Methods for cell concentration and isolation

Abstract

To provide a bioprocessing system for cellular immunotherapies that reduces contamination risk.SOLUTION: Disclosed is a method of bioprocessing that includes the steps of combining a suspension comprising 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. Collecting the target cells includes removing bead-bound cells from the isolation column with an air plug. Also disclosed is a system including a magnetic field generator, a magnetic cell isolation holder, a disposable fluidic kit and related methods of their use.SELECTED DRAWING: Figure 78

Description

Technical Field

[0001] Embodiments of the present invention generally relate to bioprocessing systems and methods, and more particularly 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 derived from either the patient's own blood (autologous) or another healthy donor (allogeneic).

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

[0004] After isolation of the target T cells, the cells are activated in a specific environment where they can proliferate actively. For example, the cells can be activated using anti-CD3 / anti-CD28 monoclonal antibodies or cell-based artificial antigen-presenting cells (aAPCs) coated with magnetic beads that 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 a lentivirus (LV) vector. The viral vector uses viral machinery to attach to the patient cells and enter the cells, and after entering the cells, the vector introduces the genetic material in the form of RNA. In the case of CAR-T cell therapy, this genetic material encodes the CAR. The RNA is reverse transcribed into DNA and permanently integrated into the patient cell genome so that CAR expression can be maintained when the cells divide and proliferate in large numbers in the bioreactor. The CAR is then transcribed and translated by the patient cells, and the CAR is expressed on the cell surface.

[0005] After the T cells are activated and transduced with the CAR-encoding viral vector, the cells are amplified until they are numerous in the bioreactor to achieve 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 manufacturing injectable doses of CAR T cells require many complex operations with numerous human touchpoints, which makes the entire manufacturing process time-consuming and increases the risk of contamination. In recent efforts to automate the manufacturing process, some human touchpoints have been eliminated, but these systems still suffer from high cost, lack of flexibility, and workflow bottlenecks. In particular, systems that utilize high levels of automation are very costly and inflexible in that customers need to adapt their processes to 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 [Summary of the Invention] [Problems to be Solved by the Invention]

[0008] In light of the above, there is a need for a bioprocessing system for cell immunotherapy that reduces the contamination risk by enhancing automation and reducing human handling. In addition, there is a need for a bioprocessing system for the manufacture of cell therapy drugs that balances the need for development flexibility and mass production consistency and further meets the various customer demands for operating different processes. [Means for Solving the Problems]

[0009] Some embodiments that are balanced with respect 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; rather, these embodiments are only intended to present an overview of possible embodiments. In fact, the present disclosure may encompass various forms that are similar to or different from the embodiments described below.

[0010] In one embodiment, a bioprocessing system includes a first module configured to concentrate and isolate a population of cells, a second module configured to activate, genetically transduce, and amplify the 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, wherein each second module is 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 that occur in parallel with each other.

[0012] In another embodiment, a method of bioprocessing comprises concentrating 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 step of activating, genetically transducing, and amplifying the population of cells is performed without removing the population of cells from the second module.

[0013] In another embodiment, an apparatus for bioprocessing comprises a housing and a drawer that is receivable within the housing. The drawer comprises a plurality of sidewalls and a bottom that define a processing chamber and a generally open top. 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 to provide 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, the method of bioprocessing includes sliding a drawer having a plurality of sidewalls, a bottom, and a generally open top from a closed position within a housing to an open position to extend the drawer from the housing and pass through the generally open top, positioning a bioreactor vessel through the generally open top onto a stationary bedplate positioned within the drawer, sliding the drawer to a closed position, and controlling a drawer engagement actuator to engage a plurality of fluid flow paths with at least one pump and a plurality of pinch valve linear actuators.

[0015] In another embodiment, a system for bioprocessing includes a housing, a first drawer receivable within the housing and having a plurality of sidewalls and a bottom defining a first processing chamber and a generally open top, at least one first bedplate positioned within the processing chamber of the first drawer and configured to receive or otherwise engage a first bioreactor vessel thereon, a second drawer receivable within the housing in a stacked relationship with the first drawer and having a plurality of sidewalls and a bottom defining a second processing chamber and a generally open top, and at least one second bedplate positioned within the processing chamber of the second drawer and configured to receive or otherwise engage a second bioreactor vessel thereon. 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 from the housing to allow access to the processing chambers through their respective open tops.

[0016] In yet another embodiment, an apparatus for bioprocessing includes a housing and a drawer that is receivable within the housing. The drawer includes a plurality of sidewalls and a bottom surface that define a processing chamber, and a generally open top. The drawer is movable between a closed position in which the drawer is received within the housing and an open position that enables access to the processing chamber through the open top of the drawer extending out of the housing. The apparatus also includes at least one bedplate positioned within the processing chamber adjacent the bottom surface, and a kit receivable within the processing chamber. The kit includes a plurality of sidewalls and a bottom surface that define an internal compartment, a generally open top, an opening formed in the bottom surface of the kit having a perimeter, and a bioreactor vessel supported by the bottom surface such that the bioreactor vessel is positioned over at least one opening within the internal compartment and a portion of the bioreactor vessel is accessible through the opening in the bottom surface. The kit is receivable within the processing chamber such that the bedplate extends through the opening in the bottom surface of the tray and supports the bioreactor vessel above the bottom surface of the kit.

[0017] In yet another embodiment, a system for bioprocessing includes a tray having a plurality of sidewalls and a bottom surface that define an internal compartment and a generally open top, at least one opening formed in the bottom surface and having a perimeter, a first tubing holder block integrated with the tray and configured to receive at least one pump tube and hold the 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 hold each of the plurality of pinch valve tubes in place for selective engagement with respective actuators of a pinch valve array, and a bioreactor vessel supported by the bottom surface such that the bioreactor vessel is positioned over at least one opening in the internal compartment and a portion of the bioreactor vessel is accessible through the opening in the bottom surface.

[0018] In yet another embodiment, a system for bioprocessing includes a processing chamber having a plurality of sidewalls, a bottom surface, and a generally open top, a bed plate positioned within the processing chamber adjacent to the bottom surface, and a tray. The tray includes a tray having a plurality of sidewalls and a bottom surface that define an internal compartment and a generally open top, and an opening within the bottom surface of the tray and having a perimeter. The perimeter of the opening has a shape and / or dimensions such that a bioreactor vessel can be supported by the bottom surface of the tray while the bioreactor vessel is positioned over the opening and a portion of the bioreactor vessel is accessible through the opening in the bottom surface. The tray is receivable within the processing chamber such that the bed plate passes through the opening in the bottom surface of the tray to support the bioreactor vessel.

[0019] In yet another embodiment, a system for bioprocessing includes a tray having a plurality of sidewalls and a bottom defining an internal compartment and a generally open top, and at least one opening in the bottom bounded by a periphery, the opening having a shape and / or dimensions such that a bioreactor vessel can be positioned over the opening and supported by the bottom of the tray within the internal compartment.

[0020] In yet another embodiment, a method for bioprocessing includes placing a bioreactor vessel in a disposable tray, the disposable tray having a plurality of sidewalls and a bottom defining an internal compartment, a generally open top, an opening formed in the bottom, and a plurality of teeth or protrusions penetrating from the bottom into the opening; arranging and configuring the bioreactor vessel within the tray such that the bioreactor vessel is supported by the plurality of teeth above the opening; and placing the tray within a processing chamber having a bedplate, the bedplate being received through the opening in the tray and supporting the bioreactor vessel.

[0021] In yet another embodiment, a tubing module for a bioprocessing system 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 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 a respective actuator of an array of pinch valves. 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 generally open top, 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 a rear portion 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, 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 an internal compartment is defined 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 installing 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 conduit connected to a first port of a first bioreactor vessel through a first bioreactor conduit of the first bioreactor vessel, the first bioreactor conduit of the first bioreactor vessel comprising a first bioreactor conduit 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 conduit connected to a second port of the first bioreactor vessel through a second bioreactor conduit of the first bioreactor vessel, the second bioreactor conduit of the first bioreactor vessel comprising a second bioreactor conduit valve for providing selective fluid communication between the second fluid assembly and the second port of the first bioreactor vessel; and an interconnecting conduit providing fluid communication between the first fluid assembly and the second fluid assembly and providing fluid communication between the second bioreactor conduit of the first bioreactor vessel and the first bioreactor conduit of the first bioreactor vessel.

[0026] In yet another embodiment, a method of bioprocessing includes providing a first fluid assembly having a first fluid assembly conduit connected to a first port of a first bioreactor vessel through a first bioreactor conduit of the first bioreactor vessel, providing a second fluid assembly having a second fluid assembly conduit connected to a second port of the first bioreactor vessel through a second bioreactor conduit of the first bioreactor vessel, and providing an interconnecting conduit between the second bioreactor conduit of the first bioreactor vessel and the first bioreactor conduit of the first bioreactor vessel, the interconnecting conduit enabling fluid communication between the first fluid assembly and the second fluid assembly and fluid communication between the second bioreactor conduit of the first bioreactor vessel and the first bioreactor conduit of the first bioreactor vessel.

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

[0028] In yet another embodiment, a bioprocessing method includes coating a bioreactor vessel with a reagent to enhance 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 vessel without removing the genetically modified cells from the bioreactor vessel.

[0029] In yet another embodiment, the bioprocessing method includes activating the cells of 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, a kit for use in a bioprocessing system comprises a process bag, a source bag, a bead addition vessel, and a process loop configured to be in fluid communication with the process bag, the source bag, and the bead addition vessel. The process loop further comprises pump tubing configured to be in fluid communication with a pump.

[0031] In yet another embodiment, an apparatus for bioprocessing is a kit comprising a process bag, a source bag, and a bead addition vessel configured to be in fluid communication with a process loop, the process loop further comprising pump tubing configured to be in fluid communication with a pump, the kit, 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 addition vessel, 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, the hooks, at least one bubble sensor, and a pump configured to be in fluid communication with the process loop.

[0032] In one embodiment, a method of bioprocessing 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 capturing target cells from the population of cells.

[0033] In one embodiment, a system includes a magnetic field generator configured to generate a magnetic field under magnetic field parameters, a first holder configured to be removably coupled to the magnetic field generator, and a second holder configured to be removably coupled to the magnetic field generator. The first holder has a passage configured to be positioned within the magnetic field in a first arrangement when the first holder is coupled to the magnetic field generator. The second holder has a passage configured to be positioned within the magnetic field in a second arrangement when the second holder is coupled to the magnetic field generator. The passage of the first holder is subjected to the action of a first magnetic field strength and a first magnetic field gradient within the magnetic field generated under the magnetic field parameters in the first arrangement. The passage of the second holder is subjected to the action of a second magnetic field strength and a second magnetic field gradient within the magnetic field under the magnetic field parameters in the second arrangement, the second magnetic field strength being different from the first magnetic field strength, and the second magnetic field gradient being different from the first magnetic field gradient or a combination thereof.

[0034] In another embodiment, a magnetic cell isolation holder includes a body configured to be removably coupled to a magnetic field generator. The body has a first passage configured to be positioned within the magnetic field of the magnetic field generator in a first arrangement when the holder is coupled to the magnetic field generator, and a second passage configured to be positioned within the magnetic field in a second arrangement when the holder is coupled to the magnetic field generator. The first passage is subjected to the action of a first magnetic field strength and a first magnetic field gradient within the magnetic field generated under the magnetic field parameters in the first arrangement, and the second passage is subjected to the action of a second magnetic field strength and a second magnetic field gradient within the magnetic field under the magnetic field parameters in the second arrangement. The second magnetic field strength is different from the first magnetic field strength, and the second magnetic field gradient is different from the first magnetic field gradient or a combination thereof.

[0035] In another embodiment, the system comprises a first kit having a first holder with a plurality of beads of a first size and a passage configured to receive the plurality of beads of the first size, and a second kit having a second holder with a plurality of beads of a second size and a passage configured to receive the plurality of beads of the second size. The passage of the first holder is positioned within the holder such that when the first holder is removably coupled to the magnetic field generator, the first holder is positioned within the magnetic field generated by the magnetic field generator in a first arrangement. The passage of the second holder is positioned within the second holder such that when the second holder is removably coupled to the magnetic field generator, the second holder is positioned within the magnetic field generated by the magnetic field generator in a second arrangement different from the first arrangement. The passage of the first holder is subjected to the action of a first magnetic field strength and a first magnetic field gradient within the magnetic field in the first arrangement, and the passage of the second holder is subjected to the action of a second magnetic field strength and a second magnetic field gradient within the magnetic field in the second arrangement. The second magnetic field strength is different from the first magnetic field strength, and the second magnetic field gradient is different from the first magnetic field gradient or a combination thereof.

[0036] In another embodiment, a method for isolating target cells includes positioning a first holder having a passage within a receiving region of a frame coupled to a magnetic field generator, and generating a first magnetic field within the receiving region by the magnetic field generator when the first holder is coupled to the magnetic field generator to apply a first magnetic field strength, a first magnetic field gradient, or both to the passage of the first holder. The method also includes positioning a second holder having a passage within the receiving region, and generating a second magnetic field within the receiving region by the magnetic field generator when the second holder is coupled to the magnetic field generator to apply a second magnetic field strength, a second magnetic field gradient, or both to the passage of the second holder. The passage of the first holder and the passage of the second holder are positioned at different locations within the receiving region.

[0037] The present invention will be better understood by reading the following description of non-limiting embodiments with reference to the accompanying drawings, which are shown below.

Brief Description of the Drawings

[0038]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

Figure 13

Figure 14

Figure 15

Figure 16

Figure 17

Figure 18

Figure 19

Figure 20

Figure 21

Figure 22

Figure 23

Figure 24

Figure 25

Figure 26

Figure 27

Figure 28A

Figure 28B

Figure 29

Figure 30

Figure 31

Figure 32

Figure 33

Figure 34

Figure 35

Figure 36

Figure 37

Figure 38

Figure 39

Figure 40

Figure 41

Figure 42

Figure 43

Figure 44

Figure 45

Figure 46

Figure 47

Figure 48

Figure 49

Figure 50

Figure 51

Figure 52

Figure 53

Figure 54

Figure 55

Figure 56

Figure 57

Figure 58

Figure 59

Figure 60

Figure 61

Figure 62

Figure 63

Figure 64

Figure 65

Figure 66

Figure 67

Figure 68

Figure 69

Figure 70

Figure 71

Figure 72

Figure 73

Figure 74

Figure 75

Figure 76

Figure 77

Figure 78

Figure 79

Figure 80

Figure 81

Figure 82

Figure 83A

Figure 83B

Figure 84A

Figure 84B

Figure 85

Figure 86

Figure 87

[0039] Exemplary embodiments of the present invention are hereinafter referred to in detail with reference to the accompanying drawings, in which examples are shown. As much as possible, the same reference characters are used throughout the drawings to refer to the same or similar parts.

[0040] 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 can also refer to a material that is compressible or expandable. An example of a flexible structure is a bag formed from a polyethylene film. The terms "rigid" and "semi-rigid" are used interchangeably herein to describe a structure that is "non-foldable", i.e., a structure that is not folded, crushed, or otherwise deformed by the application of normal forces to significantly reduce its elongate dimension. Depending on the context, "semi-rigid" can also represent a structure that is more flexible than a "rigid" element, such as a bendable tube or conduit, but still represents a structure that does not collapse longitudinally under normal conditions and forces.

[0041] As used herein, "container" means, as appropriate, a flexible bag, a flexible enclosure, a semi-rigid enclosure, a rigid enclosure, or flexible or semi-rigid tubing. The term "container" as used herein encompasses bioreactor vessels having walls or portions of walls that are semi-rigid or rigid, and further includes, for example, cell culture / purification systems, mixing systems, media / buffer preparation systems, and filtration / purification systems, such as chromatography and tangential flow filtration systems, and other enclosures or conduits commonly used in biological or biochemical processes, including their associated flow paths. As used herein, the term "bag" means a flexible or semi-rigid enclosure or container used, for example, as a containment device for various fluids and / or media.

[0042] As used herein, "fluidly coupled" or "in fluid communication" means that components of a system are capable of receiving or transferring fluid between the components. The term fluid includes gases, liquids, or combinations thereof. As used herein, "in electrical communication" 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, "operatively coupled" refers to a connection, which can be direct or indirect. The connection is not necessarily a mechanical coupling.

[0043] As used herein, the term "tray" refers to any object that can support a plurality of components, at least temporarily. The tray may be made of a variety of suitable materials. For example, the tray may be made of a cost-effective material suitable for sterile and single-use disposable products.

[0044] As used herein, the term "functionally closed system" refers to a closed fluid pathway that may have inlet and outlet ports for adding or removing fluid or air to or from the system without compromising the integrity of the closed fluid pathway (e.g., to maintain an internally sterile biomedical fluid pathway), whereby those ports may include, for example, a filter or membrane at each port to maintain sterility 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), containers, receptacles, and ports, depending on a given embodiment.

[0045] Embodiments of the present invention provide systems and methods for manufacturing a cellular immunotherapy agent from a biological sample (e.g., blood, tissue, etc.). 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 separation column, and harvesting target cells from the population of cells. Harvesting the target cells includes removing the bead-bound cells from an isolation column having an air plug.

[0046] Referring to FIG. 1, a schematic diagram of a bioprocessing system 10 according to an embodiment of the present invention is illustrated. The bioprocessing system 10 is configured to be used in the production of a cellular immunotherapy agent (e.g., an autologous cellular immunotherapy agent), and for example, human blood, fluid, tissue, or cell samples are collected, and a cell therapy agent is generated from or based on the collected samples. A chimeric antigen receptor (CAR) T cell therapy agent is one type of cellular immunotherapy agent that can be produced using the bioprocessing system 10, but other cell therapy agents can also be produced using the system of the present invention or aspects of the present invention without departing from the broader aspects of the present invention. As illustrated in FIG. 1, the production of a CAR T cell therapy agent generally begins with the collection of a patient's blood and the separation of lymphocytes through apheresis therapy. The collection / apheresis therapy may be well performed at the clinical site, and then the apheresis product is 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., white blood cells) is concentrated to produce a cell therapy agent or separated from the collected blood, and the target cells of interest are isolated from the progenitor cell mixture. Next, the target cells of interest are 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 dose is formulated. Next, the formulation is often cryopreserved, thawed, prepared, and finally delivered to the clinical site for infusion into the patient's body.

[0047] Referring further to FIG. 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 and functionally closed and scalable manner. In particular, the bioprocessing system 10 includes a first module 100 configured to perform concentration and isolation steps, a second module 200 configured to perform activation, genetic modification, and amplification steps, and a third module 300 configured to perform steps of harvesting the amplified cell population. In one embodiment, each module 100, 200, 300 may be communicatively coupled to a dedicated controller (e.g., a first controller 110, a second controller 210, and a third controller 310, respectively). The controllers 110, 210, and 310 are configured to perform substantially automated control over the manufacturing processes within each module. The first module 100, the second module 200, and the third module 300 are illustrated as each comprising a dedicated controller for controlling the operation of the respective module, although it is contemplated that a master control unit may be utilized to perform global control over the three modules. Each module 100, 200, 300 is designed to cooperate with other modules to form a single coherent bioprocessing system 10, as will be described in detail below.

[0048] By automating the processes within each module, product consistency from each module can be enhanced and the costs associated with extensive manual operations can be reduced. In addition, as will be described in detail later, each module 100, 200, 300 is substantially closed, helping to ensure patient safety by reducing the risk of external contamination, ensuring regulatory compliance, and helping to avoid the costs associated with open systems. Further, each module 100, 200, 300 is scalable and supports both development with a small number of patients and commercial manufacturing with a large number of patients.

[0049] Referring further to FIG. 1, a particular manner in which process steps are partitioned into distinct modules that each provide closed and automated bioprocessing enables an efficient use of capital equipment to a degree not previously seen in this technical field. As will be appreciated, the step of amplifying a cell population to achieve a desired cell density prior to harvesting and formulation is typically the most time-consuming step in the manufacturing process, while the concentration and isolation steps, as well as the harvesting and formulation steps, and further the activation and genetic modification steps are less time-consuming. Thus, attempts to automate the entire cell therapy drug manufacturing process can, in addition to being difficult with respect to logistics, disrupt the workflow and exacerbate bottlenecks within the process that reduce manufacturing efficiency. In particular, in a fully automated process, the steps of cell concentration, isolation, activation, and genetic modification can be carried out quite rapidly, but the amplification of genetically modified cells proceeds very slowly. Thus, the manufacture 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. When using a fully automated system, the entire process / system is occupied by the amplification equipment that performs the amplification of the cells from the first sample, and the processing of a second sample cannot be initiated until the entire system is released for use. In this regard, in a fully automated bioprocessing system, the entire system is essentially offline and not available for the processing of a second sample until the entire cell therapy drug manufacturing process from concentration to harvesting / formulation is completed in the first sample.

[0050] However, embodiments of the present invention enable the parallel processing of multiple samples (from the same or different patients) to promote more efficient utilization of capital resources. This advantage is a direct result of a particular manner in which the process steps are divided into three modules 100, 200, 300, as alluded to above. Referring particularly to FIG. 2, in one embodiment, a single first module 100 and / or a single third module 300 can be utilized in conjunction with a plurality of second modules, e.g., second modules 200a, 200b, 200c, in a 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, and the first population of target cells may then be transferred to one of the second modules, e.g., module 200a, for activation, genetic modification, and amplification under the control of a controller 210a. After the first population of target cells has been transferred out of the first module 100, the first module is also available here for use, e.g., 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, e.g., second module 200b, for activation, genetic modification, and amplification under the control of a controller 201b.

