CN111757928A - Methods for cell enrichment and isolation - Google Patents

Methods for cell enrichment and isolation Download PDF

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Publication number
CN111757928A
CN111757928A CN201880088322.2A CN201880088322A CN111757928A CN 111757928 A CN111757928 A CN 111757928A CN 201880088322 A CN201880088322 A CN 201880088322A CN 111757928 A CN111757928 A CN 111757928A
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Prior art keywords
magnetic field
bag
magnetic
holder
cells
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CN201880088322.2A
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Chinese (zh)
Inventor
W.B.格里芬
张小华
R.D.史密斯
张呈昆
K.A.谢赫
白烨
三根进
徐民风
K.R.康维
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Globegroup Life Technology Consulting America Co ltd
Global Life Sciences Solutions USA LLC
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Globegroup Life Technology Consulting America Co ltd
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Priority claimed from US15/829,615 external-priority patent/US10696961B2/en
Application filed by Globegroup Life Technology Consulting America Co ltd filed Critical Globegroup Life Technology Consulting America Co ltd
Priority to CN202310912295.9A priority Critical patent/CN117343836A/en
Publication of CN111757928A publication Critical patent/CN111757928A/en
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Abstract

A method of biological treatment is disclosed, the method comprising the steps of: combining a suspension comprising a population of cells with magnetic beads to form a bead-bound population of cells in the suspension; separating the bead-bound cell population on a magnetic separation column; and collecting the target cells from the cell population. Collecting the target cells comprises removing the bead-bound cells from the separation column using an air plug. Also disclosed is a system comprising the magnetic field generator, the magnetic cell separation holder, the disposable fluid set, and associated methods of use.

Description

Methods for cell enrichment and isolation
Technical Field
Embodiments of the present invention generally relate to biological treatment systems and methods, and more particularly to biological treatment systems and methods for generating cellular immunotherapy.
Background
Various medical therapies involve the extraction, culture, and expansion of cells for use in downstream therapeutic processes. For example, Chimeric Antigen Receptor (CAR) T cell therapy is a cell therapy that redirects the patient's T cells to specifically target and destroy tumor cells. The rationale for CAR-T cell design involves recombinant receptors that combine antigen binding and T cell activation functions. A general premise of CAR-T cells is the artificial generation of T cells that target markers found on cancer cells. Scientists can remove T cells from the body, genetically modify them, and place them back into the patient for them to attack cancer cells. CAR-T cells can be derived from the patient's own blood (autologous), or from another healthy donor (allogeneic).
The first step in the production of CAR-T cells involves the use of apheresis (e.g., leukopheresis) to remove blood from a patient and isolate leukocytes. After a sufficient amount of leukocytes have been harvested, the leukocyte apheresis product is enriched for T cells, which involves washing the cells from the leukocyte apheresis buffer. A subpopulation of T cells having a particular biomarker is then isolated from the enriched subpopulation using a particular antibody conjugate or marker.
Following isolation of the target T cells, the cells are activated in an environment in which they can actively proliferate. For example, cells may be activated using magnetic beads coated with anti-CD 3/anti-CD 28 monoclonal antibodies or cell-based artificial antigen presenting cells (aapcs), which may be removed from the culture using magnetic separation. The CAR gene is then used to transduce T cells by integration of a gamma Retrovirus (RV) or Lentivirus (LV) vector. Viral vectors attach to patient cells using viral mechanisms, and upon entering the cells, the vectors introduce genetic material in the form of RNA. In the case of CAR-T cell therapy, this genetic material encodes a CAR. RNA is reverse transcribed into DNA and permanently integrated into the genome of the patient's cells; allowing maintenance of CAR expression when cells divide and grow in bulk in the bioreactor. The CAR is then transcribed and translated by the patient cell, and the CAR is expressed on the cell surface.
After T cells are activated and transduced with the CAR-encoding viral vector, the cells are extensively expanded in a bioreactor to achieve the desired cell density. After expansion, the cells are harvested, washed, concentrated, and formulated for infusion into a patient.
Existing systems and methods for manufacturing infusible doses of CAR T cells require many complex operations involving a large number of human contact points, which increases the time of the overall manufacturing process and increases the risk of contamination. Although recent efforts to automate the manufacturing process have eliminated some human contact points, these systems still suffer from high cost, inflexibility, and workflow bottlenecks. In particular, systems utilizing added automation are very expensive and inflexible as they require the consumer to adapt their process to the particular equipment of the system.
In view of the above, there is a need for a bioprocessing system for cellular immunotherapy that reduces the risk of contamination by increasing automation and reducing manual operations. In addition, there is a need for a bioprocessing system for cell therapy manufacturing that balances the need for flexibility in development and consistency in mass production, and meets the expectations of different customers running different processes.
Disclosure of Invention
The following outlines certain embodiments commensurate with the scope of the initially claimed subject matter. These embodiments are not intended to limit the scope of the claimed subject matter, but rather these embodiments are intended only to provide a brief summary of possible embodiments. Indeed, the present disclosure may include a variety of forms that may be similar to or different from the embodiments set forth below.
In one embodiment, a bioprocessing system comprises: a first module configured for enriching and isolating a population of cells; a second module configured for activating, genetically transducing, and expanding a population of cells; and a third module configured for harvesting the expanded cell population.
In another embodiment, a bioprocessing system comprises: a first module configured for enriching and isolating cells; a plurality of second modules, each second module configured for activating, genetically transducing, and expanding a cell; and a third module configured for harvesting the cells after expansion. Each second module is configured to support activation, genetic transduction, and amplification of different cell populations in parallel with one another.
In another embodiment, a method of biological treatment includes the steps of: in a first module, enriching and isolating a population of cells; in a second module, activating, genetically transducing, and expanding a population of cells; and in a third module, harvesting the expanded cell population. The steps of activating, genetically transducing, and expanding the cell population are performed without removing the cell population from the second module.
In another embodiment, an apparatus for bioprocessing includes a housing and a drawer receivable within the housing. The drawer includes a plurality of sidewalls and a bottom defining a processing chamber and a substantially open top. The drawer is movable between a closed position in which the drawer is received within the housing and an open position in which the drawer extends from the housing to enable access to the processing chamber through the open top. The apparatus also includes at least one seat positioned within the processing chamber and configured to receive a bioreactor container.
In another embodiment, a method of biological treatment includes the steps of: sliding a drawer having a plurality of side walls, a bottom, and a generally open top from a closed position within the enclosure to an open position such that the drawer extends from the enclosure through the generally open top; positioning the bioreactor container through the substantially open top on a static seat plate located within the drawer; sliding the drawer to a closed position; and controlling the drawer engagement actuator to engage the plurality of fluid flow lines with the at least one pump and the plurality of pinch valve linear actuators.
In another embodiment, a system for bioprocessing includes: a housing; a first drawer receivable within the housing, the first drawer including a plurality of sidewalls and a bottom defining a first processing chamber and a substantially open top; at least one first seat plate positioned within the processing chamber of the first drawer and configured to receive or otherwise engage a first bioreactor container thereon; a second drawer receivable within the housing in stacked relation to the first drawer, the second drawer including a plurality of sidewalls and a bottom defining a second processing chamber and a substantially open top; and at least one second seat plate positioned within the processing chamber of the second drawer and configured to receive or otherwise engage a second bioreactor container thereon. The first drawer and the second drawer are each movable between a closed position in which the first drawer and/or the second drawer is received within the housing and an open position in which the first drawer and/or the second drawer extends from the housing to enable access to the processing chambers through the open top, respectively.
In yet another embodiment, an apparatus for bioprocessing includes: a housing; a drawer receivable within the housing, the drawer including a plurality of sidewalls and a bottom surface defining a processing chamber and a generally open top, the drawer being movable between a closed position in which the drawer is received within the housing and an open position in which the drawer extends from the housing such that the processing chamber is accessible through the open top; at least one pedestal plate positioned within the process chamber adjacent the bottom surface; and a kit receivable within the processing chamber. The kit comprises: a plurality of sidewalls and a bottom surface defining an interior compartment; and a substantially open top; an opening formed in a bottom surface of the sleeve, the opening having a perimeter; and a bioreactor vessel positioned within the interior compartment above the at least one opening and supported by the bottom surface such that a portion of the bioreactor vessel is accessible through the opening in the bottom surface. The kit is receivable within the processing chamber such that the seat plate extends through the opening in the bottom surface of the tray to support the bioreactor container above the bottom surface of the kit.
In yet another embodiment, a system for bioprocessing includes: a tray having a plurality of side walls and a bottom surface defining an interior compartment, and a generally open top; at least one opening formed in the bottom surface, the at least one opening having a perimeter; a first tubing retainer block integral with the tray and configured to receive and retain at least one pump tube in position for selective engagement with a pump; a second tube retainer block integrated with the tray and configured to receive and hold each of the plurality of pinch valve tubes in position for selective engagement with a respective actuator of the pinch valve array; and a bioreactor vessel positioned within the interior compartment above the at least one opening and supported by the bottom surface such that a portion of the bioreactor vessel is accessible through the opening in the bottom surface.
In yet another embodiment, a system for bioprocessing includes: a process chamber having a plurality of sidewalls, a bottom surface, and a substantially open top; a pedestal plate positioned within the process chamber adjacent the bottom surface; and a tray. The tray includes: a plurality of sidewalls and a bottom surface defining an interior compartment; and a substantially open top; and an opening in the bottom surface of the tray, the opening having a perimeter. The perimeter of the opening is shaped and/or sized such that the bioreactor container can be positioned over the opening and supported by the bottom surface of the tray while a portion of the bioreactor container is accessible through the opening in the bottom surface. The tray is receivable within the processing chamber such that the seat plate extends through an opening in a bottom surface of the tray to support the bioreactor container.
In yet another embodiment, a system for bioprocessing includes a tray having: a plurality of sidewalls and a bottom surface defining an interior compartment; and a substantially open top; and at least one opening in the bottom surface, the opening bounded by a peripheral edge, wherein the opening is shaped and/or sized such that the bioreactor container is positionable over the opening and supported within the interior compartment by the bottom surface of the tray.
In yet another embodiment, a method of biological treatment includes the steps of: placing the bioreactor container in a disposable tray having a plurality of sidewalls and a bottom surface defining an interior compartment, a substantially open top, an opening formed in the bottom surface, and a plurality of projections or protrusions extending from the bottom surface into the opening; disposing the bioreactor container within the tray such that the bioreactor container is supported above the opening by the plurality of projections; and placing the tray into a process chamber having a seat plate such that the seat plate is received through the opening in the tray and supports the bioreactor container.
In yet another embodiment, a pipeline module for a biological treatment system includes: a first tubing retainer block configured to receive and retain at least one pump tube in position for selective engagement with a peristaltic pump; and a second tube retainer block configured to receive and hold each of the plurality of pinch valve tubes in position for selective engagement with a respective actuator of the pinch valve array. The first and second tube holder blocks are interconnected.
In yet another embodiment, a system for bioprocessing includes: a tray having a plurality of sidewalls and a bottom surface defining an interior compartment and a substantially open top, the tray configured to receive, support or otherwise engage a bioreactor container thereon; a pump assembly positioned adjacent the rear sidewall of the tray; an array of pinch valves positioned adjacent a rear sidewall of the tray; and a duct module positioned at a rear of the tray. The piping module includes: a first tubing retainer block configured to receive and retain at least one pump tube in position for selective engagement with a pump assembly; and a second tube retainer block configured to receive and hold each of the plurality of pinch valve tubes in position for selective engagement with a respective actuator of the pinch valve array.
In yet another embodiment, a bioreactor vessel comprises: a base plate; a container body coupled to the bottom panel, the container body and the bottom panel defining an interior compartment therebetween; and a plurality of recesses formed in the base plate, each recess of the plurality of recesses configured to receive a corresponding alignment pin on the seat plate for aligning the bioreactor container on the seat plate.
In yet another embodiment, a method for bioprocessing comprises: operatively connecting a bottom panel to the container body to define an interior compartment therebetween, the bottom panel and the container body forming a bioreactor container; aligning the recess in the base plate with an alignment pin of the bioprocessing system; and placing the bioreactor vessel on a floor of a biological treatment system.
In yet another embodiment, a bioprocessing system comprises: a first fluidic component having a first fluidic component line connected to the first port of the first bioreactor vessel by the first bioreactor line of the first bioreactor vessel, the first bioreactor line of the first bioreactor vessel including a first bioreactor line valve for providing selective fluid communication between the first fluidic component and the first port of the first bioreactor vessel; a second fluid assembly having a second fluid assembly line connected to the second port of the first bioreactor vessel through the second bioreactor line of the first bioreactor vessel, the second bioreactor line of the first bioreactor vessel including a second bioreactor line valve for providing selective fluid communication between the second fluid assembly and the second port of the first bioreactor vessel; and an interconnection line providing fluid communication between the first fluid component and the second fluid component, and between the second bioreactor line of the first bioreactor vessel and the first bioreactor line of the first bioreactor vessel.
In yet another embodiment, a method of biological treatment includes: providing a first fluidic component having a first fluidic component line connected to a first port of a first bioreactor vessel through a first bioreactor line of the first bioreactor vessel; providing a second fluid assembly having a second fluid assembly line connected to the second port of the first bioreactor vessel through the second bioreactor line of the first bioreactor vessel; and providing an interconnection line between the second bioreactor line of the first bioreactor vessel and the first bioreactor line of the first bioreactor vessel, the interconnection line allowing fluid communication between the first fluid component and the second fluid component and allowing fluid communication between the second bioreactor line of the first bioreactor vessel and the first bioreactor line of the first bioreactor vessel.
In yet another embodiment, a method of biological treatment for cell therapy includes: genetically modifying the population of cells in the bioreactor vessel to produce a genetically modified population of cells; and expanding the population of genetically modified cells within the bioreactor container without removing the population of genetically modified cells from the bioreactor container to produce a number of genetically modified cells sufficient for one or more doses used in cell therapy treatment.
In yet another embodiment, a biological treatment method includes: coating a bioreactor vessel with reagents for increasing the efficiency of genetic modification of a population of cells; genetically modifying cells of a cell population to produce a genetically modified cell population; and expanding the population of genetically modified cells in the bioreactor vessel without removing the genetically modified cells from the bioreactor vessel.
In yet another embodiment, a biological treatment method includes: activating cells of a cell population in a bioreactor vessel using magnetic or non-magnetic beads to produce an activated cell population; genetically modifying activated cells in a bioreactor vessel to produce a population of genetically modified cells; washing the genetically modified cells in the bioreactor vessel to remove unwanted material; and expanding the genetically modified cell population in the bioreactor vessel to produce an expanded transduced cell population. Activation, genetic modification, washing, and amplification are performed in the bioreactor vessel without removing cells from the bioreactor vessel.
In yet another embodiment, a kit for use in a biological processing system includes a processing bag, a source bag, a bead addition container, and a processing circuit configured to be in fluid communication with the processing bag, the source bag, and the bead addition container. The treatment circuit additionally includes a pump conduit configured to be in fluid communication with the pump.
In yet another embodiment, an apparatus for bioprocessing includes: a kit comprising a processing bag, a source bag, and a bead addition container configured to be in fluid communication with a processing circuit, the processing circuit additionally comprising a pump conduit configured to be in fluid communication with a pump; a magnetic field generator configured to generate a magnetic field; a plurality of hooks for suspending a source bag, a processing bag, and a bead addition container, each hook of the plurality of hooks operatively connected to a load cell configured to sense a weight of a bag connected thereto; at least one bubble sensor; and a pump configured to be in fluid communication with the treatment circuit.
In an embodiment, a method of biological processing includes: combining a suspension comprising a population of cells with magnetic beads to form a bead-bound population of cells in the suspension; separating the bead-bound cell population on a magnetic separation column; and collecting the target cells from the cell population.
In one embodiment, a system comprises: a magnetic field generator configured to generate a magnetic field at a magnetic field parameter; a first holder configured to be removably coupled to the magnetic field generator; and a second holder configured to be removably coupled to the magnetic field generator. The first holder has a channel configured to be positioned within the magnetic field at a first location when the first holder is coupled to the magnetic field generator. The second holder has a channel configured to be positioned within the magnetic field at a second location when the second holder is coupled to the magnetic field generator. The channel of the first holder experiences a first magnetic field strength and a first magnetic field gradient within a magnetic field generated at the first location under the magnetic field parameters. The channel of the second holder experiences a second magnetic field strength and a second magnetic field gradient within a magnetic field generated under the magnetic field parameters at the second location, and the second magnetic field strength is different from the first magnetic field strength, the second magnetic field gradient is different from the first magnetic field gradient, or a combination thereof.
In another embodiment, a magnetic cell separation holder includes a body configured to be removably coupled to a magnetic field generator. The main body has: a first channel configured to be positioned within a magnetic field of the magnetic field generator at a first location when the holder is coupled to the magnetic field generator; and a second channel configured to be positioned within the magnetic field at a second location when the holder is coupled to the magnetic field generator. The first channel experiences a first magnetic field strength and a first magnetic field gradient within a magnetic field produced at magnetic field parameters at a first location, and the second channel experiences a second magnetic field strength and a second magnetic field gradient within the magnetic field at magnetic field parameters at a second location. The second magnetic field strength is different than the first magnetic field strength, the second magnetic field gradient is different than the first magnetic field gradient, or a combination thereof.
In another embodiment, a system comprises: a first kit having a plurality of first size beads and a first holder having a channel configured to receive the plurality of first size beads; and a second kit having a plurality of second-sized beads and a second holder having a channel configured to receive the plurality of second-sized beads. The channel of the first holder is positioned within the holder such that when the first holder is removably coupled to the magnetic field generator, the first holder is positioned within a magnetic field generated by the magnetic field generator at a first location. The channel of the second holder is positioned within the holder such that when the second holder is removably coupled to the magnetic field generator, the second holder is positioned within the magnetic field generated by the magnetic field generator at a second position different from the first position. The channel of the first holder experiences a first magnetic field strength and a first magnetic field gradient within the magnetic field at the first location, and the channel of the second holder experiences a second magnetic field strength and a second magnetic field gradient within the magnetic field at the second location. The second magnetic field strength is different than the first magnetic field strength, the second magnetic field gradient is different than the first magnetic field gradient, or a combination thereof.
In another embodiment, a method for isolating a target cell comprises: positioning a first holder having a channel within a receiving area of a frame coupled to a magnetic field generator; and generating, by the magnetic field generator, a first magnetic field in the receiving area when the first holder is coupled to the magnetic field generator such that the channel of the first holder experiences a first magnetic field strength, a first magnetic field gradient, or both. The method further comprises the following steps: positioning a second retainer having a channel within the receiving area; and generating, by the magnetic field generator, a second magnetic field in the receiving area when the second holder is coupled to the magnetic field generator to cause the channel of the second holder to experience a second magnetic field strength, a second magnetic field gradient, or both. The channel of the first retainer and the channel of the second retainer are positioned at different locations within the receiving area.
Drawings
The invention will be better understood by reading the following description of non-limiting embodiments with reference to the attached drawings, in which:
FIG. 1 is a schematic illustration of a bioprocessing system according to an embodiment of the present invention.
FIG. 2 is a schematic illustration of a biological processing system according to another embodiment of the invention.
Fig. 3 is a block diagram illustrating a fluid flow configuration/system of the cell activation, genetic modification, and amplification subsystems of the biological processing system of fig. 1.
Fig. 4 is a detailed view of a portion of the block diagram of fig. 3, illustrating a first fluid component of the fluid flow arrangement/system.
Fig. 5 is a detailed view of a portion of the block diagram of fig. 3, illustrating a second fluid assembly of the fluid flow arrangement/system.
Fig. 6 is a detailed view of a portion of the block diagram of fig. 3, illustrating a sampling assembly of the fluid flow arrangement/system.
Fig. 7 is a detailed view of a portion of the block diagram of fig. 3, illustrating a filtration flow path of a fluid flow arrangement/system.
Fig. 8 is a perspective view of a bioreactor vessel according to an embodiment of the present invention.
Fig. 9 is an exploded view of the bioreactor vessel of fig. 8.
Fig. 10 is an exploded cross-sectional view of the bioreactor vessel of fig. 8.
Fig. 11 is an exploded bottom perspective view of the bioreactor vessel of fig. 8.
Fig. 12 is a perspective top front view of a disposable plug-in kit of the bioprocessing system of fig. 1 according to an embodiment of the invention.
Fig. 13 is another perspective top front view of the disposable insertion set of fig. 12.
Fig. 14 is a perspective top rear view of the disposable plug-in kit of fig. 12.
Fig. 15 is a perspective view of a tray of the disposable plug-in kit of fig. 12, according to an embodiment of the present invention.
Fig. 16 is a front perspective view of a conduit module of the disposable plug-in kit of fig. 12, according to an embodiment of the present invention.
Fig. 17 is a rear perspective view of the duct module of fig. 16.
FIG. 18 is a front view of a second tube holder block of a tube module according to an embodiment of the present invention.
FIG. 19 is a cross-sectional view of the second tube holder block of FIG. 18.
Fig. 20 is another perspective front view of the plug-in kit of fig. 12, showing the flow architecture integrated therein.
Fig. 21 is a perspective rear view of the plug-in kit of fig. 12 showing the flow architecture integrated therein.
Fig. 22 is a front elevational view of the plug-in kit of fig. 12, showing the flow architecture integrated therein.
Fig. 23 is a perspective view of a biological treatment apparatus according to an embodiment of the invention.
Fig. 24 is a perspective view of a drawer of a biological treatment apparatus for receiving the plug-in kit of fig. 12, according to an embodiment of the invention.
FIG. 25 is a top plan view of the drawer of FIG. 24.
FIG. 26 is a front perspective view of the processing chamber of the drawer of FIG. 24.
Fig. 27 is a top plan view of a processing chamber of a drawer.
Fig. 28 is a top plan view of the seat plate of the bioprocessing apparatus of fig. 23.
Fig. 28A is a top plan view of hardware components housed under the seat pan of fig. 28.
Fig. 29 is a side elevational view of the bioprocessing apparatus of fig. 12.
Fig. 30 is a perspective view of a drawer engagement actuator of the bioprocessing apparatus of fig. 12.
Fig. 31 is a top plan view of a drawer of the bioprocessing apparatus illustrating clearance positions of the drawer engagement actuator, pump assembly, and solenoid array.
Fig. 32 is a top plan view of a drawer of the bioprocessing apparatus illustrating the engaged position of the drawer engagement actuator, pump assembly, and solenoid array.
FIG. 33 is a perspective view of the bioprocessing equipment illustrating the plug-in kit in place within the processing chamber of the drawer.
FIG. 34 is a top plan view of the bioprocessing apparatus illustrating the plug-in kit in place within the processing chamber of the drawer.
FIG. 35 is a perspective view of a peristaltic pump assembly of the bioprocessing apparatus.
FIG. 36 is a side elevational view of the peristaltic pump assembly and the tube holder module of the plug-in kit illustrating the relationship between the components.
FIG. 37 is a perspective view of a solenoid array and a pinch valve anvil forming a pinch valve array of a biological treatment apparatus.
FIG. 38 is another perspective view of a pinch valve array of a bioprocessing apparatus.
FIG. 39 is another perspective view of the pinch valve array illustrating positioning of a tube holder module of a plug-in kit relative to the pinch valve array in an engaged position.
FIG. 40 is a cross-sectional view of a drawer of the bioprocessing apparatus illustrating the placement of bioreactor containers on the tray.
Fig. 41 is a side elevational view of a bioreactor received on a deck illustrating a stirring/mixing mode of operation of the bioreactor system.
Fig. 42 is a side cross-sectional view of a bioreactor received on a seat deck illustrating a stirring/mixing mode of operation of the bioreactor system.
Fig. 43 is a schematic illustration of a bioreactor vessel showing fluid levels within the bioreactor vessel during a stirring/mixing mode of operation.
Fig. 44 is a cross-sectional detail view of the interface between the locating pins on the seat plate and the receiving recesses on the bioreactor container during the stirring/mixing mode of operation.
