JP2021503939A - Methods for cell enrichment and isolation - Google Patents

Abstract

Disclosed is a method of bioprocessing, which involves combining a suspension containing a population of cells with magnetic beads to form a population of bead-bound cells in the suspension and on a magnetic separation column. Includes a step of isolating a population of bead-bound cells and a step of collecting target cells from the population of cells. Collecting target cells involves removing bead-bound cells from an isolated column with an air plug. Further disclosed is a system that includes a magnetic field generator, a magnetic cell isolation holder, a disposable fluid kit, and the methods involved in its use.

Description

Embodiments of the invention generally relate to bioprocessing systems and methods, and more specifically to bioprocessing systems and methods for the production of cell immunotherapeutic agents.

Various drug therapies involve extraction, culturing, and amplification 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 with T cell activation function. A general premise of CAR-T cells is the artificial production of T cells that target markers found on cancer cells. Scientists can take T cells from humans, genetically modify them, and return them to the patient to attack the cancer cells. CAR-T cells can either come from the patient's own blood (self) 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 leukocyte apheresis therapy. After a sufficient amount of leukocytes have been harvested, the leukocyte aferesis product is concentrated on the T cells, which involves flushing the cells from the leukocyte aferesis buffer. A subset of T cells with a particular biomarker is then isolated from a subset enriched using a specific antibody conjugate or marker.

After isolation of the target T cells, the cells are activated in a particular environment in which they can proliferate actively. 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) that can be removed from the medium using magnetic separation. .. T cells are then transduced with the CAR gene by either an integration gammaretrovirus (RV) or lentivirus (LV) vector. The viral vector uses the viral mechanism to attach to the patient's cell and enter the cell, after which the vector introduces the genetic material in the form of RNA. For CAR-T cell therapy, this genetic material encodes CAR. RNA can be reverse transcribed into DNA and permanently integrated into the genome of the patient's cell so that CAR expression is maintained as the cell divides and proliferates in large numbers in the bioreactor. CAR is then transcribed and translated by the patient's cells, and CAR is expressed on the cell surface.

After T cells are activated and transduced with a CAR-encoded viral vector, the cells are amplified to a large number in the bioreactor to achieve the desired cell density. After amplification, the cells are harvested, washed, concentrated, compounded and injected into the patient's body.

Existing systems and methods for producing injectable doses of CAR T cells require many complex operations involving multiple human touch points, which makes the entire manufacturing process time consuming and contaminated. Risk increases. Although recent efforts to automate manufacturing processes have eliminated some human touchpoints, these systems still suffer from high costs, inflexibility, 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 specific equipment in the system.

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 cell immunotherapy that reduce the risk of contamination by increasing automation and reducing human handling. In addition, there is a need for a bioprocessing system for the production of cell therapies that balances the need for development flexibility with the need for mass production consistency and meets the needs of different customers who want to run different processes. ..

The subject matter originally claimed and some embodiments that are balanced in terms of scope are summarized below. These embodiments are not intended to limit the scope of the subject matter claimed, but rather these embodiments are intended only to provide an overview of possible embodiments. In fact, the present disclosure may include various embodiments that may be similar to or different from the embodiments described below.

In one embodiment, the bioprocessing system is to activate, genetically transfect, and amplify the cell population with a first module configured to concentrate and isolate the cell population. It comprises a second module that is configured and a third module that is configured to harvest an amplified population of cells.

In another embodiment, the bioprocessing system is a first module configured to concentrate and isolate cells and a plurality of second modules, each second module activating cells. It comprises a plurality of second modules configured to transform, genetically transfect and amplify, and a third module configured to harvest the amplified cells. Each second module is configured to support activation, genetic transduction, and amplification of different populations of cells that occur in parallel with each other.

In another embodiment, the method of bioprocessing involves the step of enriching and isolating the cell population in the first module and the activation and genetic transduction of the cell population in the second module. , Amplification and, in a third module, harvesting an amplified population of cells. The steps of activating, genetically transducing, and amplifying a cell population are performed without removing the cell population from the second module.

In another embodiment, the device for bioprocessing comprises a housing and a drawer that can be accommodated within the housing. The drawer comprises a plurality of side walls and bottoms that define the processing chamber, and a generally open top. The drawer is movable between a closed position where the drawer is received within the housing and an open position where the drawer extends from the housing and allows access to the processing chamber through an open top. The device also comprises at least one bed plate that is positioned within the processing chamber and is configured to accommodate a bioreactor vessel.

In another embodiment, the method of bioprocessing is to slide a drawer with multiple side walls, a bottom, and a generally open top from a closed position to an open position within the housing to extend the drawer from the housing. Sliding the drawer with a step through the generally open top and a step to position the bioreactor vessel through the generally open top onto a stationary bed plate positioned in the drawer. It includes a step of controlling the drawer engagement actuator to engage the plurality of fluid channels with at least one pump and a plurality of pinch valve linear actuators.

In another embodiment, the system for bioprocessing is typically a housing and a first drawer that is acceptable within the housing, with multiple sidewalls and bottoms defining a first processing chamber. A first drawer with an open top and is positioned within the processing chamber of the first drawer to receive or otherwise engage with the first bioreactor vessel on it. A plurality of first bed plates configured in the above and a second drawer that is acceptable in a housing that is stacked with the first drawer and defines a second processing chamber. A second drawer with a side wall and bottom, and a generally open top, located within the processing chamber of the second drawer, on which a second bioreactor vessel is received or otherwise. It comprises at least one second bed plate that is configured to engage in any way. The first drawer and the second drawer have a closed position where the first drawer and / or the second drawer is received in the housing, respectively, and the first drawer and / or the second drawer extend from the housing. It is movable to and from an open position that allows access to the processing chamber through each open top.

In yet another embodiment, the device for bioprocessing is a housing and a drawer that is acceptable within the housing, which is generally open with multiple side walls and bottom surfaces that define the processing chamber. The drawer is movable between a closed position where the drawer is received within the housing and an open position where the drawer extends from the housing and allows access to the processing chamber through the open top. It comprises a drawer, at least one bed plate positioned in a processing chamber adjacent to the bottom surface, and a kit that is acceptable within the processing chamber. The kit is a bioreactor vessel with multiple side walls and bottoms that define the internal compartment, a generally open top, an opening that is formed within the bottom of the kit and has a circumference, and within the internal compartment. It comprises a bioreactor vessel that is positioned above at least one opening of the bioreactor vessel and is supported by the bottom surface so that a portion of the bioreactor vessel is accessible through the opening in the bottom surface. The kit is acceptable in the processing chamber such that the bed plate penetrates the opening in the bottom of the tray and supports the bioreactor vessel on the bottom of the kit.

In yet another embodiment, the system for bioprocessing has a tray formed within the bottom surface and having a circumference with a plurality of side walls and bottom surfaces defining an internal compartment and a generally open top. First, integrated with the tray with at least one opening, accepting at least one pump tube and holding at least one pump tube in place for selective engagement with the pump. Tubing holder block, integrated with tray, accepts multiple pinch valve tubes, and puts each pinch valve tube out of multiple pinch valve tubes in place for selective engagement with each actuator of the pinch valve array. A second tubing holder block configured to hold in and a bioreactor vessel that is positioned above at least one opening in the internal compartment and a portion of the bioreactor vessel with an opening in the bottom surface. It comprises a bioreactor vessel supported by the bottom so that it is accessible through the section.

In yet another embodiment, the system for bioprocessing is a processing chamber having a plurality of sidewalls, a bottom surface, and a generally open top, and a bed plate positioned within a processing chamber adjacent to the bottom surface. And a tray. The tray comprises a tray having a plurality of side walls and bottoms defining an internal compartment, a generally open top, and an opening within the bottom of the tray with a perimeter. The circumference of the opening is shaped and / or so that the bioreactor vessel can be positioned above the opening and supported by the bottom of the tray while part of the bioreactor vessel is accessible through the opening in the bottom. Has dimensions. The tray can be accommodated in the processing chamber such that the bed plate penetrates the opening in the bottom of the tray and supports the bioreactor vessel.

In yet another embodiment, the system for bioprocessing is a tray with multiple side walls and bottoms defining an internal compartment and a generally open top, and within the bottom, bordered by a perimeter. With at least one opening, the opening has a shape and / or size such that the bioreactor vessel is positioned above the opening and can be supported by the bottom of the tray within the internal compartment. Be prepared.

In yet another embodiment, the method of bioprocessing is the step of placing the bioreactor container in a disposable tray, where the disposable tray has multiple side walls and bottoms that define the internal compartment and a generally open top. And a step with an opening formed in the bottom surface and a plurality of claws or protrusions penetrating into the opening from the bottom surface and a bioreactor container in the tray and a bioreactor container above the opening. Includes a step of arranging and configuring to be supported by the claws of the tray and a step of placing the tray in a processing chamber with a bed plate so that the bed plate is received through an opening in the tray and supports the bioreactor container. ..

In yet another embodiment, the tubing module for a bioprocessing system is configured to accept at least one pump tube and hold at least one pump tube in place for selective engagement with the peristaltic pump. Accepts a first tubing holder block and multiple pinch valve tubes, and holds each pinch valve tube in place for selective engagement with each actuator of the pinch valve array. It is provided with a second tubing holder block that is configured to do so. The first tubing holder block and the second tubing holder block are interconnected.

In yet another embodiment, the system for bioprocessing has multiple side walls and bottoms that define the internal compartment, and a generally open top, where the tray accepts or supports the bioreactor vessel. A tray that is configured to engage or otherwise engage on it, a pump assembly that is positioned adjacent to the rear wall of the tray, and a position that is positioned adjacent to the rear wall of the tray. It includes a pinch valve array and a tubing module positioned at the rear of the tray. The tubing module receives at least one pump tube and is configured to hold at least one pump tube in place for selective engagement with the pump assembly, with a first tubing holder block and multiple pinches. A second tubing holder block that is configured to accept the valve tubes and hold each pinch valve tube in place among multiple pinch valve tubes for selective engagement with the respective actuator of the pinch valve array. And.

In yet another embodiment, the bioreactor vessel is a bottom plate and a vessel body coupled to the bottom plate, the vessel body and the bottom plate defining an internal compartment between them. And a plurality of recesses formed within the bottom plate, each recess of which is a corresponding alignment pin on the bed plate to align the bioreactor container on the bed plate. It comprises a plurality of recesses, which are configured to accept.

In yet another embodiment, the method for bioprocessing is to operably connect the bottom plate to the container body and operably define an internal compartment between them, the bottom plate and the container body. The parts form and connect the bioreactor vessel, align the recesses in the bottom plate with the alignment pins of the bioprocessing system, and install the bioreactor vessel on the bed plate of the bioreactor system. Including.

In yet another embodiment, the bioprocessing system is a first fluid assembly conduit that is connected to the first port of the first bioreactor vessel through the first bioreactor conduit of the first bioreactor vessel. The first fluid assembly of the first bioreactor vessel is the selective that the first bioreactor conduit of the first bioreactor vessel is between the first fluid assembly and the first port of the first bioreactor vessel. A second port of the first bioreactor vessel through a first fluid assembly and a second bioreactor conduit of the first bioreactor vessel, comprising a first bioreactor conduit valve to provide fluid communication. A second fluid assembly having a second fluid assembly conduit connected to, the second bioreactor conduit of the first bioreactor vessel is the second fluid assembly and the first bioreactor vessel. Between the second fluid assembly and between the first and second fluid assemblies, with a second bioreactor conduit valve to provide selective fluid communication with the second port of the Fluid communication, and an interconnect line that provides fluid communication between the second bioreactor line of the first bioreactor container and the first bioreactor line of the first bioreactor container.

In yet another embodiment, the method of bioprocessing is a first fluid assembly tube connected to the first port of the first bioreactor vessel through the first bioreactor conduit of the first bioreactor vessel. A second fluid assembly that provides a first fluid assembly with a path and is 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 with a conduit and the interconnection between the second bioreactor conduit of the first bioreactor vessel and the first bioreactor conduit of the first bioreactor vessel. To provide a conduit, the interconnect conduit is the fluid communication between the first fluid assembly and the second fluid assembly, and the second bioreactor conduit and the second bioreactor conduit in the first bioreactor vessel. Includes providing an interconnect line that allows fluid communication to and from a first bioreactor line of one bioreactor vessel.

In yet another embodiment, bioprocessing methods for cell therapy include genetic modification of a population of cells in a bioreactor vessel to produce a population of genetically modified cells and of genetically modified cells from the bioreactor vessel. Includes 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 used in cell therapy therapy without removing the population.

In yet another embodiment, the bioprocessing method involves coating a bioreactor vessel with a reagent to increase the efficiency of genetic modification of a population of cells and cells of the population of cells to produce a population of genetically modified cells. Includes genetic modification and amplification of a population of genetically modified cells in the bioreactor vessel without removing the genetically modified cells from the bioreactor vessel.

In yet another embodiment, the bioprocessing method involves activating cells in a population of cells in a bioreactor vessel using magnetic or non-magnetic beads to produce a population of activated cells. Gene modification of activated cells in a bioreactor vessel to produce a population of genetically modified cells, washing of genetically modified cells in a bioreactor vessel to remove unwanted substances, and transfection. Includes amplifying a population of genetically modified cells in a bioreactor vessel to produce an amplified population of the cells. Activation, genetic modification, washing, and amplification are performed in the bioreactor vessel without removing the cells from the bioreactor vessel.

In yet another embodiment, the kit used in the bioprocessing system is configured to fluidly communicate with the process bag, the source bag, the bead addition container, the process bag, the source bag, and the bead addition container. It has a process loop. The process loop additionally comprises pump tubing configured to communicate fluid with the pump.

In yet another embodiment, the device for bioprocessing is a kit comprising a process bag, a source bag, and a bead addition container configured to be fluid-operated with the process loop, the process loop. In addition to that, a kit with pump tubing configured to communicate with the pump, a magnetic field generator configured to generate a magnetic field, a source bag, a process bag, and beads. Multiple hooks for suspending additional containers, each hook of the multiple hooks is operably connected to a load cell, which is configured to sense the weight of the bag connected to it. It comprises a hook, at least one bubble sensor, and a pump configured to communicate fluid with the process loop.

In one embodiment, the method of bioprocessing involves combining a suspension containing a population of cells with magnetic beads to form a population of bead-bound cells in the suspension, and simply magnetically. It involves isolating a population of bead-bound cells on a detached column and collecting target cells from the population of cells.

In one embodiment, the system comprises a magnetic field generator configured to generate a magnetic field under magnetic field parameters and a first holder configured to be detachably coupled to the magnetic field generator. It comprises a second holder configured to be detachably coupled to the magnetic field generator. The first holder has a passage that is configured to be positioned in the magnetic field in the first arrangement when the first holder is coupled to the magnetic field generator. The second holder has a passage that is configured to be positioned in the magnetic field in a second arrangement when the second holder is coupled to the magnetic field generator. The passage of the first holder is affected by the first magnetic field strength and the first magnetic field gradient within the magnetic field generated under the magnetic field parameters in the first arrangement. The passage of the second holder is affected by the second magnetic field strength and the second magnetic field gradient in the magnetic field under the magnetic field parameters in the second arrangement, the second magnetic field strength is different from the first magnetic field strength, The second magnetic field gradient is different from or a combination of the first magnetic field gradients.

In another embodiment, the magnetic cell isolation holder comprises a body that is configured to be detachably coupled to a magnetic field generator. The main body has a first passage configured to be positioned within the magnetic field of the magnetic field generator in the first arrangement when the holder is coupled to the magnetic field generator, and the holder is coupled to the magnetic field generator. It has a second passage that is configured to be positioned in the magnetic field in a second arrangement when The first passage is affected by the first magnetic field strength and the first magnetic field gradient in the magnetic field generated under the magnetic field parameters in the first arrangement, and the second passage is the magnetic field parameters in the second arrangement. Under the action of a second magnetic field strength and a second magnetic field gradient in 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 or a combination of the first magnetic field gradients.

In another embodiment, the system comprises a first kit having a first holder having a passage configured to accept a plurality of first size beads and a plurality of first size beads. It comprises a second kit having a plurality of second size beads and two holders having passages configured to accept multiple second size beads. The passage of the first holder is such that when the first holder is detachably coupled to the magnetic field generator, the first holder is positioned within the magnetic field generated by the magnetic field generator in the first arrangement. Positioned in the holder. The passage of the second holder is the magnetic field generated by the magnetic field generator in a second arrangement where the second holder is different from the first arrangement when the second holder is detachably coupled to the magnetic field generator. Positioned in the second holder so that it is positioned in. The passage of the first holder is affected by the first magnetic field strength and the first magnetic field gradient in the magnetic field in the first arrangement, and the passage of the second holder is the second magnetic field in the magnetic field in the second arrangement. It is affected by the strength 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 or a combination of the first magnetic field gradients.

In another embodiment, the method for isolating the target cell is to position a first holder having a passage within the receiving region of the frame attached to the magnetic field generator and the passage of the first holder. Generates a first magnetic field in the receiving region by the magnetic field generator when the first holder is coupled to the magnetic field generator to act on the first magnetic field strength, the first magnetic field gradient, or both. Including that. The method is also to position a second holder with a passage within the receiving region and to act on the passage of the second holder with a second magnetic field strength, a second magnetic field gradient, or both. This includes generating a second magnetic field in the receiving region by the magnetic field generator when the two holders are coupled to the magnetic field generator. The passage of the first holder and the passage of the second holder are positioned at different positions within the receiving area.

The present invention will be better understood by reading the following description of the non-limiting embodiment with reference to the accompanying drawings, shown below.

