Cell enrichment method and cell enrichment device for use in automated bioreactors

Abstract

To provide cassettes for use in automated cell engineering systems that include cell concentration filters for reducing fluid volume of a cell sample during or following automated processing, and automated cell engineering systems.SOLUTION: A cassette comprises: (a) a cell culture chamber; (b) a pump fluidly connected to the cell culture chamber; (c) a tangential flow filter fluidly connected to the pump, wherein the pump provides a retentate flow to the tangential flow filter, wherein a permeate flow of the tangential flow filter is controlled by a flow controller; (d) a satellite volume connected to the tangential flow filter, contained in the cassette; (e) a fluid pathway for recirculating the retentate flow back through the tangential flow filter; (f) a fixed volume waste collection chamber fluidly connected to the tangential flow filter; and (g) a cellular sample output fluidly connected to the tangential flow filter.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] This disclosure provides a cassette for use in an automated cell handling system, comprising a cell concentration filter for reducing the fluid volume of a cell sample during or after automated processing. The disclosure further provides a method for concentrating a cell population, and an automated cell handling system capable of utilizing the cassette and performing the method. [Background technology]

[0002] As expectations for the accelerated clinical application of advanced cell therapies grow, attention is shifting to the foundational manufacturing strategies that can enable such treatments to benefit patients worldwide. While cell therapies hold great clinical promise, their high manufacturing costs relative to rewards pose a significant barrier to commercialization. Therefore, there is a growing need for cost-effectiveness, process efficiency, and product consistency, leading to a focus on automation in many cell therapy fields.

[0003] The production of therapeutic cell populations involves the automation of various processes. Automation includes integrating cell activation, transduction, and expansion into commercially available manufacturing platforms, enabling the application of these important therapies to a wide range of patient populations.

[0004] It is often necessary to reduce the volume of a cell population either during automated processing or before the final output from an automated system. What is needed is a process that can concentrate the cell sample; that is, to reduce the sample volume either during automation or before sample output.

[0005] [Overview of the prefecture] In some embodiments, the Specified Specification provides a cassette for use in an automated cell handling system, the cassette comprising a cell culture chamber, a pump system fluid-connected to the cell culture chamber, a tangential flow filter fluid-connected to the pump system, the pump system delivering a retenate flow to the tangential flow filter, the permeate flow of the tangential flow filter being controlled by a flow controller, and a cell sample output fluid-connected to the tangential flow filter.

[0006] In further embodiments, a cassette for use in an automated cell manipulation system is provided herein, comprising a cell culture chamber, a pump system fluid-connected to the cell culture chamber, a tangential flow filter fluid-connected to the pump system, the pump system sending a concentrated flow to the tangential flow filter, the osmotic flow of the tangential flow filter being controlled by a flow controller, a satellite volume connected to the tangential flow filter, a fluid path for recirculating the concentrated flow back to and passing through the tangential flow filter, a constant volume waste collection chamber fluid-connected to the tangential flow filter, and a cell sample output fluid-connected to the tangential flow filter.

[0007] In additional embodiments, the Specified herein provides a method for reducing the volume of a cell sample during automated processing, the method comprising introducing the cell sample into a tangential flow filter having a concentration flow and an osmotic flow, the osmotic flow being controlled by a flow controller, passing the cell sample through the concentration flow of the tangential flow filter, removing a volume from the cell sample via the osmotic flow to a constant volume waste collection chamber, and collecting the reduced-volume cell sample.

[0008] In further embodiments, an automated cell handling system is provided herein, comprising a sealable housing, a cassette contained within the sealable housing, the cassette comprising a cell culture chamber, a pump system fluid-connected to the cell culture chamber, a tangential flow filter fluid-connected to the pump system, the pump system sending a concentrated flow to the tangential flow filter, the osmotic flow of the tangential flow filter being controlled by a flow controller, the cassette comprising a cell sample output fluid-connected to the tangential flow filter, and a user interface for receiving input from a user. [Brief explanation of the drawing]

[0009] [Figure 1] Figure 1 shows various steps that may be performed in a cassette of an automated cell manipulation system as described in embodiments of this specification. [Figure 2A] Figure 2A shows an exemplary cassette according to an embodiment of this specification. [Figure 2B] Figure 2B shows an exemplary tangential flow filter for use in the cassettes, systems, and methods described herein. [Figure 2C] Figure 2C shows an exemplary flow controller for use with the tangential flow filters described herein. [Figure 3] Figures 3A and 3B show images of an automated cell manipulation system according to the embodiments described herein. [Figure 4] Figure 4 shows a laboratory space including an exemplary cell manipulation system described in the embodiments of this specification. [Figure 5] Figure 5 shows the flow path for cell enrichment in a cassette of an automated cell manipulation system described in embodiments of this specification. [Figure 6] Figures 6A-6B show the effect of serum on tangential flow filtration according to embodiments of this specification. [Figure 7]Figures 7A–7C illustrate the use of permeation control to reduce clogging of a tangential flow filter, according to embodiments of this specification. [Figure 8] Figures 8A-8B show the volume reduction of peripheral blood mononuclear cells (PBMCs) using tangential flow filtration according to embodiments of this specification. [Figure 9] Figures 9A–9D illustrate the optimization of the infiltration pump during tangential flow volume reduction of PMBC according to embodiments of this specification. [Figure 10A] Figure 10A shows cell recovery after tangential flow filtration according to an embodiment of this specification. [Figure 10B] Figure 10B shows cell viability before and after tangential flow filtration according to embodiments of this specification. [Figure 11] Figure 11 shows CD4+ and CD8+ expression in the control cell suspension and TFF cell suspension. [Modes for carrying out the invention]

[0010] Please be aware that the specific embodiments shown and described herein are illustrative and not intended to limit the scope of this application in any way.

[0011] Published patents, patent applications, websites, company names, and scientific literature referenced herein are incorporated herein by reference in the same degree as each is specifically and individually indicated as being incorporated by reference. In the event of any conflict between any reference cited herein and any particular teaching herein, the latter shall prevail. Similarly, in the event of any conflict between the definition of a word or phrase as understood in the art and the definition of a word or phrase specifically taught herein, the latter shall prevail.

[0012] As used herein, the singular forms "a," "an," and "the" specifically include the plural forms of the terms to which they refer, unless the context clearly dictates otherwise. The term "about" is used herein to mean approximately, around, roughly, or generally. When the term "about" is used in conjunction with a numerical range, the term modifies the range by extending the boundaries above and below the recited numerical values. In general, the term "about" is used herein to modify a recited value above and below the value by up to a 20% difference.

[0013] Technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this application pertains, unless otherwise defined. References are made herein to various techniques and materials known to those of ordinary skill in the art.

[0014] In several embodiments, the present specification provides a cassette for use in an automated cell manipulation system. FIG. 1 shows an exemplary cassette 102, in which various processes are carried out in a sealed automated system, and various cell samples and cell populations can be produced. Such processes may include steps of activation, transduction, expansion, concentration, washing, and collection / harvesting.

[0015] As described herein, the cassette and method are utilized and implemented in a fully enclosed automated cell manipulation system 300 (see FIGS. 3A, 3B), which preferably has instructions for carrying out steps such as activation, transduction, expansion, concentration, and collection. A cell manipulation system for automated manufacturing, such as for gene-modified immune cells including CAR T cells, is described in U.S. Patent Application 16 / 119,618, filed Aug. 31, 2018 (the disclosure of which is incorporated herein by reference in its entirety), and is also referred to herein as an automated cell manipulation system, the COCOON™, or the COCOON™ system.

[0016] For example, a user can provide an automated cell manipulation system pre-filled with cell cultures and reagents (e.g., activation reagents, vectors, cell culture media, nutrients, selection reagents, etc.), and parameters for cell production (e.g., starting cell number, type of media, type of activation reagent, type of vector, number of cells or dosage to be produced, etc.). The automated cell manipulation system can perform various automated methods, including methods for producing genetically modified immune cell cultures such as CAR T cells, without requiring further input from the user. In some embodiments, a fully enclosed automated cell manipulation system minimizes contamination of the cell culture fluid by reducing exposure of the cell culture to a non-sterile environment. In additional embodiments, a fully enclosed automated cell manipulation system minimizes contamination of the cell culture by reducing the user's handling of the cells.

[0017] As described herein, the automated cell manipulation system 300 preferably includes a cassette 102. Thus, in multiple embodiments, this specification provides cassettes for use in an automated cell manipulation system. As used herein, "cassette" refers to a large, self - contained, removable and replaceable element of an automated cell manipulation system, which includes one or more chambers for performing various elements of the methods described herein, and preferably further includes one or more of cell culture media, activation reagents, and washing media.

