Methods for bioprocessing immune cells or naive cells in cell culture

Abstract

The present invention relates to a method for bioprocessing cells in a cell culture, wherein the cell culture is present in a cell container. The method comprises performing one or more unit operations on the cells in an automated manner, and expanding the cells in the cell container, wherein the expansion is performed before and / or after performing the one or more unit operations. The present invention also relates to a bioreactor module for bioprocessing cells in a cell culture. The present invention also relates to a bioprocessing system for bioprocessing cells in a cell culture. The present invention also relates to the use of the bioreactor module and the bioprocessing system.

Description

Technical Field

[0001] This invention relates to a method for bioprocessing cells in a cell culture, wherein the cell culture is located in a cell container. The method includes performing one or more unit operations on the cells in an automated manner, and expanding the cells in the cell container, wherein the expansion occurs before and / or after performing the one or more unit operations. The invention also relates to a bioreactor module for bioprocessing cells in a cell culture. Furthermore, the invention relates to a bioprocessing system for bioprocessing cells in a cell culture. Finally, the invention relates to the use of the bioreactor module and the bioprocessing system. Background Technology

[0002] The term "bioprocessing" currently refers to a biotechnological process, specifically one involving the use of immune cell cultures or primary cell cultures. One or more processing steps may be performed on each cell culture. Therefore, in this sense, bioprocessing can refer to a manufacturing process involving a series of processing steps on cell cultures that ultimately produce a final product.

[0003] Here, bioprocessing falls under the fields of cell and gene therapy, such as for the creation of autologous T cells modified to express chimeric antigen receptors (CARs). These cells can be used to treat various types of cancer, including different types of leukemia (blood cancers). Other cell therapies based on primary cells, particularly stem cells and their derivatives, are also attracting attention.

[0004] It is not yet fully clear which factors significantly contribute to the success of automated cell manufacturing for cell and gene therapy. There is a need to design a highly fault-tolerant bioprocessing system, aiming to consistently protect cells while achieving a high degree of automation to reduce labor costs and human error. A highly flexible bioprocessing system also needs to be designed, with components and workflows planned so that automation is not hindered by complex mechanisms and components. Furthermore, because the sources of success for automated bioprocessing systems are unknown, many problems remain to be solved. For example, fault-prone bioreactors may only be feasible for a portion of emerging processes, potentially leading to system failure or requiring numerous separate solutions.

[0005] In the field of cell and gene therapy, it is also important for the bioprocessing of allogeneic or autologous production of genetically modified immune cells to comply with regulatory requirements, and bioprocessing is often highly regulated and requires regulatory approval.

[0006] One specific issue requiring improvement is providing highly flexible and low-complexity methods and apparatus to reduce the number of transfers of cell cultures between cell containers, allowing for more unit operations within a single cell container, thereby accelerating the overall process while reducing the likelihood of contamination and errors. Furthermore, high flexibility is needed because many factors can influence the success of the manufacturing process, with the contribution of each individual factor still under discussion. Factors under investigation include the requirement for uniform nutrient distribution, the requirement for cell agitation, the metabolic state of the cells, the distribution of cells within the cell container, and the surface area of ​​the cell culture.

[0007] Some known methods and cell containers (e.g., Miltenyi Prodigy) ® These are centrifuge chambers, thus offering high practicality, but for a given level of flexibility, they also introduce high complexity and cost. Furthermore, the volume of cultures processed within these chambers is typically limited to the order of magnitude. Other known cell containers (e.g., G-Rex) ® It is primarily used manually, specifically for certain cell culture volumes, and lacks the features required for high automation.

[0008] One object of the present invention is to provide a method for performing various unit operations and cell expansion in a simplified and efficient manner. Another object is to provide cell containers and environments that can be used with greater flexibility and lower complexity. Summary of the Invention

[0009] The present invention addresses the aforementioned objectives. Specifically, the present invention provides a method for biologically treating cells in a cell culture, wherein the cell culture is situated in a cell container. The method includes performing one or more unit operations on the cells in an automated manner. Each such unit operation includes: i. Add the liquid composition to the cell container; ii. Incubating the cells in a cell container with the added liquid composition; and iii. Remove liquid from the cell container through at least one filter, thereby removing at least a portion of the added liquid composition.

[0010] The method also includes expanding cells in a cell container, wherein the expansion is performed before and / or after one or more unit operations.

[0011] The provided method advantageously simplifies the biological processing of cells in cell cultures by applying cell containers that enable automated one or more unit operations (e.g., activation, transfection, transduction) of cells. This is achieved by removing liquid from the cell container through at least one filter, thereby removing a portion of the added liquid composition. The cell container may include at least one filter and / or be fluidly connected to at least one filter. The filter enables flexible removal of fluid, thereby achieving separation from the cells without substantially negatively impacting sensitive cells, for example, through fluid transfer and / or centrifugation. Using this specific setup, unit operations are also performed automatically, for example, by adding liquid to the container using an active element such as a pump to incubate cells in the container with the added liquid, and removing liquid from the cell container through at least one filter, thereby removing at least a portion of the added liquid. Simultaneously, the cell container is suitable for cell expansion, thus allowing advantageous performance of unit operations and cell expansion within the same cell container. This significantly simplifies processes such as cell therapy manufacturing, compared to conventional methods in the art, as cumbersome manual processes are automated. Therefore, processing errors are avoided, thereby improving reproducibility and reducing the risk of contamination. Furthermore, the conditions applied to the cells can be tightly controlled by enabling multiple unit operations and amplifications within the same cell container. Additionally, the transfer step is avoided, resulting in fewer negative impacts on the cells overall and reduced cell loss. The embodiments disclosed herein highlight these positive effects.

[0012] According to a first aspect of the present invention, a method is provided for biologically treating cells in a cell culture, wherein the cell culture is present in a cell container, wherein the method includes...

[0013] - Performing one or more unit operations on cells in an automated manner, wherein the unit operations include: i. Add the liquid composition to the cell container; ii. Incubating the cells in a cell container with the added liquid composition; and iii. Removing liquid from the cell container through at least one filter, thereby removing at least a portion of the added liquid composition; and - Expanding cells in a cell container, wherein the expansion is performed before and / or after one or more unit operations.

[0014] According to a second aspect of the present invention, a bioreactor module for bioprocessing cells in a cell culture is provided, wherein the bioreactor module comprises

[0015] - Cellular containers, and

[0016] - At least one filter for removing liquid from the cell container through said at least one filter. - The bioreactor module is configured to perform one or more unit operations on cells in a cell container in an automated manner, and to expand the cells in the cell container.

[0017] According to a third aspect of the present invention, a biological treatment system for biologically treating cells in a cell culture is provided, wherein the biological treatment system comprises a bioreactor module according to a second aspect of the present invention.

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

[0019] According to a fifth aspect of the invention, the use of a bioreactor module according to the second aspect and / or a biological treatment system according to the third aspect in a method according to the first aspect of the invention is provided. Attached Figure Description

[0020] Embodiments of the present invention will now be explained with reference to the accompanying drawings. The drawings are shown below: Figure 1 A biological treatment system with several bioreactor modules. Figure 2 Bioreactor modules with cell containers Figure 3 Cell containers of a bioreactor module Figure 4 An internal view of the cell container. Figure 5 A schematic diagram of possible fluid piping for the bioreactor module in different implementations. Figure 6 Compared with the reference cell culture (BRO2), the total number of T cells (see 1) and the daily expansion rate (see 2) of T cells using the biological treatment system (BRO3) were compared. Figure 7 Throughout the entire culture period (see 1) and during perfusion culture only (see 2), the viable cell density using the biological treatment system (BRO3) compared to the control cell culture (BRO2) was... Figure 8 A container containing bonding units. Figure 9 Examples of preferred embodiments, which may be referred to as "sequential implementation methods" in this disclosure, are provided. Figure 10 Examples of preferred embodiments, which may be referred to as "parallel implementations," in the method of this disclosure. Figure 11 According to an example of a preferred embodiment of the method disclosed herein, which may be referred to as the "transfection short implementation method", Figure 12 According to an example of a preferred embodiment of the method disclosed herein, which may be referred to as the "transfection long-term implementation method", Figure 13 According to an example of a preferred embodiment of the method disclosed herein, which may be referred to as a "transfection and transduction implementation method", Figure 14 Using the biological processing system according to the invention, the total number of cells (see 1), total expansion fold (see 2), cell viability (see 3), percentage of activated CD4+ T cells (see 4), and percentage of activated CD8+ T cells (see 5) in the case of using T cells from two different donors (BRO1 and BRO3). Figure 15 The percentage of T cell population during the activation and culture period in accordance with the methods (BR1 and BR2) and culture flask culture according to the present invention. Figure 16 The transfection efficiency (see A) and T cell phenotype (see B) of GFP-transfected T cells transfected according to the methods of this disclosure, and Figure 17 The percentage of T cells expressing chimeric antigen receptor (CAR) and GFP after transduction according to the method of the present invention. Detailed Implementation

[0021] The following description is intended to enhance understanding of this disclosure and should be understood as supplementing the description of exemplary embodiments of this disclosure provided in the foregoing sections of this specification, and should be read in conjunction with the description of the exemplary embodiments. It should be understood that the invention is not limited to the specific embodiments, methods, procedures, and reagents described herein, as these embodiments, methods, procedures, and reagents may vary within the scope set forth in the claims. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to limit the scope of the invention, which is defined by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.

[0022] In the following description, certain elements of the invention will be described. These elements may be discussed in conjunction with specific embodiments; however, it should be understood that they can be combined in any manner and in any number to create additional embodiments. The various embodiments, features, and specific implementations described should not be construed as limiting the invention to the explicitly described embodiments or combinations of explicitly described features. This description should be understood as disclosing and covering implementations that combine the explicitly described embodiments with any number of disclosed and / or preferred elements. Furthermore, unless the context otherwise indicates, any permutation and combination of all elements described in this application should be considered as disclosed in this specification.

[0023] A method for biologically treating cells in a cell culture according to a first aspect of the present invention

[0024] According to a first aspect of the present invention, a method is provided for biologically treating cells in a cell culture, wherein the cell culture is present in a cell container, wherein the method includes...

[0025] - Performing one or more unit operations on cells in an automated manner, wherein the unit operations include: i. Add the liquid composition to the cell container; ii. Incubating the cells in a cell container with the added liquid composition; and iii. Removing liquid from the cell container through at least one filter, thereby removing at least a portion of the added liquid composition; and - Expanding cells in a cell container, wherein the expansion is performed before and / or after one or more unit operations.

[0026] The provided method advantageously simplifies the biological processing of cells in cell cultures by applying cell containers that enable automated one or more unit operations (e.g., activation, transfection, transduction) of cells. This is achieved by removing liquid from the cell container through at least one filter, thereby removing a portion of the added liquid composition. The cell container may include at least one filter and / or be fluidly connected to at least one filter. The filter enables flexible removal of fluid, thereby achieving separation from the cells without substantially negatively impacting sensitive cells, for example, through fluid transfer and / or centrifugation. Using this specific setup, unit operations are also performed automatically, for example, by adding liquid to the container using an active element such as a pump to incubate cells in the container with the added liquid, and removing liquid from the cell container through at least one filter, thereby removing at least a portion of the added liquid. Simultaneously, the cell container is suitable for cell expansion, thus allowing advantageous performance of unit operations and cell expansion within the same cell container. This significantly simplifies processes such as cell therapy manufacturing, compared to conventional methods in the art, as cumbersome manual processes are automated. Therefore, processing errors are avoided, thereby improving reproducibility and reducing the risk of contamination. Furthermore, the conditions applied to the cells can be tightly controlled by enabling multiple unit operations and expansions within the same cell container. Additionally, the transfer step is avoided, resulting in fewer negative impacts on the cells overall and reduced cell loss. The embodiments disclosed herein highlight these positive effects, demonstrating the attainment of higher cell numbers and cell viability, indicating more efficient and higher-quality cell therapy manufacturing (see [link to documentation]). Figure 6 and 7 ).

[0027] As disclosed herein, biological treatment of cells in cell cultures specifically refers to performing one or more unit operations and typically expanding the cells. Other biological treatments of cells may also be performed, including selection, washing, harvesting, or preparation of cells, as disclosed herein.

[0028] Performing one or more unit operations typically refers to unit operations involved in cell therapy manufacturing. Preferred embodiments described herein involve activation and gene modification. Those skilled in the art fully understand unit operations within the framework of biological processing of cell cultures, particularly for cell therapy manufacturing. The disclosed “unit operation” is not a dedicated cell expansion step. This does not mean that cells cannot proliferate or grow throughout the unit operation, but rather that proliferation or growth occurs as a side effect throughout the unit operation. Therefore, cells can expand throughout the unit operation, but the primary intention of the unit operation is not cell expansion.

[0029] The method relates to unit manipulation of cells in an "automated manner." Those skilled in the art are well aware of the term "automated manner" and conventional automation methods. However, in the case of this invention, automation is advantageously achieved by providing a cell container that collectively enables unit manipulation and at least one filter. As disclosed herein, other means may be provided to facilitate automation, such as active elements, like pumps, for transferring liquids.

[0030] The term "cell container" is well known in the art and understood by those skilled in the art. In this document, the term "cell container" may be used interchangeably with the terms "container" or "bag." A cell container can be a flexible bag or a rigid vessel or container. A cell container preferably comprises both rigid and flexible portions. Within the cell container, steps of the method, particularly one or more unit operations and amplifications, are performed.

[0031] In this disclosure, cells are present in cell cultures. Those skilled in the art are well aware of this terminology. It generally refers to cells dispersed in a liquid medium, thereby forming a cell culture. According to this disclosure, "liquid medium" specifically refers to compositions in which cells are kept viable to the greatest extent possible. For example, cells may be combined with a cell culture medium to provide an environment in which cells can proliferate. Furthermore, cells may be combined with a buffer or storage solution in which cells can be stored and / or kept substantially alive. In another embodiment, cells may be combined with an agent (also referred to as a "reagent") to manipulate the cells; for example, an activator may be provided to activate the cells, and / or a gene-modifying agent may be provided to genetically modify the cells. In this document, the term "liquid medium" is used interchangeably with the terms "medium" and "liquid media." Sometimes, activators and / or gene-modifying agents, particularly transducers and / or transfection agents, may also be referred to as "liquid media," and therefore the term should encompass such compositions.

[0032] The term "cell amplification" is well known in the art. This generally refers to a process in which cells multiply or proliferate, and thus increase in number. The scope of "amplification" (also referred to as a "culture procedure") in this disclosure should not be limited. In fact, those skilled in the art are well aware of various amplification methods or culture procedures sufficient to culture cells in liquid media. Amplification may include batch culture, fed-batch culture, or perfusion culture, or combinations thereof. Furthermore, amplification may have been achieved if the cells are provided with a sufficient environment in which they can proliferate or at least not undergo complete apoptosis. In one embodiment, amplification may also include adding liquid, such as cell culture medium or reagent, to cells present in a container according to this disclosure. According to a preferred embodiment, particularly suitable for manufacturing cell therapy products, amplification encompasses an initial culture phase in which cells are initially cultured, preferably in which cells are cultured primarily in a small-volume compartment (also referred to as a "reservoir") of the container; and a subsequent culture phase in which cells are subsequently cultured, preferably in which cells are cultured in both the reservoir and a larger-volume compartment (also referred to as a "shell"). Such culture allows for the scaling up of small numbers of cells, typically only obtainable from patient samples used for autologous cell therapy, within the same container.

[0033] The container is particularly suitable for culturing cells in a liquid medium during the amplification step. For amplification, cells are typically provided in a liquid medium, which enables efficient cell amplification and manipulation. However, this disclosure is not limited to cells solely in a liquid medium. For example, cells may be provided with a carrier matrix or support matrix for culture, wherein these carrier matrices or support matrices are dispersed in a liquid medium. Furthermore, the medium may partially contain a dissolved or undissolved gas phase (e.g., the culture gases disclosed herein).

[0034] Unit operations

[0035] The method includes performing one or more unit operations on cells in an automated manner, the unit operations preferably selected from activation and gene modification. In this document, unit operations specifically refer to unit operations required throughout the cell therapy manufacturing process, for example, throughout the manufacturing of therapeutic immune cells such as CAR T cells or NK cells. Unit operations within the framework of cell therapy manufacturing processes are known and may include cell enrichment, cell selection, cell activation, gene modification of cells, etc. In this document, cell selection is considered a process separate from the unit operations disclosed herein (see further disclosure below regarding cell selection). As used herein, unit operations are distinct from the step of cell amplification (although cells may also proliferate and thus expand during the unit operation process).

[0036] According to a preferred embodiment, the unit operation is selected from: - Cell activation, preferably wherein the liquid composition comprises an activator; and - Gene modification of cells, particularly transduction and / or transfection, preferably wherein the liquid composition contains a gene-modifying agent, particularly a transducer and / or transfectant.

[0037] Cell therapy products typically require both cell activation and genetic modification, particularly genetically modified cells, for example, to target specific cell types, such as cancer cells. This specifically refers to the manufacture of CAR T cells or other immune cells that are typically genetically modified to target a specific cell type. Within the framework of this disclosure, it is particularly advantageous to perform the required unit operations using the same cell container on the same bioprocessing apparatus. For example, it is particularly advantageous to perform at least one cell activation step and at least one genetic modification step on the cells, preferably both in the same cell container. This is advantageous because it eliminates the need for a cell transfer step, thereby reducing the risk of contamination and cell damage.

[0038] On the other hand, since some unit operations can also be performed prior to the method according to this disclosure, the method according to this disclosure may have only one unit operation. For example, cells may be activated before being present in a cell container, so the activated cells only require gene modification as a unit operation for the biological treatment disclosed herein, particularly for cell therapy manufacturing. Furthermore, cells may be activated and expanded according to the method of this disclosure, while gene modification may subsequently be performed in the same cell container or another cell container. While such implementations are within the scope of this disclosure, those skilled in the art will appreciate that efficiently performing as many unit operations as possible in the same container is highly advantageous in simplifying the process and avoiding the negative impacts associated with cell transfer steps to another cell container (e.g., risk of contamination, risk of cell damage).

[0039] Therefore, according to a preferred embodiment, the method includes performing more than one unit operation, preferably wherein the method includes at least two unit operations, more preferably wherein the at least two unit operations include cell activation and gene modification. A specific embodiment including the method of cell activation and gene modification is described in... Figures 9 to 12 The explanation is as follows.

[0040] According to another preferred embodiment, the method includes performing more than one unit operation, preferably wherein the method includes at least three unit operations, more preferably wherein the at least three unit operations include performing cell activation and two gene modifications, such as activation, transfection, and / or transduction. A specific embodiment of the method including performing cell activation and two gene modifications is described in... Figure 13 The explanation is as follows.

[0041] According to a preferred embodiment, one or more unit operations are repeated once or more, preferably at least once more, and in particular, different unit operations are performed when the unit operation is repeated once more.

[0042] Liquid composition

[0043] The method according to the first aspect includes performing one or more unit operations on the cells in an automated manner, wherein the unit operations include i. adding a liquid composition to a cell container; ii. incubating the cells in the cell container with the added liquid composition; and iii. removing liquid from the cell container through at least one filter, thereby removing at least a portion of the added liquid composition.

[0044] As disclosed herein, a “liquid composition” means a composition that is primarily in a liquid state when added to a cell container. This does not preclude the composition from being frozen to a solid state before or after the addition of the liquid composition in step i. Furthermore, it does not preclude the liquid composition from also containing substances in a solid or gaseous state. For example, the liquid composition may contain solid particles, such as solid particles that can be used within the framework of the cell activation step. The liquid composition may also contain air bubbles. However, as disclosed herein, the liquid composition is primarily in a liquid state when added to the cell container because this simplifies the addition to the cell container (compared to a solid or gaseous state), thereby allowing for very precise control of the volume of the added liquid composition. Furthermore, cells in cell cultures are typically present in a liquid medium, such as cell culture medium or storage / freezing buffer, so the addition of the liquid composition facilitates easy mixing and sufficient contact with the cells. The composition typically contains a solvent, such as water, in which one or more compounds are dissolved or dispersed. For example, the composition may contain water and reagents, such as reagents for cell activation and / or gene modification. Additionally, the liquid composition may contain other compounds required by or supporting the cells, such as salts, amino acids, and / or nutrients.

