Systems and methods for bioprocessing - Patents.com

Abstract

To provide a bioprocessing system for cellular immunotherapies that reduces contamination risk by increasing automation and decreasing human handling.SOLUTION: A bioprocessing system includes a first module configured for enriching and isolating a population of cells, a second module configured for activating, genetically modifying, and expanding the population of cells, and a third module configured for harvesting the expanded population of cells.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] FIELD OF THE DISCLOSURE Embodiments of the present invention relate generally to bioprocessing systems and methods, and more specifically to bioprocessing systems and methods for the production of cellular immunotherapeutics. [Background technology]

[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 an engineered receptor that combines antigen binding and T-cell activation functions. The 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 humans, genetically modify them, and inject them back into the patient to attack cancer cells. CAR-T cells can be either derived from the patient's own blood (autologous) or from another healthy donor (allogeneic).

[0003] The first step in producing CAR-T cells involves drawing blood from the patient's body and separating leukocytes using apheresis, e.g., leukapheresis. After a sufficient amount of leukocytes is harvested, the leukapheresis product is enriched for T cells, which involves washing the cells out of the leukapheresis buffer. T cell subsets with specific biomarkers are then isolated from the enriched subpopulation using specific antibody conjugates or markers.

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

[0005] After T cells are activated and transduced with a CAR-encoding viral vector, the cells are expanded to large numbers in a bioreactor to achieve the desired cell density, after which the cells are harvested, washed, concentrated, formulated, and infused into the patient.

[0006] Existing systems and methods for manufacturing injectable doses of CAR T cells require many complex operations with numerous human touchpoints, making the entire manufacturing process time-consuming and increasing the risk of contamination. While recent efforts to automate the manufacturing process have eliminated some human touchpoints, these systems still suffer from high costs, lack of flexibility, and workflow bottlenecks. In particular, systems that utilize a high degree of automation are very costly and inflexible in that they require customers to adapt their processes to the system's specific equipment. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] U.S. Patent Application Serial No. 15 / 893,336 [Patent Document 2] U.S. Patent Application Serial No. 15 / 829,615 Summary of the Invention [Problem to be solved by the invention]

[0008] In light of the above, there is a need for a bioprocessing system for cellular immunotherapies that reduces contamination risks by increasing automation and reducing human handling, as well as a bioprocessing system for cell therapy manufacturing that balances the need for development flexibility and mass production consistency, and caters to the needs of various customers who wish to run different processes. [Means for solving the problem]

[0009] Summarized below are some embodiments commensurate in scope with the originally claimed subject matter. These embodiments are not intended to limit the scope of the claimed subject matter, but rather, these embodiments are intended only to provide a brief summary of possible embodiments. Indeed, this disclosure may encompass a variety of forms that may be similar to or different from the embodiments set forth below.

[0010] In one embodiment, the bioprocessing system comprises a first module configured to enrich 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, a bioprocessing system comprises a first module configured to enrich and isolate cells, a plurality of second modules, each configured to activate, genetically transduce, and amplify cells, and a third module configured to harvest the expanded cells, each of the second modules configured to support activation, genetic transduction, and amplification of different populations of cells in parallel with each other.

[0012] In another embodiment, a method of bioprocessing includes 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, wherein the steps of activating, genetically transducing and amplifying the population of cells are performed without removing the population of cells from the second module.

[0013] In another embodiment, an apparatus for bioprocessing comprises a housing and a drawer receivable within the housing. The drawer comprises a plurality of sidewalls and a bottom defining a processing chamber, and a normally 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 allow access to the processing chamber through the open top. The apparatus also comprises at least one bed plate positioned within the processing chamber and configured to receive a bioreactor vessel.

[0014] In another embodiment, a method of bioprocessing includes sliding a drawer having a plurality of sidewalls, a bottom, and a normally open top from a closed position within a housing to an open position to extend the drawer from the housing through the normally open top, positioning a bioreactor vessel through the normally open top onto a stationary bedplate positioned within the drawer, sliding the drawer to the 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 comprises a housing, a first drawer receivable within the housing, the first drawer having a plurality of sidewalls and a bottom defining a first processing chamber and a normally 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 stacked relationship with the first drawer, the second drawer having a plurality of sidewalls and a bottom defining a second processing chamber and a normally 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 and second drawers are each movable between a closed position in which the first and / or second drawer are received within the housing and an open position in which the first and / or second drawer extend from the housing to allow access to the processing chamber through their respective open tops.

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

[0017] In yet another embodiment, a system for bioprocessing comprises a tray having a plurality of sidewalls and a bottom defining an internal compartment and a normally open top, at least one opening formed in the bottom and having a periphery, 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 pinch valve tube of the plurality of pinch valve tubes in place for selective engagement with a respective actuator of a pinch valve array, and a bioreactor vessel positioned over the at least one opening in the internal compartment and supported by the bottom such that a portion of the bioreactor vessel is accessible through the opening in the bottom.

[0018] In yet another embodiment, a system for bioprocessing comprises a processing chamber having a plurality of sidewalls, a bottom surface, and a normally open top surface, a bedplate positioned within the processing chamber adjacent the bottom surface, and a tray. The tray comprises the plurality of sidewalls and bottom surface defining an interior compartment, and the normally open top surface, and an opening in the bottom surface of the tray and having a periphery. The periphery of the opening has a shape and / or dimension such that a bioreactor vessel can be positioned over the opening and supported by the bottom surface of the tray while a portion of the bioreactor vessel is accessible through the opening in the bottom surface. The tray is receivable within the processing chamber such that the bedplate extends through the opening in the bottom surface of the tray and supports 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 normally open top and at least one opening in the bottom bounded by a periphery, the opening having a shape and / or dimension 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 of 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, as well as a normally open top, an opening formed in the bottom, and a plurality of tabs or protrusions extending from the bottom into the opening; locating the bioreactor vessel in the tray such that the bioreactor vessel is supported by the plurality of tabs over the opening; and placing the tray in a processing chamber having a bed plate such that the bed plate is received through the opening in the tray and supports the bioreactor vessel.

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

[0022] In yet another embodiment, a system for bioprocessing comprises a tray having a plurality of sidewalls and a bottom defining an interior compartment and a normally open top, the tray configured to receive, support or otherwise engage a bioreactor vessel thereon, a pump assembly positioned adjacent a rear sidewall of the tray, a pinch valve array positioned adjacent the rear sidewall of the tray, and a tubing module positioned at the rear of the tray, the tubing module comprising 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 pinch valve tube 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 comprises a bottom plate, a vessel body coupled to the bottom plate, the vessel body and the bottom plate defining an interior compartment therebetween, and a plurality of recesses formed in the bottom plate, each recess of the plurality of recesses configured to receive a corresponding alignment pin on the bedplate to align the bioreactor vessel on the bedplate.

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

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

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

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

[0029] In yet another embodiment, a bioprocessing method includes activating cells of a population of cells in a bioreactor vessel using magnetic or non-magnetic beads to produce a population of activated cells, genetically modifying the activated cells in the bioreactor vessel to produce a population of genetically modified cells, washing the genetically modified cells in the bioreactor vessel to remove undesired material, and amplifying the population of genetically modified cells in the bioreactor vessel to produce an amplified population of transduced cells. The activation, genetic modification, washing, and amplification are performed in 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 container, and a process loop configured to be in fluid communication with the process bag, the source bag, and the bead addition container, and additionally comprises pump tubing configured to be in fluid communication with a pump.

[0031] In yet another embodiment, an apparatus for bioprocessing comprises a kit comprising a process bag, a source bag, and a bead addition container configured to be in fluid communication with a process loop, the process loop additionally comprising pump tubing configured to be in fluid communication with a pump; a magnetic field generator configured to generate a magnetic field; a plurality of hooks for hanging the source bag, the process bag, and the bead addition container, each hook of the plurality of hooks operably connected to a load cell, the load cell configured to sense a weight of a bag connected thereto; at least one air 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 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 non-transitory computer readable medium is provided, the non-transitory computer readable medium comprising instructions adapted to adapt a controller to maintain a first target environment in a bioreactor vessel containing a population of cells for a first incubation period to produce a population of genetically modified cells from the population of cells, initiate a flow of medium into the bioreactor vessel, and maintain a second target environment in the bioreactor vessel for a second incubation period to produce an amplified population of genetically modified cells.

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

[0035] In yet another embodiment, a non-transitory computer readable medium is provided, the non-transitory computer readable medium including instructions configured to adapt a controller to receive data related to a mass and / or volume of a bioreactor vessel containing a population of cells suspended in a medium, to operate a first pump to pump fresh medium into the bioreactor vessel, and to operate a second pump to pump spent medium from the bioreactor vessel to a waste bag, and to control an operating set point of at least one of the first pump and the second pump in response to the data related to the mass and / or volume of the bioreactor vessel.

[0036] The invention will be better understood on reading the following description of non-limiting embodiments, with reference to the accompanying drawings, in which: [Brief description of the drawings]

[0037] [Figure 1] FIG. 1 is a schematic diagram of a bioprocessing system according to one embodiment of the present invention. [Diagram 2] FIG. 2 is a schematic diagram of a bioprocessing system according to another embodiment of the present invention. [Diagram 3] FIG. 2 is a block diagram illustrating the fluid flow configuration / system of the cell activation, genetic modification, and amplification subsystem of the bioprocessing system of FIG. 1. [Figure 4] FIG. 4 is a detailed view of a portion of the block diagram of FIG. 3 illustrating a first fluid assembly of the fluid flow configuration / system. [Diagram 5] FIG. 4 is a detailed view of a portion of the block diagram of FIG. 3 illustrating a second fluid assembly of the fluid flow configuration / system. [Figure 6] FIG. 4 is a detailed view of a portion of the block diagram of FIG. 3 illustrating a sampling assembly of the fluid flow configuration / system. [Figure 7] FIG. 4 is a detailed view of a portion of the block diagram of FIG. 3 illustrating the filtration flow path of the fluid flow configuration / system. [Figure 8] FIG. 1 is a perspective view of a bioreactor vessel according to one embodiment of the present invention. [Figure 9] FIG. 9 is an exploded view of the bioreactor vessel of FIG. [Figure 10] FIG. 9 is an exploded cross-sectional view of the bioreactor vessel of FIG. [Figure 11] FIG. 9 is an exploded bottom perspective view of the bioreactor vessel of FIG. [Figure 12] 2 is a top and front perspective view of a disposable drop-in kit for the bioprocessing system of FIG. 1 according to one embodiment of the present invention. [Figure 13]13 is another top and front perspective view of the disposable drop-in kit of FIG. 12. [Figure 14] 13 is another top and rear perspective view of the disposable drop-in kit of FIG. 12. [Figure 15] FIG. 13 is a perspective view of a tray of the disposable drop-in kit of FIG. 12 according to one embodiment of the present invention. [Figure 16] FIG. 13 is a front perspective view of a tubing module of the disposable drop-in kit of FIG. 12 in accordance with one embodiment of the present invention. [Figure 17] FIG. 17 is a rear perspective view of the tubing module of FIG. [Figure 18] FIG. 2 is an elevational view of a second tubing holder block of a tubing module according to one embodiment of the invention. [Figure 19] FIG. 19 is a cross-sectional view of the second tubing holder block of FIG. 18. [Figure 20] FIG. 13 is another front perspective view of the drop-in kit of FIG. 12 showing the flow architecture integrated therein. [Figure 21] FIG. 13 is a rear perspective view of the drop-in kit of FIG. 12 showing the flow architecture integrated therein. [Figure 22] FIG. 13 is a front elevation view of the drop-in kit of FIG. 12 showing the flow architecture integrated therein. [Figure 23] FIG. 1 is a perspective view of a bioprocessing device according to one embodiment of the present invention. [Figure 24] 13 is a perspective view of a drawer of a bioprocessing device for receiving the drop-in kit of FIG. 12, according to one embodiment of the present invention. [Diagram 25] FIG. 25 is a top view of the drawer of FIG. 24. [Figure 26] FIG. 25 is a front perspective view of the processing chamber of the drawer of FIG. [Figure 27] FIG. 2 is a top view of the processing chamber of the drawer. [Figure 28] FIG. 24 is a top view of the bed plate of the bioprocessing apparatus of FIG. 23. [Figure 28A] FIG. 29 is a top view of the hardware components housed under the bedplate of FIG. 28. [Figure 29] FIG. 13 is a side elevational view of the bioprocessing device of FIG. 12. [Diagram 30] 13 is a perspective view of a drawer engagement actuator of the bioprocessing device of FIG. 12. [Diagram 31] FIG. 1 is a top view of a drawer of a bioprocessing apparatus illustrating the clearance positions of the drawer engaging actuators, pump assemblies, and solenoid arrays. [Diagram 32] FIG. 1 is a top view of a drawer of a bioprocessing apparatus illustrating the engagement positions of the drawer engagement actuators, pump assemblies, and solenoid arrays. [Diagram 33] FIG. 1 is a perspective view of a bioprocessing apparatus illustrating the drop-in kit in position within the processing chamber of a drawer. [Diagram 34] FIG. 13 is a top view of a bioprocessing device illustrating the drop-in kit within the processing chamber of the drawer. [Diagram 35] FIG. 1 is a perspective view of a peristaltic pump assembly of a bioprocessing device. [Diagram 36] FIG. 13 is a side elevation view of the peristaltic pump assembly and tubing holder module of the drop-in kit, illustrating the relationship between the components. [Figure 37] FIG. 1 is a perspective view of a solenoid array and pinch valve anvil forming a pinch valve array of a bioprocessing device. [Figure 38] FIG. 2 is another perspective view of a pinch valve array of a bioprocessing device. [Figure 39] 13 is another perspective view of the pinch valve array illustrating the positioning of the tubing holder module of the drop-in kit relative to the pinch valve array in an engaged position. FIG. [Diagram 40] FIG. 1 is a cross-sectional view of a drawer of a bioprocessing apparatus illustrating the mounting location of a bioreactor vessel on a bed plate. [Diagram 41] FIG. 1 is a side elevation view of a bioreactor received on a bed plate illustrating the stirring / mixing mode of operation of the bioreactor system. [Diagram 42] FIG. 1 is a side cross-sectional view of a bioreactor received on a bed plate illustrating the stirring / mixing mode of operation of the bioreactor system. [Diagram 43] FIG. 1 is a schematic diagram of a bioreactor vessel showing the liquid level within the bioreactor vessel when in stirring / mixing mode of operation. [Diagram 44] FIG. 13 is a cross-sectional detailed view of the interface between locating pins on the bed plate and receiving recesses on the bioreactor vessel during the stirring / mixing mode of operation. [Diagram 45] FIG. 1 is a perspective view of a bioprocessing device having a flip-down front panel according to one embodiment of the present invention, showing the processing drawer in an open position. [Figure 46] FIG. 46 is another perspective view of the bioprocessing device of FIG. 45 showing the processing drawer in an open position. [Figure 47] FIG. 46 is an enlarged perspective view of the auxiliary compartment of the bioprocessing device of FIG. 45, showing the processing drawer in a closed position allowing access to the auxiliary compartment. [Figure 48] FIG. 46 is another enlarged perspective view of the auxiliary compartment of the bioprocessing device of FIG. 45, showing the processing drawer in a closed position allowing access to the auxiliary compartment. [Figure 49] FIG. 46 is a perspective view of the bioprocessing device of FIG. 45 showing the processing drawer in a closed position allowing access to the auxiliary compartment. [Figure 50] FIG. 46 is another perspective view of the bioprocessing device of FIG. 45 showing the processing drawer in a closed position allowing access to the auxiliary compartment. [Figure 51] FIG. 2 is a perspective view of an auxiliary compartment of a bioprocessing device, according to another embodiment of the invention. [Figure 52] FIG. 1 is a perspective view of a bioprocessing system having a waste tray, according to one embodiment of the present invention. [Diagram 53] FIG. 4 is a schematic diagram of an automated generic protocol for a bioprocessing system utilizing the fluid flow architecture of FIG. 3, according to one embodiment of the present invention. [Figure 54] FIG. 4 is a schematic diagram of an automated generic protocol for a bioprocessing system utilizing the fluid flow architecture of FIG. 3, according to one embodiment of the present invention. [Figure 55] FIG. 4 is a schematic diagram of an automated generic protocol for a bioprocessing system utilizing the fluid flow architecture of FIG. 3, according to one embodiment of the present invention. [Figure 56] FIG. 4 is a schematic diagram of an automated generic protocol for a bioprocessing system utilizing the fluid flow architecture of FIG. 3, according to one embodiment of the present invention. [Figure 57] FIG. 4 is a schematic diagram of an automated generic protocol for a bioprocessing system utilizing the fluid flow architecture of FIG. 3, according to one embodiment of the present invention. [Figure 58] FIG. 4 is a schematic diagram of an automated generic protocol for a bioprocessing system utilizing the fluid flow architecture of FIG. 3, according to one embodiment of the present invention. [Figure 59] FIG. 4 is a schematic diagram of an automated generic protocol for a bioprocessing system utilizing the fluid flow architecture of FIG. 3, according to one embodiment of the present invention. [Figure 60] FIG. 4 is a schematic diagram of an automated generic protocol for a bioprocessing system utilizing the fluid flow architecture of FIG. 3, according to one embodiment of the present invention. [Figure 61] FIG. 4 is a schematic diagram of an automated generic protocol for a bioprocessing system utilizing the fluid flow architecture of FIG. 3, according to one embodiment of the present invention. [Figure 62] FIG. 4 is a schematic diagram of an automated generic protocol for a bioprocessing system utilizing the fluid flow architecture of FIG. 3, according to one embodiment of the present invention. [Figure 63]FIG. 4 is a schematic diagram of an automated generic protocol for a bioprocessing system utilizing the fluid flow architecture of FIG. 3, according to one embodiment of the present invention. [Figure 64] FIG. 4 is a schematic diagram of an automated generic protocol for a bioprocessing system utilizing the fluid flow architecture of FIG. 3, according to one embodiment of the present invention. [Figure 65] FIG. 4 is a schematic diagram of an automated generic protocol for a bioprocessing system utilizing the fluid flow architecture of FIG. 3, according to one embodiment of the present invention. [Figure 66] FIG. 4 is a schematic diagram of an automated generic protocol for a bioprocessing system utilizing the fluid flow architecture of FIG. 3, according to one embodiment of the present invention. [Figure 67] FIG. 4 is a schematic diagram of an automated generic protocol for a bioprocessing system utilizing the fluid flow architecture of FIG. 3, according to one embodiment of the present invention. [Figure 68] FIG. 4 is a schematic diagram of an automated generic protocol for a bioprocessing system utilizing the fluid flow architecture of FIG. 3, according to one embodiment of the present invention. [Figure 69] FIG. 4 is a schematic diagram of an automated generic protocol for a bioprocessing system utilizing the fluid flow architecture of FIG. 3, according to one embodiment of the present invention. [Figure 70] FIG. 4 is a schematic diagram of an automated generic protocol for a bioprocessing system utilizing the fluid flow architecture of FIG. 3, according to one embodiment of the present invention. [Figure 71] FIG. 4 is a schematic diagram of an automated generic protocol for a bioprocessing system utilizing the fluid flow architecture of FIG. 3, according to one embodiment of the present invention. [Figure 72] FIG. 4 is a schematic diagram of an automated generic protocol for a bioprocessing system utilizing the fluid flow architecture of FIG. 3, according to one embodiment of the present invention. [Figure 73]FIG. 4 is a schematic diagram of an automated generic protocol for a bioprocessing system utilizing the fluid flow architecture of FIG. 3, according to one embodiment of the present invention. [Figure 74] FIG. 4 is a schematic diagram of an automated generic protocol for a bioprocessing system utilizing the fluid flow architecture of FIG. 3, according to one embodiment of the present invention. [Figure 75] FIG. 4 is a schematic diagram of an automated generic protocol for a bioprocessing system utilizing the fluid flow architecture of FIG. 3, according to one embodiment of the present invention. [Figure 76] FIG. 4 is a schematic diagram of an automated generic protocol for a bioprocessing system utilizing the fluid flow architecture of FIG. 3, according to one embodiment of the present invention. [Figure 77] FIG. 4 is a schematic diagram of an automated generic protocol for a bioprocessing system utilizing the fluid flow architecture of FIG. 3, according to one embodiment of the present invention. [Figure 78] FIG. 1 is a perspective view of a concentration and isolation device according to one embodiment of the present invention. [Figure 79] FIG. 79 is a process flow diagram of the concentration and isolation apparatus of FIG. 78. [Figure 80] FIG. 79 is a schematic diagram of the fluid flow architecture of the device of FIG. 78 for performing enrichment and isolation of a population of cells. [Figure 81] 2 is a flow chart of a method of bioprocessing using the system of FIG. 1 according to one embodiment of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0038] Reference will now be made in detail to the exemplary embodiments of the invention, examples of which are illustrated in the accompanying drawings. Wherever possible, the same reference characters will be used throughout the drawings to refer to the same or like parts.