[0051] Similarly, after a second population of target cells is transferred out from the first module 100, the first module is also available here for use, for example, in processing a third apheresis product from a third patient. Then, a third target population of cells produced in the first module 100 from a sample taken from the third patient can be transferred to another second module, for example, the second module 200c, for activation, genetic modification, and amplification under the control of the controller 201c. In this regard, the amplification of, for example, CAR-T cells for the first patient can occur simultaneously with the amplification of CAR-T cells for the second patient, the third patient, and so on.

[0052] With this approach, the post-processing can also be performed asynchronously as needed. In other words, not all patient cells can grow simultaneously. The cultures can reach their final density at different times, but the plurality of second modules 200 are not linked, and the third module 300 can be used as needed. According to the present invention, the samples can be processed in parallel, but these need not be performed in batch processing.

[0053] The harvesting of the amplified populations of cells from the second modules 200a, 200b, and 200c can likewise be accomplished using a single third module 300 when the preparation for harvesting each amplified population of cells is complete.

[0054] Accordingly, by dividing the activation, genetic modification, and amplification steps, which are the most time-consuming, share some operational requirements, and / or require similar culture conditions, into stand-alone, automated, functionally closed modules, the other system equipment used for concentration, isolation, harvesting, and formulation 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 carried out simultaneously, maximizing equipment and usable floor space and enhancing the efficiency of the overall process and facilities. An additional second module may be added to the bioprocessing system 10 to perform parallel processing of any number of cell populations as desired. Accordingly, the bioprocessing system of the present invention enables the use of a plug & play-like function and can be easily scaled up or down in manufacturing equipment.

[0055] In one embodiment, the first module 100 can be any system or device capable of producing a target population of concentrated and isolated cells for use in biological processes, such as the production of immunotherapeutic and regenerative medicine drugs, from an apheresis product taken from a patient. For example, the first module 100 can be a modified version of the Sefia Cell Processing System available from GE Healthcare. The configuration of the first module 100 according to some embodiments of the present invention will be described in detail later.

[0056] In one embodiment, the third module 300 may similarly be any system or device capable of harvesting and / or formulating CAR-T cells or other modified cells produced by the second module 200 for injection into a patient's body for use in adoptive cell therapy or regenerative medicine. In some embodiments, the third module 300 may similarly be the Sefia Cell Processing System, available from GE Healthcare. In some embodiments, the first module 100 is first utilized for the concentration and isolation of cells (which are then transferred to the second module 200 for activation, transduction, and amplification (and in some embodiments, harvesting)), and may then also be used at the end of the process for cell harvesting and / or formulation. In this regard, in some embodiments, the same equipment may be utilized for the front-end cell concentration and isolation steps, as well as the back-end harvesting and / or formulation steps.

[0057] First, turning to the second module 200, the ability to combine the process steps of cell activation, genetic modification, and cell amplification in a single functionally closed and automated module 200 that provides the efficiency of the workflow described above is enabled by a unique fluid architecture that results in a specific configuration of components within the second module 200 and a specific interconnectivity between such components. FIGS. 3-77, described below, illustrate various aspects of the second module 200 according to various embodiments of the present invention. Referring first to FIG. 3, a schematic diagram is shown illustrating a fluid flow architecture 400 (also broadly referred to herein as a bioprocessing subsystem 400 or a bioprocessing system 400) within the second module 200 that performs cell activation, genetic modification, and amplification (and in some cases, harvesting). The system 400 includes a first bioreactor vessel 410 and a second bioreactor vessel 420. The first bioreactor vessel includes at least a first port 412 and a first bioreactor conduit 414 in fluid communication with the first port 412, as well as a second port 416 and a second bioreactor conduit 418 in fluid communication with the second port 416. Similarly, the second bioreactor vessel includes at least a first port 422 and a first bioreactor conduit 424 in fluid communication with the first port 422, as well as a second port 426 and a second bioreactor conduit 428 in fluid communication with the second port 426. Together, the first bioreactor vessel 410 and the second bioreactor vessel 420 form a bioreactor array 430. The system 400 is shown as having two bioreactor vessels, but embodiments of the present invention may include a single bioreactor or three or more bioreactor vessels.

[0058] The first and second bioreactor conduits 414, 418, 424, 428 of the first and second bioreactor vessels 410, 420 each include respective valves for controlling the flow of fluid therethrough, as described below. In particular, the first bioreactor conduit 414 of the first bioreactor vessel 410 includes a first bioreactor conduit valve 432, and the second bioreactor conduit 418 of the first bioreactor vessel 410 includes a second bioreactor conduit valve 424. Similarly, the first bioreactor conduit 424 of the second bioreactor vessel 420 includes a first bioreactor conduit valve 436, and the second bioreactor conduit 428 of the second bioreactor vessel 420 includes a second bioreactor conduit valve 438.

[0059] Referring further to FIG. 3, system 400 also includes a first fluid assembly 440 having a first fluid assembly conduit 442, a second fluid assembly 444 having a second fluid assembly conduit 446, and a sampling assembly 448. An interconnect conduit 450 having an interconnect conduit valve 452 fluidly communicates between the first fluid assembly 440 and the second fluid assembly 444. As shown in FIG. 3, interconnect conduit 450 also fluidly communicates between the second bioreactor conduit 418 and the first bioreactor conduit 414 of the first bioreactor vessel 410, allowing fluid circulation along the first circulation loop of the first bioreactor vessel. Similarly, the interconnect conduit also fluidly communicates between the second bioreactor conduit 428 and the first bioreactor conduit 424 of the second bioreactor vessel 420, allowing fluid circulation along the second circulation loop of the second bioreactor vessel. Further, interconnect conduit 450 further fluidly communicates between the second port 416 of the first bioreactor vessel 410 and the first port 422 of the second bioreactor vessel 420 and between the second bioreactor conduit 418 and the first bioreactor conduit 424, enabling the transfer of the contents of the first bioreactor vessel 410 to the second bioreactor vessel 420 as described below. As illustrated in FIG. 3, in one embodiment, interconnect conduit 450 extends from the second bioreactor conduits 418, 428 to the intersection of the first bioreactor conduit 414 of the first bioreactor vessel 410 and the first fluid assembly conduit 442.

[0060] As illustrated in FIG. 3, the first and second fluid assemblies 440, 450 are disposed along the interconnecting conduit 450. In addition, in one embodiment, the first fluid assembly fluidly communicates 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 conduit 414 of the first bioreactor vessel and the first bioreactor conduit 424 of the second bioreactor vessel 420, respectively. The second fluid assembly 444 fluidly communicates with the second port 416 of the first bioreactor vessel 410 and the second port 426 of the second bioreactor vessel 420 via the interconnecting conduit 450.

[0061] A first pump or interconnecting conduit pump 454 capable of flowing fluid bidirectionally is disposed along the first fluid assembly conduit 442, and a second pump or circulation conduit pump 456 capable of flowing fluid bidirectionally is disposed along the interconnecting conduit 450, and their functions and purposes will be described below. In one embodiment, the pumps 454, 456 are high dynamic range pumps. As also shown in FIG. 3, a sterile air source 458 is connected to the interconnecting conduit 450 through a sterile air source conduit 460. A valve 462 positioned along the sterile air source conduit 460 provides selective fluid communication between the sterile air source 458 and the interconnecting conduit 450. Although FIG. 3 shows a sterile air source 458 connected to the interconnecting conduit 450, 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 intermediate the second bioreactor conduit valve and the first bioreactor conduit valve of either the first bioreactor or the second bioreactor without departing from the broader aspects of the present invention.

[0062] Next, in addition thereto, referring to FIGS. 4-6, detailed views of a first fluid assembly 440, a second fluid assembly 444, and a sampling assembly 448 are shown. Referring particularly to FIG. 4, the first fluid assembly 440 includes a plurality of tubing tails 464a-f, each of which is configured to selectively / removably connect to one of a plurality of first storage tanks 466a-f. Each tubing tail 464a-f of the first fluid assembly 440 includes tubing tail valves 468a-f for selectively controlling the flow of fluid to or from each one of the plurality of first storage tanks 466a-f of the first fluid assembly 440. FIG. 4 particularly shows that the first fluid assembly 440 includes six fluid storage tanks, although more or fewer storage tanks may be utilized, as desired, for the introduction or collection of various process fluids. Each tubing tail 464a-f is contemplated to be individually connectable to the storage layers 466a-f at the times required during operation of the fluid assembly 440, as described below.

[0063] Referring particularly to FIG. 5, the second fluid assembly 444 includes a plurality of tubing tails 470a-d, each of which is configured to selectively / removably connect to one of a plurality of second storage tanks 472a-d. Each tubing tail 470a-d of the second fluid assembly 444 includes tubing tail valves 474a-d for selectively controlling the flow of fluid to or from each one of a plurality of second storage tanks 472a-d of the first fluid assembly 444. FIG. 5 particularly shows that the second fluid assembly 444 includes four fluid storage tanks, although more or fewer storage tanks may be utilized as desired for the introduction or collection of various processing fluids. In one embodiment, at least one of the second storage tanks, e.g., the second storage tank 472d, is a collection storage tank for collecting an amplified population 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 contemplates that one or more of the storage tanks 472a-d may be pre-connected to their respective tails 470a-d, and each additional storage tank may be connected to its respective tail within the second fluid assembly 440 in time for its use.

[0064] 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 in which opposing panels are welded or otherwise secured together around their perimeter and support connection conduits for connection to their respective tails, as is known in the art.

[0065] In one embodiment, the storage tank / bag can be connected to the tubing tails of the first and second tubing assemblies using a sterilization welding device. In one embodiment, the welding device is positioned next to module 200 and the welding device is utilized to butt-weld one of the tubing tails so that it attaches to the tail of the tube on the bag (while maintaining sterility). Thus, the operator can provide the bag when it is needed (e.g., 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, cutting the tube with a new razor blade, and heating the cut ends while they are still molten so that the two tube ends are forced together and the razor is pulled away). Conversely, the bag can be removed by welding the tubing from the bag and cutting the two closed tubing at the weld. Thus, the storage tank / bag can be individually connected when desired, and in the present invention, it is not necessary for all storage tanks / bags to be connected at the start of the protocol since the operator has access to the appropriate tubing tails throughout the process to connect the storage tank / bag in time for its use. In fact, as will be described below, all storage tanks / bags are pre-connected, but the present invention does not require pre-connection, and one advantage of the second module 200 is that it enables the operator to access the fluid assembly / tubing during operation, whereby the used bag can be sterilized and disconnected so that other bags can be connected in a sterile state in the protocol.

[0066] As illustrated in FIG. 6, the sampling assembly 448 includes one or more sampling conduits, such as sampling conduits 476a - 476d, fluidly connected to the interconnect conduit 450. Each of the sample conduits 476a - 476d may include sample conduit valves 478a - d that are selectively operable to allow fluid to flow from the interconnect conduit 450 through the sample conduits 476a - 476d. As also shown, the distal ends of each of the sampling conduits 476a - 476d are configured to selectively connect to a sample collection device (e.g., sample collection devices 280a and 280d) for collecting fluid from the interconnect conduit 450. The sample collection device may take the form of any sampling device known in the art, such as a syringe, an immersion tube, a bag, etc. FIG. 6 illustrates the sampling assembly 448 connected to the interconnect conduit, but in other embodiments, the sampling assembly may be fluidly coupled to the first fluid assembly 440, the second fluid assembly 444, the fluid flow path intermediate the second bioreactor conduit valve 434 and the first bioreactor conduit valve 432 of the first bioreactor vessel 410, and / or the fluid flow path intermediate the second bioreactor conduit valve 438 and the first bioreactor conduit valve 436 of the second bioreactor vessel 420. The sampling assembly 448 performs sampling that is fully functionally closed for fluid at one or more points within the system 400 as needed.

[0067] Referring again to FIG. 3, in one embodiment, system 400 may also include a filtration line 482 connected at two points along interconnect line 450, defining a filtration loop along interconnect line 450. A filter 484 is positioned along filtration line 482 to remove permeate waste from the fluid passing through filtration line 482. As shown therein, filtration line 482 includes an upstream filtration line valve 486 and a downstream filtration line valve 488 positioned upstream and downstream of filter 484, respectively. Waste line 490 provides fluid communication between filter 484 and second fluid assembly 444 and, in particular, with tubing tail 470a of second fluid assembly 444, which is connected to waste storage tank 472a. In this regard, waste line 490 carries waste removed from the fluid passing through filtration line 482 by filter 484 to waste storage tank 472a. As illustrated in FIG. 3, filtration line 482 surrounds interconnect valve 452 such that the flow of fluid through interconnect line 450 can be forced through filtration line 482, as described below. A permeate pump 492 positioned along waste line 490 is operable to pump the waste removed by the filter to waste storage tank 472a. In one embodiment, filter 484 is preferably an elongate hollow fiber filter, but other crossflow or dead-end filtration means known in the art, such as flat sheet membrane filters, may also be utilized without departing from the broader aspects of the present invention.

[0068] In one embodiment, the valves of the first fluid assembly 440 and the second fluid assembly 444, and further the bioreactor line valves (i.e., valves 432, 434, 436, 438), the sterilization line valve 462, the interconnect 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 need not include a pinch valve, and the pinch valve diagrams of FIGS. 3-8 may simply represent locations where a pinch valve can operate on the line to prevent fluid flow. In particular, as described below, the pinch valves of the fluid architecture 400 operate / act on corresponding anvils while the fluid path / line is therebetween to "pinch off" the line and can be effected by respective actuators (e.g., solenoids) that prevent fluid flow from passing therethrough.

[0069] In one embodiment, pumps 454, 456, and 492 are peristaltic pumps, and these pumps are integrated into a single assembly as described below. Desirably, the operation of these valves and pumps is automatically commanded according to a protocol programmed to enable proper operation of the module 200. It is contemplated that a second controller 210 may command the operation of these valves and pumps by the module 200.

[0070] Next, referring to FIGS. 8-11, the configuration of the first bioreactor vessel 410 according to an embodiment of the present invention is illustrated. The second bioreactor vessel 420 is desirably, but not necessarily, of the same configuration as the first bioreactor vessel 410, and for simplicity, only the first bioreactor vessel 410 will be described below. In one embodiment, the bioreactor vessels 410, 420 are perfusion-compatible silicone membrane-based bioreactor vessels that support the activation, transduction, and amplification of the cell population therein. The bioreactor vessels 410, 420 may be used for cell culture, cell processing, and / or cell amplification, increasing the cell density for use in medical treatment or other processes. The bioreactor vessels are disclosed herein as being used in conjunction with specific cell types, but it should be understood that the bioreactor vessels may be used for the activation, genetic modification, and / or amplification of any suitable cell type. Further, the disclosed techniques may be used in conjunction with adherent cells, i.e., cells that adhere to and / or proliferate on the 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 Feb. 9, 2018, which is hereby incorporated by reference in its entirety.

[0071] As shown in FIGS. 8 and 9, the first bioreactor vessel 410 may include a bottom plate 502 and a vessel body portion 504 coupled to the bottom plate 502. The bottom plate 502 may be a rigid structure that supports the cell culture. However, the bottom plate may be a non-solid plate (e.g., may be open and / or porous) that allows oxygen to permeate so as to be supplied to the cell culture, as described in more detail with reference to FIG. 9. In the illustrated embodiment, the bottom plate 502 is rectangular or substantially rectangular in shape. In other embodiments, the bottom plate 502 may be any other shape that enables the use of a low-profile vessel and / or maximizes the space in which the first bioreactor vessel can be utilized or stored.

[0072] In one embodiment, the vessel body portion 504 has a rigid, generally concave structure that forms a cavity or internal compartment 506 of the first bioreactor vessel 410 when coupled to the bottom plate 502. As shown there, the vessel body portion 504 may have a peripheral shape that is similar to the peripheral shape of the bottom plate 502 such that the vessel body portion 504 and the bottom plate 502 can be coupled to each other. In addition, as in the illustrated embodiment, the vessel body portion 504 may be made of a transparent or translucent material that allows visual inspection of the contents of the first bioreactor vessel 410 and / or allows light to enter the first bioreactor vessel 410. The internal compartment formed by the bottom plate 502 and the vessel body portion 504 may contain a cell culture medium and a cell culture during use of the first bioreactor vessel for cell activation, genetic modification (i.e., transduction), and / or cell amplification.

[0073] As best shown in FIGS. 8-11, the first bioreactor vessel 410 may include a plurality of ports passing through a vessel body portion 504 that enables fluid communication between an internal compartment 506 related to several processes related to cell activation, transduction / genetic modification, and amplification, such as medium input and waste liquid removal, and the outside of the first bioreactor vessel 410. The ports may include, for example, a first port 412 and a second port 416. The port 416 may be disposed at any position within the vessel body portion 504, such as through the top surface 508 and / or the side surface 510 of the vessel body portion 504 as in the illustrated embodiment. As described in more detail herein, the specific structure of the first bioreactor vessel 410, including the specific quantity and position of the ports 412, 416, enables the first bioreactor vessel 410 to be used to support cell activation, cell genetic modification, and high cell density amplification.

[0074] FIG. 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 illustrated embodiment, the bottom plate 502 may house a grid 510 that may be structurally rigid, but further provide an opening for enabling free gas exchange through the bottom plate 502 to an internal compartment 506 that houses the cell culture. The grid 510 may include a plurality of holes 512 defined between solid regions or crossbars 514 between each of the holes 512 of the grid 510. Thus, the holes 512 may serve as openings for gas exchange, and the crossbars 514 may serve as a structural support for other structures within the internal compartment 506 of the first bioreactor vessel 410 and the cell culture.

[0075] To form an additional support for the cell culture within the inner compartment 506 of the first bioreactor vessel 410, the first bioreactor vessel 410 may comprise a membrane 516 that 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 rates, and / or high permeability to oxygen and carbon dioxide. Thus, the membrane 516 can support a high cell density (e.g., up to about 35MM / cm 2 up to) within the inner compartment 506. The gas-permeable characteristics of the membrane 516 can allow for free gas exchange to support the cell culture and / or cell amplification. As such, the membrane 516 may be a cell culture surface and / or a cell amplification surface. The membrane 516 may desirably have a relatively small thickness (e.g., 0.010 inches or 0.02 cm), which can allow the membrane 516 to be gas-permeable. Further, the membrane 516 can be formed from a gas-permeable material such as silicone or other gas-permeable materials.

[0076] The flatness of the membrane 516 can increase the surface area of the cell culture that adheres for activation, transduction, and / or amplification. To enable the membrane 516 to remain flat during use of the first bioreactor vessel 410, a mesh sheet 520 can be disposed between the bottom plate 502 and the membrane 516. The mesh sheet 520 can serve as a structural support for the membrane 516, such that the membrane 516 remains planar and cannot sag or distort under the weight of any cell culture medium added to the first bioreactor vessel 410 for cell culture and / or cell culture and / or cell amplification. Further, the mesh characteristics of the mesh sheet 520 can allow for support of the membrane 516, yet still be porous to allow for free gas exchange between the inner compartment 506 of the first bioreactor vessel 410 and the environment just outside the first bioreactor vessel 410. The mesh sheet can be a polyester mesh or any other suitable mesh material that can support the membrane and allow for free gas exchange.

[0077] As already described, the container main 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 the membrane 516 can be disposed within or at least partially within the internal compartment 506. An O-ring 522 can be used to seal the first bioreactor container 410 when the container main body 504 is coupled to the bottom plate 502. In one embodiment, the O-ring 522 can be a biocompatible O-ring (size 173, Soft Viton® Fluoroelastomer O-Ring). The O-ring 522 can fit within a groove 524 formed within the circumferential surface 526 of the container main body 504. The circumferential surface 526 faces the top surface 518 of the plate 502 when the main body 504 is fitted to the plate 502. As such, the O-ring 522 can be press-fitted within the groove 524 and press-contacted against the top surface 518 of the plate 516 and / or the bottom plate 502. Such press-fitting of the O-ring 522 desirably seals the first bioreactor container 410 without adhering with chemicals or epoxy resin. Since the first bioreactor container 410 can be used for activation, transduction, and amplification of living cells, the O-ring 522 is desirably formed from a suitable biocompatible, autoclave-processable, gamma-radiation-stable, and / or ETO-sterilization-stable material.

[0078] As described above, the first bioreactor vessel 410 may include a plurality of ports, such as a first port 412 and a second port 416. Ports 412, 416 may be disposed through the vessel body portion 504 and may enable communication between the internal compartment 506 and the exterior of the first bioreactor vessel 410 for several processes related to cell culture, cell activation, cell transfection, and / or cell amplification, such as fluid or media introduction, waste liquid removal, collection, and sampling. Each port 416 may include an opening 526 and respective fittings or tubing 528 (e.g., luer fittings, barb fittings, etc.). In some embodiments, the opening 526 may be configured to enable direct adhesion of tubing and eliminate the need for fittings (e.g., counterbores).

[0079] In one embodiment, in addition to the first port 412 and the second port 416, the first bioreactor vessel 410 may further include an air balance port 530 disposed within the top surface 508 of the vessel body portion 504. The air balance port 530 may be fabricated in a similar form to the first port 412 and the second port 416, with similar reference numerals representing similar parts. The air balance port 530 may be configured to further effect gas exchange between the internal compartment 506 and the exterior of the first bioreactor vessel 410, which is used in cell culture for amplification. Further, the air balance port 530 may help maintain the atmospheric pressure within the internal compartment 506 to provide an environment for cell culture and / or cell amplification within the internal compartment. The air balance port 530 may be disposed through the top surface 508 of the vessel body portion 504 or at any other location around the vessel body portion 504, as in the illustrated embodiment. The central location through the top surface 508 of the vessel body portion 504 may help prevent wetting of the air balance port 530 during mixing of the cell culture through the inclination of the first bioreactor vessel 410, as described in more detail below.

[0080] 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 of a material having biocompatibility, being autoclaveable, and having gamma ray and / or ETO sterilization stability. As such, each element and the first bioreactor vessel 410 as a whole may be used for activation, transduction, and amplification of living cells and / or other processes of the cell manufacturing process.