[0045] According to a preferred embodiment, the liquid composition added in step i. comprises: (i) Activator; (ii) Gene modifiers, preferably transducers or transfectants, and optionally enhancers; or (iii) Activators and gene modifiers, preferably transducers or transfection agents, and optionally enhancers.

[0046] As disclosed herein, "reagent" specifically refers to a compound that achieves a desired result, such as cell activation or genetic modification of cells. This will immediately become clear to those skilled in the art upon reading the term "reagent" within the framework of this disclosure. Relevant reagents can be added to cell containers as part of a liquid composition, preferably wherein the cell culture is present in the cell container prior to the addition of the reagent. A variety of reagents are known in the art to achieve the desired results.

[0047] For example, activators should adequately activate cells, such as T cells, for in vitro expansion. T cell activation typically requires primary-specific signals (first signal) from the T cell receptor and co-stimulatory signals (second signal) such as CD28, 4-1BB, or OX40. Transduction of CAR cDNA via retroviral vectors also requires T cell activation. Activators can be selected from cell-based activators, such as antigen-presenting cells, such as dendritic cells, which are endogenous activators of the T cell response. Another cell-based T cell activation method is through artificial antigen-presenting cells. Activators can also be selected from bead-based activators such as Invitrogen CTS Dynabeads CD3 / 28, Miltenyi MACS GMP ExpAct Treg beads, Miltenyi MACS GMP TransAct CD3 / 28 beads, and Juno Stage Expamer technology. These reagents significantly simplify the in vitro T cell activation procedure. Activators can be specifically selected from antibody-coated magnetic beads, antibody-coated nanobeads, or utilizing expamer technology (based on streptamer technology).

[0048] As disclosed herein, a gene-modifying agent is a reagent capable of genetically modifying at least a portion of cells in a cell culture. Gene-modifying agents may be required for genetic modification of cells used in cell therapies such as CAR T-cell therapy. Gene-modifying agents are preferably transducers or transfectants. Transfectants may be, for example, lipid nanoparticles (e.g., described in WO2024 / 008967 A1). 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 GFP-mRNA formulation in liposomes to achieve high transfection efficiency. Following transfection, cells can be expanded.

[0049] Transduction agents can be viral vectors, particularly lentiviral or adenoviral vectors. Current CAR-T cell therapies largely rely on stable CAR expression after delivery via viral and non-viral gene transfer systems. There are three main types of stable gene expression vectors used in clinical applications: σ retroviral vectors, lentiviral vectors, and transposon / transposase systems. Messenger RNA transfer-mediated gene expression is another method for introducing CARs into cells while avoiding long-term expression.

[0050] As disclosed herein, "enhancers" are specifically provided to improve unit operations, such as cell activation or gene modification. Such enhancers are known in the art and are recognized by those skilled in the art. For example, sodium butyrate is a well-known compound that can improve transfection.

[0051] According to a preferred embodiment, the method includes performing more than one unit operation, preferably wherein performing more than one unit operation includes repeating steps i. to iii. once or more. Such repetition generally refers to repeating the steps sequentially, i.e., performing steps i. to iii. for the first time, and then repeating steps i. to iii. once or more. This may also be included when two or more unit operations are involved. Additionally or alternatively, performing more than one unit operation includes adding a liquid composition containing reagents for performing more than one unit operation to the cell container, preferably the liquid composition containing an activator and a gene-modifying agent. In such an embodiment, two unit operations, such as activation and gene modification, are performed simultaneously. Additionally or alternatively, performing more than one unit operation includes repeating step i. before step ii. and / or before step iii., particularly wherein...

[0052] (i) The liquid composition in step i. contains an activator, and

[0053] (ii) Other liquid compositions contain gene modifiers.

[0054] Alternatively, performing more than one unit operation may include repeating step i by adding other liquid compositions to the cell container before step ii. and / or before step iii., particularly wherein

[0055] (i) The liquid composition in step i. contains a gene-modifying agent, and

[0056] (ii) Other liquid compositions contain an activator.

[0057] In such an embodiment, the liquid composition and other liquid compositions are present simultaneously, particularly when these compositions are incubated with cells in step ii. Therefore, two (or more) unit operations can be performed concurrently without separate incubation and removal steps. One embodiment of such a dual unit operation is... Figure 10 and 11 The explanation is as follows.

[0058] According to a preferred embodiment, the method includes adding other liquid compositions to the cell container after step i and before step ii, such that the other liquid compositions are present in the cell container when the cells are incubated in step ii, and removing them in step iii includes removing at least a portion of the other liquid compositions by means of a filter.

[0059] Cells in cell containers

[0060] According to the method of the first aspect, the cell culture is present in a cell container. Performing unit operations and expansion within the same cell container is advantageous because it avoids transfer steps that could expose biological processing to risks, such as those due to contamination or cell damage. The cell culture is preferably present in the cell container prior to one or more unit operations, as this simplifies the handling of the liquid composition to an automated manner. On the other hand, adding cells throughout the unit operation is not excluded. For example, after the liquid composition has been added to the cell container, cells from the cell culture can be added to the cell container, allowing for subsequent cell incubation.

[0061] Therefore, according to a preferred embodiment, cells in a cell culture are transferred to a cell container before performing the method according to this disclosure. According to an optional or additional embodiment, cells in the cell culture are added after step i. and before step ii. of one or more unit operations; specifically, the first unit operation of more than one unit operation is performed. The transfer is preferably performed automatically, for example by pumping the cell culture containing cells into the cell container, e.g., through a fluid line. This avoids human error.

[0062] Measurement

[0063] According to a preferred embodiment, the method includes measuring cell density once or multiple times, preferably at least before step i. The cell density measurement can be performed by methods known in the art. For example, cell density can be measured using microscopy-based or capacitance-based measurement methods. The measurement can be performed before step i, whether the cells are present in or outside the cell container. Preferably, the cell density is measured while the cell culture is present in the cell container.

[0064] Cell density measurement of the cell culture within the cell container can be performed manually, for example, by obtaining a cell culture sample from the cell container and measuring the cells in the obtained sample. Preferably, the measurement is performed automatically. According to one embodiment, the method includes automatically measuring the cell density of the cell culture in the cell container once or multiple times, preferably by providing a suitable sensor or measuring device for measuring cell density. For example, the cell container may contain or be connected to a sensor for measuring cell density. In a preferred alternative, the cell container is fluidly connected to a recirculation loop that contains or is coupled to a suitable sensor or measuring device for measuring cell density. In one example, a suitable sensor or measuring device for measuring cell density may be based on microscopy.

[0065] Cell density measurements can be performed once or multiple times. Measuring cell density throughout the cell expansion process to monitor the expansion process is particularly advantageous. Measuring cell density before and / or after a unit operation for monitoring and adjusting parameters of the unit operation is also advantageous. For example, when cell density is measured before and after a unit operation, it is possible to determine whether the unit operation has a positive or negative impact on the cells. Measuring cell density before a unit operation also has the advantage of allowing adjustment of the volume of the liquid composition, particularly the amount of reagent added to the cell container, based on the cell density.

[0066] Therefore, according to a preferred embodiment, the method includes, in step i, automatically adding an appropriate volume of liquid composition to a cell container based on a measured cell density, wherein the liquid composition contains an activator and / or a gene modifier, such that the volume of the liquid composition is suitable for cell activation and / or gene modification, preferably adjusted for cell activation and / or gene modification.

[0067] According to a preferred embodiment, the method further includes one or more of the following: - The cell culture is introduced into a cell container prior to performing one or more unit operations on the cells in the cell culture in an automated manner, preferably wherein the introduction is performed in an automated manner; and / or - Before step i., remove liquid from the cell container and / or add liquid to the cell container, preferably wherein the cell density is adjusted.

[0068] Automating the introduction of cell cultures into cell containers is advantageous because it minimizes processing errors. Prior to step i, and thus before performing the unit operation, liquid is removed from and / or added to the cell container to adjust the cell density, allowing it to be tailored to the requirements of the unit operation. For example, the cell density can be adjusted for cell activation and / or gene modification of the cells as a unit operation, thereby achieving sufficient or even optimized conditions in terms of cell density for the corresponding unit operation. In this context, "adjusted" does not necessarily mean optimized, but rather suitable for the corresponding unit operation.

[0069] According to the specific implementation of the method in the first aspect

[0070] According to a preferred embodiment, the method has the following characteristics: - Optionally, prior to step i, the method includes measuring cell density, preferably in an automated manner; - The liquid composition in step i. contains an activator, which is at least partially removed in step iii. - The method includes performing other unit operations, wherein the liquid composition in step i. comprises a gene-modifying agent, preferably a transducer; and - Optionally, another liquid, preferably a cell culture medium, is added after step iii.

[0071] A specific process of such an implementation is in Figure 9 Overview. Optionally, between steps in such embodiments, a washing composition may be added to a cell container, thereby adding it to the cells, and then removed to wash the cells, for example, after activation and / or after gene modification. Such washing may be performed once or multiple times.

[0072] According to a preferred embodiment, the method has the following characteristics: - Optionally, prior to step i, the method includes measuring cell density, preferably in an automated manner; - The liquid composition in step i contains o activator, o Gene modifiers, preferably transducers or transfection agents, and o Optional reinforcing agent, The above substances are at least partially removed in step iii; and - Optionally, another liquid, preferably a cell culture medium, is added after step iii.

[0073] Optionally, between the steps of such an embodiment, a washing composition may be added to the cell container, thereby adding it to the cell, and then removed to wash the cell, for example after removal step iii. Such washing may be performed once or multiple times.

[0074] According to a preferred embodiment, the method has the following characteristics: - Optionally, prior to step iii., the method includes measuring cell density, preferably in an automated manner; - The liquid composition in step i. contains an activator, which is at least partially removed in step iii. - The method includes adding other liquid compositions to the cell container (4) after step i and before step ii, such that o When the cells are incubated in step ii., other liquid compositions are present in the cell container (4), and o Step iii. removal includes removing at least a portion of the other liquid composition, Other liquid compositions include o Gene modifiers, preferably transducers or transfection agents, and o Optional enhancers, and - Optionally, another liquid, preferably a cell culture medium, is added after step iii.

[0075] A specific process of such an implementation is in Figure 10 and 11 Overview. Optionally, between the steps of such an embodiment, a washing composition may be added to the cell container, thereby adding it to the cell, and then removed to wash the cell, for example after removal step iii. Such washing may be performed once or multiple times.

[0076] According to a preferred embodiment, cells are expanded in a cell container after performing one or more unit operations, and said method includes performing one or more other unit operations after expanding cells in the cell container, preferably wherein such a method has the following characteristics: - Optionally, prior to step i, the method includes measuring cell density, preferably in an automated manner; - The liquid composition in step i. contains an activator, which is at least partially removed in step iii. - After step iii, the cells are expanded in the cell container (4); - After cell expansion, a liquid composition containing a gene modifier, preferably a transducer or transfectant, more preferably a transfectant, is added to the cell container, and the cells in the cell container (4) are incubated together with the other liquid composition; and - Harvest cells, optionally.

[0077] A specific process of such an implementation is in Figure 12 Overview. Optionally, between the steps of such an embodiment, a washing composition may be added to the cell container, thereby adding it to the cell, and then removed to wash the cell, for example after removal step iii. Such washing may be performed once or multiple times.

[0078] According to a preferred embodiment, the method has the following characteristics: - Optionally, prior to step iii., the method includes measuring cell density, preferably in an automated manner; - Perform the first unit operation, wherein the liquid composition in step i. contains an activator, which is at least partially removed in step iii. - The method includes performing a second unit operation, wherein the liquid composition in step i. contains a gene-modifying agent, preferably a transfection agent; - Optionally, another liquid, preferably cell culture medium, is added after the second unit operation; - The method includes performing a third unit operation, wherein the liquid composition in step i. contains a gene modifier, preferably a transducer; - Optionally, another liquid, preferably cell culture medium, is added after the third unit operation; - Optionally, cells are expanded in a cell container; and - Harvest cells, optionally.

[0079] A specific process of such an implementation is in Figure 13 Overview. Optionally, between the steps of such an embodiment, a washing composition may be added to the cell container, thereby adding it to the cell, and then removed to wash the cell, for example after removal step iii. Such washing may be performed once or multiple times.

[0080] Step i.

[0081] The method according to a first aspect of the invention includes performing one or more unit operations on cells in an automated manner, wherein the unit operation includes step i. adding a liquid composition to a cell container. Those skilled in the art are familiar with various means and systems for automating the "addition" of a liquid composition, and therefore specific configurations should not be limited in this disclosure. For example, those skilled in the art are familiar with pump-based systems, such as peristaltic pumps, for automating the addition of liquid compositions.

[0082] Step ii.

[0083] The method according to a first aspect of the invention includes performing one or more unit operations on cells in an automated manner, wherein the unit operation includes step ii. incubating the cells in a cell container with an added liquid composition. Incubation facilitates the corresponding unit operation and is generally performed under the corresponding desired conditions. For example, incubation may be performed at a temperature, humidity, and atmosphere sufficient for the corresponding unit operation to be performed. According to a preferred embodiment, step ii. further includes agitating the cells in the cell container, for example by shaking or wave motion.

[0084] Step iii.

[0085] The method according to a first aspect of the invention includes performing one or more unit operations on cells in an automated manner, wherein the unit operation includes step iii. removing liquid from the cell container through at least one filter, thereby removing at least a portion of the added liquid composition. Such a removal step has the advantage that the corresponding liquid composition can be removed from the cell container, thereby reducing or mitigating contact between the cells and the liquid composition. The liquid composition can be removed substantially completely from the cell container by an optional washing step. Meanwhile, the cells remain substantially within the cell container. Those skilled in the art are aware of various means and systems for automatically “removing” liquid compositions, and therefore specific configurations should not be limited to this disclosure. For example, those skilled in the art are aware of pump-based systems, such as peristaltic pumps, for automatically adding liquid compositions.

[0086] For example, an optional washing step may consist of perfusion, preferably in which at least 1, 2, 3, 5, or 10 times the volume of the cell culture is exchanged. Perfusion preferably involves adding a liquid medium, such as fresh cell culture medium, to the cell culture. It is also preferable that perfusion be performed for a sufficiently long period so that the cultured cells are not negatively affected by the perfusion action. For example, the perfusion rate may be 15 VVD or less, 10 VVD or less, 8 VVD or less, or 4 VVD or less.

[0087] Alternatively, optional washing steps may include the following: - Remove the first amount of liquid from the cell container. - Add a second amount of liquid medium to the cell container, and - Shake the cell container for a certain period of time.

[0088] The first and second quantities are preferably the same volume, for example, up to 33% or up to 50% of the cell culture volume. The time period is preferably a sufficient amount of time to adequately mix the contents of the cell container, for example, at least 1 minute, preferably at least 5 minutes or at least 10 minutes, for example, 30 minutes.

[0089] Cell cultures grown in cell containers can benefit from an optional washing step. For example, after T cell activation, washing and thus removing activating agents (e.g., activation beads) makes the cells more readily uptake transgenes, thereby leading to improved transfection or transduction rates even after a washing step has been performed. Therefore, the method of the present invention can result in improved automated transfection / transduction.

[0090] Amplification

[0091] The method according to the first aspect includes expanding cells in a cell container, wherein the expansion is performed before and / or after one or more unit operations. Preferably, the cells undergo at least one unit operation before expansion, and in some cases, more than one unit operation. In some cases, expansion occurs after one or more unit operations, but one or more other unit operations may be performed after expansion. Different embodiments are described in... Figures 9 to 13 The explanation is as follows.

[0092] According to a preferred embodiment, the step of expanding cells in a cell container involves agitating the cells, for example by shaking or wave motion.

[0093] According to a preferred embodiment, the amplification includes one or more of batch culture, fed-batch culture, and / or perfusion culture, preferably, the amplification includes at least perfusion culture.

[0094] According to a preferred embodiment, during the cell amplification step, preferably after at least one unit operation on the cells, the volume of the cell culture in the cell container increases.

[0095] According to a preferred embodiment, during the cell amplification step, the volume of the cell culture in the cell container 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%, and more preferably at least 900%.

[0096] According to a preferred embodiment, at the end of the cell amplification step, the volume of the cell culture in the cell container is at least 100 ml, preferably at least 300 ml, more preferably at least 500 ml, and even more preferably at least 1 l.

[0097] According to a preferred embodiment, before step i., the volume of the cell culture in the cell container is at least 5 ml. Preferably, the volume of the cell culture in the cell container is at most 250 ml, more preferably 200 ml, more preferably 150 ml, even more preferably 120 ml, for example 100 ml or 80 ml. According to a preferred embodiment, before and / or after step i., the volume of the cell culture in the cell container is at most 100 ml.

[0098] According to a preferred embodiment, a unit operation is performed, wherein the volume of cell culture and added fluid in the cell container is selected from the range of 1 ml to 200 ml, preferably 5 ml to 100 ml, and in particular, the cell amplification step is performed after the unit operation.

[0099] According to a preferred embodiment, the method includes removing liquid from the cell container, thereby reducing the volume of the cell culture by at least 10%, preferably at least 30%, more preferably at least 50%, particularly after performing unit operations and / or expanding the cells.

[0100] Other optional steps according to the method of the first aspect

[0101] According to a preferred embodiment, the method further includes the step of washing the cells once or multiple times after performing the unit operation, preferably wherein the washing includes: i. Add the washing liquid composition to the cell container; ii. Optionally, incubate the cells in a cell container with added washing fluid; and iii. Remove fluid from the cell container through at least one filter, thereby removing at least a portion of the added washing fluid.

[0102] According to a preferred embodiment, the method further includes harvesting cells, preferably after unit operations and cell expansion. According to a preferred embodiment, harvesting includes transferring cell culture from a cell container, preferably through a port of the cell container, optionally in an automated manner. According to a preferred embodiment, harvesting includes removing liquid from the cell container through at least one filter to increase cell density, and then transferring concentrated cell culture from the cell container, preferably through a port of the cell container, optionally in an automated manner.

[0103] According to a preferred embodiment, the method further includes formulating the cell culture into a cell therapy product. According to a preferred embodiment, formulation is performed after unit operations and cell expansion. According to a preferred embodiment, formulation includes: i. Remove liquid from the cell container through at least one filter; ii. Adding a liquid for formulating cell cultures into cell therapy products to the cell container; and iii. Optionally, the cell therapy product is transferred from the cell container, preferably through a port of the cell container, optionally in an automated manner.

[0104] biological treatment system

[0105] According to a preferred embodiment, the method includes using a biological processing system comprising a control system and at least one active element, wherein the control system implements control commands to cause the at least one active element to propel the unit operation.

[0106] According to a preferred embodiment, the control system generates control commands to cause the active element to add the liquid composition to the cell container in step i, and / or remove the liquid from the cell container through at least one filter in step iii.

[0107] According to a preferred embodiment, at least one active element includes at least one pump for adding a liquid composition to the cell container and / or for removing liquid from the cell container through at least one filter.

[0108] According to a preferred embodiment, the cell container comprises two or more compartments, preferably a small-volume compartment and distinct large-volume compartments. "Distinct" does not mean that the small-volume and large-volume compartments partially overlap or contact each other, but rather that the container is typically divided into two compartments. Typically, cells are initially cultured in one compartment, for example, when one or more unit operations are performed in the small-volume compartment, while throughout the cell expansion process, cells are typically in both compartments simultaneously. Such a cell container is advantageous for culturing cells from small-scale to large-scale. Specifically, the small-volume compartment defines a volume in which cells can undergo initial culture and / or unit operations. This volume is typically smaller than the volume of the large-volume compartment, thus allowing for small-scale culture. Simultaneously, by being arranged adjacent to the small-volume compartment, the small-volume compartment is preferably fluidly connected for fluid exchange, thereby providing access to the typically larger volume defined by the large-volume compartment. Therefore, cells can be expanded in a sufficiently large environment as volume increases, i.e., initially in the volume of the small-volume compartment and then cultured in the volume of the large-volume compartment (and the volume of the small-volume compartment). Thus, scaling up can be achieved without changing containers (e.g., transferring to larger containers). Simultaneously, the small and large volume compartments provide ample culture environments for the corresponding needs throughout the culture process. For example, cells can initially be present in a small volume compartment, requiring a small surface area to minimize evaporation. Later in the culture, more volume is needed to obtain a suitably high cell count, so the large volume compartment provides sufficient extension for the small volume compartment. This culture modality is particularly advantageous for manufacturing cell therapy products, such as autologous immune cell therapies (e.g., CAR T cells), because the initial cell count obtained from patient samples is relatively low and needs to be expanded to manufacture the cell therapy product. Therefore, despite the low cell count and small culture volume, cells can be cultured in the same container throughout the entire expansion process. This not only simplifies the overall process but also makes it less prone to errors and reduces the risk of contamination. As demonstrated in the examples below, higher daily expansion rates and cell viability can also be achieved, enabling shorter culture times and improved product quality (see, for example...). Figure 6 and 7 ).