[0039] As used herein, the terms "flexible" or "foldable" refer to structures or materials that are pliable or can bend without breaking, and may also refer to materials that are 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 structures that are "non-foldable", i.e., structures that do not fold, collapse, or otherwise deform under normal forces without significantly reducing their elongated dimensions. Depending on the context, "semi-rigid" can also refer to structures that are more flexible than "rigid" elements, e.g., tubes or conduits that are bendable, but that still do not collapse longitudinally under normal conditions and forces.

[0040] As used herein, "vessel" refers to a flexible bag, a flexible container, a semi-rigid container, a rigid container, or flexible or semi-rigid tubing, as the case may be. As used herein, the term "vessel" is intended to encompass bioreactor vessels having walls or portions of walls that are semi-rigid or rigid, as well as other containers or conduits commonly used in biological or biochemical processing, including, 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 associated flow paths thereof. As used herein, the term "bag" refers to, for example, a flexible or semi-rigid container or vessel used as a containment device for various fluids and / or media.

[0041] As used herein, "fluidly coupled" or "fluid communication" means that components of a system can receive or transfer fluid between components. The term fluid includes gas, liquid, or a combination thereof. As used herein, "electrical communication" or "electrically coupled" means that certain components are configured to communicate with each other through direct or indirect signaling with a direct or indirect electrical connection. As used herein, "operably coupled" refers to a connection, which may be direct or indirect. A connection is not necessarily a mechanical linkage.

[0042] As used herein, the term "tray" refers to any object capable of at least temporarily supporting multiple components. The tray may be made from a variety of suitable materials. For example, the tray may be made from a cost-effective material suitable for sterilization and one-time disposable products.

[0043] As used herein, the term "functionally closed system" refers to multiple components that make up a closed fluid pathway that may have inlet and outlet ports for adding or removing fluids or air from the system without compromising the integrity of the closed fluid pathway (e.g., to maintain an internal sterile biomedical fluid pathway), whereby the ports may include, for example, a filter or membrane at each port to maintain sterile integrity when fluids or air are added to or removed from the system. These components may include, but are not limited to, one or more conduits, valves (e.g., multi-point diverters), containers, receptacles, and ports, depending on a given embodiment.

[0044] Embodiments of the present invention provide systems and methods for producing cellular immunotherapeutics from biological samples (e.g., blood, tissue, etc.). In embodiments, a bioprocessing system comprises a first module configured to enrich and isolate a population of cells, a second module configured to activate, genetically modify, and amplify the population of cells, and a third module configured to harvest the amplified population of cells. In embodiments, the system may comprise multiple second modules, each second module configured to activate, genetically modify, and amplify cells. In some embodiments, the second modules are configured to support activation, genetic modification, and amplification of different populations of cells in parallel with each other.

[0045] 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 for use in the manufacture of cellular immunotherapeutics (e.g., autologous cellular immunotherapeutics), for example, a human blood, fluid, tissue, or cell sample is collected and a cellular therapy is generated from or based on the collected sample. Chimeric antigen receptor (CAR) T-cell therapy is one type of cellular immunotherapeutic that may be manufactured using the bioprocessing system 10, although other cellular therapy may be produced using the system of the present invention or embodiments of the present invention without departing from the broader aspects of the present invention. As illustrated in FIG. 1, the manufacture of CAR T-cell therapy generally begins with the collection of a patient's blood and the separation of lymphocytes through apheresis therapy. The collection / apheresis therapy may be performed at a clinical site, and the apheresis product is then sent to a laboratory or manufacturing facility for the production of CAR T cells. In particular, after the apheresis product is received for processing, the desired cell population (e.g., white blood cells) is enriched or separated from the collected blood to produce a cell therapy, and the target cells of interest are isolated from the original cell mixture. The target cells of interest are then activated, genetically modified to specifically target and destroy tumor cells, and amplified to achieve a desired cell density. After amplification, the cells are harvested and a dose is formulated. The formulation is then often stored frozen and thawed, prepared, and finally shipped to the clinical site for infusion into the patient's body.

[0046] 1, the bioprocessing system 10 of the present invention comprises a number of distinct modules or subsystems, each configured to perform a particular subset of manufacturing steps in a substantially automated, functionally closed, and scalable manner. In particular, the bioprocessing system 10 comprises a first module 100 configured to perform enrichment and isolation steps, a second module 200 configured to perform activation, genetic modification, and amplification steps, and a third module 300 configured to perform 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 provide substantially automated control over the manufacturing process within each module. Although the first module 100, the second module 200, and the third module 300 are illustrated as having dedicated controllers for controlling the operation of each module, it is contemplated that a master control unit may be utilized to provide global control to the three modules. Each module 100, 200, 300 is designed to work in cooperation with the other modules to form a single coherent bioprocessing system 10, as described in more detail below.

[0047] By automating the processes within each module, product consistency from each module may be increased and costs associated with extensive manual operations may be reduced. Additionally, as described in more detail below, each module 100, 200, 300 is substantially closed, helping to ensure patient safety by lowering the risk of external contamination, ensuring regulatory compliance, and helping to avoid the costs associated with open systems. Furthermore, each module 100, 200, 300 is scalable, supporting both low-patient development and high-patient commercial manufacturing.

[0048] With further reference to FIG. 1, the particular manner in which the process steps are partitioned into different modules each providing closed and automated bioprocessing allows for efficient use of capital equipment to a degree not previously seen in the art. As will be appreciated, the step of amplifying the cell population to achieve a desired cell density prior to harvesting and blending is typically the most time-consuming step in the manufacturing process, while the enrichment and isolation steps, as well as the harvesting and blending steps, and even the activation and genetic modification steps, are less time-consuming. Thus, attempts to automate the entire cell therapy manufacturing process, in addition to being logistically challenging, may exacerbate bottlenecks within the process that impede workflow and reduce manufacturing efficiency. In particular, in a fully automated process, the steps of enrichment, isolation, activation, and genetic modification of cells can be performed fairly quickly, while the amplification of genetically modified cells proceeds very slowly. Thus, the manufacturing of cell therapy from a first sample (e.g., blood of a first patient) proceeds quickly until the amplification step, which requires substantial time to achieve the desired cell density to harvest. When a fully automated system is used, the entire process / system is occupied by the amplification equipment performing the amplification of cells from the first sample, and processing of a second sample cannot begin until the entire system is freed up for use. In this regard, in a fully automated bioprocessing system, the entire system is essentially offline and is not available for processing of a second sample until the entire cell therapy manufacturing process, from concentration to harvesting / compounding, is completed on the first sample.

[0049] However, embodiments of the present invention allow for parallel processing of multiple samples (from the same or different patients) to facilitate more efficient utilization of capital resources. This advantage is a direct result of the particular manner in which the process steps are divided into three modules 100, 200, 300, as alluded to above. With particular reference to FIG. 2, in one embodiment, a single first module 100 and / or a single third module 300 can be utilized in conjunction with multiple second modules, e.g., second modules 200a, 200b, 200c, in the bioprocessing system 12 to perform parallel and synchronous processing of multiple samples from the same or different patients. For example, a first apheresis product from a first patient may be enriched and isolated using the first module 100, thereby producing a first population of isolated target cells, which may then be transferred to one of the second modules, e.g., module 200a, for activation, genetic modification, and amplification under the control of controller 210a. After the first population of target cells has been transferred out of the first module 100, the first module is again available for use in processing a second apheresis product, for example from a second patient. The second population of target cells produced in the first module 100 from a sample removed from the second patient can then be transferred to another second module, for example, second module 200b, for activation, genetic modification, and amplification under the control of controller 201b.

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

[0051] This approach also allows for post-processing to be performed asynchronously if desired. In other words, the patient cells may not all grow at the same time. The cultures may reach final density at different times, but multiple second modules 200 are not linked and a third module 300 can be used if desired. The present invention allows samples to be processed in parallel, but they do not have to be done in batches.

[0052] Harvesting of the amplified populations of cells from the second modules 200a, 200b, and 200c may similarly be accomplished using a single third module 300 when each amplified population of cells is ready for harvest.

[0053] Thus, by separating the activation, genetic modification, and amplification steps, which are the most time-consuming, share some operational requirements, and / or require similar culture conditions, into standalone automated and functionally closed modules, other system equipment utilized for enrichment, isolation, harvesting, and compounding is not tied down or offline while the amplification of one population of cells is being performed. As a result, the production of multiple cell therapy drugs may be performed simultaneously, maximizing equipment and floor space usage and increasing the efficiency of the entire process and facility. Additional second modules may be added to the bioprocessing system 10 for parallel processing of any number of cell populations as desired. Thus, the bioprocessing system of the present invention allows for the use of plug-and-play-like functionality, making it easy to scale up or down a manufacturing facility.

[0054] In one embodiment, the first module 100 may be any system or device capable of producing a target population of enriched and isolated cells from an apheresis product taken from a patient for use in biological processes, such as the manufacture of immunotherapeutics and regenerative medicines. For example, the first module 100 may 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 is described in detail hereinafter.

[0055] In one embodiment, the third module 300 may be any system or device capable of harvesting and / or blending the CAR-T cells or other modified cells produced by the second module 200 for injection into a patient for use in cellular immunotherapy or regenerative medicine. In some embodiments, the third module 300 may be a Sefia Cell Processing System, also available from GE Healthcare. In some embodiments, the first module 100 may be utilized first for enrichment and isolation of cells (which are then transferred to the second module 200 for activation, transduction, and expansion (and in some embodiments, harvesting)) and then also used at the end of the process for cell harvesting and / or blending. In this regard, in some embodiments, the same equipment may be utilized for the front-end cell enrichment and isolation steps as well as the back-end harvesting and / or blending steps.

[0056] Turning first 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, automated module 200 resulting in the workflow efficiencies described above is enabled by a unique flow architecture resulting in a particular configuration of components within the second module 200 and a particular interconnectivity between such components. FIGS. 3-77, described below, illustrate various aspects of the second module 200 according to various embodiments of the 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 bioprocessing system 400) within the second module 200 for cell activation, genetic modification, and amplification (and in some cases, harvesting). The system 400 comprises a first bioreactor vessel 410 and a second bioreactor vessel 420. The first bioreactor vessel comprises at least a first port 412 and a first bioreactor line 414 in fluid communication with the first port 412, and a second port 416 and a second bioreactor line 418 in fluid communication with the second port 416. Similarly, the second bioreactor vessel comprises at least a first port 422 and a first bioreactor line 424 in fluid communication with the first port 422, and a second bioreactor line 428 in fluid communication with the second port 426 and the second port 426. Together, the first bioreactor vessel 410 and the second bioreactor vessel 420 form a bioreactor array 430. Although the system 400 is shown as having two bioreactor vessels, embodiments of the invention may comprise a single bioreactor or more than two bioreactor vessels.

[0057] The first and second bioreactor lines 414, 418, 424, 428 of the first and second bioreactor vessels 410, 420 each include a respective valve for controlling the flow of fluids therethrough, as described below. In particular, the first bioreactor line 414 of the first bioreactor vessel 410 includes a first bioreactor line valve 432, and the second bioreactor line 418 of the first bioreactor vessel 410 includes a second bioreactor line valve 424. Similarly, the first bioreactor line 424 of the second bioreactor vessel 420 includes a first bioreactor line valve 436, and the second bioreactor line 428 of the second bioreactor vessel 420 includes a second bioreactor line valve 438.

[0058] 3, the system 400 also includes a first fluid assembly 440 having a first fluid assembly line 442, a second fluid assembly 444 having a second fluid assembly line 446, and a sampling assembly 448. An interconnect line 450 having an interconnect line valve 452 fluidly communicates between the first fluid assembly 440 and the second fluid assembly 444. As shown in FIG. 3, the interconnect line 450 also fluidly communicates between the second bioreactor line 418 and the first bioreactor line 414 of the first bioreactor vessel 410, allowing for circulation of fluid along the first circulation loop of the first bioreactor vessel. Similarly, the interconnect line also provides fluid communication between the second bioreactor line 428 and the first bioreactor line 424 of the second bioreactor vessel 420, allowing circulation of fluid along the second circulation loop of the second bioreactor vessel. Additionally, the interconnect line 450 provides further fluid communication between the second port 416 and the second bioreactor line 418 of the first bioreactor vessel 410 and the first port 422 and the first bioreactor line 424 of the second bioreactor vessel 420, allowing the contents of the first bioreactor vessel 410 to be transferred to the second bioreactor vessel 420, as described below. As illustrated in FIG. 3, in one embodiment, the interconnect line 450 extends from the second bioreactor line 418, 428 to the intersection of the first bioreactor line 414 and the first fluid assembly line 442 of the first bioreactor vessel 410.

