JP7350741B2 - Methods for cell enrichment and isolation - Google Patents

Description

Embodiments of the present invention generally relate to bioprocessing systems and methods, and more particularly to bioprocessing systems and methods for the production of cellular immunotherapeutics.

Various drug therapies involve extraction, culture, and expansion of cells for use in downstream therapeutic 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 recombinant receptors that combine 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 take T cells from humans, genetically modify them, and put them back into patients to attack cancer cells. CAR-T cells can be derived either from the patient's own blood (autologous) or from another healthy donor (allogeneic).

The first step in producing CAR-T cells involves removing blood from the patient's body and separating white blood cells using apheresis therapy, such as leukapheresis therapy. After a sufficient amount of leukocytes are 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.

Following isolation of targeted T cells, the cells are activated in a specific environment where they can actively proliferate. For example, cells can be activated using magnetic beads coated with anti-CD3/anti-CD28 monoclonal antibodies or cell-based artificial antigen-presenting cells (aAPCs), which can be removed from the culture medium using magnetic separation. . T cells are then transduced with the CAR gene by either integrating gammaretroviral (RV) or lentiviral (LV) vectors. Viral vectors use the viral machinery to attach to patient cells and, after entering the cells, 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's cells, ensuring that CAR expression is maintained as the cells divide and grow into large numbers in the bioreactor. The CAR is then transcribed and translated by patient cells, and the CAR is expressed on the cell surface.

After T cells are activated and transduced with the CAR-encoded viral vector, the cells are expanded to large numbers in a bioreactor to achieve the desired cell density. After amplification, the cells are harvested, washed, concentrated, formulated, and injected into the patient.

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 susceptible to contamination. The risk of Although recent efforts to automate manufacturing processes 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.

U.S. Patent Application No. 15/893,336 U.S. Patent Application No. 15/829,615

In light of the above, there is a need for bioprocessing systems for cellular immunotherapy that reduce contamination risks by increasing automation and reducing human handling. In addition, there is a need for bioprocessing systems for cell therapy manufacturing that balance the flexibility of development with the need for consistency in high-volume production, as well as meet the demands of different customers who want to run different processes. .

A number of embodiments commensurate in scope with the originally claimed subject matter are summarized below. These embodiments are not intended to limit the scope of the claimed subject matter; rather, these embodiments are intended only to present an overview of possible embodiments. Indeed, this disclosure may encompass various forms that may be similar or different from the embodiments described below.

In one embodiment, the bioprocessing system includes a first module configured to enrich and isolate a population of cells and a first module configured to activate, genetically transduce, and amplify the population of cells. and a third module configured to harvest the expanded population of cells.

In another embodiment, a bioprocessing system includes a first module configured to concentrate and isolate cells and a plurality of second modules, each second module configured to activate cells. a plurality of second modules configured to transduce, genetically transduce, and amplify the cells; and a third module configured to harvest the cells after amplification. Each second module is configured to support activation, genetic transduction, and amplification of different populations of cells in parallel to each other.

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

In another embodiment, an apparatus for bioprocessing includes a housing and a drawer receivable within the housing. The drawer includes a plurality of sidewalls and a bottom that define a processing chamber and a generally open top. The drawer is movable between a closed position in which the drawer is received within the housing and an open position in which the drawer extends from the housing and allows access to the processing chamber through an open top. The apparatus also includes at least one bed plate positioned within the processing chamber and configured to receive a bioreactor vessel.

In another embodiment, a method of bioprocessing includes sliding a drawer having a plurality of sidewalls, a bottom, and a generally open top from a closed position within a housing to an open position to extend the drawer from the housing. passing the bioreactor vessel through the generally open top and positioning the bioreactor vessel onto a stationary bed plate positioned within the drawer; and sliding the drawer. and controlling the drawer engagement actuator to engage the plurality of fluid flow paths with the at least one pump and the plurality of pinch valve linear actuators.

In another embodiment, a system for bioprocessing includes a housing, a first drawer receivable within the housing, the plurality of sidewalls and a bottom defining a first processing chamber; a first drawer positioned within the processing chamber of the first drawer to receive or otherwise engage a first bioreactor vessel thereon; and a second drawer receivable within the housing in stacked relationship with the first drawer, the plurality of drawers defining a second processing chamber. a second drawer having a sidewall and a bottom and a generally open top; a second drawer positioned within the processing chamber of the second drawer to receive a second bioreactor vessel thereon; at least one second bed plate configured to engage in some manner. The first drawer and the second drawer each have a closed position in which the first drawer and/or the second drawer are received within the housing and a closed position in which the first drawer and/or the second drawer extend from the housing. and an open position allowing access to the processing chamber through the respective open tops.

In yet another embodiment, an apparatus for bioprocessing includes a housing and a drawer receivable within the housing, the drawer having a plurality of sidewalls and a bottom surface defining a processing chamber and a generally open the drawer is movable between a closed position in which the drawer is received within the housing and an open position in which the drawer extends from the housing to provide access to the processing chamber through the open top. , a drawer, at least one bed plate positioned within the processing chamber adjacent the bottom, and a kit receivable within the processing chamber. The kit includes a bioreactor vessel having a plurality of sidewalls and a bottom surface defining an internal compartment, a generally open top, and an opening formed in the bottom surface of the kit and having a periphery. a bioreactor vessel positioned over at least one opening of the bioreactor vessel 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 bed plate passes through an opening in the bottom of the tray and supports the bioreactor vessel over the bottom of the kit.

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

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

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

In yet another embodiment, a method of bioprocessing includes placing a bioreactor container within a disposable tray, the disposable tray having a plurality of sidewalls and a bottom surface defining an internal compartment and a generally open top surface. a step having an opening formed in the bottom surface and a plurality of tabs or protrusions extending from the bottom surface into the opening; and placing the tray within a processing chamber having a bedplate such that the bedplate is received through an opening in the tray and supports the bioreactor vessel. .

In yet another embodiment, a tubing module for a bioprocessing system is 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. a first tubing holder block that receives a plurality of pinch valve tubes and holds 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; and a second tubing holder block configured to. The first tubing holder block and the second tubing holder block are interconnected.

In yet another embodiment, a system for bioprocessing has a plurality of sidewalls and a bottom surface defining an interior compartment and a generally open top, wherein the tray receives or supports a bioreactor vessel. a pump assembly positioned adjacent to the rear wall of the tray; a pump assembly positioned adjacent to the rear wall of the tray; and a tubing module positioned at the rear of the tray. The tubing module includes a first tubing holder block configured to receive at least one pump tube and hold the at least one pump tube in place for selective engagement with the pump assembly, and a plurality of pinch pins. a second tubing holder block configured to receive the 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; Equipped with.

In yet another embodiment, the bioreactor vessel includes a bottom plate and 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 having a corresponding alignment pin on the bedplate for aligning the bioreactor vessel on the bedplate. a plurality of recesses configured to receive the recesses.

In yet another embodiment, a method for bioprocessing includes operably connecting a bottom plate to a container body to define an internal compartment therebetween, the bottom plate and the container body comprising: The parts include forming and connecting a bioreactor vessel, aligning recesses in the bottom plate with alignment pins of the bioprocessing system, and mounting the bioreactor vessel on the bed plate of the bioprocessing system. include.

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

In yet another embodiment, the method of bioprocessing includes a first fluid assembly tube connected to a first port of the first bioreactor vessel through a first bioreactor conduit of the first bioreactor vessel. and a second fluid assembly connected to a second port of the first bioreactor vessel through a second bioreactor conduit of the first bioreactor vessel. providing a second fluid assembly having a conduit and an interconnection between a second bioreactor conduit of the first bioreactor vessel and a first bioreactor conduit of the first bioreactor vessel; providing a conduit, the interconnecting conduit providing fluid communication between the first fluid assembly and the second fluid assembly and the second bioreactor conduit and the second bioreactor vessel of the first bioreactor vessel; and providing an interconnecting line that allows fluid communication between one bioreactor vessel and a first bioreactor line.

In yet another embodiment, a bioprocessing method for cell therapy comprises genetically modifying a population of cells in a bioreactor vessel to produce a population of genetically modified cells; amplifying a population of genetically modified cells in a 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.

In yet another embodiment, a bioprocessing method comprises coating a bioreactor vessel with a reagent to increase the efficiency of genetic modification of a population of cells; and amplifying the population of genetically modified cells within the bioreactor vessel without removing the genetically modified cells from the bioreactor vessel.

In yet another embodiment, a bioprocessing method comprises 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 a bioreactor vessel to produce a population of genetically modified cells, washing the genetically modified cells within the bioreactor vessel to remove undesirable substances, and transduction. and amplifying the population of genetically modified cells in a bioreactor vessel to produce an expanded population of cells that have undergone genetic modification. Activation, genetic modification, washing, and amplification are performed within the bioreactor vessel without removing the cells from the bioreactor vessel.

In yet another embodiment, a kit for use in a bioprocessing system is configured to have a process bag, a source bag, a bead addition container, and in fluid communication with the process bag, the source bag, and the bead addition container. A process loop is provided. The process loop additionally includes pump tubing configured to be in fluid communication with the pump.

In yet another embodiment, an apparatus for bioprocessing is 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 kit comprising: further includes a kit comprising pump tubing configured to be in fluid communication with the pump, a magnetic field generator configured to generate a magnetic field, a source bag, a process bag, and beads. a plurality of hooks for suspending additional containers, each hook of the plurality of hooks being operably connected to a load cell, the load cell being configured to sense the weight of a bag connected thereto; A hook, at least one air bubble sensor, and a pump configured to be in fluid communication with the process loop.

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; The method includes isolating a population of bead-bound cells on a separate column and collecting target cells from the population of cells.

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

In another embodiment, the magnetic cell isolation holder comprises a body configured to be removably coupled to a magnetic field generator. The body portion has a first passage configured to be positioned within the magnetic field of the magnetic field generator in a first configuration when the holder is coupled to the magnetic field generator; a second passageway configured to be positioned within the magnetic field in a second configuration when The first passage is subjected to a first magnetic field strength and a first magnetic field gradient in a magnetic field generated under magnetic field parameters in a first configuration, and the second passage is subjected to a first magnetic field gradient in a magnetic field generated under magnetic field parameters in a first configuration. under the influence of a second magnetic field strength and a second magnetic field gradient within the magnetic field. The second magnetic field strength is different from the first magnetic field strength, and the second magnetic field gradient is different from the first magnetic field gradient, or a combination thereof.

In another embodiment, the system includes a first kit having a plurality of first size beads and a first holder having a passageway configured to receive the plurality of first size beads; a second kit having a plurality of second size beads and two holders having passageways configured to receive the plurality of second size beads. The passageway in the first holder is configured such that when the first holder is removably coupled to the magnetic field generator, the first holder is positioned within the magnetic field generated by the magnetic field generator in the first configuration. is positioned within the holder. The passageway in the second holder is arranged such that when the second holder is removably coupled to the magnetic field generator, the second holder receives the magnetic field generated by the magnetic field generator in a second configuration that is different from the first configuration. the second holder such that the second holder is positioned within the second holder; The passages of the first holder are subjected to a first magnetic field strength and a first magnetic field gradient in a magnetic field in a first configuration, and the passages of the second holder are subjected to a second magnetic field in a magnetic field in a second configuration. influenced by the intensity and the second magnetic field gradient. The second magnetic field strength is different from the first magnetic field strength, and the second magnetic field gradient is different from the first magnetic field gradient, or a combination thereof.

In another embodiment, a method for isolating target cells includes positioning a first holder having a passage within a receiving region of a frame coupled to a magnetic field generator; generating a first magnetic field within the receiving region by the magnetic field generator when the first holder is coupled to the magnetic field generator to exert a first magnetic field strength, a first magnetic field gradient, or both; Including things. The method also includes positioning a second holder having a passageway within the receiving region and applying a second magnetic field strength, a second magnetic field gradient, or both to the passageway of the second holder. generating a second magnetic field within the receiving region by the magnetic field generator when the second holder is coupled to the magnetic field generator. The first holder passage and the second holder passage are positioned at different positions within the receiving area.

The invention will be better understood on reading the following description of non-limiting embodiments, given below and with reference to the accompanying drawings, in which: FIG.

FIG. 1 is a schematic diagram of a bioprocessing system according to an embodiment of the invention. FIG. 2 is a schematic diagram of a bioprocessing system according to another embodiment of the invention. FIG. 2 is a block diagram illustrating the fluid flow configuration/system of the cell activation, genetic modification, and amplification subsystems of the bioprocessing system of FIG. 1. 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 arrangement/system. 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 arrangement/system. FIG. 4 is a detailed view of a portion of the block diagram of FIG. 3 illustrating the sampling assembly of the fluid flow arrangement/system. FIG. 4 is a detailed view of a portion of the block diagram of FIG. 3 illustrating the filtration channels of the fluid flow arrangement/system. FIG. 1 is a perspective view of a bioreactor vessel according to an embodiment of the invention. FIG. FIG. 9 is an exploded view of the bioreactor vessel of FIG. 8; 9 is an exploded cross-sectional view of the bioreactor vessel of FIG. 8. FIG. FIG. 9 is an exploded bottom perspective view of the bioreactor container of FIG. 8; 2 is a top and front perspective view of the disposable drop-in kit of the bioprocessing system of FIG. 1, according to one embodiment of the invention. FIG. 13 is another top and front perspective view of the disposable drop-in kit of FIG. 12. FIG. 13 is another top and rear perspective view of the disposable drop-in kit of FIG. 12. FIG. 13 is a perspective view of a tray of the disposable drop-in kit of FIG. 12, according to an embodiment of the invention. FIG. 13 is a front perspective view of the tubing module of the disposable drop-in kit of FIG. 12, according to one embodiment of the invention. FIG. 17 is a rear perspective view of the tubing module of FIG. 16. FIG. FIG. 3 is an elevational view of a second tubing holder block of a tubing module, according to one embodiment of the invention. 19 is a cross-sectional view of the second tubing holder block of FIG. 18. FIG. 13 is another front perspective view of the drop-in kit of FIG. 12 showing the flow architecture integrated therein; FIG. 13 is a rear perspective view of the drop-in kit of FIG. 12 showing the flow architecture integrated therein; FIG. 13 is a front elevation view of the drop-in kit of FIG. 12 showing the flow architecture integrated therein; FIG. FIG. 1 is a perspective view of a bioprocessing device according to an embodiment of the invention. 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 invention. FIG. 25 is a top view of the drawer of FIG. 24. FIG. 25 is a front perspective view of the processing chamber of the drawer of FIG. 24. FIG. FIG. 3 is a top view of the processing chamber of the drawer. 24 is a top view of the bed plate of the bioprocessing device of FIG. 23. FIG. 29 is a top view of the hardware components stored under the bed plate of FIG. 28; FIG. 13 is a side elevational view of the bioprocessing device of FIG. 12. FIG. 13 is a perspective view of a drawer engagement actuator of the bioprocessing device of FIG. 12. FIG. FIG. 3 is a top view of a drawer of a bioprocessing device illustrating clearance positions of drawer engagement actuators, pump assemblies, and solenoid arrays. FIG. 2 is a top view of a drawer of a bioprocessing device illustrating the engaged positions of a drawer engagement actuator, pump assembly, and solenoid array. 1 is a perspective view of a bioprocessing device illustrating a drop-in kit in position within a processing chamber of a drawer. FIG. 1 is a top view of a bioprocessing device illustrating a drop-in kit within a processing chamber of a drawer. FIG. FIG. 2 is a perspective view of a peristaltic pump assembly of a bioprocessing device. FIG. 3 is a side elevational view of the peristaltic pump assembly and tubing holder module of the drop-in kit illustrating the relationship between the components. FIG. 2 is a perspective view of a solenoid array and pinch valve anvil forming a pinch valve array of a bioprocessing device. FIG. 3 is another perspective view of the pinch valve array of the bioprocessing device. FIG. 7 is another perspective view of the pinch valve array illustrating the positioning of the tubing holder module of the drop-in kit with respect to the pinch valve array in an engaged position. FIG. 2 is a cross-sectional view of a drawer of a bioprocessing device illustrating the mounting location of a bioreactor vessel on a bed plate. FIG. 2 is a side elevation view of a bioreactor received on a bed plate illustrating the agitation/mixing mode of operation of the bioreactor system. FIG. 2 is a side cross-sectional view of a bioreactor received on a bed plate, illustrating the agitation/mixing mode of operation of the bioreactor system. 1 is a schematic diagram of a bioreactor vessel showing the liquid level within the bioreactor vessel when in the agitation/mixing mode of operation; FIG. FIG. 4 is a cross-sectional detail view of the interface between the locating pin on the bed plate and the receiving recess on the bioreactor vessel during the agitation/mixing mode of operation. FIG. 2 is a perspective view of a bioprocessing device with a flip-down front panel according to an embodiment of the invention showing the processing drawer in an open position. FIG. 46 is another perspective view of the bioprocessing device of FIG. 45 showing the processing drawer in an open position. 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 with access to the auxiliary compartment; 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 with access to the auxiliary compartment; FIG. 46 is a perspective view of the bioprocessing device of FIG. 45 showing the processing drawer in a closed position with access to an auxiliary compartment; FIG. 46 is another perspective view of the bioprocessing device of FIG. 45 showing the processing drawer in a closed position with access to the auxiliary compartment; FIG. FIG. 3 is a perspective view of an auxiliary compartment of a bioprocessing device according to another embodiment of the invention. 1 is a perspective view of a bioprocessing system with a waste tray, according to an embodiment of the invention. FIG. 4 is a schematic diagram of a generic protocol for automation of a bioprocessing system that utilizes the fluid flow architecture of FIG. 3, according to one embodiment of the invention. FIG. 4 is a schematic diagram of a generic protocol for automation of a bioprocessing system that utilizes the fluid flow architecture of FIG. 3, according to one embodiment of the invention. FIG. 4 is a schematic diagram of a generic protocol for automation of a bioprocessing system that utilizes the fluid flow architecture of FIG. 3, according to one embodiment of the invention. FIG. 4 is a schematic diagram of a generic protocol for automation of a bioprocessing system that utilizes the fluid flow architecture of FIG. 3, according to one embodiment of the invention. FIG. 4 is a schematic diagram of a generic protocol for automation of a bioprocessing system that utilizes the fluid flow architecture of FIG. 3, according to one embodiment of the invention. FIG. 4 is a schematic diagram of a generic protocol for automation of a bioprocessing system that utilizes the fluid flow architecture of FIG. 3, according to one embodiment of the invention. FIG. 4 is a schematic diagram of a generic protocol for automation of a bioprocessing system that utilizes the fluid flow architecture of FIG. 3, according to one embodiment of the invention. FIG. 4 is a schematic diagram of a generic protocol for automation of a bioprocessing system that utilizes the fluid flow architecture of FIG. 3, according to one embodiment of the invention. FIG. 4 is a schematic diagram of a generic protocol for automation of a bioprocessing system that utilizes the fluid flow architecture of FIG. 3, according to one embodiment of the invention. FIG. 4 is a schematic diagram of a generic protocol for automation of a bioprocessing system that utilizes the fluid flow architecture of FIG. 3, according to one embodiment of the invention. FIG. 4 is a schematic diagram of a generic protocol for automation of a bioprocessing system that utilizes the fluid flow architecture of FIG. 3, according to one embodiment of the invention. FIG. 4 is a schematic diagram of a generic protocol for automation of a bioprocessing system that utilizes the fluid flow architecture of FIG. 3, according to one embodiment of the invention. FIG. 4 is a schematic diagram of a generic protocol for automation of a bioprocessing system that utilizes the fluid flow architecture of FIG. 3, according to one embodiment of the invention. FIG. 4 is a schematic diagram of a generic protocol for automation of a bioprocessing system that utilizes the fluid flow architecture of FIG. 3, according to one embodiment of the invention. FIG. 4 is a schematic diagram of a generic protocol for automation of a bioprocessing system that utilizes the fluid flow architecture of FIG. 3, according to one embodiment of the invention. FIG. 4 is a schematic diagram of a generic protocol for automation of a bioprocessing system that utilizes the fluid flow architecture of FIG. 3, according to one embodiment of the invention. FIG. 4 is a schematic diagram of a generic protocol for automation of a bioprocessing system that utilizes the fluid flow architecture of FIG. 3, according to one embodiment of the invention. FIG. 4 is a schematic diagram of a generic protocol for automation of a bioprocessing system that utilizes the fluid flow architecture of FIG. 3, according to one embodiment of the invention. FIG. 4 is a schematic diagram of a generic protocol for automation of a bioprocessing system that utilizes the fluid flow architecture of FIG. 3, according to one embodiment of the invention. FIG. 4 is a schematic diagram of a generic protocol for automation of a bioprocessing system that utilizes the fluid flow architecture of FIG. 3, according to one embodiment of the invention. FIG. 4 is a schematic diagram of a generic protocol for automation of a bioprocessing system that utilizes the fluid flow architecture of FIG. 3, according to one embodiment of the invention. FIG. 4 is a schematic diagram of a generic protocol for automation of a bioprocessing system that utilizes the fluid flow architecture of FIG. 3, according to one embodiment of the invention. FIG. 4 is a schematic diagram of a generic protocol for automation of a bioprocessing system that utilizes the fluid flow architecture of FIG. 3, according to one embodiment of the invention. FIG. 4 is a schematic diagram of a generic protocol for automation of a bioprocessing system that utilizes the fluid flow architecture of FIG. 3, according to one embodiment of the invention. FIG. 4 is a schematic diagram of a generic protocol for automation of a bioprocessing system that utilizes the fluid flow architecture of FIG. 3, according to one embodiment of the invention. FIG. 1 is a perspective view of a concentration and isolation device according to an embodiment of the invention; FIG. 79 is a process flow diagram of the concentration and isolation device of FIG. 78. 79 is a schematic diagram of the fluid flow architecture of the device of FIG. 78 for performing enrichment and isolation of populations of cells. FIG. 2 is a block diagram of a magnetic particle-based cell selection system that may be used with a magnetic cell isolation holder, according to aspects of the present disclosure. 2 is a flowchart of a method of magnetic cell isolation, according to aspects of the present disclosure. FIG. 3 is a top view of one embodiment of a magnetic cell isolation holder in an unloaded configuration with a magnetic field generator, according to aspects of the present disclosure. FIG. 3 is a top view of one embodiment of a magnetic cell isolation holder in a loaded configuration with a magnetic field generator, according to aspects of the present disclosure. FIG. 3 is a perspective view of one embodiment of a magnetic cell isolation holder in an unloaded configuration with a magnetic field generator, according to aspects of the present disclosure. FIG. 3 is a perspective view of one embodiment of a magnetic cell isolation holder in a loaded configuration with a magnetic field generator in a loaded configuration, according to aspects of the present disclosure. 2 is a flowchart of a method of magnetic cell isolation using different magnetic cell isolation holders according to aspects of the present disclosure. FIG. 3 is a top view of one embodiment of a magnetic field generator in a magnetic cell isolation holder and loading configuration, according to aspects of the present disclosure, showing the magnetic field distribution of the magnetic field generator, according to aspects of the present disclosure.