FIG. 45 is a perspective view of a bioprocessing apparatus having a flip down front panel showing a processing drawer of the bioprocessing apparatus in an open position according to an embodiment of the present invention.
FIG. 46 is another perspective view of the bioprocessing apparatus of FIG. 45, showing the processing drawer of the bioprocessing apparatus in an open position.
FIG. 47 is an enlarged perspective view of the auxiliary compartment of the bioprocessing apparatus of FIG. 45 showing the processing drawer in a closed position with access to the auxiliary compartment.
FIG. 48 is another enlarged perspective view of the auxiliary compartment of the bioprocessing apparatus of FIG. 45 showing the processing drawer in a closed position with access to the auxiliary compartment.
FIG. 49 is a perspective view of the bioprocessing apparatus of FIG. 45 showing the processing drawer of the bioprocessing apparatus in a closed position with access to the auxiliary compartment.
FIG. 50 is another perspective view of the bioprocessing apparatus of FIG. 45, showing the processing drawer of the bioprocessing apparatus in a closed position with access to the auxiliary compartment.
FIG. 51 is a perspective view of an auxiliary compartment of a bioprocessing apparatus according to another embodiment of the present invention.
FIG. 52 is a perspective view of a biological treatment system having a waste tray according to an embodiment of the invention.
Fig. 53-77 are schematic illustrations of an automated general protocol for a biological processing system utilizing the fluid flow architecture of fig. 3, in accordance with an embodiment of the invention.
FIG. 78 is a perspective view of an enrichment and separation apparatus according to an embodiment of the invention.
FIG. 79 is a process flow diagram of the enrichment and separation apparatus of FIG. 78.
Fig. 80 is a schematic illustration of a fluid flow architecture of the device of fig. 78 for performing enrichment and separation of a cell population.
Fig. 81 is a block diagram of a magnetic particle-based cell selection system that may be used with a magnetic cell separation holder, according to aspects of the present disclosure.
Fig. 82 is a flow diagram of a magnetic cell separation method according to aspects of the present disclosure.
Fig. 83A illustrates a top view of an embodiment of a magnetic cell separation holder in an unloaded configuration relative to a magnetic field generator, in accordance with aspects of the present disclosure.
Fig. 83B illustrates a top view of an embodiment of a magnetic cell separation holder in a loaded configuration relative to a magnetic field generator, in accordance with aspects of the present disclosure.
Fig. 84A illustrates a perspective view of an embodiment of a magnetic cell isolation holder in an unloaded configuration relative to a magnetic field generator, in accordance with aspects of the present disclosure.
Fig. 84B illustrates a top view of an embodiment of a magnetic cell separation holder in a loaded configuration relative to a magnetic field generator in a loaded configuration, in accordance with aspects of the present disclosure.
Fig. 85 is a flow diagram of a magnetic cell separation method using different magnetic cell separation holders, according to aspects of the present disclosure.
Fig. 86 illustrates a top view of an embodiment of a magnetic cell separation holder and magnetic field generator in a loaded configuration, in accordance with aspects of the present disclosure.
Fig. 87 is a magnetic field distribution of a magnetic field generator according to aspects of the present disclosure.
Detailed Description
Reference will now be made in detail to exemplary embodiments of the invention, examples of which are illustrated in the accompanying drawings. Wherever possible, the same reference characters will be used throughout the drawings to refer to the same or like parts.
As used herein, the terms "flexible" or "collapsible" refer to structures or materials that are flexible or capable of bending without breaking, and may also refer to compressible or expandable materials. An example of a flexible structure is a bag formed from polyethylene film. The terms "rigid" and "semi-rigid" are used interchangeably herein to describe a "non-collapsible" structure, that is, a structure that does not fold, collapse, or otherwise deform under normal forces to significantly reduce its elongated dimension. Depending on the context, "semi-rigid" may also refer to structures that are more flexible than "rigid" elements, such as bendable tubes or catheters, but still structures that do not collapse longitudinally under normal conditions and forces.
The term "container" as used herein refers to a flexible bag, a flexible receptacle, a semi-rigid receptacle, a rigid receptacle, or a flexible or semi-rigid conduit, as the case may be. The term "vessel" as used herein is intended to include bioreactor vessels having semi-rigid or rigid walls or portions of walls, as well as other receptacles or conduits commonly used in biological or biochemical processes, including, for example, cell culture/purification systems, mixing systems, media/buffer preparation systems, and filtration/purification systems, such as chromatography and tangential flow filter systems, and their associated flow paths. As used herein, the term "bag" means a flexible or semi-rigid receptacle or container that is used, for example, as a containment device for a variety of fluids and/or media.
As used herein, "fluidly coupled" or "in fluid communication" means that the components of the system are capable of receiving or transferring fluid between the components. The term fluid includes a gas, a liquid, or a combination thereof. As used herein, "in electrical communication" or "electrically coupled" means that certain components are configured to communicate with each other by signaling, either directly or indirectly, through either a direct or indirect electrical connection. As used herein, "operatively coupled" means a connection that may be direct or indirect. The connection need not be a mechanical attachment.
As used herein, the term "tray" refers to any object capable of at least temporarily supporting a plurality of components. The tray may be made of a variety of suitable materials. For example, the tray may be made of a cost-effective material suitable for sterile and single-use disposable products.
As used herein, the term "functionally closed system" refers to a plurality of components making up a closed fluid path that may have an inlet port and an outlet port to add or remove fluid or air from the system without compromising the integrity of the closed fluid path (e.g., to maintain an internal sterile biomedical fluid path), whereby the ports may include, for example, a filter or membrane at each port to maintain sterile integrity as fluid or air is added to or removed from the system. Depending on a given embodiment, the components may include, but are not limited to, one or more conduits, valves (e.g., a multi-port shunt), containers, receptacles, and ports.
Embodiments of the present invention provide systems and methods for producing cellular immunotherapy from a biological sample (e.g., blood, tissue, etc.). In an embodiment, a method of biological processing includes: combining a suspension comprising a population of cells with magnetic beads to form a bead-bound population of cells in the suspension; separating the bead-bound cell population on a magnetic separation column; and collecting the target cells from the cell population. Collecting the target cells comprises removing the bead-bound cells from the separation column using an air plug.
Referring to FIG. 1, a schematic illustration of a biological processing system 10 is illustrated, in accordance with an embodiment of the present invention. The bioprocessing system 10 is configured for use in the manufacture of cellular immunotherapy (e.g., autologous cellular immunotherapy) in which, for example, a human blood, fluid, tissue, or cell sample is collected and cellular therapy is generated from or based on the collected sample. One type of cellular immunotherapy that may be produced using biological treatment system 10 is Chimeric Antigen Receptor (CAR) T cell therapy, although other cell therapies may also be produced using the system of the invention or aspects thereof without departing from the broader aspects of the invention. As illustrated in figure 1, the manufacture of CAR T cell therapy generally begins with the collection of a patient's blood and the isolation of lymphocytes by apheresis. Collection/apheresis may be performed in a clinical setting, and the apheresis product is then sent to a laboratory or manufacturing facility for use in generating CAR T cells. In particular, once the apheresis product is received for processing, a desired cell population (e.g., white blood cells) is enriched or isolated from the collected blood for use in the manufacture of cell therapy, and target cells of interest are isolated from the initial cell mixture. The target cells of interest are then activated, genetically modified to specifically target and destroy tumor cells, and expanded to achieve the desired cell density. After expansion, cells were harvested and a dose was formulated. The preparation is then typically cryopreserved and delivered to a clinical setting for thawing, preparation, and finally infusion into a patient.
With further reference to fig. 1, the bioprocessing system 10 of the present invention includes a plurality of different modules or subsystems that are each configured to perform a particular subset of the manufacturing steps in a substantially automated, functionally closed and scalable manner. In particular, the biological treatment system 10 includes: a first module 100 configured to perform the steps of enriching and separating; a second module 200 configured to perform the steps of activating, genetically modifying, and amplifying; and a third module 300 configured to perform the step of harvesting the expanded cell population. In an embodiment, each module 100, 200, 300 is communicatively coupled to a dedicated controller (e.g., to first controller 110, second controller 210, and third controller 310, respectively). The controllers 110, 210, and 310 are configured to provide substantially automated control of the manufacturing process within each module. Although the first, second and third modules 100, 200, 300 are illustrated as including dedicated controllers for controlling the operation of each module, it is contemplated that a master control unit may be utilized to provide global control of the three modules. Each module 100, 200, 300 is designed to work in conjunction with other modules to form a single, coherent bioprocessing system 10, as discussed in detail below.
By automating the process within each module, product consistency from each module may be improved and costs associated with extensive manual handling reduced. Additionally, as discussed in detail below, each module 100, 200, 300 is substantially enclosed, which helps ensure patient safety by reducing the risk of external contamination, ensures regulatory compliance, and helps avoid costs associated with open systems. Furthermore, each module 100, 200, 300 is scalable to support both development at low patient counts and commercial manufacturing at high patient counts.
With further reference to fig. 1, the particular manner in which the process steps are divided among the different modules each providing closed and automated biological processes allows for efficient utilization of capital equipment to a degree heretofore not seen in the art. As will be appreciated, the step of expanding the cell population prior to harvesting and formulation to achieve the desired cell density is typically the most time-consuming step in the manufacturing process, while the enrichment and isolation steps, harvesting and formulation steps, and activation and genetic modification steps are much less time-consuming. Thus, attempting to automate the entire cell therapy manufacturing process, in addition to being logistically challenging, can exacerbate bottlenecks in the process that impede workflow and reduce manufacturing efficiency. In particular, in a fully automated process, while the steps of enrichment, isolation, activation and genetic modification of cells can occur quite rapidly, the expansion of genetically modified cells occurs very slowly. Thus, the production of a cell therapy from a first sample (e.g., blood of a first patient) will proceed rapidly up to an expansion step, which takes a significant amount of time to achieve the desired cell density for harvesting. In the case of a fully automated system, the entire process/system will be monopolized by the amplification device performing cell expansion from the first sample, and processing of the second sample may not begin until the entire system is vacated for use. In this regard, in the case of a fully automated bioprocessing system, the entire system is essentially off-line and unavailable to process a second sample until the entire cell therapy manufacturing process from enrichment to harvest/formulation is completed for the first sample.
However, embodiments of the invention allow more than one sample (from the same or different patients) to be processed in parallel to provide more efficient use of capital resources. As implied above, this advantage is a direct result of the particular way in which the process steps are separated into the three modules 100, 200, 300. Referring particularly to fig. 2, in an embodiment, a single first module 100 and/or a single third module 300 may be utilized in conjunction with a plurality of second modules (e.g., second modules 200a, 200b, 200c) in the bioprocessing system 12 to provide parallel and asynchronous processing of multiple samples from the same or different patients. For example, a first module 100 may be used to enrich and isolate a first apheresis product from a first patient to produce a first population of isolated target cells, and then the first population of target cells may be transferred to one of a second module, such as module 200a, for activation, genetic modification, and amplification under the control of controller 210 a. Once the first population of target cells is transferred out of the first module 100, the first module is again available for use to process a second apheresis product from, for example, a second patient. The second population of target cells generated in the first module 100 from the sample taken from the second patient may then be transferred to another second module, such as second module 200b, for activation, genetic modification, and amplification under the control of controller 201 b.
Similarly, after the second population of target cells is transferred out of the first module 100, the first module is again available for use to process a third apheresis product from, for example, a third patient. A third target population of cells produced in the first module 100 from a sample taken from a third patient may then be transferred to another second module, such as second module 200c, for activation, genetic modification, and amplification under the control of controller 201 c. In this regard, for example, expansion of CAR-T cells for a first patient can occur simultaneously with expansion of CAR-T cells for a second patient, a third patient, and so forth.
The method also allows post-processing to occur asynchronously as needed. In other words, the patient cells may not all grow at the same time. The culture may reach final density at different times, but the plurality of second modules 200 are not linked, and the third module 300 may be used as needed. In the case of the present invention, although samples can be processed in parallel, they need not be performed in batches.
When each expanded cell population is ready for harvest, harvesting of the expanded cell populations from the second modules 200a, 200b, and 200c can also be accomplished using a single third module 300.
Thus, by separating the steps of activation, genetic modification and amplification (which is the most time consuming and shares certain operational requirements and/or requires similar culture conditions) into separate, automated and functionally closed modules, another system device for enrichment, isolation, harvesting and formulation is not occupied or taken offline when performing the amplification of one cell population. As a result, the manufacture of multicellular therapies can be performed simultaneously, thereby maximizing the use of equipment and floor space, and increasing the efficiency of the overall process and facility. It is contemplated that additional second modules may be added to the bioprocessing system 10 to provide parallel processing of any number of cell populations, as desired. Thus, the bio-processing system of the present invention allows plug-and-play like functionality, which enables manufacturing facilities to be easily scaled up or down.
In embodiments, the first module 100 may be any system or device capable of producing a target population of enriched and isolated cells for use in biological processes (such as the manufacture of immunotherapy and regenerative medicine) from an apheresis product taken from a patient. For example, the first module 100 may be a modified version of the Sefia cell processing system available from GE Healthcare. The configuration of the first module 100 according to some embodiments of the invention is discussed in detail below.
In embodiments, the third module 300 may similarly be any system or device capable of harvesting and/or formulating CAR-T cells or other modified cells produced by the second module 200 for infusion into a patient, for use in cellular immunotherapy or regenerative medicine. In some embodiments, the third module 300 may likewise be a Sefia cell processing system available from GE Healthcare. In some embodiments, the first module 100 may be used first to enrich and isolate cells (which are then transferred to the second module 200 for activation, transduction, and expansion (and in some embodiments harvesting)), and then also at the end of the process for cell harvesting and/or formulation. In this regard, in some embodiments, the same apparatus can be used for the front-end cell enrichment and isolation steps, as well as the back-end harvesting and/or formulation steps.
Focusing first on the second module 200, the ability to combine the process steps of cell activation, genetic modification, and cell expansion into a single functionally closed and automated module 200 that provides the workflow efficiencies described above is achieved through the specific configuration of the components within the second module 200 and the unique flow architecture that provides specific interconnectivity between such components. Fig. 3-77, discussed below, illustrate various aspects of the second module 200 according to various embodiments of the invention. Referring first to fig. 3, a schematic diagram illustrating a fluid flow architecture 400 (also broadly referred to herein as a bioprocessing subsystem 400 or bioprocessing system 400) within a second module 200 that provides for cell activation, genetic modification, and amplification (and in some cases harvesting) is shown. System 400 includes a first bioreactor vessel 410 and a second bioreactor vessel 420. The first bioreactor vessel includes at least a first port 412 and a first bioreactor line 414 in fluid communication with the first port 412 and a second port 416 and a second bioreactor line 418 in fluid communication with the second port 416. Similarly, the second bioreactor vessel includes at least a first port 422 and a first bioreactor line 424 in fluid communication with the first port 422 and a second port 426 and a second bioreactor line 428 in fluid communication with the second port 426. Together, first bioreactor vessel 410 and second bioreactor vessel 420 form bioreactor array 430. Although system 400 is shown with two bioreactor vessels, embodiments of the invention may include a single bioreactor or more than two bioreactor vessels.
As discussed below, first and second bioreactor lines 414 and 418 of first bioreactor vessel 410 and first and second bioreactor lines 424 and 428 of second bioreactor vessel 420 each include a respective valve for controlling fluid flow therethrough. In particular, first bioreactor line 414 of first bioreactor vessel 410 includes a first bioreactor line valve 432, and second bioreactor line 418 of first bioreactor vessel 410 includes a second bioreactor line valve 424. Similarly, first bioreactor line 424 of second bioreactor vessel 420 includes a first bioreactor line valve 436, and second bioreactor line 428 of second bioreactor vessel 420 includes a second bioreactor line valve 438.
With further reference to fig. 3, the system 400 also includes a first fluid assembly 440 having a first fluid assembly line 442, a second fluid assembly 444 having a second fluid assembly line 446, and a sampling assembly 448. An interconnect line 450 having an interconnect line valve 452 provides fluid communication between the first fluid assembly 440 and the second fluid assembly 444. As shown in fig. 3, interconnecting line 450 also provides fluid communication between second bioreactor line 418 and first bioreactor line 414 of first bioreactor vessel 410, allowing fluid to circulate along the first circulation loop of the first bioreactor vessel. Similarly, the interconnecting line also provides fluid communication between second bioreactor line 428 of second bioreactor vessel 420 and first bioreactor line 424, allowing fluid to circulate along the second circulation loop of the second bioreactor vessel. Furthermore, as discussed below, interconnecting line 450 further provides fluid communication between second port 416 and second bioreactor line 418 of first bioreactor vessel 410 and first port 422 and first bioreactor line 424 of second bioreactor vessel 420, thereby allowing transfer of the contents of first bioreactor vessel 410 to second bioreactor vessel 420. As illustrated in fig. 3, in an embodiment, interconnecting line 450 extends from second bioreactor lines 418, 428 to the intersection of first bioreactor line 414 and first fluidic component line 442 of first bioreactor vessel 410.
As illustrated by fig. 3, a first fluid assembly 440 and a second fluid assembly 450 are disposed along an interconnecting line 450. Additionally, in an embodiment, the first fluid assembly is in fluid communication with the first port 412 of the first bioreactor vessel 410 and the first port of the second bioreactor vessel 420 through the first bioreactor line 414 of the first bioreactor vessel and the first bioreactor line 424 of the second bioreactor vessel 420, respectively. Second fluid assembly 444 is in fluid communication with second port 416 of first bioreactor vessel 410 and second port 426 of second bioreactor vessel 420 via interconnecting line 450.
A first pump or interconnecting line pump 454 capable of providing bi-directional fluid flow is disposed along the first fluid assembly line 442 and a second pump or circulating line pump 456 capable of providing bi-directional fluid flow is disposed along the interconnecting line 450, the function and purpose of the pumps 454 and 456 being discussed below. In an embodiment, pumps 454, 456 are high dynamic range pumps. As also shown in fig. 3, a sterile air source 458 is connected to the interconnecting line 450 by a sterile air source line 460. A valve 462 positioned along sterile air source line 460 provides selective fluid communication between sterile air source 458 and interconnect line 450. Although fig. 3 shows a sterile air source 458 connected to the interconnection line 450, in other embodiments, the sterile air source may be connected to the first fluid assembly 440, the second fluid assembly 444, or a fluid flow path intermediate the second bioreactor line valve and the first bioreactor line valve of the first bioreactor or the second bioreactor, without departing from the broader aspects of the invention.
Referring now additionally to fig. 4-6, detailed views of the first fluid assembly 440, the second fluid assembly 444, and the sampling assembly 448 are shown. With particular reference to fig. 4, the first fluid assembly 440 includes a plurality of conduit tails 464a-f, each of the conduit tails 464a-f configured for selective/removable connection to one of a plurality of first reservoirs 466 a-f. Each of the conduit tails 464a-f of the first fluid assembly 440 includes a conduit tail valve 468a-f for selectively controlling fluid flow to or from a respective one of the plurality of first reservoirs 466a-f of the first fluid assembly 440. Although fig. 4 specifically illustrates the first fluid assembly 440 as including six fluid reservoirs, more or fewer reservoirs may be utilized to provide for the input or collection of multiple treatment fluids, as desired. As described below, it is contemplated that each conduit tail 464a-f may be individually connected to reservoirs 466a-f, respectively, at times required during operation of fluid assembly 440.
With particular reference to fig. 5, the second fluid assembly 444 includes a plurality of conduit tail portions 470a-d, each of the conduit tail portions 470a-d configured for selective/removable connection to one of a plurality of second reservoirs 472 a-d. Each of the conduit tails 470a-d of the second fluid assemblies 444 includes a conduit tail valve 474a-e for selectively controlling fluid flow to or from a respective one of the plurality of second reservoirs 472a-d of the first fluid assemblies 444. Although fig. 5 specifically illustrates the second fluid assembly 444 as including four fluid reservoirs, more or fewer reservoirs may be utilized to provide for the input or collection of multiple treatment fluids, as desired. In an embodiment, at least one of the second reservoirs (e.g., second reservoir 472d) is a collection reservoir for collecting the expanded cell population, as discussed below. In an embodiment, the second reservoir 472a is a waste reservoir, the purpose of which is discussed below. The present invention further contemplates that one or more reservoirs 472a-d may be pre-connected to their respective tails 470a-d, with each additional reservoir being connected to its respective tail in time for its use within second fluid assembly 440.
In an embodiment, the first reservoirs 466a-f and the second reservoirs 472a-d are single use/disposable flexible bags. In an embodiment, the bag is a substantially two-dimensional bag having opposing panels welded or otherwise secured together around their peripheries and supporting a connecting conduit for connection to their respective tails (as is known in the art).
In an embodiment, the reservoir/bag may be connected to the tubing tails of the first tubing assembly and the second tubing assembly using a sterile welding device. In an embodiment, a welding device may be positioned alongside the module 200, and the welding device is used to splice weld one of the pipe tails to the tail of the pipe on the bag (while maintaining sterility). Thus, the operator can provide the bag when it is needed (e.g., by grasping the tube tail and inserting its free end into a welding device, placing the free end of the tube of the bag near the end of the tube tail, cutting the tube with a new razor blade, and heating the cut end as the razor is pulled away while the two tube ends are pressed together while still molten, causing them to resolidify together). Conversely, the bag may be removed by welding the tubing from the bag and cutting at the weld to separate the two closed tubing lines. Thus, the reservoirs/bags can be connected individually when desired, and the present invention does not require that all reservoirs/bags must be connected at the beginning of the protocol, as the operator will be able to access the appropriate tubing tail during the entire procedure, and connect the reservoirs/bags for their use at that time. Indeed, while it is possible that all reservoirs/bags are pre-connected, the present invention does not require pre-connection, and one advantage of the second module 200 is that it allows an operator to access the fluid assembly/line during operation so that used bags can be connected in a sterile manner, and disconnected so that other bags can be aseptically connected during the protocol (as discussed below).
As illustrated in fig. 6, the sampling assembly 448 includes one or more sampling lines, such as sampling lines 476a-476d, that are fluidly connected to the interconnect line 450. Each of the sampling lines 476a-476d may include a sampling line valve 478a-d that is selectively actuatable to allow fluid flow from the interconnecting line 450 through the sampling lines 476a-476 d. As also shown in fig. 6, the distal end of each sampling line 476a-476d is configured for selective connection to a sample collection device (e.g., sample collection devices 280a and 280d) for collecting fluid from the interconnecting line 450. The sample collection device may take the form of any sampling device known in the art, such as, for example, a syringe, dip tube, bag, or the like. Although fig. 6 illustrates sampling assembly 448 connected to the interconnection lines, in other embodiments, the sampling assembly may be fluidly coupled to the fluid flow path intermediate the first fluid assembly 440, the second fluid assembly 444, the second bioreactor line valve 434 and the first bioreactor line valve 432 of the first bioreactor vessel 410, and/or the fluid flow path intermediate the second bioreactor line valve 438 and the first bioreactor line valve 436 of the second bioreactor vessel 420. The sampling assembly 448 provides fully functionally closed fluid sampling at one or more points in the system 400, as desired.
Referring back to fig. 3, in an embodiment, the system 400 may also include a filter line 482, the filter line 482 being connected at two points along the interconnecting line 450 and defining a filter loop along the interconnecting line 450. A filter 484 is positioned along the filter line 482 for removing permeate waste from the fluid passing through the filter line 482. As shown in fig. 3, the filter line 482 includes an upstream filter line valve 486 and a downstream filter line valve 488 positioned on the upstream and downstream sides, respectively, of the filter 484. Waste line 490 provides fluid communication between filter 484 and second fluid assembly 444 and, in particular, with conduit tail 470a of second fluid assembly 444 connected to waste reservoir 472 a. In this regard, the waste line 490 conveys waste removed from the fluid passing through the filter line 482 by the filter 484 to the waste reservoir 472 a. As illustrated in fig. 3, filter line 482 encircles interconnect line valve 452 such that fluid flow through interconnect line 450 may be forced through filter line 482 (as discussed below). An osmotic pump 492 located along the waste line 490 is operable to pump waste removed by the filter to a waste reservoir 472 a. In an embodiment, the filter 484 is desirably an elongated hollow fiber filter, however other tangential flow or cross-flow filtration devices known in the art, such as, for example, flat sheet membrane filters, may also be utilized without departing from the broader aspects of the present invention.