It is the schematic of the bioprocessing system according to one Embodiment of this invention. It is the schematic of the bioprocessing system according to another embodiment of this invention. FIG. 6 is a block diagram showing the fluid flow composition / system of the cell activation, genetic modification, and amplification subsystems of the bioprocessing system of FIG. It is a detailed view of a part of the block diagram of FIG. 3 illustrating a first fluid assembly of a fluid flow configuration / system. It is a detailed view of a part of the block diagram of FIG. 3 illustrating a second fluid assembly of a fluid flow configuration / system. It is a detailed view of a part of the block diagram of FIG. 3 exemplifying a sampling assembly of a fluid flow configuration / system. It is a detailed view of a part of the block diagram of FIG. 3 exemplifying the filtration flow path of a fluid flow configuration / system. It is a perspective view of the bioreactor container by one Embodiment of this invention. It is an exploded view of the bioreactor container of FIG. It is an exploded sectional view of the bioreactor container of FIG. It is an exploded bottom perspective view of the bioreactor container of FIG. It is a top view and a front perspective view of the disposable drop-in kit of the bioprocessing system of FIG. 1 according to one embodiment of the present invention. FIG. 12 is another top and front perspective view of the disposable drop-in kit of FIG. FIG. 12 is another top and rear perspective view of the disposable drop-in kit of FIG. It is a perspective view of the tray of the disposable drop-in kit of FIG. 12 according to one embodiment of the present invention. It is a front perspective view of the tubing module of the disposable drop-in kit of FIG. 12 according to one embodiment of the present invention. It is a rear perspective view of the tubing module of FIG. FIG. 5 is an elevational view of a second tubing holder block of a tubing module according to an embodiment of the invention. It is sectional drawing of the 2nd tubing holder block of FIG. It is another front perspective view of the drop-in kit of FIG. 12, showing the flow architecture integrated inside. It is a rear perspective view of the drop-in kit of FIG. 12 showing the flow architecture integrated inside. It is a front elevation view of the drop-in kit of FIG. 12 showing the flow architecture integrated inside. It is a perspective view of the bioprocessing apparatus according to one Embodiment of this invention. FIG. 6 is a perspective view of a drawer of a bioprocessing device for accepting the drop-in kit of FIG. 12 according to an embodiment of the present invention. It is a top view of the drawer of FIG. 24. It is a front perspective view of the processing chamber of the drawer of FIG. It is a top view of the processing chamber of a drawer. It is a top view of the bed plate of the bioprocessing apparatus of FIG. 23. It is a top view of the hardware component housed under the bed plate of FIG. 28. It is a side elevation view of the bioprocessing device of FIG. It is a perspective view of the drawer engaging actuator of the bioprocessing apparatus of FIG. It is a top view of the drawer of the bioprocessing device which illustrates the clearance position of a drawer engaging actuator, a pump assembly, and a solenoid array. Drawer Engagement Top view of a drawer of a bioprocessing device illustrating engagement positions of actuators, pump assemblies, and solenoid arrays. It is a perspective view of the bioprocessing apparatus which illustrates the drop-in kit of the position in the processing chamber of a drawer. It is a top view of the bioprocessing apparatus which illustrates the drop-in kit in the processing chamber of a drawer. It is a perspective view of the peristaltic pump assembly of a bioprocessing apparatus. FIG. 3 is a side elevation view of a peristaltic pump assembly and drop-in kit tubing holder module exemplifying the relationships between components. It is a perspective view of the solenoid array and the pinch valve anvil which form the pinch valve array of a bioprocessing apparatus. Another perspective view of a pinch valve array of a bioprocessing device. Another perspective view of a pinch valve array exemplifying the positioning of the drop-in kit tubing holder module with respect to the pinch valve array in the engaged position. It is sectional drawing of the drawer of the bioprocessing apparatus which illustrates the installation position of the bioreactor container on a bed plate. FIG. 6 is a side elevation view of a bioreactor accepted on a bed plate, illustrating a stirring / mixing mode of operation of the bioreactor system. FIG. 6 is a side sectional view of a bioreactor accepted on a bed plate, illustrating a stirring / mixing mode of operation of the bioreactor system. It is the schematic of the bioreactor container which shows the liquid level in a bioreactor container in a stirring / mixing operation mode. FIG. 5 is a detailed cross-sectional view of the interface between the positioning pin on the bed plate and the receiving recess on the bioreactor vessel in the stirring / mixing operation mode. FIG. 5 is a perspective view of a bioprocessing apparatus having a flip-down front panel according to an embodiment of the present invention, showing a processing drawer in an open position. Another perspective view of the bioprocessing apparatus of FIG. 45 showing a processing drawer in the open position. FIG. 45 is an enlarged perspective view of the auxiliary compartment of the bioprocessing apparatus of FIG. 45 showing a processing drawer in a closed position with access to the auxiliary compartment. Another enlarged perspective view of the auxiliary compartment of the bioprocessing device of FIG. 45 showing a processing drawer in a closed position with access to the auxiliary compartment. FIG. 45 is a perspective view of the bioprocessing apparatus of FIG. 45 showing a processing drawer in a closed position with access to an auxiliary compartment. Another perspective view of the bioprocessing apparatus of FIG. 45 showing a processing drawer in a closed position with access to an auxiliary compartment. FIG. 3 is a perspective view of an auxiliary compartment of a bioprocessing device according to another embodiment of the present invention. It is a perspective view of the bioprocessing system which has a waste liquid tray according to one Embodiment of this invention. FIG. 5 is a schematic representation of an automated generic protocol for a bioprocessing system utilizing the fluid flow architecture of FIG. 3 according to an embodiment of the present invention. FIG. 5 is a schematic representation of an automated generic protocol for a bioprocessing system utilizing the fluid flow architecture of FIG. 3 according to an embodiment of the present invention. FIG. 5 is a schematic representation of an automated generic protocol for a bioprocessing system utilizing the fluid flow architecture of FIG. 3 according to an embodiment of the present invention. FIG. 5 is a schematic representation of an automated generic protocol for a bioprocessing system utilizing the fluid flow architecture of FIG. 3 according to an embodiment of the present invention. FIG. 5 is a schematic representation of an automated generic protocol for a bioprocessing system utilizing the fluid flow architecture of FIG. 3 according to an embodiment of the present invention. FIG. 5 is a schematic representation of an automated generic protocol for a bioprocessing system utilizing the fluid flow architecture of FIG. 3 according to an embodiment of the present invention. FIG. 5 is a schematic representation of an automated generic protocol for a bioprocessing system utilizing the fluid flow architecture of FIG. 3 according to an embodiment of the present invention. FIG. 5 is a schematic representation of an automated generic protocol for a bioprocessing system utilizing the fluid flow architecture of FIG. 3 according to an embodiment of the present invention. FIG. 5 is a schematic representation of an automated generic protocol for a bioprocessing system utilizing the fluid flow architecture of FIG. 3 according to an embodiment of the present invention. FIG. 5 is a schematic representation of an automated generic protocol for a bioprocessing system utilizing the fluid flow architecture of FIG. 3 according to an embodiment of the present invention. FIG. 5 is a schematic representation of an automated generic protocol for a bioprocessing system utilizing the fluid flow architecture of FIG. 3 according to an embodiment of the present invention. FIG. 5 is a schematic representation of an automated generic protocol for a bioprocessing system utilizing the fluid flow architecture of FIG. 3 according to an embodiment of the present invention. FIG. 5 is a schematic representation of an automated generic protocol for a bioprocessing system utilizing the fluid flow architecture of FIG. 3 according to an embodiment of the present invention. FIG. 5 is a schematic representation of an automated generic protocol for a bioprocessing system utilizing the fluid flow architecture of FIG. 3 according to an embodiment of the present invention. FIG. 5 is a schematic representation of an automated generic protocol for a bioprocessing system utilizing the fluid flow architecture of FIG. 3 according to an embodiment of the present invention. FIG. 5 is a schematic representation of an automated generic protocol for a bioprocessing system utilizing the fluid flow architecture of FIG. 3 according to an embodiment of the present invention. FIG. 5 is a schematic representation of an automated generic protocol for a bioprocessing system utilizing the fluid flow architecture of FIG. 3 according to an embodiment of the present invention. FIG. 5 is a schematic representation of an automated generic protocol for a bioprocessing system utilizing the fluid flow architecture of FIG. 3 according to an embodiment of the present invention. FIG. 5 is a schematic representation of an automated generic protocol for a bioprocessing system utilizing the fluid flow architecture of FIG. 3 according to an embodiment of the present invention. FIG. 5 is a schematic representation of an automated generic protocol for a bioprocessing system utilizing the fluid flow architecture of FIG. 3 according to an embodiment of the present invention. FIG. 5 is a schematic representation of an automated generic protocol for a bioprocessing system utilizing the fluid flow architecture of FIG. 3 according to an embodiment of the present invention. FIG. 5 is a schematic representation of an automated generic protocol for a bioprocessing system utilizing the fluid flow architecture of FIG. 3 according to an embodiment of the present invention. FIG. 5 is a schematic representation of an automated generic protocol for a bioprocessing system utilizing the fluid flow architecture of FIG. 3 according to an embodiment of the present invention. FIG. 5 is a schematic representation of an automated generic protocol for a bioprocessing system utilizing the fluid flow architecture of FIG. 3 according to an embodiment of the present invention. FIG. 5 is a schematic representation of an automated generic protocol for a bioprocessing system utilizing the fluid flow architecture of FIG. 3 according to an embodiment of the present invention. It is a perspective view of the enrichment and isolation apparatus by one Embodiment of this invention. It is a process flow diagram of the concentration and isolation apparatus of FIG. 78. FIG. 6 is a schematic representation of the fluid flow architecture of the device of FIG. 78 for performing enrichment and isolation of cell populations. FIG. 6 is a block diagram of a magnetic particle-based cell selection system that can be used with a magnetic cell isolation holder according to aspects of the present disclosure. It is a flowchart of the method of magnetic cell isolation according to the aspect of this disclosure. FIG. 5 is a top view of an embodiment of an unloaded magnetic cell isolation holder for a magnetic field generator according to aspects of the present disclosure. It is a top view of one embodiment of the magnetic cell isolation holder of the loading configuration with respect to the magnetic field generator according to the aspect of the present disclosure. FIG. 5 is a perspective view of an embodiment of an unloaded magnetic cell isolation holder for a magnetic field generator according to aspects of the present disclosure. FIG. 5 is a perspective view of an embodiment of a loaded magnetic cell isolation holder for a loaded magnetic field generator according to an aspect of the present disclosure. It is a flowchart of the method of magnetic cell isolation using a different magnetic cell isolation holder according to the aspect of this disclosure. It is a top view of one embodiment of a magnetic cell isolation holder and a loaded configuration magnetic field generator according to the present disclosure, showing the magnetic field distribution of the magnetic field generator according to the present disclosure.

Illustrative embodiments of the invention, examples of which are shown in the accompanying drawings, are referenced in detail below. Wherever possible, the same reference characters are used to refer to the same or similar parts throughout the drawing.

As used herein, the terms "flexible" or "foldable" refer to structures or materials that are flexible or bendable without breaking, and are compressible or compressible. It can also refer to a material that is expandable. An example of a flexible structure is a bag made of polyethylene film. The terms "rigid" and "semi-rigid" are used herein to refer to structures that are "non-foldable," that is, to be folded, crushed, or deformed in some other way under normal force. It is used interchangeably to describe a structure that does not significantly reduce its elongated dimensions. Depending on the context, "semi-rigid" can also represent a structure that is more flexible than the "rigid" element, such as a bendable tube or conduit, but still collapses longitudinally under normal conditions and forces. Can represent no structure.

As used herein, "container" means a flexible bag, a flexible container, a semi-rigid container, a rigid container, or a flexible or semi-rigid tubing, as the case may be. The term "container" as used herein refers to a bioreactor vessel having a semi-rigid or rigid wall or part of a wall, as well as, for example, a cell culture / purification system, a mixing system, a medium /. Includes buffer preparation systems and filtration / purification systems, such as chromatography and tangential flow filtration systems, and other containers or conduits commonly used in biological or biochemical processing, including their associated channels. Is intended to be. As used herein, the term "bag" means, for example, a flexible or semi-rigid container or container used as a containment device for various fluids and / or media.

As used herein, "fluid-coupled" or "fluid communication" means that components of a system can accept or transfer fluid between components. The term fluid includes gases, liquids, or combinations thereof. As used herein, "telecommunications" or "electrically coupled" means that some components use direct or indirect electrical connections through direct or indirect signaling. It means that it is configured to communicate with each other. As used herein, "operably combined" refers to a connection that can be direct or indirect. The 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 of a variety of suitable materials. For example, trays may be made from cost-effective materials suitable for sterile and one-time disposable products.

As used herein, the term "functional closure system" refers to a system without compromising the integrity of the closed fluid pathway (eg, to maintain an internal sterile biomedical fluid pathway). Refers to multiple components that make up a closed fluid path that may have inlet and outlet ports for adding fluid or air or removing fluid or air from the system, whereby those ports are fluid to the system. 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 divertors), vessels, receptacles, and ports, depending on a given embodiment.

Embodiments of the present invention provide systems and methods for producing cell immunotherapeutic agents from biological samples (eg, blood, tissues, etc.). In one embodiment, the method of bioprocessing is to combine a suspension containing a population of cells with magnetic beads to form a population of bead-bound cells in the suspension and to form a population of bead-bound cells on a magnetic separation column. It involves isolating the population and collecting target cells from the population of cells. Collecting target cells involves removing bead-bound cells from an isolated column with an air plug.

With reference to FIG. 1, a schematic diagram of a bioprocessing system 10 according to an embodiment of the present invention is illustrated. The bioprocessing system 10 is configured for use in the manufacture of cell immunotherapeutic agents (eg, autologous cell immunotherapeutic agents), eg, human blood, fluid, tissue, or cell samples have been collected and collected. Cell therapy agents are produced from or based on the sample. Chimeric antigen receptor (CAR) T cell therapies are a type of cell immunotherapeutic agents that can be produced using the bioprocessing system 10, but other cell therapies also deviate from the broader aspects of the invention. Can be produced using the system of the present invention or aspects of the present invention. As illustrated in Figure 1, the production of CAR T cell therapies generally begins with the collection of patient blood and the separation of lymphocytes through apheresis therapy. Collection / apheresis therapy may be performed in the clinical setting, and then the apheresis product is sent to a laboratory or manufacturing facility for the production of CAR T cells. In particular, after the apheresis product has been received for processing, the desired cell population (eg, leukocyte cells) is noted, either concentrated to produce a cytotherapeutic agent or isolated from the collected blood. Target cells are isolated from the primordial cell mixture. The target cells of interest are then activated, genetically modified to specifically target and destroy tumor cells, and amplified to achieve the desired cell density. After amplification, the cells are harvested and dosed. The combination drug is then often cryopreserved and delivered to the clinical setting for thawing, preparation, and finally infusion infusion into the patient's body.

Further referring to FIG. 1, the bioprocessing system 10 of the present invention is configured to perform a specific subset of manufacturing steps in a substantially automated, functionally closed and scalable manner. It has a module or subsystem. In particular, the bioprocessing system 10 is configured to perform a first module 100 that is configured to perform enrichment and isolation steps and a first module that is configured to perform activation, genetic modification, and amplification steps. It comprises two 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 communicably 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. The first module 100, the second module 200, and the third module 300 are illustrated as having a dedicated controller for controlling the operation of each module, but the master control unit is broadly divided into three modules. It is intended that it may be used to perform target control. Each module 100, 200, 300 is designed to work with other modules to form a single coherent bioprocessing system 10, as detailed below.

By automating the processes within each module, product consistency from each module can be increased and the costs associated with extensive manual operation can be reduced. In addition, as will be explained in detail below, each module 100, 200, 300 is virtually closed, helping to ensure patient safety by reducing the risk of external pollution, and regulatory regulations. Helps ensure compliance and avoids costs associated with open systems. In addition, each module 100, 200, 300 is scalable, supporting both low patient development and high patient commercial manufacturing.

Further referring to Figure 1, the specific scheme in which the process steps are divided into different modules, each providing closed and automated bioprocessing, is a capital facility efficiency to a degree not previously found in this art. Make available. As will be appreciated, the step of amplifying the cell population to achieve the desired cell density prior to harvesting and compounding is typically the most time-consuming step in the manufacturing process, but concentration and isolation. The steps, as well as the harvesting and compounding steps, as well as the activation and genetic modification steps, are less time consuming. Therefore, attempts to automate the entire cytotherapeutic drug manufacturing process can be logistically difficult, as well as impede workflows and exacerbate bottlenecks within the process that reduce manufacturing efficiency. In particular, in a fully automated process, the steps of cell enrichment, isolation, activation, and genetic modification can be performed fairly quickly, but amplification of the genetically modified cells proceeds very slowly. Therefore, the production of the cytotherapeutic agent from the first sample (eg, the blood of the first patient) proceeds quickly to the amplification step, which takes substantial time to achieve the desired cell density to harvest. It takes. When using a fully automated system, the entire process / system is occupied by an amplification device that performs the amplification of cells from the first sample and the processing of the second sample is until the entire system is released for use. I can't start. In this regard, in a fully automated bioprocessing system, the entire system is essentially offline, processing the second sample until the entire cytotherapeutic drug manufacturing process from concentration to harvest / formulation is completed in the first sample. Not available.

However, embodiments of the present invention allow parallel processing of multiple samples (from the same or different patients) to facilitate more efficient use of capital resources. This advantage, as hinted at above, is a direct result of a particular scheme in which the process steps are divided into three modules 100, 200 and 300. With particular reference to FIG. 2, in one embodiment, the single first module 100 and / or the single third module 300 for parallel and synchronous processing of multiple samples from the same or different patients. In addition, in the bioprocessing system 12, it can be used in conjunction with a plurality of second modules, for example, the second modules 200a, 200b, 200c. For example, the first apheresis product from the first patient is concentrated and isolated using the first module 100, thereby producing a first population of isolated target cells. Well, a first population of target cells can then be transferred to one of the second modules, eg, 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 ejected from the first module 100, the first module is again again, for example, in the treatment of the second apheresis product from the second patient. Available for use. A second population of target cells produced in the first module 100 from the sample taken from the second patient is then separated for activation, genetic modification, and amplification under the control of controller 201b. Can be transferred to a second module of, for example, a second module 200b.

Similarly, after a second population of target cells has been transferred and exited from the first module 100, the first module is again again, for example, a third apheresis product from a third patient. It is available for use in the processing of. A third target population of cells produced in the first module 100 from a sample taken from a third patient is then separated for activation, genetic modification, and amplification under the control of controller 201c. Can be transferred to a second module of, for example, a second module 200c. In this regard, amplification of CAR-T cells for a first patient, eg, CAR-T cells, can occur at the same time as amplification of CAR-T cells for a second patient, a third patient, and so on.

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

Harvesting an amplified population of cells from the second modules 200a, 200b, and 200c also results in a single third module 300 when each amplified population of cells is ready to be harvested. Can be carried out using.

Therefore, the steps of activation, genetic modification, and amplification, which are the most time consuming, share some operational requirements, and / or require similar culture conditions, are stand-alone, automated, and functionally closed. Other system equipment utilized for enrichment, isolation, harvesting, and compounding by dividing into modules that are bound or offline while amplification of one population of cells is being performed, There is no such thing. As a result, the production of multiple cell therapies may be performed simultaneously, maximizing equipment and floor space used, and increasing the efficiency of the entire process and equipment. An additional second module may be added to the bioprocessing system 10 to perform parallel processing of any number of cell populations, as desired. Therefore, the bioprocessing system of the present invention makes it possible to use functions similar to plug and play, and it is possible to easily expand or contract the manufacturing equipment.

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

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

First, focusing on the second module 200, of cell activation, gene modification, and cell amplification in a single functionally closed, automated module 200 that provides the efficiency of the workflow described above. The ability to combine process steps is enabled by the specific configuration of the components within the second module 200, and the unique flow architecture that provides the specific interoperability between such components. 3 to 77, described below, illustrate different aspects of the second module 200 according to different embodiments of the present invention. First referring to Figure 3, the fluid flow architecture 400 (bioprocessing subsystem 400 or bioprocessing system 400) within a second module 200 that performs cell activation, genetic modification, and amplification (harvesting in some cases). A schematic diagram illustrating (widely referred to herein) is shown. The system 400 includes a first bioreactor container 410 and a second bioreactor container 420. The first bioreactor vessel has fluid communication with at least the first port 412 and the first port 412, the first bioreactor line 414, and the second port 416 and the second port 416. It is equipped with a second bioreactor line 418. Similarly, the second bioreactor vessel has at least a first bioreactor line 424 that communicates with at least the first port 422 and the first port 422, as well as a second port 426 and a second port 426. It is equipped with a second bioreactor line 428 that communicates with the fluid. Together, the first bioreactor vessel 410 and the second bioreactor vessel 420 form the bioreactor array 430. Although the system 400 is shown to have two bioreactor vessels, embodiments of the present invention may include a single bioreactor or three or more bioreactor vessels.

The first and second bioreactor lines 414, 418, 424, 428 of the first and second bioreactor vessels 410, 420 each have a flow of fluid through them, as described below. Each valve is provided for control. In particular, the first bioreactor line 414 of the first bioreactor container 410 comprises a first bioreactor line valve 432, and the second bioreactor line 418 of the first bioreactor container 410 It is equipped with a second bioreactor conduit valve 424. Similarly, the first bioreactor line 424 of the second bioreactor container 420 comprises a first bioreactor line valve 436 and the second bioreactor line 428 of the second bioreactor container 420 , Equipped with a second bioreactor conduit valve 438.

Further referring to FIG. 3, the system 400 also samples a first fluid assembly 440 with a first fluid assembly line 442 and a second fluid assembly 444 with a second fluid assembly line 446. With assembly 448. The interconnect line 450 with the interconnect line valve 452 provides fluid communication between the first fluid assembly 440 and the second fluid assembly 444. As shown in FIG. 3, the interconnect line 450 also provides fluid communication between the second bioreactor line 418 and the first bioreactor line 414 of the first bioreactor vessel 410. Then, the fluid can be circulated along the first circulation loop of the first bioreactor vessel. Similarly, the interconnect line also fluidly communicates between the second bioreactor line 428 and the first bioreactor line 424 of the second bioreactor vessel 420, the second bioreactor vessel. The fluid can be circulated along the second circulation loop of. Further, the interconnect conduit 450 is a second port 416 of the first bioreactor vessel 410 and a first port 422 and a first bio of the second bioreactor conduit 418 and the second bioreactor vessel 420. Further fluid communication between the reactor lines 424 allows 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 interconnect line 450, in one embodiment, from the second bioreactor line 418, 428 to the first bioreactor line 414 of the first bioreactor container 410. It extends to the intersection with the first fluid assembly line 442.

As illustrated in FIG. 3, the first and second fluid assemblies 440, 450 are disposed along the interconnect line 450. In addition, in one embodiment, the first fluid assembly is the first port 412 of the first bioreactor vessel 410 and the first port of the second bioreactor vessel 420, respectively, the first bioreactor. Fluids communicate through the first bioreactor line 414 of the vessel and the first bioreactor line 424 of the second bioreactor container 420. The second fluid assembly 444 communicates fluid with the second port 416 of the first bioreactor vessel 410 and the second port 426 of the second bioreactor vessel 420 via the interconnection line 450.

The first pump or interconnect pipeline pump 454 capable of flowing fluid in both directions is arranged along the first fluid assembly pipeline 442 and is a second pump capable of flowing fluid in both directions. Alternatively, the circulation line pump 456 is disposed along the interconnect 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, the sterile air source 458 is connected to the interconnect line 450 through the sterile air source line 460. Valve 462, positioned along sterile air source line 460, provides selective fluid communication between sterile air source 458 and interconnect line 450. FIG. 3 shows a sterile air source 458 connected to an interconnect line 450, but in other embodiments, the sterile air source is the first without departing from the broader aspects of the invention. First fluid assembly 440, second fluid assembly 444, or fluid between the second bioreactor line valve and the first bioreactor line valve of either the bioreactor or the second bioreactor. Can be connected to the flow path.

Then, in addition to that, with reference to FIGS. 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 has multiple tubing tails 464a-f, each of which is selectively / removable connected to one of the plurality of first storage tanks 466a-f. Is configured to. Each tubing tail 464a-f of the first fluid assembly 440 is a fluid flow to or from each one of the plurality of first storage tanks 466a-f of the first fluid assembly 440. It is equipped with tubing tail valves 468a to f for selectively controlling. FIG. 4 shows, in particular, that the first fluid assembly 440 comprises six fluid reservoirs, but as desired, more or less reservoirs for loading or collecting various processing fluids. May be used. It is contemplated that each tubing tail 464a-f may be individually connected to the storage layer 466a-f at the time required during operation of the fluid assembly 440, respectively, as described below.

With particular reference to FIG. 5, the second fluid assembly 444 has multiple tubing tails 470a-d, each of which is selectively / removable connected to one of the plurality of second storage tanks 472a-d. Is configured to. Each tubing tail 470a-d of the second fluid assembly 444 is a fluid flow to or from each one of the plurality of second storage tanks 472a-d of the first fluid assembly 444. It is equipped with tubing tail valves 474a to d for selectively controlling. FIG. 5 shows, in particular, that the second fluid assembly 444 comprises four fluid reservoirs, but as desired, more or less reservoirs for loading or collecting various processing fluids. May be used. In one embodiment, at least one of the second storage tanks, eg, the second storage tank 472d, is a collection storage for collecting an amplified population of cells, as described below. It is a tank. In one embodiment, the second storage tank 472a is a waste liquid storage tank, the purpose of which is described below. In the present invention, one or more storage tanks 472a-d may be pre-connected to their respective tails 470a-d, and each additional storage tank is in time for its use within the second fluid assembly 440, respectively. Intended to be connected to the tail of the.

