CN117343836A - Methods for cell enrichment and isolation - Google Patents
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- CN117343836A CN117343836A CN202310912295.9A CN202310912295A CN117343836A CN 117343836 A CN117343836 A CN 117343836A CN 202310912295 A CN202310912295 A CN 202310912295A CN 117343836 A CN117343836 A CN 117343836A
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- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
- C12M47/10—Separation or concentration of fermentation products
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N13/00—Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
- C12N2509/10—Mechanical dissociation
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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 population of cells that bind the beads in the suspension; separating the bead-bound cell population on a magnetic separation column; and collecting the target cells from the population of cells. Collecting the target cells includes removing the bound bead cells from the separation column using an air plug. Also disclosed is a system including a magnetic field generator, a magnetic cell separation holder, a disposable fluid kit, and associated methods of use.
Description
Technical Field
Embodiments of the present invention relate generally 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 treatment procedures. For example, chimeric Antigen Receptor (CAR) T cell therapy is a cell therapy that redirects T cells of a patient 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. The general premise of CAR-T cells is to artificially generate T cells that target markers found on cancer cells. Scientists can remove T cells from the human body, genetically engineer them, and put them back into the patient for them to attack cancer cells. The CAR-T cells may 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., white blood cell apheresis) to remove blood from a patient and isolate white blood cells. 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 specific biomarker is then isolated from the enriched subpopulation using a specific antibody conjugate or marker.
After isolation of the T cells of interest, the cells are activated in an environment in which they can actively proliferate. For example, magnetic beads coated with anti-CD 3/anti-CD 28 monoclonal antibodies or cell-based artificial antigen presenting cells (aapcs) may be used to activate cells, which may be removed from culture using magnetic separation. The CAR gene is then used to transduce T cells by integrating a gamma Retrovirus (RV) or Lentiviral (LV) vector. Viral vectors use viral mechanisms to attach to patient cells and, upon entry into the cells, the vector introduces genetic material in the form of RNA. In the case of CAR-T cell therapy, this genetic material encodes the CAR. RNA is reverse transcribed into DNA and permanently integrated into the genome of the patient's cells; allowing CAR expression to be maintained as the cells divide and grow in bulk in the bioreactor. The CAR is then transcribed and translated by the patient cells, and the CAR is expressed on the cell surface.
After T cells are activated and transduced with a viral vector encoding a CAR, the cells are expanded in bulk 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. While recent efforts to automate the manufacturing process have eliminated some of the human contact points, these systems still suffer from high cost, inflexibility, and workflow bottlenecks. In particular, systems that utilize increased automation are very expensive and inflexible as they require the consumer to adapt their process to the specific equipment of the system.
In view of the above, there is a need for a biological treatment 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 biological treatment system for cell therapy manufacturing that balances the need for flexibility of development and consistency of mass production and meets the desire of different consumers to run different processes.
Disclosure of Invention
Certain embodiments commensurate in scope with the originally claimed subject matter are summarized below. These embodiments are not intended to limit the scope of the claimed subject matter, but rather these embodiments are intended only to provide a brief overview 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 biological treatment system includes: 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 biological treatment system includes: a first module configured for enriching and isolating cells; a plurality of second modules, each second module configured for activating, genetically transducing and expanding cells; and a third module configured for harvesting the cells after expansion. Each second module is configured to support activation, genetic transduction, and expansion of different cell populations in parallel with each other.
In another embodiment, a method of biological treatment comprises the steps of: enriching and isolating a population of cells in a first module; activating, genetically transducing and expanding a population of cells in a second module; and in a third module, harvesting the expanded cell population. The steps of activating, genetically transducing and expanding the population of cells are performed without removing the population of cells from the second module.
In another embodiment, an apparatus for biological treatment 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 generally open top. The drawer is movable between a closed position in which the drawer is received within the housing and an open position in which the drawer extends from the housing, thereby enabling access to the process chamber through the open top. The apparatus also includes at least one seat plate positioned within the processing chamber and configured to receive the bioreactor container.
In another embodiment, a method of biological treatment comprises the steps of: sliding a drawer having a plurality of side walls, a bottom, and a substantially open top from a closed position within the housing to an open position such that the drawer extends from the housing through the substantially open top; positioning the bioreactor vessel through a substantially open top on a static seat plate located within a 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 biological treatment includes: a housing; a first drawer receivable within the housing, the first drawer including a plurality of sidewalls and a bottom defining a first process 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 the 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 process 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 are received within the housing and an open position in which the first drawer and/or the second drawer extend from the housing, thereby enabling access to the process chamber through the open top, respectively.
In yet another embodiment, an apparatus for biological treatment includes: a housing; a drawer receivable within the housing, the drawer including a plurality of side walls and a bottom surface defining a process 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 to enable access to the process chamber through the open top; at least one seat plate positioned within the process chamber adjacent the bottom surface; and a kit that is receivable within the processing chamber. The kit includes: a plurality of side walls 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 container positioned within the interior compartment above the at least one opening and supported by the bottom surface such that a portion of the bioreactor container is accessible through the opening in the bottom surface. The kit is receivable within the processing chamber such that the seat plate extends through an 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 biological treatment includes: a tray having a plurality of side walls and a bottom surface defining an interior compartment, and a substantially open top; at least one opening formed in the bottom surface, the at least one opening having a perimeter; a first conduit retainer block integrated with the tray and configured to receive and hold in place at least one pump tube for selective engagement with the pump; a second conduit retainer block integrated with the tray and configured to receive and hold in place each of the plurality of pinch valve tubes for selective engagement with a respective actuator of the pinch valve array; and a bioreactor container positioned within the interior compartment above the at least one opening and supported by the bottom surface such that a portion of the bioreactor container is accessible through the opening in the bottom surface.
In yet another embodiment, a system for biological treatment includes: a process chamber having a plurality of sidewalls, a bottom surface, and a substantially open top; a seat plate positioned within the processing chamber adjacent the bottom surface; and a tray. The tray includes: a plurality of side walls 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 is positionable over the opening and supported by the bottom surface of the tray while portions of the bioreactor container are 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 biological treatment includes a tray having: a plurality of side walls and a bottom surface defining an interior compartment; and a substantially open top; and at least one opening in the bottom surface, the opening being defined by a peripheral edge, wherein the opening is shaped and/or dimensioned such that the bioreactor container can be positioned 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 comprises the steps of: placing the bioreactor container in a disposable tray having a plurality of side walls and a bottom surface defining an interior compartment, a substantially open top, an opening formed in the bottom surface, and a plurality of protrusions or projections 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 protrusions; and placing the tray into a process chamber having a seat plate such that the seat plate is received through an opening in the tray and supports the bioreactor container.
In yet another embodiment, a piping module for a biological treatment system includes: a first tubing retainer block configured to receive and hold in place at least one pump tube for selective engagement with a peristaltic pump; and a second conduit retainer block configured to receive the plurality of pinch valve tubes and to retain 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 conduit retainer blocks are interconnected.
In yet another embodiment, a system for biological treatment includes: a tray having a plurality of side walls and a bottom surface defining an interior compartment and a generally 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 the rear sidewall of the tray; and a duct module positioned at a rear of the tray. The pipe module includes: a first tubing retainer block configured to receive and hold in place at least one pump tube for selective engagement with a pump assembly; and a second conduit retainer block configured to receive the plurality of pinch valve tubes and to retain 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 bottom plate; a container body coupled to the floor, the container body and the floor 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 base plate for aligning the bioreactor container on the base plate.
In yet another embodiment, a method for biological treatment includes: operatively connecting a base plate to the vessel body to define an interior compartment therebetween, the base plate and the vessel body forming a bioreactor vessel; aligning the recess in the base plate with an alignment pin of the biological treatment system; and positioning the bioreactor vessel on a floor of the biological treatment system.
In yet another embodiment, a biological treatment system includes: a first fluid assembly having a first fluid assembly line connected to the first port of the first bioreactor vessel through 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 fluid assembly 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 a 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 interconnecting lines 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 comprises: providing a first fluid assembly having a first fluid assembly line connected to a first port of a first bioreactor vessel through the first bioreactor line of the first bioreactor vessel; providing a second fluid assembly having a second fluid assembly line connected to a second port of the first bioreactor vessel through a second bioreactor line of the first bioreactor vessel; and providing an 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 biological treatment method 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 genetically modified cell population within the bioreactor vessel without removing the genetically modified cell population from the bioreactor vessel to produce a number of genetically modified cells in one or more doses sufficient for use in cell therapy treatment.
In yet another embodiment, a biological treatment method includes: coating the bioreactor container with a reagent for increasing the efficiency of genetic modification of the cell population; genetically modifying cells of the cell population to produce a genetically modified cell population; and amplifying 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 the cell population in the bioreactor container using magnetic or non-magnetic beads to produce an activated cell population; genetically modifying the activated cells in the bioreactor vessel to produce a genetically modified population of cells; washing the genetically modified cells in the bioreactor vessel to remove unwanted material; and amplifying the genetically modified cell population in the bioreactor vessel to produce an amplified transduced cell population. Activation, genetic modification, washing, and expansion 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 processing circuit additionally includes a pump conduit configured to be in fluid communication with the pump.
In yet another embodiment, an apparatus for biological treatment 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 further 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 hanging a source bag, a process bag, and a bead addition container, each hook of the plurality of hooks operatively connected to a load cell (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 processing circuit.
In an embodiment, a method of biological treatment comprises: combining a suspension comprising a population of cells with magnetic beads to form a population of cells that bind the beads in the suspension; separating the bead-bound cell population on a magnetic separation column; and collecting the target cells from the population of cells.
In one embodiment, a system includes: a magnetic field generator configured to generate a magnetic field under a magnetic field parameter; a first holder configured to be removably coupled to a 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 position 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 position 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 under the magnetic field parameters at the first location. The channel of the second holder experiences a second magnetic field strength and a second magnetic field gradient within the magnetic field generated under the magnetic field parameter 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 position 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. The first channel experiences a first magnetic field strength and a first magnetic field gradient within a magnetic field generated at the magnetic field parameter at the first location, and the second channel experiences a second magnetic field strength and a second magnetic field gradient within a magnetic field at the magnetic field parameter at the second location. 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 system includes: a first kit having a plurality of first-sized beads and a first holder having a channel configured to receive the plurality of first-sized 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 the magnetic field generated by the magnetic field generator at the first position. 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 a 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 a second location. 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 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 when the first holder is coupled to the magnetic field generator, generating a first magnetic field in the receiving region by the magnetic field generator such that the channel of the first holder experiences the first magnetic field strength, the first magnetic field gradient, or both. The method further comprises the steps of: positioning a second retainer having a channel within the receiving area; and generating a second magnetic field in the receiving region by the magnetic field generator when the second holder is coupled to the magnetic field generator such that the channel of the second holder experiences a second magnetic field strength, a second magnetic field gradient, or both. The channel of the first holder and the channel of the second holder 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 accompanying drawings, in which:
fig. 1 is a schematic illustration of a biological treatment system according to an embodiment of the invention.
Fig. 2 is a schematic illustration of a biological treatment 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 subsystem of the biological treatment 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 the 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, insert-type kit of the biological treatment system of fig. 1, in accordance with an embodiment of the present invention.
Fig. 13 is another perspective top front view of the disposable insert kit of fig. 12.
Fig. 14 is a perspective top rear view of the disposable insert kit of fig. 12.
Fig. 15 is a perspective view of the tray of the disposable insert kit of fig. 12 according to an embodiment of the invention.
Fig. 16 is a front perspective view of a tubing module of the disposable insert kit of fig. 12 according to an embodiment of the present invention.
Fig. 17 is a rear perspective view of the pipe module of fig. 16.
Fig. 18 is a front view of a second conduit retainer block of a conduit module in accordance with an embodiment of the present invention.
Fig. 19 is a cross-sectional view of the second conduit retainer block of fig. 18.
Fig. 20 is another perspective front view of the male kit of fig. 12, showing a flow architecture integrated therein.
Fig. 21 is a perspective rear view of the male kit of fig. 12, showing a flow architecture integrated therein.
Fig. 22 is a front elevation view of the male kit of fig. 12, showing a flow architecture integrated therein.
Fig. 23 is a perspective view of a biological treatment apparatus according to an embodiment of the present invention.
Fig. 24 is a perspective view of a drawer for receiving the biological treatment device of 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 a process chamber of the drawer of FIG. 24.
Fig. 27 is a top plan view of the process chamber of the drawer.
Fig. 28 is a top plan view of the seat plate of the biological treatment 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 elevation view of the biological treatment apparatus of fig. 12.
Fig. 30 is a perspective view of a drawer engagement actuator of the biological treatment apparatus of fig. 12.
FIG. 31 is a top plan view of a drawer of the biological treatment apparatus illustrating the gap positions of the drawer engagement actuator, pump assembly, and solenoid array.
FIG. 32 is a top plan view of a drawer of the biological treatment apparatus illustrating the engaged positions of the drawer engaging actuator, pump assembly and solenoid array.
FIG. 33 is a perspective view of the biological treatment apparatus illustrating the male set in place within the treatment chamber of the drawer.
Fig. 34 is a top plan view of the biological treatment apparatus illustrating the male kit in place within the treatment chamber of the drawer.
Fig. 35 is a perspective view of a peristaltic pump assembly of a biological treatment device.
Fig. 36 is a side elevation view of the peristaltic pump assembly and tubing retainer module of the male set, illustrating the relationship between the components.
FIG. 37 is a perspective view of a solenoid array and pinch valve anvil forming a pinch valve array of a biological treatment device.
FIG. 38 is another perspective view of a pinch valve array of a biological treatment apparatus.
FIG. 39 is another perspective view of the pinch valve array illustrating the positioning of the tubing retainer module of the male kit in an engaged position relative to the pinch valve array.
FIG. 40 is a cross-sectional view of a drawer of the biological treatment apparatus illustrating the placement of the bioreactor vessel on the seat plate.
Fig. 41 is a side elevation view of the bioreactor received on the seat plate, illustrating the stirring/mixing mode of operation of the bioreactor system.
Fig. 42 is a side cross-sectional view of the bioreactor received on the seat plate, illustrating the 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 vessel during the stirring/mixing mode of operation.
Fig. 45 is a perspective view of a biological treatment apparatus having a front panel flipped downward, showing a treatment drawer of the biological treatment apparatus in an open position, in accordance with an embodiment of the invention.
FIG. 46 is another perspective view of the biological treatment apparatus of FIG. 45, showing the treatment drawer of the biological treatment apparatus in an open position.
FIG. 47 is an enlarged perspective view of the auxiliary compartment of the biological treatment apparatus of FIG. 45, showing the treatment drawer in a closed position in which the auxiliary compartment is accessible.
FIG. 48 is another enlarged perspective view of the auxiliary compartment of the biological treatment apparatus of FIG. 45, showing the treatment drawer in a closed position in which the auxiliary compartment is accessible.
FIG. 49 is a perspective view of the biological treatment apparatus of FIG. 45 showing the treatment drawer of the biological treatment apparatus in a closed position in which the auxiliary compartment is accessible.
FIG. 50 is another perspective view of the biological treatment apparatus of FIG. 45 showing the treatment drawer of the biological treatment apparatus in a closed position in which the auxiliary compartment is accessible.
Fig. 51 is a perspective view of an auxiliary compartment of a biological treatment apparatus according to another embodiment of the invention.
Fig. 52 is a perspective view of a biological treatment system having a waste tray in accordance with an embodiment of the invention.
Fig. 53-77 are schematic illustrations of automated generic protocols for a biological treatment system utilizing the fluid flow architecture of fig. 3, according to an embodiment of the invention.
FIG. 78 is a perspective view of an enrichment and separation device according to an embodiment of the present 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 apparatus of FIG. 78 for performing enrichment and isolation of a cell population.
FIG. 81 is a block diagram of a magnetic particle-based cell selection system that can be used with a magnetic cell separation holder in accordance with aspects of the present disclosure.
Fig. 82 is a flow chart 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 separation 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 loading configuration relative to a magnetic field generator in a loading configuration, in accordance with aspects of the present disclosure.
Fig. 85 is a flow chart 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 stowed 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 numbers will be used throughout the drawings to refer to the same or like parts.
As used herein, the term "flexible" or "collapsible" refers to a structure or material that is pliable or capable of bending without breaking, and may also refer to a compressible or expandable material. 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 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 flexible tubes or catheters, but still structures that do not longitudinally collapse under normal conditions and forces.
The term "container" as used herein refers to a flexible bag, a flexible reservoir, a semi-rigid reservoir, a rigid reservoir, or a flexible or semi-rigid tubing, 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, for example, that serves as a containment device for a variety of fluids and/or media.
