Bioprocessing methods for cell therapy

Abstract

We provide bioprocessing systems for cell immunotherapy that reduce contamination risks by increasing automation and minimizing human handling. We offer bioprocessing systems for cell therapy drug manufacturing that balance the need for development flexibility with the consistency of mass production, and meet the diverse needs of customers who require different processes. [Solution] A bioprocessing method for cell therapy comprises the steps of: genetically modifying a population of cells in a bioreactor vessel to produce a population of genetically modified cells; and amplifying the population of genetically modified cells in the bioreactor vessel to produce a sufficient number of genetically modified cells for use in cell therapy treatment without removing the population of genetically modified cells from the bioreactor vessel. The cells are immobilized on a gas-permeable, liquid-impermeable membrane for amplification and are resuspended when or after reaching a desired cell density.

Description

Technical Field

[0001] Embodiments of the present invention generally relate to bioprocessing systems and methods, and more specifically, to bioprocessing systems and methods for the production of cell immunotherapy drugs.

Background Art

[0002] Various drug therapies involve the extraction, culture, and amplification of cells for use in downstream treatment processes. For example, chimeric antigen receptor (CAR) T cell therapy is a cell therapy that directs a patient's T cells to specifically target and destroy tumor cells. The basic principle of CAR-T cell design involves a recombinant receptor that combines antigen binding and T cell activation functions. A general premise of CAR-T cells is to artificially generate T cells that target markers found on cancer cells. Scientists can remove T cells from a human, genetically modify them, and return them to the patient's body to attack cancer cells. CAR-T cells can be either derived from the patient's own blood (autologous) or from another healthy donor (allogeneic).

[0003] The first step in producing CAR-T cells involves removing blood from a patient's body using apheresis therapy, such as leukapheresis therapy, and separating white blood cells. After a sufficient amount of white blood cells have been harvested, the leukapheresis product is concentrated against T cells, which involves washing the cells out of the leukapheresis buffer. Next, a subset of T cells having specific biomarkers is isolated from the enriched subpopulation using specific antibody conjugates or markers.

[0004] After isolating the target T cells, the cells are activated in a specific environment in which they can actively proliferate. For example, the cells can be activated using magnetic beads coated with anti-CD3 / anti-CD28 monoclonal antibodies or cell-based artificial antigen-presenting cells (aAPCs), which can be removed from the culture medium using magnetic separation. The T cells are then transduced with the CAR gene by either an integration gamma retrovirus (RV) or lentiviral (LV) vector. The viral vector uses a viral mechanism to attach to the patient cells, and after entering the cells, the vector introduces the genetic material in the form of RNA. In CAR-T cell therapy, this genetic material encodes the CAR. The RNA is reverse transcribed into DNA and permanently integrated into the genome of the patient cells, so that CAR expression is maintained as the cells divide and proliferate in large numbers in a bioreactor. The CAR is then transcribed and translated by the patient cells, and the CAR is expressed on the cell surface.

[0005] T cells are activated with a CAR-encoding viral vector and transduced. The cells are then amplified in a bioreactor until a large number of cells are produced, achieving the desired cell density. After amplification, the cells are harvested, washed, concentrated, formulated, and injected into the patient's body.

[0006] Existing systems and methods for producing injectable doses of CAR T cells require numerous complex operations involving many human touchpoints, making the entire manufacturing process time-consuming and increasing the risk of contamination. Recent efforts to automate the manufacturing process have eliminated some human touchpoints, but these systems still suffer from high costs, lack of flexibility, and workflow bottlenecks. Systems that utilize advanced automation, in particular, are very costly and inflexible, as customers need to adapt their processes to the specific equipment of the system. [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] U.S. Patent Application No. 15 / 893,336 [Patent Document 2] U.S. Patent Application No. 15 / 829,615 [Overview of the Initiative] [Problems that the invention aims to solve]

[0008] In light of the above, there is a need for a bioprocessing system for cell immunotherapy that reduces contamination risk by increasing automation and reducing human handling. In addition, there is a need for a bioprocessing system for cell therapy drug manufacturing that balances the need for development flexibility with the need for mass production consistency, and that can meet the diverse needs of customers who want to operate different processes. [Means for solving the problem]

[0009] Several embodiments that are in proportion to the originally claimed subject matter and scope are summarized below. These embodiments are not intended to limit the scope of the claimed subject matter, but rather are intended solely to provide an overview of possible embodiments. In fact, this disclosure may encompass a variety of forms that are similar to or different from the embodiments described below.

[0010] In one embodiment, the bioprocessing system comprises a first module configured to concentrate and isolate a population of cells, a second module configured to activate, genetically transduce, and amplify a population of cells, and a third module configured to harvest the amplified population of cells.

[0011] In another embodiment, the bioprocessing system comprises a first module configured to concentrate and isolate cells, a plurality of second modules, each configured to activate, genetically transduce, and amplify cells, and a third module configured to harvest the amplified cells. Each second module is configured to support the activation, genetic transduction, and amplification of different populations of cells, performed in parallel with one another.

[0012] In another embodiment, the bioprocessing method includes the steps of enriching and isolating a population of cells in a first module, activating, genetically transducing, and amplifying the population of cells in a second module, and harvesting the amplified population of cells in a third module. The steps of activating, genetically transducing, and amplifying the population of cells are performed without removing the population of cells from the second module.

[0013] In another embodiment, the apparatus for bioprocessing comprises a housing and a drawer housed within the housing. The drawer comprises a plurality of side walls and a bottom defining a processing chamber and a top that is normally open. The drawer is movable between a closed position in which the drawer is received within the housing and an open position in which the drawer extends from the housing, allowing access to the processing chamber through the open top. The apparatus also comprises at least one bed plate positioned within the processing chamber and configured to receive a bioreactor vessel.

[0014] In another embodiment, a bioprocessing method includes the steps of: sliding a drawer having a plurality of side walls, a bottom and a normally open top from a closed position to an open position within a housing to extend the drawer out of the housing through the normally open top; positioning a bioreactor vessel on a stationary bed plate positioned within the drawer through the normally open top; sliding the drawer to a closed position; and controlling a drawer engagement actuator to engage a plurality of fluid passages with at least one pump and a plurality of pinch valve linear actuators.

[0015] In another embodiment, the system for bioprocessing comprises a housing; a first drawer receivable within the housing, comprising a plurality of side walls and bottoms defining a first processing chamber and a normally open top; at least one first bed plate positioned within the processing chamber of the first drawer and configured to receive or otherwise engage with a first bioreactor container; a second drawer receivable within the housing in a stacking relationship with the first drawer, comprising a plurality of side walls and bottoms defining a second processing chamber and a normally open top; and at least one second bed plate positioned within the processing chamber of the second drawer and configured to receive or otherwise engage with a second bioreactor container. The first drawer and the second drawer are each movable between a closed position in which the first drawer and / or the second drawer are received within the housing and an open position in which the first drawer and / or the second drawer extend out of the housing, allowing access to the processing chamber through their respective open tops.

[0016] In yet another embodiment, the apparatus for bioprocessing comprises a housing, a drawer receivable within the housing, the drawer comprising a plurality of side walls and bottom surfaces defining a processing chamber, and a normally open top, the drawer movable between a closed position in which the drawer is received within the housing and an open position in which the drawer extends from the housing and allows access to the processing chamber through the open top, at least one bed plate positioned within the processing chamber adjacent to the bottom surface, and a kit receivable within the processing chamber. The kit comprises a plurality of side walls and bottom surfaces defining an internal compartment, a normally open top, an opening formed within the bottom surface of the kit and having a periphery, and a bioreactor container positioned above at least one opening within the internal compartment, with a portion of the bioreactor container supported by the bottom surface so as to be accessible through the opening in the bottom surface. The kit is receivable within the processing chamber such that the bed plate penetrates the opening in the bottom surface of the tray and supports the bioreactor container above the bottom surface of the kit.

[0017] In yet another embodiment, the system for bioprocessing comprises a tray having a plurality of side walls and a bottom surface defining an internal compartment, and a normally open top; at least one opening formed in the bottom surface and having a periphery; a first tubing holder block integrated with the tray and configured to receive at least one pump tube and to hold at least one pump tube in place for selective engagement with a pump; a second tubing holder block integrated with the tray and configured to receive a plurality of pinch valve tubes and to hold each of the plurality of pinch valve tubes in place for selective engagement with each actuator of a pinch valve array; and a bioreactor container positioned above at least one opening in the internal compartment and supported by the bottom surface such that a portion of the bioreactor container is accessible through an opening in the bottom surface.

[0018] In yet another embodiment, the system for bioprocessing comprises a processing chamber having a plurality of side walls, a bottom surface, and a normally open top surface; a bed plate positioned within the processing chamber adjacent to the bottom surface; and a tray. The tray comprises a plurality of side walls and a bottom surface defining an internal compartment, as well as a normally open top surface and an opening located within the bottom surface of the tray and having a periphery. The periphery of the opening has a shape and / or dimensions such that a bioreactor vessel can be positioned above the opening and supported by the bottom surface of the tray while a portion of the bioreactor vessel is accessible through the opening in the bottom surface. The tray is receivable into the processing chamber such that the bed plate penetrates the opening in the bottom surface of the tray and supports the bioreactor vessel.

[0019] In yet another embodiment, the system for bioprocessing comprises a tray having a plurality of side walls and a bottom surface defining an internal compartment, and a top surface that is normally open, and at least one opening in the bottom surface with a periphery boundary, wherein the opening has a shape and / or dimensions such that a bioreactor container can be positioned above the opening and supported by the bottom surface of the tray within the internal compartment.

[0020] In yet another embodiment, a bioprocessing method includes the steps of: placing a bioreactor container in a disposable tray, the disposable tray having a plurality of side walls and bottom surfaces defining an internal compartment, as well as a normally open top, an opening formed in the bottom surface, and a plurality of claws or protrusions penetrating from the bottom surface into the opening; positioning the bioreactor container in the tray such that the bioreactor container is supported by the claws above the opening; and placing the tray in a processing chamber having a bed plate such that the bed plate is received through the opening in the tray and supports the bioreactor container.

[0021] In yet another embodiment, a tubing module for a bioprocessing system comprises a first tubing holder block configured to receive at least one pump tube and hold at least one pump tube in place for selective engagement with a peristaltic pump, and a second tubing holder block configured to receive a plurality of pinch valve tubes and hold each of the plurality of pinch valve tubes in place for selective engagement with each actuator of a pinch valve array. The first tubing holder block and the second tubing holder block are interconnected.

[0022] In yet another embodiment, a system for bioprocessing has a plurality of sidewalls and a bottom surface that define an internal compartment and a top that is normally open, and a tray configured to receive, support, or otherwise engage a bioreactor vessel thereon, a pump assembly positioned adjacent a rear sidewall of the tray, a pinch valve array positioned adjacent the rear sidewall of the tray, and a tubing module positioned at the rear of the tray. The tubing module includes a first tubing holder block configured to receive at least one pump tube and hold the at least one pump tube in place for selective engagement with the pump assembly, and a second tubing holder block configured to receive a plurality of pinch valve tubes and hold each of the plurality of pinch valve tubes in place for selective engagement with a respective actuator of the pinch valve array.

[0023] In yet another embodiment, a bioreactor vessel includes a bottom plate, and a vessel body portion coupled to the bottom plate, the vessel body portion and the bottom plate defining an internal compartment therebetween, and a plurality of recesses formed in the bottom plate, each of the plurality of recesses configured to receive a corresponding alignment pin on a bed plate for aligning the bioreactor vessel on the bed plate.

[0024] In yet another embodiment, a method for bioprocessing includes operably connecting a bottom plate to a vessel body portion such that they define an internal compartment therebetween, the bottom plate and the vessel body portion forming a bioreactor vessel, aligning recesses in the bottom plate with alignment pins of a bioprocessing system, and mounting the bioreactor vessel on a bed plate of the bioprocessing system.

[0025] In yet another embodiment, the bioprocessing system includes a first fluid assembly having a first fluid assembly line connected to a first port of the first bioreactor vessel through a first bioreactor line of the first bioreactor vessel, the first bioreactor line of the first bioreactor vessel comprising a first bioreactor line valve for providing selective fluid communication between the first fluid assembly and the first port of the first bioreactor vessel; a second fluid assembly having a second fluid assembly line connected to a second port of the first bioreactor vessel through a second bioreactor line of the first bioreactor vessel, the second bioreactor line of the first bioreactor vessel comprising a second bioreactor line valve for providing selective fluid communication between the second fluid assembly and the second port of the first bioreactor vessel; and an interconnecting line providing fluid communication between the first fluid assembly and the second fluid assembly and fluid communication between the second bioreactor line of the first bioreactor vessel and the first bioreactor line of the first bioreactor vessel.

[0026] In yet another embodiment, the bioprocessing method includes providing a first fluid assembly having a first fluid assembly line connected to a first port of a first bioreactor vessel through a first bioreactor line of a first bioreactor vessel; providing a second fluid assembly having a second fluid assembly line connected to a second port of a first bioreactor vessel through a second bioreactor line of a first bioreactor vessel; and providing an interconnection line between the second bioreactor line of a first bioreactor vessel and the first bioreactor line of a first bioreactor vessel, wherein the interconnection line enables fluid communication between the first fluid assembly and the second fluid assembly, and between the second bioreactor line of a first bioreactor vessel and the first bioreactor line of a first bioreactor vessel.

[0027] In yet another embodiment, a bioprocessing method for cell therapy includes genetically modifying a population of cells in a bioreactor vessel to produce a population of genetically modified cells, and amplifying the population of genetically modified cells in the bioreactor vessel to produce a sufficient number of genetically modified cells for use in cell therapy treatment without removing the population of genetically modified cells from the bioreactor vessel.

[0028] In yet another embodiment, the bioprocessing method includes coating a bioreactor container with a reagent to increase the efficiency of genetic modification of a population of cells, genetically modifying the cells of the population of cells to produce a population of genetically modified cells, and amplifying the population of genetically modified cells within the bioreactor container without removing the genetically modified cells from the bioreactor container.

[0029] In yet another embodiment, the bioprocessing method includes activating cells in a population of cells within a bioreactor vessel using magnetic or non-magnetic beads to produce a population of activated cells; genetically modifying the activated cells within the bioreactor vessel to produce a population of genetically modified cells; washing the genetically modified cells within the bioreactor vessel to remove undesirable substances; and amplifying the population of genetically modified cells within the bioreactor vessel to produce an amplified population of transduced cells. Activation, genetic modification, washing, and amplification are performed within the bioreactor vessel without removing the cells from the bioreactor vessel.

[0030] In yet another embodiment, the kit used in the bioprocessing system comprises a process bag, a source bag, a bead dispenser, and a process loop configured to be in fluid communication with the process bag, the source bag, and the bead dispenser. The process loop further comprises pump tubing configured to be in fluid communication with a pump.

[0031] In yet another embodiment, the apparatus for bioprocessing comprises a kit comprising a process bag, a source bag, and a bead-adding container configured to be in fluid communication with a process loop, wherein the process loop further comprises a pump tubing configured to be in fluid communication with a pump; a magnetic field generator configured to generate a magnetic field; a plurality of hooks for suspending the source bag, the process bag, and the bead-adding container, each hook of the plurality of hooks being operably connected to a load cell, the load cell being configured to sense the weight of the bag to which it is connected; at least one bubble sensor; and a pump configured to be in fluid communication with the process loop.

[0032] In one embodiment, the bioprocessing method includes combining a suspension containing a population of cells with magnetic beads to form a population of bead-bound cells in the suspension, isolating the population of bead-bound cells on a magnetic isolation column, and collecting target cells from the population of cells.

[0033] In one embodiment, a non-temporary computer-readable medium is provided. The non-temporary computer-readable medium includes instructions configured to adapt a controller to maintain a first target environment in a bioreactor vessel containing a population of cells for a first incubation period to produce a population of genetically modified cells from a population of cells, to initiate a flow of culture medium into the bioreactor vessel, and to maintain a second target environment in the bioreactor vessel for a second incubation period to produce an amplified population of genetically modified cells.

[0034] In another embodiment, a non-temporary computer-readable medium is provided. The non-temporary computer-readable medium includes instructions configured to adapt a controller to maintain a first target environment within the first bioreactor vessel for a first incubation period to activate a population of cells in the first bioreactor, and to maintain a second target environment within the first bioreactor vessel for a second incubation period to produce a population of genetically modified cells from the population of cells.

[0035] In yet another embodiment, a non-temporary computer-readable medium is provided. The non-temporary computer-readable medium includes instructions configured to receive data relating to the mass and / or volume of a bioreactor container containing a population of cells suspended in a culture medium, to activate a first pump to pump fresh culture medium into the bioreactor container, to activate a second pump to pump used culture medium from the bioreactor container to a waste bag, and to adapt a controller to control the operating setpoint of at least one of the first and second pumps in accordance with the data relating to the mass and / or volume of the bioreactor container.

[0036] The present invention will be better understood by reading the following description of non-limiting embodiments with reference to the accompanying drawings shown below. [Brief explanation of the drawing]