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

In one embodiment, the storage tank / bag may be connected to the tubing tail of the first and second tubing assemblies using a sterile welding device. In one embodiment, the welding device is positioned next to the module 200 and the welding device is to butt weld one of the tubing tails (while maintaining aseptic condition) to the tail of the tube on the bag. Used. Thus, the operator can provide the bag when it is needed (eg, grab the tubing tail, insert its free end into the welding device, and adjacent the bag to the end of the tubing tail. Lay the free end of the bag tube on its side, cut the tube with a new razor blade, and pull the razor apart while the two tube ends are squeezed together while melting as it is to resolidify together. By heating the cut end when Conversely, the bag can be removed by welding the pipeline from the bag and cutting two closed pipelines at the weld. Therefore, the storage tanks / bags may be connected individually when desired, and the present invention gives the operator access to the appropriate tubing tail throughout the process to connect the storage tanks / bags in time for their use. So it is not necessary that all storage tanks / bags must be connected at the beginning of the protocol. In fact, as described below, all storage tanks / bags are pre-connected, but the present invention does not require pre-connection, and one advantage of the second module 200 is that it is operated by the operator. At times it allows access to fluid assemblies / pipelines, which allows used bags to be sterilized and connected so that other bags can be sterilized and connected in the protocol.

As illustrated in FIG. 6, the sampling assembly 448 comprises one or more sampling lines fluidly connected to the interconnect line 450, for example, sampling lines 476a-476d. Each of the sample lines 476a-476d may be provided with sample line valves 478a-d that are selectively operable to allow fluid to flow from the interconnect line 450 through the sample lines 476a-476d. As also shown there, the distal end of each sampling line 476a-476d is a sample collection device for collecting fluid from the interconnect line 450 (eg, sample collection devices 280a and 280d). ) Is configured to selectively connect. The sample collection device can take the form of any sampling device known in the art, such as a syringe, immersion tube, bag, and the like. FIG. 6 illustrates that the sampling assembly 448 is connected to an interconnect line, but in other embodiments, the sampling assembly is a first fluid assembly 440, a second fluid assembly 444, a first. An intermediate fluid flow path between the second bioreactor line valve 434 of the first bioreactor container 410 and the first bioreactor line valve 432, and / or the second bioreactor of the second bioreactor container 420. It may be fluid coupled to the fluid flow path intermediate between the line valve 438 and the first bioreactor line valve 436. Sampling assembly 448 optionally performs a fully functionally closed sampling of the fluid at one or more points within the system 400.

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, along the interconnect line 450. Define the filtration loop. The filter 484 is positioned along the filter line 482 for removing permeated effluent from the fluid passing through the filter line 482. As shown therein, the filter line 482 comprises an upstream filter line valve 486 and a downstream filter line valve 488 positioned upstream and downstream of the filter 484, respectively. The effluent line 490 provides fluid communication between the filter 484 and the second fluid assembly 444, and in particular with the tubing tail 470a of the second fluid assembly 444, which is connected to the effluent storage tank 472a. Bring. At this point, the effluent line 490 carries the effluent removed from the fluid passing through the filter line 482 by the filter 484 to the effluent storage tank 472a. As illustrated in FIG. 3, the filtration lines 482 are interconnected so that the flow of fluid through the interconnect line 450 can be forced through the filter line 482, as described below. Surround the connecting pipeline valve 452. The permeation pump 492, positioned along the effluent line 490, can operate to pump the effluent removed by the filter into the effluent storage tank 472a. In one embodiment, the filter 484 is preferably an elongated hollow fiber filter, but other tangential or orthogonal flow filtration means known in the art, such as flat sheet membrane filters, are also present invention. Can be utilized without departing from the broader aspect of.

In one embodiment, the valves of the first fluid assembly 440 and the second fluid assembly 444, as well as the bioreactor line valves (ie, valves 432, 434, 436, 438), sterile line valves 462, interconnect pipes. The road valve 452 and the filtration pipe line valves 486 and 488 are pinch valves manufactured by the method described below. In one embodiment, the conduit itself does not need to be equipped with a pinch valve, and the pinch valve diagrams of FIGS. 3-8 are simply locations where the pinch valve can operate on the conduit to prevent fluid flow. It may represent. In particular, as described below, the pinch valve of the flow architecture 400 acts / acts on the corresponding anvil while the fluid path / conduit is in between, “pinch off” the conduit and fluid flow. Can be provided by each actuator (eg, solenoid) that prevents it from passing through.

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

Next, with reference to FIGS. 8 to 11, the configuration of the first bioreactor container 410 according to one embodiment of the present invention is illustrated. The second bioreactor vessel 420 preferably does not have to be, but since it has the same configuration as the first bioreactor vessel 410, only the first bioreactor vessel 410 is below for simplicity. Explained. In one embodiment, bioreactor vessels 410, 420 are perfusion-compatible silicone membrane-based bioreactor vessels that support activation, transduction, and amplification of the cell population within. Bioreactor vessels 410, 420 may be used for cell culture, cell processing, and / or cell amplification to increase cell density for use in medical treatment or other processes. Although bioreactor vessels are disclosed herein for use in conjunction with specific cell types, bioreactor vessels activate, genetically modify, and / or amplify any suitable cell type. It should be understood that it can be used in. In addition, the disclosed techniques can be used in conjunction with adherent cells, i.e. 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. Manufactured and can function as it is.

As shown in FIGS. 8 and 9, the first bioreactor vessel 410 may include a bottom plate 502 and a vessel body 504 coupled to the bottom plate 502. The bottom plate 502 may be a rigid structure that supports the cell culture. However, the bottom plate is a non-solid plate (eg, open and / or porous) that is permeable to oxygen to be fed into the cell culture, as described in more detail with reference to FIG. It may be). In the illustrated embodiment, the bottom plate 502 is rectangular or almost rectangular in shape. In other embodiments, the bottom plate 502 may be of any other shape that allows the low profile vessel to be used and / or maximizes the space where the first bioreactor vessel can be utilized or stored. Good.

In one embodiment, the vessel body 504 comprises a rigid, generally concave structure that forms the cavity or internal compartment 506 of the first bioreactor vessel 410 when coupled to the bottom plate 502. As shown therein, the container body 504 may have a circumferential shape that resembles the circumferential shape of the bottom plate 502 so that the container body 504 and the bottom plate 502 can be coupled to each other. In addition, as in the illustrated embodiment, the container body 504 may allow visual inspection of the contents of the first bioreactor container 410 and / or the light may allow the first bioreactor container 410 to be lighted. It may be made from a transparent or translucent material that can allow it to enter. The internal compartment formed by the bottom plate 502 and the vessel body 504 is a cell medium and cells when using the first bioreactor vessel for cell activation, gene modification (ie, transduction), and / or cell amplification. Can contain cultures.

As best shown in FIGS. 8-11, the first bioreactor vessel 410 contains several cells involved in cell activation, transduction / gene modification, and amplification, such as media loading and effluent removal. It may have multiple ports through the vessel body 504 that may allow fluid communication between the internal compartment 506 and the outside of the first bioreactor vessel 410 for the process of. The port may include, for example, a first port 412 and a second port 416. The port 416 may be disposed at any position within the container body 504, such as through either the top surface 508 and / or the side surface 510 of the container body 504, as in the illustrated embodiment. As described in more detail herein, specific structures of the first bioreactor vessel 410, including specific amounts and locations of ports 412, 416, include cell activation, cellular genetic modification, and high. Allows the first bioreactor vessel 410 to be used to support cell density amplification.

FIG. 9 is an exploded view of an embodiment of the first bioreactor container 410. The bottom plate 502 of the first bioreactor container 410 may be the bottom or support of the first bioreactor container 410. As previously described, the bottom plate 502 can be formed from a non-solid structure. In the illustrated embodiment, the bottom plate 502 may contain a grid 510 which may be structurally rigid, but allows free gas exchange through the bottom plate 502 to an internal compartment 506 which houses the cell culture. An additional opening is provided. 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 hole 512 may be an opening for gas exchange and the crossbar 514 can be a structural support for other structures and cell cultures within the internal compartment 506 of the first bioreactor vessel 410.

To form an additional support for the cell culture in the inner compartment 506 of the first bioreactor vessel 410, the first bioreactor vessel 410 is a membrane that can be placed on the top surface 518 of the bottom plate 502. May have 516. The membrane 516 may be a gas permeable liquid impermeable membrane. Membrane 516 may also be selected to have properties that allow high gas permeability, high gas transport rates, and / or high permeability to oxygen and carbon dioxide. Thus, the membrane 516 can support high cell densities within the internal compartment 506 (eg, up to about 35 MM / cm ² ). The gas permeable characteristics of membrane 516 may allow free gas exchange to support cell culture and / or cell amplification. As such, the membrane 516 may be a cell culture surface and / or a cell amplification surface. The membrane 516 may have a relatively small thickness (eg, 0.010 inch or 0.02 cm), which may allow the membrane 516 to be gas permeable. In addition, the membrane 516 can be formed from a gas permeable material, such as silicone or other gas permeable material.

The flatness of the membrane 516 can increase the surface area of the cell culture that colonizes against activation, transduction, and / or amplification. A mesh sheet 520 may be placed 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. The mesh sheet 520 can be a structural support for the membrane 516, whereby the membrane 516 remains planar and is added to the first bioreactor vessel 410 for cell culture and / or cell culture and / or cell amplification. It cannot sag or distort under the weight of any cell culture medium. In addition, the mesh properties of the mesh sheet 520 may allow support for the membrane 516, but are still porous and therefore the environment immediately outside the internal compartment 506 of the first bioreactor vessel 410 and the first bioreactor vessel 410. Free gas exchange between and is possible. The mesh sheet can be a polyester mesh or any other suitable mesh material that can support the membrane and allow free gas exchange.

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

As described above, the 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 transfection, and / or cell amplification, such as fluid or media loading, effluent removal, collection, and sampling. It may allow communication between the internal compartment 506 and the outside of the first bioreactor vessel 410 for some of the processes involved. Each port 416 may include an opening 526 and a respective fitting or tubing 528 (eg, lure fitting, barb fitting, etc.). In some embodiments, the opening 526 may be configured to allow the tubing to be glued directly, eliminating the need for fittings (eg, counterbores).

In one embodiment, in addition to the first port 412 and the second port 416, the first bioreactor vessel 410 further comprises an air balance port 530 disposed within the top surface 508 of the vessel body 504. obtain. The air balance port 530 may be made in a manner similar to the first port 412 and the second port 416, with similar reference numbers representing similar parts. The air balance port 530 may further perform gas exchange between the internal compartment 506 and the outside of the first bioreactor vessel 410, which is used in cell culture for amplification. In addition, the air balance port 530 can help maintain atmospheric pressure within the internal compartment 506 so that an environment for cell culture and / or cell amplification is provided within the internal compartment. The air balance port 530 may be disposed through the top surface 508 of the container body 504 or at any other position around the container body 504, as in the illustrated embodiment. The central position of the vessel body 504 through the apex 508 prevents wetting of the air balance port 530 during cell culture mixing through the tilt 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 autoclaved. It may be made from a material that is processable and has gamma and / or ETO sterilization stability. As such, the first bioreactor vessel 410 for each element, and unit as a whole, can be used for activation, transduction, and amplification of living cells, and / or other processes of the cell production process.

The first bioreactor vessel 410 may be capable of allowing cell culture and / or cell amplification via perfusion, which provides the nutrients needed to support cell proliferation and in the cell culture. It can reduce impurities. Continuous perfusion is the addition of a fresh medium feed to the growing cell culture while simultaneously removing used medium (eg, used medium). The first port 412 and the second port 416 can be used for the perfusion process as described below. The first port 412 may allow communication between the internal compartment 506 and the outside of the first bioreactor vessel 410 and is a fresh medium (such as from the medium storage tank of the first fluid assembly 440). Can be used to add to the first bioreactor vessel 410. In some embodiments, the first port 412 may be disposed and penetrated within the vessel body 504 at any location on the surface of the cell culture and medium in the first bioreactor vessel 410. .. In some embodiments, the first port 412 may be arranged to contact or penetrate the surface of the cell culture and medium in the first bioreactor vessel 410.

The second port 416 can be located in any position within the first bioreactor vessel 410 that is completely or partially submerged under the surface of the cell culture and medium. For example, the second port 416 may be an almost lateral port disposed through one of the side 510s of the container body 504. In some embodiments, the second port 416 may be arranged so that the second port 416 does not reach the bottom of the internal compartment 506 (eg, membrane 516). In some embodiments, the second port 416 can reach the bottom of the internal compartment 506. The second port 416 may be a dual function port. As such, the second port can be used to withdraw the perfusion medium from the internal compartment 506 of the first bioreactor vessel 410 to facilitate perfusion of the cell culture. In addition, the second port 416 can also be used to remove cells from cell culture. As pointed out above, in some embodiments, the second port cannot reach the bottom surface of the internal compartment 506 of the first bioreactor vessel 410. For example, the second port 416 may be located approximately 0.5 cm away from the membrane 516. Thus, in a static planar position, the second port 416 removes used cell medium without withdrawing cells from the cell culture as cells can colonize membrane 516 (eg, 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. The first bioreactor vessel 410 is in the manner described below when it is desirable for cells to be removed from the internal compartment 506 to minimize hold-up volume, for example during harvesting of cell culture. , Can be tilted towards the second port 416, thereby allowing access to cells for cell retrieval.

In addition, in one embodiment, the second port 416 does not include a filter, so the perfusion process can be done without a filter. As such, there can be no physical inhibition that prevents cells from entering the second port 416 when the second port 416 is used for media removal. In addition, the second port 416 is such that the second port 416 is laterally disposed through the side 22 of the container body 504, but the second port 416 can tilt towards the membrane 516 and the bottom plate 502. It may be inclined as follows. The tilting feature of the second port 416 helps keep the second port 416 sealed with the first bioreactor vessel 410 in use, minimizing interference with the O-ring 522 and groove 524. However, it may be possible to position it at a relatively low position on the container body 504, which is closer to the membrane surface 36. Further, in some embodiments, the tilting feature of the second port 416 may be to reduce the flow rate of the fluid through the second port 416 when the used medium is removed. In addition, the port diameter, along with the fluid flow rate exiting the second port 416, causes the inhalation velocity through the second port 416, which is used to draw the medium from the external compartment 506, to the second port 416. The diameter may be such that the attractive force applied to the adjacent individual cells is minimized so as to be lower than the force of gravity pulling the cells toward the membrane 516. Thus, as described above, the second port 416 can be used to facilitate perfusion of the cell culture without removing the perfusion medium and substantially removing the cells from the cell culture. As the cell colonization time increases, the cell concentration in the removed medium decreases and can fall within the unmeasurable range smoothed by the location of the second port 416. In addition, the position of the internal opening 540 can be repositioned to change the recommended cell colonization time. Positions closer to the membrane 516 may be associated with longer colonization times, but locations at or near the top of the medium have shorter colonization times as cells colonize and deplete first from the top of the growth medium. is connected with.

Thus, in one embodiment, the second port 416 is used not only to remove the medium used in the perfusion process, but also to remove cells from the cell culture from the internal compartment 506, for example, when harvesting the cell culture. Can be used. To facilitate the removal of more perfusion medium and cells used, the vessel body 504 may include an angled or chevron-shaped side wall 532. Thus, the chevron-shaped side wall 532 comprises an apex, i.e. a tip point, 534. The apex 534 of the side wall 532 may further include a second port 416 through which the container body 504 is disposed near the tip point 534 when the container body 504 is coupled to the bottom plate 502. To. The angled side wall 532 and tip point 534 are media and / or cell cultures when the first bioreactor vessel 410 is tilted towards the second port 416, for example at a 5 degree angle. It may be possible to expel a larger amount of cells.

Medium within the internal compartment 506, as described in more detail with reference to FIG. 10, by using perfusions that proliferate cells, facilitated by the location of first port 412 and second port 416. It may be possible to reduce the height of the (eg 0.3-2.0 cm). The relatively low height of the medium in the internal compartment 506 allows the first bioreactor vessel 410 to be a relatively low profile vessel while allowing for the highest achievable increase in cell density. Can be. In addition, the use of perfusion by the first bioreactor vessel 410 supports cell growth by supplying fresh medium to the cells in the internal compartment 506, but also allows removal of impurities in the cell culture. Additional cell lavage within another device may not be necessary after reaching a particular cell density target within the first bioreactor vessel 410. For example, through filterless perfusion, the first bioreactor vessel 410 can be fed fresh medium at a rate of total replacement daily to reduce impurities in the cell culture (eg, as a result, as a result). Impurities are reduced at a rate of about 1 log every 2.3 days). Therefore, the structure of the first bioreactor vessel 410 allows the use of perfusion for the growth of the cell culture in the first bioreactor vessel 410, thus lowering the cell culture with impurity levels. It may be possible to amplify to high target densities. Also, through perfusion without a filter, as described below, the first bioreactor vessel 410 is used for cell seeding, rinsing, washing / residue reduction, and / or discharge / harvesting after amplification. The fresh medium may be supplied in substantially multiple doses per day (eg, in an amount greater than 2 doses per day).

A relatively low medium height can be maintained within the internal compartment 506 to facilitate the use of the low profile structure of the first bioreactor vessel 410. FIG. 10 is a cross-sectional view of the first bioreactor container 410 illustrating the height 536 of the cell culture medium 538 in the first bioreactor container 410. As previously described, the vessel body 504 can be attached to the bottom plate 502 to form an internal compartment 506 in which amplification of cell culture can be achieved through perfusion. As such, replacement or fresh medium 538 may be supplied for cell proliferation through a first port 412 disposed through the container body 504, existing or used medium 538. Can 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 the relatively low medium height 536 of medium 538 in the internal compartment 506 of the first bioreactor vessel 410. The relatively low height 536 of the perfusion medium 538 in the internal compartment 506 allows the first bioreactor vessel 410 to have a low profile structure, thus providing an overall compact cell production system. It can be possible.

The height 536 of the perfusion medium 538 in the internal compartment 506 of the first bioreactor vessel 410 may be between 0.3 cm and 2 cm, and the headroom 542, i.e. the container body in the medium 538 and the internal compartment 506. The height of the gap formed between the top surface 508 of the portion 504 may be about 2 cm. Thus, 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 the surface area of membrane 516 below a certain value. As such, the ratio of medium volume to membrane surface area may be below threshold levels or within the desired range, facilitated by the use of perfusion to proliferate cells in cell cultures. To. For example, the threshold level can be a ratio between 0.3 and 2.0. Due to the small ratio of medium volume to membrane surface area, it may be possible to make the first bioreactor vessel 410 a low profile or compact structure while still providing cell cultures with high cell density.

As previously described, the dual-function second port 416 is the vessel body so that it is completely or partially submerged under the surface 544 of medium 538 in the first bioreactor vessel 410. Can be disposed through 504. In some embodiments, the second port 416 may be arranged such that the second port 416 reaches the bottom of the internal compartment 506 (eg, membrane 516). Positioning of the second port 416 can facilitate the removal of medium and impurities from the cell culture in the internal compartment 506 without removing the cells until such removal, eg harvesting, is desired. The second port 416 without a filter, along with the first port 412, uses perfusion to feed growth medium 538 to cells for cell amplification and remove used medium 538 and other impurities or by-products. Can make it possible. The location of the first port 412 and the dual functioning second port 416 around the vessel body 504 keeps the medium height 536 in the internal compartment 506 at a relatively low level, thereby the first. The bioreactor vessel 410 of 1 facilitates a configuration that can 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 provides various features that allow the use of the bioreactor vessel as part of the broader bioprocessing system 10, especially of the bioreactor vessel 10. Provided as a second module 200. As shown therein, the bottom plate 502 comprises a plurality of recesses 550 formed within the bottom surface of the bottom plate 502, the purpose of which is described below. In one embodiment, the recess may be located adjacent to a corner of the bottom plate 502. Each recess 550 generally has a cylindrical shape and can be terminated at a dome-shaped or hemispherical inner surface. As also shown in FIG. 11, the bottom plate 502 interacts with the sensor of the second module 200 to ensure proper positioning of the first bioreactor vessel 410 within the second module 200. It may comprise a position verification structure 552 configured as such. In one embodiment, the position verification structure may be a beam break configured to block the light beam of the second module 200 when the first bioreactor vessel 410 is properly mounted therein. ..

The bottom plate 502 also comprises a pair of flat engaging surfaces 554 formed on adjacent bottom surfaces that are offset from the centerline of the bottom plate (crossing the width of the bottom plate) bottom plate. Desirably, the engaging surface 554 is spaced along the vertical centerline of the bottom plate 502 so that it is located adjacent to the opposing ends of the bottom plate 502. The bottom plate 502 may further comprise at least one opening or opening 556 that allows the contents of the first bioreactor container 410 to be sensed by a bioprocessing device that engages and operates the bioreactor container.

In one embodiment, the first and second bioreactor vessels 410, 420 and fluid architecture 400 may be integrated into the assembly or kit 600 in the manner disclosed below. In one embodiment, the kit 600 is a one-time disposable kit. As best shown in FIGS. 12-14, the first bioprocessing vessel 410 and the second bioprocessing vessel 420 are placed side by side in tray 610 of the disposable kit 600 and vary in flow architecture 400. Tubes are arranged in tray 610 according to the method described below.

