Systems, methods, and apparatus for mixing fluids in a bioprocessing system

Abstract

The rocking mechanism for a bioreactor vessel includes a base, a motor mounted to the base having an eccentric roller driven by the motor, and a rocking plate in contact with the eccentric roller and configured to receive a bioreactor vessel thereon, the motor being controllable to drive the eccentric roller and transmit a force to an underside of the rocking plate to tilt the rocking plate and the bioreactor vessel.

Description

Technical Field

[0001] Cross-reference to Related Applications This application claims the benefit of U.S. Provisional Application No. 63 / 125,858, filed December 15, 2020, which is hereby incorporated herein by reference in its entirety.

[0002] Embodiments of the present invention generally relate to bioprocessing systems and methods, and more specifically, to bioprocessing systems and methods for the production of cell immunotherapy drugs.

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 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 and T cell activation functions. 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 the patient's body and separate white blood cells. After a sufficient amount of white blood cells have been harvested, the leukapheresis product is concentrated with respect to T cells, which involves depleting unwanted cell types. Next, a subset of T cells having a specific biomarker can be isolated from the concentrated subpopulation, if desired, using a specific antibody conjugate or marker.

[0005] After the isolation of targeted T cells, the cells are activated in a specific environment in which they can actively proliferate. For example, cells can be activated using magnetic beads coated with anti-CD3 / anti-CD28 monoclonal antibodies or cell-based artificial antigen-presenting cells (aAPCs) (which can be removed from the culture using magnetic separation). The T cells are then transduced with the CAR gene by either an integration gamma retrovirus (RV) vector or a lentiviral (LV) vector. Using a viral mechanism, the viral vector attaches to patient cells and, upon entering the cells, introduces 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 genome of the patient cells, allowing CAR expression to be maintained as the cells divide and proliferate in large numbers in a bioreactor. The CAR is then transcribed and translated by the patient cells, and the CAR is expressed on the cell surface.

[0006] After T cells are activated and transduced by a CAR-encoded viral vector, the cells are amplified in a bioreactor until a large number are reached, achieving the desired cell density. Following amplification, the cells are harvested, washed, concentrated, and formulated for injection into the patient.

[0007] Existing systems and methods for producing injectable doses of CAR T cells typically require numerous complex operations involving many human touchpoints, which adds time to the overall manufacturing process and increases the risk of contamination. While recent efforts to automate the manufacturing process have eliminated some human touchpoints, 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 need to adapt their processes to the specific instruments of the system. International Publication No. 2019 / 106207 (which is incorporated herein by this reference) discloses a system and method for bioprocessing that successfully addresses many of the shortcomings of the prior art.

[0008] However, in light of the above, there is a need for a bioprocessing system and method that improves upon the teachings contained in International Publication No. 2019 / 106207 (Patent Document 1) in terms of overall functionality, flexibility, adaptability, and ease of use. [Prior art documents] [Patent Documents]

[0009] [Patent Document 1] International Publication No. 2019 / 106207 [Patent Document 2] U.S. Patent Application Publication No. 2020 / 0238282 [Overview of the project] [Means for solving the problem]

[0010] Specific embodiments corresponding to the subject matter originally claimed are summarized below. These embodiments are not intended to limit the scope of the claimed subject matter, but rather are intended solely to provide an overview of possible embodiments. In fact, this disclosure may encompass a variety of forms that may be similar to or different from the embodiments described below.

[0011] In one embodiment, a kit for magnetic cell isolation is provided. The kit includes a first stopcock manifold having at least four stopcocks, and a separation chamber configured for use with a centrifugal processing chamber of a cell processing device, wherein the separation chamber is in fluid communication with the first stopcock manifold, and a mixing bag configured for use with a heating / cooling mixing chamber of a cell processing device, wherein the mixing bag is in fluid communication with the first stopcock manifold, and a second stopcock manifold having at least four stopcocks, wherein the second stopcock manifold is in fluid communication with the first stopcock manifold, and a magnetic cell isolation holder is in fluid communication with the second stopcock manifold, wherein the magnetic cell isolation holder is configured for use with a 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 stopcock manifolds.

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

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

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

[0015] In another embodiment, an apparatus for magnetic cell isolation is provided. The apparatus includes a stopcock manifold interface, which is positioned on a base and configured to receive a stopcock manifold of a cell processing kit; a magnetic field generator positioned within the base; and a slot formed within the base, which is 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 including a centrifugation chamber, a pump assembly, a stopcock manifold interface configured to receive a stopcock manifold of a removable cell processing kit, and a heating / cooling mixing chamber. The IM includes a base, an IM stopcock manifold interface on the base, the IM stopcock manifold interface configured to receive a stopcock manifold of a removable cell processing kit, a magnetic field generator positioned within the base, and a slot formed within the base, the slot configured to removably receive a magnetic cell isolation holder and to selectively and operably contact the holder with the magnetic field generator.

[0017] In another embodiment, a method for magnetically isolating cells is provided. The method includes the steps of: inserting a magnetic cell isolation holder into a slot of an isolation apparatus; moving the magnetic field generator of the isolation apparatus 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 in such a way that it retains bead-bound cells within it, but in the engaged position, the magnetic field generated by the magnetic field generator is sufficient to retain bead-bound cells within it; and flowing a population of bead-bound cells into the magnetic cell isolation holder to trap the bead-bound cells within it.

[0018] In yet another embodiment, a method for bioprocessing is provided. The method includes the steps of 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 recombinant cells; and amplifying the population of genetically recombinant cells in the first and second bioreactor vessels.

[0019] In another embodiment, a method for bioprocessing is provided. The method includes the steps of 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 the steps of 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 recombinant cells; transferring the population of genetically recombinant cells to at least one first bioreactor vessel and a second bioreactor; and amplifying the population of genetically recombinant 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, the process drawer being receivable into the housing and movable between a closed position and an open position, and the process drawer being configured to receive at least one culture vessel therein, and a cabinet positioned in a vertical relationship stacked with respect to the housing, the cabinet including at least one vertical storage drawer slidably received inside the cabinet.

[0022] In another embodiment, a disposable kit for a bioprocessing apparatus is provided. The disposable kit includes a tray, at least one bioprocessing vessel received in the tray, a valve manifold mounted on the rear 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 in a temperature-controlled process drawer of the bioprocessing apparatus.

[0023] In another embodiment, a bioprocessing method is provided. The method includes the steps of: positioning a disposable bioprocessing kit in a process drawer of a bioprocessing apparatus, such that the culture vessel of the disposable kit is received on a rocking assembly of the bioprocessing apparatus; connecting a tubing organizer to the door of a cabinet of the bioprocessing apparatus, the tubing organizer holding a plurality of tubing tails for fluid connections to a plurality of culture medium bags and / or reagent bags mounted inside the cabinet; and fluidly connecting at least one of the plurality of tubing tails to at least one of the plurality of culture medium bags and / or reagent bags.

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

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

[0026] In another embodiment, a bioprocessing system is provided. The bioprocessing system includes a base, a fulcrum mounted to the base, a rocking plate received on the fulcrum and configured to pivot thereon, an eccentric roller in contact with the underside of the rocking plate, a motor configured to drive the eccentric roller and cause the eccentric roller to exert a force on the 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 bioprocessing method is provided. The method includes providing a bioreactor vessel having a gas-permeable and liquid-impermeable membrane, initiating a gas flow, and passing the gas flow across the bottom surface of the membrane to induce a turbulent interaction between the gas flow 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, 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 top of the pair of support legs, at least one bioreactor vessel positioned in 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 and return an atmosphere from below the bioreactor vessel upward through a first opening of the pair of openings, across the bottom surface of the gas-permeable, liquid-impermeable membrane of the bioreactor vessel, and through a second opening of the pair of openings to the bottom of the bioreactor vessel.

[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 sections, and a gas-permeable, liquid-impermeable membrane, the gas-permeable, liquid-impermeable membrane being sandwiched between the base and the lid and held in place by the plurality of heat-staking sections.

[0031] In another embodiment, a disposable kit for a bioprocessing system is provided. The disposable kit includes a tray, the tray having a pair of opposing legs and a platform extending between the legs, the platform configured to support at least one bioreactor vessel; a first bioreactor vessel, the first bioreactor vessel having a base having a plurality of through-openings, the first bioreactor vessel having 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, the 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 having an oval shape.

[0032] In another embodiment, a method is provided for assessing the integrity of a bioprocessing system. The method includes the steps of: 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 with the mass of the second container; and generating a notification indicating that leakage is present 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 is provided for assessing the integrity of a bioprocessing system. The method includes the steps of 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 leakage is present if the change in the mass of the second container exceeds a threshold.

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

[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 vessel through a first flow line; a process pump configured to circulate the fluid from the bioprocessing vessel through a circulation line and a filtration line; a waste pump configured to pump waste removed by a filter along the filtration line to a waste storage unit through a waste line; a first valve configured to isolate the bioprocessing vessel from the first flow line, the filtration line 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 / or circulation line, and monitor pressure drop in at least one of the first flow line and / or circulation line.

[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 between the front plate and the back plate, a first port fluid-communicating with the fluid channel and allowing fluid to flow into the fluid channel, and a second port fluid-communicating with the fluid channel and allowing fluid to flow out of the fluid channel. The at least one fluid channel includes a plurality of segments that enable 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 the parameters of a fluid is provided. The method includes the steps of: flowing a 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 in the tray, a flow-through sensing chamber having a front plate and a back plate, a fluid channel between the front plate and the back plate, a first port communicating with the fluid channel and allowing fluid to flow into the fluid channel, and a second port communicating with the fluid channel and allowing fluid to flow out of the fluid channel. The flow-through sensing chamber is mounted on the tray.

[0039] The present invention will be better understood by referring to the accompanying drawings and reading the following description of non-limiting embodiments. [Brief explanation of the drawing]

[0040] [Figure 1] This is a schematic diagram of a bioprocessing system according to an embodiment of the present invention. [Figure 2] This is a schematic diagram of a bioprocessing system according to another embodiment of the present invention. [Figure 3] This is a schematic diagram of a cell processing and isolation system according to an embodiment of the present invention. [Figure 4] Figure 3 is a perspective view of the isolation module of the cell processing and isolation system. [Figure 5] This is a top view of the isolation module. [Figure 6] This is a perspective view of a stopcock manifold interface of an isolated module according to an embodiment of the present invention. [Figure 7] This is an enlarged perspective view of the stopcock manifold interface. [Figure 8] Another perspective view of the isolated module. [Figure 9] This is a rear perspective view of the isolation module. [Figure 10] This is a front exploded perspective view of the isolated module. [Figure 11] This is a rear exploded perspective view of the isolated module. [Figure 12] This is an enlarged perspective view of the bubble sensor assembly in the isolated module. [Figure 13] This is a side cross-sectional view of a bubble sensor assembly. [Figure 14] This is a front perspective view of a magnetic field generator assembly for an isolation module according to an embodiment of the present invention. [Figure 15] Another front perspective view of the magnetic field generator assembly. [Figure 16] This is a rear perspective view of the magnetic field generator assembly. [Figure 17] This is a rear perspective view of a portion of the magnetic field generator assembly. [Figure 18] A simplified front perspective view of the carriage of the magnetic field generator assembly. [Figure 19] This is a simplified rear perspective view of the carriage. [Figure 20] This is a cross-sectional view of the magnetic field generator assembly in the retracted position. [Figure 21] This is a cross-sectional view of the magnetic field generator assembly with the isolation holder received in the slot of the isolation module. [Figure 22] This is a cross-sectional view of a magnetic field generator assembly in its extended position. [Figure 23] This is a cross-sectional view of a magnetic field generator assembly in an extended position within an isolation holder that is received in a slot. [Figure 24] This is a cross-sectional view of a magnetic field generator assembly in its extended position, with the isolation holder locked in the slot. [Figure 25] This is a cross-sectional view of the magnetic field generator assembly illustrating the misalignment of the isolation holder. [Figure 26] This is a perspective view of a magnetic cell isolation holder for use with the isolation module shown in Figure 4, according to an embodiment of the present invention. [Figure 27] Figure 26 is a disassembled perspective view of the magnetic cell isolation holder. [Figure 28] Figure 26 is a side view of the column of the magnetic cell isolation holder. [Figure 29] Figure 28 is an exploded view of the column. [Figure 30] This is a perspective view illustrating the insertion of a magnetic cell isolation holder into a slot within the isolation module. [Figure 31] This is a perspective view of a magnetic cell isolation holder for use with the isolation module shown in Figure 4, according to another embodiment of the present invention. [Figure 32] Figure 31 is a perspective view of the magnetic cell isolation holder. [Figure 33]Figure 31 is a top view of the magnetic cell isolation holder illustrating the magnetic field distribution of the magnetic field generator according to an embodiment of this disclosure. [Figure 34] This is a simplified perspective view of a magnetic cell isolation holder according to another embodiment of the present invention. [Figure 35] This is a simplified perspective view of a magnetic cell isolation holder according to another embodiment of the present invention. [Figure 36] This is a schematic diagram of a disposable kit for washing and concentrating cell products, to be used with the processing apparatus shown in Figure 3. [Figure 37A] This is a schematic diagram of a disposable kit for magnetic cell isolation, intended for use with the processing apparatus and isolation module shown in Figure 3, and showing its installation on top of the processing apparatus and isolation module shown in Figure 3. [Figure 37B] Figure 37A is a schematic diagram of a disposable kit for magnetic cell isolation, showing its installation on top of the processing apparatus and isolation module shown in Figure 3. [Figure 38] This flowchart illustrates the magnetic cell isolation workflow / process using the disposable kits shown in Figures 37A and 37B on the processing unit and isolation module shown in Figure 3. [Figure 39] This is a schematic diagram of a disposable kit for dosage preparation / compounding, to be used with the processing device and isolation module shown in Figure 3. [Figure 40] Figure 39 is a schematic diagram of a disposable kit for dosage preparation / compounding, showing its installation on top of the processing unit and isolation module in Figure 3. [Figure 41] This flowchart illustrates the dosage preparation workflow / process using the disposable kit shown in Figure 39 on the processing unit and isolation module shown in Figure 3. [Figure 42] This is a perspective view of a bioprocessing system / apparatus according to an embodiment of the present invention, showing a process drawer and cabinet in the closed position. [Figure 43] Another perspective view of the bioprocessing apparatus in Figure 42, showing the cabinet in the open position. [Figure 44]Figure 42 is a perspective view of the cabinet of the bioprocessing apparatus, illustrating the extended position of the vertical drawer. [Figure 45] This is a front view of the cabinet. [Figure 46] Figure 42 is a perspective view of the bioprocessing apparatus housing and process drawer, illustrating the open position of the process drawer. [Figure 47] Figure 42 is a top view of the process drawer of the bioprocessing apparatus. [Figure 48] This is a perspective view of a pair of platform rocker assemblies for process extraction according to an embodiment of the present invention. [Figure 49] Figure 42 is a perspective view of the waste drawer of the bioprocessing apparatus. [Figure 50] Figure 42 is a perspective view of a disposable bioprocessing kit for use with the bioprocessing apparatus. [Figure 51] Figure 50 is a rear perspective view of the tray of a disposable bioprocessing kit. [Figure 52] Figure 50 is a perspective view of the anchor comb of a disposable bioprocessing kit. [Figure 53] Figure 52 is a front view of the anchor comb. [Figure 54] Figure 50 is a perspective view of the tubing organizer for a disposable bioprocessing kit. [Figure 55] Figure 50 is a perspective view of the sampling card for a disposable bioprocessing kit. [Figure 56] Figure 53 is a front view of the sampling card. [Figure 57] This is a perspective view illustrating the insertion of disposable kit trays and culture vessels into the process drawer of a bioprocessing apparatus. [Figure 58] This is a side cross-sectional view illustrating the trays and culture vessels of disposable kits received in the processing drawer of a bioprocessing apparatus. [Figure 59] This is a top view of the process drawer of a bioprocessing apparatus, showing various alignment features and sensors of the bioprocessing apparatus. [Figure 60] This is an enlarged front perspective view of the peristaltic pump assembly of a bioprocessing device, showing its alignment and engagement features. [Figure 61] This is an enlarged rear perspective view of the peristaltic pump assembly of a bioprocessing device, showing its alignment and engagement features. [Figure 62] This is an enlarged perspective view of the linear actuator array of the bioprocessing device, showing its alignment and engagement features. [Figure 63] This is a perspective cross-sectional view of the process drawer of a bioprocessing apparatus. [Figure 64] Figure 50 is a perspective view of the disassembled culture vessel of a disposable bioprocessing kit. [Figure 65] Figure 64 is a plan view of the bottom of the culture vessel. [Figure 66] Figure 42 is a perspective view of a portion of the oscillating assembly of the bioprocessing apparatus. [Figure 67] This is another perspective view of the oscillating assembly shown in Figure 66. [Figure 68] Another perspective view of the rocking assembly in Figure 66, illustrating the engagement of the rocking assembly with the culture vessel. [Figure 69] Figure 66 is a schematic diagram illustrating the operation of the oscillating assembly. [Figure 70] Figure 42 is a cross-sectional view of the process drawer of the bioprocessing apparatus. [Figure 71] Another cross-sectional view of the process drawer of the bioprocessing apparatus in Figure 42, showing the recirculation air channel. [Figure 72] Another cross-sectional view of the process drawer of the bioprocessing apparatus in Figure 42, showing the turbulent recirculating air flow at the interface with the culture vessel. [Figure 73]Figure 50 is a perspective cross-sectional view of the tray of the disposable bioprocessing kit, illustrating the recirculation airflow path. [Figure 74] This is a perspective cross-sectional view of the process drawer and tray of a bioprocessing apparatus, illustrating the recirculation air passage. [Figure 75] Another perspective cross-sectional view of the process drawer and tray of the bioprocessing apparatus, illustrating the recirculation air passage. [Figure 76] Another perspective cross-sectional view of the process drawer and tray of the bioprocessing apparatus, illustrating the recirculation air passage. [Figure 77] Figure 42 is a rear perspective view of the flow-through sensing chamber of a bioprocessing apparatus according to an embodiment of the present invention. [Figure 78] Figure 77 is a front perspective view of the flow-through sensing chamber. [Figure 79] Figure 77 is a cross-sectional perspective view of the flow-through sensing chamber. [Figure 80] Figure 77 is a perspective view of the backplate of the flow-through sensing chamber. [Figure 81] Figure 50 is an enlarged perspective view of the backbone of the disposable bioprocessing kit, illustrating the location of the flow-through sensing chamber. [Figure 82] Figure 50 is another enlarged perspective view of the backbone of the disposable bioprocessing kit, illustrating the location of the flow-through sensing chamber. [Figure 83] This is a schematic diagram showing the integration of a flow-through sensing chamber with various sensing devices. [Figure 84] This is another schematic diagram showing the integration of a flow-through sensing chamber with various sensing devices. [Figure 85] This is a block diagram illustrating the fluid flow architecture of the bioprocessing apparatus shown in Figure 42, according to an embodiment of the present invention. [Figure 86]This is a detailed view of a portion of the block diagram in Figure 85, illustrating the first fluid assembly of the fluid flow architecture. [Figure 87] This is a detailed view of a portion of the block diagram in Figure 85, illustrating the second fluid assembly of the fluid flow architecture. [Figure 88] This is a detailed view of a portion of the block diagram in Figure 85, illustrating the sampling assembly of the fluid flow architecture. [Figure 89] This is a detailed view of a portion of the block diagram in Figure 85, illustrating the filtration channel of the fluid flow architecture. [Figure 90] This is a flowchart illustrating a method for bioprocessing carried out using the bioprocessing apparatus shown in Figure 42, according to an embodiment of the present invention. [Figure 91] This is a flowchart illustrating a method for bioprocessing carried out using the bioprocessing apparatus shown in Figure 42, according to an embodiment of the present invention. [Figure 92] This is a flowchart illustrating a method for bioprocessing carried out using the bioprocessing apparatus shown in Figure 42, according to an embodiment of the present invention. [Figure 93] This is a block diagram illustrating the fluid flow architecture of the bioprocessing apparatus shown in Figure 42, according to an embodiment of the present invention. [Figure 94] This is a block diagram illustrating the fluid flow architecture of the bioprocessing apparatus shown in Figure 42, according to another embodiment of the present invention. [Figure 95] This is a block diagram illustrating the fluid flow architecture of the bioprocessing apparatus shown in Figure 42, according to another embodiment of the present invention. [Figure 96] This is a block diagram illustrating the fluid flow architecture of the bioprocessing apparatus shown in Figure 42, according to another embodiment of the present invention. [Modes for carrying out the invention]