As used herein, "fluid coupling" or "fluid communication" means that components of the system are capable of receiving or transmitting fluid between the components. The term fluid includes a gas, a liquid, or a combination thereof. As used herein, "electrical communication" or "electrically coupled" means that certain components are configured to communicate with each other by direct or indirect signaling through direct or indirect electrical connection. As used herein, "operatively coupled" refers to a connection that may be direct or indirect. The connection need not be a mechanical attachment.
As used herein, the term "pallet" 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 sterilized and single-use disposable products.
As used herein, the term "functionally closed system" refers to a plurality of components that make up a closed fluid path that may have an inlet port and an outlet port to add or remove fluid or air to or from the system without compromising the integrity of the closed fluid path (e.g., to maintain an internally sterile biomedical fluid path), whereby the ports may include, for example, a filter or membrane at each port to maintain sterile integrity when adding or removing fluid or air to or from the system. Depending on the given embodiment, the components may include, but are not limited to, one or more conduits, valves (e.g., multiport shunts), containers, receptacles, and ports.
Embodiments of the present invention provide systems and methods for manufacturing cellular immunotherapy from biological samples (e.g., blood, tissue, etc.). In an embodiment, a method of biological treatment comprises: combining a suspension comprising a population of cells with magnetic beads to form a population of cells that bind the beads in the suspension; separating the bead-bound cell population on a magnetic separation column; and collecting the target cells from the population of cells. Collecting the target cells includes removing the bound bead cells from the separation column using an air plug.
Referring to FIG. 1, a schematic illustration of a biological treatment system 10 is illustrated, in accordance with an embodiment of the present invention. The biological treatment system 10 is configured for use in the manufacture of cellular immunotherapy (e.g., autologous cellular immunotherapy), wherein, for example, human blood, fluid, tissue, or cell samples are collected and cellular therapy is generated from or based on the collected samples. One type of cellular immunotherapy that may be manufactured 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 fig. 1, the manufacture of CAR T cell therapy generally begins with the collection of patient blood and the isolation of lymphocytes by apheresis. The collection/apheresis may be performed in a clinical setting, and the apheresis product is then sent to a laboratory or manufacturing facility for production of CAR T cells. In particular, 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 manufacturing cell therapies, and the 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, the cells are harvested and a dose is formulated. The formulation is then typically cryopreserved and delivered to a clinical setting for thawing, preparation and final infusion into a patient.
With further reference to FIG. 1, the biological treatment 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, 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., coupled to the first controller 110, the second controller 210, and the third controller 310, respectively). The controllers 110, 210, and 310 are configured to provide substantially automated control of the manufacturing process within each module. While 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 concert with other modules to form a single, coherent biological treatment system 10, as discussed in detail below.
By automating the process within each module, product consistency from each module can be improved and costs associated with extensive manual manipulation reduced. In addition, as discussed in detail below, each module 100, 200, 300 is substantially enclosed, which helps to ensure patient safety by reducing the risk of external contamination, ensure regulatory compliance, and help to avoid costs associated with open systems. Furthermore, each module 100, 200, 300 is scalable to support both development at low patient numbers and commercial manufacturing at high patient numbers.
With further reference to fig. 1, the particular manner in which the process steps are divided into different modules that each provide closed and automated biological processing allows for efficient utilization of capital equipment to the extent heretofore unseen in the art. As will be appreciated, the step of expanding the cell population to achieve the desired cell density prior to harvesting and formulation is typically the most time consuming step in the manufacturing process, while the enrichment and isolation step, the harvesting and formulation step, and the activation and genetic modification steps are much less time consuming. Thus, attempting to automate the entire cell therapy manufacturing process can exacerbate bottlenecks in the process that impede workflow and reduce manufacturing efficiency, in addition to being logistically challenging. In particular, in a fully automated process, although 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, manufacturing cell therapies from a first sample (e.g., the blood of a first patient) will proceed rapidly until an expansion step that requires 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 equipment performing cell amplification from the first sample, and the processing of the second sample may not begin until the entire system is emptied for use. In this regard, in the case of a fully automated biological treatment system, the entire system is essentially off-line and unavailable for processing the second sample until the entire cell therapy manufacturing process from enrichment to harvest/formulation is completed for the first sample.
However, embodiments of the present invention allow more than one sample (from the same or different patients) to be processed in parallel to provide more efficient utilization of capital resources. As implied above, this advantage is a direct consequence of the particular way in which the process steps are divided into the three modules 100, 200, 300. Referring specifically to fig. 2, in an embodiment, a single first module 100 and/or a single third module 300 may be utilized in conjunction with multiple second modules (e.g., second modules 200a, 200b, 200 c) in biological treatment system 12 to provide parallel and asynchronous processing of multiple samples from the same or different patients. For example, the first module 100 can 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 can be transferred to one of the second modules, e.g., module 200a, for activation, genetic modification, and expansion under the control of the 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 in processing a second apheresis product from, for example, a second patient. Then, a second population of target cells generated in the first module 100 from the sample taken from the second patient may be transferred to another second module, e.g., second module 200b, for activation, genetic modification, and expansion 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. Then, a third target population of cells generated in the first module 100 from the sample taken from a third patient may be transferred to another second module, such as second module 200c, for activation, genetic modification, and expansion 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, etc.
The method also allows post-processing to occur asynchronously as needed. In other words, patient cells may not all grow at the same time. The culture may reach the final density at different times, but the plurality of second modules 200 are not linked and the third module 300 may be used as desired. In the case of the present invention, although the samples may be processed in parallel, they do not have to be performed batchwise.
When each expanded cell population is ready for harvesting, harvesting of the expanded cell population from the second modules 200a, 200b, and 200c can likewise be accomplished using a single third module 300.
Thus, by separating the steps of activation, genetic modification and expansion (which are the most time consuming and share certain operational requirements and/or require similar culture conditions) into separate, automated and functionally closed modules, another system device for enrichment, isolation, harvesting and formulation is not occupied or taken off-line when performing the expansion of one cell population. As a result, the manufacture of multicellular therapy can be performed simultaneously, thereby maximizing the utilization of equipment and floor space and improving the efficiency of the overall process and facility. It is contemplated that additional second modules may be added to biological treatment system 10 to provide parallel processing of any number of cell populations, as desired. Thus, the biological treatment system of the present invention allows for 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 manufacture of immunotherapy and regenerative medicine) from a single 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 present invention is discussed in detail below.
In embodiments, the third module 300 can similarly be any system or device capable of harvesting and/or formulating the 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 for cell harvesting and/or formulation at the end of the process. In this regard, in some embodiments, the same apparatus may be used for both front-end cell enrichment and isolation steps, as well as back-end harvesting and/or formulation steps.
Focusing first on the second module 200, the ability to combine 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 specific configurations of components within the second module 200 and 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 referred to herein broadly as a biological treatment subsystem 400 or biological treatment system 400) within the second module 200 that provides cell activation, genetic modification, and expansion (and in some cases harvesting) is shown. The system 400 includes a first bioreactor vessel 410 and a second bioreactor vessel 420. The first bioreactor vessel includes at least a first port 412 and a first bioreactor 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, the first bioreactor vessel 410 and the second bioreactor vessel 420 form a 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, the first and second bioreactor lines 414, 418 of the first and second bioreactor containers 410, 420 each include a respective valve for controlling fluid flow therethrough. In particular, the first bioreactor line 414 of the first bioreactor vessel 410 includes a first bioreactor line valve 432, while the second bioreactor line 418 of the first bioreactor vessel 410 includes a second bioreactor line valve 424. Similarly, the first bioreactor line 424 of the second bioreactor vessel 420 includes a first bioreactor line valve 436, and the second bioreactor line 428 of the second bioreactor vessel 420 includes a second bioreactor line valve 438.
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 450 having an interconnect valve 452 provides fluid communication between the first fluid assembly 440 and the second fluid assembly 444. As shown in fig. 3, the interconnecting line 450 also provides fluid communication between the second bioreactor line 418 and the first bioreactor line 414 of the first bioreactor vessel 410, allowing fluid to circulate along the first circulation loop of the first bioreactor vessel. Similarly, the interconnecting lines also provide fluid communication between the second bioreactor line 428 and the first bioreactor line 424 of the second bioreactor vessel 420, allowing fluid to circulate along the second circulation loop of the second bioreactor vessel. In addition, as discussed below, the interconnecting line 450 further provides fluid communication between the second port 416 and the second bioreactor line 418 of the first bioreactor container 410 and the first port 422 and the first bioreactor line 424 of the second bioreactor container 420, thereby allowing the contents of the first bioreactor container 410 to be transferred to the second bioreactor container 420. As illustrated in fig. 3, in an embodiment, the interconnect line 450 extends from the second bioreactor line 418, 428 to the intersection of the first bioreactor line 414 and the first fluid assembly line 442 of the first bioreactor vessel 410.
As illustrated by fig. 3, a first fluid assembly 440 and a second fluid assembly 450 are disposed along the interconnect line 450. Additionally, in the embodiment, the first fluid component is in fluid communication with the first port 412 of the first bioreactor container 410 and the first port of the second bioreactor container 420 via the first bioreactor line 414 of the first bioreactor container and the first bioreactor line 424 of the second bioreactor container 420, respectively. The second fluid assembly 444 is in fluid communication with the second port 416 of the first bioreactor vessel 410 and the second port 426 of the second bioreactor vessel 420 via an 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 functions and purposes of the pumps 454 and 456 being discussed below. In an embodiment, the pumps 454, 456 are high dynamic range pumps. As also shown in fig. 3, a sterile air source 458 is connected to the interconnect 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 interconnect line 450, in other embodiments, the sterile air source may be connected to the first fluid assembly 440, the second fluid assembly 444, or a fluid flow path intermediate the first bioreactor or the second bioreactor line valve of the second bioreactor and the first bioreactor line valve 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 specific 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 466a-f. Each conduit tail 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. While fig. 4 specifically illustrates that the first fluid assembly 440 includes six fluid reservoirs, more or fewer reservoirs may be utilized to provide for the input or collection of a variety of treatment fluids as desired. As described below, it is contemplated that each conduit tail 464a-f may be individually connected to the reservoir 466a-f, respectively, at a time required during operation of the fluid assembly 440.
Referring specifically to FIG. 5, the second fluid assembly 444 includes a plurality of conduit tails 470a-d, each of the conduit tails 470a-d configured for selective/removable connection to one of a plurality of second reservoirs 472 a-d. Each conduit tail 470a-d of the second fluid assembly 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 assembly 444. Although fig. 5 specifically illustrates that the second fluid assembly 444 includes four fluid reservoirs, more or fewer reservoirs may be utilized to provide for the input or collection of a variety of treatment fluids as desired. In an embodiment, at least one of the second reservoirs (e.g., second reservoir 472 d) 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, first reservoirs 466a-f and 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 about their peripheries and supporting connecting conduits for connection to their respective tails (as is known in the art).
In an embodiment, the reservoir/bag may be connected to the conduit tails of the first and second conduit assemblies 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 tubing tails to the tail of the tube on the bag (while maintaining sterility). Thus, an operator may provide a bag when it is desired (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 so that they resolidify together). Conversely, the bag may be removed by welding the lines from the bag and cutting at the weld to separate the two closed 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 process, and connect the reservoirs/bags in time for their use. 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/lines during operation so that used bags can be connected in a sterile manner and disconnected so that other bags can be connected aseptically 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 to flow from the interconnect line 450 through the sampling lines 476a-476d. 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 280 d) for collecting fluid from the interconnect 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 the sampling assembly 448 connected to the interconnecting lines, in other embodiments the sampling assembly may be fluidly coupled to the first fluid assembly 440, the second fluid assembly 444, the fluid flow path intermediate the second bioreactor line valve 434 and the first bioreactor line valve 432 of the first bioreactor container 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 container 420. The sampling assembly 448 desirably provides a fully functionally closed fluid sample at one or more points in the system 400.
Referring back to fig. 3, in an embodiment, the system 400 may further include a filter line 482, the filter line 482 being connected at two points along the interconnect line 450 and defining a filter circuit along the interconnect 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. The waste line 490 provides fluid communication between the filter 484 and the second fluid assembly 444 and, in particular, with a conduit tail 470a of the second fluid assembly 444 that is connected to a waste reservoir 472a. In this regard, waste line 490 delivers waste removed from the fluid passing through filter line 482 by way of filter 484 to waste reservoir 472a. As illustrated in fig. 3, the filter line 482 surrounds the interconnect line valve 452 such that fluid flow through the interconnect line 450 may be forced through the filter line 482 (as discussed below). An osmotic pump 492 located along the waste line 490 is operable to pump the waste removed by the filter to a waste reservoir 472a. 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, a flat panel membrane filter, 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 the manner described below. In embodiments, the pipeline itself need not include a pinch valve, and the depiction of a pinch valve in fig. 3-8 may merely represent the location where the pinch valve may operate on the pipeline 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/function 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, as discussed below, and the pumps are integrated into a single assembly. Desirably, the operation of these valves and pumps is 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 these valves and pumps through the module 200.
Turning now to fig. 8-11, a configuration of a first bioreactor vessel 410 according to an embodiment of the present invention is illustrated. Since the second bioreactor vessel 420 is desirably (although not necessarily) identical in configuration to the first bioreactor vessel 410, only the first bioreactor vessel 410 will be described below for simplicity. In an embodiment, bioreactor containers 410, 420 are silicone membrane-based bioreactor containers that enable perfusion, which support activation, transduction, and expansion of cell populations therein. Bioreactor containers 410, 420 may be used for cell culture, cell processing, and/or cell expansion to increase cell density for use in medical treatment or other procedures. Although bioreactor containers may be disclosed herein as being used in connection with a particular cell type, it should be understood that bioreactor containers may be used for activation, genetic modification, and/or expansion of any suitable cell type. In addition, the disclosed techniques may be used in conjunction with adherent cells (i.e., cells that adhere to and/or proliferate on a cell-expanding surface). In an embodiment, the first bioreactor vessel 410 and the second bioreactor vessel 420 may be constructed and function as disclosed in U.S. patent serial No. 15/893336 filed 2/9/2018, which is incorporated herein by reference in its entirety.
As shown in fig. 8 and 9, the first bioreactor vessel 410 may include a base plate 502 and a vessel body 504 coupled to the base plate 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 for the supply of oxygen to the cell culture. In the illustrated embodiment, the base 502 is rectangular or nearly rectangular in shape. In other embodiments, the 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, the vessel body 504 includes a rigid, generally concave structure that, when coupled to the base plate 502, forms a cavity or interior compartment 506 of the first bioreactor vessel 410. As shown therein, the container body 504 may have a peripheral shape similar to that of the base plate 502 such that the container body 504 and the base plate 502 may be coupled to each other. 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 medium 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 container 410 may include a plurality of ports through the container body 504 that may enable fluid communication between the interior compartment 506 and the exterior of the first bioreactor container 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 greater detail herein, the specific structure of the first bioreactor container 410 (including the specific number and location of ports 412, 416) enables the first bioreactor container 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 a first bioreactor vessel 410. The floor 502 of the first bioreactor vessel 410 may be a bottom or support of the 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 contains a grid 510, which grid 510 may be structurally rigid, while further providing openings to enable free gas to exchange through the base plate 502 to the interior compartment 506 containing the cell culture. The grid 510 may include a plurality of holes 512 defined between solid areas or crossbars 514 between each hole 512 of the grid 510. Thus, the apertures 512 may provide openings for gas exchange, and the rails 514 may provide structural support for cell cultures and other structures within the interior compartment 506 of the first bioreactor container 410.
To provide further support for cell culture within the interior compartment 506 of the first bioreactor container 410, the first bioreactor container 410 may include a membrane 516 that may be disposed above the top surface 518 of the bottom plate 502. The membrane 516 may be a gas permeable, liquid impermeable membrane. The membrane 516 may also be selected to have properties that 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 506 2 ). The gas permeability characteristics of 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. In addition, 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 sedimentation of the cell culture for activation, transduction, and/or expansion. To enable membrane 516 to remain flat during use of first bioreactor container 410, mesh sheet 520 may be disposed between bottom 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 medium added to the first bioreactor vessel 410 for cell culture and/or cell expansion. Furthermore, the reticulated nature of the reticulated sheets 520 may enable support of the membrane 516 while still enabling free gas exchange between the interior compartment 506 of the first bioreactor container 410 and the environment immediately outside of the first bioreactor container 410. The mesh sheet may be a polyester mesh, or any other suitable mesh material that can provide support to the membrane and enable free gas exchange.