In an embodiment, the valves of the first and second fluid assemblies 440, 444 and the bioreactor line valves (i.e., valves 432, 434, 436, 438, sterile line valve 462, interconnection line valve 452, and filtration line valves 486, 488) are pinch valves configured in a manner described below. In embodiments, the pipeline itself need not include a pinch valve, and the depictions of the pinch valve in fig. 3-8 may merely represent locations on the pipeline where the pinch valve may be operated so as to prevent fluid flow. In particular, as discussed below, the pinch valves of the flow architecture 400 may be provided by respective actuators (e.g., solenoids) that operate/act against corresponding anvils when the fluid path/line is in the middle to "pinch off" the line, thereby preventing fluid flow therethrough.
In an embodiment, pumps 454, 456, and 492 are peristaltic pumps, and the pumps are integrated into a single component, as discussed below. Desirably, the operation of these valves and pumps are automatically directed according to a programmed protocol in order to achieve proper operation of the module 200. It is contemplated that the second controller 210 may direct the operation of the valves and pumps through the module 200.
Turning now to fig. 8-11, configurations of a first bioreactor vessel 410 according to embodiments of the present invention are illustrated. Since second bioreactor vessel 420 is desirably (although not necessarily) identical in configuration to first bioreactor vessel 410, for simplicity, only first bioreactor vessel 410 will be described below. In an embodiment, the bioreactor containers 410, 420 are perfusion-enabled, silicone membrane-based bioreactor containers that support activation, transduction, and expansion of a cell population therein. Bioreactor vessels 410, 420 may be used for cell culture, cell processing, and/or cell expansion to increase cell density for use in medical treatments or other procedures. Although the bioreactor vessel may be disclosed herein as being used in conjunction with a particular cell type, it should be understood that the bioreactor vessel may be used for activation, genetic modification, and/or expansion of any suitable cell type. Furthermore, the disclosed techniques can be used in conjunction with adherent cells (i.e., cells that adhere to and/or proliferate on a cell expansion surface). In an embodiment, first bioreactor vessel 410 and second bioreactor vessel 420 may be constructed and function as disclosed in U.S. patent No. 15/893336 filed 2018, 2, 9, incorporated herein by reference in its entirety.
As shown in fig. 8 and 9, first bioreactor vessel 410 may include a floor 502 and a vessel body 504 coupled to the floor 502. The base plate 502 may be a rigid structure to support the cell culture. However, as discussed in more detail with reference to fig. 9, the bottom plate may be a non-solid plate (e.g., may be open and/or porous) to allow oxygen to be provided to the cell culture. In the illustrated embodiment, the base plate 502 is rectangular or nearly rectangular in shape. In other embodiments, floor 502 may be any other shape that may enable a low profile container and/or may maximize space in a location where the first bioreactor container may be utilized or stored.
In an embodiment, vessel body 504 includes a rigid, generally concave structure that, when coupled to floor 502, forms a cavity or interior compartment 506 of first bioreactor vessel 410. As shown therein, the container body 504 may have a perimeter shape similar to the perimeter shape of the floor 502 such that the container body 504 and the floor 502 may be coupled to one another. Additionally, as in the illustrated embodiment, the container body 504 may be made of a transparent or translucent material that may enable visual inspection of the contents of the first bioreactor container 410 and/or may enable light to enter the first bioreactor container 410. The interior compartment 506 formed by the floor 502 and the container body 504 may contain cell culture media and cell culture during use of the first bioreactor container for cell activation, genetic modification (i.e., transduction), and/or cell expansion.
As best shown in fig. 8-11, the first bioreactor vessel 410 may include a plurality of ports through the vessel body 504 that enable fluid communication between the interior compartment 506 and the exterior of the first bioreactor vessel 410 for certain processes related to activation, transduction/genetic modification, and expansion of cells, such as media input and waste removal. The ports may include, for example, a first port 412 and a second port 416. As in the illustrated embodiment, the port 416 may be disposed at any location in the container body 504, including through the top surface 508 and/or any side 510 of the container body 504. As will be discussed in more detail herein, the particular configuration of the first bioreactor vessel 410, including the particular number and location of the ports 412, 416, enables the first bioreactor vessel 410 to be used to support activation of cells, genetic modification of cells, and high cell density expansion.
Fig. 9 is an exploded view of an embodiment of first bioreactor vessel 410. Floor 502 of first bioreactor vessel 410 may be the bottom or support of first bioreactor vessel 410. As previously discussed, the base plate 502 may be formed of a non-solid structure. In the illustrated embodiment, the base plate 502 includes a grid 510, which grid 510 may be structurally rigid while further providing openings to enable free gas to be exchanged through the base plate 502 to the interior compartment 506 containing the cell culture. The grid 510 may include a plurality of apertures 512 defined between solid areas or a cross-bar 514 between each aperture 512 of the grid 510. Thus, apertures 512 may provide openings for gas exchange and cross-bar 514 may provide structural support for cell cultures and other structures within interior compartment 506 of first bioreactor vessel 410.
To be a first bioreactorFurther support is provided by cell culture within the interior compartment 506 of the container 410, and the first bioreactor container 410 may include a membrane 516 that may be disposed over a top surface 518 of the base plate 502. Membrane 516 may be a gas permeable, liquid impermeable membrane. The membrane 516 may also be selected to have properties that achieve high gas permeability, high gas transmission rates, and/or high permeability to oxygen and carbon dioxide. Thus, the membrane 516 may support a high cell density (e.g., up to about 35 MM/cm) within the interior compartment 5062). The gas permeable characteristics of the membrane 516 may enable free gas exchange to support cell culture and/or cell expansion. As such, the membrane 516 may be a cell culture surface and/or a cell expansion surface. The membrane 516 may have a relatively small thickness (e.g., 0.010 inches or 0.02 cm), which may allow the membrane 516 to be gas permeable. Further, the membrane 516 may be formed of a gas permeable material, such as silicone or other gas permeable material.
The flatness of the membrane 516 may increase the surface area for settling of the cell culture for activation, transduction, and/or expansion. To enable membrane 516 to remain flat during use of first bioreactor vessel 410, a mesh sheet 520 may be disposed between base plate 502 and membrane 516. The mesh sheet 520 may provide structural support to the membrane 516 such that the membrane 516 may remain planar and may not sag or deform under the weight of the cell culture and/or any cell culture media added to the first bioreactor vessel 410 for cell culture and/or cell expansion. Furthermore, the mesh nature of mesh sheet 520 may enable support of membrane 516 while its porosity still enables free gas exchange between interior compartment 506 of first bioreactor vessel 410 and the environment immediately outside of first bioreactor vessel 410. The mesh sheet may be a polyester mesh, or any other suitable mesh material that can provide support to the membrane and allow free gas exchange.
As previously discussed, the vessel body 504 may be coupled to the floor 502 to form the interior compartment 506 of the first bioreactor vessel 410. As such, the mesh sheet 520 and the membrane 516 may be disposed within the interior compartment 506, or at least partially disposed within the interior compartment 506. O-ring 522 may be used to seal first bioreactor vessel 410 when vessel body 504 is coupled to base plate 502. In an embodiment, the O-rings 522 may be biocompatible O-rings (size 173, soft Viton @. fluoroelastomer O-rings). The O-ring 522 may fit within a groove 524 formed in a peripheral surface 526 of the container body 504. When the body 504 is mated to the plate 502, the perimeter surface 526 faces the top surface 518 of the plate 502. As such, the O-ring 522 may be compressed within the groove 524 and against the top surface 518 of the plate 516 and/or the base plate 502. Such compression of O-ring 522 desirably seals first bioreactor vessel 410 without any chemical or epoxy bonding. Because the first bioreactor vessel 410 may be used for activation, transduction, and expansion of biological cells, the O-ring 522 is desirably formed of a suitably biocompatible, autoclavable, gamma radiation stable, and/or ETO sterilization stable material.
As discussed above, first bioreactor vessel 410 may include a plurality of ports, such as first port 412 and second port 416. Ports 412, 416 may be provided through container body 504 and may enable communication between internal compartment 506 and the exterior of first bioreactor vessel 410 for certain processes related to cell culture, cell activation, cell transduction, and/or cell expansion, such as fluid or culture medium input, waste removal, collection, and sampling. Each port 416 may include an opening 526 and a corresponding fitting or tubing 528 (e.g., a luer fitting, a barb fitting, etc.). In some embodiments, the opening 526 may be configured so as to allow for direct coupling of tubing and avoid the need for fittings (e.g., counterbores).
In an embodiment, first bioreactor vessel 410 may further include an air balance port 530 disposed in a top surface 508 of vessel body 504 in addition to first port 412 and second port 416. The air balance port 530 may be configured similarly to the first port 412 and the second port 416, with like reference numbers referring to like components. Air balance port 530 may further provide for gas exchange between interior compartment 506 and the exterior of first bioreactor vessel 410 for use by cell culture for expansion. Further, the air balancing port 530 may help to maintain atmospheric pressure within the interior compartment 506 to provide an environment within the interior compartment 506 for cell culture and/or cell expansion. The air balancing port 530 may be provided through the top surface 508 of the container body 504 (as in the illustrated embodiment), or at any other location around the container body 504. As discussed in more detail below, the centered position through the top surface 508 of the container body 504 may help prevent the air balancing port 530 from becoming wet during mixing of the cell culture by tilting the first bioreactor vessel 410.
Each of the elements of the first bioreactor vessel 410, including the floor 502, the vessel body 504, the ports 412, 416, and 530, the membrane 516, the mesh sheet 520, and the O-ring 522, may be made of biocompatible, autoclavable, and gamma radiation stable and/or ETO-sterilization stable materials. As such, each element and the first bioreactor vessel 410 as an integral unit may be used for activation, transduction, and expansion of biological cells, and/or for other processes of the cell manufacturing process.
First bioreactor vessel 410 may enable cell culture and/or cell expansion via perfusion, which may provide nutrients necessary to support cell growth and may reduce impurities in the cell culture. Continuous perfusion is the addition of a fresh supply of media to a growing cell culture while the spent media (e.g., used media) is removed. As discussed below, the first port 412 and the second port 416 may be used for a perfusion process. First port 412 may enable communication between interior compartment 506 and the exterior of first bioreactor vessel 410, and may be used to add fresh media to first bioreactor vessel 410 (such as from a media reservoir of first fluidic component 440). In some embodiments, first port 412 may be disposed in container body 504 at any location above the surface of the cell culture and media within first bioreactor vessel 410 and extend through container body 504. In some embodiments, first port 412 may be positioned such that it contacts or extends through the surface of the cell culture and media within first bioreactor vessel 410.
Second port 416 may be disposed at any location completely or partially submerged below the surface of the cell culture and media within first bioreactor vessel 410. For example, the second port 416 may be an almost lateral port disposed through one of the sides 510 of the container body 504. In some embodiments, the second port 416 may be disposed such that the second port 416 does not reach the bottom of the interior compartment 506 (e.g., the membrane 516). In some embodiments, the second port 416 may reach the bottom of the interior compartment 506. The second port 416 may be a dual function port. As such, the second port may be used to pull perfusion medium out of the interior compartment 506 of the first bioreactor vessel 410 to facilitate perfusion of the cell culture. In addition, the second port 416 may also be used to remove cells of the cell culture. As noted above, in some embodiments, the second port may not reach the bottom surface of the interior compartment 506 of the first bioreactor vessel 410. For example, the second port 416 may be positioned approximately 0.5 cm away from the membrane 516. Thus, in the static planar position, the second port 416 may be used to remove used cell culture medium without pulling out cells of the cell culture, as the cells may settle to the membrane 516 (e.g., cell expansion surface) via gravity. Thus, in the static planar position, second port 416 may facilitate the perfusion process and may enable an increase in cell density of the growing cell culture within first bioreactor vessel 410. When it is desired to remove cells from the interior compartment 506 (e.g., during harvesting of the cell culture), to minimize the hold-up volume, the first bioreactor vessel 410 may be tilted toward the second port 416 in a manner described below, thereby enabling access to the cells for removal of the cells.
Additionally, in embodiments, the second port 416 may not include a filter, and thus the priming process may be filterless. As such, when the second port 416 is used to remove media, there may be no physical obstruction to the cells entering the second port 416. Further, the second port 416 may be angled such that although the second port 416 is disposed laterally through the side 22 of the container body 504, the second port 416 may be angled toward the membrane 516 and the bottom plate 502. The sloped feature of the second port 416 may enable the second port 416 to be positioned relatively low on the vessel body 504 closer to the membrane surface 36 while minimizing interference with the O-ring 522 and groove 524 to help maintain the seal of the first bioreactor vessel 410 when in use. Further, in some embodiments, the sloped feature of the second port 416 can reduce the velocity of the fluid flow through the second port 416 when the used media is removed. Additionally, the port diameter in combination with the fluid flow rate out of the second port 416 may be such that the suction rate through the second port 416 for pulling media out of the interior compartment 506 may minimize the suction force on individual cells adjacent the second port 416 such that the force is below the gravitational force that pulls the cells toward the membrane 516. Thus, as discussed above, the second port 416 may be used to remove perfusion medium to facilitate perfusion of the cell culture without substantially removing cells of the cell culture. As the cell settling time increases, the cell concentration of the removed media can decrease into an unmeasurable range facilitated by the location of second port 416. In addition, the position of the internal opening 540 can be varied to vary the recommended cell settling time. A position closer to the membrane 516 may be associated with a longer settling time, while a position at or closer to the top of the medium is associated with a shorter settling time, as the cells will settle from the top of the growth medium and be depleted first.
In embodiments, the second port 416 may thus be used not only to remove used media during the perfusion process, but also to remove cells of the cell culture from the interior compartment 506 (e.g., during harvesting of the cell culture). To facilitate greater removal of spent perfusion medium and removal of cells, the container body 504 may include angled or chevron-shaped sidewalls 532. The chevron shaped sidewall 532 thus includes an apex or point 534. The apex 534 of the sidewall 532 may further include a second port 416 therethrough, and the container body 504 is disposed proximate the apex 534 when the container body 504 is coupled to the floor 502. Angled sides 532 and tips 534 may enable a greater degree of discharge of media and/or cells of the cell culture when first bioreactor vessel 410 is tilted (e.g., at a 5 degree angle) toward second port 416.
As discussed in more detail with reference to fig. 10, the use of perfusion to grow cells facilitated by the location of the first port 412 and the second port 416 may achieve a low media height (e.g., 0.3-2.0 cm) within the interior compartment 506. The relatively low media height within interior compartment 506 may enable first bioreactor vessel 410 to be a relatively low profile vessel while enabling an increase in the maximum achievable cell density. Furthermore, the use of perfusion with first bioreactor vessel 410 may support cell growth by providing fresh media to the cells within internal compartment 506, but also enables removal of impurities in the cell culture such that once a particular cell density target is achieved within first bioreactor vessel 410, additional cell washing in a separate device may not be required. For example, by perfusion without a filter, first bioreactor vessel 410 can provide fresh media and reduce impurities within the cell culture at a rate of total volume exchange per day (e.g., resulting in a 1 log reduction of impurities at approximately every 2.3 days). Thus, the configuration of first bioreactor vessel 410 may enable the use of perfusion to grow the cell culture within first bioreactor vessel 410, which may thus enable the cell culture to expand to a high target density with reduced impurity levels. As also discussed below, by perfusion without a filter, the first bioreactor vessel 410 may provide fresh media at a rate of significantly more volume per day (e.g., greater than 2 volumes per day) for seeding, rinsing, washing/residue reduction, and/or draining/harvesting cells after expansion.
To facilitate the low profile configuration of first bioreactor vessel 410, a relatively low media level within interior compartment 506 may be maintained. Fig. 10 is a cross-sectional view of first bioreactor vessel 410, illustrating a height 536 of cell culture medium 538 within first bioreactor vessel 410. As previously discussed, the container body 504 may be coupled to the base plate 502 to form an interior compartment 506, and expansion of the cell culture may be achieved by perfusion within the interior compartment 506. As such, replacement or fresh media 538 may be provided for cell growth through the first port 412 provided through the container body 504, and existing or used media 538 may be removed through the second port 416 provided through the side 510 of the container body 504. The perfusion process may facilitate a relatively low media height 536 of the media 538 within the interior compartment 506 of the first bioreactor vessel 410. The relatively low height 536 of perfusion medium 538 within interior compartment 506 may enable first bioreactor vessel 410 to be a low-profile structure, which may thus enable a compact cell manufacturing system as a whole.
The height 536 of the perfusion medium 538 within the interior compartment 506 of the first bioreactor vessel 410 may be between 0.3cm and 2 cm, and the height of the headspace 542 (i.e., the gap formed between the medium 538 in the interior compartment 506 and the top surface 508 of the vessel body 504) may be approximately 2 cm. Therefore, per cm2Less than 2 mL of medium may be present and per cm2A total volume of less than 4 mL may be present (including culture medium, cell culture and headspace). The relatively low media height 536 may enable the ratio of media volume to surface area of the membrane 516 to be below a certain value. As such, the ratio of the volume of medium to the surface area of the membrane may be below a threshold level, or within a desirable range, which is facilitated by the use of cells that are perfused to grow the cell culture. For example, the threshold level may be a ratio between 0.3 and 2.0. The low ratio of medium volume to membrane surface area may enable first bioreactor vessel 410 to have a low profile or compact structure while still allowing for high cell density cell culture.
As previously discussed, dual function second port 416 may be provided through container body 504 such that second port 416 is fully or partially submerged below surface 544 of media 538 within first bioreactor vessel 410. In some embodiments, the second port 416 may be disposed such that the second port 416 reaches the bottom of the interior compartment 506 (e.g., the membrane 516). The positioning of the second port 416 can facilitate removal of media and impurities from the cell culture within the interior compartment 506 without removing the cells until such removal is desired, such as at the time of harvesting. The filter-less second port 416, in conjunction with the first port 412, may allow perfusion to be used to provide growth medium 538 to the cells for cell expansion and to remove used medium 538 and other impurities or byproducts. The location of the first port 412 and the dual function second port 416 about the container body 504 facilitates the following configuration: in this configuration, the height 536 of the media within the interior compartment 506 is maintained at a relatively low level and thus allows the first bioreactor vessel 410 to be a relatively low profile vessel while still allowing for the production of a high density cell culture.
With particular reference to fig. 11, the floor 502 of the bioreactor container 410 includes a variety of features that enable the use of the bioreactor container as part of a broader bioprocessing system 10, and in particular as part of the second module 200 of the bioprocessing system 10. As shown in fig. 11, the base plate 502 includes a plurality of recesses 550 formed in a bottom surface of the base plate 502, the purpose of which will be described below. In an embodiment, the recesses may be located near corners of the base plate 502. The recesses 550 may each have a generally cylindrical shape and terminate at a dome-shaped or hemispherical inner surface. As also shown in fig. 11, the base plate 502 may include a position verification structure 552 configured to interact with a sensor of the second module 200 to ensure proper positioning of the first bioreactor vessel 410 within the second module 200. In an embodiment, the position verification structure may be a beam interrupter configured to interrupt the light beam of the second module 200 when the first bioreactor vessel 410 is properly positioned in the second module 200.
The base plate 502 also includes a pair of flat engagement surfaces 554 formed on adjacent bottom surfaces that are offset from a centerline of the base plate (extending across the width of the base plate). Desirably, the engagement surfaces 554 are spaced apart along a longitudinal centerline of the base plate 502 so as to be positioned adjacent opposite ends of the base plate 502. The floor 502 may further include at least one aperture or opening 556 to allow sensing of the contents of the first bioreactor vessel 410 by engaging and operating the bioprocessing equipment of the bioreactor vessel.
In an embodiment, the first and second bioreactor vessels 410, 420 and fluidic architecture 400 may be integrated into an assembly or kit 600 in the manner disclosed below. In an embodiment, the kit 600 is a single use disposable kit. As best shown in fig. 12-14, the first and second bioprocessing containers 410 and 420 are received side-by-side within the tray 610 of the disposable kit 600, and the various tubes of the flow architecture 400 are arranged within the tray 610 in a manner described below.
With additional reference to fig. 15, the tray 610 includes a plurality of generally thin, rigid or semi-rigid side walls including a front wall 612, a rear wall 614, and opposing lateral sides 616, 618 that peripherally define a bottom surface 620 and a generally open top. The sidewalls and bottom surface 620 define an interior compartment 622 of the tray 610. In an embodiment, the open top of the tray 610 is defined by a peripheral flange 624, the peripheral flange 624 presenting the following surfaces: this surface is for receiving a removable cover (not shown) enclosing the interior compartment 622, and for desirably seating on the upper edge of a drawer of the bioprocessing equipment (as indicated below). The bottom surface 620 of the tray 610 includes 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 the first and second openings 626, 628 for purposes described below. In embodiments, the tray 610 may be thermoformed, 3D printed, or injection molded, although other manufacturing techniques and processes may also be utilized without departing from the broader aspects of the invention.
As best shown in fig. 15, each of first opening 626 and second opening 628 has a perimeter shaped and/or sized such that first bioreactor container 410 and second bioreactor container 420 may be positioned over respective openings 626, 628 and supported by bottom surface 620 of tray 610 within interior compartment 622 while still allowing portions of bioreactor containers 610, 620 to be accessible from the bottom of tray 610 through respective openings 626, 628. In an embodiment, the perimeter of the opening comprises at least one protrusion or projection for supporting the bioreactor container above the respective opening. For example, the perimeter of each opening 626, 628 may include a protrusion 632 protruding inward toward the center of the opening 626, 628 for supporting the bioreactor container 410, 420 placed thereon. As shown in fig. 12 and 15, tray 610 may also include one or more bosses extending upwardly above openings 626, 628 for inhibiting lateral movement of the bioreactor containers when they are received over the respective openings 626, 628. Thus, the bosses serve as alignment devices that facilitate proper positioning of the bioreactor containers 410, 420 within the tray 610 and help prevent accidental movement of the bioreactor containers 410, 420 during loading or positioning of the kit 600 in the second module 200 (as discussed below).
With further reference to fig. 12 and 13, the tray 610 may include one or more support ribs 636 formed on a bottom surface of the tray 610. The support ribs 636 may extend across the width and/or length of the tray 610 and impart rigidity and strength to the tray 610, thereby facilitating movement and manipulation of the kit 600. The ribs 636 may be integrally formed with the tray or may be added as an auxiliary member via attachment means known in the art (see fig. 13). In an embodiment, the tray 610 includes openings 638 for receiving engagement plates, also referred to herein as tubing modules 650, therethrough that hold fluid flow lines in an organized manner and hold them in place for engagement by pumps and pinch valves. In other embodiments, the duct module 650 may be integrally formed with the rear wall 614 of the tray 610.
Fig. 16 and 17 illustrate configurations of the pipe module 650 according to an embodiment of the present invention. As shown therein, the tubing module 650 includes a first tubing holder block 652 configured to receive the first fluid assembly line 442, the interconnect line 450, and the waste line 490 of the fluid flow system 400 and hold the first fluid assembly line 442, the interconnect line 450, and the permeate waste line 490 in place for selective engagement with respective pump heads 454, 456, 492 of the peristaltic pump assembly described below in connection with fig. 35 and 36. In an embodiment, the fluid assembly line 442, the interconnect line 450, and the waste line 490 are maintained in a horizontally extending and vertically spaced orientation by the first conduit retainer block 652. In particular, as best shown in fig. 17, first conduit retainer block 652 engages each of lines 442, 450, 490 at two spaced-apart locations 656, 658 (such as by a simple interference fit between a clip or tube and a slot in conduit retainer block 652), locations 656, 658 defining a void therebetween. As also shown in fig. 17, the first tubing holder block 652 includes a clearance opening 660, the clearance opening 660 configured to receive a shoe (not shown) of the peristaltic pump assembly. This configuration allows peristaltic compression of the lines 442, 450, 490 against the boot by respective pump heads of the peristaltic pump(s) to provide respective motive forces of fluid through the lines (as discussed below).