Further referring to FIG. 15, the tray 610 is a plurality of generally thin, rigid bodies including a front wall 612, a rear wall 614, and opposite sides 616, 618 that surround the bottom 620 and a generally open top. Or with a semi-rigid side wall. The side wall and bottom 620 define the internal compartment 622 of the tray 610. In one embodiment, the open top of the tray 610 accepts a removable cover (not shown) surrounding the internal compartment 622, as well as the upper rim of the drawer of the bioprocessing device, as shown below. It is surrounded by a peripheral flange 624 that provides a surface for installation, preferably on top. The bottom surface 620 of the tray 610 comprises a number of openings corresponding to the number of bioreactor containers in the bioprocessing system. For example, the tray 610 may include a first opening 626 and a second opening 628. The bottom surface 620 may also include additional openings 630 adjacent to the first and second openings 626, 628 for the purposes described below. In one embodiment, the tray 610 may be thermoformed, manufactured by a 3D printer, or injection molded, but other manufacturing techniques and processes also deviate from the broader aspects of the invention. Can be used without doing.

As best shown in FIG. 15, each of the first and second openings 626, 628 is the bottom of the tray 610 through which parts of the bioreactor vessels 610, 620 remain intact through the respective openings 626, 628. As the first and second bioreactor vessels 410, 420 can be positioned above the respective openings 626, 628 and supported by the bottom 620 of the tray 610 in the internal compartment 622, making them accessible from. It has a circumference of shape and / or dimensions. In one embodiment, the perimeter of the openings comprises at least one claw or protrusion to support the bioreactor vessel above each opening. For example, assuming that the perimeter of each opening 626, 628 is provided with a claw 632 that projects inward toward the center of the openings 626, 628 to support the bioreactor vessels 410, 420 placed on it. Good. As shown in FIGS. 12 and 15, the tray 610 of the openings 626, 628 to prevent lateral movement of the bioreactor vessel when received over the openings 626, 628, respectively. It may also have one or more ridges extending upwards above. Therefore, the ridge acts as an alignment device that facilitates proper positioning of the bioreactor vessels 410, 420 in tray 610, and the loading of kit 600 in the second module 200, as described below. Or help prevent the bioreactor vessels 410, 420 from inadvertently moving during positioning.

Further referring 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 traverse the width and / or length of the tray 610, providing rigidity and strength to the tray 610 and facilitating movement and operation of the kit 600. The rib 636 can be integrally formed with the tray or can be added as an auxiliary component via mounting means known in the art (see Figure 13). In one embodiment, the tray 610 holds the fluid flow lines in an organized manner and holds them in place for engagement by pumps and pinch valves, through which the tubing module 650 is also described herein. It comprises an opening 638 for receiving an engagement plate, referred to in. In other embodiments, the tubing module 650 may be integrally formed with the rear wall 614 of the tray 610.

16 and 17 illustrate the configuration of the tubing module 650 according to an embodiment of the present invention. As shown therein, the tubing module 650 accepts the first fluid assembly line 442, the interconnect line 450, and the waste liquid line line 490 of the fluid flow system 400 and the first fluid assembly line 442. , Interconnected line 450, and permeation fluid line 490 are in place so as to selectively engage the respective pump heads 454, 456, 492 of the perturbation pump assembly described below with respect to FIGS. 35 and 36. It comprises a first tubing holder block 652 configured to hold in. In one embodiment, the fluid assembly line 442, the interconnect line 450, and the effluent line 490 are maintained in a horizontally extending and vertically separated orientation by a first tubing holder block 652. In particular, as best shown in FIG. 17, the first tubing holder block 652 has two spaced arrangements 656,658 (tubes and slots within the tubing holder block 652) that define a gap between them. Engage with pipelines 442, 450, 490, respectively) (through clips between, or simple interference, etc.). As also shown in FIG. 17, the first tubing holder block 652 comprises a clearance opening 660 configured to receive a shoe (not shown) of the peristaltic pump assembly. This configuration causes peristaltic compression on the shoes of the lines 442, 450, 490 by the respective pump heads of the peristaltic pump and provides the respective propulsion force for the fluid through the line as described below. Can be done.

Further referring to FIGS. 16-18, the tubing module 650 further comprises a second tubing holder block 654 integrally formed (or otherwise coupled) with the first tubing holder block 652. The second tubing holder block 654 is configured to accommodate all of the fluid flow lines of the fluid flow system 400 with which the pinch valve is associated. For example, the second tubing holder block 654 has tubing tails 464a-f of the first fluid assembly 440, tubing tails 470a-d of the second fluid assembly 444, and a first bioreactor container 410. The bioreactor line 414 and the second bioreactor line 418, the first bioreactor line 424 and the second bioreactor line 428 of the second bioreactor container 420, and the sterile air source line 460. , The interconnection line 450 and the filtration line 482 (and, in some embodiments, the sampling lines 476a-476d) are configured to hold. Like the first tubing holder block 652, the second tubing holder block 654 may extend these tubes horizontally and maintain them in a vertically spaced orientation. In particular, the second tubing holder block 654 may include a plurality of vertically spaced, horizontally extending slots 666 configured to accommodate the pipeline in. 18 and 19 also best illustrate the configuration of slot 666, which also holds all of the flow lines that are subject to / contact with the pinch valve. Desirably, slot 666 traces the contour of block 654, but in particular traverses the planar back plate to open towards filter 484. As shown in FIG. 18, in one embodiment, the second tubing holder block 654 is a second tubing holder for holding a loop in the interconnect line 450 from which the sampling line extends. The bottom of the block 654 may have one or more narrow tubing slots 682 and a effluent line tubing slot 684 for receiving the tubing tail 470a connected to the effluent storage tank 472a.

The second tubing holder block 654 may include a planar back plate 662 having a plurality of openings 664 corresponding to the plurality of fluid flow lines held by the second tubing holder block 654. In particular, at least one opening 664 is horizontally aligned with each slot 666 and the flow conduit held therein. As best shown in FIG. 16, the second tubing holder block 654 has two clearance openings 668, 670 configured to accommodate the anvil (not shown) of the pinch valve assembly through. To be equipped. This configuration comprises 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 bioreactor vessel 410, as described below. 1 bioreactor line 414 and 2nd bioreactor line 418, 1st bioreactor line 424 and 2nd bioreactor line 428 of 2nd bioreactor container 420, and sterile air source line Allows the 460, interconnect line 450, and filter line 482 to be selectively compressed against the anvil by the respective pistons of the actuators of the pinch valve array to selectively block or allow fluid flow. to enable. As shown in FIGS. 18 and 19, the openings 664 may be configured to be arranged in first and second rows positioned side by side, and the openings in the first row of openings are openings. The openings in the first row of openings are vertically offset with respect to the openings in the second row of openings so that they do not align horizontally with the openings in the second row of openings. This configuration allows the tubing module 650, tray 610, and kit 600 to have a low profile as a whole.

In one embodiment, the filter 484 (shown as an elongated hollow fiber filter module in FIG. 16) is with the tubing module 650, such as by attaching the filter 484 to the tubing module 650 using a retaining clip 672. Can be integrated. If the filter 484 is a hollow fiber filter, the filter 484 may extend substantially over the entire length of the tubing module 650 and is the first to receive fluid input flow from the filter line 482. The charging end 674 and the holding fluid after removing the permeate / waste liquid are carried back to the filtration line 482 and the interconnection line 450, and are of the first bioreactor container 410 or the second bioreactor container 420. It may be equipped with a second output 676 for circulation to one side. The filter 484 may also include a permeate port 678 adjacent to a second output 676 that connects to a waste conduit 490 for transporting the waste liquid / permeate to the permeate / permeate storage tank 472a. Finally, the tubing module 650 receives the clip and receives the bioreactor line (eg, the first and second bioreactor lines 414, 418 and / or the second bioreactor container 420 of the first bioreactor container 410. Can have multiple features 680 for organizing the first and second bioreactor lines 424, 428)

Like the tray 610, the tubing module 650 may be thermoformed, machined by a 3D printer, or injection molded, but other manufacturing techniques and processes are also broader aspects of the invention. Can be used without deviating from. As described above, in one embodiment, the tubing module 650 may be integrally formed with the tray 610. In other embodiments, the tubing module 650 may be a separate component that is detachably accepted by the tray 610.

20 to 22 are various diagrams showing one embodiment of the kit 600, which are received by the first bioreactor container 410 and the second bioreactor container 420 and the tubing module 650 received in the tray 610. Illustrates the fluid pipeline of the flow architecture 400. As illustrated therein, instead of having an opening 630, the kit 600, as shown in FIGS. 20-22, has a solid floor and a sampling line (eg, sampling lines 476a, 476b). A sampling space 631 is provided in the tray 610 to accommodate the container that holds). Kit 600 includes a modular platform for cell treatment that is easily set up and can be discarded after use. The tubing tails of the first and second fluid assemblies 440, 444 allow the use of plug-and-play features, which makes it quick and easy to connect various media, reagents, effluents, samplings, and collection bags. It allows you to run the various processes you use on a single platform. In one embodiment, the connection and disconnection is performed by sterile cutting and welding of the tube segment, as described above, such as by a TERUMO device, or by pinching the tail segment as is known in the art. Can be accomplished by welding and cutting.

Then, referring to FIGS. 23-25, the kit 600 contains all of the hardware required to operate the kit 600 as part of the bioprocessing method (ie, controller, pump, pinch valve actuator, etc.). Specially configured to be accepted by the bioprocessing device 700. In one embodiment, the bioprocessing device 700 and the kit 600 (accommodating the flow architecture 400 and the bioreactor vessels 410, 420) are together described above in connection with FIGS. 1 and 2. Form the bioprocessing module 200 of. The bioprocessing device 700 comprises a housing 710, which has a plurality of drawers 712, 714, 716 that can be accommodated within the housing 710. FIG. 23 shows a device 700 that houses three drawers, but the device has only one, two, or more drawers with a single drawer and bioprocessing within each drawer. The operations may be executed at the same time. In particular, in one embodiment, each drawer 712, 714, 716 is described above with respect to FIG. 2 as a stand-alone bioprocessing module for performing the processes of cell activation, genetic modification, and / or amplification. It may be equivalent to the second module 200a, 200b, and 200c). In this regard, any number of drawers may be added to the 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, in one embodiment each drawer may be accommodated within a dedicated housing so that the housings can be stacked on top of each other.

As shown in FIGS. 23 and 24, each drawer, eg, drawer 712, comprises a plurality of side walls 718, a processing chamber 722 and a bottom surface 720 defining a generally open top. Drawer 712 is a processing chamber 722 through a closed position where the drawer is fully received within housing 710 and a top where drawer 712 extends from housing 710 and is open, as shown for drawers 714 and 716 in FIG. It is movable to and from the open position, as shown for the drawer 712 in FIGS. 23 and 24, which allows access to. In one embodiment, one or more of the side walls 718 are temperature controlled to control the temperature inside the processing chamber 722. For example, one or more of the side walls 718 may include an embedded heating element (not shown) or be thermally communicative with the heating element, whereby the side wall 718 and / or The processing chamber 722 can be heated to a desired temperature to maintain the processing chamber 722 to a desired temperature (eg, 37 ° C.) that is optimized for the process steps to be performed by the module 200. In some embodiments, the bottom surface 720 and the underside of the top surface of the housing (above the processing chamber when the drawer is closed) can be temperature controlled in the same manner (eg, with an embedded heating element). The hardware compartment 724 of the drawer 712 behind the processing chamber 722 may accommodate all of the hardware components of device 700, as detailed below. In one embodiment, the drawer 712 is an auxiliary compartment adjacent to a processing chamber 722 for accommodating a storage tank for containing media, reagents, etc. connected to the first fluid assembly 440 and the second fluid assembly 444. Further equipped with 730. In one embodiment, the auxiliary compartment 730 can be refrigerated.

Each drawer, eg, drawer 712, may be slidably accepted onto opposing guide rails 726 mounted inside housing 710. The linear actuator may be operably connected to the drawer 712 to selectively move the drawer 712 between the open and closed positions. The linear actuator can operate to allow the drawer 712 to move smoothly and in a controlled manner between the open and closed positions. In particular, the linear actuator provides the drawer 712 at a substantially constant speed (and with minimal acceleration and deceleration at stop and start of operation) to minimize disturbance to the contents of the bioreactor vessel. It is configured to open and close.

FIG. 25 is a top view showing the interior of the drawer showing the processing chamber 722, hardware compartment 724, and auxiliary compartment 730 of the drawer 712. As illustrated therein, the hardware compartment 724 is located behind the processing chamber 722 and is integrated with the power supply 732 and the second module controller 210 or communicates in some other way. It comprises 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. The hardware compartment 724 of the drawer 712 further comprises a pump shoe 742 and a pair of pinch valve anvils 744 for interlocking with the pump assembly 738 and solenoid array 736, respectively, as described below. In one embodiment, the pump shoe 742 and the solenoid anvil 744 are secured to the front base plate (front plate) of the processing chamber. All hardware compartments (and the components described) are mounted on the rear base plate. Both plates are slidably attached to the rails. 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 (moving the pump roller head into the pump shoe). , Thereby squeezing the pump tubing if inserted in between). As further described herein, the pump assembly selectively manipulates pipelines 442, 450, and 490 of the fluid path 400 to provide independent peristaltic propulsion. Similarly, the tubing holder block 654 of the tray 600 will be positioned between the solenoid array 736 and the anvil 744, as described further.

As illustrated in FIG. 25, two bed plates, eg, first and second bed plates 746, 748, are located within the processing chamber 722 on the bottom surface 720 and extend upwards. Or stick out from there. In one embodiment, the processing chamber 722 may accommodate a single bed plate, or three or more bed plates. Bed plates 746, 748 are configured to accept or otherwise engage the first bioreactor vessel 410 and the second bioreactor vessel 420 on it. As also shown in FIG. 25, the drawer 712 is located adjacent to the bed plates 746, 748 in the processing chamber 722 to sense the weight of the storage layer, eg, the waste liquid storage tank 472a positioned above. It also has a plate 750 that is configured with a load cell placed in.

Figures 26-28 best illustrate the configuration of bed plates 746, 748, with Figure 28A showing the hardware components positioned under the bed plate. As used herein, bed plates 746, 748 and hardware components (ie, sensors, motors, integrated with or positioned under it as shown in Figure 28A, Actuators, etc.) may be collectively referred to as bed plates. The first and second bed plates 746, 748 are substantially identical in configuration and operation, but for simplicity, the following description of bed plates 746, 748 will refer only to the first bed plate 746. .. Bed plates 746, 748 generally have a substantially flat top surface 752 having a shape and surface area corresponding to the shape and area of the bottom plate 502 of the first bioreactor vessel 410. For example, the bed plate may generally have a rectangular shape. Bed plates 746, 748 may generally also include a relief or clearance area 758 corresponding to the position of the protrusion or claw 632 of the tray 610, the purpose of which is described below. Bed plates 746, 748 are supported by a plurality of load cells 760 (eg, four load cells 760 positioned below each corner of bed plate 746). The load cell 760 is configured to sense the weight of the first bioreactor vessel 410 in bioprocessing for use by the controller 210.

In one embodiment, the bed plate 746 has an embedded heating element so that the contents of the processing chamber 722 and / or the first bioreactor vessel 410 placed on it can be maintained at the desired temperature. It may be provided or may be thermally communicated with the heating element. In one embodiment, the heating element may be the same as or different from the heating element that heats the side wall 718, the top wall, and the bottom surface.

As illustrated, the bed plate 746 comprises a plurality of positioning or alignment pins 754 protruding above the top surface 452 of the bed plate 746. The number of positioning pins 754 and the position and spacing of the positioning pins 754 may correspond to the number, position, and spacing of recesses 550 within the bottom surface of the bottom plate 502 of the bioreactor vessels 410, 420. As shown below, the positioning pin 754 allows the first bioreactor vessel 410 to ensure proper alignment of the first bioreactor vessel 410 on the first bed plate 746 in the processing chamber. Receivable in recess 550 of bottom plate 502 of first bioreactor vessel 410 when positioned within 722.

Further referring to FIGS. 26-28, the bed plate 746 is an integrated sensor for detecting the proper alignment (or misalignment) of the first bioreactor vessel 410 on the first bed plate 746. 756 may be further equipped. In one embodiment, the sensor 756 is an infrared light beam, but other sensor types, such as lever switches, can also 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 mounted 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 (ie, a flat claw), and a virtually IR opaque position verification structure 552 is used, the first bioreactor vessel. When the 410 is fully mounted on the bed plate 746, the beam break blocks the infrared light beam (ie, breaks the beam). This signals the controller 210 that the first bioreactor vessel 410 has been properly installed. If, after positioning the first bioreactor vessel 410 on the first bed plate 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 Indicates that it is not completely or properly installed 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 allow the first bioreactor vessel 410 to be horizontal on the bed plate 746 before starting bioprocessing. Make sure it is installed in the correct position (as determined by the alignment pins).

Still further referring to FIGS. 26-28A, the bed plate 746 is additionally positioned at an embedded temperature aligned with the opening 556 in the bottom plate 502 of the first bioreactor vessel 410. It is equipped with a sensor 759. The temperature sensor 759 is configured to measure or sense one or more parameters within the bioreactor vessel 410, such as the temperature level within the bioreactor vessel 410. In one embodiment, the bed plate 746 is additionally configured with a resistance temperature detector 760 to measure the temperature of the top surface 752 and carbon dioxide to measure the carbon dioxide level in the bioreactor vessel. It may be equipped with a sensor (located under the bed plate).

As further shown in FIGS. 26-28A, each bed plate 746, 748 comprises, for example, an actuator mechanism 761 (eg, a motor) with a pair of opposing cam arms 762. The cam arm 762 is housed in slot 764 of the bed plates 746, 748, with the clearance position where the cam arm 762 is positioned below the top surface 752 of the bed plate 746 and the cam arm 762 above the top surface 752 of the bed plate. Engagement that extends and contacts the opposing flat engaging surface 554 of the bottom plate 502 of the first bioreactor vessel 410 when the first bioreactor vessel 410 is received over the first bed plate 746. It is rotatable around the cam pin 766 to and from the mating position. As described in detail below, the actuator mechanism can operate to tilt and agitate the bioreactor vessel on the bed plate and / or assist in draining from the bioreactor vessel.

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

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

31 and 32 show the clearance and engagement positions of the pump assembly 738 and solenoid array 736 in a relatively easy-to-understand manner. As illustrated in FIG. 31, at the clearance position, the pump assembly 738 and solenoid array 736 are separated from the pump shoe 742 and the pinch valve anvil 744, respectively. After the lead screw 772 is activated, the drawer engagement mechanism 740 moves the pump assembly 738 and solenoid array linearly forward to the position shown in FIG. In this position, the pump head of the pump assembly 738 engages the pipelines 442, 450, 490 in the first tubing holder block 652, and the solenoid array 736 is a pinch valve anvil 744 with the piston / actuator of the solenoid array 736. Each fluid flow line of the second tubing holder block 654 can be pinched / tightened relative to the pinch valve anvil 744, which blocks the flow through the fluid flow line. ..

With reference to FIG. 24 again and further with reference to FIGS. 33-39, the drawer 712 may be controllably moved to the open position by activating the rocker switch 770 on the outside of the housing 710 during operation. The disposable drop-in kit 600, which houses the tubing module 650 (holding all tubes and tubing tails of the fluid architecture 400) and the first and second bioreactor vessels 410, 420, is lowered into place in the processing chamber 722. .. When the kit 600 is lowered into the processing chamber 722, the pump shoe 742 is positioned so that the pump tubes 442, 450, 490 are positioned between the pump shoe 742 of the peristaltic pump assembly 738 and the pump heads 454, 456, 492. Accepted through the clearance opening 660 of the first tubing holder block 652. FIG. 35 is a perspective view of the peristaltic pump assembly 738 showing the positioning of the pump heads 454, 456, 492 relative to each other. FIG. 36 illustrates the positioning of pump heads 454, 456, 492 with respect to pump tubes 442, 450, 490 when kit 600 is received within processing chamber 722. As shown therein, the pump tubes 442, 450, 490 are positioned between the pump shoe 742 and the pump heads 454, 456, 492. During operation, pump heads 454, 456, 492 start, maintain, and stop the flow of fluid through tubes 442, 450, 490 when the drawer engaging actuator 740 positions the pump assembly 738 in the engaging position. Therefore, it can be selectively operated under the control of the controller 210.

Similarly, when the kit 600 is lowered into the processing chamber 722, the pinch valve anvil 744 is held by the second tubing holder block 654, the tubing tails 464a-f of the first fluid assembly 440, the second fluid assembly. 444 tubing tails 470a-d, first bioreactor line 414 and second bioreactor line 418 of first bioreactor container 410, first bioreactor line 424 of second bioreactor container 420 And the second bioreactor line 428, sterile air source line 460, interconnect line 450, and filtration line 482 are positioned between the solenoid array 736 and the pinch valve anvil 744. Accepted through clearance openings 668, 670 of tubing holder block 654. This configuration is best illustrated in FIGS. 37-39 (FIGS. 37 and 38 show the solenoid array 736 and pinch valve anvil 744 before accepting the back plate 662 of the second tubing holder block 654 in space 776. Illustrates the relationship with).