[0081] The first bioreactor vessel 410 may be configured to enable 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 media feed to the growing cell culture while simultaneously removing the spent media (e.g., the used media). 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 enable communication between the internal compartment 506 and the exterior of the first bioreactor vessel 410 and may be used to add fresh media to the first bioreactor vessel 410 (e.g., from a media reservoir of the first fluid assembly 440). In some embodiments, the first port 412 may be disposed within the vessel body 504 at any position above the surface of the cell culture and media within the first bioreactor vessel 410 and may penetrate. In some embodiments, the first port 412 may be disposed to contact or penetrate the surface of the cell culture and media within the first bioreactor vessel 410.

[0082] The second port 416 can be disposed at any position that is completely or partially submerged below the surface of the cell culture and medium within the first bioreactor vessel 410. For example, the second port 416 can be a substantially horizontal port disposed through one of the sides 510 of the vessel body 504. In some embodiments, the second port 416 can be disposed such that the second port 416 does not reach the bottom (e.g., membrane 516) of the internal compartment 506. In some embodiments, the second port 416 can reach the bottom of the internal compartment 506. The second port 416 can be a dual-function port. As such, the second port can be used to draw perfusion medium from the internal compartment 506 of the first bioreactor vessel 410 to facilitate perfusion of the cell culture. Further, the second port 416 can also be used to remove cells from the cell culture. As pointed out above, in some embodiments, the second port does not reach the bottom surface of the internal compartment 506 of the first bioreactor vessel 410. For example, the second port 416 can be disposed at a location about 0.5 cm away from the membrane 516. Thus, in a static planar position, the second port 416 can be used to remove spent cell culture medium without removing cells from the cell culture since the cells can settle by gravity onto the membrane 516 (e.g., cell expansion surface). Thus, in a static planar position, the second port 416 can facilitate the perfusion process and allow for an increase in the cell density of the growing cell culture within the first bioreactor vessel 410. When it is desirable for the cells to be removed from the internal compartment 506, for example, during harvesting of the cell culture, to minimize the hold-up volume, the first bioreactor vessel 410 can be tilted towards the second port 416 in the manner described below, such that access to the cells for cell removal is enabled.

[0083] In addition, in one embodiment, the second port 416 does not include a filter, and thus the perfusion process can be performed without a filter. As such, there can be no physical impediment to prevent cells from entering the second port 416 when the second port 416 is used for media withdrawal. Further, the second port 416 may be inclined such that the second port 416 is disposed laterally through the side 22 of the container body 504, but the second port 416 can be inclined towards the membrane 516 and the bottom plate 502. The inclined feature of the second port 416 can 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 the groove 524 and helping to maintain the seal of the first bioreactor container 410 during use. Further, in some embodiments, the inclined feature of the second port 416 may serve to reduce the flow rate of the fluid passing through the second port 416 when the used media is withdrawn. In addition, the port diameter, in conjunction with the fluid flow rate exiting the second port 416, is such that the suction rate through the second port 416 used to draw media 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 towards the membrane 516. Thus, as described above, the second port 416 can be used to smooth the perfusion of the cell culture by removing the perfusion media without substantially removing cells from the cell culture. As the cell attachment time increases, the cell concentration of the withdrawn media decreases and can fall within an imperceptible range smoothed by the position of the second port 416. Further, the position of the internal opening 540 can be changed to vary the recommended cell attachment time. A position closer to the membrane 516 may be associated with a longer attachment time, while a position at or near the top of the media may be associated with a shorter attachment time as the cells attach and the growth media first dries up from the top.

[0084] Thus, in one embodiment, the second port 416 can be used not only for removing the medium used in the perfusion process, but also for removing cells of the cell culture from the internal compartment 506, for example, during harvesting of the cell culture. To facilitate more removal of the used perfusion medium and removal of cells, the container body 504 may be provided with angled or chevron-shaped side walls 532. Thus, the chevron-shaped side walls 532 have a vertex, i.e., a tip point, 534. The vertex 534 of the side walls 532 may further comprise the second port 416 passing therethrough, and the container body 504 is disposed near the tip point 534 when the container body 504 is coupled to the bottom plate 502. The angled side walls 532 and the tip point 534 may enable a greater amount of the medium and / or cells of the cell culture to be discharged when the first bioreactor container 410 is inclined towards the second port 416, for example, at an angle of 5 degrees.

[0085] By using perfusion to grow cells smoothed by the positions of the first port 412 and the second port 416, it may be possible to lower the height of the culture medium in the internal compartment 506 (for example, 0.3 to 2.0 cm) as described in more detail with reference to FIG. 10. The relatively low height of the culture medium in the internal compartment 506 makes it possible to make the first bioreactor vessel 410 a vessel with a relatively low profile while allowing for the highest achievable increase in cell density. Further, by using perfusion with the first bioreactor vessel 410, it supports cell growth by supplying fresh culture medium to the cells in the internal compartment 506, but also enables the removal of impurities in the cell culture, and additional cell washing in another device may not be necessary after reaching a specific cell density target in the first bioreactor vessel 410. For example, through perfusion without a filter, the first bioreactor vessel 410 can supply fresh culture medium at a rate of full volume exchange per day and reduce impurities in the cell culture (for example, as a result, impurities are reduced at a rate of about 1 log every 2.3 days). Thus, the structure of the first bioreactor vessel 410 enables the use of perfusion for the growth of the cell culture in the first bioreactor vessel 410, and thus may enable the amplification of the cell culture to a high target density while reducing the impurity level. Also, as described below, through perfusion without a filter, the first bioreactor vessel 410 may be configured to supply fresh culture medium at a rate of substantially multiple volumes per day (for example, more than two volumes per day) for seeding, rinsing, washing / reducing residues, and / or discharging / harvesting cells after amplification.

[0086] To facilitate the use of the rope profile structure of the first bioreactor vessel 410, a relatively low medium height can be maintained within the internal compartment 506. FIG. 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 explained, the vessel body portion 504 is coupled to the bottom plate 502 and can form an internal compartment 506 through which amplification of the cell culture can be achieved through perfusion. As such, replacement or fresh medium 538 may be supplied for cell growth through a first port 412 disposed through the vessel body portion 504, and existing or used medium 538 can be removed through a second port 416 disposed through the side portion 510 of the vessel body portion 504. The perfusion process can facilitate a relatively low medium height 536 of the medium 538 within the internal compartment 506 of the first bioreactor vessel 410. At a relatively low height 536 of the perfusion medium 538 within the internal compartment 506, it becomes possible to make the first bioreactor vessel 410 into a rope profile structure, and thus it may be possible to realize a compact cell manufacturing system as a whole.

[0087] The height 536 of the perfusion medium 538 within the internal compartment 506 of the first bioreactor vessel 410 may be between 0.3 cm and 2 cm, and the headroom 542, i.e., the height of the gap formed between the medium 538 and the top surface 508 of the vessel body portion 504 within the internal compartment 506, may be about 2 cm. Thus, less than 2 mL of medium per 1 cm 2 including medium, cell culture, and headspace, and less than 2 mL of medium per 1 cm 2There can be less than 4 mL of the total volume of contact. A relatively low medium height 536 can possibly make the ratio of the medium volume to the surface area of the membrane 516 lower than a specific value. As such, the ratio of the medium volume to the membrane surface area may be less than a threshold level or within a desired range, and is made convenient for use by the use of perfusion to grow the cells of the cell culture. For example, the threshold level can be a ratio between 0.3 and 2.0. Since the ratio of the medium volume to the membrane surface area is small, it may be possible to make the first bioreactor container 410 have a low-profile or compact structure while still obtaining a cell culture with a high cell density.

[0088] As already explained, the second port 416 having a dual function can be disposed through the container body portion 504 so as to be completely or partially submerged below the surface 544 of the medium 538 in the first bioreactor container 410. In some embodiments, the second port 416 can be disposed such that the second port 416 reaches the bottom (e.g., the membrane 516) of the inner compartment 506. The positioning of the second port 416 can facilitate the removal of the medium and impurities from the cell culture in the inner compartment 506 smoothly without removing the cells until such removal, e.g., harvesting, is desired. The second port 416 without a filter, together with the first port 412, can make it possible to supply the growth medium 538 to the cells for cell amplification using perfusion and remove the used medium 538 and other impurities or by-products. The positions of the first port 412 and the second port 416 having a dual function around the container body portion 504 maintain the height 536 of the medium in the inner compartment 506 at a relatively low level, thereby facilitating a configuration that can make the first bioreactor container 410 a relatively low-profile container while still enabling the production of a high-density cell culture.

[0089] Referring particularly to FIG. 11, the bottom plate 502 of the bioreactor vessel 410 comprises, as part of a more extensive bioprocessing system 10, various features that enable 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 comprises a plurality of recesses 550 formed in the bottom surface of the bottom plate 502, the purpose of which will be described below. In one embodiment, the recesses may be located adjacent to the corners of the bottom plate 502. Each of the recesses 550 generally takes a cylindrical shape and may terminate at a domed or semi-spherical inner surface. As also shown in FIG. 11, the bottom plate 502 may comprise a position verification structure 552 configured to interact with sensors of the second module 200 to 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 break configured to interrupt a light beam of the second module 200 when the first bioreactor vessel 410 is properly installed therein.

[0090] The bottom plate 502 also comprises a pair of flat engagement surfaces 554 formed on adjacent bottom surfaces that are offset from the centerline of the bottom plate (across the width of the bottom plate). Desirably, the engagement surfaces 554 are spaced along the longitudinal centerline of the bottom plate 502 such that they are located adjacent to opposite ends of the bottom plate 502. The bottom plate 502 may further comprise at least one opening or aperture 556 that engages with the bioreactor vessel and enables a bioprocessing device to sense the contents of the first bioreactor vessel 410.

[0091] 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 FIGS. 12-14, the first bioprocessing vessel 410 and the second bioprocessing vessel 420 are received adjacent to each other within a tray 610 of the disposable kit 600, and the various tubes of the fluid architecture 400 are arranged and configured within the tray 610 in the manner described below.

[0092] Referring further to FIG. 15, the tray 610 includes a plurality of generally thin, rigid, or semi-rigid side walls including a bottom surface 620 and front wall 612, rear wall 614, and opposing side surfaces 616, 618 that generally surround an open top. The side walls and bottom surface 620 define an internal compartment 622 of the tray 610. In one embodiment, the open top of the tray 610 receives a removable cover (not shown) that surrounds the internal compartment 622, and is further surrounded by a peripheral flange 624 that provides a surface for preferably mounting on the upper rim of a drawer of the bioprocessing apparatus. The bottom surface 620 of the tray 610 includes a number of openings corresponding to the number of bioreactor vessels within the bioprocessing system. For example, the tray 610 can include a first opening 626 and a second opening 628. The bottom surface 620 can also include additional openings 630 adjacent to the first and second openings 626, 628 for purposes described below. In one embodiment, the tray 610 may be thermoformed, fabricated by a 3D printer, or injection molded, although other manufacturing techniques and processes may also be utilized without departing from the broader aspects of the present invention.

[0093] As best shown in FIG. 15, each of the first and second openings 626, 628 has a perimeter of such shape and / or dimensions that the first and second bioreactor vessels 410, 420 are positioned over the respective openings 626, 628 while a portion of the bioreactor vessels 610, 620 remains accessible from the bottom of the tray 610 through the respective openings 626, 628 and can be supported by the bottom surface 620 of the tray 610 within the internal compartment 622. In one embodiment, the perimeter of the opening comprises at least one claw or protrusion for supporting the bioreactor vessel over the respective opening. For example, the perimeter of each opening 626, 628 may comprise claws 632 that project inwardly towards the center of the openings 626, 628 for supporting the bioreactor vessels 410, 420 placed thereon. As shown in FIGS. 12 and 15, the tray 610 may also comprise one or more ridges that extend upwardly over the openings 626, 628 for preventing lateral movement of the bioreactor vessels when received over the respective openings 626, 628. Thus, the ridges serve as an alignment device that facilitates proper positioning of the bioreactor vessels 410, 420 within the tray 610 and helps prevent accidental movement of the bioreactor vessels 410, 420 during loading or positioning of the kit 600 within the second module 200, as described below.

[0094] Referring further to FIGS. 12 and 13, tray 610 may comprise one or more support ribs 636 formed on the bottom surface of tray 610. Support ribs 636 traverse the width and / or length of tray 610, providing rigidity and strength to tray 610 and facilitating the movement and manipulation of kit 600. Ribs 636 may be integrally formed with the tray or added as auxiliary components via attachment means known in the art (see FIG. 13). In one embodiment, tray 610 includes an opening 638 for receiving an engagement plate, also referred to herein as a tubing module 650, which holds fluid flow conduits in an organized manner and holds them in place for engagement by pumps and pinch valves. In other embodiments, tubing module 650 may be integrally formed with the rear wall 614 of tray 610.

[0095] Figures 16 and 17 illustrate the configuration of tubing module 650 according to an embodiment of the present invention. As shown therein, tubing module 650 receives the first fluid assembly line 442, the interconnect line 450, and the waste line 490 of fluid flow system 400, and the first fluid assembly line 442, the interconnect line 450, and the permeate waste line 490 are selectively engaged with the respective pump heads 454, 456, 492 of the peristaltic pump assembly described below with respect to FIGS. 35 and 36. It includes a first tubing holder block 652 configured to hold in place. In one embodiment, the fluid assembly line 442, the interconnect line 450, and the waste line 490 are maintained in an orientation that extends horizontally and is spaced vertically by the first tubing holder block 652. In particular, as best shown in FIG. 17, the first tubing holder block 652 engages each of the lines 442, 450, 490 with two spaced-apart arrangements 656, 658 (through a clip or simple interference between the tube and the slot within the tubing holder block 652) that define a void therebetween. As also shown in FIG. 17, the first tubing holder block 652 includes a clearance opening 660 configured to receive a shoe (not shown) of the peristaltic pump assembly. This configuration causes peristaltic compression of lines 442, 450, 490 by the respective pump heads of the peristaltic pump and can provide the respective driving forces of the fluid passing through the lines, as described below.

[0096] Referring further to FIGS. 16 - 18, the tubing module 650 further includes a second tubing holder block 654 integrally formed with (or otherwise coupled to) a first tubing holder block 652. The second tubing holder block 654 is configured to receive all of the fluid flow conduits of the fluid flow system 400 to which pinch valves are 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 conduit 414 and the second bioreactor conduit 418 of the first bioreactor vessel 410, the first bioreactor conduit 424 and the second bioreactor conduit 428 of the second bioreactor vessel 420, the sterile air source conduit 460, the interconnect conduit 450, the filtration conduit 482 (and, in some embodiments, the sampling conduits 476a - 476d). Similar to the first tubing holder block 652, the second tubing holder block 654 may hold these tubes in a horizontally extending and vertically spaced orientation. In particular, the second tubing holder block 654 may include a plurality of vertically spaced and horizontally extending slots 666 configured to receive the conduits therein. FIGS. 18 and 19 also best illustrate the configuration of the slots 666 that hold all of the flow conduits that are subject to / contact the pinch valves. Desirably, the slots 666 follow the contour of the block 654, and in particular, cross a planar backplate so as to open towards the filter 484. As shown in FIG. 18, in one embodiment, the second tubing holder block 654 has one or more narrow tubing slots 682 at the bottom of the second tubing holder block 654 for holding a loop of the interconnect conduit 450 from which the sampling conduits extend, and a waste fluid conduit tubing slot 684 for receiving the tubing tail 470a connected to the waste fluid reservoir 472a.

[0097] The second tubing holder block 654 may include a planar backplate 662 having a plurality of openings 664 corresponding to a plurality of fluid flow conduits held by the second tubing holder block 654. In particular, at least one opening 664 is horizontally aligned with each slot 666 and the conduit held therein. As best shown in FIG. 16, the second tubing holder block 654 includes two clearance openings 668, 670 configured to receive an anvil (not shown) of a pinch valve assembly passing therethrough. This configuration, as described below, selectively compresses 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 conduit 414 and the second bioreactor conduit 418 of the first bioreactor vessel 410, the first bioreactor conduit 424 and the second bioreactor conduit 428 of the second bioreactor vessel 420, the sterile air source conduit 460, the interconnect conduit 450, and the filtration conduit 482 against the anvil by respective pistons of the actuator of the pinch valve array, and enables selectively obstructing or permitting fluid flow. As shown in FIGS. 18 and 19, the openings 664 may be arranged in first and second columns positioned adjacent to each other, and the openings in the first column of openings are vertically offset with respect to the openings in the second column of openings such that the openings in the first column of openings do not horizontally align with the openings in the second column of openings. This configuration enables the tubing module 650, the tray 610, and the kit 600 to have an overall low profile.

[0098] In one embodiment, the filter 484 (shown as an elongate hollow fiber filter module in FIG. 16) can be integrated with the tubing module 650, such as by using a retaining clip 672 to attach the filter 484 to the tubing module 650. When the filter 484 is a hollow fiber filter, the filter 484 may extend substantially the entire length of the tubing module 650, and may include a first inlet end 674 for receiving an input flow of fluid from the filter line 482, and a second outlet end 676 for carrying the retained fluid after removal of the permeate / effluent back to the filter line 482 and the interconnect line 450 and circulating it to one of the first bioreactor vessel 410 or the second bioreactor vessel 420. The filter 484 may also include a permeate port 678 adjacent to the second outlet end 676 that connects to a waste line 490 for carrying the waste / effluent to the permeate / effluent storage tank 472a. Finally, the tubing module 650 may include a plurality of features 680 for receiving clips and weaving bioreactor lines (e.g., 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).

[0099] Similar to the tray 610, the tubing module 650 may be thermoformed, fabricated by a 3D printer, or injection molded, although other manufacturing techniques and processes may also be utilized without departing from the 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 removably received by the tray 610.

[0100] Figures 20-22 are various views showing one embodiment of kit 600, which illustrate the fluid conduits of fluid architecture 400 received by first bioreactor vessel 410 and second bioreactor vessel 420 received within tray 610 and tubing module 650. As shown therein, instead of having an opening 630, kit 600 as shown in Figures 20-22 comprises a solid bed and provides a sampling space 631 within tray 610 for receiving receptacles that hold sampling conduits (e.g., sampling conduits 476a, 476b). Kit 600 comprises a modular platform for cell processing that can be easily set up and discarded after use. At the tubing tails of first and second fluid assemblies 440, 444, a plug & play function can be used, whereby connections to various media, reagents, waste fluids, sampling, and collection bags can be quickly and easily made, enabling the various processes used to be performed on a single platform. In one embodiment, the connections and disconnections can be accomplished by sterile cutting and welding of tube segments, as by a TERUMO device as described above, or by pinching, welding, and cutting tail segments as known in the art.

[0101] Next, referring to FIGS. 23-25, kit 600 is specifically configured to be received by a bioprocessing device 700 that houses all of the hardware (i.e., controllers, pumps, pinch valve actuators, etc.) necessary to operate kit 600 as part of a method of bioprocessing. In one embodiment, bioprocessing device 700 and kit 600 (housing fluid architecture 400 and bioreactor vessels 410, 420) together form the second bioprocessing module 200 described above in connection with FIGS. 1 and 2. Bioprocessing device 700 includes a housing 710, which has a plurality of drawers 712, 714, 716 that are receivable within housing 710. FIG. 23 shows device 700 housing three drawers, although the device may have as few as a single drawer, two drawers, or more than three drawers, and each drawer may be made to perform bioprocessing operations simultaneously. In particular, in one embodiment, each of drawers 712, 714, 716 may be a stand-alone bioprocessing module (i.e., equivalent to second modules 200a, 200b, and 200c described above in connection with FIG. 2) for performing processes of cell activation, genetic modification, and / or amplification. In this regard, any number of drawers may be added to device 700 for parallel processing of multiple samples from the same or different patients. In one embodiment, rather than each drawer sharing a common housing, in one embodiment, each drawer may be received within a dedicated housing, and the housings may be stacked on top of one another.

[0102] As shown in FIGS. 23 and 24, each drawer, e.g., drawer 712, includes a plurality of sidewalls 718 and a bottom surface 720 that defines a processing chamber 722 and generally an open top. Drawer 712 is movable between a closed position where the drawer is fully received within housing 710, as shown for drawers 714 and 716 in FIG. 23, and an open position where drawer 712 extends from housing 710 and provides access to processing chamber 722 through the open top, as shown for drawer 712 in FIGS. 23 and 24. In one embodiment, one or more of the sidewalls 718 are temperature controlled to control the temperature within processing chamber 722. For example, one or more of the sidewalls 718 may include or be in thermal communication with an embedded heating element (not shown), whereby the sidewall 718 and / or processing chamber 722 may be heated to a desired temperature (e.g., 37° C.) to maintain processing chamber 722 at a desired temperature 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 similarly be temperature controlled (e.g., with embedded heating elements). The hardware compartment 724 of drawer 712 behind processing chamber 722 may house all of the hardware components of apparatus 700, as described in detail below. In one embodiment, drawer 712 may further include an auxiliary compartment 730 adjacent to processing chamber 722 for housing a storage tank containing media, reagents, etc. connected to first fluid assembly 440 and second fluid assembly 444. In one embodiment, auxiliary compartment 730 may be refrigerated.

[0103] 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 the drawer 712 to selectively move the drawer 712 between an open position and a closed position. The linear actuator is operable to effect a smooth and controlled movement of the drawer 712 between the open position and the closed position. In particular, the linear actuator is configured to open and close the drawer 712 at a substantially constant speed (and with minimal acceleration and deceleration at the start and stop of operation) to minimize disturbance to the contents of the bioreactor vessel.