[0109] According to a preferred embodiment, the cell container comprises a pre-formed reservoir that forms small volume compartments. Through "pre-forming," the reservoir is dimensionally stable, allowing the reservoir volume to be fully formed without requiring additional devices or systems, such as a shaking platform / shaking motion platform, to form the reservoir. "Pre-forming" does not preclude the reservoir from being generally deformable, but it should be understood that the reservoir has a defined shape independent of other (external) devices or systems (e.g., a shaking platform). When the deformation forces decrease or disappear, the reservoir can return to its pre-formed form. Pre-forming allows for the repeatable and stable provision of the reservoir, making the culture process more robust. In particular, it enables the provision of the same volume (here, the reservoir volume) for each batch of cultures. Furthermore, through pre-forming, the reservoir is more resistant to deformation and material breakage than a reservoir or cavity formed only when combined with a culture platform or system, thereby reducing the risk of failure or contamination.

[0110] According to a preferred embodiment, the cell container includes a shell forming large-volume compartments, wherein the shell is preferably flexible. The term "shell" is well known to those skilled in the art. Generally, the shell of a container refers to a bag or pouch, preferably flexible, but can also be rigid or semi-rigid. Preferably, the shell in this disclosure is at least partially flexible to accommodate the increased culture volume throughout the culture process and during amplification. The container according to this disclosure may also include small-volume compartments that can be connected to or form an integral part of the large-volume compartment serving as the shell. Typically, the shell can be pre-formed. However, the shell can also be flexible, particularly acquiring its shape during the culture procedure, for example due to increased pressure or weight within the container.

[0111] The outer shell is preferably a disposable bag widely used in cell culture, such as for single-use bioreactors or for storing or transporting cell fluids, such as cell culture media, culture buffers, or cell cultures. Such a bag may contain at least one outer shell portion, which may also be referred to as a “wall.” Thus, according to a preferred embodiment, the outer shell comprises outer shell portions. Such outer shell portions can be folded and sealed together to form a bag-like structure. Typically, the bag may contain two (large) walls (here, the outer shell portions) sealed to each other. Once expanded, they have a limited volume and remain relatively thin, explaining why they are often referred to as 2D bags (D stands for dimension). 3D bags are also known and typically contain two end walls and a side wall that can be folded flat or unfolded for use, these walls being sealed to each other, where the volume can reach up to 3000 liters or more. Therefore, according to a preferred embodiment, the outer shell comprises two or more outer shell portions, such as an upper outer shell portion and a lower outer shell portion. Such outer shell portions can be joined to form a bag-like structure (also referred to as a 2D bag). Also within the scope of this disclosure, the outer shell may comprise three, four, five, or more than five outer shell portions. This makes it possible to form 3D shells (e.g., 3D bags).

[0112] The outer casing may consist of a multilayer membrane comprising a contact layer that contacts the medium filling the casing, a barrier layer, and an outer layer that contacts the external environment of the casing. These three layers are interconnected by an adhesive layer. In cases where the casing will be filled with a biopharmaceutical product, the contact layer should be made of a material capable of contacting the biopharmaceutical product without causing degradation of the membrane or the biopharmaceutical product. Furthermore, it must be self-sealing. For this purpose, the material is typically selected from polyolefins, such as polyethylene. The barrier layer provides a barrier to the passage of gases such as oxygen and carbon dioxide and is typically made of ethylene-vinyl alcohol copolymer (EVOH). The outer layer contributes to the mechanical strength of the casing. For this purpose, the outer layer must be flexible enough to withstand high mechanical stresses but not excessively stretchable to prevent deformation of the casing when it is filled.

[0113] According to a preferred embodiment, the method has the following characteristics: - During unit operations, cell cultures are located in small volume compartments; and / or - During cell amplification in the cell container, preferably after step iii, the volume of the cell culture is increased such that the cell culture is preferably located in a small volume compartment and a large volume compartment.

[0114] According to a preferred embodiment, one or more of at least two compartments include at least one filter, and / or are fluidly connected to at least one filter. The term "filter" is well known to those skilled in the art. A "filter" is generally used to remove particles from a fluid process flow and contains a porous media. The filtration process is the process of removing particles from a fluid by passing the fluid through a porous media. In this document, filters are specifically used to retain cells within a container. The remaining filtered cells may be referred to as "retained material." The fluid passing through the filter is substantially free of cells. The fluid passing through may be referred to as "permeable material." The selection of filters is within the skill of those skilled in the art. In particular, those skilled in the art are well aware of the various filters used to retain cells but allow liquid to pass through. The selection of a suitable pore size or molecular cutoff value is within the normal working scope of those skilled in the art. Therefore, this disclosure should not be limited to specific filter materials or pore sizes, provided that the material and pore size are suitable for the cell culture.

[0115] 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 that most cells should be retained. Furthermore, this does not preclude cell debris or apoptotic cells (typically smaller than living cells) from passing through the filter.

[0116] According to one embodiment, at least one filter comprises a filter medium selected from surface filters and depth filters.

[0117] The term "membrane" is known in the art. A variety of membrane materials can be used, including polyvinylidene fluoride (PVDF), polyethersulfone (PES), or polysulfone (PS). In one embodiment, the membrane is a PES membrane, optionally having a rejection value of 0.5 micrometers to 5 micrometers, for example 1.2 micrometers.

[0118] According to a preferred embodiment, - Small volume compartments contain at least one filter and / or are fluidly connected to at least one filter, and / or - The large-volume compartment contains at least one filter and / or is fluidly connected to at least one filter.

[0119] According to a preferred embodiment, the large-volume compartment includes at least one filter, which is a main membrane, preferably not used during unit operations, and / or used for perfusion during cell expansion in the cell container. According to one embodiment, at least one filter includes a main membrane and / or a reservoir membrane, preferably both. In this document, "main membrane" generally refers to a membrane disposed in a large-volume compartment, also known as a "shell". In this document, "reservoir membrane" generally refers to a membrane disposed in a small-volume compartment, also known as a "reservoir". Reservoir membranes advantageously enable larger-scale filtration processes, for example, when cell cultures are present in a shell (and typically also in a reservoir). Therefore, a main membrane can be advantageously used for medium to large-volume liquid filtration, for example, as may be required throughout the perfusion culture process.

[0120] According to a preferred embodiment, the small volume compartment includes at least one filter, which is a reservoir membrane, preferably used for unit operations, particularly for removing liquid from the cell container through the reservoir membrane in step iii.

[0121] According to a preferred embodiment, at least one filter

[0122] - Configured to allow liquid flow through, but essentially preserve cells, and

[0123] - Preferably not used for gas exchange, especially gas exchange between cell cultures in contact with the filter and gas in contact with the other side of the filter.

[0124] According to a preferred embodiment, at least one filter is a membrane, such as a planar membrane, a hollow fiber membrane, and / or a floating membrane. A floating membrane may also be referred to as a "pouch membrane," and is particularly fluidly connected to the outside of the container, especially to at least a waste container (also referred to as a "waste container").

[0125] According to a preferred embodiment, the cell container includes

[0126] - At least one filter and / or fluidly connected to at least one filter, - A tubular assembly connected to and pre-sterilized together with the cell container.

[0127] According to a preferred embodiment, the pipe assembly includes: - At least one disposable pump head; - At least one mating element for the sensor, particularly a magnetic mating element; - Recirculation loop; - Sealing and weldable fittings for media containers; and / or - Sealing and weldable connectors for waste containers.

[0128] According to a preferred embodiment, the biological treatment system further includes a bioreactor module, which contains cell containers.

[0129] According to a preferred embodiment, the biological treatment system, preferably a bioreactor module, further includes a shaking element for applying shaking motion to the cell container.

[0130] According to a preferred embodiment, the bioreactor module includes at least one sensor for sensing the characteristics of the cell culture, preferably wherein during unit operation, the control system receives sensor data from the at least one sensor and derives control commands from the sensor data.

[0131] According to a preferred embodiment, at least one sensor includes

[0132] - A sensor for measuring the characteristics of cell cultures, particularly...

[0133] o Number of cells per unit volume; o Cell size; o Cell viability; and / or o pH value of cell culture; and / or - Balance, the balance weighs o Cell cultures; o The amount of liquid added to the cell culture; and / or o The amount of liquid removed from the cell culture.

[0134] According to a preferred embodiment, the control system determines the time point at which gene modification begins from the measured cell size, generates a control instruction to control at least one pump to add a fluid containing the gene-modifying agent to the cell container in step i at said time point, and implements said control instruction.

[0135] According to a preferred embodiment, the bioreactor module further includes: - A media container connected to a cell container; - Waste container connected to the cell container; - Bypass lines used to connect the external containment of the bioreactor module to the cell container; - Control units as part of the control system; and / or - Heating elements used to heat cell containers.

[0136] According to a preferred embodiment, the bioreactor module also includes a recirculation loop that guides the cell container to the cell container.

[0137] According to a preferred embodiment, the recirculation loop has one or more of the following features: - It contains at least one sensor, specifically a sensor for cell counting; - It connects to the media container, particularly via a bypass line; - It is connected to the waste container, particularly via a recirculation loop membrane and / or bypass line.

[0138] According to a preferred embodiment, the bioreactor module includes a support structure for carrying cell containers and optional media containers.

[0139] According to a preferred embodiment, the bioreactor module includes at least one tube retainer that holds at least one tube in a defined position relative to a support structure.

[0140] According to a preferred embodiment, the tube retainer includes a tube movement mechanism for extending and / or retracting the tube relative to the tube retainer.

[0141] According to a preferred embodiment, the tube retainer is configured to interact with a connection arrangement for aseptically connecting and / or disconnecting the receiver, preferably wherein the connection arrangement includes a tube welding unit for welding and / or cutting and sealing the tube, preferably in an automated manner. The welding unit may employ thermal welding, ultrasonic welding, or other suitable welding techniques to ensure a strong and sterile seal. To verify the integrity of the sealed connection, the bioprocessing system may also include a seal testing unit capable of assessing the success of the sealing process. This test may be performed by applying pressure, vacuum, or other diagnostic methods to detect leaks or structural weaknesses in the sealed area.

[0142] According to a preferred embodiment, the biological treatment system includes a connection arrangement for aseptically connecting and / or disconnecting the container, preferably including a tube welding unit for welding tubes together and / or cutting and sealing the tubes.

[0143] According to a preferred embodiment, the method includes welding pipes to bypass lines and / or pipes connected to media containers and / or pipes connected to waste containers via a pipe welding unit.

[0144] According to a preferred embodiment, the biological treatment system has one or more of the following features: - It contains a framework; - It contains at least one amplification site where amplification operations are performed and preferably unit operations are performed, particularly with the support of a biological processing system; - It replenishes the media container; and / or - It includes a transport mechanism for transporting the bioreactor module, particularly from and / or into the amplification site.

[0145] cell

[0146] According to a preferred embodiment, the cell is a mammalian cell, preferably an immune cell or primary cell. According to one embodiment, the cell is an immune cell, preferably a T cell. The term "immune cell" is well known in the art and is readily understood by those skilled in the art. Immune cells generally refer to a type of white blood cell. Any type of white blood cell can be used herein. Combinations of different types are also contemplated. However, according to a preferred embodiment, the term "immune cell" herein refers to a single type of cell, such as a T cell or a subtype thereof, or an NK cell or a subtype thereof. Thus, the term "immune cell" includes a variety of cells, such as, but not limited to, dendritic cells; T lymphocytes, also known as T cells; B lymphocytes; natural killer cells; macrophages, etc. Immune cells may also include subtypes of immune cells, such as tumor-infiltrating lymphocytes or different types of T cells. A subtype of a certain type of immune cell can be classified based on the type of antigen present on the cell surface. Thus, the term immune cell can, for example, refer to a T cell containing the surface antigen CD4 ("CD4+ T cell").

[0147] Preferably, the T cells are genetically modified in a unit operation to express a chimeric antigen receptor (CAR). Therefore, the term "CAR-T cell" describes a T cell that has been genetically modified to express a CAR. Therefore, preferably, the genetically modified T cell is a genetically modified CAR-T cell.

[0148] According to a preferred embodiment, the cell culture comprises cells and a liquid in which the cells are present, preferably a cell culture medium.

[0149] According to one embodiment, the cells are specifically designed for cell and / or gene therapy, preferably specifically designed for cell therapy.

[0150] Cell selection

[0151] According to a preferred embodiment, the method includes cell selection, preferably prior to unit operations. Cell selection is advantageous in order to select specific cells within a group of cells, which subsequently undergo one or more unit operations. Cell selection is particularly performed in cell therapy manufacturing. Therefore, this disclosure allows cell selection to be performed within or outside the method according to the first aspect, for example by providing cells that have already undergone cell selection in a cell culture.

[0152] According to a preferred embodiment, cell selection includes: - Positive cell selection, wherein the target portion of the cell binds directly or indirectly to the binding matrix of the binding unit; or - Negative cell selection, in which the target portion of the cell does not bind directly or indirectly to the binding matrix of the binding unit.

[0153] According to a preferred embodiment, cell selection includes: - Add a mixture of cells to the cell container. - To contact a mixture of cells with a binding unit, wherein at least a portion of the cells bind directly or indirectly to the binding matrix of the binding unit, and - Remove liquid from the cell container, thereby removing at least some cells that are not directly or indirectly bound to the binding matrix.

[0154] According to a preferred embodiment, the binding matrix is ​​configured to enable it to directly or indirectly bind at least a portion of the ligands expressed by the cells.

[0155] According to a preferred embodiment, indirect binding includes adding at least one binding agent to a cell container such that a mixture of cells comes into contact with the binding agent, and the binding agent binds to at least a portion of the cells, wherein the binding agent is configured to bind to the binding matrix.

[0156] According to a preferred embodiment, the cell container

[0157] - Contains bonding units; and / or

[0158] - Fluidly connected to at least one bonding unit, The binding unit comprises a binding matrix configured to enable direct or indirect binding of a ligand expressed by at least a subset of cells. Such a binding unit is particularly suitable for cell selection. Specifically, by providing the binding unit, at least a subset of cells in a liquid medium can be sorted by expressing a ligand that binds to the binding matrix. Cells not expressing such a ligand will not bind to the binding matrix. Such binding can be described as “direct” binding of the matrix to the ligand expressed by at least a subset of cells. Alternatively, one or more binding agents can be provided that enable the binding matrix to “indirectly” bind to the ligand expressed by at least a subset of cells. In such cases, the one or more binding agents typically bind to the ligand expressed by the cells, such as an antibody or antibody fragment capable of binding to the ligand expressed by the cells. The binding agent then typically also includes a portion for binding to the binding matrix, such as biotin, avidin, or streptavidin, so that cells expressing the ligand bind indirectly (i.e., via the binding agent) to the binding matrix. When more than one binding agent is provided, a first binding agent binds to the ligand expressed by the cells, while a second binding agent binds to the binding matrix, and both the first and second binding agents bind to each other. Other configurations or chemical schemes can be applied to achieve the desired result, namely, enabling cells expressing ligands to bind directly or indirectly to the binding matrix. Those skilled in the art are well aware of suitable binding matrices and chemical schemes that enable the binding of ligands expressed by at least a portion of the cells.

[0159] The connecting unit can be fluidly connected to the container, wherein such a fluid connection can be provided in the form of a pipe. More than one fluid connection may be present, for example, preferably two, particularly on different sides or at different locations of the connecting unit. For example, there may be one fluid connection from the container to the inlet of the connecting unit, and another fluid connection from the outlet of the connecting unit to the container. Other fluid connections may be present, allowing fluid to be transferred through the connecting unit to another container, such as a waste container or storage container. The fluid connection between the container and the connecting unit may be located at the outer shell of the container and / or at the reservoir. Preferably, there is one fluid connection between the reservoir and the connecting unit, and another fluid connection between the outer shell and the connecting unit. Other configurations are also possible; for example, there may be two fluid connections between the outer shell and the connecting unit. Alternatively, there may be two fluid connections between the reservoir and the connecting unit.

[0160] After at least some cells have bound, the liquid medium present in the container can be removed, for example through a binding matrix or a fluid line connected to the container, along with any unbound cells (those not expressing the ligand at that time) removed with the liquid. Thus, only cells expressing the ligand remain in the container, i.e., positive cell selection is performed. To release cells from the binding matrix, the cells can be eluted, as is commonly known in the art (e.g., by providing a releasing agent, such as salt, biotin, avidin, or streptavidin). The eluted cells are then released into the liquid medium present in the container. Preferably, the cells can be recycled back into the container, for example by pressing liquid through a binding unit that elutes the cells, and then eluting them into a fluid line connected to the container, thus recycling the released cells back into the container. Thereafter, the binding unit can be closed or deactivated. The binding unit can also remain "open," so in principle, cells can enter. However, it is preferable not to apply force or pressure to the binding unit, thereby not actively moving cells into the binding unit. The binding unit can also be closed, for example by closing a valve or cap, isolating the binding unit from the container.

[0161] In addition, a negative cell selection step can be performed. For example, after at least a portion of the cells have bound, the liquid medium present in the container can be removed, for example through a binding matrix or a fluid line connected to the container, and unbound cells (which at that time did not express ligands) can be removed along with the liquid. These unbound cells can then be collected, for example, in a container. To release the cells from the binding matrix, the cells can be eluted, as is generally known in the art. The eluted cells are then released into the liquid medium present in the container and can then be removed through a waste port. Preferably, the cells can be released from a binding unit, for example by pressing liquid through the binding unit, which elutes the cells, and then eluting them into a fluid line connected to a waste container, thereby removing the released cells from the container. Thereafter, the collected cells can be recycled back into the container.

[0162] According to some embodiments of this disclosure, more than one cell selection step is performed, such as at least two cell selection steps. The cell selection steps may include positive and / or negative cell selection, as disclosed herein.

[0163] According to a preferred embodiment, the binding matrix is ​​provided in the form of a resin or a monolithic material. Various materials for the binding matrix are known in the art. Particularly preferred are binding matrices comprising resin beads. The binding matrix may be covered or encapsulated by one or more membranes that separate the cells from the binding matrix, provided no force is applied that would drag the cells into contact with the binding matrix.

[0164] According to a preferred embodiment, the binding unit is located within or adjacent to a small-volume compartment of the cell container, preferably arranged adjacent to a filter within the small-volume compartment. By providing a binding unit located within or adjacent to a small-volume compartment, small volumes of cell cultures or cell suspensions can be processed. Specifically, cells can be provided in the form of a mixture of cells that require a cell selection step using a binding matrix. Since cells are often present in small-volume cell cultures at such a stage, it is advantageous to provide the binding unit within a small-volume compartment of the container, which can be advantageously used for processing and culturing small-volume cell cultures. Furthermore, the small-volume compartment is preferably pre-formed, giving it greater mechanical stability than a reservoir that is formed only during interaction with other devices throughout the culture process. Therefore, the binding unit has a fixed position, enabling precise cell selection.

[0165] In one embodiment, the binding unit is located within or adjacent to a small volume compartment of the container. The binding unit can be provided in the form of a column, wherein the column is primarily internal, i.e., within the container, particularly within the small volume compartment, extending into its volume. This “internal column” configuration advantageously allows cells, respectively present in the cell container and the small volume compartment, to directly connect with the column, thus eliminating the need for additional fluid transfer steps. In such a configuration, it is advantageous to provide a binding matrix within a membrane-covered column, such that the cells are not always in contact with the binding matrix, to control cell selection. Therefore, the cells only interact with the binding matrix when a force is applied to them, dragging the cells across the membrane into the column and thus into contact with the binding matrix. Thus, cell selection occurs only when such a force is applied. According to one embodiment, an active element, such as a pump, is provided to manipulate fluid in the container to draw the fluid into the column. For example, the pump can draw fluid from the container and cells present in the fluid into the column, bringing the cells into contact with the binding matrix, thereby binding at least a portion of the cells to the binding matrix. According to one embodiment, the binding unit further includes a valve configured to close the fluid line exiting the binding unit. This advantageously shuts off the binding matrix, preventing cells from being actively drawn into the binding unit and thus avoiding contact with the binding matrix.