As shown therein, each solenoid 778 of the solenoid array 736 has an associated opening (opening) within the back plate 662 of the second tubing holder block 654 to tighten the associated tube against the pinch valve anvil 744. It is equipped with a piston 780 that can extend linearly through (of 664). In this regard, the solenoid array 736 and the anvil 744 together form a pinch valve array (which is the valve of the first fluid assembly 440 and the second fluid assembly 444, as well as the bioreactor pipeline valve, (Including valves 432, 434, 436, 438, sterile line valves 462, interconnect line valves 452, and filtration line valves 486, 488). In particular, the pinch valve of the flow architecture 400 is provided by each solenoid 778 (ie, solenoid piston) of the solenoid array 736 that operates / acts on each anvil 744 while the fluid path / conduit is in between. Be done. In particular, when the drawer engaging actuator 740 positions the solenoid array 736 in the engaging position during operation, each solenoid 778 tightens the associated fluid flow line with respect to the anvil 744 to allow fluid flow through it. It can be selectively operated under the control of controller 210 to prevent it. In the present invention, it is intended that each fluid pipeline is positioned between a planar anvil surface and a planar solenoid actuator head. Alternatively, the solenoid actuator head is optimized to apply the desired pinching force to the elastic flexible fluid conduit, such as two tapered surfaces that meet at an elongated edge similar to a Phillips screwdriver. A head may be provided. Still alternative, the anvil surface is towards each fluid line so that the planar solenoid head presses the fluid line against this laterally extending protrusion to close the line to the fluid flow through it. It may have an elongated protrusion or protrusion extending to.

With reference to FIGS. 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 placed by the circumference of the opening and In particular, it is supported over openings 626, 628 by claws / protrusions 632. When the kit is further lowered, the bed plates 746, 748 penetrate the openings 626, 628 to accept or engage the bioreactor vessels 410, 420 in some other way. The shape of the openings 626, 628 and top 752 of the bed plates 746, 748 (eg, the embossed area 758 of the bed plates 746, 748 corresponding to the claws / protrusions 632 of the tray 610) is the bottom and claws / claws of the tray 610. The bioreactor so that the protrusion 632 is installed below the top surface 752 of the bed plates 746, 748 and the bioreactor vessels 410, 420 can be supported by the bed plates 746, 748, which are spaced apart from the bottom surface 620 of the tray 610. Allows tray 610 to continue moving downwards after containers 410, 420 have been accepted by bed plates 746, 748. This ensures that the tray 610 does not interfere with the horizontal installation of the bioreactor vessels 410, 420 on the bed plates 746, 748.

When the bed plates 746, 748 penetrate the openings 726, 728 in the tray 610, the positioning pin 754 on the bed plates 746, 748 corresponds to the recess 550 in the bottom plate 502 of the bioreactor vessels 410, 420. Accepted within, this ensures that the bioreactor vessels 410, 420 are properly aligned with the bed plates 410, 420. When properly mounted on the bed plates 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 vessels 410, 420 are in the proper position. .. Since the bed plates 746, 748 and the alignment pins are at the same height, blocking the light beam of the sensor 756 by the beam break 552 also ensures that the bioreactor vessels 410, 420 are horizontal. In this properly installed position, the sensor 759 on the bed plates 746, 748 is aligned with the opening 556 in the bottom plate 502, thereby adjusting the processing parameters in the internal compartments of the bioreactor vessels 410, 420, respectively. Make it possible to sense. In addition, in the fully installed position, the cam arms 762 of the bed plates 746, 748 are aligned with the flat engagement surface 554 on the bottom plate 502 of the bioreactor vessels 410, 420, respectively.

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

As further shown in FIG. 40, in one embodiment, the side wall 718 and bottom (and housing top wall) of the drawer 712 help minimize heat loss from the cover 788, processing chamber 722. It may include an insulating foam layer 790 for, a film heater 792 for heating the walls as described above, and an internal metal plate 794. In one embodiment, the internal metal plate 794 can be formed from aluminum, but other thermally conductive materials can also be utilized without departing from the broader aspects of the invention. The drawer 712 is a brush seal 796 with one or more brush seals to help minimize heat loss from the processing chamber 722, and other components of the device 700 from the drawer 712 (housing 710 or other drawers (eg, for example). It may further be provided with an insulating layer 798 to minimize or impede the flow of thermal energy to (drawers 714, 716), etc.).

Seeing FIG. 34 again, when the kit 600 is received in 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 liquid bag 472a to the tubing tail 470a. It penetrates the opening 730 in the tray 610 so that it can be positioned on the load cell 750. As shown therein, when the kit 600 is received in the drawer 712, the second tubing holder block 654 has the tubing tails 464a-f and the second fluid assembly 444 of the first fluid assembly 440. The tubing is held so that the tubing tails 470b-d penetrate into the auxiliary compartment 730 for the connection of its storage layer. In one embodiment, the sampling lines 476a-476d also penetrate into the auxiliary compartment 730.

Next, with reference to FIGS. 41 to 44, the operation of the cam arm 762 of the bed plates 746 and 748 is illustrated. As shown there, the cam arms 762 have a retracted position where they are positioned below the top surface of the bed plates 746, 748 and they are rotated around the cam pins 766 and above the bed plates 746, 748. It can be extended to engage with the flat engagement surface 554 of the bioreactor vessels 410, 420 and move from the bed plates 746, 748 to the engagement position where the bioreactor vessels 410, 420 are lifted and pulled away. The bioreactor vessels 410, 420 are supported (and especially in the horizontal position) on the horizontal bed plates 746, 748, as the cam arm 762 is pulled under the top surface of the bed plates 746, 748 in the default state. Alignment pin 754. No power is required to keep 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 servomotors to maintain a horizontal position. In fact, in the cam arm 762 configuration of the present invention, the actuator only needs to be energized when tilting the bioreactor vessel for stirring / mixing, as described below, thereby heating the processing chamber 722. Is minimized.

As shown in FIGS. 41-43, the cam arm 762 may be capable of sequential operation 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 with the opposite end mounted on the bed plate and the positioning pin 754 on the non-raised end is the bottom plate. Lift one end of the bioreactor vessel 410 and pull it away from the bed plate 746 (and disengage it from the positioning pin 754 on the bed plate 746) while remaining accommodated in the corresponding recess 550 in the 502. Is activated. The raised cam arm is then rotated to return to the clearance position under the bed plate, and the opposing cam arm is rotated to the engaging position to lift the opposing end of the bioreactor vessel and pull it away from the bed plate and positioning pin.

In one embodiment, the cam actuation system may be designed so that the cam arm 762 can return to the home position without touching the bioreactor vessel, thereby preventing disturbance to the culture and the cam arm 762 is long. Allows you to return (or be tested) to the home position at any time during the cell processing period. Therefore, the present invention contemplates that the bioreactor vessel may be provided with other rocking or stirring means, but by having two cam arms 762 on opposite sides of the bed plate, the overall height of the mixing mechanism can be increased. It can be kept to a minimum. For example, a central actuator (located in the center of the bed plate) can achieve ± 5 degree movement, but 0-5 degree movement of the container driven by cam arms on both sides of the container of the container. Almost the same movement can be achieved, which effectively brings ± 5 degrees of movement to the vessel at half the height. In addition, the movement of the cam arm 762 (eg, the speed of cam arm rotation and the timing between the opposing cam arms) maximizes wave formation within the vessel and maximizes wave amplitude, thus (ideally) the vessel contents. It can be adjusted to maximize the homogeneity and time to achieve homogeneity. Timing can also be adjusted based on the volume in the container with a given geometry that maximizes mixing efficiency.

In one embodiment, the optical sensor 756 can be used to confirm that the first bioreactor vessel 410 has been properly repositioned after each cam agitation operation. It is further contemplated that the correct repositioning of the bioreactor vessel is checked and can be verified even during alternating cam movements. This allows misalignment to be detected quickly in virtually real time, thereby intervening the operator to re-install the bioreactor vessel without substantial deviation from the bioprocessing operation / protocol. Make it possible.

FIG. 43 is a schematic diagram showing the location of the fluid 800 in the bioreactor vessel during this agitation process. As shown in FIG. 42, in one embodiment, the homing sensor 802 integrated with 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 utilized by the controller for. This is useful in coordinating the movement of the cam arm 762 to give the desired mixing frequency within the bioreactor vessel. In one embodiment, the cam arm 762 is configured to provide a tilt angle of up to 5 degrees with respect to the bed plate 746.

With reference to FIG. 44, the interface between the positioning pin 754 of the bed plate of the bioreactor vessel 410 during mixing / stirring and the recess 550 in the bottom plate 502 is illustrated. In one embodiment, the recess 550 has a domed or hemispherical inner surface and a diameter d1 greater than the diameter d2 of the positioning pin 754. As illustrated in FIG. 44, this configuration provides a clearance between the positioning pin 754 and the recess 550, allowing the bioreactor vessel 410 to be tilted when the positioning pin 554 is received within the recess 550. it can.

In one embodiment, each drawer of the bioprocessing device 700, eg, the drawer 712, preferably comprises a flip-down front panel 810 that is hingedly attached, as shown in FIGS. 45-50. The flip-down front panel 810 allows access to the auxiliary compartment 730 without opening the drawer 712, as best shown in FIGS. 45, 49, and 50. As will be appreciated, this configuration allows for in-process sampling and medium bag replacement. In connection with the above, in one embodiment, the auxiliary compartment 730 may consist of a plurality of telescopic sliding rails 812 provided with attachment means 815 capable of suspending various storage tanks / medium bags. Rail 812 can be moved from a retracted position within the compartment 730, as shown in FIG. 48, to an extended position exiting the compartment 730, as shown in FIG. When the collection bag is full, or when the medium / fluid bag needs to be replaced, simply extend the rail 812 out and unclip the bag. New bags are attached to their respective tails and can then be hung from rails and slid back into the auxiliary compartment 730 without opening the drawer 712 or suspending processing. In one embodiment, the rail 812 can be mounted on a cross rod 814 that extends laterally. The rail 812 may thus be laterally slidable on the rod 814, extendable from the auxiliary compartment, and retractable into the auxiliary compartment. In addition, when the drawer is open (Figure 46), rail 812 is around the rear cross rod to clean the compartment 730 and allow the user to screw the tubing tail towards the front of the compartment 730. It can rotate, which gives it 3 degrees of freedom.

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

Referring to FIG. 52, in one embodiment, the bioprocessing device 700 may further comprise a low profile waste tray 816 that is accepted within each drawer, eg, housing 710 under the drawer 712. The effluent tray 816 is mounted independently on the drawer so that it can be moved between the closed and open positions. Desirably, in the closed position, the tray 816 extends in the same plane as the front surface of the drawer, and in the open position, the tray 816 exposes its chamber 819 so that the operator can access it. Chamber 819 makes it easy to store a large effluent bag connected to the fluid flow path of tray 600 that rests on it, and allows access to it without opening the drawer 712. In addition, in the closed position, the effluent tray 816 positions chamber 819 to align with the drawer and has a size and shape that can be operated to accommodate leaks from the processing chamber 722 or auxiliary compartment 730. Have.

In one embodiment, each drawer is a camera positioned above a processing chamber (eg, above each bioreactor container 410, 420) so that the interior of the drawer 712 can be visually monitored without opening the drawer 712. Can be equipped. In one embodiment, the camera (or additional camera) can be integrated with the bed plate assembly or on the side wall looking sideways inside the bioreactor vessel.

Therefore, the second module 200 of the present invention allows for automation of cell processing to a degree not previously found in the art. In particular, the fluid flow architecture 400, pump assembly 738, and pinch valve array 736 are fluid manipulations (eg,) between the bioreactor vessels 410, 420 and the bags connected to the first and second fluid assemblies 740, 744. , Fluid addition, transfer, drainage, rinsing, etc.) can be automated. As described below, this configuration also allows for hollow fiber filler concentrations and washes, filterless perfusion, and pipeline priming. The drawer engagement actuator 740 is also used for automatic engagement and engagement of the drop-in kit 600, further minimizing human touch points. In fact, human touchpoints may only be needed for source / medium bag addition and removal, sampling, and data entry (eg, sample volume, cell density, etc.).

Referring to FIGS. 53-77, automated for workflows with immobilized Ab coating by amplification in the same vessel, soluble Ab addition, gammaretrovirus vector using a second module 200 and its fluid flow architecture 400. General protocols are illustrated. This general protocol includes activation (exemplified in FIGS. 53-59), pre-transduction preparation and transduction (exemplified in FIGS. 60-71), amplification (illustrated in FIGS. 72-76), and. For some embodiments, it results in the harvesting of a population of cells in an automated, functionally closed manner (Fig. 77). 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. Therefore, after the valve is opened and a particular action is possible, when that action is complete, the valve is closed before proceeding to the next action / step.

As shown in FIG. 53, in the first step, the valves 432 and 468f are opened and the first fluid assembly pipeline pump 454 is activated to pump the antibody (Ab) coating solution to the first. From the storage tank 466f connected to the fluid assembly 440, it is sent to the first bioreactor container 410 through the first port 412. The antibody coating solution is incubated for a period of time and then drained through the interconnect line to the waste fluid storage tank 472a of the first fluid assembly 440 by opening valves 434, 474a and operating the circulation line pump 456. As described herein, the discharge of the bioreactor vessel 410 can be facilitated by tilting the bioreactor vessel 410 using the cam arm 462.

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

Referring to FIG. 55, after rinsing the first bioreactor vessel 410 in buffer, the cells in the seed bag 466d (already concentrated and isolated using the first module 100) are valve 468d. And 432 are opened and pump 454 is operated to transfer to the first bioreactor vessel. The cells are pumped through the first bioreactor line 414 of the first bioreactor vessel 410 and into the bioreactor vessel 410 through the first port 412. As shown in FIG. 56, valves 432 and 468a are opened, pump 454 is activated, and the pump pumps the second antibody (Ab) solution from the storage tank 466a connected to the first fluid assembly 440. Send to the first bioreactor vessel 410 through the first port 412.

After pumping the second antibody solution into the first bioreactor vessel, the second antibody solution storage tank 466a is rinsed and the rinse medium is pumped into the first bioreactor vessel. .. In particular, as shown in FIG. 57, valves 474b, 452, and 468a are opened and the rinsing medium from the rinsing medium storage tank / bag 472b of the second fluid assembly 444 is pump 454. It is sent to the antibody solution storage tank 466a of 2 and rinses the storage tank. After rinsing, valve 432 is opened and the rinsing medium is pumped from storage layer 466a to the first bioreactor vessel 410. In one embodiment, the second antibody solution storage tank 466a can be rinsed multiple times using this procedure.

After rinsing the second antibody solution storage tank 466a, the inoculum / seed cell bag 466d may also be optionally rinsed. In particular, as shown in FIG. 58, valves 474b, 452, and 468d are opened and the rinse medium from the rinse medium storage tank / bag 472b of the second fluid assembly 444 is inoculated using the pump 454. Sent to Material / Seed Cell Bag 466d and rinse bag. After rinsing, valve 432 is opened and the rinsing medium is pumped from bag 466d to the first bioreactor vessel 410 using pump 454. After rinsing the inoculum / seed cell bag 466d, pumping the rinsing medium into the first bioreactor vessel 410 reduces the cell density in the first bioreactor vessel 410. At this time, to measure one or more parameters of the solution in the bioreactor vessel prior to activation (eg, to ensure that the desired cell density is present prior to activation). Samples can be taken. In particular, as shown in FIG. 58, valves 434, 452, and 432 are opened, pump 456 is activated, and the first bioreactor vessel is along the first circulation loop of the first bioreactor vessel. Pump the contents of 410 (ie, from the second port 416, through the interconnect line 450, through the first bioreactor line 414 and the first port 412 of the first bioreactor vessel 410. Return to the first bioreactor vessel 410). To take a sample, a first sample container 280a (eg, immersion tube, syringe, etc.) is connected to the first sample tubing tail 476a, a valve 478a is opened, and part of the flow is interconnected pipeline 450. Bypass to the first sample container 280a for analysis.

If analysis of the sample taken shows that all solution parameters are within a predetermined range, then the solution in the first bioreactor vessel 410 is in solution, as illustrated in FIG. 59. Incubate for a predetermined period of time to activate a population of cells. For example, in one embodiment, the population of cells in the first bioreactor vessel 410 can be incubated for about 24-48 hours.

Then, referring to FIG. 60, after activation, valves 438 and 474b are opened, pump 456 is activated, and RetroNectin solution is pumped from storage tank 472b to the second bioreactor to prepare for transduction. Pump to the second bioreactor vessel 420 through the second port 426 of the vessel 420. After pumping the RetroNectin solution into the second bioreactor vessel 420 for the RetroNectin coating of the second bioreactor vessel 420, the solution is incubated in the second bioreactor vessel 420 for a predetermined period of time. Will be done. After incubation, all RetroNectin solutions are then stored from the second bioreactor vessel 420 by opening valves 438 and 474a and activating the circulation line pump 456, as further shown in FIG. It is discharged to tank 472a. Note that during these RetroNectin coating, incubation, and efflux steps (related to 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 need not be utilized in all processes.

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

Then, referring to FIG. 62, after a defined time period, all buffers in the second bioreactor vessel 420 are second by opening valves 438 and 474a and activating the interconnect pipeline pump 456. It is discharged to the waste liquid storage tank 472a of the fluid assembly 444.

At this point, the post-activation, pre-concentration sample can be taken from the cells in the first bioreactor vessel 410, as shown in FIG. As shown there, valves 434, 486, 488, and 432 are opened, pump 456 is activated, and the solution in the first bioreactor vessel 410 is circulated from the second port 434, Through the interconnect line, through the filter line 48 and the filter 484, through the first bioreactor line 414 of the first bioreactor vessel 410, through the first port 412, through the first bioreactor. Return to container 410. To collect the sample, a second sample container 280b (eg, immersion tube, syringe, etc.) is connected to the second sample tubing tail 476b, the valve 478b is opened, and part of the flow is interconnected pipeline 450. Bypass to the second sample container 280b for analysis.

Then, referring to FIG. 64, depending on the concentration obtained from the sample, the concentration can 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 be second. Circulating from port 416 of the, the solution passes through the second bioreactor line 418, the interconnect line 450, the filter line 482 and the filter 484, and the first of the first bioreactor container 410. It passes through the bioreactor line 414 and returns to the first bioreactor vessel 410 through the first port 412. As the fluid passes through the filter 484, the effluent is removed and the permeation pump 492 sends such effluent through the effluent line 490 to the effluent storage tank 472a of the second fluid assembly 444. In one embodiment, this procedure is repeated until the volume in the first bioreactor vessel 410 is concentrated to a predetermined volume.

With reference to FIG. 65, after concentration, the concentrated cell population in the activation vessel (ie, the first container 410 containing the concentrated 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 passed through the second port 416 so 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 through the interconnect line 450 through the first port 412 into the first bioreactor vessel 410. When the medium is added and removed from the container 410, the effluent may be filtered by the filter 484 and directed to the effluent storage tank 472a.

The post-wash sample can be taken from the cells in the first bioreactor vessel 410 in a manner similar to that 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 the fluid in the first bioreactor vessel 410 from the second port 434. Through the interconnect line, the filter line 48 and the filter 484, the first bioreactor line 414 of the first bioreactor container 410, and the first port 412. Return to bioreactor container 410. To collect the sample, a third sample container 280c (eg, immersion tube, syringe, etc.) is connected to the third sample tubing tail 476c, the valve 478c is opened, and part of the flow is interconnected pipeline 450. Bypass to a third sample container 280c for analysis.

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

After the second bioreactor vessel 420 is coated with the viral vector, as illustrated in FIG. 68, the washed cells from the first bioreactor vessel 410 are second for transduction / gene modification. Transferred to bioreactor container 420. In particular, valves 434, 452, and 436 are opened, the circulation line pump 456 is activated, pumping cells out of the first bioreactor vessel 420 to the second port 416 of the first bioreactor vessel 410. Through, through the interconnect line 450, to the first bioreactor line 424 of the second bioreactor container 420, and through the first port 422 of the second bioreactor container 420, the second bioreactor container 420. Send to.

The medium from the medium bag 466b is then added to the second bioreactor vessel 420 by opening the valves 468b and 436 and activating the pump 454, as illustrated in FIG. 69, to the second bioreactor. Increase the total volume of solution in container 420 to a given volume. Then, referring to FIG. 70, the pretransfection sample opens valves 438, 452, and 436, activates the circulation line pump 456, and transfers the solution in the second bioreactor vessel 420 to the second bioreactor vessel. Can be harvested by pumping along the circulation loop of (ie, from the second port 426, through the interconnect line 450, the first bioreactor line 414 and the first bioreactor line 414 of the second bioreactor vessel 420. Return to the second bioreactor vessel 420 through port 422 of 1). To take the sample, a fourth sample container 280d (eg, immersion tube, syringe, etc.) is connected to the fourth sample tubing tail 476d, the valve 478d is opened, and part of the flow is interconnected pipeline 450. Bypass to the 4th sample container 280d for analysis.

Population of cells in a second bioreactor vessel 420 when analysis of a fourth sample taken shows that all parameters are within the prescribed range required for successful transduction. Is incubated for a predetermined time period for transduction of a population of cells in solution, as shown in FIG. For example, in one embodiment, a population of cells in a second bioreactor vessel 420 can be incubated for about 24 hours for transduction.

Referring to FIG. 72, after transduction, medium 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 medium, valves 468b and 436 are opened, pump 454 is activated, and growth / perfusion medium is added from medium bag 466b to the second until a predetermined amplification volume is reached. Pump through the first port 422 of the bioreactor vessel to the second bioreactor vessel 420.

The pre-amplification sample then opens valves 438, 452, and 436, activates the circulation line pump 456, as illustrated in FIG. 73, and as shown above, the second bioreactor. The solution in vessel 420 can be harvested by pumping along the circulation loop of second bioreactor vessel 420 (ie, from second port 426 through interconnect line 450, second bioreactor. Return to the second bioreactor vessel 420 through the first bioreactor conduit 414 and the first port 422 of the vessel 420). To take the sample, a fifth sample container 280e (eg, immersion tube, syringe, etc.) is connected to the fifth sample tubing tail 476e, the valve 478e is opened, and part of the flow is interconnected pipeline 450e. Bypass to the 5th sample container 280e for analysis.