[0104] FIG. 25 is a top view showing the interior of the drawer showing the processing chamber 722, the hardware compartment 724, and the auxiliary compartment 730 of the drawer 712. As illustrated therein, the hardware compartment 724 is disposed behind the processing chamber 722 and is integrated with the power supply 732 and the operation control board and drive electronic circuit 734 that communicate with the second module controller 210 in some other form, the low power solenoid array 736, the pump assembly 738 (including pump heads for pumps 454, 456, 492), and the drawer engagement actuator 740. The hardware compartment 724 of the drawer 712 further includes 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. 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. The hardware compartment (and the components described) are all attached to the rear base plate. Both plates are slidably attached to the rails. Further, the drawer engagement actuator 740 is used to couple the two plates and move the two plates and the components mounted on the plates to the engaged position (move the pump roller head into the pump shoe, thereby compressing the pump tubing when inserted therebetween). As further described herein, the pump assembly performs selective operations on the conduits 442, 450, and 490 of the fluid path 400, resulting in independent peristaltic propulsion forces for each. Similarly, the tubing holder block 654 of the tray 600 will be positioned between the solenoid array 736 and the anvil 744, as further described.

[0105] As also illustrated in FIG. 25, two bed plates, for example, first and second bed plates 746, 748 are disposed within the processing chamber 722 on the bottom surface 720 and extend upwardly or project therefrom. In one embodiment, the processing chamber 722 can accommodate 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 thereon or engage them in some other manner. As also shown in FIG. 25, the drawer 712 also includes a plate 750 configured with load cells positioned adjacent the bed plates 746, 748 within the processing chamber 722 to sense the weight of a storage layer, for example, the waste liquid storage tank 472a positioned thereon.

[0106] Figures 26 to 28 best illustrate the configuration of the bed plates 746, 748, and Figure 28A shows the hardware components positioned under the bed plate. As used herein, the bed plates 746, 748 and the hardware components (i.e., sensors, motors, actuators, etc. that are integrated with or positioned under it as shown in Figure 28A) may be collectively referred to as the bed plate. The first and second bed plates 746, 748 have substantially the same configuration and operation, but for simplicity, only the first bed plate 746 will be referred to in the following description of the bed plates 746, 748. 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 plate may generally be rectangular in shape. The bed plates 746, 748 may generally also include relief or clearance regions 758 corresponding to the positions of the protrusions or claws 632 of the tray 610, the purpose of which will be described below. The bed plates 746, 748 are supported by a plurality of load cells 760 (e.g., 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.

[0107] In one embodiment, the bed plate 746 may comprise an embedded heating element or be in thermal communication with a heating element such that the contents of the processing chamber 722 and / or the first bioreactor vessel 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 element that heats the side wall 718, the top wall, and the bottom surface.

[0108] As illustrated, the bed plate 746 includes a plurality of positioning pins or alignment pins 754 that project above the top surface 452 of the bed plate 746. The number and the position and spacing of the positioning pins 754 may correspond to the number, position, and spacing of the recesses 550 within the bottom surface of the bottom plate 502 of the bioreactor vessels 410, 420. As shown below, the positioning pins 754 are receivable within the recesses 550 of the bottom plate 502 of the first bioreactor vessel 410 when the first bioreactor vessel 410 is positioned within the processing chamber 722 so as to ensure proper alignment of the first bioreactor vessel 410 on the first bed plate 746.

[0109] Referring further to FIGS. 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, although other sensor types such as a lever switch may be utilized without departing from the broader aspects 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 installed 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 break (i.e., a flat pawl) and a substantially IR - impermeable position verification structure 552 is used, the beam break interrupts (i.e., breaks) the infrared light beam when the first bioreactor vessel 410 is fully installed on the bed plate 746. This signals to the controller 210 that the first bioreactor vessel 410 is properly installed. After positioning the first bioreactor vessel 410 on the first bed plate 746, if the controller does not detect that the infrared light beam of the sensor 756 has been interrupted, this indicates that the first bioreactor vessel 410 is not fully or properly installed on the bed plate 746 and that adjustment is required. 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 commencing bioprocessing.

[0110] Referring still further to FIGS. 26-28A, the bed plate 746 further 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, for example, the temperature level within the bioreactor vessel 410. In one embodiment, the bed plate 746 may further include a resistance temperature detector 760 configured to measure the temperature of the top surface 752 and a carbon dioxide sensor (disposed under the bed plate) for measuring the carbon dioxide level within the bioreactor vessel.

[0111] As further shown in FIGS. 26-28A, each of the bed plates 746, 748 includes an actuator mechanism 761 (e.g., a motor) having, for example, a pair of opposing cam arms 762. The cam arms 762 are received within slots 764 in the bed plates 746, 748 and are rotatable about 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 more detail below, the actuator mechanism is operable to tilt the bioreactor vessel on the bed plate to effect stirring and / or assist in discharging from the bioreactor vessel.

[0112] Referring to FIGS. 29 - 32, a more detailed view of the linear actuator 768 and the drawer engagement actuator 740 within the hardware compartment 724 of the drawer 712 is shown. Referring to FIG. 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 outside of the housing 710 that enables user control of the movement of the drawer. The linear actuator 770 controls the movement of the drawer 712 to prevent agitation of the contents of the bioreactor vessel within the drawer 712. In one embodiment, the linear actuator 768 has a stroke of approximately 16″ and a maximum speed of approximately 2 inches per second.

[0113] Next, referring to FIG. 30, the drawer engagement actuator 740 includes a feed screw 772 and a clevis arm 774 attached to a front plate 751 within the drawer 712. The drawer engagement actuator is operably connected to the pump assembly 738 and the solenoid array 736 to be operable to move the pump assembly 738 and the solenoid array 736 between a first clearance position and an engagement position.

[0114] Figures 31 and 32 relatively clearly show the clearance position and the engagement position of the pump assembly 738 and the solenoid array 736. As illustrated in Figure 31, in the clearance position, the pump assembly 738 and the solenoid array 736 are each spaced apart from the pump shoe 742 and the pinch valve anvil 744. After the feed screw 772 is actuated, the drawer engagement mechanism 740 linearly moves the pump assembly 738 and the solenoid array forward to the position shown in Figure 32. In this position, the pump head of the pump assembly 738 engages the conduits 442, 450, 490 within the first tubing holder block 652, and the solenoid array 736 is positioned close enough to the pinch valve anvil 744 such that the pistons / actuators of the solenoid array 736 can pinch / clamp each of the fluid flow conduits of the second tubing holder block 654 against the pinch valve anvil 744, thereby obstructing the flow through those fluid flow conduits.

[0115] Referring again to FIG. 24 and further to FIGS. 33-39, during operation, the drawer 712 can be controllably moved to the open position by actuating a rocker switch 770 outside the housing 710. A disposable drop-in kit 600 that houses the tubing module 650 (which holds all the tubes and tubing tails of the fluid architecture 400) as well as the first and second bioreactor vessels 410, 420 is lowered into place within the processing chamber 722. When the kit 600 is lowered into the processing chamber 722, the pump shoe 742 is received through a clearance opening 660 in the first tubing holder block 652 such that the pump tubes 442, 450, 490 are positioned between the pump shoe 742 of the peristaltic pump assembly 738 and the pump heads 454, 456, 492. FIG. 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. FIG. 36 illustrates the positioning of the pump heads 454, 456, 492 relative to the pump tubes 442, 450, 490 when the kit 600 is received within the processing chamber 722. As shown therein, the pump tubes 442, 450, 490 are positioned between the pump shoe 742 and the pump heads 454, 456, 492. During operation, when the drawer engagement actuator 740 positions the pump assembly 738 in the engaged position, the pump heads 454, 456, 492 are selectively operable under the control of the controller 210 to initiate, maintain, and stop the flow of fluid through the tubes 442, 450, 490.

[0116] Similarly, when the kit 600 is lowered into the processing chamber 722, the pinch valve anvil 744 is configured to receive the tubing tails 464a - f of the first fluid assembly 440 held by the second tubing holder block 654, the tubing tails 470a - d of the second fluid assembly 444, the first bioreactor tubing 414 and the second bioreactor tubing 418 of the first bioreactor vessel 410, the first bioreactor tubing 424 and the second bioreactor tubing 428 of the second bioreactor vessel 420, the sterile air source tubing 460, the interconnect tubing 450, and the filtration tubing 482 through the clearance openings 668, 670 of the second tubing holder block 654 such that they are positioned between the solenoid array 736 and the pinch valve anvil 744. This configuration is best illustrated in FIGS. 37 - 39 (FIGS. 37 and 38 illustrate the relationship between the solenoid array 736 and the pinch valve anvil 744 before receiving the backplate 662 of the second tubing holder block 654 within the space 776).

[0117] As shown there, each solenoid 778 of the solenoid array 736 comprises a piston 780 that is linearly extendable through a related opening (of the openings 664) in the backplate 662 of the second tubing holder block 654 to clamp a related tube against the pinch valve anvil 744. In this regard, 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, namely, valves 432, 434, 436, 438, the sterilization line valve 462, the interconnect line valve 452, and the filtration line valves 486, 488). In particular, the pinch valves of the fluid architecture 400 are provided by respective solenoids 778 (i.e., the pistons of the solenoids) of the solenoid array 736 that operate / act against their respective anvils 744 while the fluid path / tubing is therebetween. In particular, in operation, each solenoid 778 is selectively operable under the control of the controller 210 to clamp the related fluid flow tubing against the anvil 744 to prevent the flow of fluid therethrough when the drawer engagement actuator 740 positions the solenoid array 736 in the engaged position. In the present invention, it is contemplated that each fluid tubing is positioned between a planar anvil surface and a planar solenoid actuator head. Alternatively, the solenoid actuator head may comprise a shaped head, such as two tapered surfaces intersecting at an elongate edge similar to a plus driver, that is optimized to apply a desired pinching force to an elastic flexible fluid tubing. Still alternatively, the anvil surface may comprise an elongate protrusion or projection that extends towards each fluid tubing such that the planar solenoid head presses the fluid tubing against a protrusion extending in this lateral direction to close the tubing to the fluid flow therethrough.

[0118] Referring to FIGS. 33, 34, and 40, when kit 600 is lowered into the processing chamber of the drawer, first bioreactor vessel 410 and second bioreactor vessel 420 are supported over openings 626, 628 by the perimeter of the openings and in particular by claws / projections 632. As the kit is further lowered, bed plates 746, 748 pass through openings 626, 628 and receive or otherwise engage bioreactor vessels 410, 420. The shape of openings 626, 628 and top surfaces 752 of bed plates 746, 748 (e.g., relief regions 758 of bed plates 746, 748 corresponding to claws / projections 632 of tray 610) are such that after bioreactor vessels 410, 420 are received by bed plates 746, 748 with the bottom surface of tray 610 and claws / projections 632 positioned lower than top surfaces 752 of bed plates 746, 748, and bioreactor vessels 410, 420 are supported by bed plates 746, 748 in a spaced relationship from bottom surface 620 of tray 610, tray 610 can continue to move downward. This ensures that tray 610 does not interfere with the horizontal installation of bioreactor vessels 410, 420 on bed plates 746, 748.

[0119] When the bed plates 746, 748 penetrate the openings 726, 728 in the tray 610, the positioning pins 754 on the bed plates 746, 748 are received within corresponding recesses 550 in the bottom plates 502 of the bioreactor vessels 410, 420, which ensures that the bioreactor vessels 410, 420 are properly aligned with the bed plates 410, 420. When properly installed on the bed plates 746, 748, the beam break 552 interrupts the light beam of the sensor 756 within the bed plate, indicating to the controller that the bioreactor vessels 410, 420 are in the proper position. Since the bed plates 746, 748 and the alignment pins are of the same height, the interruption of the light beam of the sensor 756 by the beam break 552 also ensures that the bioreactor vessels 410, 420 are horizontal. At this properly installed position, the sensors 759 on the bed plates 746, 748 are aligned with the openings 556 within the bottom plates 502, thereby enabling the sensing of the process parameters within the internal compartments of the bioreactor vessels 410, 420, respectively. In addition, at the fully installed position, the cam arms 762 of the bed plates 746, 748 are aligned with the flat engagement surfaces 554 on the bottom plates 502 of the bioreactor vessels 410, 420, respectively.

[0120] 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 heating pads 782 and heating modules 784 can be positioned under the bed plate 746 to heat the bed plate 746. As shown in Figure 40, the carbon dioxide sensing module 786 can also be positioned under the bed plate to sense the carbon dioxide content within the processing chamber 722.

[0121] As further shown in FIG. 40, in one embodiment, the sidewall 718 and bottom of the drawer 712 (and the top wall of the housing) may include a cover 788, an insulating foam layer 790 to help minimize heat loss from the processing chamber 722, a film heater 792 to heat the walls as described above, and an internal metal plate 794. In one embodiment, the internal metal plate 794 may be formed from aluminum, although other thermally conductive materials may also be utilized without departing from the broader aspects of the present invention. The drawer 712 may further include one or more brush seals 796 to help minimize heat loss from the processing chamber 722 and a thermal insulation layer 798 to minimize or prevent 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)).

[0122] Referring again to FIG. 34, when the kit 600 is received within the processing chamber 722, a load cell 750 within the bottom of the processing chamber 722 adjacent to the second bed plate 748 allows the waste liquid bag 472a to pass through an opening 730 within the tray 610 such that it can be connected to the tubing tail 470a and positioned over the load cell 750. As shown there, when the kit 600 is received within the drawer 712, the second tubing holder block 654 holds the tubing such 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 within its storage layer. In one embodiment, the sampling conduits 476a - 476d similarly penetrate into the auxiliary compartment 730.

[0123] Next, referring to FIGS. 41-44, the operation of the cam arms 762 of the bed plates 746, 748 is illustrated. As shown therein, the cam arms 762 are in a retracted position where they are positioned below the top surfaces of the bed plates 746, 748, and they are rotated about the cam pins 766 and extended above the bed plates 746, 748 to engage the flat engagement surfaces 554 of the bioreactor vessels 410, 420 and lift and separate the bioreactor vessels 410, 420 from the bed plates 746, 748. The cam arms 762 are retracted below the top surfaces of the bed plates 746, 748 in a default state, so the bioreactor vessels 410, 420 are supported on the horizontal bed plates 746, 748 (and in particular, the horizontal alignment pins 754. No power is required to maintain the bioreactor vessels in a horizontal position). In particular, when the bioreactor vessels 410, 420 are received on the bed plates 746, 748, they are in a horizontal position. In the event of a power outage, the bioreactor vessels 410, 420 remain installed on the horizontal bed plates 746, 748 and do not need to be constantly adjusted using the cam arms 762 to maintain a horizontal position. This is in contrast to some systems that may require the use of a servo motor to constantly adjust the bioreactor to maintain a horizontal position. In fact, with the configuration of the cam arms 762 of the present invention, the actuator only needs to be energized when tilting the bioreactor vessels for agitation / mixing, as described below, thereby minimizing the amount of heat applied to the processing chamber 722.

[0124] As shown in FIGS. 41 to 43, the cam arm 762 may be operable sequentially to stir the contents of the bioreactor vessels 410, 420. For example, when it is desired to stir the contents of the bioreactor vessel 410, 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-lifted end remains received within the corresponding recess 550 in the bottom plate 502. The raised cam arm is then rotated back to the clearance position under the bed plate and the opposing cam arm is rotated to the engaged position to lift the opposing end of the bioreactor vessel away from the bed plate and the positioning pins.

[0125] In one embodiment, the cam actuation system may be designed such that the cam arm 762 can return to the home position without touching the bioreactor vessel, thereby preventing disruption to the culture and allowing the cam arm 762 to return to (or be tested at) the home position at any point during a long cell processing period. Thus, while the present invention contemplates that other rocking or agitation means may be provided on the bioreactor vessel, having two cam arms 762 on opposite sides of the bedplate can minimize the overall height of the mixing mechanism. For example, it is possible to achieve a movement of ±5 degrees by a central actuator (disposed at the center of the bedplate), but a movement of 0 to 5 degrees of the vessel driven by cam arms on both sides of the vessel can achieve substantially the same movement of the vessel, which effectively provides a movement of ±5 degrees to the vessel at half the height. Further, 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 the formation of waves within the vessel, maximize the amplitude of the waves, and thus (ideally) maximize the time to achieve uniformity and homogeneity of the vessel contents. The timing can also be adjusted based on the volume within a vessel having a given geometry that maximizes mixing efficiency.

[0126] In one embodiment, the optical sensor 756 can be used to confirm that the first bioreactor vessel 410 has been correctly repositioned after each cam agitation operation. It is further contemplated that correct repositioning of the bioreactor vessel can be checked and verified even during alternating cam operations. This allows for quick detection of alignment misalignment, substantially in real time, thereby enabling an operator to intervene to reposition the bioreactor vessel without substantial deviation from the bioprocessing operation / protocol.

[0127] FIG. 43 is a schematic view showing the position of the fluid 800 within the bioreactor vessel in this agitation process. As shown in FIG. 42, in one embodiment, the homing sensor 802 integrated into the bed plate 746 can be utilized by the controller to determine when the cam arm 762 has returned to the clearance position below the top surface of the bed plate 746. This is useful in coordinating the movement of the cam arm 762 to provide the desired mixing frequency within the bioreactor vessel. In one embodiment, the cam arm 762 is configured to provide a tilt angle of up to 5 degrees with respect to the bed plate 746.

[0128] Referring to FIG. 44, the interface between the positioning pin 754 of the bed plate of the bioreactor vessel 410 and the recess 550 within the bottom plate 502 during mixing / stirring is illustrated. In one embodiment, the recess 550 has a domed or semi-spherical inner surface and a diameter d1 that is larger than the diameter d2 of the positioning pin 754. As illustrated in FIG. 44, this configuration provides a 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.

[0129] In one embodiment, each drawer of the bioprocessing apparatus 700, for example, drawer 712, preferably comprises a flip-down front panel 810 that is hingedly attached as shown in FIGS. 45 - 50. The flip-down front panel 810 allows access to the auxiliary compartment 730 without opening the drawer 712, as best shown in FIGS. 45, 49, and 50. As will be appreciated, this configuration allows for in-process sampling and media bag replacement. In connection with the above, in one embodiment, the auxiliary compartment 730 can be composed of a plurality of telescoping sliding rails 812 that provide attachment means 815 for suspending various storage tanks / media bags. The rails 812 are movable from a retracted position within the compartment 730, as shown in FIG. 48, to an extended position out of the compartment 730, as shown in FIG. 49. When the collection bag is full or when media / fluid bag replacement is needed, simply extend the rails 812 and remove the bag clips. New bags can be connected to their respective tails and then suspended from the rails and slid back into the auxiliary compartment 730 without opening the drawer 712 or pausing the process. In one embodiment, the rails 812 can be mounted on a laterally extending cross rod 814. The rails 812 can thus be slid laterally on the rod 814, extendable from and retractable into the auxiliary compartment. Additionally, when the drawer is open (FIG. 46), the rails 812 can rotate around the rear cross rod to clean the compartment 730 and allow the user to screw the tubing tails forward of the compartment 730, which provides three degrees of freedom.

[0130] As illustrated in FIG. 51, in another embodiment, the media / fluid bag can be mounted on a platform 820 that is rotatable out of the auxiliary compartment 730 from a stuffed position to an access position. For example, the platform 820 can be mounted to move along a guide track 822 formed within the sidewall of the auxiliary compartment 730.

[0131] Referring to FIG. 52, in one embodiment, the bioprocessing apparatus 700 can further include a low-profile waste liquid tray 816 that is received within the housing 710 under each drawer, such as drawer 712. The waste liquid tray 816 is independently mounted on the drawer so as to be movable between a closed position and an open position. Desirably, in the closed position, the tray 816 extends in the same plane as the front surface of the drawer, and in the open position, the tray 816 exposes its own chamber 819 for access by the operator. The chamber 819 enables easy storage of a large waste liquid bag connected to the fluid flow path of the tray 600 placed thereon and allows access thereto without opening the drawer 712. In addition, in the closed position, the waste liquid tray 816 positions the chamber 819 so as to be aligned below with the drawer and has a size and shape operable to contain leakage from the processing chamber 722 or the auxiliary compartment 730.

[0132] In one embodiment, each drawer can include a camera positioned above the processing chamber (e.g., above each bioreactor vessel 410, 420) so that the interior of the drawer 712 can be visually monitored without opening the drawer 712. In one embodiment, the camera (or additional cameras) can be integrated with the bedplate assembly or integrated on the sidewall looking across the interior of the bioreactor vessel.

[0133] Accordingly, the second module 200 of the present invention enables cell processing to be automated to an extent 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, drainage, 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 filler 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 the human touch points. In fact, human touch points may only be necessary for source / media bag addition and removal, sampling, and data entry (e.g., sample volume, cell density, etc.).

[0134] Referring to FIGS. 53-77, an automated general protocol for an immobilized Ab coating by amplification, soluble Ab addition, and a workflow with a gamma retrovirus vector in the same container using the second module 200 and its fluid flow architecture 400 is illustrated. This general protocol results in activation (illustrated in FIGS. 53-59), pre-transduction preparation and transduction (illustrated in FIGS. 60-71), amplification (FIGS. 72-76), and, for some embodiments, harvesting of a population of cells in an automated, functionally closed manner (FIG. 77). When explaining the operation of the pinch valve, in the following, the valve is in its closed state / position when it is not used for a particular operation. Thus, after the valve is opened and a particular operation becomes possible, when that operation is completed, the valve is closed before proceeding to the next operation / step.

[0135] As shown in FIG. 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, by opening valves 434 and 474a and operating the circulation line pump 456, it is discharged to the waste liquid storage tank 472a of the first fluid assembly 440 through the interconnecting line. As described herein, the discharge of the bioreactor vessel 410 can be facilitated by tilting the bioreactor vessel 410 using the cam arm 462.