[0166] According to a preferred embodiment, the unit is provided in the form of a column, preferably wherein...

[0167] - The opening of the column is arranged within the opening of a small volume compartment of the cell container, more preferably within an opening at the bottom of the small volume compartment, and / or

[0168] - A column and a cell container, preferably a reservoir, are fluidly connected, wherein a fluid connection is provided between the cell container and the column, and in particular, wherein the fluid connection is provided by one or more pipes, and optionally also includes a valve.

[0169] This has the following advantages: the column can be used as is known in the art for cell selection columns, but as a cell container implementation, cell selection can be performed within the same container, where cell culture and at least one unit operation, such as cell activation, cell transduction, cell transfection, and media exchange and / or volume reduction, can occur. Therefore, the relevant steps for manufacturing cell therapy products can be performed within the same container, significantly simplifying the process. Furthermore, or alternatively, the binding unit is provided in the form of a column fluidly connected to a container, preferably a reservoir, wherein a fluid connection is provided between the container and the column, particularly wherein the fluid connection is provided by one or more tubes, and optionally also includes a valve. Fluid connection between the column and the reservoir has the advantage that the binding unit is separate from the container, allowing for separate control. Simultaneously, the two are generally in close proximity, such that temperature control, for example, will effectively heat the container and the column, which is advantageous for maintaining high cell viability. Optionally, the fluid connection, such as one or more tubes, also includes a valve. This has the advantage that the fluid connection between the binding unit (particularly the column) and the container (particularly the reservoir) can be closed, which can be done after cell selection.

[0170] The connecting units are provided in the form of columns, and as disclosed herein, the term "column" should not be limited to any specific geometry as is known in the art. While columns can generally be provided in a cylindrical or columnar shape, other shapes are also known in the art. In this document, a column can also refer to a bag-like structure in which two or more walls are joined to form an outer shell. Because outer shells with a bag-like shape are typically flexible, such column configurations can also be referred to as "flexible columns."

[0171] According to a preferred embodiment, the bonding unit includes a flexible column comprising a housing, an inlet port, and an outlet port, wherein the housing contains a bonding matrix. The flexible column may also include two membranes that limit the volume of the housing containing the bonding matrix, for example, one membrane at the inlet of the flexible column and one membrane at the outlet of the flexible column. The flexible column may be fluidly connected to a container, wherein preferably such a connection includes a valve for controlling fluid flow between the container and the flexible column.

[0172] According to a preferred embodiment, the binding unit is provided in the form of a floating unit configured to float within a cell container. When the binding unit is provided in the form of a floating unit, the floating unit may be provided in the form of a pouch that can float within the fluid of the container. Such a floating unit is typically connected via a fluid line extending through the container to the outside. Thus, the floating unit can float, but is limited to a certain length of the fluid line. The floating unit has the advantage that it is present within the container, allowing contact between the floating unit and the cells within the container. To achieve interaction between the cells and the binding matrix of the binding unit, cells can be forced into or propelled into the floating binding unit by positive pressure within the container and / or negative pressure within the binding unit, for example, generated by the fluid line. As explained above, bound cells can then remain within the binding matrix, while unbound cells leave the binding unit via the fluid line.

[0173] According to a preferred embodiment, the bonding unit is fluidly connected to the cell container and / or waste port via an external loop, preferably to both. Specific configurations of such an embodiment can be found in [reference needed]. Figure 8 By providing an external loop, cells can be recycled back into the container via the binding unit, particularly into the small volume compartment, and then recycled again. This has the advantage that cells can undergo multiple rounds of binding to achieve binding of as many cells as possible that express ligands that bind to the binding matrix. Furthermore, this also has the advantage that cells bound to the binding matrix and then eluted can leave the binding unit and be recycled back into the cell container, particularly into the small volume compartment. Additionally, by connecting the binding unit to a waste port, unbound cells can be transferred to a waste container.

[0174] According to a preferred embodiment, the external loop includes at least one valve, preferably one valve for controlling fluid flow between the binding unit and the waste port, and another valve for controlling fluid flow between the cell container and the waste port. Preferably, both valves are used to control fluid flow between the binding unit and the cell container through the external loop. A specific configuration of such an embodiment can be found in [reference needed]. Figure 8 By providing valves, fluid flow can be controlled, allowing fluid to flow into waste containers and / or be recycled back into containers, particularly into small volume compartments.

[0175] According to a second aspect of the invention, a bioreactor module for bioprocessing cells in a cell culture is used.

[0176] According to a second aspect of the present invention, a bioreactor module for bioprocessing cells in a cell culture is provided, wherein the bioreactor module comprises

[0177] - Cellular containers, and

[0178] - At least one filter for removing liquid from the cell container through said at least one filter. - The bioreactor module is configured to perform one or more unit operations on cells in a cell container in an automated manner, and to expand the cells in the cell container.

[0179] According to the bioreactor module of the second aspect, the application of a cell container that enables automated processing of cells in cell cultures advantageously simplifies the biological treatment of cells. Simultaneously, the cell container of the bioreactor module is suitable for cell expansion, thus allowing advantageous performance of unit operations and cell expansion within the same cell container. This significantly simplifies processes such as cell therapy manufacturing, as cumbersome manual processes are automated compared to conventional methods in the art. Therefore, processing errors are avoided, thereby improving reproducibility and reducing the risk of contamination. Furthermore, the conditions applied to the cells can be tightly controlled by enabling multiple unit operations and expansions within the same cell container. Additionally, transfer steps are avoided, resulting in fewer negative impacts on the cells overall and reduced cell loss. The embodiments disclosed herein highlight these positive effects, demonstrating the attainment of higher cell numbers and cell viability, indicating more efficient and higher-quality cell therapy manufacturing (see [link to documentation]). Figure 6 and 7 ).

[0180] 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, provided that these features and embodiments are directly or indirectly related to the bioreactor module. Therefore, referring to the above disclosure, the above disclosure should also apply to the bioreactor module according to the second aspect. Other features will now be described in detail.

[0181] According to a preferred embodiment, the bioreactor module further comprises one or two of the following: - At least one active element for influencing cell cultures in a bioreactor module, preferably for adding fluid to and / or removing fluid from cell containers, preferably wherein the fluid is a liquid; and - At least one sensor for sensing the properties of the cell culture.

[0182] According to a preferred embodiment, at least one active element includes at least one pump for adding fluid to the cell container and / or for removing fluid from the cell container through at least one filter.

[0183] According to a preferred embodiment, the cell container comprises one or more of the following: - Two or more compartments, preferably small-volume compartments and different large-volume compartments; - Pre-formed storage containers that create small-volume compartments; - An outer shell forming a large-volume compartment, preferably wherein the outer shell is flexible; - Main membrane; and / or - A control unit and / or an interface with a control system, wherein the control unit is adapted to...

[0184] According to a preferred embodiment, the bioreactor module has the following characteristics: - During unit operations, cell cultures are located in small volume compartments; and / or - During cell expansion in cell containers, the volume of the cell culture increases, making it preferable for the cell culture to be located in small-volume compartments and large-volume compartments.

[0185] According to a preferred embodiment, the cell container includes at least two compartments, wherein at least one compartment contains at least one filter and / or is fluidly connected to at least one filter.

[0186] According to a preferred embodiment, a small-volume compartment includes at least one filter and / or is in fluid connection with at least one filter, and / or a large-volume compartment includes at least one filter and / or is in fluid connection with at least one filter.

[0187] According to a preferred embodiment, the large-volume compartment includes at least one filter, which is a main membrane, preferably not used during unit operations, and / or used for perfusion during cell expansion in a cell container.

[0188] According to a preferred embodiment, the small volume compartment includes at least one filter, which is a reservoir membrane, preferably used for unit operations, particularly for removing fluid from the cell container through the reservoir membrane.

[0189] According to a preferred embodiment, the bioreactor module includes a control unit and / or an interface with a control system, wherein the control unit is adapted to...

[0190] After introducing cell cultures into cell containers, the cells are subjected to at least one unit operation and amplification operation within the cell containers, particularly the subsequent amplification operation, and

[0191] Control commands are implemented during the unit operation and / or the amplification operation to cause at least one active element to influence the cell culture, thereby advancing the unit operation and / or the amplification operation, and / or wherein the interface with the control system is adapted to allow the control system to implement control commands to cause at least one active element to influence the cell culture, thereby advancing the unit operation and / or the amplification operation. Preferably, the control unit is adapted to receive sensor data from at least one sensor and derive control commands from the sensor data, and / or the interface with the control system is adapted to allow the control system to receive sensor data from at least one sensor.

[0192] According to a preferred embodiment, the cell container includes a tube assembly connected to the cell container; preferably, the tube assembly is pre-sterilized together with the cell container.

[0193] According to a preferred embodiment, the pipe assembly includes

[0194] - At least one disposable pump head; - At least one mating element for the sensor, particularly a magnetic mating element; - Recirculation loop; - Sealing and weldable fittings for media containers; and / or - Sealing and weldable connectors for waste containers.

[0195] A biological treatment system for biologically treating cells in a cell culture, according to a third aspect of the present invention.

[0196] According to a third aspect of the present invention, a biological treatment system for biologically treating cells in a cell culture is provided, wherein the biological treatment system comprises a bioreactor module according to a second aspect of the present invention.

[0197] According to a preferred embodiment, the biological processing system includes a control system adapted to implement control commands during unit operations and / or amplification steps to cause at least one active element to influence the cell culture, thereby advancing the unit operations and / or amplification operations.

[0198] The bioprocessing system according to the third aspect advantageously simplifies the bioprocessing of cells in cell cultures by applying the bioreactor module according to the second aspect. The embodiments disclosed herein highlight the positive effects, demonstrating the attainment of higher cell numbers and cell viability, indicating more efficient and higher-quality cell therapy manufacturing (see [link to documentation]). Figure 6 and 7 ).

[0199] The individual features and preferred embodiments of the biological treatment system according to the third aspect correspond to the individual features and embodiments of the method according to the first aspect and the bioreactor module according to the second aspect, provided that these features and embodiments are directly or indirectly related to the biological treatment system. Therefore, referring to the above disclosure, the above disclosure should also apply to the biological treatment system according to the third aspect.

[0200] Applications according to the fourth and fifth aspects of the invention

[0201] According to a fourth aspect of the invention, there is use for manufacturing cell therapy products using a bioreactor module according to the second aspect and / or a bioprocessing system according to the third aspect, preferably wherein the manufacturing comprises at least one of the following: - Optionally, cells are selected once or multiple times, particularly by binding at least a portion of cells expressing the ligand, preferably a subset of T cells; - Activates cells, preferably T cells; - Transduced cells, preferably transduced T cells; - Transfected cells, preferably T cells; and / or - Expand cells, preferably T cells, and more preferably genetically modified T cells.

[0202] According to a fifth aspect of the invention, the use of a bioreactor module according to the second aspect and / or a biological treatment system according to the third aspect in a method according to the first aspect of the invention is provided.

[0203] The uses according to the fourth and fifth aspects advantageously simplify the biological processing of cells in cell cultures to manufacture cell therapy products. Where applicable, the individual features and preferred embodiments of the uses correspond to the individual features and embodiments of other aspects of the invention. Therefore, the above disclosure should also apply to the uses, taking into account the foregoing disclosure.

[0204] Other embodiments of this disclosure

[0205] The following embodiments provide other advantages of this disclosure: 1. A method for biologically treating cells in a cell culture, wherein the cell culture is present in a cell container (4), wherein the method includes - Perform one or more unit operations on the cells in an automated manner, wherein the unit operations include: i. Add the liquid composition to the cell container (4); ii. Incubate the cells in the cell container (4) together with the added liquid composition; and iii. Removing liquid from the cell container (4) through at least one filter, thereby removing at least a portion of the added liquid composition; and - The cells are amplified in the cell container (4), wherein the amplification is performed before and / or after the one or more unit operations.

[0206] 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 activator; and - Gene modification of the cells, particularly transduction and / or transfection, preferably wherein the liquid composition comprises a gene modifier, particularly a transducer and / or transfectant.

[0207] 3. The method according to embodiment 1 or 2, wherein the method includes performing more than one unit operation, preferably wherein the method includes at least two unit operations, more preferably wherein the at least two unit operations include activating the cell and modifying the gene.

[0208] 4. The method according to one or more of embodiments 1 to 3, wherein the method includes performing more than one unit operation, preferably wherein the method includes at least three unit operations, more preferably wherein the at least three unit operations include activating the cell and two gene modifications, such as activation, transfection and / or transduction.

[0209] 5. The method according to one or more of embodiments 1 to 4, wherein the liquid composition added in step i. comprises: (i) Activator; (ii) Gene modifiers, preferably transducers or transfectants, and optionally enhancers; or (iii) Activators and gene modifiers, preferably transducers or transfection agents, and optionally enhancers.

[0210] 6. The method according to one or more of embodiments 1 to 5, wherein the method includes performing more than one unit operation, preferably wherein performing more than one unit operation includes: - Repeat steps i. to iii. once or more; - Add a liquid composition containing reagents for performing more than one unit operation to the cell container (4), preferably the liquid composition containing an activator and a gene modifier; - Before step ii. and / or before step iii., step i. is repeated by adding other liquid compositions to the cell container (4), particularly wherein (i) The liquid composition in step i. contains an activator, and (ii) The other liquid compositions contain a gene-modifying agent, and / or - Before step ii. and / or before step iii., step i. is repeated by adding other liquid compositions to the cell container (4), particularly wherein (i) The liquid composition in step i. contains a gene-modifying agent, and (ii) The other liquid compositions contain an activator.

[0211] 7. The method according to one or more of embodiments 1 to 6, wherein the method includes measuring cell density once or multiple times, preferably measuring cell density at least before step i.

[0212] 8. The method according to embodiment 7, wherein the method includes, in step i, automatically adding an appropriate volume of liquid composition to the cell container (4) based on a measured cell density, wherein the liquid composition contains an activator and / or a gene modifier, such that the volume of the liquid composition is suitable for the activation and / or gene modification of the cells, preferably being adjusted for the activation and / or gene modification of the cells.

[0213] 9. The method according to one or more of embodiments 1 to 8, wherein the method further comprises one or more of the following: - The cell culture is introduced into the cell container (4) prior to performing the one or more unit operations on the cells in the cell culture in an automated manner; and / or - Before step i, remove liquid from the cell container (4) and / or add liquid to the cell container (4), preferably wherein the cell density is adjusted.

[0214] 10. The method according to one or more of embodiments 1 to 9, wherein the one or more unit operations are repeated once or more, preferably at least once more, and in particular, different unit operations are performed when the unit operation is repeated once more.

[0215] 11. The method according to one or more of embodiments 1 to 10, wherein the method includes adding other liquid composition to the cell container (4) after step i and before step ii, such that the other liquid composition is present in the cell container (4) when the cells are incubated in step ii, and such that removal in step iii includes removing at least a portion of the other liquid composition by a filter.

[0216] 12. 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 includes measuring the cell density, preferably in an automated manner; - The liquid composition in step i. contains an activator, which is at least partially removed in step iii. - The method includes performing other unit operations, wherein the liquid composition in step i. comprises a gene-modifying agent, preferably a transducer; and - Optionally, another liquid, preferably a cell culture medium, is added after step iii.

[0217] 13. 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 includes measuring the cell density, preferably in an automated manner; - The liquid composition in step i. comprises o activator, o Gene modifiers, preferably transducers or transfection agents, and o Optional reinforcing agent, The above substances are at least partially removed in step iii; and - Optionally, another liquid, preferably a cell culture medium, is added after step iii.

[0218] 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 includes measuring the cell density, preferably in an automated manner; - The liquid composition in step i. contains an activator, which is at least partially removed in step iii. The method includes adding other liquid compositions to the cell container (4) after step i and before step ii, such that... o When the cells are incubated in step ii., other liquid compositions are present in the cell container (4), and o Step iii. removal includes removing at least a portion of the other liquid composition, The other liquid composition comprises o Gene modifiers, preferably transducers or transfection agents, and o Optional enhancers, and - Optionally, another liquid, preferably a cell culture medium, is added after step iii.

[0219] 15. The method according to one or more of embodiments 1 to 14, wherein the step of amplifying the cells in the cell container (4) involves agitating the cells, for example by shaking or wave motion.

[0220] 16. The method according to embodiment 15, wherein amplification includes one or more of batch culture, fed-batch culture and / or perfusion culture, preferably, amplification includes at least perfusion culture.

[0221] 17. The method according to one or more of embodiments 1 to 16, wherein the cells are amplified in the cell container (4) after performing one or more unit operations, and wherein the method includes performing one or more other unit operations after amplifying the cells in the cell container (4), preferably wherein such a method has the following characteristics: - Optionally, prior to step i, the method includes measuring the cell density, preferably in an automated manner; - The liquid composition in step i. contains an activator, which is at least partially removed in step iii. - After step iii, the cells are expanded in the cell container (4); - After amplifying the cells, a liquid composition containing a gene modifier, preferably a transducer or transfectant, more preferably a transfectant, is added to the cell container, and the cells in the cell container (4) are incubated together with the other liquid composition; and - Optionally, harvest the cells.

[0222] 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 includes measuring the cell density, preferably in an automated manner; - Perform the first unit operation, wherein the liquid composition in step i. contains an activator, which is at least partially removed in step iii. - The method includes performing a second unit operation, wherein the liquid composition in step i. comprises a gene-modifying agent, preferably a transfection agent; - Optionally, another liquid, preferably cell culture medium, is added after the second unit operation; - The method includes performing a third unit operation, wherein the liquid composition in step i. contains a gene modifier, preferably a transducer; - Optionally, another liquid, preferably cell culture medium, is added after the third unit operation; - Optionally, the cells are expanded in the cell container (4); and - Optionally, harvest the cells.

[0223] 19. The method according to one or more of embodiments 1 to 18, wherein step ii. further includes agitating the cells in the cell container (4), for example by shaking or wave motion.

[0224] 19a. The method according to one or more of embodiments 1 to 19, wherein step iii. further includes one or more washing steps to substantially completely remove the liquid composition from the cell container.

[0225] 19b. The method according to embodiment 19a, wherein the washing step comprises perfusion, preferably wherein a liquid volume equal to at least 1, 2, 3, 5, or 10 times the volume of the cell culture is exchanged. Preferably, the infusion rate is 15 VVD or lower, 10 VVD or lower, 8 VVD or lower, or 4 VVD or lower.

[0226] 19c. The method according to embodiment 19a, wherein the washing step includes the following steps: - Remove the first amount of liquid from the cell container. - Add a second amount of liquid medium to the cell container, and - Shake the cell container for a certain period of time.

[0227] 19d. The method according to embodiment 19c, wherein the first amount and the second amount are the same volume, for example, up to 33% or up to 50% of the volume of the cell culture. Preferably, the time period is at least 1 minute, more preferably at least 5 minutes or at least 10 minutes, such as 30 minutes.

[0228] 20. The method according to one or more of embodiments 1 to 19d, wherein during the step of amplifying the cells, preferably after the at least one unit operation is performed on the cells, the volume of the cell culture in the cell container (4) increases.

[0229] 21. The method according to embodiment 20, wherein during the step of amplifying the cells, the volume of the cell culture in the cell container (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%.

[0230] 22. The method according to embodiment 20 or 21, wherein at the end of the step of amplifying the cells, the volume of the cell culture in the cell container (4) is at least 100 ml, preferably at least 300 ml, more preferably at least 500 ml, and even more preferably at least 1 l.

[0231] 23. The method according to one or more of embodiments 1 to 22, wherein prior to step i, the volume of the cell culture in the cell container (4) is at least 5 ml.

[0232] 24. The method according to one or more of embodiments 1 to 23, wherein before and / or after step i, the volume of the cell culture in the cell container (4) is at most 100 ml.

[0233] 25. The method according to one or more of embodiments 1 to 24, wherein the unit operation is performed, wherein the volume of the cell culture and the added fluid in the cell container (4) is selected from the range of 1 ml to 200 ml, preferably 5 ml to 100 ml, and in particular, wherein the step of amplifying the cells is performed after the unit operation.

[0234] 26. The method according to one or more of embodiments 1 to 25, wherein the method includes removing liquid from the cell container (4) to reduce the volume of the cell culture by at least 10%, preferably at least 30%, more preferably at least 50%, particularly after performing the unit operation and / or expanding the cells.

