Disposable kit and culture vessel for a bioprocessing system

Abstract

A disposable kit for a bioprocessing system includes a tray having a spine with a first window and a second window, at least one bioprocessing container received within the tray, a pinch valve manifold removably received by the spine within the first window, a first tubing segment removably received by the spine within the second window, and a second tubing segment removably received by the spine within the second window.

Description

Technical Field

[0001] Cross - reference to related applications This application claims the benefit of U.S. Provisional Application No. 63 / 352,483, filed on June 15, 2022, which is hereby incorporated by reference in its entirety.

[0002] Embodiments of the present invention generally relate to bioprocessing systems and methods, and more particularly, to disposable bioprocessing kits and culture vessels for bioprocessing systems.

Background Art

[0003] Various drug therapies involve the extraction, culture, and amplification of cells for use in downstream treatment processes. For example, chimeric antigen receptor (CAR) T - cell therapy is a type of cell therapy that directs a patient's T cells to specifically target and destroy tumor cells. The basic principle of CAR - T cell design involves a recombinant receptor that combines antigen - binding function and T - cell activation function. A 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 a person, genetically modify them, and return them into the patient so that they attack cancer cells. CAR - T cells can be either derived from the patient's own blood (autologous) or from another healthy donor (allogeneic).

[0004] The first step in the production of CAR - T cells involves using apheresis (e.g., leukapheresis) to remove blood from a patient's body and separate white blood cells. After a sufficient amount of white blood cells are harvested, the leukapheresis product is concentrated with respect to T cells, which involves depleting unwanted cell types. Then, a subset of T cells having specific biomarkers can be isolated from the concentrated subpopulation, if desired, using specific antibody conjugates or markers.

[0005] After isolation of the targeted T cells, the cells are activated in a specific environment where they can actively proliferate. For example, the cells can be activated using magnetic beads coated with anti-CD3 / anti-CD28 monoclonal antibodies or cell-based artificial antigen-presenting cells (aAPCs) which can be removed from the culture using magnetic separation. The T cells are then transduced with the CAR gene by either an integration gamma-retrovirus (RV) vector or a lentivirus (LV) vector. The viral vector uses viral machinery to attach to the patient cells and enter into the cells, whereupon the vector introduces the genetic material in the form of RNA. In the case of CAR-T cell therapy, this genetic material encodes the CAR. The RNA is reverse transcribed into DNA and permanently integrated into the patient cell genome, enabling CAR expression to be maintained as the cells divide and proliferate in large numbers in the bioreactor. The CAR is then transcribed and translated by the patient cells and the CAR is expressed on the cell surface.

[0006] After the T cells are activated and transduced by the CAR-encoding viral vector, the cells are amplified until they are numerous in the bioreactor to achieve the desired cell density. After amplification, the cells are harvested, washed, concentrated, and formulated for injection into the patient.

[0007] Existing systems and methods for manufacturing injectable doses of CAR-T cells typically require many complex operations with numerous human touchpoints, which add time to the overall manufacturing process and increase the risk of contamination. Recent efforts to automate the manufacturing process have eliminated some human touchpoints, but these systems may still suffer from high costs, lack of flexibility, and workflow bottlenecks. In particular, systems that utilize advanced automation are very costly and inflexible in that customers are required to adapt their processes to specific equipment of the system. Patent Document 1 (which is incorporated herein by reference) discloses a system and method for bioprocessing that addresses many of the drawbacks of the prior art.

Prior Art Documents

Patent Documents

[0008]

Patent Document 1

Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0009] However, in light of the above, there is a need for a bioprocessing system and method that improves on the teachings contained in International Publication No. 2019 / 106207 from the viewpoints of overall functionality, flexibility, compatibility, and ease of use.

Means for Solving the Problems

[0010] Certain embodiments corresponding to the subject matter initially claimed with respect to the scope are summarized below. These embodiments are not intended to limit the scope of the claimed subject matter; rather, these embodiments are only intended to provide an overview of possible embodiments. Indeed, the present disclosure can encompass various forms that may be the same as or different from the embodiments described below.

[0011] In one embodiment, a kit for magnetic cell isolation is provided. The kit includes a first stopper manifold having at least four stoppers, a separation chamber configured to be used with the centrifugation chamber of a cell processing device and in fluid communication with the first stopper manifold, a mixing bag configured to be used with the heating / cooling mixing chamber of the cell processing device and in fluid communication with the first stopper manifold, a second stopper manifold having at least four stoppers and in fluid communication with the first stopper manifold, a magnetic cell isolation holder in fluid communication with the second stopper manifold and configured to be used with the magnetic field generator of a magnetic cell isolation device, and a plurality of cell processing bags in fluid communication with the first and / or second stopper manifolds.

[0012] In another embodiment of the present invention, a method for magnetic cell isolation using a disposable kit is provided. The method includes engaging a first stopper manifold having at least four stoppers with a stopper manifold interface of a cell processing device; placing a separation chamber into a centrifugation chamber of the cell processing device, the separation chamber being in fluid communication with the first stopper manifold; placing a mixing bag into a heating / cooling mixing chamber of the cell processing, the mixing bag being in fluid communication with the first stopper manifold; engaging a second stopper manifold with a stopper manifold interface of a magnetic cell isolation device; and inserting a magnetic cell isolation holder into a slot of the magnetic cell isolation device, the magnetic cell isolation holder being in fluid communication with the second stopper manifold. The magnetic cell isolation device is configured to generate a magnetic field for retaining bead-bound cells in the magnetic cell isolation holder when received in the slot.

[0013] In another embodiment of the present invention, a kit for cell processing is provided. The kit includes a stopper manifold having at least six stoppers, the stopper manifold being configured for use with a cell processing device; a mixing bag configured for use with a heating / cooling mixing chamber of the cell processing device, the mixing bag being in fluid communication with the stopper manifold; and a plurality of cell processing bags fluidly connected to the stopper manifold.

[0014] In another embodiment, a method for isolating target cells is provided. The method includes incubating a cell population with magnetic particles to form a cell mixture comprising bead-bound target cells; generating a magnetic field; and passing the cell mixture through a flow path in the magnetic field a plurality of times to retain the bead-bound target cells within an area of the flow path in the magnetic field.

[0015] In another embodiment, an apparatus for magnetic cell isolation is provided. The apparatus includes a stopper manifold interface positioned on a base and configured to receive a stopper manifold of a cell processing kit, a magnetic field generator positioned within the base, and a slot formed within the base configured to removably receive a magnetic cell isolation holder and to selectively and operatively contact the holder with the magnetic field generator.

[0016] In another embodiment, a system for cell processing is provided. The system includes a cell processing module having a housing and a magnetic isolation module (IM). The cell processing module includes a centrifugation chamber, a pump assembly, a stopper manifold interface configured to receive a stopper manifold of a removable cell processing kit, and a heating / cooling mixing chamber. The IM includes a base, an IM stopper manifold interface positioned on the base and configured to receive a stopper manifold of a removable cell processing kit, a magnetic field generator positioned within the base, and a slot formed within the base configured to removably receive a magnetic cell isolation holder and to selectively and operatively contact the holder with the magnetic field generator.

[0017] In another embodiment, a method for magnetically isolating cells is provided. The method includes inserting a magnetic cell isolation holder into a slot of an isolation device, moving a magnetic field generator of the isolation device from a retracted position to an engaged position, wherein in the retracted position the magnetic field generated by the magnetic field generator does not act on the magnetic cell isolation holder to retain bead-bound cells within the magnetic cell isolation holder and in the engaged position the magnetic field generated by the magnetic field generator is sufficient to retain bead-bound cells within the magnetic cell isolation holder, and flowing a population of bead-bound cells into the magnetic cell isolation holder and capturing the bead-bound cells within the magnetic cell isolation holder.

[0018] In yet another embodiment, a method for bioprocessing is provided. The method includes providing a bioprocessing system having a first bioreactor vessel and a second bioreactor vessel, activating a population of cells in the first bioreactor vessel, genetically recombining the population of cells to create a population of genetically recombined cells, and amplifying the population of genetically recombined cells in the first bioreactor vessel and the second bioreactor vessel.

[0019] In another embodiment, a method for bioprocessing is provided. The method includes providing a bioprocessing system having a first bioreactor vessel and a second bioreactor vessel, activating, genetically recombining, and amplifying a first population of cells in the first bioreactor vessel, and activating, genetically recombining, and amplifying a second population of cells in the first bioreactor vessel.

[0020] In another embodiment, a method for bioprocessing is provided. The method includes providing a bioprocessing system having a first bioreactor vessel and a second bioreactor vessel, activating a population of cells in the first bioreactor vessel, transferring the population of cells from the first bioreactor vessel, genetically recombining the population of cells to create a population of genetically recombined cells, transferring the population of genetically recombined cells to at least one of the first bioreactor vessel and the second bioreactor vessel, and amplifying the population of genetically recombined cells in the first bioreactor vessel and / or the second bioreactor vessel.

[0021] In another embodiment, a bioprocessing apparatus is provided. The apparatus includes a housing and a process drawer that is receivable within the housing and movable between a closed position and an open position and is configured to receive at least one culture vessel therein, and a cabinet positioned in a stacked vertical relationship to the housing and including at least one vertical storage drawer slidably received therein.

[0022] In another embodiment, a disposable kit for a bioprocessing apparatus is provided. The disposable kit includes a tray, at least one bioprocessing container received within the tray, a valve manifold mounted to a rear surface of the tray and configured for engagement with a linear actuator array of the bioprocessing apparatus, at least one peristaltic pump tube configured for engagement with a peristaltic pump of the bioprocessing apparatus, and a tubing organizer that holds a plurality of tubes fluidly connected to the valve manifold. The tray is configured to be received within a temperature-controlled process drawer of the bioprocessing apparatus.

[0023] In another embodiment, a bioprocessing method is provided. The method includes positioning a disposable bioprocessing kit within a process drawer of a bioprocessing apparatus such that a culture vessel of the disposable kit is received over a rocking assembly of the bioprocessing apparatus, connecting a tubing organizer to a door of a cabinet of the bioprocessing apparatus, the tubing organizer holding a plurality of tubing tails for fluid connection to a plurality of media bags and / or reagent bags mounted within the cabinet, and fluidly connecting at least one tubing tail of the plurality of tubing tails to at least one of the plurality of media bags and / or reagent bags.

[0024] In another embodiment, a rocking mechanism for a bioreactor vessel is provided, the rocking mechanism including a base, a motor having an eccentric roller mounted to the base and driven by the motor, and a rocking plate in contact with the eccentric roller and configured to receive a bioreactor vessel thereon, the motor is controllable to drive the eccentric roller to transmit a force to an underside of the rocking plate to tilt the rocking plate and the bioreactor vessel.

[0025] In another embodiment, a method of bioprocessing is provided that includes receiving a bioreactor vessel on a rocking plate and actuating a motor to cause an eccentric roller to exert a force on an underside of the rocking plate to tilt the rocking plate and the bioreactor vessel about a horizontal axis.

[0026] In another embodiment, a bioprocessing system is provided that includes a base, a fulcrum attached to the base, a rocking plate received on the fulcrum and configured to pivot on the fulcrum, an eccentric roller in contact with an underside of the rocking plate, a motor configured to drive the eccentric roller and cause the eccentric roller to exert a force on an underside of the rocking plate to pivot the rocking plate about the fulcrum, and a bioreactor vessel received on the rocking plate.

[0027] In another embodiment, a method of bioprocessing is provided that includes providing a bioreactor vessel having a gas-permeable, liquid-impermeable membrane, initiating a flow of gas, and passing the flow of gas across a bottom surface of the membrane to induce turbulent interaction between the flow of gas and the membrane.

[0028] In another embodiment, a bioprocessing system is provided. The bioprocessing system includes an incubation chamber, a support structure configured to support a culture vessel in an elevated position within the incubation chamber, and at least one fan configured to circulate the atmosphere within the incubation chamber across the bottom surface of the gas-permeable and liquid-impermeable membrane of the culture vessel when the culture vessel is supported by the support structure.

[0029] In another embodiment, a bioprocessing system is provided. The bioprocessing system includes a disposable tray having a pair of opposing support legs, a pair of openings in the tray adjacent to the upper portions of the pair of support legs, at least one bioreactor vessel positioned within the disposable tray at a vertical location corresponding to the vertical position of the pair of openings, and at least one fan configured to circulate the atmosphere upwardly from below the bioreactor vessel, through a first opening of the pair of openings, across the bottom surface of the gas-permeable and liquid-impermeable membrane of the bioreactor vessel, and back downwardly below the bioreactor vessel through a second opening of the pair of openings.

[0030] In another embodiment, a bioreactor vessel is provided. The bioreactor vessel includes a base having a plurality of through openings, a lid connected to the base via a plurality of heat-staking portions, and a gas-permeable and liquid-impermeable membrane sandwiched between the base and the lid and held in place by the plurality of heat-staking portions.

[0031] In another embodiment, a disposable kit for a bioprocessing system is provided. The disposable kit includes a tray having a pair of opposing legs and a platform extending between the legs, the platform being configured to support at least one bioreactor vessel, a first bioreactor vessel of the at least one bioreactor vessel received in the tray, the first bioreactor vessel having a base with a plurality of through openings, a lid connected to the base, and a gas-permeable liquid-impermeable membrane sandwiched between the base and the lid. The base includes a plurality of wells configured to receive corresponding support posts of a rocking platform of a bioprocessing system in which the tray is positioned, and one of the plurality of wells has an oval shape.

[0032] In another embodiment, a method for assessing the integrity of a bioprocessing system is provided. The method includes determining the mass of a first container, transferring a volume of fluid from the first container to a second container, determining the mass of the second container, comparing the mass of the first container to the mass of the second container, and generating a notification indicating that a leak exists if the difference between the mass of the first container and the mass of the second container exceeds a threshold.

[0033] In another embodiment, a method for assessing the integrity of a bioprocessing system is provided. The method includes perfusing a liquid from a first container through a second container to a third container, measuring the mass of the second container during the perfusing step, and generating a notification indicating that a leak exists if a change in the mass of the second container exceeds a threshold.

[0034] In one embodiment, a method for assessing the integrity of a bioprocessing system is provided. The method includes utilizing a pump of the bioprocessing system, pressurizing a plurality of flow lines, and measuring a decay of pressure in the plurality of flow lines over a predetermined duration.

[0035] In another embodiment, a bioprocessing system is provided. The bioprocessing system includes a source pump configured to pump a first fluid from a source to a bioprocessing container through a first flow line, a process pump configured to circulate fluid from the bioprocessing container through a circulation pipe and through a filtration pipe, a waste pump configured to pump waste removed by a filter along the filtration pipe to a waste storage through a waste line, a first valve configured to isolate the bioprocessing container from the first flow line, the filtration pipe, and the waste line, and a controller configured to control one of the source pump and the process pump, pressurize at least one of the first flow line and the circulation pipe, and monitor a decay of pressure in at least one of the first flow line and the circulation pipe.

[0036] In yet another embodiment, a sensing chamber for a bioprocessing system is provided. The sensing chamber includes a front plate, a back plate, at least one fluid channel intermediate the front plate and the back plate, a first port in fluid communication with the fluid channel and permitting flow of fluid into the fluid channel, and a second port in fluid communication with the fluid channel and permitting flow of fluid out of the fluid channel. The at least one fluid channel includes a plurality of segments enabling sensing of a plurality of parameters of the fluid by at least a first sensing device and a second sensing device. The first sensing device is configured to sense at least one parameter of the fluid using a first sensing technique, and the second sensing device is configured to sense at least one parameter of the fluid using a second sensing technique. The first sensing technique is different from the second sensing technique.

[0037] In one embodiment, a method for sensing a parameter of a fluid is provided. The method includes flowing fluid from a bioprocessing vessel into a fluid channel of a sensing assembly, electrochemically analyzing the fluid in the fluid channel via contact with at least one electrode of the fluid, and optically analyzing the fluid in the fluid channel.

[0038] In another embodiment, a disposable kit for a bioprocessing system is provided. The disposable kit includes a tray, a bioprocessing vessel received within the tray, a flow-through sensing chamber having a front plate and a back plate, a fluid channel intermediate the front plate and the back plate, a first port in fluid communication with the fluid channel and enabling flow of fluid into the fluid channel, and a second port in fluid communication with the fluid channel and enabling flow of fluid out of the fluid channel. The flow-through sensing chamber is mounted to the tray.

[0039] In another embodiment, a disposable kit for a bioprocessing system is provided. The disposable kit includes a tray having a spine with a first window and a second window, at least one bioprocessing container receivable within the tray, a pinch valve manifold removably receivable by the spine within the first window, a first tubing segment removably receivable by the spine within the second window, and a second tubing segment removably receivable by the spine within the second window.

[0040] In another embodiment, a culture vessel for a bioprocessing system is provided. The culture vessel includes a base, a lid connected to the base and having a vent opening, and a gas-permeable liquid-impermeable membrane sandwiched between the base and the lid. The lid includes a baffle adjacent to the vent port. The baffle is configured to prevent contact of fluid in the bioprocessing container with the vent port.

[0041] The present invention will be better understood from the following description of non-limiting embodiments with reference to the accompanying drawings.

Brief Description of the Drawings

[0042]

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DETAILED DESCRIPTION OF THE INVENTION

[0043] Exemplary embodiments of the present invention will be referred to in detail below, and the examples thereof are illustrated in the accompanying drawings. As far as possible, the same reference characters used throughout the drawings refer to the same or similar parts.

[0044] As used herein, the terms "flexible" or "collapsible" refer to a structure or material that is pliable or can be bent without breaking, and can also refer to a material that is compressible or expandable. An example of a flexible structure is a bag formed from a polyethylene film. The terms "rigid" and "semi-rigid" are used interchangeably herein to describe a structure that "cannot be collapsed", i.e., a structure that cannot be folded, crushed, or otherwise deformed under normal forces that would substantially reduce its elongated dimension. Depending on the context, "semi-rigid" can indicate a structure that is more flexible than a "rigid" element (e.g., a bendable tube or conduit), but can still indicate a structure that does not collapse longitudinally under normal conditions and forces.

[0045] As used herein, the term "container" optionally means a flexible bag, a flexible container, a semi-rigid container, a rigid container, or flexible or semi-rigid tubing. The term "container" as used herein is intended to encompass bioreactor containers having walls or portions of walls that are semi-rigid or rigid, as well as other containers or conduits commonly used in biological or biochemical processes (e.g., cell culture / purification systems, mixing systems, media / buffer preparation systems, and filtration / purification systems, such as chromatography and tangential flow filter systems, and including their associated flow paths). As used herein, the term "bag" means a flexible or semi-rigid container or vessel, for example, used as a containment device for various fluids and / or media.

[0046] As used herein, "fluidly connected" or "in fluid communication" means that the components of a system are capable of receiving or transferring fluid between the components. The term fluid includes gases, liquids, or combinations thereof. As used herein, "electrical communication" or "electrically connected" means that particular components are configured to communicate with each other through direct or indirect signal transmission by direct or indirect electrical connection. As used herein, "operatively connected" refers to a connection, which can be direct or indirect. The connection is not necessarily a mechanical attachment.

[0047] As used herein, the term "tray" refers to any object that can support a plurality of components, at least temporarily. Trays can be made from a variety of suitable materials. For example, trays can be made from cost-effective materials that are suitable for sterilization and single-use disposable products.

[0048] As used herein, the term "functionally closed system" refers to a plurality of components that constitute a closed fluid path, and the closed fluid path can have an inlet port and an outlet port for adding or removing fluid or air to or from the system without compromising the integrity of the closed fluid path (e.g., to maintain a sterile biomedical fluid path therein), such that those ports can include, for example, a filter or a membrane at each port to maintain sterility integrity when fluid or air is added to or removed from the system. The components can include, but are not limited to, one or more conduits, valves (e.g., multi-port diverters), containers, receptacles, and ports, depending on a given embodiment.

[0049] Embodiments of the present invention provide a system and method for manufacturing a cellular immunotherapy agent from a biological sample (e.g., blood, tissue, etc.). In certain embodiments, the method includes genetically recombining a population of cells in a bioreactor vessel to produce a population of genetically recombined cells, and amplifying the population of genetically recombined cells in the bioreactor vessel without removing the population of genetically recombined cells from the bioreactor vessel to generate a plurality of genetically recombined cells sufficient for one or more doses for use in cell therapy treatment. In particular embodiments, one or more of the methods can include using magnetic or non-magnetic beads to activate cells in the same bioreactor vessel to produce a population of activated cells prior to genetically recombining the cells, and washing the genetically recombined cells over the bioreactor vessel to remove unwanted materials.

[0050] Referring to FIG. 1, a schematic diagram of a bioprocessing system 10 according to an embodiment of the present invention is illustrated. The bioprocessing system 10 is configured to be used in the production of a cell immunotherapy agent (e.g., an autologous cell immunotherapy agent), where, for example, human blood, fluid, tissue, or cell samples are collected, and a cell therapy agent is generated from or based on the collected samples. One type of cell immunotherapy agent that can be produced using the bioprocessing system 10 is a chimeric antigen receptor (CAR)-T cell therapy agent, although other cell therapy agents can also be produced using the system of the present invention or aspects thereof without departing from the broader aspects of the present invention. As illustrated in FIG. 1, the production of a CAR-T cell therapy agent generally begins with the collection of a patient's blood and the separation of lymphocytes through apheresis. The collection / apheresis can be performed at the clinical site, and then the apheresis product is sent to a laboratory or manufacturing facility for the production of CAR-T cells. In particular, when the apheresis product is received for processing, the desired cell population (e.g., white blood cells) is concentrated with respect to the collected blood, separated from the collected blood, and the target cells of interest are isolated from the progenitor cell mixture. The target cells of interest are then activated, genetically recombined to specifically target and destroy tumor cells, and amplified to achieve the desired cell density. After amplification, the cells are harvested and a dosage is formulated. The formulated agent is then often cryopreserved and delivered to the clinical site for thawing, preparation, and finally injection into the patient.

[0051] Referring further to FIG. 1, the bioprocessing system 10 of the present invention includes a plurality of individual modules or subsystems, and the plurality of individual modules or subsystems are each configured to perform a specific subset of manufacturing steps in a substantially automated, functionally closed, and scalable manner. In particular, the bioprocessing system 10 includes a first module 100 configured to perform concentration and isolation steps, a second module 200 configured to perform activation, genetic recombination, and amplification steps, and a third module 300 configured to perform the step of harvesting the amplified cell population. In certain embodiments, each of the modules 100, 200, 300 can be communicatively coupled to a dedicated controller (e.g., a first controller 110, a second controller 210, and a third controller 310, respectively). The controllers 110, 210, and 310 are configured to provide substantially automated control over the manufacturing process within each module. The first module 100, the second module 200, and the third module 300 are illustrated as including dedicated controllers for controlling the operation of each module, but it is contemplated that a master control unit may also be utilized to provide global control over the three modules. Each of the modules 100, 200, 300 is designed to cooperate with the other modules to form a single coherent bioprocessing system 10, as described in detail below.

[0052] By automating the processes within each module, the consistency of the products from each module can be increased, and the costs associated with large-scale manual operations can be reduced. Additionally, as will be discussed in detail later, each of the modules 100, 200, 300 is substantially functionally closed, which helps to ensure patient safety by reducing the risk of external contamination, ensures regulatory compliance, and helps to avoid the costs associated with open systems. Moreover, each of the modules 100, 200, 300 is scalable and supports both development with low patient numbers and commercial manufacturing with high patient numbers.

[0053] Referring further to FIG. 1, a particular manner in which process steps are compartmentalized within individual modules that each provide a closed and automated bioprocessing enables an efficient use of capital equipment to an extent not previously seen in the art. As will be appreciated, the step of amplifying a cell population to achieve a desired cell density prior to harvesting and formulation is typically the most time-consuming step in the manufacturing process, while the concentration and isolation steps, the harvesting and formulation steps, and the activation and genetic recombination steps are much less time-consuming. Thus, attempts to automate the entire cell therapy manufacturing process are not only logistically difficult, but can exacerbate bottlenecks in the process that impede the workflow and reduce manufacturing efficiency. In particular, in a fully automated process, the steps of cell concentration, isolation, activation, and genetic recombination can be performed fairly rapidly, but the amplification of genetically recombined cells occurs very slowly. Thus, the manufacture of a cell therapy product from a first sample (e.g., the blood of a first patient) will proceed rapidly up to the amplification step, which requires a significant amount of time to achieve the desired cell density for harvesting. In a fully automated system, the entire process / system will be monopolized by the amplification equipment that performs the amplification of the cells from the first sample, and the processing of a second sample cannot be initiated until the entire system is released for use. In this regard, in a fully automated bioprocessing system, the entire system is essentially offline and not available for processing a second sample until the entire manufacturing process of the cell therapy product (from concentration to harvesting / formulation) is completed for the first sample.

[0054] However, embodiments of the present invention enable the parallel processing of two or more samples (from the same or different patients) and provide for a more efficient utilization of capital resources. This advantage is a direct result of the particular manner in which the process steps are separated into three modules 100, 200, 300, as suggested above. Referring particularly to FIG. 2, in one embodiment, a single first module 100 and / or a single third module 300 can be utilized in a bioprocessing system 12, together with a plurality of second modules (e.g., second modules 200a, 200b, 200c), to provide for the parallel and asynchronous processing of a plurality of samples from the same or different patients. For example, a first apheresis product from a first patient can be concentrated and isolated using the first module 100 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 recombination, and amplification under the control of controller 210a. Once the first population of target cells is transferred from the first module 100, the first module is again available for use, e.g., to process a second apheresis product from a second patient. A second population of target cells produced in the first module 100 from a sample taken from the second patient can then be transferred to another second module (e.g., second module 200b) for activation, genetic recombination, and amplification under the control of controller 210b.