As previously discussed, the container body 504 may be coupled to the floor 502 to form an interior compartment 506 of the first bioreactor container 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. When the container is in the mainWhen body 504 is coupled to base plate 502, O-ring 522 may be used to seal first bioreactor vessel 410. In an embodiment, O-ring 522 may be a biocompatible O-ring (size 173, softFluoroelastomer 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 peripheral 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 the O-ring 522 desirably seals the first bioreactor vessel 410 without any chemical or epoxy bonding. Since the first bioreactor vessel 410 is useful 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, the first bioreactor vessel 410 may include a plurality of ports, such as a first port 412 and a second port 416. Ports 412, 416 may be provided through the container body 504 and may enable communication between the interior compartment 506 and the exterior of the first bioreactor container 410 for certain processes related to cell culture, cell activation, cell transduction, and/or cell expansion, such as fluid or media input, waste removal, collection, and sampling. Each port 416 may include an opening 526 and a corresponding fitting or conduit 528 (e.g., luer fitting, barb fitting, etc.). In some embodiments, the opening 526 may be configured to allow direct coupling of pipes and avoid the need for fittings (e.g., counterbores).
In an embodiment, the first bioreactor vessel 410 may further comprise an air balancing port 530 disposed in the top surface 508 of the vessel body 504 in addition to the first port 412 and the second port 416. The air balancing port 530 may be configured similarly to the first port 412 and the second port 416, wherein like reference numerals designate like components. The air balancing port 530 may further provide gas exchange between the interior compartment 506 and the exterior of the first bioreactor container 410 for use by the cell culture for amplification. In addition, the air balancing ports 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 central position of the top surface 508 of the container body 504 may help prevent the air balance port 530 from becoming wet during mixing of the cell culture by tilting the first bioreactor container 410.
Each of the elements of the first bioreactor vessel 410 (including the base plate 502, the vessel body 504, the ports 412, 416, and 530, the membrane 516, the mesh sheet 520, and the O-ring 522) may be made of biocompatible, autoclavable, and gamma radiation stable and/or ETO sterilization stable materials. As such, each element and the first bioreactor container 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.
The first bioreactor vessel 410 may enable cell culture and/or cell expansion via perfusion, which may provide nutrients necessary to support cell growth, and may reduce impurities in the cell culture. Continuous infusion is the addition of a fresh supply of medium to a growing cell culture while removing spent medium (e.g., spent medium). As discussed below, the first port 412 and the second port 416 may be used for a priming process. The first port 412 may enable communication between the interior compartment 506 and the exterior of the first bioreactor container 410 and may be used to add fresh media to the first bioreactor container 410 (such as from a media reservoir of the first fluid assembly 440). In some embodiments, the first port 412 may be disposed in the container body 504 at any location above the surface of the cell culture and culture medium within the first bioreactor container 410 and extend through the 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 culture medium within first bioreactor container 410.
The second port 416 may be disposed at any location below the surface of the cell culture and culture medium that is fully or partially submerged within the first bioreactor container 410. For example, the second port 416 may be a nearly lateral port provided 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 container 410 to facilitate perfusion of the cell culture. In addition, the second port 416 may also be used to remove cells of a 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 0.5cm 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 via gravity to the membrane 516 (e.g., cell expansion surface). Thus, in the static planar position, the second port 416 may facilitate the perfusion process and may enable an increase in cell density of the growing cell culture within the 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 container 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 filter-free. As such, when the second port 416 is used to remove media, there may be no physical impediment to the entry of cells into the second port 416. Further, the second port 416 may be sloped 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 sloped 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 at a relatively low location on the container body 504 closer to the membrane surface 36 while minimizing interference with the O-ring 522 and the groove 524 to help maintain the seal of the first bioreactor container 410 when in use. Further, in some embodiments, the sloped feature of the second port 416 may reduce the velocity of fluid flow through the second port 416 when spent media is removed. In addition, 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 suction on individual cells adjacent the second port 416 such that the force is less than the force of gravity pulling 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 may decrease into an unmeasurable range facilitated by the location of the second port 416. In addition, the position of the internal opening 540 may be varied to vary the recommended cell settling time. A position closer to 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 cells will settle from the top of the growth medium and be depleted first.
In an embodiment, the second port 416 may thus be used not only to remove spent 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 pointed tip 534. The apex 534 of the sidewall 532 may further include the second port 416 therethrough, with the container body 504 disposed adjacent the apex 534 when the container body 504 is coupled to the base 502. The angled sides 532 and tips 534 may enable greater drainage of the medium and/or cells of the cell culture when the first bioreactor container 410 is tilted (e.g., at a 5 degree angle) toward the 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 in the interior compartment 506 may enable the first bioreactor vessel 410 to be a relatively low profile vessel while enabling the maximum achievable cell density to be increased. Furthermore, the use of perfusion with the first bioreactor container 410 may support cell growth by providing fresh medium to the cells within the interior compartment 506, and also enable removal of impurities in the cell culture, such that once a particular cell density target is achieved within the first bioreactor container 410, additional cell washing in a separate device may not be required. For example, by filter-free priming, the first bioreactor vessel 410 can provide fresh medium and reduce impurities within the cell culture at a rate of total volume exchange per day (e.g., resulting in a reduction of impurities at a rate of approximately 1log per 2.3 days). Thus, the structure of the first bioreactor container 410 may enable the growth of the cell culture within the first bioreactor container 410 using perfusion, which may thus enable the cell culture to expand to a high target density with reduced impurity levels. As also discussed below, by filter-free priming, the first bioreactor vessel 410 can provide fresh medium at a rate of significantly more volume per day (e.g., greater than 2 volumes per day) for seeding, rinsing, washing/reducing residues, and/or draining/harvesting cells after expansion.
To facilitate a low profile configuration of the first bioreactor vessel 410, a relatively low media height within the interior compartment 506 may be maintained. Fig. 10 is a cross-sectional view of first bioreactor container 410, illustrating height 536 of cell culture medium 538 within first bioreactor container 410. As previously discussed, the container body 504 may be coupled to the base plate 502 to form an interior compartment 506, within which interior compartment 506 expansion of the cell culture may be achieved by perfusion. As such, replacement or fresh media 538 may be provided for cell growth through a first port 412 disposed through the container body 504, and existing or used media 538 may be removed through a second port 416 disposed through the side 510 of the container body 504. The perfusion process may promote a relatively low media height 536 of the media 538 within the interior compartment 506 of the first bioreactor container 410. The relatively low height 536 of the perfusion medium 538 within the interior compartment 506 may enable the first bioreactor container 410 to be a low profile structure, which may thus enable an overall compact cell manufacturing system.
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 2cm, 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 2cm. Thus, per cm 2 Less than 2mL of medium may be present and per cm 2 A total volume (including medium, cell culture, and headspace) of less than 4mL may be present. 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 growing cells of the cell culture using perfusion. 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 the first bioreactor vessel 410 to have a low profile or compact structure while still allowing for high cell density cell culture.
As previously discussed, the dual function second port 416 may be disposed through the vessel body 504 such that the second port 416 is fully or partially submerged below the surface 544 of the culture medium 538 within the 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 may facilitate removal of media and impurities from the cell culture within the interior compartment 506 without removing cells until such removal is desired, e.g., at harvest. The filter-less second port 416, together with the first port 412, may allow for the use of perfusion to provide growth medium 538 to cells for cell expansion, and to remove spent medium 538 and other impurities or byproducts. The location of the first port 412 and the dual function second port 416 around the container body 504 facilitates the following configuration: in this configuration, the height 536 of the medium 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 high density cell cultures.
Referring specifically to fig. 11, the floor 502 of the bioreactor vessel 410 includes a variety of features that enable the use of the bioreactor vessel as part of a broader biological treatment system 10, and in particular as part of the second module 200 of the biological treatment 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 recess may be located near a corner 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 container 410 within the second module 200. In an embodiment, the location verification structure may be a beam interrupter configured to interrupt the light beam of the second module 200 when the first bioreactor container 410 is properly positioned in the second module 200.
The base plate 502 also includes a pair of planar engagement surfaces 554 formed on adjacent bottom surfaces that are offset from the center line of the base plate (extending across the width of the base plate). Desirably, the engagement surfaces 554 are spaced along the longitudinal centerline of the base plate 502 so as to be positioned adjacent opposite ends of the base plate 502. The base plate 502 may further include at least one aperture or opening 556 to allow the contents of the first bioreactor vessel 410 to be sensed by the biological treatment apparatus engaging and operating the bioreactor vessel.
In an embodiment, the first and second bioreactor containers 410 and 420 and the fluid architecture 400 may be integrated into the 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 biological treatment vessel 410 and the second biological treatment vessel 420 are received side-by-side within the tray 610 of the disposable set 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 side walls 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) that encloses the interior compartment 622, and for desirably seating on the upper edge of a drawer of the biological treatment apparatus (as indicated below). The bottom surface 620 of the tray 610 includes a plurality of openings corresponding to the number of bioreactor containers in the biological treatment system. For example, the tray 610 may include a first opening 626 and a second opening 628. The bottom surface 620 may also include additional openings 630 adjacent to the first and second openings 626, 628 for purposes described below. In embodiments, the tray 610 may be thermoformed, 3D printed, or injection molded, although other manufacturing techniques and processes may be utilized without departing from the broader aspects of the invention.
As best shown in fig. 15, each of the first and second openings 626, 628 has a perimeter shaped and/or sized such that the first and second bioreactor containers 410, 420 are positionable over the respective openings 626, 628 and supported by the bottom surface 620 of the tray 610 within the interior compartment 622, while still allowing portions of the bioreactor containers 610, 620 to be accessed from the bottom of the tray 610 through the 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 inwardly toward the center of the opening 626, 628 for supporting the bioreactor vessel 410, 420 placed thereon. As shown in fig. 12 and 15, tray 610 may further include one or more bosses extending upwardly above openings 626, 628 for inhibiting lateral movement of the bioreactor containers when received over the respective openings 626, 628. Thus, the boss serves as an alignment device that facilitates proper positioning of the bioreactor containers 410, 420 within the tray 610 and helps to prevent inadvertent 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 an opening 638 for receiving therethrough an engagement plate, also referred to herein as a tubing module 650, that holds the fluid flow lines in an organized manner and holds them in place for engagement by the pump and pinch valves. In other embodiments, the duct module 650 may be integrally formed with the rear wall 614 of the tray 610.
Fig. 16 and 17 illustrate the configuration of a pipeline module 650 according to an embodiment of the present invention. As shown therein, the tubing module 650 includes a first tubing retainer 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 to 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 apart orientation by a first conduit retainer block 652. In particular, as best shown in fig. 17, the first conduit retainer block 652 engages each of the conduits 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 the conduit retainer block 652), the locations 656, 658 defining a void therebetween. As also shown in fig. 17, the first tubing retainer block 652 includes a clearance opening 660, the clearance opening 660 configured to receive a boot (not shown) of a peristaltic pump assembly. This configuration allows peristaltic compression of the lines 442, 450, 490 against the shoes by the respective pump heads of the peristaltic pump(s) to provide respective motive forces for fluid passing through the lines (as discussed below).
With further reference to fig. 16-18, the conduit module 650 further includes a second conduit retainer block 654 integrally formed with (or otherwise coupled to) the first conduit retainer block 652. The second conduit retainer block 654 is configured to receive all of the fluid flow lines of the fluid flow system 400 associated with the pinch valve. For example, the second conduit retainer block 654 is configured to retain the conduit tails 464a-f of the first fluid assembly 440, the conduit tails 470a-d of the second fluid assembly 444, the first and second bioreactor lines 414, 418 of the first and second bioreactor containers 410, the first and second bioreactor lines 424, 428 of the second bioreactor container 420, the sterile air source line 460, the interconnect line 450, and the filter line 482 (and, in some embodiments, the sampling lines 476a-476 d). Similar to the first conduit retainer block 652, the second conduit retainer block 654 may maintain the tubes in a horizontally extending and vertically spaced apart orientation. In particular, the second conduit retainer block 654 may include a plurality or vertically spaced apart and horizontally extending slots 666, the slots 666 configured to receive a conduit therein. Fig. 18 and 19 also best illustrate the configuration of slots 666, slots 666 hold all flow lines acted upon/interfacing with the pinch valve. Desirably, the slot 666 follows the contour of the block 654, but particularly extends across the planar back plate so as to be open to the filter 484. As shown in fig. 18, in an embodiment, the second conduit retainer block 654 may have one or more narrow conduit slots 682 at the bottom of the second conduit retainer block 654, and a waste conduit slot 684, the narrow conduit slots 682 for holding a ring of interconnecting conduits 450 from which the sampling lines extend, the waste conduit slot 684 for receiving a conduit tail 470a connected to a waste reservoir 472 a.
The second conduit retainer block 654 may include a planar back plate 662 having a plurality of apertures 664, the apertures 664 corresponding to the plurality of fluid flow lines held by the second conduit retainer block 654. In particular, at least one aperture 664 is horizontally aligned with each slot 666 and the flow line held therein. As best shown in fig. 16, the second conduit retainer block 654 includes two clearance openings 668, 670 configured to receive an anvil (not shown) of a pinch valve assembly therethrough. This configuration allows the tubing tails 464a-f of the first fluid assembly 440, the tubing tails 470a-d of the second fluid assembly 444, the first and second bioreactor lines 414, 418 of the first and second bioreactor containers 410, 424, 428 of the second bioreactor container 420, the sterile air source line 460, the interconnect line 450, and the filter line 482 to be selectively compressed against an anvil by respective pistons of the 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 offset in a vertical direction 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 as an elongated hollow fiber filter module in fig. 16) may be integrated with the conduit module 650, such as by mounting the filter 484 to the conduit module 650 using the retention clips 672. Where filter 484 is a hollow fiber filter, filter 484 may extend substantially the entire length of conduit 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 interconnect line 450 for recycling to one of first bioreactor vessel 410 or second bioreactor vessel 420 after removal of permeate/waste. The filter 484 may also include a permeate port 678 adjacent the second output 676 for connection to a waste line 490 for delivering waste/permeate to the permeate/waste reservoir 472a. Finally, the conduit module 650 may include a plurality of features 680 for receiving and organizing the bioreactor lines (e.g., the first and second bioreactor lines 414, 418 and/or the first and second bioreactor lines 424, 428 of the first and/or second bioreactor containers 410, 420).
Similar to the tray 610, the conduit module 650 may be thermoformed, 3D printed, or injection molded, although other manufacturing techniques and processes may be utilized without departing from the broader aspects of the invention. As 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 member 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 containers 410, 420 received within the tray 610 and the fluid lines of the flow architecture 400 received by the tubing module 650. As shown therein, the kit 600 as shown in fig. 20-22 does not have an opening 630, but rather includes a solid floor therein to provide a sampling space 631 in the tray 610 for receiving a receptacle holding a sampling line (e.g., sampling lines 476a, 476 b). 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 fluid assemblies 440, 444 allow for plug-and-play functionality, enabling quick and easy connection of multiple media bags, reagent bags, waste bags, sampling bags, and collection bags to allow 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 pipe sections as discussed above, such as with a tert mo device, or by clamping, welding and cutting the tail section as is known in the art.
Turning now to fig. 23-25, the kit 600 is specifically configured to be received by a biological treatment device 700, the biological treatment device 700 containing all of the hardware (i.e., controller, pump, pinch valve actuator, etc.) necessary to actuate the kit 600 as part of a biological treatment method. In an embodiment, the biological treatment apparatus 700 and the kit 600 (comprising the flow architecture 400 and the bioreactor containers 410, 420) together form the second biological treatment module 200 described above in connection with fig. 1 and 2. The biological treatment apparatus 700 includes a housing 710, the housing 710 having a plurality of drawers 712, 714, 716 that can be received 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 treatment operations to be performed simultaneously within each drawer. In particular, in an embodiment, each drawer 712, 714, 716 may be a separate biological processing module for performing 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 process chamber 722, and a substantially open top. The drawer 712 is movable between a closed position, as shown for drawers 714 and 716 in fig. 23, in which the drawer is fully received within the enclosure 710, and an open position, as shown for drawer 712 in fig. 23 and 24, in which the drawer 712 extends from the enclosure 710, thereby enabling access to the process chamber 722 through an 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 an embedded heating element (not shown), or be in thermal communication with a heating element, such that the sidewalls 718 and/or the process chamber 722 may be heated to a desired temperature for maintaining the process 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) may 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 processing chamber 722 may house all hardware components of the apparatus 700. In an embodiment, drawer 712 may further include an auxiliary compartment 730 adjacent to process chamber 722 for containing a reservoir containing media, reagents, etc., connected to first fluid assembly 440 and second fluid assembly 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 is operatively connected to the drawer 712 to selectively move the drawer 712 between an open position and a closed position. The linear actuator is operable to provide a 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 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 vessel(s).