With further reference to fig. 16-18, conduit module 650 further includes a second conduit retainer block 654 integrally formed with (or otherwise coupled to) first conduit retainer block 652. Second tube holder block 654 is configured to receive all of the fluid flow lines of fluid flow system 400 associated with the pinch valve. For example, second tube holder block 654 is configured to hold tube tails 464a-f of first fluid assembly 440, tube tails 470a-d of second fluid assembly 444, first and second bioreactor lines 414, 418 of first bioreactor vessel 410, first and second bioreactor lines 424, 428 of second bioreactor vessel 420, sterile air source line 460, interconnection line 450, and filtration line 482 (and, in some embodiments, sampling line 476 a)-476d) In that respect Similar to first conduit retainer block 652, second conduit retainer block 654 may maintain the tubes in a horizontally extending and vertically spaced orientation. In particular, second tube holder block 654 may include a plurality or vertically spaced and horizontally extending slots 666, the slots 666 configured to receive a line therein. Fig. 18 and 19 also best illustrate the configuration of slots 666, slots 666 holding all flow lines acted upon/interfaced with the pinch valve. Desirably, the slot 666 follows the contour of the block 654, but particularly extends across the planar backing plate so as to open toward the filter 484. As shown in fig. 18, in an embodiment, the second tube holder block 654 may have one or more narrow tube slots 682 at the bottom of the second tube holder block 654, and a waste tube slot 684, the narrow tube slot 682 for holding the loop of interconnect tubing 450, the sample tube slot 684From which a line extends, a waste line conduit slot 684 is provided for receiving conduit tail 470a which connects to waste reservoir 472 a.
The second tube holder block 654 may include a planar backing plate 662 having a plurality of apertures 664, the apertures 664 corresponding to the plurality of fluid flow lines held by the second tube holder block 654. In particular, at least one aperture 664 is horizontally aligned with each slot 666 and the flow line retained therein. As best shown in fig. 16, second tube holder block 654 includes two clearance openings 668, 670 configured to receive an anvil (not shown) of a pinch valve assembly therethrough. This configuration allows for selective compression of tubing tails 464a-f of first fluid assembly 440, tubing tails 470a-d of second fluid assembly 444, first and second bioreactor lines 414 and 418 of first bioreactor vessel 410, first and second bioreactor lines 424 and 428 of second bioreactor vessel 420, sterile air source line 460, interconnect line 450, and filter line 482 against the anvil by respective pistons of actuators of the pinch valve array to selectively prevent or allow fluid flow (as discussed below). As shown in fig. 18 and 19, the apertures 664 may be arranged in first and second columns positioned side-by-side, with the apertures in the first column of apertures being vertically offset relative to the apertures on the second column of apertures such that the apertures in the first column of apertures are not horizontally aligned with the apertures in the second column of apertures. This configuration allows the conduit module 650, tray 610 and kit 600 as a whole to have a low profile.
In an embodiment, the filter 484 (shown in fig. 16 as an elongated hollow fiber filter module) may be integrated with the duct module 650, such as by mounting the filter 484 to the duct module 650 using the retaining clip 672. Where filter 484 is a hollow fiber filter, filter 484 may extend substantially the entire length of piping module 650 and may include a first input 674 for receiving a fluid input stream from filtration line 482 and a second output 676 for conveying retentate back to filtration line 482 and interconnection line 450 for recycling to one of first bioreactor vessel 410 or second bioreactor vessel 420 after removal of permeate/waste. The filter 484 can also include a permeate port 678 adjacent the second output 676 for connection to a waste line 490 for conveying waste/permeate to the permeate/waste reservoir 472 a. Finally, piping module 650 can include a plurality of features 680 for receiving and organizing the bioreactor lines (e.g., first bioreactor line 414 and second bioreactor line 418 of first bioreactor vessel 410 and/or first bioreactor line 424 and second bioreactor line 428 of second bioreactor vessel 420).
Similar to the tray 610, the duct module 650 may be thermoformed, 3D printed, or injection molded, although other manufacturing techniques and processes may also be utilized without departing from the broader aspects of the invention. As discussed above, in embodiments, the conduit module 650 may be integrally formed with the tray 610. In other embodiments, the conduit module 650 may be a separate component removably received by the tray 610.
Fig. 20-22 show various views of an embodiment of a kit 600 illustrating the first and second bioreactor vessels 410, 420 received within the tray 610 and the fluid lines of the flow architecture 400 received by the piping module 650. As shown therein, the kit 600 as shown in fig. 20-22 does not have an opening 630, but instead includes a solid floor therein to provide a sampling space 631 in the tray 610 for receiving a reservoir holding sampling lines (e.g., sampling lines 476a, 476 b). The kit 600 provides a modular platform for cell processing that can be easily assembled and discarded after use. The tubing tails of the first and second fluidic assemblies 440, 444 allow for plug and play functionality, enabling quick and easy connection of multiple media, reagent, waste, sampling and collection bags, allowing multiple processes to be performed on a single platform. In embodiments, the connection and disconnection may be accomplished by aseptic cutting and welding of the tube segments as discussed above, such as with a TERUMO device, or by clamping, welding, and cutting the tail segment as is known in the art.
Turning now to fig. 23-25, the kit 600 is specifically configured to be received by a bioprocessing apparatus 700, the bioprocessing apparatus 700 containing all hardware (i.e., controllers, pumps, pinch valve actuators, etc.) required to actuate the kit 600 as part of a bioprocessing process. In an embodiment, the bioprocessing apparatus 700 and the kit 600 (including the flow architecture 400 and the bioreactor vessels 410, 420) together form the second bioprocessing module 200 described above in connection with fig. 1 and 2. The bioprocessing device 700 includes a housing 710 with a plurality of drawers 712, 714, 716 receivable within the housing 710. Although fig. 23 depicts an apparatus 700 containing three drawers, the apparatus may have as few as a single drawer, two drawers, or more than three drawers to provide biological processing operations to be performed simultaneously within each drawer. In particular, in an embodiment, each drawer 712, 714, 716 may be a separate bioprocessing module for performing the processes of cell activation, genetic modification, and/or expansion (i.e., equivalent to the second modules 200a, 200b, and 200c described above in connection with fig. 2). In this regard, any number of drawers may be added to the apparatus 700 to provide parallel processing of multiple samples from the same or different patients. In embodiments, rather than each drawer sharing a common housing, in embodiments each drawer may be received within a dedicated housing, and the housings may be stacked on top of each other.
As shown in fig. 23 and 24, each drawer (e.g., drawer 712) includes a plurality of sidewalls 718 and a bottom surface 720 defining a processing chamber 722, and a generally open top. Drawer 712 is movable between a closed position, in which the drawer is fully received within housing 710, as shown for drawers 714 and 716 in fig. 23, and an open position, in which drawer 712 extends from housing 710, as shown for drawer 712 in fig. 23 and 24, enabling access to processing chamber 722 through the open top. In an embodiment, one or more of the sidewalls 718 are temperature controlled for controlling the temperature within the process chamber 722. For example, one or more of the sidewalls 718 may include embedded heating elements (not shown), or be in thermal communication with heating elements, such that the sidewalls 718 and/or the processing chamber 722 may be heated to a desired temperature for maintaining the processing chamber 722 at a desired temperature (e.g., 37 degrees celsius) as 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 process chamber when the drawer is closed) can be temperature controlled in a similar manner (e.g., embedded heating elements). As discussed in detail below, the hardware compartment 724 of the drawer 712 behind the process chamber 722 may house all of the hardware components of the apparatus 700. In an embodiment, the drawer 712 may further include an auxiliary compartment 730 adjacent the process chamber 722 for containing reservoirs containing media, reagents, etc., connected to the first and second fluid assemblies 440, 444. In an embodiment, the auxiliary compartment 730 may be refrigerated.
Each drawer (e.g., drawer 712) may be slidably received on opposing rails 726 mounted to the interior of the housing 710. A linear actuator may be operatively connected to the drawer 712 to selectively move the drawer 712 between the open and closed positions. The linear actuator is operable to provide smooth and controlled movement of the drawer 712 between the open and closed positions. In particular, the linear actuator is configured to open and close the drawer 712 at a substantially constant speed (and minimal acceleration and deceleration at the stop and start of movement) to minimize interference with the contents of the bioreactor container(s).
Fig. 25 is a top plan view of the interior of the drawer, showing the processing chamber 722, the hardware compartment 724, and the auxiliary compartment 730 of the drawer 712. As illustrated therein, the hardware compartment 724 is located behind the processing chamber 722, and includes a power source 732, motion control board and drive electronics 734 that are integrated with or otherwise in communication with the second module controller 210, the low power solenoid array 736, the pump assembly 738 (which includes pump heads for the pumps 454, 456, 492), and the drawer engagement actuator 740. The hardware compartment 724 of the drawer 712 further includes a pump shoe 742 and a pair of pinch valve anvils 744 for interfacing with the pump assembly 738 and the solenoid array 736, respectively, as described below. In an embodiment, the pump shoe 742 and the solenoid anvil 744 are secured to a front substrate (front plate) of the process chamber. The hardware compartments (and components described) are all mounted to the rear substrate. Both plates can be slidably mounted to the guide rail. In addition, a drawer engagement actuator 740 couples the two plates and is used to bring the two plates (and the components carried thereon) to an engaged position (thereby bringing the pump roller head into the pump shoe and thus squeezing the pump tubing (if interposed therebetween)). As further described herein, the pump assembly provides selective operation on the lines 442, 450, and 490 of the fluid path 400, providing independent respective peristaltic motive forces thereto. Similarly, as will be further described, the tube holder block 654 of the tray 600 will be positioned between the solenoid array 736 and the anvil 744.
As also illustrated in fig. 25, two seatplates (e.g., a first seatplate 746 and a second seatplate 748) are located within the process chamber 722 on the bottom surface 720 and extend or stand upwardly from the bottom surface 720. In embodiments, the process chamber 722 may house a single pedestal, or more than two pedestals. The seat plates 746, 748 are configured to receive or otherwise engage the first bioreactor container 410 and the second bioreactor container 420 thereon. As also shown in fig. 25, the drawer 712 also includes a plate 750, the plate 750 configured with load cells positioned adjacent the seat plates 746, 748 within the processing chamber 722 for sensing the weight of the reservoir (e.g., the waste reservoir 472a positioned thereon).
Fig. 26-28 best illustrate the configuration of the seat pans 746, 748, with fig. 28A showing the hardware components positioned beneath the seat pans. As used herein, the seat pads 746, 748 and hardware components (i.e., sensors, motors, actuators, etc. integrated with the seat pads or positioned under the seat pads as shown in fig. 28A) may be collectively referred to as seat pads. The first and second seatpads 746, 748 are substantially identical in configuration and operation, but for simplicity, the following description of the seatpads 746, 748 refers only to the first seatpad 746. The seat plates 746, 748 have a substantially planar top surface 752, the shape and surface area of the top surface 752 generally corresponding to the shape and area of the bottom plate 502 of the first bioreactor vessel 410. For example, the seat pan may be substantially rectangular in shape. The saddles 746, 748 can also include a relief or clearance area 758 that generally corresponds to the location of the protrusion or tab 632 of the tray 610, the purpose of which will be described below. The bed plates 746, 748 are supported by a plurality of load cells 760 (e.g., four load cells 760 positioned under each corner of the bed plate 746). The load cell 760 is configured to sense the weight of the first bioreactor container 410 during bioprocessing for use by the controller 210.
In embodiments, the base plate 746 can include an embedded heating element or be in thermal communication with a heating element such that the contents of the processing chamber 722 and/or the first bioreactor vessel 410 placed thereon can be maintained at a desired temperature. In embodiments, the heating elements may be the same or different than the heating elements that heat the side walls 718, top wall, and bottom surface.
As illustrated, the shoe 746 includes a plurality of dowel or alignment pins 754 protruding above the top surface 452 of the shoe 746. The number of dowel pins 754 and the location and spacing of dowel pins 754 may correspond to the number, location and spacing of recesses 550 in the bottom surface of the bottom plate 502 of the bioreactor vessel 410, 420. As indicated below, when the first bioreactor container 410 is positioned within the process chamber 722, the locating pins 754 can be received within the recesses 550 in the bottom plate 502 of the first bioreactor container 410 to ensure proper alignment of the first bioreactor container 410 on the first seat plate 746.
With further reference to fig. 26-28, the seat plate 746 may further include an integrated sensor 756 for detecting proper alignment (or misalignment) of the first bioreactor vessel 410 on the first seat plate 746. In an embodiment, the sensor 756 is an infrared light beam, however other sensor types, such as lever switches, may be utilized without departing from the broader aspects of the invention. The sensor is configured to interact with the position verification structure 552 on the base plate 502 when the first bioreactor vessel 410 is properly seated on the first seat plate 746. For example, where sensor 756 is an infrared light beam and position verification structure 552 is a beam interrupter (i.e., a flat protrusion), for a substantially infrared-opaque position verification structure 552, the beam interrupter will interrupt the infrared light beam (i.e., interrupt the beam) when first bioreactor vessel 410 is fully seated on seat plate 746. This will signal controller 210 that first bioreactor vessel 410 is properly positioned. If the controller does not detect that the infrared beam of sensor 756 is interrupted after positioning first bioreactor vessel 410 on first seat plate 746, this indicates that first bioreactor vessel 410 is not fully or properly seated on seat plate 746 and requires adjustment. Thus, the sensors 756 on the bed plate 746 and the position verification structure 552 on the bottom plate 502 of the first bioreactor container 410 ensure that the first bioreactor container 410 is positioned at an equal height (as determined by the alignment pins) on the bed plate 746 prior to beginning bioprocessing.
Still further referring to fig. 26-28A, the seat plate 746 additionally includes an embedded temperature sensor 759 positioned so as to align with the aperture 556 in the bottom plate 502 of the first bioreactor vessel 410. Temperature sensors 759 are configured to measure or sense one or more parameters within bioreactor vessel 410, such as, for example, a temperature level within bioreactor vessel 410. In an embodiment, the seat plate 746 may additionally include: a resistance temperature detector 760 configured to measure a temperature of the top surface 752; and a carbon dioxide sensor (located below the seat plate) for measuring the level of carbon dioxide within the bioreactor vessel.
As further shown in fig. 26-28A, each seat pan 746, 748 includes an actuator mechanism 761 (e.g., a motor) including, for example, a pair of opposing cam arms 762. The cam arms 762 are received within slots 764 in the seat plates 746, 748 and are rotatable about cam pins 766 between a clearance position, where the cam arms 762 are positioned below a top surface 752 of the seat plate 746, and an engaged position, where the cam arms 762 extend above the top surface 752 of the seat plate and contact the opposing flat engagement surface 554 of the bottom plate 502 of the first bioreactor container 410 when the first bioreactor container 410 is received on top of the first seat plate 746. As discussed in detail below, the actuator mechanism is operable to tilt the bioreactor container on top of the seat deck to provide agitation and/or to assist in the draining of the bioreactor container.
Referring to fig. 29-32, more detailed views of the drawer engagement actuator 740 and the linear actuator 768 in the hardware compartment 724 of the drawer 712 are shown. Referring to fig. 29, and as indicated above, the linear actuator 768 is operable to move the drawer 712 between an open position and a closed position. In an embodiment, the linear actuator 768 is electrically connected to a rocker switch 770 on the exterior of the housing 710 that allows a user to control the movement of the drawer. The linear actuator 770 provides controlled movement of the drawer 712 to prevent interference with the contents of the bioreactor container(s) within the drawer 712. In an embodiment, the linear actuator 768 has a stroke of approximately 16 ″ and has a maximum speed of approximately 2 inches per second.
Turning now to fig. 30, the drawer engagement actuator 740 includes a lead screw 772 and a hook arm 774 attached to a front plate 751 within the drawer 712. The drawer engagement actuator is operatively connected to the pump assembly 738 and the solenoid array 736 and is operable to move the pump assembly 738 and the solenoid array 736 between the first clearance position and the engaged position.
Fig. 31 and 32 better illustrate the gap and engaged positions of the pump assembly 738 and the solenoid array 736. As illustrated in fig. 31, in the gap position, the pump assembly 738 and solenoid array 736 are spaced from the pump shoe 742 and pinch valve anvil 744, respectively. Upon actuation of the lead screw 772, the drawer engagement mechanism 740 linearly moves the pump assembly 738 and solenoid array forward to the position shown in fig. 32. In this position, the pump head of pump assembly 738 engages lines 442, 450, 490 in first tubing holder block 652, and solenoid array 736 is positioned sufficiently close to pinch valve anvil 744 that the pistons/actuators of solenoid array 736 can pinch/clamp their respective fluid flow lines of second tubing holder block 654 against pinch valve anvil(s) 744, thus preventing flow through the fluid flow lines.
Referring back to fig. 24, and with additional reference to fig. 33-39, in operation, the drawer 712 may be controllably moved to the open position by actuating a rocker switch 770 on the exterior of the housing 710. The disposable plug-in kit 600 containing the tubing module 650 (which holds all the tubes and tubing tails of the flow architecture 400) and the first and second bioreactor vessels 410, 420 is then lowered to a position within the process chamber 722. When the kit 600 is lowered into the process chamber 722, the pump shoe 742 is received through the clearance opening 660 of the first tubing holder block 652 such that the pump tubes 442, 450, 490 are positioned between the pump heads 454, 456, 492 of the peristaltic pump assembly 738 and the pump shoe 742. Fig. 35 is a perspective view of peristaltic pump assembly 738 showing the positioning of pump heads 454, 456, 492 relative to one another. Fig. 36 illustrates the positioning of pump heads 454, 456, 492 relative to pump tubes 442, 450, 490 when the kit 600 is received within a process chamber 722. As shown therein, pump tubes 442, 450, 490 are positioned between a pump shoe 742 and pump heads 454, 456, 492. In operation, when the drawer engagement actuator 740 positions the pump assembly 738 in the engaged position, the pump heads 454, 456, 492 are selectively actuatable under the control of the controller 210 to start, maintain, and stop fluid flow through the tubes 442, 450, 490.
Similarly, when kit 600 is lowered into process chamber 722, pinch valve anvil 744 is received through clearance openings 668, 670 of second tube holder block 654 such that tube tails 464a-f of first fluid assembly 440, tube tails 470a-d of second fluid assembly 444, first and second bioreactor lines 414, 418 of first bioreactor vessel 410, first and second bioreactor lines 424, 428 of second bioreactor vessel 420, sterile air source line 460, interconnection line 450, and filter line 482, held by second tube holder block 654, are positioned between solenoid array 736 and pinch valve anvil 744. This configuration is best illustrated in fig. 37-39 (fig. 37 and 38 illustrate the relationship between solenoid array 736 and pinch valve anvil 744 prior to receiving back plate 662 of second tube holder block 654 within space 776).
As shown therein, each solenoid 778 of the solenoid array 736 includes a piston 780, the piston 780 being linearly extendable through an associated aperture (aperture 664) in the back plate 662 of the second tube holder block 654 to clamp an associated tube against the pinch valve anvil 744. In this regard, the solenoid array 736 and the anvil 744 together form a pinch valve array (which includes the valves of the first and second fluid assemblies 440, 444, as well as the bioreactor line valves, i.e., valves 432, 434, 436, 438, sterile line valve 462, interconnecting line valve 452, and filtrate line valves 486, 488). In particular, the pinch valves of the flow architecture 400 are provided by respective solenoids 778 (i.e., pistons of the solenoids) of the solenoid array 736, the solenoid array 736 operating/acting against its respective anvil 744 with the fluid path/line in between. In particular, in operation, when the drawer engagement actuator 740 positions the solenoid array 736 in the engaged position, each solenoid 778 can be selectively actuated under the control of the controller 210 to clamp the associated fluid flow line against the anvil 744 to prevent fluid flow therethrough. The present invention contemplates that each fluid line is positioned between a planar anvil surface and a planar solenoid actuator head. Alternatively, the solenoid actuator head may comprise a shaped head, such as two tapered surfaces (similar to a Phillips head screwdriver) meeting at an elongated edge, optimized to provide the desired clamping force on the resilient flexible fluid line. Still alternatively, the anvil surface may include an elongated ridge or protrusion that extends toward each fluid line such that the planar solenoid head may compress the fluid line against the laterally extending ridge in order to close the line to prevent fluid flow therethrough.
Referring to fig. 33, 34 and 40, when the kit 600 is lowered into the processing chamber of the drawer, the first and second bioreactor containers 410, 420 are supported over the openings 626, 628 by the perimeter of the openings (and in particular by the projections/protrusions 632). When the kit is further lowered, the seat plates 746, 748 extend through the openings 626, 628 and receive or otherwise engage the bioreactor containers 410, 420. Once the bioreactor containers 410, 420 are received by the seatpads 746, 748, the shape of the openings 626, 628 and the top surface 752 of the seatpads 746, 748 (e.g., the cushioned regions 758 of the seatpads 746, 748, which correspond to the protrusions/projections 632 of the tray 610) allow the tray 610 to continue to travel downward such that the bottom surface of the tray 610 and the protrusions/projections 632 are positioned below the top surface 752 of the seatpads 746, 748 such that the bioreactor containers 410, 420 may be supported by the seatpads 746, 748 in spaced relation to the bottom surface 620 of the tray 610. This ensures that the tray 610 does not interfere with the equal height placement of the bioreactor vessels 410, 420 on the trays 746, 748.
When the seat plates 746, 748 extend through the openings 726, 728 in the tray 610, the locating pins 754 on the seat plates 746, 748 are received in the corresponding recesses 550 in the bottom plate 502 of the bioreactor container 410, 420, ensuring that the bioreactor container 410, 420 will be properly aligned with the seat plates 410, 420. When properly seated on the seat plates 746, 748, the beam interrupter 552 interrupts the light beam from the sensor 756 in the seat plate, indicating to the controller that the bioreactor container 410, 420 is in the proper position. Because the bed plates 746, 748 and alignment pin heights are equal, interruption of the light beam by the beam interrupter 552 to the sensor 756 likewise ensures that the bioreactor containers 410, 420 are equal in height. In this properly seated position, sensors 759 on the seat plates 746, 748 are aligned with apertures 556 in the floor 502 to allow sensing of process parameters within the interior compartments of the bioreactor containers 410, 420, respectively. In addition, in the fully seated position, the cam arms 762 of the seat plates 746, 748 are aligned with the flat engagement surfaces 554 on the floor 502 of the bioreactor containers 410, 420, respectively.
Fig. 40 is a cross-sectional front view illustrating this fully seated position of first bioreactor vessel 410 on seat 746. As shown in fig. 40, heating elements in the form of a heating mat 782 and a heating module 784 may be positioned below the seat plate 746 for heating the seat plate 746. As shown in fig. 40, a carbon dioxide sensing module 786 may also be positioned below the pedestal plate for sensing the carbon dioxide content within the processing chamber 722.
As further shown in fig. 40, in an embodiment, the side walls 718 and bottom of the drawer 712 (and the top wall of the housing) may include a cover 788, a layer of insulating foam 790 to help minimize heat loss from the process chamber 722, a thin film heater 792 for heating the walls as described above, and an inner metal plate 794. In an embodiment, the inner metal plate 794 may be formed of aluminum, although other thermally conductive materials may also be utilized without departing from the broader aspects of the present invention. Drawer 712 may further include one or more brush seals 796 to help minimize heat loss from processing chamber 722 and a thermal break 798 to minimize or prevent the flow of thermal energy from drawer 712 to other components of apparatus 700, such as enclosure 710 or other drawers (e.g., drawers 714, 716).