[0059] 3, the first and second fluid assemblies 440, 450 are disposed along an interconnect line 450. Additionally, in one embodiment, the first fluid assembly is in fluid communication with a first port 412 of the first bioreactor vessel 410 and a first port of the second bioreactor vessel 420 through a first bioreactor line 414 of the first bioreactor vessel 410 and a first bioreactor line 424 of the second bioreactor vessel 420, respectively. The second fluid assembly 444 is in fluid communication with a second port 416 of the first bioreactor vessel 410 and a second port 426 of the second bioreactor vessel 420 via the interconnect line 450.

[0060] A first pump or interconnecting line pump 454 capable of bidirectional fluid flow is disposed along the first fluid assembly line 442, and a second pump or circulation line pump 456 capable of bidirectional fluid flow is disposed along the interconnecting line 450, the function and purpose of which 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 line 450 through a sterile air source line 460. A valve 462 positioned along the sterile air source line 460 provides selective fluid communication between the sterile air source 458 and the interconnecting line 450. Although FIG. 3 shows a sterile air source 458 connected to the interconnect line 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 line valve and the first bioreactor line valve of either the first bioreactor or the second bioreactor without departing from the broader aspects of the invention.

[0061] 4-6, detailed views of the first fluid assembly 440, the second fluid assembly 444, and the sampling assembly 448 are shown. With particular reference to FIG. 4, the first fluid assembly 440 comprises a plurality of tubing tails 464a-f, each of which is configured to selectively / removably connect to one of a plurality of first reservoirs 466a-f. Each tubing tail 464a-f of the first fluid assembly 440 comprises a tubing tail valve 468a-f for selectively controlling the flow of fluid to or from a respective one of the plurality of first reservoirs 466a-f of the first fluid assembly 440. Although FIG. 4 particularly illustrates the first fluid assembly 440 comprising six fluid reservoirs, more or fewer reservoirs may be utilized for input or collection of various process fluids, as desired. It is contemplated that each tubing tail 464a-f may be individually connected to a storage layer 466a-f, respectively, at times needed during operation of the fluid assembly 440, as described below.

[0062] With particular reference to FIG. 5, the second fluid assembly 444 comprises a plurality of tubing tails 470a-d, each of which is configured to selectively / removably connect to one of a plurality of second reservoirs 472a-d. Each tubing tail 470a-d of the second fluid assembly 444 comprises a tubing tail valve 474a-d for selectively controlling the flow of fluid to or from a respective one of the plurality of second reservoirs 472a-d of the first fluid assembly 444. FIG. 5 specifically illustrates the second fluid assembly 444 comprising four fluid reservoirs, although more or fewer reservoirs may be utilized for input or collection of various process fluids, as desired. In one embodiment, at least one of the second reservoirs, for example, second reservoir 472d, is a collection reservoir for collecting an amplified population of cells, as described below. In one embodiment, second reservoir 472a is a waste reservoir, the purpose of which is described below. The present invention contemplates that one or more reservoirs 472a-d may be pre-connected to their respective tails 470a-d, with each additional reservoir being connected to its respective tail in time for its use within the second fluid assembly 440.

[0063] In one embodiment, the first reservoirs 466a-f and the second reservoirs 472a-d are one-time / disposable flexible bags. In one embodiment, the bags are substantially two-dimensional bags with opposing panels welded or otherwise fastened together around their perimeter and supporting connecting conduits for connecting to the respective tails, as known in the art.

[0064] In one embodiment, the reservoir / bag may be connected to the tubing tails of the first and second tubing assemblies using a sterile welding device. In one embodiment, the welding device is positioned next to the module 200 and the welding device is utilized to butt weld one of the tubing tails (while maintaining sterility) so that it attaches to the tail of the tube on the bag. Thus, the operator may 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 the end of the tubing tail, cutting the tube with a new razor blade, and heating the cut end as the razor is pulled apart while the two tube ends are forced together while still melting so that they resolidify together). Conversely, the bag may be removed by welding a line from the bag and cutting the two closed lines at the weld. Thus, the reservoirs / bags may be individually connected when desired, and the invention does not require that all reservoirs / bags must be connected at the start of the protocol as the operator has access to the appropriate tubing tails throughout the process to connect the reservoirs / bags in time for their use. In fact, as described below, all reservoirs / bags are pre-connected, but the invention does not require pre-connection, and one advantage of the second module 200 is that it allows the operator access to the fluid assemblies / lines during operation, so that used bags can be sterilely connected and disconnected so that other bags can be sterilely connected in the protocol.

[0065] 6, the sampling assembly 448 includes one or more sampling lines, e.g., sampling lines 476a-d, fluidly connected to the interconnection line 450. Each of the sample lines 476a-d may include a sample line valve 478a-d that is selectably actuable to allow fluid flow from the interconnection line 450 through the sample line 476a-d. As also shown therein, the distal end of each sampling line 476a-d is configured to selectively connect to a sample collection device (e.g., sample collection devices 280a and 280d) for collecting fluid from the interconnection line 450. The sample collection device may take the form of any sampling device known in the art, e.g., a syringe, a dip tube, a bag, etc. 6 illustrates the sampling assembly 448 being connected to the interconnect line, in other embodiments the sampling assembly may be fluidly coupled to the first fluid assembly 440, the second fluid assembly 444, a fluid flow path intermediate the second bioreactor line valve 434 and the first bioreactor line valve 432 of the first bioreactor vessel 410, and / or a fluid flow path intermediate the second bioreactor line valve 438 and the first bioreactor line valve 436 of the second bioreactor vessel 420. The sampling assembly 448 provides fully functionally closed sampling of fluid at one or more points in the system 400, as desired.

[0066] 3, in one embodiment, system 400 may also include a filtration line 482 connected at two points along interconnection line 450 to define a filtration loop along interconnection line 450. A filter 484 is positioned along filtration line 482 for removing 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. A 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 reservoir 472a. In this regard, waste line 490 carries waste removed by filter 484 from the fluid passing through filtration line 482 to waste reservoir 472a. 3, filtration line 482 surrounds interconnect line valve 452 such that fluid flow 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 waste liquid removed by the filter to waste reservoir 472a. In one embodiment, filter 484 is desirably an elongated hollow fiber filter, although other tangential or cross-flow filtration means known in the art, such as, for example, flat sheet membrane filters, may be utilized without departing from the broader aspects of the invention.

[0067] In one embodiment, the valves of the first fluid assembly 440 and the second fluid assembly 444, as well as the bioreactor line valves (i.e., valves 432, 434, 436, 438), the sterilization line valve 462, the interconnect line valve 452, and the filtration line valves 486, 488, are pinch valves fabricated in a manner described below. In one embodiment, the lines themselves need not include pinch valves, and the pinch valve illustrations in Figures 3-8 may simply represent where pinch valves can operate on the lines to prevent fluid flow. In particular, as described below, the pinch valves of the flow architecture 400 may be effected by respective actuators (e.g., solenoids) that operate / act against corresponding anvils while the fluid path / line is in between to "pinch off" the line and prevent fluid flow therethrough.

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

[0069] 8-11, there is illustrated the configuration of a first bioreactor vessel 410 according to one embodiment of the present invention. The second bioreactor vessel 420 is preferably, but not necessarily, identical in configuration to the first bioreactor vessel 410, and therefore for simplicity, only the first bioreactor vessel 410 is described below. In one embodiment, the bioreactor vessels 410, 420 are perfusion-compatible silicone membrane-based bioreactor vessels that support the activation, transduction, and expansion of a population of cells therein. The bioreactor vessels 410, 420 may be used for cell culture, cell processing, and / or cell expansion to increase cell density for use in medical treatments or other processes. Although the bioreactor vessels are disclosed herein as being used in conjunction with certain cell types, it should be understood that the bioreactor vessels may be used for the activation, genetic modification, and / or expansion of any suitable cell type. Additionally, the disclosed techniques may be used in conjunction with adherent cells, i.e., cells that adhere and / or grow on a cell expansion surface. In one embodiment, the first and second bioreactor vessels 410, 420 may be constructed and function as disclosed in U.S. patent application Ser. No. 15 / 893,336, filed Feb. 9, 2018, which is incorporated by reference in its entirety.

[0070] As shown in Figures 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., open and / or porous) that is permeable to oxygen to be supplied to the cell culture, as described in more detail with reference to Figure 9. In the illustrated embodiment, the bottom plate 502 is rectangular or nearly rectangular in shape. In other embodiments, the bottom plate 502 may be any other shape that allows for a low profile vessel and / or maximizes the space where the first bioreactor vessel may be utilized or stored.

[0071] In one embodiment, the vessel body 504 comprises a rigid, generally concave structure that, when coupled to the bottom plate 502, forms a cavity or interior compartment 506 of the first bioreactor vessel 410. As shown therein, the vessel body 504 may have a perimeter that is similar to that of the bottom plate 502 such that the vessel body 504 and bottom plate 502 may be coupled to one another. Additionally, as in the illustrated embodiment, the vessel body 504 may be made from a transparent or translucent material that may allow visual inspection of the contents of the first bioreactor vessel 410 and / or allow light to enter the first bioreactor vessel 410. The interior compartment formed by the bottom plate 502 and the vessel body 504 may contain cell media and cell cultures during use of the first bioreactor vessel for cell activation, genetic modification (i.e., transduction), and / or cell amplification.

[0072] As best shown in FIGS. 8-11 , the first bioreactor vessel 410 may include multiple ports through the vessel body 504 that may allow fluid communication between the interior compartment 506 and the outside of the first bioreactor vessel 410 for several processes related to cell activation, transduction / genetic modification, and amplification, such as media input and waste output. The ports may include, for example, a first port 412 and a second port 416. The port 416 may be disposed anywhere within the vessel body 504, such as through either the top surface 508 and / or side surface 510 of the vessel body 504, as in the illustrated embodiment. As will be described in more detail herein, the particular structure of the first bioreactor vessel 410, including the particular amount and location of the ports 412, 416, allows the first bioreactor vessel 410 to be used to support cell activation, cell genetic modification, and high cell density amplification.

[0073] 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 previously described, the bottom plate 502 may be formed from a non-solid structure. In the illustrated embodiment, the bottom plate 502 may contain a grid 510, which may be structurally rigid, but further provides openings to allow free gas exchange through the bottom plate 502 to the interior compartment 506 that contains the cell culture. The grid 510 may include a plurality of holes 512 defined between solid regions or cross bars 514 between each hole 512 of the grid 510. Thus, the holes 512 may provide openings for gas exchange, and the cross bars 514 may provide structural support for other structures and the cell culture within the interior compartment 506 of the first bioreactor vessel 410.

[0074] To provide additional support for the cell culture within the interior compartment 506 of the first bioreactor vessel 410, the first bioreactor vessel 410 may include a membrane 516 that may be disposed on the top surface 518 of the bottom plate 502. The membrane 516 may be a gas permeable, liquid impermeable membrane. The membrane 516 may also be selected to have properties that allow for high gas permeability, high gas transport rate, and / or high permeability to oxygen and carbon dioxide. Thus, the membrane 516 may be capable of supporting a high cell density (e.g., about 35 MM / cm) within the interior compartment 506. 2 Membrane 516 may support up to 100 mm of gas permeable material. The gas permeable characteristics of membrane 516 may allow for free gas exchange to support cell culture and / or cell expansion. As such, membrane 516 may be a cell culture surface and / or cell expansion surface. Membrane 516 may have a relatively small thickness (e.g., 0.010 inches or 0.02 cm), which may allow membrane 516 to be gas permeable. Additionally, membrane 516 may be formed from a gas permeable material, such as silicone or other gas permeable material.

[0075] The flatness of the membrane 516 may increase the surface area of ​​the cell culture to settle for activation, transduction, and / or amplification. To allow the membrane 516 to remain flat during use of the first bioreactor vessel 410, a mesh sheet 520 may be disposed between the bottom plate 502 and the membrane 516. The mesh sheet 520 may provide structural support for the membrane 516, so that it remains flat and does not sag or distort under the weight of the cell culture and / or any cell media added to the first bioreactor vessel 410 for cell culture and / or cell amplification. Additionally, the mesh nature of the mesh sheet 520 may allow support for the membrane 516, yet be porous to allow free gas exchange between the interior compartment 506 of the first bioreactor vessel 410 and the environment immediately outside the first bioreactor vessel 410. The mesh sheet may be a polyester mesh or any other suitable mesh material that may support the membrane and allow free gas exchange.

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

[0077] As described above, the first bioreactor vessel 410 may include multiple ports, such as a first port 412 and a second port 416. The ports 412, 416 may be disposed through the vessel body 504 and may allow communication between the interior compartment 506 and the exterior of the first bioreactor vessel 410 for several processes related to cell culture, cell activation, cell transduction, and / or cell amplification, such as fluid or media input, waste removal, collection, and sampling. Each port 416 may include an opening 526 and a respective fitting or tubing 528 (e.g., a luer fitting, a barb fitting, etc.). In some embodiments, the openings 526 may be configured to allow for direct attachment of tubing, eliminating the need for a fitting (e.g., a counterbore).

[0078] In one embodiment, in addition to the first port 412 and the second port 416, the first bioreactor vessel 410 may further include an air balance port 530 disposed in the top surface 508 of the vessel body 504. The air balance port 530 may be fabricated in a similar manner to the first port 412 and the second port 416, with like reference numbers representing like parts. The air balance port 530 may further provide gas exchange between the internal compartment 506 and the exterior of the first bioreactor vessel 410, which may be used in cell culture for amplification. Additionally, the air balance port 530 may help maintain atmospheric pressure within the internal compartment 506 to provide an environment within the internal compartment for cell culture and / or cell amplification. The air balance port 530 may be disposed through the top surface 508 of the vessel body 504, as in the illustrated embodiment, or at any other location around the vessel body 504. The central location through the top surface 508 of the vessel body 504 may help prevent wetting of the air balance port 530 upon mixing of the cell culture through the tilt of the first bioreactor vessel 410, as described in more detail below.

[0079] Each element of the first bioreactor vessel 410, including the bottom plate 502, vessel body 504, ports 412, 416 and 530, membrane 516, mesh sheet 520, and O-ring 522, may be made from materials that are biocompatible, autoclavable, and gamma and / or ETO sterilization stable. As such, each element, and the first bioreactor vessel 410 as an entire unit, may be used for biological cell activation, transduction, and amplification, and / or other processes in cell manufacturing processes.

[0080] The first bioreactor vessel 410 may enable cell culture and / or cell expansion via perfusion, which may provide nutrients necessary to support cell growth and reduce impurities in the cell culture. Continuous perfusion is the addition of fresh media supply to a growing cell culture while simultaneously removing spent media (e.g., spent media). The first port 412 and the second port 416 may be used for the perfusion process, as described below. The first port 412 may enable communication between the interior compartment 506 and the exterior of the first bioreactor vessel 410, and may be used to add fresh media (such as from a media reservoir of the first fluid assembly 440) to the first bioreactor vessel 410. In some embodiments, the first port 412 may be disposed within and penetrate the vessel body 504 at any location above the surface of the cell culture and media in the first bioreactor vessel 410. In some embodiments, the first port 412 can be disposed to contact or penetrate a surface of the cell culture and medium within the first bioreactor vessel 410.

[0081] The second port 416 may be disposed at any location that is fully or partially submerged below the surface of the cell culture and medium in the first bioreactor vessel 410. For example, the second port 416 may be a mostly lateral port disposed through one of the sides 510 of the vessel body 504. In some embodiments, the second port 416 may 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 may reach the bottom of the internal compartment 506. The second port 416 may be a dual-function port. As such, the second port may be used to withdraw perfusion medium from the internal compartment 506 of the first bioreactor vessel 410 to facilitate perfusion of the cell culture. Additionally, the second port 416 may also be used to remove cells from the cell culture. As noted above, in some embodiments, the second port 416 may not reach the bottom of the internal compartment 506 of the first bioreactor vessel 410. For example, the second port 416 may be located about 0.5 cm away from the membrane 516. Thus, in a static flat position, the second port 416 may be used to remove spent cell medium without pulling cells out of the cell culture, as the cells may settle to the membrane 516 (e.g., cell expansion surface) by gravity. Thus, in a static flat position, the second port 416 may facilitate the perfusion process and allow for an increase in cell density of the growing cell culture in the first bioreactor vessel 410. When it is desired to remove cells from the internal compartment 506 to minimize hold-up volume, for example, at the time of harvesting the cell culture, the first bioreactor vessel 410 may be tilted toward the second port 416 in a manner described below, thereby making the cells accessible for cell removal.

