the cell vessel (4) comprises a main membrane (7), wherein the bioreactor module (3) comprises the control unit and / or an interface to the control system (2), wherein the control unit is adapted to, after a cell culture was introduced into the cell vessel (4), subject the cells to at least one unit operation and an, in particular subsequent, expansion operation inside the cell vessel (4), and, during the unit operation and / or the expansion operation, implement control instructions to cause the at least one active element (5) to influence the cell culture and thereby advance the unit operation and / or the expansion operation, and / or, wherein the interface to the control system (2) is adapted to allow the control system (2) to implement control instructions to cause the at least one active element (5) to influence the cell culture and thereby advance the unit operation and / or the expansion operation, preferably, wherein the control unit is adapted to receive sensor data from the at least one sensor

[0362] (6) and derive from the sensor data control instructions and / or, wherein the interface to the control system (2) is adapted to allow the control system (2) to receive sensor data from the at least one sensor (6).

[0363] 19. Bioreactor module according to item 18, characterized in that the cell vessel (4) comprises a tube set connected to the cell vessel (4) and pre-sterilized together with the cell vessel (4), that the tube set comprises at least one disposable pump (8) head and / or at least one, in particular magnetic, docking element for a sensor, and / or, the recirculation loop (24), and / or, a sealed and weldable connection for a media container (10) and / or a sealed and weldable connection for a waste container (18).

[0364] 20. Bioprocessing system in particular for use in the method according to one of items 1 to 17, wherein the bioprocessing system (1) comprises a control unit and a bioreactor module (3) which comprises a cell vessel (4) for containing the cell culture, at least one active element (5) for influencing the cell culture in the bioreactor module (3) and preferably at least one sensor (6) for sensing a characteristic of the cell culture, wherein the cell vessel (4) comprises a main membrane

[0365] (7), wherein the bioprocessing system (1) is adapted to, after a cell culture was introduced into the cell vessel (4), subject the cells to at least one unit operation and an, in particular subsequent, expansion operation inside the cell vessel (4), wherein the control system (2) is adapted to, during the unit operation and / or the expansion operation, implement control instructions to cause the at least one active element (5) to influence the cell culture and thereby advance the unit operation and / or the expansion operation.

[0366] In the application, where an element or component is said to be included in and / or selected from a list of recited elements or components, it should be understood, that the element or component can be any one of the recited elements or components, or the element or component can be selected from a group consisting of two or more of the recited elements or components.

[0367] Terms "a" and ''an'' and ''the'' and similar reference used in the context of describing the invention (especially in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The use of the term "include," "includes," "including," "have," "has," "having," "contain," "contains," or "containing," including grammatical equivalents thereof, should be understood generally as open-ended and non-limiting, for example, not excluding additional unrecited elements or steps, unless otherwise specifically stated or understood from the context.

[0368] Where the use of the term "about" or ''approximately'' is before a quantitative value, the present invention also includes the specific quantitative value itself, unless specifically stated otherwise. As used herein, the term "about" refers to a ±10% variation from the nominal value unless otherwise indicated or inferred.

[0369] Also as used herein, ''and / or'' refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (''or''). The use of the alternative (e.g., ''or'') should be understood to mean either one, both, or any combination thereof of the alternatives.

[0370] All citations are hereby incorporated by reference.

[0371] All individual embodiments and aspects as disclosed herein can be combined with each other within the framework and context of the present disclosure. It will be understood that the embodiments disclosed herein are only exemplary, and that any feature presented for a particular exemplary embodiment may be used with the present disclosure on its own or in combination with any feature presented for the same or another particular exemplary embodiment and / or in combination with any other feature not mentioned. It will further be understood that any feature presented for an example embodiment in a particular category may also be used in a corresponding manner in an example embodiment of any other category.