[0037] [Figure 1] This is a schematic diagram of a bioprocessing system according to one embodiment of the present invention. [Figure 2] This is a schematic diagram of a bioprocessing system according to another embodiment of the present invention. [Figure 3] Figure 1 is a block diagram showing the fluid flow configuration / system of the cell activation, gene modification, and amplification subsystems of the bioprocessing system. [Figure 4] This is a detailed view of a portion of the block diagram in Figure 3, illustrating the first fluid assembly of a fluid flow configuration / system. [Figure 5] This is a detailed view of a portion of the block diagram in Figure 3, illustrating a second fluid assembly of a fluid flow configuration / system. [Figure 6] This is a detailed view of a portion of the block diagram in Figure 3, illustrating a sampling assembly of a fluid flow configuration / system. [Figure 7] This is a detailed view of a portion of the block diagram in Figure 3, illustrating the filtration channel of a fluid flow configuration / system. [Figure 8] This is a perspective view of a bioreactor vessel according to one embodiment of the present invention. [Figure 9] Figure 8 is an exploded view of the bioreactor container. [Figure 10] Figure 8 is a disassembled cross-sectional view of the bioreactor container. [Figure 11] Figure 8 is a perspective view of the disassembled bottom of the bioreactor container. [Figure 12] Figure 1 shows top and front perspective views of a disposable drop-in kit of the bioprocessing system according to one embodiment of the present invention. [Figure 13]Figure 12 shows another top and front perspective view of the disposable drop-in kit. [Figure 14] Figure 12 shows other top and rear perspective views of the disposable drop-in kit. [Figure 15] Figure 12 is a perspective view of the tray of a disposable drop-in kit according to one embodiment of the present invention. [Figure 16] Figure 12 shows a front perspective view of the tubing module of a disposable drop-in kit according to one embodiment of the present invention. [Figure 17] Figure 16 is a rear perspective view of the tubing module. [Figure 18] This is an elevation view of a second tubing holder block of a tubing module according to one embodiment of the invention. [Figure 19] Figure 18 is a cross-sectional view of the second tubing holder block. [Figure 20] Figure 12 is another front perspective view of the drop-in kit, showing the integrated fluid architecture inside. [Figure 21] Figure 12 is a rear perspective view of the drop-in kit, showing the integrated fluid architecture inside. [Figure 22] Figure 12 is a front elevation view of the drop-in kit, showing the integrated fluid architecture inside. [Figure 23] This is a perspective view of a bioprocessing apparatus according to one embodiment of the present invention. [Figure 24] Figure 12 is a perspective view of a drawer of a bioprocessing apparatus for receiving a drop-in kit, according to one embodiment of the present invention. [Figure 25] Figure 24 is a top view of the drawer. [Figure 26] Figure 24 is a front perspective view of the drawer's processing chamber. [Figure 27] This is a top view of the drawer's processing chamber. [Figure 28A] Figure 23 is a top view of the bed plate of the bioprocessing apparatus. [Figure 28B] Figure 28A is a top view of the hardware components housed beneath the bed plate. [Figure 29] Figure 12 is a side elevation view of the bioprocessing apparatus. [Figure 30] Figure 12 is a perspective view of the drawer engagement actuator of the bioprocessing apparatus. [Figure 31] This is a top view of a drawer in a bioprocessing apparatus, illustrating the clearance positions of the drawer engagement actuator, pump assembly, and solenoid array. [Figure 32] This is a top view of a drawer in a bioprocessing apparatus, illustrating the engagement positions of the drawer engagement actuator, pump assembly, and solenoid array. [Figure 33] This is a perspective view of a bioprocessing apparatus illustrating the position of a drop-in kit within the processing chamber of a drawer. [Figure 34] This is a top view of a bioprocessing apparatus illustrating a drop-in kit in the drawer's processing chamber. [Figure 35] This is a perspective view of the peristaltic pump assembly of a bioprocessing device. [Figure 36] This is a side elevation view of the peristaltic pump assembly and the tubing holder module of the drop-in kit, illustrating the relationships between components. [Figure 37] This is a perspective view of a solenoid array and pinch valve anvil forming a pinch valve array in a bioprocessing device. [Figure 38] Another perspective view of the pinch valve array in a bioprocessing device. [Figure 39] Another perspective view of the pinch valve array illustrating the positioning of the tubing holder module of the drop-in kit relative to the pinch valve array in the engaged position. [Figure 40] This is a cross-sectional view of the drawer of a bioprocessing apparatus, illustrating the installation position of the bioreactor vessel on the bed plate. [Figure 41] This is a side elevation view of a bioreactor received on a bed plate, illustrating the agitation / mixing operation mode of the bioreactor system. [Figure 42] This is a side cross-sectional view of a bioreactor received on a bed plate, illustrating the stirring / mixing operation mode of the bioreactor system. [Figure 43] This is a schematic diagram of the bioreactor container showing the liquid level inside the container when it is in stirring / mixing mode. [Figure 44] This is a detailed cross-sectional view of the interface between the positioning pins on the bed plate and the receiving recess on the bioreactor vessel during the agitation / mixing operation mode. [Figure 45] This is a perspective view of a bioprocessing apparatus having a flip-down front panel according to one embodiment of the present invention, showing a processing drawer in the open position. [Figure 46] This is another perspective view of the bioprocessing apparatus shown in Figure 45, indicating the processing drawer in the open position. [Figure 47] Figure 45 is an enlarged perspective view of the auxiliary compartment of the bioprocessing unit, showing the processing drawer in a closed position that provides access to the auxiliary compartment. [Figure 48] Figure 45 shows another enlarged perspective view of the auxiliary compartment of the bioprocessing apparatus, indicating the processing drawer in a closed position that provides access to the auxiliary compartment. [Figure 49] Figure 45 is a perspective view of the bioprocessing apparatus, showing the processing drawer in a closed position that provides access to the auxiliary compartment. [Figure 50] Another perspective view of the bioprocessing apparatus in Figure 45, showing the processing drawer in a closed position that provides access to the auxiliary compartment. [Figure 51] This is a perspective view of an auxiliary compartment of a bioprocessing apparatus according to another embodiment of the present invention. [Figure 52] This is a perspective view of a bioprocessing system having a waste liquid tray according to one embodiment of the present invention. [Figure 53] This is a schematic diagram of an automated generic protocol for a bioprocessing system utilizing the fluid flow architecture shown in Figure 3, according to one embodiment of the present invention. [Figure 54] This is a schematic diagram of an automated generic protocol for a bioprocessing system utilizing the fluid flow architecture shown in Figure 3, according to one embodiment of the present invention. [Figure 55] This is a schematic diagram of an automated generic protocol for a bioprocessing system utilizing the fluid flow architecture shown in Figure 3, according to one embodiment of the present invention. [Figure 56] This is a schematic diagram of an automated generic protocol for a bioprocessing system utilizing the fluid flow architecture shown in Figure 3, according to one embodiment of the present invention. [Figure 57] This is a schematic diagram of an automated generic protocol for a bioprocessing system utilizing the fluid flow architecture shown in Figure 3, according to one embodiment of the present invention. [Figure 58] This is a schematic diagram of an automated generic protocol for a bioprocessing system utilizing the fluid flow architecture shown in Figure 3, according to one embodiment of the present invention. [Figure 59] This is a schematic diagram of an automated generic protocol for a bioprocessing system utilizing the fluid flow architecture shown in Figure 3, according to one embodiment of the present invention. [Figure 60] This is a schematic diagram of an automated generic protocol for a bioprocessing system utilizing the fluid flow architecture shown in Figure 3, according to one embodiment of the present invention. [Figure 61] This is a schematic diagram of an automated generic protocol for a bioprocessing system utilizing the fluid flow architecture shown in Figure 3, according to one embodiment of the present invention. [Figure 62] This is a schematic diagram of an automated generic protocol for a bioprocessing system utilizing the fluid flow architecture shown in Figure 3, according to one embodiment of the present invention. [Figure 63]This is a schematic diagram of an automated generic protocol for a bioprocessing system utilizing the fluid flow architecture shown in Figure 3, according to one embodiment of the present invention. [Figure 64] This is a schematic diagram of an automated generic protocol for a bioprocessing system utilizing the fluid flow architecture shown in Figure 3, according to one embodiment of the present invention. [Figure 65] This is a schematic diagram of an automated generic protocol for a bioprocessing system utilizing the fluid flow architecture shown in Figure 3, according to one embodiment of the present invention. [Figure 66] This is a schematic diagram of an automated generic protocol for a bioprocessing system utilizing the fluid flow architecture shown in Figure 3, according to one embodiment of the present invention. [Figure 67] This is a schematic diagram of an automated generic protocol for a bioprocessing system utilizing the fluid flow architecture shown in Figure 3, according to one embodiment of the present invention. [Figure 68] This is a schematic diagram of an automated generic protocol for a bioprocessing system utilizing the fluid flow architecture shown in Figure 3, according to one embodiment of the present invention. [Figure 69] This is a schematic diagram of an automated generic protocol for a bioprocessing system utilizing the fluid flow architecture shown in Figure 3, according to one embodiment of the present invention. [Figure 70] This is a schematic diagram of an automated generic protocol for a bioprocessing system utilizing the fluid flow architecture shown in Figure 3, according to one embodiment of the present invention. [Figure 71] This is a schematic diagram of an automated generic protocol for a bioprocessing system utilizing the fluid flow architecture shown in Figure 3, according to one embodiment of the present invention. [Figure 72] This is a schematic diagram of an automated generic protocol for a bioprocessing system utilizing the fluid flow architecture shown in Figure 3, according to one embodiment of the present invention. [Figure 73]This is a schematic diagram of an automated generic protocol for a bioprocessing system utilizing the fluid flow architecture shown in Figure 3, according to one embodiment of the present invention. [Figure 74] This is a schematic diagram of an automated generic protocol for a bioprocessing system utilizing the fluid flow architecture shown in Figure 3, according to one embodiment of the present invention. [Figure 75] This is a schematic diagram of an automated generic protocol for a bioprocessing system utilizing the fluid flow architecture shown in Figure 3, according to one embodiment of the present invention. [Figure 76] This is a schematic diagram of an automated generic protocol for a bioprocessing system utilizing the fluid flow architecture shown in Figure 3, according to one embodiment of the present invention. [Figure 77] This is a schematic diagram of an automated generic protocol for a bioprocessing system utilizing the fluid flow architecture shown in Figure 3, according to one embodiment of the present invention. [Figure 78] This is a perspective view of a concentration and isolation apparatus according to one embodiment of the present invention. [Figure 79] Figure 78 is a process flow diagram of the concentration and isolation apparatus. [Figure 80] Figure 78 is a schematic diagram of the fluid flow architecture of the apparatus for performing cell population enrichment and isolation. [Figure 81] This is a flowchart of a bioprocessing method using the system shown in Figure 1, according to one embodiment of the present invention. [Modes for carrying out the invention]

[0038] Exemplary embodiments of the present invention, for which examples are shown in the accompanying drawings, are referred to in detail below. Wherever possible, the same reference letters are used throughout the drawings to refer to the same or similar parts.

[0039] As used herein, the terms “flexible” or “foldable” refer to a structure or material that is easily bent or can be bent without breaking, and may also refer to a material that is compressible or expandable. An example of a flexible structure is a bag formed from polyethylene film. The terms “rigid” and “semi-rigid” are used herein interchangeably to describe a structure that is “non-foldable,” i.e., a structure that does not fold, collapse, or otherwise deform in any way to significantly reduce its elongation dimension when subjected to normal forces. Depending on the context, “semi-rigid” may also refer to a structure that is more flexible than a “rigid” element, such as a bendable tube or conduit, but still does not collapse longitudinally under normal conditions and forces.

[0040] As used herein, “container” means, as it may be, a flexible bag, a flexible container, a semi-rigid container, a rigid container, or flexible or semi-rigid tubing. As used herein, the term “container” is intended to encompass bioreactor vessels having walls or parts of walls that are semi-rigid or rigid, as well as other containers or conduits commonly used in biological or biochemical processes, including, for example, cell culture / purification systems, mixing systems, culture medium / buffer preparation systems, and filtration / purification systems, such as chromatography and tangential flow filtration systems, and their associated channels. As used herein, the term “bag” means, for example, a flexible or semi-rigid container or vessel used as a containment device for various fluids and / or culture media.

[0041] As used herein, “fluidically coupled” or “fluidly communicating” means that components of a system can receive or transfer fluids between components. The term “fluid” includes gases, liquids, or combinations thereof. As used herein, “electrically connected” or “electrically coupled” means that some components are configured to communicate with each other through direct or indirect signaling using direct or indirect electrical connections. As used herein, “operably coupled” refers to a connection that may be direct or indirect. A connection is not necessarily a mechanical connection.

[0042] As used herein, the term “tray” refers to any object capable of supporting multiple components, at least temporarily. A tray may be made from a variety of suitable materials. For example, a tray may be made from a cost-effective material suitable for sterile and single-use disposable products.

[0043] As used herein, the term “functional closed system” refers to a set of components constituting a closed fluid pathway that may have inlet and outlet ports for adding or removing fluid or air from a system without compromising the integrity of the closed fluid pathway (for example, to maintain an internal sterile biomedical fluid pathway), thereby including, at each port, a filter or membrane, for example, to maintain sterile integrity when fluid or air is added to or removed from the system. These components may include, but are not limited to, one or more conduits, valves (e.g., multipoint diverters), vessels, receptacles, and ports, depending on a given embodiment.

[0044] Embodiments of the present invention provide systems and methods for producing cellular immunotherapy drugs from biological samples (e.g., blood, tissue, etc.). In one embodiment, the method includes genetically modifying a population of cells in a bioreactor vessel to produce a population of genetically modified cells, and amplifying the population of genetically modified cells in the bioreactor vessel to produce a sufficient number of genetically modified cells for use in one or more doses in cellular therapy without removing the population of genetically modified cells from the bioreactor vessel. In some embodiments, the bioreactor vessel may be coated with a reagent to enhance the efficiency of genetic modification of the population of cells, and the amplification of genetically modified cells is carried out in the same reagent-coated bioreactor vessel. In some embodiments, one or more of these methods may include activating cells in the same bioreactor vessel using magnetic or non-magnetic beads to produce a population of activated cells before genetic modification, and washing the genetically modified cells on the bioreactor vessel to remove undesirable substances.

[0045] Referring to Figure 1, a schematic diagram of a bioprocessing system 10 according to one embodiment of the present invention is illustrated. The bioprocessing system 10 is configured for use in the manufacture of cellular immunotherapy drugs (e.g., autologous cellular immunotherapy drugs), for example, by collecting human blood, fluid, tissue, or cell samples, and by generating cellular therapy drugs from or based on the collected samples. Chimeric antigen receptor (CAR) T-cell therapy drugs are one type of cellular immunotherapy drug that can be manufactured using the bioprocessing system 10, but other cellular therapy drugs can also be produced using the present invention or systems of embodiments of the present invention without departing from broader embodiments of the present invention. As illustrated in Figure 1, the manufacture of CAR T-cell therapy drugs generally begins with the collection of a patient's blood and separation of lymphocytes through apheresis therapy. The collection / apheresis therapy may be performed in a clinical setting, and the apheresis product is then sent to a laboratory or manufacturing facility for the production of CAR T cells. In particular, after the apheresis product is received for processing, the desired cell population (e.g., leukocytes) is concentrated or separated from the collected blood to produce a cell therapy drug, and the target cells of interest are isolated from the progenitor cell mixture. The target cells of interest are then activated, genetically modified to specifically target and destroy tumor cells, and amplified to achieve the desired cell density. After amplification, the cells are harvested and the dosage is formulated. The formulation is then often frozen for storage, thawing, preparation, and finally delivered to the clinical site for infusion into the patient's body.

[0046] Referring further to Figure 1, the bioprocessing system 10 of the present invention comprises a plurality of distinct modules or subsystems, each configured to perform a specific subset of manufacturing steps in a substantially automated, functionally closed, and scalable manner. In particular, the bioprocessing system 10 comprises a first module 100 configured to perform enrichment and isolation steps, a second module 200 configured to perform activation, genetic modification, and amplification steps, and a third module 300 configured to perform a step of harvesting the amplified cell population. In one embodiment, each module 100, 200, and 300 may be communicably coupled to a dedicated controller (e.g., a first controller 110, a second controller 210, and a third controller 310, respectively). Controllers 110, 210, and 310 are configured to provide substantially automated control over the manufacturing processes within each module. The first module 100, the second module 200, and the third module 300 are exemplified as having dedicated controllers for controlling the operation of each module, but it is intended that a master control unit may be used to provide global control to the three modules. Each module 100, 200, and 300 is designed to work in cooperation with the other modules to form a single coherent bioprocessing system 10, as described in detail below.

[0047] Automating the processes within each module can improve the consistency of the products produced from each module and reduce the costs associated with extensive manual operations. In addition, as will be explained in detail later, each module 100, 200, and 300 is substantially closed, helping to ensure patient safety by reducing the risk of external contamination, ensuring compliance with regulations, and avoiding the costs associated with open systems. Furthermore, each module 100, 200, and 300 is scalable, supporting both development with a small number of patients and commercial production with a large number of patients.

[0048] Referring further to Figure 1, a particular scheme in which process steps are divided into distinct modules providing closed and automated bioprocessing, respectively, enables an unprecedented level of efficient use of capital equipment. As can be understood, the step of amplifying the cell population to achieve a desired cell density before harvesting and compounding is typically the most time-consuming step in the manufacturing process, while the concentration and isolation steps, as well as the harvesting and compounding steps, and even the activation and genetic modification steps, are less time-consuming. Therefore, attempts to automate the entire cell therapy drug manufacturing process, in addition to being logistically challenging, can exacerbate bottlenecks in the process that disrupt the workflow and reduce manufacturing efficiency. In particular, in a fully automated process, the steps of cell concentration, isolation, activation, and genetic modification can be performed fairly quickly, while the amplification of genetically modified cells proceeds very slowly. Thus, the production of a cell therapy drug from a first sample (e.g., the blood of a first patient) proceeds quickly up to the amplification step, which requires substantial time to achieve the desired cell density to be harvested. In a fully automated system, the entire process / system is occupied by amplification equipment that performs cell amplification from the first sample, and processing of the second sample cannot begin until the entire system is freed up for use. In this respect, a fully automated bioprocessing system is essentially offline, and the entire cell therapy drug manufacturing process, from concentration to harvesting / compounding, is not available for processing the second sample until it is completed with the first sample.

[0049] However, embodiments of the present invention enable parallel processing of multiple samples (from the same or different patients) to advance the more efficient use of capital resources. This advantage is a direct result of a particular arrangement in which the process steps are divided into three modules 100, 200, and 300, as implied above. Referring particularly to Figure 2, in one embodiment, a single first module 100 and / or a single third module 300 can be used in conjunction with a plurality of second modules, for example, second modules 200a, 200b, and 200c, in the bioprocessing system 12 to perform parallel and synchronous processing of multiple samples from the same or different patients. For example, a first apheresis product from a first patient may be concentrated and isolated using the first module 100, thereby producing a first population of isolated target cells, the first population of target cells may then be transferred to one of the second modules, for example, module 200a, for activation, genetic modification, and amplification under the control of controller 210a. After the first population of target cells is transferred out of the first module 100, the first module is again available for use, for example, in processing a second apheresis product from a second patient. The second population of target cells produced in the first module 100 from the sample taken from the second patient can then be transferred to another second module, for example, the second module 200b, for activation, genetic modification, and amplification under the control of controller 201b.

[0050] Similarly, after a second population of target cells has been transferred out of the first module 100, the first module is again available for use, for example, in processing a third apheresis product from a third patient. The third target population of cells produced in the first module 100 from a sample taken from the third patient can then be transferred to another second module, for example, the second module 200c, for activation, genetic modification, and amplification under the control of controller 201c. In this regard, amplification of CAR-T cells in the first patient, for example, may occur simultaneously with amplification of CAR-T cells in the second patient, the third patient, and so on.

[0051] This approach also allows post-processing to be performed asynchronously as needed. In other words, patient cells cannot all grow simultaneously. Cultures may reach their final density at different times, but multiple second modules 200 are not linked, and a third module 300 can be used as needed. The present invention allows samples to be processed in parallel, but these do not have to be done in batch processing.

[0052] The harvesting of amplified populations of cells from the second modules 200a, 200b, and 200c can similarly be performed using a single third module 300 when each amplified population of cells is ready for harvesting.

[0053] Therefore, by separating the activation, genetic modification, and amplification steps—which are the most time-consuming, share several operational requirements, and / or require similar culture conditions—into standalone, automated, and functionally closed modules, other system equipment used for concentration, isolation, harvesting, and compounding is not tied up or offline while the amplification of one population of cells is being performed. As a result, the production of multiple cell therapy drugs can be performed simultaneously, maximizing equipment and floor space utilization and increasing the efficiency of the overall process and equipment. An additional second module may be added to the bioprocessing system 10 to parallel process any number of cell populations as desired. Thus, the bioprocessing system of the present invention allows for the use of plug-and-play-like functionality and facilitates the scaling up or down of the manufacturing facility.

[0054] In one embodiment, the first module 100 may be any system or device capable of producing a target population of cells concentrated and isolated for use in biological processes, such as the manufacture of immunotherapy drugs and regenerative medicine drugs, from apheresis products taken from a patient. For example, the first module 100 may be a modified version of the Sefia Cell Processing System, available from GE Healthcare. Configurations of the first module 100 according to several embodiments of the present invention will be described in detail below.

[0055] In one embodiment, the third module 300 may be any system or device capable of similarly harvesting and / or compounding CAR-T cells or other modified cells produced by the second module 200 for use in cellular immunotherapy or regenerative medicine, and for injection into a patient's body. In some embodiments, the third module 300 may also be the Sefia Cell Processing System, available from GE Healthcare. In some embodiments, the first module 100 may be initially used for the enrichment and isolation of cells (which are then transferred to the second module 200 for activation, transduction, and amplification (and harvesting in some embodiments)), and then also used at the end of the process for cell harvesting and / or compounding. In this regard, in some embodiments, the same instrument may be used for the front-end cell enrichment and isolation steps, and further for the back-end harvesting and / or compounding steps.

[0056] First, focusing on the second module 200, the ability to combine the process steps of cell activation, genetic modification, and cell amplification in a single, functionally closed, automated module 200, resulting in the workflow efficiency described above, is enabled by the specific configuration of the components within the second module 200 and the unique fluid architecture that results in specific interconnectivity between such components. Figures 3 to 77, described below, illustrate various aspects of the second module 200 according to various embodiments of the present invention. Referring first to Figure 3, a schematic diagram illustrating the fluid-flow architecture 400 (also broadly referred to herein as the bioprocessing subsystem 400 or bioprocessing system 400) within the second module 200 that performs cell activation, genetic modification, and amplification (in some cases, harvesting). The system 400 comprises a first bioreactor vessel 410 and a second bioreactor vessel 420. The first bioreactor vessel comprises at least a first port 412 and a first bioreactor line 414 fluidly communicating with the first port 412, and a second port 416 and a second bioreactor line 418 fluidly communicating with the second port 416. Similarly, the second bioreactor vessel comprises at least a first port 422 and a first bioreactor line 424 fluidly communicating with the first port 422, and a second port 426 and a second bioreactor line 428 fluidly communicating with the second port 426. Together, the first bioreactor vessel 410 and the second bioreactor vessel 420 form a bioreactor array 430. Although the system 400 is shown as having two bioreactor vessels, embodiments of the present invention may comprise a single bioreactor or three or more bioreactor vessels.

[0057] The first and second bioreactor lines 414, 418, 424, and 428 of the first and second bioreactor vessels 410 and 420 are each equipped with a valve for controlling the flow of fluid passing through them, as described below. In particular, the first bioreactor line 414 of the first bioreactor vessel 410 is equipped with a first bioreactor line valve 432, and the second bioreactor line 418 of the first bioreactor vessel 410 is equipped with a second bioreactor line valve 424. Similarly, the first bioreactor line 424 of the second bioreactor vessel 420 is equipped with a first bioreactor line valve 436, and the second bioreactor line 428 of the second bioreactor vessel 420 is equipped with a second bioreactor line valve 438.

[0058] Referring further to Figure 3, the system 400 also comprises a first fluid assembly 440 having a first fluid assembly line 442, a second fluid assembly 444 having a second fluid assembly line 446, and a sampling assembly 448. An interconnection line 450 having an interconnection line valve 452 provides fluid communication between the first fluid assembly 440 and the second fluid assembly 444. As shown in Figure 3, the interconnection line 450 also provides fluid communication between a second bioreactor line 418 and the first bioreactor line 414 of the first bioreactor vessel 410, allowing fluid circulation along the first circulation loop of the first bioreactor vessel. Similarly, the interconnection line also provides fluid communication between the second bioreactor line 428 and the first bioreactor line 424 of the second bioreactor vessel 420, allowing fluid circulation along the second circulation loop of the second bioreactor vessel. Furthermore, the interconnection line 450 provides further fluid communication between the second port 416 and the second bioreactor line 418 of the first bioreactor vessel 410 and the first port 422 and the first bioreactor line 424 of the second bioreactor vessel 420, enabling the transfer of the contents of the first bioreactor vessel 410 to the second bioreactor vessel 420, as described below. As illustrated in Figure 3, in one embodiment, the interconnection line 450 extends from the second bioreactor lines 418, 428 to the intersection of the first bioreactor line 414 and the first fluid assembly line 442 of the first bioreactor vessel 410.

[0059] As illustrated in Figure 3, the first and second fluid assemblies 440 and 450 are arranged along the interconnection line 450. In addition, in one embodiment, the first fluid assembly is in fluid communication with the first port 412 of the first bioreactor vessel 410 and the first port of the second bioreactor vessel 420 through the first bioreactor line 414 of the first bioreactor vessel and the first bioreactor line 424 of the second bioreactor vessel 420, respectively. The second fluid assembly 444 is in fluid communication with the second port 416 of the first bioreactor vessel 410 and the second port 426 of the second bioreactor vessel 420 via the interconnection line 450.

[0060] A first pump or interconnection line pump 454, capable of bidirectional fluid flow, is disposed along a first fluid assembly line 442, and a second pump or circulation line pump 456, capable of bidirectional fluid flow, is disposed along an interconnection line 450, whose functions and purposes are described below. In one embodiment, pumps 454, 456 are high dynamic range pumps. As also shown in Figure 3, a sterile air source 458 is connected to the interconnection line 450 through a sterile air source line 460. A valve 462 positioned along the sterile air source line 460 provides selective fluid communication between the sterile air source 458 and the interconnection line 450. Figure 3 shows a sterile air source 458 connected to the interconnection line 450, but in other embodiments, the sterile air source may be connected to the first fluid assembly 440, the second fluid assembly 444, or the fluid flow path midway between the second bioreactor line valve and the first bioreactor line valve of either the first or second bioreactor.

[0061] Next, with reference to Figures 4–6, detailed diagrams of the first fluid assembly 440, the second fluid assembly 444, and the sampling assembly 448 are shown. Referring particularly to Figure 4, the first fluid assembly 440 comprises a plurality of tubing tails 464a–f, each configured to be selectively / removably connected to one of a plurality of first storage tanks 466a–f. Each tubing tail 464a–f of the first fluid assembly 440 comprises tubing tail valves 468a–f for selectively controlling the flow of fluid to or from each of the plurality of first storage tanks 466a–f of the first fluid assembly 440. Figure 4 specifically shows that the first fluid assembly 440 comprises six fluid storage tanks, but more or fewer storage tanks may be used for the input or collection of various treatment fluids as desired. Each tubing tail 464a to f is intended to be individually connected to a storage layer 466a to f for the duration required during the operation of the fluid assembly 440, as described below.