[0018] Figure 2A shows an exemplary cassette 102 for use in an automated cell manipulation system. In multiple embodiments, the cassette 102 includes a cell sample input 202 shown as a vial or chamber in Figure 2A, and the cell sample can be placed prior to introduction or loading into the cassette 102. In other embodiments, the cell sample input 202 can simply be a sterile lock - type tube (e.g., a luer - lock type tube connection), to which a syringe or a cell - containing bag such as a blood bag can be connected.

[0019] Cassette 102 further includes a cell culture chamber 206. Features and examples of use of the cell culture chamber 206 are described herein. Cassette 102 also includes a pump system 520 fluidly connected to the cell culture chamber 206 (see Figure 5 for an exemplary arrangement in the flow path).

[0020] As used herein, “fluidically connected” means that one or more components of a system, such as components of cassette 102, are connected via suitable elements that allow fluid (including gases and liquids) to pass between the components without leakage or volume reduction. Examples of fluid connections include various tubes, channels, and connections known in the art, such as silicone or rubber tubes and Luer lock connections. It should be understood that fluidically connected components may also include additional elements between each component while maintaining the fluid connection. That is, fluidically connected components may include additional elements, and as a result, fluid passing between components may also pass through these additional elements, although this is not mandatory.

[0021] Pump system 520 is preferably a peristaltic pump system, but other pump systems can also be used. A peristaltic pump is a type of positive displacement pump used to pump fluid. The fluid is contained within a flexible tube fitted inside the pump frame—often annular. The rotor compresses the flexible tube, along with many “rollers,” “shoes,” “wipers,” or “lobes” mounted on the outer circumference of the rotor. As the rotor rotates, the compressed section of the tube is squeezed and closed (or “blocked”), and the fluid is consequently pumped through the tube. After passing through a cam, the tube opens (“recovers” or “restores”), and the fluid flow is guided to the pump. This process is called peristalsis and is used to pass fluid through a flexible tube. Typically, there are two or more rollers or wipers that block the tube, trapping a large amount of fluid between them. The large amount of fluid is then transferred to the pump’s drain port.

[0022] Cassette 102 further includes a tangential flow filter 204 fluid-coupled to the pump system. Figure 2B shows an exemplary tangential flow filter. Figure 2C shows a schematic diagram of the inside of the tangential flow filter. Tangential flow filtration, also known as cross-flow filtration, is a filtration system or process in which a supply, injection, or inflow fluid flow (250 in Figure 2C) passes parallel to the membrane surface, with some of it being discharged through the membrane (osmotic flow - 252 in Figure 2C), and the remainder passing through the membrane (concentrating flow - 254 in Figure 2C), where it is recirculated and returned to the input for concentration, and finally moved to a storage section or output.

[0023] The tangential flow filter 204 is preferably composed of a series of hollow fiber membranes (single fibers may also be used), into which a solution is supplied. The concentrated flow passes through the hollow fibers, and the cells are retained in the solution inside the fiber membrane. Meanwhile, the excess volume passes through the fiber membrane and is discharged into the osmotic flow. This reduces the total volume of the cell sample and concentrates the cell sample. The membranes are preferably supplied in the form of a self-contained device, which may include a flow controller 258.

[0024] As described herein, in relation to Figure 2C, the pump system 520 delivers a concentrated flow 254 to the tangential flow filter 204 while controlling the permeation flow 252 of the tangential flow filter with a flow controller 258. As used herein, “flow controller” refers to a fluid engineering or other mechanism, including valves, shrinkage devices, flow dividers, pump mechanisms, various tubing configurations, for controlling the amount of fluid flowing from the fiber membrane of the tangential flow filter into the permeation flow. The flow controller 258 in Figure 2C is presented simply to illustrate the integration of a mechanism for controlling the amount of permeation flow 252 and does not represent the structure of this mechanism.

[0025] In exemplary embodiments, the flow controller 258 is a flow restrictor 260. “Flow restrictor” refers to a valve, a gradually narrowing tube, or a shrinking device for controlling the amount and rate of seepage flow 252 exiting the tangential flow filter. The flow restrictor 260 is located downstream of the tangential flow filter 204. As a result, control of the seepage flow occurs after it has exited the membrane of the tangential flow filter 204. The flow restrictor 260 is shown in Figure 2C for illustrative purposes only, and the arrangement and mechanism of the flow restrictor 260 are not limited to the representation in Figure 2C. Those skilled in the art will readily recognize various ways in which a flow restrictor can be used to control the amount and rate of seepage flow 252. The flow restrictor 260 is preferably located adjacent to the end 262 of the tangential flow filter 204 (see Figure 2B) to limit the amount and rate of seepage flow 252.

[0026] In a further embodiment, the flow controller 258 is an additional pump system that can be configured to control and limit (or increase) the amount and rate of the seepage flow 252.

[0027] In further embodiments, the flow controller 258 is a system having multiple tubes that can be directed and arranged within the cassette 102, providing desired control (limitation or increase) with respect to the amount and velocity of the seepage flow 252.

[0028] In several embodiments, the cassette 102 further includes one or more fluid pathways appropriately connected to the cell culture chamber (see the interior of the cassette 102 in Figure 2A). The cassette 102 also includes a cell sample output 208 fluid-connected to the cell culture chamber. Preferably, the cassette 102 further includes a cell sample output 208 fluid-connected to a tangential flow filter 204.

[0029] As described herein, the cell sample output 208 can be used to collect cells after various automated procedures for further processing, storage, or potentially use in patients. The cell sample output 208 may also be the sample port 220 as described herein, which allows cell samples to be removed from the cassette for transduction, such as by electroporation, and then returned to the cassette for further automated processing. Examples of fluid pathways include various tubes, channels, capillaries, and microfluidic elements that provide nutrients or solutions to elements of the cassette, as described herein. Alternatively, the cell sample output 208 may simply be the output of a tangential flow filter, which is then fluidically connected to another section or part of the cassette 102 as described herein.

[0030] In several embodiments, cassette 102 explicitly eliminates centrifugation before or after the tangential flow filter 204. Using the various cell separation filters and methods described herein, it has been determined that additional cell separation via centrifugation steps or the use of a centrifuge is not essential. However, in several embodiments, additional filtration systems, such as column filtration and / or magnetic filtration systems, may be utilized.

[0031] In exemplary embodiments, the tangential flow filter 204 includes a membrane having a pore size of approximately 0.40 μm to approximately 0.80 μm and a fiber diameter of approximately 0.5 mm to approximately 0.9 mm. In some embodiments, the pore size of the tangential flow filter 204 is approximately 0.2 μm to approximately 1.0 μm, or approximately 0.3 μm to approximately 0.9 μm, approximately 0.4 μm to approximately 0.8 μm, approximately 0.5 μm to approximately 0.7 μm, approximately 0.6 μm to approximately 0.7 μm, or approximately 0.40 μm, approximately 0.45 μm, approximately 0.50 μm, approximately 0.55 μm, approximately 0.60 μm, approximately 0.65 μm, approximately 0.70 μm, approximately 0.75 μm, or approximately 0.80 μm. In several embodiments, the fiber diameter is approximately 0.30 mm to approximately 1.2 mm, preferably approximately 0.40 mm to approximately 1.0 mm, approximately 0.50 mm to approximately 0.90 mm, approximately 0.60 mm to approximately 0.80 mm, approximately 0.70 mm to approximately 0.80 mm, or approximately 0.60 mm, approximately 0.65 mm, approximately 0.70 mm, approximately 0.75 mm, approximately 0.80 mm, approximately 0.85 mm, or approximately 0.90 mm.

[0032] The tangential flow filter 204 preferably contains about 15 to 20 fibers, preferably 18 fibers, with a total fiber lumen length of about 10 to 20 cm, preferably about 10 to 15 cm, or about 13 cm. The surface area of ​​the fibers is about 40 to 70 cm². 2 A width of approximately 50-60 cm is more preferable. 2 With a width of approximately 57 cm. 2 In several embodiments, films with a relatively large surface area and large pore size are desirable for use in the tangential flow filter 204.

[0033] Examples of materials for use in the tangential flow filter 204 include, but are not limited to, polymers such as poly(ethersulfone), poly(acrylonitrile), and poly(vinylidene difluoride), cellulose esters, and poly(sulfone). Examples of tangential flow filters include filters available from SPECTRUM LABS®, including MICROKROS® and MIDIKROS® filters, as well as modifications thereof to fit inside a desired cassette. In several embodiments, the material is modified poly(ethersulfone).