Referring again to fig. 34, when the kit 600 is received in the process chamber 722, the load cell 750 adjacent the second bed plate 748 in the bottom of the process chamber 722 extends through the opening 730 in the tray 610 so that a waste bag 472a can be connected to the plumbing tail 470a and positioned over the load cell 750. As shown therein, when the kit 600 is received within the drawer 712, the second tube holder block 654 holds the tubes such that the tube tails 464a-f of the first fluid assembly 440 and the tube tails 470b-d of the second fluid assembly 444 extend into the auxiliary compartment 730 for connecting the reservoir to the auxiliary compartment 730. In an embodiment, sampling lines 476a-476d also extend into the auxiliary compartment 730.
Turning now to fig. 41-44, the operation of the cam arms 762 of the seat plates 746, 748 is illustrated. As shown therein, the cam arms 762 are movable between a retracted position, in which the cam arms 762 are positioned below the top surface of the seat plates 746, 748, and an engaged position, in which the cam arms 762 rotate about the cam pins 766 and extend above the seat plates 746, 748 to engage the flat engagement surfaces 554 of the bioreactor containers 410, 420 to lift the bioreactor containers 410, 420 off of the seat plates 746, 748. Because in the default state the cam arms 762 are retracted below the top surfaces of the saddles 746, 748 and the bioreactor containers 410, 420 are supported on the saddles 746, 748 of equal height (and in particular the alignment pins 754 of equal height), no power is required to maintain the bioreactor containers in an equal height position. In particular, when the bioreactor vessels 410, 420 are received on the seat plates 746, 748, they are in a level position. In the event of a power outage, the bioreactor vessels 410, 420 remain seated on the contoured seat plates 746, 748 and do not require any continuous adjustment using cam arms 762 to maintain the contoured position. This is in contrast to some systems, which may require the use of servo motors to continuously adjust the bioreactor to maintain a high elevation. Indeed, with the configuration of the cam arm 762 of the present invention, as discussed below, the actuator need only be energized when tilting the bioreactor container for stirring/mixing, which minimizes the thermal contribution to the process chamber 722.
As shown in fig. 41-43, cam arm 762 may be sequentially operable to stir the contents of bioreactor vessels 410, 420. For example, when it is desired to stir the contents of bioreactor container 410, one of the cam arms will be actuated to lift one end of bioreactor container 410 off of seat plate 746 (and out of engagement with dowel pin 754 on seat plate 746), while the opposite end remains seated on the seat plate and dowel pin 754 on the non-raised end remains received in corresponding recess 550 in bottom plate 502. The raised cam arm will then be rotated back to the clearance position under the seat plate and the opposing cam arm will be rotated to the engaged position to raise the opposing end of the bioreactor container off of the seat plate and alignment pins.
In an embodiment, the cam actuation system may be designed such that the cam arm 762 may be parked without contacting the bioreactor container, thereby preventing damage to the culture and allowing the cam arm 762 to be parked (or tested) at any point during a long cell processing cycle. Thus, while the present invention contemplates that other rocking or stirring devices may be provided for the bioreactor vessel, by having two cam arms 762 on opposite sides of the seat plate, the overall height of the mixing mechanism may be minimized. For example, a central actuator (located in the center of the seat plate) may be used to achieve +/-5 degrees of movement, but 0-5 degrees of movement of the container driven by cam arms on both sides of the container may be used to achieve nearly the same movement of the container, effectively imparting +/-5 degrees of movement to the container at half height. In addition, the movement of the cam arm 762 (e.g., the speed at which the cam arm rotates and the timing between opposing cam arms) may be adjusted to maximize wave formation in the vessel, thereby maximizing wave amplitude, and thus (ideally) the uniformity of the vessel contents and the time to achieve uniformity. The timing may also be adjusted based on the volume in the vessel with a given geometry to maximize mixing efficiency.
In an embodiment, optical sensor 756 may be used to confirm that first bioreactor vessel 410 has been properly repositioned after each cam agitation movement. It is further contemplated that the proper repositioning of the bioreactor container may be checked and verified even between alternating cam movements. This enables rapid detection of misalignment in substantially real time, allowing operator intervention to relocate the bioreactor vessel without significant deviation from the bioprocessing operation/protocol.
Fig. 43 is a schematic illustration showing the location of fluid 800 within a bioreactor vessel during this agitation process. As shown in fig. 42, in an embodiment, a home sensor 802 integrated with the seat plate 746 may be utilized by the controller to determine when the cam arm 762 has returned to a clearance position below the top surface of the seat plate 746. This is useful in coordinating the movement of the cam arm 762 to provide a desired mixing frequency in the bioreactor vessel. In an embodiment, the cam arms 762 are configured to provide a maximum 5 degree angle of inclination with respect to the seat plate 746.
Referring to fig. 44, the interface between dowel pin 754 of the seat plate and recess 550 in bottom plate 502 of bioreactor vessel 410 during mixing/agitation is illustrated. In an embodiment, recess 550 has a dome-shaped or hemispherical inner surface, and diameter d1 is greater than diameter d2 of locating pin 754. As illustrated in fig. 44, this configuration provides a gap between the locating pin 754 and the recess 550, which allows the bioreactor container 410 to tilt when the locating pin 554 is received in the recess 550.
In an embodiment, as shown in fig. 45-50, each drawer (e.g., drawer 712) of the bioprocessing equipment 700 desirably includes a flip-down front panel 810 hingedly mounted thereto. As best shown in fig. 45, 49 and 50, the flip down front panel 810 allows access to the auxiliary compartment 730 without having to open the drawer 712. As will be appreciated, this configuration allows for sampling and replacement of media bags during the process. In conjunction with the above, in an embodiment, the auxiliary compartment 730 may be configured with a plurality of telescoping slide rails 812, the telescoping slide rails 812 providing an attachment device 815 from which a variety of reservoir/media bags may be suspended. The track 812 is movable between a retracted position within the compartment 730 as depicted in fig. 48 and an extended position out of the compartment 730 as depicted in fig. 49. When the collection bag is full or the media/fluid bag needs to be replaced, the tracks 812 can simply be extended outward and the bag released. A new bag may be attached to its respective tail and then hung from the rail and slid back into the auxiliary compartment 730 without having to open the drawer 712 or pause the process. In an embodiment, the track 812 may be mounted on a laterally extending crossbar 814. Thus, the track 812 may be able to slide laterally on the rod 814 and be able to extend from and retract into the auxiliary compartment. Additionally, when the drawer is open (fig. 46), the track 812 may rotate about the rear cross bar such that it passes over the compartment 730 to allow a user to thread the duct tail toward the front of the compartment 730, providing a third degree of freedom.
As illustrated in fig. 51, in another embodiment, the media/fluid bag may be mounted on a platform 820, the platform 820 being able to rotate away from the auxiliary compartment 730 from a stowed position to an entry position. For example, the platform 820 may be mounted for movement along a rail 822 formed in a side wall of the auxiliary compartment 730.
Referring to fig. 52, in an embodiment, the biological treatment apparatus 700 can further include a low profile waste tray 816 received within the housing 710 beneath each drawer (e.g., drawer 712). The waste tray 816 is independently mounted on its drawer to be movable between a closed position and an open position. In the closed position, the tray 816 desirably extends flush with the front surface of the drawer, while in the open position, the tray 816 exposes its own compartment 819 to enable access by an operator. The chamber 819 provides easy storage of a large waste bag connected to the fluid path of the tray 600 thereon and enables access to the waste bag without having to open the drawer 712. In addition, in the closed position, the waste tray 816 positions the chamber 819 in underlying alignment with its drawer and is sized and shaped so as to be operable to contain any leaks from the processing chamber 722 or the auxiliary compartment 730.
In an embodiment, each drawer may include a camera positioned above the process chamber (e.g., above each bioreactor container 410, 420) to allow visual monitoring of the interior of the drawer 712 without having to open the drawer 712. In embodiments, the camera (or additional cameras) may be integrated with the seat plate assembly, or on the side wall looking laterally into the bioreactor container(s).
Thus, the second module 200 of the present invention provides automation of cell processing to a degree heretofore unseen in the art. In particular, fluid flow architecture 400, pump assembly 738, and pinch valve array 736 allow for automated fluid manipulation (e.g., fluid addition, transfer, draining, flushing, etc.) between bioreactor vessels 410, 420 and bags connected to first fluid assembly 740 and second fluid assembly 744. This configuration also allows for hollow fiber filter concentration and washing, no filter priming, and line priming, as discussed below. The use of the drawer engagement actuator 740 also provides for automatic engagement and disengagement of the plug-in kit 600, thereby further minimizing human contact points. Indeed, only human contact points may be required to add and remove source/media bags, sample and enter data (e.g., sample volume, cell density, etc.).
Referring to fig. 53-77, an automated general protocol for immobilized Ab coating, soluble Ab addition, gamma-retroviral vector amplification in the same container using the second module 200 and its fluid flow architecture 400 is illustrated. The general protocol provides for activation of cell populations (illustrated in fig. 53-59), pre-transduction preparation and transduction (illustrated in fig. 60-71), expansion (fig. 72-76), and for some embodiments harvesting (fig. 77) in an automated and functionally closed manner. When the operation of the pinch valve is described below, the valve is in its closed state/position when the valve is not used for a particular operation. Thus, after the valve is opened to allow a particular operation to be performed, and once the operation is complete, the valve is closed before the next operation/step is performed.
As shown in fig. 53, in a first step, valves 432 and 468f are opened and the first fluidic assembly line pump 454 is actuated to pump the antibody (Ab) coating solution from the reservoir 466f connected to the first fluidic assembly 440 to the first bioreactor vessel 410 through the first port 412 of the first bioreactor vessel 410. The antibody coating solution is incubated for a period of time and then drained through the interconnecting line to the waste reservoir 472a of the first fluidic component 440 by opening the valves 434, 474a and activating the circulating line pump 456. As described herein, the discharge of bioreactor container 410 may be facilitated by tilting bioreactor container 410 using cam arm 462.
After the antibody coating solution is drained, valves 432 and 468e are opened and pump 454 is actuated to pump the flush buffer from reservoir 466e connected to the first fluidic assembly 440 to the first bioreactor vessel 410 through the first bioreactor line. The flush buffer is then discharged through the interconnecting line 450 to the waste reservoir 472a by activating the recycle line pump 456 and opening the valve 474 a. In an embodiment, this flushing and draining procedure may be repeated multiple times to adequately flush first bioreactor vessel 410.
Turning to fig. 55, after the first bioreactor container 410 is flushed with buffer, the cells in the seed bag 466d (which have been previously enriched and separated using the first module 100) are transferred to the first bioreactor container by opening valves 468d and 432 and actuating the pump 454. The cells are pumped through first bioreactor line 414 of first bioreactor vessel 410 and enter bioreactor vessel 410 through first port 412. As shown in fig. 56, valves 432 and 468a are then opened and pump 454 is actuated to pump the second antibody (Ab) solution from reservoir 466a connected to first fluid assembly 440 to first bioreactor vessel 410 through first port 412.
After pumping the second antibody solution into the first bioreactor vessel, the second antibody solution reservoir 466a is then flushed and the flush medium is pumped to the first bioreactor vessel. In particular, as shown in fig. 57, valves 474b, 452, and 468a are opened and flush medium from flush medium reservoir/bag 472b of second fluid assembly 444 is pumped into second antibody solution reservoir 466a using pump 454 to flush the reservoirs. After flushing, valve 432 is opened and flush medium is pumped from reservoir 466a to first bioreactor vessel 410. In an embodiment, the procedure may be used to flush the second antibody solution reservoir 466a multiple times.
After flushing the second antibody solution reservoir 466a, the inoculum/seed cell bag 466d may also be optionally flushed. In particular, as shown in fig. 58, valves 474b, 452, and 468d are opened and flush medium from flush medium reservoir/bag 472b of second fluid assembly 444 is pumped into inoculum/seed cell bag 466d using pump 454 to flush the bag. After flushing, valve 432 is opened and flush medium is pumped from bag 466d to first bioreactor vessel 410 using pump 454. By pumping the flush medium to the first bioreactor vessel 410 after flushing the inoculum/seed cell bag 466d, the cell density in the first bioreactor vessel 410 is reduced. At this point, a sample may be taken prior to activation to measure one or more parameters of the solution in the first bioreactor vessel (e.g., to ensure that a desired cell density exists prior to activation). In particular, as shown in fig. 58, valves 434, 452, and 432 are opened and pump 456 is actuated to pump the contents of first bioreactor vessel 410 along the first circulation loop of the first bioreactor vessel (i.e., out of second port 416, through interconnecting line 450, and back to first bioreactor vessel 410 through first bioreactor line 414 and first port 412 of first bioreactor vessel 410). To obtain a sample, a first sample container 280a (e.g., dip tube, syringe, etc.) is connected to the first sample line tail 476a and a valve 478a is opened to divert some of the flow through the interconnecting line 450 to the first sample container 280a for analysis.
If analysis of the obtained sample indicates that all solution parameters are within a predetermined range, the solution within the first bioreactor vessel 410 is incubated for a predetermined period of time for activating the cell population in the solution (as illustrated in fig. 59). For example, in an embodiment, the cell population in first bioreactor vessel 410 may be incubated for approximately 24-48 hours.
Referring now to fig. 60, after activation, to prepare for transduction, valves 438 and 474b may be opened and pump 456 operated to pump RetroNectin solution from reservoir 472b to second bioreactor vessel 420 through second port 426 of second bioreactor vessel 420. After pumping the RetroNectin solution to the second bioreactor container 420 for RetroNectin coating of the second bioreactor container 420, the solution is incubated in the second bioreactor container 420 for a predetermined period of time. As further shown in fig. 60, after incubation, all RetroNectin solution is then drained from the second bioreactor vessel 420 to waste reservoir 472a by opening valves 438 and 474a and actuating circulation line pump 456. During these RetroNectin coating, incubation and draining steps (involving the second bioreactor vessel 420), it should be noted that the activated cell population remains in the first bioreactor vessel 410. It should be noted that it is not necessary to utilize RetroNectin or other agents for improving the efficiency of the genetic modification in all processes.
As shown in fig. 61, after RetroNectin coating, flush buffer bag 472b is connected to second fluid assembly 444 (or it may already be present and connected to one of the tubing tails), and valves 474b and 438 are opened and pump 456 is actuated to pump buffer from bag 472b to second bioreactor container 420. As discussed above, alternatively, the buffer fluid may be pumped through the first port 422 of the second bioreactor vessel 420 by opening valves 452 and 436 instead.
Turning now to fig. 62, after a defined period of time, all of the buffer in the second bioreactor tank 420 is drained to the waste reservoir 472a of the second fluid assembly 444 by opening valves 438 and 474a and actuating the interconnecting line pump 456.
At this point, as shown in fig. 63, a post-activation preconcentrated sample may be taken from the cells in the first bioreactor vessel 410. As shown therein, valves 434, 486, 488, and 432 are opened and pump 456 is actuated to circulate the solution in first bioreactor vessel 410 as follows: from second port 434, through the interconnecting lines, through filter line 48 and filter 484, through first bioreactor line 414 of first bioreactor vessel 410, and back to first bioreactor vessel 410 through first port 412. To obtain a sample, a second sample container 280b (e.g., dip tube, syringe, etc.) is connected to the second sample tubing tail 476b, and a valve 478b is opened to divert some of the flow through the interconnecting line 450 to the second sample container 280b for analysis.
Referring now to fig. 64, and depending on the concentration obtained from the sample, concentration may be performed by circulating the contents of first bioreactor vessel 410 through filter 484. As discussed above, this is accomplished by opening valves 434, 486, 488, and 432 and actuating pump 456, which causes the solution in first bioreactor vessel 410 to circulate as follows: from second port 416, through second bioreactor line 418, through interconnecting line 450, through filter line 482 and filter 484, through first bioreactor line 414 of first bioreactor vessel 410, and back to first bioreactor vessel 410 through first port 412. As the fluid passes through filter 484, waste is removed and osmotic pump 492 pumps such waste through waste line 490 to waste reservoir 472a of second fluid assembly 444. In an embodiment, the procedure is repeated until the volume in the first bioreactor vessel 410 is concentrated to a predetermined volume.
Turning to fig. 65, after concentration, the concentrated cell population in the activation container (i.e., the first container 410 containing the concentrated cell population) is washed at a constant volume by perfusion. In particular, as shown therein, media from media bag 466b of first fluidic assembly 440 is pumped into first bioreactor vessel 410 through first port 412 via interconnecting line 450 while media is pumped out of first bioreactor vessel 410 through second port 416 such that a constant volume is maintained in first bioreactor vessel 410. As media is added to container 410 and removed from container 410, waste may be filtered out by filter 484 and directed to waste reservoir 472 a.
The washed sample may be taken from the cells in the first bioreactor vessel 410 in a manner similar to that described above for pre-concentrated sampling. In particular, as shown in fig. 66, valves 434, 486, 488, and 432 are opened and pump 456 is actuated to circulate fluid in first bioreactor vessel 410 as follows: from second port 434, through the interconnecting lines, through filter line 48 and filter 484, through first bioreactor line 414 of first bioreactor vessel 410, and back to first bioreactor vessel 410 through first port 412. To obtain a sample, a third sample container 280c (e.g., dip tube, syringe, etc.) is connected to the third sample tubing tail 476c, and a valve 478c is opened to divert some of the flow through the interconnecting line 450 to the third sample container 280c for analysis.
As shown in fig. 67, the bag containing thawed viral vectors is connected to the first fluidic component 440 (such as by the conduit tail 464 c). Valves 468c and 436 are then opened and pump 454 is actuated to transfer the viral vector coating solution from bag 466c to second bioreactor vessel 420 through first port 422. Incubation is then performed for a predetermined period of time for viral coating of the second bioreactor vessel 420. After incubation, the viral vector coating solution is drained from the second bioreactor vessel 420 to waste reservoir 472a by opening valves 438 and 474a and actuating circulation line pump 456. In embodiments, viral and non-viral vectors may be used as reagents for transduction/genetic modification.
As illustrated in fig. 68, after the second bioreactor vessel 420 is coated with a viral vector, the washed cells from the first bioreactor vessel 410 are transferred to the second bioreactor vessel 420 for transduction/genetic modification. In particular, valves 434, 452, and 436 are opened and circulation line pump 456 is actuated to pump cells out of first bioreactor vessel 420 through second port 416 of first bioreactor vessel 410, into first bioreactor line 424 of second bioreactor vessel 420 through interconnection line 450, and into second bioreactor vessel 420 through first port 422 of second bioreactor vessel 420.
Media from media bag 466b is then added to second bioreactor vessel 420 by opening valves 468b and 436 and actuating pump 454 to increase the total volume of solution in second bioreactor vessel 420 to a predetermined volume (as illustrated in fig. 69). Referring to fig. 70, a pre-divert sample may then be obtained by: valves 438, 452, and 436 are opened and circulation line pump 456 is actuated to pump the solution in second bioreactor vessel 420 along the circulation loop of the second bioreactor vessel (i.e., out of second port 426, through interconnecting line 450, and back to second bioreactor vessel 420 through first port 422 and first bioreactor line 414 of second bioreactor vessel 420). To obtain a sample, a fourth sample container 280d (e.g., dip tube, syringe, etc.) is connected to the fourth sample tubing tail 476d, and a valve 478d is opened to divert some of the flow through the interconnecting line 450 to the fourth sample container 280d for analysis.
If analysis of the obtained fourth sample indicates that all parameters are within the predetermined range required for successful transduction, the cell population within the second bioreactor vessel 420 is incubated for a predetermined period of time for transduction of the cell population in solution (as illustrated in fig. 71). For example, in an embodiment, the cell population in second bioreactor vessel 420 may be incubated for 24 hours for transduction.
Referring to fig. 72, after transduction, media is added to second bioreactor vessel 420 to achieve a predetermined amplification volume in second bioreactor vessel 420. As shown therein, to add media, valves 468b and 436 are opened and pump 454 is actuated to pump growth/perfusion media from media bag 466b through first port 422 of the second bioreactor container to second bioreactor container 420 until a predetermined amplification volume is reached.
As illustrated in fig. 73, the pre-amplified sample may then be obtained by: valves 438, 452, and 436 are opened and circulation line pump 456 is actuated to pump the solution in second bioreactor vessel 420 along the circulation loop of second bioreactor vessel 420 as indicated above (i.e., out of second port 426, through interconnection line 450, and back to second bioreactor vessel 420 through first port 422 and first bioreactor line 414 of second bioreactor vessel 420). To obtain a sample, a fifth sample container 280e (e.g., dip tube, syringe, etc.) is connected to the fifth sample tubing tail 476e, and a valve 478e is opened to divert some of the flow through the interconnecting line 450 to the fifth sample container 280e for analysis.
If analysis of the obtained fifth sample indicates that all parameters are within a predetermined range required for successful expansion of the cell population, the cell population within the second bioreactor vessel 420 is incubated for a predetermined period of time, for example 4 hours, to allow the cells to settle.
As shown in fig. 74, after this incubation period or at a predetermined time thereafter, perfusion is performed at a rate of 1 volume/day (1x perfusion) by pumping media from media bag 466b into second bioreactor container 420 through first port 422, while at the same time spent/used media is pumped out of second bioreactor container 420 through second port 426 (and to waste reservoir 472a through interconnect line 450). This priming is accomplished by opening valves 468b, 436, 438, and 474a and actuating first pump 454 and circulation line pump 456. During such 1x perfusion, media from media bag 466b is introduced into second bioreactor vessel 420 at substantially the same rate as used media is removed from second bioreactor vessel 420 and sent to waste to maintain a substantially constant volume within second bioreactor vessel 420.
Sampling can then be performed as needed/desired to monitor the amplification process and/or to determine when a desired cell density is reached. As discussed above, the sample may be obtained by: valves 438, 452, and 436 are opened and circulation line pump 456 is actuated to pump the solution in second bioreactor vessel 420 along the circulation loop of second bioreactor vessel 420 as indicated above (i.e., out of second port 426, through second bioreactor line 428, through interconnection line 450, and back to second bioreactor vessel 420 through first port 422 and first bioreactor line 424 of second bioreactor vessel 420). To obtain a sample, another sample container 280x (e.g., dip tube, syringe, etc.) is connected to the sample tubing tail of the sample assembly 448 and the valve of the tubing tail is opened to divert some of the flow through the interconnecting line 450 to the sample container 280x for analysis (as shown in fig. 75). After each sampling operation, an incubation without perfusion is performed for a predetermined period of time (e.g. 4 hours) to allow the cells to settle before the perfusion is resumed.
As shown in fig. 76, after this incubation period, perfusion is performed at a rate of 1 volume/day (1x perfusion) by pumping media from media bag 466b into second bioreactor container 420 through first port 422, while at the same time, used/used media is pumped out of second bioreactor container 420 through second port 426 (and to waste reservoir 472a through interconnect line 450) (as shown in fig. 74). This priming is accomplished by opening valves 468b, 436, 438, and 474a and actuating first pump 454 and circulation line pump 456.
As shown in fig. 76, when the sampling indicates a predetermined threshold (e.g., 5 MM/mL) Viable Cell Density (VCD), perfusion is performed at a rate of 2 volumes/day (2x perfusion) by pumping media from media bag 466b through first port 422 into second bioreactor container 420, while at the same time, used/used media is pumped out of second bioreactor container 420 through second port 426 (and through interconnect line 450 to waste reservoir 472 a). This priming is accomplished by opening valves 468b, 436, 438, and 474a and actuating first pump 454 and circulation line pump 456. During such 2x perfusion, media from media bag 466b is introduced into second bioreactor vessel 420 at substantially the same rate as used media is removed from second bioreactor vessel 420 and sent to waste to maintain a substantially constant volume within second bioreactor vessel 420.