Fig. 25 is a top plan view of the interior of the drawer showing the process chamber 722, hardware compartment 724, and auxiliary compartment 730 of the drawer 712. As illustrated therein, the hardware compartment 724 is located behind the process chamber 722, including a power source 732, a motion control board, and drive electronics 734, which 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 pumps 454, 456, 492) and the drawer engagement actuator 740. As described below, 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. In an embodiment, pump shoe 742 and solenoid anvil 744 are secured to a front substrate (front plate) of the process chamber. The hardware compartment (and the components described) is mounted to the rear substrate. Both plates are slidably mounted to the rail. Further, the drawer engagement actuator 740 couples the two plates and serves to bring the two plates (and the components carried on the plates) to an engaged position (thereby bringing the pump roller belt into the pump shoe and thus squeezing the pump tubing (if interposed therebetween)). As further described herein, the pump assembly provides selective operation on lines 442, 450, and 490 of the fluid path 400, thereby providing independent respective peristaltic motive forces thereto. Similarly, as will be further described, the conduit retainer 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 seat plates (e.g., a first seat plate 746 and a second seat plate 748) are located within the process chamber 722 on the bottom surface 720 and extend or stand up from the bottom surface 720. In embodiments, the process chamber 722 may house a single seat plate, or more than two seat plates. The seat plates 746, 748 are configured to receive or otherwise engage the first bioreactor vessel 410 and the second bioreactor vessel 420 thereon. As also shown in fig. 25, drawer 712 further includes a plate 750, plate 750 being configured with load cells positioned adjacent to seat plates 746, 748 within process chamber 722 for sensing the weight of a reservoir (e.g., waste reservoir 472a positioned thereon).
Fig. 26-28 best illustrate the configuration of the seat pan 746, 748, with fig. 28A showing the hardware components positioned underneath the seat pan. As used herein, the seat pan 746, 748 and hardware components (i.e., sensors, motors, actuators, etc. integrated with the seat pan or positioned underneath the seat pan as shown in fig. 28A) may be collectively referred to as a seat pan. The first seat plate 746 and the second seat plate 748 are substantially identical in configuration and operation, but for simplicity, the following description of the seat plates 746, 748 refers only to the first seat plate 746. The seat plates 746, 748 have a substantially planar top surface 752, the shape and surface area of the top surface 752 substantially corresponding to the shape and area of the bottom plate 502 of the first bioreactor container 410. For example, the seat pan may be generally rectangular in shape. The seat plates 746, 748 may also include a cushioning or clearance area 758 that generally corresponds to the location of the protrusion or ledge 632 of the tray 610, the purpose of which will be described below. The seat pan 746, 748 is supported by a plurality of load cells 760 (e.g., four load cells 760 positioned beneath each corner of the seat pan 746). Load cell 760 is configured to sense the weight of first bioreactor container 410 during a biological process for use by controller 210.
In embodiments, the seat plate 746 may include or be in thermal communication with a heating element such that the process chamber 722 placed thereon and/or the contents of the first bioreactor container 410 may be maintained at a desired temperature. In embodiments, the heating element may be the same as or different from the heating element that heats the side wall 718, top wall, and bottom surface.
As illustrated, the seat plate 746 includes a plurality of locating or alignment pins 754 that protrude above the top surface 452 of the seat plate 746. The number of locating pins 754 and the location and spacing of the locating pins 754 may correspond to the number, location and spacing of the recesses 550 in the bottom surface of the bottom plate 502 of the bioreactor containers 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 container 410 on the first seat plate 746. In an embodiment, the sensor 756 is an infrared beam, however other sensor types such as a lever switch may be utilized without departing from the broader aspects of the present invention. The sensor is configured to interact with the position verification feature 552 on the base plate 502 when the first bioreactor container 410 is properly positioned on the first base plate 746. For example, where the sensor 756 is an infrared beam and the position verification structure 552 is a beam interrupter (i.e., a flat tab), for a position verification structure 552 that is substantially opaque to infrared light, the beam interrupter will interrupt the infrared beam (i.e., interrupt the beam) when the first bioreactor container 410 is fully seated on the seat plate 746. This will signal the controller 210 that the first bioreactor vessel 410 is properly positioned. If the controller does not detect that the infrared light beam of the sensor 756 is interrupted after positioning the first bioreactor container 410 on the first seat plate 746, this indicates that the first bioreactor container 410 is not fully or properly positioned on the seat plate 746 and that an adjustment is required. Thus, the sensor 756 on the seat 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 placed in a contour position on the seat plate 746 (as determined by the alignment pins) before starting the biological process.
With still further reference to fig. 26-28A, the seat plate 746 additionally includes an embedded temperature sensor 759 positioned so as to be aligned with the aperture 556 in the bottom plate 502 of the first bioreactor container 410. The temperature sensor 759 is configured to measure or sense one or more parameters within the bioreactor container 410, such as, for example, a temperature level within the bioreactor container 410. In an embodiment, the seat plate 746 may additionally include: a resistive temperature detector 760 configured to measure the temperature of the top surface 752; and a carbon dioxide sensor (located below the seat plate) for measuring the carbon dioxide level within the bioreactor vessel.
As further shown in fig. 26-28A, each seat plate 746, 748 includes an actuator mechanism 761 (e.g., a motor) including, for example, a pair of opposing cam arms 762. Cam arm 762 is received within slot 764 in seat plates 746, 748 and is rotatable about cam pin 766 between a clearance position in which cam arm 762 is positioned below top surface 752 of seat plate 746, and an engaged position in which cam arm 762 extends above top surface 752 of seat plate and contacts opposing flat engagement surface 554 of bottom plate 502 of first bioreactor container 410 when first bioreactor container 410 is received on top of first seat plate 746. As discussed in detail below, the actuator mechanism is operable to tilt the bioreactor vessel on top of the seat pan to provide agitation and/or assist in draining the bioreactor vessel.
Referring to fig. 29-32, more detailed views of drawer engagement actuator 740 and linear actuator 768 in hardware compartment 724 of drawer 712 are shown. Referring to fig. 29, and as indicated above, the linear actuator 768 is operable to move the drawer 712 between the open and closed positions. In an embodiment, the linear actuator 768 is electrically connected to a rocker switch 770 external to the housing 710 that allows a user to control the movement of the drawer. The linear actuator 770 provides for controlled movement of the drawer 712 to prevent the contents of the bioreactor vessel(s) within the drawer 712 from being disturbed. In an embodiment, linear actuator 768 has a stroke of approximately 16 "and has a maximum velocity of approximately 2 inches per second.
Turning now to fig. 30, drawer engagement actuator 740 includes lead screw 772 and hanger arm 774 attached to front plate 751 within drawer 712. The drawer engagement actuator is operatively connected to the pump assembly 738 and the solenoid array 736 and is operable to move the pump assembly 738 and the solenoid array 736 between a first clearance position and an engaged position.
Figures 31 and 32 better illustrate the clearance and engagement positions of the pump assembly 738 and the solenoid array 736. As illustrated in fig. 31, in the clearance position, the pump assembly 738 and the solenoid array 736 are spaced apart from the pump shoe 742 and the pinch valve anvil 744, respectively. Upon actuation of lead screw 772, drawer engagement mechanism 740 moves pump assembly 738 and the solenoid array linearly forward to the position shown in fig. 32. In this position, the pump head of the pump assembly 738 engages the lines 442, 450, 490 in the first tubing retainer block 652, and the solenoid array 736 is positioned sufficiently close to the pinch valve anvil 744 such that the piston/actuator of the solenoid array 736 can pinch/clamp its respective fluid flow line of the second tubing retainer block 654 against the pinch valve anvil(s) 744, thus preventing flow through that fluid flow line.
Referring back to fig. 24, and with additional reference to fig. 33-39, in operation, the drawer 712 may be controllably moved to an open position by actuating a rocker switch 770 external to the housing 710. The disposable insert kit 600 containing the tubing module 650 (which holds all of the tubing and tubing tails of the flow architecture 400) and the first bioreactor container 410 and the second bioreactor container 420 is then lowered into position within the process chamber 722. When the sleeve 600 is lowered into the process chamber 722, the pump shoe 742 is received through the clearance opening 660 of the first tubing retainer block 652 such that the pump tubes 442, 450, 490 are positioned between the pump heads 454, 456, 492 and the pump shoe 742 of the peristaltic pump assembly 738. 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 the pump heads 454, 456, 492 relative to the pump tubes 442, 450, 490 when the kit 600 is received within the process chamber 722. As shown therein, the pump tubes 442, 450, 490 are positioned between the pump shoe 742 and the pump heads 454, 456, 492. In operation, when the drawer engagement actuator 740 positions the pump assembly 738 in the engaged position, the pump heads 454, 456, 492 are selectively actuatable under the control of the controller 210 to start, maintain, and stop fluid flow through the tubes 442, 450, 490.
Similarly, when the kit 600 is lowered into the processing chamber 722, the pinch valve anvil 744 is received through the clearance openings 668, 670 of the second conduit retainer block 654 such that the conduit tails 464a-f of the first fluid assembly 440, the conduit tails 470a-d of the second fluid assembly 444, the first and second bioreactor lines 414, 418 of the first and second bioreactor containers 410, 424, 428, the sterile air source line 460, the interconnect line 450, and the filter line 482 of the first fluid assembly 444 held by the second conduit retainer block 654 are positioned between the solenoid array 736 and the pinch valve anvil 744. This configuration is best illustrated in fig. 37-39 (fig. 37 and 38 illustrate the relationship between the solenoid array 736 and the pinch valve anvil 744 prior to receiving the back plate 662 of the second conduit retainer block 654 within the space 776).
As shown therein, each solenoid 778 of the solenoid array 736 includes a piston 780, the piston 780 being capable of extending linearly through an associated aperture (aperture 664) in the back plate 662 of the second conduit retainer block 654 to clamp an associated tube against the pinch valve anvil 744. In this regard, solenoid array 736 and anvil 744 together form a pinch valve array (which includes the valves of first and second fluid assemblies 440, 444, as well as bioreactor line valves, i.e., valves 432, 434, 436, 438, sterile line valve 462, interconnection line valve 452, and filtration line valves 486, 488). In particular, the pinch valves of the flow architecture 400 are provided by respective solenoids 778 (i.e., plungers of the solenoids) of the solenoid array 736, the solenoid array 736 operating against its respective anvil 744, while the fluid path/line is 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 an associated fluid flow line against the anvil 744, thereby preventing fluid flow therethrough. The present invention contemplates that each fluid line is positioned between the planar anvil surface and the planar solenoid actuator head. Alternatively, the solenoid actuator head may comprise a shaped head, such as two tapered surfaces that meet at an elongated edge (similar to a Phillips head screwdriver), optimized to provide a desired clamping force on a resiliently flexible fluid line. Still alternatively, the anvil surface may include an elongate ridge or protrusion extending toward each fluid line such that the planar solenoid head may compress the fluid line against the laterally extending ridge to close the line against 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 bioreactor container 410 and the second bioreactor container 420 are supported above the openings 626, 628 by the perimeter of the openings (and in particular by the protrusions/projections 632). As 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 seat plates 746, 748, the shape of the openings 626, 628 and the top surfaces 752 of the seat plates 746, 748 (e.g., the cushioning regions 758 of the seat plates 746, 748, which correspond to the protrusions/projections 632 of the tray 610) allow the tray 610 to continue traveling downward such that the bottom surface of the tray 610 and the protrusions/projections 632 are disposed below the top surfaces 752 of the seat plates 746, 748 such that the bioreactor containers 410, 420 may be supported by the seat plates 746, 748 in spaced relationship from the bottom surface 620 of the tray 610. This ensures that the tray 610 does not interfere with the contoured placement of the bioreactor containers 410, 420 on the seat plates 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, thereby ensuring that the bioreactor container 410, 420 will be properly aligned with the seat plates 410, 420. When properly positioned on the seat plates 746, 748, the beam interrupter 552 interrupts the light beam of the sensor 756 in the seat plate, thereby indicating to the controller that the bioreactor vessel 410, 420 is in the proper position. Because the seat plates 746, 748 and alignment pins are of equal height, interruption of the light beam to the sensor 756 by the beam interrupter 552 also ensures that the bioreactor vessels 410, 420 are of equal height. In this properly positioned position, the sensors 759 on the seat plates 746, 748 are aligned with the apertures 556 in the bottom plate 502 to allow for 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 planar engagement surfaces 554 on the bottom plate 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 plate 746. As shown in fig. 40, heating elements in the form of heating pads 782 and heating modules 784 may be positioned below seat plate 746 for heating seat plate 746. As shown in fig. 40, a carbon dioxide sensing module 786 may also be positioned below the seat pan for sensing the carbon dioxide content within the process 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 lid 788, an insulating foam layer 790 that helps minimize heat loss from the process chamber 722, a 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 be utilized without departing from the broader aspects of the invention. The drawer 712 can further include one or more brush seals 796 that help minimize or prevent heat loss from the process chamber 722 as well as thermal breaks 798 that minimize or prevent heat energy from flowing from the drawer 712 to other components of the apparatus 700, such as the 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 in the bottom of the process chamber 722 adjacent the second seat plate 748 extends through the opening 730 in the tray 610 such that the waste bag 472a can be connected to the conduit tail 470a and positioned over the load cell 750. As shown therein, when the kit 600 is received within the drawer 712, the second conduit retainer block 654 retains the conduit such that the conduit tails 464a-f of the first fluid assembly 440 and the conduit 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, the 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, cam arm 762 is movable between a retracted position in which cam arm 762 is positioned below the top surfaces of seat plates 746, 748 and an engaged position in which cam arm 762 rotates about cam pin 766 and extends above seat plates 746, 748 to engage flat engagement surface 554 of bioreactor containers 410, 420 to lift bioreactor containers 410, 420 off of seat plates 746, 748. Because cam arm 762 is retracted below the top surfaces of seat plates 746, 748 in the default state and bioreactor vessels 410, 420 are supported on equal height seat plates 746, 748 (and in particular, alignment pins 754) no power is required to maintain the bioreactor vessels in the 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 contoured positions. This is in contrast to systems that may require the use of a servo motor to continuously adjust the bioreactor to maintain the contour position. Indeed, with the cam arm 762 configuration of the present invention, as discussed below, the actuator need only be energized when tilting the bioreactor vessel for agitation/mixing, which minimizes thermal contribution to the process chamber 722.
As shown in fig. 41-43, cam arm 762 may be sequentially operable to agitate the contents of bioreactor containers 410, 420. For example, when it is desired to agitate the contents of the bioreactor container 410, one of the cam arms will be actuated to lift one end of the bioreactor container 410 off of the seat plate 746 (and out of engagement with the locating pins 754 on the seat plate 746) while the opposite end remains seated on the seat plate and the locating pins 754 on the non-raised end remain received in the corresponding recesses 550 in the 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 ends of the bioreactor vessel away from the seat plate and the locating pins.
In embodiments, the cam actuation system may be designed such that cam arm 762 can home without contacting the bioreactor vessel, thereby preventing damage to the culture and allowing cam arm 762 to home (or test) at any point during long cell processing cycles. Thus, while the present invention contemplates that other rocking or agitation means may be provided for the bioreactor vessel, by having two cam arms 762 on opposite sides of the seat plate, the overall height of the mixing mechanism may be minimized. For example, a central actuator (centered on the seat plate) may be utilized to achieve +/-5 degrees of motion, but 0-5 degrees of motion of the container driven by cam arms on both sides of the container may be utilized to achieve nearly the same motion of the container, effectively imparting +/-5 degrees of motion to the container over 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 the opposing cam arms) can be adjusted to maximize wave formation in the container, thereby maximizing wave amplitude and thus (desirably) the uniformity of the container contents and the time to achieve uniformity. Timing can also be adjusted based on the volume in a container of a given geometry to maximize mixing efficiency.
In an embodiment, the optical sensor 756 may be used to confirm that the first bioreactor container 410 has been properly repositioned after each cam stirring motion. It is further contemplated that even between alternating camming movements, proper repositioning of the bioreactor vessel may be checked and verified. This enables a quick detection of misalignment in substantially real time, allowing operator intervention to relocate the bioreactor vessel without significant deviation from the biological treatment operation/protocol.
Fig. 43 is a schematic illustration showing the location of a fluid 800 within a bioreactor vessel during this agitation process. As shown in fig. 42, in an embodiment, a homing sensor 802 integrated with the seat plate 746 can be utilized by the controller to determine when the cam arm 762 has returned to a clearance position below the top surface of the seat plate 746. This is useful in coordinating the movement of cam arm 762 to provide a desired mixing frequency in the bioreactor vessel. In an embodiment, cam arm 762 is configured to provide a tilt angle of up to 5 degrees with respect to seat plate 746.