[0235] 27. The method according to one or more of embodiments 1 to 25, wherein the method further comprises the step of washing the cells once or more after performing the unit operation, preferably wherein the washing includes: i. Add the washing liquid composition to the cell container (4); ii. Optionally, the cells are incubated in the cell container (4) with added washing fluid; and iii. Remove fluid from the cell container (4) through at least one filter, thereby removing at least a portion of the added washing fluid.

[0236] 28. The method according to one or more of embodiments 1 to 27, wherein the method further includes harvesting the cells, preferably after performing the unit operation and expanding the cells.

[0237] 29. The method according to embodiment 28, wherein harvesting includes transferring the cell culture from the cell container (4), preferably through a port of the cell container, optionally in an automated manner.

[0238] 30. The method according to embodiment 28 or 29, wherein harvesting includes removing liquid from the cell container (4) through the at least one filter to increase the cell density, and then transferring concentrated cell culture from the cell container (4), preferably through the port of the cell container, optionally in an automated manner.

[0239] 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.

[0240] 32. The method according to embodiment 31, wherein the preparation is performed after the unit operation and the cell amplification.

[0241] 33. The method according to embodiment 31 or 32, wherein the preparation includes: i. Remove liquid from the cell container (4) through the at least one filter; ii. Adding a liquid for formulating the cell culture into a cell therapy product to the cell container (4); and iii. Optionally, the cell therapy product is transferred from the cell container (4), preferably through a port of the cell container, optionally in an automated manner.

[0242] 34. The method according to one or more of embodiments 1 to 33, wherein the method includes using a biological processing system (1) comprising a control system (2) and at least one active element (5), wherein the control system (2) implements control commands to cause the at least one active element (5) to advance the unit operation.

[0243] 35. The method according to embodiment 34, wherein the control system (2) generates control commands to cause the active element (5) to add the liquid composition to the cell container (4) in step i, and / or remove the liquid from the cell container (4) through the at least one filter in step iii.

[0244] 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 container (4) and / or for removing liquid from the cell container (4) through the at least one filter.

[0245] 37. The method according to one or more of embodiments 1 to 36, wherein the cell container (4) comprises two or more compartments, preferably small volume compartments (13) and different large volume compartments (14).

[0246] 38. The method according to one or more of embodiments 1 to 37, wherein the cell container (4) comprises a pre-formed reservoir (15) forming a small volume compartment (13).

[0247] 39. The method according to one or more of embodiments 1 to 38, wherein the cell container (4) comprises a shell (16) forming a large volume compartment (14), wherein the shell is preferably flexible.

[0248] 40. The method according to one or more of embodiments 37 to 39, wherein the method has the following characteristics: - During the unit operation, the cell culture is located in the small volume compartment (13); and / or - During the amplification of the cells in the cell container (4), preferably after step iii, the volume of the cell culture is increased such that the cell culture is preferably located in the small volume compartment (13) and the large volume compartment (14).

[0249] 41. The method according to one or more of embodiments 37 to 40, wherein one or more of the at least two compartments comprises at least one filter and / or is fluidly connected to at least one filter.

[0250] 42. The method according to one or more of embodiments 37 to 41, wherein

[0251] - The small volume compartment (13) contains at least one filter and / or is fluidly connected to at least one filter, and / or

[0252] - The large volume compartment (14) contains at least one filter and / or is fluidly connected to at least one filter.

[0253] 43. The method according to embodiment 42, wherein the large volume compartment (14) comprises at least one filter, the filter being a main membrane (7), preferably not used during the unit operation, and / or used for perfusion during cell amplification in the cell container (4).

[0254] 44. The method according to embodiment 42 or 43, wherein the small volume compartment (13) comprises at least one filter, the filter being a reservoir membrane (17), preferably the reservoir membrane (17) for performing the unit operation, particularly for removing liquid from the cell container (4) through the reservoir membrane in step iii.

[0255] 45. The method according to one or more of embodiments 1 to 44, wherein the at least one filter

[0256] - Configured to allow liquid flow through, but essentially preserve the cells, and

[0257] - Preferably not used for gas exchange, especially gas exchange between the cell culture in contact with the filter and the gas in contact with the other side of the filter.

[0258] 46. ​​The method 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 a floating membrane.

[0259] 47. The method according to one or more of embodiments 1 to 46, wherein the cell container (4) comprises

[0260] - At least one filter and / or fluidly connected to at least one filter, - A tube assembly connected to the cell container (4) and pre-sterilized together with the cell container (4).

[0261] 48. The method according to embodiment 47, wherein the pipe assembly comprises: - At least one disposable head for the pump (8); - At least one mating element for the sensor, particularly a magnetic mating element; - Recirculation loop (24); - A sealing and weldable connection for the media container (10); and / or - A sealed and weldable connector for the waste container (18).

[0262] 49. The method according to one or more of embodiments 34 to 48, wherein the biological treatment system further comprises a bioreactor module (3) comprising the cell container (4).

[0263] 50. The method according to one or more of embodiments 34 to 49, wherein the biological treatment system (1), preferably the bioreactor module (3), further comprises a shaking element (9) for applying shaking motion to the cell container (4).

[0264] 51. The method according to embodiment 49 or 50, wherein the bioreactor module (3) includes at least one sensor (6) for sensing the characteristics of the cell culture, preferably wherein during the unit operation, the control system (2) receives sensor data from the at least one sensor (6) and derives the control command from the sensor data.

[0265] 52. The method according to embodiment 51, wherein the at least one sensor (6) comprises

[0266] - A sensor for measuring the properties of the cell culture, particularly

[0267] o Number of cells per unit volume; o Cell size; o Cell viability; and / or o The pH value of the cell culture; and / or - Balance, the balance weighs o The cell culture; o The amount of liquid added to the cell culture; and / or o The amount of liquid removed from the cell culture.

[0268] 53. The method according to embodiment 51 or 52, wherein the control system (2) determines the time point at which the gene modification begins from the measured cell size, generates a control instruction to control the at least one pump (8) to add the fluid containing the gene modifier to the cell container (4) in step i. at the time point, and implements the control instruction.

[0269] 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 container (4); - Waste container (18) connected to the cell container (4); - Bypass line (22) for connecting the container outside the bioreactor module (3) to the cell container (4); - A control unit that is part of the control system (2); and / or - Heating element for heating the cell container (4).

[0270] 55. The method according to one or more of embodiments 49 to 54, wherein the bioreactor module (3) further includes a recirculation loop (24) leading from the cell container (4) to the cell container (4).

[0271] 56. The method according to embodiment 55, wherein the recirculation loop (24) has one or more of the following features: - It contains at least one sensor (6), particularly a sensor for cell counting; - It is connected to the medium container (10), in particular via a bypass line (22); - It is connected to the waste container (18), particularly via the recirculation loop membrane (12) and / or the bypass line (22).

[0272] 57. The method according to one or more of embodiments 49 to 56, wherein the bioreactor module (3) includes a support structure (19) for carrying the cell container (4) and optionally the media container (10).

[0273] 58. The method according to one or more of embodiments 49 to 57, wherein the bioreactor module (3) includes at least one tube retainer (38) that holds at least one tube in a defined position relative to the support structure (19).

[0274] 59. The method according to embodiment 58, wherein the tube retainer (38) includes a tube movement mechanism for extending and / or retracting the tube relative to the tube retainer (38).

[0275] 60. The method according to embodiment 58 or 59, wherein the tube retainer is configured to interact with a connection arrangement (36) for aseptic connection and / or disconnection of the receiver, preferably wherein the connection arrangement (36) includes a tube welding unit (37) for welding and / or cutting and sealing the tube.

[0276] 61. The method according to one or more of embodiments 34 to 60, wherein the biological treatment system (1) includes a connection arrangement (36) for aseptically connecting and / or disconnecting the container, preferably, the connection arrangement (36) includes a tube welding unit (37) for welding tubes together and / or cutting and sealing the tubes.

[0277] 62. The method according to embodiment 61, wherein the method includes welding pipes to the bypass line (22) and / or to the medium container (10) and / or to the waste container (18) via the pipe welding unit (37).

[0278] 63. The method according to one or more of embodiments 34 to 62, wherein the biological treatment system (1) has one or more of the following features: - It contains a frame (32); - It contains at least one amplification site (33) where the amplification operation is performed and preferably the unit operation is performed, particularly with the support of the biological processing system (1); - It replenishes the medium container (10); and / or - It includes a transport mechanism (34) that transports the bioreactor module (3), particularly from and / or into the amplification site (33).

[0279] 64. The method according to one or more of embodiments 1 to 63, wherein the cell is a mammalian cell, preferably an immune cell or a primary cell.

[0280] 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.

[0281] 66. The method according to one or more of embodiments 1 to 65, wherein the method includes cell selection, preferably prior to performing the unit operation.

[0282] 67. The method according to embodiment 66, wherein the cell selection includes: - Positive cell selection, wherein the target portion of the cell binds directly or indirectly to the binding matrix of the binding unit; or - Negative cell selection, wherein the target portion of the cell does not bind directly or indirectly to the binding matrix of the binding unit.

[0283] 68. The method according to embodiment 66 or 67, wherein cell selection includes: - Add a mixture of cells to the cell container (4). - The mixture of cells is brought into contact with the binding unit, wherein at least a portion of the cells bind directly or indirectly to the binding matrix of the binding unit, and - Remove liquid from the cell container (4) to remove at least some cells that are not directly or indirectly bound to the binding matrix.

[0284] 69. The method according to embodiment 68, wherein the binding matrix is ​​configured to enable direct or indirect binding of at least a portion of the ligands expressed by the cells.

[0285] 70. The method according to embodiment 68 or 69, wherein indirect binding comprises adding at least one binding agent to the cell container (4) such that the mixture of cells is contacted with the binding agent and the binding agent binds to at least a portion of the cells, and wherein the binding agent is configured to bind to the binding matrix.

[0286] 71. The method according to one or more of embodiments 66 to 70, wherein the cell container (4)

[0287] - Contains bonding units; and / or

[0288] - Fluidly connected to at least one bonding unit, The binding unit includes a binding matrix configured to enable direct or indirect binding of at least a portion of the ligands expressed by the cells.

[0289] 72. The method according to embodiment 71, wherein the bonding matrix is ​​provided in the form of a resin or a monolithic material.

[0290] 73. The method according to embodiment 71 or 72, wherein the binding unit is located within or adjacent to the small volume compartment (13) of the cell container, preferably wherein the binding unit is arranged adjacent to the filter of the small volume compartment (13).

[0291] 74. The method according to one or more of embodiments 71 to 73, wherein the connecting unit is provided in the form of a column, preferably wherein

[0292] - The opening of the column is arranged within the opening of the small volume compartment (13) of the cell container, more preferably within the opening at the bottom of the small volume compartment (13), and / or

[0293] - The column is fluidly connected to the cell container (4), preferably a reservoir, wherein a fluid connection is provided between the cell container (4) and the column, and in particular, wherein the fluid connection is provided by one or more pipes, and optionally also includes a valve.

[0294] 75. The method according to one or more of embodiments 71 to 74, wherein the binding unit is provided in the form of a floating unit configured to float within the cell container (4).

[0295] 76. The method according to one or more of embodiments 71 to 75, wherein the binding unit is fluidly connected to the cell container (4) and / or waste port via an external circuit, preferably to both.

[0296] 77. The method according to embodiment 76, wherein the external circuit includes at least one valve, preferably one valve for controlling fluid flow between the binding unit and the waste port, and another valve for controlling fluid flow between the cell container (4) and the waste port, preferably both valves for controlling fluid flow between the binding unit and the cell container (4) through the external circuit.

[0297] 78. A bioreactor module for bioprocessing cells in a cell culture, wherein the bioreactor module (3) comprises

[0298] - Cell containers (4), and

[0299] - At least one filter for removing liquid from the cell container (4) through the at least one filter. The bioreactor module is configured to perform one or more unit operations on the cells in the cell container (4) in an automated manner, and to expand the cells in the cell container (4).

[0300] 79. The bioreactor module according to embodiment 78, wherein the bioreactor module further comprises one or two of the following: - At least one active element (5) for influencing the cell culture in the bioreactor module (3), preferably for adding fluid to and / or removing fluid from the cell container (4), preferably wherein the fluid is a liquid; and - At least one sensor (6) for sensing the characteristics of the cell culture.

[0301] 80. The bioreactor module according to embodiment 79, wherein the at least one active element (5) comprises at least one pump (8) for adding fluid to the cell container (4) and / or for removing fluid from the cell container (4) through the at least one filter.

[0302] 81. A bioreactor module according to one or more of embodiments 78 to 80, wherein the cell container (4) comprises one or more of the following: - Two or more compartments, preferably small volume compartments (13) and different large volume compartments (14); - A pre-formed reservoir (15) forming a small volume compartment (13); - An outer shell (16) forming a large-volume compartment (14), preferably wherein the outer shell is flexible; - Main membrane (7); and / or - A control unit and / or an interface with a control system (2), wherein the control unit is adapted to...

[0303] 82. The bioreactor module according to embodiment 81, wherein the bioreactor module (3) has the following characteristics: - During the unit operation, the cell culture is located in the small volume compartment (13); and / or - During the amplification of the cells in the cell container (4), 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).

[0304] 83. A bioreactor module according to one or more of embodiments 78 to 82, wherein the cell container (4) comprises at least two compartments, wherein at least one of the compartments comprises the at least one filter and / or is in fluid connection with the at least one filter.

[0305] 84. A bioreactor module according to one or more of embodiments 81 to 83, wherein

[0306] - The small volume compartment (13) contains at least one filter and / or is fluidly connected to at least one filter, and / or

[0307] - The large volume compartment (14) contains at least one filter and / or is fluidly connected to at least one filter.

[0308] 85. The bioreactor module according to embodiment 84, wherein the large volume compartment (14) includes the at least one filter, the filter being a main membrane (7), preferably not used during the unit operation, and / or used for perfusion during cell amplification in the cell container (4).

[0309] 86. The bioreactor module according to embodiment 84 or 85, wherein the small volume compartment (13) comprises at least one filter, the filter being a reservoir membrane (17), preferably the reservoir membrane (17) for performing the unit operation, particularly for removing fluid from the cell container (4) through the reservoir membrane.

[0310] 87. A bioreactor module according to one or more of embodiments 78 to 86, wherein the bioreactor module (3) includes a control unit and / or an interface with a control system (2), wherein the control unit is adapted to

[0311] After the cell culture is introduced into the cell container (4), the cells are subjected to at least one unit operation and amplification operation within the cell container (4), particularly the subsequent amplification operation, and

[0312] Control commands are implemented during the unit operation and / or the amplification operation to cause the at least one active element (5) to affect the cell culture, thereby advancing the unit operation and / or the amplification operation, and / or wherein the interface with the control system (2) is adapted to allow the control system (2) to implement control commands to cause the at least one active element (5) to affect the cell culture, thereby advancing the unit operation and / or the amplification operation. Preferably, the control unit is adapted to receive sensor data from the at least one sensor (6) and derive control commands from the sensor data, and / or the interface with the control system (2) is adapted to allow the control system (2) to receive sensor data from the at least one sensor (6).

[0313] 88. A bioreactor module according to one or more of embodiments 78 to 87, wherein the cell container (4) includes a tube assembly connected to the cell container (4), preferably wherein the tube assembly is pre-sterilized together with the cell container (4).

[0314] 89. The bioreactor module according to embodiment 88, wherein the tubing comprises

[0315] - At least one disposable pump head (8); - At least one mating element for the sensor, particularly a magnetic mating element; - Recirculation loop (24); - A sealing and weldable connection for the media container (10); and / or - A sealed and weldable connector for the waste container (18).

[0316] 90. A bioprocessing system for biologically processing cells in a cell culture, wherein the bioprocessing system (1) comprises a bioreactor module (3) according to one or more of embodiments 78 to 89.

[0317] 91. The biological processing system according to embodiment 90, wherein the biological processing system (1) includes a control system (2) adapted to implement control commands during the unit operation and / or the amplification step to cause the at least one active element (5) to influence the cell culture, thereby advancing the unit operation and / or the amplification operation.

[0318] 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 cell therapy products, preferably wherein the manufacturing comprises at least one of the following: - Optionally, cells are selected once or multiple times, particularly by binding at least a portion of cells expressing the ligand, preferably a subset of T cells; - Activates cells, preferably T cells; - Transduced cells, preferably transduced T cells; - Transfected cells, preferably T cells; and / or - Expand cells, preferably T cells, and more preferably genetically modified T cells.

[0319] 93. Use of the bioreactor module (3) according to one or more of embodiments 78 to 89 and / or the biological treatment system according to embodiment 90 or 91 in the method according to one or more of embodiments 1 to 77.

[0320] Other aspects and features of this disclosure

[0321] The cell container may include a membrane that can be used for at least one amplification operation supported by the control system of the bioprocessing system, as well as another unit operation, particularly prior to the amplification operation. To achieve this automation, active elements may be advantageously included as part of a bioreactor module containing the cell container. These active elements enable the active advancement of the unit operation or amplification operation. Active advancement of the bioprocessing is not merely the control of ambient temperature, which only provides the necessary conditions for cell culture but does not actively advance the bioprocessing. For example, changing the parameters of the shaking motion based on cell density measurements or changing the temperature could also be envisioned as actively advancing the bioprocessing. By combining multiple unit operations within the cell container, the number of cell transfers can be reduced. This simplifies the process and also reduces, for example, cell damage or cell migration to environments unfavorable to growth during transfer.

[0322] Biological treatment systems can use sensor feedback to advance biological treatment.

[0323] Preferably, the unit operation is activation or gene modification. In a preferred embodiment, activation, as well as transduction and / or transfection, are performed in cell containers, thereby reducing the number of containers required for cell cultures throughout the bioprocess. This also simplifies the overall control system.

[0324] Another embodiment relates to a preferred active element, which includes a pump or a shaking element. The pump can be controlled by a control system to automatically add reagents to the cell container for activation and / or gene modification, thereby advancing the unit operation. Furthermore, preferred sensors are also disclosed herein.

[0325] A preferred implementation involves determining cell size to pinpoint the correct time point from activation to gene modification. Cell size can indicate the activation state.

[0326] The bioreactor module preferably allows for increasing and / or decreasing the volume of cell cultures, preferably by a large factor. The main membrane also typically allows for adjustment of cell concentration, which enables cell concentration to be adjusted prior to unit operations.

[0327] This document describes a preferred method for achieving these large volume variations in cell cultures. According to this embodiment, the cell container comprises two compartments for different culture volumes, the compartments being interconnected and adjacent to each other. Specifically, the smaller volume compartment is designed to allow handling small culture volumes, for example, as low as 5 ml, within a confined space, while the larger volume compartment handles culture volumes greater than, for example, 100 ml. The smaller volume compartment may be located at the bottom of the larger volume compartment; therefore, when the larger volume compartment is used, the smaller volume compartment is also filled, but it has been shown that approximately 100 ml within the smaller volume compartment is acceptable when using the larger volume compartment. The main membrane may be part of the larger volume compartment.

[0328] A preferred method for providing small-volume compartments is a pre-formed reservoir. Alternatively, a second membrane may be provided within the small-volume compartment. It should be noted that the terms "main" membrane and "reservoir" membrane are used only to describe preferred embodiments. The main membrane may be located within the reservoir, and everything described for the reservoir also applies to the main membrane. Thus, there may be only one membrane, used as described herein for a reservoir membrane, or the main membrane may be used as described herein for a reservoir membrane, and another membrane may be present outside or even within the reservoir.

[0329] One of the membranes can be used to remove activators and / or gene modifiers from cell containers, particularly under the control of a control system.

[0330] Another implementation relates to a bioreactor module, which may include a media container, a waste container, a bypass line, a control unit, and / or a heating element.

[0331] Another preferred embodiment provides a recirculation loop that guides from one cell container to another. The recirculation loop can be used for sensors and / or for adding or removing liquid from the cell container.

[0332] Another implementation describes a biological treatment system, which also relates to integrating a bioreactor module into a broader biological treatment system, and providing support functions for the bioreactor module.

[0333] A highly preferred feature of a biological treatment system is a connection arrangement that enables automated aseptic connections, particularly through pipe welding units.

[0334] To enable tube welding to be performed with low complexity, the bioreactor module preferably includes at least one tube retainer that holds the tube in a defined position and allows the tube welding unit to interact with the tube retainer.

[0335] This article also describes a preferred tube assembly.