[0082] Additionally, in one embodiment, the second port 416 does not include a filter, and therefore the perfusion process may occur without a filter. As such, there may be no physical impediment preventing cells from entering the second port 416 when the second port 416 is used for medium removal. Furthermore, the second port 416 may be angled such that the second port 416 is disposed laterally through the side 22 of the vessel body 504, but may be angled toward the membrane 516 and bottom plate 502. The angled feature of the second port 416 may allow the second port 416 to be positioned relatively lower on the vessel body 504 closer to the membrane surface 36 while minimizing interference with the O-ring 522 and groove 524, helping to maintain a seal of the first bioreactor vessel 410 during use. Further, in some embodiments, the sloped feature of the second port 416 may reduce the flow rate of fluid through the second port 416 as spent media is removed. Additionally, the port diameter may be such that the suction rate through the second port 416 used to draw media from the exterior compartment 506, in combination with the fluid flow rate out of the second port 416, is minimized such that the suction force on an individual cell adjacent to the second port 416 is lower than the force of gravity pulling the cell toward the membrane 516. Thus, as explained above, the second port 416 may be used to facilitate perfusion of the cell culture without removing the perfusion media and substantially removing the cells from the cell culture. As the cell settlement time increases, the cell concentration of the removed media may decrease and fall within an immeasurable range facilitated by the position of the second port 416. Additionally, the position of the interior opening 540 may be altered to alter the recommended cell settlement time. Locations closer to the membrane 516 may be associated with longer settlement times, while locations at or near the top of the medium are associated with shorter settlement times as cells settle and are depleted from the top of the growth medium first.

[0083] Thus, in one embodiment, the second port 416 can be used to remove not only spent medium in the perfusion process, but also cells of the cell culture from the internal compartment 506, for example, during harvesting of the cell culture. To facilitate greater removal of spent perfusion medium and removal of cells, the vessel body 504 can include angled or chevron-shaped sidewalls 532. Thus, the chevron-shaped sidewalls 532 include an apex, or tip point, 534. The apex 534 of the sidewalls 532 can further include the second port 416 therethrough, with the vessel body 504 disposed near the tip point 534 when the vessel body 504 is coupled to the bottom plate 502. The angled sidewalls 532 and tip point 534 can allow for greater discharge of medium and / or cells of the cell culture when the first bioreactor vessel 410 is tilted toward the second port 416, for example, at a 5 degree angle.

[0084] The use of perfusion to grow cells, facilitated by the location of the first port 412 and the second port 416, may allow for a low medium height (e.g., 0.3-2.0 cm) within the internal compartment 506, as described in more detail with reference to FIG. 10. The relatively low medium height within the internal compartment 506 may allow the first bioreactor vessel 410 to be a relatively low profile vessel while allowing for an increase in the highest achievable cell density. Additionally, the use of perfusion with the first bioreactor vessel 410 supports cell growth by providing fresh medium to the cells within the internal compartment 506, but also allows for the removal of impurities in the cell culture, and additional cell washing in a separate device may not be necessary after a particular cell density goal is reached within the first bioreactor vessel 410. For example, through filterless perfusion, the first bioreactor vessel 410 may provide fresh medium at a rate of full volume change per day, reducing impurities in the cell culture (e.g., resulting in approximately 1 log reduction in impurities every 2.3 days). Thus, the structure of the first bioreactor vessel 410 may allow for the use of perfusion for the growth of the cell culture in the first bioreactor vessel 410, and thus allowing the cell culture to be amplified to a high target density with reduced impurity levels. Also, as described below, through filterless perfusion, the first bioreactor vessel 410 may provide fresh medium at a rate of substantially multiple doses per day (e.g., more than two doses per day) for cell seeding, rinsing, cleaning / residue reduction, and / or draining / harvesting after amplification.

[0085] To facilitate the low-profile construction of the first bioreactor vessel 410, a relatively low medium height may 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 previously described, the vessel body 504 may be coupled to the bottom plate 502 to form the internal compartment 506 in which expansion of the cell culture may be accomplished 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 504, and existing or used medium 538 may be removed through a second port 416 disposed through the side 510 of the vessel body 504. The perfusion process may facilitate the relatively low medium height 536 of the medium 538 within the internal compartment 506 of the first bioreactor vessel 410. The relatively low height 536 of the perfusion medium 538 within the internal compartment 506 may enable the first bioreactor vessel 410 to have a low profile structure, thus enabling an overall compact cell manufacturing system to be achieved.

[0086] The height 536 of the perfusion medium 538 in the internal compartment 506 of the first bioreactor vessel 410 may be between 0.3 cm and 2 cm, and the headroom 542, i.e., the height of the gap formed between the medium 538 and the top surface 508 of the vessel body 504 in the internal compartment 506, may be approximately 2 cm. Thus, the height of the headroom 542, i.e., the height of the gap formed between the medium 538 and the top surface 508 of the vessel body 504 in the internal compartment 506, may be approximately 1 cm. 2 Less than 2 mL of medium per cm 2There may be less than 4 mL of total volume per membrane. The relatively low medium height 536 may allow the ratio of medium volume to membrane 516 surface area to be lower than a certain value. As such, the ratio of medium volume to membrane surface area may be below a threshold level or within a desired range that is amenable to the use of perfusion to grow cells of the cell culture. For example, the threshold level may be a ratio between 0.3 and 2.0. The low ratio of medium volume to membrane surface area may allow the first bioreactor vessel 410 to have a low profile or compact structure while still allowing a cell culture with high cell density to be obtained.

[0087] As previously described, the dual-function second port 416 may be disposed through the vessel body 504 such that it is fully or partially submerged below the surface 544 of the medium 538 in the first bioreactor vessel 410. In some embodiments, the second port 416 may be disposed such that it reaches the bottom (e.g., membrane 516) of the internal compartment 506. The positioning of the second port 416 may facilitate removal of medium and impurities from the cell culture in the internal compartment 506 without removing the cells until such removal, e.g., harvesting, is desired. The strainer-less second port 416, in conjunction with the first port 412, may allow for perfusion to be used to supply growth medium 538 to the cells for cell expansion and to remove spent medium 538 and other impurities or by-products. The location of the first port 412 and the dual-function second port 416 about the vessel body 504 facilitates a configuration in which the medium height 536 within the internal compartment 506 is maintained at a relatively low level, thereby allowing the first bioreactor vessel 410 to be a relatively low-profile vessel while still allowing for the production of high density cell cultures.

[0088] With particular reference to FIG. 11 , the bottom plate 502 of the bioreactor vessel 410 includes various features that enable the use of the bioreactor vessel as part of the broader bioprocessing system 10, and in particular as the second module 200 of the bioprocessing system 10. As shown therein, the bottom plate 502 includes a plurality of recesses 550 formed in a bottom surface of the bottom plate 502, the purpose of which will be explained below. In one embodiment, the recesses may be located adjacent corners of the bottom plate 502. The recesses 550 may each be generally cylindrical in shape and terminate in a domed or hemispherical inner surface. As also shown in FIG. 11 , the bottom plate 502 may include a position verification structure 552 configured to interact with a sensor 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 the light beam of the second module 200 when the first bioreactor vessel 410 is properly seated therein.

[0089] The bottom plate 502 also includes a pair of flat engagement surfaces 554 formed on adjacent bottom surfaces that are offset from a 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 so as to be located adjacent opposite ends of the bottom plate 502. The bottom plate 502 may further include at least one aperture or opening 556 to allow sensing of the contents of the first bioreactor vessel 410 by a bioprocessing device that engages and operates the bioreactor vessel.

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

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

[0092] 15, each of the first and second openings 626, 628 has a perimeter shaped and / or sized such that the first and second bioreactor vessels 410, 420 may be positioned over the respective openings 626, 628 and supported by the bottom surface 620 of the tray 610 within the interior compartment 622 while a portion of the bioreactor vessels 610, 620 remains accessible from the bottom of the tray 610 through the respective openings 626, 628. In one embodiment, the perimeter of the openings includes at least one tab or protrusion for supporting the bioreactor vessel over the respective opening. For example, the perimeter of each opening 626, 628 may include a tab 632 protruding inwardly toward the center of the opening 626, 628 for supporting the bioreactor vessel 410, 420 placed thereover. 12 and 15, the tray 610 may also include one or more ridges extending upwardly above the openings 626, 628 for inhibiting lateral movement of the bioreactor vessels when received over the respective openings 626, 628. The ridges thus act as alignment devices to facilitate proper positioning of the bioreactor vessels 410, 420 within the tray 610 and help prevent inadvertent movement of the bioreactor vessels 410, 420 during loading or positioning of the kit 600 within the second module 200, as described below.

[0093] 12 and 13, the tray 610 may include one or more support ribs 636 formed on the bottom surface of the tray 610. The support ribs 636 traverse the width and / or length of the tray 610, providing rigidity and strength to the tray 610 and facilitating movement and manipulation of the kit 600. The ribs 636 may be integrally formed with the tray or may be added as an auxiliary component via attachment means known in the art (see FIG. 13). In one embodiment, the tray 610 includes an opening 638 for receiving an engagement plate, also referred to herein as a tubing module 650, passing therethrough that holds the fluid flow lines in an organized manner and holds them in place for engagement by the pump and pinch valves. In other embodiments, the tubing module 650 may be integrally formed with the rear wall 614 of the tray 610.

[0094] 16 and 17 illustrate the configuration of a tubing module 650 according to one embodiment of the present invention. As shown therein, the tubing module 650 comprises a first tubing holder block 652 configured to receive the first fluid assembly lines 442, the interconnect lines 450, and the waste lines 490 of the fluid flow system 400 and hold the first fluid assembly lines 442, the interconnect lines 450, and the permeate waste lines 490 in position for selective engagement with respective pump heads 454, 456, 492 of a peristaltic pump assembly described below with respect to FIGS. 35 and 36. In one embodiment, the fluid assembly lines 442, the interconnect lines 450, and the waste lines 490 are maintained in a horizontally extending, vertically spaced orientation by the first tubing holder block 652. In particular, as best shown in FIG. 17, a first tubing holder block 652 engages the lines 442, 450, 490, respectively (such as through clips or simple interference between tubes and slots in the tubing holder block 652) at two spaced apart locations 656, 658 that define a gap therebetween. As also shown in FIG. 17, the first tubing holder block 652 includes a clearance opening 660 that is configured to receive a shoe (not shown) of a peristaltic pump assembly. This configuration can cause peristaltic compression of the lines 442, 450, 490 against the shoe by the respective pump heads of the peristaltic pumps to provide a respective driving force for fluid through the lines, as described below.

[0095] 16-18, the tubing module 650 further comprises a second tubing holder block 654 integrally formed with (or otherwise coupled to) the first tubing holder block 652. The second tubing holder block 654 is configured to receive all of the fluid flow lines of the fluid flow system 400 with which the pinch valves are associated. For example, the second tubing holder block 654 is configured to hold tubing tails 464a-f of the first fluid assembly 440, tubing tails 470a-d of the second fluid assembly 444, the first and second bioreactor lines 414 and 418 of the first bioreactor vessel 410, the first and second bioreactor lines 424 and 428 of the second bioreactor vessel 420, the sterile air source line 460, the interconnect line 450, and the filtration line 482 (as well as, in some embodiments, the sampling lines 476a-d). Like the first tubing holder block 652, the second tubing holder block 654 may maintain these tubes in a horizontally extending, vertically spaced orientation. In particular, the second tubing holder block 654 may include a plurality of vertically spaced, horizontally extending slots 666 configured to receive the lines therein. FIGS. 18 and 19 also best illustrate the configuration of the slots 666 that hold all of the flow lines that are acted upon / contacted by the pinch valve. Desirably, the slots 666 follow the contours of the block 654, but in particular traverse the planar backplate to open toward the strainer 484. As shown in FIG. 18, in one embodiment, the second tubing holder block 654 may have one or more narrow tubing slots 682 at the bottom of the second tubing holder block 654 to hold the loop of interconnect line 450 from which the sampling line extends, and a waste line tubing slot 684 to receive the tubing tail 470a that is connected to the waste reservoir 472a.

[0096] 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 lines held by the second tubing holder block 654. In particular, at least one opening 664 is horizontally aligned with each slot 666 and the flow lines held therein. As best shown in FIG. 16, the second tubing holder block 654 includes two clearance openings 668, 670 configured to receive anvils (not shown) of a pinch valve assembly therethrough. This configuration allows tubing tails 464a-f of the first fluid assembly 440, tubing tails 470a-d of the second fluid assembly 444, first bioreactor line 414 and second bioreactor line 418 of the first bioreactor vessel 410, first bioreactor line 424 and second bioreactor line 428 of the second bioreactor vessel 420, sterile air source line 460, interconnect line 450, and filtration line 482 to be selectively compressed against the anvil by respective pistons of actuators of the pinch valve array to selectively prevent or allow fluid flow, as described below. 18 and 19, the apertures 664 may be arranged in first and second rows positioned side-by-side, with the apertures in the first row of apertures vertically offset with respect to the apertures on the second row of apertures such that the apertures in the first row of apertures are not horizontally aligned with the apertures in the second row of apertures. This configuration allows the tubing module 650, tray 610, and kit 600 as a whole to have a low profile.

[0097] In one embodiment, the filter 484 (shown in FIG. 16 as an elongated hollow fiber filter module) may be integrated with the tubing module 650, such as by attaching the filter 484 to the tubing module 650 using a retaining clip 672. 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 input end 674 for receiving an input flow of fluid from the filtration line 482 and a second output end 676 for carrying the retentate, after removal of the permeate / effluent, back to the filtration line 482 and the interconnecting line 450 for circulation 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 the second output end 676 that connects to a waste line 490 for carrying the waste / permeate to the permeate / effluent storage tank 472a. Finally, the tubing module 650 may include a number of features 680 for receiving clips and organizing the 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).

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

[0099] 20-22 are various views of an embodiment of a kit 600 illustrating the first and second bioreactor vessels 410, 420 received in a tray 610 and the fluid lines of the flow architecture 400 received by a tubing module 650. As shown therein, instead of having an opening 630, the kit 600 as shown in FIGS. 20-22 includes a solid bed and provides a sampling space 631 in the tray 610 to receive a container that holds the sampling line (e.g., sampling lines 476a, 476b). The kit 600 provides a modular platform for cell processing that can be easily set up and discarded after use. The tubing tails of the first and second fluid assemblies 440, 444 allow for plug-and-play functionality, which allows for quick and easy connection of various media, reagents, waste, sampling, and collection bags, allowing the various processes used to be performed on a single platform. In one embodiment, connection and disconnection can be accomplished by sterile cutting and welding of the tube segments, such as with a TERUMO device, as described above, or by pinching, welding, and cutting the tail segment as known in the art.

[0100] 23-25, the kit 600 is specifically configured to be received by a bioprocessing apparatus 700 that houses all of the hardware (i.e., controllers, pumps, pinch valve actuators, etc.) necessary to operate the kit 600 as part of a method of bioprocessing. In one embodiment, the bioprocessing apparatus 700 and the kit 600 (containing the flow architecture 400 and the bioreactor vessels 410, 420) together form the second bioprocessing module 200 described above in connection with FIGS. 1 and 2. The bioprocessing apparatus 700 comprises a housing 710 having a number of drawers 712, 714, 716 that are receivable within the housing 710. Although FIG. 23 shows the apparatus 700 containing three drawers, the apparatus may have as little as a single drawer, two drawers, or more than three drawers, with bioprocessing operations being performed simultaneously within each drawer. In particular, in one embodiment, each drawer 712, 714, 716 may be a stand-alone bioprocessing module (i.e., equivalent to the second modules 200a, 200b, and 200c described above with respect to FIG. 2) for performing processes of cell activation, genetic modification, and / or amplification. In this regard, any number of drawers may be added to the 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 each other.