Referring to fig. 44, an interface between the locating pins 754 of the seat plate and the recesses 550 in the bottom plate 502 of the bioreactor container 410 during mixing/stirring is illustrated. In an embodiment, recess 550 has a dome-shaped or hemispherical inner surface and a diameter d1 that is greater than a diameter d2 of dowel 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 of the biological treatment apparatus 700 (e.g., drawer 712) desirably includes a flip-down front panel 810 hingedly mounted thereto. As best shown in fig. 45, 49 and 50, the downward flipped 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 the media bag in the process. In combination 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 attachment means 815 from which a variety of reservoirs/media bags may be suspended. The rail 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 rail 812 can simply be extended outward and the bag released. The new bag may be connected 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 rail 812 may be mounted on a laterally extending rail 814. Thus, the rail 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 rail 812 may be rotated about the rear rail such that it passes over the compartment 730 to allow the user to thread the duct tail toward the front of the compartment 730, thereby 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 rotatable from a stowed position to an entry position out of the auxiliary compartment 730. For example, the platform 820 may be mounted for movement along rails 822 formed in the side walls of the auxiliary compartment 730.
Referring to fig. 52, in an embodiment, the biological treatment apparatus 700 may further include a low profile waste tray 816 received within the housing 710 under each drawer (e.g., drawer 712). The waste tray 816 is independently mounted on its drawers 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 chamber 819 to enable operator access. The chamber 819 provides for easy storage of large waste bags connected to the fluid path of the tray 600 above it and enables access to the waste bags without having to open the drawer 712. Additionally, 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 leakage from the processing chamber 722 or 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 a side wall looking laterally into the bioreactor vessel(s).
Thus, the second module 200 of the present invention provides automation of cell processing to an extent heretofore not seen in the art. In particular, the fluid flow architecture 400, pump assembly 738, and pinch valve array 736 allow for automated fluid handling (e.g., fluid addition, transfer, draining, flushing, etc.) between the bioreactor vessels 410, 420 and bags connected to the first and second fluid assemblies 740, 744. As discussed below, this configuration also allows for hollow fiber filter concentration and washing, filter-less priming and line priming. The use of drawer engagement actuators 740 also serves to automatically engage and disengage the male assembly 600, thereby further minimizing human contact points. Indeed, only human contact points may be required to add and remove source/media bags, sample and input data (e.g., sample volume, cell density, etc.).
Referring to fig. 53-77, an automated generic protocol for immobilized Ab coating, soluble Ab addition, gamma-retroviral vector amplification in the same vessel using the second module 200 and its fluid flow architecture 400 is illustrated. This generic protocol provides activation of the cell population (illustrated in fig. 53-59), pre-transduction preparation and transduction (illustrated in fig. 60-71), amplification (fig. 72-76), and harvesting (fig. 77) for some embodiments 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 completed, 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 first fluid assembly line pump 454 is actuated to pump antibody (Ab) coating solution from reservoir 466f connected to first fluid assembly 440 to first bioreactor container 410 through first port 412 of first bioreactor container 410. The antibody coating solution is incubated for a period of time and then discharged through the interconnect line to the waste reservoir 472a of the first fluid assembly 440 by opening the valves 434, 474a and activating the circulation line pump 456. As described herein, the draining of the bioreactor vessel 410 may be facilitated by tilting the bioreactor vessel 410 using the cam arm 462.
After draining the antibody coating solution, valves 432 and 468e are opened and pump 454 is actuated to pump flush buffer from reservoir 466e connected to first fluid assembly 440 to first bioreactor vessel 410 through the first bioreactor line. Then, by actuating the circulation line pump 456 and opening the valve 474a, the flush buffer is discharged through the interconnection line 450 to the waste reservoir 472a. In an embodiment, this flushing and draining procedure may be repeated multiple times to fully flush the first bioreactor vessel 410.
Turning to fig. 55, after flushing the first bioreactor vessel 410 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 vessel by opening valves 468d and 432 and actuating pump 454. Cells are pumped through the first bioreactor line 414 of the first bioreactor container 410 and into the bioreactor container 410 through the 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 through first port 412 from reservoir 466a, which is connected to first fluid assembly 440, to first bioreactor vessel 410.
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 the rinse medium from rinse medium reservoir/bag 472b of second fluid assembly 444 is pumped into second antibody solution reservoir 466a using pump 454 to rinse the reservoirs. After flushing, valve 432 is opened and flushing medium is pumped from reservoir 466a to first bioreactor vessel 410. In an embodiment, this procedure may be used to flush second antibody solution reservoir 466a multiple times.
After flushing the second antibody solution reservoir 466a, the inoculum/seed cell bag 466d may also optionally be flushed. In particular, as shown in fig. 58, valves 474b, 452 and 468d are opened and rinse medium from rinse medium reservoir/bag 472b of second fluid assembly 444 is pumped into inoculum/seed cell bag 466d using pump 454 to rinse the bag. After flushing, valve 432 is opened and flushing medium is pumped from bag 466d to first bioreactor vessel 410 using pump 454. By pumping the flush medium into the first bioreactor container 410 after flushing the inoculum/seed cell bag 466d, the cell density in the first bioreactor container 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 is present 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 interconnect 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 tube tail 476a and a valve 478a is opened to divert some of the flow through the interconnect line 450 to the first sample container 280a for analysis.
If analysis of the acquired samples indicates that all solution parameters are within a predetermined range, the solution within the first bioreactor container 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 population of cells in the 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 the RetroNectin solution is pumped 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 discharged from the second bioreactor vessel 420 to the waste reservoir 472a by opening valves 438 and 474a and actuating the circulation line pump 456. During these RetroNectin coating, incubation, and draining steps (involving the second bioreactor container 420), it should be noted that the activated cell population remains in the first bioreactor container 410. It should be noted that it is not necessary to utilize RetroNectin or other agents for improving the efficiency of genetic modification in all processes.
As shown in fig. 61, after RetroNectin coating, a flush buffer bag 472b is connected to the second fluid assembly 444 (or it may already be present and connected to one of the conduit tails), and valves 474b and 438 are opened and pump 456 is actuated to pump buffer from bag 472b to the second bioreactor container 420. Alternatively, as discussed above, buffer may instead be pumped through the first port 422 of the second bioreactor vessel 420 by opening valves 452 and 436.
Turning now to fig. 62, after a defined period of time, all buffer in the second bioreactor vessel 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 pre-concentrated sample may be obtained from the cells in the first bioreactor container 410. As shown therein, valves 434, 486, 488, and 432 are open and pump 456 is actuated to circulate the solution in first bioreactor vessel 410 as follows: from the second port 434, through the interconnecting lines, through the filter line 48 and the filter 484, through the first bioreactor line 414 of the first bioreactor vessel 410, and back to the first bioreactor vessel 410 through the first port 412. To obtain a sample, a second sample container 280b (e.g., dip tube, syringe, etc.) is connected to the second sample tube tail 476b, and a valve 478b is opened to divert some of the flow through the interconnect 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 the first bioreactor vessel 410 through a 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 the second port 416, through the second bioreactor line 418, through the interconnecting line 450, through the filter line 482 and the filter 484, through the first bioreactor line 414 of the first bioreactor vessel 410, and back to the first bioreactor vessel 410 through the first port 412. As the fluid passes through the filter 484, the waste is removed and the osmotic pump 492 pumps such waste through the waste line 490 to the waste reservoir 472a of the 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 vessel (i.e., the first vessel 410 containing the concentrated cell population) is washed at a constant volume by perfusion. In particular, as shown therein, culture medium from culture medium bag 466b of first fluid assembly 440 is pumped into first bioreactor container 410 through first port 412 via interconnecting line 450 while at the same time, culture medium is pumped out of first bioreactor container 410 through second port 416 such that a constant volume is maintained in first bioreactor container 410. As media is added to vessel 410 and removed from vessel 410, waste may be filtered out by filter 484 and directed to waste reservoir 472a.
The washed sample may be obtained from the cells in the first bioreactor vessel 410 in a similar manner as described above for pre-concentrate sampling. In particular, as shown in fig. 66, valves 434, 486, 488, and 432 are open and pump 456 is actuated to circulate the fluid in first bioreactor vessel 410 as follows: from the second port 434, through the interconnecting lines, through the filter line 48 and the filter 484, through the first bioreactor line 414 of the first bioreactor vessel 410, and back to the first bioreactor vessel 410 through the first port 412. To obtain a sample, a third sample container 280c (e.g., dip tube, syringe, etc.) is connected to the third sample tube tail 476c and a valve 478c is opened to divert some of the flow through the interconnect line 450 to the third sample container 280c for analysis.
As shown in fig. 67, a bag containing thawed viral vectors is connected to first fluid assembly 440 (such as by tubing 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. Then, incubation is performed for a predetermined period of time for virus coating of the second bioreactor container 420. After incubation, the viral vector coating solution is discharged from the second bioreactor container 420 to the waste reservoir 472a by opening valves 438 and 474a and actuating the 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 container 420 is coated with viral vectors, the washed cells from the first bioreactor container 410 are transferred to the second bioreactor container 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, through interconnection line 450, to first bioreactor line 424 of second bioreactor vessel 420, and into second bioreactor vessel 420 through first port 422 of second bioreactor vessel 420.
Then, by opening valves 468b and 436 and actuating pump 454, medium from medium bag 466b is added to second bioreactor vessel 420 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-transduction 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 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 fourth sample container 280d (e.g., dip tube, syringe, etc.) is connected to the fourth sample tube tail 476d and a valve 478d is opened to divert some of the flow through the interconnect line 450 to the fourth sample container 280d for analysis.
If analysis of the fourth sample obtained indicates that all parameters are within the predetermined range required for successful transduction, the cell population within the second bioreactor container 420 is incubated for a predetermined period of time for the cell population in the transduction solution (as illustrated in fig. 71). For example, in an embodiment, the population of cells in the second bioreactor vessel 420 may be incubated for 24 hours for transduction.
Referring to fig. 72, after transduction, a culture medium is added to the second bioreactor container 420 to achieve a predetermined amplification volume in the second bioreactor container 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 vessel to second bioreactor vessel 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 tube tail 476e and a valve 478e is opened to divert some of the flow through the interconnect line 450 to the fifth sample container 280e for analysis.
If analysis of the fifth sample obtained indicates that all parameters are within the predetermined range required for successful expansion of the cell population, then the cell population within the second bioreactor container 420 is incubated for a predetermined period of time, such as 4 hours, to allow the cells to settle.
As shown in fig. 74, after this incubation period or at a subsequent predetermined time, priming (1 x priming) is performed at a rate of 1 volume/day by pumping medium from medium bag 466b through first port 422 into second bioreactor container 420, while at the same time, pumping used/spent medium out of second bioreactor container 420 through second port 426 (and into 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, medium from medium bag 466b is introduced into second bioreactor vessel 420 at substantially the same rate as used medium is removed from second bioreactor vessel 420 and sent to waste to maintain a substantially constant volume within second bioreactor vessel 420.
Sampling may then be performed as needed/desired to monitor the amplification process and/or to determine when the 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 interconnect 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 shunt some flow through the interconnect line 450 to the sample container 280x for analysis (as shown in fig. 75). After each sampling operation, incubation without perfusion is performed for a predetermined period of time (e.g., 4 hours) to allow the cells to settle before restarting perfusion.
As shown in fig. 76, after this incubation period, priming (1 x priming) is performed at a rate of 1 volume/day by pumping medium from medium bag 466b through first port 422 into second bioreactor container 420, while at the same time, pumping spent/used medium out of second bioreactor container 420 through second port 426 (and into 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 sampling indicates a predetermined threshold (e.g., 5 MM/mL) of Viable Cell Density (VCD), priming (2 x priming) is performed at a rate of 2 volumes/day by pumping medium from medium bag 466b through first port 422 into second bioreactor container 420 while at the same time, used/used medium 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 2x perfusion, medium from medium bag 466b is introduced into second bioreactor vessel 420 at substantially the same rate as used medium 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 may be harvested by opening valves 438 and 474d and actuating circulation line pump 456. The expanded cell population is then pumped out of the second bioreactor vessel 420 through the second port 426, through the interconnecting line 450, and to a collection bag 472d connected to the conduit tail 470d of the second conduit 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 biological treatment system 10, as well as the flow architecture 400 and the bioreactor vessels 410, 420 thereof, provide a flexible platform upon which a variety of biological treatment operations can be performed in a substantially automated and functionally enclosed manner. In particular, although fig. 53-77 illustrate an exemplary general protocol that may be performed using biological treatment system 10 of the present invention (and in particular, second module 200 thereof), the system is not so limited in this respect. Indeed, a variety of automation protocols may be implemented by the system of the present invention, including many customer-specific protocols.
In contrast to prior systems, the second module 200 of the biological treatment system 10 is a functionally closed automated system that houses the first and second bioreactor containers 410, 420 and the fluid handling 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 implements all the steps of immune cell activation, transduction, amplification, sampling, priming and washing in a single system. It also provides the user with 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 an embodiment, a single amplification 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 biological treatment system of the present invention include a robust and scalable manufacturing technique that enables product commercialization by simplifying workflow, reducing labor intensity, reducing the burden of clean room infrastructure, reducing failure nodes, reducing costs, and increasing the ability to operate on a large scale.
As discussed above in connection with the general workflow, the flow architecture 400 and bioreactor containers 410, 420 of the system, biological treatment system 10, and second module 200 of the present invention enable the 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, the pump 456 on the interconnect line 450 may be used to circulate fluid from one of the ports of the bioreactor through the filter line 482 and the filter 484 and then back to the other port on the bioreactor while the osmotic pump 492 is operated (typically at a percentage of the circulation pump 456, such as, for example, about 10%) in the concentration step. The concentration may be operated in an open loop or may be stopped based on the measured volume removed from the bioreactor or the measured volume accumulated in the waste. In an embodiment, the filter, pump speed, filter area, lumen number, etc. are all sized appropriately for the total number of cells and target cell density to limit fouling and excessive cell loss due to shear.
In embodiments, and as discussed above, the systems of the invention may also be used for washing, e.g., to remove residues, such as viral vectors remaining after incubation. Washing involves the same steps described above for concentration, except that pump 454 on first fluid assembly line 442 is used to pump additional media to replace fluid pumped from osmotic waste pump 492. The rate of introduction of new media may correspond to the rate of removal of fluid 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 thoroughly mixed (circulation may be sufficient). In an embodiment, this same process may be used for in situ hollow fiber filtration based cell suspension washing after activation to remove residues. Elution of the soluble activating agent may also be accomplished via filter-based priming for coated and uncoated surfaces.
As also discussed above, during priming, the pump 454 on the first fluid assembly line 442 can be used to add culture medium to a given bioreactor vessel, while the pump 456 on the interconnect line 450 is used to move spent culture medium to a waste bag in the second fluid assembly. In an embodiment, 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 operated at a set rate, and the rate of the other pump may be adjusted based on the mass/volume of the bioreactor vessel or the mass/volume of the waste bag (or the measured mass/volume of the source bag).
In conjunction with the above, it is contemplated that pump control may be based on weight measurements of the bioreactor vessel (using feedback from the weighing sensor 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 biological treatment system allows for cyclic priming 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 perfuse cells within first bioreactor container 410 in conjunction with appropriate pinch valve conditions. The perfusion of cells within the first bioreactor vessel 410 may then be stopped or paused, and the circulation pump 456 and pump 454, as well as appropriate pinch valves, may then be actuated to perfuse cells within the second bioreactor vessel 420. In this regard, the infusion of the multiple bioreactors may be performed sequentially (i.e., infuse the first bioreactor vessel 410 for a period of time and then infuse the second bioreactor vessel 420 for a period of time in a repeated and alternating manner). This allows any number of bioreactor vessels in the perfusion system without the need to use more pumps, media bags or waste bags.
In the case of cyclic priming, the pumps may be run continuously, may be run together intermittently (duty cycle), or may be run sequentially (source, then waste, repeated) in order 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 together intermittently and waiting for a period of time) will also allow multiple containers to be primed using the same two pumps. Furthermore, even if the pump does not have a large low-end dynamic range, the cyclic priming allows for a lower effective exchange rate (such as about 1 volume/day). Furthermore, the cyclic priming also allows each vessel to be primed with a different medium as controlled by the valve in the first fluidic component 440.
Additionally, in embodiments, rapid priming may be used for residue removal (e.g., for post-activation Ab removal and/or post-transduction residue removal). In rapid infusion processes, the infusion process described above may run much faster than 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 1log reduction in about a few minutes to a few hours. In an embodiment, the perfusion rate is balanced with cell loss. In some embodiments, rapid priming may allow for elimination of the hollow filter 484 and still meet the biological requirements of rapid removal of residue after certain steps.