[0336] In another teaching, it is conceivable to omit the membrane, particularly to add a membrane in the recirculation loop connected to the cell container. The membrane in the recirculation loop can be used to remove reagents and / or add media, especially reagents, and / or for perfusion.

[0337] Another equally important teaching relates to a bioreactor module comprising a cell container for containing cell cultures, at least one active element for influencing the cell cultures within the bioreactor module, and preferably at least one sensor for sensing properties of the cell cultures, wherein the cell container comprises a main membrane. The bioreactor module includes a control unit and / or an interface with a control system, wherein the control unit is adapted to subject cells to at least one unit operation and amplification operation, particularly a subsequent amplification operation, within the cell container after the cell culture is introduced into the cell container, and to implement control commands during the unit operation and / or the amplification operation to cause at least one active element to influence the cell culture, thereby advancing the unit operation and / or the amplification operation, and / or wherein the interface with the control system is adapted to allow the control system to implement control commands to cause at least one active element to influence the cell culture, thereby advancing the unit operation and / or the amplification operation, preferably wherein the control unit is adapted to receive sensor data from at least one sensor and derive control commands from the sensor data, and / or wherein the interface with the control system is adapted to allow the control system to receive sensor data from at least one sensor.

[0338] All explanations given regarding the proposed method are fully applicable to the bioreactor module.

[0339] The bioreactor module may be equipped with the tubing groups disclosed herein.

[0340] Another equally important teaching relates to a biological processing system comprising a control unit and a bioreactor module, the bioreactor module comprising a cell container for containing cell cultures, at least one active element for influencing the cell cultures within the bioreactor module, and preferably at least one sensor for sensing characteristics of the cell cultures, wherein the cell container comprises a main membrane, and wherein the biological processing system is adapted to subject the cells to at least one unit operation and an amplification operation, particularly a subsequent amplification operation, within the cell container after the cell cultures have been introduced into the cell container, wherein the control system is adapted to implement control commands during the unit operation and / or the amplification operation to cause the at least one active element to influence the cell cultures, thereby advancing the unit operation and / or the amplification operation.

[0341] All explanations given regarding the proposed method and the proposed bioreactor module are fully applicable to biological treatment systems.

[0342] Attached Figure Description

[0343] Figure 1 A biological processing system 1 suitable for performing the proposed method is shown. The biological processing system 1 is used for performing several parallel biological treatments on immune cells or primary cells. The cells are preferably specifically designed for cell and / or gene therapy.

[0344] The term "immune cell" can refer to different types of white blood cells. Therefore, the term "immune cell" includes a variety of cells, such as, but not limited to, dendritic cells; T lymphocytes, also known as T cells; B lymphocytes; natural killer cells; macrophages, etc. Immune cells can also include subtypes of immune cells, such as tumor-infiltrating lymphocytes or different types of T cells. A subtype of a particular type of immune cell can be classified based on the type of antigen present on the cell surface. Therefore, the term "immune cell" can, for example, refer to T cells containing the surface antigen CD4 ("CD4+ T cells").

[0345] Preferably, the cell is a genetically modified T cell, or a T cell modified to become a genetically modified T cell. Preferably, the T cell is genetically modified to express a chimeric antigen receptor (CAR). Therefore, the term "CAR-T cell" describes a T cell that has been genetically modified to express a CAR. Therefore, preferably, the genetically modified T cell is a genetically modified CAR-T cell.

[0346] This paper also discloses a method for biologically treating immune cells or primary cells in cell cultures using a biological treatment system 1. The biological treatment system 1 includes a control system 2 and a bioreactor module 3. Figure 1 A biological treatment system 1 is shown, which preferably includes multiple bioreactor modules 3.

[0347] Figure 2 An embodiment of a bioreactor module 3 is shown, the bioreactor module 3 comprising a cell container 4 for containing cell cultures, at least one active element 5 for influencing cell cultures within the bioreactor module 3, and preferably at least one sensor 6 for sensing characteristics of cell cultures.

[0348] Figure 3 A top view of cell container 4 is shown. Figure 4 A view inside cell container 4 is shown, with the top portion removed. (See diagram.) Figure 4 As can be seen, the cell container 4 contains a main membrane 7, and preferably even contains two membranes, as will be explained later.

[0349] As part of the biological treatment, cell cultures are introduced into cell container 4. Cell cultures can be introduced into cell container 4 automatically, manually by the user, or semi-automatically with the support of the biological treatment system 1.

[0350] After the cell culture is introduced into the cell container 4, the cells are subjected to at least one unit operation and an amplification operation within the cell container 4, particularly the subsequent amplification operation. The biological processing system 1 preferably performs the unit operation and amplification operation automatically, and preferably the transition from the unit operation to the amplification operation. The term "automatic" means that no user intervention is required at the level of interaction between the components and the cell culture, other liquids used for the cell culture, tubes, etc., but does not preclude the user from enabling the biological processing to continue through, for example, a user interface.

[0351] During unit operation and / or amplification operation, control system 2 preferably receives sensor data from at least one sensor 6, for example by measuring analog sensor signals or reading digital sensor signals, and preferably derives and implements control commands from said sensor data, particularly the derived control commands, to cause at least one active element 5 to influence the cell culture, particularly the cell culture within the cell container 4, thereby advancing the unit operation and / or amplification operation. The control commands are preferably dependent on the sensor signals and may depend on other factors, such as time. "Propelling unit" operation refers to the action of the active element 5 causing the next step of the biological process to be performed, for example, initiating shaking motion or pumping liquid. Simply maintaining a steady state, such as a stable temperature, is not considered advancing the biological process. Amplification may be performed partially or entirely in a batch and / or fed-batch and / or perfusion manner, or any combination thereof. For example, amplification may encompass an initial batch culture followed by fed-batch and / or perfusion culture.

[0352] According to one embodiment, a bioreactor module 3 is proposed to include at least one sensor 6 for sensing the characteristics of cell cultures, wherein during unit operation and / or amplification operation, a control system 2 receives sensor data from the at least one sensor 6 and derives control commands from the sensor data.

[0353] Preferably, at least one unit operation includes activation, particularly with an activator, and / or gene modification, particularly transduction and / or transfection, particularly with a gene-modifying agent, particularly a transducer and / or transfectant. The appropriate reagent is added to the cell culture, preferably to the cell container 4. The transfection agent can be, for example, lipid nanoparticles (e.g., described in WO2024 / 008967 A1). The transduction agent can be a viral vector, particularly a lentiviral or adenoviral vector. Activation is preferably performed within the cell container 4, and preferably at different cell culture volumes, followed by gene modification. Selection can also be performed within the cell container 4, particularly if selection is performed via reagents and / or membranes rather than magnetically. Enrichment can also be performed within the cell container 4. Preferably, the cell culture does not leave the cell container 4 between the start of the unit operation and the end of the amplification operation. This provides several advantages, such as fewer pumping operations, where liquid transfer itself can be error-prone, and the elimination of the need to reposition the cells to different environments, which could trigger shearing.

[0354] At least one unit operation, particularly activation and gene modification, as well as amplification, can be performed fully automatically in the cell container 4 via the biological processing system 1. Other possible unit operations, particularly those that can be performed after amplification, are concentration and / or media exchange, especially for subsequent freezing.

[0355] Observe active element 5, for example Figure 3 As shown, at least one active element 5 may include at least one pump 8 for adding liquid to the cell culture, and / or at least one pump 8 for removing liquid from the cell container 4, preferably a pump 8 adapted to perform both, and / or Figure 2 The shaking element 9 shown is used to apply shaking motion to the cell container 4. The bioreactor module 3 preferably includes the shaking element 9 and / or one or more pumps 8. Figure 2 (Not shown in the image).

[0356] Preferably, the control system 2 controls at least one pump 8 to add an activator to the cell culture when it is located in the cell container 4, and / or to add a gene modifier and / or culture medium to the cell culture when it is located in the cell container 4. A media container 10 containing the appropriate reagents may be connected to the cell container 4 prior to the unit operation, and the control system 2 preferably controls the addition of reagents without human intervention. The media container 10 may be automatically connected by the biological processing system 1, as disclosed herein.

[0357] At least one sensor 6 may include a sensor for measuring characteristics of the cell culture, particularly the number of cells per unit volume, and / or cell size, and / or cell size distribution, and / or cell viability, and / or the pH of the cell culture, and / or the concentration of metabolites such as glucose in the cell culture, and / or the concentration of cellular metabolic byproducts such as lactate in the cell culture. Additionally or optionally, at least one sensor 6 may include a balance that weighs the cell culture, and / or the amount of liquid added to the cell culture, and / or the amount of liquid removed from the cell culture. The control system 2 may determine parameters for the next step in a unit operation or amplification operation based on the characteristics of the cells, particularly the number of cells per unit volume, and / or cell size, and / or cell size distribution, and / or cell viability, and / or the pH of the cell culture, and / or the concentration of metabolites such as glucose in the cell culture, and / or the concentration of cellular metabolic byproducts such as lactate in the cell culture, and / or the elapsed time. The next step may also be the end of the unit operation and the transition to amplification. Cell size may include cell size distribution and / or average cell size and / or median cell size.

[0358] Specifically, the control system 2 can determine the time point for initiating gene modification based on the measured cell size, generate control instructions for at least one pump 8 to add gene-modifying agents to the cell container 4 at said time point, and implement said control instructions. The control system 2 can determine the time point based on the cell size reaching a predetermined value and can automatically implement gene modification. Several reagents can be added together to combine the steps. Cell size can indicate the state of activation.

[0359] More typically, the control system 2 can determine the time point for initiating activation and / or transduction and / or transfection and / or amplification from the measured characteristics of the cell culture, generate control instructions for at least one pump 8 to add activator and / or transducer and / or transfection agent and / or culture medium to the cell container 4 at said time point, and implement said control instructions. The time point may also depend on a predefined minimum elapsed time.

[0360] Furthermore, the control system 2 can determine the amount of activator and / or gene modifier, particularly transducer and / or transfectant, and / or culture medium from the measured cell characteristics of the cell culture, generate control instructions to control at least one pump 8 to add the determined amount of activator and / or transducer and / or transfectant and / or culture medium to the cell container 4, and implement the control instructions.

[0361] Preferably, the volume of the cell culture is actively altered, rather than simply by adding reagents. Preferably, between the start of the unit operation and the end of the amplification operation, the volume of the cell culture 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%. Alternatively, at the start of the unit operation, the volume of the cell culture in the cell container 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, and / or at the end of the amplification operation at at least 100 ml, preferably at least 300 ml, more preferably at least 500 ml, more preferably at least 1 L. Between the start of the unit operation and the end of the amplification operation, the biological processing system 1 may also reduce the volume of the cell culture by at least 10%, preferably at least 30%, more preferably at least 50%. Volume reduction can be achieved via the main membrane 7, via a sampling port (e.g., one of the supply ports 11 described below), or via the recirculation loop membrane 12 described below.

[0362] Specifically, activation can be performed with one of the specified volumes of less than 100 ml. For gene modification, the volume can be increased by, for example, by adding, particularly an automated feed medium, one of the specified percentages.

[0363] In particular, when combined with shaking motion, a wide range of live cell counts can be achieved; for example, the live cell count of the cell culture can be at least 10⁻⁶ between the start of the unit operation and the end of the expansion. 10^6 live cells / ml to 20 Variations between 10^6 live cells / ml, preferably within 5 10^6 live cells / ml to 25 Variations between 10^6 live cells / ml, more preferably within 1 10^6 live cells / ml to 30 Variations between 10^6 live cells / ml.

[0364] Figure 4 This illustrates the processing of different culture volumes in a preferred embodiment. The preferred cell container 4 comprises small-volume compartments 13 and different large-volume compartments 14. The term "different" means that the volume compartments are identifiable and functionally distinct. The boundaries between the compartments cannot be arbitrarily set, but must be functional and visible boundaries.

[0365] During unit operations, particularly throughout the entire unit operation, the volume of the cell culture is preferably fitted into the small volume compartment 13 and remains within the small volume compartment 13 during periods without shaking or other mixing. During shaking, liquid may splash out of the small volume compartment 13. However, the large volume compartment 14 is here, and preferably arranged, such that liquid returns to the small volume compartment 13 by gravity during periods without shaking.

[0366] During the amplification operation, at least at some point, the volume of the cell culture is preferably too large to fit into the small volume compartment 13, and therefore preferably located in both the small volume compartment 13 and the large volume compartment 14, especially during periods without shaking or other mixing. Therefore, the amplification operation is preferably performed in a perfusion mode, wherein the cells are retained within the cell container 4 by the main membrane 7.

[0367] As can be seen, preferably, the bulk compartment 14 contains the main membrane 7. Therefore, it is preferable that the main membrane 7 is not used during unit operations. The main membrane 7 can be used for perfusion during amplification operations.

[0368] Figure 4 It is also shown that, preferably, the cell container 4 includes a pre-formed reservoir 15 forming small volume compartments 13, and a particularly flexible outer shell 16 forming large volume compartments 14. In addition, or independently, the small volume compartments 13 may include a reservoir membrane 17 for use during unit operation.

[0369] The terms "main" and "reservoir" preceding "membrane" are used to distinguish membranes according to their preferred use, but are not limiting. For example, the main membrane 7 may be located at the lowest point of the cell container 4, regardless of whether the cell container 4 has a reservoir 15.

[0370] For both membranes, a variety of uses are conceivable, such as the choices already explained. Furthermore, reservoir membrane 17 can be used for perfusion during unit operations and / or expansion operations. The membrane has pore sizes that retain cells within it and are adapted to the corresponding function. One or more membranes may also be functionalized.

[0371] In a preferred embodiment, activators and / or gene-modifying agents are removed from the cell container 4 through one of the membranes, particularly reservoir membrane 17. For this purpose, the control system 2 preferably generates control data to cause pump 8 to remove the activators and / or gene-modifying agents from the cell container 4. Feed media or other neutral media can be added via pump 8, and permeate can be drawn through the membrane by the same pump or another pump 8. The addition of media and the removal of permeate can be carried out continuously or in parallel. This can be repeated, for example, two or three times, to reduce the concentration of the corresponding reagent in the cell container 4.

[0372] It is also conceivable to remove the culture medium, especially cell-free culture medium, from the cell container 4 by allowing the cells to settle to the bottom and then extracting the medium from above the settled cells through the corresponding port.

[0373] like Figure 2 As shown, the preferred bioreactor module 3 includes a media container 10 and / or a waste container 18 connected to the cell container 4. The bioreactor module 3 may include a support structure 19 that supports the cell container 4 and the media container 10. Therefore, due to the support structure 19, the bioreactor module 3 can be processed as a unit together with the cell container 4 and the media container 10. If manual processing is required, the bioreactor module 3 can be lifted by its support structure 19, which then supports the cell container 4 and the media container 10. The bioreactor module 3 may provide temperature control for the media container 10, particularly for cooling activators and / or gene-modifying agents.

[0374] However, the media container 10 may not contain sufficient media and / or may not contain all the media required for the biological processing steps within the cell container 4, particularly the amplification operation. To overcome this shortage, the liquid media within the media container 10 is replenished, particularly automatically by the biological processing system 1.

[0375] One way to achieve replenishment is to replace the media container 10 with another media container 10. According to one embodiment, a bioreactor module 3 is proposed to include a life support container 20, which comprises a media container 10 and a waste container 18. Preferably, the life support container 20 containing the media container 10 and / or the waste container 18 is replaced with a second life support container 20 containing a second media container 10 and / or a second waste container 18, particularly automatically by the biological treatment system 1 during the replacement process.

[0376] In a different implementation or even as an alternative, during the refilling process, the biological treatment system 1 can automatically connect the refill container 21 to the media container 10 and automatically refill the media container 10, and preferably automatically disconnect the refill container 21 from the media container 10.

[0377] Furthermore, the bioreactor module 3 may include a bypass line 22 for connecting a container outside the bioreactor module 3 to the cell container 4, and / or the bioreactor module 3 may include a control unit as part of the control system 2, and / or the bioreactor module 3 may include a heating element for heating the cell container 4. The bypass line 22 bypasses the media container 10 and the waste container 18.

[0378] The life support container 20 and / or the media container 10 and / or the waste container 18 can be weighed by a balance, which is one of at least one of the sensors 6, in particular for determining the amount of liquid added to or removed from the cell container 4.

[0379] According to one embodiment, a bioreactor module 3 is proposed to include a cover 23 for providing a controlled environment for a cell container 4, and / or a heating unit for the cell container 4.

[0380] Figure 3 The upper right corner shows that the bioreactor module 3 may include a recycling loop 24 guiding from the cell container 4 to the cell container 4. The recycling loop 24 may connect to a supply port 11 of the cell container 4, which is also connected to a media container 10 and / or a waste container 18. Preferably, the recycling loop 24 includes at least one branch 25. Figure 3 In the schematic recirculation loop 24, the tubes are arranged in the same order as those shown at cell container 4. For example, the tube shown leading from supply port 11 may include a branch 25, preferably leading to pump 8 and / or sensor, and from there returning to another branch 25 of the tube, for example, connected to another supply port 11 (only when...). Figure 5 In this embodiment, the recirculation loop 24 returns to the supply port 11. In a preferred embodiment shown, the recirculation loop 24 is a bidirectional recirculation loop 24. Thus, the recirculation loop 24 originates from one port, specifically via branch 25, and reaches a loop destination 26, such as a sensor. From there, the recirculation loop 24 continues to a gas source, specifically another gas port 27 of the cell container 4. However, samples drawn through the recirculation loop 24 do not traverse the entire recirculation loop 24, but instead reach the loop destination 26 and return. The portion of the recirculation loop 24 not used for liquid is used for air to push the liquid back and allow it to flow forward. The cell container 4 may also include a gas inlet port 28, a gas outlet port 29, and / or a waste port 30, as shown.

[0381] Preferably, the recirculation loop 24 includes at least one of the sensors 6, particularly a sensor for cell counting, especially at the loop destination 26. Figure 5 One embodiment of a fluid pipeline is shown, which can be connected to the cell container 4 and also includes a recirculation loop 24. It can be seen therein that the recirculation loop 24 is preferably connected to the media container 10 and / or the waste container 18, particularly via the recirculation loop membrane 12 and / or bypass line 22. Additional connections between the media container 10 and the waste container 18 and the cell container 4 may also be present. Figure 5 In the middle, reagent source 31 is connected to bypass line 22. Figure 5 The fluid pipelines in the text do not completely correspond to Figures 2 to 4 Fluid pipelines in the system.

[0382] The recirculation loop 24 typically allows for non-destructive sensing of the characteristics of cell cultures, particularly cells, preferably non-contact sensing. Destructive sampling or probes in or connected to the reservoir 15 are also conceivable. Examples of suitable probes may include capacitance-based sensors, microscopic devices, etc.

[0383] During the amplification step, culture medium from the medium container 10 may be added to the cell container 4, and / or liquid removed from the cell container 4 may be introduced into the waste container 18.

[0384] Volume adjustment during unit operations and / or amplification operations can be performed by: obtaining the desired cell characteristics from the formulation; measuring the cell characteristics using at least one sensor 6; determining the current volume in the cell container 4, for example by a balance, flow sensor, or calculation, thereby determining the required volume adjustment, optionally by adding media, particularly from the media container 10, preferably monitored by a balance. Alternatively, the media container 10 may be pre-filled with a predefined amount. Cell numbers can be adjusted based on the characteristics of the cell culture before adding reagents, by introducing or removing media from the cell container 4.

[0385] Turn Figure 1 The biological treatment system 1 in the image preferably includes a frame 32. The frame 32 may include a user interface and may typically house up to all components of the biological treatment system 1.

[0386] The biological processing system 1 preferably includes at least one amplification site 33 where amplification operations are performed, and preferably unit operations are performed, particularly with the support of the biological processing system 1. The biological processing system 1 preferably supplements the media container 10.

[0387] Preferably, the biological treatment system 1 includes a transport mechanism 34, such as a movable pallet mounted on a track (located in...). Figure 1 (Outside the visible area on the right) or a robotic arm, for automatically moving the bioreactor module 3. A transport mechanism 34 is used for automatically transporting the bioreactor module 3 containing the cell container 4 and the media container 10, and for automatically transporting several bioreactor modules 3. Here, the transport mechanism 34 is an elevator and / or loads the bioreactor module 3 from and / or into the amplification position 33. Several such amplification positions 33 can be arranged in a row, as shown. The transport mechanism 34 simultaneously transports the cell container 4 and the media container 10 together because they are fluidly connected.