In the second bioreactor vessel 420, where analysis of the fifth sample taken shows that all parameters are within the predetermined range required for successful amplification of the cell population. Populations of cells are incubated for a predetermined period of time, eg, 4 hours, to colonize the cells.

After this incubation period, or at a given time thereafter, as shown in Figure 74, a single daily dose of perfusion (1x perfusion) is used / used medium from medium bag 466b. The soaked medium is pumped out of the second bioreactor vessel 420 through the second port 426 (and through the interconnect line 450 to the waste liquid storage tank 472a) and at the same time the second bio through the first port 422. It is performed by pumping into the reactor vessel 420. This perfusion is achieved by opening valves 468b, 436, and 474a and activating the first pump 454 and the circulation line pump 456. In this 1x perfusion, the medium from the medium bag 466b is placed in the second bioreactor vessel 420 at substantially the same rate as the medium used is removed from the second bioreactor vessel 420 and sent to the waste liquid section. Introduced into the second bioreactor vessel 420 to maintain a substantially constant capacity.

Sampling can then be performed as needed / when 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 the circulation line pump 456, and as shown above, the solution in the second bioreactor vessel 420. Can be harvested by pumping along the circulation loop of the second bioreactor vessel 420 (ie, from the second port 426, through the second bioreactor line 428, through the interconnect line 450. Return to the second bioreactor vessel 420 through the first bioreactor conduit 424 and the first port 422 of the second bioreactor vessel 420). To take the sample, another sample container 280x (eg, immersion tube, syringe, etc.) was connected to the sample tubing tail of sample assembly 448 and the tubing tail valve was opened as shown in Figure 75. , Part of the flow is passed through the interconnect line 450 and diverted to the sample container 280x for analysis. After each sampling operation, incubation without perfusion can be performed for a predetermined time period, eg, 4 hours, to allow cells to settle before resuming perfusion.

After this incubation period, as shown in FIG. 76, a once-daily rate of perfusion (1x perfusion), as shown in FIG. 74, used the medium from the medium bag 466b. / The used medium is pumped out of the second bioreactor vessel 420 through the second port 426 (and through the interconnect line 450 to the waste liquid storage tank 472a) and at the same time through the first port 422. It is carried out by pumping into the bioreactor vessel 420 of. This perfusion is achieved by opening valves 468b, 436, and 474a and activating the first pump 454 and the circulation line pump 456.

When sampling showed a predetermined threshold (eg, 5 MM / mL) of viable cell density (VCD), two doses of permeation per day (2x perfusion) were shown in Figure 76. , Medium from medium bag 466b, used / used medium is pumped out of the second bioreactor vessel 420 through the second port 426 (and through the interconnect line 450 to the effluent storage tank 472a). At the same time, it is carried out by pumping through the first port 422 into the second bioreactor vessel 420. This perfusion is achieved by opening valves 468b, 436, 438, and 474a and activating the first pump 454 and the circulation line pump 456. In this 2x perfusion, the medium from the medium bag 466b is placed in the second bioreactor vessel 420 at substantially the same rate as the medium used is removed from the second bioreactor vessel 420 and sent to the waste liquid section. Introduced into the second bioreactor vessel 420 to maintain a substantially constant capacity.

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

Therefore, the second module 200 of the bioprocessing system 10, as well as its flow architecture 400 and bioreactor vessels 410, 420, can perform various bioprocessing operations in a substantially automated and functionally closed manner. Providing a flexible platform. In particular, FIGS. 53-77 illustrate exemplary general protocols that can be implemented using the bioprocessing system 10 of the present invention (particularly using its second module 200). , The system is not limited to that in this regard. In fact, various automated protocols can be enabled by the systems of the invention, including a number of customer-specific protocols.

In contrast to existing systems, the second module 200 of the bioprocessing system 10 is a functionally closed automation that houses the first and second bioreactor vessels 410, 420 and the fluid manipulation and fluid containment system. Systems, all of which are maintained in cell culture friendly environmental conditions (ie, within temperature and gas controlled environments) to enable cell activation, transfection, and amplification. As described above, the system has automation kit loading and closed sampling capabilities. In this configuration, the system enables all steps of immune cell activation, transduction, amplification, sampling, perfusion, and lavage in a single system. Also, combine all steps within a single bioreactor vessel (eg, 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 amplified bioreactor vessel (eg, bioreactor vessel 420) can reliably produce billions of T cells for a single dose. Either single or multiple doses can be produced in situ with high recovery and high viability. In addition, the system is designed to give the end user the flexibility to execute different protocols for the production of genetically modified immune cells.

Some of the commercial benefits offered by the bioprocessing system of the present invention are simplified workflow, reduced labor intensity, reduced burden on clean room infrastructure, reduced fault nodes, and cost. Includes robust and scalable manufacturing technology to commercialize products with the ability to reduce and expand sales.

As described above with respect to the general workflow, the system of the present invention, the bioprocessing system 10, and the flow architecture 400 and bioreactor vessels 410, 420 of the second module 200 are automated and functionally closed. It provides a process of culture concentration, washing, slow perfusion, fast perfusion, and "round robin" perfusion to be performed in a fashion. For example, as explained above, the pump 456 on the interconnect line 450 has a permeation pump 492 in the enrichment step (typically at the rate of the circulation pump 456, for example about 10%). While in operation, it can be used to circulate fluid from one of the bioreactor ports through filtration line 482 and filter 484 and then back to another port on the bioreactor. it can. Concentration can be performed in an open loop or stopped based on the measured volume taken from the bioreactor or the measured volume accumulated in the effluent. In one embodiment, the filter, pump speed, filter area, number of lumens, etc. are all appropriately sized for the total number of cells and target cell density to limit shear contamination and excess cell loss. Is done.

In one embodiment, as described above, the system of the invention can also be used to clean, eg, remove residues such as residual viral vectors after incubation. Washing is described above for concentration, except that pump 454 on the first fluid assembly line 442 is used to pump additional medium and replace the fluid sent from permeation effluent pump 492. With the same steps. The rate of introduction of the new medium may correspond to the rate of fluid removal by the permeation pump 492. This makes it possible to maintain a constant volume within the bioreactor vessel, and the residue is exponential with respect to time as long as the contents in the bioreactor are well mixed (circulation can be adequate). Can be taken out. In embodiments, this same process can be utilized after activation for in-situ hollow fiber filtration-based washes of cell suspensions 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 the perfusion process, pump 454 on the first fluid assembly line 442 can be used to add medium to a given bioreactor vessel, interconnect line 450. The pump 456 above is used to move the used medium to a waste liquid bag in the second fluid assembly. In one embodiment, gravity may be used to colonize the cells and the used medium can be pumped at such a rate without significantly disturbing the cells in the bioreactor vessel. This process can involve running open loops of pumps 454 and 456 at the same speed. In one embodiment, one pump (454 or 456) may operate at a set speed, the speed of the other pump being the mass / capacity of the bioreactor container or the mass / capacity (or measured) of the effluent bag. It can be adjusted based on the mass / capacity of the source bag.

In connection with the above, it is contemplated that pump control may be based on the weight measurement results of the bioreactor vessel (using feedback from load cell 760). For example, the system configuration enables 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. In addition, this method can be used for closed-loop control over the rate of change in mass (volume) as the bioreactor vessel is emptied or filled.

In another embodiment, the bioprocessing system uses a flow architecture 400 to allow 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, as described above, are cells in the first bioreactor vessel 410 in conjunction with the proper pinch valve condition. Is used to perfuse. Perfusion of cells in the first bioreactor vessel 410 may then be stopped or suspended, and the circulation pump 456 and pump 454 and the appropriate pinch valve are then in the second bioreactor vessel 420. It may be actuated to perfuse the cells. In this regard, perfusion of the various bioreactors can be performed sequentially (ie, perfusion of the first bioreactor vessel 410 over a period of time, then perfusion of the second bioreactor vessel 420 over a period of time, alternating repeatedly. Will be done). This allows perfusion of any number of bioreactor vessels in the system without the use of more pumps, medium bags, or effluent bags.

For round-robin perfusion, the pumps can be operated continuously, intermittently together (duty cycle), or sequentially (source). , Then effluent, and so on), which allows the volume / mass in various bioreactor vessels to remain at about the same level. For round-robin perfusion (a pair of pumps running together intermittently and waiting at regular intervals), this also uses the same two pumps for multiple pumps, as shown. Allows perfusion of the container. In addition, round robin perfusion allows for low effective exchange rates (such as about 1 volume / day) even when the pump does not have a large low dynamic range. In addition, round robin perfusion also allows each container to be perfused with a different medium as controlled by the valve in the first fluid assembly 440.

In addition, in one embodiment, fast perfusion may be used for residue removal (eg, post-activation Ab removal and / or post-transduction residue removal). In the fast perfusion process, the perfusion process described above is significantly faster than a typical 1-5 volume / day, for example between about 8-20 volume / day, or only minutes to hours. It can operate at a value exceeding about 20 capacity / day that achieves 1 log reduction. In one embodiment, the perfusion rate is balanced against cell loss. In some embodiments, fast perfusion may allow the hollow filter 484 to be eliminated and still meet the biological requirements for rapid removal of residues after several steps.

As further described above, the system of the present invention uses a pump 454 on the first fluid assembly line 442 from another bag / storage tank connected to the second fluid assembly 444. Use a rinse buffer or fluid to facilitate the rinse of the bag / storage tank connected to the first fluid assembly 440. In addition, the fluid pipeline of Flow Architecture / System 400 is cleaned with sterile air from sterile air source 458, which prevents cells from sitting in the pipeline and dying, or media or reagents. It is possible to prevent the person from staying in the pipeline and deteriorating or becoming useless. The sterile air source 458 can also be used to expel reagents from the conduit to ensure that more reagents than intended are not pumped into the bioreactor vessels 410, 420. The sterile air source 458 also cleans the inside of the pipeline leading 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 using a sterile air source 458 to clean the pipeline, or in addition, the pipeline does not sink the port through which air is drawn in, and the bioreactor vessel has an air balance port 530. As long as it is, it can be cleaned using air drawn from one of the bioreactor vessels.

As described above, the system allows sampling of the contents of the bioreactor vessel within the closed drawer process. During sampling, the container from which the sample is drawn uses the cam arm 762, the circulation line pump 456 is used to circulate the contents of the container, and the sampling assembly 448 is used to pull the sample out of the interconnect line 450. It may be agitated. In one embodiment, only non-bead-bound cells can be agitated.

Also, as explained above, the system of the invention allows a population of cells to be harvested after the target cell density has been achieved. In one embodiment, collecting an amplified population of transduced cells is performed by using a pump 456 on the interconnect line 450 to connect the cells to a second fluid assembly 444. It may include moving to one of them, or circulating the cells by an interconnect pump 456 to move the cells to a bag attached 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 (ie, the syringe or bag in the first fluid assembly 440). By connecting to and 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 towards the syringe / bag). In such cases, the circulation pump 456 and valve can then be used to clean the fluid / cell circulation line. In addition, the pump 454 on the first fluid assembly line 442 uses the air in the line to transfer the sample to the vessel with no perceptible amount of cells left in the line. Upon completion, all of the aliquoted sample volumes can be used to continue pushing into the sample container.

In the embodiment described above, a workflow in which cell activation is performed in a first bioreactor vessel and the activated cells are transferred to a second bioreactor vessel for transfection and amplification. However, in one embodiment, the system of the present invention performs activation and transfection actions in a first bioreactor vessel and amplifies genetically modified cells in a second bioreactor vessel. It can be made possible to be executed within. Further, in one embodiment, the system of the invention may be such as to allow in-situ treatment of isolated T cells, with activation, transduction, and amplification unit operation all in a single bioreactor vessel. Executed within. In one embodiment, therefore, the invention simplifies existing protocols by enabling 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, activation (and isolation) of T cells may be performed in one of the bioreactor vessels 410 using Dynabead in the manner shown above. The activated cells are then transduced by the virus for genetic modification, such as as described above in relation to FIGS. 60-71. After activation and viral transduction, the virus is then washed from the bioreactor vessel 410 using the filterless perfusion method described above for retaining cells and micron-sized Dynabead in the bioreactor vessel 410. It may be issued. This allows cell amplification within the same bioreactor vessel 410 used for activation and transduction. The filterless perfusion method also allows the culture wash to be performed without the need to first immobilize the activated beads that need to be retained with the cells during amplification. Especially when the virus is washed out, the micron-sized Dynabead is not fluidized at low perfusion rates and is retained in the container. Nanometer-sized virus particles and residual macromolecules are fluidized and washed out during slow 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 Dynabead from the collected cells. In other embodiments, the step of harvesting an amplified population of cells and the step of removing beads from the cells are performed simultaneously using perfusion, whereby the medium is introduced into the bioreactor vessel through the feed port. The cell culture medium containing the amplified population of cells is removed from the bioreactor vessel through a discharge port within the bioreactor vessel. Filterless perfusion to perform bead removal of micron-sized beads by taking advantage of the difference in cell weight and cell Dynabead complex weight, especially when final bead removal of the culture is required. Can be used. To remove the beads 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 described above). After mixing / stirring, the heavy Dynabead sinks within 10-15 minutes and settles on the silicone membrane 516. In contrast, cells take more than 4 hours to settle on membrane 516. After a retention period of 10-15 minutes after mixing / stirring, the cell suspension can be slowly withdrawn using perfusion without disturbing the colonized Dynabead. The medium entry conduit can be used to maintain medium height within the bioreactor vessel. Therefore, the invention described herein simplifies the current Dynabead protocol by eliminating the need for some process intermediate cell transfer and careful washing and bead removal steps, minimizing costs and potential risks. Keep to the limit. Debeading the culture at the same time as harvesting the cells can eliminate the need for additional magnetic devices or disposable parts that were typically required in the past.

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 (eg, up to 35 mm / cm ² ) and therefore. 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 process intermediate cell transfer and careful washing steps, minimizing costs and potential risks resulting from multiple human touch points. Keep it to the limit.

In one embodiment, the two bioreactor vessels 410, 420 of the system are either the same starting culture or two co-divided cultures, eg, CD4 + cells in one bioreactor vessel 410, and the other bioreactor. It can operate on CD8 + cells in container 420. The split culture allows parallel independent processing and amplification of two cell types that can be combined prior to injection 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, 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, although the system of the invention is TCR-T cells and NK cells. Also suitable for the production of other immune cells such as. Further, in embodiments of the present invention, the use of two bioreactor vessels 410, 420 is added to the second bioreactor vessel 420 for the product of the first bioreactor vessel 410 for an additional processing step 2 Although disclosed in a step-by-step process (eg, activation in a first bioreactor vessel and characterization and amplification in a second bioreactor vessel), in some embodiments, two bioreactor vessels. Can be used for the same workflow as a duplicate. An exemplary reason for using the second bioreactor vessel sequentially is a residual chemical modification that cannot be washed out of the first bioreactor, which is harmful if cell overexposure occurs in later or earlier steps (eg,). , The presence of coated or immobilized reagents), or the need to precoat the bioreactor surface prior to the addition of cells (eg, RetroNectin coating).

Additional examples of potential single bioreactor vessel workflows enabled by the systems of the invention are: (1) soluble activator activation, viral transduction, filters in a single bioreactor vessel. None perfusion and amplification, (2) Dynabead-based activation in a single bioreactor vessel, viral transduction, filterless perfusion, and amplification, and (3) TransAct-based activation in a single vessel. , Includes viral transduction, filterless perfusion, and amplification.

In addition, further examples of the potential multiple bioreactor vessel workflows enabled by the systems of the invention are: (1) soluble activator activation, viral characterization, filtration within the first bioreactor vessel 410. Instrumentless perfusion and amplification, and soluble activator activation in a second bioreactor vessel 420, lentivirus transfection, filterless perfusion, and amplification, the same in these two bioreactor vessels. Using the same cell type or split culture, (2) Dynabead-based activation in first bioreactor vessel 410, viral transfection, filterless perfusion, and amplification, and in second bioreactor vessel 420. Dynabead-based activation, lentivirus transfection, filterless perfusion, and amplification, which use the same cell type or split culture in these two bioreactor vessels, (3) first. TransAct bead-based activation, virus transfection, filterless perfusion, and amplification in bioreactor vessel 410, and TransAct-based activation, lentivirus transfection, filterless perfusion, and in second bioreactor vessel 420. Amplification, which uses the same cell type or split culture in these two bioreactor vessels, (4) soluble activator activation in the first bioreactor vessel 410, and second. RetroNectin coating, transfection, and amplification in bioreactor vessel 420, (5) Immobilization activator activation in first bioreactor vessel 410, and RetroNectin coating, transfection in second bioreactor vessel 420. , And amplification, (6) Dynabead activation in the first bioreactor vessel 410, and RetroNectin coating, transfection, and amplification in the second bioreactor vessel 420, (7) in the first bioreactor vessel 410. Dynabead activation and lentivirus transfection, and amplification in the second bioreactor vessel 420, (8) TransAct activation in the first bioreactor vessel 410, and RetroNectin coating in the second bioreactor vessel 420. , Transfection, and amplification, (9) Soluble activator activation in first bioreactor vessel 410, and second bioreactor Amplification of electroporation-treated cells or other non-virus-modified cells in laboratory vessel 420, (10) TransAct activation in first bioreactor vessel 410, and experimentation in second bioreactor vessel 420 In-house electroporation treatment Amplification of cells or other non-virus-modified cells, (11) Dynabead activation in first bioreactor vessel 410, and laboratory electroporation treatment in second bioreactor vessel 420. Amplification of cells or other non-viral modified cells, (12) Amplification of allogeneic NK cells in a first bioreactor vessel 410, and amplification of allogeneic NK cells in a second bioreactor vessel 420 (small molecule based amplification). , (No genetic modification), (13) Amplification of allogeneic NK cells in the first bioreactor vessel 410, and amplification of allogeneic NK cells in the second bioreactor vessel 420 (feeder cell-based amplification, gene modification (No), and (14) Soluble activator activation of allogeneic CAR-NK or CAR-NK 92 cells in first and / or first and second bioreactor vessels 410, 420, Includes viral transfection, filterless perfusion and amplification (without RetroNectin coating, polybrene is used to aid in transfection).

The embodiments described above are integrated with the bioreactor vessel and / or bed plate (eg, on the membrane, in the membrane, on the side wall of the vessel, etc.) Process monitoring sensor. However, in other embodiments, it is contemplated that additional sensors may be added to the fluid architecture 400, for example, along the fluid flow line itself. These sensors may be disposable compatible sensors for monitoring parameters such as pH, dissolved oxygen, density / turbidity (optical sensor) conductivity, and viability in the circulating fluid. By arranging the sensors within 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 can be simplified. In addition, in some embodiments, sensors along the circulation loop can provide a more accurate representation of the container contents when circulated (measured when the cells are stationary in the container). not). In addition, 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 shown above, the first and third modules 100, 300 can be any system known in the art capable of cell enrichment and isolation, as well as harvesting and / or formulation. It can take any form of device. FIG. 78 is used as the first module 100 in the bioprocessing system 10 for cell enrichment and isolation using various magnetically isolated bead types, including, for example, Miltenyi beads, Dynabead, and StemCell EasySep beads. Illustrates a possible configuration of a possible device / device 900. As shown therein, device 900 includes a centrifuge chamber 912, a high dynamic range peristaltic pump assembly 914, a pump tube 916 with a suitable inner diameter accepted by the peristaltic pump assembly, a stopcock manifold 918, and optics. It comprises a base 910 that houses a sensor 920 and a heating / cooling mixing chamber 922. As shown below, the Stopcock Manifold 918 provides a simple and reliable means of joining multiple fluid or gas pipelines together using, for example, luer fitting. In one embodiment, the pump 914 is rated to output a low flow rate of about 3 mL / min and a high flow rate of about 150 mL / min.

As further shown in FIG. 78, the device 900 extends from the base 910 and is generally T-shaped with multiple processing and / or multiple hooks 926 for suspending the source container or bag. It may include a hanger assembly 924. In one embodiment, the number of hooks may be six. Each hook is equipped with an integrated weight sensor for detecting the weight of each container / bag. In one embodiment, the bags are sample source bag 930, process bag 932, isolation buffer bag 934, cleaning bag 936, first storage bag 938, second storage bag 940, post-isolation effluent bag 942. , Cleaning effluent bag 944, medium bag 946, release bag 948, and collection bag 950.

The device 900 is used or configured to be used or provided with a magnetic cell isolation holder 960 as presented herein. The magnetic cell isolation holder 960 can be detachably attached to a magnetic field generator 962 (eg, magnetic field plates 964, 966 in FIG. 80). The magnetic cell isolation holder 960 houses a magnetic holding element or material 968, such as a separation column, matrix, or tube. In one embodiment, the Magnetic Cell Isolation Holder 960 is described in more detail below, which is incorporated herein by reference in its entirety, US Patent Application No. 15 /, filed December 1, 2017. It can be manufactured as disclosed in No. 829,615. The device 900 may be under the control of a controller (eg, controller 110) and operates according to instructions executed by the processor and stored in memory. Such instructions may include magnetic field parameters. In one embodiment, device 900 may further comprise a syringe 952 available for bead addition, as described below.