[0136] After discharging the antibody coating solution, valves 432 and 468e are opened, pump 454 is activated, and the pump pumps the rinse buffer from the storage tank 466e connected to the first fluid assembly 440 through the first bioreactor line to the first bioreactor vessel 410. Then, by operating the circulation line pump 456 and opening valve 474a, the rinse buffer is discharged to the waste liquid storage tank 472a through the interconnecting line 450. In one embodiment, this rinsing and discharging procedure may be repeated a plurality of times to sufficiently rinse the first bioreactor vessel 410.

[0137] Referring to FIG. 55, after flushing the first bioreactor vessel 410 with the buffer, the cells in the 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 operating pump 454. The cells are passed by the pump into the first bioreactor conduit 414 of the first bioreactor vessel 410 and enter the bioreactor vessel 410 through the first port 412. As shown in FIG. 56, valves 432 and 468a are opened, pump 454 is operated, and the pump sends the second antibody (Ab) solution from the storage tank 466a connected to the first fluid assembly 440 through the first port 412 into the first bioreactor vessel 410.

[0138] After the second antibody solution is pumped into the first bioreactor vessel, the second antibody solution storage tank 466a is flushed and the flushing medium is pumped into the first bioreactor vessel. In particular, as shown in FIG. 57, valves 474b, 452, and 468a are opened, and the flushing medium from the flushing medium storage tank / bag 472b of the second fluid assembly 444 is sent to the second antibody solution storage tank 466a using pump 454 to flush the storage tank. After flushing, valve 432 is opened and the flushing medium is pumped from the storage layer 466a into the first bioreactor vessel 410. In one embodiment, the second antibody solution storage tank 466a can be flushed multiple times using this procedure.

[0139] After rinsing the second antibody solution storage tank 466a, the inoculum / species cell bag 466d may optionally also be rinsed. In particular, as shown in FIG. 58, valves 474b, 452, and 468d are opened, and the rinse medium from the rinse medium storage tank / bag 472b of the second fluid assembly 444 is sent to the inoculum / species cell bag 466d using pump 454 to rinse the bag. After rinsing, valve 432 is opened and the rinse medium is sent from bag 466d to the first bioreactor vessel 410 using pump 454. By sending the rinse medium to the first bioreactor vessel 410 by pump after rinsing the inoculum / species cell bag 466d, the cell density in the first bioreactor vessel 410 is decreased. At this time, a sample may be taken to measure one or more parameters of the solution in the bioreactor vessel before activation (e.g., to confirm that the desired cell density is present before activation). In particular, as shown in FIG. 58, valves 434, 452, and 432 are opened, pump 456 is activated, and the contents of the first bioreactor vessel 410 are pumped along the first circulation loop of the first bioreactor vessel (i.e., from the second port 416, through the interconnecting line 450, through the first bioreactor line 414 and the first port 412 of the first bioreactor vessel 410, and back to the first bioreactor vessel 410). To take a sample, a first sample container 280a (e.g., a dipping tube, syringe, etc.) is connected to the first sample tubing tail 476a, valve 478a is opened, and a portion of the flow is diverted through the interconnecting line 450 and into the first sample container 280a for analysis.

[0140] If the analysis of the collected sample indicates that all solution parameters are within a predetermined range, the solution in the first bioreactor vessel 410 is incubated for a predetermined time period to activate the population of cells in the solution, as illustrated in FIG. 59. For example, in one embodiment, the population of cells in the first bioreactor vessel 410 may be incubated for about 24 to 48 hours.

[0141] Next, referring to FIG. 60, after activation, to prepare for transduction, valves 438 and 474b are opened and pump 456 is operated to pump the RetroNectin solution from storage tank 472b through the second port 426 of the second bioreactor vessel 420 into the second bioreactor vessel 420. After pumping the RetroNectin solution into the second bioreactor vessel 420 for RetroNectin coating of the second bioreactor vessel 420, the solution is incubated within the second bioreactor vessel 420 for a predetermined time period. As further shown in FIG. 60, after incubation, all of the RetroNectin solution is then discharged from the second bioreactor vessel 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 discharge steps (relating to the second bioreactor vessel 420), the activated cell population remains within the first bioreactor vessel 410. Note that RetroNectin or other reagents for enhancing the efficiency of gene modification need not be utilized in all processes.

[0142] As shown in FIG. 61, after RetroNectin coating, the rinse buffer bag 472b is connected to the second fluid assembly 444 (or may be already present and connected to one of the tubing tails), valves 474b and 438 are opened, and pump 456 is operated to pump the buffer from the bag 472b into the second bioreactor vessel 420. Alternatively, as described above, the buffer may instead be passed by the pump through the first port 422 of the second bioreactor vessel 420 by opening valves 452 and 436.

[0143] Next, referring to FIG. 62, after a defined time period, all of the buffer in the second bioreactor vessel 420 is discharged to the waste liquid storage tank 472a of the second fluid assembly 444 by opening valves 438 and 474a and operating the interconnecting line pump 456.

[0144] At this point, as shown in FIG. 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 operated to circulate the solution in the first bioreactor vessel 410 from the second port 434 through the interconnecting line, through the filtration line 48 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. To take a sample, a second sample container 280b (e.g., an immersion tube, syringe, etc.) is connected to the second sample tubing tail 476b, valve 478b is opened, and a portion of the flow is diverted through the interconnecting line 450 and into the second sample container 280b for analysis.

[0145] Next, referring to FIG. 64, depending on the concentration obtained from the sample, concentration can be performed 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, whereby the solution in the first bioreactor vessel 410 circulates from the second port 416, the solution passes through the second bioreactor conduit 418, through the interconnect conduit 450, through the filtration conduit 482 and filter 484, through the first bioreactor conduit 414 of the first bioreactor vessel 410, and back to the first bioreactor vessel 410 through the first port 412. As the fluid passes through the filter 484, waste fluid is removed and the permeate pump 492 sends such waste fluid through the waste fluid conduit 490 to the waste fluid storage tank 472a of the second fluid assembly 444. In one embodiment, this procedure is repeated until the volume in the first bioreactor vessel 410 is concentrated to a predetermined volume.

[0146] Referring to FIG. 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 there, the medium from the medium bag 466b of the first fluid assembly 440 is pumped into the first bioreactor vessel 410 through the first port 412 through the interconnect conduit 450 at the same time as the medium is pumped out of the first bioreactor vessel 410 through the second port 416 so that a constant volume is maintained within the first bioreactor vessel 410. When medium is added and removed from the vessel 410, the waste fluid may be filtered by the filter 484 and directed to the waste fluid storage tank 472a.

[0147] The washed sample can be collected from the cells in the first bioreactor vessel 410 in the same manner as already described for the sample before concentration. In particular, as shown in FIG. 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 interconnecting line, through the filtration line 48 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. To collect a sample, a third sample container 280c (e.g., an immersion tube, syringe, etc.) is connected to the third sample tubing tail 476c, valve 478c is opened, and a portion of the flow is diverted through the interconnecting line 450 and into the third sample container 280c for analysis.

[0148] As shown in FIG. 67, a bag containing the thawed viral vector is connected to the first fluid assembly 440 through tubing tail 464c, etc. Then, valves 468c and 436 are opened and pump 454 is activated to transfer the viral vector coating solution from the bag 466c through the first port 422 to the second bioreactor vessel 420. Then, an incubation is performed for a predetermined time period 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 the 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 can be utilized as agents for transduction / gene modification.

[0149] As illustrated in FIG. 68, after the second bioreactor vessel 420 is coated with the viral vector, the washed cells from the first bioreactor vessel 410 are transferred to the second bioreactor vessel 420 for transduction / gene modification. In particular, valves 434, 452, and 436 are opened, the circulation line pump 456 is activated, and the cells are pumped out from the first bioreactor vessel 420, passed through the second port 416 of the first bioreactor vessel 410, through the interconnecting line 450, sent to the first bioreactor line 424 of the second bioreactor vessel 420, and fed into the second bioreactor vessel 420 through the first port 422 of the second bioreactor vessel 420.

[0150] Next, as illustrated in FIG. 69, the medium from the medium bag 466b is added to the second bioreactor vessel 420 by opening valves 468b and 436 and activating pump 454, increasing the total volume of the solution in the second bioreactor vessel 420 to a predetermined volume. Then, referring to FIG. 70, the pre-transduction sample can be taken by opening valves 438, 452, and 436 and activating the circulation line pump 456 to pump the solution in the second bioreactor vessel 420 along the circulation loop of the second bioreactor vessel (i.e., from the second port 426, through the interconnecting line 450, through the first bioreactor line 414 and the first port 422 of the second bioreactor vessel 420, and back to the second bioreactor vessel 420). To take a sample, a fourth sample container 280d (e.g., dip tube, syringe, etc.) is connected to the fourth sample tubing tail 476d, valve 478d is opened, and a portion of the flow is passed through the interconnecting line 450 and diverted to the fourth sample container 280d for analysis.

[0151] When the analysis of the fourth sample taken indicates that all parameters are within the predetermined ranges necessary for successful transduction, the population of cells in the second bioreactor vessel 420 is incubated for a predetermined time period for transduction of the population of cells in solution, as shown in FIG. 71. For example, in one embodiment, the population of cells in the second bioreactor vessel 420 can be incubated for about 24 hours for transduction.

[0152] Referring to FIG. 72, after transduction, media is added to the second bioreactor vessel 420 to achieve a predetermined amplification volume within the second bioreactor vessel 420. As shown therein, to add media, valves 468b and 436 are opened and pump 454 is activated to pump growth / perfusion media from media bag 466b through the first port 422 of the second bioreactor vessel into the second bioreactor vessel 420 until the predetermined amplification volume is reached.

[0153] Next, as illustrated in FIG. 73, a pre-amplification sample can be taken by opening valves 438, 452, and 436 and activating circulation line pump 456 to pump the solution within the second bioreactor vessel 420 along the circulation loop of the second bioreactor vessel 420 (i.e., from the second port 426, through the interconnect line 450, through the first bioreactor line 414 of the second bioreactor vessel and the first port 422 back into the second bioreactor vessel 420). To take a sample, a fifth sample container 280e (e.g., dip tube, syringe, etc.) is connected to the fifth sample tubing tail 476e, valve 478e is opened, and a portion of the flow is diverted through the interconnect line 450 and routed to the fifth sample container 280e for analysis.

[0154] When the analysis of the fifth sample taken indicates that all parameters are within a predetermined range necessary for successful amplification of the cell population, the cell population in the second bioreactor vessel 420 is incubated for a predetermined time period, e.g., 4 hours, to allow the cells to settle.

[0155] After this incubation period, or at a predetermined time thereafter, as shown in FIG. 74, perfusion at a rate of one volume per day (1x perfusion) is performed by pumping medium from the medium bag 466b into the second bioreactor vessel 420 through the first port 422 while pumping the used medium out of the second bioreactor vessel 420 through the second port 426 (and through the interconnecting line 450 to the waste liquid storage tank 472a). This perfusion is achieved by opening valves 468b, 436, and 474a and operating the first pump 454 and the circulation line pump 456. In this 1x perfusion, the medium from the medium bag 466b is introduced into the second bioreactor vessel 420 at substantially the same rate as the used medium is removed from the second bioreactor vessel 420 and sent to the waste section, maintaining a substantially constant volume within the second bioreactor vessel 420.

[0156] Next, the amplification process can be monitored and / or sampling can be performed as needed / desired to determine when the desired cell density is reached. As described above, a sample can be taken by opening valves 438, 452, and 436, operating circulation line pump 456, and pumping the solution within second bioreactor vessel 420 along the circulation loop of second bioreactor vessel 420 (i.e., from second port 426, through second bioreactor line 428, through interconnect line 450, through first bioreactor line 424 and first port 422 of second bioreactor vessel 420 and back to second bioreactor vessel 420). To take a sample, another sample container 280x (e.g., dip tube, syringe, etc.) is connected to the sample tubing tail of sample assembly 448, and as shown in FIG. 75, the valve of the tubing tail is opened to divert a portion of the flow through interconnect line 450 and into sample container 280x for analysis. After each sampling operation, an incubate without perfusion can be performed for a predetermined time period, e.g., 4 hours, to allow the cells to settle before restarting perfusion.

[0157] As shown in FIG. 76, after this incubation period, as shown in FIG. 74, perfusion at a rate of one volume per day (1x perfusion) is performed by pumping medium from media bag 466b into second bioreactor vessel 420 through first port 422 simultaneously with pumping used medium out of second bioreactor vessel 420 through second port 426 (and through interconnect line 450 to waste liquid storage tank 472a). This perfusion is accomplished by opening valves 468b, 436, and 474a and operating first pump 454 and circulation line pump 456.

[0158] When sampling indicates a viable cell density (VCD) at a predetermined threshold (e.g., 5 MM / mL), perfusion at a rate of two volumes per day (2x perfusion) is performed by pumping medium from the medium bag 466b into the second bioreactor vessel 420 through the first port 422 while pumping used / spent medium out of the second bioreactor vessel 420 through the second port 426 (and through the interconnecting line 450 to the waste liquid storage tank 472a), as shown in FIG. 76. This perfusion is achieved by opening valves 468b, 436, 438, and 474a and operating the first pump 454 and the circulation line pump 456. In this 2x perfusion, the medium from the medium bag 466b is introduced into the second bioreactor vessel 420 at substantially the same rate as the used medium is removed from the second bioreactor vessel 420 and sent to the waste section, maintaining a substantially constant volume within the second bioreactor vessel 420.

[0159] Finally, referring to FIG. 77, after the desired viable cell density is achieved, the cells can be harvested by opening valves 438 and 474d and operating the circulation line pump 456. The amplified cell population is then pumped out of the second bioreactor vessel 420, passed through the second port 426, through the interconnecting 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 and delivered to and injected into a patient in a manner known heretofore in the art.

[0160] 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 on which various bioprocessing operations can be substantially automated and executed in a functionally closed manner. In particular, FIGS. 53-77 illustrate exemplary general protocols that can be executed using the bioprocessing system 10 of the present invention (in particular, using its second module 200), but the system is not limited thereto in this regard. Indeed, various automated protocols, including numerous customer-specific protocols, can be made available for use by the system of the present invention.

[0161] In contrast to existing systems, the second module 200 of the bioprocessing system 10 is a functionally closed automated system that houses the first and second bioreactor vessels 410, 420 and the fluid handling and fluid containment systems, all of which are maintained in cell culture-friendly environmental conditions (i.e., within a temperature and gas control environment) that enable cell activation, transduction, and amplification. As described above, the system includes automated kit loading and closed sampling capabilities. In this configuration, the system enables all steps of immune cell activation, transduction, amplification, sampling, perfusion, and washing to be performed 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 a single dose or multiple doses can be generated in situ with high recovery and high viability. In addition, the system is designed to provide the end user with the flexibility to execute different protocols for the production of genetically modified immune cells.

[0162] Some of the commercial advantages provided by the bioprocessing system of the present invention include simplifying the workflow, reducing labor intensity, reducing the burden on cleanroom infrastructure, reducing failure nodes, reducing costs, and enabling product commercialization through a robust and scalable manufacturing technology with the ability to expand the scale of operations.

[0163] As described above with respect to the general workflow, the system of the present invention, bioprocessing system 10, and the fluid architecture 400 and bioreactor vessels 410, 420 of the second module 200 result in a process of culture concentration, washing, low-speed perfusion, high-speed perfusion, and "round-robin" perfusion that should be performed in an automated, functionally closed manner. For example, as described above, pump 456 on interconnecting conduit 450 operates permeate pump 492 (typically, for example, at a rate of the circulation pump 456 such as about 10%) while circulating fluid from one of the ports of the bioreactor through filtration conduit 482 and filter 484 and then back to another port on the bioreactor for use. Concentration can be performed in an open loop or stopped based on a measured volume withdrawn from the bioreactor or a measured volume accumulated in the waste liquid. In one embodiment, the filter, pump speed, filter area, number of lumens, etc. are all appropriately sized relative to the total number of cells and the target cell density to limit shear-induced fouling and excessive cell loss.

[0164] In one embodiment, as described above, the system of the present invention can also be used to wash, for example, to remove residues such as residual viral vectors after incubation. Washing involves the same steps as described above for concentration, except that the pump 454 on the first fluid assembly line 442 is used to pump additional medium to replace the fluid sent from the permeate waste liquid pump 492. The introduction rate of the new medium may correspond to the removal rate of the fluid by the permeate pump 492. This makes it possible to maintain a constant volume within the bioreactor vessel, and the residue can be removed exponentially with respect to time as long as the contents within the bioreactor are well mixed (sufficient circulation can be achieved). In an embodiment, this same process can be utilized after activation for in-situ hollow fiber filtration-based washing of the cell suspension to remove residues. For the coated and non-coated surfaces, soluble activator reagent washing removal can also be performed via filter-based perfusion.

[0165] As also described above, in the perfusion process, the pump 454 on the first fluid assembly line 442 can be used to add medium to a given bioreactor vessel, and the pump 456 on the interconnecting line 450 is used to move the spent medium to the waste liquid bag within the second fluid assembly. In one embodiment, gravity may be used to settle the cells, and the spent medium can be pumped out at such a rate as not to significantly disturb the cells within the bioreactor vessel. This process may involve operating the open loops of pumps 454 and 456 at the same rate. In one embodiment, one pump (454 or 456) may be operated at a set rate, and the rate of the other pump can be adjusted based on the mass / volume of the bioreactor vessel or the mass / volume of the waste liquid bag (or the measured mass / volume of the supply bag).

[0166] In connection with the above, it is contemplated that pump control may be based on the weight measurement results of the bioreactor vessel (using feedback from load cell 760). For example, the system configuration enables the use of on-the-fly pump calibration based on load cell readings, which allows the system to automatically adapt to changes in tube / pump performance that occur over time. Further, this method can be used for closed-loop control of the mass (volume) change rate when emptying or filling the bioreactor vessel.

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

[0168] In the case of round-robin perfusion, can the pumps operate continuously together, intermittently together (duty cycle), or sequentially (source, then waste, repeating this), thereby maintaining the volume / mass in various bioreactor vessels at approximately the same level. In the case of round-robin perfusion (operating a set of pumps intermittently together and waiting at regular intervals), this also enables perfusion of multiple vessels using the same two pumps, as shown. Furthermore, round-robin perfusion allows for a low effective exchange rate (such as about 1 volume / day) even when the pumps do not have a large low dynamic range. Additionally, round-robin perfusion also allows each vessel to be perfused with a different medium such that it is controlled by a valve within 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 a high-speed perfusion process, the perfusion process described above can operate at a rate significantly faster than a typical 1 - 5 volume / day, such as between about 8 - 20 volume / day, or at a value exceeding about 20 volume / day that achieves a 1-log reduction in a matter of minutes to a few hours. In one embodiment, the perfusion rate is balanced against cell loss. In some embodiments, high-speed perfusion may enable the elimination of the hollow filter 484 and still meet the biological requirement of quickly removing residues after several steps.

[0170] As further described above, the system of the present invention uses a pump 454 on the first fluid assembly conduit 442 and a rinse buffer or fluid from another bag / reservoir connected to the second fluid assembly 444 to facilitate the rinsing of the bag / reservoir connected to the first fluid assembly 440. In addition, the fluid conduits of the fluid architecture / system 400 are cleaned with sterilized air from a sterilized air source 458, thereby preventing cells from sitting and dying in the conduits, or preventing the medium or reagent from sitting and degrading or becoming ineffective in the conduits. The sterilized air source 458 can also be used to expel reagents from the conduits to ensure that more reagents than intended are not pumped into the bioreactor vessels 410, 420. The sterilized air source 458 can similarly be used to clean the conduits up to the connected bag (of the first or second fluid assemblies 440, 444) and to clean the sterilized tube welds to limit residues. Instead of, or in addition to, using the sterilized air source 458 to clean the conduits, the conduits can be cleaned using air drawn from one of the bioreactor vessels as long as the ports through which the air is drawn are not submerged and the bioreactor vessel has an air balance port 530.

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

[0172] Also as described above, the system of the present invention enables the population of cells to be harvested after the target cell density has been achieved. In one embodiment, harvesting the amplified population of transduced cells can involve using pump 456 on interconnected conduit 450 to move the cells into one of the bags connected to second fluid assembly 444, or circulating the cells by interconnected pump 456 to move the cells into the bag connected to first fluid assembly 440. This process can be used for final harvesting or for use with large sample volumes, or can be used to fully automate the sampling process (i.e., connecting a syringe or bag to first fluid assembly 440, circulating the contents of the bioreactor vessel, withdrawing a desired sample volume portion from the circulating contents by fluid assembly pump 454, and moving it towards the syringe / bag). Then, in such cases, circulation pump 456 and valves can be used to clean the fluid / cell circulation conduits. In addition, pump 454 on first fluid assembly conduit 442 can be used to continue pushing all of the aliquoted sample volume into the sample container by completing sample transfer to the container using the air within the conduit with no perceivable amount of cells remaining within the conduit.

[0173] In the embodiments described above, a workflow is disclosed in which cell activation is performed within a first bioreactor vessel and the activated cells are transferred to a second bioreactor vessel for transduction and amplification. However, in one embodiment, the system of the present invention may enable activation and transduction operations to be performed within a first bioreactor vessel and amplification of the genetically modified cells to be performed within a second bioreactor vessel. Further, in one embodiment, the system of the present invention may be well-suited to enable in-situ processing of isolated T cells, and all of the activation, transduction, and amplification unit operations are performed within a single bioreactor vessel. In one embodiment, therefore, the present invention simplifies existing protocols by enabling the use of a simplified automation-friendly "one-pot" activation, transduction, and amplification vessel.