[0034] In further embodiments, the surface of the tangential flow filter may be coated. Preferably, the coating can help reduce or eliminate fouling on the surface of the tangential flow filter 204. Examples of non-fouling coatings include phospholipid coatings, polymer coatings such as poly(vinyl alcohol) (PVA) and poly(ethylene glycol) coatings. Additional surface coatings may be applied to the tangential flow filter to provide stability, increased or decreased flow, or other desirable properties.

[0035] In further embodiments, additional pre-filters and post-filters (i.e., before or after tangential flow filters) may be used in the cassettes and methods described herein. For example, a magnetic separation process may be used to further remove and separate undesirable cells and residues from the cell population. In such embodiments, magnetic beads or other structures can be bound to biomolecules (e.g., antibodies, antibody fragments, etc.) and interact with target cells. The target cell population may then be separated from any undesirable cells, residues, etc. that may be present in the cell sample using various magnetic separation methods, including the use of filters, columns, flow tubes, or channels with a magnetic field. For example, the target cell population may flow through a tube or other structure and be exposed to a magnetic field, thereby holding or retaining the target cell population with the magnetic field, allowing undesirable cells and residues to pass through the tube. The magnetic field may then be turned off, allowing the target cell population to pass through a further holding chamber or other area of ​​the cassette for further automated processing. Additional filtration may include conventional column filtration or the use of other filtration membranes or filtration structures.

[0036] In a further embodiment, the cassette 102 further includes a constant-volume waste collection chamber 510 fluid-connected to the tangential flow filter 204. The constant-volume waste collection chamber 510 is used to collect the osmotic flow 252 that has exited the tangential flow filter. By utilizing a constant volume, the constant-volume waste collection chamber is permitted to hold only a predetermined amount of collected osmotic flow 252. Once this predetermined amount of osmotic flow 252 is reached, no additional osmotic flow 252 is permitted to exit the tangential flow filter 204, and consequently, the volume of the cell sample does not decrease further. As a result, a cell concentration and cell sample volume with predetermined and known values ​​are obtained, for example, a predetermined value to meet the final objective or a predetermined value for further processing of a predetermined volume. Examples of the constant-volume waste collection chamber 510 include various rigid plastics, metals, etc., that do not expand and therefore only hold a constant volume. In addition, bags or flexible plastics may be used, which are placed inside a rigid plastic container or between immovable walls (e.g., plastic walls) so that when the bag reaches a predetermined volume, it hits the immovable wall or container and stops expanding. When the constant volume wastewater collection chamber 510 is filled to its maximum volume, no further seepage flow 252 can escape, and thereafter the concentrated flow 254 simply recirculates through a tangential flow filter until collection is desired. This recirculation preferably occurs via a fluid path (i.e., collectively shown as 540 in the flow path of Figure 5). The constant volume wastewater collection chamber 510 may further include a level monitor that guides and directs the stopping of the seepage flow 252 and the recirculation of the concentrated flow 254.

[0037] In an additional embodiment, the satellite volume 550 is fluid-coupled to the tangential flow filter 204, which can provide additional storage capacity to the cassette to increase the overall volume of the automated process, or provide additional volumetric flow to the tangential flow filter. An exemplary arrangement of the satellite volume 550 is shown in the flow path of Figure 5.

[0038] The cassette may further include one or more fluid pathways (collectively 540) that provide recirculation, waste removal, uniform gas phase exchange, and nutrient distribution to various parts of the cassette, including the cell culture chamber, without disturbing the cells in the cell culture chamber. The cassette 102 further includes one or more valves 522 or 552 that control the flow-through of the various fluid pathways (see Figure 5 for an exemplary arrangement in the flow paths).

[0039] In exemplary embodiments, as shown in Figure 2A, the cell culture chamber 206 is a flat, inflexible chamber (i.e., made from a substantially inflexible material such as plastic, for example) and does not easily bend or flex. By using an inflexible chamber, the cells are maintained in a substantially undisturbed state. As shown in Figure 2A, the cell culture chamber 206 is oriented so that the immunotherapy cell culture is spread across the entire bottom of the cell culture chamber. As shown in Figure 2A, it is preferable that the cell culture chamber 206 is maintained in a position parallel to the floor or table so that the cell culture is maintained in an undisturbed state and can be spread across a large area of ​​the bottom of the cell culture chamber. In some embodiments, the overall thickness (i.e., the height of the chamber) of the cell culture chamber 206 is small, with a width of about 0.5 cm to about 5 cm. The cell culture chamber preferably has a volume of about 0.50 ml to about 300 ml, more preferably about 50 ml to about 200 ml, or the cell culture chamber has a volume of about 180 ml. A low chamber height (less than 5 cm, preferably less than 4 cm, less than 3 cm, or less than 2 cm) allows for efficient exchange of culture medium and gases close to the cells. The ports are configured to allow mixing via fluid recirculation without disturbing the cells. A higher fixed vessel height generates a concentration gradient, which can result in limited oxygen and fresh nutrients in areas close to the cells. By controlling the flow dynamics, culture medium can be exchanged without disturbing the cells. Culture medium can be removed from an additional chamber (where no cells are present) without the risk of cell loss.

[0040] As described herein, in exemplary embodiments, the cassette is pre-filled with one or more of the following, including any combination thereof: cell culture, culture medium, preferably cell washing medium, activation reagent, and / or vector. In further embodiments, these various elements may be added later via a suitable injection port or the like.

[0041] As described herein, in several embodiments, the cassette preferably further includes one or more of the following: pH sensor 524, glucose sensor (not shown), oxygen sensor 526, carbon dioxide sensor (not shown), lactate sensor / monitor (not shown), and / or optical density sensor (not shown). See Figure 5 for an exemplary arrangement in the flow path. The cassette may also include one or more sampling ports and / or injection ports. Examples of such sampling ports 220 and injection ports 222 are shown in Figure 2A, and an exemplary arrangement in the flow path is shown in Figure 5, and may include an access port to connect the cartridge to an external device, such as an electroporation unit or an additional medium supply source. Figure 2A further shows the arrangement of an input 202, a reagent warming bag 224 which may be used to warm cell medium, etc., and a second chamber 230.

[0042] In several embodiments, the cassette 102 preferably includes a low-temperature chamber containing a refrigerated area 226 suitable for storing cell culture media, and preferably includes a high-temperature chamber suitable for performing activation, transduction, and / or expansion of cell cultures. The high-temperature chamber is preferably separated from the low-temperature chamber by a heat-shielding wall. As used herein, “low-temperature chamber” refers to a chamber maintained below room temperature, more preferably about 4°C to about 8°C, for the maintenance of cell culture media and the like at refrigerated temperatures. The low-temperature chamber may include a bag or other holder for the medium containing about 1 L, about 2 L, about 3 L, about 4 L, or about 5 L of fluid. Additional medium bags or other fluid sources may be externally connected to the cassette and connected to the cassette via an access port.

[0043] As used herein, “high-temperature chamber” refers to a chamber preferably maintained at a temperature higher than room temperature, more preferably at a temperature at which cells can proliferate and grow, i.e., about 35-40°C, and more preferably about 37°C. In some embodiments, the high-temperature chamber preferably includes a cell culture chamber 206 (also referred to throughout the specification as a proliferation chamber or cell proliferation chamber).

[0044] In several embodiments, it is preferable that the tangential flow filters 204 are appropriately arranged in the cassette 102 at an angle of approximately 3° to approximately 20° with respect to the horizontal, more preferably at an angle of approximately 5° to approximately 15° or approximately 10° with respect to the horizontal (the outlet end of the tangential flow filter 204 is positioned above / higher than the inlet end). Arranging the tangential flow filters 204 at an angle with respect to the horizontal, with the outlet end (i.e., 262) of the tangential flow filter above the inlet end, is desirable for providing desirable flow characteristics for improving volume reduction and cell enrichment through the tangential flow filter 204.

[0045] By arranging tangential flow filters at an angle of approximately 3° to 20° relative to the horizontal, the benefit of potentially reducing or avoiding cell priming (or gravity sedimentation) is also offered. Using such angles allows cells to roll off the suspension as they flow down the tangential flow filters.

[0046] In several embodiments, the cassette 102 may further include a cell washing system 512, which is contained within the cassette 102 (i.e., within the structure shown in Figure 2A) and is preferably fluid-coupled to the tangential flow filter 204, or coupled to other sections within the cassette depending on whether cell washing is desired. In several embodiments, the cell washing system 512 is a container or bag contained within the cassette 102 and preferably contains a cell washing medium. It is preferable to use the cell washing medium to clean a desired cell population, remove any undesirable waste cells or contaminants, and then transfer the cell population inside or outside the cassette for further processing or use. The cell washing system 512 may be contained outside the cassette 102.