[0062] Referring particularly to Figure 5, the second fluid assembly 444 comprises a plurality of tubing tails 470a-d, each configured to be selectively / removably connected to one of the plurality of second storage tanks 472a-d. Each tubing tail 470a-d of the second fluid assembly 444 comprises tubing tail valves 474a-d for selectively controlling the flow of fluid of the first fluid assembly 444 to or from each of the plurality of second storage tanks 472a-d. Figure 5 shows in particular that the second fluid assembly 444 comprises four fluid storage tanks, but more or fewer storage tanks may be used for the input or collection of various treatment fluids as desired. In one embodiment, at least one of the second storage tanks, for example, the second storage tank 472d, is a collection storage tank for collecting amplified populations of cells, as described below. In one embodiment, the second storage tank 472a is a waste liquid storage tank, the purpose of which is described below. The present invention may involve one or more storage tanks 472a-d being pre-connected to their respective tails 470a-d, with each additional storage tank being intended to be connected to its respective tail within a second fluid assembly 440 in time for use.

[0063] In one embodiment, the first storage tanks 466a-f and the second storage tanks 472a-d are single-use / disposable flexible bags. In one embodiment, the bags are substantially two-dimensional bags, as known in the art, with opposing panels welded together or fixed together at their periphery and supporting connecting conduits for connecting to their respective tails.

[0064] In one embodiment, the storage tank / bag may be connected to the tubing tails of the first and second tubing assemblies using a sterile welding device. In one embodiment, the welding device is positioned next to module 200 and is used to butt weld one of the tubing tails to the tail of the tubing on the bag (while maintaining sterility). Thus, the operator can provide the bag when it is needed (for example, by grasping the tubing tail, inserting its free end into the welding device, laying the free end of the bag tube adjacent to the end of the tubing tail on the bag, cutting the tube with a new razor blade, and heating the cut end as the razor is pulled away while the two tube ends are forcibly brought together while they remain melted to re-solidify together). Conversely, the bag may be removed by welding a line from the bag and cutting the two closed lines at the weld. Therefore, the storage tanks / bags may be connected individually as desired, and the present invention does not require all storage tanks / bags to be connected at the start of the protocol, as the operator has access to the appropriate tubing tails throughout the process to connect the storage tanks / bags in time for their use. In fact, all storage tanks / bags are pre-connected as described below, but the present invention does not require pre-connection, and one advantage of the second module 200 is that it allows the operator to access the fluid assembly / line during operation, so that used bags can be sterilized and connected and other bags can be disconnected so that they can be connected in a sterile state throughout the protocol.

[0065] As illustrated in Figure 6, the sampling assembly 448 comprises one or more sampling lines, e.g., sampling lines 476a–476d, fluidly connected to the interconnection line 450. Each of the sampling lines 476a–476d may be equipped with sampling line valves 478a–476d that are selectively operable to allow fluid to flow from the interconnection line 450 through the sampling lines 476a–476d. As also shown there, the distal end of each sampling line 476a–476d is configured to selectively connect to a sampling device (e.g., sampling devices 280a and 280d) for collecting fluid from the interconnection line 450. The sampling device may take the form of any sampling device known in the art, such as a syringe, immersion tube, or bag. Figure 6 illustrates that the sampling assembly 448 is connected to an interconnection line, but in other embodiments, the sampling assembly may be fluid-coupled to a first fluid assembly 440, a second fluid assembly 444, an intermediate fluid passage between the second bioreactor line valve 434 and the first bioreactor line valve 432 of the first bioreactor vessel 410, and / or an intermediate fluid passage between the second bioreactor line valve 438 and the first bioreactor line valve 436 of the second bioreactor vessel 420. The sampling assembly 448 performs a fully functionally closed sampling of the fluid at one or more points within the system 400, as needed.

[0066] Referring again to Figure 3, in one embodiment, the system 400 may also include a filtration line 482 connected at two points along the interconnection line 450, defining a filtration loop along the interconnection line 450. A filter 484 is positioned along the filtration line 482 to remove permeate waste from the fluid passing through the filtration line 482. As shown therein, the filtration line 482 includes an upstream filtration line valve 486 and a downstream filtration line valve 488, positioned upstream and downstream of the filter 484, respectively. A waste line 490 provides fluid communication between the filter 484 and the second fluid assembly 444, and in particular, with the tubing tail 470a of the second fluid assembly 444, which is connected to the waste storage tank 472a. In this respect, the waste line 490 carries the waste removed by the filter 484 from the fluid passing through the filtration line 482 to the waste storage tank 472a. As illustrated in Figure 3, the filtration line 482 surrounds an interconnection line valve 452 so that the fluid flow through the interconnection line 450 can be forced through the filtration line 482, as described below. A permeate pump 492 positioned along the wastewater line 490 is operable to pump the wastewater removed by the filter to the wastewater storage tank 472a. In one embodiment, the filter 484 is preferably an elongated hollow fiber filter, but other tangential flow or direct AC filtration means known in the art, such as a flat sheet membrane filter, can also be used without departing from a broader aspect of the present invention.

[0067] In one embodiment, the valves of the first fluid assembly 440 and the second fluid assembly 444, as well as the bioreactor line valves (i.e., valves 432, 434, 436, 438), the sterilization line valve 462, the interconnection line valve 452, and the filtration line valves 486, 488 are pinch valves fabricated in the manner described below. In one embodiment, the line itself does not need to have pinch valves, and the pinch valve diagrams in Figures 3 to 8 may simply represent locations on the line where pinch valves can operate to prevent fluid flow. In particular, as described below, pinch valves in the fluid architecture 400 may be provided by respective actuators (e.g., solenoids) that act / activate on the corresponding anvil while the fluid path / line is in between, “pinching off” the line and preventing fluid flow from passing through it.

[0068] In one embodiment, pumps 454, 456, and 492 are peristaltic pumps, and these pumps are integrated into a single assembly as described below. Preferably, the operation of these valves and pumps is automatically commanded according to a protocol programmed to enable proper operation of module 200. A second controller 210 may be intended to command the operation of these valves and pumps by module 200.

[0069] Referring next to Figures 8 to 11, the configuration of a first bioreactor vessel 410 according to one embodiment of the present invention is illustrated. A second bioreactor vessel 420 is preferably, but not necessarily, identical in configuration to the first bioreactor vessel 410, so for simplicity, only the first bioreactor vessel 410 will be described below. In one embodiment, the bioreactor vessels 410, 420 are perfusion-responsive silicone membrane-based bioreactor vessels that support the activation, transduction, and amplification of a population of cells contained therein. The bioreactor vessels 410, 420 may be used for cell culture, cell processing, and / or cell amplification to increase cell density for use in medical treatment or other processes. Although the bioreactor vessels are disclosed herein as being used in conjunction with specific cell types, it should be understood that the bioreactor vessels may be used for the activation, genetic modification, and / or amplification of any suitable cell types. Furthermore, the disclosed techniques may be used in conjunction with adherent cells, i.e., cells that adhere to and / or proliferate on a cell amplification surface. In one embodiment, the first and second bioreactor vessels 410, 420 may be fabricated and function as disclosed in U.S. Patent Application No. 15 / 893,336 filed on 9 February 2018, which is incorporated herein by reference in its entirety.

[0070] As shown in Figures 8 and 9, the first bioreactor vessel 410 may comprise a bottom plate 502 and a vessel body 504 coupled to the bottom plate 502. The bottom plate 502 may be a rigid structure supporting the cell culture. However, the bottom plate may be a non-solid plate (e.g., open and / or porous) that is permeable to oxygen to be supplied to the cell culture, as described in more detail with reference to Figure 9. In the embodiments illustrated, the bottom plate 502 is rectangular or nearly rectangular in shape. In other embodiments, the bottom plate 502 may be any other shape that allows the use of a low-profile vessel and / or maximizes the space in which the first bioreactor vessel may be used or stored.

[0071] In one embodiment, the container body 504 comprises a rigid, generally concave structure that, when coupled to the bottom plate 502, forms a cavity or internal compartment 506 of the first bioreactor container 410. As shown therein, the container body 504 may have a circumferential shape similar to the circumferential shape of the bottom plate 502 so that the container body 504 and the bottom plate 502 can be coupled to each other. In addition, as in the embodiment illustrated, the container body 504 may be made of a transparent or translucent material that allows for visual inspection of the contents of the first bioreactor container 410 and / or allows light to enter the first bioreactor container 410. The internal compartment formed by the bottom plate 502 and the container body 504 may contain cell media and cell cultures when the first bioreactor container is used for cell activation, genetic modification (i.e., transduction), and / or cell amplification.

[0072] As best illustrated in Figures 8–11, the first bioreactor vessel 410 may have a number of ports through the vessel body 504 that can enable fluid communication between the internal compartment 506 and the outside of the first bioreactor vessel 410 for several processes related to cell activation, transduction / genetic modification, and amplification, such as culture medium loading and wastewater removal. The ports may include, for example, a first port 412 and a second port 416. Port 416 may be located at any position within the vessel body 504, such as through either the top surface 508 and / or side surface 510 of the vessel body 504, as in the illustrated embodiment. Specific structures of the first bioreactor vessel 410, including the specific number and location of ports 412, 416, as will be explained in more detail herein, enable the first bioreactor vessel 410 to be used to support cell activation, cell genetic modification, and high cell density amplification.

[0073] Figure 9 is an exploded view of one embodiment of the first bioreactor vessel 410. The bottom plate 502 of the first bioreactor vessel 410 may be the bottom or support of the first bioreactor vessel 410. As already described, the bottom plate 502 may be formed from a non-solid structure. In the embodiment shown, the bottom plate 502 may accommodate a grid 510 which is structurally rigid, but further provided with openings to allow free gas exchange through the bottom plate 502 to the internal compartment 506 which contains the cell culture. The grid 510 may comprise a plurality of holes 512 defined between solid regions or crossbars 514 between each hole 512 of the grid 510. Thus, the holes 512 may be openings for gas exchange, and the crossbars 514 may be structural supports for other structures and cell cultures within the internal compartment 506 of the first bioreactor vessel 410.

[0074] To form an additional support for the cell culture in the internal compartment 506 of the first bioreactor vessel 410, the first bioreactor vessel 410 may be equipped with a membrane 516 which can be disposed on the top surface 518 of the bottom plate 502. The membrane 516 may be a gas-permeable, liquid-impermeable membrane. The membrane 516 may also be selected to have properties that allow for high gas permeability, high gas transport rate, and / or high permeability to oxygen and carbon dioxide. Thus, the membrane 516 can be used to support high cell densities (e.g., about 35 mm / cm³) in the internal compartment 506. 2 It may support (up to). The gas permeability of membrane 516 may allow free gas exchange to support cell cultures and / or cell amplification. As such, membrane 516 may be a cell culture surface and / or cell amplification surface. Membrane 516 may have a relatively small thickness (e.g., 0.010 inches or 0.02 cm), which may allow membrane 516 to be gas permeable. Furthermore, membrane 516 may be formed from a gas permeable material such as silicone or other gas permeable material.

[0075] The flatness of the membrane 516 can increase the surface area of ​​the cell culture to be immobilized for activation, transduction, and / or amplification. To allow the membrane 516 to remain flat during use of the first bioreactor vessel 410, a mesh sheet 520 may be placed between the bottom plate 502 and the membrane 516. The mesh sheet 520 can act as a structural support for the membrane 516, thereby keeping the membrane 516 planar and preventing it from sagging or distorting under the weight of the cell culture and / or any cell medium added to the first bioreactor vessel 410 for cell culture and / or cell amplification. Furthermore, the mesh properties of the mesh sheet 520 may allow support for the membrane 516, but it is still porous, allowing free gas exchange between the internal compartment 506 of the first bioreactor vessel 410 and the environment just outside the first bioreactor vessel 410. The mesh sheet may be a polyester mesh or any other suitable mesh material that can support the membrane and allow free gas exchange.

[0076] As already described, the container body 504 can be coupled to the bottom plate 502 to form an internal compartment 506 of the first bioreactor container 410. As such, the mesh sheet 520 and membrane 516 can be disposed within, or at least partially within, the internal compartment 506. An O-ring 522 may be used to seal the first bioreactor container 410 when the container body 504 is coupled to the bottom plate 502. In one embodiment, the O-ring 522 may be a biocompatible O-ring (size 173, Soft Viton® Fluoroelastomer O-Ring). The O-ring 522 may be fitted into a groove 524 formed in the circumferential surface 526 of the container body 504. The circumferential surface 526 faces the top surface 518 of the plate 502 when the body 504 is fitted to the plate 502. As such, the O-ring 522 may be press-fitted into the groove 524 and pressed against the top surface 518 of the plate 516 and / or bottom plate 502. Such press-fitting of the O-ring 522 preferably seals the first bioreactor vessel 410 without bonding with chemicals or epoxy resins. Since the first bioreactor vessel 410 may be used for activation, transduction, and amplification of living cells, the O-ring 522 is preferably formed from a suitable biocompatible, autoclavable, gamma radiation stable, and / or ETO sterilization stable material.

[0077] As described above, the first bioreactor vessel 410 may have multiple ports, such as a first port 412 and a second port 416. The ports 412, 416 may be arranged through the vessel body 504 and may allow communication between the internal compartment 506 and the outside of the first bioreactor vessel 410 for several processes related to cell culture, cell activation, cell transduction, and / or cell amplification, such as fluid or culture medium infusion, wastewater removal, collection, and sampling. Each port 416 may have an opening 526 and its respective fitting or tubing 528 (e.g., a Luer fitting, a barb fitting, etc.). In some embodiments, the opening 526 may be configured to allow direct bonding of the tubing, eliminating the need for a fitting (e.g., a counterbore).

[0078] In one embodiment, in addition to the first port 412 and the second port 416, the first bioreactor vessel 410 may further comprise an air balance port 530 disposed within the top surface 508 of the vessel body 504. The air balance port 530 may be fabricated in a similar manner to the first port 412 and the second port 416, and similar reference numbers represent similar parts. The air balance port 530 may further facilitate gas exchange between the internal compartment 506 and the outside of the first bioreactor vessel 410, which is used for cell culture for amplification. Furthermore, the air balance port 530 may help maintain atmospheric pressure within the internal compartment 506 to provide an environment within the internal compartment for cell culture and / or cell amplification. The air balance port 530 may be disposed through the top surface 508 of the vessel body 504, as in the embodiment illustrated, or at any other location around the vessel body 504. The central position passing through the top surface 508 of the container body 504 can help prevent wetting of the air balance port 530 when mixing the cell culture through the incline of the first bioreactor container 410, as will be described in more detail below.

[0079] Each element of the first bioreactor vessel 410, including the bottom plate 502, the vessel body 504, the ports 412, 416, and 530, the membrane 516, the mesh sheet 520, and the O-ring 522, may be made from a material that is biocompatible, autoclavable, and has gamma-ray and / or ETO sterilization stability. As such, each element, and the first bioreactor vessel 410 as a whole, may be used for the activation, transduction, and amplification of living cells, and / or other processes in the cell manufacturing process.

[0080] The first bioreactor vessel 410 may be configured to allow cell culture and / or cell amplification via perfusion, which can provide the nutrients necessary to support cell growth and reduce impurities in the cell culture. Continuous perfusion is the addition of a fresh medium supply to the growing cell culture while simultaneously removing used medium (e.g., used medium). The first port 412 and the second port 416 may be used in the perfusion process as described below. The first port 412 may be configured to allow communication between the internal compartment 506 and the outside of the first bioreactor vessel 410, and may be used to add fresh medium to the first bioreactor vessel 410 (e.g., from a medium storage tank of the first fluid assembly 440). In some embodiments, the first port 412 may be located within the vessel body 504 at any position above the surface of the cell culture and medium within the first bioreactor vessel 410 and may penetrate it. In some embodiments, the first port 412 may be positioned to contact or penetrate the surface of the cell culture and culture medium within the first bioreactor vessel 410.

[0081] The second port 416 may be positioned at any location where it is fully or partially submerged below the surface of the cell culture and culture medium within the first bioreactor vessel 410. For example, the second port 416 may be a nearly lateral port positioned through one of the sides 510 of the vessel body 504. In some embodiments, the second port 416 may be positioned so that it does not reach the bottom of the internal compartment 506 (e.g., the membrane 516). In some embodiments, the second port 416 may reach the bottom of the internal compartment 506. The second port 416 may be a dual-function port. As such, the second port may be used to draw the perfusion medium from the internal compartment 506 of the first bioreactor vessel 410 to facilitate perfusion of the cell culture. Furthermore, the second port 416 may also be used to remove cells from the cell culture. As noted above, in some embodiments, the second port may not reach the bottom of the internal compartment 506 of the first bioreactor vessel 410. For example, the second port 416 may be located about 0.5 cm away from the membrane 516. Thus, in a static planar position, the second port 416 may be used to remove used cell medium without pulling the cells out of the cell culture, as the cells may adhere to the membrane 516 (e.g., the cell amplification surface) by gravity. Thus, in a static planar position, the second port 416 may facilitate the perfusion process and allow for an increase in the cell density of the growing cell culture in the first bioreactor vessel 410. When it is desirable for the cells to be removed from the internal compartment 506, for example during cell culture harvesting, in order to minimize the hold-up volume, the first bioreactor vessel 410 may be tilted toward the second port 416 in the manner described below, thereby allowing access to the cells for cell removal.

[0082] In addition, in one embodiment, the second port 416 does not include a filter, and therefore the perfusion process can be carried out without a filter. In such a case, there can be no physical obstruction preventing cells from entering the second port 416 when the second port 416 is used for culture medium removal. Furthermore, the second port 416 may be positioned laterally through the side 22 of the container body 504, but inclined so that the second port 416 can tilt toward the membrane 516 and bottom plate 502. The inclined feature of the second port 416 may allow the second port 416 to be positioned at a relatively low position on the container body 504, closer to the membrane surface 36, while minimizing interference with the O-ring 522 and groove 524 and helping to maintain the seal of the first bioreactor container 410 during use. Furthermore, in some embodiments, the inclined feature of the second port 416 may be such that it reduces the fluid velocity through the second port 416 when the used medium is withdrawn. In addition, the port diameter may be such that, together with the fluid velocity exiting the second port 416, the aspiration velocity through the second port 416 used to withdraw medium from the external compartment 506 is minimized so that the suction force applied to individual cells adjacent to the second port 416 is lower than the gravitational force pulling the cells toward the membrane 516. Thus, as described above, the second port 416 may be used to withdraw the perfusion medium and facilitate the perfusion of the cell culture without substantially removing the cells from the cell culture. As the cell settlement time increases, the cell concentration in the withdrawn medium decreases and may fall within an immeasurable range facilitated by the position of the second port 416. Furthermore, the position of the internal opening 540 may be changed to modify the recommended cell settlement time. Locations closer to membrane 516 may be associated with longer settlement times, while locations at or near the top of the culture medium are associated with shorter settlement times because cells settle and deplete the growth medium first from the top.

[0083] Therefore, in one embodiment, the second port 416 may be used not only for removing the culture medium used in the perfusion process, but also, for example, for removing cells from the cell culture from the internal compartment 506 during cell culture harvesting. To facilitate the removal of more of the perfused medium used and the cells, the container body 504 may be provided with angled or chevron-shaped sidewalls 532. Thus, the chevron-shaped sidewall 532 has a vertex, or tip, 534. The vertex 534 of the sidewall 532 may further provide a second port 416 passing through it, and the container body 504 is positioned near the tip 534 when the container body 504 is coupled to the bottom plate 502. The angled sidewall 532 and tip 534 may allow for the discharge of larger quantities of the culture medium and / or cells from the cell culture when the first bioreactor container 410 is tilted toward the second port 416, for example at an angle of 5 degrees.

[0084] By using perfusion to promote cell growth, facilitated by the positions of the first port 412 and the second port 416, it may be possible to reduce the height of the culture medium within the internal compartment 506 (e.g., 0.3–2.0 cm), as described in more detail with reference to Figure 10. The relatively low height of the culture medium within the internal compartment 506 may allow the first bioreactor vessel 410 to be a relatively low-profile vessel while enabling an increase in the highest possible cell density. Furthermore, using perfusion with the first bioreactor vessel 410 supports cell growth by supplying fresh culture medium to the cells in the internal compartment 506, but also allows for the removal of impurities from the cell culture, and additional cell washing in another device may not be necessary after a specific cell density target has been reached within the first bioreactor vessel 410. For example, through filterless perfusion, the first bioreactor vessel 410 may be supplied with fresh medium at a rate of total replacement every day, thereby reducing impurities in the cell culture (e.g., resulting in a reduction of impurities at a rate of approximately 1 log every 2.3 days). Thus, the structure of the first bioreactor vessel 410 may allow the use of perfusion for the growth of the cell culture within the first bioreactor vessel 410, and thus allow the cell culture to be amplified to a high target density while reducing impurity levels. Also, as described below, through filterless perfusion, the first bioreactor vessel 410 may be supplied with fresh medium at a rate of substantially multiple times per day (e.g., more than twice per day) for cell seeding, rinsing, washing / residue reduction, and / or discharge / harvesting after amplification.

[0085] To facilitate the low-profile structure of the first bioreactor vessel 410, a relatively low culture medium height can be maintained within the internal compartment 506. Figure 10 is a cross-sectional view of the first bioreactor vessel 410 illustrating the height 536 of the cell culture medium 538 within the first bioreactor vessel 410. As already described, the vessel body 504 is connected to the bottom plate 502 and can form an internal compartment 506 in which the amplification of the cell culture can be achieved through perfusion. For example, replacement or fresh culture medium 538 may be supplied for cell proliferation through a first port 412 located through the vessel body 504, and existing or used culture medium 538 may be removed through a second port 416 located through the side 510 of the vessel body 504. The perfusion process can facilitate the relatively low culture medium height 536 of the culture medium 538 within the internal compartment 506 of the first bioreactor vessel 410. The relatively low height 536 of the perfusion medium 538 within the internal compartment 506 allows for a low-profile structure of the first bioreactor vessel 410, and thus, it may be possible to realize a compact cell manufacturing system overall.