[0101] 23 and 24, each drawer, for example drawer 712, includes a number of sidewalls 718 and a bottom 720 that defines a processing chamber 722 and a normally open top. The drawer 712 is movable between a closed position in which the drawer is fully received within the housing 710, as shown for drawers 714 and 716 in FIG. 23, and an open position in which the drawer 712 extends from the housing 710 to allow access to the processing chamber 722 through the open top, as shown for drawer 712 in FIG. 23 and 24. In one embodiment, one or more of the sidewalls 718 are temperature controlled to control the temperature within the 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), such that the sidewalls 718 and / or the processing chamber 722 may be heated to a desired temperature to maintain the processing chamber 722 at a desired temperature (e.g., 37° C.) as optimized for the process step to be performed by the module 200. In some embodiments, the bottom surface 720 of the housing (above the processing chamber when the drawer is closed) and the underside of the top surface may be temperature controlled in a similar manner (e.g., with an embedded heating element). A hardware compartment 724 of the drawer 712 behind the processing chamber 722 may house all of the hardware components of the device 700, as described in more detail below. In one embodiment, the drawer 712 may further include an auxiliary compartment 730 adjacent the processing chamber 722 for housing reservoirs containing media, reagents, etc., that are connected to the first and second fluid assemblies 440 and 444. In one embodiment, the auxiliary compartment 730 may be refrigerated.

[0102] Each drawer, e.g., drawer 712, may be slidably received on opposing guide rails 726 mounted inside the housing 710. A linear actuator may be operatively connected to the drawer 712 to selectively move the drawer 712 between the open and closed positions. The linear actuator is operable to effect smooth and controlled movement of the drawer 712 between the open and closed positions. 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 when stopping and starting operation) to minimize disturbance to the contents of the bioreactor vessel.

[0103] 25 is a top view of the interior of the drawer showing the process chamber 722, hardware compartment 724, and auxiliary compartment 730 of the drawer 712. As illustrated therein, the hardware compartment 724 is disposed rearward of the process chamber 722 and includes a power supply 732, an operational control board and drive electronics 734 that is integrated with or in other ways in communication with the second module controller 210, a low power solenoid array 736, a pump assembly 738 (including pump heads for pumps 454, 456, 492), and a 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 interfacing with the pump assembly 738 and the solenoid array 736, respectively, as described below. In one embodiment, the pump shoe 742 and the solenoid anvil 744 are secured to the front base plate (front plate) of the process chamber. The hardware compartments (and components described) are all mounted to the rear base plate. Both plates are slidably mounted to rails. Additionally, a drawer engagement actuator 740 is used to couple the two plates and move the two plates and the components loaded thereon to an engaged position (moving the pump roller head into the pump shoe, thereby compressing the pump tubing when inserted therebetween). As further described herein, the pump assembly selectively operates the lines 442, 450, and 490 of the fluid path 400 to provide independent respective peristaltic propulsive forces. 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.

[0104] As also illustrated in FIG. 25, two bedplates, e.g., first and second bedplates 746, 748, are disposed within the processing chamber 722 on the bottom surface 720 and extend upwardly or protrude therefrom. In one embodiment, the processing chamber 722 may accommodate a single bedplate, or three or more bedplates. The bedplates 746, 748 are configured to receive or otherwise engage the first bioreactor vessel 410 and the second bioreactor vessel 420 thereon. As also illustrated in FIG. 25, the drawer 712 also includes a plate 750 configured with a load cell positioned adjacent the bedplates 746, 748 within the processing chamber 722 to sense the weight of a storage layer, e.g., a waste storage tank 472a positioned thereon.

[0105] 26-28 best illustrate the configuration of the bedplates 746, 748, with FIG. 28A showing the hardware components positioned below the bedplates. As used herein, the bedplates 746, 748 and the hardware components (i.e., sensors, motors, actuators, etc., integrated therewith or positioned below as shown in FIG. 28A) may be collectively referred to as a bedplate. The first and second bedplates 746, 748 are substantially identical in configuration and operation, but for simplicity, the following description of the bedplates 746, 748 will only refer to the first bedplate 746. The bedplates 746, 748 have a substantially planar top surface 752 having a shape and surface area generally corresponding to the shape and area of ​​the bottom plate 502 of the first bioreactor vessel 410. For example, the bedplates may be generally rectangular in shape. The bedplates 746, 748 may also include reliefs or clearance areas 758 that generally correspond to the locations of the protrusions or tabs 632 of the tray 610, the purpose of which is explained below. The bedplates 746, 748 are supported by a number of load cells 760 (e.g., four load cells 760 positioned under each corner of the bedplate 746). The load cells 760 are configured to sense the weight of the first bioreactor vessel 410 in bioprocessing for use by the controller 210.

[0106] In one embodiment, the bedplate 746 may include an embedded heating element or be in thermal communication with a heating element such that the processing chamber 722 and / or the contents of the first bioreactor vessel 410 placed thereon may be maintained at a desired temperature. In one embodiment, the heating element may be the same or different from the heating elements that heat the sidewalls 718, top wall, and bottom surface.

[0107] As illustrated, the bedplate 746 includes a number of locating or alignment pins 754 that protrude above the top surface 452 of the bedplate 746. The number of locating pins 754 as well as the location and spacing of the locating pins 754 may correspond to the number, location, and spacing of the recesses 550 in the bottom surface of the bottom plate 502 of the bioreactor vessel 410, 420. As shown below, the locating 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 to ensure proper alignment of the first bioreactor vessel 410 on the first bedplate 746.

[0108] 26-28, the bedplate 746 may further include an integrated sensor 756 for detecting proper alignment (or misalignment) of the first bioreactor vessel 410 on the first bedplate 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 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 seated on the first bedplate 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 tab), and a substantially IR-opaque position verification structure 552 is used, the beam break will interrupt the infrared light beam (i.e., break the beam) when the first bioreactor vessel 410 is fully seated on the bedplate 746. This signals to the controller 210 that the first bioreactor vessel 410 is properly seated. If, after positioning the first bioreactor vessel 410 on the first bedplate 746, the controller does not detect that the infrared light beam of the sensor 756 is broken, this indicates that the first bioreactor vessel 410 is not fully or properly seated on the bedplate 746 and that adjustment is required. Thus, the sensor 756 on the bedplate 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 seated in a horizontal position on the bedplate 746 (as determined by the alignment pins) before bioprocessing begins.

[0109] 26-28A, the bedplate 746 additionally includes an embedded temperature sensor 759 positioned to align with the 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 bedplate 746 may additionally include a resistance temperature detector 760 configured to measure the temperature of the top surface 752, and a carbon dioxide sensor (located below the bedplate) for measuring the carbon dioxide level within the bioreactor vessel.

[0110] 26-28A, each bedplate 746, 748 includes an actuator mechanism 761 (e.g., a motor) including, for example, a pair of opposing cam arms 762. The cam arms 762 are received within slots 764 in the bedplates 746, 748 and are rotatable about cam pins 766 between a clearance position in which the cam arms 762 are positioned below the top surface 752 of the bedplate 746 and an engagement position in which the cam arms 762 extend above the top surface 752 of the bedplate 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 bedplate 746. As described in more detail below, the actuator mechanism is operable to tilt the bioreactor vessel over the bedplate to provide agitation and / or assist in draining the bioreactor vessel.

[0111] 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 on the outside of the housing 710 that allows for user control of the movement of the drawer. The linear actuator 770 controls the movement of the drawer 712 to prevent disturbance of the contents of the bioreactor vessel within the drawer 712. In one embodiment, the linear actuator 768 has a stroke of about 16" and a maximum speed of about 2 inches per second.

[0112] 30, the drawer engagement actuator 740 includes a lead screw 772 and a clevis arm 774 that is attached to a front plate 751 in the drawer 712. The drawer engagement actuator is operatively connected to the pump assembly 738 and the solenoid array 736 and is operable to move the pump assembly 738 and the solenoid array 736 between a first clearance position and an engaged position.

[0113] 31 and 32 provide a relatively clear view of the clearance and engagement positions of the pump assembly 738 and solenoid array 736. As illustrated in FIG. 31, in the clearance position, the pump assembly 738 and solenoid array 736 are spaced apart from the pump shoe 742 and pinch valve anvil 744, respectively. After the lead screw 772 is actuated, the drawer engagement mechanism 740 moves the pump assembly 738 and solenoid array linearly forward to the position shown in FIG. In this position, the pump head of the pump assembly 738 engages with the lines 442, 450, 490 in the first tubing holder block 652 and the solenoid array 736 is positioned sufficiently close to the pinch valve anvil 744 that the pistons / actuators of the solenoid array 736 can pinch / clamp each fluid flow line of the second tubing holder block 654 against the pinch valve anvil 744, thereby preventing flow through that fluid flow line.

[0114] 24, and with further reference to FIGS. 33-39, in operation, the drawer 712 can be controllably moved to an open position by actuating a rocker switch 770 on the exterior of the housing 710. The disposable drop-in kit 600 containing the tubing module 650 (holding all the tubes and tubing tails of the flow architecture 400) and the first and second bioreactor vessels 410, 420 is lowered into place within the processing chamber 722. Once the kit 600 is lowered into the processing chamber 722, the pump shoe 742 is received through the clearance opening 660 of the first tubing holder block 652 such that the pump tubes 442, 450, 490 are positioned between the pump shoe 742 and the pump heads 454, 456, 492 of the peristaltic pump assembly 738. FIG. 35 is a perspective view of the peristaltic pump assembly 738 showing the positioning of the pump heads 454, 456, 492 relative to one another. 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. In operation, when the drawer engagement actuator 740 positions the pump assembly 738 in the engaged position, the pump heads 454, 456, 492 are selectively actuable under the control of the controller 210 to initiate, maintain, or stop fluid flow through the tubes 442, 450, 490.

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

[0116] As shown therein, each solenoid 778 of the solenoid array 736 includes a piston 780 that is linearly extendable through an associated opening (of openings 664) in the backplate 662 of the second tubing holder block 654 to pinch an associated 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, i.e., 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 flow architecture 400 are provided by the respective solenoids 778 (i.e., the pistons of the solenoids) of the solenoid array 736 that act / act against the respective anvils 744 while the fluid paths / lines are therebetween. In particular, in operation, when the drawer engagement actuator 740 positions the solenoid array 736 in the engaged position, each solenoid 778 is selectively operable under the control of the controller 210 to clamp an associated fluid flow line against the anvil 744 to prevent fluid flow therethrough. The present invention contemplates that each fluid line is positioned between a planar anvil face and a planar solenoid actuator head. Alternatively, the solenoid actuator head may comprise a shaped head, such as two tapered surfaces meeting at an elongated edge resembling a Phillips head screwdriver, optimized to apply a desired pinching force to a resiliently flexible fluid line. Still alternatively, the anvil face may comprise an elongated protrusion or projection extending towards each fluid line such that the planar solenoid head presses the fluid line against the laterally extending protrusion to close the line to fluid flow therethrough.

[0117] 33, 34, and 40, when the kit 600 is lowered into the processing chamber of the drawer, the first bioreactor vessel 410 and the second bioreactor vessel 420 are supported over the openings 626, 628 by the periphery of the openings, and in particular by the tabs / projections 632. As the kit is lowered further, the bedplates 746, 748 penetrate the openings 626, 628 and receive or otherwise engage the bioreactor vessels 410, 420. The shape of the openings 626, 628 and top surfaces 752 of the bedplates 746, 748 (e.g., the raised areas 758 of the bedplates 746, 748 corresponding to the tabs / projections 632 of the tray 610) allows the tray 610 to continue to move downward after the bioreactor vessels 410, 420 are received by the bedplates 746, 748 such that the bottom surface and tabs / projections 632 of the tray 610 are seated lower than the top surface 752 of the bedplates 746, 748 and the bioreactor vessels 410, 420 can be supported by the bedplates 746, 748 in spaced apart relationship with the bottom surface 620 of the tray 610. This ensures that the tray 610 does not interfere with the horizontal seating of the bioreactor vessels 410, 420 on the bedplates 746, 748.

[0118] When the bedplates 746, 748 pass through the openings 726, 728 in the tray 610, the alignment pins 754 on the bedplates 746, 748 are received in corresponding recesses 550 in the bottom plate 502 of the bioreactor vessels 410, 420, ensuring that the bioreactor vessels 410, 420 are properly aligned with the bedplates 410, 420. When properly seated on the bedplates 746, 748, the beam break 552 breaks the light beam of the sensor 756 in the bedplate, indicating to the controller that the bioreactor vessels 410, 420 are properly positioned. Because the bedplates 746, 748 and the alignment pins are at 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 level. In this properly seated position, the sensors 759 on the bedplates 746, 748 are aligned with the openings 556 in the bottom plate 502, thereby enabling them to sense process parameters within the interior compartments of the bioreactor vessels 410, 420, respectively. Additionally, in the fully seated position, the cam arms 762 of the bedplates 746, 748 are aligned with the flat engagement surfaces 554 on the bottom plates 502 of the bioreactor vessels 410, 420, respectively.

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

[0120] 40, in one embodiment, the side walls 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 for heating 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 be utilized without departing from broader aspects of the invention. The drawer 712 may further include one or more brush seals 796 to help minimize heat loss from the processing chamber 722, and an insulating 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).

[0121] 34, when the kit 600 is received in the processing chamber 722, a load cell 750 in the bottom of the processing chamber 722 adjacent the second bed plate 748 penetrates an opening 730 in the tray 610 so that the waste bag 472a can be connected to the tubing tail 470a and positioned on the load cell 750. As shown therein, when the kit 600 is received in 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 of their storage layers. In one embodiment, the sampling lines 476a-d similarly penetrate into the auxiliary compartment 730.

[0122] 41-44, the operation of the cam arms 762 of the bedplates 746, 748 is illustrated. As shown therein, the cam arms 762 are movable between a retracted position in which they are positioned below the top surfaces of the bedplates 746, 748, and an engaged position in which they are rotated about the cam pins 766 and extend above the bedplates 746, 748 to engage the flat engagement surfaces 554 of the bioreactor vessels 410, 420 and lift and pull the bioreactor vessels 410, 420 off the bedplates 746, 748. The cam arms 762 are retracted below the top surfaces of the bedplates 746, 748 in a default state such that the bioreactor vessels 410, 420 are supported on the horizontal bedplates 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 seated on the horizontal bed plates 746, 748 and do not require constant adjustment using the cam arm 762 to maintain a horizontal position. This is in contrast to some systems that may require constant adjustment of the bioreactor using a servo motor to maintain a horizontal position. In fact, with the configuration of the cam arm 762 of the present invention, the actuator need only be energized when tilting the bioreactor vessel for stirring / mixing, as described below, thereby minimizing the amount of heat added to the process chamber 722.

[0123] 41-43, the cam arms 762 may be sequentially operable to agitate the contents of the bioreactor vessel 410, 420. For example, when it is desired to agitate the contents of the bioreactor vessel 410, one of the cam arms is actuated to lift one end of the bioreactor vessel 410 away from the bed plate 746 (and out of engagement with the locating pins 754 on the bed plate 746) while the opposing end remains seated on the bed plate and the locating pins 754 on the non-raised end remain received within corresponding recesses 550 in the bottom plate 502. The raised cam arm is then rotated back to a clearance position below the bed plate and the opposing cam arm is rotated to an engaged position to lift the opposing end of the bioreactor vessel away from the bed plate and the locating pins.

[0124] In one embodiment, the cam actuation system may be designed to allow the cam arm 762 to return to the home position without touching the bioreactor vessel, thereby preventing disturbance to the culture and allowing the cam arm 762 to return to the home position (or be tested) at any point during a long cell processing period. Thus, while the present invention contemplates that other rocking or agitation means may be provided for the bioreactor vessel, by having two cam arms 762 on opposing sides of the bed plate, the overall height of the mixing mechanism can be minimized. For example, a ±5 degree movement can be achieved with a central actuator (located in the center of the bed plate), but approximately the same movement of the vessel can be achieved with 0-5 degree movement of the vessel driven by cam arms on either side of the vessel, which effectively provides a ±5 degree movement of the vessel at half the height. Additionally, the movement of the cam arm 762 (e.g., the speed of cam arm rotation and the timing between opposing cam arms) can be adjusted to maximize wave formation within the vessel, maximize the amplitude of the waves, and therefore maximize the time to achieve (ideally) uniformity and homogeneity of the vessel contents. The timing can also be adjusted based on the volume within a vessel having a given geometry to maximize mixing efficiency.

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

[0126] Figure 43 is a schematic diagram showing the position of fluid 800 within the bioreactor vessel during this mixing process. As shown in Figure 42, in one embodiment, a homing sensor 802 integrated into the bedplate 746 can be utilized by the controller to determine when the cam arm 762 has returned to a clearance position below the top surface of the bedplate 746. This is useful in coordinating the movement of the cam arm 762 to provide a desired mixing frequency within the bioreactor vessel. In one embodiment, the cam arm 762 is configured to provide a maximum 5 degree tilt angle with respect to the bedplate 746.

[0127] 44, the interface between the locating pin 754 of the bed plate of the bioreactor vessel 410 and the recess 550 in the bottom plate 502 during mixing / agitation is illustrated. In one embodiment, the recess 550 has a domed or hemispherical inner surface and a diameter d1 that is greater than the diameter d2 of the locating pin 754. As illustrated in FIG. 44, this configuration provides clearance between the locating pin 754 and the recess 550, allowing the bioreactor vessel 410 to tilt when the locating pin 554 is received within the recess 550.