[0041] Exemplary embodiments of the present invention are referenced in detail below, and such examples are illustrated in the accompanying drawings. Wherever possible, the same reference letters used throughout the drawings refer to the same or similar parts.

[0042] As used herein, the terms “flexible” or “crumblable” refer to structures or materials that are pliable or can be bent without breaking, and can also refer to materials that are compressible or expandable. An example of a flexible structure is a bag formed from polyethylene film. The terms “rigid” and “semi-rigid” are used herein interchangeably to describe structures that are “crumblable,” i.e., structures that do not fold, crush, or otherwise deform under normal forces to substantially reduce their elongated dimensions. Depending on the context, “semi-rigid” can refer to a structure that is more flexible than a “rigid” element (e.g., a bendable tube or conduit), but can still refer to a structure that does not collapse longitudinally under normal conditions and forces.

[0043] When the term is used herein, “vessel” means, as it may be, a flexible bag, a flexible container, a semi-rigid container, a rigid container, or a flexible or semi-rigid tubing. The term “vessel” as used herein is intended to encompass bioreactor vessels 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, e.g., chromatography and tangential flow filter systems, and their associated channels). The term “bag” as used herein means, for example, a flexible or semi-rigid container or vessel used as a containment device for various fluids and / or media.

[0044] As used herein, “fluidically connected” or “fluidly communicating” means that components of a system are capable of receiving or transferring fluids between them. The term fluid includes gases, liquids, or combinations thereof. As used herein, “electrically communicating” or “electrically connected” means that certain components are configured to communicate with each other through direct or indirect signaling by direct or indirect electrical connections. As used herein, “operably connected” refers to a connection that can be direct or indirect. A connection is not necessarily a mechanical attachment.

[0045] As used herein, the term “tray” refers to any object capable of supporting multiple components, at least temporarily. Trays can be made from a variety of suitable materials. For example, trays can be made from cost-effective materials suitable for sterilization and single-use disposable products.

[0046] As used herein, the term “functionally closed system” refers to a set of components constituting a closed fluid pathway, which may have inlet and outlet ports to add or remove fluid or air from the system without compromising the integrity of the closed fluid pathway (for example, to maintain a sterile biomedical fluid pathway inside), thereby allowing each port to include, for example, a filter or membrane to maintain sterile integrity when fluid or air is added to or removed from the system. The components may include, but are not limited to, one or more conduits, valves (e.g., multi-port diverters), vessels, receptacles, and ports, depending on a given embodiment.

[0047] Embodiments of the present invention provide systems and methods for producing cellular immunotherapy drugs from biological samples (e.g., blood, tissue, etc.). In one embodiment, the method includes the steps of: genetically recombining a population of cells in a bioreactor vessel to produce a population of genetically recombinant cells; and amplifying the population of genetically recombinant cells in the bioreactor vessel without removing the population of genetically recombinant cells from the bioreactor vessel to generate a number of genetically recombinant cells sufficient to produce one or more doses for use in cellular therapy. In certain embodiments, one or more of the methods may include the steps of: activating cells in the same bioreactor vessel using magnetic or non-magnetic beads to produce a population of activated cells before genetically recombining the cells; and washing the genetically recombinant cells over the bioreactor vessel to remove unwanted material.

[0048] Referring to Figure 1, a schematic diagram of a bioprocessing system 10 according to an embodiment of the present invention is shown. The bioprocessing system 10 is configured for use in the manufacture of cellular immunotherapy drugs (e.g., autologous cellular immunotherapy drugs), for example, by collecting human blood, fluid, tissue, or cell samples, and generating cellular immunotherapy drugs from or based on the collected samples. One type of cellular immunotherapy drug that can be manufactured using the bioprocessing system 10 is a chimeric antigen receptor (CAR) T-cell therapy drug, but other cellular immunotherapy drugs can also be produced using the present invention or a system of its embodiments without departing from a broader embodiment of the present invention. As illustrated in Figure 1, the manufacture of a CAR T-cell therapy drug generally begins with the collection of a patient's blood and the separation of lymphocytes through apheresis. Collection / apheresis can be performed in a clinical setting, and the apheresis product is then sent to a laboratory or manufacturing facility for the production of CAR T cells. In particular, once the apheresis product is received for processing, the desired cell population (e.g., leukocytes) is concentrated or separated from the collected blood to produce a cell therapy drug, 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 the dosage is formulated. The formulation is then often cryopreserved and delivered to the clinical site for thawing, preparation, and finally, injection into the patient.

[0049] Referring further to Figure 1, the bioprocessing system 10 of the present invention comprises a plurality of separate modules or subsystems, 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 the steps of concentration and isolation, a second module 200 configured to perform the steps of activation, genetic recombination, and amplification, and a third module 300 configured to perform the step of harvesting the amplified cell population. In one embodiment, each of the modules 100, 200, and 300 can be communicably connected to a dedicated controller (for example, 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 processes within their respective modules. 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 intended that a master control unit may also be used to provide global control across the three modules. Each module 100, 200, and 300 is designed to work in cooperation with the other modules to form a single coherent bioprocessing system 10, as will be described in detail below.

[0050] Automating the processes within each module can increase the consistency of products from each module and reduce the costs associated with extensive manual operations. In addition, as discussed in detail below, each module 100, 200, and 300 is substantially functionally closed, which helps ensure patient safety by reducing the risk of external contamination, guarantees compliance with regulations, and avoids the costs associated with open systems. Furthermore, each module 100, 200, and 300 is scalable, supporting both development at low patient populations and commercial manufacturing at high patient populations.

[0051] Referring further to Figure 1, the particular configuration in which process steps are compartmentalized within separate modules providing closed and automated bioprocessing, respectively, enables the efficient use of capital equipment to a degree previously unseen in the art. As will be recognized, the step of amplifying the cell population to achieve the desired cell density before harvesting and compounding is typically the most time-consuming step in the manufacturing process, while the concentration and isolation steps, as well as the harvesting and compounding steps, and the activation and recombination steps, are not as time-consuming. Therefore, attempts to automate the entire cell therapy drug manufacturing process are not only logistically challenging but also likely to exacerbate bottlenecks in the process that disrupt the workflow and reduce manufacturing efficiency. In particular, in a fully automated process, the steps of cell concentration, isolation, activation, and recombination can be carried out fairly rapidly, while the amplification of genetically modified cells is carried out very slowly. Thus, the production of a cell therapy drug from a first sample (e.g., blood from a first patient) proceeds rapidly up to the amplification step, which requires a considerable amount of time to achieve the desired cell density for harvesting. In a fully automated system, the entire process / system is monopolized by amplification equipment that carries out the amplification of cells from the first sample, and processing of the second sample cannot be started until the entire system is freed up for use. In this respect, in a fully automated bioprocessing system, the entire system is essentially offline and is not available for processing the second sample until the entire cell therapy drug manufacturing process, from concentration to harvesting / compounding, is completed for the first sample.

[0052] However, embodiments of the present invention enable parallel processing of two or more samples (from the same or different patients) to provide a more efficient use of capital resources. This advantage is a direct result of the particular configuration in which the process steps are separated into three modules 100, 200, and 300, as suggested above. Referring particularly to Figure 2, in one embodiment, a single first module 100 and / or a single third module 300 are utilized in conjunction with a plurality of second modules (e.g., second modules 200a, 200b, and 200c) in the bioprocessing system 12 to provide parallel and asynchronous processing of multiple samples from the same or different patients. For example, a first 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, which can then be transferred to one of the second modules (e.g., module 200a) for activation, recombination, and amplification under the control of controller 210a. Once the first population of target cells has been transferred out of the first module 100, the first module is again available for use, for example, to process a second apheresis product from a second patient. The 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 (for example, the second module 200b) for activation, recombination, and amplification under the control of controller 210b.

[0053] Similarly, after a second population of target cells has been transferred out of the first module 100, the first module is again available for use, for example, to process 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 a third patient can be transferred to another second module (e.g., a second module 200c) for activation, recombination, and amplification under the control of controller 210c. In this regard, for example, amplification of CAR-T cells for a first patient can occur simultaneously with amplification of CAR-T cells for a second patient, a third patient, and so on.

[0054] Furthermore, this approach allows post-processing to occur asynchronously as needed. In other words, patient cells may not all grow simultaneously. Cultures may reach their final density at different times, but multiple second modules 200 are not linked together, and a third module 300 can be used as needed. The present invention allows samples to be processed in parallel, but the samples do not need to be processed in batches.

[0055] The harvesting of 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 harvest.

[0056] Therefore, by separating the activation, recombination, and amplification steps (which are the most time-consuming and share specific operating requirements and / or require similar culture conditions) into standalone, automated, and functionally closed modules, other system equipment used for concentration, isolation, harvesting, and compounding is not idle or offline while amplification of one population of cells is being carried out. As a result, the production of multiple cell therapy drugs can be performed simultaneously, maximizing equipment and floor space utilization and improving overall process and facility efficiency. It is assumed 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. Thus, the bioprocessing system of the present invention enables plug-and-play-like functionality, allowing the manufacturing facility to easily scale up or down.

[0057] In one embodiment, the first module 100 can be any system or device capable of producing a target population of enriched and isolated cells from apheresis products taken from a patient for use in biological processes (e.g., the manufacture of immunotherapy drugs and regenerative medicine drugs). The third module 300 can be any system or device capable of harvesting and / or compounding CAR-T cells or other recombinant cells produced by the second module 200 for injection into a patient for use in cellular immunotherapy or regenerative medicine. In certain embodiments, the first module 100 and the third module 300 are configured similarly or identically, and the first module 100 is initially utilized for cell enrichment and isolation (which is then transferred to the second module 200 for activation, transduction, and amplification (and, in some embodiments, harvesting)), and is then also available at the end of the process for cell harvesting and / or compounding. In this regard, in some embodiments, the same instrument can be used for the front-end cell enrichment and isolation steps, as well as for the back-end harvesting and / or compounding steps.

[0058] Referring here to Figure 3, an exemplary configuration of the first module 100 (and, in some embodiments, a third module 300) is illustrated. In some embodiments, the first module 100 (and, the third module 300) includes a processing unit 102 and an isolation module 104. In some embodiments, the processing unit 102 and the isolation module 104 can be mechanically interconnected with each other, for example, via brackets 105 mounted on the bottom of each device. The processing unit 102 can be, for example, a Sefia S-2000 cell processing device available from Cytiva. In some embodiments, the processing unit can be configured in the same way as (or substantially the same way as) the device 900 disclosed in International Publication No. 2019 / 106207. Accordingly, the processing apparatus 102 includes a base 106, which houses a centrifugal chamber 108, a high dynamic range peristaltic pump assembly 111, a stopcock manifold interface 112, and a heating-cooling-mixing chamber (thermal mixer) 114. As shown below, the stopcock manifold interface 112 is configured to accept single-use disposable kits 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 of interfaceing multiple fluid or gas lines together, for example, using Luer fittings. Inside the base 106 is a motor, which is drivably connected to multiple (four in this case) output shafts, which, under the control of a controller, are operable to move the stopcock of the disposable kit between open and closed positions. In one embodiment, the pump assembly 111 is rated to provide low flow rates of about 3 mL / min and high flow rates of about 150 mL / min.The processing unit 102 may further include a series of sensors configured to monitor various parameters of the device 102 itself and of various fluids handled by the device 102.