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

As used herein, the terms "flexible" or "foldable" refer to a structure or material that is flexible or capable of being bent without breaking, compressible or It can also refer to materials that are expandable. An example of a flexible structure is a bag formed from polyethylene film. The terms "rigid" and "semi-rigid" are used herein to refer to structures that are "non-foldable," i.e., capable of folding, collapsing, or otherwise deforming under the application of normal forces. Used interchangeably to describe a structure without significantly reducing its elongated dimensions. Depending on the context, "semi-rigid" can also describe a structure that is more flexible than a "rigid" element, such as a tube or conduit that can be bent, but still collapses longitudinally under normal conditions and forces. can represent a structure that does not exist.

As used herein, "container" means a flexible bag, flexible container, semi-rigid container, rigid container, or flexible or semi-rigid tubing, as the case may be. The term "vessel" as used herein refers to bioreactor vessels having walls or portions of walls that are semi-rigid or rigid, as well as, for example, cell culture/purification systems, mixing systems, media/ Buffer preparation systems, and filtration/purification systems, including other vessels or conduits commonly used in biological or biochemical processing, including chromatography and tangential flow filtration systems and their associated flow paths. is intended. As used herein, the term "bag" refers to a flexible or semi-rigid container or container used, for example, as a containment device for various fluids and/or media.

As used herein, "fluidically coupled" or "fluidic communication" means that components of a system are capable of receiving or transferring fluids between the components. The term fluid includes gases, liquids, or combinations thereof. As used herein, "telecommunications" or "electrically coupled" means that the components of the means configured to communicate with each other. As used herein, "operably coupled" refers to a connection, which may be direct or indirect. A connection is not necessarily a mechanical connection.

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

As used herein, the term "functionally closed system" refers to a system that does not compromise the integrity of the closed fluid pathway (e.g., to maintain an internal sterile biomedical fluid pathway). Refers to a plurality of components that constitute a closed fluid pathway that may have inlet and outlet ports for adding fluid or air to or removing fluid or air from the system, such that the ports Alternatively, each port may include, for example, a filter or membrane to maintain sterile integrity when air is added or fluid or air is removed from the system. These components may include, but are not limited to, one or more conduits, valves (eg, multipoint diverters), containers, receptacles, and ports, depending on the given embodiment.

Embodiments of the invention provide systems and methods for producing cellular immunotherapeutics from biological samples (eg, blood, tissue, etc.). In one embodiment, a method of bioprocessing comprises combining a suspension containing a population of cells with magnetic beads to form a population of bead-bound cells in suspension, and forming a population of bead-bound cells on a magnetic separation column. The method includes isolating the population and collecting target cells from the population of cells. Collecting target cells involves removing bead-bound cells from an isolation column with an air plug.

Referring to FIG. 1, a schematic diagram of a bioprocessing system 10 according to one embodiment of the invention is illustrated. The bioprocessing system 10 is configured for use in the manufacture of a cellular immunotherapy (e.g., an autologous cellular immunotherapy), e.g., in which a human blood, fluid, tissue, or cell sample is collected and collected. A cell therapy agent is produced from or based on the sample. Although chimeric antigen receptor (CAR) T cell therapy is one type of cellular immunotherapy that can be produced using bioprocessing system 10, other cell therapies also depart from the broader aspects of the invention. may be produced using the system of the present invention or embodiments of the present invention. As illustrated in Figure 1, the manufacture of CAR T cell therapy generally begins with collection of patient blood and isolation of lymphocytes through apheresis therapy. Collection/apheresis therapy may be performed in a clinical setting, and the apheresis product is then sent to a laboratory or manufacturing facility for production of CAR T cells. In particular, after the apheresis product is received for processing, the desired cell population (e.g., white blood cells) is concentrated or isolated from the collected blood to produce a cell therapy agent and is of interest. Target cells are isolated from the progenitor cell mixture. The target cells of interest are then activated, genetically modified to specifically target and destroy tumor cells, and amplified to achieve the desired cell density. After amplification, cells are harvested and doses are formulated. The combination is then often stored frozen, thawed, prepared, and finally delivered to a clinical site for infusion into a patient.

With further reference to FIG. 1, the bioprocessing system 10 of the present invention includes a plurality of different Comprises a module or subsystem. In particular, bioprocessing system 10 includes a first module 100 configured to perform enrichment and isolation steps and a first module 100 configured to perform activation, genetic modification, and amplification steps. 2 modules 200 and a third module 300 configured to perform the steps of harvesting the amplified cell population. In one embodiment, each module 100, 200, 300 may be communicatively coupled to a dedicated controller (eg, first controller 110, second controller 210, and third controller 310, respectively). 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 to control the operation of each module, the master control unit provides global control over the three modules. It is contemplated that the system may be used for physical control. Each module 100, 200, 300 is designed to work in conjunction with other modules to form a single coherent bioprocessing system 10, as described in detail below.

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. In addition, as detailed below, each module 100, 200, 300 is substantially closed to help ensure patient safety by lowering the risk of external contamination and to comply with regulatory requirements. Help ensure compliance and avoid costs associated with open systems. Additionally, each module 100, 200, 300 is scalable, supporting both low patient volume development and high patient volume commercial manufacturing.

With further reference to Figure 1, the particular manner in which process steps are partitioned into distinct modules that provide for closed and automated bioprocessing, respectively, increases the efficiency of capital equipment to a degree not previously seen in this art. make available. As will be appreciated, the step of amplifying cell populations to achieve the desired cell density prior to harvest and formulation is typically the most time-consuming step in the manufacturing process, whereas enrichment and isolation The steps, as well as the harvesting and compounding steps, as well as the activation and genetic modification steps, do not take much time. Therefore, attempts to automate the entire cell therapy manufacturing process, in addition to being logistically difficult, can exacerbate bottlenecks within the process that impede workflow and reduce manufacturing efficiency. In particular, in a fully automated process, the cell enrichment, isolation, activation, and genetic modification steps can be performed fairly quickly, whereas the amplification of genetically modified cells proceeds very slowly. Therefore, the production of cell therapy from a first sample (e.g., the blood of a first patient) proceeds quickly up to the amplification step, which takes a substantial amount of time to achieve the desired cell density to harvest. It takes. When using a fully automated system, the entire process/system is occupied by the amplification equipment performing the amplification of cells from the first sample and the processing of the second sample until the entire system is released for use. Unable to start. In this regard, in fully automated bioprocessing systems, the entire system is essentially off-line and the entire cell therapy drug manufacturing process, from concentration to harvest/formulation, is completed in the first sample until the second sample is processed. Not available.

However, embodiments of the present invention enable parallel processing of multiple samples (from the same or different patients) to promote more efficient utilization of capital resources. This advantage is a direct result of 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 are used for parallel and synchronous processing of multiple samples from the same or different patients. Furthermore, in the bioprocessing system 12, it can be used in conjunction with a plurality of second modules, for example, second modules 200a, 200b, and 200c. For example, a first apheresis product from a first patient is enriched and isolated using a first module 100, thereby producing a first population of isolated target cells. Often, the first population of target cells may then be transferred to one of the second modules, e.g., module 200a, for activation, genetic modification, and amplification under the control of controller 210a. . After the first population of target cells has been transferred and exited the first module 100, the first module is again configured to process a second apheresis product, for example from a second patient. available for use. The second population of target cells produced in the first module 100 from the sample removed from the second patient is then separately processed for activation, genetic modification, and amplification under the control of the controller 201b. , for example, second module 200b.

Similarly, after the second population of target cells has been transferred and exited from the first module 100, the first module is again configured to collect a third apheresis product, for example from a third patient. It can be used for processing. The third target population of cells produced in the first module 100 from the sample removed from the third patient is then separately processed for activation, genetic modification, and amplification under the control of the controller 201c. , for example, second module 200c. In this regard, for example, expansion of CAR-T cells to a first patient may occur simultaneously to expansion of CAR-T cells to a second patient, a third patient, and so on.

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

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

Therefore, the activation, genetic modification, and amplification steps, which are most time-consuming, share some operational requirements, and/or require similar culture conditions, are closed to stand-alone automated functionally. By dividing the system into modules that are used for enrichment, isolation, harvesting, and formulation, other system equipment is tied down or offline while amplification of one population of cells is performed. There is no such thing. As a result, manufacturing of multiple cell therapy drugs may be performed simultaneously, maximizing equipment and floor space usage and increasing overall process and facility efficiency. Additional second modules may be added to bioprocessing system 10 to provide parallel processing of any number of cell populations as desired. Thus, the bioprocessing system of the present invention allows the use of plug-and-play-like functionality, allowing for easy expansion or contraction of manufacturing facilities.

In one embodiment, the first module 100 concentrates and isolates cells from an apheresis product taken from a patient for use in biological processes, such as the manufacture of immunotherapeutic and regenerative medicine drugs. Any system or device capable of producing a target population of. 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 invention will be described in detail below.

In one embodiment, the third module 300 similarly collects CAR-T cells or other cells produced by the second module 200 for infusion into the patient for use in cellular immunotherapy or regenerative medicine. It may be any system or device that can harvest and/or compound modified cells. In some embodiments, third module 300 may be a Sefia Cell Processing System, also available from GE Healthcare. In some embodiments, the first module 100 is used to store cells, which are then transferred to the second module 200 for activation, transduction, and amplification (and in some embodiments, harvesting). It may be utilized initially for enrichment and isolation and then also used at the end of the process for cell harvesting and/or formulation. In this regard, in some embodiments, the same equipment may be utilized for front-end cell enrichment and isolation steps as well as back-end harvesting and/or formulation steps.

Focusing first on the second module 200, cell activation, genetic modification, and cell amplification in a single functionally closed automated module 200 yields the efficiency of the workflow described above. The ability to combine process steps is enabled by the specific configuration of components within the second module 200 and the unique flow architecture that provides specific interconnectivity between such components. 3-77, discussed below, illustrate various aspects of the second module 200 according to various embodiments of the invention. Referring first to Figure 3, a fluid flow architecture 400 (bioprocessing subsystem 400 or bioprocessing system 400) within a second module 200 that performs cell activation, genetic modification, and amplification (in some cases, harvesting) (broadly referred to herein) is shown as a schematic diagram illustrating the invention. System 400 includes a first bioreactor vessel 410 and a second bioreactor vessel 420. The first bioreactor vessel is in fluid communication with 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 the second port 416. A second bioreactor line 418 is provided. Similarly, the second bioreactor vessel includes at least a first port 422 and a first bioreactor line 424 in fluid communication with the first port 422, and a second port 426 and a second port 426. A second bioreactor line 428 is provided in fluid communication with the second bioreactor line 428. Together, first bioreactor vessel 410 and second bioreactor vessel 420 form bioreactor array 430. Although system 400 is shown as having two bioreactor vessels, embodiments of the invention may include a single bioreactor or more than two bioreactor vessels.

The first and second bioreactor lines 414, 418, 424, 428 of the first and second bioreactor vessels 410, 420 each direct fluid flow therethrough, as described below. with respective valves for control. 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 A second bioreactor line valve 424 is provided. 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 first bioreactor line valve 436. , a second bioreactor line valve 438.

With further reference to FIG. 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 interconnecting line 450 having an interconnecting line valve 452 provides fluid communication between the first fluid assembly 440 and the second fluid assembly 444. As shown in FIG. 3, the interconnecting line 450 also provides fluid communication between the second bioreactor line 418 and the first bioreactor line 414 of the first bioreactor vessel 410. and fluid circulation may occur along the first circulation loop of the first bioreactor vessel. Similarly, the interconnecting line also provides fluid communication between the second bioreactor line 428 and the first bioreactor line 424 of the second bioreactor vessel 420 and the second bioreactor vessel 420. Circulation of the fluid may occur along a second circulation loop. Further, the interconnecting line 450 connects the second port 416 and the second bioreactor line 418 of the first bioreactor vessel 410 to the first port 422 of the second bioreactor vessel 420 and the first Further fluid communication is provided between the reactor lines 424 to enable 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, the interconnecting line 450, in one embodiment, connects the second bioreactor line 418, 428 to the first bioreactor line 414 of the first bioreactor vessel 410. It extends to an intersection with the first fluid assembly conduit 442.

As illustrated in FIG. 3, first and second fluid assemblies 440, 450 are disposed along an interconnecting conduit 450. Additionally, in one embodiment, the first fluid assembly is connected to the first port 412 of the first bioreactor vessel 410 and the first port of the second bioreactor vessel 420, respectively. Fluid communication is through a first bioreactor line 414 of the vessel and a first bioreactor line 424 of the second bioreactor vessel 420. Second fluid assembly 444 is in fluid communication with second port 416 of first bioreactor vessel 410 and second port 426 of second bioreactor vessel 420 via interconnecting conduit 450.

A first pump or interconnect line pump 454 capable of bidirectional flow of fluid is disposed along first fluid assembly line 442 and a second pump capable of bidirectional flow of fluid. Alternatively, a circulation line pump 456 is disposed along interconnecting line 450, the function and purpose of which is described below. In one embodiment, pumps 454, 456 are high dynamic range pumps. As also shown in FIG. 3, a sterile air source 458 is connected to the interconnect line 450 through a sterile air source line 460. A valve 462 positioned along sterile air source line 460 provides selective fluid communication between sterile air source 458 and interconnecting line 450. Although FIG. 3 shows a sterile air source 458 connected to interconnecting conduit 450, in other embodiments, the sterile air source may be connected to the first The first fluid assembly 440, the second fluid assembly 444, or the fluid intermediate the second bioreactor line valve and the first bioreactor line valve of either the bioreactor or the second bioreactor. It can be connected to a flow path.

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 includes a plurality of tubing tails 464a-f, each of which is selectively/removably connected to one of a plurality of first reservoirs 466a-f. configured to do so. Each tubing tail 464a-f of the first fluid assembly 440 directs fluid flow to or from a respective one of the plurality of first reservoirs 466a-f of the first fluid assembly 440. Tubing tail valves 468a-f are provided for selectively controlling the FIG. 4 specifically shows that the first fluid assembly 440 includes six fluid reservoirs, but more or fewer reservoirs for inputting or collecting various process fluids as desired. may be used. It is contemplated that each tubing tail 464a-f may be individually connected to a storage layer 466a-f, respectively, at any time required during operation of fluid assembly 440, as described below.

With particular reference to FIG. 5, the second fluid assembly 444 includes a plurality of tubing tails 470a-d, each of which is selectively/removably connected to one of a plurality of second reservoirs 472a-d. configured to do so. Each tubing tail 470a-d of the second fluid assembly 444 directs fluid flow to or from a respective one of the plurality of second reservoirs 472a-d of the first fluid assembly 444. Tubing tail valves 474a to 474d are provided for selectively controlling. FIG. 5 specifically shows that the second fluid assembly 444 includes four fluid reservoirs, but more or fewer reservoirs for inputting or collecting various process fluids as desired. may be used. In one embodiment, at least one of the second reservoirs, e.g., second reservoir 472d, is a collection reservoir for collecting an expanded population of cells, as described below. It's a tank. In one embodiment, the second storage tank 472a is a waste liquid storage tank, the purpose of which is explained below. The present invention provides that one or more reservoirs 472a-d may be pre-connected to their respective tails 470a-d, with each additional reservoir being adapted for use within the second fluid assembly 440, respectively. It is intended to be connected to the tail of the

In one embodiment, first reservoirs 466a-f and second reservoirs 472a-d are one-time/disposable flexible bags. In one embodiment, the bag has opposing panels welded or secured together at its periphery and supports connecting conduits for connection to the respective tails, as is known in the art. It is essentially a two-dimensional bag.

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, a welding device is positioned next to the module 200 and the welding device is positioned to butt weld one of the tubing tails (while maintaining sterility) to the tail of the tubing on the bag. used. Thus, the operator can provide the bag when it is needed (e.g., by grasping the tubing tail, inserting its free end into the welding device, and placing the bag adjacent to the end of the tubing tail). Lay the free end of the bag tube on its side and cut the tube with a new razor blade, pulling the razor apart while the two tube ends are forced together while still melting together to re-solidify. (by heating the cut end when cutting). Conversely, the bag can be removed by welding the conduit from the bag and cutting the two closed conduits at the weld. Thus, the reservoirs/bags may be connected individually when desired, and the present invention provides the operator with access to the appropriate tubing tails throughout the process to connect the reservoirs/bags in time for their use. so it does not require that all reservoirs/bags must be connected at the beginning of the protocol. In fact, as explained below, all reservoirs/bags are pre-connected, but the present invention does not require pre-connection and one advantage of the second module 200 is that the operator can The point is that the used bag can be sterilely connected and disconnected so that other bags can be sterilely connected in the protocol.