Next, referring to FIG. 79, the general protocol 1000 of the device 700 is shown. As illustrated therein, in the first step 1010 concentration is performed by reducing platelets and plasma in the sample. Then, in an embodiment in which Dynabead is utilized as magnetically isolated beads, washing step 1012 may be performed to remove residues in the Dynabead suspension. After concentration, the cells are then transferred to process bag 932 in step 1014. In some embodiments, some of the enriched cells can be stored in the first storage bag 938 in step 1016 before being transferred to the process bag 932. In step 1018, the magnetically isolated beads are injected into the process bag, for example, by using a syringe 952 in step 1020. In one embodiment, the magnetically isolated beads are Miltenyi beads or Stem Cell Easy Sep beads. If Dynabead is utilized, the washed Dynabead from step 1012 is resuspended in process bag 932. In one embodiment, instead of utilizing a syringe, the magnetically isolated beads may be housed in a bag or container connected to the system, which can be pumped into the system by pump 914.

The beads and cells in the process bag 932 are then incubated for a period of time in step 1020. This step involves circulating fluid out of the process bag 932, through a loop, and then back into the bag. In an embodiment in which the magnetically isolated beads are Miltenyi nanosized beads, sedimentation washing is performed in step 1022 to remove excess nanosized beads and in step 1024 some of the incubated bead-bound cells are second preserved. Stored in the bag 940. After incubation, bead-bound cells are isolated in step 1026 using a magnet, eg, magnetic field plates 964, 966 in a magnetic cell isolation holder 960. Residual bead-bound cells are then rinsed and isolated in step 1028. Finally, in an embodiment in which Miltenyi or Dynabead is utilized, in step 1030, the isolated bead-bound cells are collected in a collection bag 950. In embodiments where StemCell EasySep beads are utilized, an additional step 1032 is performed to release the cells from the beads and remove the beads, and an optional step 1034 to wash / concentrate the collected cells.

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

As disclosed above, in some embodiments where Dynabead is utilized as magnetically isolated beads, a washing step (step 1012) is performed to remove residues in the bead suspension buffer. .. The bead is circulated in the process loop 1110 (for example, from the process bag 932 through the peristaltic pump tubing 914, through the manifold 918 and back to the process bag 932) using the injector 952. Injecting, cleaning process loop 1110, and then while the magnetic field generator 962 is "ON", i.e., while the holder is magnetically coupled to the active magnetic field generator 962 for the permanent magnets. , Or, in each case, beading, including capturing the beads by flushing the process bag 932 through an isolated effluent bag 942, when the electromagnetic field is actively generated by using electromagnets. Is captured in the "ON" state. In embodiments where cleaning is not desired, the process bag 932 is flushed into the isolated effluent bag 942 to ensure that the process bag 932 is clean. For permanent magnets, as used herein, ON means that the magnetic holding element or material 968 (eg, separation column, matrix, or tube) is in the proper position in the magnetic field. .. OFF means that the tubing section has been removed from the magnetic field.

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

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

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

After adding the magnetically isolated beads, as described above, the cells can be incubated for a period of time (step 1020). In one embodiment, prior to incubation, the contents of process back 932 may be transferred to a second storage bag 940, which is agitated (using a heating / cooling mixing chamber 922). And so on). The contents of the second storage bag 940 are then transferred back to the process bag 932. The buffer from the isolated buffer bag 934 is then drawn into the processing chamber 912 and the contents of the chamber are discharged into the second storage bag 940 and then transferred to the process bag 932 for the second storage. Rinse bag 940.

In either embodiment, the cells are then incubated with magnetically isolated beads by circulating the cells along process loop 1110 over a defined incubation time. After incubation, process loop 1110 is cleaned.

As explained above, after incubation, an optional step of washing out excess beads (eg, nano-sized beads) can be performed (step 1022). Washing out excess nano-sized beads simply initiates the flow from process bag 932 to the second storage bag 940 and pulls the contents of the second storage bag 940 into the processing chamber 912. Transferring the buffer from the release buffer bag 934 to the process bag 932, transferring the contents of the process bag 932 to the second storage bag 940, and transferring the contents of the second storage bag 940 to the processing chamber. Including pulling 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 needed to wash out excess beads. In one embodiment, chamber 912 may then be filled with buffer from isolated buffer bag 934, initiate rotation of chamber 912, and then discharge the supernatant into waste liquid bag 742. These steps can be repeated as needed. In one embodiment, the cells in the chamber are discharged into process bag 932, the buffer from isolation buffer bag 934 is drawn into chamber 932, and then the chamber is discharged into process bag 932. This process can also be repeated as needed. The process loop mixing and process loop cleaning are then performed.

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

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

In one embodiment, rinsing without resuspension involves pumping buffer from isolated buffer bag 934 to process bag 932, rinsing process loop 1110, cleaning process loop 1110, and generating a magnetic field. It can be performed by flowing the vessel 962 from the process bag 932 to the effluent bag 942 in the "ON" state.

In another embodiment, rinsing via resuspension pumps buffer from isolated buffer bag 934 to process bag 932 with the magnetic field generator 962 "OFF" and circulates in process loop 1110. It can be done by cleaning the process loop and flowing the magnetic field generator 962 from the process bag 932 to the effluent bag 942 in the "ON" state.

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

The residual bead-bound cells are then rinsed to isolate the residual bead-bound cells, after which the isolated bead-bound cells are collected (step 1028). In one method, the medium from the medium bag 946 is simply in a state where the magnetic field generator 962 is "OFF", if the bead-bound cells should be collected without releasing the cells from the beads. Pumped through column 968 to collection bag 950. Alternatively, the buffer from isolated buffer bag 934 is pumped to process bag 932, which is then pumped to collection bag 950 with the magnetic field generator 962 "OFF". Sent. This second method provides post-isolation washing. In the third method, medium from medium bag 946 is pumped through column 966 to process bag 932 (if cleaning is not required after isolation). Alternatively, buffers from isolation buffer bag 934 are pumped through column 966 to process bag 932 (if post-isolation cleaning is desired). Then, in either process, the contents of process bag 932 circulate within process loop 1110, process loop 1110 is cleaned by returning to process bag 932, and the contents of process bag 932 are pumped. It is sent to bag 950 to collect bead-binding cells.

If bead-bound cells should be harvested after releasing the cells from the beads, a number of potential processes may be performed. For example, in one embodiment, the cell / bead pumps a release buffer from bag 948 into process bag 932 through a column with the magnet "OFF" and circulates in process loop 1110, then processes the fluid. It can be resuspended by cleaning the process loop by returning it to bag 932. Incubation and collection are then incubated within process loop 1110 with the magnet "ON", cleaning process loop 1110 and pumping from process bag 932 through column 966 to collection bag 950. Collect the cells released in, pump the buffer from buffer bag 934 to process bag 932, pump the contents of process bag 932 through column 966 and send the residue to collection bag 950. Performed by collecting. The released beads (step 1032) are then pumped from isolated buffer bag 934 through column 966 to process bag 932 with the magnet "OFF" and circulated in process loop 1110 for process. It can be discarded by cleaning loop 1110 and pumping the contents of process bag 932 to waste bag 942.

In connection with the above, in one embodiment, cleaning / concentration (step 1034) pumps the contents of collection bag 950 into processing chamber 912 and pumps buffer from isolation buffer bag 934 to process bag 932. It can be carried out by feeding and transferring the buffer from the process bag 932 to the processing chamber 912. The wash cycle is then performed by filling the processing chamber 912 with buffer from the isolated buffer bag 934, rotating the chamber 912, discharging the supernatant into the waste liquid bag 942, and repeating the rotation and discharge steps as needed. Can be done. Finally, transferring the cells to the collection bag after washing / concentrating transfers the medium from the medium bag 946 to the collection bag 950, pumps the contents of the collection bag to the processing chamber 912, and the processing chamber 912. This can be achieved by discharging the contents of the shavings into the collection bag 950 and then manually cleaning the conduit between the processing chamber 912 and the collection bag 950.

In one embodiment, one of the bags, eg, process bag 932, has sterile air in the system to clean the pipeline, as needed, in the various process steps described above. It may be equipped with a top port 1118 with a filter so that it can be introduced (when the process bag 932 is empty). Cleaning the pipeline can be performed as a first step in the concentration / isolation process and / or at the time of process execution. In one embodiment, the air from the collection bag 950 can be used to clean any of the lines of the system (eg, the air from the collection bag 950 is used to clean the process line 1112). The air in the process line 1112 is then used to clean the desired tubing line (ie, line 1114, 1116, etc.), thereby filling the process line 1112 with the liquid from the process bag 932. Finally, the air from the collection bag 950 can be used again to clean the process line 1112).

In one embodiment, the processing bag 932 is blow molded and flanked (higher than liquid level) to limit micron-sized beads from sticking to the sidewalls, especially during long accelerated mixing in a circulation-based incubation. Has a large angle on) (has a 3D shape with a defined air pocket in).

In one embodiment, the syringe 952 allows a small amount (such as a bead suspension aliquot) to be added to the circulation-based flow loop 1110. In addition, the fluid from flow loop 1110 is drawn into the syringe 952, which allows further removal and cleaning of the residue in the syringe 952.

In one embodiment, one of the sensors 920 may be configured to measure fluid flow. For example, one of the sensors 920 is a load cell integrated with the hook 926, as well as a bubble or optical detector that can be used as a secondary confirmation measure to ensure accurate flow control. Good. This can actually be used in isolation when it is desirable to allow the volume in the process bag to flow through the magnet without introducing air into the column. The load cell indicates that the process bag is close to empty within some expected tolerance of load cell variability, and the bubble detector 920 then identifies the liquid / air interface that follows to stop the flow. Thus, the sensor 920 can enter the loop by removing the cells or generating slag that exposes the cells to a dry environment, or by inadvertently pulling material into the waste bag in situations where the pump does not stop after the process bag is completely drained. Can be used by the controller to prevent the drawing of air. Therefore, in one embodiment, the bubble detector 920 may be used in combination with a load cell that is integrated with the hook, thereby improving capacity control accuracy, thereby reducing cell loss, and /. Alternatively, air can be prevented from entering the column tubing and column.

As implicitly mentioned above, in one embodiment, air is in the loop due to the deliberate generation of air slag that can be used to remove bead-bound cells in the isolated column / tube for collection. Can be drawn in. In one embodiment, the buffer solution can be circulated through the isolation column to elute the bead-bound cells from the isolation column, either by alternative to using air slag or in addition. ..

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

In some embodiments, System 900 can be used to elute the isolated / captured bead cell complex. In particular, it is contemplated that the air-liquid interface can be used to help remove the complex from the tube sidewall or column interstitial space. Air can circulate through the column / tube or be shuffled back and forth through the column / tube. In the absence of an air / liquid interface, the bead / bead-bound cell packed bed can be difficult to remove by flow control alone, without significantly increasing shear rate (which has a potential adverse effect 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 elution of positively selected bead cell complexes directly into the selected medium (based on downstream steps). This eliminates the buffer replacement / cleaning step. In one embodiment, it is also envisioned that the incubation is initiated by elution directly into the medium and viral vector. This concept may also allow viral vectors to be added to the final bag. In one embodiment, instead of elution of bead-bound cells in buffer, medium can be used as the elution fluid. Similarly, the release buffer can be used to elute Stem Cell beads for subsequent release of cells from the beads. Dilution can be minimized by replacing the buffer in the portion of System 900 with medium.

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

Next, one embodiment of the magnetic cell isolation holder (960 in FIG. 78) will 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 target-binding cell surface molecules to antibodies or ligands of magnetic beads (eg, beads of the type described above). .. After binding, the cells bound to the magnetic beads can be separated from the unbound population of cells. For example, a cell mixture containing bound and unbound cells can pass 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 can incorporate nano-sized beads (eg beads less than about 50 nm in diameter), while others can use larger beads (eg beads about 2 μm or larger in diameter). .. For example, similar beads may be desirable, as smaller bead sizes can avoid receptor activation on target cells. In addition, downstream steps can skip bead removal, as nano-sized beads may have little effect on downstream processing or cellular function. However, smaller nano-sized magnetic beads can be separated using a magnetic cell isolation procedure that involves amplifying the applied magnetic field gradient using a magnetic field gradient enhancer. In contrast, larger beads have a higher magnetic moment. Therefore, isolation of some larger beads cannot be accompanied by a magnetic field gradient increasing device. However, larger beads can nevertheless be used in conjunction with additional cell bead separation steps. Therefore, depending on the size and / or type of magnetic beads used, the workflow, appropriate magnetic parameters, and / or isolation device itself may vary, which makes magnetic bead-based cell isolation techniques. It's complicated.

In particular, since beads can differ in material and magnetic properties (including, but not limited to, size, magnetic permeability, saturation magnetization, resistivity, surface properties, and mass density), separation conditions also affect the bead properties. It may vary depending and may be accompanied by magnetic fields of different intensities 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 approach of the present invention 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, the magnetic cell isolation holder is an arrangement in which the magnetic cell isolation holder is associated with the desired magnetic field properties for cell separation when used with beads of appropriate size. It is configured to be used in conjunction with a magnetic field generator to position the beads within the magnetic field of. The magnetic field generator can apply a magnetic field using preset (eg, fixed) magnetic field parameters or static magnetic field generator elements. In this manner, the operator can avoid the complexity of changing the magnetic field parameters according to the selected beads. Instead, the magnetic field acting on the cells is suitable for separation by selecting the appropriate magnetic cell isolation holder. In addition, different magnetic cell isolation holders that position the beads in their respective arrangements within the applied magnetic field associated with each desired magnetic field characteristic when using beads of different sizes and / or with different desired magnetic field characteristics. Can be selected.

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

In one embodiment, a suitable magnetic retention material, such as a column matrix carried within a separation tube, is coupled to or positioned in the passage of the magnetic cell isolation holder and used in the magnetic cell isolation procedure. It is positioned in a magnetic field arrangement within the magnetic field generator that corresponds to the desired magnetic field characteristics (ie, magnetic field strength and magnetic field gradient) for the bead type to be made. The set of magnetic cell isolation holders and accompanying magnetizable beads as the beads described above may be provided in the kit, which may include disposable or one-off components. The kit may also include multiple sets of beads or different types of beads and / or multiple magnetic cell isolation holders, such as holders optimized or designed for each set of beads.

In another embodiment, a magnetic cell isolation holder with multiple passages for use with each different size bead may be provided and the user has the appropriate passage associated with the desired bead type. You can choose. For example, a magnetic cell isolation holder has a first arrangement passage (eg, configured to accommodate a first cell separation column) for use with beads having a first diameter, and a second. It may have a second arrangement passage (eg, configured to accommodate a second cell separation column) for use with beads having a larger diameter of. When the magnetic cell isolation holder is inserted into the magnetic cell isolation device and a magnetic field is generated, the passage of the first arrangement has a higher magnetic field than the passage of the second arrangement within the magnetic cell isolation holder. It may be in a position to receive strength.

In another embodiment, the magnetic cell isolation holder can be prefilled with the 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 the fluid manipulation system of the magnetic isolation system or can be functionally attached to one or more fluid manipulation systems. The magnetic cell isolation system may also include a controller that is configured to automatically perform the magnetic cell isolation procedure. The magnetic isolation system can be configured as a functionally closed system.

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

System 2100 is configured to be used with a magnetic cell isolation holder 2134 equivalent to the magnetic cell isolation holder 960 described above with reference to FIG. 78. The magnetic cell isolation holder 2134 can be detachably attached to a magnetic field generator 2121 (eg, magnetic field plates 2122 and 2124 equivalent to plates 964 and 966 in FIG. 80) (eg, loaded and positioned with respect to it). ). The system 2100 may be under the control of the controller 2150, which is executed by the processor 2152 and operates according to the instructions stored in the memory 2154. Such instructions may include magnetic field parameters. System 2100 may include any or all of the illustrated components.

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

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

83A and 83B are top views of different configurations of the magnetic cell isolation holder 2302 (eg, the magnetic cell isolation holder 2134 of FIG. 81) positioned within the magnetic isolation device 2300 of FIGS. 83A and 83B. is there. FIG. 83A shows the unloaded configuration of the magnetic cell isolation holder 2302 in the magnetic cell isolation device 2300. The magnetic cell isolation holder 2302 may comprise a body portion 2301, which may be configured to contain the cell isolate and be formed from a suitable non-magnetic material attached to the magnetic isolation device 2300. .. The magnetic cell isolation holder 2302 comprises one or more passages formed within or through the body 2301 through which the cell mixture can flow. Although Figure 83A shows two separate passages 2303 and 2305, it will be appreciated that the magnetic cell isolation holder 2302 may have only one passage, two or more passages, and so on. With reference to passage 2303, passage 2303 may be configured to hold cells bound to magnetic beads and contain a magnetic holding material 2304 that is configured to pass unbound cells under a magnetic field. .. Similarly, the passage 2305 may also accommodate the magnetic holding material 2306. The magnetic holding materials 2304 and 2306 may be the same or different. It is also possible to omit the holding material, but the efficiency will be inferior as a result, for example, the passage 2305 can be a hollow tube. Further, the passages 2303 and 2305 may be of different sizes and positions with respect to the end face 2307 of the main body 2301. For example, the distance 2315 between the end face 2307 and the center point of the passage 2303 may differ from the distance between the other passages of the body 2301 with respect to the end face 2307. In this manner, the passage is subject to the action of a magnetic field that correlates with its position within the body 2301.

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

The magnetic isolation device 2300 may also include doors or other features that are configured to reduce the leakage of magnetic fields to the outside of the receiving region 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 magnetize in the presence of a magnetic field and degauss when the magnetic 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 gaps with the help of a compressed spring attached to either door. Closes, thereby surrounding the magnetic flux in the steel backing 2308 and door 2318. This prevents magnetic flux leakage into passages 2303, 2305 when degaussing is desired for some processes such as elution.

FIG. 83B shows the magnetic cell isolation holder 2302 in a loaded configuration in the magnetic isolation device 2300. When the magnetic cell isolation holder 2302 is completely inserted into the receiving region 2316 of the magnetic field generator 2313, the positions of passages 2303, 2305 are geometric of the magnetic cell isolation holder 2302 and the magnetic isolation device 2300. Determined by shape. Part of the backing 2308 (eg, 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. Can abut. In addition, the magnetic cell isolation holder 2302 has the tapered shape of FIGS. 3A and 3B, but any suitable shape of the magnetic cell isolation holder 2302 can be used.

The door 2318 of the magnetic field generator 2313 opens, allowing the insertion of the magnetic cell isolation holder 2302 between the magnetic field plates 2312 and 2314 of the magnetic field generator 2313. For example, the location of passage 2303 in the magnetic field generator 2313 may be targeted for placement in the magnetic field with the highest magnetic field strength (ie, 0.5T). In another example, the location of passage 2305 within the magnetic field generator 2313 provides the highest magnetic field gradient (ie 50 T / m) placement in the magnetic field while satisfying the magnetic bead magnetic field strength requirements (ie 0.15 T). It may be the target range.

To elute the retained beads (eg, beads or bead-bound cells) from the magnetic retention material (eg, magnetic retention materials 2304, 2306), an external magnetic field disengages the isolation holder 2302 (ie, figure). It can be removed by retracting into the 83A unloaded configuration). When no external magnetic field is required near passages 2303, 2305, the door closes to ensure that there is no leakage of magnetic flux affecting passages 2303, 2305. A high flow rate of fluid then flows through passages 2303, 2305, which creates a large shear force on the retained beads. When the viscous force is greater than the holding force (ie, the magnetic force due to the residual magnetic field), the beads are washed off and collected from the magnetic holding material in passages 2303, 2305. However, in other embodiments, the applied magnetic field can be censored under the control of controller 2150.

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

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

It is understood that some disclosed techniques relate to positioning magnetic cell isolation holders such as those disclosed within a fixed position magnetic field generator, but other implementations may also be contemplated. Will be done. For example, the magnetic field generator can move relative to a magnetic isolation holder loaded within a fixed position frame.

FIG. 85 shows a flow chart for method 2500 of magnetic cell isolation that can be used with magnetic isolation devices. In step 2502, the first cell mixture is prepared by incubating the cell mixture with a set of magnetic beads that have the desired properties (eg, size, type, ligand, etc.). After a sufficient period of time has passed to ensure that the target cells are labeled with magnetic beads, the excess incubation mixture is removed. In another embodiment, a portion of the incubated mixture may be withdrawn and evaluated for quality control purposes, i.e., an excess incubated mixture is evaluated and thereby assessing binding properties. May be. In step 2504, the first magnetic cell isolation holder 2302 may be bound within the receiving region 2316 of the magnetic field generator 2313. In step 2506, the magnetic field generator 2313 generates a magnetic field within the receiving region 2316 of the magnetic field generator 2313. In step 2508, the first cell mixture flows through a passage within the first magnetic cell isolation holder 2302 (eg, one or more of passages 2303 or 2305). The magnetic bead-labeled cells in the cell mixture are the magnetic retention material of the first magnetic cell isolation holder 2302 (eg, while the rest of the cell mixture material is flowing through the passage of the first magnetic cell isolation holder 2302). It is held in the passage by one or more of the magnetic holding 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 region 2316 of the magnetic field generator 2313 (or by discontinuing the application of the magnetic field), resulting in a single magnetic cell. The release holder 2302 is demagnetized. In step 2512, the retained or isolated cells and beads from the first magnetic cell isolation holder 2302 are magnetically retained in the passage of the magnetic cell isolation holder 2302 in a fluid with a high flow rate. It is collected by elution of the beads or cells that have been made, or by another suitable method. At step 2514, the magnetic cell isolation holder 2302 can optionally be disposed of. Steps 2522 to 2534 reflect steps 2502 to 2514, but instead the cells labeled with different sized sets of beads in the second cell mixture are the second (ie, different) magnetic cells. It may be passed through a passage within the release holder 2302 or through a different passage within the first magnetic cell isolation holder 2302. Steps 2522 to 2534 illustrate a method of using two different magnetic cell isolation holders, where the two magnetic cell isolation holders instead have the same magnetic cell unit with different pathways for each cell mixture. It will be understood that it can be a release holder. In addition, the second cell mixture may be the cell mixture obtained as a result of passing through the first magnetic cell isolation holder 2302 from step 2508 without retained bead labeled cells.