[0059] Furthermore, as shown in Figure 3, the processing unit 102 of the first and / or third modules 100, 300 also includes a hanger assembly 116, which extends from the base 106 and includes a plurality of hooks 118 for suspending a plurality of bags for containing or receiving fluids used in the bioprocessing operations performed by the first or third module. In one embodiment, six hooks may be present. Each hook may include an integrated weight sensor or load cell (not shown) for monitoring the weight of the respective vessel / bag. In one embodiment, the bags may 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, culture medium bags, release bags, and / or collection bags, depending on the specific process being performed. The processing unit 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.

[0060] Referring further to Figure 3, and more specifically to Figures 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 stopcock manifold interface 132, and a vertical aperture or slot 134 in the base, the stopcock manifold interface 132 being positioned on the base 130 and configured to receive a stopcock manifold of a single-use disposable cell processing kit, and the vertical aperture or slot 134 being configured to removably receive a magnetic cell isolation holder 136 of the isolation module 104, the purpose of which will be described later. The isolation module 104 further includes a support pole 138 having one or more hooks 140 or pegs for suspending a fluid bag or vessel from there. In one embodiment, the hooks 140 can be configured with or connected to a load cell for real-time mass monitoring of the contents of the bag. Figure 3 illustrates the isolation module 104 as having two hooks 140, but it is possible for there to be more or fewer hooks than two. For example, in one embodiment, the isolation module 104 has four hooks 140. In one embodiment, the inner surface of the housing 130 and / or its base structure can be coated or covered with a conductive paint or coating to shield, for example, from EMC perturbations. In one embodiment, the housing 130 can be manufactured from plastic, and the base structure supporting the housing can be made of metal, but in certain embodiments, both the base structure and the housing can be formed from plastic or a similar non-conductive material.

[0061] In one embodiment, the isolation module 104 includes a drip chamber holder 113 for inserting and holding the drip chamber of a disposable bioprocessing kit (for example, for washing, preparing, compounding, and / or isolating cells), as described below. In one embodiment, the drip chamber holder can accommodate different diameters or shapes to fit different versions of the disposable kit drip chambers (for example, the drip chamber 380 of kit 350 shown in Figure 36, and / or the drip chamber 829 of kit 800 shown in Figures 37A and 37B). In one embodiment, the drip chamber holder can include one or more spring plungers to improve the grip of the drip chamber when inserted.

[0062] As best illustrated in Figures 5–7, the stopcock manifold interface 132 includes one or more latches or clamps 142, 143, which can be selectively deployed to hold the cell processing kit in a suitable position on the interface 132, as described below. The interface 132 further includes an array of stopcock pins or keyed output shafts 144 that are drivably connected to at least one stopcock motor 146 housed in a base 130. In one embodiment, there are six output shafts configured to interface with one stopcock each of the six stopcock manifolds of a disposable cell processing kit, although it is also conceivable that more or fewer than six stopcock pins can be utilized without departing from broader aspects of the 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, under the control of the controller, move the stopcocks of the disposable cell processing kits received on interface 132 between the open and closed positions, as described below. In particular, the six stopcock interfaces shown in Figure 4 can interface with four or six stopcock manifolds of disposable cell processing kits.

[0063] Referring specifically to Figures 4–6, 12, and 13, the isolation module 104 can include multiple sensors for monitoring various operating parameters of the isolation module 104, as well as parameters or conditions of the fluid in the flow line and / or therein. For example, in one embodiment, the isolation module 104 can include a line pressure sensor assembly 148 having an interface to which a pressure sensor is positioned below, and a bubble sensor / detector assembly 150, both forming part of a stopcock manifold interface 132 for monitoring the pressure and presence of bubbles in one or more fluid flow lines connected to the module 104, respectively. As shown therein, the bubble sensor assembly 150 includes a housing 152 and a cover 156, the housing 152 having an upward-facing channel 154 or passage therein, the bubble sensor being associated with the channel 154 or passage, and the cover 156 pivotally connected to the housing 152. The channel 154 is sized and dimensioned to accommodate the length of the tubing, and the cover 156 is selectively movable between an open and closed position relative to the housing 152 to capture and hold the length of the tubing within the channel 154. In one embodiment, the housing 152 and cover 156 are formed from a material having poor electrical conductivity (e.g., anodized aluminum or plastic) so that any present current does not pass through the housing 130 of the isolation module 104 and through the bubble sensor 150 (which could adversely affect its operation). In one embodiment, the housing 130 includes an air inlet with an integrated filter, through which air can be drawn into the housing 130 to cool its internal components during operation.

[0064] Referring to Figures 10, 11, and 14–19, the isolation module 104 additionally includes a magnetic field generator assembly 160 housed within the base housing 130. In one embodiment, the magnetic field generator assembly 160 includes a pair of opposing permanent magnets 162, 164 (with space between them) mounted on a movable carriage 166. Although a pair of magnets 162, 164 are illustrated, it is intended that, with respect to the same final height, each illustrated magnet 162 and 164 could be made from a single long magnet or from a stack of several shorter magnets, without departing from broader embodiments of the invention. As described in detail below, the carriage 166 is movable between an extended position and a retracted position. In the extended position, magnets 162 and 164 are positioned on opposing sides of the slot 134 to generate a magnetic field within the slot 134. In the retracted position, magnets 162 and 164 are moved behind the slot 134 to not generate (or generate only a small or negligible) magnetic field within the slot 134. The carriage 166 is slidably connected to upper and lower shafts 168 and 170, which are received by bushings or bearings 172 within the carriage assembly 166, and is supported by the upper and lower shafts 168 and 170, and is operably connected to a lead screw 174, which is received through a central bushing 176 of the carriage 166. The lead screw 174 is rotatable to move the carriage 166 slidably between its extended position and its retracted position, as disclosed in detail below.

[0065] As best illustrated in Figures 14 and 16, the magnetic field generator assembly 160 includes a motor 178, which is drivably connected to the lead screw 174 via a gearbox 180 and a belt 182 (which links the timing pulley 183 of the gearbox 180 to the 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 there, the magnetic field generator assembly 160 further includes an array of sensors, which are used 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, the first and second sensors 186, 188 being used to detect and confirm the movement of the carriage 166, and the third sensor 190 being used to detect the presence of a magnetic cell isolation holder 136 in a slot 134 in the housing. In one embodiment, the sensors 186, 188, 190, 192 are inductive proximity sensors, but other types of sensors known in the art may also be used without departing from 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), the flange 196 being configured to engage with the rear surface of the carriage 166 adjacent to its upper edge. The locking pin 194 also includes a coil spring 198, which is configured to bias the locking pin 194 toward the front of the isolated module 104 (i.e., toward the slot 134), the purpose of which is described below.

[0066] Looking at Figures 20 to 25, the operation of the magnetic field generator assembly 160 and the positioning of its carriage 166 will be described here. Referring to Figure 20, the presence or absence of the magnetic cell isolation holder 136 in the slot 134 is detected using the second sensor 188 and the third sensor 190. At the beginning of the process, the carriage 166 is in its retracted position, in which it is sensed by sensors 188 and 186. In this position, the locking pin 194 is also in its retracted position (because 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, against the biasing force of the spring 198, holds the locking pin 194 in its retracted position and releases the slot 134 so that the magnetic cell isolation holder 136 can be inserted.

[0067] As shown in Figure 21, the magnetic cell isolation holder 136 is inserted here. When the motor 178 rotates the lead screw 168, the carriage 166 is driven forward toward 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 which propels the locking pin 196 forward. As shown there, when the flange 196 or disk of the locking pin 194 is propelled forward, it is detected by the sensor 190 (as well as the first and second sensors, which 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, which is engaged with the slot 134.

[0068] As shown in Figure 22, the carriage 166 is then driven to its furthest forward position by the motor 178 and lead screw 168 until the opposing magnets 162, 164 are aligned with the opposing sides 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 therefore 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 away from them, and therefore the presence of the carriage 166 is not detected by these sensors. Therefore, the detection of the flange 196 by the sensor 190 indicates that the isolation holder 136 is received in 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 a forward working position, where a magnetic field can be generated in the slot 134.

[0069] Looking at Figure 23, we see that the carriage 166 and magnets 162, 164 are moved forward toward the extended position, but when the isolation holder 136 is not received in the slot 134 in the housing 130, the locking pin 194 can move freely forward with the carriage 166 under the bias 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 the end of its range of motion. In this position, the distal end of the locking pin 194 obstructs the slot 134, preventing the insertion of the isolation holder 136, and the flange 196 is in front of the sensor 190, and the flange 196 is not detected by it, indicating that the isolation holder 136 is not present. As shown in Figure 23, the absence of the isolation holder 136 can be detected even when the carriage is not in its foremost position (i.e., sensor 186 detects the presence of carriage 166, while sensor 188 does not).

[0070] Referring to Figure 24 and as shown above, if the isolation holder 136 is properly inserted into the slot 134, the locking pin 194 moves forward with the carriage 166 until it is seated in the recess or seat portion within the isolation holder 136. In this position, the locking pin 194 prevents the isolation holder 136 from being removed from the slot 134. However, as shown in Figure 25, if 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. Therefore, the locking pin 194 is prevented from traveling far enough forward to allow the flange 196 to align 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, the sensor 190 does not detect the flange 196 of the locking pin 194, which indicates that the isolation holder 136 is not properly received in the slot 134. At the position shown in Figure 24, with the locking pin 194 holding the isolation holder 136 in the proper position in the slot 134, and with the magnets 162, 164 aligned with the opposing sides of the slot 134, it is possible to generate a magnetic field to capture the bead-bound cells in the isolation holder 136 in a manner known in the art and discussed in more detail thereafter.

[0071] Referring again to Figures 9, 11, 15, and 16, in one embodiment, the isolation module 104 further includes a manual crank 171 operably connected to a linear screw 174. The crank 171 is operable to manually move the carriage 166 and magnets 162, 164 to a retracted position in an emergency or in the event of a power loss. The crank 171 has a pivotable handle, which remains closed when not in use, but can be extended outward when needed. A ball detent screwed into the handle holds the handle in the closed position. In one embodiment, the crank 171 may include a pawl and ratchet mechanism such that, when the crank is closed, a pin, due to the force of a spring, separates the pawl from the ratchet. 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 spring force and pin retraction. With the pawl and ratchet now in contact, the crank 171 can be rotated and engage 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 to prevent manual forward movement of the carriage 166 (thereby preventing inadvertent or accidental activation of the magnetic circuit).

[0072] Referring back to Figure 9, the rear surface of the isolation module 104 may include a connector 151 for connecting to a power supply unit for powering the isolation module 104, a switch 153 for turning the isolation module 104 on and off, a communication connector 155 for communicatingly connecting the isolation module 104 to a controller, and a plurality of openings 157, through which an internal fan 159 can expel 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 may be a USB connector, but other wired or wireless means known in the art may also be used. In one embodiment, the isolation module 104 is communicatively connected to a processing unit 102, which is controlled by its controller 110. In this regard, all information acquired by the various sensors of the isolation module 104 (for example, 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 fluids passing through the various flow lines, etc.) is communicated to the controller of the processing unit 102, where it is analyzed and then used by the controller to control the operation of the isolation module 104, generate alarms, etc. Thus, the isolation module 104 does not need to be equipped with a separate processor and memory (which would increase cost and complexity).

[0073] In relation to the operation of the isolation module 104, the front of the isolation module 104 may include an array of indicator lights to inform the operator of the status / position of the magnetic field generator assembly, as shown in Figure 4. For example, the indicator lights 161 may include a green indicator light to indicate that the carriage 166 and magnets 162, 164 are in their retracted position, a flashing yellow indicator light to indicate that the carriage 166 is moving, and a continuous yellow indicator light to indicate that the carriage and magnets are in their extended position for the magnetic retention of bead-bound cells passing through the isolation holder 136. In another embodiment, the front of the isolation module 104 may instead or in addition include pictograms, for example, a first pictogram, a second pictogram, and a third pictogram in the form of a lock or other icon, wherein the first pictogram, when lit, indicates that the isolation holder 136 can be inserted into the slot 134 in the isolation module 104; the second pictogram, when lit, indicates that the application / process has been successfully completed and the magnetic circuit is turned off (and that the isolation holder 136 can be removed from the isolation module 104); and the third pictogram, when lit, indicates that the isolation holder 136 is properly locked in the appropriate place.

[0074] As will be discussed in detail thereafter, the isolation module 104 provides an amplified array of bioprocessing functions to be carried out in a single, easy-to-use system. These processes can include, for example, cell enrichment and magnetic isolation, washing, and dosage 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 via targeted binding of cell surface molecules to antibodies or ligands of magnetic particles (e.g., beads). Once bound, cells bound to magnetic particles can be separated from a population of unbound cells. For example, a cell mixture containing bound and unbound cells can be passed through a separation column positioned in a magnetic field generator (e.g., the magnetic field generator assembly 160 of the isolation module 104), which captures the magnetic particles and, consequently, the associated bound cells. Unbound cells pass through the column without being captured. In embodiments, the magnetic cell isolation holder 136 and / or isolation module 104 can be specifically configured for cell enrichment and isolation using various magnetic isolation bead types (e.g., Miltenyi beads, Dynabeads, and StemCell EasySep beads). An exemplary configuration of the isolation holder 136 is provided below.

[0075] 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). Referring to Figures 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 which receives and holds a vertical column 280 therein, and the handle 276 which is connected to the body portion 174 to allow for easy operation by the user (e.g., for setting the isolation holder 136 into a slot 134 in the isolation module 104 and removing it therefrom). In one embodiment, the body portion 274 and the handle portion 276 can be integral and formed from molded halves 277, 278 that sandwich the column 280. As best shown in Figure 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 Figures 28 and 29, in one exemplary embodiment, the column shell can be a stock extruded aluminum shell, anodized and further machined as necessary for dimensional tolerances. The column 280 has a pair of identical end caps 282 connected to the column 280 at its opposing ends, which include a female glue port for directly interface connection to the lengths of the PVC tubing 284, 286, an O-ring (for forming a fluid seal), and a heat-sealed-on piece of mesh (useful in the process of holding beads before encapsulating agent is added). In one embodiment, the column is filled with a magnetic retaining element and an encapsulating agent, the magnetic retaining element being, in one embodiment, an array of ferromagnetic spheres or beads.The encapsulating agent used can be a biocompatible epoxy. To apply the encapsulating agent, the column is packed with ferromagnetic spheres or beads, the encapsulating agent is added to completely wet the beads, and then any excess encapsulating agent is removed by centrifugation, and the encapsulating agent is cured.

[0076] As best illustrated in Figures 26 and 27, a first-length PVC tubing 284 enters vertically from above into the upper end of the column 280, forming an inlet flow channel for bead-bound cells into the column 280 when the isolation holder 136 is received in the slot 134 of the isolation module 104. A second-length PVC tubing 286 exits the lower end of the column 280, providing an exit channel for fluid from the column 280 while the bead-bound cells are retained in the column, as is known in the art. In one embodiment, the second-length PVC tubing 286 is routed through a handle 276, at which point it exits vertically from the isolation holder 136. Not shown, the first and second-length tubings 284, 286 include connectors for integrating the column 280 with the flow channels of a magnetocellular isolation kit or cassette, which are received above the interface 132 of the isolation module, as described below. Figure 30 illustrates the installation of the magnetic cell isolation holder 136 into slot 134 within the isolation module (i.e., by sliding the magnetic cell isolation holder 136 into slot 134 from above). Removal of the magnetic cell isolation holder 136 is performed by sliding the holder upward within slot 134.