[0128] In one embodiment, each drawer of the bioprocessing device 700, for example drawer 712, desirably includes a hinged flip-down front panel 810 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 exchange. In connection with the above, in one embodiment, the auxiliary compartment 730 may be configured with a number of telescoping sliding rails 812 that provide mounting means 815 from which various reservoirs / media bags may be suspended. 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 the media / fluid bag needs replacing, the rail 812 is simply extended out and the bag unclipped. A new bag is connected to its respective tail and can then be hung from the rail and slid back into the auxiliary compartment 730 without having to open the drawer 712 or pause processing. In one embodiment, the rail 812 can be mounted on a laterally extending cross rod 814. The rail 812 can thus be laterally slidable on the rod 814 and extendable from and retractable into the auxiliary compartment. Additionally, when the drawer is open (FIG. 46), the rail 812 can rotate about the rear cross rod to clear the compartment 730 and allow the user to thread the tubing tail towards the front of the compartment 730, providing three degrees of freedom.

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

[0130] 52, in one embodiment, the bioprocessing device 700 may further include a low-profile waste tray 816 received within the housing 710 beneath each drawer, e.g., drawer 712. The waste tray 816 is independently mounted on the drawer such that it is movable between a closed position and an open position. Desirably, in the closed position, the tray 816 extends flush with the front of the drawer, and in the open position, the tray 816 exposes its own chamber 819 for operator access. The chamber 819 facilitates the storage of large waste bags connected to the fluid flow paths of the overlying tray 600 and allows access thereto without opening the drawer 712. Additionally, in the closed position, the waste tray 816 has a size and shape operable to position the chamber 819 in alignment beneath the drawer and to contain any leakage from the processing chamber 722 or auxiliary compartment 730.

[0131] In one embodiment, each drawer may include a camera positioned above the processing chamber (e.g., above each bioreactor vessel 410, 420) to allow visual monitoring of the interior of the drawer 712 without opening the drawer 712. In one embodiment, the camera (or additional cameras) may be integrated with the bedplate assembly or integrated on a sidewall looking sideways into the bioreactor vessel.

[0132] Thus, the second module 200 of the present invention allows for the automation of cell processing to a degree not previously seen in the art. In particular, the fluid flow architecture 400, pump assembly 738, and pinch valve array 736 can automate fluid operations (e.g., fluid addition, transfer, drain, rinse, 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 allows for hollow fiber filler concentration and washing, strainer-less perfusion, and line priming. The drawer engagement actuator 740 is also used for automatic engagement and disengagement of the drop-in kit 600, further minimizing human touch points. In fact, human touch points may only be required for source / media bag addition and removal, sampling, and data entry (e.g., sample volume, cell density, etc.).

[0133] With reference to Figures 53-77, an automated general protocol for a workflow of immobilized Ab coating, soluble Ab addition, gamma retroviral vector with amplification in the same vessel using the second module 200 and its fluid flow architecture 400 is illustrated. This general protocol results in activation (illustrated in Figures 53-59), pre-transduction preparation and transduction (illustrated in Figures 60-71), amplification (Figures 72-76), and for some embodiments, harvesting of a population of cells in an automated, functionally closed manner (Figure 77). In describing the operation of the pinch valve, hereinafter, the valve is in its closed state / position when it is not used for a particular operation. Thus, after the valve is opened to allow a particular operation, once that operation is completed, the valve is closed before proceeding to the next operation / step.

[0134] 53, in a first step, valves 432 and 468f are opened and the first fluid assembly line pump 454 is actuated to pump antibody (Ab) coating solution from a reservoir 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 period of time and then drained through the interconnecting lines to the waste reservoir 472a of the first fluid assembly 440 by opening valves 434, 474a and running the circulation line pump 456. As described herein, draining of the bioreactor vessel 410 can be facilitated by tilting the bioreactor vessel 410 using the cam arm 762.

[0135] After draining the antibody coating solution, valves 432 and 468e are opened and pump 454 is activated, pumping the rinse buffer from reservoir 466e, which is connected to first fluid assembly 440, through the first bioreactor line and into the first bioreactor vessel 410. The rinsing buffer is then drained through interconnect line 450 to waste reservoir 472a by activating circulation line pump 456 and opening valve 474a. In one embodiment, this rinse and drain procedure may be repeated multiple times to thoroughly rinse the first bioreactor vessel 410.

[0136] 55, after rinsing the first bioreactor vessel 410 with 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 activating pump 454. The cells are pumped through the first bioreactor line 414 of the first bioreactor vessel 410 and enter the bioreactor vessel 410 through the first port 412. As shown in FIG. 56, valves 432 and 468a are opened and pump 454 is activated to pump a second antibody (Ab) solution from a reservoir 466a connected to the first fluidic assembly 440 through the first port 412 into the first bioreactor vessel 410.

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

[0138] After rinsing the second antibody solution reservoir 466a, the inoculum / seed cell bag 466d may also be optionally rinsed. In particular, as shown in FIG. 58, valves 474b, 452, and 468d are opened and rinsing medium from the rinsing medium reservoir / bag 472b of the second fluid assembly 444 is pumped to the inoculum / seed cell bag 466d using pump 454 to rinse the bag. After rinsing, valve 432 is opened and rinsing medium is pumped from bag 466d to the first bioreactor vessel 410 using pump 454. Pumping the rinsing medium to the first bioreactor vessel 410 after rinsing the inoculum / seed cell bag 466d reduces the cell density in the first bioreactor vessel 410. At this time, samples may be taken to measure one or more parameters of the solution in the bioreactor vessel prior to activation (e.g., to verify that a desired cell density is present prior to activation). In particular, as shown in Figure 58, valves 434, 452, and 432 are opened and pump 456 is actuated to pump the contents of first bioreactor vessel 410 along the first circulation loop of the first bioreactor vessel (i.e., from second port 416, through interconnect line 450, through first bioreactor line 414 and first port 412 of first bioreactor vessel 410, and back to first bioreactor vessel 410). To take a sample, first sample container 280a (e.g., dip tube, syringe, etc.) is connected to first sample tubing tail 476a and valve 478a is opened to divert a portion of the flow through interconnect line 450 and into first sample container 280a for analysis.

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

[0140] 60, after activation, to prepare for transduction, valves 438 and 474b are opened and pump 456 is operated to pump the RetroNectin solution from reservoir 472b into the second bioreactor vessel 420 through the second port 426 of 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 period of time. As further shown in FIG. 60, after incubation, all of the RetroNectin solution is then drained from the second bioreactor vessel 420 to the waste reservoir 472a by opening valves 438 and 474a and operating the circulation line pump 456. Note that during these RetroNectin coating, incubation, and discharge steps (involving second bioreactor vessel 420), the activated cell population remains within first bioreactor vessel 410. Note that RetroNectin or other reagents to increase the efficiency of genetic modification need not be utilized in all processes.

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

[0142] Referring now to FIG. 62, after a determined period of time, all buffer in the second bioreactor vessel 420 is drained into the waste reservoir 472a of the second fluid assembly 444 by opening valves 438 and 474a and activating the interconnecting line pump 456.

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

[0144] 64, depending on the concentration obtained from the sample, concentration may be performed by circulating the contents of the first bioreactor vessel 410 through the filter 484. As explained above, this is done by opening valves 434, 486, 488, and 432 and activating pump 456, which circulates the solution in the first bioreactor vessel 410 out of the second port 416, passing the solution through the second bioreactor line 418, through the interconnect line 450, through the filtration line 482 and the filter 484, through the first bioreactor line 414 of the first bioreactor vessel 410, and back through the first port 412 to the first bioreactor vessel 410. Once the fluid has passed through the filter 484, effluent is removed and a permeate pump 492 pumps such effluent through the effluent line 490 to the effluent reservoir 472a of the second fluid assembly 444. In one embodiment, this procedure is repeated until the volume within the first bioreactor vessel 410 is concentrated to a predetermined volume.

[0145] 65, after enrichment, the enriched cell population in the activation vessel (i.e., the first vessel 410 containing the enriched cell population) is washed to a constant volume through perfusion. In particular, as shown therein, medium from the medium bag 466b of the first fluid assembly 440 is pumped through the first port 412 through the interconnecting line 450 into the first bioreactor vessel 410 at the same time that medium is pumped out of the first bioreactor vessel 410 through the second port 416 such that a constant volume is maintained in the first bioreactor vessel 410. As medium is added and removed from the vessel 410, effluent may be filtered by the filter 484 and directed to a waste reservoir 472a.

[0146] A post-wash sample may be taken from the cells in the first bioreactor vessel 410 in a manner similar to that already described for the pre-concentration sample. In particular, as shown in FIG. 66, valves 434, 486, 488, and 432 are opened and pump 456 is actuated to circulate fluid in the first bioreactor vessel 410 from the second port 434, through the interconnect 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 third sample vessel 280c (e.g., a dip tube, syringe, etc.) is connected to the third sample tubing tail 476c and valve 478c is opened to divert a portion of the flow through the interconnect line 450 and into the third sample vessel 280c for analysis.

[0147] As shown in FIG. 67, a bag containing thawed viral vector is connected to the first fluid assembly 440 through tubing tail 464c or the like. Valves 468c and 436 are then opened and pump 454 is actuated to transfer the viral vector coating solution from bag 466c through first port 422 to second bioreactor vessel 420. Incubation is then performed for a predetermined period of time for viral coating of second bioreactor vessel 420. After incubation, the viral vector coating solution is drained from second bioreactor vessel 420 to waste reservoir 472a by opening valves 438 and 474a and actuating circulation line pump 456. In an embodiment, viral and non-viral vectors may be utilized as agents for transduction / genetic modification.

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

[0149] Then, as illustrated in Figure 69, media from media bag 466b is added to the second bioreactor vessel 420 by opening valves 468b and 436 and activating pump 454 to increase the total amount of solution in the second bioreactor vessel 420 to a predetermined volume. Then, referring to Figure 70, a pre-transduction sample may be taken by opening valves 438, 452, and 436, activating circulation line pump 456, and pumping the solution in the second bioreactor vessel 420 around the circulation loop of the second bioreactor vessel (i.e., from the second port 426, through the interconnect line 450, through the first bioreactor line 414 and first port 422 of the second bioreactor vessel 420, and back to the second bioreactor vessel 420). To collect a sample, a fourth sample container 280d (e.g., a dip tube, syringe, etc.) is connected to the fourth sample tubing tail 476d and valve 478d is opened, diverting a portion of the flow through the interconnection line 450 to the fourth sample container 280d for analysis.

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

[0151] 72, after transduction, media is added to the second bioreactor vessel 420 to achieve a predetermined amplification volume in 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 first port 422 of the second bioreactor vessel into the second bioreactor vessel 420 until the predetermined amplification volume is reached.

[0152] 73, a pre-amplification sample may then be taken by opening valves 438, 452, and 436, activating circulation line pump 456, and pumping the solution in second bioreactor vessel 420 along the circulation loop of second bioreactor vessel 420 as shown above (i.e., from second port 426, through interconnect line 450, through first bioreactor line 414 and first port 422 of second bioreactor vessel 420, and back to second bioreactor vessel 420). To take the sample, a fifth sample container 280e (e.g., a dip tube, syringe, etc.) is connected to fifth sample tubing tail 476e, and valve 478e is opened to divert a portion of the flow through interconnect line 450 and into fifth sample container 280e for analysis.

[0153] If analysis of the taken fifth sample indicates that all parameters are within the predetermined ranges required for successful amplification of the population of cells, the population of cells in the second bioreactor vessel 420 is incubated for a predetermined period of time, for example, four hours, to allow the cells to settle.

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

[0155] Sampling may then be performed as necessary / desired to monitor the amplification process and / or determine when a desired cell density has been reached. As explained above, samples may be taken by opening valves 438, 452, and 436, activating circulation line pump 456, and pumping the solution in second bioreactor vessel 420 along the circulation loop of second bioreactor vessel 420 as shown above (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 back to second bioreactor vessel 420). To collect a sample, a separate sample container 280x (e.g., dip tube, syringe, etc.) is connected to the sample tubing tail of the sample assembly 448, and a valve in the tubing tail is opened to divert a portion of the flow through the interconnect line 450 to the sample container 280x for analysis, as shown in Fig. 75. After each sampling operation, an incubation without perfusion is performed for a predetermined period of time, e.g., 4 hours, to allow the cells to settle before resuming perfusion.

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

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

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

[0159] Thus, the second module 200 of the bioprocessing system 10, as well as its flow architecture 400 and bioreactor vessels 410, 420, provide a flexible platform on which a variety of bioprocessing operations may be performed in a substantially automated and functionally closed manner. In particular, although Figs. 53-77 illustrate exemplary general protocols that may be performed using the bioprocessing system 10 of the present invention (particularly using its second module 200), the system is not limited thereto in this respect. Indeed, a variety of automated protocols may be enabled by the system of the present invention, including numerous customer-specific protocols.

[0160] 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 fluid handling and fluid containment systems, all maintained in cell culture friendly environmental conditions (i.e., in a temperature and gas controlled environment) to enable cell activation, transduction, and amplification. As explained 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 in a single system. It also provides the user with the flexibility to combine all steps in a single bioreactor vessel (e.g., the first bioreactor vessel 410) or use both bioreactor vessels 410, 420 for end-to-end activation and washing. In one embodiment, a single amplification bioreactor vessel (e.g., the bioreactor vessel 420) can reliably generate billions of T cells for a single dose. Either single or multiple doses can be produced in situ with high recovery and high viability. In addition, the system is designed to give the end user the flexibility to implement different protocols for the manufacturing of genetically modified immune cells.

[0161] Some of the commercial advantages offered by the bioprocessing system of the present invention include simplified workflow, reduced labor intensity, reduced strain on cleanroom infrastructure, fewer failure nodes, reduced costs, and robust and scalable manufacturing technology for product commercialization with the ability to scale up operations.

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

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

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

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

[0166] 81 illustrates an exemplary embodiment of a method 480 utilizing the second module 200 in a perfusion process. The method 480 includes activating a first pump 454 to pump fresh medium into a bioreactor vessel 410 housing a population of genetically modified cells at 482, activating a second pump 456 to pump spent medium from the bioreactor vessel 410 to a waste bag 472a at 484, obtaining mass data related to a mass of the bioreactor vessel (e.g., the bioreactor vessel 410) using a load cell associated with the bed plate at 486, determining whether the mass of the bioreactor vessel 410 has changed or has remained substantially constant at 488, and, if the mass of the bioreactor vessel has changed, adjusting operating parameters of at least one of the first pump and the second pump to maintain a substantially constant mass of the bioreactor vessel 410 at 490. For example, if it is determined that the mass of the bioreactor vessel 410 has decreased, this indicates that spent medium is being removed from the bioreactor vessel at a rate that is faster than the rate of fresh medium being added to the bioreactor vessel. Thus, the flow rate of the first pump may be increased and / or the flow rate of the second pump may be decreased accordingly to maintain a substantially constant mass (and volume) within the bioreactor vessel 410. Further mass data may then be acquired and further adjustments to the pump operation may be made, if necessary, to maintain a substantially constant mass / volume within the bioreactor vessel 410. If the mass is determined to be substantially constant after operation of the first and second pumps has continued for a period of time, the pump may be maintained at its current operating set point (e.g., flow rate), as shown at 492.

[0167] In another embodiment, the bioprocessing system uses the flow architecture 400 to allow for round-robin perfusion of various bioreactor vessels in the system. For example, the circulation pump 456 and pump 545 along the first fluid assembly line 442 are used to perfuse cells in the first bioreactor vessel 410 in conjunction with appropriate pinch valve states, as described above. The perfusion of cells in the first bioreactor vessel 410 may then be stopped or paused, and the circulation pump 456 and pump 454 and appropriate pinch valves may be activated to perfuse cells in the second bioreactor vessel 420. In this regard, the perfusion of the various bioreactors may be performed sequentially (i.e., perfusion of the first bioreactor vessel 410 for a period of time, then perfusion of the second bioreactor vessel 420 for a period of time, repeatedly alternating). This allows for the perfusion of any number of bioreactor vessels in the system without the use of more pumps, media bags, or waste bags.