[0047] Cassette 102 may optionally include a cell retention chamber 516 (located inside cassette 102 and therefore not visible in Figure 2). Figure 5 shows an exemplary arrangement of the cell retention chamber 516 in the flow path of the cassette. The cell retention chamber 516 is preferably a container or suitable chamber located within the cassette, in which a population of cells may be retained before or after tangential flow filtration as described herein.

[0048] In an additional embodiment, a cassette 102 for use in an automated cell handling system 300 is provided herein, which preferably comprises a cell culture chamber 206, a pump system 520 fluid-coupled to the cell culture chamber, and a tangential flow filter 204 fluid-coupled to the pump system. As described herein, the pump system delivers a concentrated flow to the tangential flow filter, and the osmotic flow of the tangential flow filter is controlled by a flow controller. The cassette further includes a satellite volume 550 coupled to the tangential flow filter, a fluid path 540 for recirculating the concentrated flow back to and passing through the tangential flow filter, a constant volume waste collection chamber 510 fluid-coupled to the tangential flow filter, and a cell sample output 208 fluid-coupled to the tangential flow filter.

[0049] Examples of pore sizes and fiber diameters for use in the tangential flow filter 204 are described herein. In several embodiments, the tangential flow filter has pore sizes ranging from about 0.40 μm to about 0.80 μm and fiber diameters ranging from about 0.5 mm to about 0.9 mm, including pore sizes ranging from about 0.60 μm to about 0.70 μm and fiber diameters ranging from about 0.70 mm to about 0.80 mm.

[0050] Suitable materials for use in tangential flow filters include, but are not limited to, polymers such as poly(ethersulfone), poly(acrylonitrile), and poly(vinylidene difluoride).

[0051] In exemplary embodiments, the cassette 102 further includes one or more fluid pathways, in which case the fluid pathways provide recirculation, wastewater removal and uniform gas phase exchange, and nutrient distribution to the cell culture chamber without disturbing the cells in the cell culture chamber. In some embodiments, the cell culture chamber is a flat, inflexible chamber with a low chamber height.

[0052] As described herein, the cassette 102 may further include one or more of a pH sensor, a glucose sensor, an oxygen sensor, a carbon dioxide sensor, and / or an optical density sensor, and may include one or more sampling ports.

[0053] In several embodiments, the tangential flow filter is positioned with the cassette 102 at an angle of approximately 3° to 20° with respect to the horizontal.

[0054] As described herein, a flow controller may be a flow restrictor, an additional pump system, a system having multiple tubes, or a combination of such controllers.

[0055] Figures 3A–3B show the COCOON® automated cell manipulation system 300, with the cassette 102 located inside (in Figure 3B, the lid of the automated cell manipulation system is open). An exemplary user interface is also shown, which may include the ability to receive input using a barcode reader and a touchpad or other similar device.

[0056] The automated cell manipulation systems and cassettes described herein preferably have three related volumes: cell culture chamber volume, working volume, and total volume. The working volume used in the cassette is preferably in the range of 180 mL to 460 mL depending on the processing step, and may be increased to a maximum of about 500 mL, about 600 mL, about 700 mL, about 800 mL, about 900 mL, or about 1 L. In some embodiments, the cassette can easily be 4 × 10 9 cells ~10×10 9 Cells can be obtained. The cell concentration during the process is 0.3 × 10⁻⁶. 6 Cells / ml ~ approximately 10 x 10 6The volume changes to cells / ml. The cells are placed in the cell culture chamber, but the culture medium is continuously recirculated through additional chambers (e.g., cross-flow vessels and satellite volumes) to increase the working volume as described herein.

[0057] Fluid pathways, including gas exchange lines, may be fabricated from gas-permeable materials such as silicone. In some embodiments, the automated cell handling system recirculates oxygen throughout the chamber, which is substantially unproductive during the cell preparation method. Therefore, in some embodiments, the oxygen level of the cell culture in the automated cell handling system is higher than that of the cell culture in a flexible gas-permeable bag. Higher oxygen levels may be important in the cell culture expansion process, and higher oxygen levels may support greater cell growth and proliferation.

[0058] In further embodiments, a method for reducing the volume of a cell sample during automated processing is provided herein. The method provided herein is described in relation to the flow path in Figure 5. The figure is for illustrative purposes only and should not be considered to limit the means by which the method may be carried out. For example, the cell sample may be introduced into the cassette 102 via the input 202. In other embodiments, the cell sample may already be in the cassette 102 after, for example, a transduction or cell expansion step in the cell culture chamber 206. The cell sample is introduced into the tangential flow filter 204 by passing, for example, through the valve V11 250. The tangential flow filter has a concentration flow 254 and an osmotic flow 252 (see Figure 2C). As described herein, the osmotic flow 252 is controlled by a flow controller 258 to provide a desired cell concentration and volume reduction. The cell sample passes through the concentration flow 254, while volume is removed from the cell sample via the osmotic flow 252. The osmotic flow 252 is preferably removed to a constant volume waste collection chamber 510 by passing through valves v1 and v13 (valve v13 may be removed if necessary). Once the desired volume reduction is achieved, the reduced volume cell sample is collected by moving it to output 208, preferably through valves V1 and V10. In other embodiments, the reduced volume cell sample may be collected, for example, in a cell retention chamber 516, after which further automated processing may be performed, or it may be removed from the cassette.

[0059] As described herein, it is preferable to recirculate the concentration flow 254 after the volume removal step and to repeatedly pass the cell sample through the concentration flow 254. For example, the concentration flow 254 may be discharged from the tangential flow filter 204, pass through valves V1, V12 and V11, and return to the tangential flow filter 204.

[0060] In embodiments utilizing a constant-volume waste collection chamber 510, when a certain volume of waste is reached, the cell sample returns to and passes through the tangential flow filter (for example, through valves V14, V12, and V11). However, no additional volume is removed because volume removal stops appropriately when the desired volume enters the constant-volume waste collection chamber.

[0061] In an additional embodiment, after the initial collection of cell samples, the samples may be washed using a cell washing system 512, and then the volume reduction method may be repeated. The cell washing system 512 is connected to a cell holding chamber 516, for example via valve V4, and the washing solution can be pushed into the holding chamber by closing valves V12 and V11.

[0062] The methods described herein may further include additional steps, such as electroporating the cell sample after collection following tangential flow filtration. This additional step may be performed internally (i.e., using cassette 102) or via an external electroporation system. Additional transduction steps may be performed after collection following tangential flow filtration.

[0063] When described herein, the method preferably utilizes a flow controller, which may be a flow restrictor, an additional pump system, a system having multiple tubes, or a combination of such controllers.

[0064] In several embodiments, the methods and cartridges described herein are utilized on the COCOON® platform (Octane Biotech, Kingston, Ontario), which incorporates multiple unit operations in a single turnkey platform. Multiple cell protocols with highly specialized cell processing targets are provided. To provide efficient and effective automated conversion, the described methods utilize the concept of application-specific / sponsor-specific disposable cassettes that combine multiple unit operations—all focused on the core requirements of the final cell therapy product. Multiple automated cell processing systems 300 may be combined and integrated into a large multi-unit operation to produce large quantities of cell samples for individual patients or multiple diverse cell samples (see Figure 4).

[0065] Figure 5 also shows an exemplary arrangement of one or more fluid paths 540, appropriately including various sensors (e.g., pH sensor 524, dissolved oxygen sensor 526), ​​as well as sampling / sample ports and various valves (including bypass check valve 552), and silicone tubular components connecting the components. As described herein, the use of silicone tubular components enables oxygenation through the tubular components, promoting gas transport and optimal oxygenation for cell culture. Figure 5 also shows the use of one or more hydrophobic filters 554 or hydrophilic filters 556 in the cassette's flow path.

[0066] In additional embodiments, an automated cell manipulation system 300 is provided herein. As shown in Figures 3A and 3B, the automated cell manipulation system 300 preferably includes a sealable housing 302 and a cassette 102 contained within the sealable housing. As used herein, “sealable housing” means a structure that can be opened and closed, in which the cassette 102 described herein may be placed and may be integrated with various components such as fluid supply lines, gas supply lines, power supplies, cooling connections, and heating connections. As shown in Figures 3A and 3B, the sealable housing can be opened to allow insertion of the cassette (Figure 3B) and can be closed to maintain a closed, sealed environment so that the various automated processes described herein can be carried out using the cassette (Figure 3A).

[0067] As described herein, the cassette 102 preferably includes a cell culture chamber 206, a pump system 520 fluid-coupled to the cell culture chamber, and a tangential flow filter 204 fluid-coupled to the pump system. As described herein, the pump system delivers a concentrated flow to the tangential flow filter, and the osmotic flow of the tangential flow filter is controlled by a flow controller. The cassette 102 preferably further includes a cell sample output 208 fluid-coupled to the tangential flow filter.