[0077] Looking at Figures 31-35, various other exemplary configurations of the magnetic cell isolation holder 136 for use with the isolation module 104 are illustrated. As disclosed above, certain magnetic cell isolation techniques can incorporate nano-sized particles (e.g., beads with a diameter of approximately 50 nm or less), while other techniques can use larger particles (e.g., beads with a diameter of approximately 2 μm or more). For example, smaller particle sizes may be desirable because they can avoid receptor activation on target cells. Furthermore, since nano-sized particles have little effect on downstream processing or cellular function, downstream steps may omit particle removal. However, smaller nano-sized magnetic particles can be isolated using magnetic cell isolation procedures that involve the use of a magnetic field gradient enhancer to amplify the applied magnetic field gradient. In contrast, larger particles have a higher magnetic moment. Therefore, isolation of certain larger particles can be done without a magnetic field gradient enhancer. However, with larger particles, the isolation column in the magnetic field generator can reach its capacity before a sufficient number of bead-bound cells are captured. In particular, bead-bound cells accumulate inside the channel until there is no longer a region with a gradient high enough to overcome the viscous resistance that propels additional bead-bound cells to be captured through the channel. Therefore, 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 herein, certain configurations of magnetic cell isolation holders can prevent the need for multiple cycles (i.e., by passing the cell mixture through a nonlinear channel in a magnetic field, circulating the cell mixture through or in a magnetic field, and / or passing through the magnetic field multiple times). As used herein, nonlinear means not a straight line through the magnetic field.For example, the flow path can be shaped spirally or helically, or it can have one or more curves or contours within the magnetic field.

[0078] As shown in Figures 31–33, the 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 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 in the slot 134 (also referred herein as the receiving area 134). The receiving area 134 and the magnetic field have a major axis (defining the longitudinal extension of the magnetic field) and a minor axis, thereby 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 the cross-sectional area perpendicular to the major axis (see, for example, Figure 32), the gradient is substantially constant along a line running in and out of the page.

[0079] The magnetic cell isolation holder 250 is configured to be removablely connected to a magnetic field generator 160, for example, by being received in a receiving area / slot 134 of the magnetic field generator 160. As illustrated in Figures 31 and 32, in one embodiment the magnetic cell isolation holder 250 includes a body 252, which can be formed from any suitable non-magnetic material configured to accommodate cell isolations and is connectable to the magnetic field generator 160. In one embodiment the body 252 is generally rectangular in shape (it has a longitudinal extension along the long axis of the magnetic field (vertically in Figure 31), which is greater than the width or thickness of the body), and includes a plurality of channels or laces 254, which extend along the body 252 to receive and hold a tube 256. The tube 256 can be configured, with respect to part, as known in the art, to hold cells bound to magnetic particles under a magnetic field and allow unbound cells to pass through. For example, the magnetic particles can be Dynabeads or SCT beads, but other magnetic particle / bead types may also be used without departing from a broader aspect of the present invention.

[0080] Tube 256 is routed along and / or through the main body 252 via a race 254, defining a flow passage for the flow of a fluid (e.g., a cell mixture). The race 254 and tube 256 are positioned such that the flow passage defined by tube 256 is positioned in the magnetic field when the magnetic cell isolation holder 250 is connected to the magnetic field generator 160 (i.e., received in the slot 134). Furthermore, the race 254 (and thus tube 356 and the flow passage defined thereby) is configured such that, when the magnetic cell isolation holder 250 is connected to the magnetic field generator 160, the direction of fluid flow in the flow passage (i.e., through the tube) at a first location in the magnetic field is different from the direction of fluid flow in the flow passage at a second location in the magnetic field.

[0081] For example, as illustrated in Figures 31 to 33, in one embodiment, the main body 252 may include eight generally vertical channels or races 254, two of which are adjacent to each longitudinal corner of the main body 252. Tubes 256 are routed through the races 254 in such a manner that they form multiple series and fluidly interconnected loops. If the main body contains eight races 254, four series loops are formed by routing the tubes 256 through the races 254. It is intended that the main body 252 may be formed by more or fewer races than eight to accommodate more or fewer loops than four, as desired. Positioning tube 256 within the loop provides an increase in the residence time of the cell mixture in the magnetic field (the total time the cell mixture passes through the high-gradient magnetic field) without reducing the flow velocity (by reducing the flow rate or increasing the cross-sectional area of ​​the flow passage), thus enabling better capture of bead-bound cells compared to a single vertical path through the magnetic field (at the same flow velocity and the same longitudinal length of the magnetic field generator).

[0082] Figure 32 shows the tubing loops of the magnetic cell isolation holder 250 more clearly. As shown therein, the multiple loops of the tubing each include a first section 258, which extends substantially linearly along the longitudinal extension of the main body 252, the longitudinal extension of the main body 252 is aligned with the major axes and magnetic fields of the magnets 162, 164, and the magnetic fields have a substantially constant gradient along a line parallel to the major axes of the magnets and, consequently, parallel (and ultimately collinear) to the tubing path running along the longitudinal axis of the holder. The loop of the tube further includes a second section 260, a third section 262, and a fourth section 264, the second section 260 extending from the first section 258 and forming a first return bend, the third section 262 extending substantially linearly and parallel to the first section 258, and the fourth section 264 extending from the second section and forming a second return bend. As shown above, the loops are connected in series with each other, and the fourth section / bend 264 of the first loop of the multiple loops is fluidly connected to the first section 258 of the second loop, providing a fluid interconnection between the first loop and the second loop for the circulation of fluid between the loops in a magnetic field. Fluid in one of the loops, for example, first passes through the generally vertical first section 258, enters the first return bend 260, and then enters the generally vertical third section 262. The fluid then enters the fourth section / bend 264 and the subsequent downstream loop. In one embodiment, the first return bend 260 and the second return bend 264 are bends of approximately 180 degrees, so that the fluid flows in the first section 258 and the third section 262 are generally parallel but opposite in direction, respectively. Although not shown, tube 256 has an inlet end for connection to a source (e.g., a process bag) and for receiving the cell mixture from the source, and an outlet end for selective connection to a waste bag and / or collection bag. Multiple loops of tube 256 are located between the inlet and outlet ends.In some embodiments, the flow passage may have an even number of lengths (e.g., vertical portions), with the inlet and outlet located at the same end of the magnetic cell isolation holder 250. In other embodiments, the flow passage may have an odd number of vertical portions, with the inlet and outlet located at opposing ends of the magnetic cell isolation holder 250.

[0083] In one embodiment, the magnetic cell isolation holder 250 may include a handle 266 or finger grip portion that allows the user to grasp the magnetic cell isolation holder 250 and position it in or remove it from the receiving area 134. As best illustrated in Figure 31, the magnetic cell isolation holder 250 is inserted between the magnetic field plates 162 and 164 of the magnetic field generator 160. For example, the position of the lace 254, and thus the position of the longitudinal paths 258, 262 of the tube 256 in the magnetic field generator 160, can cover locations in the magnetic field with the highest magnetic field strength. In another example, the position of the lace 254, and thus the position of the vertical paths 258, 262 of the tube 256 in the magnetic field generator 160, can cover locations in the magnetic field with the highest magnetic field gradient while satisfying the magnetic field strength requirements for the magnetic particles. Figure 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 magnets 162 and 164. As illustrated therein, the body 252 and the location of the lace 254 of the magnetic cell isolation holder 250, as well as the magnets 162 and 164, are configured and dimensional so that the vertical path of the tube 256 is positioned within the high-gradient region 268 of the magnetic field generator 160 when the magnetic cell isolation holder 302 is connected to the magnetic field generator 350.

[0084] Looking at Figure 34, in one embodiment, the magnetic cell isolation holder 300 may include a ferromagnetic core 270, which extends substantially over the entire length of the magnetic field and is surrounded by a tube 256. In one embodiment, the ferromagnetic core 270 may be an integral part of the main body 252 of the isolation holder 250, or it may be an additional component. The use of the ferromagnetic core 270 allows for the creation of higher gradients over longer lengths compared to systems without a ferromagnetic core. In particular, the ferromagnetic core generates additional parallel high-gradient regions along the longitudinal axis of the magnetic plate, thereby allowing longer lengths 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 may be formed from various ferromagnetic materials (e.g., iron).

[0085] Figure 35 is a simplified diagram of another configuration for a magnetic cell isolation holder according to another embodiment of the present invention. As illustrated therein, rather than being formed into multiple longitudinal loops, the tube 256 is wound or wrapped in a substantially spiral or helical configuration. As shown, the multiple loops 272 extend substantially perpendicular to the longitudinal direction, so that the flow through each loop 272 is generally perpendicular to the longitudinal direction (e.g., horizontal rather than vertical) in the magnetic field. Similar to the tube configurations shown in Figures 31–34, the multiple loops 272 in the magnetic field provide longer travel for the fluid in the flow passages of the tube 256 compared to a single column extending linearly through the magnetic field. In some embodiments, the horizontal or spiral loops 272 of the tube 256 can surround a ferromagnetic core 270.

[0086] Figures 31–35 illustrate tubes 306 arranged in loops extending substantially vertically and horizontally (i.e., parallel or perpendicular to the longitudinal axis of the magnetic plate and receiving area), but it is intended that the tubes can be arranged in any configuration to provide an increased length / distance flow path in the magnetic field generated by the magnetic field generator compared to a single linear path through the magnetic field. This can be achieved through the use of multiple loops of any orientation / direction (so that the cell mixture passes through the magnetic field multiple times) and / or through the use of one or more nonlinear paths through the magnetic field (for example, the tubing has curved or arc-shaped portions in the magnetic field).

[0087] In one embodiment, the tubing can be arranged to form multiple loops, which loop around the outside of the receiving area 134 (i.e., outside the magnetic field), so that all flows within the magnetic field area run in the same direction (for example, from top to bottom or from bottom to top). Furthermore, it is intended that all tubes within the magnetic field can run in the same direction, and the system can include manifolds at the top and bottom to enable parallel flows.

[0088] Furthermore, the magnetic isolation holder may be configured with flow passages / tubes, which are intended to be repurposed into multiple passages through the magnetic field and then rejoin. Moreover, in one embodiment, the main body 252 of the magnetic cell isolation holder 250 may be configured as a fluid device having an integrated flow passage (i.e., without requiring a separate tube 256). In particular, it is intended that the flow passages and / or ferromagnetic core may be manufactured entirely from metal. This would allow for further utilization of regions with higher gradients in the magnetic field. In yet another embodiment, it is intended that the flow passages may be injection molded into an insert. To add more gradient regions, it is intended that they may be insert molded by a metal framework. Similarly, it is intended that the flow passages may be additionally manufactured / printed from a suitable non-ferrous material (e.g., plastic).

[0089] While it has been disclosed above that a magnetic field generator can be composed of two opposing magnetic plates forming a permanent magnet, the present invention is not limited in this respect. In particular, it is intended that the magnetic field generator can be an electromagnet that generates a magnetic field substantially similar to that produced by a permanent magnet.

[0090] As disclosed above, the processing unit 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 unit 102 and the isolation module 104 can be controlled to perform various operations associated with these processes in sequence with minimal or no human intervention, according to a set of instructions (which are executed by the controller of the processing unit 102 (e.g., controller 110 or 310) and also stored in the memory of the processing unit 102). In one embodiment, the processing unit 102 is configured and operable to perform either the protocol described in International Publication No. 2019 / 106207 and the protocol performed by the apparatus 900 disclosed therein, with the isolation module 104 providing additional functionality and possible workflows as described below. In fact, the processing apparatus 102 and isolation module, as disclosed above and more specifically described below, provide, for example, fluid control, centrifugation, temperature control, cell isolation, cell washing, cell concentration, cell preparation, and formulation.

[0091] In relation to the processing apparatus 102 and the isolation module 104, embodiments of the present invention provide a variety of single-use disposable / consumable kits designed to be used with the processing apparatus 102 and / or the isolation module 104 to assist in carrying out processes and / or workflows associated with the isolation, harvesting, and final formulation of cell products. Referring to Figure 36, a disposable washing kit 350 for use with the processing apparatus 102 is shown. The washing kit 350 is a single-use disposable kit that is used in conjunction with the apparatus 102 to wash and concentrate fresh or thawed input products after a discretionary temperature-controlled initial dilution. As shown in Figure 36, the kit 350 includes a cassette or manifold 352 having four stopcocks 354, 356, 358, and 360; an input line 362 fluidly connected to stopcock 354; a final product / collection container or bag 364 fluidly connected to stopcock 356 via line 366; a washing solution line 368 and a resuspension solution line 370 fluidly connected to stopcock 358 via line 372; and a waste container or bag 374 fluidly connected to stopcock 360 via line 376. As shown therein, the input line 362, the washing solution line 368, and the resuspension solution line 370 may be equipped with end caps 378, which preserve the sterility of the lines during transport and storage, and which may be removed or cut off immediately before use, and which may be connected to the lines via any means known in the art (e.g., sterile welding, etc.)

[0092] Furthermore, as shown in Figure 36, the input line 362 includes an in-line drip chamber 380 having an integrated filter. The kit 350 further includes a separation chamber 382 fluidly connected to a cassette 352 via a flow line 384, and a branch tubing tail 386 fluidly connected to the cassette 352 via line 384. The tubing tail 386 and a second tubing tail connected to a stopcock 358 of the cassette are equipped with a hydrophobic filter 388. In one embodiment, the hydrophobic filter is a 2-micrometer hydrophobic filter. In one embodiment, the kit 250 can be sterilized by means known in the art (e.g., ethylene oxide sterilization) and sealed in a blister pack for transport to the end user and for storage.

[0093] During use, the manifold 352 is mounted on the stopcock manifold interface 112 of the processing unit 102, and each motor output shaft of the interface 112 is configured to engage with the respective stopcocks 354, 356, 358, and 360 to control the position of the stopcocks. The separation chamber 382 is received inside the centrifugal processing chamber 108. The input line 362 is connected to a bag containing the input product to be washed, the washing solution line 372 is connected to a bag containing the washing solution, and the resuspension solution line 370 is connected to a bag containing the resuspension solution. These bags, along with the waste bag 374 and the collection bag 364, are suspended from the hook 118 of the processing unit 102. The washing process and optional concentration process are then carried out according to a set of pre-programmed instructions, which are stored in memory and utilized by the controller 110 of the processing unit 102. In one embodiment, a washing process utilizing the processing apparatus 102 and a disposable kit 350 optionally includes initial dilution of the input product, concentration of the input product (to reduce its volume), washing of the input product, and then resuspending the input product and collecting the resuspended input product into a collection bag.

[0094] During the initial dilution step, parameters (e.g., temperature, post-dilution mixing, dilution mixing time, and dilution mixing rate) can be entered or retrieved from memory, and the washing solution from the bag connected to the washing solution line 372 is used to carry out the initial dilution. During the concentration / volume reduction step, parameters such as priming of the flow line with the input product (if any), input bag rinsing during the final volume reduction cycle, input bag rinsing volume, and input bag manual mixing (during the input bag rinsing step) can be selected and / or entered, and / or enabled or disabled. Furthermore, the number of washing cycles performed during the washing phase can be entered and selected. Finally, during the resuspension phase, prompts instructing the user to switch between washing clamps and resuspension clamps after the washing phase can be enabled or disabled, and the volume of the final product at the end of the resuspension phase can be entered and / or selected. In one embodiment, a washing process carried out using the processing apparatus 102 and kit 350 can be used to wash and concentrate the input product before and / or after activation, transduction, and amplification.