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

Referring again to FIG. 3, in one embodiment, the system 400 may also include a filtration line 482 connected at two points along the interconnect line 450; Define a filtration loop. A filter 484 is positioned along the filtration line 482 for removing permeate waste from the fluid passing through the filtration line 482. As shown therein, filtration line 482 includes an upstream filtration line valve 486 and a downstream filtration line valve 488, positioned upstream and downstream of filter 484, respectively. Waste conduit 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. bring. At this point, waste line 490 conveys waste removed from the fluid passing through filtration line 482 by filter 484 to waste storage tank 472a. As illustrated in FIG. 3, filtration lines 482 are interconnected such that fluid flow through interconnection line 450 can be forced through filtration line 482, as described below. Surrounding the connecting line valve 452. A permeate pump 492 positioned along waste line 490 is operable to pump waste removed by the filter to waste storage tank 472a. In one embodiment, filter 484 is preferably an elongated hollow fiber filter, although other tangential flow or cross flow filtration means known in the art, such as, for example, flat sheet membrane filters, are also contemplated by the present invention. may be utilized without departing from the broader aspects of.

In one embodiment, the valves of first fluid assembly 440 and second fluid assembly 444, as well as bioreactor line valves (i.e., valves 432, 434, 436, 438), sterile line valves 462, interconnecting pipes Line valve 452 and filtration line valves 486, 488 are pinch valves constructed in the manner described below. In one embodiment, the conduit itself need not be equipped with a pinch valve, and the pinch valve illustrations of FIGS. 3-8 are simply locations where the pinch valve can operate on the conduit to prevent fluid flow. It may be assumed that it represents In particular, as explained below, the pinch valve of the flow architecture 400 operates/activates against a corresponding anvil to "pinch off" the fluid path/conduit while the fluid path/conduit is between the fluid may be provided by a respective actuator (eg, a solenoid) that prevents the passage of the

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

Referring now to FIGS. 8-11, a configuration of a first bioreactor vessel 410 is illustrated in accordance with 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, so for simplicity only the first bioreactor vessel 410 will be described below. explained. In one embodiment, bioreactor vessels 410, 420 are perfusion-compatible silicone membrane-based bioreactor vessels that support activation, transduction, and amplification of populations of cells therein. Bioreactor vessels 410, 420 may be used for cell culture, cell processing, and/or cell expansion to increase cell density for use in medical therapy or other processes. Although bioreactor vessels are disclosed herein for use in conjunction with specific cell types, bioreactor vessels can be used for the activation, genetic modification, and/or amplification of any suitable cell type. It should be understood that it can be used for Additionally, the disclosed technology can be used in conjunction with adherent cells, ie, cells that adhere to and/or proliferate on cell amplification surfaces. In one embodiment, the first and second bioreactor vessels 410, 420 are disclosed in U.S. Patent Application No. 15/893,336, filed February 9, 2018, which is incorporated herein by reference in its entirety. It can be made and function as described.

As shown in FIGS. 8 and 9, the first bioreactor vessel 410 can include a bottom plate 502 and a vessel body 504 coupled to the bottom plate 502. Bottom plate 502 may be a rigid structure that supports cell cultures. 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. may be). 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 may enable the use of low profile vessels and/or maximize the space where the first bioreactor vessel may be utilized or stored. good.

In one embodiment, vessel body 504 comprises a rigid, generally concave structure that, when coupled to bottom plate 502, forms a cavity or interior compartment 506 of first bioreactor vessel 410. As shown therein, container body 504 may have a circumferential shape similar to that of bottom plate 502 such that container body 504 and bottom plate 502 may be coupled together. In addition, as in the illustrated embodiment, the vessel body 504 may allow for visual inspection of the contents of the first bioreactor vessel 410 and/or the light may allow for visual inspection of the contents of the first bioreactor vessel 410. It may be made of a transparent or translucent material that may allow light to enter. The internal compartment formed by the bottom plate 502 and vessel body 504 contains cell culture medium and cells during use of the first bioreactor vessel for cell activation, genetic modification (i.e., transduction), and/or cell expansion. May contain cultures.

As best shown in Figures 8-11, the first bioreactor vessel 410 is used for several purposes related to cell activation, transduction/genetic modification, and amplification, such as media input and waste removal. A plurality of ports may be provided through the vessel body 504 that may allow fluid communication between the interior compartment 506 and the exterior of the first bioreactor vessel 410 for the process of. The ports may include, for example, a first port 412 and a second port 416. Port 416 may be disposed anywhere within container body 504, such as through either the top surface 508 and/or side 510 of container body 504, as in the illustrated embodiment. As 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, can facilitate cell activation, genetic modification of the cells, and Enables the first bioreactor vessel 410 to be used to support cell density amplification.

FIG. 9 is an exploded view of one embodiment of the first bioreactor vessel 410. The bottom plate 502 of the first bioreactor vessel 410 may be the bottom or support of the first bioreactor vessel 410. As previously discussed, bottom plate 502 may be formed from a non-solid structure. In the illustrated embodiment, the bottom plate 502 may house a grid 510, which may be structurally rigid, but allow free gas exchange through the bottom plate 502 to the interior compartment 506 containing the cell culture. Further, an opening is provided for the purpose of The grid 510 may include a plurality of holes 512 defined between solid regions or a crossbar 514 between each hole 512 of the grid 510. Thus, the holes 512 may provide openings for gas exchange and the crossbars 514 may provide structural support for other structures and cell cultures within the interior compartment 506 of the first bioreactor vessel 410.

To form further support for the cell culture within the interior compartment 506 of the first bioreactor vessel 410, the first bioreactor vessel 410 includes a membrane that may be disposed on the top surface 518 of the bottom plate 502. 516. Membrane 516 may be a gas permeable liquid impermeable membrane. Membrane 516 may also be selected to have properties that allow for high gas permeability, high gas transport rates, and/or high permeability to oxygen and carbon dioxide. Thus, membrane 516 can support high cell densities (eg, up to about 35 MM/cm ² ) within interior compartment 506. The gas permeable characteristics of membrane 516 may allow free gas exchange to support cell culture and/or cell expansion. As such, membrane 516 may be a cell culture surface and/or cell amplification surface. Membrane 516 may have a relatively small thickness (eg, 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.

The flatness of membrane 516 may increase the surface area of the cell culture to settle for activation, transduction, and/or amplification. A mesh sheet 520 may be disposed between the bottom plate 502 and the membrane 516 to allow the membrane 516 to remain flat during use of the first bioreactor vessel 410. Mesh sheet 520 can provide structural support for membrane 516 so that membrane 516 remains planar and can be added to first bioreactor vessel 410 for cell culture and/or cell culture and/or cell amplification. It cannot sag or buckle under the weight of any cell culture medium that is placed on it. Additionally, the mesh properties of the mesh sheet 520 may allow for support of the membrane 516, but are still porous and therefore the interior compartment 506 of the first bioreactor vessel 410 and the environment immediately outside the first bioreactor vessel 410. Free gas exchange between the two is possible. The mesh sheet may be a polyester mesh or any other suitable mesh material that can support the membrane and allow free gas exchange.

As previously discussed, 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, mesh sheet 520 and membrane 516 may be disposed within, or at least partially within, 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, O-ring 522 may be a biocompatible O-ring (size 173, Soft Viton® Fluoroelastomer O-Ring). O-ring 522 may fit within a groove 524 formed in circumferential surface 526 of container body 504. Circumferential surface 526 faces top surface 518 of plate 502 when body portion 504 is fitted to plate 502. As such, O-ring 522 may be press-fit into groove 524 and pressed against top surface 518 of plate 516 and/or bottom plate 502. Such press fit of O-ring 522 desirably seals first bioreactor vessel 410 without adhesion with chemicals or epoxies. Since the first bioreactor vessel 410 may be used for activation, transduction, and amplification of biological cells, the O-ring 522 is preferably suitably biocompatible, autoclavable, gamma radiation stable, and/or Constructed from ETO sterile stable material.

As explained above, first bioreactor vessel 410 may include multiple ports, such as first port 412 and second port 416. Ports 412, 416 may be disposed through the vessel body 504 for cell culture, cell activation, cell transduction, and/or cell amplification, such as fluid or media input, waste removal, collection, and sampling. Communication may be allowed between the interior compartment 506 and the exterior of the first bioreactor vessel 410 for several processes involved. Each port 416 may include an opening 526 and a respective fitting or tubing 528 (eg, luer fitting, barb fitting, etc.). In some embodiments, opening 526 may be configured to allow tubing to be glued directly, eliminating the need for a fitting (eg, a counterbore).

In one embodiment, in addition to the first port 412 and the second port 416, the first bioreactor vessel 410 further includes an air balance port 530 disposed within the top surface 508 of the vessel body 504. obtain. Air balance port 530 may be made similar to first port 412 and second port 416, with like reference numbers representing like parts. Air balance port 530 may further provide gas exchange between interior compartment 506 and the exterior of first bioreactor vessel 410, which is used in cell culture for amplification. Additionally, air balance port 530 may help maintain atmospheric pressure within interior compartment 506 to provide an environment within the interior compartment for cell culture and/or cell expansion. Air balance port 530 may be disposed through the top surface 508 of container body 504, as in the illustrated embodiment, or at any other location around container body 504. The central location through the top surface 508 of the vessel body 504 prevents wetting of the air balance port 530 during mixing of the cell culture through the slope of the first bioreactor vessel 410, as described in more detail below. can help.

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, is biocompatible and autoclavable. It may be made from materials that are processable and have gamma and/or ETO sterilization stability. As such, each element, and the first bioreactor vessel 410 as a whole, may be used for activation, transduction, and amplification of biological cells and/or other processes of cell manufacturing processes.

The first bioreactor vessel 410 may enable cell culture and/or cell expansion via perfusion, which provides the necessary nutrients to support cell growth and Impurities can be reduced. Continuous perfusion is the addition of a fresh medium supply to a growing cell culture while simultaneously removing spent medium (eg, spent medium). First port 412 and second port 416 may be used for perfusion processes, as described below. The first port 412 may allow communication between the interior compartment 506 and the exterior of the first bioreactor vessel 410 to provide 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 through the vessel body 504 at any location above the surface of the cell culture and media within the first bioreactor vessel 410. . In some embodiments, the first port 412 may be disposed to contact or penetrate the surface of the cell culture and media within the first bioreactor vessel 410.

The second port 416 may be disposed at any location where it is fully or partially submerged below the surface of the cell culture and media within the first bioreactor vessel 410. For example, the second port 416 may be a nearly lateral port disposed through one of the sides 510 of the container body 504. In some embodiments, second port 416 may be arranged such that second port 416 does not reach the bottom of interior compartment 506 (eg, membrane 516). In some embodiments, second port 416 may reach the bottom of interior compartment 506. Second port 416 may be a dual function port. As such, the second port may be used to draw perfusion medium from the internal compartment 506 of the first bioreactor vessel 410 to facilitate perfusion of the cell culture. Additionally, second port 416 may also be used to remove cells from cell culture. As noted above, in some embodiments, the second port cannot reach the bottom of the interior compartment 506 of the first bioreactor vessel 410. For example, second port 416 may be located approximately 0.5 cm from membrane 516. Thus, in a static planar position, the second port 416 removes spent cell medium without pulling cells from the cell culture as cells may settle to the membrane 516 (e.g., a cell amplification surface) by gravity. can be used for Thus, in a static planar position, the second port 416 may facilitate the perfusion process and allow an increase in cell density of the growing cell culture within the first bioreactor vessel 410. When it is desired that cells be removed from the internal compartment 506, for example during harvest of the cell culture, to minimize hold-up volume, the first bioreactor vessel 410 is removed in the manner described below. , may be tilted toward the second port 416, thereby providing access to the cells for cell removal.

Additionally, in one embodiment, the second port 416 does not include a filter, so the perfusion process can be performed without a filter. As such, there can be no physical obstruction to prevent cells from entering the second port 416 when the second port 416 is used for medium removal. Further, the second port 416 may be disposed laterally through the side 22 of the container body 504, but the second port 416 may be angled toward the membrane 516 and the bottom plate 502. It may be inclined as shown. The angled feature of the second port 416 minimizes interference with the O-ring 522 and groove 524, helping the second port 416 maintain a seal on the first bioreactor vessel 410 during use. However, it can be positioned relatively low on the container body 504 closer to the membrane surface 36. Additionally, in some embodiments, the sloped feature of the second port 416 may reduce the rate of fluid flow through the second port 416 when spent medium is removed. In addition, the port diameter, in conjunction with the fluid flow rate exiting the second port 416, determines that the suction rate through the second port 416 used to draw medium from the external compartment 506 is It may be a diameter that minimizes the suction force exerted on adjacent individual cells to be less than the force of gravity pulling the cells toward the membrane 516. Thus, as explained above, the second port 416 may be used to facilitate perfusion of the cell culture without removing perfusion medium and substantially removing cells from the cell culture. As the cell settling time increases, the cell concentration of the removed medium decreases and may fall within a non-measurable range facilitated by the position of the second port 416. Additionally, the position of internal opening 540 can be changed to change the recommended cell fixation time. Positions closer to the membrane 516 may be associated with longer settlement times, whereas positions at or near the top of the medium may be associated with shorter settlement times as cells settle and deplete from the top of the growth medium first. is connected with.

Thus, in one embodiment, the second port 416 is used not only for removing the medium used in the perfusion process, but also for removing the cells of the cell culture from the internal compartment 506, for example, during harvest of the cell culture. can be used. To facilitate removal of more of the spent perfusion medium and removal of cells, the container body 504 may include angled or chevron-shaped side walls 532. Thus, the chevron-shaped sidewall 532 includes an apex, or apex point, 534. The apex 534 of the sidewall 532 may further include a second port 416 therethrough, and the container body 504 may be disposed proximate the apex point 534 when the container body 504 is coupled to the bottom plate 502. Ru. The angled sidewall 532 and tip point 534 allow media and/or cell culture to be deposited when the first bioreactor vessel 410 is tilted toward the second port 416, e.g., at a 5 degree angle. cells may be expelled in larger quantities.

Using perfusion to proliferate the cells facilitated by the location of the first port 412 and the second port 416, the culture medium within the internal compartment 506, as described in more detail with reference to FIG. It may be possible to reduce the height (eg, from 0.3 to 2.0 cm). The relatively low height of the medium within the internal compartment 506 allows the first bioreactor vessel 410 to be a relatively low profile vessel while allowing for the highest achievable cell density increase. It can be. Additionally, the use of perfusion by 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. , additional cell washing in a separate device may not be necessary after reaching a particular cell density goal in the first bioreactor vessel 410. For example, through filterless perfusion, the first bioreactor vessel 410 may provide fresh medium at a rate of full change every day, reducing impurities within the cell culture (e.g., resulting in impurities are reduced at a rate of approximately 1 log every 2.3 days). The structure of the first bioreactor vessel 410 therefore allows for the use of perfusion for the growth of the cell culture within the first bioreactor vessel 410, thus allowing the cell culture to be grown while reducing the level of impurities. It may be possible to amplify to high target densities. As also described below, through filterless perfusion, the first bioreactor vessel 410 is used for cell seeding, rinsing, washing/residue reduction, and/or draining/harvesting after amplification. Fresh medium may be provided at substantially multiple doses per day (eg, more than two doses per day).

To facilitate the low profile structure of the first bioreactor vessel 410, a relatively low medium height may be maintained within the interior 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 discussed, the vessel body 504 may be coupled to the bottom plate 502 to form an internal compartment 506 in which cell culture amplification may be accomplished through perfusion. As such, replacement or fresh medium 538 may be provided for cell growth through a first port 412 disposed through the container body 504, and existing or used medium 538 may be provided for cell growth through a first port 412 disposed through the container body 504. may be removed through a second port 416 disposed through the side 510 of the container body 504. The perfusion process may facilitate the use of a relatively low medium height 536 of the medium 538 within the interior compartment 506 of the first bioreactor vessel 410. The relatively low height 536 of the perfusion medium 538 within the internal compartment 506 allows the first bioreactor vessel 410 to have a low profile structure, thus providing an overall compact cell manufacturing system. It could be possible.

The height 536 of the perfusion medium 538 within the internal compartment 506 of the first bioreactor vessel 410 may be between 0.3 cm and 2 cm, providing headroom 542, i.e. between the medium 538 and the vessel body within the internal compartment 506. The height of the gap formed between the portion 504 and the top surface 508 may be approximately 2 cm. Therefore, there can be less than 2 mL of medium per cm ² and less than 4 mL of total volume per cm ² , including medium, cell culture, and headspace. A relatively low medium height 536 may allow the ratio of medium volume to surface area of membrane 516 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 desirable range, facilitated by the use of perfusion to propagate cells in a cell culture. Ru. For example, the threshold level may be a ratio between 0.3 and 2.0. Because of the small ratio of medium volume to membrane surface area, it may be possible to provide the first bioreactor vessel 410 with a low profile or compact structure while still providing a cell culture with high cell density.

As previously described, the second port 416, which has dual functionality, is connected to the vessel body so that it is fully or partially submerged below the surface 544 of the medium 538 within the first bioreactor vessel 410. 504. In some embodiments, second port 416 may be arranged such that second port 416 reaches the bottom of interior compartment 506 (eg, membrane 516). The positioning of the second port 416 may facilitate removal of media and impurities from the cell culture within the interior compartment 506 without removing the cells until such removal, eg, harvesting, is desired. A second port 416 without a filter, along with the first port 412, uses perfusion to supply growth medium 538 to cells for cell amplification and remove spent medium 538 and other impurities or byproducts. can be made possible. The location of the first port 412 and the dual-function second port 416 around the container body 504 ensures that the medium height 536 within the internal compartment 506 is maintained at a relatively low level, thereby 1 facilitates a configuration that allows the bioreactor vessel 410 of the present invention to be a relatively low-profile vessel while still allowing the production of high-density cell cultures.

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 a broader bioprocessing system 10, and in particular, the bottom plate 502 of the bioreactor vessel 410. It is provided as a second module 200. As shown therein, the bottom plate 502 includes a plurality of recesses 550 formed in the bottom surface of the bottom plate 502, the purpose of which will be explained below. In one embodiment, the recesses may be located adjacent the corners of the bottom plate 502. The recesses 550 are each generally cylindrical in shape and may terminate in a domed or hemispherical inner surface. As also shown in FIG. 11, the bottom plate 502 interacts with the sensors of the second module 200 to ensure proper positioning of the first bioreactor vessel 410 within the second module 200. A location verification structure 552 may be provided that is configured to. In one embodiment, the position verification structure may be a beam break that is configured to interrupt the light beam of the second module 200 when the first bioreactor vessel 410 is properly installed therein. .

The bottom plate 502 also includes a pair of flat engagement surfaces 554 formed on adjacent bottom surfaces that are offset from the centerline of the bottom plate (across the width of the bottom plate). Desirably, the engagement surfaces 554 are spaced apart along the longitudinal centerline of the bottom plate 502 such that they are located adjacent opposite ends of the bottom plate 502. The bottom plate 502 may further include at least one aperture or opening 556 that allows sensing of the contents of the first bioreactor vessel 410 by a bioprocessing device that engages and manipulates the bioreactor vessel.

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

With further reference to FIG. 15, the tray 610 includes a plurality of generally thin, rigid bodies including a front wall 612, a rear wall 614, and opposing sides 616, 618 surrounding a bottom surface 620 and a generally open top. or semi-rigid side walls. Sidewalls and bottom surface 620 define an interior compartment 622 of tray 610. In one embodiment, the open top of the tray 610 receives a removable cover (not shown) that surrounds the interior compartment 622, as shown below, and also the upper rim of the bioprocessing device drawer. It is surrounded by a peripheral flange 624 which preferably provides a mounting surface thereon. The bottom surface 620 of the tray 610 includes a number of openings corresponding to the number of bioreactor vessels in the bioprocessing system. For example, tray 610 may include 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 depart from the broader aspects of the invention. It can be used without doing anything.