[0086] The height 536 of the perfusion medium 538 in the internal compartment 506 of the first bioreactor vessel 410 may be between 0.3 cm and 2 cm, and the height of the headroom 542, i.e., the gap formed between the medium 538 and the top surface 508 of the vessel body 504 in the internal compartment 506, may be about 2 cm. Thus, including the medium, cell culture, and headspace, 1 cm 2 Less than 2 mL of culture medium and 1 cm 2The total volume per unit may be less than 4 mL. A relatively low medium height 536 may allow the ratio of medium volume to the surface area of ​​the membrane 516 to be lower than a specific value. In such a case, the ratio of medium volume to membrane surface area may be below a threshold level or within a desirable range, making it easier to use by perfusion for growing cells in the cell culture. For example, the threshold level may be a ratio between 0.3 and 2.0. Because the ratio of medium volume to membrane surface area is small, it may be possible to make the first bioreactor vessel 410 low-profile or compact in structure while still obtaining cell cultures with high cell density.

[0087] As already described, a dual-function second port 416 may be positioned through the container body 504 so as to be fully or partially submerged below the surface 544 of the culture medium 538 in the first bioreactor container 410. In some embodiments, the second port 416 may be positioned so as to reach the bottom of the internal compartment 506 (e.g., the membrane 516). Positioning of the second port 416 may facilitate the removal of culture medium and impurities from the cell culture in the internal compartment 506 without removing the cells until such removal, e.g., harvesting, is desired. A second port 416 without a filter, together with the first port 412, may allow the use of perfusion to supply the growth medium 538 to cells for cell amplification and to remove the used culture medium 538 and other impurities or by-products. The positions of the first port 412 and the dual-function second port 416 around the container body 504 facilitate a configuration in which the height 536 of the culture medium in the internal compartment 506 is maintained at a relatively low level, thereby enabling the first bioreactor container 410 to be a relatively low-profile container while still allowing for the production of high-density cell cultures.

[0088] Referring particularly to Figure 11, the bottom plate 502 of the bioreactor vessel 410 is provided as part of a broader bioprocessing system 10, which includes various features enabling the use of the bioreactor vessel, and in particular, as a second module 200 of the bioprocessing system 10. As shown therein, the bottom plate 502 includes a plurality of recesses 550 formed within the bottom surface of the bottom plate 502, the purpose of which is described below. In one embodiment, the recesses may be located adjacent to the corners of the bottom plate 502. Each recess 550 generally takes a cylindrical shape and may terminate at a dome-shaped or hemispherical inner surface. As also shown in Figure 11, the bottom plate 502 may include a position verification structure 552 configured to interact with sensors of the second module 200 and ensure proper positioning of the first bioreactor vessel 410 within the second module 200. In one embodiment, the position verification structure may be a beam interruption configured to block the light beam of the second module 200 when the first bioreactor container 410 is properly installed inside it.

[0089] The bottom plate 502 also includes a pair of flat engaging surfaces 554 formed on adjacent bottom surfaces, offset from the centerline of the bottom plate (across the width of the bottom plate). Preferably, the engaging surfaces 554 are spaced apart along the longitudinal centerline of the bottom plate 502 so as to be positioned adjacent to opposing ends of the bottom plate 502. The bottom plate 502 may further include at least one opening or opening 556 that engages with and allows a bioprocessing device operating the bioreactor vessel to sense the contents of the first bioreactor vessel 410.

[0090] In one embodiment, the first and second bioreactor vessels 410, 420 and the fluid architecture 400 can be integrated into an assembly or kit 600 in the manner disclosed below. In one embodiment, the kit 600 is a single-use, disposable kit. As best shown in Figures 12 to 14, the first bioprocessing vessel 410 and the second bioprocessing vessel 420 are received side by side in a tray 610 of the disposable kit 600, and the various tubes of the fluid architecture 400 are arranged and configured in the tray 610 in the manner described below.

[0091] Referring further to Figure 15, the tray 610 comprises a number of generally thin, rigid, or semi-rigid side walls, including a front wall 612, a rear wall 614, and opposing sides 616, 618, encircling the bottom surface 620 and the normally open top. The side walls and bottom surface 620 define the internal compartment 622 of the tray 610. In one embodiment, the open top of the tray 610 is surrounded by a circumferential flange 624 that receives a removable cover (not shown) encircling the internal compartment 622, as shown below, and further provides a surface for preferably mounting on the upper rim of a drawer of a bioprocessing apparatus. The bottom surface 620 of the tray 610 comprises a number of openings corresponding to the number of bioreactor vessels in the bioprocessing system. For example, the tray 610 may have a first opening 626 and a second opening 628. The bottom surface 620 may also include an additional opening 630 adjacent to the first and second openings 626, 628 for the purposes described below. In one embodiment, the tray 610 may be thermoformed, 3D printed, or injection molded, but other manufacturing techniques and processes may also be used without departing from a broader aspect of the present invention.

[0092] As best shown in Figure 15, each of the first and second openings 626, 628 has a perimeter of a shape and / or dimensions such that the first and second bioreactor containers 410, 420 are positioned above the respective openings 626, 628 and supported by the bottom surface 620 of the tray 610 within the internal compartment 622, while a portion of the bioreactor containers 610, 620 remains accessible from the bottom of the tray 610 through the respective openings 626, 628. In one embodiment, the perimeter of an opening is provided with at least one claw or projection for supporting the bioreactor container above the respective opening. For example, the perimeter of each opening 626, 628 may be provided with a claw 632 projecting inward toward the center of the opening 626, 628 for supporting the bioreactor container 410, 420 placed thereon. As shown in Figures 12 and 15, the tray 610 may also include one or more protrusions extending upward over the respective openings 626, 628 to prevent lateral movement of the bioreactor containers when they are received over the openings 626, 628. Thus, the protrusions act as alignment devices to facilitate the proper positioning of the bioreactor containers 410, 420 within the tray 610 and help prevent accidental movement of the bioreactor containers 410, 420 when loading or positioning the kit 600 within the second module 200, as described below.

[0093] Referring further to Figures 12 and 13, the tray 610 may comprise one or more support ribs 636 formed on the bottom surface of the tray 610. The support ribs 636 traverse the width and / or length of the tray 610, providing rigidity and strength to the tray 610 and facilitating the movement and operation of the kit 600. The ribs 636 may be integrally formed with the tray or may be added as auxiliary components via mounting means known in the art (see Figure 13). In one embodiment, the tray 610 comprises an opening 638 for receiving an engagement plate, a tubing module 650, which passes through it, holding fluid flow lines in an organized manner and holding them in place for engagement by the pump and pinch valve. In another embodiment, the tubing module 650 may be integrally formed with the rear wall 614 of the tray 610.

[0094] Figures 16 and 17 illustrate the configuration of a tubing module 650 according to one embodiment of the present invention. As shown therein, the tubing module 650 includes a first tubing holder block 652 configured to receive and hold in place the first fluid assembly line 442, interconnect line 450, and waste line 490 of a fluid flow system 400, so as to selectively engage with the respective pump heads 454, 456, and 492 of a peristaltic pump assembly described below with respect to Figures 35 and 36. In one embodiment, the fluid assembly line 442, interconnect line 450, and waste line 490 are maintained by the first tubing holder block 652 in an orientation that extends horizontally and is spaced apart vertically. In particular, as best shown in Figure 17, the first tubing holder block 652 engages with lines 442, 450, and 490 respectively through two spaced-apart arrangements 656 and 658 (through clips or simple interference between the tubes and slots within the tubing holder block 652, etc.) that define a gap between them. Also shown in Figure 17, the first tubing holder block 652 includes a clearance opening 660 configured to receive the shoe (not shown) of a peristaltic pump assembly. This configuration causes peristaltic compression of the shoe of lines 442, 450, and 490 by the respective pump heads of the peristaltic pumps, which can provide the respective propulsion force for the fluid through the lines, as described below.

[0095] Referring further to Figures 16-18, the tubing module 650 further comprises a second tubing holder block 654 which is integrally formed with (or otherwise somehow coupled with) the first tubing holder block 652. The second tubing holder block 654 is configured to receive all of the fluid flow lines of the fluid flow system 400 to which the pinch valve is associated. For example, the second tubing holder block 654 is configured to hold the tubing tails 464a-f of the first fluid assembly 440, the tubing tails 470a-d of the second fluid assembly 444, the first bioreactor line 414 and the second bioreactor line 418 of the first bioreactor container 410, the first bioreactor line 424 and the second bioreactor line 428 of the second bioreactor container 420, the sterile air source line 460, the interconnection line 450, and the filtration line 482 (and, in some embodiments, the sampling lines 476a-476d). Similar to the first tubing holder block 652, the second tubing holder block 654 may be configured to maintain these tubes in an orientation that extends horizontally and is spaced apart vertically. In particular, the second tubing holder block 654 may have a plurality of vertically spaced and horizontally extending slots 666 configured to receive lines into it. Figures 18 and 19 also best illustrate a configuration of slots 666 that hold all flow lines that are acted upon by / contact with a pinch valve. Preferably, the slots 666 follow the contour of the block 654, but in particular traverse the planar backplate so as to open toward the filter 484. As shown in Figure 18, in one embodiment, the second tubing holder block 654 may have one or more narrow tubing slots 682 at the bottom of the second tubing holder block 654 to hold a loop of interconnection lines 450 from which sampling lines extend, and a wastewater line tubing slot 684 for receiving a tubing tail 470a connected to a wastewater storage tank 472a.

[0096] The second tubing holder block 654 may comprise a planar backplate 662 having a plurality of openings 664 corresponding to a plurality of fluid flow lines held by the second tubing holder block 654. In particular, at least one opening 664 is horizontally aligned with each slot 666 and the flow line held therein. As best shown in Figure 16, the second tubing holder block 654 comprises two clearance openings 668, 670 configured to receive an anvil (not shown) of a pinch valve assembly through which it passes. This configuration allows the tubing tails 464a-f of the first fluid assembly 440, the tubing tails 470a-d of the second fluid assembly 444, the first bioreactor line 414 and the second bioreactor line 418 of the first bioreactor vessel 410, the first bioreactor line 424 and the second bioreactor line 428 of the second bioreactor vessel 420, the sterile air source line 460, the interconnection line 450, and the filtration line 482 to be selectively compressed against the anvil by the respective pistons of the actuators of the pinch valve array, thereby selectively obstructing or allowing fluid flow. As shown in Figures 18 and 19, the openings 664 may be configured to be arranged in first and second adjacent rows, with the openings in the first row being vertically offset with respect to the openings on the second row such that the openings in the first row are not horizontally aligned with the openings in the second row. This configuration allows the tubing module 650, tray 610, and kit 600 to have a low profile as a whole.

[0097] In one embodiment, the filter 484 (shown as an elongated hollow fiber filter module in Figure 16) may be integrated with the tubing module 650, for example, by attaching the filter 484 to the tubing module 650 using retaining clips 672. If the filter 484 is a hollow fiber filter, the filter 484 may extend substantially over the entire length of the tubing module 650 and may comprise a first input end 674 for receiving the inlet flow of fluid from the filtration line 482 and a second output end 676 for carrying and returning the retained fluid after removing the permeate / waste liquid to the filtration line 482 and interconnection line 450 and circulating it to one of the first bioreactor vessel 410 or the second bioreactor vessel 420. The filter 484 may also comprise a permeate port 678 adjacent to the second output end 676, which connects to a waste liquid line 490 for carrying the waste liquid / permeate to the permeate / waste liquid storage tank 472a. Finally, the tubing module 650 may have multiple features 680 for receiving clips and organizing bioreactor lines (for example, the first and second bioreactor lines 414, 418 of the first bioreactor vessel 410 and / or the first and second bioreactor lines 424, 428 of the second bioreactor vessel 420).

[0098] Similar to the tray 610, the tubing module 650 may be thermoformed, 3D printed, or injection molded, but other manufacturing techniques and processes may also be used without departing from broader aspects of the present invention. As described above, in one embodiment, the tubing module 650 may be integrally formed with the tray 610. In other embodiments, the tubing module 650 may be a separate component that is removably received by the tray 610.

[0099] Figures 20–22 are various diagrams illustrating one embodiment of Kit 600, which illustrate the first bioreactor container 410 and the second bioreactor container 420, which are received in a tray 610, as well as the fluid lines of the fluid architecture 400, which are received by the tubing module 650. Instead of having an opening 630 as illustrated therein, Kit 600 as shown in Figures 20–22 has a solid bed and a sampling space 631 in the tray 610 to receive containers that hold sampling lines (e.g., sampling lines 476a, 476b). Kit 600 features a modular platform for cell processing that can be easily set up and discarded after use. The tubing tails of the first and second fluid assemblies 440, 444 allow for plug-and-play functionality, enabling quick and easy connection of various media, reagents, waste liquids, sampling, and collection bags, and allowing various processes to be performed on a single platform. In one embodiment, connection and disconnection may be performed by sterilizing and cutting and welding the tube segments using a TERUMO device or the like, as described above, or by pinching, welding, and cutting the tail segments as known in the art.

[0100] Referring next to Figures 23-25, the kit 600 is specifically configured to be received by a bioprocessing apparatus 700 that houses all the hardware (i.e., controller, pump, pinch valve actuator, etc.) necessary to operate the kit 600 as part of a bioprocessing method. In one embodiment, the bioprocessing apparatus 700 and the kit 600 (housing the fluid architecture 400 and bioreactor vessels 410, 420) together form a second bioprocessing module 200 as described above in relation to Figures 1 and 2. The bioprocessing apparatus 700 comprises a housing 710, which has several drawers 712, 714, 716 that can be received within the housing 710. Figure 23 shows the apparatus 700 housing three drawers, but the apparatus may have as few as one single drawer, two drawers, or more than three drawers, allowing bioprocessing operations to be performed simultaneously within each drawer. In particular, in one embodiment, each drawer 712, 714, 716 may be a standalone bioprocessing module for performing processes of cell activation, gene modification, and / or amplification (i.e., equivalent to the second modules 200a, 200b, and 200c described above with respect to Figure 2). In this regard, any number of drawers may be added to the apparatus 700 to process multiple samples from the same or different patients in parallel. In one embodiment, rather than each drawer sharing a common housing, each drawer may be housed in a dedicated housing, and the housings may be stacked on top of each other.

[0101] As shown in Figures 23 and 24, each drawer, for example, drawer 712, comprises a plurality of side walls 718 and a bottom surface 720 defining the processing chamber 722 and a normally open top. Drawer 712 is movable between a closed position in which the drawer is fully received within the housing 710, as shown for drawers 714 and 716 in Figure 23, and an open position in which the drawer 712 extends from the housing 710 and allows access to the processing chamber 722 through the open top, as shown for drawer 712 in Figures 23 and 24. In one embodiment, one or more of the side walls 718 are temperature-controlled to control the temperature inside the processing chamber 722. For example, one or more of the side walls 718 may be equipped with or thermally communicate with an embedded heating element (not shown), so that the side walls 718 and / or the processing chamber 722 may be heated to a desired temperature to maintain the processing chamber 722 at a desired temperature (e.g., 37°C) optimized for the process steps to be performed by module 200. In some embodiments, the bottom surface 720 and the underside of the top surface of the housing (above the processing chamber when the drawer is closed) may be temperature controlled in a similar manner (e.g., with an embedded heating element). The hardware compartment 724 of the drawer 712 behind the processing chamber 722 may house all of the hardware components of the apparatus 700, as described in detail below. In one embodiment, the drawer 712 may further include an auxiliary compartment 730 adjacent to the processing chamber 722 for housing storage tanks containing culture media, reagents, etc., connected to a first fluid assembly 440 and a second fluid assembly 444. In one embodiment, the auxiliary compartment 730 can be refrigerated.

[0102] Each drawer, for example, drawer 712, can be slidably received on opposing guide rails 726 mounted inside the housing 710. A linear actuator can be operably connected to drawer 712 to selectively move drawer 712 between an open position and a closed position. The linear actuator is operable to cause smooth and controlled movement of drawer 712 between the open and closed positions. In particular, the linear actuator is configured to open and close drawer 712 at a substantially constant speed (and with minimal acceleration and deceleration when stopping and starting operation) to minimize disturbance to the contents of the bioreactor vessel.

[0103] Figure 25 is a top view showing the interior of the drawer 712, including the processing chamber 722, hardware compartment 724, and auxiliary compartment 730. As illustrated therein, the hardware compartment 724 is located behind the processing chamber 722 and comprises a power supply 732, an operation control board and drive electronics 734 which are integrated with or otherwise communicate with a second module controller 210, a low-power solenoid array 736, a pump assembly 738 (including pump heads for pumps 454, 456, and 492), and a drawer engagement actuator 740. The hardware compartment 724 of the drawer 712 further comprises a pump shoe 742 and a pair of pinch valve anvils 744 for interlocking with the pump assembly 738 and the solenoid array 736, respectively, as described below. In one embodiment, the pump shoe 742 and the solenoid anvils 744 are fixed to the front base plate (front plate) of the processing chamber. All hardware compartments (and components described herein) are mounted to the rear base plate. Both plates are slidably mounted to rails. Furthermore, a drawer engagement actuator 740 is used to connect the two plates and move the two plates and the components mounted on them to the engagement position (moving the pump roller head into the pump shoe, thereby compressing the pump tubing when inserted between them). As further described herein, the pump assembly performs selective operation on lines 442, 450, and 490 of the fluid path 400, resulting in independent peristaltic thrusts for each. Similarly, the tubing holder block 654 of tray 600 will be positioned between the solenoid array 736 and the anvil 744, as further described herein.

[0104] As illustrated in Figure 25, two bed plates, for example, first and second bed plates 746, 748, are positioned within the processing chamber 722 on the bottom surface 720 and extend upward or protrude from there. In one embodiment, the processing chamber 722 may house a single bed plate or three or more bed plates. The bed plates 746, 748 are configured to receive the first bioreactor vessel 410 and the second bioreactor vessel 420 on top of them or to engage them in any other way. As also shown in Figure 25, the drawer 712 also includes a plate 750 configured with load cells positioned adjacent to the bed plates 746, 748 in the processing chamber 722 to sense the weight of a storage layer, for example, a waste liquid storage tank 472a positioned above it.

[0105] Figures 26–28 best illustrate the configurations of bed plates 746, 748, and Figure 28A shows hardware components positioned beneath the bed plates. As used herein, the bed plates 746, 748 and hardware components (i.e., sensors, motors, actuators, etc., integrated with or positioned beneath them as shown in Figure 28A) may be collectively referred to as the bed plates. The first and second bed plates 746, 748 are substantially identical in configuration and operation, but for simplicity, the following description of bed plates 746, 748 will refer only to the first bed plate 746. The bed plates 746, 748 generally have a substantially planar top surface 752 having a shape and surface area corresponding to the shape and area of ​​the bottom plate 502 of the first bioreactor vessel 410. For example, the bed plates may generally be rectangular in shape. The bed plates 746, 748 also typically include raised or clearance areas 758 corresponding to the positions of protrusions or tabs 632 of the tray 610, the purpose of which is described below. The bed plates 746, 748 are supported by a plurality of load cells 760 (for example, four load cells 760 positioned under each corner of the bed plate 746). The load cells 760 are configured to sense the weight of the first bioreactor vessel 410 in bioprocessing for use by the controller 210.

[0106] In one embodiment, the bed plate 746 may be equipped with an embedded heating element or be thermally in communication with a heating element so that the contents of the processing chamber 722 and / or the first bioreactor container 410 placed thereon can be maintained at a desired temperature. In one embodiment, the heating element may be the same as, or different from, the heating elements that heat the side walls 718, the top wall, and the bottom surface.

[0107] As illustrated, the bed plate 746 is provided with a plurality of positioning pins or alignment pins 754 protruding from the top surface 452 of the bed plate 746. The number of positioning pins 754 and their positions and spacing may correspond to the number, positions, and spacing of recesses 550 in the bottom surface of the bottom plate 502 of the bioreactor vessels 410, 420. As shown below, the positioning pins 754 are receivable in the recesses 550 of the bottom plate 502 of the first bioreactor vessel 410 when the first bioreactor vessel 410 is positioned in the processing chamber 722 to ensure proper alignment of the first bioreactor vessel 410 on the first bed plate 746.

[0108] Referring further to Figures 26-28, the bed plate 746 may further comprise an integrated sensor 756 for detecting proper alignment (or misalignment) of the first bioreactor vessel 410 on the first bed plate 746. In one embodiment, the sensor 756 is an infrared light beam, but other sensor types, such as a lever switch, may also be used without departing from a broader aspect of the present invention. The sensor is configured to interact with a position verification structure 552 on the bottom plate 502 when the first bioreactor vessel 410 is properly seated on the first bed plate 746. For example, if the sensor 756 is an infrared light beam and the position verification structure 552 is a beam interruption (i.e., a flat claw), and a substantially IR-impermeable position verification structure 552 is used, the beam interruption will block (i.e., interrupt the beam) the infrared light beam when the first bioreactor vessel 410 is fully seated on the bed plate 746. This signals to the controller 210 that the first bioreactor vessel 410 is properly installed. If, after positioning the first bioreactor vessel 410 on the first bed plate 746, the controller does not detect an interruption in the infrared light beam of the sensor 756, this indicates that the first bioreactor vessel 410 is not fully or properly installed on the bed plate 746 and requires adjustment. Thus, the sensor 756 on the bed plate 746 and the position verification structure 552 on the bottom plate 502 of the first bioreactor vessel 410 ensure that the first bioreactor vessel 410 is installed in a horizontal position on the bed plate 746 (as determined by the alignment pins) before starting bioprocessing.

[0109] Referring further to Figures 26 to 28A, the bed plate 746 also includes an embedded temperature sensor 759 positioned to align with an opening 556 in the bottom plate 502 of the first bioreactor vessel 410. The temperature sensor 759 is configured to measure or sense one or more parameters within the bioreactor vessel 410, such as the temperature level within the bioreactor vessel 410. In one embodiment, the bed plate 746 may also include a resistance temperature detector 760 configured to measure the temperature of the top surface 752 and a carbon dioxide sensor (located below the bed plate) for measuring the carbon dioxide level within the bioreactor vessel.