[0068] As shown in Figures 3A-3B, the automated cell manipulation system 300 further includes a user interface 304 for receiving input from the user. The user interface 304 may be a touchpad, tablet, keyboard, computer terminal, or other suitable interface, and the user interface allows the user to input desired controls or criteria into the automated cell manipulation system and control the automated process and flow path. Preferably, the user interface is connected to a computer control system and issues commands to the automated cell manipulation system to control the overall activity of the automated cell manipulation system. Such commands may include opening and closing various valves, providing culture medium or cell populations, raising or lowering the temperature, etc.

[0069] Exemplary features of the pore size and fiber diameter of the tangential flow filter 204 for use in automated cell manipulation systems are described herein, and in several embodiments, the tangential flow filter has a pore size of about 0.40 μm to about 0.80 μm and a fiber diameter of about 0.5 mm to about 0.9 mm, preferably a pore size of about 0.60 μm to about 0.70 μm and a fiber diameter of about 0.70 mm to about 0.80 mm. Suitable polymers for use in tangential flow filters are described herein, including poly(ethersulfone), poly(acrylonitrile), and poly(vinylidene difluoride).

[0070] In several embodiments, the cassette of the automated cell manipulation system further comprises a constant-volume waste collection chamber 510 fluid-coupled to the tangential flow filter 204. In several embodiments, the cassette 102 of the automated cell manipulation system 300 further comprises one or more fluid pathways 540, in which case the fluid pathways provide recirculation, waste removal and uniform gas phase exchange, and nutrient distribution to the cell culture chamber 206 without disturbing the cells in the cell culture chamber. In several embodiments, the cell culture chamber is a flat, inflexible chamber with a low chamber height. Fluid pathways may be included for the concentrated flow to recirculate and pass back to the tangential flow filter. The cassette may also include a satellite volume 550 fluid-coupled to the tangential flow filter.

[0071] In several embodiments of the automated cell handling system, the cassette 102 is pre-filled with culture medium, cell washing medium, etc. In several embodiments as described herein, the cassette of the automated cell handling system may further include one or more of a pH sensor 524, a glucose sensor, an oxygen sensor 526, a carbon dioxide sensor, and / or an optical density sensor, and in a preferred embodiment, it may also include one or more sampling ports.

[0072] Examples of flow controllers are described herein, including flow restrictors, additional pumping systems, and systems having multiple tubes. In several embodiments, the tangential flow filter is located within a cassette at an angle of about 3° to about 20° with respect to the horizontal.

[0073] Automating unit operations in the manufacturing of cell therapy products offers an opportunity to deliver universal benefits to allogeneic and autologous cell therapy applications. The advantages of automation are particularly compelling in the unique circumstances of patient-specific autologous cell products, and, more importantly, in the significant micro-lot complexity related to GMP compliance, economics, patient traceability, and early detection of processing deviations in small batches. While the emergence of associated complex manufacturing protocols is noteworthy, the thorough integration of automated unit operations in micro-lot cell product manufacturing has not been a primary research topic. However, given the anticipated demand for these therapies immediately following approval, implementing a fully enclosed, end-to-end system can provide many much-needed solutions to manufacturing bottlenecks, such as workload and installation space.

[0074] Developers of advanced medical treatments are encouraged to consider automation early in the development of clinical interpretations and the scaling up of clinical trial protocols. Early automation impacts protocol development, eliminates the need for comparability testing when switching from manual to automated processes later, and deepens understanding of long-term commercialization plans.

[0075] In exemplary embodiments, the automated cell manipulation system described herein comprises a plurality of chambers, in which each step of the various methods described herein is carried out in a different chamber of the plurality of chambers of the automated cell manipulation system, and before the method is started, the activating reagent, vector, and cell culture medium are each contained in a different chamber of the plurality of chambers, in which at least one of the plurality of chambers is maintained at a cell growth temperature (e.g., about 37°C), and at least one of the plurality of chambers is maintained at a refrigeration temperature (e.g., about 4-8°C).

[0076] In several embodiments, the automated cell manipulation systems described herein are monitored using temperature sensors, pH sensors, glucose sensors, oxygen sensors, carbon dioxide sensors, and / or optical density sensors. Thus, in some embodiments, the automated cell manipulation system includes one or more of the temperature sensors, pH sensors, glucose sensors, oxygen sensors, carbon dioxide sensors, and / or optical density sensors. In additional embodiments, the automated cell manipulation system is configured to adjust the temperature, pH, glucose, oxygen level, carbon dioxide level, and / or optical density of the cell culture according to a predetermined culture size. For example, if the automated cell manipulation system detects that the current oxygen level of the cell culture is too low to reach the growth required for the desired cell culture size, the automated cell manipulation system automatically increases the oxygen level of the cell culture, for example, by introducing oxygenated cell culture medium, by exchanging the cell culture medium with oxygenated cell culture medium, or by circulating the cell culture medium through an oxygenation component (i.e., a silicone tube). In another embodiment, if the automated cell manipulation system detects that the current temperature of the cell culture is too high and that the cells are growing rapidly (for example, if the cells are potentially overcrowded, which may result in undesirable properties), the automated cell manipulation system automatically lowers the temperature of the cell culture to maintain a constant cell growth rate (or a logarithmic growth rate, if desired). In yet another embodiment, the automated cell manipulation system automatically adjusts the cell feeding schedule (i.e., providing fresh medium and / or nutrients to the cell culture) based on the cell growth rate and / or cell number, or other monitoring factors such as pH, oxygen, glucose, etc. The automated cell manipulation system may be configured to store the medium (and other reagents, such as washing solutions, etc.) in a low-temperature chamber (e.g., 4°C or -20°C), and then warm the medium in a room-temperature chamber or a high-temperature chamber (e.g., 25°C or 37°C, respectively) before introducing the warmed medium into the cell culture.

[0077] Additional exemplary embodiments Embodiment 1 is a cassette for use in an automated cell manipulation system, the cassette comprising a cell culture chamber, a pump system fluid-connected to the cell culture chamber, a tangential flow filter fluid-connected to the pump system, the pump system sending a concentrated flow to the tangential flow filter, the osmotic flow of the tangential flow filter being controlled by a flow controller, and a cell sample output fluid-connected to the tangential flow filter.

[0078] Embodiment 2 includes the cassette described in Embodiment 1, wherein the tangential flow filter has a pore size of about 0.40 μm to about 0.80 μm and a fiber diameter of about 0.5 mm to about 0.9 mm.

[0079] Embodiment 3 includes the cassette described in Embodiment 2, wherein the tangential flow filter has a pore size of about 0.60 μm to about 0.70 μm and a fiber diameter of about 0.70 mm to about 0.80 mm.

[0080] Embodiment 4 includes a cassette according to any one of Embodiments 1 to 3, wherein the tangential flow filter comprises a polymer selected from the group consisting of poly(ethersulfone), poly(acrylonitrile), and poly(vinylidene difluoride).

[0081] Embodiment 5 includes the cassette according to any one of Embodiments 1 to 4, further comprising a constant volume waste liquid collection chamber fluidly connected to the tangential flow filter.

[0082] Embodiment 6 includes the cassette according to any one of Embodiments 1 to 5, further comprising a fluid path for recirculating the concentrated flow back to the tangential flow filter and passing it through.

[0083] Embodiment 7 includes a cassette according to any one of Embodiments 1 to 6, further comprising a satellite volume fluid-connected to the tangential flow filter.

[0084] Embodiment 8 further comprises one or more fluid pathways, in which case the fluid pathways include a cassette according to any one of Embodiments 1 to 7, which provides recirculation, wastewater removal, and homogeneous gas phase exchange and nutrient distribution to the cell culture chamber without disturbing the cells in the cell culture chamber.

[0085] Embodiment 9 includes a cassette according to any one of Embodiments 1 to 8, wherein the cell culture chamber is a flat, inflexible chamber with a low chamber height.

[0086] Embodiment 10 includes a cassette according to any one of Embodiments 1 to 9, further comprising one or more of a pH sensor, a glucose sensor, an oxygen sensor, a carbon dioxide sensor, and / or an optical density sensor.

[0087] Embodiment 11 includes the cassette described in any one of Embodiments 1 to 10, further comprising one or more sampling ports.

[0088] Embodiment 12 includes a cassette according to any one of the embodiments, wherein the tangential flow filter is at an angle of about 3° to about 20° with respect to the horizontal.

[0089] Embodiment 13 includes a cassette according to any one of Embodiments 1 to 12, wherein the flow controller is a flow restrictor.