As further described above, the system of the present invention facilitates flushing a bag/reservoir connected to the first fluid assembly 440 with a pump 454 on the first fluid assembly line 442, with a flushing buffer or fluid from another bag/reservoir connected to the second fluid assembly 444. In addition, the fluid lines of the flow architecture/system 400 may be purged with sterile air from the sterile air source 458 to prevent cells from residing in the lines and dying, or to prevent media or reagents from residing in the lines and degrading or not being used. A sterile air source 458 may also be used to purge reagents from the lines to ensure that no more reagents than desired are pumped to the bioreactor containers 410, 420. The sterile air source 458 may also be used to purge lines leading to connected bags (of either the first fluid assembly 440 or the second fluid assembly 444) to purge sterile tube welds, thereby limiting carryover. Alternatively, or in addition to purging the lines using a sterile air source 458, the lines may be purged using air drawn from one of the bioreactor vessels, so 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-type 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 drawing the sample from the interconnect line 450 using the sampling assembly 448. In an embodiment, only cells that are not bound to the beads may be stirred.
As also discussed above, the system of the present invention allows for the collection of cell populations after achieving a target cell density. In an embodiment, collecting the expanded population of transduced cells may include: a pump 456 on the interconnect line 450 is used to move the cells to one of the bags connected to the second fluid assembly 444; or circulate cells using an interconnection pump 456 to move cells to a bag connected to the first fluid assembly 440. This process may be used to ultimately collect 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 fluid assembly 440, circulating the contents of the bioreactor vessel, and withdrawing a portion of the desired sample volume from the circulating contents and moving toward the syringe/bag using the fluid assembly pump 454). In such a case, the circulation pump 456 and valve may then be used to purge the circulation line of fluid/cells. In addition, the pump 454 on the first fluid assembly line 442 may be used to continue pushing all aliquots of the sample volume into the sample receptacles, thereby using the air in the line to complete the transfer of the sample to the receptacles without an appreciable number of cells remaining in the line.
While the embodiments described above disclose a workflow that performs cell activation in a first bioreactor vessel and transfers the activated cells to a second bioreactor vessel for transduction and expansion, in embodiments, the system of the present invention may allow for performing activation and transduction operations in the first bioreactor vessel and performing expansion of genetically modified cells in the second bioreactor vessel. Furthermore, in embodiments, the system of the present invention may allow for in situ processing of isolated T cells, wherein activation, transduction, and expansion unit operations are all performed within a single bioreactor vessel. In an embodiment, the present invention thus simplifies existing protocols by enabling simplified and automated friendly "one-pot" activation, transduction, and amplification vessels.
In such embodiments, the T cell activator may be a micron-sized Dynabead, and the lentiviral vector is used for transduction. In particular, micron-sized Dynabead is used for the dual purpose of isolating and activating T cells, as disclosed herein. In an embodiment, activation (and isolation) of T cells may be performed in one of the bioreactor vessels 410 using Dynabead 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 after virus transduction, the virus may then be washed out of the bioreactor container 410 using the filter-less priming method described above, which retains cells and micron-sized Dynabead in the bioreactor container 410. This enables the cells to be expanded in the same bioreactor vessel 410 used for activation and transduction. The filter-free perfusion method additionally enables culture washing to be performed without first fixing the activation beads that need to be held with the cells during expansion. In particular, when the virus is washed away, the micron-sized Dynabead is not fluidized at a slow infusion rate, but is held in the container. The nano-sized virus particles and residual macromolecules are fluidized and washed away during slow perfusion.
In an embodiment, after expansion, the cells may be harvested in the manner described above in connection with fig. 77. After harvesting, dynabeads can be removed from the harvested cells using a magnetic bead removal process. In other embodiments, the steps of harvesting the expanded cell population and debulking 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 bead removal of the culture is desired, filter-free infusion can be used to remove micron-sized beads by taking advantage of the difference in cell weight and cell-Dynabead complex weight. To debulk the culture, the entire contents of the bioreactor vessel will be mixed (using cam arm 762, e.g., an actuator mechanism, in the manner described above). After mixing/stirring, the heavy Dynabead will sink and settle on the silicon film 516 within 10-15 minutes. In contrast, cells take more than 4 hours to settle on membrane 516. After a holding period of 10-15 minutes after mixing/stirring, the cell suspension can be slowly withdrawn using perfusion without disturbing the settling Dynabead. 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 transfer and careful washing and bead removal steps, and minimizes costs and potential risks. By debulking 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, perfusion-free culture systems, the gas-permeable membrane-based bioreactor vessel 410 of the present invention supports high density cell culture (e.g., up to 35mm/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 biological site of the present inventionThe management system simplifies current protocols by eliminating the need for intermediate process cell transfer and careful washing steps, and minimizes costs and potential risks caused by multiple human contact points.
In an embodiment, the two bioreactor vessels 410, 420 of the system may operate with the same starting culture or two simultaneously dividing cultures (e.g., cd4+ cells in one bioreactor vessel 410 and cd8+ cells in the other bioreactor vessel 420). Split cultures allow for parallel independent processing and expansion of two cell types that can be combined prior to infusion into a patient.
While many possible CAR-T workflows for generating and expanding genetically modified cells using the biological treatment system of the present invention have been described above, the workflows described herein are not intended to be comprehensive, as the system of the present invention also implements other CAR-T workflows. In addition, while the system of the invention and particularly 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. Furthermore, 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 a first bioreactor vessel 410 is added to a 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 repeated for the same workflow. Exemplary reasons for sequentially using the second bioreactor vessel may include: residual chemical modifications (e.g., coating or immobilization reagents) that cannot be washed out of the first bioreactor, which are detrimental in later steps (or in earlier steps if overexposure of cells occurs); or it may be desirable to pre-coat the bioreactor surface (e.g., retroNectin coating) prior to adding cells.
Additional examples of potential single bioreactor vessel workflows implemented by the system of the present invention include: (1) Soluble activator activation, viral transduction, filter-free priming and amplification in a single bioreactor vessel; (2) Dynabead-based activation, viral transduction, filter-free priming and amplification in a single bioreactor vessel; and (3) transactant bead-based activation, viral transduction, filter-free priming, and amplification in a single vessel.
Further, additional examples of potential multiple bioreactor vessel workflows implemented by the system of the present invention include: (1) Soluble activator activation, viral transduction, no filter priming and amplification in the first bioreactor vessel 410, and soluble activator activation, lentivirus transduction, no filter priming 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 priming and amplification in the first bioreactor vessel 410, and Dynabead-based activation, lentiviral transduction, filter-less priming and amplification in the second bioreactor vessel 420, the same cell type or dividing culture was used 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 dividing culture was used in both bioreactor vessels; (4) Soluble activator activation in first bioreactor vessel 410, and RetroNectin coating, transduction, and amplification in second bioreactor vessel 420; (5) Immobilized activator activation in first bioreactor vessel 410, and RetroNectin coating, transduction, and amplification in second bioreactor vessel 420; (6) Dynabead activation in first bioreactor vessel 410, and RetroNectin coating, transduction, and amplification in 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 first bioreactor vessel 410, and retroNectin coating, transduction, and amplification in second bioreactor vessel 420; (9) Activation of the soluble activator in the first bioreactor vessel 410 and expansion of the ectopic electroporated cells or other non-virus modified cells in the second bioreactor vessel 420; (10) TransAct activation in the first bioreactor vessel 410, and expansion of ectopic electroporation cells or other non-virus modified cells in the second bioreactor vessel 420; (11) Dynabead activation in the first bioreactor vessel 410 and expansion of ectopic electroporation cells or other non-virus modified cells in the second bioreactor vessel 420; (12) Expansion of allogeneic NK cells in the first bioreactor vessel 410, and expansion of allogeneic NK cells in the second bioreactor vessel 420 (based on expansion of small molecules, without genetic modification); (13) Expansion of allogeneic NK cells in the first bioreactor vessel 410, and expansion of allogeneic NK cells in the second bioreactor vessel 420 (feeder cell-based expansion, without genetic modification); and (14) soluble activator activation, viral transduction, no filter priming 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 (without RetroNectin coating, and wherein the polybrene is used to aid transduction).
While the above-described embodiments illustrate process monitoring sensors integrated with the bioreactor vessel and/or the seat plate (e.g., on the membrane, integrated in the membrane, on the vessel sidewall, etc.), in other embodiments, it is contemplated that additional sensors may be added to the fluid 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 configuration can be simplified. Additionally, in some embodiments, sensors along the circulation loop may provide a more accurate representation of the container contents as they circulate (rather than measuring when the cells are stationary within the container). 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 algorithms as necessary to correct pumping parameters.
As indicated above, the first module 100 and the third module 300 may employ any form of any system(s) or device known in the art that is capable of cell enrichment and isolation, as well as harvesting and/or compounding. Fig. 78 illustrates one possible configuration of an apparatus/device 900 that may be used as the first module 100 in the biological treatment system 10 for cell enrichment and separation using a variety of magnetic separation bead types, including, for example, miltenyi beads, dynabead, and StemCell EasySep beads. As shown therein, the 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 docking multiple fluid or gas lines together using, for example, luer fittings. 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 extending from the base 910 and including a plurality of hooks 926 for hanging a plurality of process 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 media bag 946, a release bag 948, and a collection bag 950.
The apparatus 900 is configured for use with a magnetic cell separation holder 960 as provided herein or includes a magnetic cell separation holder 960. 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 1-12-2017, 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., the controller 110) according to instructions executed by a processor and stored in memory. Such instructions may include magnetic field parameters. In an embodiment, as discussed below, the apparatus 900 may further include a syringe 952, which may be used to add beads.
Turning now to fig. 79, a generic protocol 1000 of 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 Dynabead is used as the magnetic separation beads, a washing step 1012 may then be performed to remove residues in the Dynabead 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 the first storage bag 938 at step 1016 prior to transfer into the processing bag 932. At step 1018, magnetic separation beads are injected into the processing bag, such as by using a syringe 952 at step 1020. In embodiments, the magnetic separation beads are Miltenyi beads or stescell EasySep beads. In the case of Dynabeads, the washed Dynabeads from step 1012 are resuspended in the processing bag 932. In an embodiment, instead of using a syringe, the magnetic separation 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 process bag 932, through the circuit, and back into the bag. In embodiments where the magnetic separation beads are Miltenyi nanosized beads, a precipitation wash is performed at step 1022 to remove excess nanosized beads, and the portion of the incubated bead-bound cells are stored in a second storage bag 940 at step 1024. After incubation, at step 1026, the bead-bound cells are separated using a magnet (e.g., magnetic field plates 964, 966 of magnetic cell separation 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 a collection bag 950 at step 1030. In an embodiment utilizing StemCell EasySep beads, an additional step 1032 of releasing the cells from the beads to remove the beads is performed, as well as an optional step 1034 of washing/concentrating the collected cells.
A more detailed description of the generic protocol of fig. 79 using device 900 is described in more detail below with particular reference to fig. 80, fig. 80 being a schematic illustration of a flow architecture 1100 of device 900. First, the enrichment process (step 1010) begins with transferring the apheresis product contained in the source bag 930 and the wash buffer from the wash buffer bag 936 into the chamber 912 for washing with the wash buffer 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 magnetic cell separation holder 960 is primed by initiating a flow of buffer from separation buffer bag 934 through manifold 918 and through the column (to prime the column) to processing bag 932.
As disclosed above, in certain embodiments, such as where Dynabead is used as the magnetic separation beads, a washing step (step 1012) is performed to remove any residue in the bead suspension buffer. The washing step comprises the following steps: the beads are injected using a syringe 952 while circulating in the processing circuit 1110 (e.g., from the processing bag 932, through peristaltic pump tubing 914, through the manifold 918, and back to the processing bag 932); cleaning the processing loop 1110; and then the beads are captured by flowing from the processing bag 932 to the separation waste bag 942 while the magnetic field generator 962 is in an "on" state, i.e. while the holder is magnetically coupled to the active magnetic field generator 962 for the permanent magnet or alternatively in case the electromagnetic field is actively generated by using an electromagnet (in each case, the beads are captured in an "on" state). In embodiments where washing is not desired, flow from the disposal bag 932 to the separation waste bag 942 is to ensure that the disposal bag 932 is clean. As used herein, "open" in the case of a permanent magnet means that the magnetic holding element or material 968 (e.g., a separation column, matrix, or tube) is in place within the magnetic field. By "closed" it is meant that the pipe section is removed from the magnetic field.
Next, the enriched cells in the process chamber 912 are transferred to the process bag 932 (step 1014), and separation buffer from the separation buffer bag 934 is drawn into the process chamber 912 to flush any remaining cells in the chamber 912. After flushing, the fluid is discharged to the processing bag 932. The flushing process may be repeated as desired. After all cells have been transferred to the processing bag 932, the chamber 912 is cleaned by sucking buffer from the separation buffer bag 934 into the chamber 912 and draining 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 purged by returning the entire contents to the processing bag 932. As indicated above, in an embodiment, the enriched portion of cells may be stored at this point by transferring the portion of the contents of the processing bag 932 to the first storage bag 938 (step 1016). The processing line 1112 and the first pouch line 1114 may then be purged.
In embodiments that do not utilize a bead washing step, the beads are then injected into the processing circuit 1110 using the syringe 952, and the processing circuit 1110 is cleaned (step 1018). In embodiments utilizing a bead washing step, the beads are resuspended and circulated through treatment loop 1110 (step 1018) and column 968, and the treatment loop is purged 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, the contents of the processing bag 932 may be transferred to the second storage bag 940 prior to incubation, and the second storage bag 940 is agitated (such as using a heat-cool-mixing chamber 922). The contents of the second storage bag 940 are then transferred back to the processing bag 932. Buffer from the separation buffer bag 934 is then drawn into the process chamber 912, and the chamber contents are discharged to the second storage bag 940, and then transferred to the process 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 the processing circuit 1110 for a prescribed incubation time. After incubation, the treatment loop 1110 is cleared.
As discussed above, after incubation, an optional step of washing away excess beads (e.g., nanosized beads) may be performed (step 1022). Washing off excess nano-sized beads includes: initiating flow from the processing bag 932 to the second storage bag 940; sucking the contents of the second storage bag 940 into the process chamber 912; transferring buffer from separation buffer bag 934 to processing bag 932; transferring the contents of the processing bag 932 to a second storage bag 940; and sucking the contents of the second storage bag 940 into the process chamber. The step 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, chamber 912 may then be filled with buffer from separation buffer bag 934, rotation of chamber 912 initiated, and then supernatant discharged to waste bag 742. These steps may be repeated as desired. In an embodiment, cells in the chamber are expelled into the processing bag 932, buffer from the separation buffer bag 934 is drawn into the chamber 932, and then expelled from the chamber into 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, portions of the incubated cell population may be stored in a second storage bag 940 (step 1024). To this end, 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 purged.
In any of the processes described above, after incubation, the cells bound to the beads 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 separation buffer bag 934 to processing bag 932 and then pumping buffer from processing bag 932 to waste bag 942 with magnetic field generator 962 "on".
In an embodiment, flushing without re-suspension may be performed by pumping buffer from separation buffer bag 934 to processing bag 932, flushing processing circuit 1110, cleaning processing circuit 1110, and flowing from processing bag 932 to waste bag 942 with magnetic field generator 962 "on".
In another embodiment, flushing via resuspension may be performed by pumping buffer from separation buffer bag 934 to processing bag 932 with magnetic field generator 962 "off", circulating in processing circuit 1110, clearing the processing circuit, and flowing from processing bag 932 to waste bag 942 with magnetic field generator 962 "on".
In an embodiment, residual waste may be removed by pumping buffer from separation buffer bag 934 to processing bag 932 and flowing from processing bag 932 to waste bag 942 with 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, where the bead-bound cells are to be collected without releasing the cells from the beads, the media from media bag 946 is simply pumped through column 968 to collection bag 950 with magnetic field generator 962 "off. In another approach, buffer from separation buffer bag 934 is pumped to processing bag 932 and then pumped from processing bag 932 to collection bag 950 with magnetic field generator 962 "off. This second method provides for post-separation washing. 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 separation buffer bag 934 is pumped through column 966 to processing bag 932 (if post-separation washing is desired). In either process, the contents of the processing bag 932 are then circulated through the processing circuit 1110, the processing circuit 1110 is purged 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 cells bound to the beads are to be collected after releasing the cells from the beads, a number of potential processes can be performed. For example, in an embodiment, 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 circuit 1110, and then clearing the processing circuit by returning fluid to the processing bag 932. Then, by incubating in the processing circuit 1110, the processing circuit 1110 is cleared, released cells are collected by pumping from the processing bag 932 through the column 966 to the collection bag 950, buffer is pumped from the separation buffer bag 934 to the processing bag 932, and residues are collected by pumping the contents of the processing bag 932 through the column 966 to the collection bag 950, thereby performing incubation and collection with the magnet "on". The released beads (step 1032) may then be discarded by pumping buffer from the separation buffer bag 934 through the column 966 to the processing bag 932 with the magnet "off", circulating in the processing circuit 1110, clearing 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 process chamber 912, pumping buffer from the separation buffer bag 934 to the process bag 932, and transferring buffer from the process bag 932 to the process chamber 912 (step 1034). Then, by filling the process 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, transferring cells to the collection bag after washing/concentrating can be accomplished by transferring the culture medium from the culture medium bag 946 to the collection bag 950, pumping the collection bag contents into the process chamber 912, draining the contents of the process chamber 912 to the collection bag 950, and then manually purging the line between the process chamber 912 and the collection bag 950.