[0110] As further shown in Figures 26 to 28A, each bed plate 746, 748 includes an actuator mechanism 761 (e.g., a motor) comprising, for example, a pair of opposing cam arms 762. The cam arms 762 are received in slots 764 of the bed plates 746, 748 and are rotatable around a cam pin 766 between a clearance position where the cam arms 762 are positioned below the top surface 752 of the bed plate 746 and an engagement position where the cam arms 762 extend above the top surface 752 of the bed plate and contact opposing flat engagement surfaces 554 of the bottom plate 502 of the first bioreactor vessel 410 when the first bioreactor vessel 410 is received on the first bed plate 746. As described in detail below, the actuator mechanism is operable to tilt the bioreactor vessel on the bed plate to agitate and / or assist in discharging from the bioreactor vessel.

[0111] Referring to Figures 29 to 32, more detailed diagrams of the linear actuator 768 and the drawer engagement actuator 740 within the hardware compartment 724 of the drawer 712 are shown. Referring to Figure 29, as shown above, the linear actuator 768 is operable to move the drawer 712 between an open position and a closed position. In one embodiment, the linear actuator 768 is electrically connected to a rocker switch 770 on the outside of the housing 710, which allows user control of the drawer's movement. The linear actuator 770 controls the movement of the drawer 712 to prevent disturbance of the contents of the bioreactor container within the drawer 712. In one embodiment, the linear actuator 768 has a stroke of about 16 inches and a maximum speed of about 2 inches per second.

[0112] Referring next to Figure 30, the drawer engagement actuator 740 comprises a feed screw 772 and a clevis arm 774 attached to a front plate 751 in the drawer 712. The drawer engagement actuator is operably connected to the pump assembly 738 and the solenoid array 736 and is operable to move the pump assembly 738 and the solenoid array 736 between a first clearance position and an engagement position.

[0113] Figures 31 and 32 show the clearance and engagement positions of the pump assembly 738 and solenoid array 736 in a relatively clear manner. As illustrated in Figure 31, in the clearance position, the pump assembly 738 and solenoid array 736 are separated from the pump shoe 742 and pinch valve anvil 744, respectively. After the feed screw 772 is actuated, the drawer engagement mechanism 740 moves the pump assembly 738 and solenoid array linearly forward to the position shown in Figure 32. In this position, the pump head of the pump assembly 738 engages with lines 442, 450, and 490 in the first tubing holder block 652, and the solenoid array 736 is positioned close enough that the piston / actuator of the solenoid array 736 can pinch / tighten each of the fluid flow lines of the second tubing holder block 654 against the pinch valve anvil 744, thereby obstructing the flow through that fluid flow line.

[0114] Referring again to Figure 24, and further to Figures 33–39, during operation, the drawer 712 can be controllably moved to the open position by activating a rocker switch 770 on the outside of the housing 710. A disposable drop-in kit 600 containing the tubing module 650 (holding all the tubing and tubing tails of the fluid architecture 400) and the first and second bioreactor vessels 410, 420 is lowered into place in the processing chamber 722. Once the kit 600 is lowered into the processing chamber 722, the pump shoe 742 is received through the clearance opening 660 of the first tubing holder block 652 so that the pump tubing 442, 450, 490 are positioned between the pump shoe 742 and the pump heads 454, 456, 492 of the peristaltic pump assembly 738. Figure 35 is a perspective view of the peristaltic pump assembly 738 showing the positioning of the pump heads 454, 456, 492 relative to each other. Figure 36 illustrates the positioning of pump heads 454, 456, and 492 relative to pump tubes 442, 450, and 490 when the kit 600 is received into the processing chamber 722. As shown therein, the pump tubes 442, 450, and 490 are positioned between the pump shoe 742 and the pump heads 454, 456, and 492. During operation, when the drawer engagement actuator 740 positions the pump assembly 738 in the engagement position, the pump heads 454, 456, and 492 are selectively actuated under the control of the controller 210 to start, maintain, and stop the flow of fluid through the tubes 442, 450, and 490.

[0115] Similarly, as the kit 600 is lowered into the processing chamber 722, the pinch valve anvil 744 is received through the clearance openings 668, 670 of the second tubing holder block 654 so that the tubing tails 464a-f of the first fluid assembly 440, the tubing tails 470a-d of the second fluid assembly 444, the first bioreactor line 414 and the second bioreactor line 418 of the first bioreactor container 410, the first bioreactor line 424 and the second bioreactor line 428 of the second bioreactor container 420, the sterile air source line 460, the interconnection line 450, and the filtration line 482 are positioned between the solenoid array 736 and the pinch valve anvil 744. This configuration is best illustrated in Figures 37-39 (Figures 37 and 38 illustrate the relationship between the solenoid array 736 and the pinch valve anvil 744 before the second tubing holder block 654 receives the back plate 662 in space 776).

[0116] As shown therein, each solenoid 778 of the solenoid array 736 comprises a piston 780 that is linearly extendable through the associated opening (of the opening 664) in the backplate 662 of the second tubing holder block 654 to clamp the associated tube to the pinch valve anvil 744. In this respect, the solenoid array 736 and the anvil 744 together form a pinch valve array (which includes the valves of the first fluid assembly 440 and the second fluid assembly 444, as well as the bioreactor line valves, i.e., valves 432, 434, 436, 438, sterilization line valve 462, interconnection line valve 452, and filtration line valves 486, 488). In particular, the pinch valves of the fluid architecture 400 are provided by the respective solenoids 778 of the solenoid array 736 (i.e., the pistons of the solenoids) that act on the respective anvils 744 while the fluid paths / lines are in between. In particular, when the drawer engagement actuator 740 positions the solenoid array 736 in the engagement position during operation, each solenoid 778 can be selectively actuated under the control of the controller 210 to clamp the associated fluid flow line against the anvil 744, thereby obstructing the flow of fluid through it. In this invention, each fluid line is intended to be positioned between the planar anvil surface and the planar solenoid actuator head. Alternatively, the solenoid actuator head may comprise a molded head, such as two tapered surfaces that intersect at elongated edges resembling a Phillips screwdriver, optimized to apply a desired pinching force to the elastically flexible fluid line. Still alternative, the anvil surface may comprise elongated projections or protrusions extending toward each fluid line, such that the planar solenoid head presses the fluid line against these laterally extending projections, thereby closing the line to the fluid flow through it.

[0117] Referring to Figures 33, 34, and 40, as the kit 600 is lowered into the drawer's processing chamber, the first bioreactor vessel 410 and the second bioreactor vessel 420 are supported over the openings 626, 628 by the circumference of the openings and, in particular, by the claws / projections 632. As the kit is lowered further, the bed plates 746, 748 penetrate the openings 626, 628 and receive or otherwise engage the bioreactor vessels 410, 420. The shape of the openings 626, 628 and top surface 752 of the bed plates 746, 748 (for example, the raised area 758 of the bed plates 746, 748 corresponding to the claws / projections 632 of the tray 610) allows the tray 610 to continue moving downward after the bioreactor containers 410, 420 have been received by the bed plates 746, 748, so that the bottom surface and claws / projections 632 of the tray 610 are positioned lower than the top surface 752 of the bed plates 746, 748, and the bioreactor containers 410, 420 can be supported by the bed plates 746, 748 which are spaced apart from the bottom surface 620 of the tray 610. This ensures that the tray 610 does not interfere with the horizontal mounting of the bioreactor containers 410, 420 on the bed plates 746, 748.

[0118] As the bed plates 746, 748 pass through the openings 726, 728 in the tray 610, the positioning pins 754 on the bed plates 746, 748 are received in corresponding recesses 550 in the bottom plates 502 of the bioreactor vessels 410, 420, ensuring that the bioreactor vessels 410, 420 are properly aligned with the bed plates 410, 420. When properly seated on the bed plates 746, 748, the beam interruption 552 interrupts the light beam of the sensor 756 in the bed plate, indicating to the controller that the bioreactor vessels 410, 420 are in the correct position. Since the bed plates 746, 748 and the positioning pins are at the same height, the interruption of the light beam of the sensor 756 by the beam interruption 552 also ensures that the bioreactor vessels 410, 420 are horizontal. In this properly installed position, the sensors 759 on the bed plates 746 and 748 are aligned with the openings 556 in the bottom plate 502, thereby enabling them to sense the processing parameters in the internal compartments of the bioreactor vessels 410 and 420, respectively. In addition, in the fully installed position, the cam arms 762 of the bed plates 746 and 748 are aligned with the flat engaging surfaces 554 on the bottom plate 502 of the bioreactor vessels 410 and 420, respectively.

[0119] Figure 40 is a front cross-sectional view illustrating this fully installed position of the first bioreactor vessel 410 on the bed plate 746. As shown in Figure 40, heating elements in the form of a heating pad 782 and a heating module 784 may be positioned below the bed plate 746 to heat the bed plate 746. As shown in Figure 40, a carbon dioxide sensing module 786 may also be positioned below the bed plate to sense the carbon dioxide content in the processing chamber 722.

[0120] As further shown in Figure 40, in one embodiment, the side walls 718 and bottom (and top wall of the housing) of the drawer 712 may comprise a cover 788, an insulating foam layer 790 to help minimize heat loss from the processing chamber 722, a film heater 792 for heating the wall as described above, and an internal metal plate 794. In one embodiment, the internal metal plate 794 may be formed from aluminum, but other thermally conductive materials may also be used without departing from a broader aspect of the invention. The drawer 712 may further comprise one or more brush seals 796 to help minimize heat loss from the processing chamber 722 and an insulating layer 798 to minimize or obstruct the flow of thermal energy from the drawer 712 to other components of the apparatus 700 (such as the housing 710 or other drawers (e.g., drawers 714, 716)).

[0121] Referring again to Figure 34, when the kit 600 is received into the processing chamber 722, the load cell 750 in the bottom of the processing chamber 722 adjacent to the second bed plate 748 penetrates the opening 730 in the tray 610 so that the waste liquid bag 472a can be connected to the tubing tail 470a and positioned on the load cell 750. As shown there, when the kit 600 is received into the drawer 712, the second tubing holder block 654 holds the tubing so that the tubing tails 464a-f of the first fluid assembly 440 and the tubing tails 470b-d of the second fluid assembly 444 penetrate into the auxiliary compartment 730 for connection to their storage layers. In one embodiment, the sampling lines 476a-476d also penetrate into the auxiliary compartment 730.

[0122] Next, referring to Figures 41 to 44, the operation of the cam arms 762 of the bed plates 746, 748 is illustrated. As shown therein, the cam arms 762 are movable between a retracted position in which they are positioned below the top surface of the bed plates 746, 748 and an engaged position in which they are rotated around the cam pin 766 and extended above the bed plates 746, 748 to engage with the flat engaging surface 554 of the bioreactor vessels 410, 420, lifting and pulling the bioreactor vessels 410, 420 away from the bed plates 746, 748. In the default state, the cam arms 762 are retracted below the top surface of the bed plates 746, 748 so that the bioreactor vessels 410, 420 are supported on the horizontal bed plates 746, 748 (and in particular the horizontal alignment pin 754; no power is required to maintain the bioreactor vessels in the horizontal position). In particular, when the bioreactor vessels 410 and 420 are received on the bed plates 746 and 748, they are in a horizontal position. In the event of a power outage, the bioreactor vessels 410 and 420 remain installed on the horizontal bed plates 746 and 748 and do not require constant adjustment using the cam arm 762 to maintain the horizontal position. This is in contrast to some systems that may require constant adjustment of the bioreactor using a servo motor to maintain the horizontal position. In fact, in the configuration of the cam arm 762 of the present invention, the actuator only needs to be energized when the bioreactor vessel is tilted for agitation / mixing, as described below, thereby minimizing the amount of heat applied to the processing chamber 722.

[0123] As shown in Figures 41 to 43, the cam arms 762 may be sequentially operable to agitate the contents of the bioreactor vessels 410, 420. For example, when agitation of the contents of bioreactor vessel 410 is desired, one of the cam arms is operated to lift one end of the bioreactor vessel 410 away from the bed plate 746 (and disengage from the positioning pin 754 on the bed plate 746), while the opposing end remains mounted on the bed plate and the positioning pin 754 on the non-raised end remains received in the corresponding recess 550 in the bottom plate 502. The raised cam arm is then rotated back to a clearance position below the bed plate, and the opposing cam arm is rotated to an engaged position to lift the opposing end of the bioreactor vessel away from the bed plate and the positioning pin.

[0124] In one embodiment, the cam actuation system may be designed so that the cam arm 762 can return to its home position without touching the bioreactor vessel, thereby preventing disturbance to the culture and allowing the cam arm 762 to return to (or be tested for) its home position at any point during a long cell processing period. Thus, although the present invention intends that other rocking or stirring means may be provided in the bioreactor vessel, the overall height of the mixing mechanism can be minimized by having two cam arms 762 on opposing sides of the bed plate. For example, while it is possible to achieve a movement of ±5 degrees with a central actuator (located in the center of the bed plate), approximately the same movement of the vessel can be achieved with a movement of 0 to 5 degrees of the vessel driven by the cam arms on both sides of the vessel, which effectively brings the movement of ±5 degrees to the vessel at half the height. Furthermore, the movement of the cam arms 762 (e.g., the speed of cam arm rotation and the timing between opposing cam arms) can be adjusted to maximize wave formation within the vessel, maximize the amplitude of the waves, and thus maximize the time to achieve (ideally) uniformity and homogeneity of the vessel contents. The timing can also be adjusted based on the volume in a container having a given geometric shape that maximizes mixing efficiency.

[0125] In one embodiment, the optical sensor 756 may be used to verify that the first bioreactor vessel 410 has been correctly repositioned after each cam agitation operation. It is further intended that the correct repositioning of the bioreactor vessel may be checked and verified even between alternating cam operations. This allows for the rapid detection of misalignment in substantially real time, thereby enabling the operator to intervene to reposition the bioreactor vessel without substantial deviation from the bioprocessing operation / protocol.

[0126] Figure 43 is a schematic diagram showing the position of the fluid 800 in the bioreactor vessel during this stirring process. As shown in Figure 42, in one embodiment, a homing sensor 802 integrated with the bed plate 746 may be used by a controller to determine when the cam arm 762 has returned to a clearance position below the top surface of the bed plate 746. This is useful in coordinating the movement of the cam arm 762 to give a desired mixing frequency in the bioreactor vessel. In one embodiment, the cam arm 762 is configured to result in a tilt angle of up to 5 degrees with respect to the bed plate 746.

[0127] Referring to Figure 44, the interface between the positioning pin 754 on the bed plate of the bioreactor vessel 410 and the recess 550 in the bottom plate 502 is illustrated during mixing / stirring. In one embodiment, the recess 550 has a dome-shaped or hemispherical inner surface and a diameter d1 that is larger than the diameter d2 of the positioning pin 754. As illustrated in Figure 44, this configuration provides clearance between the positioning pin 754 and the recess 550, allowing the bioreactor vessel 410 to be tilted when the positioning pin 554 is received within the recess 550.

[0128] In one embodiment, each drawer of the bioprocessing apparatus 700, for example, drawer 712, is preferably equipped with a hinged flip-down front panel 810, as shown in Figures 45-50. The flip-down front panel 810 allows access to an auxiliary compartment 730 without opening the drawer 712, as best shown in Figures 45, 49, and 50. As can be seen therefrom, this configuration allows for in-process sampling and replacement of culture medium bags. In connection with the above, in one embodiment, the auxiliary compartment 730 may consist of a plurality of retractable sliding rails 812 providing mounting means 815 from which various storage tanks / culture medium bags can be suspended. The rails 812 are movable from a retracted position within the compartment 730, as shown in Figure 48, to an extended position out of the compartment 730, as shown in Figure 49. When the collection bag is full or when the culture medium / fluid bag needs to be replaced, one simply needs to extend the rail 812 outwards and release the bag clips. A new bag is attached to its respective tail and can then be suspended from the rail and slid back into the auxiliary compartment 730 without opening the drawer 712 or pausing the process. In one embodiment, the rail 812 may be mounted on a laterally extending cross rod 814. The rail 812 may thus be laterally slidable on the rod 814, extendable from the auxiliary compartment, and retractable into the auxiliary compartment. In addition, when the drawer is open (Figure 46), the rail 812 can rotate around the rear cross rod so that the compartment 730 can be cleared and the user can screw the tubing tails forward into the compartment 730, which provides a degree of freedom 3.

[0129] As illustrated in Figure 51, in another embodiment, the culture medium / fluid bag may be mounted on a platform 820 that is rotatable out of the auxiliary compartment 730 from a packing position to an access position. For example, the platform 820 may be mounted to move along a guide track 822 formed within the side wall of the auxiliary compartment 730.

[0130] Referring to Figure 52, in one embodiment, the bioprocessing apparatus 700 may further comprise low-profile waste trays 816 that are received in a housing 710 below each drawer, for example, drawer 712. The waste trays 816 are mounted independently on the drawer so as to be movable between a closed position and an open position. Preferably, in the closed position, the tray 816 extends coplanar with the front surface of the drawer, and in the open position, the tray 816 exposes its chamber 819 so that it is accessible to the operator. The chamber 819 allows for easy storage of a large waste bag connected to the fluid passage of the tray 600 that sits on top of it, and allows access to it without opening the drawer 712. In addition, in the closed position, the waste tray 816 positions the chamber 819 so as to be aligned with the drawer and below, and has a size and shape that allows it to operate to accommodate leaks from the processing chamber 722 or auxiliary compartment 730.

[0131] In one embodiment, each drawer may be equipped with a camera positioned above the processing chamber (for example, above each bioreactor vessel 410, 420) so that the inside of the drawer 712 can be visually monitored without opening the drawer 712. In one embodiment, the camera (or additional camera) may be integrated with the bed plate assembly or integrated on the side wall for a lateral view inside the bioreactor vessel.

[0132] Therefore, the second module 200 of the present invention enables the automation of cell processing to a degree not previously seen in the art. In particular, the fluid flow architecture 400, pump assembly 738, and pinch valve array 736 can automate fluid operations (e.g., fluid addition, transfer, discharge, rinsing, etc.) between the bioreactor vessels 410, 420 and the bags connected to the first and second fluid assemblies 740, 744. As described below, this configuration also enables hollow fiber packing concentration and washing, filterless perfusion, and line priming. The drawer engagement actuator 740 is also used for the automatic engagement and disengagement of the drop-in kit 600, further minimizing human touchpoints. In fact, human touchpoints may only be required for adding and removing source / culture bag, sampling, and data entry (e.g., sample volume, cell density, etc.).

[0133] Referring to Figures 53–77, an automated general protocol for a workflow involving immobilized Ab coating, soluble Ab addition, and gamma retroviral vectors by amplification in the same vessel is illustrated using the second module 200 and its fluid flow architecture 400. This general protocol results in activation (illustrated in Figures 53–59), transduction preparation and transduction (illustrated in Figures 60–71), amplification (Figures 72–76), and, in some embodiments, harvesting of cell populations in an automated, functionally closed manner (Figure 77). When describing the operation of the pinch valve, it is stated below that the valve is in its closed state / position when it is not used for a particular operation. Therefore, after the valve is opened and a particular operation becomes possible, and once that operation is complete, the valve is closed before proceeding to the next operation / step.

[0134] As shown in Figure 53, in the first step, valves 432 and 468f are opened and the first fluid assembly line pump 454 is activated to pump the antibody (Ab) coating solution from the storage tank 466f connected to the first fluid assembly 440 through the first port 412 to the first bioreactor vessel 410. The antibody coating solution is incubated for a certain period of time and then discharged through the interconnection line to the waste liquid storage tank 472a of the first fluid assembly 440 by opening valves 434 and 474a and activating the circulation line pump 456. As described herein, discharge from the bioreactor vessel 410 can be facilitated by tilting the bioreactor vessel 410 using the cam arm 762.

[0135] After the antibody coating solution is discharged, valves 432 and 468e are opened, and pump 454 is activated to pump the rinse buffer from storage tank 466e, which is connected to the first fluid assembly 440, through the first bioreactor line to the first bioreactor vessel 410. The circulating line pump 456 is then activated, opening valve 474a, which discharges the rinse buffer through the interconnection line 450 to the waste liquid storage tank 472a. In one embodiment, this rinsing and discharge procedure may be repeated multiple times to thoroughly rinse the first bioreactor vessel 410.

[0136] Referring to Figure 55, after rinsing the first bioreactor vessel 410 with buffer, the cells in seed bag 466d (already concentrated and isolated using the first module 100) are transferred to the first bioreactor vessel by opening valves 468d and 432 and activating pump 454. The cells are pumped through the first bioreactor line 414 of the first bioreactor vessel 410 and enter the bioreactor vessel 410 through the first port 412. As shown in Figure 56, valves 432 and 468a are opened and pump 454 is activated, pumping the second antibody (Ab) solution from storage tank 466a, connected to the first fluid assembly 440, through the first port 412 to the first bioreactor vessel 410.

[0137] After the second antibody solution is pumped into the first bioreactor vessel, the second antibody solution storage tank 466a is rinsed, and the rinsing medium is pumped into the first bioreactor vessel. In particular, as shown in Figure 57, valves 474b, 452, and 468a are opened, and the rinsing medium from the rinsing medium storage tank / bag 472b of the second fluid assembly 444 is pumped into the second antibody solution storage tank 466a using pump 454 to rinse the storage tank. After rinsing, valve 432 is opened, and the rinsing medium is pumped from the storage tank 466a into the first bioreactor vessel 410. In one embodiment, the second antibody solution storage tank 466a may be rinsed multiple times using this procedure.

[0138] After rinsing the second antibody solution storage tank 466a, the inoculum / seed cell bag 466d may also be optionally rinsed. In particular, as shown in Figure 58, valves 474b, 452, and 468d are opened and the rinsing medium from the rinsing medium storage tank / bag 472b of the second fluid assembly 444 is sent to the inoculum / seed cell bag 466d using pump 454 to rinse the bag. After rinsing, valve 432 is opened and the rinsing medium is sent from the bag 466d to the first bioreactor container 410 using pump 454. By pumping the rinsing medium to the first bioreactor container 410 after rinsing the inoculum / seed cell bag 466d, the cell density in the first bioreactor container 410 is reduced. At this time, a sample may be taken to measure one or more parameters of the solution in the bioreactor container before activation (for example, to confirm that a desired cell density is present before activation). In particular, as shown in Figure 58, valves 434, 452, and 432 are opened, and pump 456 is activated to pump the contents of the first bioreactor container 410 along the first circulation loop of the first bioreactor container (i.e., from the second port 416, through the interconnection line 450, and back to the first bioreactor container 410 through the first bioreactor line 414 and the first port 412 of the first bioreactor container 410). To collect a sample, a first sample container 280a (e.g., an immersion tube, syringe, etc.) is connected to the first sample tubing tail 476a, and valve 478a is opened, allowing a portion of the flow to pass through the interconnection line 450 and be diverted to the first sample container 280a for analysis.