[0090] Embodiment 14 includes a cassette according to any one of Embodiments 1 to 13, wherein the flow controller is an additional pump system.

[0091] Embodiment 15 includes a cassette according to any one of Embodiments 1 to 14, wherein the flow controller is a system having a plurality of tubes.

[0092] Embodiment 16 is a cassette for use in an automated cell manipulation system, the cassette comprising a cell culture chamber, a pump system fluid-connected to the cell culture chamber, a tangential flow filter fluid-connected to the pump system, the pump system sending a concentrated flow to the tangential flow filter, the osmotic flow of the tangential flow filter being controlled by a flow controller, a satellite volume connected to the tangential flow filter, a fluid path for recirculating the concentrated flow back to and passing through the tangential flow filter, a constant volume waste collection chamber fluid-connected to the tangential flow filter, and a cell sample output fluid-connected to the tangential flow filter.

[0093] Embodiment 17 includes the cassette described in Embodiment 16, wherein the tangential flow filter has a pore size of about 0.40 μm to about 0.80 μm and a fiber diameter of about 0.5 mm to about 0.9 mm.

[0094] Embodiment 18 includes the cassette described in Embodiment 17, wherein the tangential flow filter has a pore size of about 0.60 μm to about 0.70 μm and a fiber diameter of about 0.70 mm to about 0.80 mm.

[0095] Embodiment 19 includes a cassette according to any one of Embodiments 16 to 18, wherein the tangential flow filter comprises a polymer selected from the group consisting of poly(ethersulfone), poly(acrylonitrile), and poly(vinylidene difluoride).

[0096] Embodiment 20 further comprises one or more fluid pathways, in which case the fluid pathways provide recirculation, wastewater removal, and homogeneous gas phase exchange and nutrient distribution to the cell culture chamber without disturbing the cells in the cell culture chamber, and includes the cassette described in any one of Embodiments 16 to 19.

[0097] Embodiment 21 includes a cassette according to any one of Embodiments 16 to 20, wherein the cell culture chamber is a flat, inflexible chamber with a low chamber height.

[0098] Embodiment 22 includes a cassette according to any one of Embodiments 16 to 21, further comprising one or more of a pH sensor, a glucose sensor, an oxygen sensor, a carbon dioxide sensor, and / or an optical density sensor.

[0099] Embodiment 23 includes a cassette according to any one of embodiments 16 to 22, further comprising one or more sampling ports.

[0100] Embodiment 24 includes a cassette according to any one of Embodiments 16 to 23, wherein the tangential flow filter is at an angle of approximately 3° to approximately 20° with respect to the horizontal.

[0101] Embodiment 25 includes a cassette according to any one of Embodiments 16 to 24, wherein the flow controller is a flow restrictor.

[0102] Embodiment 26 includes a cassette according to any one of Embodiments 16 to 25, wherein the flow controller is an additional pump system.

[0103] Embodiment 27 includes a cassette according to any one of Embodiments 16 to 26, wherein the flow controller is a system having a plurality of tubes.

[0104] Embodiment 28 is a method for reducing the volume of a cell sample during automated processing, the method comprising introducing the cell sample into a tangential flow filter having a concentration flow and an osmotic flow, the osmotic flow being controlled by a flow controller, passing the cell sample through the concentration flow of the tangential flow filter, removing a volume from the cell sample via the osmotic flow to a constant volume waste collection chamber, and collecting the reduced-volume cell sample.

[0105] Embodiment 29 further includes the method of Embodiment 28, wherein after the volume removal step, the concentrated flow is recirculated to repeatedly pass the cell sample through the concentrated flow.

[0106] Embodiment 30 includes a method according to any one of Embodiments 28 to 29, wherein the volume removal stops when a desired volume is entered into a certain volume waste liquid collection chamber.

[0107] Embodiment 31 further includes the method according to any one of Embodiments 28 to 30, further comprising washing the cell sample after collection and repeating steps (a) to (d) of the method.

[0108] Embodiment 32 further comprises the method of any one of Embodiments 28 to 31, further comprising electroporating the cell sample after collection.

[0109] Embodiment 33 includes the method according to any one of Embodiments 28 to 32, wherein the flow controller is a flow restrictor.

[0110] Embodiment 34 includes the method according to any one of Embodiments 28 to 33, wherein the flow controller is an additional pump system.

[0111] Embodiment 35 includes the method according to any one of Embodiments 28 to 34, wherein the flow controller is a system having a plurality of tubes.

[0112] Embodiment 36 is an automated cell manipulation system comprising a sealable housing, a cassette contained within the sealable housing, the cassette comprising a cell culture chamber, a pump system fluid-connected to the cell culture chamber, a tangential flow filter fluid-connected to the pump system, the pump system sending a concentrated flow to the tangential flow filter, the osmotic flow of the tangential flow filter being controlled by a flow controller, the cassette comprising a cell sample output fluid-connected to the tangential flow filter, and a user interface for receiving input from a user.

[0113] Embodiment 37 includes the automated cell manipulation system described in Embodiment 36, wherein the tangential flow filter has a pore size of about 0.40 μm to about 0.80 μm and a fiber diameter of about 0.5 mm to about 0.9 mm.

[0114] Embodiment 38 includes the automated cell manipulation system described in Embodiment 37, wherein the tangential flow filter has a pore size of about 0.60 μm to about 0.70 μm and a fiber diameter of about 0.70 mm to about 0.80 mm.

[0115] Embodiment 39 includes an automated cell manipulation system according to any one of Embodiments 36 to 38, wherein the tangential flow filter comprises a polymer selected from the group consisting of poly(ethersulfone), poly(acrylonitrile), and poly(vinylidene difluoride).

[0116] Embodiment 40 includes an automated cell manipulation system according to any one of embodiments 36 to 39, further comprising a constant-volume waste collection chamber fluidly connected to the tangential flow filter.

[0117] Embodiment 41 includes an automated cell manipulation system according to any one of Embodiments 36 to 40, further comprising a fluid path for recirculating the concentrated flow back to and passing through the tangential flow filter.

[0118] Embodiment 42 includes an automated cell manipulation system according to any one of Embodiments 36 to 41, further comprising a satellite volume fluidly connected to the tangential flow filter.

[0119] Embodiment 43 further comprises one or more fluid pathways, in which case the fluid pathways provide recirculation, wastewater removal, and homogeneous gas phase exchange and nutrient distribution to the cell culture chamber without disturbing the cells in the cell culture chamber, and includes the automated cell manipulation system according to any one of Embodiments 36 to 42.

[0120] Embodiment 44 includes an automated cell handling system according to any one of Embodiments 36 to 43, wherein the cell culture chamber is a flat, inflexible chamber with a low chamber height.

[0121] Embodiment 45 includes an automated cell manipulation system according to any one of Embodiments 36 to 44, further comprising one or more of a pH sensor, a glucose sensor, an oxygen sensor, a carbon dioxide sensor, and / or an optical density sensor.

[0122] Embodiment 46 includes an automated cell manipulation system according to any one of embodiments 36 to 45, further comprising one or more sampling ports.

[0123] Embodiment 47 includes an automated cell manipulation system according to any one of Embodiments 36 to 46, wherein the tangential flow filter is at an angle of about 3° to about 20° with respect to the horizontal.

[0124] Embodiment 48 further comprises a computer control system, the user interface of which is connected to the computer control system and issues commands to the automated cell manipulation system, the automated cell manipulation system according to any one of embodiments 36 to 47.

[0125] Embodiment 49 includes an automated cell manipulation system according to any one of Embodiments 36 to 48, wherein the flow controller is a flow restrictor.

[0126] Embodiment 50 includes an automated cell manipulation system according to any one of Embodiments 36 to 48, wherein the flow controller is an additional pump system.

[0127] Embodiment 51 includes an automated cell manipulation system according to any one of Embodiments 36 to 48, wherein the flow controller is a system having a plurality of tubes. [Examples]

[0128] Example 1 - Tangential flow filtration in the COCOON® system Tangential flow filtration (TFF) can be used for cell therapy applications to separate, purify, recover, and collect cells from a harvested suspension for subsequent formulation. The conventional TFF process consists of two steps: 1) volume reduction and 2) diafiltration. During the volume reduction step, the bulk volume (cells in collection reagent and culture medium) is repeatedly removed via filtration through the permeation surface of the filter until the desired cell concentration is reached in the processing bag. During diafiltration, the solution of the concentrated cell suspension is replaced with formulation buffer, and undesirable residual proteins and contaminants in the final solution are reduced to acceptable levels. The final cell suspension has a formulation-ready cell concentration and buffer. Tangential flow filters are preferred over standard filters because they can reduce fluid volume while preventing clogging and cell damage. Cell recovery is also easier because the cells are not pressed against the filter.