Finally, referring to fig. 77, after the desired viable cell density is achieved, the cells can be harvested by opening valves 438 and 474d and actuating the circulation line pump 456. The expanded cell population is then pumped out of second bioreactor container 420 through second port 426, through interconnecting line 450, and to collection bag 472d connected to tubing tail 470d of second tubing assembly 444. These cells may then be formulated in a manner heretofore known in the art for delivery and infusion into a patient.
Thus, the second module 200 of the bioprocessing system 10, as well as its flow architecture 400 and bioreactor vessels 410, 420, provide a flexible platform on which a variety of bioprocessing operations may be performed in a substantially automated and functionally closed manner. In particular, although fig. 53-77 illustrate exemplary general protocols that may be performed using the bioprocessing system 10 of the present invention (and in particular, using the second module 200 thereof), the system is not so limited in this regard. Indeed, a variety of automated protocols, including many customer-specific protocols, may be implemented by the system of the present invention.
In contrast to existing systems, the second module 200 of the bioprocessing system 10 is a functionally closed, automated system housing the first and second bioreactor vessels 410 and 420 and the fluid processing and fluid containment systems, all maintained under cell culture-friendly environmental conditions (i.e., within a temperature and gas controlled environment) to effect cell activation, transduction, and expansion. As discussed above, the system includes automated kit loading and closed sampling capabilities. In this configuration, the system enables all steps of immune cell activation, transduction, expansion, sampling, perfusion, and washing to be performed in a single system. It also provides the user with the flexibility to combine all steps in a single bioreactor vessel (e.g., first bioreactor vessel 410) or to use both bioreactor vessels 410, 420 for end-to-end activation and washing. In embodiments, a single expansion bioreactor vessel (e.g., bioreactor vessel 420) is capable of robustly producing doses of billions of T cells. Single or multiple doses can be generated in situ with high recovery and high viability. In addition, the system is designed to give the end user the flexibility to run different protocols for the manufacture of genetically modified immune cells.
Some of the commercial advantages provided by the bioprocessing system of the present invention include robust and scalable manufacturing techniques that enable product commercialization through the ability to simplify workflow, reduce labor intensity, reduce the burden on clean room infrastructure, reduce failed nodes, reduce cost, and increase operational scale.
As discussed above in connection with the general workflow, the flow architecture 400 and bioreactor vessels 410, 420 of the system, bioprocessing system 10 and second module 200 of the present invention enable culture concentration, washing, slow perfusion, fast perfusion and "cycling" perfusion processes to be performed in an automated and functionally closed manner. For example, as discussed above, pump 456 on interconnection line 450 may be used to circulate fluid from one of the ports of the bioreactor through filtration line 482 and filter 484, and then back to the other port on the bioreactor, while operating osmotic pump 492 (typically at a percentage of circulation pump 456, such as, for example, about 10%) in the concentration step. The concentration may be run open loop or may be stopped based on the measured volume removed from the bioreactor or the measured volume accumulated in the waste. In embodiments, the filter, pump speed, filter area, number of lumens, etc. are appropriately sized for the total number of cells and the target cell density to limit fouling and excessive cell loss due to shearing.
In embodiments, and as discussed above, the systems of the invention can also be used for washing, e.g., to remove residues, such as viral vectors remaining after incubation. The wash involves the same steps described above for concentration, except that the pump 454 on the first fluidic module line 442 is used to pump in additional media to replace the fluid pumped from the osmotic waste pump 492. The rate of introduction of fresh media may correspond to the rate of fluid removal by the osmotic pump 492. This allows a constant volume to be maintained in the bioreactor vessel and the residue can be removed exponentially over time as long as the contents of the bioreactor are well mixed (circulation may be sufficient). In an embodiment, this same process can be used for in situ hollow fiber filtration based cell suspension washing after activation to remove residues. Elution of soluble activating reagents can also be accomplished via filter-based perfusion for coated and uncoated surfaces.
As also discussed above, during perfusion, the pump 454 on the first fluidic component line 442 may be used to add media to a given bioreactor container, while the pump 456 on the interconnecting line 450 is used to move spent media to a waste bag in the second fluidic component. In embodiments, gravity may be used to settle the cells, and the spent media may be pumped out at a rate that does not significantly interfere with the cells within the bioreactor vessel. This process may involve operating pumps 454 and 456 open-loop at the same rate. In an embodiment, one pump (454 or 456) may be run at a set rate, and the rate of the other pump may be adjusted based on the mass/volume of the bioreactor container or the mass/volume of the waste bag (or the measured mass/volume of the source bag).
In conjunction with the above, it is contemplated that pump control may be based on weight measurements of the bioreactor vessel (using feedback from load cell 760). For example, the configuration of the system enables dynamic pump calibration based on load cell readings, allowing the system to automatically adapt to changes in tube/pump performance over time. Furthermore, the method can be used for closed loop control of the rate of change of mass (volume) when emptying or filling a bioreactor vessel.
In another embodiment, the bioprocessing system allows for the cyclical filling of a variety of bioreactor vessels in the system using the flow architecture 400. For example, as described above, circulation pump 456 and pump 545 along first fluid assembly line 442 are used to prime the cells within first bioreactor vessel 410 in conjunction with the appropriate pinch valve status. The perfusion of the cells within the first bioreactor vessel 410 may then be stopped or paused, and the circulation pump 456 and pump 454, and appropriate pinch valves, may then be actuated to perfuse the cells within the second bioreactor vessel 420. In this regard, the priming of the various bioreactors may be performed sequentially (i.e., first bioreactor vessel 410 is primed for a period of time and then second bioreactor vessel 420 is primed for a period of time in a repeated and alternating manner). This allows any number of bioreactor vessels in the system to be filled without the need to use more pumps, media bags or waste bags.
In the case of cyclic perfusion, the pumps may run continuously, may run intermittently together (duty cycle), or may run sequentially (source, then waste, repeat) to still maintain the volume/mass in the various bioreactor vessels at about the same level. As indicated, cyclic priming (running a set of pumps intermittently together and waiting for a time interval) will also allow multiple containers to be primed using the same two pumps. In addition, even if the pump does not have a large low end dynamic range, cyclic perfusion allows for a lower effective exchange rate (such as about 1 volume/day). In addition, cyclic 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 embodiments, rapid priming may be used for residue removal (e.g., for post-activation Ab removal and/or post-transduction residue removal). In a rapid perfusion process, the perfusion process described above may run much faster than a typical 1-5 volumes/day, such as, for example, between about 8-20 volumes/day, or greater than about 20 volumes/day, to achieve a 1 log reduction in about a few minutes to a few hours. In embodiments, the perfusion rate is balanced with cell loss. In some embodiments, rapid priming may allow for elimination of hollow filter 484 and still meet the biological requirements of rapidly removing residue after certain steps.
As further described above, the system of the present invention facilitates flushing the bag/reservoir connected to the first fluidic component 440 using the pump 454 on the first fluidic component line 442 using a flush buffer or fluid from another bag/reservoir connected to the second fluidic component 444. Additionally, the fluid lines of the flow architecture/system 400 may be purged with sterile air from the sterile air source 458 to prevent cells from settling in the lines and dying, or to prevent media or reagents from settling in the lines and degrading or not being used. A sterile air source 458 may also be used to purge reagent from the lines to ensure that no more reagent is pumped to the bioreactor vessels 410, 420 than is desired. The sterile air source 458 may also be used to clean lines leading up to the connected bag (of either the first or second fluid assemblies 440, 444) to clean the sterile tube welds to limit carryover. Alternatively, or in addition to cleaning the lines using the sterile air source 458, the lines may be cleaned using air drawn from one of the bioreactor vessels, as long as the port through which air is drawn is not submerged and the bioreactor vessel has an air balance port 530.
As discussed above, the system allows for closed drawer in-process sampling of the contents of the bioreactor vessel(s). During sampling, the cam arm 762 may be used to agitate the container from which the sample is to be drawn, thereby circulating the contents of the container using the circulation line pump 456, and using the sampling assembly 448 to draw the sample from the interconnecting line 450. In an embodiment, only cells that are not bound to beads may be agitated.
As also discussed above, the system of the present invention allows for collection of a cell population after a target cell density is achieved. In embodiments, collecting the expanded population of transduced cells can comprise: using a pump 456 on the interconnect line 450 to move the cell into one of the bags connected to the second fluid assembly 444; or the cells are circulated using the interconnect pump 456 to move the cells to a bag connected to the first fluidic component 440. This process may be used for final collection or large sample volumes, or may be used to fully automate the sampling process (i.e., by connecting a syringe or bag to the first fluidic assembly 440, circulating the contents of the bioreactor container, and using the fluidic assembly pump 454 to draw a portion of the desired sample volume from the circulated contents and move toward the syringe/bag). In such a case, the circulation pump 456 and valves may then be used to clear the circulation lines of the fluid/cells. In addition, the pump 454 on the first fluidic component line 442 may be used to continue to push all of the aliquot sample volume into the sample reservoir, thereby using the air in the line to complete the transfer of the sample to the reservoir without an appreciable number of cells remaining in the line.
While the above-described embodiments disclose workflows for performing cell activation in a first bioreactor vessel and transferring the activated cells to a second bioreactor vessel for transduction and expansion, in embodiments, the system of the present invention may allow activation and transduction operations to be performed in the first bioreactor vessel and expansion of the genetically modified cells to be performed in the second bioreactor vessel. Furthermore, in embodiments, the systems of the present invention may allow for in situ processing of isolated T cells, wherein the activation, transduction, and amplification unit operations are all performed within a single bioreactor vessel. In an embodiment, the present invention thus simplifies existing protocols by enabling a simplified and automation friendly "one pot" activation, transduction, and amplification vessel.
In such embodiments, the T cell activator can be micrometer-sized dynabeads, and a lentiviral vector is used for transduction. In particular, as disclosed herein, micrometer-sized dynabeads serve the dual purpose of isolating and activating T cells. In an embodiment, activation (and isolation) of T cells may be performed in one of bioreactor containers 410 using dynabeads in the manner described above. Subsequently, the activated cells are transduced by the virus for genetic modification, such as in the manner described above in connection with fig. 60-71. After activation and virus transduction, the virus may then be washed out of bioreactor container 410 using the filter-less perfusion method described above, which holds the cells and micron-sized dynabeads in bioreactor container 410. This enables the cells to be expanded in the same bioreactor vessel 410 used for activation and transduction. The filter-less perfusion method additionally enables culture washing without the need to first immobilize the activated beads that need to be held with the cells during amplification. In particular, when the virus is washed away, the micron-sized dynabeads are not fluidized at a slow perfusion rate, but are held in the container. The nano-sized virus particles and residual macromolecules are fluidized and washed away during slow perfusion.
In embodiments, after amplification, the cells may be harvested in the manner described above in connection with fig. 77. After harvesting, dynabeads can be removed from the collected cells using a magnetic debeading process. In other embodiments, the steps of harvesting the expanded cell population and debeading the cells are performed simultaneously using perfusion, whereby the culture medium is introduced through a feed port in the bioreactor vessel while the cell culture medium comprising the expanded cell population is removed from the bioreactor vessel through a drain port in the bioreactor vessel. In particular, when final debeading of the culture is required, by taking advantage of the difference in cell weight and cell-Dynabead complex weight, no filter perfusion can be used to remove micron-sized beads. To debeade the culture, the entire contents of the bioreactor vessel will be mixed (using, for example, the cam arm 762 of the actuator mechanism in the manner described above). After mixing/stirring, heavy dynabeads will sink and settle on the silica film 516 in 10-15 minutes. In contrast, cells take more than 4 hours to settle on the membrane 516. After a 10-15 minute hold period following mixing/stirring, the cell suspension can be slowly withdrawn using perfusion without disturbing the settled dynabeads. The input media line may be used to maintain the media bed height within the bioreactor vessel. Thus, the invention described herein simplifies the current Dynabead protocol by eliminating the need for several intermediate process cell transfers and careful washing and debeading steps, and minimizes costs and potential risks. By debeading the culture while harvesting the cells, the need for additional magnetic devices or disposables, which are typically necessary, can be eliminated.
In contrast to other static non-perfusion culture systems, the gas-permeable membrane-based bioreactor vessel 410 of the present invention supports high density cell culture (e.g., up to 35 mm/cm)2). Thus, all four unit processes using Dynabead activation, transduction, washing and amplification can be performed in the same bioreactor vessel in a fully automated and functionally closed manner. Thus, the bioprocessing system of the present invention simplifies current protocols by eliminating the need for intermediate process cell transfer and careful washing steps, and makes contact with a plurality of human contactsThe resulting cost and potential risk are minimized.
In an embodiment, the two bioreactor vessels 410, 420 of the system may be operated with the same starting culture or two cultures that divide simultaneously (e.g., CD4+ cells in one bioreactor vessel 410 and CD8+ cells in the other bioreactor vessel 420). Dividing cultures allow for parallel independent processing and expansion of two cell types, which can be combined prior to infusion into a patient.
Although many possible CAR-T workflows for generating and expanding genetically modified cells using the biological processing system of the invention have been described above, the workflows described herein are not intended to be comprehensive, as the system of the invention also enables other CAR-T workflows. In addition, although the system of the invention, and in particular the second module 200 of the system, has been described in connection with the manufacture of CAR-T cells, the system of the invention is also compatible with the manufacture of other immune cells such as TCR-T cells and NK cells. Further, while embodiments of the present invention disclose the use of two bioreactor vessels 410, 420 in a two-step sequential process, wherein the output of the first bioreactor vessel 410 is added to the second bioreactor vessel 420 for additional processing steps (e.g., activation in the first bioreactor vessel and transduction and amplification in the second bioreactor vessel), in some embodiments, both bioreactor vessels may be reused for the same workflow. Exemplary reasons for sequentially using the second bioreactor vessel may include: residual chemical modifications (e.g., coating or immobilized reagents) that are not washable from the first bioreactor that are detrimental in later steps (or earlier steps if over-exposure of the cells occurs); or it may be desirable to pre-coat the bioreactor surface (e.g., RetroNectin coating) prior to adding the cells.
Additional examples of potential single bioreactor vessel workflows enabled by the system of the present invention include: (1) soluble activator activation, viral transduction, filter-less perfusion, and amplification in a single bioreactor vessel; (2) dynabead-based activation, viral transduction, filter-less perfusion, and amplification in a single bioreactor vessel; and (3) TransAct bead-based activation, viral transduction, filter-free perfusion, and amplification in a single vessel.
Further, additional examples of potential multiple bioreactor vessel workflows enabled by the system of the present invention include: (1) soluble activator activation, viral transduction, no filter perfusion, and amplification in the first bioreactor vessel 410, and soluble activator activation, lentiviral transduction, no filter perfusion, and amplification in the second bioreactor vessel 420, using the same cell type or dividing culture in both bioreactor vessels; (2) dynabead-based activation, viral transduction, filter-less perfusion, and amplification in the first bioreactor vessel 410, and Dynabead-based activation, lentiviral transduction, filter-less perfusion, and amplification in the second bioreactor vessel 420, using the same cell type or split culture in both bioreactor vessels; (3) TransAct bead-based activation, viral transduction, filter-free perfusion and amplification in the first bioreactor vessel 410, and TransAct bead-based activation, lentiviral transduction, filter-free perfusion and amplification in the second bioreactor vessel 420, the same cell type or split culture being used in both bioreactor vessels; (4) soluble activator activation in the first bioreactor vessel 410, and RetroNectin coating, transduction, and amplification in the second bioreactor vessel 420; (5) activation of immobilized activator in the first bioreactor vessel 410, and RetroNectin coating, transduction, and amplification in the second bioreactor vessel 420; (6) dynabead activation in the first bioreactor vessel 410, and RetroNectin coating, transduction, and amplification in the second bioreactor vessel 420; (7) dynabead activation and lentiviral transduction in the first bioreactor vessel 410, and amplification in the second bioreactor vessel 420; (8) TransAct activation in the first bioreactor vessel 410, and RetroNectin coating, transduction, and amplification in the second bioreactor vessel 420; (9) activation of soluble activator in the first bioreactor vessel 410, and expansion of ectopically electroporated cells or other non-virally modified cells in the second bioreactor vessel 420; (10) (ii) TransAct activation in the first bioreactor vessel 410, and expansion of ectopically electroporated cells or other non-virally modified cells in the second bioreactor vessel 420; (11) dynabead activation in the first bioreactor vessel 410, and expansion of ectopically electroporated cells or other non-virally modified cells in the second bioreactor vessel 420; (12) expansion of allogeneic NK cells in first bioreactor vessel 410, and expansion of allogeneic NK cells in second bioreactor vessel 420 (based on small molecule expansion, without genetic modification); (13) expansion of allogeneic NK cells in first bioreactor vessel 410, and expansion of allogeneic NK cells in second bioreactor vessel 420 (feeder cells-based expansion, without genetic modification); and (14) soluble activator activation, viral transduction, filter-free perfusion, and expansion of allogeneic CAR-NK or CAR-NK 92 cells in the first bioreactor vessel 410 and/or in the first bioreactor vessel 410 and the second bioreactor vessel 420 (no RetroNectin coating, and wherein polybrene is used to aid in transduction).
While the embodiments described above illustrate process monitoring sensors integrated with the bioreactor vessel and/or seat deck (e.g., on the membrane, integrated in the membrane, on the vessel sidewall, etc.), in other embodiments it is contemplated that additional sensors may be added to the fluidic architecture 400 (e.g., along the fluid flow line itself). These sensors may be disposable compatible sensors for monitoring parameters within the circulating fluid such as pH, dissolved oxygen, density/turbidity (optical sensors), conductivity and viability. By arranging the sensor in the circulation loop (e.g. the circulation loop of the first bioreactor vessel and/or the circulation loop of the second bioreactor vessel), the vessel construction can be simplified. Additionally, in some embodiments, sensors along the circulation loop may provide a more accurate representation of the contents of the vessel as it circulates (rather than measuring when the cells are stationary within the vessel). Still further, a flow rate sensor (e.g., ultrasonic based) may be added to the flow circuit to measure pumping performance and used in conjunction with an algorithm to correct pumping parameters as necessary.
As indicated above, the first module 100 and the third module 300 may employ any form(s) of system or device known in the art capable of cell enrichment and isolation, as well as harvesting and/or formulation. Fig. 78 illustrates one possible configuration of a device/apparatus 900 that may be used as first module 100 in biological processing system 10 for cell enrichment and separation using a variety of magnetic separation bead types, including, for example, Miltenyi beads, dynabeads, and StemCell EasySep beads. As shown therein, apparatus 900 includes a base 910 housing a centrifugal processing chamber 912, a high dynamic range peristaltic pump assembly 914, a suitable inner diameter pump tube 916 received by the peristaltic pump assembly, a stopcock manifold 918, an optical sensor 920, and a heating-cooling-mixing chamber 922. As indicated below, the stopcock manifold 918 provides a simple and reliable means of wiring multiple fluid or gas lines together using, for example, a luer fitting. In an embodiment, pump 914 is rated to provide a flow rate as low as about 3mL/min and as high as about 150 mL/min.
As further shown in fig. 78, the apparatus 900 may include a generally T-shaped hanger assembly 924, the hanger assembly 924 extending from the base 910 and including a plurality of hooks 926 for hanging a plurality of processing and/or source containers or bags. In an embodiment, there may be six hooks. Each hook may include an integrated weight sensor for detecting the weight of each container/bag. In an embodiment, the bags may include a sample source bag 930, a processing bag 932, a separation buffer bag 934, a wash bag 936, a first storage bag 938, a second storage bag 940, a post-separation waste bag 942, a wash waste bag 944, a culture medium bag 946, a release bag 948, and a collection bag 950.
The apparatus 900 is configured for use with or includes a magnetic cell separation holder 960 as provided herein. The magnetic cell separation holder 960 may be removably coupled to a magnetic field generator 962 (e.g., magnetic field plates 964, 966 of fig. 80). The magnetic cell separation holder 960 houses a magnetic holding element or material 968, such as a separation column, matrix, or tube. In an embodiment, the magnetic cell separation holder 960 may be constructed as disclosed in U.S. patent application serial No. 15/829615 filed on 12/1/2017, which is incorporated by reference herein in its entirety and described in more detail below. The device 900 may operate under the control of a controller (e.g., controller 110) according to instructions executed by a processor and stored in memory. Such instructions may include magnetic field parameters. In embodiments, as discussed below, the device 900 may further include a syringe 952, which may be used to add beads.
Turning now to fig. 79, a generic protocol 1000 for a device 700 is shown. As illustrated therein, in a first step 1010, enrichment is performed by reducing platelets and plasma in a sample. In embodiments where dynabeads are used as the magnetic separation beads, a wash step 1012 may then be performed to remove residue from the dynabeads suspension. After enrichment, the cells are then transferred to a processing bag 932 at step 1014. In some embodiments, the enriched portion of cells may be stored in a first storage bag 938 at step 1016 prior to transfer into the processing bag 932. At step 1018, the magnetic separation beads are injected into the processing bag, such as by using a syringe 952 at step 1020. In an embodiment, the magnetic separation beads are Miltenyi beads or StemCell EasySep beads. In the case of dynabeads, the washed dynabeads from step 1012 are resuspended in processing bag 932. In an embodiment, instead of using a syringe, the magnetically separable beads may be contained in a bag or container connected to the system, and the beads may be drawn into the system by pump 914.
Then, at step 1020, the beads and cells in the processing bag 932 are incubated for a period of time. This step includes circulation of fluid out of the processing bag 932, through the loop, and back into the bag. In embodiments where the magnetic separation beads are Miltenyi nano-sized beads, a pellet wash is performed at step 1022 to remove excess nano-sized beads, and the incubated bead-bound cell fraction is stored in second storage bag 940 at step 1024. After incubation, at step 1026, the bead-bound cells are isolated using a magnet (e.g., magnetic field plates 964, 966 of magnetic cell isolation holder 960). Then, at step 1028, the remaining bead-bound cells are washed and isolated. Finally, in embodiments utilizing Miltenyi or Dynabead, the isolated bead-bound cells are collected in collection bag 950 at step 1030. In embodiments utilizing StemCellEasySep beads, an additional step 1032 of releasing cells from the beads to remove beads is performed, as well as an optional step 1034 of washing/concentrating the collected cells.
A more detailed description of the general protocol of fig. 79 using the device 900 is described in more detail below with specific reference to fig. 80, fig. 80 being a schematic illustration of a flow architecture 1100 of the device 900. First, the enrichment process (step 1010) begins by transferring the apheresis product contained in the source bag 930 and the wash buffer from the wash buffer bag 936 to the chamber 912 for washing with the wash buffer in order to reduce the amount of platelets and serum. At this point, the enriched source material is located in chamber 912. To begin the separation process, the separation column received by the magnetic cell separation holder 960 is primed by initiating flow of buffer from the separation buffer bag 934 through the manifold 918 and through the column (to prime the column) to the processing bag 932.
As disclosed above, in certain embodiments, such as where dynabeads are used as magnetic separation beads, a washing step (step 1012) is performed to remove any residue in the bead suspension buffer. The washing step comprises: injecting the beads using a syringe 952 while circulating in the processing circuit 1110 (e.g., from the processing bag 932, through the peristaltic pump tubing 914, through the manifold 918, and back to the processing bag 932); a cleaning process loop 1110; and then capture the beads by flowing from the processing bag 932 to a separation waste bag 942 while the magnetic field generator 962 is in an "on" state, i.e., while the holder is magnetically coupled with the active magnetic field generator 962 for a permanent magnet, or alternatively, while the electromagnetic field is actively generated by using an electromagnet (in each case, capturing the beads in an "on" state). In embodiments where washing is not desired, flow is from the processing bag 932 to a separate waste bag 942 to ensure that the processing bag 932 is clean. As used herein, "activated," in the case of a permanent magnet, means that a magnetic holding element or material 968 (e.g., a separation column, matrix, or tube) is in place within the magnetic field. "closed" means that the pipe section is removed from the magnetic field.