The magnetic selection of target cells can be either positive or negative selection. In positive selection, magnetic beads are used to label the target cells and the target cells are collected as a labeled fraction. In negative selection or cell depletion, 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 the magnetic cell isolation holder 2602 relative to the permanent magnets 2612, 2614 of the magnetic field generator 2600. In one embodiment, the distance between the permanent magnets 2612, 2614 is 0.37 inches (10 mm) or so. However, other distances between the permanent magnets can also be used depending on the configuration of the isolated device, such as the physical properties of the magnet, the aspect ratio of the cross section, and the design of the instrument that holds the magnet. In the illustrated embodiment, the magnetic retention material may be, for example, a column matrix 2604 for use with Miltenyi microbead-labeled cells, the magnetic retention material 2606, for example, a tube for use with Dynabead-labeled cells. It may be. Either a column matrix or a tube can be used for any individual magnetic cell isolation procedure.

FIG. 87 shows the magnetic field distribution by the permanent magnets and backing steel of the magnetic field generator showing different magnetic field characteristics of the magnetic fields in different arrangements. 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 forces of equal magnitude when compared to smaller beads with 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 with an external magnetic field strength (ie 0.15 T 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 techniques can be used to isolate cells for chimeric antigen receptor cell therapy (or CAR-T). CAR-T involves the isolation of several types of leukocyte cells from peripheral blood mononuclear cells (PBMCs), T cells. Target cells (T cells) are modified with receptors that allow them to recognize and attack cancer. In addition, the disclosed techniques can be used in conjunction with any suitable type of beads, such as Miltenyi nanosized microbeads (50 nm in diameter) and Dynabead (4.5 μm in diameter). Miltenyi microbeads are nano-sized superparamagnetic beads, which require a magnetizable column matrix to hold from the flow field. The magnetizable column matrix is made of soft magnetic material spheres (eg, stainless steel 400 series balls with a diameter of 0.4 mm). Stainless steel 400 series balls do not rust. The magnetic properties of the material of a sphere are associated with increased magnetization when exposed to an external magnetic field and little residual magnetism when the external magnetic field is removed. The process of manufacturing a column matrix of magnetic retention material is to spherically pack the column matrix using a vibrator, apply lacquer to the column matrix, gravitationally expel the lacquer, and centrifuge to remove residual lacquer. It involves air blowing and re-centrifugation. The air blow and centrifuge steps are repeated several times until all residual lacquer has been removed. The column matrix is then placed in the oven at a temperature of about 100 ° C. for 3 days. After the column matrix is completely cured, the column matrix is organized by the applied lacquer. The magnetizable column matrix can act as a magnetic enhancer that is filled with spheres and enhances the magnetic field gradient by about 10,000 times. The increased magnetic field gradient helps attract nano-sized 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, which allows the nanosized bead-labeled cells to be released from the column matrix. The nanosized bead-labeled cells are then eluted with a stream of rinse fluid through the column matrix.

Dynabeads are larger superparamagnetic beads made from synthetic polymers. Since Dynabead is considerably larger than Miltenyi nanosized beads, Dynabead has a much higher magnetic moment than Miltenyi nanosized beads when placed in a magnetic field. Therefore, the use of Dynabead in magnetic cell isolation does not necessarily require a magnetic augmentation device such as a magnetic column matrix. Tube-based systems are typically used with Dynabead-labeled cells, where permanent magnets are placed near the tube. Dynabead-labeled target cells are attracted to the wall of the tube, and unlabeled cells are then removed with buffer or medium. Other beads of different sizes are also trademarked in addition to Miltenyi nanosized microbeads and Dynabead.

The magnetic isolation device is a magnetic field generator, eg, a pair of permanent magnets with a magnetic cell isolation holder and associated magnetic cell retention material, fluid tubing, collection and preparation vessel, and other components of the disclosed system 2100. Can be used with. In addition, some of these components may be offered as one-off components, disposable components, and / or package kits.

In one embodiment, a dedicated kit may be provided to achieve magnetic isolation for a particular bead type. Kits optimized for one or more bead sizes may be provided for any particular isolation event. The kit may include a suitable magnetic retention material, which can be preloaded in a properly constructed magnetic cell isolation holder. This method prevents the user from inadvertently loading or coupling a rogue magnetic holding material into the passage of the magnetic isolation holder. In one embodiment for use with Miltenyi microbeads, the magnetic retention column in the passage of the magnetic isolation holder is positioned to be at the center of the gap or space between the permanent magnets of the magnetic field generator when loaded. It can be associated with the highest or higher magnetic field strength (ie, higher than 0.45T). In another embodiment, the magnetic retention tubing for Dynabead can be positioned within the highest gradient region between the permanent magnets. The Dynabead isolation was positioned relative to the magnetic field generator to be affected by both a magnetic field strength suitable for retention (ie, greater than 0.1T) and a magnetic field gradient (ie, greater than 40T / m). It can be performed in conjunction with a magnetic isolation holder that has a passage.

When used in conjunction with the disclosed techniques, the average recovery and average purity of CD3 + using a magnetic isolation device, respectively, is greater than approximately 80% for Miltenyi nanosized beads. At Dynabead, the average recovery of CD3 + using a magnetic isolation device is about 60%, and the average purity of CD3 + using a magnetic isolation device is more than approximately 70%.

The technical effect of the present disclosure is a holder for magnetic isolation of cells used 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 for each of the different sized beads, eliminating or manipulating the user's interaction and manipulation with the raw material. Can be reduced.

As used herein, elements or steps that are listed in the singular form in the English source text and are preceded by the article "a" or "an" are said elements unless the exclusion is explicitly stated. Or it should be understood as not excluding multiple steps. Furthermore, reference to "one embodiment" of the present invention is not intended to be construed as excluding the existence of additional embodiments that also incorporate the listed features. Further, unless otherwise specified, embodiments that "contain," "compare," or "have" an element or plurality of elements that have a particular property may include additional such elements that do not have that property.

The present specification uses examples to disclose some embodiments of the invention, including the best embodiments, as well as to manufacture and use any device or system, and any method incorporated. Allows one of ordinary skill in the art to implement embodiments of the invention, including carrying out. The patentable scope of the present invention is defined by the claims and may include other examples that can be conceived by those skilled in the art. Other such examples include equivalent structural elements where these examples have structural elements that do not differ from the wording of the claim, or where these examples differ slightly from the wording of the claim. In some cases, it is intended to be within the claims.

10 Bio-processing system
100 1st module
110 1st controller
200 Second module
200a, 200b, 200c second module
210 Second controller
280a, 280d sample collection device
300 Third module
310 3rd controller
400 fluid flow architecture
400 Bio-processing subsystem
400 Bio-processing system
410 First bioreactor vessel
412 1st port
414 First bioreactor pipeline
416 2nd port
418 Second bioreactor pipeline
420 Second bioreactor vessel
422 first port
424 First bioreactor pipeline
426 Second port
428 Second bioreactor pipeline
430 bioreactor array
432 First bioreactor pipeline valve
436 First bioreactor pipeline valve
438 Second bioreactor pipeline valve
440 First fluid assembly
442 First fluid assembly pipeline
444 Second fluid assembly
446 Second fluid assembly pipeline
448 sampling assembly
450 interconnect pipeline
452 Interconnect pipeline valve
454 Interconnect Pipeline Pump
456 Second pump or circulation line pump
458 Sterilized air source
460 Sterilized air source pipeline
462 valve
464a ~ f Tubing tail
466a ~ f 1st storage tank
468a ~ f Tubing tail valve
470a ~ d Tubing tail
472a ~ d 1st storage tank
474a ~ d Tubing tail valve
476a ~ 476d Sampling pipeline
478a ~ d Sample pipeline valve
482 Filtration line
484 filter
486 Upstream filtration line valve
488 Downstream filtration line valve
490 Waste liquid pipeline
492 Penetration pump
502 bottom plate
504 Container body
506 internal compartment
508 top
510 side
510 grid
512 holes
514 crossbar
516 membrane
518 top
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 vertices, tip points
536 height
538 Cell culture medium
542 Headroom
544 surface
550 recess
552 Position verification structure
554 Flat engaging surface
556 Aperture or opening
600 kit
610 tray
612 front wall
614 rear wall
616, 618 sides
622 internal compartment
620 bottom
622 internal compartment
624 Peripheral flange
626, 628 1st and 2nd openings
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 placement
660 clearance opening
662 Flat back plate
664 Aperture
666 Vertically spaced, horizontally extending slots
668, 670 clearance opening
672 retention clip
674 1st input end
676 Second output
680 Features
682 Narrow tubing slot
684 Waste liquid pipeline tubing slot
700 bio-processing device
710 housing
712, 714, 716 Drawer
718 side wall
720 bottom
722 Processing chamber
724 Hardware compartment
730 auxiliary compartment
732 power supply
734 Operation control board and drive electronic circuit
736 Low power solenoid array
738 Pump assembly
740 Drawer engagement actuator
742 Pump shoe
744 Pinch valve anvil
746 1st bed plate
748 Second bed plate
750 plate
752 top
754 Positioning pin or alignment pin
756 Integrated sensor
759 Embedded temperature sensor
760 load cell
760 Resistance temperature detector
761 Actuator mechanism
762 cam arm
764 slots
766 cam pin
768 Linear actuator
770 Rocker switch
770 Linear actuator
772 Lead screw
774 clevis arm
776 space
778 solenoid
780 piston
782 heating pad
784 Heating module
786 Carbon dioxide sensing module
788 cover
790 Insulation foam layer
792 film heater
794 Internal metal plate
798 Insulation layer
800 fluid
810 flip down front panel
812 Telescopic sliding rail
814 Cross rod that extends laterally
815 Mounting method
816 Low profile waste liquid tray
819 Own chamber
820 platform
822 Guide track
900 devices / equipment
910 base
912 Centrifugal processing 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 Isolated buffer bag
936 wash bag
938 First storage bag
940 Second storage bag
942 Waste liquid bag after isolation
944 Cleaning 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 retaining element or material
1000 general protocols
1110 process loop
1112 Process pipeline
1114 1st storage bag pipeline
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 Beaded 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 body
2302 Magnetic cell isolation holder
2303, 2305 passage
2304, 2306 Magnetic retention material
2307 End face
2308 Steel backing
2311 Stop or surface
2312, 2314 Magnetic field plate
2313 Magnetic field generator
2315 distance
2316 Receiving area
2318 door
2319 frame
2330 Guide plate
2500 methods
2600 magnetic field generator
2602 Magnetic Cell Isolation Holder
2604 Column Matrix
2606 Magnetic retention material
2612, 2614 Permanent magnet

Claims (54)

It ’s a bio-processing method,
A step of combining a suspension containing a population of cells with magnetic beads to form a population of bead-bound cells in the suspension.
In the step of isolating the population of bead-bound cells on a magnetic isolation column,
Including the step of collecting target cells from said population of cells.
The step of collecting the target cells includes a step of removing the bead-bound cells from the isolated column having an air plug. The method of claim 1, further comprising the step of circulating the buffer solution through the isolation column to elute the bead-bound cells from the isolation column. The method of claim 2, wherein the buffer solution comprises an isolation buffer, a cell culture medium, or a cell release buffer. The method of claim 1, further comprising combining the magnetic beads with an isolation buffer and further isolating the magnetic beads on the isolation column prior to combining with the population of cells. The steps of collecting the target cells include collecting the bead-bound cells using a release buffer to produce the target cells, incubating the target cells to release them from the magnetic beads, and on an isolated column. The method of claim 1, comprising the step of isolating the magnetic beads and collecting the released target cells. The method of claim 1, further comprising washing the target cells and exchanging the suspension. The method of claim 1, further comprising the step of concentrating the target cells. The method of claim 1, further comprising the step of washing and concentrating the target cells. The method of claim 1, wherein the step of isolating the target cells further comprises the step of flowing the isolation buffer through the isolation column and rinsing the isolated cell population. The steps of isolating the target cells include elution of the isolated cell population from the isolation column in an isolation buffer for rinsing, followed by the above-mentioned bead-bound cells on a magnetic isolation column. The method of claim 1, further comprising the step of isolating the swept population. The method of claim 1, wherein the magnetic beads are nano-sized beads and the suspension is washed to remove excess magnetic beads after incubation and before isolating the target cells with the magnet. The method according to claim 1, wherein the magnetic beads are Dynabead, Miltenyi beads, or StemCell EasySep beads. The step of drawing air into the process bag through its port,
The method of claim 1, further comprising the step of cleaning the process line by circulating the air through a process line that communicates fluid with the process bag. Further including the step of detecting the air in the process loop.
The method of claim 1, wherein the detection of air is an indication that the process bag is empty. The method of claim 1, further comprising the step of concentrating a desired cell population from a biological sample. The steps of concentrating the desired cell population include transferring the biological sample and the buffer to the treatment chamber and washing the biological sample in the processing chamber to reduce the amount of platelets and serum in the biological sample. The method of claim 15, including with steps. After concentration, the step of transferring the concentrated cell population to a processing bag and
16. The method of claim 16, further comprising the step of circulating the concentrated cell population through the process loop. It's a system
With a magnetic field generator configured to generate a magnetic field under magnetic field parameters,
A first holder that is configured to be detachably coupled to the magnetic field generator, wherein the first holder is the first when the first holder is coupled to the magnetic field generator. A first holder comprising a first passage configured to be positioned in said magnetic field in an arrangement.
A second holder that is configured to be detachably coupled to the magnetic field generator, wherein the second holder is the second when the second holder is coupled to the magnetic field generator. With a second holder, with a second passage configured to be positioned in said magnetic field in the arrangement,
The first passage is affected by the first magnetic field strength and the first magnetic field gradient within the magnetic field generated under the magnetic field parameters in the first arrangement, and the second passage is the first passage. Under the magnetic field parameters in the arrangement 2, the second magnetic field strength is affected by the second magnetic field strength and the second magnetic field gradient in the magnetic field, and the second magnetic field strength is different from the first magnetic field strength, or A system in which the second magnetic field gradient is different from or a combination of the first magnetic field gradient. 18. The system of claim 18, wherein the magnetic field generator is coupled to a frame, the frame transmitting magnetic flux and forming a receiving region configured to receive the first holder or the second holder. 19. The system of claim 19, wherein the receiving area is sized to accept only one of the first holder or the second holder at a given time point. 19. The system of claim 19, wherein the receiving area is configured to receive only a portion of the first holder or the second holder. 21. The system of claim 21, wherein said portion comprises the first passage of the first holder or the second passage of the second holder. 19. The frame comprises a retractable portion that reduces the magnetic flux generated by the magnetic field generator as it moves beyond the receiving region while the first holder is separated from the magnetic field generator. Described system. 23. The system of claim 23, wherein the retractable portion comprises a spring that allows the first holder to enter the receiving region of the frame when compressed. 18. The system of claim 18, wherein the first passage is unaffected by the first magnetic field strength when the first holder is not coupled to the magnetic field generator. 19. The system of claim 19, wherein the first holder comprises a first end face configured to abut the stop portion of the frame when the first holder is coupled to the magnetic field generator. .. The second holder comprises a second end face that is configured to abut the stop portion of the frame when the second holder is coupled to the magnetic field generator, the first end face. 26. The system of claim 26, wherein the first distance between the first passage and the first passage is different from the second distance between the second end face and the second passage. The system of claim 18, wherein the first passage and the second passage are of different sizes. The first holder is configured to be fluid-coupled to a first source of beads of first size, and the second holder is fluid to a second source of beads of second size. The system according to claim 18, which is configured to be combined. 29. The system of claim 29, wherein the first size beads have a diameter of less than 1 μm and the second size beads have a diameter of more than 2 μm. 29. The first size bead is bound to a target cell in a first cell mixture and the second size bead is bound to a target cell in a second cell mixture. system. 18. The system of claim 18, wherein the first passage or the second passage comprises a plurality of magnetizable spheres and other magnetic augmentation devices. A magnetic cell isolation holder
A main body that is configured to be detachably coupled to the magnetic field generator, wherein the main body is of the magnetic field generator in a first arrangement when the holder is coupled to the magnetic field generator. A first passage configured to be positioned in the magnetic field and a second passage configured to be positioned in the magnetic field in a second arrangement when the holder is coupled to the magnetic field generator. With 2 passages, with a main body,
The first passage is affected by the first magnetic field strength and the first magnetic field gradient within the magnetic field generated under the magnetic field parameters in the first arrangement.
The second passage is affected by the second magnetic field strength and the second magnetic field gradient within the magnetic field generated under the magnetic field parameter in the second arrangement, and the second magnetic field strength is: A magnetic cell isolation holder that differs from or is a combination of the first magnetic field strength or the second magnetic field gradient. It's a system
The first kit,
With multiple first size beads,
A first holder comprising a first passage configured to receive the plurality of first-sized beads, wherein the first passage generates a magnetic field in which the first holder generates a magnetic field. A first holder comprising a first holder that, when detachably coupled to the vessel, is positioned within the first holder such that the first holder is positioned in the magnetic field in a first arrangement. 1 kit and
The second kit,
With multiple second size beads,
A second holder comprising a second passage configured to receive the plurality of second sized beads, wherein the second holder generates the magnetic field. When detachably coupled to the magnetic field generator, the second holder is positioned in the second holder such that it is positioned in the magnetic field in a second arrangement different from the first arrangement. , With a second holder, with a second kit,
The first passage is affected by the first magnetic field strength and the first magnetic field gradient in the magnetic field in the first arrangement.
The second passage is affected by the second magnetic field strength and the second magnetic field gradient in the magnetic field in the second arrangement, and is the second magnetic field strength different from the first magnetic field strength? , Or the system in which the second magnetic field gradient is different from or a combination thereof from the first magnetic field gradient. A method for isolating target cells
With the step of positioning the first holder with the first passage within the receiving area of the frame coupled to the magnetic field generator,
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 on the first passage. Steps to generate a first magnetic field inside,
A step of positioning a second holder having a second passage in the receiving area, wherein the first passage and the second passage are positioned in different arrangements in the receiving area. ,
The receiving region by the magnetic field generator when the second holder is coupled to the magnetic field generator to act on the second passage with a second magnetic field strength, a second magnetic field gradient, or both. A method that includes a step of generating a second magnetic field within. 35. The method of claim 35, wherein the first magnetic field and the second magnetic field are generated under the same magnetic field parameters. When the first holder is positioned within the receiving area of the frame, the second holder is not positioned within the receiving area of the frame and the second holder is of the frame. 35. The method of claim 35, wherein the first holder is not positioned within the receiving area of the frame when positioned within the receiving area. The first cell mixture is labeled with the first size beads, then the second cell mixture is labeled with the second size beads, and the target cells in the first cell mixture are labeled with the first size beads. And the step of incubating the target cells in the second cell mixture so that they are labeled with the second size beads.
35. The method of claim 35, further comprising: passing the first cell mixture through the first passage and then passing the second cell mixture through the second passage. A kit for use within a bioprocessing system
With a process bag
Source bag and
With a bead addition container,
It comprises the process bag, the source bag, and a process loop configured to communicate with the bead addition container.
The process loop is a kit with pump tubing configured to communicate fluid with the pump. Isolation column and
With a waste liquid bag,
With a buffer bag
With a collection bag
39. The kit of claim 39, wherein the process loop is configured to communicate fluidly with the isolation column, the effluent bag, the buffer bag, and the collection bag. 39. The kit of claim 39, further comprising a valve manifold that is capable of operating the source bag, the process bag, the bead addition container, and the process loop to selectively allow fluid communication. 39. The kit of claim 39, further comprising a storage bag configured to communicate fluidly with the process loop. 39. The kit of claim 39, further comprising a processing chamber configured to communicate fluid with the process loop. The kit according to claim 39, wherein the bead addition container is a syringe for injecting magnetically isolated beads into the process loop. 39. The kit of claim 39, further comprising a release buffer bag configured to communicate fluidly with the process loop. 39. The kit of claim 39, further comprising a medium bag configured to communicate fluidly with the process loop. A device for bioprocessing
A kit comprising a process bag, a source bag, and a beading additional container configured to communicate fluid with the process loop, wherein the process loop additionally communicates fluidly with the pump. With a kit and a pump tubing that is configured in
A magnetic field generator that is configured to generate a magnetic field,
A plurality of hooks for suspending the source bag, the process bag, and the bead addition container, each hook of the plurality of hooks is operably connected to a load cell, and the load cell is connected to the load cell. With multiple hooks configured to completely heal the weight of the bag,
With at least one bubble sensor,
A device comprising the process loop and a pump configured to communicate fluid. The process bag has an air port and
47. The device of claim 47, wherein the pump can operate to draw air into the system through the air port. The device according to claim 48, wherein the air port is located in the top of the processing bag at a position higher than the liquid level with the processing bag. 47. The apparatus of claim 47, further comprising a second effluent bag that selectively communicates with the valve manifold. 47. The effluent bag comprises a first effluent bag and a second effluent bag, wherein the first effluent bag and the second effluent bag are in fluid communication with the valve manifold. apparatus. A claim comprising a first storage bag and a second storage bag, wherein the first storage bag and the second storage bag are configured to be in fluid communication with the valve manifold. Item 47. 50. The apparatus of claim 50, further comprising a release media bag that selectively communicates with the valve manifold. A first pump tube with a first inner diameter and
Further comprising a second pump tube having a second inner diameter that is larger than the first pump tube.
47. The device of claim 47, wherein the first pump tube and the second pump tube are selectively engageable with the pump so as to enable a range of flow rates for the pump.