[0129] Because the TFF filter is single-use and disposable, it can be easily mounted in a cassette and operated in a sealed and automated manner. Since cell therapy products cannot ultimately be sterilized or filtered, a completely sealed system allows the process to be carried out in a sterile condition. Being a completely disposable system eliminates the risk of secondary contamination and reduces the need for washing. To enhance the functionality of COCOON®, the cassette is provided with an integrated tangential flow filter. This embodiment details the development of a TFF system for cell concentration and washing in an automated system for cell therapy applications.

[0130] method Tangential flow filtration in COCOON® cassettes TFF systems for cell enrichment often have two pumps: one that controls the feed flow rate and another that controls the osmotic (i.e., waste) flow rate. The flow rate of each pump is typically determined based on optimizing the intermembrane pressure. If the pressure difference is too large or too small, the system may be useless, with nothing passing through the filter, or clogging may occur. COCOON® generally operates with a single pump and does not have a pressure sensor, so conventional filtration methods via TFF are not applicable.

[0131] The experiments were conducted using a TFF filter mounted in either a COCOON® cassette or a cassette-like pathway. As described herein, the cassette pathway includes an expansion chamber for cell culture, a satellite bag or L-type chamber for cell processing, TFF for removing excess medium, and a waste liquid bag for collecting excess medium. The COCOON® cassette preferably recirculates up to 450 mL of culture medium in its culture chamber. 260 cm 2Additional culture medium volumes exceeding the maximum 180 mL volume of the growth chamber are supplied from various satellite containers in the COCOON® cassette. These additional culture media from satellite containers can be recycled within the culture area of ​​the disposable cassette to provide fresh nutrients and remove waste products originating from the cells in the growth chamber.

[0132] Floral restrictors were used in the infiltration line to create a pressure difference. Based on experimental optimization, an ideal infiltration flow rate was selected to avoid clogging, maximize cell recovery, and minimize the time for volume reduction. In parallel, a wide range of filters were examined to understand the effects of fiber diameter, fiber area, fiber number, total surface area, cell type, concentration flow rate, pore size, and filter material.

[0133] Fixed volume waste liquid container In some experiments, a fixed-volume waste container was also used. Typically, the cassette has a flexible waste bag placed inside the fluid container. This bag expands to have a maximum volume that can completely drain the satellite bag and, under certain circumstances, completely drain the TFF. By completely draining the filter, cells are trapped on the membrane of the filter, resulting in irreversible loss of cells. To limit the maximum volume of the drain bag, the waste bag may be held between two rigid layers of fixed, separated plastic in the fluid container. The bag is filled to a certain volume, at which point the pressure inside the bag continues recirculation through the satellite bag / TFF, and no further fluid is sent to the waste container. Custom filters for enriching peripheral blood mononuclear cells (PBMCs)

[0134] Early cell enrichment experiments revealed desirable characteristics for tangential flow filters, such as large surface area and large pore size. The Spectrum Labs P-OCTA01-04-N filter is a custom-designed filter that meets these requirements and fits inside the Cocoon cassette. Its characteristics include:

[0135] mPES membrane

[0136] Fiber diameter = 0.75

[0137] Pore size = 0.65 μm

[0138] Number of fibers = 18

[0139] Lumen = 13 cm in total length

[0140] Surface area = 57 cm 2

[0141] This filter was evaluated, optimized, and then used in a proof-of-concept electroporation integration experiment.

[0142] Reduction of TFF volume using a custom filter Initial experiments with the custom filter were conducted without using COCOON™. Using a KrosFlo® Research 2i TFF System (Spectrum Labs), the feed, concentration, permeation, and transmembrane pressures during cell processing were monitored. Only one pump was utilized to control the flow rate of the feed line (unless otherwise stated) to mimic the function of the COCOON™ device. A 20-gauge, 0.024” I.D. / 0.036” O.D. Nordson EFD flow restrictor was attached to the end of the permeation line to mimic a previously optimized TFF procedure. By using this system, a 100 mL cell suspension was concentrated to 10 - 20 mL. TFF was performed at room temperature on the bench. The transmembrane pressure is defined as follows:

[0143]

Number

[0144] PBMC culture 1x10 8 individual PBMCs were at 1x10 8Cells were stimulated with individual CD3+:CD28+ Dynabeads (Invitrogen) and expanded for up to 10 days in complete T cell medium consisting of X-VIVO 15 medium (Lonza) supplemented with 5% human serum A / B (Sigma) and 10 ng / mL IL-2 (Peprotech) using multiple GREX 100 (Wilson Wolf) culture tubes. To accommodate the high viscosity serum that could clog filters, the serum concentration was first reduced using a pre-washing protocol with COCOON®, followed by volume reduction using the TFF process. Cells at validation concentrations were transferred to 250 mL conical vials and centrifuged or incubated in a 37°C incubator with 5% CO2 and humidified air for 2–4 hours. The supernatant of the incubated cell suspension was reduced to 10 mL, and the excess supernatant was discarded. Appropriate medium was added to the concentrated cell suspension to a final volume of 100 mL.

[0145] analysis Dual counting was performed using a Nucleocounter NC-200 (Chemometec) on pre-diluted cell cultures, diluted cultures, and final concentration cell suspensions. Volume was measured before and after TFF using a serum pipette and KrosFlo scale. Remaining validation samples were obtained from the initial cultures before dilution, the supernatant before TFF, and the final concentrated cell suspension after TFF. The percentage of serum remaining after dilution and concentration was determined using a human serum ELISA kit (Bethyl Laboratories). FACS analysis was performed on control cells and TFF concentrated cell suspensions for CD4+ and CD8+ expression.

[0146] The experimental success of the TFF volume reduction was defined as follows:

[0147] Cell recovery rate after TFF ≥ 85%

[0148] Cell viability after TFF decreased by ≤10%.

[0149] The residual human serum after initial enrichment following TFF (for electroporation experiments) is ≤10%.

[0150] result Evaluation of tangential flow filters A wide variety of filters were tested to understand the effects of various filter parameters. Fiber diameter, fiber area, fiber number, total surface area, concentration flow rate, pore size, and filter material all affected the filter's effectiveness in reducing the volume of the cell suspension. The results were also influenced by the solution (e.g., type of medium and type of serum) as well as the cell type (i.e., size), cell number, cell concentration, and the final target volume. Hydrostatic pressure also had an effect, so the flora restrictor had to be adjusted according to the hydrostatic pressure. Human mesenchymal stem cells (hMSCs) were used as the cell type tested in most experiments.

[0151] To accommodate variability in the amount of permeation flow, a fixed-volume, inflexible waste container was used. For example, if 100 mL needed to be removed from the total volume, a waste container of exactly 100 mL was used. The flow duration could be set based on the slowest permeation flow. A bypass loop was placed on either side of the pump tubing along with an in-line high-pressure check valve. If the waste container filled before the pumping time was complete, the bypass line was moved to force the fluid into the circle, thus terminating the TFF process. This method resulted in a very consistent flow rate into the waste container, as shown in Table 1. For further control, a level sensor may be incorporated into COCOON® to monitor the fluid level in the inflexible container.

[0152] [Table 1]

[0153] Evaluation and optimization of custom tangential flow filters The desired conditions were revealed through the verification results of various tangential flow filters. The custom filter Spectrum Labs P-OCTA01-04-N met these specifications, but verification and optimization were necessary. The inventors wanted to confirm that the filter functioned correctly and to first isolate all limitations of the COCOON® system. Therefore, the inventors evaluated the filter using the Spectrum Labs TFF system.

[0154] Cell-free experiments were initiated to obtain the initial operating parameters of the filter. When the volume of RPMI medium decreased, a constant intermembrane pressure (TMP) and flow through the filter were present (Figure 6A). However, when serum was added to RMMI, the TMP increased and the flow decreased over time (Figure 6B). This indicates that the filter was becoming clogged with proteins in the serum.

[0155] To control clogging by serum, either an automated back pressure valve (Figures 7A and 7B) or a second pump (Figure 7C) was added to the osmosis line. The automated back pressure valve could control the osmotic pressure after 3 minutes of volume reduction. The second pump controlled the osmotic flow rate initially to 20 ml / min and then to 10 ml / min after 5.5 minutes. In both cases of osmotic control, the flow, osmotic pressure, and TMP remained nearly constant. These results suggest that filter clogging can be controlled in COCOON® by controlling the pressure on the TFF osmosis line.

[0156] A similar trend was observed in the volume reduction of serum-free PBMC suspensions (Figures 8A and 8B). Adding a back pressure control valve to the osmotic flow helped stabilize the flow, osmotic pressure, and TMP. This further confirmed the need for osmotic flow control.