[0372] EXAMPLES

[0373] It should be understood that the following example is for illustrative purpose only and not to be construed as limiting this invention in any manner. The following example demonstrates that the method according to the present disclosure advantageously allows performing a bioprocesses on cells in a cell culture, specifically on an immune cell culture comprising T cells, including at least one unit operations. In particular, it is shown that T cells can be transferred into the cell vessel, followed by activation and expansion in the very same cell vessel, allowing for simple, precise and robust production of a cell therapy product. Example 1

[0374] Peripheral blood mononuclear cells (PBMCs) were thawed and isolated using CD3+ StemCell Technologies Kit according to the manufacturer's instructions. Part of the cells were activated in flask for reference (herein referred to as "BRO2") according to the manufacturer's protocols and part were used for performing the method according to the present disclosure (herein referred to as ''BRO3"). In addition, cells derived from a different donor were activated by the method according to the present disclosure (herein referred to as "BROl").

[0375] In BRO l and BRO3, cells were inoculated and activated in the cell vessel mounted on a bioreactor module (50mL at a cell density of 1.43xlO6 / mL). In BRO2, cells were activated in T- flask at 1.43xl06(in 20mL).

[0376] For culture according to the present disclosure (BRO1 / BRO3), cells were shaken for 1 h to ensure that the added activation reagent and cell culture were evenly uniform (rate 16rpm angle- 4 / +4). The fluidic lines were primed with media 6 mL media to ensure volume was representative. After 24 h, cell activation was completed and the cells were rocked for 30 min to ensure homogenous distribution (rate: 16 rpm angle-4 / +4), then returned to static position. After 48h, cells were rocked at a rate of 16 rpm angle-4 / +4.

[0377] Post 65 h of activation the cells went through three cycles of perfusion to wash off the activation reagent (0.34mL / min). For this purpose, the culture liquid comprising left over activation reagent was retrieved as permeate through a membrane comprised in the cell vessel (here the reservoir of the cell vessel). Afterwards, fresh cell culture medium was added through the fluidic lines. This process was accordingly repeated until the activation reagent has been essentially completely removed. From day 3 on, cell density was set to lxlO6 / mL by dilution and the cells were cultured in the cell vessel up to day 7 (BRO2, BRO3) or up to day 6 (BROl).

[0378] On day 6 or 7, respectively, cell density was set to lxlO6 / mL again by dilution with fresh cell culture medium, and the cells were cultured in the cell vessel for another 2 h interval. Then, perfusion started and carried on until day 13. This was advantageously done using the reservoir membrane and / or the main membrane disclosed herein. At the end of day 13, the cells were harvested. As shown in Fig. 6, BRO3 led not only to a larger total number of cells compared to BRO2 (p=0.0002, graph 1) - which would be expected due to the different starting volumes - but also to a higher daily fold expansion. Hence, by applying the method according to the present disclosure and performing a step of expanding the cells, greater cell numbers were achieved in shorter time, allowing for more efficient production of activated T cells, and thus better manufacturing of a cell therapy product.

[0379] In addition, as shown in Fig. 7, the expansion of BRO3 had higher a viable cell density (VCD) compared to BRO2 (p=0.01 graph 1). No significant difference was seen in the VCD during-the batch culture (p=0.84), but a significant difference was observed the moment perfusion culture was initiated. Hence, by applying the method according to the present disclosure and performing a step of expanding the cells, not only higher cell density but also more viable cells were obtained. Thus, not only was production more efficient with higher yields but also of higher quality, which is an important parameter in the manufacture of a cell therapy product.

[0380] As shown in Fig. 14, the expansion of BRO1 and BRO3 led to comparable cell numbers (graph 1) and fold expansions (graph 2), with both experiments leading to an expansion of above 300-fold. Cell Viability was also measured during the expansion period (graph 3). Following cell activation between days 0 and 3, a marked decline in viability was observed, reaching approximately 50% in both runs. However, from day 3 onward, cell viability showed a recovery trend, progressively increasing until stabilizing at around 95% from day 6 onwards, where it remained consistent for the duration of the observation period. Assessing the activation count (graphs 4 and 5) at the end of the activation period showed that more than 80% CD4+and more than 80% CD8+T cells were activated in each run.

[0381] The example shows that the method according to the present disclosure shows improved results for cell activation and expansion over known flask activation protocols. The results obtained by the method according to the present invention are consistent over different donors, and result in a high percentage of viable, activated CD4+and CD8+T cells.