In an embodiment, one of the bags (e.g., processing bag 932) may include a top port 1118 with a filter so that sterile air may be introduced into the system (when processing bag 932 is empty) for cleaning the line as needed (such as in the various process steps discussed above). The cleaning of the line 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 desired piping lines (i.e., lines 1114, 1116, etc.), thereby filling the process line 1112 with liquid from the process bag 932, and finally purging the process line 1112 again with 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 surface) to limit adhesion of micron-sized beads to the sidewalls (particularly during long-term promoting mixing during cycle-based dosing).
In an embodiment, the syringe 952 allows for the addition of a small volume (such as an aliquot of bead suspension) to the circulation-based flow loop 1110. In addition, fluid from the flow loop 1110 may be drawn into the syringe 952 to further clean up any residue from the syringe 952.
In an embodiment, one of the sensors 920 may be configured to measure the flow of fluid. For example, one of the sensors 920 may be a bubble detector or an optical detector that may be used as an auxiliary validation 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 have the volume in the processing bag flow through the magnet without introducing air into the column. The load cell indicates that the process bag is approaching empty within some expected tolerance of load cell variability and then the bubble detector 920 identifies a trailing liquid/air interface to stop flow. Thus, the sensor 920 can be used by the controller to prevent air from being drawn into the circuit (which can create an air plug to dislodge cells, or expose cells to a dry environment) or by accidentally drawing material into the waste bag without stopping the pump after the processing bag is completely emptied. In an embodiment, the bubble detector 920 may thus be used in combination with a weighing sensor integrated with a hook to improve 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 remove cells of the bound beads within the separation column/tube for collection. In an embodiment, instead of or in addition to using an air plug, a buffer solution may be circulated through the separation column to elute the bead-bound cells from the separation column.
In embodiments, two or more peristaltic pump tubes with different inner diameters connected in series may be employed to achieve an expanded range of flow rates 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 may be used for elution of isolated/captured bead-cell complexes. In particular, it is contemplated that an air-liquid interface may be used to assist in removing the composite from the tube sidewall or column void 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 the bead/bead-bound cell packed bed 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 removal from the magnet.
In conjunction with the above, system 900 supports the concept of eluting the bead-cell complex being selected directly into the medium of choice (based on downstream steps). This eliminates the buffer exchange/washing step. In an embodiment, it is also envisaged to elute directly into the medium and viral vector to initiate incubation. The concept may also enable the addition of viral vectors to the final bag. In an embodiment, instead of eluting cells bound to the beads with a buffer, a medium may be used as an elution fluid. Similarly, release buffers can be used to elute the stescell beads for subsequent release of cells from the beads. Dilution can be minimized by replacing the buffer in portions of system 900 with media.
As disclosed above, the apparatus 900 of the first module 100 is a single kit that provides reduced enrichment of platelets and plasma, followed by magnetic separation of target cells. The apparatus 900 is automated to allow the enrichment, isolation and collection steps, as well as all intervening steps, to be performed with minimal human intervention. Similar to the second module 200, the first module 100 and its apparatus 900 are functionally closed to minimize the risk of contamination and flexible to handle a variety of therapeutic volumes/doses/cell concentrations and can support a variety of cell types in addition to CAR-T cells.
One embodiment of the magnetic cell separation holder (960 of FIG. 78) will now be described in more detail with particular reference to FIGS. 81-87. Magnetic bead-based cell selection involves the separation of certain cells from a mixture of cells via targeted binding of cell surface molecules to antibodies or ligands of the magnetic beads (e.g., beads of the types 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 cells and unbound cells may pass through a separation column positioned within a magnetic field generator that captures magnetic beads and thus the associated bound cells. Unbound cells pass through the column without being captured.
Some magnetic cell separation techniques may incorporate nanosized beads (e.g., beads about 50nm or less in diameter), while other techniques may use larger beads (e.g., beads about 2 μm or more in diameter). For example, smaller beads may be desirable because smaller bead sizes may avoid receptor activation on target cells. In addition, downstream steps may skip bead removal, as nanosized beads may have little effect on downstream processing or cell function. However, smaller nano-sized magnetic beads can be separated using a magnetic cell separation procedure that involves the use of a magnetic field gradient booster 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 may still be used in combination 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 increases the complexity of the magnetic bead-based cell separation technique.
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) can vary, the separation conditions can also vary depending on the properties of the beads, and can 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 may vary. The present method eliminates the workflow step of adjusting the magnetic field generator or its parameters between magnetic cell separation procedures using beads of different sizes. In the embodiments provided herein, the magnetic cell separation holder is configured for use in conjunction with a magnetic field generator such that when used with properly sized beads, the magnetic cell separation holder positions the beads within the magnetic field at a location associated with desired magnetic field characteristics for 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 beads selected. Instead, by selecting an appropriate magnetic cell separation holder, the magnetic field experienced by the cells is adapted for separation. Furthermore, when beads of different sizes and/or involving different desired magnetic field characteristics are used, 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 may 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 includes a channel or other cell receptacle, and when the magnetic cell separation holder is loaded into a magnetic bead-based cell separation system that includes a magnetic field generator, cells in the magnetic field separation holder are positioned within the magnetic field at locations having characteristics suitable for separating certain types of magnetic beads (e.g., based on bead material, shape, size, and/or size range) from the 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 setting 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 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 in the magnetic cell separation procedure. The magnetic cell separation holder and accompanying set of magnetizable beads as described above may be provided in a kit, which may comprise a disposable or single-use component. 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 the desired bead type. For example, the magnetic cell separation holder may have a channel at a first location for use with a bead having a first diameter (e.g., configured to accommodate a first cell separation column) and a channel at a second location for use with a bead having a second and larger diameter (e.g., configured to accommodate a second cell separation column). When the magnetic cell separation holder is inserted into the magnetic cell separation device and the magnetic field is generated, the channel at the first position may be in a position experiencing a higher magnetic field strength than the channel at the second position 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 the appropriate location associated with the 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 further include a controller configured to automatically perform the magnetic cell separation procedure. The magnetic separation system may be configured as a functional 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 a magnetic bead-based cell separation system. The system 2100 includes a Source Pump (SP) 2112, a Process Pump (PP) 2114, and a magnetic separator pump (MP) 2116. The system 2100 also includes a collection pinch valve (PV-C) 2126, a waste pinch valve (PV-W) 2128, a bead addition syringe (SG 1) 2118, and a check valve (CV 1) 2120. In an embodiment, check valve 2120 is rated, for example, at a 3psi cracking pressure. The system 2100 may also include suitable processing and/or source containers, such as a sample Source Bag (SB) 2104, a Processing Bag (PB) 2106, a Buffer Bag (BB) 2108, a Media Bag (MB) 2110, a Collection Bag (CB) 2130, and a Waste Bag (WB) 2132. The incubation remover 2102 may also be a bag or may be another collection container suitable for containing and/or disposing of waste material from the 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. The magnetic cell separation retainer 2134 may be removably coupled to (e.g., loaded into, positioned relative thereto) a magnetic field generator 2121 (e.g., magnetic field plates 2122 and 2124 equivalent to plates 964 and 966 in fig. 80). The system 2100 may operate under the control of the controller 2150 according to instructions executed by the processor 2152 and stored in the 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 chart of a method 2200 for magnetic bead-based cell separation that can be used with a magnetic separation system (e.g., 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 bead-based cell separation workflows. In step 2202, source bag 2104, medium bag 2110, buffer bag 2108, and bead addition syringe 2118 are prepared for use with the magnetic separation system. In step 2204, source bag 2104, medium bag 2110, buffer bag 2108 and bead addition syringe 2118 are loaded into a magnetic separation system. The source bag 2104 is fluidly coupled to the source pump 2112. Medium 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, the magnetic cell separation retainer 2134 is coupled to (e.g., positioned adjacent to, inserted into, loaded into) a magnetic field generator 2121 (e.g., magnetic field plates 2122 and 2124) of the magnetic separation system 2100. In step 2208, the bags 2104, 2110, 2108 and syringe 2118 are aseptically welded to the magnetic separation device. In step 2210, source material, such as a cell mixture, from source bag 2104 is transferred to processing bag 2106 via source pump 2112.
In step 2212, magnetic beads (e.g., beads) within bead addition syringe 2118 are added to processing bag 2106. In step 2214, the magnetic beads are incubated with the cell mixture in a processing bag 2106. Incubation material (e.g., cell mixture and magnetic beads) may be circulated into and out of the processing bag 2106 via the processing pump 2114 in order to promote adequate binding between the target cells and the magnetic beads. In step 2216, source bag 2104 is disengaged from source pump 2112 and incubation remover 2102 is fluidly coupled to source pump 2112. Excess incubation material is then removed from processing bag 2106 via source pump 2112 and deposited in incubation remover 2102. In step 2218, magnetic cell separation is performed on the bead-labeled cell mixture. The magnetic cell separation holder 2134 is coupled to the magnetic field generator 2121, and then the magnetic field generator 2121 generates a magnetic field under predetermined magnetic field parameters. The bead labeled cell mixture from processing bag 2106 flows through magnetic cell separation holder 2134 via magnetic separation pump 2116. In an embodiment, the magnetic cell separation retainer 2134 houses a magnetic retaining 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 non-held material 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 a collection bag 2130. In step 2224, the bags (e.g., collection bag 2130, waste bag 2132, buffer bag 2108, and media bag 2110) are sealed, and in one embodiment, the magnetic cell separation holder 2134 may then be disposed of.
Fig. 83A and 83B illustrate top views of different configurations of magnetic cell separation holders 2302 (e.g., magnetic cell separation holder 2134 of fig. 81) positioned within magnetic separation device 2300 in fig. 83A and 83B. Fig. 83A depicts magnetic cell separation holder 2302 in an unloaded configuration in magnetic cell separation device 2300. The 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 the magnetic separation device 2300. The magnetic cell separation holder 2302 can include one or more channels formed within the body 2301 or through the body 2301, and the cell mixture can flow through the channels. While fig. 83A shows two separate channels 2303 and 2305, it is to be understood that the magnetic cell separation holder 2302 may include only one channel, two or more channels, and the like. Turning to channel 2303, channel 2303 may be configured to receive 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 contain magnetically held material 2306. The magnetic holding materials 2304, 2306 may be the same or different. It is also possible to omit the holding material but provide less efficient results, for example the channel 2305 may be a hollow tube. Furthermore, the channels 2303, 2305 may have different sizes and different positions relative to the end surface 2307 of the body 2301. For example, the distance 2315 between the end surface 2307 and the center point of the channel 2303 may be different than the distance between other channels of the body 2301 relative to the end surface 2307. In this way, the channel may experience a magnetic field that is related to its position within the body 2301.
The end surface 2307 may be configured to abut a stop portion or surface 2311 of the frame 2319. The frame 2319 may be configured to conduct magnetic flux. While the body 2301 is shown as terminating at a point at the 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 urging end surface 2307 toward stop surface 2311 until end surface 2307 abuts the stop surface. In the stowed configuration, portions of the body 2301 may still remain outside of the receiving area 2316. Thus, in an embodiment, one or more channels of the body 2301 may be positioned within the receiving area 2316 when loaded.
The magnetic separation device 2300 may also include a door or other feature configured to reduce leakage of the magnetic field outside of the receiving area 2316. The steel backing 2308 of the magnetic separator 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 separation holder 2302 is not inserted into the receiving area 2316 of the magnetic field generator 2313, the doors 2318 of the magnetic field generator 2313 close the gap by means of a compressed spring attached to either door, thereby encapsulating the magnetic flux within the steel backing 2308 and the doors 2318. This prevents magnetic flux from leaking into the channels 2303, 2305 when some processes, such as elution, desire demagnetization.
Fig. 83B depicts magnetic cell separation holder 2302 in a loaded configuration in magnetic separation device 2300. When the magnetic cell separation holder 2302 is fully inserted into the receiving area 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 (e.g., the stop surface 2311) of the backing 2308 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. 83A and 83B, any suitable shape of magnetic cell separation holder 2302 may be used.
The door 2318 of the magnetic field generator 2313 is opened to allow the magnetic cell separation holder 2302 to be inserted between the magnetic field plates 2312, 2314 of the magnetic field generator 2313. For example, the location of the channel 2303 within the magnetic field generator 2313 may cover the location of 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 of 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 the retained beads (e.g., beads or bead-bound cells) from the magnetic retention material (e.g., magnetic retention materials 2304, 2306), the external magnetic field may 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. Fluid with high flow rates then flows through channels 2303, 2305, which creates large shear forces on the held beads. When the adhesive 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 terminate under the control of controller 2150.
As discussed, the magnetic cell separation holder 2302 may have one or more channels, where each channel corresponds to the type and/or size of beads used in the magnetic cell separation procedure. For example, magnetic cell separation holder 2302 may have three channels: a tube for beads having a diameter of 4.5 μm, a tube for beads having a diameter of 3 μm, and a tube for beads having 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 illustrates 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 magnetic cell separation holder 2302 after magnetic cell separation holder 2302 is engaged in magnetic field generator 2313 for magnetic separation. Frame 2319 may include opposing guide plates 2330 spaced apart from one another to allow magnetic cell separation holder 2302 to pass therebetween and facilitate proper positioning within receiving area 2316.
While certain disclosed techniques involve positioning a magnetic cell separation holder within a magnetic field generator in a fixed position as disclosed, it should be understood that other embodiments are contemplated. For example, the magnetic field generator may be movable relative to a magnetically separate holder loaded into a frame in a fixed position.
FIG. 85 depicts a flow chart 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 desired characteristics (e.g., size, type, ligand, etc.). After a sufficient time to ensure that the target cells have been labeled with magnetic beads, the excess incubation mixture is removed. In another embodiment, a portion of the incubated mixture may be removed and evaluated for quality control purposes, i.e., excess incubation mixture may be evaluated to evaluate binding characteristics. In step 2504, a first magnetic cell separation holder 2302 may be coupled within a receiving region 2316 of a magnetic field generator 2313. In step 2506, the magnetic field generator 2313 generates a magnetic field in a receiving area 2316 of the magnetic field generator 2313. In step 2508, a first cell mixture flows through channels (e.g., one or more of channels 2303 or 2305) in 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 area 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 with a fluid having a high flow rate or another suitable method. In step 2514, optionally, magnetic cell separation holder 2302 may be disposed of. Steps 2522 through 2534 mirror steps 2502 through 2514, but may instead pass cells in the second cell mixture labeled with a set of different 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 through 2534 illustrate a method using two different magnetic cell separation holders, it should be appreciated that the two magnetic cell separation holders may instead have the same magnetic cell separation holder for different channels of each cell mixture. Additionally, the second cell mixture may be the resulting cell mixture from step 2508 that passed through the first magnetic cell separation holder 2302 without the held bead labeled cells.