[0139] If analysis of the collected sample indicates that all solution parameters are within a predetermined range, the solution in the first bioreactor container 410 is incubated for a predetermined period of time to activate the cell population in the solution, as illustrated in Figure 59. For example, in one embodiment, the cell population in the first bioreactor container 410 may be incubated for about 24 to 48 hours.

[0140] Next, referring to Figure 60, after activation, in order to prepare for transduction, valves 438 and 474b are opened and pump 456 is operated to pump the RetroNectin solution from storage tank 472b to the second bioreactor container 420 through the second port 426 of the second bioreactor container 420. After pumping the RetroNectin solution to the second bioreactor container 420 for RetroNectin coating of the second bioreactor container 420, the solution is incubated in the second bioreactor container 420 for a predetermined period of time. After incubation, as further shown in Figure 60, all of the RetroNectin solution is then discharged from the second bioreactor container 420 to the waste liquid storage tank 472a by opening valves 438 and 474a and operating the circulation line pump 456. Note that during these RetroNectin coating, incubation, and efflux steps (related to the second bioreactor vessel 420), the activated cell population remains in the first bioreactor vessel 410. Note that RetroNectin or other reagents to enhance the efficiency of genetic modification do not need to be used in all processes.

[0141] As shown in Figure 61, after the RetroNectin coating, the rinse buffer bag 472b is connected to the second fluid assembly 444 (or may already be present and connected to one of the tubing tails), valves 474b and 438 are opened, and pump 456 is activated to pump the buffer from bag 472b to the second bioreactor vessel 420. Alternatively, as described above, the buffer may be pumped through the first port 422 of the second bioreactor vessel 420 by opening valves 452 and 436 instead.

[0142] Next, referring to Figure 62, after a predetermined period of time, all buffers in the second bioreactor container 420 are discharged into the waste liquid storage tank 472a of the second fluid assembly 444 by opening valves 438 and 474a and operating the interconnection line pump 456.

[0143] At this point, as shown in Figure 63, a post-activation, pre-concentration sample can be taken from the cells in the first bioreactor vessel 410. As shown there, valves 434, 486, 488, and 432 are opened and pump 456 is activated to circulate the solution in the first bioreactor vessel 410 from the second port 434, through the interconnection line, through the filtration line 48 and filter 484, through the first bioreactor line 414 of the first bioreactor vessel 410, and back into the first bioreactor vessel 410 through the first port 412. To take a sample, a second sample container 280b (e.g., immersion tube, syringe, etc.) is connected to the second sample tubing tail 476b, valve 478b is opened, and a portion of the flow is passed through the interconnection line 450 and diverted to the second sample container 280b for analysis.

[0144] Next, referring to Figure 64, depending on the concentration obtained from the sample, concentration may be carried out by circulating the contents of the first bioreactor vessel 410 through the filter 484. As described above, this is done by opening valves 434, 486, 488, and 432 and operating pump 456, which causes the solution in the first bioreactor vessel 410 to circulate from the second port 416, and the solution passes through the second bioreactor line 418, through the interconnection line 450, through the filter line 482 and filter 484, through the first bioreactor line 414 of the first bioreactor vessel 410, and back to the first bioreactor vessel 410 through the first port 412. Once the fluid has passed through filter 484, wastewater is removed and permeate pump 492 sends such wastewater through wastewater line 490 to wastewater storage tank 472a of the second fluid assembly 444. In one embodiment, this procedure is repeated until the volume in the first bioreactor container 410 is concentrated to a predetermined volume.

[0145] Referring to Figure 65, after concentration, the concentrated cell population in the activation vessel (i.e., the first vessel 410 containing the concentrated cell population) is washed to a constant volume through perfusion. In particular, as shown therein, the culture medium from the culture medium bag 466b of the first fluid assembly 440 is pumped out of the first bioreactor vessel 410 through the second port 416 and simultaneously pumped into the first bioreactor vessel 410 through the interconnection line 450 via the first port 412, so that a constant volume is maintained in the first bioreactor vessel 410. When the culture medium is added and removed from the vessel 410, the waste liquid may be filtered by a filter 484 and directed to a waste liquid storage tank 472a.

[0146] The washed sample can be collected from cells in the first bioreactor vessel 410 in a manner similar to that already described for the pre-concentration sample. In particular, as shown in Figure 66, valves 434, 486, 488, and 432 are opened and pump 456 is activated to circulate the fluid in the first bioreactor vessel 410 from the second port 434, through the interconnection line, through the filtration line 48 and filter 484, through the first bioreactor line 414 of the first bioreactor vessel 410, and back into the first bioreactor vessel 410 through the first port 412. To collect the sample, a third sample container 280c (e.g., immersion tube, syringe, etc.) is connected to the third sample tubing tail 476c, valve 478c is opened, and a portion of the flow is passed through the interconnection line 450 and diverted to the third sample container 280c for analysis.

[0147] As shown in Figure 67, a bag containing the thawed viral vector is connected to a first fluid assembly 440 via a tubing tail 464c, etc. Valves 468c and 436 are then opened, and pump 454 is activated to transfer the viral vector coating solution from bag 466c through a first port 422 to a second bioreactor vessel 420. Incubation is then performed for a predetermined period of time for viral coating of the second bioreactor vessel 420. After incubation, the viral vector coating solution is discharged from the second bioreactor vessel 420 to a waste liquid storage tank 472a by opening valves 438 and 474a and activating the circulation line pump 456. In embodiments, viral vectors and non-viral vectors may be used as active ingredients for transduction / genetic modification.

[0148] As illustrated in Figure 68, after the second bioreactor vessel 420 is coated with a viral vector, the washed cells from the first bioreactor vessel 410 are transferred to the second bioreactor vessel 420 for transduction / genetic modification. Specifically, valves 434, 452, and 436 are opened and the circulation line pump 456 is activated, pumping the cells out of the first bioreactor vessel 420, through the second port 416 of the first bioreactor vessel 410, through the interconnection line 450, to the first bioreactor line 424 of the second bioreactor vessel 420, and then into the second bioreactor vessel 420 through the first port 422 of the second bioreactor vessel 420.

[0149] Next, as illustrated in Figure 69, the culture medium from culture bag 466b is added to the second bioreactor container 420 by opening valves 468b and 436 and operating pump 454, thereby increasing the total volume of solution in the second bioreactor container 420 to a predetermined volume. Then, referring to Figure 70, the pre-transduction sample can be collected by opening valves 438, 452, and 436 and operating circulation line pump 456, thereby pumping the solution in the second bioreactor container 420 along the circulation loop of the second bioreactor container (i.e., from the second port 426, through the interconnection line 450, and back to the second bioreactor container 420 through the first bioreactor line 414 and first port 422 of the second bioreactor container 420). To collect a sample, a fourth sample container 280d (e.g., an immersion tube, syringe, etc.) is connected to a fourth sample tubing tail 476d, a valve 478d is opened, and a portion of the flow is passed through the interconnection line 450 and diverted to the fourth sample container 280d for analysis.

[0150] If analysis of the collected fourth sample indicates that all parameters are within a predetermined range necessary for successful transduction, the cell population in the second bioreactor container 420 is incubated for a predetermined period of time for transduction of the cell population in solution, as shown in Figure 71. For example, in one embodiment, the cell population in the second bioreactor container 420 may be incubated for about 24 hours for transduction.

[0151] Referring to Figure 72, after transduction, the culture medium is added to the second bioreactor container 420 to achieve a predetermined amplification volume in the second bioreactor container 420. As shown therein, in order to add the culture medium, valves 468b and 436 are opened and pump 454 is activated to pump the growth / perfusion medium from the culture medium bag 466b through the first port 422 of the second bioreactor container to the second bioreactor container 420 until a predetermined amplification volume is reached.

[0152] Next, as illustrated in Figure 73, the pre-amplified sample can be collected by opening valves 438, 452, and 436 and activating the circulation line pump 456, and pumping the solution in the second bioreactor vessel 420 along the circulation loop of the second bioreactor vessel 420, as shown above (i.e., from the second port 426, through the interconnection line 450, and back to the second bioreactor vessel 420 through the first bioreactor line 414 and the first port 422 of the second bioreactor vessel 420). To collect the sample, a fifth sample container 280e (e.g., immersion tube, syringe, etc.) is connected to the fifth sample tubing tail 476e, and valve 478e is opened to allow a portion of the flow to pass through the interconnection line 450 and be diverted to the fifth sample container 280e for analysis.

[0153] If analysis of the collected fifth sample indicates that all parameters are within the predetermined range necessary for successful amplification of the cell population, the cell population in the second bioreactor container 420 is incubated for a predetermined period of time, for example, 4 hours, to establish the cells.

[0154] After this incubation period, or at a predetermined time thereafter, as shown in Figure 74, a perfusion at a rate of once per day (1x perfusion) is performed by pumping culture medium from culture bag 466b into the second bioreactor container 420 through the first port 422 at the same time that used / used culture medium is pumped out of the second bioreactor container 420 through the second port 426 (and through the interconnection line 450 to the waste liquid storage tank 472a). This perfusion is achieved by opening valves 468b, 436, and 474a and activating the first pump 454 and the circulation line pump 456. In this 1x perfusion, culture medium from culture bag 466b is introduced into the second bioreactor container 420 at substantially the same rate as used culture medium is removed from the second bioreactor container 420 and sent to the waste liquid section, maintaining a substantially constant volume within the second bioreactor container 420.

[0155] Next, sampling may be performed as needed / desired to monitor the amplification process and / or determine when the desired cell density is reached. As described above, the sample may be taken by opening valves 438, 452, and 436, activating the circulation line pump 456, and pumping the solution in the second bioreactor vessel 420 along the circulation loop of the second bioreactor vessel 420, as shown above (i.e., from the second port 426, through the second bioreactor line 428, through the interconnection line 450, and back to the second bioreactor vessel 420 through the first bioreactor line 424 and first port 422 of the second bioreactor vessel 420). To collect a sample, another sample container 280x (e.g., immersion tube, syringe, etc.) is connected to the sample tubing tail of the sample assembly 448, and as shown in Figure 75, the valve on the tubing tail is opened, allowing a portion of the flow to pass through the interconnection line 450 and be diverted to the sample container 280x for analysis. After each sampling operation, an incubation without perfusion is performed for a predetermined period, for example, 4 hours, to allow the cells to settle before perfusion is resumed.

[0156] As shown in Figure 76, after this incubation period, as shown in Figure 74, perfusion at a rate of once per day (1x perfusion) is performed by pumping culture medium from culture bag 466b into the second bioreactor vessel 420 through the first port 422, at the same time that used / used culture medium is pumped out of the second bioreactor vessel 420 through the second port 426 (and through the interconnection line 450 to the waste liquid storage tank 472a). This perfusion is achieved by opening valves 468b, 436, 438, and 474a and activating the first pump 454 and the circulation line pump 456.

[0157] When sampling indicates a viable cell density (VCD) of a predetermined threshold (e.g., 5 mm / mL), a perfusion of two doses per day (2x perfusion) is performed by pumping medium from medium bag 466b into the second bioreactor vessel 420 through the first port 422, at the same time that used / used medium is pumped from the second bioreactor vessel 420 through the second port 426 (and through the interconnection line 450 to the waste liquid storage tank 472a), as shown in Figure 76. This perfusion is achieved by opening valves 468b, 436, 438, and 474a and activating the first pump 454 and the circulation line pump 456. In this 2x perfusion, the culture medium from culture bag 466b is introduced into the second bioreactor container 420 at substantially the same rate as the used culture medium is removed from the second bioreactor container 420 and sent to the waste liquid section, maintaining a substantially constant volume within the second bioreactor container 420.

[0158] Finally, referring to Figure 77, after the desired viable cell density is achieved, the cells can be harvested by opening valves 438 and 474d and activating the circulation line pump 456. The amplified cell population is then pumped out of the second bioreactor vessel 420, through the second port 426, through the interconnection line 450, and into a collection bag 472d connected to the tubing tail 470d of the second tubing assembly 444. These cells can then be formulated in a manner known in the art and delivered to and injected into the patient.

[0159] Accordingly, the second module 200 of the bioprocessing system 10, as well as its fluid architecture 400 and bioreactor vessels 410, 420, provide a flexible platform in which various bioprocessing operations can be performed in a substantially automated and functionally closed manner. In particular, Figures 53 to 77 illustrate exemplary general protocols that can be performed using the bioprocessing system 10 of the present invention (particularly using its second module 200), but the system is not limited thereto. In fact, a variety of automated protocols, including numerous customer-specific protocols, can be made available by the system of the present invention.

[0160] In contrast to existing systems, the second module 200 of the bioprocessing system 10 is a functionally closed automated system housing the first and second bioreactor vessels 410, 420 and a fluid handling and fluid containment system, all of which are maintained under cell culture-friendly environmental conditions (i.e., within a temperature and gas-controlled environment) to enable cell activation, transduction, and amplification. As described above, the system features automated kit loading and closed sampling capabilities. In this configuration, the system makes all steps of immune cell activation, transduction, amplification, sampling, perfusion, and washing available in a single system. It also provides the user with the flexibility to combine all steps within a single bioreactor vessel (e.g., the first bioreactor vessel 410) or to use both bioreactor vessels 410, 420 for end-to-end activation and washing. In one embodiment, a single amplification bioreactor vessel (e.g., bioreactor vessel 420) can reliably generate billions of T cells in a single dose. Either single-dose or multi-dose formulations can be generated in situ with high recovery rates and high viability. In addition, the system is designed to give end-users the flexibility to implement different protocols for the production of genetically modified immune cells.

[0161] Some of the commercial advantages provided by the bioprocessing system of the present invention include a robust and scalable manufacturing technology that enables product commercialization by simplifying workflows, reducing labor intensity, easing the burden on cleanroom infrastructure, reducing failure nodes, lowering costs, and scaling up operations.

[0162] As described above with respect to the general workflow, the system of the present invention, the bioprocessing system 10, and the fluid architecture 400 and bioreactor vessels 410, 420 of the second module 200 bring to a process of culture concentration, washing, slow perfusion, fast perfusion, and “round-robin” perfusion that should be performed in an automated, functionally closed manner. For example, as described above, pump 456 on interconnect line 450 can be used to circulate fluid from one of the ports of the bioreactor through filtration line 482 and filter 484 and then back to another port on the bioreactor while the permeate pump 492 is operating in the concentration step (typically at a rate of circulating pump 456, such as about 10%). Concentration can be performed in an open loop or stopped based on a measured volume taken out of the bioreactor or a measured volume accumulated in the wastewater. In one embodiment, the filter, pump speed, filter area, number of lumens, etc., are all appropriately sized with respect to the total number of cells and target cell density in order to limit contamination by shear and excess cell loss.

[0163] In one embodiment, as described above, the system of the present invention can also be used for washing, for example, to remove residues such as residual viral vectors after incubation. Washing involves the same steps as described above for concentration, except that a pump 454 on the first fluid assembly line 442 is used to pump in additional medium and replace the fluid delivered from the permeate waste pump 492. The rate of introduction of new medium may correspond to the rate of fluid removal by the permeate pump 492. This allows for the maintenance of a constant volume in the bioreactor vessel, and residues can be removed exponentially with respect to time, as long as the contents in the bioreactor are well mixed (sufficient circulation can be made). In embodiments, this same process can be used post-activation for in-situ hollow fiber filtration-based washing of cell suspensions to remove residues. For coated and uncoated surfaces, soluble activating reagent washing removal can also be performed via filter-based perfusion.

[0164] As described above, in the perfusion process, pump 454 on the first fluid assembly line 442 can be used to add culture medium to a given bioreactor vessel, and pump 456 on the interconnection line 450 is used to move the spent culture medium to a waste bag in the second fluid assembly. In one embodiment, gravity may be used to fix the cells, and the spent culture medium may be pumped out at such a rate so as not to significantly disturb the cells in the bioreactor vessel. This process may involve operating pumps 454 and 456 in open loop at the same rate. In one embodiment, one pump (454 or 456) may operate at a set rate, and the rate of the other pump may be adjusted based on the mass / volume of the bioreactor vessel or the mass / volume of the waste bag (or the mass / volume of the measured source bag).

[0165] In relation to the above, pump control may be based on the gravimetric results of the bioreactor vessel (using feedback from load cell 760). For example, the system configuration allows for on-the-fly pump calibration based on load cell readings, which enables the system to automatically adapt to changes in tubing / pump performance that occur over time. Furthermore, this method can be used for closed-loop control over the rate of change in mass (volume) when emptying or filling the bioreactor vessel.

[0166] Figure 81 shows an exemplary embodiment of Method 480, which utilizes the second module 200 in a perfusion process. Method 480 includes, 482, activating a first pump 454 to pump fresh culture medium into a bioreactor vessel 410 containing a population of genetically modified cells; 484, activating a second pump 456 to pump used culture medium from the bioreactor vessel 410 into a waste bag 472a; 486, using a load cell associated with a bed plate to obtain mass data relating to the mass of the bioreactor vessel (e.g., bioreactor vessel 410); 488, determining whether the mass of the bioreactor vessel 410 has changed or remains substantially constant; and 490, if the mass of the bioreactor vessel has changed, adjusting the operating parameters of at least one of the first and second pumps to maintain a substantially constant mass of the bioreactor vessel 410. For example, if it is determined that the mass of the bioreactor container 410 has decreased, this indicates that the used culture medium is being removed at a faster rate than the rate at which fresh culture medium is added from one bioreactor container to the other. Accordingly, the flow rate of the first pump may be increased and / or the flow rate of the second pump may be decreased to maintain a substantially constant mass (and volume) in the bioreactor container 410. Further mass data may then be obtained and, if necessary, further adjustments to the pump operation may be made to maintain a substantially constant mass / volume in the bioreactor container 410. If it is determined that the mass is substantially constant after the operation of the first and second pumps has continued for a period of time, the pumps may be maintained at their current operating setpoint (e.g., flow rate), as shown in 492.

[0167] In another embodiment, the bioprocessing system uses the fluid architecture 400 to enable round-robin perfusion of various bioreactor vessels within the system. For example, circulation pump 456 and pump 545 along the first fluid assembly line 442 are used to perfuse cells in the first bioreactor vessel 410 in conjunction with the state of appropriate pinch valves, as described above. Perfusion of cells in the first bioreactor vessel 410 may then be stopped or paused, and circulation pump 456 and pump 454, as well as appropriate pinch valves, may be activated to perfuse cells in the second bioreactor vessel 420. In this regard, perfusion of the various bioreactors may be performed sequentially (i.e., perfusion of the first bioreactor vessel 410 over a period of time, followed by perfusion of the second bioreactor vessel 420 over a period of time, repeated alternately). This enables perfusion of any number of bioreactor vessels in the system without the use of many more pumps, culture bags, or waste bags.

[0168] In a round-robin perfusion system, the pumps can operate continuously, intermittently together (duty cycle), or sequentially (source, then waste, and so on), thereby maintaining the volume / mass in various bioreactor vessels at approximately the same level. A round-robin perfusion system (operating a pair of pumps intermittently together with standby at regular intervals) also allows for the perfusion of multiple vessels using the same two pumps, as shown. Furthermore, round-robin perfusion allows for low effective exchange rates (such as approximately 1 volume / day) even if the pumps do not have a large low dynamic range. Additionally, round-robin perfusion also allows each vessel to be perfused with a different culture medium, controlled by a valve in the first fluid assembly 440.

[0169] In addition, in one embodiment, high-speed perfusion may be used for residue removal (e.g., post-activation Ab removal and / or post-transduction residue removal). In the high-speed perfusion process, the perfusion process described above may be considerably faster than the typical 1–5 volumes / day, for example, between about 8–20 volumes / day, or may operate at values ​​greater than about 20 volumes / day, achieving a 1-log reduction in just a few minutes to a few hours. In one embodiment, the perfusion rate is balanced with cell loss. In some embodiments, high-speed perfusion may allow for the elimination of the hollow filter 484 while still satisfying the biological requirement to quickly remove residue after several steps.

[0170] As further described above, the system of the present invention uses a pump 454 on the first fluid assembly line 442 to facilitate rinsing of a bag / storage tank connected to the first fluid assembly 440 using a rinsing buffer or fluid from another bag / storage tank connected to the second fluid assembly 444. In addition, the fluid lines of the fluid architecture / system 400 are cleaned with sterile air from a sterile air source 458, which can prevent cells from settling in the line and dying, or culture media or reagents from settling in the line and degrading or becoming useless. The sterile air source 458 may also be used to flush reagents out of the line to ensure that no more reagents than intended are pumped into the bioreactor containers 410, 420. The sterile air source 458 may also be used to clean the inside of the line up to the connected bags (of the first or second fluid assemblies 440, 444) and to clean the sterile tube welds to limit residue. Instead of, or in addition to, cleaning the line using a sterile air source 458, the line may be cleaned using air drawn in from one of the bioreactor vessels, provided that the port through which the air is drawn in is not submerged and the bioreactor vessel has an air balance port 530.

[0171] As described above, the system enables in-closed-drawer process sampling of the contents of the bioreactor vessel. During sampling, the vessel from which the sample is drawn may be agitated by using a cam arm 762, circulating the contents of the vessel using a circulation line pump 456, and drawing the sample from the interconnection line 450 using a sampling assembly 448. In one embodiment, only non-bead-bound cells may be agitated.

[0172] As described above, the system of the present invention also allows for the collection of a population of cells after a target cell density has been achieved. In one embodiment, collecting an amplified population of transduced cells may involve using a pump 456 on an interconnection line 450 to move the cells into one of a group of bags connected to a second fluid assembly 444, or circulating the cells by the interconnection pump 456 to move them into a bag connected to a first fluid assembly 440. This process can be used for final collection or for large sample volumes, or it can be used to fully automate the sampling process (i.e., by connecting a syringe or bag to the first fluid assembly 440, circulating the contents of the bioreactor container, and drawing a portion of the desired sample volume from the circulating contents by the fluid assembly pump 454 and moving it towards the syringe / bag). In such cases, the circulation pump 456 and valve can then be used to clean the fluid / cell circulation line. In addition, the pump 454 on the first fluid assembly line 442 can be used to continue pushing all of the aliquot-divided sample volume into the sample container by using the air in the line to complete the sample transfer to the container when no detectable amount of cells remain in the line.