[0055] Similarly, after a second population of target cells has been transferred from the first module 100, the first module is reusable, for example, for the use of processing a third apheresis product from a third patient. Then, a third target population of cells produced in the first module 100 from a sample taken from the third patient can be transferred to another second module (e.g., the second module 200c) for activation, genetic recombination, and amplification under the control of the controller 210c. In this regard, for example, the amplification of CAR-T cells for a first patient can occur simultaneously with the amplification of CAR-T cells for a second patient, a third patient, and so on.

[0056] Also, this approach allows for post-processing to occur asynchronously as needed. In other words, patient cells are not required to all grow simultaneously. Cultures can reach their final density at different times, and the plurality of second modules 200 are not linked, and a third module 300 can be used as needed. In the present invention, samples can be processed in parallel, but samples do not need to be processed in batches.

[0057] Harvesting of the amplified cell populations from the second modules 200a, 200b, and 200c can similarly be achieved using a single third module 300 when each amplified cell population is ready for harvesting.

[0058] Accordingly, by separating the steps of activation, genetic recombination, and amplification (which are the most time-consuming, share specific operating requirements, and / or require similar culture conditions) into stand-alone, automated, and functionally closed modules, the other system equipment utilized for concentration, isolation, harvesting, and formulation is not constrained or taken offline while the amplification of a population of cells is being performed. As a result, the production of multiple cell therapy drugs can be carried out simultaneously, maximizing the utilization of equipment and floor space and increasing the overall process and facility efficiency. It is envisioned that an additional second module can be added to the bioprocessing system 10 to provide parallel processing of any number of cell populations as desired. Accordingly, the bioprocessing system of the present invention enables functionality such as plug-and-play, which allows the manufacturing facility to be easily scaled up or down.

[0059] In certain embodiments, the first module 100 can be any system or device capable of producing a targeted population of concentrated and isolated cells for use in biological processes (such as the manufacture of immunotherapeutic and regenerative medicine products) from an apheresis product collected from a patient. The third module 300 can be any system or device capable of harvesting and / or formulating CAR-T cells or other engineered cells produced by the second module 200 for injection into a patient for use in a cell immunotherapy or regenerative medicine product. In certain embodiments, the first module 100 and the third module 300 are similarly or identically configured such that the first module 100 can first be utilized for the concentration and isolation of cells (which are then transferred to the second module 200 for activation, transduction, and amplification (and, in some embodiments, harvesting)) and then also be used at the end of the process for the harvesting and / or formulation of cells. In this regard, in some embodiments, the same equipment can be utilized for both the front-end cell concentration and isolation steps as well as for the back-end harvesting and / or formulation steps.

[0060] Referring now to FIG. 3, an exemplary configuration of the first module 100 (and, in some embodiments, the third module 300) is illustrated. In certain embodiments, the first module 100 (and the third module 300) includes a processing device 102 and an isolation module 104. In certain embodiments, the processing device 102 and the isolation module 104 can be mechanically interconnected with each other, for example, via a bracket 105 mounted to the respective bottom of the device. The processing device 102 can be, for example, a Sefia S-2000 cell processing instrument available from Cytiva. In certain embodiments, the processing device can be configured in the same (or substantially the same) manner as the device 900 disclosed in Patent Document 1. Accordingly, the processing device 102 includes a base 106, and the base 106 houses a centrifugation chamber 108, a high dynamic range peristaltic pump assembly 111, a stopper manifold interface 112, and a heating-cooling-mixing chamber (thermal mixer) 114. As shown below, the stopper manifold interface 112 is configured to receive a single-use disposable kit specifically configured to perform cell concentration, platelet removal and density gradient-based separation, washing, and / or final formulation, and also provides a simple and reliable means for interfacing a plurality of fluid or gas pipes together, for example, using a luer fitting. Inside the base 106 is a motor, which is drivably connected to a plurality of (in this case, four) output shafts that are operable, under the control of a controller, to move the stopper of the disposable kit between an open position and a closed position. In certain embodiments, the pump assembly 111 is rated to provide a low flow rate of about 3 mL / min and a high flow rate of about 150 mL / min. The processing device 102 can further include a series of sensors configured to monitor various parameters of the device 102 itself and the various fluids handled by the device 102.

[0061] As further shown in FIG. 3, the processing apparatus 102 of the first and / or third modules 100, 300 generally also includes a T-shaped hanger assembly 116 that extends from the base 106 and includes a plurality of hooks 118 for suspending a plurality of bags that contain or receive fluids used in the bioprocessing operations performed by the first or third module. In certain embodiments, it is possible to have six hooks. Each hook can include an integrated weight sensor or load cell (not shown) for monitoring the weight of each container / bag. In certain embodiments, the bags can be, for example, sample source bags, process bags, isolation buffer bags, wash bags, one or more storage bags, post-isolation waste bags, wash waste bags, media bags, release bags, and / or collection bags, depending on the particular process being performed. The processing apparatus 102 also includes a centralized control unit (e.g., controller 110) for performing one or more bioprocessing operations according to algorithms stored in memory in an automated or semi-automated manner.

[0062] Referring further to FIG. 3, and more specifically to FIGS. 4 - 11, the isolation module 104 of the first and / or third modules 100, 300 is shown. The isolation module 104 includes a base / housing 130, a stopper manifold interface 132, and a vertical aperture or slot 134 in the base. The stopper manifold interface 132 is positioned on top of the base 130 and is configured to receive the stopper manifold of a single - use disposable cell processing kit. The vertical aperture or slot 134 is configured to removably receive the magnetic cell isolation holder 136 of the isolation module 104, the purpose of which will be described hereinafter. The isolation module 104 further includes a support pole 138 having one or more hooks 140 or pegs for suspending a fluid bag or container therefrom. In certain embodiments, the hook 140 is configured to be or connectable to a load cell for real - time mass monitoring of the contents of the bag. FIG. 3 illustrates the isolation module 104 as having two hooks 140, although more or fewer than two hooks may be present. For example, in certain embodiments, the isolation module 104 has four hooks 140. In certain embodiments, the inner surface of the housing 130 and / or its base structure is coated or covered by a conductive paint or coating, for example, to shield from EMC perturbations. In certain embodiments, the housing 130 can be manufactured from plastic, while the base structure supporting the housing can be made of metal. However, in certain specific embodiments, both the base structure and the housing can be formed from plastic or a similar non - conductive material.

[0063] In certain embodiments, the isolation module 104 includes a drip chamber holder 113 for inserting and holding a drip chamber of a disposable bioprocessing kit (e.g., for washing, dosing preparation, formulation, and / or isolation of cells), as described hereinafter. In certain embodiments, the drip chamber holder can accommodate different diameters or shapes to fit different versions of the disposable kit drip chamber (e.g., drip chamber 380 of kit 350 shown in FIG. 36 and / or drip chamber 829 of kit 800 shown in FIGS. 37A and 37B). In certain embodiments, the drip chamber holder can include one or several spring plungers to improve the grip of the drip chamber when inserted.

[0064] As best shown in FIGS. 5-7, the stopper manifold interface 132 includes one or more latches or clamps 142, 143, and the one or more latches or clamps 142, 143 can be selectively deployed to hold the cell processing kit in place on the interface 132 as described hereinafter. The interface 132 further includes an array of stopper pins or keyway output shafts 144 that are drivably connected to at least one stopper motor 146 housed within the base 130. In one embodiment, there are six output shafts configured to interface with one stopper each of the six stopper manifolds of a disposable cell processing kit, although more or fewer stopper pins may be utilized without departing from the broader aspects of the present invention and depending on the particular configuration of the disposable kit. In one embodiment, each output shaft 144 has a dedicated motor 146. The motor 146 is configured to rotate the output shaft 144 and move the stoppers of the disposable cell processing kit received on the interface 132 between an open position and a closed position under the control of a controller, as described hereinafter. In particular, the six stopper interfaces shown in FIG. 4 can interface with four or six stopper manifolds of a disposable cell processing kit.

[0065] Referring specifically to FIGS. 4-6, 12, and 13, isolation module 104 can include various operating parameters of isolation module 104, as well as a plurality of sensors for monitoring parameters or conditions of the flow line and / or the fluid therein. For example, in one embodiment, isolation module 104 can include a piping pressure sensor assembly 148 having an interface under which a pressure sensor is positioned, and a bubble sensor / detector assembly 150, both of which form part of the plug manifold interface 132 for respectively monitoring the pressure and the presence of bubbles in one or more of the fluid flow lines connected to module 104. As shown there, bubble sensor assembly 150 includes a housing 152 and a cover 156, housing 152 having an upwardly facing channel 154 or passage therein, the bubble sensor being associated with channel 154 or passage, and cover 156 being pivotally connected to housing 152. Channel 154 is sized and dimensioned to receive the length of tubing, and cover 156 is selectively movable between an open position and a closed position relative to housing 152 to capture and hold the length of tubing within channel 154. In one embodiment, housing 152 and cover 156 are formed from a material having poor electrical conductivity (such as anodized aluminum or plastic, etc.), such that any current present will pass through housing 130 of isolation module 104 and not through bubble sensor 150 (which could potentially affect its operation). In one embodiment, housing 130 includes an air inlet having an integrated filter, and air can be drawn into housing 130 through the filter to cool its internal components during operation.

[0066] Referring to FIGS. 10, 11, and 14-19, the isolation module 104 additionally includes a magnetic field generator assembly 160 housed within the base housing 130. In certain embodiments, the magnetic field generator assembly 160 includes a pair of opposing permanent magnets 162, 164 (having a space therebetween) mounted to a movable carriage 166. Although a pair of magnets 162, 164 are shown, for the same final height, each of the illustrated magnets 162 and 164 is contemplated to be capable of being made from a single long magnet or from a stack of several shorter magnets, without departing from the broader aspects of the present invention. As will be described in detail below, the carriage 166 is movable between an extended position and a retracted position, in which the magnets 162, 164 are positioned on opposing sides of the slot 134 to generate a magnetic field within the slot 134 in the extended position, and in which the magnets 162, 164 are moved behind the slot 134 and are configured not to generate (or to generate only a small or negligible magnetic field) a magnetic field within the slot 134 in the retracted position. The carriage 166 is slidably connected to upper and lower shafts 168, 170 received by bushings or bearings 172 within the carriage assembly 166, supported by the upper and lower shafts 168, 170, and operably connected to a lead screw 174 received through a central bushing 176 of the carriage 166. The lead screw 174 is rotatable to slidably move the carriage 166 between its extended position and its retracted position, as will be disclosed in detail hereinafter.

[0067] As best shown in FIGS. 14 and 16, the magnetic field generator assembly 160 includes a motor 178, which is drivingly connected to a lead screw 174 via a gearbox 180 and a belt 182, which links a timing pulley 183 of the gearbox 180 to a timing pulley 184 of the lead screw 174. Thus, the motor 178 is configured to rotate the lead screw 174 and extend or retract the carriage 166 and magnets 162, 164. As also shown therein, the magnetic field generator assembly 160 further includes an array of sensors, which are utilized to detect the movement of the carriage 166, the position of the carriage 166 (and thus, the magnets 162, 164), and the presence of the magnetic isolation holder 136 in the slot 134. For example, the magnetic field generator assembly 160 includes first and second sensors 186, 188, a third sensor 190, and a crank sensor 192, where the first and second sensors 186, 188 are utilized to detect and confirm the movement of the carriage 166, and the third sensor 190 is utilized to detect the presence of the magnetic cell isolation holder 136 in the slot 134 within the housing. In one embodiment, the sensors 186, 188, 190, 192 are inductive proximity sensors, although other types of sensors known in the art may also be utilized without departing from the broader aspects of the present invention. In connection with the detection of the magnetic cell isolation holder 136, the magnetic field generator assembly 160 also includes a slidable locking pin 194 having a flange 196 (or washer), where the flange 196 is configured to engage the rear face of the carriage 166 adjacent its upper edge. Further, the locking pin 194 includes a coil spring 198, which is configured to bias the locking pin 194 towards the front face of the isolation module 104 (i.e., towards the slot 134), the purpose of which will be explained hereinafter.

[0068] Referring now to FIGS. 20 - 25, the operation of the magnetic field generator assembly 160 and the positioning of its carriage 166 will be described herein. Referring to FIG. 20, the detection of the presence or absence of the magnetic cell isolation holder 136 in the slot 134 is performed using the second sensor 188 and the third sensor 190. At the start of the process, the carriage 166 is in its retracted position, in which it is sensed by the sensor 188 and the sensor 186. In this position, the locking pin 194 is in its retracted position (the reason being that it is prevented from sliding forward due to the engagement of the flange 196 with the rear surface of the carriage 166). In particular, the carriage 166 holds the locking pin 194 in its retracted position against the biasing of the spring 198, releasing the slot 134 to allow the magnetic cell isolation holder 136 to be inserted.

[0069] As shown in FIG. 21, the magnetic cell isolation holder 136 is inserted herein. When the motor 178 rotates the lead screw 168, the carriage 166 is driven forward towards the slot 134 and the isolation holder 136. The locking pin 194 and its flange 196 move forward with the carriage 166 due to the biasing of the spring 198 that urges the locking pin 196 forward. As shown there, when the flange 196 or disk of the locking pin 194 is urged forward, it is detected by the sensor 190 (and the first and second sensors also continue to detect the presence of the carriage 166). In this position, the distal end of the locking pin 194 contacts the magnetic cell isolation holder 136 engaged with the slot 134.

[0070] As shown in FIG. 22, the carriage 166 is then driven to its most forward position by the motor 178 and lead screw 168 until the opposing magnets 162, 164 are aligned with the opposing side portions of the slot 134. As shown there, the locking pin 194 is prevented from moving further forward due to its seating engagement with the inserted isolation holder 136 (i.e., it is in contact with the seat portion of the isolation holder 136), and thus the flange 196 continues to be detected by the sensor 190. However, in this position, the carriage 166 is in front of the sensors 186, 188 and is away from the sensors 186, 188, and thus the presence of the carriage 166 is not detected by these sensors. Thus, as will be recognized, the detection of the flange 196 by the sensor 190 indicates that the isolation holder 136 is received within the slot 134, and the absence of detection of the carriage 166 by either the first sensor 186 or the second sensor 188 indicates that the carriage 166 and its magnets 162, 164 are in the forward working position, where a magnetic field can be generated within the slot 134.

[0071] Referring now to FIG. 23, when the carriage 166 and magnets 162, 164 are moved forward toward the extended position, but the isolation holder 136 is not received in the slot 134 in the housing 130, the locking pin 194 can freely move forward with the carriage 166 under the biasing of the spring 198 (i.e., its forward movement does not contact the seat portion in the isolation holder 136). Thus, the locking pin 194 slides forward until its end reaches the bottom and reaches the end of its movement range. In this position, the distal end of the locking pin 194 obstructs the slot 134 and prevents the insertion of the isolation holder 136, and the flange 196 is in front of the sensor 190 and is not detected thereby, indicating the absence of the isolation holder 136. As shown in FIG. 23, the absence of the isolation holder 136 can be detected even when the carriage is not in its most forward position (i.e., the sensor 186 detects the presence of the carriage 166 and the sensor 188 does not).

[0072] Referring to FIG. 24, and as shown above, when the isolation holder 136 is correctly inserted into the slot 134, the locking pin 194 moves forward with the carriage 166 until it seats in a recess or seat portion within the isolation holder 136. In this position, the locking pin 194 prevents removal of the isolation holder 136 from the slot 134. However, as shown in FIG. 25, when the isolation holder 136 is not properly positioned within the slot 134, the seat portion 199 within the isolation holder 136 is not aligned with the distal end of the locking pin 194. This misalignment prevents the locking pin 194 from entering the recess / seat portion 199. Thus, the locking pin 194 is prevented from traveling far enough forward to align the flange 196 with the sensor 190. In this position, the sensor 188 does not detect the carriage 166, indicating that the carriage 166 has been moved forward. However, at this position of the carriage 166, since the sensor 190 does not detect the flange 196 of the locking pin 194, it indicates that the isolation holder 136 has not been properly received within the slot 134. When in the position shown in FIG. 24, with the locking pin 194 holding the isolation holder 136 in place within the slot 134 and the magnets 162, 164 aligned with opposite side portions of the slot 134, a magnetic field can be generated in a manner known in the art and discussed in more detail hereinafter to capture the bead-bound cells within the isolation holder 136.

[0073] Referring once again to FIGS. 9, 11, 15, and 16, in one embodiment, the isolation module 104 further includes a manual crank 171 operably connected to the linear screw 174. The crank 171 is operable to manually move the carriage 166 and the magnets 162, 164 to a retracted position in the event of an emergency or power loss. The crank 171 has a pivotable handle that remains closed when not in use but can be spread out when needed. A ball detent screwed into the handle holds the handle in the closed position. In one embodiment, the crank 171 can include a pawl and ratchet mechanism such that when the crank is closed, the pin separates the pawl from the ratchet due to the force of a spring. In this position, the pawl and ratchet are not in contact so the crank can rotate freely. To open the crank 171, the operator must spread the crank arm lever, which presses the pawl against the ratchet by the force of the spring and the withdrawal of the pin. Since the pawl and ratchet are now in contact, the crank 171 can be rotated and engaged with the lead screw 174 in a clockwise direction, which corresponds to the rearward movement of the carriage 166. As suggested above, a sensor 192 is provided to detect the open position of the crank 171. In one embodiment, the crank 171 is configured to prevent rotation in the opposite direction and prevent manual forward movement of the carriage 166 (thereby preventing inadvertent or accidental activation of the magnetic circuit).

[0074] Referring back to FIG. 9, the rear surface of the isolation module 104 includes a connector 151 for connection to a power supply for powering the isolation module 104, a switch 153 for turning the isolation module 104 on and off, a communication connector 155 for communicatively connecting the isolation module 104 to a controller, and a plurality of openings 157. The internal fan 159 can discharge air through the plurality of openings 157 to maintain the isolation module 104 at an optimal operating temperature. In one embodiment, the communication connector 155 can be a USB connector, although other wired or wireless communication means known in the art can also be utilized. In one embodiment, the isolation module 104 is communicatively coupled to the processing device 102 and is controlled by its controller 110. In this regard, all information obtained by various sensors of the isolation module 104 (e.g., information regarding the position and status of the magnetic field generator assembly 160, the cell processing kit on the interface 132, and / or the parameters of the fluid passing through various flow lines) is communicated to the controller of the processing device 102, where it is analyzed and then utilized by the controller to control the operation of the isolation module 104 and to generate alerts and the like. Thus, the isolation module 104 need not be equipped with a separate processor and memory, which would increase cost and complexity.

[0075] In connection with the operation of the isolation module 104, the front face of the isolation module 104 can include an array of indicator lights for communicating the status / position of the magnetic field generator assembly to the operator, as shown in FIG. 4. For example, the indicator light 161 can include a green indicator light indicating that the carriage 166 and magnets 162, 164 are in their retracted positions, a flashing yellow indicator light indicating that the carriage 166 is moving, and a steady yellow indicator light indicating that the carriage and magnets are in their extended positions for magnetic retention of bead-bound cells passing through the isolation holder 136. In another embodiment, the front face of the isolation module 104 can instead or in addition include pictograms, which can include, for example, a first pictogram, a second pictogram, and a third pictogram in the form of a lock or other icon, where the first pictogram indicates, when lit, that the isolation holder 136 can be inserted into the slot 134 in the isolation module 104, the second pictogram indicates, when lit, that the application / process has been successfully completed and the magnetic circuit is off (and that the isolation holder 136 can be removed from the isolation module 104), and the third pictogram indicates, when lit, that the isolation holder 136 is properly locked in place.

[0076] As will be discussed in more detail below, the isolation module 104 provides an amplified array of bioprocessing functions to be implemented in a single, user-friendly system. These processes can include, for example, cell concentration and magnetic isolation, washing, and dosing preparation (including cell harvesting and final formulation). As is known in the art, magnetic particle-based cell selection or isolation involves isolating specific cells from a cell mixture through target-specific binding of cell surface molecules to antibodies or ligands on magnetic particles (e.g., beads). Once bound, the cells linked to the magnetic particles can be separated from the population of unbound cells. For example, a cell mixture containing bound and unbound cells can be passed through a separation column positioned within a magnetic field generator (e.g., the magnetic field generator assembly 160 of the isolation module 104), which captures the magnetic particles and, by extension, the associated bound cells. The unbound cells pass through the column without being captured. In embodiments, the magnetic cell isolation holder 136 and / or the isolation module 104 can be specifically configured for cell concentration and isolation using various magnetic isolation bead types (including, for example, Miltenyi beads, Dynabeads, and StemCell EasySep beads). An exemplary configuration of the isolation holder 136 is provided below.

[0077] As shown above, the magnetic cell isolation holder 136 can be designed and configured for use with a variety of different magnetic bead sizes and types. For example, in one embodiment, the magnetic cell isolation holder 136 can be specifically designed for use with nano-sized magnetic beads (e.g., Miltenyi beads, etc.). Referring to FIGS. 26 - 29, in one embodiment, the magnetic cell isolation holder 136 of the isolation module 104 can include a body portion 274 and a handle 276. The body portion 274 receives and holds a vertical column 280 therein, and the handle 276 is connected to the body portion 174, enabling easy operation by the user (e.g., to install and remove the isolation holder 136 into the slot 134 in the isolation module 104). In one embodiment, the body portion 274 and the handle portion 276 can be integrally formed from molded half - bodies 277, 278 that sandwich the column 280. As best shown in FIG. 26, a recess 199 for receiving the locking pin 194 of the magnetic field generator assembly 160 is formed in the front face of the body portion 274. Referring to FIGS. 28 and 29, in one exemplary embodiment, the column shell can be a stock extruded aluminum shell that is anodized and further machined as needed for dimensional tolerances. The column 280 has a pair of identical end caps 282 connected to the column 280 at its opposing ends, which each include a female - type glue port for directly interfacing with the lengths of PVC tubing 284, 286, an O - ring (for forming a fluid seal), and a mesh heat - sealed - on piece (useful in the process for holding the beads before the encapsulating agent is added). In one embodiment, the column is filled with a magnetic retention element and an encapsulating agent, and the magnetic retention element is, in one embodiment, an array of ferromagnetic spheres or beads.The encapsulating agent utilized can be a biocompatible epoxy. To apply the encapsulating agent, the column is filled with ferromagnetic spheres or beads, the encapsulating agent is added to completely wet the beads, then the excess encapsulating agent is removed via centrifugation, and the encapsulating agent is cured.

[0078] As best illustrated in FIGS. 26 and 27, a first length of PVC tubing 284 enters the upper end of column 280 vertically from above and forms an inlet flow path for bead-bound cells into column 280 when isolation holder 136 is received into slot 134 of isolation module 104. A second length of PVC tubing 286 exits the lower end of column 280 and provides an exit path for fluid from column 280 while bead-bound cells are retained within the column, as is known in the art. The second length of PVC tubing 286 is routed through handle 276 in one embodiment, where it exits isolation holder 136 vertically. Although not shown, the first and second lengths of tubing 284, 286 include connectors for integration of column 280 with the flow path of a magnetic cell isolation kit or cassette that is received onto interface 132 of the isolation module, as described hereinafter. FIG. 30 illustrates the installation of magnetic cell isolation holder 136 into slot 134 within the isolation module (i.e., by sliding magnetic cell isolation holder 136 into slot 134 from above). Removal of magnetic cell isolation holder 136 is effected by sliding the holder upward within slot 134.

[0079] Referring to FIGS. 31 - 35, various other exemplary configurations of a magnetic cell isolation holder 136 for use with the isolation module 104 are illustrated. As disclosed above, certain magnetic cell isolation techniques are capable of incorporating nano - sized particles (e.g., beads having a diameter of about 50 nm or less), while other techniques are capable of using larger particles (e.g., beads having a diameter of about 2 μm or more). For example, smaller particle sizes may be desirable as they can avoid receptor activation on target cells. Additionally, nano - sized particles are likely to have little impact on downstream processing or cell function, so downstream steps may be able to omit particle removal. However, smaller nano - sized magnetic particles can be separated using magnetic cell isolation procedures involving the use of a magnetic field gradient enhancer to amplify the applied magnetic field gradient. In contrast, larger particles have a higher magnetic moment. Thus, isolation of certain larger particles can be possible without a magnetic field gradient enhancer. However, with larger particles, the isolation column in the magnetic field generator can reach capacity before a sufficient number of bead - bound cells are captured. In particular, bead - bound cells accumulate inside the passage until the additional bead - bound cells to be captured are no longer in a region with a gradient high enough to overcome the viscous drag force that urges them through the passage. Thus, using larger particles may require multiple capture and elution cycles to obtain the desired yield, which adds complexity to magnetic particle - based cell isolation techniques. As disclosed hereinafter, certain configurations of the magnetic cell isolation holder can prevent the need for multiple cycles to be performed (i.e., by passing the cell mixture through a non - linear flow path in a magnetic field, circulating the cell mixture through or in the magnetic field, and / or passing the magnetic field multiple times). As used herein, non - linear means not in a straight line passing through the magnetic field.For example, the flow path can be shaped spirally or helically, or can have one or more curves or contours in a magnetic field.

[0080] As shown in FIGS. 31-33, a magnetic cell isolation holder 250 for use with the isolation module 104 is shown coupled to the magnetic field generator assembly 160 (i.e., received between the magnets 162 and 164 of the magnetic field generator assembly 160). As suggested above, the magnetic field generator 160 is configured to generate a magnetic field within the slot 134 (also referred to herein as the receiving area 134). The receiving area 134 and the magnetic field have a major axis (defining the longitudinal extent of the magnetic field) and a minor axis, whereby the gradient and magnetic field strength are substantially constant along a line parallel to the major axis (and can decrease at the extremes of the major axis). Looking at a cross-sectional area perpendicular to the major axis (e.g., see FIG. 32), the gradient is substantially constant along lines running into and out of the page.