The magnetic selection of target cells may be positive or negative. Magnetic beads are being selected 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 permanent magnets 2612, 2614 is around 0.37 inches (10 mm). However, other distances between 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 restraint fixture design. In the depicted embodiment, the magnetic holding material may be a column matrix 2604 for use with cells labeled with, for example, miltenyi microbeads, and the magnetic holding material 2606 may be a tube for use with cells labeled with, for example, dynabead. 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 used for separation are different for different sized beads (e.g., beads). Larger beads have higher magnetic momentum and therefore require lower magnetic field gradients to produce an equivalent amount of force when compared to smaller beads having lower magnetic momentum. The magnetic force can be expressed as: Wherein M is magnetic momentum, anIs a magnetic field gradient. To ensure the highest magnetic momentum, the magnetic material must be saturated with the external magnetic field strength (i.e., 0.15T for Dynabead 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, i.e., 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 the cancer cells. In addition, the disclosed techniques may be used in combination with any suitable type of beads, such as, by way of example, miltenyi nanosized microbeads (50 nm diameter) and Dynabead (4.5 μm diameter). Miltenyi microbeads are nano-sized superparamagnetic beads that require a magnetizable pillar matrix to prevent them from entering the flow field. The magnetizable pillar matrix consists of spheres of soft magnetic material (e.g. stainless steel 400 series spheres with a diameter of 0.4 mm). Stainless steel 400 series balls are rust resistant. The magnetic properties of the sphere material relate to the strong magnetization when it is exposed to an external magnetic field and the small remanence when the external magnetic field is removed. The process of manufacturing a column matrix of magnetically held material involves ball filling of the column matrix using a vibrator, applying paint to the column matrix, gravity draining the paint, centrifuging to remove any remaining paint, blowing air, 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. The magnetizable pillar matrix filled with balls may be used as a magnetic enhancer to enhance the magnetic field gradient up to 10000 times. The enhanced magnetic field gradient helps attract the nanosized bead-labeled cells to the sphere in the presence of an external magnetic field. After removal of the external magnetic field, the column matrix demagnetizes, which allows the release of the nanosized bead-labeled cells from the column matrix. The nanosized bead-labeled cells are then eluted with a flow of wash fluid through the column matrix.
Dynabead is a larger superparamagnetic bead made of synthetic polymer. Since Dynabeads are much larger than Miltenyi nano-sized beads, dynabeads have much higher magnetic momentum than Miltenyi nano-sized beads when placed in a magnetic field. Thus, magnetic cell separation using Dynabeads does not necessarily involve magnetic intensifiers, such as magnetic column matrices. Tube-based systems are typically used with Dynabead-labeled cells, where a permanent magnet is placed close to the tube. Target cells labeled with Dynabead are attracted to the wall of the tube and unlabeled cells can then be removed with buffer or culture medium. In addition to Miltenyi nanosized microbeads and Dynabead, other differently sized beads 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 tubing, collection and preparation containers, and other components of the disclosed system 2100. In addition, some of these components may be provided and/or packaged as a kit as single-use components, disposable components.
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 include suitable magnetic holding materials that may be preloaded into a suitably configured magnetic cell separation holder. In this way, a user cannot accidentally load or couple incorrect magnetic holding material into the channels 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 Dynabead may be positioned in the highest gradient region in the middle of the permanent magnet. Dynabead separation may 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 a magnetic separation device were each approximately greater than 80% for Miltenyi nanosized 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 magnetic beads of different sizes without the need to adjust magnetic field parameters between procedures. In addition, the magnetic separation device may 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. Moreover, 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 embodiments of the invention, including the best mode, and also to enable any person skilled in the art to practice 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 skilled in the art. Such other examples are intended to be within the scope of the claims if they have structural elements that do not differ from the literal language of the claims, or if they include equivalent structural elements with insubstantial differences from the literal languages of the claims.
Claims (37)
1. A system, comprising:
a magnetic field generator configured to generate a magnetic field under 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 position when the first holder is coupled to the magnetic field generator; and
a second holder configured to be removably coupled to the magnetic field generator, the second holder comprising a second channel configured to be positioned within the magnetic field at a second position when the second holder 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 generated at the first location under the magnetic field parameters; and is also provided with
The second channel experiences a second magnetic field strength and a second magnetic field gradient within the magnetic field at the second location under the magnetic field parameter, 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.
2. The system of claim 1, wherein the magnetic field generator is coupled to a frame that conducts magnetic flux and forms a receiving area configured to receive the first retainer or the second retainer.
3. The system of claim 2, wherein the receiving area is sized to receive only one of the first retainer or the second retainer at a given time.
4. The system of claim 2, wherein the receiving area is configured to receive only a portion of the first retainer or the second retainer.
5. The system of claim 4, wherein the portion comprises the first channel of the first retainer or the second channel of the second retainer.
6. The system of claim 2, wherein the frame includes 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.
7. The system of claim 6, wherein the retractable portion comprises a spring that compresses to allow the first retainer to enter the receiving area of the frame.
8. The system of claim 1, wherein the first channel does not experience the first magnetic field strength when the first holder is not coupled to the magnetic field generator.
9. The system of claim 2, 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.
10. The system of claim 9, 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.
11. The system of claim 1, wherein the first channel and the second channel have different sizes.
12. The system of claim 1, wherein the first holder is configured to be fluidly coupled to a first source of first size beads, and wherein the second holder is configured to be fluidly coupled to a second source of second size beads.
13. The system of claim 12, wherein the first size beads have a diameter of less than 1 μιη and the second size beads have a diameter of greater than 2 μιη.
14. The system of claim 12, wherein the first size beads bind to target cells within a first cell mixture and the second size beads bind to target cells within a second cell mixture.
15. The system of claim 1, wherein the first channel or the second channel comprises a magnetic booster such as a plurality of magnetizable balls.
16. 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 position 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 position 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 generated under a magnetic field parameter at the first location; and is also provided with
The second channel experiences a second magnetic field strength and a second magnetic field gradient within the magnetic field at the second location under the magnetic field parameter, 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.
17. A system, comprising:
a first kit, comprising:
a plurality of first size 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 when the first holder is removably coupled to a magnetic field generator that generates a magnetic field, the first holder is positioned within the magnetic field at a first location; and
a second kit, comprising:
a plurality of second size 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 also provided with
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.
18. 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, such that the first channel experiences 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 by the magnetic field generator in the receiving region to cause the second channel to experience a second magnetic field strength, a second magnetic field gradient, or both.
19. The method of claim 18, wherein the first magnetic field and the second magnetic field are generated at the same magnetic field parameter.
20. The method of claim 18, 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 wherein 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.
21. The method as recited in claim 18, further comprising:
incubating a first cell mixture with first size beads and then 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
the first cell mixture is passed through the first channel and then the second cell mixture is passed through the second channel.
22. A kit for use in a biological treatment system, comprising:
A processing bag;
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 processing circuit includes a pump conduit configured to be in fluid communication with a pump.
23. The kit of claim 22, 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.
24. The kit of claim 22, further comprising:
a valve manifold operable to selectively place the source bag, the processing bag, the bead addition vessel, and the processing circuit in fluid communication.
25. The kit of claim 22, further comprising:
a storage bag configured to be in fluid communication with the processing circuit.
26. The kit of claim 22, further comprising:
a process chamber configured to be in fluid communication with the process circuit.
27. The kit as claimed in claim 22, wherein:
The bead addition vessel is a syringe for injecting magnetic separation beads into the processing circuit.
28. The kit of claim 22, further comprising:
a release buffer bag configured to be in fluid communication with the processing circuit.
29. The kit of claim 22, further comprising:
a media bag configured to be in fluid communication with the processing circuit.
30. An apparatus for biological treatment, comprising:
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 hanging 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 processing circuit.
31. The apparatus according to claim 30, wherein:
The processing bag includes an air port;
wherein the pump is operable to draw air into the system through the air port.
32. The apparatus according to claim 31, wherein:
the air port is in a top portion of the processing bag that is above a fluid level of the processing bag.
33. The apparatus as recited in claim 30, further comprising:
a second waste bag in selective fluid communication with the valve manifold.
34. The apparatus according to claim 30, wherein:
the waste bag comprises a first waste bag and a second waste bag; wherein the first and second waste bags are in fluid communication with the valve manifold.
35. The apparatus as recited in claim 30, further comprising:
a first storage bag and a second storage bag configured to be in fluid communication with the valve manifold.
36. The apparatus as recited in claim 33, further comprising:
a release medium bag is in selective fluid communication with the valve manifold.
37. The apparatus as recited in claim 30, further comprising:
A first pump tube having a first inner diameter;
a second pump tube having a second inner diameter greater than the inner diameter of the first pump tube;
wherein the first pump tube and the second pump tube are selectively engageable with the pump to achieve a range of flow rates for the pump.
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US15/829,615 US10696961B2 (en) | 2017-12-01 | 2017-12-01 | Magnetic cell isolation techniques |
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Families Citing this family (23)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN114144510A (en) * | 2019-06-17 | 2022-03-04 | 德卡产品有限公司 | Systems and methods for centralized fluid management and culture control |
US11000780B1 (en) * | 2019-12-06 | 2021-05-11 | AnuCell Biosystems Limited | Torus reactor for a combined cell isolator and bioreactor |
CA3168941A1 (en) * | 2020-02-06 | 2021-08-12 | Cutiss Ag | Cell isolation device and method |
JP2023546534A (en) * | 2020-10-12 | 2023-11-02 | ライフ・テクノロジーズ・アクシェセルスカプ | Magnetic particle processing systems and related methods for use with biological cells |
WO2022132751A1 (en) | 2020-12-15 | 2022-06-23 | Global Life Sciences Solutions Usa Llc | System and method for heat and mass transfer for a bioprocessing system |
US20240102882A1 (en) | 2020-12-15 | 2024-03-28 | Global Life Sciences Solutions Usa Llc | System and methods for verifying the integrity of a bioprocessing system using mass balancing techniques |
EP4263797A1 (en) | 2020-12-15 | 2023-10-25 | Global Life Sciences Solutions USA LLC | Bioprocessing system and methods |
EP4263786A1 (en) | 2020-12-15 | 2023-10-25 | Global Life Sciences Solutions Usa Llc | Apparatus and method for in-line monitoring of a bioprocess fluid |
US20240002775A1 (en) | 2020-12-15 | 2024-01-04 | Global Life Sciences Solutions Usa Llc | System and methods for verifying the integrity of a bioprocessing system using pressurization |
WO2022132770A1 (en) | 2020-12-15 | 2022-06-23 | Global Life Sciences Solutions Usa Llc | System, method and apparatus for mixing a fluid in a bioprocessing system |
JP2024503215A (en) | 2020-12-15 | 2024-01-25 | グローバル・ライフ・サイエンシズ・ソリューションズ・ユーエスエー・エルエルシー | Methods for cell activation, transduction and amplification |
EP4263785A1 (en) | 2020-12-15 | 2023-10-25 | Global Life Sciences Solutions USA LLC | Disposable kits for cell washing, magnetic isolation and dosing preparation |
CA3202594A1 (en) | 2020-12-15 | 2022-06-23 | Global Life Sciences Solutions Usa Llc | Systems and methods for cell enrichment, isolation and formulation |
JP2024503210A (en) | 2020-12-15 | 2024-01-25 | グローバル・ライフ・サイエンシズ・ソリューションズ・ユーエスエー・エルエルシー | Culture vessels for bioprocessing systems |
WO2022214558A1 (en) * | 2021-04-07 | 2022-10-13 | Norgren LLC | Bioprocessing systems |
WO2023110682A2 (en) * | 2021-12-15 | 2023-06-22 | Norgren LLC | Bioprocessing system and associated sensor manifold |
CN114563331B (en) * | 2022-03-22 | 2023-12-19 | 重庆市公共卫生医疗救治中心 | Automatic counting and detecting system for CD4+T lymphocyte based on micro-fluidic chip |
CN114574323B (en) * | 2022-03-31 | 2023-04-07 | 江苏莱尔生物医药科技有限公司 | Cell separation device |
WO2023215876A1 (en) * | 2022-05-06 | 2023-11-09 | Tissue Genesis International Llc | Method for isolation of pancreatic islets |
WO2023244647A1 (en) | 2022-06-15 | 2023-12-21 | Global Life Sciences Solutions Usa Llc | Disposable kit and culture vessel for a bioprocessing system |
WO2024047359A1 (en) * | 2022-08-31 | 2024-03-07 | Cellular Origins Limited | Automated fill / finish system |
WO2024091900A1 (en) * | 2022-10-24 | 2024-05-02 | National Resilience, Inc. | Systems, methods, and compositions for selecting or isolating cells |
WO2024215466A1 (en) * | 2023-04-12 | 2024-10-17 | University Of Pittsburgh - Of The Commonwealth System Of Higher Education | Methods and systems for tissue perfusion |
Family Cites Families (28)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7217565B2 (en) * | 1999-02-12 | 2007-05-15 | Stemcells California, Inc. | Enriched central nervous system stem cell and progenitor cell populations, and methods for identifying, isolating and enriching for such populations |
US6159378A (en) | 1999-02-23 | 2000-12-12 | Battelle Memorial Institute | Apparatus and method for handling magnetic particles in a fluid |
AU777180B2 (en) | 1999-07-19 | 2004-10-07 | Organon Teknika B.V. | Device and method for mixing magnetic particles with a fluid |
AU5967100A (en) * | 1999-07-21 | 2001-02-13 | Britta Christensen | A novel method for the improved isolation of a target cell population |
DE102004040785B4 (en) * | 2004-08-23 | 2006-09-21 | Kist-Europe Forschungsgesellschaft Mbh | Microfluidic system for the isolation of biological particles using immunomagnetic separation |
JP2009511001A (en) * | 2005-09-15 | 2009-03-19 | アルテミス ヘルス,インク. | Device and method for magnetic concentration of cells and other particles |
CN101172664A (en) * | 2006-11-01 | 2008-05-07 | 中国民航大学 | Ultrasound wave auxiliary hydrothermal synthesis technique for magnetic magnetic iron oxide nano ultra-tiny grain |
JP5456689B2 (en) | 2007-12-07 | 2014-04-02 | ミルテンイ バイオテック ゲーエムベーハー | A centrifuge that separates a sample into at least two components |
WO2011089603A1 (en) * | 2010-01-21 | 2011-07-28 | Biocep Ltd. | Magnetic separation of rare cells |
EP2412802A1 (en) | 2010-07-29 | 2012-02-01 | TXCell | IL-13 producing TR1-like cells and use thereof |
CN102174465A (en) * | 2011-01-12 | 2011-09-07 | 武汉格蓝丽富科技有限公司 | Method for separating enriched target cells from tissues |
CN103298922B (en) * | 2011-03-29 | 2014-11-19 | 南京新诺丹生物技术有限公司 | Multifunctional bioreactor system and methods for cell sorting and culturing |
EP2737317A2 (en) * | 2011-07-28 | 2014-06-04 | The Trustees Of The University Of Pennsylvania | Methods for diagnosing cancer by characterization of tumor cells associated with pleural or serous fluids |
CN102504991B (en) * | 2011-10-11 | 2013-12-04 | 中国人民解放军军事医学科学院卫生装备研究所 | Device and method for non-specific rapid enrichment and purification of bacteria in liquid sample |
ES2602500T3 (en) * | 2011-11-25 | 2017-02-21 | Miltenyi Biotec Gmbh | Cell separation method |
JP2013223820A (en) | 2012-04-20 | 2013-10-31 | Hitachi High-Technologies Corp | Magnetic separator, automatic analyzer with the same, and separation method |
US9797817B2 (en) * | 2012-05-03 | 2017-10-24 | The Regents Of The University Of Michigan | Multi-mode separation for target detection |
GB201208547D0 (en) * | 2012-05-15 | 2012-06-27 | Life Technologies As | Sample holder |
CN102690786B (en) * | 2012-06-05 | 2014-02-12 | 武汉格蓝丽富科技有限公司 | Cell enriching, separating and extracting method and instrument and single cell analysis method |
FR3009082B1 (en) * | 2013-07-26 | 2016-10-21 | Centre Nat Rech Scient | MICROFLUIDIC SYSTEM AND METHOD FOR ISOLATING AND QUANTIFYING AT LEAST ONE SUB-POPULATION OF CELLS FROM A POPULATION OF CELLS |
ES2708584T3 (en) * | 2014-04-24 | 2019-04-10 | Miltenyi Biotec Gmbh | Method for the automated generation of genetically modified T cells |
US20170268037A1 (en) * | 2014-05-15 | 2017-09-21 | Fluxion Biosciences, Inc. | Methods and systems for cell separation using magnetic-and size-based separation |
JP6700282B2 (en) * | 2014-12-19 | 2020-05-27 | ビオセフ エス・アー | Continuous treatment of biological liquids |
EP3037170A1 (en) * | 2014-12-27 | 2016-06-29 | Miltenyi Biotec GmbH | Multisort cell separation method |
EP3061529A1 (en) * | 2015-02-24 | 2016-08-31 | AdnaGen GmbH | Apparatus and method for the analysis, isolation and/or enrichment of target structures in a fluid sample |
EP3341893A4 (en) * | 2015-08-24 | 2019-03-06 | GPB Scientific, LLC | Methods and devices for multi-step cell purification and concentration |
CN105606795B (en) * | 2015-12-31 | 2018-04-24 | 上海白泽医疗器械有限公司 | A kind of cellular immunity magnetic bead sorting system |
CN105651680A (en) * | 2016-02-04 | 2016-06-08 | 关节动力安达(天津)生物科技有限公司 | Method for identifying cell sorting efficiency of different immunomagnetic bead cell separators |
-
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