Next, the enriched cells in the processing chamber 912 are transferred to the processing bag 932 (step 1014), and separation buffer from the separation buffer bag 934 is drawn into the processing chamber 912 to flush any remaining cells in the chamber 912. After flushing, the fluid is drained to the processing bag 932. The rinsing process may be repeated as desired. After all cells have been transferred to the processing bag 932, the chamber 912 is cleaned by drawing buffer from the separation buffer bag 934 into the chamber 912 and expelling the fluid to the source bag 930. The cleaning process may be repeated as desired.
The contents of the processing bag 932 may then be mixed by circulating the contents along the processing circuit 1110 before the processing circuit 1110 is cleaned by returning all of the contents to the processing bag 932. As indicated above, in an embodiment, by transferring a portion of the contents of the processing bag 932 to the first storage bag 938, a portion of the enriched cells may be stored at this point (step 1016). The process line 1112 and the first pouch line 1114 may then be cleaned.
In embodiments that do not utilize a bead wash step, the beads are then injected into the processing loop 1110 using the syringe 952, and the processing loop 1110 is cleaned (step 1018). In embodiments utilizing a bead wash step, beads are resuspended and circulated through processing loop 1110 (step 1018) and column 968, and the processing loop is cleaned through column 968.
As discussed above, after the addition of the magnetic separation beads, the cells may be incubated for a period of time (step 1020). In an embodiment, prior to incubation, the contents of the processing bag 932 may be transferred to the second storage bag 940, and the second storage bag 940 is agitated (such as using the heat-cool-mixing chamber 922). The contents of the second storage bag 940 are then transferred back to the disposal bag 932. Buffer from the separation buffer bag 934 is then drawn into the processing chamber 912 and the chamber contents are discharged to the second storage bag 940 and then transferred to the processing bag 932 to flush the second storage bag 940.
In either embodiment, the cells are then incubated with the magnetic separation beads by cycling the cells along processing circuit 1110 for a prescribed incubation time. After incubation, processing circuit 1110 is cleaned.
As discussed above, after incubation, an optional step of washing off excess beads (e.g., nano-sized beads) may be performed (step 1022). Washing away excess nano-sized beads includes: initiating flow from the processing bag 932 to the second storage bag 940; drawing the contents of the second storage bag 940 into the processing chamber 912; transferring buffer from the separation buffer bag 934 to the processing bag 932; transferring the contents of the processing bag 932 to a second storage bag 940; and drawing the contents of the second storage bag 940 into the processing chamber. The steps of flowing from the separation buffer bag 934 to the processing bag 932 and then to the second storage bag 940 may be repeated as desired to wash away excess beads. In an embodiment, the chamber 912 may then be filled with buffer from the separation buffer bag 934, rotation of the chamber 912 is initiated, and the supernatant is then discharged to the waste bag 742. These steps may be repeated as desired. In an embodiment, cells in the chamber are drained to the processing bag 932, buffer from the separation buffer bag 934 is aspirated into the chamber 932, and then drained from the chamber to the processing bag 932. The process may also be repeated as desired. Mixing of the processing circuits and cleaning of the processing circuits are then performed.
In some embodiments, the portion of the incubated cell population may be stored in second storage bag 940 (step 1024). To do so, a portion of the contents of the processing bag 932 may be transferred to the second storage bag 940, and then the processing line and the second storage line 1116 are cleaned.
In any of the procedures described above, after incubation, the bead-bound cells are isolated using magnets 964, 966 (step 1026). This is accomplished by flowing from the disposal bag 932 to the waste bag 942 when the magnetic field generator 962 is "on". The residual waste is then cleaned by pumping buffer from the separation buffer bag 934 to the processing bag 932 and then from the processing bag 932 to the waste bag 942 with the magnetic field generator 962 "on".
In an embodiment, a flush without resuspension may be performed by pumping buffer from the separation buffer bag 934 to the processing bag 932, flushing the processing circuit 1110, clearing the processing circuit 1110, and flowing from the processing bag 932 to the waste bag 942 with the magnetic field generator 962 "on".
In another embodiment, flushing via resuspension can be performed by pumping buffer from the separation buffer bag 934 to the processing bag 932 with the magnetic field generator 962 "off," circulating in the processing circuit 1110, purging the processing circuit, and flowing from the processing bag 932 to the waste bag 942 with the magnetic field generator 962 "on.
In an embodiment, residual waste may be removed by pumping buffer from the separation buffer bag 934 to the processing bag 932 and flowing from the processing bag 932 to the waste bag 942 with the magnetic field generator 962 "on".
After washing and isolating the remaining bead-bound cells, the isolated bead-bound cells are then collected (step 1028). In one approach, in the event that bead-bound cells are to be collected without releasing the cells from the beads, media from the media bag 946 is simply pumped through the column 968 to the collection bag 950 with the magnetic field generator 962 "off". In another method, buffer from the separation buffer bag 934 is pumped to the processing bag 932, and then from the processing bag 932 to the collection bag 950 with the magnetic field generator 962 "off". This second method provides a post-separation wash. In a third method, media from media bag 946 is pumped through column 966 to processing bag 932 (if post-separation washing is not required). Alternatively, buffer from the separation buffer bag 934 is pumped through the column 966 to the processing bag 932 (if post-separation washing is desired). In either process, the contents of the processing bag 932 are then circulated in the processing circuit 1110, the processing circuit 1110 is cleaned by returning to the processing bag 932, and the contents of the processing bag 932 are pumped to the collection bag 950 to collect the bead-bound cells.
In the case where the bead-bound cells are to be collected after releasing the cells from the beads, a number of potential processes may be performed. For example, in an embodiment, the cells/beads may be resuspended with the magnet "off" by pumping release buffer from the bag 948 through the column to the processing bag 932, circulating in the processing loop 1110, and then clearing the processing loop by returning fluid to the processing bag 932. Then, incubation and collection is performed with the magnet "on" by incubating in the processing circuit 1110, purging the processing circuit 1110, collecting the released cells by pumping from the processing bag 932 through the column 966 to the collection bag 950, pumping buffer from the separation buffer bag 934 to the processing bag 932, and collecting the residue by pumping the contents of the processing bag 932 through the column 966 to the collection bag 950. The released beads (step 1032) can then be discarded by pumping buffer from the separation buffer bag 934 to the processing bag 932 through the column 966 with the magnet "off", circulating in the processing circuit 1110, purging the processing circuit 1110, and pumping the contents of the processing bag 932 to the waste bag 942.
In conjunction with the above, in an embodiment, washing/concentration may be performed by pumping the contents of the collection bag 950 to the processing chamber 912, pumping buffer from the separation buffer bag 934 to the processing bag 932, and transferring buffer from the processing bag 932 to the processing chamber 912 (step 1034). Then, by filling the processing chamber 912 with buffer from the separation buffer bag 934, rotating the chamber 912, discharging the supernatant to the waste bag 942, and repeating the rotating and discharging steps as desired, a washing cycle may be performed. Finally, the transfer of cells to the collection bag after washing/concentration can be accomplished by transferring media from the media bag 946 to the collection bag 950, pumping the collection bag contents into the processing chamber 912, draining the contents of the processing chamber 912 to the collection bag 950, and then manually clearing the line between the processing chamber 912 and the collection bag 950.
In an embodiment, one of the bags (e.g., the processing bag 932) may include a top port 1118 with a filter so that sterile air may be introduced into the system (when the processing bag 932 is empty) for cleaning the line as needed (such as in the various process steps discussed above). The cleaning of the lines may be done as a first step in the enrichment/separation process and/or during the process. In an embodiment, air from the collection bag 950 may be used to purge any lines of the system (e.g., air from the collection bag 950 may be used to purge the process line 1112, then air in the process line 1112 may be used to purge the desired tubing lines (i.e., lines 1114, 1116, etc.), filling the process line 1112 with liquid from the process bag 932, and finally purging the process line 1112 again using air from the collection bag 950).
In an embodiment, the processing bag 932 is blow molded and has a high angle on the sides (with a 3D shape with defined air pockets above the liquid level) to limit micron-sized beads from sticking to the sidewalls (particularly during long-term facilitated mixing during cycle-based rendering).
In an embodiment, the syringe 952 allows for the addition of small volumes (such as bead suspension aliquots) to the cycle based flow loop 1110. Additionally, fluid from the flow circuit 1110 may be drawn into the syringe 952 to further clean any residue from the syringe 952.
In an embodiment, one of the sensors 920 may be configured to measure the flow rate of the fluid. For example, one of the sensors 920 may be a bubble detector or an optical detector that may be used as a secondary confirmation measure to ensure accurate flow control (in addition to the load cell integrated with the hook 926). This may be practical during separation, where it is desirable to flow the volume in the processing bag past the magnet without introducing air into the column. The load cell indicates that the processing bag is approaching empty within some expected tolerance of the load cell variability, and then the bubble detector 920 identifies a trailing liquid/air interface to stop the flow. Thus, the sensor 920 can be used by the controller to prevent air from being drawn into the circuit (which can create a plug of air to dislodge cells, or expose cells to a dry environment) or by accidentally drawing material into the waste bag without the pump stopping after the processing bag is completely emptied. In an embodiment, the bubble detector 920 may thus be used in combination with a hook-integrated load cell to improve the volume control accuracy, thereby reducing cell loss and/or preventing air from entering the column tubing and column.
As alluded to above, in embodiments, air may be drawn into the circuit for purposefully creating an air plug that may be used to dislodge bead-bound cells within the separation column/tube for collection. In embodiments, instead of or in addition to using an air plug, a buffer solution may be circulated through the separation column to elute bead-bound cells from the separation column.
In embodiments, two or more peristaltic pump tubes having different inner diameters connected in series may be employed in order to achieve an extended flow rate range for a single pump. To switch between tubes, the pump cap is opened, the existing tube is physically removed, the desired tube is physically inserted, and then the pump head is closed.
In some embodiments, the system 900 can be used for elution of the separated/captured bead-cell complexes. In particular, it is contemplated that an air-liquid interface may be used to aid in the removal of the composite from the tube sidewall or column interstitial space. Air may be circulated through or shuttled back and forth through the column/tube. Without an air/liquid interface, it is difficult to remove a packed bed of beads/bead-bound cells using flow rate control alone without significantly increasing the shear rate (which has a potential negative impact on cell viability). In combination with the flow rate, it is therefore possible to remove the bead-cell complexes without removing them from the magnet.
In conjunction with the above, system 900 supports the concept of eluting positively selected bead-cell complexes directly into the media of choice (based on downstream steps). This eliminates the buffer exchange/wash step. In the examples, elution directly into the culture medium and viral vector to begin incubation is also contemplated. This concept also enables the addition of viral vectors to the final bag. In embodiments, instead of using a buffer to elute the bead-bound cells, a culture medium may be used as the elution fluid. Similarly, a release buffer may be used to elute the StemCell beads for subsequent release of cells from the beads. Dilution can be minimized by replacing the buffer in portions of the system 900 with media.
As disclosed above, the device 900 of the first module 100 is a single kit that provides for platelet and plasma reduced enrichment followed by magnetic separation of target cells. The apparatus 900 is automated so as to allow the enrichment, separation, and collection steps, as well as all the intervening steps, to be performed with minimal human intervention. Similar to the second cartridge 200, the first cartridge 100 and its device 900 are functionally closed to minimize the risk of contamination, and flexible to handle multiple treatment volumes/doses/cell concentrations and to be able to support multiple cell types in addition to CAR-T cells.
One embodiment of the magnetic cell separation holder (960 of fig. 78) is now described in more detail with particular reference to fig. 81-87. Magnetic bead-based cell selection involves the separation of certain cells from a mixture of cells via targeted binding of cell surface molecules to antibodies or ligands of magnetic beads (e.g., beads of the type described above). Once bound, cells coupled to the magnetic beads can be separated from the unbound cell population. For example, a cell mixture comprising bound and unbound cells may be passed through a separation column positioned within a magnetic field generator that captures magnetic beads and thus associated bound cells. Unbound cells pass through the column without being captured.
Certain magnetic cell separation techniques may incorporate nano-sized beads (e.g., beads about 50 nm in diameter or smaller), while other techniques may employ larger beads (e.g., beads about 2 μm in diameter or larger). For example, smaller beads may be desirable because smaller bead sizes may avoid receptor activation on target cells. Furthermore, downstream steps may skip bead removal, as nano-sized beads may have little effect on downstream processing or cell function. However, smaller nanometer-sized magnetic beads can be separated using a magnetic cell separation procedure that involves the use of a magnetic field gradient enhancer to amplify the applied magnetic field gradient. In contrast, larger beads have a higher magnetic moment. Thus, the separation of certain larger beads may not involve a magnetic field gradient enhancer. However, larger beads can still be used in conjunction with additional cell-bead separation steps. Thus, depending on the size and/or type of magnetic beads used, the workflow, appropriate magnetic parameters, and/or the separation device itself may vary, which adds to the complexity of magnetic bead-based cell separation techniques.
In particular, because the material and magnetic properties of the beads (including but not limited to size, permeability, saturation magnetization, resistivity, surface properties, and mass density) may vary, the separation conditions may also vary depending on the nature of the beads, and may involve magnetic fields of different strengths and/or different gradients. In other words, for magnetic cell separation procedures using beads with different materials and magnetic properties, the magnetic field parameters of the magnetic field generator can be varied. The method eliminates the workflow steps of using different sized beads to adjust the magnetic field generator or its parameters between magnetic cell separation procedures. In embodiments provided herein, a magnetic cell separation holder is configured for use in conjunction with a magnetic field generator such that, when used with appropriately sized beads, the magnetic cell separation holder positions the beads within a magnetic field at a location associated with a desired magnetic field characteristic of cell separation. The magnetic field generator may apply the magnetic field using preset (e.g., fixed) magnetic field parameters or static magnetic field generator elements. In this way, the operator can avoid the complexity of varying the magnetic field parameters according to the selected beads. Instead, by selecting an appropriate magnetic cell separation holder, the magnetic field experienced by the cells is suitable for separation. Furthermore, when using beads of different sizes and/or involving different desired magnetic field characteristics, different magnetic cell separation holders may be selected that position the beads at respective locations within the applied magnetic field associated with the respective desired magnetic field characteristics.
For example, different magnetic cell separation holders can be sized and shaped according to the desired positioning of cells (e.g., target cells in a cell mixture) in a magnetic field generated by a magnetic field generator. In one embodiment, each magnetic cell separation holder comprises a channel or other cell receptacle, and when the magnetic cell separation holder is loaded into a magnetic bead-based cell separation system comprising a magnetic field generator, the cells in the magnetic field separation holder are positioned at locations within the magnetic field having characteristics suitable for separating a type of magnetic beads (e.g., based on bead material, shape, size, and/or size range) from a cell mixture. By selecting a magnetic cell separation holder associated with a particular bead type, proper separation can be achieved without changing the magnetic separation device or the settings of the magnetic field generator of the magnetic separation device.
In an embodiment, a suitable magnetic holding material (such as a column matrix supported in a separation tube) is coupled to or positioned within the channel of the magnetic cell separation holder and is positioned at a location in the magnetic field within the magnetic field generator that corresponds to the desired magnetic field characteristics (i.e., magnetic field strength and magnetic field gradient) for the bead type used in the magnetic cell separation procedure. A magnetic cell separation holder as described above and an accompanying set of magnetisable beads may be provided in a kit which may comprise disposable or single-use components. The kit may also include multiple sets of beads or different types of beads and/or multiple magnetic cell separation holders, e.g., holders optimized or designed for each set of beads.
In another embodiment, a magnetic cell separation holder having multiple channels may be provided for use with corresponding different sized beads, and a user may select the appropriate channel associated with a desired bead type. For example, a magnetic cell separation holder may have a channel (e.g., configured to receive a first cell separation column) for use with beads having a first diameter at a first location, and a channel (e.g., configured to receive a second cell separation column) for use with beads having a second and larger diameter at a second location. When the magnetic cell separation holder is inserted into the magnetic cell separation device and a magnetic field is generated, the channel at the first location may be at a location that experiences a higher magnetic field strength than the channel at the second location in the magnetic cell separation holder.
In another embodiment, the magnetic cell separation holder may be pre-filled with a magnetic bead-cell mixture at a suitable location associated with a desired bead type. In addition, the magnetic cell separation device may be part of a fluid handling system of the magnetic separation system, or may be functionally attached to one or more fluid handling systems. The magnetic cell separation system may also include a controller configured to automatically perform the magnetic cell separation procedure. The magnetic separation system may be configured as a functionally closed system.
Fig. 81 depicts an alternative magnetic separation system 2100 that may be used in the alternative and in conjunction with the techniques disclosed herein for magnetic bead-based cell separation systems. The system 2100 includes a Source Pump (SP) 2112, a treatment pump (PP)2114, and a magnetic separation pump (MP) 2116. System 2100 further includes a collection pinch valve (PV-C) 2126, a waste pinch valve (PV-W) 2128, a bead addition syringe (SG1) 2118, and a check valve (CV1) 2120. In an embodiment, check valve 2120 is rated at a cracking pressure of 3 psi, for example. The system 2100 may also include suitable processing and/or source containers, such as sample Source Bag (SB) 2104, Processing Bag (PB) 2106, Buffer Bag (BB) 2108, Medium Bag (MB) 2110, Collection Bag (CB)2130, and Waste Bag (WB) 2132. Incubation remover 2102 can also be a bag or can be another collection container suitable for containing and/or disposing of waste material from system 2100.
The system 2100 is configured for use with a magnetic cell separation holder 2134 identical to the magnetic cell separation holder 960 described above with reference to fig. 78. Magnetic cell separation holder 2134 can be removably coupled to (e.g., loaded into, positioned relative to) magnetic field generator 2121 (e.g., magnetic field plates 2122 and 2124 equivalent to plates 964 and 966 in fig. 80). System 2100 is operable under the control of controller 2150 in accordance with instructions executed by processor 2152 and stored in memory 2154. Such instructions may include magnetic field parameters. The system 2100 may include any or all of the depicted components.
Fig. 82 depicts a flow diagram of a method 2200 for magnetic bead-based cell separation that can be used with a magnetic separation system (e.g., the system 2100 of fig. 81). It should be understood that the depicted method 2200 is by way of example, and that the techniques disclosed herein may be used in conjunction with other magnetic bead-based cell separation workflows. In step 2202, a source bag 2104, a culture medium bag 2110, a buffer bag 2108, and a bead addition syringe 2118 are prepared for use with the magnetic separation system. In step 2204, the source bag 2104, the culture medium bag 2110, the buffer bag 2108 and the bead addition syringe 2118 are loaded into the magnetic separation system. The source bag 2104 is fluidly coupled to a source pump 2112. Media bag 2110 and buffer bag 2108 are fluidly coupled to check valve 2120. The bead addition syringe 2118 is fluidly coupled to the processing bag 2106. In step 2206, magnetic cell separation holder 2134 is coupled to (e.g., positioned adjacent to, inserted into, loaded into) magnetic field generator 2121 (e.g., magnetic field plates 2122 and 2124) of magnetic separation system 2100. In step 2208, the bags 2104, 2110, 2108 and the syringes 2118 are aseptically welded to the magnetic separation apparatus. In step 2210, source material, such as a cell mixture, from the source bag 2104 is transferred to the processing bag 2106 via the source pump 2112.
In step 2212, magnetic beads (e.g., beads) within the bead addition syringe 2118 are added to the processing bag 2106. In step 2214, the magnetic beads are incubated with the cell mixture in the processing bag 2106. The incubation material (e.g., cell mixture and magnetic beads) can be circulated into and out of the processing bag 2106 via the processing pump 2114 to promote adequate binding between the target cells and the magnetic beads. In step 2216, the source bag 2104 is disengaged from the source pump 2112 and the incubation remover 2102 is fluidly coupled to the source pump 2112. Excess incubation material is then removed from the processing bag 2106 via source pump 2112 and deposited in the incubation remover 2102. In step 2218, magnetic cell separation is performed on the bead-labeled cell mixture. Magnetic cell separation holder 2134 is coupled to magnetic field generator 2121, and magnetic field generator 2121 then generates a magnetic field at predetermined magnetic field parameters. The bead-labeled cell mixture from the processing bag 2106 flows through the magnetic cell separation holder 2134 via the magnetic separation pump 2116. In an embodiment, magnetic cell separation holder 2134 houses a magnetic holding element or material, such as a separation column, matrix, or tube. The bead labeled cells are then magnetically held in the tube or column matrix of the magnetic cell separation holder 2134, and any material not held flows through the magnetic cell separation holder 2134 to the waste bag 2132. In an optional step, the buffer or culture medium may rinse the processing bag and the magnetic cell separation procedure may be repeated. In step 2220, the magnetic cell separation holder 2134 is removed from the magnetic separation device. In step 2222, the retained cells are then eluted by flushing the magnetic cell separation holder 2134 with a fluid at a high flow rate such that the viscous force of the fluid overcomes any residual magnetic force on the retained magnetic beads. The fluid and bead labeled cells are then collected in collection bag 2130. In step 2224, the bags (e.g., collection bag 2130, waste bag 2132, buffer bag 2108, and culture medium bag 2110) are sealed, and in one embodiment, magnetic cell separation holder 2134 can then be disposed of.
Fig. 83A and 83B illustrate top views of different configurations of a magnetic cell separation holder 2302 (e.g., magnetic cell separation holder 2134 of fig. 81) positioned within the magnetic separation device 2300 in fig. 83A and 83B. Fig. 83A depicts a magnetic cell separation holder 2302 in an unloaded configuration in a magnetic cell separation device 2300. Magnetic cell separation holder 2302 can include a body 2301, which can be formed of any suitable non-magnetic material configured to accommodate cell separation, and coupled to magnetic separation device 2300. Magnetic cell separation holder 2302 can include one or more channels formed within body 2301 or through body 2301 through which a cell mixture can flow. Although fig. 83A shows two separate channels 2303 and 2305, it is to be understood that magnetic cell separation holder 2302 can include only one channel, two or more channels, and the like. Turning to channel 2303, channel 2303 can be configured to contain magnetic holding material 2304, magnetic holding material 2304 configured to hold cells bound to magnetic beads under a magnetic field and allow unbound cells to pass through. Similarly, the channel 2305 may also house a magnetic retaining material 2306. The magnetic holding materials 2304, 2306 may be the same or different. It is also possible to omit the retaining material but provide less efficient results, for example, the channel 2305 may be a hollow tube. Further, channels 2303, 2305 can have different sizes and different positions relative to end surface 2307 of body 2301. For example, distance 2315 between end surface 2307 and a center point of channel 2303 may be different than the distance between other channels of body 2301 relative to end surface 2307. In this manner, the channel may experience a magnetic field associated with its position within body 2301.
End surface 2307 may be configured to abut a stop portion or surface 2311 of frame 2319. The frame 2319 may be configured to conduct magnetic flux. While body 2301 is shown as terminating at a point at end surface 2307, it should be understood that other configurations are contemplated. Fig. 83B shows a loading configuration in which magnetic cell separation holder 2302 is positioned within receiving area 2316 of magnetic field generator 2313. Loading may include advancing end surface 2307 toward stop surface 2311 until end surface 2307 abuts the stop surface. In the stowed configuration, portions of body 2301 may remain outside of receiving area 2316. Thus, in embodiments, one or more channels of body 2301 may be positioned within receiving area 2316 when loaded.
The magnetic separation device 2300 may also include a gate or other feature configured to reduce magnetic field leakage outside of the receiving area 2316. The steel backing 2308 of the magnetic separation device 2300 and the door 2318 of the frame 2319 are made of a soft magnetic material (e.g., 1018 steel). They are magnetized in the presence of a magnetic field and demagnetized when the magnetic field is removed. When the magnetic cell isolation holder 2302 is not inserted into the receiving area 2316 of the magnetic field generator 2313, the gate 2318 of the magnetic field generator 2313 closes the gap by means of a compressed spring attached to either gate, thereby enclosing the magnetic flux within the steel backing 2308 and the gate 2318. This prevents magnetic flux from leaking into the channels 2303, 2305 when certain processes, such as elution, are expected to demagnetize.