[0173] While the embodiments described above disclose a workflow in which cell activation is performed in a first bioreactor vessel and the activated cells are transferred to a second bioreactor vessel for transduction and amplification, in one embodiment, the system of the present invention may allow the activation and transduction operations to be performed in the first bioreactor vessel and the amplification of genetically modified cells to be performed in the second bioreactor vessel. Furthermore, in one embodiment, the system of the present invention may enable in-situ processing of isolated T cells, in which the activation, transduction, and amplification unit operations are all performed in a single bioreactor vessel. In one embodiment, the present invention thus simplifies existing protocols by making a simplified, automation-friendly "one-pot" activation, transduction, and amplification vessel available.

[0174] In one such embodiment, the T cell activator may be a micron-sized Dynabead, and a lentiviral vector is used for transduction. In particular, as disclosed herein, the micron-sized Dynabead serves the dual purpose of isolating and activating T cells. In one embodiment, T cell activation (and isolation) may be performed in one of the bioreactor vessels 410 using the Dynabead in the manner shown above. The activated cells are then transduced by a virus for genetic modification, such as in the manner described above in relation to Figures 60–71. Subsequently, after activation and viral transduction, the virus may be washed out of the bioreactor vessel 410 using a filterless perfusion method described for retaining the cells and micron-sized Dynabead in the bioreactor vessel 410. This allows for cell amplification in the same bioreactor vessel 410 used for activation and transduction. The filterless perfusion method also allows for culture washing without the need to first immobilize the activating beads, which need to be retained with the cells during amplification. In particular, when viruses are washed away, micron-sized Dynabead particles are not fluidized at low perfusion rates and remain in the container. Nanometer-sized virus particles and residual polymers are fluidized and washed away at slow perfusion rates.

[0175] In one embodiment, after amplification, cells may be harvested in the manner described above in relation to Figure 77. After harvesting, a magnetic bead removal process may be used to remove Dynabeads from the collected cells. In another embodiment, the steps of harvesting the amplified population of cells and removing the beads from the cells are performed simultaneously using perfusion, thereby introducing the culture medium into the bioreactor vessel through a supply port, and removing the cell medium containing the amplified population of cells from the bioreactor vessel through a discharge port in the bioreactor vessel. In particular, when final bead removal of the culture is required, filterless perfusion may be used to remove micron-sized beads by utilizing the difference in weight between the cells and the cell-Dynabead complex. To remove the beads from the culture, the entire contents of the bioreactor vessel are mixed (for example, by using the cam arm 762 of the actuator mechanism in the manner described above). After mixing / stirring, the heavier Dynabeads sink and settle on the silicone membrane 516 within 10-15 minutes. In contrast, cells require more than 4 hours to settle on the membrane 516. After mixing / stirring and a 10-15 minute retention period, the cell suspension can be slowly withdrawn using perfusion without disturbing the immobilized Dynabead. The inlet line can be used to maintain the culture height in the bioreactor vessel. Thus, the present invention described herein simplifies current Dynabead protocols by eliminating several process intermediate cell transfer and careful washing and bead removal steps, minimizing costs and potential risks. By performing bead removal of the culture simultaneously with cell harvesting, the need for additional magnetic devices or disposable parts, which were typically required until now, can be eliminated.

[0176] In contrast to other culture systems without static perfusion, the gas-permeable membrane-based bioreactor vessel 410 of the present invention supports high-density cell culture (e.g., up to 35 mm / cm²). 2Therefore, all four unit processes using Dynabead—activation, transduction, washing, and amplification—can be performed in the same bioreactor vessel in a fully automated and functionally closed manner. Thus, the bioprocessing system of the present invention simplifies current protocols by eliminating the need for process intermediate cell transfer and careful washing steps, minimizing costs and the potential risks resulting from multiple human touchpoints.

[0177] In one embodiment, the system's two bioreactor vessels 410, 420 can operate with either the same starting culture or two simultaneously divided cultures, for example, CD4+ cells in one bioreactor vessel 410 and CD8+ cells in the other bioreactor vessel 420. The divided cultures allow for parallel independent processing and amplification of two cell types that can be combined before injection into a patient.

[0178] While numerous possible CAR-T workflows for generating and amplifying genetically modified cells using the bioprocessing system of the present invention are described above, the workflows described herein are not intended to be exhaustive, as other CAR-T workflows are also usable by the system of the present invention. In addition, while the system of the present invention, and in particular the second module 200 of the system, are described in relation to the production of CAR-T cells, the system of the present invention is also suitable for the production of other immune cells such as TCR-T cells and NK cells. Furthermore, while embodiments of the present invention disclose the use of two bioreactor vessels 410, 420 in a two-step sequential process in which the product of the first bioreactor vessel 410 is added to the second bioreactor vessel 420 for additional processing steps (e.g., activation in the first bioreactor vessel and transduction and amplification in the second bioreactor vessel), in some embodiments, the two bioreactor vessels can be used in the same workflow as replication. Exemplary reasons for sequentially using a second bioreactor vessel include the presence of residual chemical modifications (e.g., coatings or immobilized reagents) that cannot be washed out of the first bioreactor and would be detrimental if excessive cell exposure occurs in a later or earlier step, or the need to pre-coat the bioreactor surface before adding cells (e.g., RetroNectin coating).

[0179] Additional examples of potential single bioreactor vessel workflows made available by the system of the present invention include (1) soluble activator activation, viral transduction, filterless perfusion, and amplification in a single bioreactor vessel; (2) Dynabead-based activation, viral transduction, filterless perfusion, and amplification in a single bioreactor vessel; and (3) TransAct-based activation, viral transduction, filterless perfusion, and amplification in a single vessel.

[0180] Furthermore, further examples of multiple potential bioreactor vessel workflows made available by the system of the present invention include: (1) soluble activator activation, viral transduction, filterless perfusion, and amplification in a first bioreactor vessel 410 and soluble activator activation, lentiviral transduction, filterless perfusion, and amplification in a second bioreactor vessel 420, using the same cell type or divided culture in these two bioreactor vessels; and (2) Dynabead ba (3) TransAct bead-based activation, lentiviral transduction, filterless perfusion, and amplification in the first bioreactor container 410, and TransAct-based activation, lentiviral transduction, filterless perfusion, and amplification in the second bioreactor container 420, wherein the same cell type or divided culture is used in these two bioreactor containers, (3) TransAct bead-based activation, lentiviral transduction, filterless perfusion, and amplification in the first bioreactor container 410, and TransAct-based activation, lentiviral transduction, filterless perfusion in the second bioreactor container 420 Perfusion without overflow, and amplification, using the same cell type or divided culture in these two bioreactor vessels; (4) Activation of soluble activator in the first bioreactor vessel 410, and RetroNectin coating, transduction, and amplification in the second bioreactor vessel 420; (5) Activation of immobilized activator in the first bioreactor vessel 410, and RetroNectin coating, transduction, and amplification in the second bioreactor vessel 420; (6) D in the first bioreactor vessel 410 (7) Dynabead activation and lentiviral transduction in the second bioreactor container 420, transduction and amplification, (8) TransAct activation in the first bioreactor container 410, and retroNectin coating, transduction and amplification in the second bioreactor container 420, (9) soluble activator activation in the first bioreactor container 410,(10) Amplification of in-house electroporation-treated cells or other non-virally modified cells in the second bioreactor container 420, (11) Activation of TransAct in the first bioreactor container 410, and amplification of in-house electroporation-treated cells or other non-virally modified cells in the second bioreactor container 420, (12) amplification of allogeneic NK cells in a first bioreactor vessel 410 and amplification of allogeneic NK cells in a second bioreactor vessel 420 (small molecule-based amplification, no genetic modification), (13) amplification of allogeneic NK cells in a first bioreactor vessel 410 and amplification of allogeneic NK cells in a second bioreactor vessel 420 (feeder cell-based amplification, no genetic modification), and (14) soluble activator activation, viral transduction, filterless perfusion and amplification of allogeneic CAR-NK or CAR-NK 92 cells in the first bioreactor vessel 410 and / or the first and second bioreactor vessels 410 and 420 (without RetroNectin coating, and polybrene used to assist transduction).

[0181] The embodiments described above illustrate process monitoring sensors integrated with the bioreactor vessel and / or bed plate (e.g., on the membrane, integrated within the membrane, on the vessel sidewall, etc.), but in other embodiments, additional sensors may be added to the fluid architecture 400, for example, along the fluid flow line itself. These sensors may be disposable sensors suitable for monitoring parameters such as pH, dissolved oxygen, density / turbidity (optical sensor), conductivity, and viability in the circulating fluid. By arranging the sensors within the circulation loop (e.g., the circulation loop of a first bioreactor vessel and / or the circulation loop of a second bioreactor vessel), the vessel structure can be simplified. In addition, in some embodiments, sensors along the circulation loop may provide a more accurate representation of the vessel contents when circulated (rather than when cells are stationary in the vessel). Furthermore, if necessary, flow sensors (e.g., ultrasonic-based) may be added to the flow loop to measure pump performance and used in conjunction with algorithms to compensate for pump parameters.

[0182] As shown above, the first and third modules 100, 300 can take any form of any system or device known in the art capable of cell enrichment and isolation, as well as harvesting and / or compounding. Figure 78 illustrates one possible configuration of a device / apparatus 900 that can be used as the first module 100 in a bioprocessing system 10 for cell enrichment and isolation using various magnetic isolation bead types (including, for example, Miltenyi beads, Dynabead, and StemCell EasySep beads). As shown therein, the apparatus 900 comprises a base 910 housing a centrifugal processing chamber 912, a high dynamic range peristaltic pump assembly 914, a pump tube 916 with a small inner diameter that is accepted by the peristaltic pump assembly, a stopcock manifold 918, an optical sensor 920, and a heating / cooling mixing chamber 922. As shown below, the stopcock manifold 918 provides a simple and reliable means of joining multiple fluid or gas lines together, for example, using Luer fittings. In one embodiment, the pump 914 has ratings to output low flow rates of about 3 mL / min and high flow rates of about 150 mL / min.

[0183] As further shown in Figure 78, the apparatus 900 may comprise a generally T-shaped hanger assembly 924 extending from the base 910 and having a plurality of hooks 926 for suspending a plurality of processing and / or supply source containers or bags. In one embodiment, there may be six hooks. Each hook is equipped with an integrated weight sensor for detecting the weight of each container / bag. In one embodiment, the bags may include a sample supply source bag 930, a process bag 932, an isolation buffer bag 934, a wash bag 936, a first storage bag 938, a second storage bag 940, a post-isolation waste liquid bag 942, a wash waste liquid bag 944, a culture medium bag 946, a release bag 948, and a collection bag 950.

[0184] The apparatus 900 is used with or configured to include a magnetic cell isolation holder 960, as presented herein. The magnetic cell isolation holder 960 may be detachably coupled to a magnetic field generator 962 (e.g., magnetic field plates 964, 966). The magnetic cell isolation holder 960 houses magnetic holding elements or materials 968, such as isolation columns, matrices, or tubes. In one embodiment, the magnetic cell isolation holder 960 may be manufactured as disclosed in U.S. Patent Application No. 15 / 829,615, filed December 1, 2017, which is incorporated herein by reference in its entirety. The apparatus 900 is often under the control of a controller (e.g., controller 110) and operates according to instructions executed by a processor and stored in memory. Such instructions may include magnetic field parameters. In one embodiment, the apparatus 900 may further include a syringe 952 available for bead addition, as described below.

[0185] Referring next to Figure 79, a general protocol 1000 for the apparatus 700 is shown. As illustrated therein, in a first step 1010, concentration is performed by reducing the platelets and plasma in the sample. Then, in embodiments where Dynabead is used as magnetic isolation beads, a washing step 1012 may be performed to remove any residue in the Dynabead suspension. After concentration, the cells are then transferred to a process bag 932 in step 1014. In some embodiments, a portion of the concentrated cells may be stored in a first storage bag 938 in step 1016 before being transferred to the process bag 932. In step 1018, magnetic isolation beads are injected into the process bag, for example, by using a syringe 952 in step 1020. In one embodiment, the magnetic isolation beads are Miltenyi beads or StemCell EasySep beads. If Dynabead is used, the washed Dynabead from step 1012 is resuspended in the process bag 932. In one embodiment, instead of using a syringe, the magnetic isolation beads may be housed in a bag or container connected to the system, and the beads may be drawn into the system by a pump 914.

[0186] Next, the beads and cells in the process bag 932 are incubated for a set period of time in step 1020. In embodiments where the magnetic isolation beads are Miltenyi nanosize beads, a sedimentation wash is performed in step 1022 to remove excess nanosize beads, and in step 1024, a portion of the incubated bead-bound cells are stored in a second storage bag 940. After incubation, the bead-bound cells are isolated in step 1026 using a magnet, for example, magnetic field plates 964, 966 of a magnetic cell isolation holder 960. The remaining bead-bound cells are then rinsed and isolated in step 1028. Finally, in embodiments where Miltenyi or Dynabead are utilized, in step 1030, the isolated bead-bound cells are collected in a collection bag 950. In embodiments where StemCell EasySep beads are utilized, an additional step 1032 is performed to release the cells from the beads and remove the beads, and an optional step 1034 is performed to wash / concentrate the collected cells.

[0187] A more detailed description of the general protocol of Figure 79 using the apparatus 900 is provided below, with particular reference to Figure 80, which is a schematic diagram of the fluid architecture 1100 of the apparatus 900. First, the concentration process (step 1010) is initiated by transferring the apheresis product and wash buffer contained in the source bag 930 from the wash buffer bag 936 to the chamber 912, where it is washed with the wash buffer to reduce the volume of platelets and serum. At this point, the concentrated raw materials are placed in the chamber 912. To initiate the isolation process, the isolation column, which is to be received by the magnetic cell isolation holder 960, is primed by initiating a buffer flow from the isolation buffer bag 934, through the manifold 918, through the column, and into the process bag 932.

[0188] As disclosed above, in some embodiments where Dynabead is used as magnetic isolation beads, a washing step (step 1012) is performed to remove any residue in the bead suspension buffer. The washing step is captured by injecting the beads using syringe 952 while they circulate in a process loop 1110 (e.g., a loop from process bag 932, through peristaltic pump tubing 914, through manifold 918, and back to process bag 932), cleaning the process loop 1110, and then flowing process bag 932 into isolation waste bag 942 while magnetic field generator 962 is "ON". In embodiments where washing is not desired, process bag 932 is flowed into isolation waste bag 942 to ensure that process bag 932 is clean. As used herein, for permanent magnets, ON means that the magnetic holding element or material 968 (e.g., isolation column, matrix, or tube) is in the correct position within the magnetic field. OFF means that the tubing section is removed from the magnetic field.

[0189] Next, the concentrated cells in the processing chamber 912 are transferred to the process bag 932 (step 1014), and the isolation buffer from the isolation buffer bag 934 is drawn into the processing chamber 912 to rinse the chamber 912 and remove any remaining cells. After rinsing, the fluid is discharged into the process bag 932. This rinsing process may be repeated as needed. After all the cells have been transferred to the process bag 932, the chamber 912 is cleaned by drawing the buffer from the isolation buffer bag 934 into the chamber 912 and discharging the fluid into the supply bag 930. This cleaning process may be repeated as needed.

[0190] Next, the contents of process bag 932 may be mixed by circulating the contents along process loop 1110 before cleaning process loop 1110 by returning the entire contents to process bag 932. As shown above, in one embodiment, a portion of the concentrated cells may be stored at this point by transferring a portion of the contents of process bag 932 to the first storage bag 938 (step 1016). Process line 1112 and first storage bag line 1114 are then cleaned.

[0191] Next, in embodiments where the bead washing step is not used, the beads are injected into the process loop 1110 using syringe 952, and the process loop 1110 is cleaned (step 1018). In embodiments where the bead washing step is used, the beads are resuspended and circulated through the process loop 1110 (step 1018) and column 968, and the process loop is cleaned through column 968.

[0192] As described above, after adding magnetic isolation beads, the cells may be incubated for a period of time (step 1020). In one embodiment, before incubation, the contents of process bag 932 may be transferred to a second storage bag 940, which is then agitated (for example, by using a heating / cooling mixing chamber 922). The contents of the second storage bag 940 are then transferred back to process bag 932. Next, the buffer from isolation buffer bag 934 is drawn into processing chamber 912, and the contents of the chamber are discharged into the second storage bag 940, then transferred to process bag 932, and the second storage bag 940 is rinsed.

[0193] In any embodiment, the cells are then incubated with magnetic isolation beads by circulating the cells along the process loop 1110 for a specified incubation time. After incubation, the process loop 1110 is cleaned.

[0194] As described above, after incubation, an optional step may be performed to wash out excess beads (e.g., nano-sized beads) (step 1022). Washing out excess nano-sized beads includes initiating a flow from process bag 932 to a second storage bag 940, drawing the contents of the second storage bag 940 into the processing chamber 912, transferring the buffer from isolation buffer bag 934 to process bag 932, transferring the contents of process bag 932 to the second storage bag 940, and drawing the contents of the second storage bag 940 into the processing chamber. The step of flowing from isolation buffer bag 934 to process bag 932 and then to the second storage bag 940 may be repeated as necessary to wash out excess beads. In one embodiment, the chamber 912 may then be filled with buffer from isolation buffer bag 934, the rotation of the chamber 912 may be initiated, and then the supernatant may be discharged into waste bag 742. These steps may be repeated as necessary. In one embodiment, cells in the chamber are discharged into a process bag 932, buffer from isolation buffer bag 934 is drawn into the chamber 932, and then the chamber is discharged into the process bag 932. This process, likewise, can be repeated as needed. Mixing and cleaning of the process loop are then performed.

[0195] In some embodiments, a portion of the incubated cell population may be stored in a second storage bag 940 (step 1024). To do so, a portion of the contents of the process bag 932 may be transferred to the second storage bag 940, and then the process line and the second storage line 1116 are cleaned.

[0196] In any of the processes described above, after incubation, the bead-bound cells are isolated using magnets 964, 966 (step 1026). This is achieved by flowing from process bag 932 to waste bag 942 while magnetic field generator 962 is "ON". The residual waste is then cleaned by pumping the buffer from isolation buffer bag 934 to process bag 932, and then pumping it from process bag 932 to waste bag 942 while magnetic field generator 962 is "ON".

[0197] In one embodiment, rinsing without resuspension can be performed by pumping the buffer from the isolation buffer bag 934 to the process bag 932, rinsing the process loop 1110, cleaning the process loop 1110, and allowing the process to flow from the process bag 932 to the waste liquid bag 942 with the magnetic field generator 962 in the "ON" position.

[0198] In another embodiment, rinsing via resuspension may be performed by pumping the buffer from the isolation buffer bag 934 to the process bag 932 with the magnetic field generator 962 in the "OFF" position, circulating it through the process loop 1110, cleaning the process loop, and then having the buffer flow from the process bag 932 to the waste liquid bag 942 with the magnetic field generator 962 in the "ON" position.

[0199] In one embodiment, residual waste liquid can be cleaned by pumping the buffer from the isolation buffer bag 934 to the process bag 932, and then flowing from the process bag 932 to the waste liquid bag 942 with the magnetic field generator 962 "ON".

[0200] Next, after rinsing and isolating any remaining bead-bound cells, the isolated bead-bound cells are collected (step 1028). If the bead-bound cells should be collected without releasing the cells from the beads, in one method, the culture medium from culture bag 946 is simply pumped through column 968 to collection bag 950 with the magnetic field generator 962 in the "OFF" position. In another method, the buffer from isolation buffer bag 934 is pumped to process bag 932, and then process bag 932 is pumped to collection bag 950 with the magnetic field generator 962 in the "OFF" position. This second method provides post-isolation washing. In a third method, the culture medium from culture bag 946 is pumped through column 966 to process bag 932 (if post-isolation washing is not required). Alternatively, the buffer from isolation buffer bag 934 is pumped through column 966 to process bag 932 (if post-isolation washing is desired). Next, in either process, the contents of the process bag 932 are circulated within the process loop 1110, which is then cleaned by returning to the process bag 932. The contents of the process bag 932 are then pumped to the collection bag 950 to collect the bead-bound cells.

[0201] If bead-bound cells are to be collected after the cells have been released from the beads, a number of potential processes may be performed. For example, in one embodiment, the cells / beads may be resuspended by pumping the release buffer from bag 948 through a column to process bag 932 with the magnet "OFF", circulating it within process loop 1110, and then cleaning the process loop by returning the fluid to process bag 932. Incubation and collection are then performed by incubating within process loop 1110 with the magnet "ON", cleaning process loop 1110, collecting the released cells by pumping the process bag 932 through column 966 to collection bag 950, and collecting the residue by pumping the buffer from isolation buffer bag 934 to process bag 932 with the contents of process bag 932 through column 966 to collection bag 950. Next, the released beads (step 1032) can be disposed of by pumping the buffer from the isolation buffer bag 934 through the column 966 to the process bag 932 with the magnet in the "OFF" position, circulating it within the process loop 1110 to clean the process loop 1110, and pumping the contents of the process bag 932 into the waste bag 942.

[0202] In connection with the above, in one embodiment, washing / concentration (step 1034) may be performed by pumping the contents of collection bag 950 to processing chamber 912, pumping buffer from isolation buffer bag 934 to processing bag 932, and transferring buffer from processing bag 932 to processing chamber 912. The washing cycle may then be performed by filling processing chamber 912 with buffer from isolation buffer bag 934, rotating chamber 912, discharging the supernatant into waste bag 942, and repeating the rotation and discharging steps as needed. Finally, transferring cells to the collection bag after washing / concentration may be achieved by transferring culture medium from culture bag 946 to collection bag 950, pumping the contents of the collection bag to processing chamber 912, discharging the contents of processing chamber 912 into collection bag 950, and then manually cleaning the line between processing chamber 912 and collection bag 950.