[0168] For round-robin perfusion, the pumps can run continuously, intermittently together (duty cycle), or sequentially (source, then effluent, repeat), thereby keeping the volumes / masses in the various bioreactor vessels at approximately the same levels. Round-robin perfusion (a set of pumps running intermittently together and waiting at regular intervals) also allows for perfusion of multiple vessels using the same two pumps, as shown. Furthermore, round-robin perfusion allows for low effective exchange rates (such as about 1 volume / day) even if the pumps do not have a large low dynamic range. Furthermore, round-robin perfusion also allows for each vessel to be perfused with a different medium as controlled by the valves in the first fluidic assembly 440.

[0169] Additionally, in one embodiment, fast perfusion may be used for retentate removal (e.g., post-activation Ab removal and / or post-transduction retentate removal). In a fast perfusion process, the perfusion process described above may be run much faster than the typical 1-5 volumes / day, for example, between about 8-20 volumes / day, or greater than about 20 volumes / day achieving a 1 log reduction in only a few minutes to hours. In one embodiment, the perfusion rate is balanced against cell loss. In some embodiments, fast perfusion may allow for the elimination of hollow filter 484 and still meet the biological requirement of quickly removing retentate after some steps.

[0170] As further described above, the system of the present invention uses a pump 454 on the first fluid assembly line 442 to facilitate rinsing of the bag / reservoir connected to the first fluid assembly 440 using a rinse buffer or fluid from another bag / reservoir connected to the second fluid assembly 444. In addition, the fluid lines of the flow architecture / system 400 can be cleared with sterile air from a sterile air source 458 to prevent cells from staying in the lines and dying, or media or reagents from staying in the lines and deteriorating or becoming useless. The sterile air source 458 can also be used to purge reagents from the lines to ensure that more reagents than intended are not pumped into the bioreactor vessels 410, 420. The sterile air source 458 can also be used to clear the lines leading up to the connected bags (of the first or second fluid assemblies 440, 444) and to clear sterile tubing welds to limit residue. Instead of, or in addition to, clearing the lines using a sterile air source 458, the lines can be cleared using air drawn from one of the bioreactor vessels, as long as the port through which the air is drawn is not submerged and the bioreactor vessel has an air balance port 530.

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

[0172] As also described above, the system of the present invention allows the population of cells to be harvested after the target cell density is achieved. In one embodiment, harvesting the amplified population of transduced cells may include using pump 456 on interconnect line 450 to move the cells to one of the bags connected to second fluidic assembly 444, or circulating the cells with interconnect pump 456 to move the cells to a bag connected to first fluidic assembly 440. This process can be used for final harvest or large sample volumes, or can be used to fully automate the sampling process (i.e., by connecting a syringe or bag to first fluidic assembly 440, circulating the contents of the bioreactor vessel, and drawing a portion of the desired sample volume from the circulating contents with fluidic assembly pump 454 and moving it towards the syringe / bag). In such a case, circulation pump 456 and valves can then be used to clear the fluid / cell circulation line. Additionally, pump 454 on the first fluid assembly line 442 can be used to continue to push all of the aliquoted sample volume into the sample container by using air in the line to complete sample transfer to the container with no appreciable amounts of cells remaining in the line.

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

[0174] In one such embodiment, the T cell activators may be micron-sized Dynabeads and a lentiviral vector is used for transduction. Notably, as disclosed herein, the micron-sized Dynabeads serve the dual purpose of isolating and activating the T cells. In one embodiment, activation (and isolation) of the 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 with a virus for genetic modification, such as in the manner described above in connection with Figures 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 that retains the cells and the micron-sized Dynabeads in the bioreactor vessel 410. This allows for cell amplification in the same bioreactor vessel 410 used for activation and transduction. The filterless perfusion method additionally allows for culture washing to be performed without the need to first immobilize the activation beads that need to be retained with the cells during amplification. In particular, the micron-sized Dynabeads are not fluidized at low perfusion rates and are retained in the vessel as the virus is washed out, whereas the nanometer-sized virus particles and residual macromolecules are fluidized and washed out during slow perfusion.

[0175] In one embodiment, after amplification, the cells may be harvested in the manner described above in connection with FIG. 77. After harvesting, a magnetic bead removal process may be utilized to remove the Dynabeads from the collected cells. In another embodiment, the steps of harvesting the amplified population of cells and removing the beads from the cells are performed simultaneously using perfusion, whereby media is introduced into the bioreactor vessel through a feed port and the cell media containing the amplified population of cells is removed from the bioreactor vessel through an outlet port in the bioreactor vessel. In particular, when final bead removal of the culture is required, filterless perfusion may be used to perform bead removal of micron-sized beads by utilizing the difference in weight of 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 previously described). After mixing / agitation, the heavy Dynabeads sink and settle onto the silicone membrane 516 within 10-15 minutes. In contrast, cells require more than 4 hours to settle onto the membrane 516. After a 10-15 minute holding period after mixing / agitation, the cell suspension can be slowly withdrawn using perfusion without disturbing the settled Dynabeads. An inlet medium line can be used to maintain medium height within the bioreactor vessel. Thus, the invention described herein simplifies current Dynabead protocols by eliminating the need for several mid-process cell transfers and careful washing and bead removal steps, minimizing costs and potential risks. By performing bead removal of the culture at the same time as 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 non-perfusion 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 2Thus, all four unit processes of activation, transduction, washing, and amplification using Dynabeads can be carried out in the same bioreactor vessel in a fully automated and functionally closed manner. The bioprocessing system of the present invention thus simplifies current protocols by eliminating the need for mid-process cell transfer and careful washing steps, minimizing costs and potential risks resulting from multiple human touch points.

[0177] In one embodiment, the two bioreactor vessels 410, 420 of the system can run either with the same starting culture or two simultaneous split cultures, for example, 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 before infusion 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 are described above, the workflows described herein are not intended to be comprehensive, as other CAR-T workflows can also be used by 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, are described in connection with the production of CAR-T cells, the system of the present invention is also compatible with the production of other immune cells, such as TCR-T cells and NK cells. Furthermore, although embodiments of the present invention disclose the use of two bioreactor vessels 410, 420 in a two-step sequential process in which the output of the first bioreactor vessel 410 is added to the second bioreactor vessel 420 for additional processing steps (e.g., activation in the first bioreactor vessel and transduction and amplification in the second bioreactor vessel), in some embodiments, the two bioreactor vessels can be used for the same workflow as duplicates. Exemplary reasons for using a second bioreactor vessel sequentially include residual chemical modifications (e.g., coatings or immobilized reagents) that cannot be washed out of the first bioreactor that are detrimental in later steps or in case of overexposure of cells in previous steps, or the need to pre-coat the bioreactor surface prior to addition of cells (e.g., RetroNectin coating).

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

[0180] Further examples of potential multiple bioreactor vessel workflows enabled by the system of the present invention include (1) soluble activator activation, viral transduction, filterless perfusion, and amplification in a first bioreactor vessel 410, and soluble activator activation, lentiviral transduction, filterless perfusion, and amplification in a second bioreactor vessel 420, using the same cell type or split culture in both bioreactor vessels; (2) Dynabead-based amplification in the first bioreactor vessel 410, and (3) soluble activator activation, lentiviral transduction, filterless perfusion, and amplification in a second bioreactor vessel 420, using the same cell type or split culture in both bioreactor vessels; (2) Dynabead-based activation, viral transduction, filterless perfusion, and amplification in a first bioreactor vessel 410, and Dynabead-based activation, lentiviral transduction, filterless perfusion, and amplification in a second bioreactor vessel 420, using the same cell type or split culture in the two bioreactor vessels; (3) TransAct bead-based activation, viral transduction, filterless perfusion, and amplification in a first bioreactor vessel 410, and TransAct-based activation, lentiviral transduction, filterless perfusion, and amplification in a second bioreactor vessel 420. (4) soluble activator activation in the first bioreactor vessel 410 and RetroNectin coating, transduction, and amplification in the second bioreactor vessel 420; (5) immobilized activator activation in the first bioreactor vessel 410 and RetroNectin coating, transduction, and amplification in the second bioreactor vessel 420; (6) D in the first bioreactor vessel 410. (7) Dynabead activation and lentiviral transduction in the first bioreactor vessel 410 and amplification in the second bioreactor vessel 420; (8) TransAct activation in the first bioreactor vessel 410 and RetroNectin coating, transduction and amplification in the second bioreactor vessel 420; (9) soluble activator activation in the first bioreactor vessel 410;and amplification of in situ electroporated or other non-virally modified cells in the second bioreactor vessel 420; (10) TransAct activation in the first bioreactor vessel 410 and amplification of in situ electroporated or other non-virally modified cells in the second bioreactor vessel 420; (11) Dynabead activation in the first bioreactor vessel 410 and amplification of in situ electroporated or other non-virally modified cells in the second bioreactor vessel 420; (12) expansion of allogeneic NK cells in the first bioreactor vessel 410 and expansion of allogeneic NK cells in the second bioreactor vessel 420 (small molecule based expansion, no genetic modification); (13) expansion of allogeneic NK cells in the first bioreactor vessel 410 and expansion of allogeneic NK cells in the second bioreactor vessel 420 (feeder cell based expansion, no genetic modification); and (14) soluble activator activation, viral transduction, filterless perfusion and expansion of allogeneic CAR-NK or CAR-NK 92 cells in the first bioreactor vessel 410 and / or the first and second bioreactor vessels 410, 420 (no RetroNectin coating, polybrene is used to assist transduction).

[0181] While the embodiments described above illustrate process monitoring sensors integrated with the bioreactor vessel and / or bed plate (e.g., on the membrane, integrated in the membrane, on the vessel sidewall, etc.), in other embodiments, it is contemplated that additional sensors may be added to the fluidic architecture 400, for example, along the fluid flow lines themselves. These sensors may be disposable compatible sensors for monitoring parameters such as pH, dissolved oxygen, density / turbidity (optical sensors), conductivity, and viability in the circulating fluid. By arranging sensors in the circulation loop (e.g., the circulation loop of the first bioreactor vessel and / or the circulation loop of the second bioreactor vessel), the construction of the vessel may be simplified. Additionally, in some embodiments, sensors along the circulation loop may provide a more accurate representation of the vessel contents as circulated (rather than measuring when the cells are stationary in the vessel). Furthermore, if desired, flow sensors (e.g., ultrasonic-based) may be added to the flow loop to measure pump performance and used with algorithms to correct pump parameters.

[0182] As shown above, the first and third modules 100, 300 may take any form of any system or device known in the art capable of cell enrichment and isolation, as well as harvesting and / or compounding. FIG. 78 illustrates one possible configuration of a device / apparatus 900 that may be used as the first module 100 in a bioprocessing system 10 for cell enrichment and isolation using various magnetic isolation bead types (including, for example, Miltenyi beads, Dynabeads, and StemCell EasySep beads). As shown therein, the apparatus 900 comprises a base 910 housing a centrifuge processing chamber 912, a high dynamic range peristaltic pump assembly 914, a small inner diameter pump tubing 916 received by the peristaltic pump assembly, a stopcock manifold 918, an optical sensor 920, and a heating and cooling mixing chamber 922. As shown below, the stopcock manifold 918 provides a simple and reliable means of joining multiple fluid or gas lines together using, for example, Luer fittings. In one embodiment, the pump 914 is rated to output 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 may include a generally T-shaped hanger assembly 924 extending from the base 910 and including a number of hooks 926 for hanging a number of processing and / or source containers or bags. In one embodiment, there may be six hooks. Each hook includes an integrated weight sensor for detecting the weight of each container / bag. In one embodiment, the bags may include a sample 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] The device 900 is configured to be used with or include a magnetic cell isolation holder 960, as presented herein. The magnetic cell isolation holder 960 may be removably coupled to a magnetic field generator 962 (e.g., magnetic field plates 964, 966). The magnetic cell isolation holder 960 houses a magnetic retention element or material 968, such as a separation column, matrix, or tube. In one embodiment, the magnetic cell isolation holder 960 may be fabricated as disclosed in U.S. Patent Application No. 15 / 829,615, filed December 1, 2017, which is incorporated herein by reference in its entirety. The device 900 may be under the control of a controller (e.g., controller 110), and operates according to instructions executed by a processor and stored in memory. Such instructions may include magnetic field parameters. In one embodiment, the device 900 may further include a syringe 952, which may be utilized for bead addition, as described below.

[0185] 79, a general protocol 1000 of the device 700 is shown. As illustrated therein, in a first step 1010, enrichment is performed by reducing platelets and plasma in the sample. In embodiments where Dynabeads are utilized as the magnetic isolation beads, a washing step 1012 may then be performed to remove any residual material in the Dynabead suspension. After enrichment, the cells are then transferred to a process bag 932 in step 1014. In some embodiments, a portion of the enriched cells may be stored in a first storage bag 938 in step 1016 before being transferred to the process bag 932. In step 1018, magnetic isolation beads are injected into the process bag in step 1020, such as by using a syringe 952. In one embodiment, the magnetic isolation beads are Miltenyi beads or StemCell EasySep beads. If Dynabeads are utilized, the washed Dynabeads from step 1012 are resuspended in the process bag 932. In one embodiment, instead of utilizing a syringe, the magnetically isolated beads may be contained in a bag or container that is connected to the system, and the beads may be drawn into the system by pump 914.

[0186] The beads and cells in the process bag 932 are then incubated for a period of time in step 1020. In embodiments where the magnetic isolation beads are Miltenyi nano-sized beads, a sedimentation wash is performed in step 1022 to remove excess nano-sized beads, and a portion of the incubated bead-bound cells are stored in a second storage bag 940 in step 1024. After incubation, the bead-bound cells are isolated in step 1026 using a magnet, e.g., the magnetic field plates 964, 966 of the 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 captured in a capture bag 950 in step 1030. In embodiments where StemCell EasySep beads are utilized, an additional step 1032 of releasing the cells from the beads and removing the beads, and an optional step 1034 of washing / concentrating the captured cells are performed.

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

[0188] In some embodiments, such as those in which Dynabeads are utilized as magnetic isolation beads, as disclosed above, a wash step (step 1012) is performed to remove any residuals in the bead suspension buffer. The wash step is captured by injecting beads using a syringe 952 while circulating in the process loop 1110 (e.g., from the process bag 932, through the peristaltic pump tubing 914, through the manifold 918, and back to the process bag 932), clearing the process loop 1110, and then flushing the process bag 932 to the isolation waste bag 942 while the magnetic field generator 962 is "ON". In embodiments in which washing is not desired, the process bag 932 is flushed to the isolation waste bag 942 to ensure that the process bag 932 is clean. As used herein, in the case of permanent magnets, ON means that the magnetic retention element or material 968 (e.g., separation column, matrix, or tube) is in the proper position within the magnetic field. OFF means that the tubing section is removed from the magnetic field.

[0189] The concentrated cells in the processing chamber 912 are then transferred to the process bag 932 (step 1014) and isolation buffer from the isolation buffer bag 934 is drawn into the processing chamber 912 to rinse the chamber 912 and remove any residual cells. After rinsing, the fluid is dispensed into the process bag 932. This rinsing process can be repeated as necessary. After all the cells have been transferred to the process bag 932, the chamber 912 is cleaned by drawing buffer from the isolation buffer bag 934 into the chamber 912 and dispensing the fluid into the source bag 930. This cleaning process can be repeated as necessary.

[0190] The contents of the process bag 932 may then be mixed by circulating the contents along the process loop 1110 before clearing the process loop 1110 by returning the entire contents back to the process bag 932. As indicated above, in one embodiment, a portion of the concentrated cells may 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). The process line 1112 and the first storage bag line 1114 are then cleared.

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

[0192] After adding the magnetic isolation beads, as described above, the cells may 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 a second storage bag 940, which is agitated (such as by using the heating and cooling mixing chamber 922). The contents of the second storage bag 940 are then transferred back to the process bag 932. Buffer from the isolation buffer bag 934 is then drawn into the processing chamber 912, and the contents of the chamber are dispensed into the second storage bag 940, which is then transferred to the process bag 932, rinsing the second storage bag 940.

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

[0194] As described above, after incubation, an optional step of washing out excess beads (e.g., nano-sized beads) may be performed (step 1022). Washing out excess nano-sized beads includes starting a 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 excess beads. In one embodiment, the chamber 912 may then be filled with buffer from the isolation buffer bag 934, start rotating the chamber 912, and then expel the supernatant into the waste bag 742. These steps may be repeated as necessary. In one embodiment, the cells in the chamber are discharged into the process bag 932, buffer from the isolation buffer bag 934 is drawn into the chamber 932, and the chamber is then discharged into the process bag 932. This process can also be repeated as necessary. Mixing of the process loop and clearing of the process loop are then performed.

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

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

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

[0198] In another embodiment, rinsing via resuspension can be performed by pumping buffer from the isolation buffer bag 934 to the process bag 932 with the magnetic field generator 962 in the "OFF" state, circulating it in the process loop 1110, clearing the process loop, and flowing from the process bag 932 to the waste bag 942 with the magnetic field generator 962 in the "ON" state.