[0388] The transport mechanism 34 may include transport elements adapted to hold the bioreactor module 3 and movable in at least one linear direction. The transport elements may be movable precisely in one linear direction, particularly vertically, as explained above regarding the embodiment of the elevator. Control strategies for controlling the cell containers 4 within the bioreactor module 3, such as agitation or media supply, particularly those executed by the control units contained within the bioreactor module 3, may depend on the location of the bioreactor module 3. Location information may be provided by the biological processing system 1. The biological processing system 1 may include dedicated unit operation locations 35 for unit operations or preferably for individual unit operations, such as selection or enrichment.

[0389] When describing the interaction between biological treatment system 1 and the bioreactor, the term biological treatment system 1 refers to the other components in biological treatment system 1 besides the bioreactor module 3.

[0390] Another relevant aspect of the biological treatment system 1 is that it preferably includes a connection arrangement 36 for aseptic connection and / or disconnection from the container. The connection arrangement 36 may include a tube welding unit 37 for welding tubes together and / or cutting and sealing the tubes. A preferred connection arrangement 36 is described in EP 4 342 975 A1. The tube welding unit 37 can weld tubes to bypass lines 22 and / or to tubes connected to media containers 10 and / or to waste containers 18. The tube welding unit 37 can be used to replace media containers 10 and / or waste containers 18, and / or refill media containers 10 and / or waste containers 18.

[0391] The tube welding unit 37 is preferably capable of automatically gripping tubes. Preferably, the bioreactor module 3, particularly the life support container 20, includes at least one tube retainer 38 that holds at least one tube in a defined position relative to the support structure 19 and / or the life support container 20, and the tube welding unit 37 interacts with the tube retainer 38 to weld and / or cut and seal the tube. Figure 1 A close-up of one embodiment of such a tube retainer 38 is shown. Preferably, the tube retainer 38 includes a tube movement mechanism for extending and / or retracting the tube relative to the tube retainer 38. In any case, the tube retainer 38 enables precise handling of the tube without requiring the tube welding unit 37 to position the tube itself in a specific manner. Positioning the tube retainer 38 is sufficient, as it has a more clearly defined location and orientation. It is also preferred that the tube welding unit 37 performs an integrity test on the newly established weld to determine whether the weld has been correctly established. If the weld fails the integrity test, it is further preferred that the tube welding unit 37 can cut the tube and establish a new weld.

[0392] Preferably, the tube is cut by a blade. For this purpose, the biological treatment system 1 may provide a blade supply unit, particularly a blade holder, and a blade removal unit, particularly a waste bin, for supplying and disposing of the blades. The blade supply and removal may be provided as a combined unit within the biological treatment system 1 (details not shown). The tube welding unit 37 may be automatically moved to the blade supply / removal unit for blade supply and / or removal.

[0393] Preferably, the bioreactor module 3 includes a drive unit for moving the tube holder 38, particularly for linear movement along a track of the bioreactor module 3. The track may also be... Figure 1 Seen in the middle.

[0394] Preferably, each bioreactor module 3 in the group includes a receiving interface having a connector for receiving electrical energy and / or a connector for receiving wired signals and / or a connector for receiving gas and / or a connector for an optical sensor, and the receiving interface can be connected to the providing interface of the frame 32.

[0395] Preferably, frame 32 includes interfaces for providing electrical power and / or wired signal and / or gas and / or optical sensor connections at different locations, particularly at amplification location 33. Bioreactor module 3 is partially self-sufficient and may include a battery. With the aid of media container 10 and preferably waste container 18, bioreactor module 3 can be self-sufficient for at least 12 hours, preferably at least 24 hours. This protects the cells, even during power outages or similar events.

[0396] Different supply interfaces can provide different subsets of connections to the receiving interface. For example, gas can be supplied only at amplification position 33, while energy can be supplied by each supply interface.

[0397] observe Figure 3 and 5 The cell container 4 may also include a tubular assembly connected to and pre-sterilized together with the cell container 4, the tubular assembly including at least one disposable head for the pump 8, and / or at least one docking element for a sensor, particularly a magnetic docking element, and / or a recirculation loop 24, and / or a sealed and weldable connection for the media container 10, and / or a sealed and weldable connection for the waste container 18. The actuator for the pump head may be included in the bioreactor module 3.

[0398] Another equally important teaching relates to a bioreactor module 3 for the proposed method, wherein the bioreactor module 3 includes a cell container 4 for containing cell cultures, at least one active element 5 for influencing the cell cultures within the bioreactor module 3, and preferably at least one sensor 6 for sensing the properties of the cell cultures, wherein the cell container 4 includes a host membrane 7, and wherein the bioreactor module 3 includes a control unit and / or an interface with a control system 2, wherein the control unit is adapted to subject the cells to at least one unit operation and amplification operation within the cell container 4 after the cell cultures are introduced into the cell container 4, particularly a subsequent amplification operation, and during the unit operation and / or amplification operation, preferably to receive sensor data from at least one sensor 6, and preferably to derive control commands from the sensor data, and to implement the control commands, particularly the control commands, to cause at least one active element 5 to influence the cell cultures, thereby advancing the unit operation and / or amplification operation, and / or wherein the interface with the control system 2 is adapted to allow the control system 2 to implement control commands to cause at least one active element 5 to influence the cell cultures, thereby advancing the unit operation and / or amplification operation. Preferably, the interface is adapted to allow the control system 2 to receive sensor data from at least one sensor 6.

[0399] It is conceivable that bioreactor module 3 can also be used to produce reagents required in the manufacturing processes of cell and gene therapies. In particular, bioreactor module 3 can also be used to produce viral vectors required for the gene modification step in the system of the present invention. In this case, cells used for vector production, such as HEK293 cells, can be transiently transfected as a unit operation followed by amplification, or amplification can be performed in HEK293 cells followed by transfection for gene modification.

[0400] According to one embodiment, a cell container 4 is provided to include a tubing assembly connected to and pre-sterilized together with the cell container 4, the tubing assembly including at least one disposable pump head 8, and / or at least one docking element for a sensor, particularly a magnetic docking element, and / or a recirculation loop 24, and / or a sealed and weldable connector for a media container 10, and / or a sealed and weldable connector for a waste container 18.

[0401] Another equally important teaching relates to a bioprocessing system 1 for the proposed method, wherein the bioprocessing system 1 includes a control unit and a bioreactor module 3, the bioreactor module 3 including a cell container 4 for containing cell cultures, at least one active element 5 for influencing the cell cultures within the bioreactor module 3, and at least one sensor 6 preferably for sensing the characteristics of the cell cultures, wherein the cell container 4 includes a host membrane 7, wherein the bioprocessing system 1 is adapted to subject the cells to at least one unit operation and amplification operation within the cell container 4 after the cell cultures are introduced into the cell container 4, particularly the subsequent amplification operation, wherein the control system 2 is adapted to implement control commands during the unit operation and / or amplification operation to cause the at least one active element 5 to influence the cell cultures, thereby advancing the unit operation and / or the amplification operation.

[0402] Another implementation of cell container 4 is in Figure 8 The diagram illustrates that the cell container 4 includes a shell 16 and a reservoir 15, and may also include a binding unit 101. The binding unit shown is located at the bottom of the reservoir, adjacent to the reservoir membrane 17. The binding unit typically includes a binding matrix (see the gray portion in 101), which is configured to bind directly or indirectly to cells for a positive or negative cell selection step. The binding unit 101 may be provided in the form of a column, such as... Figure 8 As shown in the figure. Alternatively, the connecting unit 101 may be provided as a floating unit (not shown), wherein the connecting unit 101 may float in the fluid present in the container 1. When provided as a column, the connecting unit 101 may be provided directly in the reservoir or adjacent to the reservoir (see Figure 1). Figure 8 Alternatively, the connecting unit 101 may be fluidly connected to the container 4, preferably to the reservoir 15, for example, via a pipe (not shown). Figure 8 As shown, the bonding unit 101 is positioned adjacent to the reservoir membrane 17. An opening may be provided in the bottom of the reservoir 15 to allow the liquid medium to enter the bonding unit 101.

[0403] like Figure 8 The connecting unit 101 shown may include an external loop fluidly connected to, preferably to, both the reservoir 15 and / or the waste port. The external loop may be provided by a pipe that allows fluid to be transported out of or into the connecting unit. Such a pipe preferably includes a valve, such as... Figure 8As shown in the diagram. The external circuit includes at least one valve, preferably one valve for controlling fluid flow between the binding unit 101 and the waste port, and another valve for controlling fluid flow between the reservoir 15 and the waste port. Preferably, both valves are used to control fluid flow between the binding unit 101 and the reservoir 15 through the external circuit. The waste port is preferably connected to a pump to allow fluid transfer operations. Alternatively, fluid transfer can also be achieved by pressurizing the cell container 4 to push fluid through the binding matrix 101. An additional inlet may also be provided between the reservoir 5 and the binding matrix 101, for example, through a tube, when the binding matrix 101 is fluidly connected to the reservoir 15, such that another tube is connected to provide positive or negative pressure to the binding matrix 101 for fluid transfer operations.

[0404] Figure 9 A preferred embodiment of the method according to this disclosure is described, which may be referred to as a "sequential embodiment". As disclosed herein, it illustrates an embodiment in which an activation step occurs, followed by gene modification, in this case, transduction. System setup and steps to be performed are also shown, which are preferably performed in an automated manner.

[0405] Figure 10 A preferred embodiment of the method according to this disclosure is described, which may be referred to as a "parallel embodiment." As disclosed herein, it illustrates an embodiment in which the activation step and gene modification, here transduction, occur substantially in parallel. System setup and steps to be performed are also shown, preferably in an automated manner.

[0406] Figure 11 A preferred embodiment of the method according to this disclosure is described, which may be referred to as the "transfection short embodiment". As disclosed herein, it illustrates an embodiment in which the activation step and gene modification, in this case, transfection, occur substantially in parallel. System setup and steps to be performed are also shown, which are preferably performed in an automated manner.

[0407] Figure 12 A preferred embodiment of the method according to this disclosure is described, which may be referred to as the "transfection long embodiment". As disclosed herein, it illustrates an embodiment in which an activation step occurs, followed by amplification, and then gene modification, in this case, transfection. System setup and steps to be performed are also shown, which are preferably performed in an automated manner.

[0408] Figure 13A preferred embodiment of the method according to this disclosure is described, which may be referred to as the "transfection and transduction embodiment". As disclosed herein, it illustrates an embodiment in which an activation step occurs, followed by gene modification, here transfection. Subsequently, the cells undergo transduction and then expansion. The system setup and the steps to be performed are also shown, which are preferably performed in an automated manner.

[0409] According to the entries of this disclosure

[0410] The following entries provide other advantageous embodiments of this disclosure: 1. A method for biologically treating immune cells or primary cells in a cell culture using a biological treatment system (1), wherein the biological treatment system (1) comprises a control system (2) and a bioreactor module (3), the bioreactor module (3) comprising a cell container (4) for containing the cell culture and at least one active element (5) for influencing the cell culture within the bioreactor module (3), wherein the cell container (4) comprises a main membrane (7). The cell culture is introduced into the cell container (4), and after the cell culture is introduced into the cell container (4), the cells are subjected to at least one unit operation and an amplification operation within the cell container (4), particularly the subsequent amplification operation. During the unit operation and / or the amplification operation, the control system (2) implements control commands to cause the at least one active element (5) to influence the cell culture, thereby advancing the unit operation and / or the amplification operation.

[0411] 2. The method according to item 1, characterized in that the bioreactor module (3) includes at least one sensor (6) for sensing the characteristics of the cell culture, wherein during the unit operation and / or the amplification operation, the control system (2) receives sensor data from the at least one sensor (6) and derives the control command from the sensor data.

[0412] 3. The method according to item 1 or 2, characterized in that the at least one unit operation includes activation, particularly with an activator, and / or gene modification, particularly transduction and / or transfection, particularly with a gene modifier, particularly a transducer and / or transfectant.

[0413] 4. The method according to any one of the preceding entries, characterized in that the at least one active element (5) comprises at least one pump (8) for adding liquid to the cell culture, and / or at least one pump (8) for removing liquid from the cell culture, and / or a shaking element (9) for applying a shaking motion to the cell container (4), preferably, the control system (2) controls the at least one pump (8) to add the activator to the cell culture when the cell culture is in the cell container (4), and / or to add the gene modifier to the cell culture when the cell culture is in the cell container (4).

[0414] 5. The method according to any one of the preceding entries, characterized in that the at least one sensor (6) comprises a sensor for measuring the characteristics of the cell culture, particularly the number of cells per unit volume and / or cell size and / or cell viability and / or pH of the cell culture, and / or the at least one sensor (6) comprises a balance that weighs the cell culture and / or the amount of liquid added to the cell culture and / or the amount of liquid removed from the cell culture.

[0415] 6. The method according to items 4 and 5, characterized in that the control system (2) determines the time point at which the gene modification begins from the measured cell size, generates a control instruction to control the at least one pump (8) to add the gene modifier to the cell container (4) at the time point, and implements the control instruction.

[0416] 7. The method according to any one of the preceding entries, characterized in that, between the start of the unit operation and the end of the amplification operation, the volume of the cell culture 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%, and / or at the start of the unit operation, the volume of the cell culture in the cell container (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, and / or at the end of the amplification operation is at least 100 ml, preferably at least 300 ml, more preferably at least 500 ml, more preferably at least 1 l, and / or between the start of the unit operation and the end of the amplification operation, the biological processing system (1) reduces the volume of the cell culture by at least 10%, preferably at least 30%, more preferably at least 50%.

[0417] 8. The method according to any one of the preceding entries, characterized in that the cell container (4) comprises a small volume compartment (13) and different large volume compartments (14), preferably, during the unit operation, the volume of the cell culture is fitted into the small volume compartment (13) and is located in the small volume compartment (13) during periods without shaking or other mixing, and during the amplification operation, the volume of the cell culture is too large to fit into the small volume compartment (13), and is preferably located in both the small volume compartment (13) and the large volume compartments (14), particularly during periods without shaking or other mixing.

[0418] 9. The method according to item 8, characterized in that the large volume compartment (14) contains the main membrane (7), preferably the main membrane (7) is not used during the unit operation, and / or the main membrane (7) is used for perfusion during the amplification operation.

[0419] 10. The method according to any one of the preceding entries, characterized in that the cell container (4) comprises a pre-formed reservoir (15) forming the small volume compartment (13), and a particularly flexible outer shell (16) forming the large volume compartment (14), and / or the small volume compartment (13) comprises a reservoir membrane (17), preferably used during the unit operation.

[0420] 11. The method according to one of the preceding entries, characterized in that the activator and / or the gene modifier is removed from the cell container (4) by means of a membrane, particularly the reservoir membrane (17), preferably the control system (2) generates control data that causes the pump (8) to remove the activator and / or the gene modifier from the cell container (4).

[0421] 12. The method according to any one of the preceding entries, characterized in that the bioreactor module (3) includes a media container (10) and / or a waste container (18) connected to the cell container (4), and / or the bioreactor module (3) includes a bypass line (22) for connecting a container outside the bioreactor module (3) to the cell container (4), and / or the bioreactor module (3) includes a control unit as part of the control system (2), and / or the bioreactor module (3) includes a heating element for heating the cell container (4).

[0422] 13. The method according to one of the preceding entries, characterized in that the bioreactor module (3) includes a recirculation loop (24) leading from the cell container (4) to the cell container (4), preferably the recirculation loop (24) includes one of the at least one sensor (6), particularly a sensor for cell counting, and / or the recirculation loop (24) is connected to the media container (10) and / or the waste container (18), particularly through the recirculation loop membrane (12) and / or bypass line (22).

[0423] 14. The method according to any one of the preceding entries, characterized in that the biological processing system (1) comprises a frame (32), and / or the biological processing system (1) comprises at least one amplification site (33) at which the amplification operation is performed and preferably the unit operation is performed, particularly with the support of the biological processing system (1), and / or the biological processing system (1) replenishes the medium container (10), and / or the biological processing system (1) comprises a transport mechanism (34) that transports the bioreactor module (3), particularly from and / or into the amplification site (33).

[0424] 15. The method according to any one of the preceding entries, characterized in that the biological treatment system (1) includes a connection arrangement (36) for aseptically connecting and / or disconnecting the container, preferably, the connection arrangement (36) includes a pipe welding unit (37) for welding pipes together and / or cutting and sealing the pipes, more preferably, the pipe welding unit (37) welds pipes to the bypass line (22) and / or to the media container (10) and / or to the waste container (18).

[0425] 16. The method according to item 15, characterized in that the bioreactor module (3) includes at least one tube retainer (38) holding at least one tube in a defined position relative to the support structure (19), and the tube welding unit (37) interacts with the tube retainer (38) to weld and / or cut and seal the tube, preferably, the tube retainer (38) includes a tube movement mechanism for extending and / or retracting the tube relative to the tube retainer (38).

[0426] 17. The method according to any one of the preceding entries, characterized in that the cell container (4) comprises a tubing assembly connected to and pre-sterilized together with the cell container (4), the tubing assembly comprising at least one disposable head for the pump (8), and / or at least one docking element for the sensor, particularly a magnetic docking element, and / or the recirculation loop (24), and / or a sealed and weldable connector for the media container (10), and / or a sealed and weldable connector for the waste container (18).

[0427] 18. A bioreactor module, particularly a bioreactor module for use in the method according to any one of the preceding entries, wherein the bioreactor module (3) comprises a cell container (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 the properties of the cell culture, wherein the cell container (4) comprises a main membrane (7). The bioreactor module (3) includes a control unit and / or an interface with the control system (2), wherein the control unit is adapted to After the cell culture is introduced into the cell container (4), the cells are subjected to at least one unit operation and amplification operation within the cell container (4), particularly the subsequent amplification operation, and Control commands are implemented during the unit operation and / or the amplification operation to cause the at least one active element (5) to affect the cell culture, thereby advancing the unit operation and / or the amplification operation, and / or wherein the interface with the control system (2) is adapted to allow the control system (2) to implement control commands to cause the at least one active element (5) to affect the cell culture, thereby advancing the unit operation and / or the amplification operation. Preferably, the control unit is adapted to receive sensor data from the at least one sensor (6) and derive control commands from the sensor data, and / or the interface with the control system (2) is adapted to allow the control system (2) to receive sensor data from the at least one sensor (6).

[0428] 19. The bioreactor module according to item 18, characterized in that the cell container (4) comprises a tubing assembly connected to and pre-sterilized together with the cell container (4), the tubing assembly comprising at least one disposable pump (8) head, and / or at least one docking element for a sensor, particularly a magnetic docking element, and / or the recirculation loop (24), and / or a sealed and weldable connector for the media container (10), and / or a sealed and weldable connector for the waste container (18).

[0429] 20. A biological treatment system, particularly a biological treatment system for use according to any one of entries 1 to 17, wherein the biological treatment system (1) comprises a control unit and a bioreactor module (3), the bioreactor module (3) comprising a cell container (4) for containing the cell culture, at least one active element (5) for influencing the cell culture in the bioreactor module (3), and at least one sensor (6) preferably for sensing the properties of the cell culture, wherein the cell container (4) comprises a main membrane (7), wherein the biological treatment system (1) is adapted to

[0430] After the cell culture is introduced into the cell container (4), the cells are subjected to at least one unit operation and an amplification operation within the cell container (4), particularly the subsequent amplification operation. The control system (2) is adapted to implement control commands during the unit operation and / or the amplification operation to cause the at least one active element (5) to affect the cell culture, thereby advancing the unit operation and / or the amplification operation.

[0431] In this application, when an element or component is described as being included in and / or selected from the list of elements or components described, it should be understood that the element or component may be any one of the elements or components described, or the element or component may be selected from two or more of the elements or components described.

[0432] Unless otherwise indicated herein or the context clearly contradicts it, the terms “a” and “an” as well as “the” and similar pronouns used in the context of describing the invention (especially in the context of the claims) should be interpreted to cover both the singular and plural forms.

[0433] Unless otherwise specifically stated or understood from the context, the use of the terms “comprising,” “including,” “having,” or “containing” in their grammatical equivalents should generally be understood as open-ended and non-restrictive, for example, not excluding other undescribed elements or steps.

[0434] Unless otherwise specified, the invention also includes the specific quantitative value itself when the terms "about" or "approximately" are used before a quantitative value. As used herein, unless otherwise indicated or inferred, the term "about" means a variation of ±10% relative to the nominal value.