[0203] In one embodiment, one of the bags, for example, process bag 932, may be equipped with a top port 1118 having a filter so that sterile air can be introduced into the system (when process bag 932 is empty) to clean the line as needed in the various process steps described above. Cleaning the line may be performed as a first step in the concentration / isolation process and / or during process execution. In one embodiment, air from collection bag 950 may be used to clean any of the lines in the system (for example, air from collection bag 950 may be used to clean process line 1112, and then the air in process line 1112 may be used to clean a desired tubing line (i.e., lines 1114, 1116, etc.), thereby filling process line 1112 with liquid from process bag 932, and finally cleaning process line 1112 again using air from collection bag 950).

[0204] In one embodiment, the processing bag 932 is blow-molded and has a large angle on its sides (having a 3D shape with defined air pockets located above the liquid level) to limit micron-sized beads from adhering to the sidewalls, especially when performing long accelerated mixing in a circulation-based incubation.

[0205] In one embodiment, the syringe 952 allows a small amount (such as a bead suspension aliquot) to be added to the circulation-based flow loop 1110. Furthermore, fluid from the flow loop 1110 is drawn into the syringe 952, thereby further removing and cleaning any residue from the syringe 952.

[0206] In one embodiment, one of the sensors 920 may be configured to measure fluid flow. For example, one of the sensors 920 may be a bubble detector or optical detector that can be used as a secondary confirmation measuring means to ensure accurate flow control in addition to a load cell integrated with the hook 926. This can be practically used during isolation when it is desirable to flow the volume in the process bag through a magnet without introducing air into the column. The load cell indicates that the process bag is nearly empty within a certain expected tolerance of load cell variation, and then the bubble detector 920 identifies the subsequent liquid / air interface to stop the flow. Thus, the sensor 920 may be used by the controller to prevent the introduction of air into the loop by accidentally drawing material into the waste bag in situations where the pump does not stop after the complete discharge of the process bag, which may generate slag that removes cells or exposes cells to a dry environment. Thus, in one embodiment, the bubble detector 920 may be used in combination with a load cell integrated with the hook, thereby improving the accuracy of volume control, thereby reducing cell loss, and / or preventing air from entering the column tubing and column.

[0207] As implicitly stated above, in one embodiment, air may be drawn into the loop for the intentional generation of air slag, which can be used for collection to remove bead-bound cells in the isolation column / tube. In one embodiment, a buffer solution may be circulated through the isolation column to elute bead-bound cells from the isolation column, either instead of using air slag or in addition to it.

[0208] In one embodiment, two or more peristaltic pump tubes with different inner diameters connected in series may be used, thereby enabling a wide range of flow rates for a single pump. To switch the tubes, the pump cover is opened, the existing tube is physically removed, the desired tube is physically inserted, and then the pump head is closed.

[0209] In some embodiments, system 900 can be used for elution of isolated / captured bead cell complexes. In particular, an air-liquid interface is intended to be used to help remove the complexes from the tube sidewall or column interstitial space. Air can be circulated through the column / tube or shuffled back and forth through the column / tube. In the absence of an air-liquid interface, the packed bed of beads / bead-bound cells may be difficult to remove by flow control alone without significantly increasing the shear rate (which has a potentially adverse effect on cell viability). Therefore, in relation to flow rate, it is possible to remove the bead cell complexes without removing them from the magnet.

[0210] In relation to the above, System 900 supports the concept of directly eluting positively selected bead cell complexes into a selected culture medium (based on downstream steps). This eliminates the buffer exchange / washing step. In one embodiment, it is also conceivable to initiate incubation by directly eluting into the culture medium and viral vector. This concept may also allow the viral vector to be added to the final bag. In one embodiment, instead of eluting bead-bound cells with a buffer, the culture medium may be used as the eluting fluid. Similarly, a release buffer can be used to elute StemCell beads for subsequent cell release from the beads. Dilution can be minimized by exchanging the buffer in a portion of System 900 with the culture medium.

[0211] As disclosed above, the instrument 900 of the first module 100 is a single kit that performs platelet and plasma reduction enrichment, followed by magnetic isolation of target cells. The instrument 900 enables the enrichment, isolation, and collection steps and is automated so that all intervening steps are performed with minimal human intervention. Like the second module 200, the first module 100 and its instrument 900 are functionally closed to minimize the risk of contamination, are flexible to handle various therapeutic doses / doses / cell concentrations, and can support multiple cell types in addition to CAR-T cells.

[0212] It will be understood that the system of the present invention may include necessary electronic equipment, software, memory, storage devices, databases, firmware, logic / state machines, microprocessors, communication links, displays or other visual or audio user interfaces, printing devices, and any other input / output interfaces for performing the functions described herein and / or achieving the results described herein. For example, the system may comprise at least one processor and a system memory / data storage structure which may include random access memory (RAM) and read-only memory (ROM). At least one processor in the system may include one or more conventional microprocessors and one or more auxiliary coprocessors such as numerical coprocessors or similar. The data storage structure described herein may include a suitable combination of magnetic, optical, and / or semiconductor memory, and may include, for example, RAM, ROM, flash drives, optical discs such as compact discs, and / or hard disks or drives.

[0213] In addition, software applications that adapt controllers, for example, controllers 110, 210, and / or 310, to perform the methods disclosed herein may be read from a computer-readable medium into the main memory of at least one processor. The term “computer-readable medium” as used herein means any medium that provides or is involved in providing instructions for execution to at least one processor of the system (or any other processor of the devices described herein). Such mediums can take many forms, including, but are not limited to, non-volatile and volatile media. Non-volatile media include, for example, optical, magnetic, or magneto-optical disks, such as memory. Volatile media include dynamic random-access memory (DRAM), which typically constitutes main memory. Common forms of computer-readable media include, for example, floppy disks, flexible disks, hard disks, magnetic tapes, any other magnetic media, CD-ROMs, DVDs, any other optical media, RAM, PROMs, EPROMs or EEPROMs (electronically erasable programmable read-only memory), FLASH-EEPROMs, any other memory chips or cartridges, or any other media that a computer can read.

[0214] In embodiments, the execution of a sequence of instructions within a software application causes at least one processor to perform the methods / processes described herein, while hard-wired circuits may be used instead of, or in combination with, software instructions for implementing the methods / processes of the present invention. Therefore, the implementation of the present invention is not limited to any particular combination of hardware and / or software. Furthermore, all methods, protocols, and workflows described herein can be executed via software, which may be a single or multiple applications, programs, etc.

[0215] Furthermore, the software is intended to be configured to execute methods, protocols, and / or workflows in fully autonomous mode, semi-autonomous mode, or gated mode. In fully autonomous mode, the software includes commands configured to adapt the system's controller to execute substantially the entire operation, method, protocol, or workflow from start to finish automatically after being initiated by a user or operator (i.e., without operator intervention and without requiring human touchpoints). In semi-autonomous operation mode, the software includes commands configured to adapt the system's controller to execute substantially the entire operation, protocol, or workflow from start to finish after being initiated by a user or operator, except that the software may give the controller commands prompting the user or operator to perform certain actions necessary to execute an operation method, protocol, or workflow, such as pausing the operation of the bioprocessing system or its components, or connecting or disconnecting a collection, waste liquid, culture medium, cells, or other bag or reservoir for sampling, etc. In gated mode of operation, the software includes instructions configured to adapt the system's controller to generate a series of prompts that instruct the user or operator to perform a given operating method, protocol, or workflow, such as connecting or disconnecting collectibles, waste liquids, culture media, cells, or other bags or reservoirs for sample collection, and to perform several specific actions necessary to autonomously control system operation between each separate operator intervention. In gated mode of operation, the bioprocessing system is largely dependent on the operator, so that the controller simply executes pre-programmed bioprocessing steps after being initiated by the operator.

[0216] As used herein, elements or steps listed in the singular in the original English text and preceded by the article "a" or "an" should be understood not to exclude any plural of such elements or steps unless an exclusion is explicitly stated. Furthermore, references to “one embodiment” of the present invention are not intended to be construed as excluding the existence of additional embodiments that also incorporate the listed features. Moreover, unless otherwise specified, embodiments that “include,” “equip,” or “have” an element or plurality of elements having a particular characteristic may include additional such elements that do not possess that characteristic.

[0217] This specification discloses several embodiments of the invention, including best modes, by means of examples, and enables those skilled in the art to practice embodiments of the invention, including fabricating and using any device or system and performing any incorporated methods. The patentable scope of the invention is defined by the claims and may include other examples that a person skilled in the art would conceive. Such other examples are intended to be within the scope of the claims if they have structural elements that are not different from the language of the claims, or if they include equivalent structural elements that differ only slightly from the language of the claims. [Explanation of symbols]

[0218] 10 Bioprocessing Systems 100 First module 110 First Controller 200 Second module 200a, 200b, 200c: Second module 210 Second Controller 280a, 280d Sample Collection Device 300 Third Module 310 Third Controller 400 Fluid Flow Architectures 400 Bioprocessing Subsystems 400 Bioprocessing Systems 410 First bioreactor container 412 First port 414 First Bioreactor Line 416 Second port 418 Second Bioreactor Line 420 Second bioreactor container 422 First port 424 First Bioreactor Line 426 Second port 428 Second Bioreactor Line 430 Bioreactor Array 432 First bioreactor line valve 436 First bioreactor line valve 438 Second bioreactor line valve 440 First fluid assembly 442 First fluid assembly line 444 Second fluid assembly 446 Second fluid assembly line 448 Sampling Assembly 450 interconnection lines 452 Interconnection line valve 454 First pump or interconnection line pump 456 Second pump or circulation line pump 458 Sterilized air source 460 Sterilized Air Source Line 462 valves 464a~f Tubing Tail 466a~f First storage tank 466b Culture medium bag 468a~f Tubing Tail Valve 470a~d Tubing Tail 472a~d First storage tank 472a Waste liquid storage tank, waste liquid bag 474a~d Tubing Tail Valve 476a~476d Sampling lines 478a~d Sample line valve 480 Method 482 Filtering Line 484 Filter 486 Upstream Filtering Line Valve 488 Downstream Filtering Line Valve 490 Waste Liquid Line 492 Osmotic Pump 502 Bottom Plate 504 Container Body Part 506 Internal Compartment 508 Top Surface 510 Side Surface 510 Grid 512 Hole 514 Crossbar 516 Membrane 518 Top Surface 520 Mesh Sheet 522 O-ring 524 Groove 526 Peripheral Surface 526 Opening 528 Fitting or Tubing 530 Air Balance Port 532 Side Wall 534 Vertex, Tip 536 Height 538 Cell Culture Medium 542 Headroom 544 Surface 550 Concave Part​​​​​​​​​​​​​​​​​​​​​​​​​​​​​636 Support rib 638 Opening 650 Tubing module 652 First tubing holder block 654 Second tubing holder block 656, 658 Arrangement 660 Clearance opening 662 Planar backplate 664 Opening 666 Slots spaced vertically and extending horizontally 668, 670 Clearance openings 672 Retaining clip 674 First input end 676 Second output end 680 Feature[[ID=2​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​ 760 load cell 760 Resistance Temperature Detector 761 Actuator mechanism 762 Cam arm 764 slots 766 Kampin 768 Linear Actuator 770 Rocker Switch 770 Linear Actuator 772 Lead screw 774 Clevis Arm 776 Space 778 Solenoid 780 Pistons 782 Heating Pad 784 Heating Module 786 Carbon Dioxide Detection Module 788 Cover 790 Insulating foam layer 792 Film Heater 794 Internal metal plate 798 Insulation layer 800 fluid 810 Flip-Down Front Panel 812 Extendable sliding rail 814 Cross rod extending laterally 815 Mounting means 816 Low-profile waste liquid tray 819 Self-chamber 820 Platforms 822 Guide Track 900 devices / equipment 910 base 912 Centrifugation Chamber 914 High Dynamic Range Peristaltic Pump Assembly 916 Pump tube with suitable inner diameter 918 Stopcock Manifold 920 Optical Sensor 922 Heating and Cooling Mixing Chamber 924 Generally T-shaped hanger assembly 926 Hook 930 Sample Source Bag 932 Process Bags 934 Isolation Buffer Bag 936 Washing Bag 938 First storage bag 940 Second storage bag 942 Waste liquid bag after isolation 944 Washing waste liquid bag 946 Culture Bags 948 Release Bag 950 Collection Bags 952 Syringe 960 Magnetic Cell Isolation Holder 962 Magnetic field generator 964, 966 Magnetic Plates 968 Magnetic holding element or material 1000 General Protocols 1110 Process Loop 1112 process line 1114 First line of storage bags 1116 Second storage line 1118 Top port

Claims

1. A bioprocessing method for cell therapy, The process involves a step of genetically modifying a population of cells in a bioreactor container in order to produce a population of genetically modified cells, A bioprocessing method comprising the step of amplifying the population of genetically modified cells in the bioreactor vessel to produce a sufficient number of genetically modified cells for use in cell therapy treatment in one or more doses, without removing the population of genetically modified cells from the bioreactor vessel.

2. The method further includes the step of activating the cell population in the bioreactor container before genetically modifying the cells, The bioprocessing method according to claim 1, wherein the steps of activation, gene modification, and amplification are performed without removing the cells from the bioreactor vessel.

3. The step of washing the population of cells before the step of genetic modification, The process further includes, after the activation step and before genetic modification, a step of concentrating the cells in the bioreactor by fluid removal, The bioprocessing method according to claim 1, wherein the washing step and the concentration step are performed without removing the cells from the bioreactor vessel.

4. The bioprocessing method according to claim 1, further comprising the step of perfusing the population of cells together with the culture medium via filterless perfusion in the amplification step.

5. The bioprocessing method according to claim 1, comprising the step of genetically modifying a population of cells with a genetically modified substance selected from viruses, viral vectors, and non-viral vectors.

6. The bioprocessing method according to claim 5, further comprising the step of washing the genetically modified cells in the bioreactor container to remove the genetically modified agent and / or undesirable soluble substances via filterless perfusion.

7. The bioprocessing method according to claim 5, wherein the viral vector is a lentivirus, a retrovirus, or an adeno-associated virus.

8. The aforementioned population of cells is the first population of cells, The steps include: genetically modifying a second population of cells in a second bioreactor vessel in order to produce a second population of genetically modified cells; The bioprocessing method according to claim 1, further comprising the step of amplifying the second population of genetically modified cells in the second bioreactor without removing the second population of genetically modified cells from the second bioreactor vessel.

9. The bioreactor vessels are interconnected first and second bioreactor vessels that are interconnected through at least one fluid flow line. The steps include activating the cells in the first bioreactor container which is interconnected with the second bioreactor container, The bioprocessing method according to claim 8, further comprising the step of transferring the cells to the second container through at least one fluid flow line.

10. The bioprocessing method according to claim 8, wherein the first population of cells and the second population of cells are derived from the same donor source.

11. The bioprocessing method according to claim 8, wherein the first population of cells and the second population of cells are derived from different donor sources.

12. The bioprocessing method according to claim 8, wherein the first population of cells is enriched with respect to a first subpopulation of cells, the second population of cells is enriched with respect to a second subpopulation of cells, and the first subpopulation of cells is different from the second subpopulation of cells.

13. The bioprocessing method according to claim 1, wherein the cells are selected from immune cells, T cells, B cells, dendritic cells, CAR-T cells, TCR-T cells, NK cells, and combinations thereof.

14. A bioprocessing method, The steps include coating the bioreactor container with reagents to increase the efficiency of genetic modification of a population of cells, The steps include: genetically modifying cells in a population of cells in order to produce a population of genetically modified cells, A bioprocessing method comprising the step of amplifying the population of genetically modified cells in the bioreactor container without removing the genetically modified cells from the bioreactor container.

15. The bioprocessing method according to claim 14, wherein the bioreactor container is coated at a temperature of approximately 37°C.

16. The bioprocessing method according to claim 14, wherein the reagent is selected from retronectin, fibronectin, fibronectin fragments, reagents having a heparin-binding region and a cell-binding region, and combinations thereof.

17. The bioprocessing method according to claim 14, wherein the step of amplifying the population of genetically modified cells comprises the step of culturing them for a predetermined duration on retronectin or fibronectin or fibronectin fragments or a reagent having a heparin-binding region and a cell-binding region.

18. A bioprocessing method, The process involves activating cells in a population of cells within a bioreactor vessel using magnetic or non-magnetic beads to produce a population of activated cells, The steps include: genetically modifying the activated cells in the bioreactor container in order to produce a population of genetically modified cells; The steps include washing the genetically modified cells in the bioreactor container to remove undesirable substances, The process includes the step of amplifying the population of genetically modified cells in the bioreactor vessel to produce an amplified population of transduced cells, A bioprocessing method in which activation, genetic modification, washing, and amplification are performed within the bioreactor vessel without removing the cells from the bioreactor vessel.

19. The bioprocessing method according to claim 18, wherein the beads are held in the bioreactor container during the step of washing the genetically modified cells.

20. The bioprocessing method according to claim 18, wherein the washing step is performed without using magnets to immobilize the magnetic isolation beads.

21. The bioprocessing method according to claim 18, further comprising the step of washing the genetically modified cells via a filterless perfusion.

22. The steps include harvesting the amplified population of genetically modified cells from the bioreactor container, The bioprocessing method according to claim 18, further comprising the step of removing beads from the amplified population of genetically modified cells.

23. The bioprocessing method according to claim 22, wherein the steps of harvesting the amplified population of genetically modified cells and removing beads from the amplified population of genetically modified cells are performed simultaneously.

24. The steps of harvesting the amplified population of genetically modified cells and removing beads from the amplified population of genetically modified cells are performed using perfusion. The bioprocessing method according to claim 22, wherein the magnetic beads remain in the bioreactor container during perfusion.

25. The bioprocessing method according to claim 18, wherein the cells are one or more of immune cells, T cells, T cell subsets, B cells, NK cells, and dendritic cells.

26. The bioprocessing method according to any one of claims 1, 14, or 18, wherein the bioreactor container comprises a first bioreactor container and a second bioreactor container disposed within the processing chamber of the bioprocessing apparatus.

27. The bioprocessing method according to claim 26, wherein the activation step and the gene modification step are performed in the first bioreactor vessel, and the amplification step is performed in the second bioreactor vessel.

28. A bioprocessing method according to any one of claims 1 to 27, which can be optionally selected. A step of introducing a suspension containing cells suspended in cell culture medium into a cavity of a cell culture vessel by passing it through at least a supply port or an outlet port, wherein the amount of the suspension is sufficient to cover a gas-permeable, liquid-impermeable membrane positioned at the bottom of the cell culture vessel, the supply port is configured to allow additional cell culture medium to enter the cavity, and the outlet port is configured to allow the cells, cell culture medium, and used cell culture medium to be removed from the cavity, and A step that allows the cells to settle on the gas-permeable, liquid-impermeable membrane by gravity, The steps include removing the used cell medium through the discharge port and introducing the additional cell medium through the supply port, wherein the removal and introduction steps are performed after the step that allows the cells to settle on the gas-permeable, liquid-impermeable membrane. A step of resuspending the cells in the cell culture medium in the cell culture vessel, wherein the resuspending step is performed until the desired cell density is achieved. A bioprocessing method comprising the step of removing the resuspended cells and the cell medium through the discharge port.

29. The method according to claim 28, wherein the step of removing the used cell medium includes the step of removing impurities, cell culture by-products, or a combination thereof from the cell culture vessel.

30. The method according to claim 28 or 29, wherein the step of enabling the cells to settle on the gas-permeable, liquid-impermeable membrane, the step of removing the used cell medium, and the step of introducing the additional cell medium are further comprising the step of positioning the cell culture vessel such that the gas-permeable, liquid-impermeable membrane is flat to allow for an even distribution of cells.

31. The method according to claim 28, 29, or 30, wherein the step of resuspending the cells in the cell medium comprises the step of vibrating the cell culture vessel at a defined angle for a certain period of time.

32. The method according to any one of claims 28 to 31, wherein the step of removing the resuspended cells and the cell medium through the discharge port includes the step of tilting the cell culture vessel at a low angle that minimizes the hold-up volume while removing them while maintaining the low overall shape of the cell culture vessel.

33. A bioprocessing method, A step of introducing a suspension containing cells suspended in cell culture medium into a cavity of a cell culture vessel by passing it through at least a supply port or an outlet port, wherein the amount of the suspension is sufficient to cover a gas-permeable, liquid-impermeable membrane positioned at the bottom of the cell culture vessel, the supply port is disposed through the surface of the cell culture vessel and configured to allow additional cell culture medium to enter the cavity, and the outlet port is disposed through the surface of the cell culture vessel and configured to allow the removal of the cells, cell culture medium, and used cell culture medium from the cavity, and A step that allows the cells to settle on the gas-permeable, liquid-impermeable membrane by gravity, A step of perfusing the cells together with the cell medium, the perfusing step comprising introducing the additional cell medium through the supply port while simultaneously or substantially simultaneously removing the used cell medium from the cell culture vessel through the discharge port to maintain a constant volume in the cell culture vessel until the cells have amplified to a desired cell density, the perfusing step being performed after a step that enables the cells to settle on the gas-permeable, liquid-impermeable membrane, The steps include sampling the cells through the discharge port to determine whether the desired cell density has been reached, After determining that the desired cell density has not been reached, the step of repeating the perfusion of the cells until the desired cell density is reached, A step of resuspending the cells in the cell culture medium in the cell culture vessel, wherein the resuspending step is carried out until the desired cell density is reached. A bioprocessing method comprising the step of removing the resuspended cells and the cell medium through the discharge port.

34. A cell culture vessel for amplifying cell density, A bottom plate having a grid surface configured to provide support and gas exchange, The container body is connected to the bottom plate and has a rigid concave structure, The cavity formed by the container body and the bottom plate, A gas-permeable, liquid-impermeable membrane is disposed on the bottom plate within the cavity, A supply port is provided, which penetrates the surface of the container body and is configured to allow cell culture medium to be added to the cavity. The container body is provided with an outlet, which is disposed through the wall of the container body and is configured to allow used cell culture medium to be removed from the cavity, The cell culture vessel is configured to hold a suspension containing cells suspended in the cell culture medium, wherein the cell culture medium in the cell culture vessel contains a cell culture volume in a certain ratio per surface area of ​​a gas-permeable, liquid-impermeable membrane that is below a predetermined threshold.

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