As best shown in FIG. 15, each of the first and second openings 626, 628 allows a portion of the bioreactor vessel 610, 620 to pass through the bottom of the tray 610 through the respective opening 626, 628. The first and second bioreactor vessels 410, 420 are positioned over their respective openings 626, 628 and may be supported by the bottom surface 620 of the tray 610 within the internal compartment 622 while being accessible from the Having a circumference of shape and/or dimensions. In one embodiment, the perimeter of the openings includes at least one tab or protrusion for supporting the bioreactor vessel above the respective opening. For example, the periphery of each opening 626, 628 may include a tab 632 projecting inwardly toward the center of the opening 626, 628 for supporting a bioreactor vessel 410, 420 placed thereon. good. As shown in FIGS. 12 and 15, the tray 610 has openings 626, 628 to prevent lateral movement of the bioreactor vessel when received over the respective openings 626, 628. It may also include one or more ridges extending upwardly on the top. The ridges thus act as alignment devices to facilitate proper positioning of the bioreactor vessels 410, 420 within the tray 610 and loading of the kit 600 within the second module 200, as described below. or to help prevent inadvertent movement of the bioreactor vessels 410, 420 during positioning.

With further reference to FIGS. 12 and 13, the tray 610 may include one or more support ribs 636 formed on the bottom surface of the tray 610. Support ribs 636 cross the width and/or length of tray 610 and provide rigidity and strength to tray 610 and facilitate movement and manipulation of kit 600. Ribs 636 may be integrally formed with the tray or added as an auxiliary component via attachment means known in the art (see FIG. 13). In one embodiment, the tray 610 has a tubing module 650 therethrough that holds fluid flow conduits in an organized manner and holds them in place for engagement by pumps and pinch valves. An opening 638 is provided for receiving an engagement plate, referred to as . In other embodiments, tubing module 650 may be integrally formed with rear wall 614 of tray 610.

16 and 17 illustrate the configuration of a tubing module 650 according to one embodiment of the invention. As shown therein, tubing module 650 receives first fluid assembly line 442, interconnect line 450, and waste liquid line 490 of fluid flow system 400, and includes first fluid assembly line 442, interconnect line 450, and waste liquid line 490. , interconnecting conduit 450, and permeate waste conduit 490 in position for selective engagement with respective pump heads 454, 456, 492 of the peristaltic pump assembly described below with respect to FIGS. 35 and 36. a first tubing holder block 652 configured to hold the tubing holder block 652; In one embodiment, fluid assembly conduit 442, interconnecting conduit 450, and waste conduit 490 are maintained in a horizontally extending and vertically spaced orientation by first tubing holder block 652. In particular, as best shown in FIG. 17, the first tubing holder block 652 has two spaced apart arrangements 656, 658 (tubes and slots within the tubing holder block 652) defining an air gap between them. 442, 450, 490, respectively) (such as through clips or simple interference between them). 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 causes peristaltic compression of the shoes of conduits 442, 450, 490 by the respective pump heads of the peristaltic pump to provide respective propulsion of fluid through the conduits, as described below. Can be done.

With further reference to FIGS. 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 conduits of the fluid flow system 400 with which the pinch valve is associated. For example, the second tubing holder block 654 holds the tubing tails 464a-f of the first fluid assembly 440, the tubing tails 470a-d of the second fluid assembly 444, and the first tubing tail of the first bioreactor vessel 410. bioreactor line 414 and second bioreactor line 418; first bioreactor line 424 and second bioreactor line 428 of second bioreactor vessel 420; sterile air source line 460; , an interconnecting line 450, and a filtration line 482 (as well as, in some embodiments, sampling lines 476a-476d). Similar to the first tubing holder block 652, the second tubing holder block 654 may maintain the tubes in a horizontally extending and 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 conduits therein. 18 and 19 also best illustrate the configuration of the slot 666 that holds all of the flow conduits that are subject to/in contact with the pinch valve. Desirably, the slot 666 follows the contour of the block 654, but specifically extends across the planar backplate as it opens toward the filter 484. As shown in FIG. 18, in one embodiment, the second tubing holder block 654 includes a second tubing holder block 654 to hold the loop of interconnecting conduit 450 from which the sampling conduit extends. The bottom of block 654 may have one or more narrow tubing slots 682 and a waste line tubing slot 684 for receiving tubing tail 470a connected to waste storage tank 472a.

The second tubing holder block 654 may include a planar backplate 662 having a plurality of openings 664 corresponding to the plurality of fluid flow conduits held by the second tubing holder block 654. In particular, at least one aperture 664 is horizontally aligned with each slot 666 and the flow conduit held therein. As best shown in FIG. 16, the second tubing holder block 654 has two clearance openings 668, 670 configured to receive an anvil (not shown) of a pinch valve assembly therethrough. Equipped with. This configuration includes tubing tails 464a-f of first fluid assembly 440, tubing tails 470a-d of second fluid assembly 444, and tubing tails 470a-d of first bioreactor vessel 410, as described below. 1 bioreactor line 414 and a second bioreactor line 418; a first bioreactor line 424 and a second bioreactor line 428 of a second bioreactor vessel 420; and a sterile air source line. 460, interconnecting conduit 450, and filtration conduit 482 are selectively compressed relative to the anvil by respective pistons of actuators of the pinch valve array to selectively impede or allow fluid flow. enable. As shown in FIGS. 18 and 19, the apertures 664 may be arranged in first and second rows positioned next to each other, with the apertures in the first row of apertures are vertically offset with respect to the apertures in the second column of apertures such that the apertures in the first column of apertures are not horizontally aligned with the apertures in the second column of apertures. This configuration allows tubing module 650, tray 610, and kit 600 to have an overall low profile.

In one embodiment, filter 484 (shown as an elongated hollow fiber filter module in FIG. 16) is attached to tubing module 650, such as by attaching filter 484 to tubing module 650 using retaining clips 672. Can be integrated. If the filter 484 is a hollow fiber filter, the filter 484 may extend substantially the entire length of the tubing module 650 and include a first input end 674 and conveys the retentate after permeate/waste removal back to the filtration line 482 and the interconnect line 450 to either the first bioreactor vessel 410 or the second bioreactor vessel 420. and a second output end 676 for circulation to one of the two. Filter 484 may also include a permeate port 678 adjacent second output end 676 that connects to waste line 490 for conveying waste/permeate to permeate/waste storage tank 472a. Finally, the tubing module 650 accepts the clips and connects the bioreactor conduits (e.g., the first and second bioreactor conduits 414, 418 of the first bioreactor vessel 410 and/or the second bioreactor vessel 420). can include a plurality of features 680 for organizing the first and second bioreactor conduits 424, 428) of

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

20-22 are various views illustrating one embodiment of a kit 600, which is received by a first bioreactor vessel 410 and a second bioreactor vessel 420 received within a tray 610 and a tubing module 650. 4 illustrates fluid conduits of a flow architecture 400. Instead of having an opening 630 as illustrated therein, the kit 600 as shown in FIGS. 20-22 may include a solid bed and sampling conduits (e.g., sampling conduits 476a, 476b ) A sampling space 631 is provided within the tray 610 to receive a container holding the sample. Kit 600 comprises a modular platform for cell processing that can be easily set up and discarded after use. The tubing tails of the first and second fluidic assemblies 440, 444 allow plug-and-play functionality, which allows for quick and easy connection of various media, reagents, waste, sampling, and collection bags. This allows the various processes used to run on a single platform. In one embodiment, connecting and disconnecting is accomplished by sterile cutting and welding of the tube segments as described above, such as with a TERUMO device, or by pinching the tail segments as known in the art. , can be accomplished by welding and cutting.

23-25, the kit 600 contains 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. The bioprocessing device 700 is specifically configured to be received by the bioprocessing device 700. In one embodiment, the bioprocessing device 700 and the kit 600 (containing the flow architecture 400 and the bioreactor vessels 410, 420) together combine the second form a bioprocessing module 200. Bioprocessing device 700 includes a housing 710 having a plurality of drawers 712, 714, 716 receivable within housing 710. Although FIG. 23 shows an apparatus 700 containing three drawers, the apparatus may have a single drawer, two drawers, or more than three drawers, with bioprocessing within each drawer. The operations may be executed simultaneously. In particular, in one embodiment, each drawer 712, 714, 716 is a stand-alone bioprocessing module (i.e., as described above with respect to FIG. 2) for performing cell activation, genetic modification, and/or amplification processes. (equivalent to the second modules 200a, 200b, and 200c). In this regard, any number of drawers may be added to device 700 to process multiple samples from the same or different patients in parallel. In one embodiment, rather than each drawer sharing a common housing, each drawer may be received within a dedicated housing, and the housings may be stacked on top of each other in one embodiment.

As shown in FIGS. 23 and 24, each drawer, eg, drawer 712, includes a plurality of sidewalls 718 and a bottom surface 720 that defines a processing chamber 722 and a generally open top. Drawer 712 can be arranged in a closed position, where the drawer is fully received within housing 710, as shown for drawers 714 and 716 in FIG. 23 and 24 for drawer 712, allowing access to the drawer 712. 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), thereby causing the sidewalls 718 and/or Processing chamber 722 may be heated to a desired temperature to maintain processing chamber 722 at a desired temperature (eg, 37° C.) as optimized for the process steps to be performed by module 200. In some embodiments, the bottom surface 720 and underside of the top surface of the housing (above the processing chamber when the drawer is closed) may be temperature controlled in a similar manner (eg, with embedded heating elements). A hardware compartment 724 in drawer 712 behind processing chamber 722 may house all of the hardware components of apparatus 700, as described in detail below. In one embodiment, the drawer 712 includes an auxiliary compartment adjacent to the processing chamber 722 for housing reservoirs containing media, reagents, etc., connected to the first fluid assembly 440 and the second fluid assembly 444. 730 may further be provided. In one embodiment, auxiliary compartment 730 may be refrigerated.

Each drawer, eg, drawer 712, may be slidably received on opposing guide rails 726 mounted within the housing 710. A linear actuator may be operably connected to drawer 712 to selectively move drawer 712 between open and closed positions. The linear actuator is operable to effect smooth and controlled movement of drawer 712 between open and closed positions. In particular, the linear actuator operates the drawer 712 at a substantially constant speed (and with minimal accelerations and decelerations when stopping and starting motion) to minimize disturbance to the contents of the bioreactor vessel. Configured to open and close.

FIG. 25 is a top view of the interior of drawer 712 showing processing chamber 722, hardware compartment 724, and auxiliary compartment 730. As illustrated therein, a hardware compartment 724 is located at the rear of the processing chamber 722 and is integrated with or otherwise in communication with a power supply 732 and a second module controller 210. a motion control board and drive electronics 734, a low power solenoid array 736, a pump assembly 738 (including pump heads for pumps 454, 456, 492), and a drawer engagement actuator 740. Hardware compartment 724 of drawer 712 further includes a pump shoe 742 and a pair of pinch valve anvils 744 for interfacing with pump assembly 738 and solenoid array 736, respectively, as described below. In one embodiment, the pump shoe 742 and solenoid anvil 744 are secured to the front base plate (front plate) of the processing chamber. All hardware compartments (and components described) are attached to the rear baseplate. Both plates are slidably attached to the rail. In addition, the drawer engagement actuator 740 is used to join the two plates and move the two plates and the components mounted on the plates to the engagement position (moves the pump roller head into the pump shoe). , thereby compressing the pump tubing if inserted between them). As further described herein, the pump assembly selectively operates on conduits 442, 450, and 490 of fluid path 400 to provide independent respective peristaltic propulsion forces. Similarly, tubing holder block 654 of tray 600 will be positioned between solenoid array 736 and anvil 744, as further described.

As also illustrated in FIG. 25, two bed plates, e.g., first and second bed plates 746, 748, are positioned within the processing chamber 722 on the bottom surface 720 and extend upwardly or Or stick out from there. In one embodiment, processing chamber 722 may house a single bedplate, or three or more bedplates. The bed plates 746, 748 are configured to receive or otherwise engage the first bioreactor vessel 410 and the second bioreactor vessel 420 thereon. As also shown in FIG. 25, the drawer 712 is positioned adjacent the bed plates 746, 748 within the processing chamber 722 to sense the weight of a storage layer, e.g., a waste liquid reservoir 472a positioned above. It also includes a plate 750 that is configured with a load cell placed on the plate 750.

26-28 best illustrate the configuration of the bed plates 746, 748, with FIG. 28A showing the hardware components positioned beneath the bed plates. As used herein, the bedplates 746, 748 and hardware components (i.e., sensors, motors, actuators, etc.) may be collectively referred to as bed plates. Although the first and second bedplates 746, 748 are substantially identical in construction and operation, for simplicity, the following description of the bedplates 746, 748 will refer only to the first bedplate 746. . Bed plates 746, 748 have a substantially planar top surface 752 having a shape and surface area that generally corresponds to the shape and area of bottom plate 502 of first bioreactor vessel 410. For example, the bedplate may be generally rectangular in shape. The bed plates 746, 748 may also include a relief or clearance area 758 that generally corresponds to the location of the protrusion or pawl 632 on the tray 610, the purpose of which will be explained below. Bed plates 746, 748 are supported by a plurality of load cells 760 (eg, four load cells 760 positioned under each corner of bed plate 746). Load cell 760 is configured to sense the weight of first bioreactor vessel 410 in bioprocessing for use by controller 210.

In one embodiment, the bed plate 746 includes embedded heating elements so that the contents of the processing chamber 722 and/or the first bioreactor vessel 410 disposed thereon can be maintained at a desired temperature. or may be in thermal communication with a heating element. In one embodiment, the heating elements may be the same or different heating elements that heat the sidewalls 718, top wall, and bottom surface.

As illustrated, the bed plate 746 includes a plurality of locating pins or alignment pins 754 that project above the top surface 452 of the bed plate 746. The number of locating pins 754 and the location and spacing of the locating pins 754 may correspond to the number, location, and spacing of recesses 550 in the bottom surface of the bottom plate 502 of the bioreactor vessel 410, 420. As shown below, the locating pins 754 ensure proper alignment of the first bioreactor vessel 410 on the first bed plate 746 so that the first bioreactor vessel 410 is in the processing chamber. 722 is receivable within the recess 550 of the bottom plate 502 of the first bioreactor vessel 410.

With further reference to FIGS. 26-28, the bed plate 746 includes an integrated sensor for detecting proper alignment (or misalignment) of the first bioreactor vessel 410 on the first bed plate 746. 756 may further be provided. In one embodiment, sensor 756 is an infrared light beam, although other sensor types such as lever switches may be utilized without departing from the broader aspects of the invention. The sensor is configured to interact with the position verification structure 552 on the bottom plate 502 when the first bioreactor vessel 410 is properly installed on the first bed plate 746. For example, if the sensor 756 is an infrared light beam, the position verification structure 552 is a beam break (i.e., a flat claw), and a substantially IR-opaque position verification structure 552 is used, the first bioreactor vessel When 410 is fully seated on bed plate 746, the beam break blocks the infrared light beam (ie, breaks the beam). This signals controller 210 that first bioreactor vessel 410 is properly installed. 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 interrupted, this means that the first bioreactor vessel 410 is Indicates that it is not fully or properly seated on the bed plate 746 and that adjustment is required. Therefore, the sensor 756 on the bed plate 746 and the position verification structure 552 on the bottom plate 502 of the first bioreactor vessel 410 ensure that the first bioreactor vessel 410 is horizontal on the bed plate 746 before starting bioprocessing. (as determined by the alignment pins).

With still further reference to FIGS. 26-28A, bed plate 746 is additionally positioned to align with opening 556 in bottom plate 502 of first bioreactor vessel 410. Equipped with sensor 759. Temperature sensor 759 is configured to measure or sense one or more parameters within bioreactor vessel 410, such as, for example, a temperature level within bioreactor vessel 410. In one embodiment, the bed plate 746 additionally includes a resistance temperature sensor 760 configured to measure the temperature of the top surface 752 and a carbon dioxide level for measuring the carbon dioxide level within the bioreactor vessel. a sensor (located below the bed plate).

As further shown in FIGS. 26-28A, each bed plate 746, 748 includes an actuator mechanism 761 (eg, a motor) that includes, for example, a pair of opposing cam arms 762. The cam arm 762 is received within the slot 764 of the bed plates 746, 748, with a clearance position in which the cam arm 762 is positioned below the top surface 752 of the bed plate 746 and a clearance position in which the cam arm 762 is positioned above the top surface 752 of the bed plate. an engagement that extends and contacts an opposing flat engagement surface 554 of the bottom plate 502 of the first bioreactor vessel 410 when the first bioreactor vessel 410 is received onto the first bed plate 746. It is rotatable about the cam pin 766 between the mating position and the mating position. As described in detail below, the actuator mechanism is operable to tilt the bioreactor vessel over the bed plate to provide agitation and/or to assist in evacuation from the bioreactor vessel.

29-32, more detailed views of linear actuator 768 and drawer engagement actuator 740 within hardware compartment 724 of drawer 712 are shown. Referring to FIG. 29, as shown above, linear actuator 768 is operable to move drawer 712 between open and closed positions. In one embodiment, linear actuator 768 is electrically connected to a rocker switch 770 on the outside of housing 710 that allows user control of drawer movement. Linear actuator 770 controls movement of drawer 712 to prevent disturbance of the contents of the bioreactor vessel within drawer 712. In one embodiment, linear actuator 768 has a stroke of about 16" and a maximum speed of about 2 inches per second.

Referring now to FIG. 30, drawer engagement actuator 740 includes a lead screw 772 and a clevis arm 774 that attaches to front plate 751 within drawer 712. The drawer engagement actuator is operably connected to the pump assembly 738 and the solenoid array 736 and is operable to move the pump assembly 738 and the solenoid array 736 between the first clearance position and the engaged position. .

31 and 32 relatively clearly illustrate the clearance and engagement positions of pump assembly 738 and solenoid array 736. As illustrated in FIG. 31, in the clearance position, pump assembly 738 and solenoid array 736 are spaced apart from pump shoe 742 and pinch valve anvil 744, respectively. After actuation of lead screw 772, drawer engagement mechanism 740 linearly moves pump assembly 738 and solenoid array forward to the position shown in FIG. 32. In this position, the pump head of the pump assembly 738 engages the conduits 442, 450, 490 in the first tubing holder block 652, and the solenoid array 736 connects the piston/actuator of the solenoid array 736 to the pinch valve anvil 744. is positioned sufficiently close to the pinch valve anvil 744 to be able to pinch/tighten the respective fluid flow conduit of the second tubing holder block 654 against and thereby prevent flow through that fluid flow conduit. .

Referring again to FIG. 24, and with further reference to FIGS. 33-39, in operation, the drawer 712 may be controllably moved to the open position by actuating a rocker switch 770 on the outside of the housing 710. A disposable drop-in kit 600 containing a tubing module 650 (holding all the tubes and tubing tails of the flow architecture 400) and first and second bioreactor vessels 410, 420 is lowered into position within the processing chamber 722. . When kit 600 is lowered into processing chamber 722, pump shoes 742 are positioned such that pump tubes 442, 450, 490 are positioned between pump shoes 742 and pump heads 454, 456, 492 of peristaltic pump assembly 738. Received through clearance opening 660 in first tubing holder block 652. FIG. 35 is a perspective view of peristaltic pump assembly 738 showing the positioning of pump heads 454, 456, 492 with respect to each other. FIG. 36 illustrates the positioning of pump heads 454, 456, 492 with respect to pump tubes 442, 450, 490 when kit 600 is received within processing chamber 722. As shown there, pump tubes 442, 450, 490 are positioned between pump shoe 742 and pump heads 454, 456, 492. In operation, the pump heads 454, 456, 492 initiate, maintain, and stop fluid flow through the tubes 442, 450, 490 when the drawer engagement actuator 740 positions the pump assembly 738 in the engaged position. is selectively operable under the control of controller 210 to

Similarly, when the kit 600 is lowered into the processing chamber 722, the pinch valve anvil 744 connects the tubing tails 464a-f of the first fluid assembly 440, held by the second tubing holder block 654, to the second fluid assembly. 444 tubing tails 470a-d, first bioreactor line 414 and second bioreactor line 418 of first bioreactor vessel 410, first bioreactor line 424 of second bioreactor vessel 420 and a second bioreactor line 428, a sterile air source line 460, an interconnect line 450, and a filtration line 482 are positioned between the solenoid array 736 and the pinch valve anvil 744. Received through clearance openings 668, 670 in tubing holder block 654. This configuration is best illustrated in FIGS. 37-39, which show the solenoid array 736 and pinch valve anvil 744 before receiving the back plate 662 of the second tubing holder block 654 within the space 776. ).