[0095] In one embodiment, the processing apparatus 102, isolation module 104, and kit 350 use an algorithm to control the filling of the resuspension medium into the isolation chamber 382 in order to avoid volume overshoot, thereby enabling a precise small final product volume to be achieved during resuspension. A method for resuspending an intermediate volume to achieve a desired final volume is performed concurrently with rinsing the cell product isolation chamber and includes the steps of: firstly, extracting the contents of the isolation chamber intermediate volume into the final bag line; secondly, calculating the number of rinsing cycles and associated filling volume required to reach the final volume; thirdly, filling the isolation chamber 382 until 10 mL of the final target rinsing cycle volume is achieved; fourthly, incrementally filling 1 mL volume increments with a 2-second pause between increments until the target rinsing cycle volume is reached; fifthly, extracting the rinsing volume toward the final / collection bag; and repeating these steps 3 to 5 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 toward the final bag, air intake between filling steps is taken into account and the filling volume of the next rinsing cycle is adjusted to ensure that the total final volume subsequently reaches the target value effectively. 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 by any desired smaller incremental volume, with pauses of a selected duration occurring between smaller volume increments (i.e., the volumes and pause durations specified above can be modified as desired).

[0096] Referring to Figures 37A and 37B, a single-use, disposable magnetic cell isolation kit 800 for use with the processing unit 102 and isolation module 104 is shown (Figure 37B more clearly shows the installation of the various components on the processing unit 102 and isolation module 104, respectively). The magnetic cell isolation kit 800, and the associated protocols enabled by the use of such a kit under the control of the controller 110 of the processing unit 100, allow for initial dilution, volume reduction, washing, incubation, post-incubation washing, magnetic isolation, and final resuspension of the cell population, as disclosed below. In one embodiment, the magnetic cell isolation kit 800 includes a cassette or manifold 802 having four stopcocks 804, 806, 808, and 810. The kit 800 also includes line 811, line 812, collection bag 813, tubing tail 815, and tubing tail 816, wherein line 811 is fluidized to a first stopcock 804 and configured for fluidized connection to a fitting on the magnetic cell isolation holder 136; line 812 is fluidized to a second stopcock 806 and configured for fluidized connection to a second fitting on the magnetic cell isolation holder 136; collection bag 813 is fluidized to a fourth stopcock 810 via line 814; tubing tail 815 is fluidized to a third stopcock 808 for fluidized connection to a resuspension buffer bag (not shown) containing a suspension medium used to resuspend the positive fraction of cells after bead isolation; and tubing tail 816 is fluidized to a third stopcock 808 for fluidized connection to a bag (not shown) containing a release buffer used to release cells from the magnetic beads in the magnetic cell isolation holder 136. Furthermore, as shown in Figures 37A and 37B, the magnetic cell isolation kit 800 additionally includes a pair of tubing tails 817, 818, which are fluid-connected to second and fourth stopcocks 804, 810, respectively, and are equipped with hydrophobic filters of the type described above.The kit 800 also includes a negative fraction bag 820 that is fluidly connected to a first stopcock 804 via line 819. As shown in Figure 37A, the manifold 802 is configured for mounting the isolation module 104 onto the manifold interface 132.

[0097] Referring further to Figures 37A and 37B, the kit 800 further includes a second manifold 821, which has four stopcocks 822, 823, 824, and 825 and is configured to be received on the manifold interface 112 of the processing apparatus 102. The kit 800 further includes a final collection / transfer bag 826 fluidly connected to a second stopcock 823, a process bag / incubation bag 827 similarly fluidly connected to the second stopcock 823, and a line 828, which is fluidly connected to a first stopcock 822 and has an in-line drip chamber 829 with a 200 micrometer filter. The line 828 further includes a branch line 830 having a tubing tail and a branch line 831 having a sampling pillow. As illustrated, kit 800 further includes line 832, which is fluidly connected to a first stopcock 822 for connection to a platelet-free buffer bag used for platelet depletion. Line 832 includes a branch line 833 with a filter. Kit 800 also includes line 834, which is fluidly connected to a fourth stopcock 825 and has a branch line 835 with a filter. Line 834 is configured for fluid connection to a bag containing isolation buffer used to carry out washing cycles during bead incubation and during optional post-incubation washing cycles to remove excess beads. As illustrated, kit 800 includes a waste bag 836 fluidly connected to a fourth stopcock 825 and a spare bag 837 fluidly connected to a third stopcock 824 (which is not used during the isolation process). Certain of the lines are equipped with a sampling pillow 838 and / or filter 839, as illustrated. Furthermore, the kit 800 includes a separation chamber 840 configured to be received inside the centrifugal processing chamber 108 of the processing apparatus 102.Line 841 includes a section of peristaltic pump tubing 842 configured to interconnect manifold 802 on isolation module 104 with manifold 821 on processing unit 102 for fluid flow between them, and to engage with peristaltic pump assembly 111 of processing unit 102, and a drip chamber 843. Kit 800 additionally includes a further tubing tail having a sterile air filter 844. Line 845 is also fluid-connected to a third stopcock 824, the opposite end of which is configured for fluid connection to a bottom port in process bag 846, which also forms part of disposable kit 800. In one embodiment, kit 800 can be sterilized by means known in the art (e.g., ethylene oxide sterilization) and can be sealed in a blister pack for transport to end users and storage.

[0098] Looking at Figure 38, an exemplary protocol 850 for magnetic isolation of cells using the magnetic cell isolation kit 800, the processing unit 102, and the isolation module 104 is illustrated. As shown above, the magnetic cell isolation kit 800, when used in conjunction with the processing unit 102 and the isolation module 104, enables initial dilution, volume reduction, washing, incubation, post-incubation washing, magnetic isolation, and final resuspension of a cell population. In one embodiment, the protocol 850 illustrated in Figure 38 performs an optional initial dilution of the apheresis product, enriches the cells, depletes the platelets, isolates (e.g.) CD3+ cells using magnetic beads in the isolation holder 136, and resuspends the cells in a pre-selected solution for downstream use (e.g., activation, transduction, and amplification, as well as, finally, for compounding and dosage preparation). As shown therein, in step 852, magnetic cell isolation beads (e.g., Miltenyi beads, Dynabeads, and StemCell EasySep beads) are inserted into the process bag 846 before initiation. In step 854, a kit test may be performed. Initial dilution is performed in a further step. Then, in step 856, volume reduction is performed, after which the cells are transferred in step 858 to a process bag positioned with the thermal mixing chamber 114 of the processing apparatus 102. The cells and beads are then incubated in the thermal mixing chamber 114 in step 860, and a post-incubation wash is performed in step 862 to remove excess beads. In one embodiment, 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 using three-dimensional process bags is improved fluid transfer (for example, during the isolation and rinsing steps from bag to bag, the bottom and top surfaces of the bags remain separated, making it more difficult to capture or retain cells).During the incubation step, volume control, temperature control, and mixed carrier transfer control are enabled for incubation (to a specific target cell density).

[0099] Following incubation and washing, magnetic isolation of bead-bound cells is then carried out in step 864 by inserting a magnetic cell isolation holder 136 into a slot 134 in the isolation module 104, and optionally by applying a magnetic field to keep the bead-bound cells in the column or flow passage of the magnetic cell isolation holder. Rinsing and isolation are carried out in step 866, and thereafter, in step 868, the target cells are collected with resuspension buffer. In one embodiment, step 868 may include exchanging the isolation buffer with culture medium and carrying out an elution cycle in three steps: (1) detaching a major amount of cells from the column and collecting the volume, (2) carrying out an elution cycle with fresh medium and collecting the volume, and (3) rinsing the tubing / bag and collecting the volume. Alternatively, an optional volume reduction step may be carried out before the resuspension of the target cells. In one embodiment, an air plug may be used to assist in removing bead-bound cells from an isolation holder / column, as specifically disclosed in International Publication No. 2019 / 106207.

[0100] In one embodiment, the processing apparatus 102, isolation module 104, and magnetic cell isolation kit 800 can be used to circulate a bead-bound population of cells back and forth through a magnetic field to isolate / capture bead-bound cells (rather than creating a single pass through the magnetic field). For example, a population of bead-bound cells after incubation can be pumped (via pump 111) from a first bag to a second bag through a magnetic cell isolation holder 136 positioned in the magnetic field in a slot 134 in the isolation module 104. As the cell mixture passes through the magnetic field generated by the magnetic field generator, the bead-bound cells, extending through the magnetic cell isolation holder 136 in the manner described above, are held / captured in a portion of the fluid path positioned between opposing plates of the magnetic field generator's magnets, while the population of unbound cells, uncaptured bead-bound cells, and other contents of the cell mixture pass through the magnetic field generator into a second bag on the other side of the magnetic field generator. Next, the pump 111 of the processing unit 102 is operated in reverse, pumping the cell mixture back from the second bag through the magnetic cell isolation holder 136 to the first bag. As the cell mixture passes through the magnetic field generated by the magnetic field generator again, additional bead-bound cells are retained / captured in the portion of the fluid pathway of the magnetic cell isolation holder 136, which is 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 in the fluid pathway (or its magnetic cell isolation holder). The round-trip transfer of the cell mixture between the first and second bags, so as to be recognized, causes the cell mixture to pass through the magnetic field multiple times, improving the system's capture efficiency.

[0101] As described above, circulating the cell mixture back and forth between bags on opposing sides of the magnetic field generator has essentially the same effect as increasing the travel distance of the cell mixture in the magnetic field by using multiple loops, paths, or nonlinear channels through the magnetic field, as disclosed above in relation to Figures 31-35. In particular, by circulating the cell mixture back and forth, the total "distance" 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 not retained on the first path through the magnetic field can be captured in the subsequent path before collection. In relation to the above, it is intended that the fluid pathways in the area of ​​the magnetic field generator (e.g., flow pathways in a magnetic cell isolation holder) can take any form of the embodiments described above. For example, the flow pathways 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 intended that the flow pathway configurations shown in Figures 31-35 can be used in relation to the isolation sequence (bag-to-bag circulation) described above. In other embodiments, a straight path through a magnetic field can be used to capture bead-bound cells.

[0102] As suggested above, Kit 800 enables and allows for the collection of both positive and negative fractions resulting from isolation (into collection bag 826 and negative fraction bag 820, respectively). In particular, rather than discarding the negative fraction, it can be collected in negative fraction bag 820 for other potential uses. While the embodiments disclosed above discuss the collection of bead-bound cells using magnetic isolation, Kit 800 additionally enables negative selection, thereby allowing a desired cell population to be unlabeled by the magnetic beads, while other cells are labeled by such beads, and the undesirable cell population is trapped in the magnetic cell isolation holder, after the bead-bound cell population has been trapped in the magnetic cell isolation holder, allowing the unlabeled desired cell population to pass through the isolation holder and be collected.

[0103] Referring here to Figure 39, a single-use disposable dose preparation kit 500 for use with the processing unit 102 and isolation module 104 is shown. The dose 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 unit 100, enable the automation of volume division, dilution, mixing, cryopreparation, and administration of cell products, as described below. The dosage preparation kit 500 includes a cassette or manifold 502 having six stopcocks 504, 506, 508, 510, 512, 514; a process bag 516 fluidly connected to stopcock 504 via peristaltic pump tubing 518; several tubing lines 520, 522, 524 fluidly connected to cassette 502 for fluid connection (e.g., sterile welding) to one or more culture bags (not shown); a final formulation / collection bag 526 fluidly connected to stopcock 508 via line 528; and a bag 530 fluidly connected to stopcock 508 via line 532 (for containing the initial / intermediate products from which the final dose / formulation is produced). In one embodiment, the process bag 516 is a three-dimensional process bag. As further shown therein, kit 500 further includes a waste bag 534 fluidly connected to a stopcock 514 via line 536, and a plurality of freezer bag connection lines 538, 540, 542, 544 fluidly connected to stopcocks 510, 512, 514 (for connection to a plurality of freezer bags utilizing sterile welding or other connection means). Finally, line 518 is equipped with a pair of hydrophobic filters 546, 547 on opposing sides of the peristaltic pump tubing section, and kit 500 further includes an air inlet line 548, which is fluidly connected to a stopcock 510 and has a hydrophobic filter 549. In one embodiment, the hydrophobic filter is a 2-micrometer hydrophobic filter.In one embodiment, the kit 500 can be sterilized by means known in the art (e.g., ethylene oxide sterilization) and sealed in a blister pack for transport to the end user and for storage.

[0104] Figure 40 illustrates the integration / installation of the dosage preparation kit 500 on the processing unit 102 and isolation module 104. As illustrated therein, on the isolation module side, the stopcock manifold / cassette 502 is installed on the stopcock manifold interface 132 of the isolation module 104, and each motor output shaft 144 of the motor 146 is configured to engage with the respective six stopcocks 504, 506, 508, 510, 512, and 514 to control the position of the stopcocks. 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, and 544 are sterile welded (or connected by other means) to the corresponding freezer bag, 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 line pressure sensor 148 of the isolation module 104, and a portion of the line connecting the 3D process bag 516 to the stopcock manifold 502 is engaged with the bubble sensor assembly 150 of the isolation module 104.

[0105] On the processing unit 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 unit 102 (which has an integrated load cell or weight sensor for sensing the weight of the bags suspended therefrom). Culture bags (not shown) are aseptically welded (or connected by other means) to culture line 520, 522, 524 and suspended from another hook 118 of the hanger assembly 116 of the processing unit 102 (which similarly has an integrated load cell or weight sensor for sensing the weight of the bags suspended therefrom). The kit's 3D process bag 516 is placed inside the thermal mixing chamber 114 of the processing unit 102. Finally, a section of peristaltic tubing 518 that fluidly interconnects the process bag 516 with the stopcock manifold 502 is engaged with the peristaltic pump assembly 111 of the processing apparatus 102, and the tubing tail with an air filter 546 is connected to a pressure sensor (not shown) of the processing apparatus 102.

[0106] Figure 41 illustrates a method 550 for preparing a dose of a cell product using a processing unit 102, an isolation module 104, and a dosage preparation kit 500. As shown above, the dosage protocol is carried out in an automated manner by a controller 110 of the processing module 102, which controls both the processing module 102 and the isolation module 104 through data connections between them. Method 550 includes, in step 552, a step of testing and priming Kit 500, which in one embodiment may include the steps of: purging air from 3D process bag 516, freezer bag, and freezer bag lines 538, 540, 542, 544 to minimize air in the bag at the end of the process; priming 3D mixing bag 534 (to equalize the amount of air inside 3D process bag 534); priming culture bag lines 520, 522, 524; and calibrating pump 111 by flowing culture medium from culture bag connected to line 520 (to calibrate the pump speed by the exact weight drawn from the bag on the load cell / hook). Next, in step 554, the initial product in bag 530 is divided. In one embodiment, this involves the steps of transferring the entire input product from bag 530 to process bag 516 positioned in the thermal mixing chamber 114 of the processing apparatus 102, and mixing the input product in the thermal mixing chamber 114 for a pre-selected or pre-set duration. The pre-set or pre-selected volume of the product is then transferred from process bag 516 to compounding bag 526. The remaining volume of the product is transferred back from process bag 516 to the initial input bag 530. In one embodiment, in step 556, the 3D process bag 516 is then rinsed with culture medium from a culture bag connected to line 520, and the rinse volume is pumped into input bag 530. In one embodiment, the rinse volume and rinse mixing time can be selected by the user.As further shown therein, in step 558, formulation preparation is then carried out. In one embodiment, this includes the steps of transferring a predetermined volume of culture medium from a culture medium bag connected to line 520 to a process bag 516 in a thermal mixer 114, and transferring such culture medium to a formulation bag 526.