[0081] The magnetic cell isolation holder 250 is configured to removably couple with the magnetic field generator 160 and is, for example, received within the receiving area / slot 134 of the magnetic field generator 160. As illustrated in FIGS. 31 and 32, in one embodiment, the magnetic cell isolation holder 250 includes a body portion 252, which can be formed from any suitable non-magnetic material configured to house cell isolation and can be coupled to the magnetic field generator 160. In one embodiment, the body portion 252 is generally rectangular in shape (it has a longitudinal extent along the major axis of the magnetic field (in the vertical direction in FIG. 31) that is greater than the width or thickness of the body portion) and includes a plurality of channels or raceways 254 that extend along the body portion 252 for receiving and holding a tube 256. The tube 256, in part, can be configured to hold cells bound to magnetic particles and allow unbound cells to pass under a magnetic field, as is known in the art. For example, the magnetic particles can be Dynabeads or SCT beads, although other magnetic particle / bead types can be utilized without departing from the broader aspects of the present invention.

[0082] The tube 256 is routed along and / or through the body portion 252 via the race 254, defining a flow passage for the flow of a fluid (e.g., a cell mixture). The race 254 and the tube 256 are positioned such that the flow passage defined by the tube 256 is positioned within the magnetic field when the magnetic cell isolation holder 250 is coupled to the magnetic field generator 160 (i.e., received within the slot 134). Moreover, the race 254 (and thus the tube 356 and the flow passage defined thereby) is configured such that when the magnetic cell isolation holder 250 is coupled to the magnetic field generator 160, as discussed hereinafter, the direction of fluid flow within the flow passage at a first location within the magnetic field (i.e., through the tube) is different from the direction of fluid flow within the flow passage at a second location within the magnetic field.

[0083] For example, as illustrated in FIGS. 31-33, in one embodiment, the body portion 252 can include eight generally vertical channels or races 254, two of which are adjacent to each longitudinal corner of the body portion 252. The tube 256 is routed through the race 254 in such a manner as to form a plurality of serially and fluidly interconnected loops. If the body portion includes eight races 254, routing the tube 256 through the race 254 forms four series loops. It is contemplated that the body portion 252 can be formed by more or fewer than eight races so as to accommodate more or fewer than four loops, as desired. Positioning the tube 256 within the loops provides an increase in the residence time of the cell mixture within the magnetic field (the total time the cell mixture passes through the high gradient magnetic field) without reducing the flow rate (by reducing the flow rate or increasing the cross-sectional area of the flow passage), and thus enables better capture of bead-bound cells as compared to a single vertical pass through the magnetic field (at the same flow rate and the same longitudinal length of the magnetic field generator).

[0084] Figure 32 shows the tube loops of the magnetic cell isolation holder 250 more clearly. As shown therein, the multiple loops of the tube each include a first portion 258, and the first portion 258 extends substantially linearly along the longitudinal extension of the main body portion 252, and the longitudinal extension of the main body portion 252 is aligned with the long axes of the magnets 162, 164 and the magnetic field, and the magnetic field has a substantially constant gradient along a line parallel to (and ultimately collinear with) the long axis of the magnet and thus parallel to the tubing path running along the longitudinal axis of the holder. The tube loops further include a second portion 260, a third portion 262, and a fourth portion 264. The second portion 260 extends from the first portion 258 and forms a first return bend. The third portion 262 extends substantially linearly and parallel to the first portion 258. The fourth portion 264 extends from the second portion and forms a second return bend. As shown above, the loops are connected in series with each other, and the fourth portion / bend 264 of the first loop of the multiple loops is fluidly connected to the first portion 258 of the second loop, providing a fluid interconnect with the second loop of the first loop for the circulation of fluid between the loops in the magnetic field. The fluid in one of the loops first passes through, for example, the generally vertical first portion 258, enters the first return bend 260, and then enters the generally vertical third portion 262. The fluid then enters the fourth portion / bend 264 and into the next downstream loop. In certain embodiments, the first return bend 260 and the second return bend 264 are approximately 180-degree bends such that the fluid flow in the first portion 258 and the third portion 262 is generally parallel but in opposite directions, respectively. Although not shown, the tube 256 has an inlet end for connection to a source (e.g., a process bag) and for receiving a cell mixture from the source, and an outlet end for selective connection to a waste bag and / or a collection bag. The multiple loops of the tube 256 are intermediate the inlet end and the outlet end.In some embodiments, the flow path can have an even length (e.g., a vertical portion), and the inlet and outlet are arranged to be at the same end of the magnetic cell isolation holder 250. In other embodiments, the flow path can have an odd number of vertical portions, and the inlet and outlet are positioned at opposite ends of the magnetic cell isolation holder 250.

[0085] In one embodiment, the magnetic cell isolation holder 250 can include a handle 266 or a finger grip portion that enables a user to grasp the magnetic cell isolation holder 250 and position it into or remove it from the receiving area 134. As best illustrated in FIG. 31, the magnetic cell isolation holder 250 is inserted between the magnetic plates 162 and 164 of the magnetic field generator 160. For example, the position of the race 254, and thus the longitudinal paths 258, 262 of the tube 256 in the magnetic field generator 160, can cover a location within the magnetic field having the highest magnetic field strength. In another example, the position of the race 254, and thus the vertical paths 258, 262 of the tube 256 in the magnetic field generator 160, can cover a location within the magnetic field having the highest magnetic field gradient while meeting the magnetic field strength requirements of the magnetic particles. FIG. 33 illustrates the position of the vertical path of the tube 256 in the magnetic field when the magnetic cell isolation holder 250 is received in the slot 134 between the magnets 162 and 164. As shown therein, the body portion 252 of the magnetic cell isolation holder 250, the location of the race 254, and the magnets 162, 164 are configured and dimensioned such that when the magnetic cell isolation holder 302 is coupled to the magnetic field generator 350, the vertical path of the tube 256 is positioned within the high gradient region 268 of the magnetic field generator 160.

[0086] Referring to FIG. 34, in one embodiment, the magnetic cell isolation holder 300 can include a ferromagnetic core 270 that extends substantially the entire length of the magnetic field and is surrounded by the tube 256. In one embodiment, the ferromagnetic core 270 can be an integral part of the body 252 of the isolation holder 250 or it can be an additional component. The use of the ferromagnetic core 270 allows for a higher gradient to be created over a longer length compared to a system without a ferromagnetic core. In particular, the ferromagnetic core creates an additional parallel high-gradient region along the longitudinal axis of the magnetic plate, thereby allowing a longer length of tubing to be routed within the same magnetic field volume compared to the case without a ferromagnetic core. In one embodiment, the ferromagnetic core 270 can be formed from various ferromagnetic materials (e.g., iron, etc.).

[0087] FIG. 35 is a simplified diagram of another configuration for a magnetic cell isolation holder according to another embodiment of the present invention. As shown therein, rather than being formed into a plurality of longitudinal loops, the tube 256 is wound or wrapped in a substantially spiral or helical configuration. As shown, the plurality of loops 272 extend in a direction substantially perpendicular to the longitudinal direction, and the flow through each loop 272 is generally perpendicular (e.g., horizontal rather than vertical) to the longitudinal direction within the magnetic field. Similar to the tube configurations shown in FIGS. 31 - 34, the plurality of loops 272 in the magnetic field provide a longer length of travel for the fluid within the flow path of the tube 256 compared to a single column extending linearly through the magnetic field. In one embodiment, the horizontal or spiral loops 272 of the tube 256 can surround the ferromagnetic core 270.

[0088] Figures 31 - 35 each illustrate a tube 306 disposed within loops extending substantially vertically and horizontally (i.e., parallel or perpendicular to the longitudinal direction / major axis of the magnetic plate and the receiving area), although the tube can be arranged in any configuration that provides an increased length / distance flow path through the magnetic field generated by the magnetic field generator as compared to a single linear path through the magnetic field. This can be accomplished through the use of multiple loops in any orientation / direction (such that the cell mixture passes through the magnetic field multiple times), and / or through the use of single or multiple non - linear paths through the magnetic field (e.g., the tubing has curved or arcuate portions within the magnetic field).

[0089] In one embodiment, the tubing can be arranged to form multiple loops, the tubing loops around the outside of the receiving area 134 (i.e., outside the magnetic field), and all flow within the magnetic field area is made to run in the same direction (e.g., from top to bottom, or from bottom to top). Moreover, it is contemplated that all of the tubes within the magnetic field can run in the same direction and the system can include a manifold at the top and a manifold at the bottom to allow for parallel flow.

[0090] Furthermore, the magnetic isolation holder can be configured to include a flow passage / tube, and the flow passage / tube is intended to be diverted into a plurality of passages passing through a magnetic field and then re-converge. Moreover, in certain embodiments, the main body 252 of the magnetic cell isolation holder 250 can be configured as a fluid device having an integral flow passage (i.e., without the need for separate tubes 256). In particular, it is contemplated that the flow passage and / or the ferromagnetic core can be made entirely of metal. This will enable further utilization of regions of higher magnetic field gradients. In yet another embodiment, it is contemplated that the flow passage can be injection molded into an insert. It is contemplated that insert molding can be performed with a metal framework to add more regions of gradients. Similarly, it is contemplated that the flow passage can be additionally manufactured / printed from a suitable non-ferrous material (e.g., plastic, etc.).

[0091] Although it has been disclosed above that the magnetic field generator can be composed of two opposing magnetic plates forming a permanent magnet, the present invention is not so limited in this regard. In particular, it is contemplated that the magnetic field generator can be an electromagnet that generates a magnetic field substantially similar to the magnetic field generated by a permanent magnet.

[0092] As disclosed above, the processing device 102 and the isolation module 104 are intended to be used in combination with each other to perform various functions, protocols, and / or workflows associated with the isolation, harvesting, and final formulation of cell products in an automated or semi-automated manner. In particular, the processing device 102 and the isolation module 104 can be controlled to perform the various operations associated with these processes in sequence with minimal or no human intervention according to a set of instructions (which are executed by a controller (e.g., controller 110 or 310) of the processing device 102 and stored in the memory of the processing device 102). In certain embodiments, the processing device 102 is configured and operable to perform either the protocol described in Patent Document 1 or the protocol implemented by the device 900 disclosed therein, and the isolation module 104 provides additional functionality and possible workflows as described below. Indeed, the processing device 102 and the isolation module, as disclosed above and as described in more detail below, provide, for example, fluid management, centrifugation, temperature control, cell isolation, cell washing, cell concentration, cell preparation, and formulation.

[0093] In connection with the processing device 102 and the isolation module 104, embodiments of the present invention provide various single-use disposable / consumable kits that are designed to be used with the processing device 102 and / or the isolation module 104 to assist in performing processes and / or workflows associated with the isolation, harvesting, and final formulation of cell products. Referring to FIG. 36, a disposable cleaning kit 350 for use with the processing device 102 is shown. The cleaning kit 350 is a single-use disposable kit that is utilized in conjunction with the device 102 to wash and concentrate fresh or thawed input product after optional temperature-controlled initial dilution. As shown in FIG. 36, the kit 350 includes a cassette or manifold 352 having four plugs 354, 356, 358, 360, an input pipe 362 fluidly connected to the plug 354, a final product / collection container or bag 364 fluidly connected to the plug 356 via a pipe 366, a cleaning solution pipe 368 and a resuspension solution pipe 370 fluidly connected to the plug 358 via a pipe 372, and a waste container or bag 374 fluidly connected to the plug 360 via a pipe 376. As shown therein, the input pipe 362, the cleaning solution pipe 368, and the resuspension solution pipe 370 can be equipped with end caps 378 that preserve the sterility of the lines during transport and storage and that can be removed or cut off immediately prior to use, such that bags containing fresh / thawed input product, cleaning solution, and resuspension solution, respectively, can be connected to the lines via any means known in the art (e.g., aseptic welding, etc.).

[0094] As further shown in FIG. 36, the input pipe 362 includes an in-line drip chamber 380 having an integral filter. The kit 350 further includes a separation chamber 382 fluidly connected to the cassette 352 via a flow pipe 384, and a branched tubing tail 386 fluidly connected to the cassette 352 via the pipe 384. The tubing tail 386 and a second tubing tail connected to the cassette plug 358 are equipped with a hydrophobic filter 388. In certain embodiments, the hydrophobic filter is a 2 micrometer hydrophobic filter. In certain embodiments, the kit 250 is sterilized by means known in the art (e.g., ethylene oxide sterilization, etc.) and can be sealed in a blister pack for transport to and storage by an end user.

[0095] In use, the manifold 352 is mounted on the stopper manifold interface 112 of the processing device 102, and each motor output shaft of the interface 112 is engaged with each of the four stoppers 354, 356, 358, 360 to control the position of the stoppers. The separation chamber 382 is received within the centrifugation chamber 108. The input pipe 362 is connected to a bag containing the input product to be washed, the wash solution pipe 372 is connected to a bag containing the wash solution, the resuspension solution pipe 370 is connected to a bag containing the resuspension solution, and these bags are suspended from the hook 118 of the processing device 102 together with the waste bag 374 and the collection bag 364. Then, the washing process and an optional concentration process are performed according to a set of pre-programmed instructions that are stored in the memory and utilized by the controller 110 of the processing device 102. In certain embodiments, the washing process that utilizes the processing device 102 and the disposable kit 350 optionally includes an initial dilution of the input product, concentration of the input product (so as to reduce its volume), washing of the input product with the wash solution over one or more wash cycles, and then resuspending the input product and collecting the resuspended input product in the collection bag.

[0096] During the initial dilution step, parameters (such as temperature, post-dilution mixing, dilution mixing time, and dilution mixing rate, etc.) can be input or retrieved from memory, and the cleaning solution from the bag connected to the cleaning solution pipe 372 is used to perform the initial dilution. During the concentration / volume reduction step, parameters such as priming (or not) of the flow line by the input product, input bag rinsing during the last volume reduction cycle, input bag rinsing volume, and input bag manual mixing (during the input bag rinsing step) can be selected and / or input, and / or can be enabled or disabled. Moreover, the number of cleaning cycles performed during the cleaning phase can be input and selected. Finally, during the resuspension phase, the prompt instructing the user to switch the cleaning clamp and the resuspension clamp after the cleaning phase can be enabled or disabled, and the volume of the final product at the end of the resuspension phase can be input and / or selected. In certain embodiments, the cleaning process implemented using the processing device 102 and the kit 350 can be used to clean and concentrate the input product before and / or after activation, transduction, and amplification.

[0097] In one embodiment, the processing device 102, the isolation module 104, and the kit 350 use an algorithm for controlling the filling of the resuspension medium into the separation chamber 382 to avoid overshooting the volume, enabling an accurate small final product volume to be achieved during resuspension. A method of resuspending an intermediate volume to achieve a desired final volume is carried out simultaneously with the rinsing of the separation chamber of the cell product, and in a first step, extracting the contents of the separation chamber intermediate volume into the final bag line; in a second step, calculating the number of rinsing cycles and the associated filling volumes required to reach the final volume; in a third step, filling the separation chamber 382 until 10 mL is achieved from the final target of the rinsing cycle volume; in a fourth step, incrementally filling in 1 mL volume increments with a 2 - second pause between increments until the target rinsing cycle volume is reached; in a fifth step, extracting the rinsing volume towards the final / collection bag; and repeating those steps three to five times until the number of rinsing cycles is completed and the final volume of the output bag is reached. During the extraction of the rinsing volume towards the final bag, the air intake during the filling step is factored into the calculation and the filling volume for the next rinsing cycle is then adjusted to ensure that the total final volume effectively reaches the target value. The general process steps disclosed above can be modified as desired, for example, the initial filling of the separation chamber is carried out to any desired volume, and then the separation chamber is incrementally filled with any desired smaller increment volume, and pauses of a selected duration are made between the smaller volume increments (i.e., the volumes and pause durations specified above can be modified as desired).

[0098] Referring to FIGS. 37A and 37B, a single-use disposable magnetic cell isolation kit 800 for use with a processing device 102 and an isolation module 104 is shown (FIG. 37B more clearly shows the placement of various components on the processing device 102 and the isolation module 104, respectively). The magnetic cell isolation kit 800, and the associated protocol enabled by the use of such a kit under the control of the controller 110 of the processing device 100, enable the initial dilution, volume reduction, washing, incubation, post-incubation washing, magnetic isolation, and final resuspension of a cell population, as disclosed hereinafter. In one embodiment, the magnetic cell isolation kit 800 includes a cassette or manifold 802 having four stoppers 804, 806, 808, 810. The kit 800 also includes a tubing 811, a tubing 812, a collection bag 813, a tubing tail 815, and a tubing tail 816. The tubing 811 is fluidly connected to the first stopper 804 and is configured for fluid connection to a fitting on the magnetic cell isolation holder 136. The tubing 812 is fluidly connected to the second stopper 806 and is configured for fluid connection to a second fitting on the magnetic cell isolation holder 136. The collection bag 813 is fluidly connected to the fourth stopper 810 via a tubing 814. The tubing tail 815 is fluidly connected to the third stopper 808 for fluid connection to a resuspension buffer bag (not shown) containing a suspension medium used to resuspend the positive fraction of cells after bead isolation. The tubing tail 816 is fluidly connected to the third stopper 808 for fluid connection to a bag (not shown) containing a release buffer used to release cells from magnetic beads in the magnetic cell isolation holder 136. Further, as shown in FIGS. 37A and 37B, the magnetic cell isolation kit 800 additionally includes a pair of tubing tails 817, 818, which are fluidly connected to the second and fourth stoppers 804, 810, respectively, and are equipped with hydrophobic filters of the type described above. The kit 800 also includes a negative fraction bag 820 fluidly connected to the first stopper 804 via a tubing 819.As shown in FIG. 37A, the manifold 802 is configured for mounting onto the manifold interface 132 of the isolation module 104.

[0099] Referring further to FIGS. 37A and 37B, kit 800 further includes a second manifold 821 having four stoppers 822, 823, 824, 825 and is configured to be received on manifold interface 112 of processing device 102. Kit 800 additionally includes a final collection / transfer bag 826 fluidly connected to second stopper 823, a process bag / incubation bag 827 also fluidly connected to second stopper 823, and a tubing 828 fluidly connected to first stopper 822 and having an in-line drip chamber 829 with a 200 micrometer filter. Tubing 828 additionally includes a branched tubing 830 with a tubing tail and a branched tubing 831 with a sampling pipette. As shown, kit 800 further includes a tubing 832 fluidly connected to first stopper 822 for connection to a platelet-free buffer bag used for platelet depletion. Tubing 832 includes a branched tubing 833 with a filter. Kit 800 also includes a tubing 834 fluidly connected to fourth stopper 825 and having a branched tubing 835 with a filter. Tubing 834 is configured for fluid connection to a bag containing an isolation buffer used to perform a wash cycle during bead incubation and during an optional post-incubation wash cycle to remove excess beads. As shown, kit 800 includes a waste bag 836 fluidly connected to fourth stopper 825 and a spare bag 837 (which is not used during the isolation process) fluidly connected to third stopper 824. Certain of the lines are equipped with a sampling pipette 838 and / or a filter 839 as shown. Further, kit 800 includes a separation chamber 840 configured to be received in centrifugation chamber 108 of processing device 102.The piping 841 interconnects the manifold 802 above the isolation module 104 with the manifold 821 above the processing device 102 for fluid flow therebetween and includes a section of peristaltic pump tubing 842 configured to engage the peristaltic pump assembly 111 of the processing device 102 and a drip chamber 843. The kit 800 additionally includes an additional tubing tail having a sterile air filter 844. The piping 845 is also fluidly connected to the third stopper 824 and its opposite end is configured for fluid connection to a bottom port in the process bag 846, which also forms part of the disposable kit 800. In certain embodiments, the kit 800 can be sterilized by means known in the art (e.g., ethylene oxide sterilization, etc.) and can be sealed in a blister pack for transport to and storage by an end user.

[0100] Referring now to FIG. 37C, a single-use disposable magnetic cell isolation kit 1800 for use with a processing device 102 and an isolation module 104, according to another embodiment of the present invention, is shown. The magnetic cell isolation kit 1800, and the associated protocol enabled by the use of such a kit under the control of the controller 110 of the processing device 100, enable initial dilution, volume reduction, washing, incubation, post-incubation washing, magnetic isolation, and final resuspension of a cell population, as disclosed hereinafter. In certain embodiments, the magnetic cell isolation kit 1800 is generally similar to the kit 800 shown in FIGS. 37A and 37B and includes a cassette or manifold 1802 having four stoppers 1804, 1806, 1808, 1810. The kit 1800 also includes tubing 1811, tubing 1812, a transfer bag 1813, a tubing tail 1815, and tubing 1816. Tubing 1811 is fluidly connected to the first stopper 1804 and is connected (or configured for fluid connection) to one side of the magnetic cell isolation holder / column 136. Tubing 1812 is fluidly connected to the second stopper 1806 and is connected (or configured for fluid connection) to the opposite side of the magnetic cell isolation holder / column 136. The transfer bag 1813 is fluidly connected to the fourth stopper 1810 via line 1814. The tubing tail 1815 is fluidly connected to the third stopper 1808 for fluid connection to a resuspension buffer bag (not shown) containing a suspension medium used to resuspend the positive fraction of cells after bead isolation. Tubing 1816 fluidly connects tubing 1812 to the third stopper 1808. Tubing 1816 can include a drip chamber strainer 1817 (e.g., a 40 micrometer drip chamber strainer) such that cells flow from the source bag, through the drip chamber strainer 1817, through the magnetic cell isolation holder / column 136, and into the negative fraction bag 1820.Further, as shown in FIG. 37C, the magnetic cell isolation kit 1800 additionally includes a tubing tail 1818 that is fluidly connected to a fourth stopper 1810, which is equipped with a hydrophobic filter of the type described above. The kit 1800 also includes a pipe 1819 that is fluidly connected to a first stopper 1804 for fluid connection to a negative fraction bag 1820. Similar to the embodiments of FIGS. 37A and 37B, the manifold 1802 is configured for mounting onto the manifold interface 132 of the isolation module 104.

[0101] Referring further to FIG. 37C, kit 1800 further includes a second manifold 1821 having four stoppers 1822, 1123, 1824, 1825 and is configured to be received on manifold interface 112 of processing device 102. Kit 1800 additionally includes a line fluidly connected to first stopper 1822 for connection to first transfer bag 1832 in addition to transfer bag (i.e., second transfer bag 1813), and further includes final collection / transfer bag 1826 also fluidly connected to first stopper 1822. As further shown in FIG. 37C, pipes 1827 and 1828 are fluidly connected to second stopper 1823 and are each configured for fluid connection to a source bag (not shown) and a process / incubation bag (not shown). As shown, pipe 1827 can have an in-line drip chamber. As shown, kit 1800 further includes pipes 1833 and 1834 which are each fluidly connected to fourth stopper 1825 for connection to a platelet-free buffer bag (not shown) used for platelet depletion and for connection to an isolation buffer bag (not shown) used to perform a wash cycle during bead incubation and during an optional post-incubation wash cycle to remove excess beads. Waste bag 1836 is fluidly connected to third stopper 1824. Pipe 1841 includes a section of peristaltic pump tubing 1842 configured to interconnect manifold 1802 on isolation module 104 and manifold 1821 on processing device 102 for fluid flow therebetween and to engage peristaltic pump assembly 111 of processing device 102, and a drip chamber 1843. In one embodiment, pipe 1841 is equipped with a large drip chamber 1844. Kit 1800 additionally includes a further tubing tail having a sterile air filter 844.In one embodiment, the kit 1800 can be sterilized by means known in the art (e.g., ethylene oxide sterilization, etc.) and can be sealed in a blister pack for transportation to an end user and for storage. In contrast to the kit 800, the kit 1800 includes a magnetic cell isolation holder / column 136 that is already attached to / integrated with the kit 1800.

[0102] Referring now to FIG. 38A, an exemplary protocol 850 for magnetic isolation of cells using the magnetic cell isolation kit 800, processing device 102, and isolation module 104 of FIGS. 37A and 37B is illustrated. As shown above, the magnetic cell isolation kit 800 enables initial dilution, volume reduction, washing, incubation, post-incubation washing, magnetic isolation, and final resuspension of a cell population when utilized in conjunction with the processing device 102 and isolation module 104. In certain embodiments, the protocol 850 illustrated in FIG. 38A performs an optional initial dilution of an apheresis product, concentrates the cells, depletes platelets, isolates (e.g.) CD3+ cells using magnetic beads in the isolation holder 136, and resuspends the cells in a preselected solution for downstream use (e.g., for activation, transduction, and amplification, and ultimately for formulation and administration preparation). As shown therein, at step 852, magnetic cell isolation beads (e.g., Miltenyi beads, Dynabeads, and StemCell EasySep beads) are inserted into the process bag 846 prior to initiation. At step 854, a kit test can be performed. An initial dilution is performed in a further step. Next, at step 856, volume reduction is performed, after which the cells are transferred to a process bag positioned with the thermal mixing chamber 114 of the processing device 102 at step 858. Next, the cells and beads are incubated in the thermal mixing chamber 114 at step 860, and a post-incubation wash is performed at step 862 to remove excess beads. In certain embodiments, the process bag containing the cells and beads is a three-dimensional process bag, which provides improved thermal control as a result of the flat bottom surface of the bag (although the upper surface remains flexible). Another advantage of utilizing a three-dimensional process bag is improved fluid transfer (e.g., during isolation and rinsing steps from bag to bag, the bottom and top surfaces of the bag remain separated, making it difficult to capture or retain cells).During the incubation step, control of the volume, temperature, and mixing carrier movement for incubation (to a specific target cell density) is enabled.