As shown therein, each solenoid 778 of the solenoid array 736 has an associated opening in the back plate 662 of the second tubing holder block 654 for clamping the associated tube against the pinch valve anvil 744. 664). In this regard, solenoid array 736 and anvil 744 together form a pinch valve array (which includes the valves of first fluid assembly 440 and second fluid assembly 444, as well as the bioreactor line valves). namely, valves 432, 434, 436, 438, sterile line valve 462, interconnect line valve 452, and filtration line valves 486, 488). In particular, the pinch valves of the flow architecture 400 are provided by respective solenoids 778 (i.e., pistons of the solenoid) of the solenoid array 736 that operate/act on the respective anvils 744 while the fluid path/conduit is interposed therebetween. It will be done. In particular, in operation, each solenoid 778 clamps an associated fluid flow conduit against anvil 744 to direct fluid flow therethrough when drawer engagement actuator 740 positions solenoid array 736 in an engaged position. is selectively operable under control of controller 210 to block. The present invention contemplates that each fluid conduit is positioned between a planar anvil surface and a planar solenoid actuator head. Alternatively, the solenoid actuator head can be shaped, such as two tapered surfaces that meet at an elongated edge similar to a Phillips screwdriver, that are optimized to apply the desired pinching force to the resiliently flexible fluid conduit. It may also include a head. Still alternatively, the anvil surface is oriented toward each fluid conduit such that the planar solenoid head urges the fluid conduit against this laterally extending protrusion, closing the conduit to fluid flow therethrough. It may include an elongated protrusion or protrusion extending to the ridge.

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 separated by the perimeter of the opening and In particular, it is supported over the apertures 626, 628 by claws/protrusions 632. As the kit is lowered further, the bedplates 746, 748 pass through the openings 626, 628 and receive or otherwise engage the bioreactor vessels 410, 420. The shape of the openings 626, 628 and top surface 752 of the bed plates 746, 748 (e.g., the raised area 758 of the bed plate 746, 748 that corresponds to the tab/protrusion 632 of the tray 610) is similar to the bottom surface of the tray 610 and the tab/protrusion 632 of the tray 610. The bioreactor is configured such that the protrusion 632 is mounted below the top surface 752 of the bed plate 746, 748 such that the bioreactor vessel 410, 420 can be supported by the bed plate 746, 748 in spaced relationship with the bottom surface 620 of the tray 610. Allowing tray 610 to continue moving downward after containers 410, 420 are received by bed plates 746, 748. This ensures that the tray 610 does not interfere with the horizontal mounting of the bioreactor vessels 410, 420 on the bed plates 746, 748.

When the bed plates 746, 748 pass through the openings 726, 728 in the tray 610, the locating pins 754 on the bed plates 746, 748 lock into the corresponding recesses 550 in the bottom plate 502 of the bioreactor vessels 410, 420. This ensures that the bioreactor vessels 410, 420 are properly aligned with the bed plates 410, 420. When properly seated on the bed plate 746, 748, the beam break 552 breaks the light beam of the sensor 756 in the bed plate, indicating to the controller that the bioreactor vessel 410, 420 is in the proper position. . Since bed plates 746, 748 and alignment pins are at the same height, blocking of the light beam of sensor 756 by beam break 552 also ensures that bioreactor vessels 410, 420 are horizontal. In this properly installed position, the sensors 759 on the bed plates 746, 748 are aligned with the apertures 556 in the bottom plate 502, thereby controlling process parameters within the internal compartments of the bioreactor vessels 410, 420, respectively. make it possible to sense. Additionally, in the fully seated position, the cam arms 762 of the bed plates 746, 748 are aligned with the flat engagement surfaces 554 on the bottom plates 502 of the bioreactor vessels 410, 420, respectively.

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

As further shown in FIG. 40, in one embodiment, the sidewalls 718 and bottom of the drawer 712 (and the top wall of the housing) help minimize heat loss from the cover 788 and the processing chamber 722. an insulating foam layer 790 for heating, a film heater 792 for heating the walls as described above, and an internal metal plate 794. In one embodiment, the inner metal plate 794 may be formed from aluminum, although other thermally conductive materials may be utilized without departing from the broader aspects of the invention. Drawer 712 includes one or more brush seals 796 to help minimize heat loss from processing chamber 722 and other components of apparatus 700 from drawer 712 (e.g., housing 710 or other drawers). and a thermal insulation layer 798 to minimize or prevent the flow of thermal energy to the drawers 714, 716), etc.).

Referring again to FIG. 34, when the kit 600 is received into the processing chamber 722, the load cell 750 in the bottom of the processing chamber 722 adjacent to the second bed plate 748 connects the waste bag 472a to the tubing tail 470a. and through an opening 730 in tray 610 so that it can be positioned over load cell 750. As shown therein, when the kit 600 is received within the drawer 712, the second tubing holder block 654 connects the tubing tails 464a-f of the first fluid assembly 440 and the second fluid assembly 444. The tubing is held such that the tubing tails 470b-d of the storage layer extend into the auxiliary compartment 730 for connection of the storage layer. In one embodiment, sampling conduits 476a-476d extend into auxiliary compartment 730 as well.

41-44, the operation of the cam arms 762 of the bed plates 746, 748 is illustrated. As shown therein, the cam arms 762 have a retracted position in which they are positioned below the top surface of the bed plates 746, 748 and a retracted position in which they are rotated about the cam pin 766 and above the bed plates 746, 748. is movable between an engaged position in which it extends to engage the flat engagement surface 554 of the bioreactor vessel 410, 420 and lift the bioreactor vessel 410, 420 away from the bed plate 746, 748. The cam arms 762 are retracted under the top surface of the bed plates 746, 748 by default so that the bioreactor vessels 410, 420 are supported on the horizontal bed plates 746, 748 (and in particular Dowel pin 754; no power is required to maintain the bioreactor vessel 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 mounted 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 servo motors to maintain a horizontal position. In fact, with the cam arm 762 configuration of the present invention, the actuator only needs to be energized when tilting the bioreactor vessel for agitation/mixing, as described below, thereby providing heat to the processing chamber 722. The extent to which this is added is kept to a minimum.

As shown in FIGS. 41-43, the cam arms 762 may be operable sequentially to agitate the contents of the bioreactor vessels 410, 420. For example, when it is desired to agitate the contents of the bioreactor vessel 410, one of the cam arms remains mounted with the opposite end on the bed plate and the locating pin 754 on the non-raised end is placed on the bottom plate. While remaining received within a corresponding recess 550 in 502, one end of bioreactor vessel 410 is lifted away from bed plate 746 (and disengaged from locating pin 754 on bed plate 746). is activated. The raised cam arm is then rotated back to a clearance position under the bed plate and the opposing cam arm is rotated to an engaged position to lift the opposite end of the bioreactor vessel away from the bed plate and the locating pin.

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 be long. Allow cells to return to their home location (or be tested) at any time during the treatment period. Therefore, although the present invention contemplates that the bioreactor vessel may be provided with other rocking or agitation means, the overall height of the mixing mechanism is reduced by having two cam arms 762 on opposite sides of the bed plate. can be kept to a minimum. For example, it is possible to achieve ±5 degrees of movement with a central actuator (located in the center of the bed plate), but with a 0-5 degree movement of the container driven by cam arms on each side of the container. Approximately the same movement can be achieved, which effectively gives the vessel ±5 degrees of movement at half its height. Additionally, the movement of the cam arm 762 (e.g., the speed of cam arm rotation and the timing between opposing cam arms) maximizes wave formation within the container, maximizing the amplitude of the waves, and thus (ideally) can be adjusted to maximize uniformity and time to achieve homogeneity. Timing can also be adjusted based on the volume within the vessel with a given geometry to maximize mixing efficiency.

In one embodiment, an optical sensor 756 may be used to confirm that the first bioreactor vessel 410 has been correctly repositioned after each cam agitation operation. It is further contemplated that correct repositioning of the bioreactor vessel can be checked and verified even during alternating cam movements. This allows for rapid detection of misalignment in virtually real-time, allowing operator intervention to reposition the bioreactor vessel without substantial deviation from the bioprocessing operation/protocol. make it possible.

Figure 43 is a schematic diagram showing the position of fluid 800 within the bioreactor vessel during this agitation process. As shown in FIG. 42, in one embodiment, a homing sensor 802 integrated into the bed plate 746 determines when the cam arm 762 returns to a clearance position below the top surface of the bed plate 746. can be used by the controller for This is useful in coordinating the movement of the cam arms 762 to provide the desired mixing frequency within the bioreactor vessel. In one embodiment, cam arm 762 is configured to provide a tilt angle of up to 5 degrees with respect to bed plate 746.

Referring to FIG. 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 semi-spherical 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 to allow the bioreactor vessel 410 to tilt when the locating pin 554 is received within the recess 550. can.

In one embodiment, each drawer of bioprocessing device 700, such as drawer 712, desirably includes a flip-down front panel 810 that is hingedly mounted, as shown in FIGS. 45-50. Flip-down front panel 810 allows access to auxiliary compartment 730 without opening drawer 712, as best shown in FIGS. 45, 49, and 50. As will be seen, this configuration allows for in-process sampling and exchange of media bags. In connection with the above, in one embodiment, the auxiliary compartment 730 may be comprised of a plurality of telescoping sliding rails 812 providing attachment means 815 from which various reservoirs/medium bags can be suspended. Rail 812 is movable from a retracted position within compartment 730, as shown in FIG. 48, to an extended position out of compartment 730, as shown in FIG. 49. When the collection bag is full or when the media/fluid bag needs to be replaced, simply extend the rails 812 out and unclip the bag. A new bag is connected to each tail and can then be hung from the rail and slid back into the auxiliary compartment 730 without opening the drawer 712 or pausing processing. In one embodiment, rail 812 may be mounted on a laterally extending cross rod 814. Rail 812 may thus be laterally slidable on rod 814 and extendable from and retractable into the auxiliary compartment. In addition, when the drawer is open (Figure 46), the rail 812 runs around the rear cross rod to clear the compartment 730 and allow the user to screw the tubing tail towards the front of the compartment 730. It can be rotated, which results in 3 degrees of freedom.

As illustrated in FIG. 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 stowage position to an access position. For example, platform 820 may be mounted to move along a guide track 822 formed in a sidewall of auxiliary compartment 730.

Referring to FIG. 52, in one embodiment, bioprocessing device 700 may further include a low profile waste tray 816 received within housing 710 below each drawer, eg, drawer 712. A waste tray 816 is independently mounted on the drawer so that it is movable between closed and open positions. 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 chamber 819 for access by the operator. Chamber 819 allows easy storage and access to a large waste bag connected to the fluid flow path of overlying tray 600 without opening drawer 712. In addition, in the closed position, waste tray 816 positions chamber 819 in alignment with the drawer below and is sized and shaped to be operable to accommodate leaks from processing chamber 722 or auxiliary compartment 730. have

In one embodiment, each drawer includes a camera positioned above the processing chamber (e.g., above each bioreactor vessel 410, 420) to visually monitor the interior of the drawer 712 without having to open the drawer 712. can be provided. In one embodiment, the camera (or additional cameras) can be integrated with the bedplate assembly or on the sidewall looking laterally into the bioreactor vessel.

Accordingly, the second module 200 of the present invention allows for automation of cell processing to a degree previously unseen in the art. In particular, the fluid flow architecture 400, pump assembly 738, and pinch valve array 736 provide fluid handling (e.g., , fluid addition, transfer, draining, rinsing, etc.) can be automated. As explained below, this configuration also allows for hollow fiber filler concentration and cleaning, filterless perfusion, and line priming. Drawer engagement actuator 740 is also used for automatic engagement and disengagement of drop-in kit 600, further minimizing human touch points. In fact, human touch points may only be required for source/medium bag addition and removal, sampling, and data entry (eg, sample volume, cell density, etc.).

Referring to FIGS. 53-77, an automated approach for workflows with immobilized Ab coating, soluble Ab addition, and gammaretroviral vectors with amplification in the same vessel using a second module 200 and its fluid flow architecture 400. A general protocol is illustrated. This general protocol covers activation (illustrated in Figures 53-59), pre-transduction preparation and transduction (illustrated in Figures 60-71), amplification (Figures 72-76), and For some embodiments, the harvesting of a population of cells (FIG. 77) is provided in an automated and functionally closed manner. In describing the operation of a pinch valve, in the following the valve is in its closed state/position when it is not used for a particular operation. Thus, after a valve is opened to enable a particular operation, once that operation is complete, the valve is closed before proceeding to the next operation/step.

As shown in FIG. 53, in a first step, valves 432 and 468f are opened and first fluid assembly line pump 454 is activated to pump the antibody (Ab) coating solution into the first A reservoir 466f connected to fluid assembly 440 is routed through first port 412 to first bioreactor vessel 410. The antibody coating solution is incubated for a period of time and then drained through the interconnecting line to the waste reservoir 472a of the first fluid assembly 440 by opening the valves 434, 474a and activating the circulation line pump 456. As described herein, evacuation of bioreactor vessel 410 may be facilitated by tilting bioreactor vessel 410 using cam arm 462.

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

Referring to Figure 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 removed from the valve 468d. and 432 and is transferred to the first bioreactor vessel by opening 432 and activating pump 454. 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, pumping a second antibody (Ab) solution from reservoir 466a connected to first fluid assembly 440. through first port 412 to first bioreactor vessel 410;

After pumping the second antibody solution into the first bioreactor vessel, the second antibody solution reservoir 466a is rinsed and the rinsing medium is pumped into the first bioreactor vessel. . In particular, as shown in FIG. 2 to the antibody solution storage tank 466a, and rinses the storage tank. After rinsing, valve 432 is opened and the rinsing medium is pumped from reservoir 466a to first bioreactor vessel 410. In one embodiment, the second antibody solution reservoir 466a may be rinsed multiple times using this procedure.

After rinsing the second antibody solution reservoir 466a, the inoculum/seed cell bag 466d may also optionally be rinsed. In particular, as shown in FIG. Material/seed cells are sent to bag 466d and rinse the bag. After rinsing, valve 432 is opened and the rinsing medium is pumped from bag 466d to first bioreactor vessel 410 using pump 454. By pumping rinsing medium into the first bioreactor vessel 410 after rinsing the inoculum/seed cell bag 466d, the cell density within the first bioreactor vessel 410 is reduced. At this time, to measure one or more parameters of the solution in the bioreactor vessel prior to activation (e.g. to ensure that the desired cell density is present prior to activation) A sample may be taken. In particular, as shown in FIG. 58, valves 434, 452, and 432 are opened and pump 456 is activated, causing the first bioreactor vessel to flow along the first circulation loop of the first bioreactor vessel. 410 (i.e., from second port 416, through interconnecting line 450, and through first bioreactor line 414 and first port 412 of first bioreactor vessel 410). and return to the first bioreactor vessel 410). To collect a sample, a first sample container 280a (e.g., dip tube, syringe, etc.) is connected to a first sample tubing tail 476a, and valve 478a is opened, directing a portion of the flow to interconnecting conduit 450. and is diverted to the first sample container 280a for analysis.

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

Referring now to Figure 60, after activation, to prepare for transduction, valves 438 and 474b are opened and pump 456 is operated to pump RetroNectin solution from reservoir 472b to second bioreactor. A second bioreactor vessel 420 is delivered through a second port 426 of 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. be done. After incubation, all RetroNectin solution is then removed from the second bioreactor vessel 420 to waste storage by opening valves 438 and 474a and activating circulation line pump 456, as further shown in FIG. It is discharged into tank 472a. Note that during these RetroNectin coating, incubating, and draining steps (associated with the second bioreactor vessel 420), the activated cell population remains within the first bioreactor vessel 410. Note that RetroNectin or other reagents to increase the efficiency of genetic modification do not need to be utilized in all processes.

As shown in Figure 61, after RetroNectin coating, a rinse buffer bag 472b is connected to a second fluid assembly 444 (or one already 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 described above, the buffer may alternatively be pumped into the first port 422 of the second bioreactor vessel 420 by opening valves 452 and 436 instead. .

Referring now to FIG. 62, after a defined period of time, all buffer in the second bioreactor vessel 420 is drained into the second bioreactor vessel 420 by opening valves 438 and 474a and activating interconnected line pump 456. waste liquid storage tank 472a of fluid assembly 444.

At this point, a post-activation pre-concentration sample can be taken from the cells in the first bioreactor vessel 410, as shown in FIG. 63. As shown therein, valves 434, 486, 488, and 432 are opened and pump 456 is activated to circulate solution within first bioreactor vessel 410 through second port 434; through the interconnecting conduit, through the filtration conduit 48 and the filter 484, through the first bioreactor conduit 414 of the first bioreactor vessel 410, and through the first port 412 to the first bioreactor. Return to container 410. To collect a sample, a second sample container 280b (e.g., dip tube, syringe, etc.) is connected to the second sample tubing tail 476b, and valve 478b is opened, directing a portion of the flow to interconnecting conduit 450. and is diverted to the second sample container 280b for analysis.

Referring now to FIG. 64, depending on the concentration obtained from the sample, concentration may be performed by circulating the contents of the first bioreactor vessel 410 through a filter 484. As explained above, this is done by opening valves 434, 486, 488, and 432 and activating pump 456, which causes the solution in the first bioreactor vessel 410 to flow into the second bioreactor vessel 410. The solution circulates from port 416 of the first bioreactor vessel 410 through the second bioreactor line 418, through interconnect line 450, through filtration line 482 and filter 484, and through the first bioreactor vessel 410. It passes through bioreactor line 414 and returns to first bioreactor vessel 410 through first port 412 . As the fluid passes through filter 484, waste fluid is removed and permeate pump 492 directs such waste fluid through waste conduit 490 to waste reservoir 472a of 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.

Referring to FIG. 65, after enrichment, the enriched cell population in the activation container (ie, the first container 410 containing the enriched cell population) is washed to a constant volume through perfusion. In particular, as shown therein, the medium from the medium bag 466b of the first fluid assembly 440 is routed through the second port 416 such that a constant volume is maintained within the first bioreactor vessel 410. At the same time that the medium is pumped out of the first bioreactor vessel 410, the medium is pumped into the first bioreactor vessel 410 through the interconnect line 450 and through the first port 412. As media is added and removed from container 410, waste liquid may be filtered by filter 484 and directed to waste liquid storage tank 472a.

A post-wash sample may be collected from cells in the first bioreactor vessel 410 in a similar manner as previously 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 activated to circulate fluid within first bioreactor vessel 410 through second port 434. through the interconnecting line, through the filtration line 48 and the filter 484, through the first bioreactor line 414 of the first bioreactor vessel 410, through the first port 412 and through the first 410 into the bioreactor vessel 410. To collect a sample, a third sample container 280c (e.g., dip tube, syringe, etc.) is connected to the third sample tubing tail 476c, and valve 478c is opened, directing a portion of the flow to interconnecting conduit 450. and is diverted to a third sample container 280c for analysis.

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

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

Medium from medium bag 466b is then added to second bioreactor vessel 420 by opening valves 468b and 436 and activating pump 454, as illustrated in FIG. The total amount of solution in container 420 is increased to a predetermined volume. Referring to FIG. 70, the pre-transduction sample is then transferred by opening valves 438, 452, and 436 and activating circulation line pump 456 to transfer the solution in second bioreactor vessel 420 to second bioreactor vessel 420. (i.e., from the second port 426 through the interconnecting line 450 to the first bioreactor line 414 and the second bioreactor vessel 420). 1 back to the second bioreactor vessel 420 through port 422). To collect a sample, a fourth sample container 280d (e.g., dip tube, syringe, etc.) is connected to the fourth sample tubing tail 476d, and valve 478d is opened, directing a portion of the flow to interconnecting conduit 450. and is diverted to a fourth sample container 280d for analysis.