[0382] Example 2

[0383] On Day 0, CD3+ T cells were inoculated in the cell vessel at approximately 1.43 million cells / mL in 50mL (total of -70 million cells) using Nutri-T media (4Cell® Nutri-T GMP PRF, Biological Industries), along with IL-2 (lOOlU / mL) and TransAct (1:100 dilution) for in-bag T cell activation. During the activation process, the cell vessel was static for the first -60 hours, with intermittent rocking at the 24 and 48 hours. At -60 hours, the activation reagents were automatically washed out of the cell vessel via perfusion media exchange. On day 3, the media within the bioreactor was reduced to achieve a cell concentration of -3 million cells / mL, after which the transfection reagents (GFP-mRNA liposomes) were introduced into the cell vessel. The system was static for the first 4 hours of transfection, after which media was added, to achieve a cell concentration of 0.75 million cells / mL. The system was rocked overnight, and after -8 hours, the transfection reagents were washed out of the cell vessel via perfusion media exchange. From days 4-11 the cells underwent the following expansion process: From days 4-7 media was added daily via fed- batch processes to achieve a cell concentration of 1 million cells / mL until IL volume was reached in the cell vessel, from days 7-9 media was exchanged via perfusion processes at 0.5 Vessel Volumes per Day (VVD), and at 1 VVD from days 9-11. Finally, on day 11, the cells were harvested from the cell vessel. The following analytics were performed: flow cytometry for transfection efficiency and cell phenotyping, Nucleocounterfor viability and cell counts and Cedex Bio HT for metabolite analysis (glucose, lactate, LDH, ammonia).

[0384] A transfection efficiency of -60% 1 day post-transfection was achieved, and a 70-fold expansion from 70 million to 5 billion CD3+ T cells over 11 days, with a viability of -94%.

[0385] Example 3

[0386] Peripheral blood mononuclearcells (PBMCs) from a single donorwere thawed and isolated using CD3+ StemCell Technologies Kit according to the manufacturer's instructions. Prior to and after T cell isolation, an aliquot was aspirated and subjected to flow cytometric analysis of immune cell markers CD4 and CD8. Part of the isolated cells were activated in flask for reference (herein referred to as "Flask") according to the manufacturer's protocols and part were used for performing the method according to the present disclosure (herein referred to as "BRI" and "BR2").

[0387] In BRI and BR2, the cells were inoculated and activated in the cell vessel mounted on a bioreactor module (50mL at a cell density of 1.43xlO6 / mL). In Flask, cells were activated in a T- flask at 1.43xl06(in 20mL).

[0388] For culture according to the present disclosure (BR1 / BR2), cells were shaken for 1 h to ensure that the added activation reagent and cell culture were evenly uniform (rate 16rpm angle-4 / +4). The fluidic lines were primed with media 6 mL media to ensure volume was representative. After 24 h, cell activation was completed and the cells were rocked for 30 min to ensure homogenous distribution (rate: 16 rpm angle-4 / +4), then returned to static position. After 48h, cells were rocked at a rate of 16 rpm angle-4 / +4.

[0389] After 65 h of activation, an aliquot was taken from each culture for flow cytometric analysis of immune cell markers. In the culture, the cells went through three cycles of perfusion to wash off the activation reagent (0.34mL / min). For this purpose, the culture liquid comprising left over activation reagent was retrieved as permeate through a membrane comprised in the cell vessel (here the reservoir of the cell vessel). Afterwards, fresh cell culture medium was added through the fluidic lines. This process was repeated until the activation reagent was essentially completely removed. From day 3 on, cell density was set to lxlO6 / mL by dilution and the cells were cultured in the cell vessel up to day 6.

[0390] On day 6, cell density was set to lxlO6 / mL again by dilution with fresh cell culture medium. At this stage, aliquots from all cultures were subjected to immunostaining and flow cytometry analysis for immune cell markers. The Flask culture was discontinued at this point, and the BRI and BR2 cells were cultured in the cell vessel for another 2 h period. Then, perfusion culture was initiated. From days 6-8 media was exchanged via perfusion processes at 0.5 Vessel Volumes per Day (VVD), and at 1 VVD from days 8-12. On day 12, the cells were harvested from the cell vessel, and aliquots from both cultures were subjected to immunostaining and flow cytometry analysis.