Fig. 83B depicts a magnetic cell separation holder 2302 in a loaded configuration in a magnetic separation device 2300. When the magnetic cell separation holder 2302 is fully inserted into the receiving region 2316 of the magnetic field generator 2313, the position of the channels 2303, 2305 is defined by the geometry of the magnetic cell separation holder 2302 and the magnetic separation device 2300. When the magnetic cell separation holder 2302 is fully inserted into the magnetic field generator 2313, a portion of the backing 2308 (e.g., stop surface 2311) of the magnetic separation device 2300 may abut a portion of the magnetic cell separation holder 2302. In addition, although the magnetic cell separation holder 2302 has a tapered shape in fig. 3A and 3B, any suitable shape of the magnetic cell separation holder 2302 may be used.
Door 2318 of magnetic field generator 2313 is opened to allow magnetic cell separation holder 2302 to be inserted between magnetic field plates 2312, 2314 of magnetic field generator 2313. For example, the location of channel 2303 within magnetic field generator 2313 may cover the location in the magnetic field having the highest magnetic field strength (i.e., 0.5T). In another example, the location of the channel 2305 within the magnetic field generator 2313 may cover the location in the magnetic field with the highest magnetic field gradient (i.e., 50T/m) while meeting the magnetic field strength requirements of the magnetic beads (i.e., 0.15T).
To elute retained beads (e.g., beads or bead-bound cells) from the magnetic retaining material (e.g., magnetic retaining material 2304, 2306), the external magnetic field can be removed by retracting the separation holder 2302 to the disengaged position (i.e., the unloaded configuration of fig. 83A). The gates are closed to ensure that no magnetic flux leaks out to affect the channels 2303, 2305 when no external magnetic field is required near the channels 2303, 2305. Then, fluid with a high flow velocity flows through the channels 2303, 2305, which creates large shear forces on the retained beads. When the viscous force is greater than the holding force (i.e., the magnetic force due to the residual magnetic field), the beads are washed off the magnetic holding material of the channels 2303, 2305 and collected. However, in other embodiments, the applied magnetic field may be terminated under the control of controller 2150.
As discussed, magnetic cell separation holder 2302 can have one or more channels, where each channel corresponds to a type and/or size of beads used in a magnetic cell separation procedure. For example, magnetic cell separation holder 2302 may have three channels: 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 channels in each magnetic cell separation holder 2302 may also be of different sizes or the same size.
Fig. 84A and 84B illustrate isometric views of different configurations of the magnetic cell separation holder and magnetic separation device of fig. 83A and 83B. FIG. 84A shows the position of magnetic cell separation holder 2302 prior to engagement of magnetic cell separation holder 2302 in magnetic separation device 2300 for magnetic separation. Fig. 84B shows the position of the magnetic cell separation holder 2302 after engagement of the magnetic cell separation holder 2302 in the magnetic field generator 2313 for magnetic separation. Frame 2319 can include opposing guide plates 2330, guide plates 2330 spaced a distance from each other to allow passage of magnetic cell separation holder 2302 therebetween and to facilitate proper positioning within receiving region 2316.
While certain disclosed techniques involve positioning a magnetic cell separation holder within a fixed position magnetic field generator as disclosed, it is to be understood that other embodiments are contemplated. For example, the magnetic field generator may be movable relative to a magnetic breakaway keeper loaded into a fixed position frame.
Fig. 85 depicts a flow diagram of a method 2500 of magnetic cell separation that can be used with a magnetic separation device. In step 2502, a first cell mixture is prepared by incubating the cell mixture with a set of magnetic beads having a desired characteristic (e.g., size, type, ligand, etc.). After a sufficient period of time has elapsed to ensure that the target cells have been labeled with magnetic beads, the excess incubation mixture is removed. In another embodiment, portions of the incubated mixture may be removed and evaluated for quality control purposes, i.e., excess incubation mixture may be evaluated to assess binding characteristics. In step 2504, first magnetic cell separation holder 2302 can be coupled within receiving region 2316 of magnetic field generator 2313. In step 2506, magnetic field generator 2313 generates a magnetic field in receiving area 2316 of magnetic field generator 2313. In step 2508, the first cell mixture flows through a channel (e.g., one or more of channels 2303 or 2305) in the first magnetic cell separation holder 2302. The magnetic bead labeled cells in the cell mixture are held in the channels by the magnetic holding material (e.g., one or more of magnetic holding materials 2304 or 2306) of the first magnetic cell separation holder 2302 while the remainder of the cell mixture material flows through the channels of the first magnetic cell separation holder 2302. In step 2510, the generation of the magnetic field is stopped by removing the first magnetic cell separation holder 2302 from the receiving region 2316 of the magnetic field generator 2313 (or by terminating the application of the magnetic field), which causes demagnetization of the magnetic cell separation holder 2302. In step 2512, the retained or separated cells and beads from the first magnetic cell separation holder 2302 are collected by eluting the magnetically retained beads or cells in the channels of the magnetic cell separation holder 2302 using a fluid with a high flow rate or another suitable method. In step 2514, optionally, magnetic cell separation holder 2302 can be disposed. Steps 2522 to 2534 mirror steps 2502 to 2514, but may instead pass cells in the second cell mixture labeled with a set of differently sized beads through a channel in the second (i.e., different) magnetic cell separation holder 2302 or a different channel of the first magnetic cell separation holder 2302. Although steps 2522 to 2534 illustrate a method using two different magnetic cell separation holders, it should be appreciated that the two magnetic cell separation holders may instead be the same magnetic cell separation holder with different channels for each cell mixture. Additionally, the second cell mixture may be the resulting cell mixture from step 2508 passed through the first magnetic cell separation holder 2302 without the retained bead-labeled cells.
The magnetic selection of the target cells may be positive or negative selection. The positive selection uses magnetic beads to label target cells, and the target cells are collected as a labeling moiety. Negative selection or depletion uses magnetic beads to label unwanted cells, and target cells are collected as unlabeled fractions.
Fig. 86 illustrates a top view of the position of the magnetic cell separation holder 2602 relative to the permanent magnets 2612, 2614 of the magnetic field generator 2600. In one embodiment, the distance between the permanent magnets 2612, 2614 is about 0.37 inches (10 mm). However, other distances between the permanent magnets may be used depending on the configuration of the separation device, such as the physical properties of the magnets, the aspect ratio of the cross-sectional area, and the magnet restraining fixture design. In the depicted embodiment, the magnetic holding material can be a column matrix 2604 for use with, for example, Miltenyi microbead-labeled cells, and the magnetic holding material 2606 can be a tube for use with, for example, Dynabead-labeled cells. For any individual magnetic cell separation procedure, a column matrix or tube may be used.
Fig. 87 depicts the magnetic field distribution of the permanent magnets and backing steel of the magnetic field generator, showing different magnetic field characteristics of the magnetic field at different locations. As disclosed, the magnetic field parameters for separation are different for different sized beads (e.g., beads). Larger beads have a higher magnetic momentum and therefore require a lower magnetic field gradient to produce an equivalent amount of force when compared to smaller beads having a lower magnetic momentum. The magnetic force can be expressed as: fmag = M · ∇ B, where M is the magnetic momentum and ∇ B is the magnetic field gradient. To ensure the highest magnetic momentum, the magnetic material must be saturated by the external magnetic field strength (i.e., 0.15T for dynabeads discussed herein). When the magnetic force is greater than the viscous force in the flow field, the magnetic beads will move in the direction of the magnetic force until they reach the sphere of the tube wall or column matrix.
In certain 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 certain types of leukocytes, T cells, from Peripheral Blood Mononuclear Cells (PBMCs). The target cells (T-cells) are modified by receptors that enable the target cells to recognize and attack cancer cells. Furthermore, the disclosed techniques may be used in conjunction with any suitable type of bead, such as, by way of example, Miltenyi nanometer-sized microbeads (50 nm in diameter) and dynabeads (4.5 μm in diameter). Miltenyi microbeads are nano-sized superparamagnetic beads that require a magnetizable column matrix to prevent them from entering the flow field. The magnetizable pillar matrix is composed of spheres of a soft magnetic material (e.g., stainless steel 400 series spheres having a diameter of 0.4 mm). Stainless steel 400 series balls are rust resistant. The magnetic properties of the spherical material relate to a strong magnetization when it is exposed to an external magnetic field and a small remanence when the external magnetic field is removed. The manufacturing process of the column matrix of magnetic holding material involves ball packing of the column matrix using a shaker, applying lacquer to the column matrix, gravity draining the lacquer, centrifuging to remove any remaining lacquer, air blowing and re-centrifuging. The steps of blowing and centrifuging may be repeated several times until all residual lacquer is removed. The column matrix was then placed in an oven at about 100 degrees celsius for three days. After the column matrix is fully cured, the column matrix is held together by the applied lacquer. A magnetizable pillar matrix filled with balls can be used as a magnetic booster to boost the magnetic field gradient up to 10000 times. In the presence of an external magnetic field, the enhanced magnetic field gradient helps attract the nano-sized bead-labeled cells to the sphere. After removal of the external magnetic field, the column matrix is demagnetized, which allows the nano-sized bead-labeled cells to be released from the column matrix. The nano-sized bead labeled cells are then eluted using a flow of washing fluid through the column matrix.
Dynabeads are large superparamagnetic beads made of synthetic polymers. Because dynabeads are much larger than Miltenyi nanometer-sized beads, dynabeads have a much higher magnetic momentum when placed in a magnetic field than Miltenyi nanometer-sized beads. Thus, magnetic cell separation using dynabeads does not necessarily involve a magnetic enhancer, such as a magnetic column matrix. Tube-based systems are typically used with Dynabead labeled cells, where a permanent magnet is placed near the tube. Target cells labeled with dynabeads are attracted to the wall of the tube, and unlabeled cells can then be removed with buffer or culture medium. In addition to Miltenyi nano-sized microbeads and dynabeads, other beads of different sizes are also commercially available.
The magnetic separation device may use a magnetic field generator, such as a pair of permanent magnets, as well as a magnetic cell separation holder and accompanying magnetic cell holding material, flow conduits, collection and preparation vessels, and other components of the disclosed system 2100. Further, some of these components may be provided as single-use components, disposable components, and/or packaged as a kit.
In embodiments, a dedicated kit may be provided to achieve magnetic separation for a particular bead type. For any particular separation event, a kit optimized for one or more bead sizes may be provided. The kit may comprise a suitable magnetic holding material, which may be pre-loaded into a suitably configured magnetic cell separation holder. In this way, a user cannot accidentally load or couple incorrect magnetic retaining material into the channel of the magnetic separation holder. In embodiments for use with Miltenyi microbeads, the magnetic retention posts in the channels of the magnetic separation holder, when loaded, may be positioned in the center of the gap or space between the permanent magnets of the magnetic field generator and associated with the highest or higher magnetic field strength (i.e., greater than 0.45T). In another embodiment, the magnetic holding tube for dynabeads may be positioned in the highest gradient region in the middle of the permanent magnet. Dynabead separation can be performed in conjunction with a magnetic separation holder having a channel positioned relative to a magnetic field generator to experience both a magnetic field strength (i.e., greater than 0.1T) and a magnetic field gradient (i.e., greater than 40T/m) suitable for holding.
When used in conjunction with the disclosed technology, the average recovery and average purity of CD3+ using the magnetic separation device was each approximately greater than 80% for Miltenyi nanometer-sized beads. For Dynabeads, the average recovery of CD3+ using the magnetic separation device was approximately 60%, and the average purity of CD3+ using the magnetic separation device was approximately greater than 70%.
Technical effects of the present disclosure include providing a holder for magnetic separation of cells for use with a magnetic field generator to enable cell separation using different sized magnetic beads without the need to adjust magnetic field parameters between procedures. In addition, the magnetic separation device can automatically perform methods of cell preparation, magnetic cell separation, and cell elution for each of the different sized beads to eliminate or reduce user interaction and manipulation of the source material.
As used herein, an element or step recited in the singular and proceeded with the word "a" or "an" should be understood as not excluding plural said elements or steps, unless such exclusion is explicitly recited. Furthermore, references to "one embodiment" of the present invention are not intended to be interpreted as excluding the existence of additional embodiments that also incorporate the recited features. Furthermore, unless explicitly stated to the contrary, embodiments "comprising," "including," or "having" an element or a plurality of elements having a particular property may include additional such elements not having that property.
This written description uses examples to disclose several embodiments of the invention, including the best mode, and also to enable any person skilled in the art to practice the embodiments of the invention, including making and using any devices or systems and performing any incorporated methods. The patentable scope of the invention is defined by the claims, and may include other examples that occur to those of ordinary skill in the art. Such other examples are intended to be within the scope of the claims if they have structural elements that do not differ from the literal language of the claims, or if they include equivalent structural elements with insubstantial differences from the literal languages of the claims.

Claims (54)

1. A method of biological treatment, the method comprising the steps of:
combining a suspension comprising a population of cells with magnetic beads to form a bead-bound population of cells in the suspension;
separating the bead-bound cell population on a magnetic separation column; and
collecting target cells from the cell population;
wherein collecting the target cells comprises removing the bead-bound cells from the separation column using an air plug.
2. The method of claim 1, further comprising the steps of:
circulating a buffer solution through the separation column to elute the bead-bound cells from the separation column.
3. The method of claim 2, wherein:
the buffer solution comprises a separation buffer, a cell culture medium or a cell release buffer.
4. The method of claim 1, further comprising the steps of:
combining the magnetic beads with a separation buffer and separating the magnetic beads on the separation column prior to combining with the population of cells.
5. The method of claim 1, wherein:
collecting target cells includes collecting bead-bound cells using a release buffer to generate the target cells, incubating to release the target cells from the magnetic beads, and separating the magnetic beads on a separation column and collecting the released target cells.
6. The method of claim 1, further comprising the steps of:
washing the target cells to replace the suspension liquid.
7. The method of claim 1, further comprising the steps of:
concentrating the target cells.
8. The method of claim 1, further comprising the steps of:
the target cells are washed and concentrated.
9. The method of claim 1, wherein:
isolating the target cells further comprises flowing a separation buffer through the separation column to wash the isolated population of cells.
10. The method of claim 1, wherein:
isolating the target cells further comprises eluting the isolated cell population from the separation column with a separation buffer to effect washing, and then separating the washed bead-bound cell population on a magnetic separation column.
11. The method of claim 1, wherein:
the magnetic beads are nano-sized beads, and after incubation and prior to isolating the target cells with a magnet, the suspension is washed to remove excess magnetic beads.
12. The method of claim 1, wherein the magnetic beads are dynabeads, Miltenyi beads, or StemCell EasySep beads.
13. The method of claim 1, further comprising the steps of:
drawing air into a treatment bag through a port in the treatment bag; and
circulating the air through a treatment line in fluid communication with the treatment bag to clear the treatment line.
14. The method of claim 1, further comprising the steps of:
detecting air within the processing loop;
wherein detection of the air indicates that the processing bag is empty.
15. The method of claim 1, further comprising the steps of:
enriching a desired cell population from a biological sample.
16. The method of claim 15, wherein:
enriching the desired cell population includes transferring the biological sample and buffer to a processing chamber and washing the biological sample in the processing chamber to reduce the amount of platelets and serum in the biological sample.
17. The method of claim 16, further comprising the steps of:
after enrichment, transferring the enriched cell population to a processing bag; and
circulating the enriched population of cells through the processing loop.
18. A system, comprising:
a magnetic field generator configured to generate a magnetic field at a magnetic field parameter;
a first holder configured to be removably coupled to the magnetic field generator, the first holder comprising a first channel configured to be positioned within the magnetic field at a first location when the first holder is coupled to the magnetic field generator; and
a second retainer configured to be removably coupled to the magnetic field generator, the second retainer including a second channel configured to be positioned within the magnetic field at a second location when the second retainer is coupled to the magnetic field generator,
wherein:
the first channel experiences a first magnetic field strength and a first magnetic field gradient within the magnetic field produced under the magnetic field parameters at the first location; and is
The second channel experiences a second magnetic field strength and a second magnetic field gradient within the magnetic field at the magnetic field parameters at the second location, and wherein the second magnetic field strength is different from the first magnetic field strength, or the second magnetic field gradient is different from the first magnetic field gradient, or a combination thereof.
19. The system of claim 18, wherein the magnetic field generator is coupled to a frame that conducts magnetic flux and forms a receiving area configured to receive the first holder or the second holder.
20. The system of claim 19, wherein the receiving area is sized to receive only one of the first holder or the second holder at a given time.
21. The system of claim 19, wherein the receiving area is configured to receive only a portion of the first holder or the second holder.
22. The system of claim 21, wherein the portion comprises the first channel of the first holder or the second channel of the second holder.
23. The system of claim 19, wherein the frame comprises a retractable portion that reduces the magnetic flux generated by the magnetic field generator from exceeding the receiving area when the first holder is disengaged from the magnetic field generator.
24. The system of claim 23, wherein the retractable portion comprises a spring that compresses to allow the first holder to enter the receiving area of the frame.
25. The system of claim 18, wherein the first channel does not experience the first magnetic field strength when the first holder is not coupled to the magnetic field generator.
26. The system of claim 19, wherein the first holder comprises a first end surface configured to abut a stop portion of the frame when the first holder is coupled to the magnetic field generator.
27. The system of claim 26, wherein the second retainer comprises a second end surface configured to abut the stop portion of the frame when the second retainer is coupled to the magnetic field generator, and wherein a first distance between the first end surface and the first channel is different than a second distance between the second end surface and the second channel.
28. The system of claim 18, wherein the first channel and the second channel have different dimensions.
29. The system of claim 18, wherein the first holder is configured to be fluidly coupled to a first source of first-sized beads, and wherein the second holder is configured to be fluidly coupled to a second source of second-sized beads.
30. The system of claim 29, wherein the first size beads are less than 1 μm in diameter and the second size beads are greater than 2 μm in diameter.
31. The system of claim 29, wherein the first sized beads bind to target cells within a first cell mixture and the second sized beads bind to target cells within a second cell mixture.
32. The system of claim 18, wherein the first channel or the second channel comprises a magnetic booster, such as a plurality of magnetizable balls.
33. A magnetic cell separation holder, comprising:
a body configured to be removably coupled to a magnetic field generator, the body comprising a first channel configured to be positioned within a magnetic field of the magnetic field generator at a first location when the retainer is coupled to the magnetic field generator and a second channel configured to be positioned within the magnetic field at a second location when the retainer is coupled to the magnetic field generator,
wherein:
the first channel experiences a first magnetic field strength and a first magnetic field gradient within the magnetic field produced under magnetic field parameters at the first location; and is
The second channel experiences a second magnetic field strength and a second magnetic field gradient within the magnetic field at the magnetic field parameters at the second location, and wherein the second magnetic field strength is different from the first magnetic field strength, or the second magnetic field gradient is different from the first magnetic field gradient, or a combination thereof.
34. A system, comprising:
a first kit, comprising:
a plurality of first-sized beads; and
a first holder comprising a first channel configured to receive the plurality of first-sized beads, the first channel positioned within the first holder such that the first holder is positioned within a magnetic field that generates the magnetic field at a first location when the first holder is removably coupled to the magnetic field generator; and
a second kit of parts comprising:
a plurality of second-sized beads; and
a second holder comprising a second channel configured to receive the plurality of second-sized beads, the second channel positioned within the second holder such that, when the second holder is removably coupled to the magnetic field generator that generates the magnetic field, the second holder is positioned within the magnetic field at a second location different from the first location;
wherein:
the first channel experiences a first magnetic field strength and a first magnetic field gradient within the magnetic field at the first location; and is
The second channel experiences a second magnetic field strength and a second magnetic field gradient within the magnetic field at the second location, and wherein the second magnetic field strength is different from the first magnetic field strength, or the second magnetic field gradient is different from the first magnetic field gradient, or a combination thereof.
35. A method for isolating a target cell, comprising:
positioning a first holder having a first channel within a receiving area of a frame coupled to a magnetic field generator;
generating, by the magnetic field generator, a first magnetic field in the receiving region when the first holder is coupled to the magnetic field generator to cause the first channel to experience a first magnetic field strength, a first magnetic field gradient, or both;
positioning a second retainer having a second channel within the receiving area, wherein the first channel and the second channel are positioned at different locations within the receiving area; and
when the second holder is coupled to the magnetic field generator, a second magnetic field is generated in the receiving area by the magnetic field generator to cause the second channel to experience a second magnetic field strength, a second magnetic field gradient, or both.
36. The method of claim 35, wherein the first magnetic field and the second magnetic field are generated at the same magnetic field parameters.
37. The method of claim 35, wherein the second retainer is not positioned within the receiving area of the frame when the first retainer is positioned within the receiving area of the frame, and the first retainer is not positioned within the receiving area of the frame when the second retainer is positioned within the receiving area of the frame.
38. The method of claim 35, further comprising:
incubating a first cell mixture with first size beads and subsequently incubating a second cell mixture with second size beads such that target cells in the first cell mixture are labeled with the first size beads and target cells in the second cell mixture are labeled with the second size beads; and
passing the first cell mixture through the first channel and subsequently passing the second cell mixture through the second channel.
39. A kit for use in a bioprocessing system, comprising:
processing the bags;
a source bag;
a bead addition vessel;
a processing circuit configured to be in fluid communication with the processing bag, the source bag, and the bead addition container;
wherein the treatment circuit comprises a pump conduit configured to be in fluid communication with a pump.
40. The kit of claim 39, further comprising:
a separation column;
a waste bag;
a buffer solution bag; and
a collection bag;
wherein the processing circuit is configured to be in fluid communication with the separation column, the waste bag, the buffer bag and the collection bag.
41. The kit of claim 39, further comprising:
a valve manifold operable to selectively place the source bag, the processing bag, the bead addition container, and the processing circuit in fluid communication.
42. The kit of claim 39, further comprising:
a storage bag configured to be in fluid communication with the processing circuit.
43. The kit of claim 39, further comprising:
a processing chamber configured to be in fluid communication with the processing circuit.
44. The kit of claim 39, wherein:
the bead addition vessel is a syringe for injecting the magnetically separable beads into the processing circuit.
45. The kit of claim 39, further comprising:
a release buffer pocket configured to be in fluid communication with the processing circuit.
46. The kit of claim 39, further comprising:
a media bag configured to be in fluid communication with the processing circuit.
47. An apparatus for bioprocessing, comprising:
a kit comprising a processing bag configured to be in fluid communication with a processing circuit, the processing circuit additionally comprising a pump conduit configured to be in fluid communication with a pump, a source bag, and a bead addition container;
a magnetic field generator configured to generate a magnetic field;
a plurality of hooks for suspending the source bag, the processing bag, and the bead addition container, each hook of the plurality of hooks operatively connected to a load cell configured to sense a weight of the bag connected thereto;
at least one bubble sensor; and
a pump configured to be in fluid communication with the treatment circuit.
48. The apparatus of claim 47, wherein:
the processing bag includes an air port;
wherein the pump is operable to draw air into the system through the air port.
49. The apparatus of claim 48, wherein:
the air port is in a top portion of the processing bag, the top portion being above a fluid level of the processing bag.
50. The apparatus of claim 47, further comprising:
a second waste bag in selective fluid communication with the valve manifold.
51. The apparatus of claim 47, wherein:
the waste bags comprise a first waste bag and a second waste bag; wherein the first and second waste bags are in fluid communication with the valve manifold.
52. The apparatus of claim 47, further comprising:
a first storage bag and a second storage bag configured to be in fluid communication with the valve manifold.
53. The apparatus of claim 50, further comprising:
a release medium bag in selective fluid communication with the valve manifold.
54. The apparatus of claim 47, further comprising:
a first pump tube having a first inner diameter;
a second pumping tube having a second inner diameter greater than the inner diameter of the first pumping tube;
wherein the first and second pump tubes are selectively engageable with the pump to achieve a range of flow rates for the pump.
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