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

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

The pre-amplification sample is then transferred to the second bioreactor by opening valves 438, 452, and 436 and activating circulation line pump 456, as illustrated in FIG. 73. The solution in vessel 420 may be harvested by pumping it along the circulation loop of second bioreactor vessel 420 (i.e., from second port 426, through interconnecting conduit 450, and into second bioreactor vessel 420). (through first bioreactor line 414 and first port 422 of vessel 420 and back to second bioreactor vessel 420). To collect a sample, a fifth sample container 280e (e.g., dip tube, syringe, etc.) is connected to the fifth sample tubing tail 476e, and valve 478e is opened, directing a portion of the flow to interconnecting conduit 450. and is diverted to the fifth sample container 280e for analysis.

in the second bioreactor vessel 420 when analysis of the fifth sample taken indicates that all parameters are within the predetermined ranges necessary for successful amplification of the population of cells. The population of cells is incubated for a predetermined period of time, eg, 4 hours, to allow the cells to settle.

After this incubation period, or at a predetermined time thereafter, perfusion at the rate of one dose per day (1x perfusion) removes the spent/used medium from the medium bag 466b, as shown in FIG. The culture medium is pumped from the second bioreactor vessel 420 through the second port 426 (and through the interconnect line 450 to the waste reservoir 472a) at the same time that the second bioreactor is pumped through the first port 422. This is carried out by pumping into the reactor vessel 420. 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, the medium from the medium bag 466b is transferred into the second bioreactor vessel 420 at substantially the same rate as the spent medium is removed from the second bioreactor vessel 420 and sent to waste. is introduced into the second bioreactor vessel 420 to maintain a substantially constant volume within the second bioreactor vessel 420.

Sampling may then be performed as needed/desired to monitor the amplification process and/or determine when the desired cell density is reached. As described above, the sample opens valves 438, 452, and 436, activates circulation line pump 456, and removes the solution in second bioreactor vessel 420, as shown above. along the circulation loop of the second bioreactor vessel 420 (i.e., from the second port 426, through the second bioreactor line 428, and through the interconnect line 450). , back to the second bioreactor vessel 420 through the first bioreactor line 424 and first port 422 of the second bioreactor vessel 420). To collect a sample, another sample container 280x (e.g., dip tube, syringe, etc.) is connected to the sample tubing tail of sample assembly 448, and the valve on the tubing tail is opened, as shown in FIG. , a portion of the flow is passed through interconnect line 450 and diverted to sample container 280x for analysis. After each sampling operation, incubation without perfusion can be performed for a predetermined period of time, eg, 4 hours, to allow cells to settle before restarting perfusion.

After this incubation period, as shown in FIG. 76, perfusion at the rate of one dose per day (1x perfusion) removes the medium from the medium bag 466b, as shown in FIG. / Spent medium is pumped from the second bioreactor vessel 420 through the second port 426 (and through the interconnect line 450 to the waste reservoir 472a) through the first port 422 and at the same time into the bioreactor vessel 420. This perfusion is accomplished by opening valves 468b, 436, and 474a and activating first pump 454 and circulation line pump 456.

When sampling shows a viable cell density (VCD) of a predetermined threshold (e.g., 5 MM/mL), perfusion at a rate of two doses per day (2x perfusion) is performed as shown in Figure 76. , media from media bag 466b, and spent/spent media is pumped from second bioreactor vessel 420 through second port 426 (and through interconnect line 450 to waste reservoir 472a). This is accomplished by simultaneously pumping into the second bioreactor vessel 420 through the first port 422. 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, the medium from the medium bag 466b is transferred into the second bioreactor vessel 420 at substantially the same rate as the spent medium is removed from the second bioreactor vessel 420 and sent to waste. is introduced into the second bioreactor vessel 420 to maintain a substantially constant volume within the second bioreactor vessel 420.

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

Accordingly, the second module 200 of the bioprocessing system 10, and its flow architecture 400 and bioreactor vessels 410, 420, allow various bioprocessing operations to be performed in a substantially automated and functionally closed manner. Provide a flexible platform. In particular, FIGS. 53-77 illustrate exemplary general protocols that can be performed using the bioprocessing system 10 of the present invention (particularly using its second module 200). , the system is not limited in this respect. In fact, a variety of automation protocols may be enabled by the system of the present invention, including numerous customer-specific protocols.

In contrast to existing systems, the second module 200 of the bioprocessing system 10 is a functionally closed automation housing the first and second bioreactor vessels 410, 420 and fluid handling and fluid containment systems. systems, all of which are maintained in cell culture friendly environmental conditions (i.e., within a temperature and gas controlled environment) to enable cell activation, transduction, and amplification. As described above, the system 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. Alternatively, combine all steps in a single bioreactor vessel (e.g., first bioreactor vessel 410) or use both bioreactor vessels 410, 420 for end-to-end activation and cleaning. Provide flexibility to users. In one embodiment, a single amplification bioreactor vessel (eg, bioreactor vessel 420) can reliably produce a single dose of billions of T cells. Either single doses or multiple doses can be produced in situ with high recovery and high survivability. In addition, the system is designed to provide end users with the flexibility to implement different protocols for the production of genetically modified immune cells.

Some of the commercial advantages provided by the bioprocessing system of the present invention are: simplifying workflow, reducing labor intensity, reducing strain on cleanroom infrastructure, reducing failed nodes, cost including robust and scalable manufacturing technologies to commercialize products by reducing manufacturing costs and the ability to scale operations.

As described above with respect to the general workflow, the system of the present invention, the bioprocessing system 10, and the flow architecture 400 of the second module 200 and the bioreactor vessels 410, 420 are arranged in an automated, functionally closed The process of culture concentration, washing, slow perfusion, fast perfusion, and "round-robin" perfusion is to be performed in a fashion. For example, as described above, pump 456 on interconnecting line 450 may displace permeate pump 492 in the concentration step (typically at a percentage of circulation pump 456, such as, for example, about 10%). During operation, it can be used to circulate fluid from one of the ports on the bioreactor through filtration line 482 and filter 484 and then back to another port on the bioreactor. can. Concentration can be performed in open loop or stopped based on the measured volume removed from the bioreactor or accumulated in the waste liquid. In one embodiment, the filter, pump speed, filter area, number of lumens, etc. are all appropriately sized for the total number of cells and target cell density to limit shear contamination and excessive cell loss. will be done.

In one embodiment, as explained above, the system of the invention can also be used to wash, eg, remove residues such as residual viral vectors after incubation. Washing is as described above for enrichment, except that pump 454 on first fluid assembly line 442 is used to pump additional medium to replace fluid delivered from permeate waste pump 492. involves the same steps. The rate of introduction of new medium may correspond to the rate of fluid removal by permeate pump 492. This makes it possible to maintain a constant volume within the bioreactor vessel, and the residue grows exponentially with time as long as the contents within the bioreactor are well mixed (enough circulation can be made). can be taken out. In embodiments, this same process can be utilized for in-situ hollow fiber filtration-based washing of cell suspensions after activation to remove residues. For coated and uncoated surfaces, soluble activation reagent wash removal can also be performed via filter-based perfusion.

As also explained above, in a perfusion process, the pump 454 on the first fluid assembly line 442 can be used to add culture medium to a given bioreactor vessel, and the interconnecting line 450 The upper pump 456 is used to move the spent medium to a waste bag in the second fluid assembly. In one embodiment, gravity may be used to settle the cells, and the spent medium may be pumped out at such a rate that it does not significantly disturb the cells within the bioreactor vessel. This process may involve running pumps 454 and 456 open loop at the same speed. In one embodiment, one pump (454 or 456) may run at a set speed, and the speed of the other pump is determined by the bioreactor vessel mass/volume or waste bag mass/volume (or measured (mass/volume of the source bag).

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

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

For round-robin perfusion, the pumps can be run continuously, together intermittently (duty cycle), or sequentially (depending on the source , then waste, and repeat), thereby making it possible to keep the volumes/mass in the various bioreactor vessels at approximately the same level. Round-robin perfusion (a set of pumps running together intermittently and waiting at regular intervals) also means that the same two pumps can be used to perform multiple Allow perfusion of the vessel to occur. Additionally, round-robin perfusion allows for low effective exchange rates (such as about 1 volume/day) even if the pump does not have a large low dynamic range. Additionally, round-robin perfusion also allows each container to be perfused with a different medium as controlled by a valve within the first fluid assembly 440.

Additionally, in one embodiment, high-speed perfusion may be used for residue removal (eg, post-activation Ab removal and/or post-transduction residue removal). In a fast perfusion process, the perfusion process described above is much faster than the typical 1-5 volumes/day, for example between about 8-20 volumes/day, or on the order of only minutes to hours. It is possible to operate at values in excess of approximately 20 capacity/day, achieving a 1 log reduction. In one embodiment, perfusion rate is balanced against cell loss. In some embodiments, fast perfusion may allow the elimination of hollow filters 484 and still meet the biological need for quick removal of residue after several steps.

As further explained above, the system of the present invention utilizes a pump 454 on a first fluid assembly line 442 to pump fluid from another bag/reservoir connected to a second fluid assembly 444. A rinse buffer or fluid is used to facilitate rinsing of the bag/reservoir connected to the first fluid assembly 440. In addition, the fluid lines of the flow architecture/system 400 are purged with sterile air from a sterile air source 458 to prevent cells from becoming lodged in the lines and dying, or to prevent media or reagents from escaping. This can prevent them from sitting in the pipes and deteriorating or becoming useless. The sterile air source 458 may also be used to force reagents out of the lines to ensure that no more reagent than intended is pumped into the bioreactor vessels 410, 420. The sterile air source 458 also cleans the conduit to the connected bag (of the first or second fluid assembly 440, 444) and cleans the sterile tube welds to limit residue. can be used for Instead of, or in addition to, using a sterile air source 458 to clear the line, the line may be constructed such that the port through which the air is drawn is not submerged and the bioreactor vessel has an air balance port 530. can be cleaned using air drawn from one of the bioreactor vessels.

As described above, the system allows for closed drawer in-process sampling of the contents of a bioreactor vessel. During sampling, the container from which the sample is drawn uses cam arm 762 to circulate the contents of the container using circulation line pump 456 and draws the sample from interconnecting line 450 using sampling assembly 448. It may be stirred by stirring. In one embodiment, only non-bead-bound cells may be agitated.

Also as explained above, the system of the invention allows a population of cells to be harvested after a target cell density is achieved. In one embodiment, collecting the expanded population of transduced cells uses a pump 456 on the interconnecting conduit 450 to move the cells into a bag connected to the second fluidic assembly 444. or circulating the cells by an interconnected pump 456 to transfer the cells to a bag connected to the first fluid assembly 440. This process can be used for final collection or large sample volumes, or can be used to fully automate the sampling process (i.e., the syringe or bag is connected to the first fluidic assembly 440). by connecting the bioreactor to the syringe/bag, circulating the contents of the bioreactor vessel, and drawing a portion of the desired sample volume from the circulating contents by the fluid assembly pump 454 and moving it toward the syringe/bag). In such cases, the circulation pump 456 and valve can then be used to clear the fluid/cell circulation line. In addition, the pump 454 on the first fluid assembly line 442 uses air in the line to transfer the sample to the container without appreciable amounts of cells remaining in the line. Once complete, the entire aliquoted sample volume can be used to continue pushing into the sample container.

In the embodiments described above, the workflow is such that cell activation is performed in a first bioreactor vessel and the activated cells are transferred to a second bioreactor vessel for transduction and amplification. Although disclosed, in one embodiment, the system of the invention provides that the activation and transduction operations are performed in a first bioreactor vessel and the amplification of the genetically modified cells is carried out in a second bioreactor vessel. may be allowed to be executed within. 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. executed within. In one embodiment, the invention thus simplifies existing protocols by enabling the use of simplified, automation-friendly "one-pot" activation, transduction, and amplification vessels.

In one such embodiment, the T cell activator may be a micron-sized Dynabead and a lentiviral vector is used for transduction. In particular, as disclosed herein, micron-sized Dynabeads serve the dual purpose of isolating and activating T cells. In one embodiment, T cell activation (and isolation) may be performed in one of the bioreactor vessels 410 using Dynabead 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 FIGS. 60-71. After activation and viral transduction, the virus is then washed out of the bioreactor vessel 410 using the filterless perfusion method described above that retains the cells and micron-sized Dynabeads within the bioreactor vessel 410. Good for serving. This allows cell expansion within the same bioreactor vessel 410 used for activation and transduction. The filterless perfusion method additionally allows culture washing to be performed without first immobilizing activated 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 within the container as the virus is washed out. Nanometer-sized virus particles and residual macromolecules are fluidized and washed out during low-speed perfusion.

In one embodiment, after amplification, cells can be harvested in the manner described above in connection with FIG. 77. After harvesting, a magnetic bead removal process can be utilized to remove Dynabeads from the captured cells. In other embodiments, the steps of harvesting the expanded population of cells and removing the beads from the cells are performed simultaneously using perfusion, whereby the medium is introduced into the bioreactor vessel through the supply port. The cell culture medium containing the expanded population of cells is removed from the bioreactor vessel through an exhaust port within the bioreactor vessel. In particular, filterless perfusion can be used to perform bead removal of micron-sized beads by exploiting the difference in cell weight and cell Dynabead complex weight when final bead removal of the culture is required. can be used. To effect bead removal from the culture, the entire contents of the bioreactor vessel are mixed (eg, by using the cam arm 762 of the actuator mechanism in the manner previously described). After mixing/agitation, the heavy Dynabead sinks and settles onto the silicone membrane 516 within 10-15 minutes. In contrast, cells require more than 4 hours to settle on membrane 516. After a 10-15 minute holding period after mixing/agitation, the cell suspension can be slowly withdrawn using perfusion without disturbing the established Dynabeads. Media entry conduits may be used to maintain media height within the bioreactor vessel. Therefore, the invention described herein simplifies current Dynabead protocols by eliminating the need for several process-intermediate cell transfer and careful washing and bead removal steps, minimizing costs and potential risks. Keep it within limits. By performing bead removal of the culture at the same time as harvesting the cells, the need for additional magnetic devices or disposable parts typically required in the past can be eliminated.

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 ² ); All four unit processes of activation, transduction, washing, and amplification using Dynabead can be performed in the same bioreactor vessel in a fully automated and functionally closed manner. Therefore, the bioprocessing system of the present invention simplifies current protocols by eliminating the need for process-intermediate cell transfer and careful washing steps, minimizing cost and potential risks resulting from multiple human touchpoints. Keep it within limits.

In one embodiment, the two bioreactor vessels 410, 420 of the system contain either the same starting culture or two co-split cultures, e.g., CD4+ cells in one bioreactor vessel 410 and the other bioreactor vessel 410, 420. It can operate with CD8+ cells within container 420. Split cultures allow parallel independent processing and amplification of two cell types that can be combined before infusion into the patient.

Although a number of possible CAR-T workflows for the generation and amplification of genetically modified cells using the bioprocessing system of the invention are described above, other CAR-T workflows can also be used by the system of the invention. As such, the workflow described herein is not intended to be comprehensive. In addition, although the system of the invention, and in particular the second module 200 of the system, has been described in connection with the production of CAR-T cells, the system of the invention is suitable for both TCR-T cells and NK cells. It is also suitable for the production of other immune cells such as. Additionally, embodiments of the present invention incorporate the use of two bioreactor vessels 410, 420, where the output of the first bioreactor vessel 410 is added to a second bioreactor vessel 420 for additional processing steps. Although disclosed in a sequential process of steps (e.g., activation in a first bioreactor vessel and transduction and amplification in a second bioreactor vessel), in some embodiments, two bioreactor vessels can be used in the same workflow as a duplicate. Exemplary reasons for sequential use of second bioreactor vessels include residual chemical modifications that cannot be washed out of the first bioreactor that could be harmful in later steps or if overexposure of cells occurs in earlier steps (e.g. , coated or immobilized reagents) or the need to pre-coat the bioreactor surface before adding cells (e.g. RetroNectin coating).

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

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

The embodiments described above provide process monitoring sensors that are integrated with the bioreactor vessel and/or bedplate (e.g., on the membrane, integrated within the membrane, on the vessel sidewall, etc.). Although illustrated, it is contemplated that in other embodiments, additional sensors may be added to fluidic architecture 400, such as along the fluid flow conduit itself. These sensors may be disposable, compatible sensors for monitoring parameters such as pH, dissolved oxygen, density/turbidity (optical sensors) conductivity, and viability within circulating fluids. By arranging the sensor in a circulation loop (eg, the circulation loop of the first bioreactor vessel and/or the circulation loop of the second bioreactor vessel), the structure of the vessel may be simplified. Additionally, in some embodiments, sensors along the circulation loop may yield a more accurate representation of the vessel contents when circulated (versus measuring when the cells are stationary within the vessel). not). Additionally, if desired, a flow sensor (eg, ultrasound-based) can be added to the flow loop to measure pump performance and used with algorithms to correct pump parameters.

As indicated above, the first and third modules 100, 300 may be any system or system known in the art capable of performing cell enrichment and isolation, as well as harvesting and/or compounding. It can take any form of device. Figure 78 shows the use as a first module 100 in a bioprocessing system 10 for cell enrichment and isolation using various magnetic isolation bead types (including, for example, Miltenyi beads, Dynabead, and StemCell EasySep beads). 9 illustrates one possible configuration of a device/apparatus 900 that may be used. As shown therein, the apparatus 900 includes a centrifugal processing chamber 912, a high dynamic range peristaltic pump assembly 914, a pump tube 916 of a suitable inner diameter to be received by the peristaltic pump assembly, a stopcock manifold 918, and an optical A base 910 is provided that houses a sensor 920 and a heating and cooling mixing chamber 922. As shown below, 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, pump 914 is rated to output a flow rate as low as about 3 mL/min and as high as about 150 mL/min.

As further shown in FIG. 78, the apparatus 900 includes a generally T-shaped structure extending from the base 910 and comprising a plurality of hooks 926 for suspending a plurality of process and/or source containers or bags. A hanger assembly 924 may be included. In one embodiment, there may be six hooks. Each hook is equipped with an integrated weight sensor to detect the weight of each container/bag. In one embodiment, the bags 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, and a post-isolation waste bag 942. , a wash waste bag 944, a media bag 946, a release bag 948, and a collection bag 950.

Device 900 is configured for use with or comprises a magnetic cell isolation holder 960, as presented herein. Magnetic cell isolation holder 960 can be removably coupled to a magnetic field generator 962 (eg, magnetic field plates 964, 966 in FIG. 80). Magnetic cell isolation holder 960 houses a magnetic retention element or material 968, such as a separation column, matrix, or tube. In one embodiment, the magnetic cell isolation holder 960 is provided in U.S. Pat. No. 829,615. Device 900 may be under the control of a controller (eg, 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 that is available for bead addition, as described below.

Referring now to FIG. 79, a general protocol 1000 for 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 magnetic isolation beads, a washing step 1012 may then be performed to remove residues in the Dynabead suspension. After concentration, the cells are then transferred to a process bag 932 in step 1014. In some embodiments, a portion of the enriched cells may be stored in a first storage bag 938 at step 1016 before being transferred to the process bag 932. At step 1018, magnetic isolation beads are injected into the process bag, such as by using syringe 952 at step 1020. In one embodiment, the magnetic isolation beads are Miltenyi beads or StemCell EasySep beads. If Dynabeads are utilized, the cleaned Dynabeads from step 1012 are resuspended within process bag 932. In one embodiment, instead of utilizing a syringe, the magnetic isolation beads may be contained within a bag or container connected to the system, and the beads may be drawn into the system by a pump 914.

The beads and cells within process bag 932 are then incubated for a period of time at step 1020. This step involves cycling the fluid out of the process bag 932, through a loop, and then back into the bag. In embodiments where the magnetically isolated beads are Miltenyi nanosized beads, a sedimentation wash is performed in step 1022 to remove excess nanosized beads, and a portion of the incubated bead-bound cells are stored in a second storage in step 1024. storage bag 940. After incubation, bead-bound cells are isolated at step 1026 using a magnet, eg, magnetic field plates 964, 966 of a magnetic cell isolation holder 960. Residual bead-bound cells are then rinsed and isolated in step 1028. Finally, in embodiments where Miltenyi or Dynabead are utilized, the isolated bead-bound cells are collected in a collection bag 950 at 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 is performed.