[0391] Fig. 15 shows that the percentages of immune cells of each kind (CD4+, CD8+, CD47CD8", CD4+ / CD8+) were almost identical between flask-activated and bioreactor-activated cells, indicating that the method according to the present invention does not introduce any specific bias in terms of survival of any particular population of T cells. As expected, CD8+ cells outgrew the CD4+ population by day 12 of culturing, agreeing with common knowledge in this respect. Collectively, the experiments show that T cell activation by the method according to the present invention leads to advantageous increases in activated cell number while keeping with the known ratios of activated T cell populations.

[0392] Example 4

[0393] On Day 0, CD3+ T cells were inoculated in different cell vessels (BRI and BR2) at approximately 1.43 million cells / mL in 50mL each (total of -70 million cells) using Nutri-T media (4Cell® Nutri-T GMP PRF, Biological Industries), along with IL-2 (lOOlU / mL) and TransAct (1:100 dilution) for in-bag T cell activation. During the activation process, the cell vessel was static for the first -60 hours, with intermittent rocking at the 24 and 48 hours. At -60 hours, the activation reagents were automatically washed out of the cell vessel via media exchange. In BRI, the exchange occurred by perfusing at a rate of 0.5 mL per minute for 10 hours (total exchange: 300 mL) according to a preferred embodiment of the present invention. In BR2, the exchange occurred by running six exchange cycles, wherein each exchange cycle consisted of first removing 0.5 mL per minute for 50 minutes (total amount removed: 25 mL), then adding 25 mL of fresh culture media, and then rocking for 30 minutes (total amount exchanged: 150 mL), according to an embodiment of the present invention.

[0394] In parallel, a control batch was inoculated and activated underthe same conditions in a T25 flask according to the manufacturer's protocols.

[0395] On day 3, the media within the vessel / flask was reduced to achieve a cell concentration of -3 million cells / mL, after which the transfection reagents (GFP-mRNA liposomes, 0.27 pg mRNA per million cells) were introduced into the cell vessel. The vessel was constantly rocked for the first 4 hours of transfection, after which media was added to achieve a cell concentration of 0.75 million cells / mL. The system was rocked overnight, and after -8 hours, the transfection reagents were washed out of the cell vessel via perfusion media exchange. From days 4-7 media was added daily via fed-batch processes to achieve a cell concentration of 1 million cells / mL until IL volume was reached in the cell vessel. On days 0, 4 and 7, flow cytometry for transfection efficiency and cell phenotyping was performed.

[0396] Fig. 16A showsthat the cell vessel BRI contained >80% GFP-expressing cells on day 4, indicating a transfection efficiency of >80% in the cell vessel, which is suitable and preferred for biotechnological production processes. The cell vessel BR2 contained >50% GFP-expressing cells on day 4, which indicates a transfection efficiency of >50% which is adequate for many processes. The control sample of flask-transfected cells contained approx. 100% GFP-positive cells, and the non-transfected negative control (cells in flask) expectedly contained no GFP- positive cells. On day 7, the percentage of GFP-positive cells was determined only for BRI, and showed to be approx. 70%.

[0397] Fig. 16B shows that the percentages of T cell populations (CD47CD8", CD47CD8+, CD47CD8", CD4+ / CD8+) were highly similar between the different approaches (BRI, BR2, cells transfected in flask, untransfected cells in flask), further underscoring that the methods of the invention do not adversely affect any particular population of T cells. The experiment shows that the methods according to the invention achieve a high transfection efficiency when transfecting directly inside a perfusion vessel of the invention. In certain embodiments of the present invention, the transfection efficiency can be improved even further.

[0398] Example 5

[0399] CD3+ T cells were inoculated and activated in a bioreactor as described above under Example 2. 24 hours after activating the cells, cells were transduced to express a recombinant chimeric antigen receptor (CAR) by adding Lentivirus and transduction enhancer to the culture (MOI: 10). A non-transduced culture was cultured in parallel. From day 2 until day 7, fresh cell culture media was added daily via fed-batch processes to the culture to achieve a cell concentration of 1 million cells / mL until IL volume was reached in the cell vessel. Starting on day 7, media was exchanged via perfusion processes at 1 VVD. Starting on day 4 (i.e., 72 h post transfection), an aliquot was taken daily and analyzed for total cell number and viability. It was found that the total number of cells and the cell viability was comparable between transduced and non-transduced cells, with transduced cells achieving an approx. 180-fold expansion over the entire culture period. Moreover, it was found that approximately 30% of T cells expressed a CAR, and the number of CAR expressing T cells was in ratio with non-CAR expressing cells (see Fig. 17).