[0107] If selected / desired, the preparation and administration of the freezer bag may then be carried out in steps 560 and 562, respectively, to prepare an additional bag (which may be a freezer bag for cryopreservation purposes). In such a case, in step 560, a selected volume of the divided product from the input bag 530 is then transferred to the process bag 516 in the thermal mixer 114 (with any excess divided 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 performed to a predetermined / pre-selected temperature (over a period of time calculated by the controller 110, which is required to adjust it down to the target temperature). Next, medium from the medium bag connected to line 520 is transferred (after prompting) to 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 pre-selected volume to a first frozen bag connected to line 538, a second frozen bag connected to line 540, a third frozen bag connected to line 542, and / or a fourth frozen bag connected to line 544, as desired. Precise control of the volume transferred is enabled by ensuring accurate peristaltic pump flow rates and by controlling the flow timing. The peristaltic pump flow rate setpoint is calibrated during the initial priming step to account for possible deviations from the nominal baseline of the peristaltic pump tubing 518 and / or peristaltic pump 111. Thus, this protocol allows for 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 / bag of user-selected volumes of up to four components (initial product plus three media) are enabled by such a system and method of the present invention.

[0108] Looking at Figures 42 to 45, exemplary embodiments of a second module 600 (also referred to herein as bioprocessing apparatus 600) for activation, transduction, and amplification of cells (e.g., cells enriched and isolated using the first module 100) are illustrated. The second module 600 can be, for example, an apparatus / system configured to carry out the workflows and methods described above in relation to the second module 200 and can be configured to operate similarly to module 200 disclosed in International Publication No. 2019 / 106207. 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, the waste bag drawer 606 located below the process drawer 604 and similarly 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 to insert and remove various components of the second module 600, as disclosed below. As 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., an RJ45 port and an RS485 port), at least one inlet for receiving a supply of carbon dioxide, air oxygen, and / or nitrogen, one or more outlet / discharge ports, and / or multiple (e.g., three) USB ports. The drawer 604 may also include a status indicator light 605, multiple USB or other ports 607 for data transfer, and an input terminal 609.

[0109] The second module 600 also includes a cabinet 608 positioned in a vertically stacked relationship with respect to the housing 602 (for example, mounted on top of the housing 602). The cabinet 608 includes a pair of latch-engageable doors 610, 612 that are hinged around 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). The cabinet 608 and the doors 610, 612 may also include an interlock mechanism (for example, a pneumatic latch or pin) used to keep the doors 610, 612 in the closed position while bioprocessing operations are underway. In one embodiment, the cabinet 608 further includes a plurality of vertically oriented storage drawers 614, 616 that are slidably received inside the cabinet 608. Two vertical storage drawers 614, 616 are illustrated in Figures 43 and 44, but it is possible that there are more or fewer drawers than two. In one embodiment, the storage drawers 614, 616 are slidably mounted on upper and / or lower tracks within the cabinet 608, allowing the drawers 614, 616 to move easily between a retracted position and an extended position (shown in Figures 43 and 44), in which case the drawers are retracted into the cabinet 608 and the doors 610, 612 can be closed, and in the extended position the drawers 614, 616 extend out of the cabinet 608, allowing easy access to components and accessories mounted on the left and right vertical sides of the drawers 614, 616.

[0110] As best shown in Figure 45, the interior surfaces of the doors 610, 612 may include a mechanism (e.g., a specific array of pegs or pins 618) for releasably connecting tubing organizer cards and / or sampling cards to the doors 610, 612, as described below. For example, in one embodiment, the left door 610 may include an array of pegs for holding a sampling card for a disposable kit, while the right door 612 may include an array of pegs for holding a tubing organizer card for a disposable kit. In one embodiment, both tubing organizer cards and sampling cards may be mounted in the right door 612. As shown in Figures 43 and 45, one or both of the vertical storage drawers 614, 616 may include a hook 620 on one or each of its surfaces for receiving culture media, reagents, and / or other fluid / solution bags for use in various bioprocessing operations performed by the apparatus 600. Each hook 620 can be operably connected to or integrated with a load cell for monitoring the weight of the bag to which it is connected. In one embodiment, a first vertical drawer 614 is configured to receive one or more culture medium bags 622, while a 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 culture medium 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 culture medium drip tray 626 on its opposite side to capture leakage or drip from culture medium bags suspended from the hook 620, while the second vertical drawer 616 is equipped with a reagent drip tray 628 on its opposite side to capture leakage or drip from reagent bags 624 suspended from the hook 620.In one embodiment, the drip trays 626 and 628 are removable from the drawers 614 and 616, respectively.

[0111] In one embodiment, one or more of the vertical drawers 614, 616 can be housed in a refrigerated compartment forming 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 status indicator lights 634. Figures 42–45 illustrate the waste bag drawer 606 as part of the lower housing 602, but it is intended that the waste bag drawer can alternatively be housed in the cabinet 608 (for example, as a horizontally oriented drawer or as a vertically mounted drawer). As best shown in Figures 43 and 46, the process drawer 604 includes an upward-facing slot 630 configured to receive an anchor comb 632, which facilitates the routing of tubing from the cabinet 608 into the process drawer 604. In one embodiment, the entire apparatus 600 is sized and dimensional to be supported by a table or benchtop, and process drawers 604 and cabinets 608 are easily accessible to the user. Control of the apparatus 600 and its functions is performed by an onboard controller (e.g., controller 210), as disclosed below.

[0112] Now, looking at Figures 46 and 47, a detailed view of the process drawer 604 is illustrated. As best shown in Figures 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 located behind the first internal space 636, the second internal space 638 housing the functional components of the apparatus 606. 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 perform the functions of the apparatus 600. In one embodiment, the peristaltic pump assembly 641, and the other components and devices can be configured as disclosed in International Publication No. 2019 / 106207. As shown in Figure 47, the first and second platform rocker assemblies 640, 642 are mounted in a first internal space 636, and the first and second platform rocker assemblies 640, 642 are configured to support culture vessels (also referred to herein as bioreactor vessels) of disposable bioprocessing kits on them in the manner disclosed herein. Each platform rocker assembly 640, 642 has a cover 644, and a plurality of culture vessel support or mounting posts 646 extend through the cover 644 to support the culture vessels of disposable kits. In one embodiment, each platform rocker assembly 640, 642 includes four support posts 646, as clearly shown in Figure 48. As also shown there, a sensor assembly 648 associated with each platform rocker assembly 640, 642 is provided for detecting the presence of culture vessels and / or for measuring the temperature inside the culture vessels.In other embodiments, the sensor assembly 648 can be used to measure various additional parameters of the culture in the culture vessels received on the respective platform rocker assemblies 640, 642 (e.g., temperature, carbon dioxide concentration, oxygen concentration, etc.) and / or to determine whether the culture vessels are properly positioned and seated on the rocker assemblies. As discussed below, each platform rocker assembly 640, 642 includes a plurality of load cells 658, 660, 662 for sensing the weight / mass of the culture vessels supported by mounting posts 646.

[0113] Referring again to Figure 47, the process drawer 604 includes several features configured to contain leakage and to prevent or block any fluid from accumulating in the process drawer 604. For example, the process drawer 604 includes a seal element 650, which forms a fluid-sealing seal between each platform rocker assembly 640, 642 and the bottom of the process drawer 604 (which extends around the perimeter of each rocker assembly), and between the rocker assemblies 640, 642 themselves. In addition, each culture vessel support post 646 is equipped with a seal element in the form of a flexible bellows 652, which forms a seal between the support post 646 and the cover 644. The seal elements 650 and bellows 652 prevent any fluid from entering the space under the cover 644 of the platform rocker assembly 640, 642. Furthermore, the bottom of the process drawer 604 is formed with a peripheral channel 654, which collects spilled or leaked fluid. A drain hole 656 in the channel 654 provides a means for the fluid collected in the channel 654 of the process drawer 604 to escape. The drain hole 656 is in fluid communication with a waste drawer 606 below the process drawer 604, so that any fluid spilled or leaked into the process drawer 604 is directly drained into the waste drawer 606, preventing damage to the electromechanics inside the process drawer 604.

[0114] Figure 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 in a housing 602 below the process drawer 604 and is configured to receive a waste bag. In one embodiment, tubing connecting to the waste bag is routed from the process drawer along a groove behind the front panel of the process drawer, allowing it to escape from the process drawer and then freely route downward to the waste drawer. Furthermore, as shown above, the waste drawer 606 is configured to directly receive fluid that has leaked into the process drawer through a drain hole 656 in the process drawer 604.

[0115] Referring here to Figure 50, a single-use, disposable bioprocessing kit 700 for use with the bioprocessing apparatus 600 is illustrated. The bioprocessing kit 700 includes a generally rectangular tray 702, sized and dimensioned to fit into a first internal space 636 of a process drawer 604, and a pair of culture vessels 704, 706 that fit into the tray 702. The tray 702 has a pair of openings or windows beneath the culture vessels 704, 706 that support the culture vessels 704, 706 in a raised position, and the culture vessels 704, 706 are lifted out of the tray 702 when the tray 702 is positioned in the first internal space 636 of the process drawer 604 and engaged with the support posts 646 of the platform rocker assemblies 640, 642. As shown in Figures 50 and 51, the tray 702 includes a pair of legs 708, 710, which are positioned on its front and rear surfaces, supporting the tray 702 above the bottom of the process drawer 604. The supporting legs 708, 710 are hollow and form a low point on the tray 702. Thus, in the event of leakage or spillage in the tray 702 (as opposed to that in the process drawer 604), the fluid will be collected and contained within the bottom of the legs 708, 710.

[0116] Referring further to Figures 50 and 51, the tray 702 further includes first and second windows 709, 711 on the rear surface of the tray 702, and up to three segments 714, 716, 718 of the valve manifold 712 and peristaltic pump tubing are positioned within the tray 702 for engagement with a peristaltic pump assembly 641 mounted in a process drawer 604 on the rear surface of the tray 702. The valve manifold 712 can be a fluid vessel, for example, as disclosed in U.S. Patent Application Publication No. 2020 / 0238282 (Patent Document 2), which is configured to interface with a plurality of linear actuators having plungers of a linear actuator array 643, which is also mounted in a process drawer 604 on the rear surface of the tray 702. Alternatively, the valve manifold 712 can be formed from multiple fluid flow lines configured to be actuated by multiple pinch valves of a pinch valve array, as disclosed in International Publication No. 2019 / 106207. The valve manifold 712 is fluidically interconnected with culture vessels 704, 706, culture and reagent bags in cabinet 608, waste bags in waste drawer 606, and sampling lines, forming a fluid network or architecture similar to or as disclosed in Publication No. WO2019 / 106207. Figure 50 illustrates various tubing connections with the valve manifold 712.

[0117] Therefore, as further illustrated in Figure 50, the disposable kit 700 further includes a tubing organizer card 720 and a sampling card 722, the tubing organizer card 720 holding multiple tubing tails 726 which are fluidly connected to a valve manifold 712 and are also configured for connection to various culture medium bags and reagent bags housed in a cabinet 608, and the sampling card 722 holding multiple sampling tubing tails which are also fluidly connected to a valve manifold 712. Finally, the disposable kit 700 also includes an anchor comb 632 which is received in a slot 630 in a process drawer 604 and also facilitates the routing of tubing from the cabinet 608 (for example, from the tubing organizer 720 and sampling card 722) into the process drawer 604 and to the valve manifold 712. As discussed hereafter, the anchor comb 632, tubing organizer 720, and sampling card 722 provide means for organizing all tubing tails during or after the installation of the kit 700 and the connection of various media, reagents, and other bags / containers. In one embodiment, a disposable kit 700 (including all the elements described above in relation to Figure 50) can be sterilized by means known in the art (e.g., ethylene oxide sterilization, gamma sterilization, etc.) and can be sealed in a blister pack for transport to the end user and for storage.

[0118] As illustrated in Figures 52 and 53, the anchor comb 632 includes a body portion 730, the body portion 730 having a passage 732 through which it passes. Within the passage 732 are a plurality of tubing retaining elements 734, which function to maintain and keep 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, facilitating the routing of various paths of the tubing from the cabinet 608 into the process drawer 604, where they are fluidly connected to the valve manifold 712.

[0119] Referring to Figure 54, a detail view of a tubing organizer 720 according to an embodiment of the present invention is shown. The tubing organizer 720 comprises a generally rigid plate body 736 and a plurality of tubing retaining channels 738, the plurality of tubing retaining channels 738 being molded into or otherwise connected to the rigid plate body 736 and configured to receive and hold therein a plurality of corresponding tubing tails 726. In one embodiment, the channels 738 extend upward along the right-hand side from the lower right-hand corner of the plate body 736, fold back on themselves, extend generally downward at a predetermined angle toward the lower right-hand corner of the plate body 736, fold back on themselves again, and extend upward along the left-hand side from the lower left-hand corner of the plate body 736. Thus, the tubing tails 726 received in these channels 738 follow the same winding path. Therefore, this meandering configuration of channel 738 maximizes the length of the tubing tail 726 that can be held by the tubing organizer, and a considerable degree of play allows for the connection of the tubing tail 726 to various bags and / or containers contained within the cabinet 608 of the bioprocessing apparatus 600. Thus, the tubing organizer 720 keeps the tubing tail 726 in an organized and easily accessible manner, which helps minimize setup time.

[0120] Furthermore, as shown in Figure 54, the plate body 736 includes features that allow the tubing organizer 720 to be removably mounted on the inside of the door 612 of the cabinet 608 or suspended from it, as shown in Figure 43. Such features may include, for example, a mounting and / or positioning aperture 740 through which a peg 618 or hook on the door 612 is received. When the tubing organizer 720 is mounted on the inside surface of the door 612 in use, the user can easily grasp the end of the tubing tail 726 extending into the clearance or relaxation area 742 of the plate body 736 and remove it from its seating position along with its corresponding channel 738. The tubing tail 726 can then be connected to culture medium bags, reagent bags, or other vessels contained within the cabinet 608 by a sterile technique (e.g., sterile tube welding). This process can be repeated until all fluid connections are made between the bag housed in cabinet 608 and the valve manifold 712 housed in drawer 604.

[0121] Referring to Figures 55 and 56, a detailed diagram of a 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, the body portion 744 having a manifold 746 and a plurality of sampling tubing tails 748 fluidly connected to the manifold 746. The sampling card 722 also includes a feed line 750 fluidly connected to a first end of the manifold 746 and a return line 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 allow the sampling card 722 to be removablely mounted inside the door 612 of a cabinet 608 or suspended from there. Such features may include, for example, a mounting and / or positioning aperture 754 through which a peg 618 or hook on the door 612 is received. When the sampling card 722 is mounted on the inner surface of the door 612 during use, the user can withdraw a sample from one of the culture vessels 704, 706 using one of the sampling tubing tails 748 which is easily accessible on the sampling card 722. Thus, the sample can be easily withdrawn during the bioprocessing operation without the need to open the process drawer 604 and without pausing the operation.

[0122] Looking at Figures 57 to 63, the installation and seating of tray 702 in process drawer 604 of the bioprocessing apparatus 600 are illustrated. As shown there, tray 702 is received into the first internal space 636 of process drawer 604 by opening process drawer 604 and lowering tray 702 into process drawer 604 from above, so that the culture vessels 704 and 706 of disposable kit 700 are in a front-to-back relationship within process drawer 604. In this position, valve manifold 712 is positioned immediately in front of linear actuator array 643 and aligned with linear actuator array 643, and the three segments 714, 716 and 718 of peristaltic pump tubing are positioned immediately in front of peristaltic pump assembly 641 and aligned with peristaltic pump assembly 641. As shown above, when tray 702 is lowered into process drawer 604, culture vessels 704, 706 are received on support / mounting posts 646 of their respective platform rocker assemblies 640, and culture vessels 704, 706 are lifted from their seating engagement with tray 702 and instead supported by support posts 646.