A more detailed description of the general protocol of FIG. 79 using apparatus 900 is discussed in more detail below, with particular reference to FIG. 80, which is a schematic diagram of flow architecture 1100 of apparatus 900. First, the process of concentration (step 1010) includes transferring the apheresis product and wash buffer contained in the source bag 930 from the wash buffer bag 936 to the chamber 912 and washing it using the wash buffer; It begins by reducing the amount of platelets and serum. At this point, the concentrated raw material is placed in chamber 912. To begin the isolation process, the separation column received by the magnetic cell isolation holder 960 begins the flow of buffer from the isolation buffer bag 934, through the manifold 918, through the column, and into the process bag 932. It is primed by priming the column.

In some embodiments, such as when Dynabeads are utilized as magnetic isolation beads, as disclosed above, a washing step (step 1012) is performed to remove residues within the bead suspension buffer. . The wash step involves using a syringe 952 to circulate the beads within a process loop 1110 (e.g., from process bag 932, through peristaltic pump tubing 914, through manifold 918, and back to process bag 932). injecting, cleaning the process loop 1110, and then while the magnetic field generator 962 is “ON”, i.e., while the holder is magnetically coupled with the active magnetic field generator 962 to the permanent magnet. , or alternatively, capturing the beads by flushing the process bag 932 into an isolation waste bag 942 when the electromagnetic field is actively generated by using an electromagnet, in each case capturing the beads. is captured in the “ON” state. In embodiments where cleaning is not desired, process bag 932 is flushed to isolated waste bag 942 to ensure process bag 932 is clean. As used herein, in the case of a permanent magnet, ON means that the magnetic retention element or material 968 (e.g., separation column, matrix, or tube) is in position within a magnetic field. . OFF means the tubing section is removed from the magnetic field.

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

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

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

After adding the magnetic isolation beads, the cells may be incubated for a period of time (step 1020), as described above. 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 (using a heated and cooled mixing chamber 922). etc.). The contents of second storage bag 940 are then transferred back to 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 expelled into a second storage bag 940, which is then transferred to a process bag 932 and transferred to the second storage bag 932. Rinse bag 940.

In either embodiment, the cells are then incubated with the magnetic isolation beads by cycling the cells along process loop 1110 for a defined incubation period. After incubation, process loop 1110 is cleaned.

As explained above, after incubation, an optional step of washing out excess beads (eg, nano-sized beads) may be performed (step 1022). Washing out excess nano-sized beads includes initiating flow from process bag 932 to second storage bag 940 and drawing the contents of second storage bag 940 into processing chamber 912. transferring the buffer from the separate buffer bag 934 to the process bag 932; transferring the contents of the process bag 932 to a second storage bag 940; and transferring the contents of the second storage bag 940 to the processing chamber. This includes drawing in. The steps of flowing from isolation buffer bag 934 to process bag 932 and then to second storage bag 940 can be repeated as necessary to wash out excess beads. In one embodiment, chamber 912 may then be filled with buffer from isolation buffer bag 934 and chamber 912 may begin to rotate, then expel the supernatant into waste bag 742. These steps may be repeated as necessary. In one embodiment, cells within the chamber are ejected into a process bag 932, buffer from an isolation buffer bag 934 is drawn into the chamber 932, and the chamber is then ejected into the process bag 932. This process may also be repeated as necessary. Process loop mixing and process loop cleaning are then performed.

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

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

In one embodiment, rinsing without resuspension includes pumping buffer from isolation buffer bag 934 to process bag 932, rinsing process loop 1110, cleaning process loop 1110, and generating a magnetic field. This can be performed by flowing from the process bag 932 to the waste bag 942 with the device 962 in the “ON” state.

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

In one embodiment, residual waste is cleaned by pumping buffer from isolation buffer bag 934 into process bag 932 and flowing from process bag 932 to waste bag 942 with magnetic field generator 962 "ON". obtain.

The isolated bead-bound cells are then collected after rinsing to isolate residual bead-bound cells (step 1028). If 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 removed with the magnetic field generator 962 turned "OFF". is pumped through column 968 to collection bag 950. Alternatively, buffer from isolation buffer bag 934 is pumped to process bag 932, which is then pumped to collection bag 950 with magnetic field generator 962 "OFF". Sent. This second method provides for post-isolation washing. In a third method, media from media bag 946 is pumped through column 966 to process bag 932 (if no post-isolation wash is required). Alternatively, buffer from isolation buffer bag 934 is pumped through column 966 to process bag 932 (if post-isolation washing is desired). Then, in either process, the contents of process bag 932 are circulated within process loop 1110, process loop 1110 is cleaned by returning to process bag 932, and the contents of process bag 932 are collected by a pump. into bag 950 to collect bead-bound cells.

If 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, cells/beads are pumped from bag 948 through a column to process bag 932 with release buffer circulated within process loop 1110 with the magnet "OFF" and then the fluid is It can be resuspended by cleaning the process loop by returning it to bag 932. The incubate and collect is then incubated in the process loop 1110 with the magnet "ON", cleaning the process loop 1110, and pumping it from the process bag 932 through the column 966 to the collection bag 950. Collects the released cells, pumps buffer from isolation buffer bag 934 to process bag 932, and pumps the contents of process bag 932 through column 966 to collection bag 950 to remove the residue. This is done by collecting. The released beads (step 1032) are then circulated in process loop 1110 by pumping buffer from isolation buffer bag 934 through column 966 and into process bag 932 with the magnet "OFF". Loop 1110 may be cleared and discarded by pumping the contents of process bag 932 to waste bag 942.

In connection with the above, in one embodiment, washing/concentrating (step 1034) includes pumping the contents of collection bag 950 into processing chamber 912 and pumping buffer from isolation buffer bag 934 into process bag 932. This can be performed by transferring the buffer from the process bag 932 to the processing chamber 912. A wash cycle is then performed by filling the processing chamber 912 with buffer from the isolation buffer bag 934, rotating the chamber 912, dispensing the supernatant into the waste bag 942, and repeating the rotation and dispensing steps as many times as necessary. can be done. Finally, transferring the cells to the collection bag after washing/concentration involves transferring the medium from the media bag 946 to the collection bag 950, pumping the contents of the collection bag to the processing chamber 912, and transferring the cells to the collection bag 950. may be accomplished by discharging the contents of the collection bag 950 into the collection bag 950 and then manually clearing the conduit between the processing chamber 912 and the collection bag 950.

In one embodiment, one of the bags, e.g., process bag 932, is configured to allow sterile air to enter the system ( A top port 1118 may be provided with a filter so that it can be introduced (when the process bag 932 is empty). Clearing the line can be accomplished as a first step in the concentration/isolation process and/or during process execution. In one embodiment, air from collection bag 950 may be used to clear any of the system lines (e.g., air from collection bag 950 may be used to clear process line 1112). , the air within process line 1112 is then used to clear the desired tubing lines (i.e., lines 1114, 1116, etc.), thereby filling process line 1112 with liquid from process bag 932. , and finally the air from the collection bag 950 can be used again to clear the process line 1112).

In one embodiment, the processing bag 932 is blow molded to the sides (higher than the liquid level) to limit micron-sized beads from sticking to the side walls, especially during long accelerated mixing in circulation-based incubations. It has a 3D shape with a defined air pocket (which has a large angle on top).

In one embodiment, the syringe 952 allows for the addition of small volumes (such as bead suspension aliquots) to the circulation-based flow loop 1110. Additionally, fluid from flow loop 1110 may be drawn into syringe 952, thereby further removing and cleaning syringe 952 of debris.

In one embodiment, one of the sensors 920 may be configured to measure fluid flow. For example, one of the sensors 920 may be a bubble detector or an optical detector that can be used as a secondary verification measurement means to ensure accurate flow control in addition to the load cell integrated with the hook 926. good. This can be used in practice during isolation when it is desired 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 near empty within some expected tolerance of load cell variation, and then the bubble detector 920 identifies the liquid/air interface that follows to stop the flow. Thus, the sensor 920 may dislodge cells or create a slug that exposes the cells to a dry environment, or inadvertently draws material into the waste bag in situations where the pump does not stop after the process bag is completely emptied. may be used by the controller to prevent air entrainment. Accordingly, in one embodiment, bubble detector 920 may be used in conjunction with a load cell that is integrated with a hook, thereby improving volume control accuracy, thereby reducing cell loss, and/or Or air can be prevented from entering the column tubing and into the column.

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

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

In some embodiments, system 900 can be used to elute isolated/captured bead cell complexes. In particular, it is contemplated that the air-liquid interface can be used to assist in removing complexes from tube sidewalls or column interstitial spaces. 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, packed layers of beads/bead-bound cells can be difficult to remove by flow control alone without significantly increasing the shear rate (which has a potentially negative impact on cell viability). Therefore, in relation to the flow rate, it is possible to remove the bead cell complex without removing it from the magnet.

In connection with the above, system 900 supports the concept of eluting positively selected bead cell complexes directly into the selected medium (based on downstream steps). This eliminates buffer exchange/wash steps. In one embodiment, it is also envisaged to elute directly into the medium and viral vector to start the incubation. This concept may also allow adding viral vectors to the final bag. In one embodiment, instead of eluting bead-bound cells with buffer, culture medium can be used as the elution fluid. Similarly, release buffer can be used to elute StemCell beads for subsequent release of cells from the beads. By replacing the buffer with media within parts of the system 900, dilution can be minimized.

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

One embodiment of the magnetic cell isolation holder (960 of FIG. 78) will now be described in more detail with particular reference to FIGS. 81-87. Magnetic bead-based cell selection involves isolating specific cells from a cell mixture by targeted binding of cell surface molecules to antibodies or ligands on magnetic beads (e.g., beads of the types described above). . After binding, cells bound to the magnetic beads can be separated from the unbound population of cells. For example, a cell mixture comprising bound and unbound cells may be passed through a separation column positioned within a magnetic field generator that captures the magnetic beads and thus the associated bound cells. Unbound cells pass through the column without being captured.

Some magnetic cell isolation techniques may incorporate nano-sized beads (e.g., beads about 50 nm in diameter or less), whereas other techniques may use larger beads (e.g., beads about 2 μm or more in diameter). . Similar beads may be desirable because, for example, smaller bead sizes may avoid receptor activation on target cells. Additionally, downstream steps may skip bead removal since nanosized beads may have little impact on downstream processing or cell function. However, smaller nanosized magnetic beads can be separated using magnetic cell isolation procedures that involve amplifying the applied magnetic field gradient using a magnetic field gradient intensifier. In contrast, larger beads have a higher magnetic moment. Therefore, isolation of some larger beads may not involve magnetic field gradient enhancement devices. However, larger beads may nevertheless be used in conjunction with an additional cell bead separation step. Therefore, depending on the size and/or type of magnetic beads used, the workflow, the appropriate magnetic parameters, and/or the isolation device itself may vary, and this makes magnetic bead-based cell isolation techniques It makes it complicated.

In particular, since beads can differ in material and magnetic properties (including, but not limited to, size, permeability, saturation magnetization, resistivity, surface properties, and mass density), separation conditions also affect bead properties. and may involve magnetic fields of different strengths and/or different gradients. In other words, the magnetic field parameters of the magnetic field generator can be different for magnetic cell isolation procedures using beads with different materials and magnetic properties. The present approach eliminates the workflow step of adjusting the magnetic field generator or its parameters between magnetic cell isolation procedures using beads of different sizes. In one embodiment provided herein, a magnetic cell isolation holder is arranged such that the magnetic cell isolation holder is associated with desirable magnetic field properties for cell separation when used with appropriately sized beads. The magnetic field generator is configured to be used in conjunction with a magnetic field generator to position the beads within a magnetic field. The magnetic field generator may apply the magnetic field using preset (eg, fixed) magnetic field parameters or static magnetic field generator elements. In this way, the operator may avoid the hassle of changing the magnetic field parameters according to the selected beads. Instead, by selecting an appropriate magnetic cell isolation holder, the magnetic field acting on the cells is suitable for separation. Furthermore, when using beads of different sizes and/or with different desired magnetic field properties, different magnetic cell isolation holders position the beads in respective positions within the applied magnetic field associated with the respective desired magnetic field properties. can be selected.

For example, different magnetic cell isolation holders can be sized and shaped according to the desired positioning of cells (eg, target cells within a cell mixture) within the magnetic field generated by the magnetic field generator. In one embodiment, each magnetic cell isolation holder is configured such that when the magnetic cell isolation holder is loaded into a magnetic bead-based cell isolation system comprising a magnetic field generator, the cells in the magnetic field isolation holder are in a cell mixture. a channel or other cell receptacle positioned within a magnetic field that has properties suitable for the separation of magnetic beads of a particular type (e.g., based on bead material, shape, size, and/or size range) from Including the body. By selecting a magnetic cell isolation holder associated with a particular bead type, appropriate separation can be achieved without changing the settings for the magnetic isolation device or the magnetic field generator of the magnetic isolation device.

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

In another embodiment, a magnetic cell isolation holder may be provided with multiple passageways for use with beads of different sizes, and the user can select the appropriate passageway associated with the desired bead type. You can choose. For example, a magnetic cell isolation holder may include a first arrangement of passageways (e.g., configured to accommodate a first cell separation column) for use with beads having a first diameter; and a second configuration of passageways (eg, configured to accommodate a second cell separation column) for use with beads having a larger diameter. When the magnetic cell isolation holder is inserted into the magnetic cell isolation device and a magnetic field is generated, the passages in the first configuration have a higher magnetic field compared to the passages in the second configuration within the magnetic cell isolation holder. It may be placed in a position where it receives strength.

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

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

System 2100 is configured to be used with magnetic cell isolation holder 2134, which is equivalent to magnetic cell isolation holder 960 described above with reference to FIG. Magnetic cell isolation holder 2134 may be removably coupled (e.g., loaded, positioned with respect to ). System 2100 may be under the control of controller 2150 and operates according to instructions executed by processor 2152 and stored in memory 2154. Such instructions may include magnetic field parameters. System 2100 may include any or all of the illustrated components.

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

At step 2212, magnetic beads (eg, beads) in bead addition syringe 2118 are added to process bag 2106. At step 2214, magnetic beads are incubated with the cell mixture within process bag 2106. Incubating materials (eg, cell mixtures and beads) may be circulated in and out of process bag 2106 via process pump 2114 to facilitate sufficient binding between target cells and magnetic beads. At step 2216, source bag 2104 is decoupled from source pump 2112 and incubate removal 2102 is fluidly coupled to source pump 2112. Excess incubate material is then removed from the process bag 2106 via source pump 2112 and deposited at incubate removal 2102. At step 2218, magnetic cell isolation is performed on the bead-labeled cell mixture. The magnetic cell isolation holder 2134 is then coupled to a magnetic field generator 2121 that generates a magnetic field under predetermined magnetic field parameters. The bead-labeled cell mixture from process bag 2106 flows through magnetic cell isolation holder 2134 via magnetic isolation pump 2116. In one embodiment, magnetic cell isolation holder 2134 houses a magnetic retention element or material, such as a separation column, matrix, or tube. The bead-labeled cells are then magnetically retained within the tube or column matrix of the magnetic cell isolation holder 2134 and unretained material flows through the magnetic cell isolation holder 2134 to the waste bag 2132. In an optional step, the buffer or medium can rinse the process bag and the magnetic cell isolation procedure can be repeated. At step 2220, magnetic cell isolation holder 2134 is removed from the magnetic isolation device. Then, in step 2222, the retained cells are eluted by flushing the magnetic cell isolation holder 2134 with a high flow rate of fluid, such that the viscous forces of the fluid act on the retained magnetic beads. Overcoming magnetic force. The fluid and bead-labeled cells are then collected into collection bag 2130. In step 2224, the bags (eg, collection bag 2130, waste bag 2132, buffer bag 2108, and media bag 2110) are sealed, and the magnetic cell isolation holder 2134 is then disposed of, in one embodiment.

83A and 83B are top views of different configurations of a magnetic cell isolation holder 2302 (e.g., magnetic cell isolation holder 2134 of FIG. 81) positioned within the magnetic isolation device 2300 of FIGS. 83A and 83B. be. FIG. 83A shows magnetic cell isolation holder 2302 in an unloaded configuration in magnetic cell isolation device 2300. Magnetic cell isolation holder 2302 may include a body portion 2301, which may be formed from a suitable non-magnetic material configured to house a cell isolate and coupled to magnetic isolation device 2300. . Magnetic cell isolation holder 2302 includes one or more passageways formed in or through body portion 2301 through which a cell mixture may flow. Although FIG. 83A shows two separate passages 2303 and 2305, it will be appreciated that the magnetic cell isolation holder 2302 may include only one passage, two or more passages, etc. Referring to passageway 2303, passageway 2303 can be configured to house a magnetic retention material 2304 that is configured to retain cells bound to magnetic beads and allow unbound cells to pass through under a magnetic field. . Similarly, passageway 2305 may also contain magnetically retentive material 2306. The magnetically retentive materials 2304, 2306 may be the same or different. It is also possible to omit the retaining material, but this would result in less efficiency; for example, the passageway 2305 could be a hollow tube. Additionally, passageways 2303, 2305 may be of different sizes and locations with respect to end surface 2307 of body portion 2301. For example, the distance 2315 between the end surface 2307 and the center point of the passageway 2303 may be different from the distance between other passageways of the body portion 2301 with respect to the end surface 2307. In this manner, the passageway may be subjected to a magnetic field that is correlated to its position within the body portion 2301.

End surface 2307 may be configured to abut a stop portion or surface 2311 of frame 2319. Frame 2319 may be configured to conduct magnetic flux. Although body portion 2301 is shown terminating at a point on end face 2307, it will be appreciated that other configurations are also contemplated. FIG. 83B shows a loaded configuration in which the magnetic cell isolation holder 2302 is positioned within the receiving area 2316 of the magnetic field generator 2313. Loading may include advancing end surface 2307 toward stop surface 2311 until end surface 2307 abuts the stop surface. Nevertheless, in the loaded configuration, a portion of body portion 2301 remains outside receiving area 2316. Thus, in one embodiment, one or more passageways of body portion 2301 may be positioned to be within receiving area 2316 when loaded.

Magnetic isolation device 2300 may also include doors or other features configured to reduce leakage of magnetic fields outside of receiving area 2316. The steel backing 2308 and door 2318 of the frame 2319 of the magnetic isolation device 2300 are made from a soft magnetic material (eg, 1018 steel). They become magnetized in the presence of a magnetic field and demagnetize when the field is removed. When the magnetic cell isolation holder 2302 is not inserted into the receiving area 2316 of the magnetic field generator 2313, the door 2318 of the magnetic field generator 2313 closes the gap with the help of a compressed spring attached to either door. , thereby surrounding the steel backing 2308 and the magnetic flux within the door 2318. This prevents leakage of magnetic flux into passageways 2303, 2305 when demagnetization is desired for some processes such as elution.

FIG. 83B shows magnetic cell isolation holder 2302 in a loaded configuration in magnetic isolation device 2300. When the magnetic cell isolation holder 2302 is fully inserted into the receiving area 2316 of the magnetic field generator 2313, the positions of the passageways 2303, 2305 are aligned with the geometry of the magnetic cell isolation holder 2302 and the magnetic isolation device 2300. Determined by shape. A portion of the backing 2308 (e.g., stop surface 2311) of the magnetic isolation device 2300 becomes part of the magnetic cell isolation holder 2302 when the magnetic cell isolation holder 2302 is fully inserted into the magnetic field generator 2313. It can come into contact with you. Additionally, although the magnetic cell isolation holder 2302 has the tapered shape of FIGS. 3A and 3B, any suitable shape of the magnetic cell isolation holder 2302 may be used.