[0174] In such an embodiment, the T cell activator may be micron-sized Dynabeads, and a lentiviral vector is used for transduction. In particular, as disclosed herein, the micron-sized Dynabeads serve the dual purpose of isolating and activating T cells. In one embodiment, the activation (and isolation) of T cells may be performed in one of the bioreactor vessels 410 using Dynabeads 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 connection with FIGS. 60-71. Then, after activation and viral transduction, the virus may be washed out of the bioreactor vessel 410 using the filterless perfusion method described above for retaining cells and micron-sized Dynabeads within the bioreactor vessel 410. This allows for cell expansion within 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 activation beads that need to be retained with the cells during expansion. In particular, when the virus is washed out, the micron-sized Dynabeads do not fluidize at low perfusion rates and are retained within the vessel. Nanometer-sized virus particles and residual macromolecules fluidize and are washed out during low-speed perfusion.

[0175] In one embodiment, after amplification, the cells can be harvested in the manner described above in connection with FIG. 77. After harvesting, a magnetic bead removal process can be utilized to remove Dynabeads from the collected cells. In other embodiments, the step of harvesting the amplified population of cells and the step of removing beads from the cells are performed simultaneously using perfusion, whereby the medium is introduced into the bioreactor vessel through a supply port and the cell culture medium containing the amplified population of cells is removed from the bioreactor vessel through a discharge port within the bioreactor vessel. In particular, when final bead removal of the culture is required, filterless perfusion can be used to perform bead removal of micron-sized beads by taking advantage of the difference in weight between the cells and the weight of the cell Dynabead complex. To perform bead removal from the culture, the entire contents of the bioreactor vessel are mixed (e.g., by using the cam arm 762 of the actuator mechanism in the manner described previously). After mixing / stirring, the heavy Dynabeads sink and settle on the silicone membrane 516 within 10 - 15 minutes. In contrast, the cells take over 4 hours to settle on the membrane 516. After a holding period of 10 - 15 minutes after mixing / stirring, the cell suspension can be slowly withdrawn using perfusion without disturbing the settled Dynabeads. The inlet medium line can be used to maintain the medium height within the bioreactor vessel. Thus, the present invention described herein simplifies the current Dynabead protocol by eliminating several process intermediate cell transfers and elaborate washing and bead removal steps, minimizing cost and potential risks. By performing bead removal of the culture simultaneously with harvesting the cells, the need for additional magnetic devices or disposable parts, which were typically required heretofore, can be eliminated.

[0176] In contrast to other static, perfusion-free culture systems, the gas permeable membrane-based bioreactor vessel 410 of the present invention supports high density cell culture (e.g., up to 35 mm / cm 2Therefore, all four unit processes of activation, transduction, washing, and amplification using Dynabeads can be performed within the same bioreactor vessel in a fully automated and functionally closed manner. Thus, the bioprocessing system of the present invention simplifies the current protocol by eliminating the need for process intermediate cell transfer and elaborate washing steps, minimizing cost and potential risks resulting from multiple human touchpoints.

[0177] In one embodiment, the two bioreactor vessels 410, 420 of the system can operate with either the same starting culture or two simultaneous split cultures, e.g., CD4+ cells in one bioreactor vessel 410 and CD8+ cells in the other bioreactor vessel 420. The split cultures allow for parallel independent processing and amplification of two cell types that can be combined prior to injection into the patient.

[0178] Although a number of possible CAR-T workflows for the generation and amplification of genetically modified cells using the bioprocessing system of the present invention have been described above, the workflows described herein are not intended to be comprehensive as other CAR-T workflows may also be used with the system of the present invention. In addition, although the system of the present invention, and in particular, the second module 200 of the system, has been described in connection with the manufacture of CAR-T cells, the system of the present invention is also suitable for the manufacture of other immune cells such as TCR-T cells and NK cells. Further, in embodiments of the present invention, the use of two bioreactor vessels 410, 420 is disclosed 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), but in some embodiments, the two bioreactor vessels can be used in the same workflow as replicates. Exemplary reasons for using the second bioreactor vessel sequentially include the presence of residual chemical modifications (e.g., coated or immobilized reagents) that cannot be washed out of the first bioreactor that are harmful if there is overexposure of the cells at a later step or an earlier step, or the need to precoat the bioreactor surface (e.g., RetroNectin coating) prior to addition of the cells.

[0179] Additional examples of potential single bioreactor vessel workflows enabled for use with the system of the present invention include (1) soluble activator activation, viral transduction, perfusion without a filter, and amplification within a single bioreactor vessel, (2) Dynabead-based activation, viral transduction, perfusion without a filter, and amplification within a single bioreactor vessel, and (3) TransAct-based activation, viral transduction, perfusion without a filter, and amplification within a single vessel.

[0180] Furthermore, additional examples of potential multiple bioreactor vessel workflows that can be used by the system of the present invention are: (1) soluble activator activation, viral transduction, perfusion without a filter, and amplification in a first bioreactor vessel 410, and soluble activator activation, lentiviral transduction, perfusion without a filter, and amplification in a second bioreactor vessel 420, where the same cell type or split culture is used in these two bioreactor vessels; (2) Dynabead-based activation, viral transduction, perfusion without a filter, and amplification in a first bioreactor vessel 410, and Dynabead-based activation, lentiviral transduction, perfusion without a filter, and amplification in a second bioreactor vessel 420, where the same cell type or split culture is used in these two bioreactor vessels; (3) TransAct bead-based activation, viral transduction, perfusion without a filter, and amplification in a first bioreactor vessel 410, and TransAct-based activation, lentiviral transduction, perfusion without a filter, and amplification in a second bioreactor vessel 420, where the same cell type or split culture is used in these two bioreactor vessels; (4) soluble activator activation in a first bioreactor vessel 410, and RetroNectin coating, transduction, and amplification in a second bioreactor vessel 420; (5) immobilized activator activation in a first bioreactor vessel 410, and RetroNectin coating, transduction, and amplification in a second bioreactor vessel 420; (6) Dynabead activation in a first bioreactor vessel 410, and RetroNectin coating, transduction, and amplification in a second bioreactor vessel 420; (7) Dynabead activation and lentiviral transduction in a first bioreactor vessel 410, and amplification in a second bioreactor vessel 420; (8) TransAct activation in a first bioreactor vessel 410, and RetroNectin coating, transduction, and amplification in a second bioreactor vessel 420; (9) soluble activator activation in a first bioreactor vessel 410,and amplification of in - facility electroporation - treated cells or other non - virus - modified cells in the second bioreactor vessel 420, (10) TransAct activation in the first bioreactor vessel 410, and amplification of in - facility electroporation - treated cells or other non - virus - modified cells in the second bioreactor vessel 420, (11) Dynabead activation in the first bioreactor vessel 410, and amplification of in - facility electroporation - treated cells or other non - virus - modified cells in the second bioreactor vessel 420, (12) amplification of allogeneic NK cells in the first bioreactor vessel 410, and amplification of allogeneic NK cells in the second bioreactor vessel 420 (small - molecule - based amplification, no gene modification), (13) amplification of allogeneic NK cells in the first bioreactor vessel 410, and amplification of allogeneic NK cells in the second bioreactor vessel 420 (feeder - cell - based amplification, no gene modification), and (14) soluble activator activation, viral transduction, filter - free perfusion and amplification of allogeneic CAR - NK or CAR - NK92 cells in the first bioreactor vessel 410 and / or in the first and second bioreactor vessels 410, 420 (without RetroNectin coating, polybrene is used to assist transduction), including.,

[0181] The embodiments described above illustrate process monitoring sensors integrated with the bioreactor vessel and / or the bedplate (e.g., on the membrane, integrated within the membrane, on the vessel sidewall, etc.), but in other embodiments, additional sensors are contemplated to 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 configuring the sensor within the circulation loop (e.g., the circulation loop of the first bioreactor vessel and / or the circulation loop of the second bioreactor vessel), the structure of the vessel can be simplified. In addition, in some embodiments, sensors along the circulation loop can provide a more accurate representation of the vessel contents when circulated (rather than when the cells are stationary within the vessel). Further, if desired, a flow sensor (e.g., ultrasonic-based) can be added to the flow loop to measure pump performance and used with an algorithm to correct 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 that can perform cell concentration and isolation, as well as harvesting and / or formulation. FIG. 78 illustrates a possible configuration of a device / apparatus 900 that can be used as the first module 100 in a bioprocessing system 10 for cell concentration and isolation using various magnetic isolation bead types (including, for example, Miltenyi beads, Dynabeads, and StemCell EasySep beads). As shown therein, the device 900 includes a base 910 that houses a centrifugation chamber 912, a high dynamic range peristaltic pump assembly 914, a pump tube 916 of a suitable inner diameter received 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 together multiple fluid or gas conduits, for example, using luer fittings. In one embodiment, the pump 914 has a rating that outputs a low flow rate of about 3 mL / min and a high flow rate of about 150 mL / min.

[0183] As further shown in FIG. 78, the device 900 can include a generally T-shaped hanger assembly 924 that extends from the base 910 and includes a plurality of hooks 926 for hanging a plurality of processing and / or supply source containers or bags. In one embodiment, there can be six hooks. Each hook includes an integral weight sensor for detecting the weight of each container / bag. In one embodiment, the bags can 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 bag 942, a wash waste bag 944, a media bag 946, a release bag 948, and a collection bag 950.

[0184] Device 900 is configured to be used with or to include magnetic cell isolation holder 960, as presented herein. Magnetic cell isolation holder 960 can be removably coupled to a magnetic field generator 962 (e.g., magnetic field plates 964, 966 of FIG. 80). Magnetic cell isolation holder 960 houses a magnetic retention element or material 968, such as a separation column, matrix, or tube. In one embodiment, magnetic cell isolation holder 960 can be fabricated as disclosed in U.S. Patent Application No. 15 / 829,615, filed Dec. 1, 2017, which is hereby incorporated by reference in its entirety and described in more detail below. Device 900 may be under the control of a controller (e.g., controller 110) and operate in accordance with instructions executed by a processor and stored in a memory. Such instructions may include magnetic field parameters. In one embodiment, device 900 may further include a syringe 952 available for bead addition, as described below.

[0185] Next, referring to FIG. 79, the general protocol 1000 of the apparatus 700 is shown. As illustrated therein, in a first step 1010, concentration is performed by reducing platelets and plasma in the sample. Next, in an embodiment where Dynabeads are utilized as magnetic isolation beads, a washing step 1012 may be performed to remove residues in the Dynabead suspension. Next, after concentration, the cells are transferred to the 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, the magnetic isolation beads are injected into the process bag, such as by using the syringe 952 in step 1020. In one embodiment, the magnetic isolation beads are Miltenyi beads or StemCell EasySep beads. When Dynabeads are utilized, the washed Dynabeads from step 1012 are resuspended within the process bag 932. In one embodiment, instead of using a syringe, the magnetic isolation beads may be housed within a bag or container connected to the system, and the beads may be drawn into the system by the pump 914.

[0186] Next, the beads and cells within process bag 932 are incubated for a period of time in step 1020. This step includes circulating the fluid out of process bag 932, through the loop, and then back into the bag. In embodiments where the magnetic isolation beads are Miltenyi nano-sized beads, sedimentation washing is performed in step 1022 to remove excess nano-sized beads, and in step 1024, a portion of the incubated bead-bound cells are stored within a second storage bag 940. After incubation, the bead-bound cells are isolated in step 1026 using a magnet, such as magnetic field plates 964, 966 of magnetic cell isolation holder 960. The remaining bead-bound cells are then rinsed and isolated in step 1028. Finally, in embodiments where Miltenyi or Dynabeads are utilized, the isolated bead-bound cells are collected within collection bag 950 in step 1030. In embodiments where StemCell EasySep beads are utilized, additional steps 1032 to release the cells from the beads and remove the beads, and optional step 1034 to wash / concentrate the collected cells are performed.

[0187] A more detailed description of the general protocol of FIG. 79 using apparatus 900 is described in further detail below, with particular reference to FIG. 80 which is a schematic diagram of the fluid architecture 1100 of apparatus 900. First, the concentration process (step 1010) is initiated by transferring the apheresis product and wash buffer contained within source bag 930 from wash buffer bag 936 to chamber 912, washing using the wash buffer, and reducing the amounts of platelets and serum. At this point, the concentrated raw material is disposed within chamber 912. To initiate the isolation process, the separation column received by magnetic cell isolation holder 960 is primed by starting the flow of buffer from isolation buffer bag 934, through manifold 918, through the column, and into process bag 932 to prime the column.

[0188] As disclosed above, in some embodiments where Dynabeads are utilized as magnetic isolation beads, a wash step (step 1012) is performed to remove residues within the bead suspension buffer. The wash step includes injecting the beads using syringe 952 while circulating within 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), flushing process loop 1110, and then capturing the beads by flowing process bag 932 into isolation waste bag 942 while magnetic field generator 962 is “ON,” i.e., while the holder is magnetically coupled to active magnetic field generator 962 against a permanent magnet, or alternatively, while an electromagnetic field is actively generated by using an electromagnet, with the beads being captured in the “ON” state in each case. 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, in the case of a permanent magnet, ON means that the magnetic retention element or material 968 (e.g., a separation column, matrix, or tube) is in the appropriate position within the magnetic field. OFF means that the tubing section has been removed from the magnetic field.

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

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

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

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

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

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

[0195] In some embodiments, a portion of the incubated cell population may be stored in the 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 conduit and the second storage conduit 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 accomplished by flowing from process bag 932 to waste liquid bag 942 while magnetic field generator 962 is "ON". Next, the residual waste liquid is cleaned by pumping buffer from isolation buffer bag 934 to process bag 932 and then pumping from process bag 932 to waste liquid bag 942 while magnetic field generator 962 is "ON".

[0197] In one embodiment, a wash without resuspension can be performed by pumping buffer from isolation buffer bag 934 to process bag 932, flushing process loop 1110, cleaning process loop 1110, and flowing from process bag 932 to waste liquid bag 942 while magnetic field generator 962 is "ON".

[0198] In another embodiment, a wash via resuspension can be performed by pumping buffer from isolation buffer bag 934 to process bag 932 while magnetic field generator 962 is "OFF", circulating in process loop 1110, cleaning the process loop, and flowing from process bag 932 to waste liquid bag 942 while magnetic field generator 962 is "ON".

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

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

[0201] If the bead-bound cells are to be harvested after releasing the cells from the beads, a number of potential processes may be performed. For example, in one embodiment, with the magnet in the "OFF" state, the cell / bead is pumped from the bag 948 through the column into the process bag 932 with the release buffer, circulated within the process loop 1110, and then resuspended by flushing the process loop by returning the fluid to the process bag 932. Then, incubation and harvesting are performed with the magnet in the "ON" state by incubating within the process loop 1110, flushing the process loop 1110, pumping the released cells from the process bag 932 through the column 966 into the collection bag 950 for collection, pumping the buffer from the isolation buffer bag 934 into the process bag 932, and pumping the contents of the process bag 932 through the column 966 into the collection bag 950 to collect the residue. Then, the released beads (step 1032) can be discarded with the magnet in the "OFF" state by pumping the buffer from the isolation buffer bag 934 through the column 966 into the process bag 932, circulating within the process loop 1110, flushing the process loop 1110, and pumping the contents of the process bag 932 into the waste fluid bag 942.

[0202] In connection with the above, in one embodiment, the washing / concentration (step 1034) can be performed by pumping the contents of the collection bag 950 into the processing chamber 912, pumping buffer from the isolation buffer bag 934 into the process bag 932, and transferring the buffer from the process bag 932 to the processing chamber 912. Next, the washing cycle can be performed by filling the processing chamber 912 with buffer from the isolation buffer bag 934, rotating the chamber 912, discharging the supernatant into the waste liquid bag 942, and repeating the rotation and discharge steps as many times as necessary. Finally, transferring the cells to the collection bag after washing / concentration can be accomplished by transferring medium from the medium bag 946 to the collection bag 950, pumping the contents of the collection bag into the processing chamber 912, discharging the contents of the processing chamber 912 into the collection bag 950, and then manually cleaning the pipeline between the processing chamber 912 and the collection bag 950.

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

[0204] In one embodiment, the processing bag 932 is blow molded and has a large angle on the side (with a 3D shape having a defined air pocket at a position higher than the liquid level) to limit the adhesion of micron-sized beads to the side wall, particularly when long accelerating mixing is performed with a circulation-based ink incubation.

[0205] In one embodiment, the syringe 952 enables adding a small amount (such as a bead suspension aliquot) to the circulation-based flow loop 1110. Further, the fluid from the flow loop 1110 can be drawn into the syringe 952, thereby further removing and cleaning the residue in the syringe 952.

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

[0207] As mentioned somewhat above, in one embodiment, air can be drawn into the loop for the intentional generation of an air slug that can be used to remove bead-bound cells within the isolation column / tube for collection. In one embodiment, the buffer solution can be circulated through the isolation column to elute the bead-bound cells either instead of or in addition to using an air slug.

[0208] In one embodiment, two or more peristaltic pump tubes having different inner diameters in series connection may be used, thereby enabling a wide range of flow rates to be used for a single pump. To switch 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 the elution of isolated / captured bead-cell complexes. In particular, it is contemplated that the air-liquid interface can be used to help remove the complex 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, it can be difficult to remove the packed bed of beads / bead-bound cells by flow rate control alone without significantly increasing the shear rate (which can potentially have an adverse effect on cell viability). Thus, it is possible to remove the bead-cell complex without removing it from the magnet in relation to the flow rate.

[0210] In connection with the above, system 900 supports the concept of directly eluting the positively selected bead-cell complexes into the selected medium (based on downstream steps). This eliminates the buffer exchange / washing step. In one embodiment, it is also contemplated to directly elute into the medium and virus vector and initiate incubation. This concept may also enable the addition of the virus vector to the final bag. In one embodiment, instead of eluting the bead-bound cells with a buffer, the medium can be used as the elution fluid. Similarly, the release buffer can be used to elute the StemCell beads for subsequent release of the cells from the beads. By exchanging the buffer within the parts of system 900 with the medium, dilution can be minimized.

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

[0212] Next, an embodiment of the magnetic cell isolation holder (960 in FIG. 78) will be described in more detail with particular reference to FIGS. 81 to 87. Magnetic bead-based cell selection involves isolating specific cells from a cell mixture by target binding of cell surface molecules to an antibody or ligand of magnetic beads (e.g., beads of the type described above). After binding, the cells bound to the magnetic beads can be separated from the unbound population of cells. For example, a cell mixture containing bound and unbound cells can be passed through a separation column positioned within a magnetic field generator that captures the magnetic beads and thus the associated bound cells. The unbound cells pass through the column without being captured.

[0213] Some magnetic cell isolation techniques can incorporate nano-sized beads (e.g., beads with a diameter of about 50 nm or less), while other techniques can use larger beads (e.g., beads with a diameter of about 2 μm or more). For example, a smaller bead size may be desirable as it can avoid receptor activation on target cells. Additionally, downstream steps may skip bead removal as nano-sized beads may have little impact on downstream processing or cell function. However, smaller nano-sized magnetic beads can be separated using magnetic cell isolation procedures that involve amplifying the applied magnetic field gradient using a magnetic field gradient increasing device. In contrast, larger beads have a higher magnetic moment. Thus, the isolation of some larger beads may not involve a magnetic field gradient increasing device. However, larger beads can still be used in conjunction with additional cell-bead separation steps. Therefore, depending on the size and / or type of magnetic beads used, the workflow, appropriate magnetic parameters, and / or the isolation device itself may vary, which complicates magnetic bead-based cell isolation techniques.

[0214] In particular, since the beads can differ in terms of material and magnetic properties (including but not limited to size, permeability, saturation magnetization, resistivity, surface properties, and mass density), the separation conditions can also vary depending on the bead properties and can involve different intensities and / or different gradients of magnetic fields. In other words, the magnetic field parameters of the magnetic field generator can differ for magnetic cell separation procedures using beads with different materials and magnetic properties. In the approach of the present invention, there is eliminated a workflow step of adjusting the magnetic field generator or its parameters during magnetic cell separation procedures using beads of different sizes. In one embodiment provided herein, the magnetic cell isolation holder is configured to be used in conjunction with a magnetic field generator to position the beads within a magnetic field in an arrangement where the magnetic cell isolation holder is associated with the magnetic field properties desirable for cell separation when used with beads of an appropriate size. The magnetic field generator can apply a magnetic field using preset (e.g., fixed) magnetic field parameters or static magnetic field generator elements. In this way, the operator can avoid the complication of changing the magnetic field parameters according to the selected beads. Instead, by selecting an appropriate magnetic cell isolation holder, the magnetic field acting on the cells is suitable for separation. Further, when using beads of different sizes and / or with different desirable magnetic field properties, different magnetic cell isolation holders can be selected that position the beads in respective arrangements within the applied magnetic field associated with their respective desirable magnetic field properties.

[0215] For example, different magnetic cell isolation holders can be sized and shaped according to the desired positioning of cells (e.g., target cells within a cell mixture) within a magnetic field generated by a magnetic field generator. In one embodiment, each magnetic cell isolation holder includes a passageway or other cell receptor that, when the magnetic cell isolation holder is loaded into a magnetic bead-based cell isolation system that includes a magnetic field generator, positions the cells within the magnetic field isolation holder in an arrangement within the magnetic field that has properties suitable for the separation of a particular type (e.g., based on bead material, shape, size, and / or size range) of magnetic beads from the cell mixture. By selecting the magnetic cell isolation holder associated with a particular bead type, appropriate separation can be achieved without changing the settings for the magnetic isolation device or the magnetic field generator of the magnetic isolation device.