[0435] As used herein, “and / or” refers to and covers any and all possible combinations of one or more of the relevant listed items, as well as combinations that are not combined when interpreted as optional (or). The use of optional words (e.g., “or”) should be understood to refer to one, two, or any combination of optional items.

[0436] All cited references are hereby incorporated into this paper by way of citation.

[0437] All individual implementations and aspects disclosed herein can be combined with each other within the framework and context of this disclosure. It should be understood that the implementations disclosed herein are merely exemplary, and any feature presented for a particular exemplary implementation can be used alone in this disclosure, or in combination with any feature presented for the same or another particular exemplary implementation, and / or in combination with any other features not mentioned. It should also be understood that any feature presented for an example implementation in a particular category can also be used in a corresponding manner in any other category of example implementations.

[0438] Example

[0439] It should be understood that the following examples are for illustrative purposes only and should not be construed as limiting the invention in any way. The following examples demonstrate that the methods according to this disclosure advantageously allow for the biological treatment of cells in cell cultures, particularly immune cell cultures containing T cells, including at least one unit operation. Specifically, it is shown that T cells can be transferred into a cell container and then activated and expanded within the same cell container, thereby enabling the simple, precise, and robust production of cell therapy products.

[0440] Example 1

[0441] Peripheral blood mononuclear cells (PBMCs) were thawed and isolated using a CD3+ StemCell Technologies kit, following the manufacturer's instructions. A subset of cells was activated in a culture flask for reference (referred to herein as "BRO2"), and another subset was used for the procedure according to this disclosure (referred to herein as "BRO3"). Additionally, cells derived from different donors (referred to herein as "BRO1") were activated according to the procedure of this disclosure.

[0442] In BRO1 and BRO3, cells were seeded in cell containers (50 mL, cell density 1.43 × 10⁻⁶) mounted on the bioreactor module. 6 / mL) and activated. In BRO2, cells were loaded at 1.43 × 10 6 Activate in a T-shaped culture flask (20 mL).

[0443] For the cultures (BRO1 / BRO3) according to this disclosure, the cells were agitated for 1 hour to ensure uniformity of the added activating agents and cell culture (rate 16 rpm, angle -4 / +4). The fluid lines were pre-filled with 6 mL of medium to ensure a representative volume. After 24 hours, cell activation was complete; the cells were agitated for 30 minutes to ensure uniform distribution (rate: 16 rpm, angle -4 / +4), then returned to a stationary position. After 48 hours, the cells were agitated at a rate of 16 rpm and an angle of -4 / +4.

[0444] Sixty-five hours after activation, the cells underwent three perfusion cycles to wash away the activating reagent (0.34 mL / min). For this purpose, the culture medium containing residual activating reagent was recovered as permeate through a membrane contained in the cell container (here, the reservoir within the cell container). Fresh cell culture medium was then added via a fluid line. This process was repeated accordingly until the activating reagent was substantially completely removed. From day 3 onwards, the cell density was set to 1 × 10⁻⁶ cells / year by dilution. 6 / mL, and cultured the cells in cell containers for 7 days (BRO2, BRO3) or 6 days (BRO1).

[0445] On day 6 or 7, the cell density was reset to 1×10⁶ by dilution with fresh cell culture medium. 6 / mL, and then culture the cells in the cell container for a period of 2 hours. Perfusion was then initiated and continued until day 13. This was advantageously accomplished using the reservoir membrane and / or host membrane disclosed herein. At the end of day 13, the cells were harvested.

[0446] like Figure 6 As shown, compared to BRO2, BRO3 not only resulted in a larger total cell number (p=0.0002, Figure 1) – as expected due to the different starting volume – but also resulted in a higher daily expansion fold. Therefore, by applying the method according to this disclosure and performing the cell expansion step, a larger cell number can be achieved in a shorter time, enabling more efficient production of activated T cells and thus better manufacturing of cell therapy products.

[0447] In addition, such as Figure 7As shown, BRO3 expansion exhibited a higher viable cell density (VCD) compared to BRO2 (p=0.01, Figure 1). No significant difference in VCD was observed during batch culture (p=0.84), but a significant difference was observed at the start of perfusion culture. Therefore, by applying the method according to this disclosure and performing the cell expansion steps, higher cell density and more viable cells were obtained. Consequently, production is not only more efficient and yields higher quantities, but also of higher quality, a crucial parameter in the manufacture of cell therapy products.

[0448] like Figure 14 As shown, amplification of BRO1 and BRO3 resulted in comparable cell numbers (Graph 1) and fold increases (Graph 2), with both experiments resulting in over 300-fold increases. Cell viability was also measured during the amplification period (Graph 3). Following cell activation, a significant decrease in viability was observed between day 0 and day 3, reaching approximately 50% in both runs. However, from day 3 onwards, cell viability showed a recovering trend, gradually increasing until stabilizing at approximately 95% from day 6, and remaining consistent throughout the observation period. Assessment of activation counts at the end of the activation period (Graphs 4 and 5) showed that over 80% of CD4 cells were activated in each run. + T cells and over 80% of CD8 + T cells.

[0449] This embodiment demonstrates that the method according to this disclosure exhibits improved cell activation and expansion results compared to known culture flask activation protocols. The results obtained by the method according to the invention are consistent across different donors and result in a high percentage of live, activated CD4. + T cells and CD8 + T cells.

[0450] Example 2

[0451] On day 0, Nutri-T medium (4 Cell) was used. ®The Nutri-T GMP PRF (Biological Industries) along with IL-2 (100 IU / mL) and TransAct (1:100 dilution) for in-bag T cell activation was used to seed CD3+ T cells at a concentration of approximately 1.43 million cells / mL (total approximately 70 million cells) in 50 mL cell containers. During activation, the cell containers were stationary for the first approximately 60 hours, with intermittent shaking at 24 and 48 hours. At approximately 60 hours, the activation reagent was automatically washed out of the cell containers via perfusion media exchange. On day 3, the media in the bioreactor was reduced to achieve a cell concentration of approximately 3 million cells / mL, and then the transfection reagent (GFP-mRNA liposomes) was introduced into the cell containers. The system was stationary for the first 4 hours of transfection, then media was added to achieve a cell concentration of 750,000 cells / mL. The system was shaken overnight, and at approximately 8 hours, the transfection reagent was washed out of the cell containers via perfusion media exchange. From day 4 to day 11, the cells underwent the following expansion process: from day 4 to day 7, the medium was added daily via a feed-by-batch process to achieve a cell concentration of 1 million cells / mL until the cell container reached a volume of 1 L; from day 7 to day 9, the medium was exchanged via a perfusion process at 0.5 container volumes / day (VVD), and from day 9 to day 11, it was exchanged at 1 VVD. Finally, on day 11, the cells were harvested from the cell containers. The following analyses were performed: flow cytometry for transfection efficiency and cell phenotyping, Nucleocounter for viability and cell counting, and Cedex Bio HT for metabolite analysis (glucose, lactate, LDH, ammonia).

[0452] One day after transfection, a transfection efficiency of approximately 60% was achieved, and within 11 days, a 70-fold expansion was achieved from 70 million CD3+ T cells to 5 billion CD3+ T cells, with a viability of approximately 94%.

[0453] Example 3

[0454] Peripheral blood mononuclear cells (PBMCs) from a single donor were thawed and isolated using the CD3+ StemCell Technologies kit, following the manufacturer's instructions. Aliquots were aspirated and analyzed by flow cytometry for the immune cell markers CD4 and CD8 before and after T cell isolation. A portion of the isolated cells was activated in a culture flask for reference (referred to herein as a "culture flask"), and another portion was used for the methods according to this disclosure (referred to herein as "BR1" and "BR2").

[0455] In BR1 and BR2, cells were seeded in cell containers (50 mL, cell density 1.43 × 10⁻⁶) mounted on the bioreactor module. 6 / mL) and activated. In culture flasks, cells were introduced at a rate of 1.43 × 10⁹ / mL. 6 Activate in a T-shaped culture flask (20 mL).

[0456] For cultures (BR1 / BR2) according to this disclosure, cells were agitated for 1 hour to ensure uniformity of the added activating agent and cell culture (rate 16 rpm, angle -4 / +4). Fluid lines were pre-filled with 6 mL of medium to ensure representative volume. After 24 hours, cell activation was complete; cells were agitated for 30 minutes to ensure uniform distribution (rate: 16 rpm, angle -4 / +4), then returned to a stationary position. After 48 hours, cells were agitated at 16 rpm, angle -4 / +4.

[0457] Sixty-five hours after activation, aliquots were taken from each culture for flow cytometry analysis of immune cell markers. In the culture, cells underwent three perfusion cycles to wash away the activation reagent (0.34 mL / min). For this purpose, the culture medium containing residual activation reagent was recovered as permeate through a membrane contained in the cell container (here, the reservoir within the cell container). Fresh cell culture medium was then added via a fluid line. This process was repeated until the activation reagent was substantially completely removed. From day 3 onwards, the cell density was set to 1 × 10⁻⁶ cells / year by dilution. 6 / mL, and the cells were cultured in cell containers for 6 days.

[0458] On day 6, the cell density was reset to 1×10⁶ by dilution with fresh cell culture medium. 6 / mL. At this stage, aliquots of all cultures were subjected to immunostaining and flow cytometry analysis of immunocellular markers. Culture in the flasks was then stopped, and BR1 and BR2 cells were cultured in cell containers for another 2 hours. Perfusion culture was then initiated. From day 6 to day 8, the medium was exchanged via perfusion at 0.5 container volumes / day (VVD), and from day 8 to day 12, at 1 VVD. On day 12, cells were harvested from the cell containers, and aliquots from both cultures were subjected to immunostaining and flow cytometry analysis.

[0459] Figure 15 This illustrates the various types (CD4+) of cells between those activated in culture flasks and those activated in bioreactors. + CD8 + CD4 - / CD8 - CD4 + / CD8 + The percentage of immune cells was almost identical, indicating that the method according to the invention does not introduce any particular bias in terms of the survival of any particular T cell population. As expected, by day 12 of culture, the growth of CD8+ cells exceeded that of the CD4+ population, consistent with common knowledge in this regard. Overall, the experiments demonstrate that T cell activation performed according to the method of the invention results in a favorable increase in the number of activated cells while maintaining the known ratio of activated T cell populations.

[0460] Example 4

[0461] On day 0, Nutri-T medium (4 Cell) was used. ® Nutri-T GMP PRF (Biological Industries) along with IL-2 (100 IU / mL) and TransAct (1:100 dilution) for in-bag T cell activation, seeded CD3+ T cells at 50 mL, approximately 1.43 million cells / mL (total approximately 70 million cells), in separate cell containers (BR1 and BR2). During the activation process, the cell containers were stationary for the first approximately 60 hours, with intermittent shaking at 24 and 48 hours. At approximately 60 hours, the activation reagents were automatically washed out of the cell containers via media exchange. In BR1, according to a preferred embodiment of the invention, exchange was performed by perfusion at a rate of 0.5 mL per minute for 10 hours (total exchange: 300 mL). In BR2, according to one embodiment of the invention, the exchange is performed by running six exchange cycles, each of which consists of the following: first, 0.5 mL is removed per minute for 50 minutes (total removal: 25 mL), then 25 mL of fresh culture medium is added, followed by shaking for 30 minutes (total exchange volume: 150 mL).

[0462] In parallel, the control batch was inoculated into T25 culture flasks and activated under the same conditions, according to the manufacturer's operating procedures.

[0463] On day 3, the medium in the container / culture flask was reduced to achieve a cell concentration of approximately 3 million cells / mL, and then the transfection reagent (GFP-mRNA liposome, 0.27 µg mRNA / million cells) was introduced into the cell container. The container was continuously shaken for the first 4 hours of transfection, and then medium was added to achieve a cell concentration of 750,000 cells / mL. The system was shaken overnight, and after approximately 8 hours, the transfection reagent was washed out of the cell container via perfusion medium exchange. From day 4 to day 7, medium was added daily via a feed-batch process to achieve a cell concentration of 1 million cells / mL until a volume of 1 L was reached in the cell container. Flow cytometry was performed on days 0, 4, and 7 to assess transfection efficiency and cell phenotyping.

[0464] Figure 16 A shows that on day 4, cell container BR1 contained >80% GFP-expressing cells, indicating a transfection efficiency of >80%, which is suitable and preferred for biotechnology production processes. On day 4, cell container BR2 contained >50% GFP-expressing cells, indicating a transfection efficiency of >50%, which is sufficient for many processes. The control sample of cells transfected in culture flasks contained approximately 100% GFP-positive cells, while the untransfected negative control (cells in culture flasks) did not contain GFP-positive cells as expected. On day 7, only the percentage of GFP-positive cells in BR1 was determined and shown as approximately 70%.

[0465] Figure 16 B shows the T cell population (CD4+) across different methods (BR1, BR2, cells transfected in culture flasks, and cells not transfected in culture flasks). + / CD8 - CD4 - / CD8 + CD4 - / CD8 - CD4 + / CD8 + The percentages are highly similar, which further highlights that the method of the present invention will not adversely affect any particular T cell population.

[0466] Experiments have shown that the method according to the invention achieves high transfection efficiency when transfected directly within the infusion container of the invention. In some embodiments of the invention, the transfection efficiency can be further improved.

[0467] Example 5

[0468] As described in Example 2 above, CD3+ T cells were seeded in a bioreactor and activated. Twenty-four hours after activation, the cells were transduced to express recombinant chimeric antigen receptor (CAR) by adding lentivirus and a transduction enhancer (MOI: 10) to the culture. Untransduced cultures were cultured in parallel. From day 2 to day 7, fresh cell culture medium was added daily via a feed-batch process to achieve a cell concentration of 1 million cells / mL until a volume of 1 L was reached in the cell container. Starting from day 7, the medium was exchanged at 1 VVD via a perfusion process. Starting from day 4 (i.e., 72 hours post-transfection), aliquots were taken daily for analysis of total cell count and viability. It was found that the total cell count and cell viability were comparable between transduced and untransduced cells, with transduced cells achieving approximately 180-fold expansion throughout the culture period. Furthermore, approximately 30% of the T cells expressed CAR, and the ratio of CAR-expressing T cells to non-CAR-expressing cells remained consistent (see [link to relevant documentation]). Figure 17 ).

[0469] This embodiment illustrates that the method according to the invention allows for the reliable production of large quantities of CAR T cells. The method enables the transition from artificial plate culture settings to a large-scale process that is at least partially automated.

[0470] While the embodiments represent an implementation of this disclosure using activation as a unit operation and subsequent amplification steps, different other steps or different implementations, such as gene modification, can also be implemented in an automated manner. Therefore, at least one unit operation and amplification step can be performed in various different sequences.

Claims

1. A method for biologically treating cells in a cell culture, wherein the cell culture is present in a cell container (4), wherein the method includes - Perform one or more unit operations on the cells in an automated manner, wherein the unit operations include: i. Add the liquid composition to the cell container (4); ii. Incubate the cells in the cell container (4) together with the added liquid composition; as well as iii. Remove liquid from the cell container (4) through at least one filter, thereby removing at least a portion of the added liquid composition; as well as - The cells are amplified in the cell container (4), wherein the amplification is performed before and / or after the one or more unit operations.

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 activator; and - Gene modification of the cells, particularly transduction and / or transfection, preferably wherein the liquid composition comprises a gene modifier, particularly a transducer and / or transfectant.

3. The method according to claim 1 or 2, wherein the method includes performing more than one unit operation, preferably wherein... - The method includes at least two unit operations, more preferably wherein the at least two unit operations include activating the cell and modifying its genes, or - The method includes at least three unit operations, more preferably wherein the at least three unit operations include activating the cell and two gene modifications, 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) Activator; (ii) Gene modifiers, preferably transducers or transfectants, and optionally enhancers; or (iii) Activators and gene modifiers, preferably transducers or transfection agents, and optionally enhancers.

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

6. The method of claim 5, wherein the washing step comprises perfusion, preferably wherein at least 1, 2, 3, 5, or 10 times the volume of the cell culture is exchanged, and more preferably wherein the perfusion rate 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 includes performing more than one unit operation, preferably wherein performing more than one unit operation includes: - Repeat steps i. to iii. once or more; - Add a liquid composition containing reagents for performing more than one unit operation to the cell container (4), preferably the liquid composition containing an activator and a gene modifier; - Before step ii. and / or before step iii., step i. is repeated by adding other liquid compositions to the cell container (4), particularly wherein (i) The liquid composition in step i. contains an activator, and (ii) The other liquid compositions contain a gene-modifying agent, and / or - Before step ii. and / or before step iii., step i. is repeated by adding other liquid compositions to the cell container (4), particularly wherein (i) The liquid composition in step i. contains a gene-modifying agent, and (ii) The other liquid compositions contain an activator.

8. The method according to one or more of claims 1 to 7, wherein the method comprises measuring cell density once or multiple times, preferably at least before step I, and optionally, wherein the method comprises, in step i, automatically adding an appropriate volume of liquid composition to the cell container (4) based on the measured cell density, particularly wherein the liquid composition comprises an activator and / or a gene modifier such that the volume of the liquid composition is suitable for the activation and / or gene modification of the cells, preferably being adjusted for the activation and / or gene modification of the cells.

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

10. The method according to one or more of claims 1 to 9, wherein the method further comprises washing the cells one or more times after performing the unit operation, preferably wherein washing includes: i. Add the washing liquid composition to the cell container (4); ii. Optionally, the cells are incubated in the cell container (4) with added washing fluid; as well as iii. Remove fluid from the cell container (4) through at least one filter, thereby removing at least a portion 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 and expanding the cells; and / or - Formulate the cell culture into a cell therapy product.

12. The method according to one or more of claims 1 to 11, wherein the method comprises using a biological processing 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 container (4) in step i. and / or remove the liquid from the cell container (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 container (4) comprises two or more compartments, preferably small-volume compartments (13) and different large-volume compartments (14), optionally wherein - The cell container (4) comprises a pre-formed reservoir (15) forming small volume compartments (13); and / or - The cell container (4) includes a shell (16) forming large volume compartments (14), wherein the shell is preferably flexible.

14. The method according to claim 13, wherein the method has the following characteristics: - During the unit operation, the cell culture is located in the small volume compartment (13); and / or - During the amplification of the cells in the cell container (4), preferably after step iii, the volume of the cell culture is increased such that the cell culture is preferably located in the small volume compartment (13) and the large volume compartment (14).

15. The method of claim 13 or 14, wherein one or more of the at least two compartments comprises at least one filter, and / or is fluidly connected to at least one filter, optionally wherein - The small volume compartment (13) contains at least one filter and / or is fluidly connected to at least one filter, and / or - The large volume compartment (14) contains at least one filter and / or is fluidly connected to at least one filter.

16. The method according to one or more of claims 1 to 15, wherein the cell is a mammalian cell, preferably an immune cell or a primary cell.

17. The method according to one or more of claims 1 to 16, wherein the method includes cell selection, preferably prior to performing the unit operation, optionally wherein the cell selection includes: - Add a mixture of cells to the cell container (4). - The mixture of cells is brought into contact with the binding unit, wherein at least a portion of the cells bind directly or indirectly to the binding matrix of the binding unit, and - Remove liquid from the cell container (4) to remove at least some cells that are not directly or indirectly bound to the binding matrix.

18. A bioreactor module for bioprocessing cells in a cell culture, wherein the bioreactor module (3) comprises - Cell containers (4), and - At least one filter for removing liquid from the cell container (4) through the at least one filter. The bioreactor module is configured to perform one or more unit operations on the cells in the cell container (4) in an automated manner, and to amplify the cells in the cell container (4), optionally wherein the bioreactor module further comprises one or two of the following: - At least one active element (5) for influencing the cell culture in the bioreactor module (3), preferably for adding fluid to and / or removing fluid from the cell container (4), preferably wherein the fluid is a liquid; and - At least one sensor (6) for sensing the characteristics of the cell culture.

19. A bioprocessing system for biologically processing cells in a cell culture, wherein the bioprocessing system (1) comprises a 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 cell therapy products, preferably wherein the manufacturing comprises at least one of the following: - Optionally, cells are selected once or multiple times, particularly by binding at least a portion of cells expressing the ligand, preferably a subset of T cells; - Activates cells, preferably T cells; - Transduced cells, preferably transduced T cells; - Transfected cells, preferably T cells; and / or - Expand cells, preferably T cells, and more preferably genetically modified T cells.

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

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