[0400] The example shows that the method according to the present invention allows for the reliable production of high numbers of CAR T cells. The method enables translating from a manual plate culturing setup to a large-scale process which is at least partially automated.

[0401] While the Examples are representative for embodiments of the present disclosure using activation as unit operation and a subsequent expansion step, different further steps or different embodiments, such genetic modification an automated manner can be achieved. Hence, various different sequences of at least one unit operation and an expansion step can be performed.

Claims

CLAIMS1. A method for performing a bioprocess on cells in a cell culture, wherein the cell culture is present in a cell vessel (4), wherein the method comprises performing one or more unit operation(s) on the cells in an automated manner, wherein a unit operation comprises: i. adding a liquid composition to the cell vessel (4); ii. incubating the cells in the cell vessel (4) with the added liquid composition; and iii. removing liquid from the cell vessel (4) through at least one filter, and thereby removing at least a part of the added liquid composition; and expanding the cells in the cell vessel (4), wherein expanding is performed prior to and / or after performing the one or more unit operation(s).

2. The method according to claim 1, wherein the unit operation is selected from: activation of the cells, preferably wherein the liquid composition comprises an activation agent; and genetic modification of the cells, in particular transduction and / or transfection, preferably wherein the liquid composition comprises a genetic modification agent, in particular a transduction agent and / or a transfection agent.

3. The method according to claim 1 or 2, wherein the method comprises performing more than one unit operation, preferably, wherein the method comprises at least two unit operations, more preferably wherein the at least two unit operations comprise performing activation and genetic modification of the cells, or the method comprises at least three unit operations, more preferably wherein the at least three unit operations comprise performing activation and two genetic modifications of the cells, such as activation, transfection and / or transduction.

4. The method according to one or more of claims 1 to 3, wherein the liquid composition added in step i. comprises:(i) an activation agent;(ii) a genetic modification agent, preferably a transduction agent or a transfection agent, and optionally an enhancer; or(iii) an activation agent and a genetic modification agent, preferably a transduction agent or a transfection agent, and optionally an enhancer.

5. The method according to any one of claims 1 to 4, wherein step iii. further comprises one or more washing steps to essentially completely remove the liquid composition from the cell vessel.

6. The method according to claim 5, wherein the washing step consists of perfusing, preferably wherein an amount of liquid equal to at least lx, 2x, 3x, 5xor lOx the volume of the cell culture is exchanged, further preferably wherein the perfusion speed is 15 VVD or less, 10 VVD or less, 8 VVD or less, or 4 VVD or less.

7. The method according to one or more of claims 1 to 6, wherein the method comprises performing more than one unit operation, preferably wherein performing more than one unit operation comprises: repeating steps i., to iii., one or more times; adding a liquid composition to the cell vessel (4) comprising agents for performing more than one unit operation, preferably the liquid composition comprises an activation agent and a genetic modification agent; repeating step i. prior to step ii. and / or prior to step iii. by adding a further liquid composition to the cell vessel (4), in particular wherein(i) the liquid composition in step i. comprises an activation agent, and(ii) the further liquid composition comprises a genetic modification agent; and / or repeating step i. prior to step ii. and / or prior to step iii. by adding a further liquid composition to the cell vessel (4), in particular wherein(i) the liquid composition in step i. comprises a genetic modification agent, and(ii) the further liquid composition comprises an activation agent.

8. The method according to one of more of claims 1 to 7, wherein the method comprises measuring the cell density one or more time(s), preferably measuring the cell density at least priorto step I, optionally, wherein the method comprises on basis of the measured cell density automatically adding a suitable volume of liquid composition to the cell vessel (4) in step i., in particular wherein the liquid composition comprises an activation agent and / or genetic modification agent, such that the volume of the liquid composition is suitable, preferably adjusted, for activation and / or genetic modification of the cells.

9. The method according to one or more of claims 1 to 8, wherein a volume of the cell culture in the cell vessel (4) increases during the step of expanding the cells, preferably after performing the at least one unit operation on the cells.