[0103] After incubation and washing, magnetic isolation of bead-bound cells is then performed in step 864 by inserting a magnetic cell isolation holder 136 into slot 134 in isolation module 104 and, optionally, applying a magnetic field to hold the bead-bound cells in the column or flow path of the magnetic cell isolation holder. Rinsing and isolation are performed in step 866, after which, in step 868, the target cells are collected with a resuspension buffer. In certain embodiments, step 868 can include an exchange of the isolation buffer with a medium and performing an elution cycle in three steps: (1) detaching a major amount of cells from the column and collecting the volume, (2) performing an elution cycle with fresh medium and collecting the volume, and (3) rinsing the tubing / bag and collecting the volume. Also, an optional volume reduction step can be performed prior to resuspension of the target cells. In certain embodiments, an air plug can be utilized to assist in removing bead-bound cells from the isolation holder / column as specifically disclosed by Patent Document 1.

[0104] In one embodiment, the processing device 102, the isolation module 104, and the magnetic cell isolation kit 800 can be utilized to circulate the bead-bound cell population back and forth through the magnetic field (rather than creating a single pass through the magnetic field) to isolate / capture the bead-bound cells. For example, the population of bead-bound cells after incubation can be pumped (via pump 111) from the first bag through the magnetic cell isolation holder 136 positioned in the slot 134 in the isolation module 104 and within the magnetic field into the second bag. When the cell mixture passes through the magnetic field generated by the magnetic field generator, the bead-bound cells extend through the magnetic cell isolation holder 136 in the manner described above and are retained / captured within the portion of the fluid path positioned between the opposing plates of the magnet of the magnetic field generator, while the population of unbound cells, the bead-bound cells that were not captured, and the other contents of the cell mixture pass through the magnetic field generator and into the second bag on the other side of the magnetic field generator. Then, the pump 111 of the processing device 102 is operated in reverse to pump the cell mixture back from the second bag through the magnetic cell isolation holder 136 into the first bag. When the cell mixture passes through the magnetic field generated by the magnetic field generator again, additional bead-bound cells are retained / captured within the portion of the fluid path of the magnetic cell isolation holder 136 positioned within the magnetic field. This process (circulation / transfer of the cell mixture from bag to bag) can be repeated until a sufficient number of bead-bound cells are retained within the fluid path (or its magnetic cell isolation holder). As will be appreciated, shuttling the cell mixture back and forth between the first bag and the second bag causes the cell mixture to pass through the magnetic field multiple times, improving the capture efficiency of the system.

[0105] As described above, circulating the cell mixture back and forth between the bags on the opposing sides of the magnetic field generator essentially has the same effect of increasing the travel distance of the cell mixture in the magnetic field by means of multiple loops, by means of paths, or by using a non-linear flow path through the magnetic field, as disclosed above in connection with FIGS. 31-35. In particular, by circulating the cell mixture back and forth, the total "distance" that the cell mixture travels through the magnetic field is increased compared to a single linear path through the magnetic field. This ensures that bead-bound cells that are not retained on the first path through the magnetic field can be captured in subsequent paths before collection. Thus, in connection with the above, it is contemplated that the fluid path in the area of the magnetic field generator (e.g., the flow passage in the magnetic cell isolation holder) can take any of the forms of the embodiments described above. For example, the flow passage in the magnetic field can include multiple loops, paths, spirals, contours, turns, etc. to increase the residence distance in the magnetic field. In particular, it is contemplated that the flow passage configurations shown in FIGS. 31-35 can be used in connection with the isolation sequence (circulation from bag to bag) described above. In other embodiments, a straight path through the magnetic field can be utilized to capture bead-bound cells.

[0106] Also, as suggested above, the kit 800 enables and permits the collection of both the positive fraction and the negative fraction resulting from isolation (in the collection bag 826 and the negative fraction bag 820, respectively). In particular, rather than flowing the negative fraction to waste, it can be collected in the negative fraction bag 820 for other potential uses. The embodiments disclosed above discuss the collection of bead-bound cells using magnetic isolation, but the kit 800 additionally enables negative selection, whereby a desired cell population is not labeled by magnetic beads, while other cells are labeled by such beads and an undesired cell population is adapted to be captured in the magnetic cell isolation holder, and after the bead-bound cell population is captured in the magnetic cell isolation holder, the desired unlabeled cell population is allowed to pass through the isolation holder and be collected.

[0107] Referring to FIG. 38B, an exemplary protocol 1850 for magnetic isolation of cells using the magnet cell isolation kit 1800, the processing device 102, and the isolation module 104 of FIG. 37C is illustrated. Protocol 1850 is generally similar to protocol 1800, however, it includes additional isolation steps to maximize capture of bead-bound cells, as disclosed hereinafter. As shown therein, in step 1852, magnetic cell isolation beads (e.g., Miltenyi beads, Dynabeads, and StemCell EasySep beads) are inserted into a process bag that is connected to the tubing 1828 prior to initiation. Next, the thermal mixing chamber 114 (i.e., thermal mixer) can be configured for protocol 1850. In step 1854, a kit test can be performed. An initial dilution is performed in a further step 1855. Next, in step 1856, a volume reduction is performed, after which the cells are transferred to a process bag that is positioned with the thermal mixing chamber 114 of the processing device 102 in step 1858. Next, the cells and beads are incubated in the thermal mixing chamber 114 in step 1860, and a post-incubation wash is performed in step 1862 to remove excess beads.

[0108] After incubation and washing, magnetic isolation of bead-bound cells is then performed in a two-step process. In a first isolation or batch capture step 1864, the bead-bound cells are passed through the magnetic cell isolation holder / column 136 in step 1866 to collect the target cells and eluted in step 1868. In a second isolation or batch capture step 1870, the fluid / media is passed through the magnetic cell isolation holder / column 136 a second time in step 1872 to collect target cells that were not captured during the first batch capture step 1864 and eluted in step 1874.

[0109] In particular, in the first isolation stage or batch capture step 1864, the magnetic column 136 is turned on and a portion of the isolation wash solution is transferred directly from an isolation buffer bag (not shown) through the pipe 1834 into the negative fraction bag 1820, and through the pipe 1834 through the pipe 1812 through the magnetic column 136 through the pipe 1811 (and finally into the negative fraction bag 1820), thereby being primed. Next, a portion of the contents of the source bag is transferred through the pipe 1828, through the manifold 1821, through the pipe 1841 through the manifold 1802, through the pipes 1816 and the drip chamber strainer 1817, through the magnetic column 136, into the negative fraction bag 1820. Thus, a portion of the cells is captured inside the column 136 and the supernatant is inside the negative fraction bag. The isolation wash solution from the isolation buffer bag (not shown) is used to rinse the column 136 and is transferred into the negative fraction bag after passing through the column. Next, the remainder of the contents of the source bag is transferred through the pipe 1828, through the manifold 1821, through the pipe 1841 through the manifold 1802, through the pipes 1816 and the drip chamber strainer 1817, through the magnetic column 136, into the negative fraction bag 1820. Thus, additional cells are captured inside the column 136 and the supernatant is inside the negative fraction bag. The isolation wash solution from the isolation buffer bag (not shown) is again used to rinse the column 136 and is transferred into the negative fraction bag after passing through the column. At this point, all of the cells should be captured inside the column 136. This completes step 1866 as described above.

[0110] During elution step 1868, the magnet in column 136 is turned off, and the first primary flush is performed by transferring the isolation wash solution from the isolation buffer bag (not shown) through tubing 1841 to the second transfer bag 1813 for storage. Then, the contents of the second transfer bag 1813 are passed from bag 1813, through manifold 1802, through isolation column 136, through tubing 1841, through manifold 1821, and into the first transfer bag 1832 to elute column 136. Thus, elution of column 136 is performed by the stored isolation wash solution from the second transfer bag 1813 to the first transfer bag 1823. During this elution step, a subset of the cells is transferred from column 136 to the first transfer bag 1832; however, some of the cells may remain inside column 136 after elution. The separation chamber 840 is then filled with the eluted cells from the first transfer bag 1832 via manifold 1821 (the cells are arranged to be split between the separation chamber 840 and column 136).

[0111] Next, the isolated wash solution is transferred from the isolation buffer bag (not shown) through line 1841, through manifold 1802 to line 1812, and through lines 1811 and 1814 to a second transfer bag 1813 and stored again, thereby performing a second flush. This step elutes the cells from column 136 and transfers them to the second transfer bag 1813. Next, the contents of the second transfer bag 1813 (including the eluted cells) are passed from bag 1813 through manifold 1802, through line 1841 to the first transfer bag 1832. Next, the contents of the first transfer bag 1832 are circulated back to the second transfer bag 1813 by passing such contents through line 1841, through line 1812, through column 136, through line 1811, and finally to line 1814. Thus, the eluted cells are transferred from the first transfer bag 1832 to the second transfer bag 1813. In certain embodiments, this second flush can be performed any desired number of times to ensure that substantially all of the cells are eluted. Next, all of the eluted product is transferred from the second transfer bag 1813 to the first transfer bag 1832. Also, cells from separation chamber 840 from the first flush are transferred to the first transfer bag 1832 such that all of the eluted cells are inside the first transfer bag 1832. This completes the first isolation phase or batch capture step 1864.

[0112] In the second isolation stage or batch capture step 1870, the contents of the first transfer bag 1832 are passed through the pipe 1841, through the manifold 1802, the pipe 1816, and the drip chamber strainer 1817, through the magnetic column 136, and into the negative fraction bag 1820. Next, a portion of the isolation wash solution from an isolation buffer bag (not shown) is passed through the pipe 1834, through the manifold 1802, the pipe 1816, and the drip chamber strainer 1817, through the magnetic column 136, and into the negative fraction bag 1820. Next, the separation chamber 840 is filled with the isolation wash solution from the isolation buffer bag. Then, this solution is transferred from the separation chamber 840 to the first transfer bag 1832. Next, the solution from the first transfer bag 1832 is passed through the pipe 1841, through the manifold 1802, the pipe 1816, and the drip chamber strainer 1817, through the magnetic column 136, and into the negative fraction bag 1820. Next, the isolation buffer solution is exchanged with the resuspension medium by passing the resuspension solution from a resuspension buffer bag (not shown) through line 1815, through the manifold 1802, through the pipe 1841, through the manifold 1821, and into the first transfer bag 1832 (i.e., a portion of the resuspension solution is transferred from the resuspension buffer bag to the first transfer bag 1832). Then, the resuspension solution from the first transfer bag 1832 is transferred through the column 136 to the negative fraction bag 1820. At this point, the bead-bound cells are captured inside the magnetic column 136.

[0113] During elution step 1874, the magnet in column 136 is turned off, and the resuspension solution from the resuspension buffer bag is passed through manifold 1802, through tubing 1811, through magnetic column 136, through tubing 1812, through manifold 1802 again, through tubing 1841, through manifold 1821, and into final collection bag 1826. Next, this first portion of the cells from collection bag 1826 is transferred to separation column 840. Next, a portion of the resuspension solution from the resuspension bag is transferred through tubing 1841 to final collection bag 1826. Then, this resuspension solution is passed back through tubing 1841, through column 1836, and into second transfer bag 1813, and another flush is performed by flushing the remaining cells from column 136. Next, the contents of this second transfer bag 1813 are transferred to final collection bag 1826. Then, the contents of final collection bag 1826 are circulated back through the column and into the second transfer bag. Thus, the eluted cells are transferred from final collection bag 1826 to second transfer bag 1813. In certain embodiments, this second flush can be performed any desired number of times to ensure that substantially all of the cells are eluted. Then, all of the eluted product is transferred from second transfer bag 1813 to final collection bag 1826. Then, the eluted cells are transferred from final collection bag 1826 to separation chamber 840 and mixed with the previously eluted cells. Next, the resuspension solution from a resuspension buffer bag (not shown) is passed into first transfer bag 1832, and separation chamber 840 is topped off by transferring the resuspension solution from first transfer bag 1832 to separation chamber 840. This ends elution step 1874, and all of the eluted cells are contained within separation chamber 840.

[0114] Subsequently, the final product formulation can be carried out via centrifugation within the separation chamber 840 and by extracting the supernatant from the separation chamber 840 to the waste bag 1836. Subsequently, the cells within the separation chamber 840 are passed to the final collection bag 1826. Finally, the resuspension solution from the first transfer bag 1832 is passed to the separation chamber 840, which is then extracted to the final collection bag 1826.

[0115] Accordingly, this protocol 1850 concentrates cells from the apheresis product, depletes platelets (PlateletFree scenario), isolates CD3+ cells using magnetic beads, and resuspends the cells in a preselected solution. As disclosed above, after the first isolation and collection of the isolated cell product, the isolation step is repeated (i.e., the cell product is isolated through the column a second time), which ensures that the target cell elution and collection are maximized.

[0116] Referring now to FIG. 39, a single-use disposable administration preparation kit 500 for use with a processing device 102 and an isolation module 104 is shown. The administration preparation kit 500, and the associated protocols enabled by the use of such a kit under the control of the controller 110 of the processing device 100, enable the automation, dilution, mixing, cryopreparation, and administration of the volumetric division of cell products, as described hereinafter. The administration preparation kit 500 includes a cassette or manifold 502 having six stoppers 504, 506, 508, 510, 512, 514, a process bag 516 fluidly connected to the stopper 504 via a peristaltic pump tubing 518, a plurality of tubing lines 520, 522, 524 fluidly connected to the cassette 502 for fluid connection (e.g., aseptic welding) to one or more media bags (not shown), a final formulation / collection bag 526 fluidly connected to the stopper 508 via a pipe 528, and a bag 530 (for containing the initial / intermediate product from which the final dose / formulation is produced) fluidly connected to the stopper 508 via a pipe 532. In certain embodiments, the process bag 516 is a three-dimensional process bag. As further shown therein, the kit 500 further includes a waste bag 534 fluidly connected to the stopper 514 via a pipe 536, and a plurality of cryogenic bag connection pipes 538, 540, 542, 544 fluidly connected to the stoppers 510, 512, 514 (for connection to a plurality of cryogenic bags using aseptic welding or other connection means). Finally, the line 518 is equipped with a pair of hydrophobic filters 546, 547 on opposite sides of the peristaltic pump tubing section, and the kit 500 further includes an air inlet pipe 548, which is fluidly connected to the stopper 510 and has a hydrophobic filter 549. In certain embodiments, the hydrophobic filter is a 2-micrometer hydrophobic filter. In certain embodiments, the kit 500 can be sterilized by means known in the art (e.g., ethylene oxide sterilization, etc.) and can be sealed in a blister pack for transportation to and storage by an end user.

[0117] Figure 40 illustrates the integration / installation of the dosing preparation kit 500 on the processing device 102 and the isolation module 104. As shown therein, on the isolation module side, the stopper manifold / cassette 502 is installed on the stopper manifold interface 132 of the isolation module 104, and each motor output shaft 144 of the motor 146 is engaged with each of the six stoppers 504, 506, 508, 510, 512, 514 to control the position of the stopper. The waste bag 534 is suspended from one of the hooks 140 on the pole 138 of the isolation module, and one or more of the lines 538, 540, 542, 544 are aseptically welded to the corresponding freezer bag (or connected via other means), which is then suspended from one or more of the hooks 140 on the pole 138 of the isolation module 104. Finally, the tubing tail with the air filter 547 is connected to the piping pressure sensor 148 of the isolation module 104, and a portion of the line connecting the 3D process bag 516 to the stopper manifold 502 is engaged with the bubble sensor assembly 150 of the isolation module 104.

[0118] On the processing device side, the initial product bag 530 and the final formulation bag 526 are suspended from a single hook 118 of the hanger assembly 116 of the processing device 102 (which has an integrated load cell or weight sensor for sensing the weight of the bag suspended therefrom). The media bag (not shown) is aseptically welded (or connected via other means) to the media pipes 520, 522, 524 and is suspended from another hook 118 of the hanger assembly 116 of the processing device 102 (which likewise has an integrated load cell or weight sensor for sensing the weight of the bag suspended therefrom). The 3D process bag 516 of the kit is installed inside the thermal mixing chamber 114 of the processing device 102. Finally, a section of the peristaltic tubing 518 that fluidly interconnects the process bag 516 with the stopper manifold 502 is engaged with the peristaltic pump assembly 111 of the processing device 102, and a tubing tail with an air filter 546 is connected to a pressure sensor (not shown) of the processing device 102.

[0119] Referring to FIG. 41, a method 550 for preparing a dose of a cell product using a processing device 102, an isolation module 104, and an administration preparation kit 500 is illustrated. As shown above, the administration protocol is implemented in an automated manner by a controller 110 of the processing module 102, controlling both the processing module 102 and the isolation module 104 through a data connection therebetween. Method 550 includes, at step 552, testing and priming the kit 500, which, in certain embodiments, includes venting air from the 3D process bag 516, the freezer bag, and the freezer bag lines 538, 540, 542, 544 to minimize air in the bag at the end of the process, priming the 3D mixing bag 534 (to equalize the amount of air inside the 3D process bag 534), priming the media bag lines 520, 522, 524, and calibrating the pump 111 by flowing media from the media bag connected to line 520 (to calibrate the pump speed by the exact weight withdrawn from the bag on the load cell / hook). Next, at step 554, the initial product in bag 530 is divided. In certain embodiments, this involves transferring the entire input product from bag 530 into the process bag 516 positioned in the thermal mixing chamber 114 of the processing device 102 and mixing the input product in the thermal mixing chamber 114 for a preselected or preset duration. Then, a preset or preselected volume of the product is transferred from the process bag 516 to the formulation bag 526. The remaining volume of the product is transferred back from the process bag 516 to the initial input bag 530. In certain embodiments, at step 556, the 3D process bag 516 is then rinsed with media from the media bag connected to line 520, and the rinse volume is pumped into the input bag 530. In certain embodiments, the rinse volume and the rinse mixing time can be selected by the user. As further shown therein, at step 558, formulation preparation is then performed.In one embodiment, this includes transferring a predetermined volume of media from a media bag connected to line 520 to process bag 516 in thermal mixer 114 and transferring such media to formulation bag 526.

[0120] If selected / desired, the preparation and administration of the frozen bags can then be carried out in steps 560 and 562, respectively, to incorporate additional bags (which can be frozen bags for cryopreservation purposes). In such cases, in step 560, a selected volume of the split product from the input bag 530 is then transferred to the process bag 516 in the thermal mixer 114 (with the excess split product remaining in the input bag 530). A predetermined volume of medium from the medium bag connected to line 524 and / or the medium bag connected to line 522 is pumped into the process bag 516 in the thermal mixer 114, where temperature adjustment is then carried out at a predetermined / preselected temperature (for a period of time calculated by the controller 110 required to adjust it down to the target temperature). Next, the medium from the medium bag connected to line 520 is transferred (after a prompt) into the process bag 516 in the thermal mixer, and the volume in the process bag 516 is mixed with such medium. The administration of the frozen bags is carried out by transferring a preselected volume to the first frozen bag connected to line 538, the second frozen bag connected to line 540, the third frozen bag connected to line 542, and / or the fourth frozen bag connected to line 544, as desired. Precise control of the volume transferred is enabled by ensuring the correct peristaltic pump flow rate and by controlling the flow timing. The peristaltic pump flow rate setting is calibrated during an initial priming step to account for possible deviations from the nominal baseline of the peristaltic pump tubing 518 and / or the peristaltic pump 111. Thus, this protocol enables the formulation / production of one formulation bag 526 and up to four frozen bags (connected to lines 538, 540, 542, and 544, respectively). Thus, one to five doses / bags of a user-selected volume of up to four components (the initial product plus three media) are enabled by such a system and method of the present invention.

[0121] Referring now to FIGS. 42-45, illustrated are exemplary embodiments of a second module 600 (also referred to herein as bioprocessing device 600) for the activation, transduction, and amplification of cells (e.g., cells concentrated and isolated using the first module 100). The second module 600 can be, for example, a device / system configured to implement the workflows and methods described above in connection with the second module 200 and can be configured to operate similarly to the module 200 disclosed in Patent Document 1. As shown therein, in one embodiment, the second module 600 includes a housing 602, a latch-engageable process drawer 604 slidably received within the housing 602, and a latch-engageable waste bag drawer 606, with the waste bag drawer 606 positioned below the process drawer 604 and also slidably received within the housing 602. Both the process drawer 604 and the waste bag drawer 606 are movable between a closed position and an open position for inserting and removing various components of the second module 600, as will be disclosed hereinafter. As will be described in detail below, the process drawer 604 is configured to receive a disposable cell processing kit having one or more culture / bioreactor vessels therein. In one embodiment, the rear surface of the housing 602 includes a power connection port or cable, one or more communication ports (e.g., RJ45 port and RS485 port), at least one inlet for receiving a supply such as carbon dioxide, air oxygen, and / or nitrogen, one or more outlet / discharge ports, and / or a plurality (e.g., three) of USB ports. Also, the drawer 604 can include a status indicator light 605, a plurality of USB or other ports 607 for data transfer, and an input terminal 609.

[0122] Further, the second module 600 includes a cabinet 608 positioned in a stacked vertical relationship with respect to the housing 602 (e.g., mounted on top of the housing 602). The cabinet 608 includes a pair of latch-engageable doors 610, 612 that are hingedly mounted about a vertical axis and are configured to move between a closed position (preventing access to the interior of the cabinet 608) and an open position (allowing access to the interior of the cabinet 608). Also, the cabinet 608 and the doors 610, 612 can include an interlock mechanism (e.g., a pneumatic latch or pin), and the interlock mechanism is utilized to maintain the doors 610, 612 in the closed position while the bioprocessing operation is in progress. In certain embodiments, the cabinet 608 further includes a plurality of vertically oriented storage drawers 614, 616 that are slidably received within the cabinet 608. Two vertical storage drawers 614, 616 are illustrated in FIGS. 43 and 44, but it is also possible to have more or fewer than two drawers. In certain embodiments, the storage drawers 614, 616 are slidably mounted to upper and / or lower tracks within the cabinet 608, enabling the drawers 614, 616 to be easily moved between a retracted position and an extended position (shown in FIGS. 43 and 44), where in the retracted position, the drawers are received within the cabinet 608 and the doors 610, 612 can be closed, and in the extended position, the drawers 614, 616 extend from the cabinet 608, allowing easy access to components and accessories mounted to the left and right vertical sides of the drawers 614, 616.

[0123] As best shown in FIG. 45, the inner faces of doors 610, 612 include a mechanism (e.g., a specific array of pegs or pins 618) for releasably connecting a tubing organizer card and / or a sampling card to doors 610, 612, as described below. For example, in one embodiment, the left door 610 can include an array of pegs for holding a sampling card of a disposable kit, while the right door 612 can include an array of pegs for holding a tubing organizer card of the disposable kit. In one embodiment, both the tubing organizer card and the sampling card can be mounted to the right door 612. As shown in FIGS. 43 and 45, one or both of the vertical storage drawers 614, 616 can include hooks 620 on one or each of their faces for receiving media, reagents, and / or other fluid / solution bags for use in various bioprocessing operations performed by the apparatus 600. The hooks 620 can be operably connected to or integrated with load cells for monitoring the weight of the bags connected thereto. In one embodiment, the first vertical drawer 614 is configured to receive one or more media bags 622, while the second vertical drawer 616 is configured to receive one or more reagent bags 624. In this regard, the first vertical drawer 614 can be referred to as a media tray or compartment, while the second vertical drawer 616 can be referred to as a reagent tray or compartment. The first vertical drawer 614 is equipped with a media drip tray 626 on its opposing face to catch leaks or drips from the media bags suspended from the hooks 620, while the second vertical drawer 616 is equipped with a reagent drip tray 628 on its opposing face to catch leaks or drips from the reagent bags 624 suspended from the hooks 620.In one embodiment, the drip trays 626, 628 are removable from the drawers 614, 616, respectively.

[0124] In one embodiment, one or more of the vertical drawers 614, 616 can be housed in a refrigerated compartment that forms part of the cabinet 608 to maintain a fluid or solution contained in one of the bags 622, 624 at a predetermined temperature. Similar to the housing 604, the cabinet 608 can also include a status indicator light 634. FIGS. 42-45 illustrate the waste bag drawer 606 as part of the lower housing 602, but it is contemplated that the waste bag drawer can alternatively be housed in the cabinet 608 (e.g., as a horizontally oriented drawer or as a vertically mounted drawer). As best shown in FIGS. 43 and 46, the process drawer 604 includes an upward-facing slot 630 configured to receive an anchor comb 632, which facilitates routing of tubing from the cabinet 608 into the process drawer 604. In one embodiment, the entire device 600 is sized and dimensioned to be supported by a table or bench top such that the process drawer 604 and the cabinet 608 can be easily accessed by a user. Control of the device 600 and its functions is implemented by an on-board controller (e.g., controller 210) as disclosed hereinafter.