Door 2318 of magnetic field generator 2313 opens to allow insertion of magnetic cell isolation holder 2302 between magnetic field plates 2312, 2314 of magnetic field generator 2313. For example, the location of the passageway 2303 within the magnetic field generator 2313 may be such that it covers placement within the magnetic field of the highest field strength (ie, 0.5T). In another example, the location of the passageway 2305 within the magnetic field generator 2313 allows for placement in the magnetic field of the highest magnetic field gradient (i.e., 50T/m) while meeting the magnetic field strength requirements of the magnetic beads (i.e., 0.15T). It may be considered as a target range.

To elute retained beads (e.g., beads or bead-bound cells) from magnetic retention materials (e.g., magnetic retention materials 2304, 2306), an external magnetic field moves isolation holder 2302 into an unlatched position (i.e., 83A (unloaded configuration)). When no external magnetic field is needed near the passageways 2303, 2305, the door closes to ensure that there is no leakage of magnetic flux affecting the passageways 2303, 2305. A high flow rate of fluid then flows through the passageways 2303, 2305, which creates large shear forces on the retained beads. When the viscous force is greater than the retentive force (ie, the magnetic force due to the residual magnetic field), the beads are washed away from the magnetically retentive material in channels 2303, 2305 and are collected. However, in other embodiments, the applied magnetic field may be truncated under control of controller 2150.

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

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

Although some disclosed techniques involve positioning a magnetic cell isolation holder, such as the disclosed one, within a fixed position magnetic field generator, it is understood that other implementations may also be contemplated. will be done. For example, the magnetic field generator may move relative to a magnetic isolation holder loaded in a fixed position frame.

FIG. 85 shows a flowchart for a method 2500 of magnetic cell isolation that can be used with a magnetic isolation device. In step 2502, a first cell mixture is prepared by incubating the cell mixture with a set of magnetic beads having desired characteristics (eg, size, type, ligand, etc.). After a sufficient period of time has elapsed to ensure that target cells are labeled with magnetic beads, excess incubation mixture is removed. In another embodiment, a portion of the incubated mixture may be removed and evaluated for quality control purposes, i.e., excess incubated mixture may be evaluated, thereby assessing binding properties. may be used as In step 2504, the first magnetic cell isolation holder 2302 may be coupled within the receiving area 2316 of the magnetic field generator 2313. In step 2506, the magnetic field generator 2313 generates a magnetic field within the receiving area 2316 of the magnetic field generator 2313. In step 2508, the first cell mixture flows through a passageway (eg, one or more of passageways 2303 or 2305) in first magnetic cell isolation holder 2302. The magnetic bead-labeled cells within the cell mixture are transferred to the magnetic retention material of the first magnetic cell isolation holder 2302 (e.g., one or more of magnetically retaining materials 2304 or 2306). In step 2510, the generation of the magnetic field is stopped by removing the first magnetic cell isolation holder 2302 from the receiving area 2316 of the magnetic field generator 2313 (or by terminating the application of the magnetic field), so that the magnetic cell isolation Demagnetization of the separation holder 2302 is performed. In step 2512, the retained or isolated cells and beads from the first magnetic cell isolation holder 2302 are magnetically retained within the passageway of the magnetic cell isolation holder 2302 with a fluid having a high flow rate. The beads or cells that have been exposed are collected by elution or by another suitable method. At step 2514, magnetic cell isolation holder 2302 may optionally be disposed of. Steps 2522 to 2534 mirror steps 2502 to 2514, but instead transfer cells labeled with a set of differently sized beads in a second cell mixture to a second (i.e., different) magnetic cell monomer. It may be passed through a passageway within separate holder 2302 or through a different passageway of first magnetic cell isolation holder 2302. Steps 2522 to 2534 illustrate how to use two different magnetic cell isolation holders, but the two magnetic cell isolation holders are instead the same magnetic cell isolation holder with different passages for each cell mixture. It will be appreciated that it can be a separate holder. In addition, the second cell mixture may be the resulting cell mixture that passes through the first magnetic cell isolation holder 2302 from step 2508 without retained bead-labeled cells.

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

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

Figure 87 shows the magnetic field distribution due to the permanent magnet and backing steel of the magnetic field generator showing different magnetic field characteristics of the magnetic field in different configurations. As disclosed, the magnetic field parameters for separation are different for beads of different sizes (eg, beads). Larger beads have a higher magnetic moment and therefore require a lower magnetic field gradient to generate an equal amount of force when compared to smaller beads that have a lower magnetic moment. The magnetic force can be expressed as Fmag=M·∇B, where M is the magnetic moment and ∇B is the magnetic field gradient. To ensure the highest magnetic moment, the magnetic material must be saturated at the external magnetic field strength (ie 0.15T for Dynabead as described herein). When the magnetic force is greater than the viscous force in the flow field, the magnetic beads move in the direction of the magnetic force until they reach the wall of the tube or the sphere of the column matrix.

In some embodiments, the disclosed technology can be used to isolate cells for chimeric antigen receptor cell therapy (or CAR-T). CAR-T involves isolating several types of white blood cells from peripheral blood mononuclear cells (PBMCs), ie, T cells. Target cells (T cells) are modified with receptors that allow them to recognize and attack cancer. Additionally, the disclosed technology can be used in conjunction with any suitable type of beads, such as Miltenyi nanosized microbeads (50 nm diameter) and Dynabeads (4.5 μm diameter). Miltenyi microbeads are nanosized superparamagnetic beads that require a magnetizable column matrix to retain them from the flow field. The magnetizable column matrix is made of soft magnetic material spheres (eg, 0.4 mm diameter stainless steel 400 series balls). Stainless steel 400 series balls will never rust. The magnetic properties of the sphere's material involve a strong magnetization when exposed to an external magnetic field and little residual magnetism when the external magnetic field is removed. The manufacturing process of the column matrix of magnetic retention material consists of sphere packing the column matrix using a vibrator, applying lacquer to the column matrix, draining the lacquer by gravity, and removing the residual lacquer by centrifugation. This involves air blowing and recentrifugation. The air blowing and centrifugation steps are repeated several times until all residual lacquer is removed. The column matrix is then placed in an oven at a temperature of approximately 100° C. for 3 days. After the column matrix has completely cured, it is held together by the applied lacquer. A magnetizable column matrix can be filled with spheres and act as a magnetic intensifier that enhances the magnetic field gradient by as much as 10,000 times. The enhanced magnetic field gradient helps attract nanosized bead-labeled cells to the sphere in the presence of an external magnetic field. The column matrix is demagnetized after the external magnetic field is removed, allowing the nanosized bead-labeled cells to be released from the column matrix. The nanosized bead-labeled cells are then eluted with a flow of rinse fluid through the column matrix.

Dynabeads are larger superparamagnetic beads made from synthetic polymers. Because Dynabead is significantly larger compared to Miltenyi nanosized beads, Dynabead has a significantly higher magnetic moment compared to Miltenyi nanosized beads when placed in a magnetic field. Therefore, using the Dynabead in magnetic cell isolation does not necessarily require a magnetic enhancement device such as a magnetic column matrix. Tube-based systems are typically used with Dynabead-labeled cells, and a permanent magnet is placed near the tube. Target cells labeled with Dynabead are attracted to the walls of the tube, and unlabeled cells are then removed with buffer or medium. Other beads of different sizes are also available under trademark besides Miltenyi nano-sized microbeads and Dynabead.

The magnetic isolation device includes a magnetic field generator, such as a pair of permanent magnets, a magnetic cell isolation holder and associated magnetic cell retention material, flow tubing, collection and preparation vessels, and other components of the disclosed system 2100. Can be used with Additionally, some of these components may be provided as one-time components, disposable components, and/or packaged kits.

In one embodiment, specialized kits may be provided to accomplish magnetic isolation for specific bead types. Kits can be provided that are optimized for one or more bead sizes for any particular isolation event. The kit may include suitable magnetic retention material, which may be preloaded into an appropriately configured magnetic cell isolation holder. This approach prevents the user from inadvertently loading or bonding incorrect magnetic retention material into the passageway of the magnetic isolation holder. In one embodiment for use with Miltenyi microbeads, the magnetic retention column in the passageway of the magnetic isolation holder is positioned so that, when loaded, it is centered in the gap or space between the permanent magnets of the magnetic field generator; May be associated with the highest or higher magnetic field strength (ie, greater than 0.45T). In another embodiment, the magnetic retention tubing for the Dynabead may be positioned within the highest slope region between the permanent magnets. The Dynabead isolate was positioned relative to the magnetic field generator to be subjected to both a magnetic field strength (i.e., greater than 0.1 T) and a magnetic field gradient (i.e., greater than 40 T/m) suitable for retention. It can be carried out in conjunction with a magnetic isolation holder with passages.

When used in conjunction with the disclosed technology, the average recovery and average purity of CD3+ using the magnetic isolation device are each approximately greater than 80% for Miltenyi nanosized beads. At Dynabead, the average recovery of CD3+ using the magnetic isolation device is approximately 60%, and the average purity of CD3+ using the magnetic isolation device is approximately over 70%.

The technical effect of the present disclosure is that the holder for magnetic isolation of cells is used in conjunction with a magnetic field generator to allow cell isolation without adjustment of magnetic field parameters between procedures using magnetic beads of different sizes. Including providing. In addition, the magnetic isolation device automatically performs cell preparation, magnetic cell isolation, and cell elution methods on each of the beads of different sizes, eliminating user interaction and manipulation of the raw materials or Can be reduced.

As used herein, an element or step listed in the singular in the English text and preceded by the article "a" or "an" refers to said element or step, unless the exclusion is explicitly stated. or more than one step. Furthermore, references to "one embodiment" of the present invention are not intended to be interpreted as excluding the existence of additional embodiments that also incorporate the recited features. Furthermore, unless otherwise specified, an embodiment that "includes," "comprises," or "has" an element or elements with a particular characteristic may include additional such elements without that characteristic.

This specification uses examples to disclose some embodiments of the invention, including the best mode, and to describe any devices or systems that may be made and used, and any methods that may be incorporated. It enables those skilled in the art to make, including to carry out, embodiments of the invention. 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 considered if these examples have structural elements that do not differ from the claim language, or if these examples contain equivalent structural elements that differ only insignificantly from the claim language. is intended to be within the scope of the claims.

10 Bioprocessing system
100 1st module
110 1st controller
200 second module
200a, 200b, 200c second module
210 2nd controller
280a, 280d sample collection device
300 3rd module
310 3rd controller
400 Fluid Flow Architecture
400 Bioprocessing Subsystem
400 Bioprocessing System
410 First bioreactor vessel
412 1st port
414 First bioreactor line
416 second port
418 Second bioreactor line
420 Second bioreactor vessel
422 1st port
424 First bioreactor line
426 second port
428 Second bioreactor line
430 Bioreactor Array
432 First bioreactor line valve
436 First bioreactor line valve
438 Second bioreactor line valve
440 First fluid assembly
442 First fluid assembly line
444 Second Fluid Assembly
446 Second Fluid Assembly Line
448 Sampling Assembly
450 Interconnect Conduit
452 Interconnect Pipe Valve
454 Interconnected 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 1st storage tank
468a-f tubing tail valve
470a-d tubing tail
472a-d First storage tank
474a-d tubing tail valve
476a-476d Sampling pipeline
478a-d Sample line valve
482 Filtration line
484 Filter
486 Upstream filtration line valve
488 Downstream filtration line valve
490 Waste liquid pipe line
492 Osmosis 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 Fitting or tubing
530 air balance port
532 Side wall
534 Vertex, tip point
536 height
538 cell culture medium
542 Headroom
544 Surface
550 Recess
552 Position verification structure
554 Flat engagement surface
556 opening or opening
600 kits
610 tray
612 Front wall
614 Back wall
616, 618 side
622 Internal compartment
620 Bottom
622 Internal compartment
624 Peripheral flange
626, 628 1st and 2nd opening
631 sampling space
632 Claw
636 Support Rib
638 opening
650 tubing module
652 1st tubing holder block
654 Second tubing holder block
656, 658 arrangement
660 Clearance opening
662 Planar back plate
664 Aperture
666 Vertically spaced, horizontally extending slots
668, 670 Clearance opening
672 retaining clip
674 1st input end
676 Second production end
680 Features
682 narrow tubing slot
684 Waste line tubing slot
700 Bioprocessing equipment
710 housing
712, 714, 716 drawer
718 Side wall
720 Bottom
722 Processing Chamber
724 Hardware Compartment
730 Auxiliary compartment
732 Power supply
734 Motion Control Board and Drive Electronics
736 Low Power Solenoid Array
738 pump assembly
740 Drawer Engagement Actuator
742 Pump shoe
744 Pinch valve anvil
746 1st bed plate
748 Second bed plate
750 plates
752 Top surface
754 Locating pin or alignment pin
756 Integrated Sensor
759 Embedded temperature sensor
760 load cell
760 Resistance Temperature Detector
761 Actuator mechanism
762 cam arm
764 slots
766 Cam Pin
768 linear actuator
770 rocker switch
770 linear actuator
772 Feed screw
774 Clevis Arm
776 Space
778 Solenoid
780 piston
782 heating pad
784 heating module
786 Carbon dioxide sensing module
788 cover
790 Insulating foam layer
792 Film heater
794 Internal metal plate
798 Heat insulation layer
800 fluid
810 flip-down front panel
812 Expandable sliding rail
814 Laterally extending cross rod
815 Attachment means
816 Low Profile Waste Tray
819 own chamber
820 platform
822 Guide track
900 devices/equipment
910 base
912 Centrifugation Chamber
914 High Dynamic Range Peristaltic Pump Assembly
916 Pump tube with suitable inner diameter
918 Stopcock Manifold
920 optical sensor
922 Heating/Cooling Mixing Chamber
924 Generally T-shaped hanger assembly
926 Hook
930 Sample Source Bag
932 Process bag
934 Isolation Buffer Bag
936 Washing bag
938 1st storage bag
940 Second storage bag
942 Post-isolation waste bag
944 Washing waste bag
946 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 Retention Element or Material
1000 general protocols
1110 Process loop
1112 Process line
1114 1st storage bag line
1116 Second storage line
1118 Top Port
2100 Alternative Magnetic Isolation System
2102 Incubate removal
2104 Sample Source Bag (SB)
2106 Process bag (PB)
2108 Buffer bag (BB)
2110 Medium bag (MB)
2112 Source Pump (SP)
2114 Process pump (PP)
2116 Magnetic Isolation Pump (MP)
2118 Bead addition syringe (SG1)
2120 Check valve
2122, 2124 magnetic field plate
2126 Collection pinch valve (PV-C)
2128 Waste liquid pinch valve (PV-W)
2120 Check valve (CV1)
2121 Magnetic field generator
2130 Collection bag (CB)
2132 Waste liquid bag (WB)
2134 Magnetic Cell Isolation Holder
2150 controller
2152 processor
2154 memory
2200 method
2300 Magnetic Isolation Device
2301 Main body
2302 Magnetic Cell Isolation Holder
2303, 2305 aisle
2304, 2306 Magnetic retention material
2307 End face
2308 steel backing
2311 Stopping part or surface
2312, 2314 magnetic field plate
2313 Magnetic field generator
2315 distance
2316 Reception Area
2318 door
2319 frame
2330 Guide plate
2500 ways
2600 magnetic field generator
2602 Magnetic Cell Isolation Holder
2604 Column matrix
2606 Magnetic Retention Material
2612, 2614 Permanent magnet

Claims (14)

A device (900) used in a bioprocessing system, comprising:
a magnetic field generator (962) configured to generate a magnetic field under magnetic field parameters;
a first configured to be removably coupled to said magnetic field generator (962);magnetic cell isolationA holder,Applicable1stmagnetic cell isolationa first passageway configured to be positioned within the magnetic field in a first configuration when the holder is coupled to the magnetic field generator (962);attitudeThe firstmagnetic cell isolationholder and
a second configured to be removably coupled to said magnetic field generator (962);magnetic cell isolationA holder,Applicablesecondmagnetic cell isolationa second passageway configured to be positioned within the magnetic field in a second configuration when the holder is coupled to the magnetic field generator (962);attitudesecondmagnetic cell isolationholder and
In an apparatus (900) for use in a bioprocessing system, comprising:
The first passage is subject to a first magnetic field strength and a first magnetic field gradient within the magnetic field occurring under the magnetic field parameters in the first configuration, and the second passage 2, the second magnetic field strength is different from the first magnetic field strength, or the second magnetic field strength is different from the first magnetic field strength, or An apparatus (900) for use in a bioprocessing system, characterized in that the second magnetic field gradient is different from the first magnetic field gradient or a combination thereof. The magnetic field generator (962) is coupled to a frame, the frame transmitting a magnetic flux and defining a receiving area configured to receive the first magnetic cell isolation holder or the second magnetic cell isolation holder. 2. The apparatus (900) of claim 1, forming an apparatus. 3. The apparatus of claim 2, wherein the receiving area is sized to receive only one of the first magnetic cell isolation holder or the second magnetic cell isolation holder at a given time. 900). 4. The apparatus (900) of claim 2 or 3, wherein the receiving area is configured to receive only a portion of the first magnetic cell isolation holder or the second magnetic cell isolation holder. 5. The apparatus (900) of claim 4, wherein the portion includes the first passageway of the first magnetic cell isolation holder or the second passageway of the second magnetic cell isolation holder. The frame comprises a retractable portion that reduces magnetic flux generated by the magnetic field generator from movement beyond the receiving area while the first magnetic cell isolation holder is separated from the magnetic field generator. 6. The device (900) according to claim 2 or any one of claims 3 to 5, which refers to claim 2. 7. The apparatus (900) of claim 6, wherein the retractable portion comprises a spring that, when compressed, allows the first magnetic cell isolation holder to enter the receiving area of the frame. . 8. Any one of claims 1 to 7, wherein when the first magnetic cell isolation holder is not coupled to the magnetic field generator, the first passageway is not affected by the first magnetic field strength. Apparatus described in Section (900). The first magnetic cell isolation holder has a first end surface configured to abut a stop portion of the frame when the first magnetic cell isolation holder is coupled to the magnetic field generator. Apparatus (900) according to claim 2 or any one of claims 3 to 8 reciting claim 2, comprising: The second magnetic cell isolation holder has a second end surface configured to abut the stop portion of the frame when the second magnetic cell isolation holder is coupled to the magnetic field generator. 3. The first distance between the first end surface and the first passageway is different from the second distance between the second end surface and the second passageway. The device described in 9 (900). 11. The apparatus (900) of any preceding claim, wherein the first passageway and the second passageway are of different sizes. The first magnetic cell isolation holder is configured to be fluidically coupled to a first source of beads of a first size, and the second magnetic cell isolation holder is configured to be fluidically coupled to a first source of beads of a second size. beads of the first size are coupled to target cells within the first cell mixture, and the beads of the second size are configured to be fluidically coupled to a second source of 12. A device (900) according to any one of claims 1 to 11, wherein the device (900) is bound to a target cell within a cell mixture. 13. The apparatus (900) of claim 12, wherein the first size beads have a diameter less than 1 μm and the second size beads have a diameter greater than 2 μm. A method for isolating target cells, the method comprising:
positioning a first holder having a first passage within a receiving area of a frame coupled to a magnetic field generator (962);
the magnetic field generator (962) when the first holder is coupled to the magnetic field generator (962) to apply a first magnetic field strength, a first magnetic field gradient, or both to the first passageway; 962) generating a first magnetic field within the receiving region;
positioning a second holder having a second passage within the receiving area, the first passage and the second passage being positioned at different locations within the receiving area; ,
the magnetic field generator (962) when the second holder is coupled to the magnetic field generator (962) to apply a second magnetic field strength, a second magnetic field gradient, or both to the second passageway; 962) generating a second magnetic field within the receiving region;
a first cell mixture with beads of a first size; a second cell mixture with beads of a second size; target cells in said first cell mixture labeled with beads of said first size; and incubating such that the target cells in the second cell mixture are labeled with the second size beads;
passing the first cell mixture through the first passage and then passing the second cell mixture through the second passage;
A method for isolating target cells, comprising:

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