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

[0200] The isolated bead-bound cells are then collected (step 1028) after rinsing to isolate the remaining bead-bound cells. 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 to the collection bag 950 with the magnetic field generator 962 "OFF". In another method, the buffer from the isolation buffer bag 934 is pumped to the process bag 932, which is then pumped to the collection bag 950 with the magnetic field generator 962 "OFF". 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 to the process bag 932 (if a post-isolation wash is not required). Alternatively, the buffer from the isolation buffer bag 934 is pumped through the column 966 to the process bag 932 (if a post-isolation wash is desired). In either process, the contents of the process bag 932 are then circulated in the process loop 1110, which is cleared by returning to the process bag 932, and the contents of the process bag 932 are pumped to the collection bag 950 to collect the bead-bound cells.

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

[0202] In relation to the above, in one embodiment, washing / concentration (step 1034) may 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 buffer from the process bag 932 into the processing chamber 912. A washing cycle may then be performed by filling the processing chamber 912 with buffer from the isolation buffer bag 934, spinning the chamber 912, dispensing the supernatant into the waste bag 942, and repeating the spin and dispense steps as necessary. Finally, transferring the cells to the collection bag after washing / concentration may be accomplished by transferring media from the media bag 946 into the collection bag 950, pumping the contents of the collection bag into the processing chamber 912, dispensing the contents of the processing chamber 912 into the collection bag 950, and then manually clearing the line between the processing chamber 912 and the collection bag 950.

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

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

[0205] In one embodiment, the syringe 952 allows for small volumes (such as bead suspension aliquots) to be added to the circulation-based flow loop 1110. Additionally, fluid from the flow loop 1110 can be drawn into the syringe 952, thereby further clearing the syringe 952 of any residue.

[0206] In one embodiment, one of the sensors 920 may be configured to measure the flow of the fluid. For example, one of the sensors 920 may be an air bubble detector or an optical detector that can be used as a secondary confirmation measurement to ensure accurate flow control in addition to the load cell integrated with the hook 926. This can be used in practice 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 close to empty within some expected tolerance of the load cell variation, and then the air bubble detector 920 identifies the liquid / air interface that follows to stop the flow. Thus, the sensor 920 can be used by the controller to prevent drawing air into the loop, which can dislodge cells or create slugs that expose cells to a dry environment, or inadvertently drawing material into the waste bag in situations where the pump does not stop after the process bag is fully drained. Thus, in one embodiment, the air bubble detector 920 may be used in combination with the load cell integrated with the hook to improve volume control accuracy, thereby reducing cell loss and / or preventing air from entering the column tubing and column.

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

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

[0209] In some embodiments, the system 900 can be used for elution of isolated / captured bead-cell complexes. In particular, it is contemplated that the air-liquid interface can be used to help remove the complexes from the tube sidewalls 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, a packed bed of beads / bead-bound cells can be difficult to remove with flow control alone without significantly increasing shear rates (potentially having adverse effects on cell viability). Thus, in conjunction with flow rate, it is possible to remove the bead-cell complexes without removal from the magnet.

[0210] In relation to the above, system 900 supports the concept of eluting the positively selected bead-cell complex directly into the selected medium (based on downstream steps). This eliminates the buffer exchange / wash step. In one embodiment, it is also envisioned to elute directly into the medium and viral vector to start the incubation. This concept may also allow the viral vector to be added to the final bag. In one embodiment, instead of eluting the bead-bound cells with a buffer, the medium may be used as the elution fluid. Similarly, a release buffer may be used to elute the StemCell beads for subsequent release of the cells from the beads. By exchanging the buffer in parts of system 900 with medium, dilution may be minimized.

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

[0212] It will be understood that the systems of the present invention may include the necessary electronics, software, memory, storage, databases, firmware, logic / state machines, microprocessors, communication links, displays or other visual or audio user interfaces, printing devices, and any other input / output interfaces to perform the functions described herein and / or achieve the results described herein. For example, the system may include at least one processor and a system memory / data storage structure, which may include random access memory (RAM) and read-only memory (ROM). The at least one processor of the system may include one or more conventional microprocessors and one or more auxiliary coprocessors, such as a mathematical coprocessor or the like. The data storage structure described herein may include an appropriate combination of magnetic, optical, and / or semiconductor memory, and may include, for example, RAM, ROM, flash drives, optical disks such as compact disks, and / or hard disks or drives.

[0213] In addition, software applications that adapt a controller, e.g., controller 110, 210, and / or 310, to execute the methods disclosed herein may be loaded from a computer-readable medium into the main memory of at least one processor. As used herein, the term "computer-readable medium" refers to any medium that provides or participates in providing instructions to at least one processor of a system (or any other processor of a device described herein) for execution. Such media may take many forms, including, but not limited to, non-volatile media and volatile media. Non-volatile media include, for example, optical, magnetic, or magneto-optical disks, such as memory. Volatile media include dynamic random access memory (DRAM), which typically constitutes the main memory. Common forms of computer readable media include, for example, a floppy disk, a flexible disk, a hard disk, a magnetic tape, any other magnetic medium, a CD-ROM, a DVD, any other optical medium, a RAM, a PROM, EPROM or EEPROM (Electronically Erasable Programmable Read Only Memory), a FLASH-EEPROM, any other memory chip or cartridge, or any other medium from which a computer can read.

[0214] In embodiments, execution of a sequence of instructions in a software application causes at least one processor to perform the methods / processes described herein, although hard-wired circuitry may be used in place of or in combination with software instructions to implement the methods / processes of the present invention. Thus, implementation of the present invention is not limited to any particular combination of hardware and / or software. Furthermore, it is contemplated that all methods, protocols, and workflows described herein may be performed via software, which may be a single or multiple applications, programs, and the like.

[0215] It is further contemplated that the software may be configured to execute the method, protocol, and / or workflow in a fully autonomous mode, a semi-autonomous mode, or in a gated manner. In a fully autonomous mode, the software comprises instructions configured to adapt the controller of the system to execute substantially the entire operation, method, protocol, or workflow from start to finish automatically after being initiated by a user or operator (i.e., without operator intervention and without the need for human touch points). In a semi-autonomous mode of operation, the software comprises instructions configured to adapt the controller of the system to execute substantially the entire operation, method, protocol, or workflow from start to finish after being initiated by a user or operator, except that the software may provide instructions to the controller prompting the user or operator to perform some specific actions required to execute the operation method, protocol, or workflow, such as pausing operation of the bioprocessing system or components thereof, connecting or disconnecting collection, waste, media, cell, or other bags or reservoirs to take samples, etc. In a gated mode of operation, the software comprises instructions configured to adapt the controller of the system to generate a series of prompts instructing a user or operator to execute a given operating method, protocol, or workflow, such as connecting or disconnecting collection, waste, media, cell, or other bags or reservoirs to take samples, etc., and to perform some specific action required to autonomously control the system operation during each separate operator intervention. In a gated mode of operation, the bioprocessing system relies quite heavily on the operator, such that the controller only executes pre-programmed bioprocessing steps after being initiated by the operator.

[0216] As used herein, elements or steps recited in the singular and preceded by the article "a" or "an" should be understood as not excluding a plurality of said elements or steps, unless the exclusion is expressly stated. Furthermore, references to "one embodiment" of the invention are not intended to be interpreted as excluding the existence of additional embodiments that also incorporate the recited features. Furthermore, unless expressly stated, embodiments "including," "comprising," or "having" an element or elements having a particular characteristic may include additional such elements that do not have that characteristic.

[0217] This specification uses examples to disclose some embodiments of the invention, including the best mode, and also to enable those skilled in the art to practice the embodiments of the invention, including making and using any device or system, and performing any methods incorporated therein. The patentable scope of the invention is defined by the claims, and may include other examples that occur to those skilled 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 claim language or if they include equivalent structural elements that differ insignificantly from the claim language. [Explanation of symbols]

[0218] 10 Bioprocessing Systems 100 1st module 110 First Controller 200 Second Module 200a, 200b, 200c Second Module 210 Second Controller 280a, 280d Sample collection device 300 3rd Module 310 Third Controller 400 Fluid Flow Architecture 400 Bioprocessing Subsystem 400 Bioprocessing System 410 first bioreactor vessel 412 first port 414 First Bioreactor Line 416 Secondary Port 418 Second Bioreactor Line 420 Second bioreactor vessel 422 first port 424 First Bioreactor Line 426 Secondary Port 428 Second Bioreactor Line 430 Bioreactor Array 432 First bioreactor line valve 436 First bioreactor line valve 438 Second Bioreactor Line Valve 440 First Fluid Assembly 442 First Fluid Assembly Line 444 Second Fluid Assembly 446 Second Fluid Assembly Line 448 Sampling Assembly 450 Interconnecting Lines 452 Interconnecting Line Valve 454 First pump or interconnecting line pump 456 Second Pump or Circulation Line Pump 458 Sterile Air Source 460 Sterile Air Source Line 462 Valve 464a~f Tubing tail 466a-f First storage tank 466b Culture medium bag 468a~f Tubing tail valve 470a~d Tubing tail 472a~d First storage tank 472a Waste liquid storage tank, waste liquid bag 474a~d Tubing tail valve 476a~476d Sampling lines 478a~d Sample line valve 480 method 482 Filtration Line 484 Filter 486 Upstream Filtration Line Valve 488 Downstream Filtration Line Valve 490 Waste liquid line 492 Osmotic Pump 502 Bottom plate 504 Container body 506 Internal Compartment 508 Top surface 510 Side 510 Grid 512 holes 514 Crossbar 516 Membrane 518 Top surface 520 Mesh sheet 522 O-ring 524 Groove 526 Circumferential surface 526 Opening 528 Fittings or Tubing 530 Air Balance Port 532 Side wall 534 Vertex, tip point 536 Height 538 Cell Culture Media 542 Headroom 544 Surface 550 Depression 552 Position Verification Structure 554 Flat engagement surface 556 Aperture or opening 600 Kits 610 Tray 612 Front wall 614 Back wall 616, 618 Side 622 Internal Compartment 620 Bottom 624 Circumferential flange 626, 628 First and second openings 631 Sampling Space 632 Claw 636 Support Rib 638 Opening 650 Tubing Module 652 1st Tubing Holder Block 654 2nd Tubing Holder Block 656, 658 arrangement 660 Clearance Opening 662 Flat Back Plate 664 Aperture 666 Vertically spaced, horizontally extending slots 668, 670 Clearance opening 672 Retaining Clip 674 First input terminal 676 Second Birth Point 680 Features 682 Narrow Tubing Slot 684 Waste Line Tubing Slot 700 Bioprocessing Equipment 710 Housing 712, 714, 716 Drawers 718 Side wall 720 Bottom 722 Processing Chamber 724 Hardware Compartment 730 Auxiliary Compartment 732 Power supply 734 Motion control board and driving electronic circuit 736 Low Power Solenoid Array 738 Pump Assembly 740 Drawer Engagement Actuator 742 Pump shoe 744 Pinch Valve Anvil 746 First Bedplate 748 Second Bedplate 750 Plate 752 Top surface 754 Locating or Aligning Pin 756 Integrated Sensor 759 Embedded Temperature Sensor 760 Load Cell 760 Resistance Temperature Detector 761 Actuator Mechanism 762 Cam Arm 764 Slots 766 Campin 768 Linear Actuator 770 Rocker Switch 770 Linear Actuator 772 Lead 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 Insulation Layer 800 fluid 810 Flip-down front panel 812 Expandable sliding rail 814 Laterally extending cross rod 815 Attachment means 816 Low Profile Waste Tray 819 My chamber 820 Platform 822 Guide Truck 900 devices / equipment 910 base 912 Centrifugal Processing Chamber 914 High Dynamic Range Peristaltic Pump Assembly 916 Pump tube with suitable inner diameter 918 stop cock manifold 920 Optical Sensor 922 Heating and cooling mixing chamber 924 Generally T-shaped hanger assembly 926 Hook 930 Sample source bag 932 Process Bag 934 Isolation Buffer Bag 936 Cleaning Bag 938 First Storage Bag 940 Secondary Storage Bag 942 Post-isolation waste bag 944 Washing waste 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 Retaining Elements or Materials 1000 General Protocol 1110 Process Loop 1112 Process Line 1114 No. 1 Storage Bag Line 1116 Second Storage Line 1118 Top Port

Claims

1. a single first module configured to enrich and isolate a population of cells; a plurality of second modules configured to activate, genetically modify, and expand the population of cells, respectively; a single third module configured to harvest the amplified population of cells; and Equipped with 1. A bioprocessing system, wherein each of the first module, the second module, and the third module is a functionally closed system, Each second module is configured to support the activation, genetic modification, and amplification of a different population of cells in parallel with each other; the first module is configured to enrich and isolate each of the distinct populations of cells prior to transfer to one of the plurality of second modules for activation, genetic modification, and amplification; The bioprocessing system is configured to perform enrichment and isolation of one of the distinct cell populations in the first module while simultaneously activating, genetically modifying, and amplifying another of the distinct cell populations in the second module.

2. the second module comprises a bioreactor vessel configured to effect the genetic modification and amplification of the population of cells without removing the population of cells from the bioreactor vessel; or 2. The bioprocessing system of claim 1, wherein said second module comprises a bioreactor vessel configured to effect said activation, genetic modification and amplification of said population of cells without removing said population of cells from the bioreactor vessel.

3. 10. The bioprocessing system of claim 1, wherein said second module comprises a first bioreactor vessel and a second bioreactor vessel in fluid interconnection with said first bioreactor vessel.

4. the first bioreactor vessel is configured for activation and genetic modification of the population of cells; 4. The bioprocessing system of claim 3, wherein said second bioreactor vessel is configured to perform amplification of said population of cells.

5. 5. The bioprocessing system of claim 4, wherein the bioprocessing system is configured to autonomously activate and genetically modify the cells in the first bioreactor vessel, transfer the cells from the first bioreactor vessel to the second bioreactor vessel, and amplify the cells in the second bioreactor vessel.

6. the first bioreactor vessel is configured to perform activation of the population of cells; 4. The bioprocessing system of claim 3, wherein said second bioreactor vessel is configured to perform genetic modification and amplification of said population of cells.

7. 5. The bioprocessing system of claim 4, wherein the bioreactor vessel is configured for perfusion without a filter.

8. the population of cells is a first population of cells; the bioprocessing system is configured to support the activation, genetic modification, and amplification of the first population of cells simultaneously with the enrichment and isolation of the second population of cells in the first module; 10. The bioprocessing system of claim 1, wherein said first population of cells is different from said second population of cells.

9. 1. A bioprocessing method for cell therapy comprising: enriching and isolating each of the distinct populations of cells in a single first module; activating, genetically modifying, and amplifying each of the distinct populations of cells in parallel with one another in a plurality of second modules; harvesting the amplified population of cells in a single third module; Including, 1. A bioprocessing method, wherein each of the first module, the second module, and the third module is a functionally closed system, comprising: A bioprocessing method comprising concentrating and isolating one of the distinct populations of cells in the first module simultaneously with activating, genetically modifying and amplifying another of the distinct populations of cells in the second module.

10. 10. The bioprocessing method of claim 9, wherein the steps of activating, genetically modifying, and amplifying each of the distinct populations of cells in parallel with one another in the second module comprises activating and genetically modifying the population of cells in a first bioreactor vessel of the second module, transferring the population of cells to a second bioreactor vessel of the second module, and amplifying the population of cells in the second bioreactor vessel.

11. 10. The bioprocessing method of claim 9, wherein the steps of activating, genetically modifying, and amplifying each of the distinct populations of cells in parallel with one another in the second module include activating the population of cells in a first bioreactor vessel of the second module, transferring the population of cells to a second bioreactor vessel of the second module, and genetically modifying and amplifying the population of cells in the second bioreactor vessel.

12. 10. The bioprocessing method of claim 9, wherein the step of activating, genetically modifying, and amplifying each of the distinct populations of cells in parallel with one another in the second module comprises activating, genetically modifying, and amplifying the populations of cells in a first bioreactor vessel and a second bioreactor vessel of the second module.

13. the population of cells is a first population of cells; The bioprocessing method includes the steps of: transferring the first population of cells from the first module to the second module after enrichment and isolation of the first population of cells in the first module for activation, genetic modification, and amplification of the first population of cells in the second module; introducing a second population of cells into the first module simultaneously with the activation, genetic modification, and amplification of the first population of cells in the second module for enrichment and isolation of the second population of cells in the first module; 10. The bioprocessing method of claim 9, wherein the first population of cells is distinct from the second population of cells.

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