[0125] Referring now to FIGS. 46 and 47, a detailed view of the process drawer 604 is illustrated. As best shown in FIGS. 46 and 47, the process drawer 604 includes a first internal space 636 configured to receive a disposable bioprocessing kit, and a second internal space 638 positioned behind the first internal space 636. The functional components of the device 606 are mounted within the second internal space 638. For example, in one embodiment, the second internal space 638 houses a peristaltic pump assembly 641, a pinch valve array or linear actuator array 643 (for controlling the flow of fluid through an array of fluid flow lines), and other components and devices necessary to implement the functions of the device 600. In one embodiment, the peristaltic pump assembly 641, as well as other components and devices, can be configured as disclosed in Patent Document 1. As shown in FIG. 47, first and second platform rocker assemblies 640, 642 are mounted within the first internal space 636, and the first and second platform rocker assemblies 640, 642 are configured to support a culture vessel (also referred to herein as a bioreactor vessel) of the disposable bioprocessing kit thereon in the manner disclosed hereinafter. The platform rocker assemblies 640, 642 each have a cover 644, and a plurality of culture vessel support or mounting posts 646 extend through the cover 644 to support the culture vessel of the disposable kit. In one embodiment, each platform rocker assembly 640, 642 includes four support posts 646, as more clearly shown in FIG. 48. Also shown there, a sensor assembly 648 associated with each platform rocker assembly 640, 642 is provided to detect the presence of the culture vessel and / or to measure the temperature within the culture vessel.In other embodiments, the sensor assembly 648 can be used to measure various additional parameters of the culture (e.g., temperature, carbon dioxide concentration, oxygen concentration, etc.) in the culture vessel received on each of the platform rocker assemblies 640, 642, and / or to determine whether the culture vessel is properly positioned and seated on the rocker assembly. As discussed below, each of the platform rocker assemblies 640, 642 includes a plurality of load cells 658, 660, 662 for sensing the weight / mass of the culture vessel supported by the mounting posts 646.

[0126] Referring once again to FIG. 47, process drawer 604 includes a plurality of features configured to contain leaks and prevent or inhibit any fluid from collecting within process drawer 604. For example, process drawer 604 includes seal elements 650 that form a fluid-tight seal between each platform rocker assembly 640, 642 and the bottom of process drawer 604, which extends around the perimeter of each rocker assembly, and between the rocker assemblies 640, 642 themselves. Additionally, each culture vessel support post 646 is equipped with a seal element in the form of a flexible bellows 652 that forms a seal between support post 646 and cover 644. Seal elements 650 and bellows 652 prevent any fluid from entering the space below cover 644 of platform rocket assemblies 640, 642. Further, the bottom of process drawer 604 is formed with a peripheral channel 654 that collects spilled or leaked fluid. Drain holes 656 in channel 654 provide a means of escape for fluid collected in channel 654 of process drawer 604. Drain holes 656 are in fluid communication with waste drawer 606 below process drawer 604 such that any fluid that spills or leaks into process drawer 604 is drained directly into waste drawer 606, preventing damage to the electromechanics within process drawer 604.

[0127] FIG. 49 illustrates the configuration of the waste drawer 606, which includes a plurality of load cells 664 as shown therein. In one embodiment, there are four load cells positioned adjacent to the four corners of the waste drawer 606. As shown above, the waste drawer is slidably received within the housing 602 below the process drawer 604 and is configured to receive a waste bag. In one embodiment, the tubing connecting to the waste bag is routed from the process drawer along a groove behind the front panel of the process drawer, escaping the process drawer and then freely routing downward to the waste drawer. Further, as shown above, the waste drawer 606 is configured to directly receive fluid that has leaked into the process drawer through the drain hole portion 656 in the process drawer 604.

[0128] Referring now to FIG. 50, a single-use disposable bioprocessing kit 700 for use with a bioprocessing apparatus 600 is illustrated. The bioprocessing kit 700 includes a generally rectangular tray 702 sized and dimensioned to be received within a first internal space 636 of a process drawer 604, and a pair of culture vessels 704, 706 received within the tray 702. The tray 702 has a pair of openings or windows beneath the culture vessels 704, 706, supports the culture vessels 704, 706 in a raised position, and is configured such that when the tray 702 is positioned within the first internal space 636 of the process drawer 604 and engaged with support posts 646 of platform rocker assemblies 640, 642, the culture vessels 704, 706 are lifted out of the tray 702. As shown in FIGS. 50 and 51, the tray 702 includes a pair of legs 708, 710 positioned on its front and rear faces, which support the tray 702 on the bottom of the process drawer 604. The support legs 708, 710 are hollow and form the low points of the tray 702. Thus, in the event of a leak or spill within the tray 702 (as opposed to within the process drawer 604), fluid will collect and be contained within the bottoms of the legs 708, 710.

[0129] Referring further to FIGS. 50 and 51, tray 702 further includes first and second windows 709, 711 on the rear surface of tray 702, and for engagement with a peristaltic pump assembly 641 mounted within process drawer 604 on the rear surface of tray 702, valve manifold 712 and up to three segments 714, 716, 718 of peristaltic pump tubing are positioned within tray 702. Valve manifold 712 can be, for example, a fluid container as disclosed in Patent Document 2, which is configured to interface with a plurality of linear actuators having plungers of linear actuator array 643, which is likewise mounted within process drawer 604 on the rear surface of tray 702. Alternatively, valve manifold 712 can be formed from a plurality of fluid flow lines configured to be actuated by a plurality of pinch valves of a pinch valve array as disclosed in Patent Document 1. Valve manifold 712 is fluidly interconnected with culture vessels 704, 706, the media bag and reagent bag within cabinet 608, the waste bag within waste drawer 606, and the sampling line, forming a fluid network or architecture as disclosed in Patent Document 1 or similar to that disclosed in Patent Document 1. FIG. 50 illustrates the various tube connections to valve manifold 712.

[0130] Accordingly, as further illustrated in FIG. 50, the disposable kit 700 further includes a tubing organizer card 720 and a sampling card 722. The tubing organizer card 720 holds a plurality of tubing tails 726, the plurality of tubing tails 726 are fluidly connected to the valve manifold 712, and they are configured for connection to various media bags and reagent bags housed within the cabinet 608. The sampling card 722 holds a plurality of sampling tubing tails, and the plurality of sampling tubing tails are likewise fluidly connected to the valve manifold 712. Finally, the disposable kit 700 also includes an anchor comb 632, the anchor comb 632 is received within a slot 630 within the process drawer 604, and it facilitates routing of tubing from within the cabinet 608 (e.g., from the tubing organizer 720 and the sampling card 722) into the process drawer 604 and to the valve manifold 712. As discussed hereinafter, the anchor comb 632, the tubing organizer 720, and the sampling card 722 provide means for organizing all of the tubing tails during and after installation of the kit 700 and connection of various media, reagents, and other bags / containers. In one embodiment, the disposable kit 700 (including all of the elements described above in relation to FIG. 50) can be sterilized by means known in the art (e.g., ethylene oxide sterilization, gamma sterilization, etc.) and can be sealed within a blister pack for transport to and storage by the end user.

[0131] As shown in FIGS. 52 and 53, the anchor comb 632 includes a body portion 730, and the body portion 730 has a passage 732 therethrough. Within the passage 732 are a plurality of tubing retaining elements 734 that function to hold and maintain the length of the tubing in an organized manner. As disclosed above, during installation, the anchor comb 632 is received into the slot 630 of the process drawer 604 to facilitate routing of various paths of tubing from the cabinet 608 into the process drawer 604, where they are fluidly connected to the valve manifold 712.

[0132] Referring to FIG. 54, a detailed view of the tubing organizer 720 according to an embodiment of the present invention is shown. The tubing organizer 720 generally includes a rigid plate body portion 736 and a plurality of tubing holding channels 738. The plurality of tubing holding channels 738 are formed into or otherwise connected to the rigid plate body portion 736 and are configured to receive and hold corresponding plural tubing tails 726 therein. In certain embodiments, the channels 738 extend upward along the right side thereof from the lower right hand corner of the plate body portion 736, fold back on themselves, and generally extend downward at a predetermined angle toward the lower right hand corner of the plate body portion 736, and fold back on themselves again and extend upward along the left side thereof from the lower left hand corner of the plate body portion 736. Accordingly, the tubing tails 726 received within these channels 738 follow the same convoluted path. Thus, this serpentine configuration of the channels 738 maximizes the length of the tubing tails 726 that can be retained by the tubing organizer and allows for a significant amount of play to facilitate connection of the tubing tails 726 to the various bags and / or containers contained within the cabinet 608 of the bioprocessing apparatus 600. Accordingly, the tubing organizer 720 maintains the tubing tails 726 in an organized and easily accessible manner, which helps to minimize setup time.

[0133] Also, as shown in FIG. 54, the plate body portion 736 includes features that enable the tubing organizer 720 to be removably mounted or suspended inside the door 612 of the cabinet 608, as shown in FIG. 43. Such features can include, for example, mounting and / or positioning apertures 740 through which pegs 618 or hooks on the door 612 can be received. In use, when the tubing organizer 720 is attached to the inner face of the door 612, the user can easily grip the end of the tubing tail 726 extending into the clearance or relief area 742 of the plate body portion 736 and remove it from its seated position together with its corresponding channel 738. The tubing tail 726 can then be connected, by aseptic techniques (such as aseptic tube welding), to a media bag, reagent bag, or other container contained within the cabinet 608. This process can be repeated until all fluid connections between the bags contained within the cabinet 608 and the valve manifold 712 contained within the drawer 604 are made.

[0134] Referring to FIGS. 55 and 56, a detailed view of the sampling card 722 according to an embodiment of the present invention is illustrated. As shown therein, the sampling card / device 722 includes a body portion 744, and the body portion 744 has a manifold 746 and a plurality of sampling tubing tails 748 fluidly connected to the manifold 746. Also, the sampling card 722 includes a feed pipe 750 fluidly connected to a first end of the manifold 746 and a return pipe 752 fluidly connected to a second end of the manifold 746. Similar to the tubing organizer 720, the body portion 744 of the sampling card 722 includes features that enable the sampling card 722 to be removably mounted or suspended inside the door 612 of the cabinet 608. Such features can include, for example, mounting and / or positioning apertures 754 through which pegs 618 or hooks on the door 612 can be received. In use, when the sampling card 722 is attached to the inner surface of the door 612, the user can use one of the sampling tubing tails 748 that is easily accessible on the sampling card 722 to draw a sample from one of the culture vessels 704, 706. Thus, the sample can be easily drawn during the bioprocessing operation without the need to open the process draw 604 and without the need to pause the operation.

[0135] Referring now to FIGS. 57-63, the installation and seating of tray 702 within process drawer 604 of bioprocessing apparatus 600 are illustrated. As shown therein, tray 702 is received within first internal space 636 of process drawer 604 by opening process drawer 604 and lowering tray 702 therein from above, with culture vessels 704, 706 of disposable kit 700 being in a front-to-back relationship within process drawer 604. In this position, valve manifold 712 is positioned immediately forward of linear actuator array 643 and is aligned therewith, and three segments 714, 716, 718 of peristaltic pump tubing are positioned immediately forward of peristaltic pump assembly 641 and are aligned therewith. As shown above, when tray 702 is lowered into process drawer 604, culture vessels 704, 706 are received onto support / mounting posts 646 of respective platform rocker assemblies 640 such that culture vessels 704, 706 are lifted from their seating engagement with tray 702 and are instead supported by support posts 646.

[0136] As most clearly shown in FIGS. 59-62, the tray 702 and the process drawer 604 of the disposable kit 700 have a plurality of cooperating features that facilitate proper positioning of the tray 702 within the process drawer 604 and enable verification of proper positioning. For example, as shown in FIG. 59, the tray 702 and the process drawer 604 include a plurality of engagement features / surfaces 756 that cooperate with each other when the tray 702 is properly positioned within the process drawer 604. The process drawer 604 within the second internal space 636 includes a plurality of sensors 758 associated with the drawer engagement features 756, and the plurality of sensors 758 can detect when the cooperating engagement features 756 on the tray 702 and the process drawer 604 are engaged with each other and indicate proper positioning of the tray 702. In certain embodiments, the engagement features 756 associated with the tray 702 are positioned on the tray backbone as best shown in FIG. 59, while the corresponding engagement features 756 (and sensors 758) associated with the process drawer 604 are positioned adjacent to the linear actuator array 643 and the peristaltic pump assembly 641, respectively. In certain embodiments, the engagement features 756 associated with the process drawer 604 are pins of the sensors 758. As shown above, in addition to detecting proper alignment and positioning of the tray 702 within the process drawer 604, the platform rocker assemblies 640, 642 include sensors 648 that are configured to detect proper positioning of the culture vessels 704, 706.

[0137] In addition to the engagement features and sensors disclosed above, the peristaltic pump assembly 641 also includes upper and lower engagement structures 760 and a pivoting pump shoe 762, which facilitate proper engagement of the peristaltic pump assembly 641 with the backbone of the tray 602. Also, these features minimize tolerance stack-up issues with respect to the engagement and operation of the peristaltic pump assembly 641 and the segments 714, 716, 718 of the peristaltic pump tubing and the valve manifold 712 of the linear actuator array 643, respectively.

[0138] In certain embodiments, the solenoid actuators of the peristaltic pump assembly 641 and the valve manifold 712 are configured to move toward and physically engage corresponding features of the disposable kit when the disposable kit is positioned within the process drawer and the drawer is closed. In particular, with specific reference to FIG. 59, the module 600 includes an electric engagement mechanism that physically moves the assembly including the peristaltic pump assembly 641 and the solenoid array 643 toward corresponding features (segments 714, 716, 718 of the peristaltic pump tubing and the valve manifold 712) within the disposable kit 700 by a fixed travel distance that is limited by features that prevent further movement. Disengagement simply involves operating this electric engagement mechanism in reverse.

[0139] Referring now to FIGS. 64 and 65, the configuration of the bioreactor / culture vessels 704, 706 of the disposable bioprocessing kit 700 is shown. For ease of illustration, only culture vessel 704 is shown (culture vessel 706 is an exact duplicate). As shown therein, in one embodiment, culture vessel 704 includes a base 764, a lid 766 connected to the base 764, a gas-permeable liquid-impermeable membrane 768 sandwiched between the base 764 and the lid 766, and a gasket 770 sandwiched between the membrane 768 and the lid 766. In one embodiment, the base 764 and the lid 766 are formed from polycarbonate, although other materials known in the art may also be utilized without departing from the broader aspects of the present invention. As shown in FIG. 64, the lid 766 includes a plurality of reinforcing supports 772 or gaskets, which reinforce the lid 766 and provide increased strength and durability. Also, the lid 766 includes inlet and outlet ports 774, 776 to which tubing can be connected. As shown therein, the inlet and outlet ports 774, 776 are molded into the lid such that the tubing extends (at least initially) vertically from the lid 766. This configuration of the ports 774, 776 facilitates setup as the tubing can be more easily connected to the culture vessel 704 from above. Further shown, a vent port 777 is provided at the top of the lid 766. In one embodiment, the lid 766 includes rounded corners (e.g., corner 778), which eliminate / prevent any stagnant zones.

[0140] Referring further to FIG. 64, the membrane 768 can be formed from a material with suitable gas permeability (e.g., silicone and / or polystyrene), or a porous material having a pore size that does not allow the passage of water or microorganisms, although other materials known in the art can also be utilized without departing from the broader aspects of the present invention. The membrane 768 includes a plurality of locations / holding holes 780 along the periphery of the membrane 768, the purpose of which will be described hereinafter. The gasket 770 can be formed from various materials known in the art (e.g., silicone, etc.) with respect to a part thereof, and includes a corresponding plurality of locations / holding holes 782, which are positioned along the periphery of the gasket 770 and are aligned with the holes 780 in the membrane 768.

[0141] In one embodiment, the lid 766 and the base 764 are connected to each other via heat staking along the periphery of the lid 766 and the base 764. In one embodiment, the heat staking portion 781 extends through the respective location / holding holes 780, 782 of the membrane 768 and the gasket 770, respectively, and functions to anchor the membrane 768 and the gasket 770 between the base 764 and the lid 766. In one embodiment, the lid 766 can be configured to include heat staking pins 784 that extend downwardly from its lower side, and during assembly, the heat staking pins 784 extend through the corresponding location / holding holes 780, 782 of the membrane 768 and the gasket 770, respectively, and are received into the corresponding holes 786 at the periphery of the base 764 and are heat staked to the base 764. In one embodiment, the lid 766 is joined to the base 764 using from about 20 to about 40 heat staking portions (more preferably, approximately 34 heat staking portions). The embodiments described herein utilize heat staking to connect the lid to the base, but it is contemplated that other connection means (such as fasteners and snap-fit connections, etc.) can also be utilized without departing from the broader aspects of the present invention.

[0142] In one embodiment, the upper surface of the base 764 has a textured surface that allows air flow and eliminates the need for a mesh (which was conventional in previous designs). As shown in FIG. 65, the flange region 788 of the base 764 includes a plurality of ribs 790 that provide increased rigidity and strength as well as a more robust interconnect with the lid 766, which additionally provides a more reliable and robust anchor fixation for the membrane 768 and gasket 770. The lower corners of the base 764 each include pin wells 791, 792, 793, 794 that are configured to receive the mounting / support posts 646 of the platform rocker assembly 640 or 642 that supports the culture vessel 704. In one embodiment, one of the pin wells (e.g., well 794) is oval in shape, which provides for improved positional tolerance when positioning the culture vessel 704 on the platform rocker assembly 640. The base 764 further provides an IR sensor window 796 and a sensor well 798, where the IR sensor window 796 is for measuring the temperature of a gas or fluid in the culture vessel 704 using a sensor positioned below the culture vessel 704 in the process drawer 604, and the sensor well 798 is utilized by the sensor 648 of the platform rocker assembly 640 or 642 to determine whether the culture vessel 704 is present in and / or properly positioned within the process drawer. Finally, as illustrated in FIG. 65, the base 764 includes an array of small openings 799 that provide fluid communication between the atmosphere in the process drawer 604 and the underside of the membrane 768 for gas transfer during bioprocessing. In one embodiment, several hundred small openings 799 are present in the base 764.

[0143] As shown above, culture vessels 704, 706 are configured to be received on platform rocker assemblies 640, 642 when tray 702 is received within process extraction 604. Various rocking mechanisms known in the art (including the mechanism disclosed in Patent Document 1) can be utilized to provide mixing of the fluid within culture vessels 704, 706 and support the bioprocessing operations therein. FIGS. 66 - 68 illustrate the configuration of platform rocker assemblies 640, 642 according to another embodiment of the present invention (rocker assembly 640 is depicted for simplicity and ease of understanding). As shown therein, platform rocker assembly 640 includes a base 870, a fulcrum 872 defining a central pivot axis 873 received on base 870, a motor 874 mounted to base 870 and having an eccentric roller 876 driven by motor 874, a rocking plate 878 received in contact with eccentric roller 876 on fulcrum 872 and pivotable about fulcrum axis 873, and a compression spring 880 configured to maintain rocking plate 878 in contact with eccentric roller 876. In certain embodiments, fulcrum 872 and motor 874 are connected to base 872 via frame 875. In certain embodiments, eccentric roller 876 is a circular roller configured to rotate along an eccentric path. In yet further embodiments, it is also possible to use a cam-shaped roller instead of a circular roller that moves along an eccentric path.

[0144] As shown in FIGS. 67 and 68, the rocking plate 878 includes four support posts 646, and the four support posts 646 are received by pin wells 791, 792, 793, 794 in the base 764 of the culture vessel 704. The motor 874 drives the eccentric roller 876 and, depending on the position of the eccentric roller 876, transmits or removes force to / from the lower side of the rocking plate 878, so as to tilt the rocking plate 878 and the culture vessel 704 received thereon upwardly and / or downwardly, and is controllable (e.g., under the control of the controller 210 of the second module 200 (i.e., the apparatus 600)). The motor 874 can be controllable by a master controller, but the platform rocker assemblies 640, 642 can alternatively have a dedicated controller positioned on the base plate 872 under the rocking plate 878. As a result of the force (or its absence) from the eccentric roller 876, the rocking plate 878 and the culture vessel 704 supported thereon pivot about the pivot axis 873 of the fulcrum 872.

[0145] In certain embodiments, each of the support posts 646 can be configured to include a load cell for measuring the mass of the culture vessel 704. Alternatively or additionally, the base 870 of the rocker assembly 640 can include a plurality of (e.g., three) load cells 882 that extend through the rocking plate 878 and engage the lower side of the culture vessel 704 for measuring the mass of the culture vessel 704. Further, as shown in FIG. 67, the rocking plate 878 can be equipped with an inclination sensor 884 that is configured to measure the degree of inclination of the rocking plate 878 (and thus the culture vessel 704) for use by the controller during the rocking / mixing process.

[0146] As shown above, when the motor 874 is operating, the eccentric roller 876 transmits a force to the bottom surface of the rocking plate 878, causing it to move upward or downward depending on the direction of rotation of the motor 874. During a constant operation, the circular profile of the eccentric roller 876 imparts a continuous sinusoidal rocking profile to the contents of the culture vessel 704. This rocking motion is illustrated in FIG. 69. Monitoring of the rocking plate 878 using the tilt sensor 884 enables closed-loop control for the angle of tilt, homing, and drain operation, and similarly enables detection of fault event conditions. The use of the support posts 646 for supporting the culture vessel 704 on the rocking plate 878 allows the entire bottom of the culture vessel 704 to remain unobstructed, enabling better ventilation, heat transfer, and other functionality as discussed later. The use of the eccentric roller 876 allows the tilt mechanism to be compact / low profile and provides a low friction and highly reliable interface with the rocking plate 878. As will be appreciated, mammalian cells in particular are very sensitive to shear forces induced by small-scale vortices on top of a highly turbulent fluid regime. Thus, excessive turbulence, bubble formation, or strong vibrations, shocks, or other mechanical stimuli leading to spillage are potentially harmful. Thus, the continuous sinusoidal rocking profile of the platform rocker assemblies 640, 642 minimizes the presence of such small-scale vortices by removing any high-frequency mechanical stimuli, providing safer and gentler mixing conditions, which is particularly beneficial for mammalian cell culture.

[0147] As shown above, ventilation and heat transfer through the bases 764 and membranes 768 of the culture vessels 704, 706 are important for various bioprocessing operations. Typically, certain cell cultures (e.g., mammalian cell cultures) must be surrounded by a sterile, homogeneous incubation atmosphere at a temperature and CO2 concentration appropriate for cell growth. The manner in which such physiochemical conditions are provided depends on the application, cell type specificity, and how they are adapted to grow in suspension or adherent. In some cases, the process may require the cells to grow in a monolayer on a gas-permeable membrane. In this case, heat and mass transfer occur by passive diffusion based on local gradients across the regions immediately adjacent to both sides of the membrane. Embodiments of the present invention optimize such phenomena by inducing a turbulent interaction between the gas-permeable membrane 768 on the bottom of the culture vessels 704, 706 and the incubation atmosphere recirculation flow.

[0148] Figures 70-72 present a cross-sectional view of a portion of the process drawer 604 of the bioprocessing apparatus 600 with the tray 702 and the culture vessels 704, 706 of the disposable bioprocessing kit 700 positioned therein. The process drawer 604 forms an incubation chamber 902, and as disclosed above, the tray 702 and the culture vessels 704, 706 are positioned within the incubation chamber 902. As shown therein, the culture vessels 704, 706 are supported by the support posts 646 of the platform rocker assemblies 640, 642. A heating element / device 904 is within the process drawer 604 (e.g., positioned above and below each culture vessel). For example, the heater 904 can be positioned adjacent to the upper part of the process drawer 604 and below each culture vessel 704, 706 to heat the incubation chamber 902 and the culture vessels 704, 706. Further, the process drawer 604 includes a pair of fans or blowers 906, 908 within the covers 644 of the rocker assemblies 640, 642 adjacent to its front and rear wall portions. As further shown therein, the cover 644 can include a pair of opposing louvers or air passages 910, 912, with the blowers 906, 908 positioned nearby, allowing air to exit from the space within the cover 644 adjacent to the rear of the process drawer 604 (defining the incubation atmosphere recirculation chamber 915) and re-enter the recirculation chamber 915 from the front of the process drawer 604. Also, temperature sensors 914 and carbon dioxide sensors 916 are positioned at at least one location along the recirculation air flow path to measure the temperature of the recirculation air flow and the carbon dioxide concentration of the recirculation air flow, as discussed below. Additionally shown therein, a carbon dioxide supply 918 is selectively in fluid communication with the process drawer 604 (e.g., via a carbon dioxide inlet port at the rear of the housing 602 of the bioprocessing apparatus 600) and a valve 920.In addition, process outlet 604 includes a gas port 922 that enables fluid communication between the interior of process outlet 604 and the atmosphere (removing the need for a dedicated separate oxygen supply). The components described above form a system 900 for direct mass transfer from the liquid of bioprocess system 600 to the atmosphere, and its operation will be described hereinafter.

[0149] Referring further to FIG. 70, temperature sensor 914 and carbon dioxide sensor 916 are electrically connected to or otherwise communicate with a controller (e.g., master controller 210 of apparatus 600, although a dedicated controller for performing the recirculating air flow process is also contemplated) for receiving information regarding the temperature and carbon dioxide concentration of the recirculating air flow. Also, controller 210 is electrically connected to or otherwise communicates with valves 920, fans 906, 908, and heater 904 to control its operation in response to sensor readings and specific setpoints.

[0150] Referring now to FIG. 71, controller 210 is operable to control fans 906, 908 to create a recirculating air flow 924. As discussed below, trays 702 and process outlet 604 each include various ducting features 926 that ensure that recirculating air flow 924 exits the recirculation chamber 915 through louver 912 adjacent to the rear of process outlet 604, travels upward to the level of culture vessels 704, 706, travels generally horizontally across the bottoms of culture vessels 704, 706, travels downward near the front of process outlet 604, and re-enters the recirculation chamber 915 through louver 910. In this regard, fan 908 pushes the recirculating air flow 924 outward from the recirculation chamber 915, while fan 906 draws the recirculating air flow 924 into the recirculation chamber 915.

[0151] Referring to FIG. 72, fan 908 pushes the incubation atmosphere through the incubation atmosphere recirculation chamber 915, and the ducting feature 926 directs the recirculation air flow 924 across the bottoms of the culture vessels 704, 706. In doing so, the ducting feature and the configuration below the base 764 of the culture vessels 704, 706 induce the formation of local turbulence 928, which helps to maintain a constant supply of oxygen and carbon dioxide in contact with the gas permeable membranes 768 of the culture vessels 704, 706.