[0216] In one embodiment, a suitable magnetic retention material, such as a column matrix carried within a separation tube, is coupled to or positioned within the passageway of the magnetic cell isolation holder and positioned in an arrangement of the magnetic field within a magnetic field generator that corresponds to the desired magnetic field properties (i.e., magnetic field strength and magnetic field gradient) for the bead type used in the magnetic cell isolation procedure. The magnetic cell isolation holder and an accompanying set of magnetizable beads as the beads described above may be provided as a kit, which may comprise disposable or single-use components. The kit may also comprise multiple sets of beads or different types of beads and / or multiple magnetic cell isolation holders, e.g., holders optimized or designed for each set of beads.

[0217] In another embodiment, a magnetic cell isolation holder may be provided having a plurality of passageways for use with beads of different sizes, respectively, and a user may select an appropriate passageway associated with a desired bead type. For example, the magnetic cell isolation holder may have a passageway of a first arrangement for use with beads having a first diameter (e.g., configured to accommodate a first cell separation column) and a passageway of a second arrangement for use with beads having a second larger diameter (e.g., configured to accommodate a second cell separation column). When the magnetic cell isolation holder is inserted into a magnetic cell isolation device and a magnetic field is generated, the passageway of the first arrangement may be in a position that receives a higher magnetic field strength compared to the passageway in the second arrangement within the magnetic cell isolation holder.

[0218] In another embodiment, the magnetic cell isolation holder may be pre-filled with a magnetic bead cell mixture in an appropriate arrangement associated with a desired bead type. In addition, the magnetic cell isolation device may be part of a fluid handling system of a magnetic isolation system or may be operatively attached to one or more fluid handling systems. The magnetic cell isolation system may also include a controller configured to automatically perform a magnetic cell isolation procedure. The magnetic isolation system may be configured as a functionally closed system.

[0219] FIG. 81 shows an alternative magnetic isolation system 2100 that can be used in place of, and in conjunction with, the techniques disclosed herein for a magnetic bead-based cell isolation system. System 2100 includes a source pump (SP) 2112, a process pump (PP) 2114, and a magnetic isolation pump (MP) 2116. System 2100 also includes a collection pinch valve (PV-C) 2126, a waste pinch valve (PV-W) 2128, a bead addition syringe (SG1) 2118, and a check valve (CV1) 2120. In one embodiment, check valve 2120 is rated, for example, at a cracking pressure of 3 psi. System 2100 may also include suitable processing and / or source containers, such as a sample source bag (SB) 2104, a process bag (PB) 2106, a buffer bag (BB) 2108, a media bag (MB) 2110, a collection bag (CB) 2130, and a waste bag (WB) 2132. Incubate removal 2102 may also be a bag or another collection container suitable for containing and / or disposing of waste from system 2100.

[0220] System 2100 is configured to be used with a magnetic cell isolation holder 2134 equivalent to the magnetic cell isolation holder 960 described above with reference to FIG. 78. The magnetic cell isolation holder 2134 can be removably coupled (e.g., loaded, positioned therewith) to a magnetic field generator 2121 (e.g., magnetic field plates 2122 and 2124 equivalent to plates 964 and 966 of FIG. 80). System 2100 may be under the control of a controller 2150 and operates in accordance with instructions executed by a processor 2152 and stored in a memory 2154. Such instructions may include magnetic field parameters. System 2100 may include any or all of the illustrated components.

[0221] FIG. 82 shows a flowchart of a magnetic bead-based cell isolation method 2200 that can be used with a magnetic isolation system, such as the system 2100 of FIG. 81. The illustrated method 2200 is an example, and it will be understood that the techniques disclosed herein can be used in conjunction with other magnetic bead-based cell isolation workflows. In step 2202, the source bag 2104, the media bag 2110, the buffer bag 2108, and the bead addition syringe 2118 are prepared for use with the magnetic isolation system. In step 2204, the source bag 2104, the media bag 2110, the buffer bag 2108, and the bead addition syringe 2118 are loaded into the magnetic isolation system. The source bag 2104 is fluidly coupled to the source pump 2112. The media bag 2110 and the buffer bag 2108 are fluidly coupled to the check valve 2120. The bead addition syringe 2118 is fluidly coupled to the process bag 2106. In step 2206, the magnetic cell isolation holder 2134 is coupled to (e.g., positioned adjacent to, inserted into, loaded into) the magnetic field generator 2121 (e.g., magnetic field plates 2122 and 2124) of the magnetic isolation system 2100. In step 2208, the bags 2104, 2110, 2108 and the syringe 2118 are aseptically welded to the magnetic isolation device. In step 2210, a raw material substance, such as a cell mixture, from the source bag 2104 is transferred to the process bag 2106 via the source pump 2112.

[0222] In step 2212, magnetic beads (e.g., beads) within the bead addition syringe 2118 are added to the process bag 2106. In step 2214, the magnetic beads are incubated with the cell mixture within the process bag 2106. The incubation material (e.g., cell mixture and beads) may circulate in and out of the process bag 2106 via the process pump 2114 to facilitate sufficient binding between the target cells and the magnetic beads. In step 2216, the source bag 2104 is decoupled from the source pump 2112, and the incubation removal 2102 is fluidly coupled to the source pump 2112. Then, excess incubation material is removed from the process bag 2106 via the source pump 2112 and deposited at the incubation removal 2102. In step 2218, magnetic cell isolation is performed on the bead-labeled cell mixture. The magnetic cell isolation holder 2134 is then coupled to a magnetic field generator 2121 that generates a magnetic field under predetermined magnetic field parameters. The bead-labeled cell mixture from the process bag 2106 flows through the magnetic cell isolation holder 2134 via the magnetic isolation pump 2116. In one embodiment, the magnetic cell isolation holder 2134 houses a magnetic retention element or material such as a separation column, matrix, or tube. Thereafter, the bead-labeled cells are magnetically retained within the tube or column matrix of the magnetic cell isolation holder 2134, and the non-retained material flows through the magnetic cell isolation holder 2134 to the waste bag 2132. Optionally, a buffer or medium may rinse the process bag, and the magnetic cell isolation procedure may be repeated. In step 2220, the magnetic cell isolation holder 2134 is removed from the magnetic isolation device. Then, in step 2222, the retained cells are eluted by flushing the magnetic cell isolation holder 2134 with a high-flow fluid, whereby the viscous force of the fluid overcomes the residual magnetic force acting on the retained magnetic beads. Then, the fluid and the bead-labeled cells are collected within the collection bag 2130.In step 2224, the bags (e.g., collection bag 2130, waste liquid bag 2132, buffer bag 2108, and medium bag 2110) are sealed, and the magnetic cell isolation holder 2134 is, in one embodiment, subsequently disposed of.

[0223] Figures 83A and 83B are top views of different configurations of a magnetic cell isolation holder 2302 (e.g., magnetic cell isolation holder 2134 of FIG. 81) positioned within the magnetic isolation device 2300 of FIGS. 83A and 83B. FIG. 83A shows the magnetic cell isolation holder 2302 in an unloaded configuration in the magnetic cell isolation device 2300. The magnetic cell isolation holder 2302 may be provided with a body portion 2301, which may be formed of a suitable non-magnetic material configured to house the cell isolate and coupled to the magnetic isolation device 2300. The magnetic cell isolation holder 2302 includes one or more passages formed within or through the body portion 2301 through which a cell mixture may flow. FIG. 83A shows two separate passages 2303 and 2305, but it will be understood that the magnetic cell isolation holder 2302 may include only one passage, two or more passages, etc. Referring to passage 2303, passage 2303 may be configured to house a magnetic retention material 2304 configured to hold cells bound to magnetic beads and allow unbound cells to pass under a magnetic field. Similarly, passage 2305 may also be configured to house a magnetic retention material 2306. The magnetic retention materials 2304, 2306 may be the same or different. It is also possible to omit the retention material, but the result will be inferior efficiency. For example, passage 2305 may be a hollow tube. Further, passages 2303, 2305 may be of different sizes and in different positions with respect to the end face 2307 of the body portion 2301. For example, the distance 2315 between the end face 2307 and the center point of passage 2303 may be different from the distances between the other passages of the body portion 2301 with respect to the end face 2307. In this way, the passages may be subject to the action of the magnetic field correlated to their position within the body portion 2301.

[0224] The end face 2307 can be configured to abut against a stop portion or surface 2311 of the frame 2319. The frame 2319 can be configured to transmit magnetic flux. Although the body portion 2301 is illustrated as terminating at a point on the end face 2307, it will be understood that other configurations are contemplated. FIG. 83B shows a loaded configuration in which the magnetic cell isolation holder 2302 is positioned within the receiving region 2316 of the magnetic field generator 2313. Loading can include advancing the end face 2307 toward the stop surface 2311 until the end face 2307 abuts the stop surface. Nevertheless, in the loaded configuration, a portion of the body portion 2301 remains outside the receiving region 2316. Thus, in one embodiment, one or more passages of the body portion 2301 can be positioned to be within the receiving region 2316 when loaded.

[0225] The magnetic isolation device 2300 can also include a door or other feature configured to reduce leakage of the magnetic field outside the receiving region 2316. The steel backing 2308 and the door 2318 of the frame 2319 of the magnetic isolation device 2300 are made of a soft magnetic material (e.g., 1018 steel). These are magnetized in the presence of a magnetic field and demagnetized when the magnetic field is removed. When the magnetic cell isolation holder 2302 is not inserted into the receiving region 2316 of the magnetic field generator 2313, the door 2318 of the magnetic field generator 2313 closes the gap with the aid of a compressed spring attached to either door, thereby surrounding the magnetic flux within the steel backing 2308 and the door 2318. This prevents leakage of magnetic flux into the passages 2303, 2305 when demagnetization is desirable for some processes such as elution.

[0226] FIG. 83B shows the magnetic cell isolation holder 2302 of the loading configuration in the magnetic isolation device 2300. When the magnetic cell isolation holder 2302 is fully inserted into the receiving region 2316 of the magnetic field generator 2313, the positions of the passages 2303, 2305 are defined by the geometric shapes of the magnetic cell isolation holder 2302 and the magnetic isolation device 2300. A part of the backing 2308 (e.g., the stop surface 2311) of the magnetic isolation device 2300 may abut against a part of the magnetic cell isolation holder 2302 when the magnetic cell isolation holder 2302 is fully inserted into the magnetic field generator 2313. In addition, although the magnetic cell isolation holder 2302 has the tapered shape of FIGS. 3A and 3B, any suitable shape of the magnetic cell isolation holder 2302 may be used.

[0227] The door 2318 of the magnetic field generator 2313 opens to allow insertion of the magnetic cell isolation holder 2302 between the magnetic field plates 2312, 2314 of the magnetic field generator 2313. For example, the position of the passage 2303 in the magnetic field generator 2313 may be targeted for placement within a magnetic field of the highest magnetic field strength (i.e., 0.5 T). In another example, the position of the passage 2305 in the magnetic field generator 2313 may be targeted for placement within a magnetic field of the highest magnetic field gradient (i.e., 50 T / m) while meeting the magnetic field strength requirement of the magnetic beads (i.e., 0.15 T).

[0228] To elute the retained beads (e.g., beads or bead-bound cells) from the magnetic retention material (e.g., magnetic retention materials 2304, 2306), the external magnetic field can be removed by retracting the isolation holder 2302 to the disengaged position (i.e., the unloaded configuration of FIG. 83A). When an external magnetic field is not required near the passages 2303, 2305, the door is closed to ensure that there is no leakage of magnetic flux that would affect the passages 2303, 2305. Then, a high flow rate of fluid is passed through the passages 2303, 2305, which generates a large shear force on the retained beads. When the viscous force is greater than the holding force (i.e., the magnetic force due to the residual magnetic field), the beads are washed off from the magnetic retention material of the passages 2303, 2305 and collected. However, in other embodiments, the applied magnetic field can be switched off under the control of the controller 2150.

[0229] As described, the magnetic cell isolation holder 2302 may be configured to have one or more passages, each passage corresponding to the type and / or size of beads used in the magnetic cell isolation procedure. For example, the magnetic cell isolation holder 2302 may have three passages, namely, a tube for beads with a diameter of 4.5 μm, a tube for beads with a diameter of 3 μm, and a tube for beads with a diameter of 2 μm. The passages within each magnetic cell isolation holder 2302 may be of different sizes or the same size.

[0230] FIGS. 84A and 84B show isometric views of different configurations of the magnetic cell isolation holder and magnetic isolation device of FIGS. 83A and 83B. FIG. 84A shows the position of the magnetic cell isolation holder 2302 before engagement of the magnetic cell isolation holder 2302 in the magnetic isolation device 2300 for magnetic isolation. FIG. 84B shows the position of the magnetic cell isolation holder 2302 after engagement of the magnetic cell isolation holder 2302 in the magnetic field generator 2313 for magnetic isolation. The frame 2319 may include opposing guide plates 2330 that are spaced apart from each other by a distance that allows the magnetic cell isolation holder 2302 to pass therebetween and facilitates proper positioning within the receiving region 2316.

[0231] Some of the disclosed techniques relate to positioning a magnetic cell isolation holder as disclosed within a fixed position magnetic field generator, although it will be understood that other implementations are also contemplated. For example, the magnetic field generator may move relative to a magnetic isolation holder loaded within a fixed position frame.

[0232] FIG. 85 shows a flowchart for a method 2500 of magnetic cell isolation that can be used with a magnetic isolation device. In step 2502, a first cell mixture is prepared by incubating the cell mixture with a set of magnetic beads having desired properties (e.g., size, type, ligand, etc.). After a sufficient period has elapsed to ensure that the target cells are labeled with the magnetic beads, the excess incubated mixture is removed. In another embodiment, a portion of the incubated mixture may be removed and evaluated for quality control purposes, i.e., the excess incubated mixture may be evaluated to thereby assess the binding properties. In step 2504, the first magnetic cell isolation holder 2302 may be coupled within the receiving region 2316 of the magnetic field generator 2313. In step 2506, the magnetic field generator 2313 generates a magnetic field within the receiving region 2316 of the magnetic field generator 2313. In step 2508, the first cell mixture flows through a passage (e.g., one or more of passages 2303 or 2305) within the first magnetic cell isolation holder 2302. The magnetic bead-labeled cells within the cell mixture are retained within the passage by the magnetic retention material (e.g., one or more of magnetic retention materials 2304 or 2306) of the first magnetic cell isolation holder 2302 while the remainder of the cell mixture material flows through the passage of the first magnetic cell isolation holder 2302. In step 2510, the generation of the magnetic field is stopped by removing the first magnetic cell isolation holder 2302 from the receiving region 2316 of the magnetic field generator 2313 (or by terminating the application of the magnetic field), resulting in demagnetization of the magnetic cell isolation holder 2302. In step 2512, the retained or isolated cells and beads from the first magnetic cell isolation holder 2302 are collected by eluting the magnetically retained beads or cells within the passage of the magnetic cell isolation holder 2302 with a fluid having a high flow rate or by another suitable method. In step 2514, optionally, the magnetic cell isolation holder 2302 may be disposed of.Steps 2522 through 2534 mirror steps 2502 through 2514, but instead of that, cells labeled with a set of beads of different sizes in the second cell mixture may be passed through the passageways within the second (i.e., different) magnetic cell isolation holder 2302, or routed into different passageways within the first magnetic cell isolation holder 2302. Steps 2522 through 2534 illustrate a method of using two different magnetic cell isolation holders, although it will be understood that the two magnetic cell isolation holders could instead be the same magnetic cell isolation holder having different passageways for each cell mixture. In addition, the second cell mixture may be the cell mixture obtained as a result of passing through the first magnetic cell isolation holder 2302 from step 2508 without retaining bead-labeled cells.

[0233] Magnetic selection of target cells can be either positive or negative selection. In positive selection, magnetic beads are used to label the target cells, and the target cells are collected as the labeled fraction. In negative selection or cell depletion, magnetic beads are used to label the unwanted cells, and the target cells are collected as the unlabeled fraction.

[0234] FIG. 86 is a top view of the arrangement of the magnetic cell isolation holder 2602 relative to the permanent magnets 2612, 2614 of the magnetic field generator 2600. In one embodiment, the distance between the permanent magnets 2612, 2614 is about 0.37 inches (10 mm). However, other distances between the permanent magnets can also be used depending on the configuration of the isolation device, such as the physical characteristics of the magnets, the aspect ratio of the cross-sectional area, and the design of the apparatus holding the magnets. In the illustrated embodiment, the magnetic retention material may be, for example, a column matrix 2604 for use with Miltenyi microbead-labeled cells, and the magnetic retention material 2606 may be, for example, a tube for use with Dynabead-labeled cells. Either a column matrix or a tube can be used for any individual magnetic cell isolation procedure.

[0235] FIG. 87 shows the magnetic field distribution by the permanent magnet and backing steel of a magnetic field generator showing different magnetic field characteristics of magnetic fields in different arrangements. As disclosed, the magnetic field parameters for separation are different for beads of different sizes (e.g., beads). The larger the beads, the higher the magnetic moment, and thus, a lower magnetic field gradient is required to generate an equal magnitude of force when compared to smaller beads having a lower magnetic moment. The magnetic force can be expressed as Fmag = M·∇B, where M is the magnetic moment and ∇B is the magnetic field gradient. To ensure the highest magnetic moment, the magnetic material must saturate at an external magnetic field strength (i.e., 0.15 T for Dynabeads as described herein). When the magnetic force is greater than the viscous force in the flow field, the magnetic beads move in the direction of the magnetic force until they reach the wall of the tube or the spheres of the column matrix.

[0236] In some embodiments, the disclosed technology can be used to isolate cells for chimeric antigen receptor cell therapy (or CAR-T). CAR-T involves isolating several types of white blood cells from peripheral blood mononuclear cells (PBMCs), namely T cells. The target cells (T cells) are modified with receptors that enable them to recognize and attack cancer. Additionally, the disclosed technology can be used in conjunction with any suitable type of beads, such as Miltenyi nanosize microbeads (50 nm in diameter) and Dynabeads (4.5 μm in diameter). Miltenyi's microbeads are nanosize superparamagnetic beads, which require a magnetizable column matrix for retention from a flowing field. The magnetizable column matrix is fabricated from soft magnetic material spheres (e.g., stainless steel 400 series balls 0.4 mm in diameter). Stainless steel 400 series balls do not rust. The magnetic properties of the sphere material involve magnetization strengthening when exposed to an external magnetic field and little residual magnetism when the external magnetic field is removed. The manufacturing process of the magnetic retention material column matrix involves filling the column matrix with spheres using a vibrator, applying lacquer to the column matrix, gravity-draining the lacquer, removing residual lacquer by centrifugation, air-blowing, and re-centrifuging. The air-blowing and centrifugation steps are repeated several times until all residual lacquer is removed. The column matrix is then placed in an oven at a temperature of about 100 °C for three days. After the column matrix is fully cured, the column matrix is held together by the applied lacquer. The magnetizable column matrix can function as a magnetic enhancement device that fills the spheres and enhances the magnetic field gradient by about 10,000 times. The enhanced magnetic field gradient helps attract nanosize bead-labeled cells to the spheres in the presence of an external magnetic field. The column matrix demagnetizes after the external magnetic field is removed, thereby enabling the release of nanosize bead-labeled cells from the column matrix. The nanosize bead-labeled cells are then eluted by the flow of the rinsing fluid through the column matrix.

[0237] Dynabeads are larger superparamagnetic beads made from synthetic polymers. Since Dynabeads are considerably larger than Miltenyi nanosized beads, Dynabeads have a considerably higher magnetic moment than Miltenyi nanosized beads when placed in a magnetic field. Thus, using Dynabeads for magnetic cell isolation does not necessarily require a magnetic enhancement device such as a magnetic column matrix. A tube-based system is typically used with Dynabead-labeled cells, and a permanent magnet is placed near the tube. The target cells labeled with Dynabeads are attracted to the wall of the tube, and the unlabeled cells are then removed along with the buffer or medium. Other beads of different sizes are also commercially available in addition to Miltenyi nanosized microbeads and Dynabeads.

[0238] Magnetic isolation devices can be used with a magnetic field generator, such as a pair of permanent magnets, a magnetic cell isolation holder and associated magnetic cell retention material, flow tubing, collection and preparation containers, and other components of the disclosed system 2100. Further, some of these components can be provided as single-use components, disposable components, and / or in a package kit.

[0239] In one embodiment, a dedicated kit can be provided to achieve magnetic isolation for a specific bead type. For any given isolation event, a kit optimized for one or more bead sizes can be provided. The kit may include a suitable magnetic retention material, which can be pre-loaded into a properly configured magnetic cell isolation holder. In this way, the user is prevented from accidentally loading or binding an improper magnetic retention material into the passage of the magnetic isolation holder. In one embodiment for use with Miltenyi microbeads, the magnetic retention column within the passage of the magnetic isolation holder is positioned at the center of the gap or space between the permanent magnets of the magnetic field generator upon loading and can be associated with the highest or a higher magnetic field strength (i.e., greater than 0.45 T). In another embodiment, the magnetic retention tubing for Dynabeads can be positioned within the highest gradient region between the permanent magnets. Dynabead isolation can be performed in conjunction with a magnetic isolation holder having a passage positioned relative to the magnetic field generator such that it is subject to the action of both a magnetic field strength suitable for retention (i.e., greater than 0.1 T) and a magnetic field gradient (i.e., greater than 40 T / m).

[0240] When used in conjunction with the disclosed technology, the average recovery and average purity of CD3+ using a magnetic isolation device are each greater than approximately 80% for Miltenyi nano-sized beads. For Dynabeads, the average recovery of CD3+ using a magnetic isolation device is approximately 60%, and the average purity of CD3+ using a magnetic isolation device is greater than approximately 70%.

[0241] The technical effect of the present disclosure includes providing a holder for magnetic isolation of cells for use with a magnetic field generator to enable cell isolation without adjustment of magnetic field parameters between procedures using magnetic beads of different sizes. In addition, the magnetic isolation device can automatically perform methods of cell preparation, magnetic cell isolation, and cell elution for each of the different sized beads, eliminating or reducing the user's interaction and manipulation of the raw materials.