10. The method according to one or more of claims 1 to 9, wherein the method further com prises a step of washing the cells one or more time(s) after performing the unit operation(s), preferably wherein washing comprises: i. adding a washing liquid composition to the cell vessel (4);ii. optionally, incubating the cells in the cell vessel (4) with the added washing fluid; and iii. removing fluid from the cell vessel (4) through at least one filter, and thereby removing at least a part of the added washing fluid.

11. The method according to one or more of claims 1 to 10, wherein the method further comprises harvesting the cells, preferably after performing the unit operation(s) and expanding the cells; and / or formulating the cell culture into a cell therapy product.

12. The method according to one of more of claims I to 11, wherein the method comprises using a bioprocessing system (1) comprising a control system (2) and at least one active element (5), wherein the control system (2) implements control instructions to cause the at least one active element (5) to advance the unit operation, optionally wherein the control system (2) generates control instructions to cause the active element (5) to add the liquid composition to the cell vessel (4) in step i. and / or remove the liquid from the cell vessel (4) through the at least one filter in step iii.

13. The method according to one or more of claims 1 to 12, wherein the cell vessel (4) comprises two or more compartments, preferably a small volume compartment (13) and a distinct large volume compartment (14), optionally wherein the cell vessel (4) comprises a pre-shaped reservoir (15) forming a small volume compartment (13); and / or cell vessel (4) comprises a casing (16) forming a large volume compartment (14), wherein the casing is preferably flexible.

14. The method according to claim 13, wherein the method has following characteristics: during performing the unit operation(s), the cell culture is located in the small volume compartment (13); and / or during expanding the cells in the cell vessel (4), preferably after step iii., the volume of the cell culture increases such that the cell culture is preferably located in the small volume compartment (13) and the large volume compartment (14).

15. The method according to claim 13 or 14, wherein the one or more of the at least two compartments comprise at least one filter and / or is / are fluid ical ly connected to at least one filter, optionally wherein the small volume compartment (13) comprises at least one filter and / or is fluidically connected to at least one filter, and / or the large volume compartment (14) comprises at least one filter and / or is fluidically connected to at least one filter.

16. The method according to one or more of claims 1 to 15, wherein the cells are mammalian cells, preferably immune or naive cells.

17. The method according to one or more of claims 1 to 16, wherein the method comprises a cell selection, preferably prior to performing the unit operation(s), optionally wherein cell selection comprises: adding a mixture of cells to the cell vessel (4), contacting the mixture of cells with a binding unit, wherein at least a fraction of the cells directly or indirectly binds to a binding matrix of the binding unit, and removing liquid from the cell vessel (4), thereby removing at least part of the cells, which do not directly or indirectly bind to the binding matrix.

18. A bioreactor module for performing a bioprocess on cells in a cell culture, wherein the bioreactor module (3) comprises a cell vessel (4), and at least one filter for removing liquid from the cell vessel (4) through the at least one filter, wherein the bioreactor module is configured for performing in an automated manner one or more unit operations on the cells in the cell vessel (4), and for expanding the cells in the cell vessel (4), optionally, wherein the bioreactor module further comprises one or both of: at least one active element (5) for influencing the cell culture in the bioreactor module (3), preferably for adding a fluid to and / or removing a fluid from the cell vessel (4), preferably wherein the fluid is a liquid; and at least one sensor (6) for sensing a characteristic of the cell culture.

19. A bioprocessing system for performing a bioprocess on cells in a cell culture, wherein the bioprocessing system (1) comprises the bioreactor module (3) according to claim 18.

20. Use of the bioreactor module (3) according to claim 18 and / or the bioprocessing system according to claim 19 for manufacturing a cell therapy product, preferably wherein manufacturing comprises at least one of the following: optionally, selecting cells one or more times, in particular by binding at least a fraction of the cells expressing a ligand, preferably selecting a subpopulation of T cells; activating cells, preferably activating T cells; transducing cells, preferably transducing T cells; transfecting cells, preferably transfecting T cells; and / or expanding cells, preferably T cells, more preferably genetically modified T cells.

21. Use of the bioreactor module (3) according to claim 18 and / or the bioprocessing system according to claim 19 in a method according to one or more of claims 1 to 17.

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