[0152] Figures 73-76 more clearly illustrate the ducting features of system 900, which allows recirculation air flow 924 to be directed back into recirculation chamber 915 across the bottoms of culture vessels 704, 706 under the influence of fans 906, 908. As shown there, the sides facing the inside of legs 708, 710 of tray 702 are formed with depressions or recessed areas 930, which, optionally, allow recirculation air 924 exiting / entering recirculation chamber 915 to travel upward or downward along the inner surfaces of legs 708, 710. Figures 73-76 specifically illustrate how recirculation air 924 present in recirculation chamber 915 is directed upward by recessed area 930 of leg 710 of tray 702. Thus, recessed areas 930 of legs 708, 710, and the outer surface of the recirculation chamber form a vertical air passage for the flow of recirculation air 924. When air exiting louver 912 travels upward through recessed area 930 of leg 710, it is obstructed at the point where leg 710 meets the bottom of tray 702. As best shown in FIGS. 73 and 74, tray 702 includes a pair of lateral vent openings 932 at a height generally corresponding to the vertical height of the bottoms of culture vessels 704, 706. Thus, vent openings 932 redirect recirculation air flow 924 laterally through such openings 932 toward culture vessels 704, 706, where recirculation air flow 924 interacts with the bottom geometries of culture vessels 704, 706 and their corresponding gas-permeable membranes, leading to the formation of local turbulence 928. Recirculation air flow 924 moves across the bottoms of culture vessels 704, 706, where it enters the opposing vent opening, travels downward through recessed area 930 of leg 708, and re-enters recirculation chamber 915 through louver 910.

[0153] As disclosed above, the formation of local turbulence 928 in the recirculation air flow 924 helps to maintain a constant supply of oxygen and carbon dioxide in contact with the gas-permeable membranes 768 of the culture vessels 704, 706. At the same time, the overall recirculation air flow 924, along with the control actions provided by the control unit 210, temperature sensor 914, carbon dioxide sensor 916, heater 904, and carbon dioxide control valve 920, enables homogenization of the volume inside the incubation chamber 902. Thus, system 900 provides optimization of heat and mass transfer. As will be recognized, a constant availability of oxygen, just a few tens of microns away from the cell monolayer, supports higher cell concentrations and minimizes the physiochemical gradients across the surface of the membranes 768 of the culture vessels 704, 706.

[0154] As described above, apparatus 600 includes a plurality of sensors and monitoring devices for monitoring the bioprocessing operations (including monitoring various parameters of the cell culture in the culture vessels 704, 706) when the bioprocessing operations are being performed. This can include, for example, periodically withdrawing samples from the culture vessels 704, 706 using the sampling tubing tails 748 of the sampling cards 722, and / or using sensors for sensing various parameters of the culture in the vessels. For example, the sensor assembly 648 houses an IR sensor for temperature measurement and for detecting the presence of the culture vessels in the process draw. The windows 796 in the bases 764 of the culture vessels 704, 706 enable IR-based temperature measurement of the membranes in the culture vessels and, thus, the liquid temperature in the culture vessels.

[0155] Referring to FIGS. 77 - 84, in one embodiment, the apparatus 600 can additionally include a flow - through sensing chamber 950 (also referred to herein as the flow - through sensing device 950), and the flow - through sensing chamber 950 can be utilized to measure or monitor various parameters of the fluid (e.g., the cultures in the culture vessels 704, 706) within the apparatus 600 using various different sensing / measurement devices and without withdrawing any fluid from the system. As best shown in FIGS. 77 - 80, the flow - through sensing chamber 950 includes a first plate 952, a second plate 954 connected in a facing relationship to the first plate 952, and a fluid channel 956 intermediate the first plate 952 and the second plate 954. In one embodiment, the fluid channel 956 is formed from a relief area on the inner surface of at least one or both of the first plate 952 and / or the second plate 954. In one embodiment, the fluid channel 956 can be between about 0.1 mm and about 1 mm in height. The chamber 950 further includes a first port 958 and a second port 960, where the first port 958 is in fluid communication with the fluid channel 956 to facilitate the flow of fluid into the chamber 950 and its fluid channel 956, and the second port 960 is in fluid communication with the fluid channel 956 to facilitate the flow of fluid out of the chamber 950 and its fluid channel 956. In one embodiment, the ports 958, 960 are in fluid communication with opposite ends of the fluid channel 956.

[0156] As shown in FIG. 78, in certain embodiments, plates 952, 954 can have features that facilitate alignment and connection of the plates to each other. For example, one of the plates (e.g., plate 952) can have a pair of notches 957 that receive corresponding tabs 959 of the other of the plates (e.g., plate 954). As discussed below, backplate / first plate 952 includes a plurality of mounting and positioning holes 961 that extend therethrough, which facilitate mounting of chamber 950 to tray 702 of disposable kit 700. In certain embodiments, first / backplate 952 and second / front plate 954 are generally rectangular in shape, transparent, and made of biocompatible plastic, glass, or a combination of plastic and glass, although the invention is not intended to be so limited in this regard.

[0157] As best shown in FIGS. 78 and 79, fluid channel 956 includes a plurality of segments or sensing locations 962, 964, 966 that permit or facilitate interrogation or monitoring of fluid within fluid channel 956 by a plurality of sensing devices and techniques. In certain embodiments, fluid within fluid channel 956 can be interrogated by a variety of different sensing devices associated with each of the plurality of sensing locations 962, 964, 966. In certain embodiments, segment 966 has one or more sensors 968 positioned within fluid channel 956, and the one or more sensors 968 are configured to remain in continuous contact with fluid passing through fluid channel 956. As shown in FIG. 77, second plate 954 includes a plurality of electrodes 970 that extend into fluid channel 956 and are accessible from flange 972 that extends laterally of second plate 954. In certain embodiments, electrodes 970 are gold-plated electrodes.

[0158] As shown above, the flow-through sensing chamber 950 utilizes various different sensing devices to enable interrogation of the fluid in fluid channel 956 in order to measure various different parameters of the fluid. For example, in one embodiment, sensing location 962 can be configured as a reflected light interrogation segment, which is configured with a gold-plated mirror 974 behind fluid channel 956, which reflects the light emitted by the sensing device. Thus, sensing location 962 can be suitable for various techniques for monitoring / sensing biological variables (e.g., optical density sensing, turbidimetry, digital holographic microscopy, dynamic light scattering, and / or optical interferometry, etc.). In one embodiment, sensing location 964 can be configured as a transmitted light and backscattered light interrogation segment, which uses a transmitted light or backscattered light sensing instrument to enable interrogation of the fluid in fluid channel 956. Sensing location 966 can be configured as a fluorescence sensor interrogation segment having various sensors 968 in contact with the fluid in fluid channel 956 for a part thereof, enabling monitoring or sensing of various parameters of the fluid (e.g., dissolved oxygen, pH, carbon dioxide, analyte, etc.). Electrodes 970 face rearward (opposite ports 958, 960) and are configured to be contacted by spring-biased pins of one or more measurement devices suitable for various electrochemical measurement techniques (e.g., electrical impedance spectroscopy, galvanometry, amperometry, and / or polarography, etc.).

[0159] Figures 81 and 82 illustrate the positioning of the flow-through sensing chamber 950 on the backbone of the tray 702 of the disposable bioprocessing kit 700. As shown above, the chamber 950 can be connected to the tray 702 by receiving the snap pin 976 positioned on the backbone of the tray 700 into the corresponding mounting aperture 961 of the chamber 950. As shown therein, in certain embodiments, the chamber 950 can be mounted to the tray 700 intermediate the valve manifold 712 and the peristaltic pump tubing segments 714, 716, 718. The pin 976 is shown as being utilized to mount the chamber 950 to the tray 700, but it is contemplated that other connection means (e.g., clipping, clamping, fasteners, snap fittings, and press-fits, etc.) can be utilized without departing from the broader aspects of the present invention. In certain embodiments, the chamber 950 can form part of the disposable bioprocessing kit 700.

[0160] Figures 83 and 84 present schematic views of the flow-through sensing chamber 950 and various sensing instruments / devices for monitoring various parameters of the fluid in the fluid channel 956. As shown in FIG. 83, for example, the first and second electrochemical sensing instruments 978, 980 mounted to the device 600 can interface with the electrodes 970 via the spring-biased pins 982. As shown in FIG. 84, the reflected light instrument 984 can be positioned and configured to interrogate the fluid in the first sensing location, the first and second fluorescence instruments 986, 988 can be positioned and configured to interrogate the fluid in the second sensing location 964, and the transmitted light / backscattered light instrument 990 can be positioned and configured to interrogate the fluid in the third sensing location 966.

[0161] Accordingly, embodiments of the present invention provide an in-line sensing chamber 950, which provides various optical and electrical measurements of the fluid within the fluid channel 956 of the chamber 950, eliminating the need to directly interrogate either of the culture vessels 704, 706. In use, when it is desired to monitor or measure various parameters of the culture within either of the culture vessels 704, 706, the fluid is pumped through the sensing chamber 950 using the peristaltic pump assembly 641, where it can be interrogated by a series of sensor instruments / devices. That is, the chamber 950 disclosed herein facilitates the use of electrochemical and optical sensing techniques on a single fluid channel, enabling multi-parametric monitoring of the physiochemical growth conditions of cell cultures within the culture vessels 704, 706, the metabolic activity of the cell types (such as lactate, glucose, etc.), and the viable cell density and total cell number measurements.

[0162] Although the components of the bioprocessing apparatus 600 (also referred to as the second module 200) have been disclosed in detail, embodiments of the fluid flow architecture or system 200 within the apparatus 600 are illustrated with reference to FIGS. 85-89. As disclosed above and as will be described in more detail hereinafter, the configurations of the bioprocessing apparatus 600 and kit 700, together with the fluid flow architecture 200 provided thereby, enable cell activation, genetic recombination and amplification of cell products, and auxiliary or related protocols, workflows, and methods in an automated and functionally closed manner. In certain embodiments, the flow architecture or system 400 can be configured or arranged as disclosed in FIGS. 3-7 of Patent Document 1, although other configurations are possible. As shown in FIG. 85, the system 400 includes a first bioreactor vessel (e.g., culture vessel 704) and a second bioreactor vessel 420 (e.g., culture vessel 706). The first bioreactor vessel includes at least a first port 412, a first bioreactor pipe 414 in fluid communication with the first port 412, a second port 416, and a second bioreactor pipe 418 in fluid communication with the second port 416. Similarly, the second bioreactor vessel includes at least a first port 422, a first bioreactor pipe 424 in fluid communication with the first port 422, a second port 426, and a second bioreactor pipe 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. The system 400 is shown as having two bioreactor vessels, although embodiments of the present invention can include a single bioreactor or three or more bioreactor vessels.

[0163] The first and second bioreactor pipes 414, 418, 424, 428 of the first and second bioreactor vessels 410, 420 each include respective valves for controlling the flow of fluid therethrough, as discussed hereinafter. In particular, the first bioreactor pipe 414 of the first bioreactor vessel 410 includes a first bioreactor pipe valve 432, while the second bioreactor pipe 418 of the first bioreactor vessel 410 includes a second bioreactor pipe valve 434. Similarly, the first bioreactor pipe 424 of the second bioreactor vessel 420 includes a first bioreactor pipe valve 436, while the second bioreactor pipe 428 of the second bioreactor vessel 420 includes a second bioreactor pipe valve 438.

[0164] Referring further to FIG. 85, system 400 also includes a first fluid assembly 440 having a first fluid manifold pipe 442, a second fluid assembly 444 having a second fluid manifold pipe 446, and a sampling assembly 448. A connection pipe interconnecting pipe 450 having a connection pipe interconnecting pipe valve 452 provides fluid communication between the first fluid assembly 440 and the second fluid assembly 444. As shown in FIG. 85, the connection pipe interconnecting pipe 450 also provides fluid communication between the second bioreactor pipe 418 and the first bioreactor pipe 414 of the first bioreactor vessel 410, enabling circulation of fluid along the first circulation loop of the first bioreactor vessel. Similarly, the connection pipe interconnecting pipe also provides fluid communication between the second bioreactor pipe 428 and the first bioreactor pipe 424 of the second bioreactor vessel 420, enabling circulation of fluid along the second circulation loop of the second bioreactor vessel. Moreover, the connection pipe interconnecting pipe 450 further provides fluid communication between the second port 416 and the second bioreactor pipe 418 of the first bioreactor vessel 410 and the first port 422 and the first bioreactor pipe 424 of the second bioreactor vessel 420, enabling transfer of the contents of the first bioreactor vessel 410 to the second bioreactor vessel 420, as discussed hereinafter. As illustrated in FIG. 85, in one embodiment, the connection pipe interconnecting pipe 450 extends from the second bioreactor pipes 418, 428 to the intersection of the first bioreactor pipe 414 of the first bioreactor vessel 410 and the first fluid manifold pipe 442.

[0165] As illustrated by FIG. 85, the first and second fluid assemblies 440, 444 are disposed along the connecting pipe interconnecting pipe 450. Additionally, in certain embodiments, the first fluid assembly fluidly communicates with the first port 412 of the first bioreactor vessel 410 and the first port of the second bioreactor vessel 420 through the first bioreactor pipe 414 of the first bioreactor vessel and the first bioreactor pipe 424 of the second bioreactor vessel 420, respectively. The second fluid assembly 444 fluidly communicates with the second port 416 of the first bioreactor vessel 410 and the second port 426 of the second bioreactor vessel 420 via the connecting pipe interconnecting pipe 450.

[0166] The first pump 454 of the peristaltic pump assembly 641 capable of providing bidirectional fluid flow is disposed along the first fluid collecting pipe 442, and the second pump or circulation pipe pump 456 of the peristaltic pump assembly 641 capable of providing bidirectional fluid flow is disposed along the connecting pipe interconnecting pipe 450, and its functions and purposes will be discussed below. Also, as shown in FIG. 85, a sterile air source 458 is connected to the connecting pipe interconnecting pipe 450 through a sterile air source pipe 460. A valve 462 positioned along the sterile air source pipe 460 provides selective fluid communication between the sterile air source 458 and the connecting pipe interconnecting pipe 450. FIG. 85 shows a sterile air source 458 connected to the connecting pipe interconnecting pipe 450, but in other embodiments, the sterile air source can be connected to an intermediate fluid flow path between the first fluid assembly 440, the second fluid assembly 444, or any of the second bioreactor pipe valves and the first bioreactor pipe valves of the first or second bioreactor without departing from the broader aspects of the present invention.

[0167] Referring additionally to FIGS. 86 - 88 here, detailed views of the first fluid assembly 440, the second fluid assembly 444, and the sampling assembly 448 are shown. Referring specifically to FIG. 86, the first fluid assembly 440 includes a plurality of tubing tails 464a - f, each of which is configured for selective / removable connection to one of a plurality of first storage portions 466a - f. Each tubing tail 464a - f of the first fluid assembly 440 includes tubing tail valves 468a - f for selectively controlling the flow of fluid to or from each respective one of the plurality of first storage portions 466a - f of the first fluid assembly 440. FIG. 86 specifically shows that the first fluid assembly 440 includes six fluid storage portions, although more or fewer storage portions may be utilized, as desired, to provide for the input or collection of various process fluids. Each tubing tail 464a - f is contemplated to be individually connectable to the storage portions 466a - f at times required during the operation of the fluid assembly 440, as described below.

[0168] Referring specifically to FIG. 87, the second fluid assembly 444 includes a plurality of tubing tails 470a - d, each of which is configured for selective / removable connection to one of a plurality of second storage portions 472a - d. Each tubing tail 470a - d of the second fluid assembly 444 includes tubing tail valves 474a - e for selectively controlling the flow of fluid to or from each one of the plurality of second storage portions 472a - d of the second fluid assembly 444. FIG. 87 specifically shows that the second fluid assembly 444 includes four fluid storage portions, but more or fewer storage portions may be utilized as desired to provide for the input or collection of various processing fluids. In certain embodiments, at least one of the second storage portions (e.g., second storage portion 472d) is a collection storage portion housed within the cabinet 608 of the apparatus 600 for collecting an amplified cell population, as discussed hereinafter. In certain embodiments, the second storage portion 472a is a waste storage portion or a bag housed within the waste drawer 606 of the apparatus 600, the purpose of which is discussed below. It is contemplated that the waste bag can be housed within the drawer 606 of the apparatus 600, but it is also envisioned that the waste bag can be housed within the media cabinet 608. For example, in certain embodiments, the waste bag can be suspended from a hook within the media cabinet. The hook can be associated with a pressure sensor to enable both waste mass measurement and leak and blockage detection. In such an arrangement where the waste bag is housed within the cabinet 608, the waste drawer 606 can be omitted.

[0169] In one embodiment, the first storage units 466a - f and the second storage units 472a - d are single - use / disposable flexible bags, which are housed within the cabinet 608 of the device 600 and are fluidly connected to the manifold 712 via the tubing tails of the tubing organizer 720. In one embodiment, the bags are substantially two - dimensional bags having opposing panels, which are welded or secured together around the perimeter thereof and support connection conduits for connection to their respective tails, as is known in the art.

[0170] In one embodiment, the storage unit / bag can be connected to the tubing tails of the first and second tubing assemblies using a sterile welding device. In one embodiment, the welding device can be positioned adjacent to the device 600, and the welding device can be utilized (while maintaining sterility) to butt-weld one of the tubing tails to the tail of the tube over the bag. Thus, the operator can provide the bag when needed (e.g., grasping the tubing tail from the tubing organizer 720, inserting its free end into the welding device, placing the free end of the bag tube adjacent to the end of the tubing tail, cutting the tube with a new razor blade, and heating the cut ends when the camisole is pulled away while the two tube ends are still molten and forced together (so that they re-solidify together)). Conversely, the bag can be removed by heat-sealing the line from the bag and cutting at the heat-seal to separate the two closed lines. Thus, the storage unit / bag can be connected individually when desired, and the present invention does not require that all storage units / bags must be connected at the start of the protocol. The reason is that the operator will have access to the appropriate tubing tail throughout the process in order to connect the storage unit / bag at the appropriate time for its use. In fact, it is also possible for all storage units / bags to be pre-connected, but the present invention does not require pre-connection, and one benefit of the second module 200 is that, as discussed below, it enables the operator to access the fluid assembly / line during operation and allows used bags to be connected in a sterile manner and other bags to be disconnected so that they can be connected sterilely during the protocol.

[0171] As shown in FIG. 88, sampling assembly 448 includes one or more sampling lines (e.g., sampling lines 476a - 476d, which can be the sampling tubing tails 748 of sampling card 722) fluidly connected to connection pipe interconnecting pipe 450. Each of sampling lines 476a - 476d can include sample pipe valves 478a - d that are selectively operable to allow fluid to flow from connection pipe interconnecting pipe 450 through sampling lines 476a - 476d. As also shown there, the distal ends of each sampling line 476a - 476d are configured for selective connection to a sample collection device (e.g., sample collection devices 280a and 280d) for collection of fluid from connection pipe interconnecting pipe 450. The sample collection device can take the form of any sampling device known in the art (e.g., syringe, dip tube, bag, etc.). FIG. 88 shows sampling assembly 448 connected to the connection pipe interconnecting pipe, but in other embodiments, the sampling assembly can be fluidly coupled to an intermediate fluid flow path between first fluid assembly 440, second fluid assembly 444, second bioreactor pipe valve 434 and first bioreactor pipe valve 432 of first bioreactor vessel 410, and / or an intermediate fluid flow path between second bioreactor pipe valve 438 and first bioreactor pipe valve 436 of second bioreactor vessel 420. Sampling assembly 448 provides a fully functionally closed sampling of fluid at one or more points in system 400 as desired.

[0172] Returning to FIG. 85, in one embodiment, system 400 can also include filtration piping 482, which is connected at two points along connection piping interconnecting piping 450 and defines a filtration loop along connection piping interconnecting piping 450. Filter 484 is positioned along filtration piping 482 to remove permeate waste from the fluid passing through filtration piping 482. As shown therein, filtration piping 482 includes an upstream filtration piping valve 486 and a downstream filtration piping valve 488 positioned upstream and downstream of filter 484, respectively. Waste line 490 provides fluid communication between filter 484 and second fluid assembly 444 and, in particular, between the tubing tail 470a of second fluid assembly 444 (which is connected to waste storage 472a). In this regard, waste line 490 conveys the waste removed from the fluid passing through filtration piping 482 by filter 484 to waste storage 472a. As illustrated in FIG. 85, filtration piping 482 surrounds connection piping interconnecting piping valve 452 such that the flow of fluid through connection piping interconnecting piping 450 can be forced through filtration piping 482 as discussed hereinafter. Permeate pump 492 positioned along waste line 490 is operable to pump the waste removed by the filter to waste storage 472a. In one embodiment, filter 484 is preferably an elongate hollow fiber filter, although other tangential flow or cross flow filtration means known in the art (e.g., flat sheet membrane filters, etc.) can also be utilized without departing from the broader aspects of the present invention.

[0173] In one embodiment, the valves of the first fluid assembly 440 and the second fluid assembly 444, as well as the bioreactor piping valves (i.e., valves 432, 434, 436, 438), the sterile piping valve 462, the connection piping interconnecting piping valve 452, and the filtration piping valves 486, 488 are formed by engagement with a valve manifold 712 of one of the linear actuators of the linear actuator array 643 to block or permit a specific flow of fluid therethrough. In one embodiment, the operation of the valves and pumps disclosed above (i.e., the linear actuators of the linear actuator array 643 and the three peristaltic pumps 454, 456, 492 of the peristaltic pump assembly 641) is automatically performed according to a programmed protocol to enable proper operation of the module 200 / device 600. It is contemplated that the second controller 210 mounted on the second module 200 / device 600 can direct the operation of these valves (linear actuators) and pumps.

[0174] As shown above, the bioprocessing apparatus 600 is configured to perform the activation, transduction, and amplification phases of cell processing in combination with a disposable bioprocessing kit 700. In one embodiment, the activation phase includes six steps, each of which includes a plurality of user-controllable / selectable parameters and is executed by the controller 210. During the activation phase, it is possible to use two pre-seeding reagents and two post-seeding reagents. The cell input to the activation phase is cells that are ready to receive activation. Following activation, it is possible to concentrate and wash the cells to remove any residual reagent components that are undesirable for subsequent process steps. The transduction phase similarly includes six steps, each of which includes a plurality of user-controllable / selectable parameters and is executed by the controller 210. During the transduction phase, it is possible to use two pre-seeding reagents and two post-seeding reagents. The cell input to the transduction phase is cells that were activated in the previous phase. Following transduction, it is possible to concentrate and wash the cells to remove any residual reagent components that are undesirable for subsequent process steps. The amplification phase, in part, includes three steps (seeding, cell culture, and harvest), each of which includes a plurality of user-controllable / selectable parameters and is executed by the controller 210. During the seeding step, the system will add media into the transduction vessel to dilute the contents to the desired cell density for amplification. During the cell culture step, the user can select the sampling frequency and define the feeding strategy used to amplify the cells in the culture vessels 704, 706. During the harvest step, the harvest can be performed at a preset time point or initiated by the user when the target cell dose is achieved.

[0175] In one embodiment, parameters that can be controlled or selected by a user include pre-seeding and post-seeding reagent parameters, input cell volume, incubation, volume reduction, washing, target seeding, and cell culture. The pre-seeding or post-seeding reagent steps include parameters for up to two reagents that can be added to the culture vessel before seeding the cells, and parameters for up to two reagents that can be added to the culture vessel after seeding the cells. Before transferring the reagent to the culture vessel, the user can transfer air or liquid through the system. After incubation of the reagent, the culture vessel can be rinsed before seeding the cells. The input cell volume parameter defines the parameter for adding source cells into the culture vessel. Before adding the cells to the culture vessel, the user can manually mix the cells in the source bag. Additionally, the source bag can be rinsed to maximize the transfer of input cells. The incubation parameter defines the parameter during the incubation of the cells in the culture vessels 704, 706. The user can set the target seeding density and the volume of activation, similar to the parameters related to sampling. The volume reduction parameter defines the parameter for concentrating the cells after activation. The cells are concentrated by using a hollow fiber filter (HFF) or by volume reduction by siphoning the liquid out of the culture vessel without disturbing the cells (i.e., perfusion without adding medium to the inlet so that the volume in the culture vessel decreases) (also referred to as high-speed perfusion (HSP)).

[0176] The washing parameters define the parameters for washing the cells after volume reduction in order to prepare the cells for transduction. The cells are washed using either a hollow fiber filter (HFF) or high speed perfusion (HSP). In certain embodiments, the HSP washing protocol includes the following process steps: 1) Initial sedimentation phase - the activation vessel remains stable over a fixed amount of time, allowing the cells to sediment onto the vessel membrane; 2) Optionally, very slow activation vessel mixing is enabled to enhance the homogeneity of the supernatant without disturbing the sedimenting cells; 3) Media addition and supernatant removal are performed simultaneously while maintaining the activation vessel volume in a stable state; 4) Optionally, very slow activation vessel mixing is enabled to enhance the homogeneity of the supernatant without disturbing the sedimenting cells during the wash duration; 5) Media addition and supernatant removal are performed simultaneously while maintaining the activation vessel volume in a stable state until the wash target duration has elapsed or the wash target media volume has been consumed; 6) Optionally, the activation vessel contents are diluted with media to the target vessel volume; 7) Optionally, very slow activation vessel mixing is enabled to enhance the homogeneity of the supernatant without disturbing the sedimenting cells; and, 8) Low flow removal of the supernatant is performed without disturbing the sedimenting cells to the target activation vessel volume.