[0123] As most clearly shown in Figures 59 to 62, the tray 702 and process drawer 604 of the disposable kit 700 have multiple cooperative features, which facilitate the proper positioning of the tray 702 within the process drawer 604 and allow for verification of proper positioning. For example, as shown in Figure 59, the tray 702 and process drawer 604 include multiple engagement features / surfaces 756, which 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 multiple sensors 758 associated with the engagement features 756 of the drawer, which can detect when the cooperating engagement features 756 on the tray 702 and process drawer 604 are engaged with each other, indicating proper positioning of the tray 702. In one embodiment, the engagement feature 756 associated with tray 702 is positioned on the tray's backbone, as best shown in Figure 59, while the corresponding engagement feature 756 (and sensor 758) associated with process drawer 604 is positioned adjacent to the linear actuator array 643 and peristaltic pump assembly 641, respectively. In one embodiment, the engagement feature 756 associated with process drawer 604 is a pin of sensor 758. In addition to detecting the proper alignment and positioning of tray 702 within process drawer 604, as shown above, platform rocker assemblies 640, 642 include a sensor 648, which is configured to detect the proper positioning of culture vessels 704, 706.

[0124] Furthermore, 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 pivot pump shoe 762, which facilitate proper engagement of the peristaltic pump assembly 641 with the backbone of the tray 602. These features also minimize tolerance stack-up problems with respect to engagement and operation of the peristaltic pump assembly 641 with segments 714, 716, 718 of the peristaltic pump tubing and the valve manifold 712 of the linear actuator array 643, respectively.

[0125] In one embodiment, the solenoid actuators of the peristaltic pump assembly 641 and the valve manifold 712 are configured to move toward and physically engage with corresponding features of the disposable kit when the disposable kit is positioned in a process drawer and the drawer is closed. Referring particularly to Figure 59, module 600 includes an electric engagement mechanism which physically moves the assembly containing the peristaltic pump assembly 641 and the solenoid array 643 toward corresponding features in the disposable kit 700 (segments 714, 716, 718 of the peristaltic pump tubing and the valve manifold 712) by a fixed travel distance limited by features that prevent further movement. Disengaging simply involves operating this electric engagement mechanism in the reverse direction.

[0126] Referring here to Figures 64 and 65, the configurations of the bioreactor / culture vessels 704 and 706 of the disposable bioprocessing kit 700 are shown. For ease of illustration, only the culture vessel 704 is illustrated (culture vessel 706 is an exact replica). As shown therein, in one embodiment, the 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, but other materials known in the art may also be utilized without departing from broader aspects of the present invention. As shown in Figure 64, the lid 766 includes a plurality of reinforcing supports 772 or gussets, which reinforce the lid 766 and provide increased strength and durability. Furthermore, the lid 766 includes inlet and outlet ports 774, 776 to which tubing can be connected. As illustrated therein, the inlet and outlet ports 774, 776 are molded into the lid so that the tubing extends vertically from the lid 766 (at least initially). This configuration of ports 774, 776 facilitates setup as the tubing can be more easily connected to the culture vessel 704 from above. As further illustrated, a vent port 777 is provided on 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.

[0127] Referring further to Figure 64, the membrane 768 can be formed from a suitable gas-permeable material (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 may also be used without departing from broader embodiments of the present invention. The membrane 768 includes a plurality of location / retaining holes 780 along its periphery, the purpose of which will be described below. The gasket 770 can be formed, in part, from a variety of materials known in the art (e.g., silicone) and includes a corresponding plurality of location / retaining holes 782, which are positioned along the periphery of the gasket 770 and aligned with the holes 780 in the membrane 768.

[0128] 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 / retaining holes 780, 782 of the membrane 768 and gasket 770, respectively, and functions to anchor the membrane 768 and gasket 770 between the base 764 and the lid 766. In one embodiment, the lid 766 may be configured with a heat staking pin 784, which extends downward from its underside, and during assembly, the heat staking pin 784 extends through the corresponding location / retaining holes 780, 782 of the membrane 768 and gasket 770, respectively, and is received in the corresponding hole 786 around the base 764, and heat staking to the base 764. In one embodiment, the lid 766 is joined to the base 764 using about 20 to about 40 heat staking sections (more preferably about 34 heat staking sections). While the embodiments described herein utilize heat staking to connect the lid to the base, it is intended that other means of connection (e.g., fasteners and snap-fit ​​connections) may also be used without departing from a broader aspect of the invention.

[0129] In one embodiment, the upper surface of the base 764 has a textured surface, which allows airflow and eliminates the need for mesh (which was conventional in previous designs). As shown in Figure 65, the flange region 788 of the base 764 includes a plurality of ribs 790, which provide increased rigidity and strength as well as a more robust interconnection with the lid 766 (which additionally provides more reliable and robust anchoring of the membrane 768 and gasket 770). The lower corners of the base 764 include pinwells 791, 792, 793, and 794, respectively, which are configured to receive mounting / support posts 646 for platform rocker assemblies 640 or 642 supporting the culture vessel 704. In one embodiment, one of the pinwells (e.g., well 794) is oval in shape, which provides improved positional tolerance when the culture vessel 704 is mounted on the platform rocker assembly 640. The base 764 further provides an IR sensor window 796 and a sensor well 798, the IR sensor window 796 for measuring the temperature of the gas or fluid in the culture vessel 704 using a sensor positioned beneath the culture vessel 704 in the process drawer 604, and the sensor well 798 is utilized by a sensor 648 of the platform rocker assembly 640 or 642 to determine whether the culture vessel 704 is present in and / or properly positioned therein. Finally, as illustrated in Figure 65, the base 764 includes an array of small openings 799, which 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 within the base 764.

[0130] As shown above, the culture vessels 704, 706 are configured to be received on the platform rocker assemblies 640, 642 when the tray 702 is received in the process drawer 604. Various rocking mechanisms known in the art (including the mechanism disclosed in International Publication No. 2019 / 106207) can be utilized to provide fluid mixing in the culture vessels 704, 706 and to support the bioprocessing operations therein. Figures 66–68 illustrate the configuration of the platform rocker assemblies 640, 642 according to another embodiment of the present invention (rocker assembly 640 is drawn for simplicity and ease of understanding). As shown therein, the platform rocker assembly 640 includes a base 870, a pivot point 872 defining a central pivot axis 873 received on the base 870, a motor 874 mounted on the base 870 having an eccentric roller 876 driven by the motor 874, a rocking plate 878 received on the pivot point 872 in contact with the eccentric roller 876, the rocking plate 878 pivoting about the pivot axis 873, and a compression spring 880 configured to maintain the rocking plate 878 in contact with the eccentric roller 876. In one embodiment, the pivot point 872 and the motor 874 are connected to the base 872 via a frame 875. In one embodiment, the eccentric roller 876 is a circular roller configured to rotate along an eccentric path. In yet another embodiment, a cam-shaped roller may be used instead of a circular roller moving along an eccentric path.

[0131] As illustrated in Figures 67 and 68, the rocking plate 878 includes four support posts 646, which are received by pinwells 791, 792, 793, and 794 in the base 764 of the culture vessel 704. The motor 874 drives an eccentric roller 876 and, depending on the position of the eccentric roller 876, transmits or removes force from the underside of the rocking plate 878, thereby tilting the rocking plate 878 and the culture vessel 704 received thereon upward and / or downward, controllable (for example, under the control of the controller 210 of the second module 200 (i.e., apparatus 600)). The motor 874 may be controllable by a master controller, but the platform rocker assemblies 640, 642 may alternatively have dedicated controllers positioned on the base plate 872 beneath the rocking plate 878. As a result of the force (or lack thereof) from the eccentric roller 876, the rocking plate 878 and culture vessel 704 supported thereon pivot around the pivot axis 873 of the pivot point 872.

[0132] In one embodiment, each of the support posts 646 may be configured to include a load cell for measuring the mass of the culture vessel 704. Alternatively or in addition, the base 870 of the rocker assembly 640 may include a plurality (e.g., three) of load cells 882, which extend through the rocking plate 878 and engage with the underside of the culture vessel 704 to measure its mass. Furthermore, as shown in Figure 67, the rocking plate 878 may be equipped with a tilt sensor 884, which is configured to measure the degree of tilt of the rocking plate 878 (and thus the culture vessel 704) for use by a controller when performing the rocking / mixing process.

[0133] As shown above, when the motor 874 is operating, the eccentric circular roller 876 transmits force to the bottom surface of the oscillating plate 878, causing it to move upward or downward depending on the direction of rotation of the motor 874. When in constant motion, the circular profile of the eccentric circular roller 876 imparts a continuous sinusoidal oscillating profile to the contents of the culture vessel 704. This oscillating motion is illustrated in Figure 69. Monitoring of the oscillating plate 878 using the tilt sensor 884 enables closed-loop control of the tilt angle, homing, and drain operation, as well as enabling detection of failure event conditions. The use of support posts 646 to support the culture vessel 704 on the oscillating plate 878 allows the entire bottom of the culture vessel 704 to remain unobstructed, enabling better aeration, heat transfer, and other functionalities, as discussed later. The use of the eccentric circular roller 876 allows the tilting mechanism to be compact / low profile and provides a low-friction and highly reliable interface with the rocking plate 878. Mammalian cells, among other things, are highly sensitive to shear forces induced by small vortices on a highly turbulent fluid regime, as will be recognized. Therefore, strong vibrations, shocks, or other mechanical stimuli that lead to excessive turbulence, bubble formation, or spillage are potentially harmful. Thus, the continuous sinusoidal rocking profile of the platform rocker assemblies 640, 642 minimizes the presence of such small vortices by eliminating any high-frequency mechanical stimuli, providing safer and gentler mixing conditions, which is particularly beneficial for mammalian cell culture.

[0134] As described above, aeration and heat transfer through the base 764 and membrane 768 of the culture vessels 704, 706 are crucial 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 physiological and chemical conditions are provided depends on the application, the specificity of the cell type, and how they are adapted to grow in a suspension or adhesive. 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 occurs by passive diffusion based on a habitual gradient across immediate regions on both sides of the membrane. Embodiments of the present invention optimize such phenomena by inducing turbulent interactions between the gas-permeable membrane 768 above the bottom of the culture vessels 704, 706 and the recirculating flow of the incubation atmosphere.

[0135] Figures 70-72 show a cross-sectional view of a portion of the process drawer 604 of the bioprocessing apparatus 600 with the tray 702 and culture vessels 704, 706 of the disposable bioprocessing kit 700 positioned therein. The process drawer 604 forms an incubation chamber 902, and the tray 702 and culture vessels 704, 706 are positioned within the incubation chamber 902 as disclosed above. As shown therein, the culture vessels 704, 706 are supported by support posts 646 of platform rocker assemblies 640, 642. A heating element / device 904 is located within the process drawer 604 (for example, positioned above and below each culture vessel). For example, a heater 904 can be positioned below each culture vessel 704, 706 and adjacent to the top of the process drawer 604 to heat the incubation chamber 902 and the culture vessels 704, 706. Furthermore, the process drawer 604 includes a pair of fans or blowers 906, 908 within the cover 644 of the rocker assemblies 640, 642, adjacent to its front and rear walls. As further shown therein, the cover 644 may include a pair of opposing louvers or air passages 910, 912, with the blowers 906, 908 positioned nearby to allow 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 to re-enter the recirculation chamber 915 from the front of the process drawer 604. Additionally, a temperature sensor 914 and a carbon dioxide sensor 916 are positioned at least one location along the recirculation airflow path to measure the temperature of the recirculation airflow and the carbon dioxide concentration of the recirculation airflow, as discussed below.As further shown therein, the carbon dioxide supply unit 918 is selectively fluid-communicated with the process drawer 604 (for example, via a carbon dioxide inlet port located on the rear surface of the housing 602 of the bioprocessing unit 600) and the valve 920. The process drawer 604 may also include a gas port 922, which allows fluid communication between the inside of the process drawer 604 and the atmosphere (eliminating the need for a separate dedicated oxygen supply unit). The components described above form a system 900 for the direct mass transfer of the bioprocess system 600 from liquid to atmosphere, the operation of which will be described below.

[0136] Referring further to Figure 70, the temperature sensor 914 and the carbon dioxide sensor 916 are electrically connected to, or otherwise communicating with, a controller (for example, the master controller 210 of the apparatus 600, but a dedicated controller for running the recirculating air flow process is also conceivable) for receiving information on the temperature and carbon dioxide concentration of the recirculating air flow. The controller 210 is also electrically connected to, or otherwise communicating with, the valves 920, fans 906 and 908, and heater 904 in order to control their operation in response to sensor readings and specific setpoints.

[0137] Referring here to Figure 71, the controller 210 is operable to control fans 906, 908 to create the recirculating airflow 924. As discussed below, the tray 702 and process drawer 604 each include various ducting features 926, which ensure that the recirculating airflow 924 exits the recirculation chamber 915 through louvers 912 adjacent to the rear surface of the process drawer 604, travels upward to the level of the culture vessels 704, 706, travels generally horizontally across the bottom of the culture vessels 704, 706, travels downward near the front surface of the process drawer 604, and re-enters the recirculation chamber 915 through louvers 910. In this regard, fan 908 pushes the recirculating airflow 924 outward from the recirculation chamber 915, while fan 906 draws the recirculating airflow 924 into the recirculation chamber 915.

[0138] Referring to Figure 72, the fan 908 pushes the incubation atmosphere through the incubation atmosphere recirculation chamber 915, and the ducting feature 926 directs the recirculating air flow 924 across the bottom of the culture vessels 704, 706. In doing so, the ducting feature and the configuration on the underside of the base 764 of the culture vessels 704, 706 induce the formation of localized turbulence 928, which helps maintain a constant supply of oxygen and carbon dioxide in contact with the gas permeable membrane 768 of the culture vessels 704, 706.

[0139] Figures 73–76 more clearly illustrate the ducting features of system 900, which, under the influence of fans 906, 908, allow the recirculating air flow 924 to be directed from the recirculation chamber 915 across the bottom of the culture vessels 704, 706 and back into the recirculation chamber 915. As shown therein, the sides of the legs 708, 710 of tray 702 facing the interior are formed with recessed or concave areas 930, which allow the recirculating air 924 exiting / entering the recirculation chamber 915 to travel upward or downward along the interior surfaces of the legs 708, 710, as applicable. Figures 73–76 specifically illustrate how the recirculating air 924 present in the recirculation chamber 915 is directed upward by the concave areas 930 of the legs 710 of tray 702. Therefore, the recessed areas 930 of the legs 708, 710 and the outer surface of the recirculation chamber form vertical air passages for the flow of recirculated air 924. As the air exiting from the louvers 912 travels upward through the recessed areas 930 of the legs 710, it is obstructed at the point where the legs 710 meet the bottom of the tray 702. As best shown in Figures 73 and 74, the tray 702 includes a pair of lateral vent openings 932 at a height generally corresponding to the vertical height of the bottoms of the culture vessels 704, 706. Therefore, the vent opening 932 redirects the recirculating airflow 924 laterally through such opening 932 toward the culture vessels 704, 706, where the recirculating airflow 924 interacts with the bottom geometry of the culture vessels 704, 706 and their corresponding gas-permeable membranes, leading to the formation of localized turbulence 928. The recirculating airflow 924 travels across the bottom of the culture vessels 704, 706, where it enters the opposing vent opening, travels downward through the recessed area 930 of the leg 708, and re-enters the recirculation chamber 915 through the louvers 910.