[0242] As used herein, an element or step recited in the singular and preceded with the article "a" or "an" should be understood as not excluding a plurality of said elements or steps, unless explicitly stated otherwise. Further, reference to "one embodiment" of the present invention is not intended to be construed as excluding the existence of additional embodiments that also incorporate the recited features. Further, unless otherwise specified, an embodiment "comprising," "including," or "having" an element or elements with a particular property may include additional such elements that do not have that property.

[0243] In this specification, examples are used to disclose several embodiments of the invention, including the best mode, and to enable those of ordinary skill in the art to practice the embodiments of the invention, including making, using, and executing any incorporated methods. The patentable scope of the present invention is defined by the claims and may include other examples that occur to those of ordinary skill in the art. Such other examples are intended to be within the scope of the claims if they have structural elements that do not differ from the literal language of the claims, or if they include equivalent structural elements with only insubstantial differences from the literal language of the claims.

[0244] [Item 1] A method of bioprocessing, comprising: combining a suspension comprising 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; collecting target cells from the population of cells; and wherein the step of collecting the target cells comprises removing the bead-bound cells from the isolation column having an air plug. [Item 2] The method according to item 1, further comprising circulating a buffer solution through the isolation column to elute the bead-bound cells from the isolation column. [Item 3] The method according to item 2, wherein the buffer solution comprises an isolation buffer, a cell culture medium, or a cell release buffer. [Item 4] The method according to item 1, further comprising the step of combining the magnetic beads with an isolation buffer and isolating the magnetic beads on the isolation column before combining with the population of cells. [Item 5] The step of collecting the target cells comprises collecting the bead-bound cells using a release buffer to produce the target cells, incubating to release the target cells from the magnetic beads, isolating the magnetic beads on an isolation column, and collecting the released target cells. The method according to item 1. [Item 6] The method according to item 1, further comprising the step of washing the target cells to exchange the suspension. [Item 7] The method according to item 1, further comprising the step of concentrating the target cells. [Item 8] The method according to item 1, further comprising the step of washing and concentrating the target cells. [Item 9] The step of isolating the target cells further comprises the step of flowing an isolation buffer through the isolation column to rinse the isolated cell population. The method according to item 1. [Item 10] The step of isolating the target cells further comprises eluting the isolated cell population from the isolation column with an isolation buffer to perform rinsing, and then isolating the rinsed population of bead-bound cells on a magnetic isolation column. The method according to item 1. [Item 11] The magnetic beads are nano-sized beads, and the suspension is washed to remove excess magnetic beads after incubation and before isolating the target cells with the magnet. The method according to item 1. [Item 12] The method according to claim 1, wherein the magnetic beads are Dynabead, Miltenyi beads, or StemCell EasySep beads. [Claim 13] drawing air into the process bag through its port; and circulating the air through a process pipeline in fluid communication with the process bag to clean the process pipeline, the method according to claim 1, further comprising. [Claim 14] further comprising detecting air in the process loop, wherein the detection of the air indicates that the process bag is empty, the method according to claim 1. [Claim 15] further comprising concentrating a desired cell population from a biological sample, the method according to claim 1. [Claim 16] The step of concentrating the desired cell population includes transferring the biological sample and a buffer to a processing chamber, and washing the biological sample in the processing chamber to reduce the amounts of platelets and serum in the biological sample, the method according to claim 15. [Claim 17] after concentration, transferring the concentrated cell population to a processing bag; and circulating the concentrated cell population through the process loop, the method according to claim 16, further comprising. [Claim 18] A system comprising: a magnetic field generator configured to generate a magnetic field under magnetic field parameters; a first holder configured to be removably coupled to the magnetic field generator, the first holder comprising a first passage configured to be positioned within the magnetic field in a first arrangement when the first holder is coupled to the magnetic field generator; A second holder configured to be removably coupled to the magnetic field generator, the second holder comprising a second passage configured to be positioned within the magnetic field in a second arrangement when the second holder is coupled to the magnetic field generator, The first passage is subjected to the action of a first magnetic field strength and a first magnetic field gradient within the magnetic field generated under the magnetic field parameters in the first arrangement, and the second passage is subjected to the action of a second magnetic field strength and a second magnetic field gradient within the magnetic field under the magnetic field parameters in the second arrangement, wherein the second magnetic field strength is different from the first magnetic field strength, or the second magnetic field gradient is different from the first magnetic field gradient, or a combination thereof. [Item 19] The system according to item 18, wherein the magnetic field generator is coupled to a frame, the frame transmits magnetic flux, and forms a receiving region configured to receive the first holder or the second holder. [Item 20] The system according to item 19, wherein the receiving region is sized to receive only one of the first holder or the second holder at a given time. [Item 21] The system according to item 19, wherein the receiving region is configured to receive only a portion of the first holder or the second holder. [Item 22] The system according to item 21, wherein the portion includes the first passage of the first holder or the second passage of the second holder. [Item 23] The system according to item 19, wherein the frame comprises a retractable portion that reduces the magnetic flux generated by the magnetic field generator as the first holder moves beyond the receiving region while the first holder is separated from the magnetic field generator. [Item 24] The system according to item 23, wherein the retractable portion comprises a spring that allows the first holder to enter the receiving region of the frame when compressed. [Item 25] The system according to claim 18, wherein when the first holder is not coupled to the magnetic field generator, the first passage is not subjected to the action of the first magnetic field strength. [Item 26] The system according to claim 19, wherein the first holder includes a first end face configured to abut against a stop portion of the frame when the first holder is coupled to the magnetic field generator. [Item 27] The system according to claim 26, wherein the second holder includes a second end face configured to abut against the stop portion of the frame when the second holder is coupled to the magnetic field generator, and a first distance between the first end face and the first passage is different from a second distance between the second end face and the second passage. [Item 28] The system according to claim 18, wherein the first passage and the second passage are of different sizes. [Item 29] The system according to claim 18, wherein the first holder is configured to be fluidly coupled to a first source of beads of a first size, and the second holder is configured to be fluidly coupled to a second source of beads of a second size. [Item 30] The system according to claim 29, wherein the beads of the first size have a diameter of less than 1 μm, and the beads of the second size have a diameter of more than 2 μm. [Item 31] The system according to claim 29, wherein the beads of the first size are bound to target cells in a first cell mixture, and the beads of the second size are bound to target cells in a second cell mixture. [Item 32] The system according to claim 18, wherein the first passage or the second passage includes a magnetic enhancement device such as a plurality of magnetizable spheres. [Item 33] A magnetic cell isolation holder, A main body configured to be removably coupled to a magnetic field generator, the main body comprising a first passage configured to be positioned within the magnetic field of the magnetic field generator in a first arrangement when the holder is coupled to the magnetic field generator, and a second passage configured to be positioned within the magnetic field in a second arrangement when the holder is coupled to the magnetic field generator. The first passage is subjected to the action of a first magnetic field strength and a first magnetic field gradient within the magnetic field generated under the magnetic field parameters in the first arrangement. The second passage is subjected to the action of a second magnetic field strength and a second magnetic field gradient within the magnetic field generated under the magnetic field parameters in the second arrangement, wherein the second magnetic field strength is different from the first magnetic field strength, or the second magnetic field gradient is different from the first magnetic field gradient, or a combination thereof, a magnetic cell isolation holder. [Claim 34] A system, A first kit, A plurality of beads of a first size, A first holder comprising a first passage configured to receive the plurality of beads of the first size, the first passage being positioned within the first holder such that the first holder is positioned within the magnetic field in a first arrangement when the first holder is removably coupled to a magnetic field generator that generates a magnetic field, the first kit. A second kit, A plurality of beads of a second size, A second holder comprising a second passage configured to receive the plurality of beads of the second size, the second passage being positioned within the second holder such that the second holder is positioned within the magnetic field in a second arrangement different from the first arrangement when the second holder is removably coupled to the magnetic field generator that generates the magnetic field, the second kit. The first passage is subjected to the action of a first magnetic field strength and a first magnetic field gradient in the magnetic field in the first arrangement. The second passage is subjected to the action of a second magnetic field strength and a second magnetic field gradient in the magnetic field in the second arrangement, and the second magnetic field strength is different from the first magnetic field strength, or the second magnetic field gradient is different from the first magnetic field gradient, or a system that is a combination thereof. [Item 35] A method for isolating target cells, comprising: positioning a first holder having a first passage in a receiving region of a frame coupled to a magnetic field generator; generating a first magnetic field in the receiving region by the magnetic field generator when the first holder is coupled to the magnetic field generator to apply a first magnetic field strength, a first magnetic field gradient, or both to the first passage; positioning a second holder having a second passage in the receiving region, wherein the first passage and the second passage are positioned in different arrangements in the receiving region; generating a second magnetic field in the receiving region by the magnetic field generator when the second holder is coupled to the magnetic field generator to apply a second magnetic field strength, a second magnetic field gradient, or both to the second passage. [Item 36] The method according to item 35, wherein the first magnetic field and the second magnetic field are generated under the same magnetic field parameters. [Item 37] The method according to item 35, wherein when the first holder is positioned in the receiving region of the frame, the second holder is not positioned in the receiving region of the frame, and when the second holder is positioned in the receiving region of the frame, the first holder is not positioned in the receiving region of the frame. [Item 38] Incubating a first cell mixture with beads of a first size and then a second cell mixture with beads of a second size such that target cells in the first cell mixture are labeled with the beads of the first size and target cells in the second cell mixture are labeled with the beads of the second size; The method according to claim 35, further comprising passing the first cell mixture through the first passageway and then passing the second cell mixture through the second passageway. [Claim 39] A kit for use within a bioprocessing system, a process bag, a source bag, a bead addition container, and a process loop configured to be in fluid communication with the process bag, the source bag, and the bead addition container; The kit, wherein the process loop comprises pump tubing configured to be in fluid communication with a pump. [Claim 40] an isolation column, a waste bag, a buffer bag, and a collection bag; The kit according to claim 39, wherein the process loop is configured to be in fluid communication with the isolation column, the waste bag, the buffer bag, and the collection bag. [Claim 41] The kit according to claim 39, further comprising a valve manifold operable to selectively place the source bag, the process bag, the bead addition container, and the process loop in fluid communication. [Claim 42] The kit according to claim 39, further comprising a storage bag configured to be in fluid communication with the process loop. [Claim 43] The kit according to claim 39, further comprising a processing chamber configured to be in fluid communication with the process loop. [Claim 44] The kit according to claim 39, wherein the bead addition container is a syringe for injecting magnetic isolation beads into the process loop. [Claim 45] The kit according to claim 39, further comprising a release buffer bag configured to be in fluid communication with the process loop. [Claim 46] The kit according to claim 39, further comprising a medium bag configured to be in fluid communication with the process loop. [Claim 47] An apparatus for bioprocessing, A kit comprising a process bag, a source bag, and a bead addition container configured to be in fluid communication with a process loop, wherein the process loop further comprises pump tubing configured to be in fluid communication with a pump, the kit, 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 addition container, each hook of the plurality of hooks being operably connected to a load cell, the load cell being configured to compensate for the weight of the bag connected thereto, the plurality of hooks, At least one bubble sensor, An apparatus comprising a pump configured to be in fluid communication with the process loop. [Claim 48] The process bag comprises an air port, The apparatus according to claim 47, wherein the pump is operable to draw air into the system through the air port. [Claim 49] The apparatus according to claim 48, wherein the air port is within the top of the process bag at a position higher than the liquid level with the process bag. [Claim 50] The apparatus according to claim 47, further comprising a second waste liquid bag selectively in fluid communication with the valve manifold. [Claim 51] The waste liquid bag includes a first waste liquid bag and a second waste liquid bag, and the first waste liquid bag and the second waste liquid bag are in fluid communication with the valve manifold. The apparatus according to claim 47. [Claim 52] The apparatus according to claim 47, further comprising a first storage bag and a second storage bag, wherein the first storage bag and the second storage bag are configured to be in fluid communication with the valve manifold. [Claim 53] The apparatus according to claim 50, further comprising a release media bag that is selectively in fluid communication with the valve manifold. [Claim 54] A first pump tube having a first inner diameter, and a second pump tube having a second inner diameter that is larger than the first inner diameter of the first pump tube, wherein the first pump tube and the second pump tube are selectively engageable with the pump to enable a range of flow rates to the pump. The apparatus according to claim 47.

Description of Reference Numerals

[0245] 10 Bioprocessing system 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 architecture 400 Bioprocessing subsystem 400 Bioprocessing system 410 First bioreactor vessel 412 First port 414 First bioreactor pipeline 416 Second port 418 Second bioreactor pipeline 420 Second bioreactor vessel 422 First port 424 First bioreactor pipeline 426 Second port 428 Second bioreactor pipeline 430 Bioreactor array 432 First bioreactor pipeline valve 436 First bioreactor pipeline valve 438 Second bioreactor pipeline valve 440 First fluid assembly 442 First fluid assembly pipeline 444 Second fluid assembly 446 Second fluid assembly pipeline 448 Sampling assembly 450 Interconnection pipeline 452 Interconnection pipeline valve 454 Interconnection pipeline pump 456 Second pump or circulation pipeline pump 458 Sterilized air source 460 Sterilized air source pipeline 462 Valve 464a~f Tubing tail 466a~f First storage tank 468a~f Tubing tail valve 470a~d Tubing tail 472a~d First storage tank 474a~d Tubing tail valve 476a~476d Sampling pipeline 478a~d Sample pipeline valve 482 Filtration pipeline 484 Filter 486 Upstream filtration pipeline valve 488 Downstream filtration pipeline valve 490 Waste liquid pipeline 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 552 Position verification structure 554 Flat engagement surface 556 Opening or aperture 600 Kit 610 Tray 612 Front wall 614 Rear wall 616, 618 Side surfaces 622 Internal compartment 620 Bottom surface 622 Internal compartment 624 Peripheral surface flange 626, 628 First and second openings 631 Sampling space 632 Claw 636 Support rib 638 Opening 650 Tubing module 652 First tubing holder block 654 Second tubing holder block 656, 658 configurations 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 Features 682 Narrow tubing slot 684 Waste liquid line tubing slot 700 Bioprocessing device 710 Housing 712, 714, 716 Drawers 718 Side wall 720 Bottom surface 722 Processing chamber 724 Hardware compartment 730 Auxiliary compartment 732 Power supply 734 Operation control board and drive electronic circuit 736 Low power solenoid array 738 Pump assembly 740 Drawer engagement actuator 742 Pump shoe 744 Pinch valve anvil 746 First bed plate 748 Second bed plate 750 Plate 752 Top surface 754 Positioning pin or alignment pin 756 Integrated sensor 759 Embedded temperature sensor 760 Load cell 760 Resistance temperature detector 761 Actuator mechanism 762 Cam arm 764 Slot 766 Cam Pin 768 Linear Actuator 770 Rocker Switch 770 Linear Actuator 772 Feed Screw 774 Clevis Arm 776 Space 778 Solenoid 780 Piston 782 Heating Pad 784 Heating Module 786 Carbon Dioxide Sensing Module 788 Cover 790 Insulating Foam Layer 792 Film Heater 794 Internal Metal Plate 798 Heat Insulation Layer 800 Fluid 810 Flip-Down Front Panel 812 Telescoping Slide Rail 814 Horizontally Extending Cross Rod 815 Mounting Means 816 Rope Profile Waste Liquid Tray 819 Self-Chamber 820 Platform 822 Guide Track 900 Device / Apparatus 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 Supply Source Bag 932 Process Bag 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 medium bag 948 Release bag 950 Collection bag 952 Syringe 960 Magnetic cell isolation holder 962 Magnetic field generator 964, 966 Magnetic field plate 968 Magnetic holding element or material 1000 General protocol 1110 Process loop 1112 Process pipeline 1114 First storage bag pipeline 1116 Second storage pipeline 1118 Top port 2100 Alternative magnetic isolation system 2102 Incubate removal 2104 Sample supply source bag (SB) 2106 Process bag (PB) 2108 Buffer bag (BB) 2110 Culture medium bag (MB) 2112 Supply source pump (SP) 2114 Process pump (PP) 2116 Magnetic isolation pump (MP) 2118 Bead addition syringe (SG1) 2120 Check valve 2122, 2124 Magnetic field plate 2126 Collection pinch valve (PV-C) 2128 Waste liquid pinch valve (PV-W) 2120 Check valve (CV1) 2121 Magnetic field generator 2130 Collection bag (CB) 2132 Waste liquid bag (WB) 2134 Magnetic cell isolation holder 2150 Controller 2152 Processor 2154 Memory 2200 Method 2300 Magnetic isolation device 2301 Main body part 2302 Magnetic cell isolation holder 2303, 2305 Passageway 2304, 2306 Magnetic holding material 2307 End face 2308 Steel backing 2311 Stop part or surface 2312, 2314 Magnetic field plate 2313 Magnetic field generator 2315 Distance 2316 Receiving area 2318 Door 2319 Frame 2330 Guide plate 2500 Method 2600 Magnetic field generator 2602 Magnetic cell isolation holder 2604 Column matrix 2606 Magnetic holding material 2612, 2614 Permanent magnet

Claims

1. A magnetic cell isolation holder, comprising: A main body configured to be removably coupled to a magnetic field generator, the main body comprising a first passage configured to be positioned within the magnetic field of the magnetic field generator in a first arrangement when the magnetic cell isolation holder is coupled to the magnetic field generator, and a second passage configured to be positioned within the magnetic field in a second arrangement when the magnetic cell isolation holder is coupled to the magnetic field generator. The first passage is subjected to the action of a first magnetic field strength and a first magnetic field gradient in the first arrangement. The second passage is subjected to the action of a second magnetic field strength and a second magnetic field gradient within the magnetic field generated under magnetic field parameters in the second arrangement, wherein the second magnetic field strength is different from the first magnetic field strength, or the second magnetic field gradient is different from the first magnetic field gradient, or a combination thereof. A magnetic cell isolation holder characterized by this.

2. The magnetic field generator is coupled to a frame of the magnetic cell isolation holder, the frame transmits magnetic flux and forms a receiving region configured to receive the main body. The magnetic cell isolation holder according to claim 1.

3. The receiving region is configured to receive only a part of the main body. The magnetic cell isolation holder according to claim 2.

4. The part comprises a first passage of the main body or a second passage of the main body. The magnetic cell isolation holder according to claim 3.

5. The frame comprises a retractable portion that reduces the magnetic flux generated by the magnetic field generator because the main body moves beyond the receiving region while separated from the magnetic field generator. The magnetic cell isolation holder according to claim 2.

6. The retractable portion comprises a spring that allows the first holder to enter the receiving region of the frame when compressed. The magnetic cell isolation holder according to claim 5.

7. When the main body is not coupled to the magnetic field generator, the first passage is not subjected to the action of the first magnetic field strength. The magnetic cell isolation holder according to claim 1.

8. The first passage and the second passage are of different sizes. The magnetic cell isolation holder according to claim 1.

9. The first passage is configured to be fluidly coupled to a first source of beads of a first size, and the second passage is configured to be fluidly coupled to a second source of beads of a second size. The magnetic cell isolation holder according to claim 1.

10. The magnetic cell isolation holder according to claim 9, wherein the beads of the first size have a diameter of less than 1 μm, and the beads of the second size have a diameter exceeding 2 μm.

11. The magnetic cell isolation holder according to claim 9, wherein the beads of the first size are bound to target cells in a first cell mixture, and the beads of the second size are bound to target cells in a second cell mixture.

12. The magnetic cell isolation holder according to claim 1, wherein the first passage or the second passage is provided with a magnetic enhancement device.

13. A kit comprising: a plurality of beads of a first size; a plurality of beads of a second size; a main body portion having a first passage configured to receive the plurality of beads of the first size, wherein the first passage is positioned within the main body portion such that when the main body portion is removably coupled to a magnetic field generator that generates a magnetic field in a first orientation, the first passage is positioned within the magnetic field in a first arrangement, and a second passage configured to receive the plurality of beads of the second size is positioned within the main body portion such that when the main body portion is removably coupled to a magnetic field generator that generates a magnetic field in a second orientation, the second passage is positioned within the magnetic field in a second arrangement different from the first arrangement; In a kit comprising: The first passage is subjected to the action of a first magnetic field strength and a first magnetic field gradient within the magnetic field in the first arrangement. The second passage is subjected to the action of a second magnetic field strength and a second magnetic field gradient within the magnetic field in the second arrangement, and the second magnetic field strength is different from the first magnetic field strength, or the second magnetic field gradient is different from the first magnetic field gradient, or a combination thereof. A kit characterized by this.

14. A method for isolating target cells, comprising: Positioning a body portion having a first passage and a second passage within a receiving region of a frame coupled to a magnetic field generator, the first passage and the second passage being disposed at different positions within or outside the receiving region, the step; Generating a magnetic field within the receiving region by the magnetic field generator when the body portion is coupled to the magnetic field generator to apply a first magnetic field strength, a first magnetic field gradient, or both to the first passage and a second magnetic field strength, a second magnetic field gradient, or both to the second passage; A method for isolating target cells, characterized by comprising the steps of:

15. The method according to claim 14, wherein a second magnetic field is generated from the magnetic field generated by the magnetic field generator.

16. The method according to claim 14, wherein when the first passage is positioned within the receiving region of the frame, the second passage is not positioned within the receiving region of the frame, and when the second passage is positioned within the receiving region of the frame, the first passage is not positioned within the receiving region of the frame.

17. Incubating a first cell mixture with beads of a first size and a second cell mixture with beads of a second size such that target cells in the first cell mixture are labeled with the beads of the first size and target cells in the second cell mixture are labeled with the beads of the second size; Passing the first cell mixture through the first passage and passing the second cell mixture through the second passage; The method according to claim 14, further comprising the steps of:

Images