[0177] In certain embodiments, with respect to the transduction phase, the steps are similar to the description of the activation phase provided above. In certain embodiments, transfer cell parameters are provided, and the transfer cell parameters define the parameters for transferring the activated cells from the activation vessel into the transduction vessel. Prior to transferring the cells to the culture vessel, the system is capable of mixing the cells in the activation vessel. A portion or the entire contents of the activation vessel can be transferred to the transduction vessel. Additionally, the activation vessel can be rinsed to maximize the transfer of cells.

[0178] Finally, the target seeding general parameters define the parameters for setting the starting conditions for the cells during amplification. The cell culture parameters define the feeding strategy used to culture the cells during amplification. The user can define the feeding period based on user - settable parameters. Exemplary feeding strategies include single - shot media addition (fed - batch) or continuous media addition (perfusion). The harvest parameters define the parameters that enable cell harvest. The user can define the volume of cells to harvest and can initiate the harvest either at a defined time point or whenever desired. As will be recognized, the selection and setting of these parameters can be performed using interface 609 or through an off - board user interface or terminal in communication with device 600 (e.g., through a data port behind device 600), but wireless communication means are also possible.

[0179] As shown above, device 600 and flow architecture 400 also enable sampling of the contents of culture vessels 704, 706, for example, using sampling tubing tails 748 of sampling card 722.

[0180] In one embodiment, the sampling sequence includes tilting platform lockers 640, 642 to mix and homogenize the contents in the culture vessel (the mixing rate depends on the vessel volume), operating process pump 456, circulating the vessel contents from the vessel outlet port (416 or 426) in the sampling tubing back to the vessel inlet port (412 or 422), prompting the user to take a sample, stopping the circulation and mixing, and finally clearing the sampling tubing.

[0181] In certain embodiments, the use of two culture vessels 704, 706 within the processing drawer 604 of the apparatus enables parallel processing to be performed in the manner disclosed below. In certain embodiments, all activation steps can be performed within the first culture vessel 704, after which the cells are transferred to the second culture vessel 706 where transduction and amplification of the cells are performed. In another embodiment, during activation in the first culture vessel 704, transduction reagent actions can be performed within the second culture vessel 706 (e.g., adding seeding reagent to the second culture vessel 706, incubating, and rinsing the culture vessel 706) before adding post-activation cells from the first culture vessel 704 to the second culture vessel 706 for transduction and amplification steps. In another embodiment, the activation, transduction, and amplification steps can be performed within a single culture vessel (e.g., the first or second culture vessel 704, 706).

[0182] Referring to FIG. 90, another workflow 1000 enabled by the bioprocessing apparatus 600 is illustrated. As shown therein, the workflow or method 1000 includes performing a series of activation steps 1002 and a series of transduction steps 1004 within the first culture vessel 704, and amplifying a population of genetically recombinant cells (post-transduction) in parallel amplification steps 1006 using both the first and second culture vessels 704, 706. This involves transferring a fraction of the genetically recombinant cells from the first culture vessel 704 to the second culture vessel such that parallel amplification 1006 can be performed simultaneously using both culture vessels 704, 706.

[0183] Referring to FIG. 91, yet another workflow 1100 enabled by the bioprocessing apparatus 600 is illustrated. As shown therein, the workflow or method 1100 is a parallel (but independent) workflow that includes performing activation, transduction, and amplification steps. This includes, for example, steps of performing an activation step 1102, a transduction step 1104, and an amplification step 1106 for a first population of cells in a first culture vessel 704, and steps of performing parallel activation step 1108, transduction step 1110, and amplification step 1112 for a second population of cells in a second culture vessel 706. In certain embodiments, the first and second populations of cells can be supplied from a single cell population that is split between the first and second culture vessels 704, 706 during the input stage of the activation phase. In another embodiment, the first and second populations of cells can be different (e.g., from different sources).

[0184] Looking now at FIG. 92, yet another workflow 1200 enabled by the bioprocessing apparatus 600 is illustrated. As shown therein, the workflow or method 1200 includes steps of performing an activation step 1202 in a first culture vessel 704 for a population of cells, and then, in step 1204, completely transferring the activated population of cells out of the first culture vessel 704 and out of the bioprocessing apparatus 600 for off-board transduction. After transduction outside of the module / device 600, the cell volume is transferred into a second culture vessel 706 of the bioprocessing apparatus 600 for post-transduction volume reduction and post-transduction wash steps 1206 in the second culture vessel 706. Also shown therein, an amplification step 1208 is also performed in the second culture vessel 706.

[0185] In one embodiment, the bioprocessing apparatus 600, disposable bioprocessing kit 700, and flow architecture of the present invention enable washing (e.g., using a hollow fiber filter) to be performed both after activation and after transfection.

[0186] In connection with the use of the bioprocessing apparatus 600 to perform activation, transfection, and amplification of a cell population in the manner described above, to ensure batch quality and product safety, it is a common requirement that all disposable devices used in cell culture and bioprocesses be sterile during their operation, functionally closed, and highly reliable. Accordingly, embodiments of the present invention also provide a leak tightness verification and occlusion detection check to be performed on the disposable bioprocessing kit 700 (including culture vessels 704, 706 and associated tubing) prior to use. Referring to FIG. 93, the flow architecture 1300 used by the apparatus 600 and disposable kit 700 according to embodiments of the present invention is illustrated. The flow architecture 1300 is generally similar to the flow architecture 400 disclosed above.

[0187] As shown therein, the flow architecture / system 1300 includes a plurality of pneumatic interfaces (e.g., two pneumatic interfaces 1302, 1304, or four pneumatic interfaces 1302, 1304, 1306, 1308), which allow air to be drawn into the system 1300. The pneumatic interfaces 1302, 1304, 1306, 1308 enable leak-tight connections with respect to the sterile air filters 1310, 1312, 1314, 1316 (which form part of the disposable kit 700) associated with each interface. The system 1300 further includes a three-way valve 1322 and a three-way valve 1323 in addition to the three-way valve 1320, and the three-way valve 1322 is capable of switching the air flow path so as to connect the kit passing through the sterile air filters 1310, 1312 to the ambient atmosphere outside the kit in the process draw 604 or to the pressure monitoring sensor 1324. The system 1300 includes two peristaltic pumps 1326, 1328 (e.g., the process pump 456 and the source pump 454 of the peristaltic pump assembly 641) intended to act as pressurizing means and pinch valves during the leak-tight verification process and as liquid management means during normal operation, as disclosed above, and a set of up to 20 pinch valves 1330 (#1 to #20) (e.g., formed by the valve manifold 712 and the linear actuator array 643), intended to act as pinch valves during leak-tight verification and as liquid management means during normal operation, and further includes one peristaltic pump 1332 (e.g., the waste pump 492 of the peristaltic pump assembly 641).

[0188] Air can be drawn into the system via a peristaltic pump through a pneumatic interface that allows for selective connection of the flow path to the atmosphere via a sterile air filter. In certain embodiments, there are two main uses for this interface: (1) it allows for pressurizing a disposable kit 700 during a kit integrity check, as discussed below; and (2) drawing in sterile air to clear fluid from the line during various automated workflows.

[0189] FIG. 94 illustrates another flow architecture / system 1400 that can be used by apparatus 600 and disposable kit 700, instead of architecture 1300, according to another embodiment of the present invention. Flow architecture / system 1400 is similar to flow architecture / system 1300, where like reference numerals designate like parts. As shown therein, system 1440 has four pneumatic interfaces 1302, 1304, 1306, 1308, one of which (pneumatic interface 1306) is connected to a three-way valve 1318 that switches between the atmosphere and a pressure sensor. An advantage of flow architecture / system 1400 is that the culture vessel can be pressurized independently of the remainder of the disposable kit 700 (before initiating the bioprocessing operation). This allows for checking the culture vessel at one pressure and the remainder of the kit at another pressure (potentially higher than the culture vessel can withstand). Also, this allows for testing the remainder of the kit 700 and its flow line under negative pressure, which is typically avoided within the culture vessel as the membrane can be removed or displaced.

[0190] FIG. 95 illustrates another flow architecture / system 1402 that can be used by apparatus 600 and disposable kit 700 instead of architecture 1300 or 1400, according to another embodiment of the present invention. The flow architecture / system 1402 is similar to the flow architecture / system 1400, where like reference numerals designate like parts. However, as shown therein, the flow architecture 1402 of FIG. 95 omits the hollow fiber filter (HFF) and the waste pump. The flow architecture 1402 of FIG. 95 is operable in a manner similar to that described above in connection with the flow architecture 1400 of FIG. 94.

[0191] FIG. 96 illustrates yet another flow architecture / system 1410 that can be used by apparatus 600 and disposable kit 700 instead of architecture 1300, 1400, or 1402, according to another embodiment of the present invention. The flow architecture / system 1410 is similar to the flow architecture / system 1402, where like reference numerals designate like parts. However, as shown therein, there is an additional pressure sensor 1412 fluidly connected to the three-way valve 1320 (instead of the flow line running from the three-way valve 1320 to the pressure sensor 1324 in FIG. 95). In particular, it has been recognized that utilizing two or more pressure sensors can provide certain advantages depending on the particular architecture and application (as opposed to the single pressure sensor used in the architecture of FIG. 95). In one embodiment, the first pressure sensor 1324 and the second pressure sensor 1412 can have different pressure ranges suitable for their particular use. However, it should be recognized that in certain embodiments, the first and second pressure sensors 1324, 1412 can have the same or similar pressure ranges.

[0192] In one embodiment, the flow architecture / system 1410 can further include an accumulator 1414. The accumulator 1414 functions as a volume buffer and can be constructed as a storage section or tubing length. Regardless of the particular construction or configuration, the accumulator 1414 has a volume greater than or equal to the total volume of the fluid flow path between and from the sterile air filter 1316 and the second pressure sensor 1412. In use, in the event of a blockage, the presence and location of the accumulator 1414 ensures that the volume of fluid accumulates within the accumulator 1414 without contacting the sterile air filter. In one embodiment, the accumulator can be mounted to the tray of the disposable kit 700 (such as via a snap-fit connection). As shown, the accumulator 1414 is positioned along the vent line from the pinch valve manifold to prevent the sterile air filter from getting wet under dead head.

[0193] In many of the components, systems, devices, and architectures disclosed above, mention has been made of the use (or employment) of a sterile air filter. In one embodiment, one or more (or all) of these sterile air filters can be hydrophobic and are adapted to be exposed to (or come into contact with) a fluid and still be able to maintain their integrity and function as intended. In still other embodiments, depending on the particular system or architecture layout and application, a non-hydrophobic filter can be used with or without an accumulator or similar device.

[0194] In one embodiment, the leak tightness verification referred to above is performed independently for three differentiated segments of the disposable culture kit. In one embodiment, the first segment includes the entire disposable kit (i.e., its entire fluid flow path) except for the tubing segment between the source pump 1328 / 454 and the tubing tails 1334a - d (e.g., the tubing tails of the tubing organizer 720). The test for the first segment is performed in two phases (a pressurization phase and a pressure decay monitoring phase). In one embodiment, the second segment includes the two culture vessels, the tubing from the inlet port to the supply pump, and the tubing segment between the source pump 1328 / 454 and the tubing tails 1334a - d. The test for the second segment is performed in two phases (a pressurization phase and a pressure decay monitoring phase). In one embodiment, the third segment includes the entire disposable kit (i.e., its entire fluid flow path) except for the T / U loop (sensor bypass) and the tubing segment between the source pump 1328 / 454 and the tubing tails 1334a - d. The test for the third segment is performed in three phases (a pressurization phase, a pressure decay monitoring, and a pressure release phase).

[0195] As shown above, the leak tightness verification and blockage detection methods disclosed above enable the end - user to perform an automated integrity test on the entire disposable kit 700 before initiating the bioprocessing operation. This enables the end - user to detect possible leaks in the disposable kit and / or blocked / pinched lines (which would affect the ability to execute the automated workflow and ultimately the batch quality).

[0196] As indicated above, mammalian cell culture processes can require significantly complex liquid transfer management operations that must be performed in an accurate and safe manner. Thus, the ability to detect leak events is an important function that should be continuously performed to sound an alarm when the viability of a batch is potentially compromised. In light of the foregoing, embodiments of the present invention also contemplate using real-time monitoring of the mass involved in a bioprocess to verify the leak tightness and detect blockages within the disposable kit 700. In (if not all) most of the bioprocessing operations disclosed herein, four situations are habitually always present or occurring: (1) a fluid is held within a closed container, (2) a fluid is transferred from a source container to a destination container, (3) a fluid is perfused from a source container to a destination container through an intermediate container, and / or (4) a fluid is recycled from and returned to the same container or vessel. Thus, as long as the source container, intermediate container, and / or destination container includes means / mechanisms for measuring the mass of such containers (e.g., one or more load cells associated with each container, as disclosed above), means for pumping fluid from one container to another or for looping fluid from and to the same container in a leak-tight manner (e.g., using the peristaltic pump assembly 641), and a control unit (e.g., the controller 210) for monitoring fluctuations in the mass of each container, multiple leak and / or blockage detection processes can be implemented as disclosed below. The load cell can include, for example, a bedplate that supports various containers (e.g., culture vessels 704, 706, waste bags, etc.), or a peg or hook having an integrated load cell (e.g., a hook 620 on the vertical storage drawers 614, 616 of the cabinet 608 for suspending media bags, reagent bags, and other bags).As disclosed below, the controller (e.g., controller 210) monitors the mass variations of each container, actuates pumping means for transferring fluid between containers, executes a mass balance equation, and generates an alarm or alert if the solution of the mass balance equation does not indicate the absence of leaks or blockages.

[0197] In one embodiment, no pumping action is performed and the control unit 210 simply verifies that the mass of the first container remains generally constant (e.g., within a predetermined or preset change threshold over a predetermined duration). If the change in mass is below a predetermined threshold amount, this indicates that the volume of fluid in the first container remains constant, which indicates the absence of leaks. However, if the change in mass exceeds the threshold, this indicates that fluid has leaked from the container and the controller 210 generates an alert to the user.

[0198] In another embodiment, a method for detecting leakage or blockage involves monitoring the masses of a first source container and a second destination container, and transferring fluid from the first container to the second container. For example, the pump of apparatus 600 is controlled by controller 210 to pump fluid from the first container to the second container while monitoring the mass of each container using associated load cells. In particular, in such an embodiment, the mass of the first container (and thus the mass of the volume of fluid therein) is first determined. Next, the volume of fluid from the first container is transferred to the second container. Then, the mass of the second container (and thus the mass of the volume of fluid in the second container) is determined. The controller 210 then compares the original mass of the volume of fluid in the first container with the mass of the volume of fluid in the second container, which should be approximately equal if there is no leakage or blockage. If the difference between the original mass of the volume of fluid in the first container and the mass of the transferred volume of fluid in the second container exceeds a threshold, the controller 210 generates a notification or alarm. In some embodiments, the controller can also perform the above leakage detection process without requiring that the entire volume of fluid be transferred between containers. In particular, in some embodiments, the controller 210 verifies whether the absolute mass-volume variation of the source container is below the sum of a specific leakage rate detection threshold and the transfer flow rate (and remains below), and whether the absolute mass-volume variation of the destination container is above the difference between the transfer flow rate and a specific leakage rate detection threshold (and remains above). Otherwise, a leakage alarm will be triggered by the controller 210.

[0199] In yet another embodiment of leak tightness verification using real-time mass balance monitoring, the aim is to keep the mass of an intermediate (e.g., third) container constant. Accordingly, the simultaneous action of two pumps of the peristaltic pump assembly 641 is required, where the liquid transfer from the source to the intermediate container must be controlled based on source container variations with respect to a specific flow setpoint, and the liquid transfer from the intermediate to the destination container must be controlled based on destination container variations with respect to a specific flow setpoint. The control unit 210 verifies whether the absolute mass-volume variation of the source container is below (or remains below) the sum of the transfer flow rate and a specific leak rate detection threshold, whether the absolute volume variation of the intermediate container is below (remains below) a specific leak rate detection threshold, and whether the absolute mass-volume variation of the destination container is above (remains above) the transfer flow rate minus a specific leak rate detection threshold. If any of these conditions do not exist, then the controller 210 is configured to generate an alarm.

[0200] In yet another embodiment, leak tightness verification is performed by the controller 210 by recirculating fluid from a first container and controlling the pump to pump out from and back into the first container. Accordingly, the pumping of the fluid is done in an open loop, and the control unit 210 is configured to verify that the absolute mass-volume variation of the first container is below (remains below) a specific leak rate detection threshold. Otherwise, a leak alarm will be triggered by the controller 210. A variation to this process is when a sample volume is withdrawn from the recirculation loop. In this case, the controller 210 verifies whether the absolute mass-volume variation of the first container remains below the sum of the specific leak rate detection threshold and the sampling flow rate.

[0201] Accordingly, embodiments of the present invention utilize real-time mass balance calculations to check the leak tightness and / or detect blockages in kit 700 prior to using kit 700 in a bioprocess operation. However, the methods disclosed herein are not limited to determining leaks prior to use of kit 700 in a bioprocess and can also be utilized during a bioprocess for real-time leak verification or blockage detection. Accordingly, it may be possible to take corrective action regarding any blockages or leaks detected in order to save or salvage a batch.

[0202] In the embodiments disclosed above, the first source bag / container can be a media bag, the second destination bag / container can be a waste bag, and the third intermediate bag / container can be a culture vessel or a bioreactor vessel. However, the present invention is not intended to be limited in this regard, and various bags / containers can be used as the first, second, and third containers as long as fluid can be transferred between and / or through such containers. Moreover, the mass balancing process for verifying leak tightness and detecting blockages has been described as being implemented on bioprocessing device 600 and disposable bioprocessing kit 700, but the present invention is not intended to be limited in this regard. In particular, it is contemplated that the mass balancing technique can be implemented on various systems and devices, including the processing device 102 and isolation module (and disposable kit therefor) disclosed above in connection with the first and third modules 100, 300.

[0203] As disclosed above, the bioprocessing system 10 of the present invention includes a first module 100, a second module 200, and a third module 300 (which can be configured the same or similar to the first module 100), which are configured to perform a plurality of discrete automated bioprocessing operations including concentration and isolation, activation, genetic recombination, and amplification, and harvesting. In certain embodiments, the first module 100 (including the processing device 102 and the isolation module 104) is used to isolate targeted cells (e.g., using the kits 800 of FIGS. 37A and 37B) prior to transferring the targeted cells from the first module 100 to the second module 200 for activation, transduction, and amplification of the transduced targeted cells. Optionally, the present invention contemplates that the first module 100 (including the processing device 102 and the isolation module 104) can be used to perform splitting and dosing preparation operations, cryopreservation preparation operations, and washing operations (e.g., using the kit 350 without any further bioprocessing operations (e.g., cell activation, transduction, or amplification) using, for example, the second module 200).

[0204] Also, the processing device 102 of the first module 100 can be utilized downstream as a stand-alone device (i.e., without the need or use of the isolation module 104) to perform harvesting and washing of the amplified cell population (e.g., using the kit 350 described in FIG. 36 and above). For such operations, different tube kits (e.g., standard Sefia disposable tube kits) can be used and can be operated by the processing device 102 alone.

[0205] In a further embodiment, the first module 100 (including the processing device 102 and the isolation module 104) can also be utilized downstream to perform splitting and dosing preparation operations, as well as cryopreservation preparation operations, as part of the formulation process. In this case, the first module 100 can be referred to as the third module 300, however, as described above, it can be configured identically or substantially similarly to the first module 100 and includes a processing device (e.g., the processing device 102 (e.g., Sefia S-2000 cell processing instrument, etc.) and the isolation module 104).

[0206] Thus, the first module 100 or its processing device 102 can be utilized on both sides of the second module 200 (i.e., before cell activation using the second module 200 and after amplification using the second module 200) to perform automated cell isolation, splitting and dosing preparation, cryopreservation preparation, and / or harvesting and washing as desired. Therefore, since the first module 100 can perform multiple functions in conjunction with different second modules 200, the versatility of the first module 100 is demonstrated. That is, when the second module 200 is processing a target cell population, each module 100 can freely begin processing cells intended for or taken from other second modules 200 operating within the production facility. Therefore, cell therapy manufacturers can utilize the first module 100 or the third module 300 simultaneously without limiting their production capacity or requiring additional equipment that would otherwise go unused if it were dedicated to a single second module 200.

[0207] As disclosed above, in one embodiment, the processing device 102 of the first module 100 is used with the washing kit 350 of FIG. 36 and is capable of providing automated dilution, washing, and concentration of cell products, for example, both upstream and downstream of a typical CAR-T manufacturing process. Thus, the kit 350 and the first module 100 are capable of processing thawed cell products through temperature control during the dilution phase or during the wash harvest. For example, the washing kit 350 (in conjunction with the first module 100) can be utilized upstream of the second module 200 to process fresh or thawed cell materials (e.g., PBMCs, T-cells). The thawed PBMCs and T-cells will be the output stored from the isolation step (e.g., cryopreserved backup material). Downstream of the second module 200, the washing kit 350 (in conjunction with the first module 100) is applicable, for example, to the harvest after T-cell expansion. Additionally, the kit 350 and the module 100 can be used to wash cell materials.

[0208] Accordingly, as described above, the module 100 and kit 350 and related software (used either upstream or downstream from the second module 200) are configured to provide primary dilution (i.e., optional dilution of the initial input source material with the wash solution), sedimentation and initial product wash (i.e., volume reduction and wash by user selection of the number of wash cycles), supernatant extraction (i.e., removal of the supernatant from the wash solution), cell resuspension (i.e., post-wash resuspension of the cells in the wash solution or a dedicated resuspension solution), and final product resuspension (which includes extraction of the cells into the final product bag). In certain embodiments, the initial dilution can be completely omitted or can be limited to a limited number of process steps. It can simply provide volume transfer or can provide volume transfer with temperature preconditioning and / or automated mixing. Moreover, the wash cycles can be completely omitted or can be composed of a limited number of wash cycles.

[0209] Referring to FIG. 97, another configuration of a single-use disposable bioprocessing kit 1500 for use with the bioprocessing apparatus 600 is shown. The bioprocessing kit 1500 is generally similar to the bioprocessing kit 700 of FIG. 50 with respect to its configuration and function, as will become apparent from a comparison of the respective figures. The bioprocessing kit 1500 includes a generally rectangular tray 1502 sized and dimensioned to be received within a first internal space 636 of the process drawer 604, and a pair of culture vessels 1504, 1506 (also referred to as bioprocessing vessels or bioreactor vessels) received within the tray 1502. As best shown in FIGS. 99 and 100, the tray 1502 has a pair of openings or windows 1508, 1510 in the floor of the tray 1502 beneath the culture vessels 1504, 1506, and the tray 1502 supports the culture vessels 1504, 1506 in an elevated position such that when the tray 1502 is positioned within the first internal space 636 of the process drawer 604 and engaged with the support posts 646 of the platform rocker assemblies 640, 642, the culture vessels 1504, 1506 are lifted out of the tray.

[0210] As shown in FIG. 98, tray 1502 includes a spine portion 1512 at one of its ends, the purpose of which will be described hereinafter. Further, tray 1502 is equipped with a pair of handles 1514, 1516 at its opposing front and rear ends to facilitate positioning and removal of tray 1502 within process drawer 604. In certain embodiments, handles 1514, 1516 can be removably connected to tray 1502, such as by snap fit or other means known in the art. As best shown in FIG. 99, tray 1502 includes a longitudinally extending wall portion 1518, which defines a tubing corridor 1520 between such wall portion 1518 and the side wall portions of tray 1502, and the tubing is routed and contained through tubing corridor 1520. As shown in FIGS. 99 and 100, the floor of tray 1502 includes a plurality of passage openings 1522 for angled platform pins / posts (which enable the angled platform pins to engage culture vessels 1504, 1506) and a plurality of positioning protrusions 1524, which cooperate with corresponding features on the underside of culture vessels 1504, 1506 to ensure proper positioning of culture vessels 1504, 1506 within tray 1502 above openings 1508, 1510. A plurality of tube routing clips 1526 are provided adjacent to the opposing lateral sides of tray 1502, which enable tubing (e.g., tubing from / to culture vessels) to be neatly stored, routed, and organized. Tray 1502 further includes a pair of snap fit connectors 1528 and an air duct cover 1530 at its spine end for receiving spine portion 1512.

[0211] Referring now to FIG. 101, the spine portion 1512 of the tray 1502 is shown. The spine portion 1512 includes a first generally rectangular opening 1532 for receiving a valve manifold 1534 (which may be the same or similar to valve manifold 712), a second opening 1536 for pump tubing segments 714, 716 (to be engaged by a peristaltic pump), and a third opening 1538 intended to function as a gripping area to facilitate disengagement of the spine portion 1512 and / or the valve manifold 1534. Side guides 1540 having tactile feedback elements assist in the insertion of the tray 1502, provide coarse registration with pinch valve hardware behind the tray 1502, and provide tactile feedback that the tray 1502 is properly positioned within the process drawer. In some embodiments, the side guides provide an angled or funnel shape to facilitate and guide the connection of the spine portion to the tray. The first and second openings 1532, 1536 each have a plurality of snap-fit wells 1542, 1544, which enable removable and reliable connection of the valve manifold 1534 and the pump tubing (e.g., pump tubing connectors 714, 716). Although snap-fit connections are contemplated, other connection means known in the art may also be utilized without departing from the broader aspects of the present invention. As shown, the tray 1502 includes only two tubing segments, rather than three (as shown in FIG. 81). This is because no separate waste pump (enabled by the architecture shown in FIG. 96) is present, and thus no tubing segment for a waste pump is required. In some embodiments, the first opening 1532 may include chamfered corners 1546 or other poka-yoke elements to ensure that the valve manifold 1534 is properly oriented within the first opening 1532.That is, the chamfered corner or other mechanism ensures that the pinch valve manifold 1534 can be connected to the spine portion 1512 only in a single orientation of the pinch valve manifold 1534.