[0157] Process parameters are optimized to obtain the maximum cell recovery rate without significantly losing viability. The first parameter examined is osmotic pressure via the osmotic flow control pump. While concentrating a PMBC+0% serum suspension, the recirculation pump was set to 60 mL / min and the osmotic flow control pump was set to either 0, 5, 10, or 15 mL / min (Figures 9A-9D). The speed of the osmotic flow pump appeared to have little effect on the flow rate or osmotic pressure. 15 mL / min was chosen for subsequent experiments as it resulted in the shortest TFF time.

[0158] The recirculation flow rate was also investigated. PBMCs in 0% serum suspension were concentrated by TFF using an osmotic flow pump at 15 mL / min and either a recirculation flow rate of 60 mL / min or 70 mL / min (Table 2). Cell recovery was higher at a flow rate of 70 mL / min.

[0159] [Table 2]

[0160] PBMCs in 0% serum suspension were concentrated by TFF using a flora restrictor on the osmosis line and a recirculation flow rate of 70 mL / min (Table 3). The average recovery rate was approximately 89%, and the viability was higher than 80%.

[0161] [Table 3]

[0162] Many cell therapies use serum, and in some cases, it may not be possible to remove serum before TFF. PBMCs in a 5% serum suspension were concentrated by TFF using a flora restrictor on the osmosis line and a recirculation flow rate of 70 mL / min (Table 4). The average recovery rate was approximately 86%, and viability was higher than 80%.

[0163] [Table 4]

[0164] Cell enrichment in COCOON® cassette via TFF for electroporation Cell washing and concentration are useful not only before downstream processes of the product, but can also be utilized in semi-automated processes for specific unit operations, such as electroporation. It is preferable that cells be concentrated to <10 mL and any residues washed away before being added to the electroporation unit. For proof of concept, cells derived from two donors were measured in 4.4 x 10⁶ units. 8 and 4.2x10 8 Cell viability was concentrated by precipitation in a 10 mL volume using a total viable cell sample. These two cell suspensions were then diluted with 90 mL of supplemental Nucleofector® solution (NFS) and concentrated to 10 mL using TFF. Cell recovery rates after TFF concentration were 92% and 87% (Figure 10A). Cell viability before transfection was 92% and 74%, and decreased to less than 5% after TFF (Figure 10B).

[0165] In both experiments, 6% and 8% of the initial culture supernatant were detected in the final TFF-enriched cell suspension (Table 5).

[0166] [Table 5]

[0167] There was no difference in the CD4+:CD8+ profile after TFF compared to the control culture that was not enriched by TFF (Figure 11).

[0168] These results demonstrate the efficacy of TFF in washing and concentrating cells prior to transfection in the production process.

[0169] conclusion Washing and concentration via tangential flow filtration may preferably be carried out using the COCOON® system. The TFF allows the process to be performed in a sealed state, automatically, and within the area of ​​a COCOON® disposable cassette. The TFF can concentrate the cell suspension to less than 20 ml through the system, and cells can be recovered with a concentration of over 85%.

[0170] Those skilled in the art will see that other suitable modifications and adaptations to the methods and applications described herein can be made without departing from any scope of these embodiments.

[0171] While specific embodiments are illustrated and described herein, it will be understood that the claims are not limited to any particular form or arrangement of the described and shown portions. Explanatory embodiments are disclosed herein. Certain terminology is used, but it is used in a general and descriptive sense and not for limiting purposes. In consideration of the above teachings, modifications and changes to the embodiments are possible. Thus, it will be understood that embodiments may be carried out in ways other than those specifically described.

[0172] All published documents, patents, and patent applications referenced herein are incorporated herein by reference, as indicated by the fact that each individual published document, patent, or patent application is specifically and individually referred to.

Claims

1. A cassette for use in an automated cell manipulation system, (a) Cell culture chamber, (b) A pump fluidly connected to the cell culture chamber, (c) A tangential flow filter fluid-connected to the pump, wherein the pump sends a concentrated flow to the tangential flow filter, the seepage flow of the tangential flow filter is controlled by a flow controller, and the tangential flow filter is angled with respect to the horizontal such that the outlet end of the tangential flow filter is positioned above the inlet end of the tangential flow filter. (d) A satellite volume connected to the tangential flow filter, which is included in the cassette, (e) A fluid path for returning the concentrated flow from the satellite volume to the tangential flow filter and recirculating it, (f) A constant volume waste liquid collection chamber fluidly connected to the tangential flow filter, and (g) A cassette comprising a cell sample output fluid-connected to the tangential flow filter.

2. The cassette according to claim 1, wherein the tangential flow filter has a pore size of 0.40 μm to 0.80 μm and a fiber diameter of 0.5 mm to 0.9 mm.

3. The cassette according to claim 1 or 2, wherein the cell culture chamber is maintained at a temperature that enables cell proliferation and growth.

4. The cassette according to any one of claims 1 to 3, wherein the tangential flow filter comprises a polymer selected from the group consisting of poly(ethersulfone), poly(acrylonitrile), and poly(vinylidene difluoride).

5. The cassette according to any one of claims 1 to 4, further comprising a pH sensor, a glucose sensor, an oxygen sensor, a carbon dioxide sensor, and / or an optical density sensor.

6. The cassette according to any one of claims 1 to 5, further comprising one or more sampling ports.

7. The cassette according to any one of claims 1 to 6, wherein the flow controller is a flow restrictor.

8. The cassette according to any one of claims 1 to 7, wherein the flow controller is an additional pump.

9. The cassette according to any one of claims 1 to 8, wherein the flow controller is a system having a plurality of tubes for limiting or increasing the amount and velocity of seepage flow.

10. An automated cell manipulation system, (a) A sealed enclosure, (b) A cassette contained within the sealable housing, i. Cell culture chamber, ii. A pump fluidly connected to the cell culture chamber, iii. A tangential flow filter fluid-connected to the pump, wherein the pump sends a concentrated flow to the tangential flow filter, and the permeation flow of the tangential flow filter is controlled by a flow controller, iv. The cell sample output fluid-connected to the tangential flow filter, and v. A cassette comprising a satellite volume for receiving a concentrated flow from the tangential flow filter, (c) A user interface for receiving input from the user, and (d) A fluid path for returning the concentrated flow from the satellite volume to the tangential flow filter and recirculating it, Equipped with, An automated cell manipulation system in which the tangential flow filter is angled with respect to the horizontal such that the outlet end of the tangential flow filter is positioned above the inlet end of the tangential flow filter.

11. The automated cell manipulation system according to claim 10, wherein the tangential flow filter has a pore size of 0.40 μm to 0.80 μm and a fiber diameter of 0.5 mm to 0.9 mm.

12. The automated cell manipulation system according to claim 10 or 11, wherein the cell culture chamber is maintained at a temperature that enables cell proliferation and growth.

13. The automated cell manipulation system according to any one of claims 10 to 12, wherein the tangential flow filter comprises a polymer selected from the group consisting of poly(ethersulfone), poly(acrylonitrile), and poly(vinylidene difluoride).

14. The automated cell manipulation system according to any one of claims 10 to 13, wherein the cassette further comprises one or more sampling ports.

15. The automated cell manipulation system according to any one of claims 10 to 14, wherein the flow controller is a flow restrictor.

16. The automated cell manipulation system according to any one of claims 10 to 15, wherein the flow controller is an additional pump.

17. The automated cell manipulation system according to any one of claims 10 to 16, wherein the flow controller is a system having a plurality of tubes for limiting or increasing the amount and velocity of the osmotic flow.

18. An automated cell manipulation system according to any one of claims 10 to 17, further comprising a pH sensor, a glucose sensor, an oxygen sensor, a carbon dioxide sensor, and / or an optical density sensor.

19. The automated cell manipulation system according to any one of claims 10 to 18, further comprising a constant-volume waste liquid collection chamber which is fluidly connected to the tangential flow filter and located within the sealable housing.

20. An automated cell manipulation system, (a) A sealed enclosure, (b) A cassette contained within the sealable housing, i. Cell culture chamber, ii. A pump fluidly connected to the cell culture chamber, iii. A tangential flow filter fluid-connected to the pump, wherein the pump sends a concentrated flow to the tangential flow filter, the seepage flow of the tangential flow filter is controlled by a flow controller, and the tangential flow filter is angled at 20° or less with respect to the horizontal such that the outlet end of the tangential flow filter is positioned above the inlet end of the tangential flow filter. iv. The cell sample output fluid-connected to the tangential flow filter, and v. A cassette comprising a satellite volume for receiving a concentrated flow from the tangential flow filter, and (c) An automated cell manipulation system comprising a user interface for receiving input from a user.

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