[0212] The space 1548, when utilized, is provided for the equipment of the biosensor. The spine portion 1512 further includes a protective flange 1550 that extends horizontally at the upper portion of the spine portion 1512. The opening 1552 enables a snap - fit connection of the handle 1516.

[0213] FIG. 102 shows the valve manifold 1534 connected to the spine portion 1512 within the first opening 1532 and the pump tubing connectors 714, 716 connected to the spine portion 1512 within the second opening 1536. On the one hand, FIG. 103 similarly shows the valve manifold 1534 connected to the spine portion 1512. In certain embodiments, the tubing connectors / tubing segments 714, 716 each include connectors (such as, for example, BarbLock connector 1554 and right - angle connector 1556) at their opposing ends (where the right - angle connector 1556 has means for interconnecting with the snap - fit well 1544). The valve manifold 1534 is similar to the valve manifold 712 disclosed above and can be, for example, a fluid container configured to interface with a plurality of linear actuators of the linear actuator array 643. The valve manifold 1534 is fluidly interconnected with the culture vessels 1504, 1506, the media bag and reagent bag within the cabinet 608, the waste bag within the waste drawer 606, and the sampling line, forming a fluid network or architecture as disclosed above.

[0214] Figures 104 to 107 more specifically show the configuration of the valve manifold 1534 according to an embodiment of the present invention. The valve manifold 1534 includes a non-fluid contact backing plate 1558 having a plurality of valve bowls 1560, a membrane 1562, and a fluid plate 1564. Among the plurality of valve bowls 1560, the plunger of the linear actuator of the actuator array 643 acts to allow or restrict / prevent fluid flow through the flow path of the valve manifold 1534. The membrane 1562 generates a fluid seal and a valve function when the plunger operates. The fluid plate 1564 includes fluid paths, valve bowls, sealing ridges, and interface points (e.g., group ports with various tubing lines). The membrane 1562 is sandwiched between the backing plate 1558 and the fluid plate 1564. As shown therein, the backing plate 1558 and the fluid plate 1564 each include a plurality of vent interfaces 1566 and vent ports 1568, and also include a plurality of alignment openings 1570 for receiving alignment pins in the process drawer to ensure proper alignment and positioning of the tray 1502 in the process drawer. The valve manifold 1534 further includes a plurality of snap-fit connectors 1572. The plurality of snap-fit connectors 1572 are configured to engage with snap-fit wells 1542 on the spine portion 1512 to hold the valve manifold 1534 in the opening 1532 in the spine portion 1512. As shown in FIGS. 104 and 106, the valve manifold 1534 includes chamfered corners 1574 corresponding to the chamfered corners 1546 in the opening 1532 to facilitate proper orientation of the valve manifold 1534 with respect to the spine portion 1512. As shown in FIG. 104, the backing plate 1558 includes a plurality of ports 1576 that function as interface ports for leak testing of the manifold 1534. In certain embodiments, the surface of the fluid plate 1564 includes a plurality of fluid port identifiers and vent port identifiers (e.g., numbers or letters that identify the various ports to facilitate proper architecture assembly).

[0215] FIG. 107 best illustrates the fluid paths in valve manifold 1534 formed by fluid plate 1564. In one embodiment, port 1578 is fluidly connected to a tubing tail bundle, port 1580 is fluidly connected to a pump (via pump tubing segments 712, 714), port 1582 is fluidly connected to culture vessels 1504, 1506, port 1584 leads to a vent filter, ports 1586 are fluidly connected to each other, and ports 1588 are fluidly connected to each other. In one embodiment, pinch valve manifold 1534 has four fluid sub-networks, three of which are shown by three sets of dark / thick lines and the fourth is shown by the absence of dark / thick lines. FIG. 108 shows fluid plate 1564 and membrane 1562 mounted on backbone 1512 (with backing plate 1558 removed).

[0216] Referring back to FIG. 97, the disposable kit 1500 includes, in addition to the tray 1502 and the culture vessels 1504, 1506, additional left and right tubing tail cards / organizers 1590, 1592 respectively, and a sample card / organizer 1594. FIGS. 109-112 show the construction of the tubing tail cards 1590, 1592 in more detail. The cards 1590, 1592 serve the same purpose as the tubing organizer 720, i.e., to organize and contain a plurality of tubing tails that are fluidly connected to the valve manifold 1534 (and which is configured for connection to the various media and reagent bags housed within the cabinet 608 as described above). As shown therein, both the left tail card 1590 and the right tail card 1592 include a plurality of tail wrap spindles 1596 around which the tubing tails are wound, and a plurality of clips 1598 for securing the tubing tails running across the bottom of the card. In one embodiment, the left tail card 1590 includes three tail wrap spindles 1596 and the right tail card 1592 includes six tail wrap spindles 1596, although more or fewer spindles can be used without departing from the broader aspects of the present invention. The tubing cards 1590, 1592 further include respective tail bundle inlet shoots 1600 at one corner of the card 1590, and a plurality of clips 1602 for securing the tail ends when not in use. In certain embodiments, the cards 1590, 1592 can also include a plurality of bottom snap clips 1604. Each card 1590, 1592 further includes a plurality of hanging clips 1606 for mounting the tubing cards 1590, 1592 as desired (e.g., on the drip tray at the bottom of the cabinet 608). Either card can additionally include a plurality of slalom clips 1608 for routing and holding the tubing in place.FIG. 112 shows left and right tubing cards 1590, 1592 attached to opposite side portions of a drip tray 1610 (which can be the same or similar to drip trays 626 or 628).

[0217] FIGS. 113 and 114 show the configuration of a sample card 1594 according to an embodiment of the present invention. As shown therein, the sample card 1594 is generally rectangular in shape and has a plurality of keyhole openings 1612 for suspending the sample card 1594 from hooks or pegs inside the door 610 or 612 of the cabinet 608. Also, the front surface side of the sample card 1594 includes a pair of opposing wells 1614 that receive a sample manifold 1616. A plurality of slalom hooks / clips 1618 for two tail bundles are provided along the left side of the card, and the upper end portion of the card includes a semi-circular tail bundle track 1620. In one embodiment, the rear side portions of the card 1594 can be provided with waffling or ribs to strengthen and stiffen the card and to prevent or inhibit warping.

[0218] Referring to FIG. 114, the sample manifold 1616 includes a pair of inlet / outlet ports 1622, 1624, a pair of opposing end caps 1626 that define a manifold 1628 therebetween, and a plurality of sample tail ports 1630 that are in fluid communication with the manifold 1628. A handle 1632 in the form of a flange is provided to facilitate insertion, positioning, and removal of the manifold 1616 and to facilitate connection of tubing to the sample tail ports 1630.

[0219] Returning once again to FIG. 97 and referring to FIGS. 115-118, the disposable kit 1500 further includes a base anchor 1634 and a pivot anchor 1636. As shown in FIGS. 115 and 116, the base anchor 1634 includes a passage 1638 for receiving a tube bundle therethrough, a slot or guide 1640 on an opposing side of the anchor 1634, a first set of clips 1642, a second set of clips 1644, and a foolproofing element 1646. As shown in FIGS. 117 and 118, the pivot anchor 1636 includes a flanged raceway 1648, a plurality of tube wrap recesses 1650 along the raceway, and a foolproofing element 1652. FIG. 119 illustrates the disposable kit 1500 with tubing installed and shows the positions of the base anchor 1634 and the pivot anchor 1636.

[0220] Figures 120 and 121 show the configuration of the culture vessel 1504 (culture vessel 1506 is the same). Culture vessels 1504 and 1506 are substantially the same as culture vessels 704 and 706, and include a base 1656, a lid 1658 connected to the base 1656, a gas-permeable and liquid-impermeable membrane 1660 sandwiched between the base 1656 and the lid 1658, and a gasket 1662 sandwiched between the membrane 1660 and the lid 1658. As best shown in FIG. 120, the opposing ends of the lid have group ports 1664 for connection of inlet and outlet tubing, as disclosed above in connection with FIGS. 64 and 65. A vent port 1666 is provided at the upper center of the lid 1658. Also, as shown in FIGS. 120 and 121, a sensor flag 1668 is provided in a receiving portion within the base 1656, which is configured for detection by a sensor associated with the process draw to ensure that the culture vessels 1504 and 1506 are properly positioned and seated. In certain embodiments, the sensor flag 1668 can be connected to the base within the receiving portion via a snap-fit connection. As illustrated in FIGS. 122 and 123, it is contemplated that various types of vent ports for connection to vent tubing can be used. For example, FIG. 122 illustrates a barb vent port 1670, while FIG. 123 illustrates a female group port vent port 1672.

[0221] Referring further to FIG. 122, in one embodiment, the culture vessel 1504 can include an annular wall or baffle 1674, regardless of the particular configuration, which surrounds the vent port in the lid 1658 of the culture vessel 1504 and extends downwardly from the lid into the culture vessel. The baffle is shown as being annular in shape, but other shapes such as square or rectangular are also contemplated. Additionally, instead of having a continuous baffle, it is contemplated that a pair of opposing linear baffles can be provided on the opposite side of the vent port at the top of the culture vessel. The baffle 1674 is configured to prevent and / or inhibit the fluid or medium in the culture vessel from exiting the culture vessel through the vent line (and thus prevent and / or inhibit the fluid or medium from entering the vent line and contacting the air filter located therein). In particular, during the rocking of the culture vessel 1504, the medium in the vessel moves from end to end in a wave-like motion. This rocking action can cause the fluid to contact the lid of the culture vessel adjacent to the vent port. However, the presence of the baffle 1674 prevents this wave of fluid from contacting the lid in the area of the vent port and thus prevents and / or inhibits such fluid from entering the vent line and / or contacting the filter. The baffles described herein are further contemplated to be able to be present on any of the culture or bioprocessing vessels described herein and are not limited to the embodiment shown in FIG. 122.

[0222] As shown above, the disposable kit 1500 can be used with the apparatus 600 (instead of, or in place of, the kit 500).

[0223] As used herein, elements or steps recited in the singular and preceded by the word "a" or "an" are to be understood as not excluding a plurality of such elements or steps unless such exclusion is explicitly stated. Moreover, reference to "one embodiment" of the present invention is not intended to be construed as excluding the existence of additional embodiments that also incorporate the recited features. Moreover, unless explicitly stated to the contrary, embodiments "comprising," "including," or "having" an element or elements with a particular property may include additional such elements that do not have that property.

[0224] This written description uses examples to disclose several embodiments of the invention, including the best mode, and also enables those 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 only minor differences from the literal language of the claims.

Description of Reference Numerals

[0225] 10 Bioprocessing System 12 Bioprocessing System 100 First Module 102 Processing Device 104 Isolation Module 105 Bracket 106 Base 108 Centrifugation Chamber 110 First Controller 111 High Dynamic Range Peristaltic Pump Assembly 112 Plug Manifold Interface 113 Drip Chamber Holder 114 Heating-Cooling-Mixing Chamber (Thermal Mixer) 116 Hanger Assembly 118 Hook 130 Base / Housing 132 Plug Manifold Interface 134 Slot 136 Magnetic Cell Isolation Holder 138 Support Pole 140 Hook 142 Latch, Clamp 143 Latch, Clamp 144 Output Shaft 146 Plug Motor 148 Piping Pressure Sensor Assembly 150 Bubble Sensor Assembly 151 Connector 152 Housing 153 Switch 154 Channel 155 Communication Connector 156 Cover 157 Opening 159 Internal Fan 160 Magnetic Field Generator Assembly 161 Indicator Light 162 Permanent Magnet 164 Permanent Magnet 166 Carriage 168 Upper Shaft 170 Lower Shaft 171 Crank 172 Bushing, Bearing 174 Lead Screw 176 Central Bushing 178 Motor 180 Gearbox 182 Belt 183 Timing Pulley 184 Timing Pulley 186 First sensor 188 Second sensor 190 Third sensor 192 Crank sensor 194 Locking pin 196 Flange 198 Coil spring 199 Sheet part, recess 200 Second module 200a, 200b, 200c Second module 210 Second controller, control unit 210a, 210b, 210c Controller 250 Magnetic cell isolation holder 252 Body part 254 Channel, race 256 Tube 258 First part 260 Second part 262 Third part 264 Fourth part 266 Handle 268 High - gradient region 270 Ferromagnetic core 272 Loop 274 Body part 276 Handle 277 Semi - split body 278 Semi - split body 280 Column 280a, 280d Sample collection device 282 End cap 284 PVC tubing of the first length 286 PVC tubing of the second length 300 Third module 310 Third controller 350 Cleaning kit 352 Cassette, manifold 354 Plug 356 Plug 358 Plug 360 Plug 362 Input pipe 364 Final product / collection container or bag 366 Pipe 368 Cleaning solution pipe 370 Resuspension solution pipe 372 Pipe 374 Waste container or bag 376 Pipe 378 End cap 380 In-line drip chamber 382 Separation chamber 384 Pipe 386 Tubing tail 388 Hydrophobic filter 400 System 410 First bioreactor container 412 First port 414 First bioreactor pipe 416 Second port 418 Second bioreactor pipe 420 Second bioreactor container 422 First port 424 First bioreactor pipe 426 Second port 428 Second bioreactor pipe 430 Bioreactor array 432 First bioreactor pipe valve 434 Second bioreactor pipe valve 436 First bioreactor pipe valve 438 Second bioreactor pipe valve 440 First fluid assembly 442 First fluid collection pipe 444 Second fluid assembly 446 Second fluid collection pipe 448 Sampling assembly 448 Sampling assembly 450 Connection pipe interconnecting pipe 452 Connection pipe interconnecting pipe valve 454 First pump, peristaltic pump, source pump 456 Second pump, circulation piping pump, process pump 458 Sterile air source 460 Sterile air source piping 462 Piping valve 464a~f Tubing tail 466a~f First storage section 468a~f Tubing tail valve 470a~d Tubing tail 472a~d Second storage section 474a~e Tubing tail valve 476a~476d Sampling line 478a~d Sample piping valve 482 Filtration piping 484 Filter 486 Upstream filtration piping valve 488 Downstream filtration piping valve 490 Waste line 492 Waste pump 500 Administration preparation kit 502 Plug manifold 504 Plug 506 Plug 508 Plug 510 Plug 512 Plug 514 Plug 516 Process bag 518 Peristaltic pump tubing 520 Medium piping 522 Medium piping 524 Medium piping 526 Final formulation / collection bag 528 Piping 530 Initial product bag 532 Piping 534 Waste bag 536 Piping 538 Freezing bag connection piping 540 Freezing bag connection piping 542 Freezing bag connection piping 544 Refrigeration Bag Connecting Pipe 546 Hydrophobic Filter 547 Hydrophobic Filter 548 Air Inlet Pipe 549 Hydrophobic Filter 600 Second Module 602 Housing 604 Process Drawout 605 Status Indicator Light 606 Waste Bag Drawout 607 USB or Other Port 608 Cabinet 609 Input Terminal 610 Door 612 Door 614 First Vertical Drawout 616 Second Vertical Drawout 618 Peg, Pin 620 Hook 622 Medium Bag 624 Reagent Bag 626 Medium Drip Tray 628 Reagent Drip Tray 630 Slot 632 Anchor Comb 634 Status Indicator Light 636 First Internal Space 638 Second Internal Space 640 First Platform Rocker Assembly 641 Peristaltic Pump Assembly 642 Second Platform Rocker Assembly 643 Linear Actuator Array 644 Cover 646 Support Post 648 Sensor 650 Seal Element 652 Bellows 654 Peripheral Channel 656 Drain Hole Part 658 Load Cell 660 Load cell 662 Load cell 664 Load cell 700 Bioprocessing kit 702 Tray 704 Culture vessel 706 Culture vessel 708 Leg 709 First window 710 Leg 711 Second window 712 Valve manifold 714 Peristaltic pump tubing segment 716 Peristaltic pump tubing segment 718 Peristaltic pump tubing segment 720 Tubing organizer card 722 Sampling card 726 Tubing tail 730 Body part 732 Passageway 734 Tubing holding element 736 Plate body part 738 Tubing holding channel 740 Mounting and / or positioning aperture 742 Clearance, relaxation area 744 Body part 746 Manifold 748 Sampling tubing tail 750 Feed pipe 752 Return pipe 754 Mounting and / or positioning aperture 756 Engagement feature / surface 758 Sensor 760 Engagement structure 762 Pivoting pump shoe 764 Base 766 Lid 768 Membrane 770 Gasket 772 Reinforcing support 774 Inlet port 776 Outlet Port 777 Vent Port 778 Corner 780 Location / Holding Hole 781 Heat Staking 782 Location / Holding Hole 784 Heat Staking Pin 786 Hole 788 Flange Area 790 Rib 791 Pin Well 792 Pin Well 793 Pin Well 794 Pin Well 796 IR Sensor Window 798 Sensor Well 799 Opening 800 Magnetic Cell Isolation Kit 802 Cassette, Manifold 804 First Plug 806 Second Plug 808 Third Plug 810 Fourth Plug 811 Pipe 812 Pipe 813 Collection Bag 814 Pipe 815 Tubing Tail 816 Tubing Tail 817 Tubing Tail 818 Tubing Tail 819 Pipe 820 Negative Fraction Bag 821 Second Manifold 822 First Plug 823 Second Plug 824 Third Plug 825 Fourth Plug 826 Final Collection / Transfer Bag 827 Process Bag / Incubation Bag 828 Pipe 829 Inline Drip Chamber 830 Branch Pipe 831 Branch Pipe 832 Pipe 833 Branch Pipe 834 Pipe 835 Branch Pipe 836 Waste Bag 837 Spare Bag 838 Sampling Pillow 839 Filter 840 Separation Chamber 841 Pipe 842 Peristaltic Pump Tubing 843 Drip Chamber 844 Sterile Air Filter 845 Pipe 846 Process Bag 870 Base 872 Fulcrum 873 Fulcrum Axis 874 Motor 875 Frame 876 Eccentric Roller 878 Oscillating Plate 880 Compression Spring 882 Load Cell 884 Tilt Sensor 900 System 902 Incubation Chamber 904 Heater 906 Fan, Blower 908 Fan, Blower 910 Louver, Air Passage 912 Louver, Air Passage 914 Temperature Sensor 915 Recirculation Chamber 916 Carbon Dioxide Sensor 918 Carbon Dioxide Supply Unit 920 Carbon Dioxide Control Valve 922 Gas Port 924 Recirculated Air Flow 926 Ducting Feature 928 Local Turbulence 930 Concave area 932 Vent opening 950 Flow-through sensing chamber 952 First plate 954 Second plate 956 Fluid channel 957 Notch 958 First port 959 Tab 960 Second port 961 Mounting and positioning hole, mounting aperture 962 Sensing location 964 Sensing location 966 Sensing location 968 Sensor 970 Electrode 972 Flange 974 Mirror 976 Snap pin 978 First electrochemical sensing device 980 Second electrochemical sensing device 982 Pin 984 Reflective light device 986 First fluorescence device 988 Second fluorescence device 990 Transmitted light / backscattered light device 1300 Flow architecture 1302 Pneumatic interface 1304 Pneumatic interface 1306 Pneumatic interface 1308 Pneumatic interface 1310 Sterile air filter 1312 Sterile air filter 1314 Sterile air filter 1316 Sterile air filter 1318 Three-way valve 1320 Three-way valve 1322 Three-way valve 1323 Three-way valve 1324 First pressure sensor 1326 Peristaltic pump 1328 Peristaltic pump 1330 Pinch valve 1332 Peristaltic pump 1334a~d Tubing tail 1400 Flow architecture / system 1402 Flow architecture / system 1410 Flow architecture / system 1412 Second pressure sensor 1414 Accumulator 1500 Bioprocessing kit 1502 Tray 1504 Culture vessel 1506 Culture vessel 1508 Opening 1510 Opening 1512 Vertebral part 1514 Handle 1516 Handle 1518 Wall part 1520 Tubing corridor 1522 Passing opening 1524 Positioning protrusion 1526 Tube routing clip 1528 Snap fit connector 1530 Air duct cover 1532 First opening 1534 Valve manifold 1536 Second opening 1538 Third opening 1540 Side guide 1542 Snap fit well 1544 Snap fit well 1546 Chamfered corner 1548 Space 1550 Protection flange 1552 Opening 1554 BarbLock connector 1556 Right-angle Connector 1558 Backing Plate 1560 Valve Bowl 1562 Membrane 1564 Fluid Plate 1566 Vent Interface 1568 Vent Port 1570 Alignment Opening 1572 Snap-fit Connector 1574 Chamfered Corner 1576 Port 1578 Port 1580 Port 1582 Port 1584 Port 1586 Port 1588 Port 1590 Tubing Tail Card / Organizer 1592 Tubing Tail Card / Organizer 1594 Sample Card / Organizer 1596 Tail Wrap Spindle 1598 Clip 1600 Tail Bundle Inlet Shoot 1602 Clip 1604 Bottom Snap Clip 1606 Suspension Clip 1608 Slalom Clip 1610 Drip Tray 1612 Keyhole Opening 1614 Well 1616 Sample Manifold 1618 Slalom Hook / Clip 1620 Tail Bundle Track 1622 Inlet Port 1624 Outlet Port 1626 End Cap 1628 Manifold 1630 Sample Tail Port 1632 Handle 1634 Base Anchor 1636 Pivot Anchor 1638 Passageway 1640 Slot, Guide 1642 First Set of Clips 1644 Second Set of Clips 1646 Poka - Yoke Element 1648 Flanged Raceway 1650 Tubular Wrap Recess 1652 Poka - Yoke Element 1656 Base 1658 Lid 1660 Membrane 1662 Gasket 1664 Group Port 1666 Vent Port 1668 Sensor Flag 1670 Barb - Fitted Vent Port 1672 Female Group Port Vent Port 1674 Annular Wall Portion or Baffle 1800 Magnetic Cell Isolation Kit 1802 Cassette, Manifold 1804 First Plug 1806 Second Plug 1808 Third Plug 1810 Fourth Plug 1811 Pipe 1812 Pipe 1813 Transfer Bag 1814 Pipe 1815 Tubing Tail 1816 Pipe 1817 Drip Chamber Strainer 1818 Tubing Tail 1819 Pipe 1820 Negative Fraction Bag 1821 Second Manifold 1822 First Plug 1823 Second Plug 1824 Third Plug 1825 Fourth Plug 1826 Final collection / transfer bag 1827 Pipe 1828 Pipe 1832 First transfer bag 1833 Pipe 1834 Pipe 1836 Waste bag 1841 Pipe 1842 Peristaltic pump tubing 1843 Drip chamber 1844 Large drip chamber

Claims

1. A disposable kit for a bioprocessing system, comprising: a tray having a spine with a first window and a second window; at least one bioprocessing container receivable within the tray; a pinch valve manifold removably receivable within the spine in the first window; a first tubing segment removably receivable within the spine in the second window; a second tubing segment removably receivable within the spine in the second window; and a disposable kit including the same.

2. The disposable kit of claim 1, further comprising a tubing organizer card having a plurality of spindles for holding the length of tubing in a coiled configuration.

3. The disposable kit of claim 2, wherein the tubing organizer card has a plurality of clips for attaching the tubing organizer card to a drip tray of the bioprocessing system.

4. The disposable kit of claim 2, wherein the tubing organizer card has a plurality of clips on a surface side of the tubing organizer card for holding at least one tube.

5. The disposable kit of claim 1, further comprising the sample card having a sample manifold removably receivable on the sample card, the sample manifold having a plurality of ports for fluid connection to a sample tube.

6. The disposable kit of claim 5, wherein the sample card includes a semi-circular raceway for routing a sample tube to the sample manifold.

7. The disposable kit of claim 2, further comprising a tubing corridor for containing tubing extending from the tubing organizer card to the pinch valve manifold, the tubing corridor extending within the tray and defined between an inner wall portion and an outer wall portion of the tray.

8. The disposable kit according to claim 1, further comprising a pair of handles configured to facilitate positioning of the tray during process withdrawal of the bioprocessing system.

9. The disposable kit according to claim 1, wherein the first tubing segment and the second tubing segment are configured for engagement with a peristaltic pump of the bioprocessing system.

10. The disposable kit according to claim 1, wherein the tray is configured to be received within a temperature-controlled process withdrawal of the bioprocessing system.

11. The disposable kit according to claim 1, wherein the pinch valve manifold is a fluid container having four fluid sub-networks.

12. The disposable kit according to claim 1, wherein the pinch valve manifold is removably received by the spine portion via a snap-fit connection.

13. The disposable kit according to claim 1, wherein the pinch valve manifold includes elements configured to enable connection of the pinch valve manifold to the spine portion in only a single orientation.

14. At least one of the bioprocessing containers includes a base and a lid, a vent port in the lid, and a baffle adjacent to the vent port, The disposable kit according to claim 1, wherein the baffle is configured to prevent contact of fluid in the bioprocessing container with the vent port.

15. The disposable kit according to claim 14, wherein the baffle is annular in shape.

16. The disposable kit according to claim 14, wherein the vent port includes one of a barb connector and a female group port.

17. A culture container for a bioprocessing system, comprising: a base; a lid connected to the base, the lid having a vent opening; and a gas-permeable and liquid-impermeable membrane sandwiched between the base and the lid, wherein the lid includes a baffle adjacent to the vent port, and the baffle is configured to prevent contact of fluid in the bioprocessing container with the vent port.

18. The culture container according to claim 17, wherein the baffle is annular in shape. ​

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