[0140] As disclosed above, the formation of localized turbulence 928 in the recirculating air flow 924 helps maintain a constant supply of oxygen and carbon dioxide in contact with the gas-permeable membrane 768 of the culture vessels 704, 706. At the same time, the overall recirculating 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 the homogenization of the volume inside the incubation chamber 902. Thus, the system 900 provides optimization of heat and mass transfer. As will be recognized, the constant availability of oxygen just tens of microns away from the cell monolayer supports higher cell concentrations and minimizes the physiochemical gradient across the surface of the membrane 768 of the culture vessels 704, 706.

[0141] As described above, the apparatus 600 includes multiple sensors and monitoring devices for monitoring bioprocessing operations while they are being performed (including monitoring various parameters of cell culture in culture vessels 704, 706). This can include, for example, periodically drawing samples from culture vessels 704, 706 using the sampling tubing tail 748 of the sampling card 722, and / or using sensors to sense various parameters of the culture in the vessels. For example, sensor assembly 648 houses an IR sensor for temperature measurement and for detecting the presence of culture vessels in process drawouts. A window 796 in the base 764 of culture vessels 704, 706 allows for IR-based temperature measurement of the membrane in the culture vessels, and thus allows for the measurement of the liquid temperature in the culture vessels.

[0142] Referring to Figures 77–84, in one embodiment, the apparatus 600 may additionally include a flow-through sensing chamber 950 (also referred herein as a flow-through sensing device 950), which can be used to measure or monitor various parameters of a fluid in the apparatus 600 (e.g., a culture in culture vessels 704, 706) using a variety of different sensing / measuring devices and without drawing any fluid out of the system. As best shown in Figures 77–80, the flow-through sensing chamber 950 includes a first plate 952, a second plate 954 connected in a relationship opposite to the first plate 952, and a fluid channel 956 between the first plate 952 and the second plate 954. In one embodiment, the fluid channel 956 is formed from a relaxation 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 approximately 0.1 mm and approximately 1 mm in height. The chamber 950 further includes a first port 958 and a second port 960, the first port 958 being 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 being in fluid communication with the fluid channel 956 to facilitate the flow of fluid from the chamber 950 and its fluid channel 956. In one embodiment, ports 958 and 960 are in fluid communication with opposing ends of the fluid channel 956.

[0143] As shown in Figure 78, in some embodiments, plates 952, 954 can have features that facilitate the 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 the corresponding tabs 959 of the other plate (e.g., plate 954). As discussed below, the back plate / first plate 952 includes a plurality of mounting and positioning holes 961 extending through it, which facilitate the mounting of the chamber 950 onto the tray 702 of the disposable kit 700. In some embodiments, the first / back plate 952 and the second / front plate 954 are generally rectangular in shape, transparent, and manufactured from biocompatible plastic, glass, or a combination of plastic and glass, but the present invention is not intended to be so limited in this respect.

[0144] As best illustrated in Figures 78 and 79, the fluid channel 956 includes multiple segments or sensing locations 962, 964, 966, which allow or facilitate interrogation or monitoring of the fluid in the fluid channel 956 by multiple sensing devices and techniques. In one embodiment, the fluid in the fluid channel 956 can be interrogated by various different sensing devices associated with each of the multiple sensing locations 962, 964, 966. In one embodiment, a segment 966 has one or more sensors 968 positioned within the fluid channel 956, and one or more sensors 968 are configured to remain in continuous contact with the fluid passing through the fluid channel 956. As shown in Figure 77, the second plate 954 includes multiple electrodes 970, which extend into the fluid channel 956 and are accessible from a flange 972 extending laterally from the second plate 954. In one embodiment, the electrodes 970 are gold-plated electrodes.

[0145] As shown above, the flow-through sensing chamber 950 allows interrogation of the fluid in the fluid channel 956 by utilizing various different sensing devices 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 the fluid channel 956 that reflects 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). In one embodiment, sensing location 964 can be configured as a transmitted and backscattered light interrogation segment, which allows interrogation of the fluid in the fluid channel 956 using transmitted or backscattered light sensing instruments. The sensing location 966 can, in part, be configured as a fluorescence sensor interrogation segment having various sensors 968 in contact with the fluid in the fluid channel 956, enabling monitoring or sensing of various parameters of the fluid (e.g., dissolved oxygen, pH, carbon dioxide, sample, etc.). The electrode 970 faces backward (opposite to ports 958, 960) and is configured to be in contact by a spring-driven pin of one or more measuring devices suitable for various electrochemical measurement techniques (e.g., electrical impedance spectroscopy, galvanometry, amperometry, and / or polarography, etc.).

[0146] Figures 81 and 82 illustrate the positioning of a flow-through sensing chamber 950 on the backbone of tray 702 of a disposable bioprocessing kit 700. As shown above, the chamber 950 can be connected to tray 702 by receiving a snap pin 976, positioned on the backbone of tray 700, into the corresponding mounting aperture 961 of the chamber 950. As shown therein, in one embodiment, the chamber 950 can be mounted on tray 700 between the valve manifold 712 and peristaltic pump tubing segments 714, 716, and 718. While the pin 976 is illustrated as being used to mount the chamber 950 on tray 700, it is intended that other means of connection (e.g., clipping, clamping, fasteners, snap fittings, and press-fits) may also be utilized without departing from broader aspects of the invention. In one embodiment, the chamber 950 can form part of a disposable bioprocessing kit 700.

[0147] Figures 83 and 84 present schematic diagrams of a 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 Figure 83, for example, first and second electrochemical sensing instruments 978, 980 mounted on the apparatus 600 can interface with the electrode 970 via spring-driven pins 982. As shown in Figure 84, a reflected light instrument 984 can be positioned and configured to interrogate the fluid in a first sensing location, first and second fluorescent instruments 986, 988 can be positioned and configured to interrogate the fluid in a second sensing location 964, and a transmitted / backscattered light instrument 990 can be positioned and configured to interrogate the fluid in a third sensing location 966.

[0148] Accordingly, embodiments of the present invention provide an inline sensing chamber 950 which provides various optical and electrical measurements of the fluid in the fluid channel 956 of the chamber 950, eliminating the need to directly interrogate either of the culture vessels 704, 706. When it is desired to monitor or measure various parameters of the culture in either of the culture vessels 704, 706 during use, the fluid is pumped through the sensing chamber 950 using a peristaltic pump assembly 641, where it can be interrogated by a series of sensor instruments / devices. In other words, the chamber 950 disclosed herein facilitates the use of electrochemical and optical sensing techniques over a single fluid channel, enabling multiparametric monitoring of the physiological and chemical growth conditions of cell cultures in the culture vessels 704, 706, the metabolic activity of cell types (lactic acid, glucose, etc.), as well as viable cell density and total cell number measurements.

[0149] While 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 Figures 85–89. As disclosed above and as described in more detail hereafter, the configuration of the bioprocessing apparatus 600 and kit 700, together with the fluid flow architecture 200 provided thereby, enables, in an automated and functionally closed manner, cell activation, genetic recombination and amplification of cell products, as well as auxiliary or related protocols, workflows, and methods. In one embodiment, the flow architecture or system 400 can be configured or arranged as disclosed in Figures 3–7 of International Publication No. 2019 / 106207, but other configurations are also possible. As illustrated in Figure 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 and a first bioreactor line 414 in fluid communication with the first port 412, as well as a second port 416 and a second bioreactor line 418 in fluid communication with the second port 416. Similarly, the second bioreactor vessel includes at least a first port 422 and a first bioreactor line 424 in fluid communication with the first port 422, as well as a second port 426 and a second bioreactor line 428 in fluid communication with the second port 426. Together, the first bioreactor vessel 410 and the second bioreactor vessel 420 form a bioreactor array 430. Although the system 400 is shown as having two bioreactor vessels, embodiments of the present invention may include a single bioreactor or three or more bioreactor vessels.

[0150] The first and second bioreactor lines 414, 418, 424, and 428 of the first and second bioreactor vessels 410 and 420 each include a valve for controlling the flow of fluid through them, as discussed below. In particular, the first bioreactor line 414 of the first bioreactor vessel 410 includes a first bioreactor line valve 432, while the second bioreactor line 418 of the first bioreactor vessel 410 includes a second bioreactor line valve 434. Similarly, the first bioreactor line 424 of the second bioreactor vessel 420 includes a first bioreactor line valve 436, while the second bioreactor line 428 of the second bioreactor vessel 420 includes a second bioreactor line valve 438.

[0151] Referring further to Figure 85, the system 400 also includes a first fluid assembly 440 having a first fluid assembly line 442, a second fluid assembly 444 having a second fluid assembly line 446, and a sampling assembly 448. An interconnection line 450 having an interconnection line valve 452 provides fluid communication between the first fluid assembly 440 and the second fluid assembly 444. As shown in Figure 85, the interconnection line 450 also provides fluid communication between the second bioreactor line 418 and the first bioreactor line 414 of the first bioreactor vessel 410, enabling fluid circulation along the first circulation loop of the first bioreactor vessel. Similarly, the interconnection line also provides fluid communication between the second bioreactor line 428 of the second bioreactor vessel 420 and the first bioreactor line 424, enabling fluid circulation along the second circulation loop of the second bioreactor vessel. Furthermore, the interconnection line 450 provides further fluid communication between the second port 416 and the second bioreactor line 418 of the first bioreactor vessel 410 and the first port 422 and the first bioreactor line 424 of the second bioreactor vessel 420, enabling the transfer of the contents of the first bioreactor vessel 410 to the second bioreactor vessel 420, as discussed below. As illustrated in Figure 85, in one embodiment, the interconnection line 450 extends from the second bioreactor lines 418, 428 to the intersection of the first bioreactor line 414 and the first fluid assembly line 442 of the first bioreactor vessel 410.

[0152] As illustrated in Figure 85, the first and second fluid assemblies 440 and 444 are arranged along the interconnection line 450. Additionally, in one embodiment, the first fluid assembly is in fluid communication with the first port 412 of the first bioreactor vessel 410 and the first port of the second bioreactor vessel 420, respectively, through the first bioreactor line 414 of the first bioreactor vessel and the first bioreactor line 424 of the second bioreactor vessel 420. The second fluid assembly 444 is in fluid communication with the second port 416 of the first bioreactor vessel 410 and the second port 426 of the second bioreactor vessel 420 via the interconnection line 450.

[0153] A first pump 454 of the peristaltic pump assembly 641, capable of providing bidirectional fluid flow, is arranged along the first fluid assembly line 442, and a second pump or circulation line pump 456 of the peristaltic pump assembly 641, capable of providing bidirectional fluid flow, is arranged along the interconnection line 450, the function and purpose of which will be discussed below. Also, as shown in Figure 85, a sterile air source 458 is connected to the interconnection line 450 through a sterile air source line 460. A valve 462 positioned along the sterile air source line 460 provides selective fluid communication between the sterile air source 458 and the interconnection line 450. Figure 85 shows a sterile air source 458 connected to the interconnection line 450, but in other embodiments, the sterile air source may be connected to the first fluid assembly 440, the second fluid assembly 444, or to an intermediate fluid flow path between the second bioreactor line valve and the first bioreactor line valve of either the first or second bioreactor.

[0154] Referring further to Figures 86–88, detail diagrams of the first fluid assembly 440, the second fluid assembly 444, and the sampling assembly 448 are shown. Referring specifically to Figure 86, the first fluid assembly 440 includes a plurality of tubing tails 464a–f, each configured for a selective / removable connection to one of the plurality of first reservoirs 466a–f. Each of the tubing tails 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 of the plurality of first reservoirs 466a–f of the first fluid assembly 440. While Figure 86 specifically shows that the first fluid assembly 440 includes six fluid reservoirs, more or fewer reservoirs may be utilized to provide input or collection of various processing fluids as desired. It is intended that each tubing tail 464a to f can be individually connected to the storage units 466a to f, respectively, for the time required during the operation of the fluid assembly 440, as described below.

[0155] Referring specifically to Figure 87, the second fluid assembly 444 includes a plurality of tubing tails 470a-d, each configured for selective / removable connection to one of the plurality of second storage units 472a-d. Each tubing tail 470a-d of the second fluid assembly 444 includes a tubing tail valve 474a-e for selectively controlling the flow of fluid to or from each of the plurality of second storage units 472a-d of the second fluid assembly 444. While Figure 87 specifically shows that the second fluid assembly 444 includes four fluid storage units, more or fewer storage units may be utilized to provide input or collection of various processing fluids as desired. In one embodiment, at least one of the second storage units (e.g., second storage unit 472d) is a collection storage unit housed in the cabinet 608 of the apparatus 600 for collecting a population of amplified cells, as discussed below. In one embodiment, the second storage section 472a is either a waste storage section or a bag housed in the waste drawer 606 of the device 600, the purpose of which is discussed below.

[0156] In one embodiment, the first storage compartments 466a-f and the second storage compartments 472a-d are single-use / disposable flexible bags, which are housed in a cabinet 608 of the apparatus 600 and are fluidly connected to a manifold 712 via tubing tails of a tubing organizer 720. In one embodiment, the bag is a substantially two-dimensional bag having opposing panels, as known in the art, the panels welded or fixed together around their periphery and supporting connecting conduits for connection to their respective tails.

[0157] 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 next to the apparatus 600 and can also be used to butt weld one of the tubing tails to the tail of the tube on top of the bag (while maintaining sterility). Thus, the operator can provide the bag when it is needed (for example, by grasping a 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 end as the razor 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-sealed section to separate the two closed lines. Therefore, the storage units / bags can be connected individually as desired, and the present invention does not require all storage units / bags to be connected at the beginning of the protocol. This is because the operator will have access to the appropriate tubing tails throughout the process to connect the storage units / bags at the appropriate time for their 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 allows the operator to access the fluid assembly / line during operation, so that used bags can be connected in a sterile manner and other bags can be disconnected so that they can be connected sterilely during the protocol.

[0158] As illustrated in Figure 88, the sampling assembly 448 includes one or more sampling lines (e.g., sampling lines 476a-476d (which can be the sampling tubing tail 748 of the sampling card 722)) fluidly connected to the interconnection line 450. Each of the sampling lines 476a-476d may include selectively operable sampling line valves 478a-476d to allow fluid to flow from the interconnection line 450 through the sampling lines 476a-476d. As also shown there, the distal end of each sampling line 476a-476d is configured for selective connection to a sample collection device (e.g., sample collection devices 280a and 280d) for collecting fluid from the interconnection line 450. The sample collection device can take the form of any sampling device known in the art (e.g., a syringe, immersion tube, bag, etc.). Figure 88 illustrates that the sampling assembly 448 is connected to an interconnection line, but in other embodiments, the sampling assembly can be fluidically connected to the fluid passages between the first fluid assembly 440, the second fluid assembly 444, the second bioreactor line valve 434 and the first bioreactor line valve 432 of the first bioreactor vessel 410, and / or the fluid passages between the second bioreactor line valve 438 and the first bioreactor line valve 436 of the second bioreactor vessel 420. The sampling assembly 448 provides a fully functionally closed sampling of the fluid at one or more points in the system 400 as desired.

[0159] Referring back to Figure 85, in one embodiment, the system 400 may also include a filtration line 482, which is connected at two points along the interconnection line 450, defining a filtration loop along the interconnection line 450. A filter 484 is positioned along the filtration line 482 to remove permeate waste from the fluid passing through the filtration line 482. As shown therein, the filtration line 482 includes an upstream filtration line valve 486 and a downstream filtration line valve 488, which are positioned upstream and downstream of the filter 484, respectively. A waste line 490 provides fluid communication between the filter 484 and a second fluid assembly 444, and, in particular, fluid communication with the tubing tail 470a of the second fluid assembly 444 (which is connected to the waste storage unit 472a). In this regard, the waste line 490 transports the waste removed by the filter 484 from the fluid passing through the filtration line 482 to the waste storage unit 472a. As illustrated in Figure 85, the filtration line 482 surrounds the interconnection line valve 452, allowing the fluid flow through the interconnection line 450 to be forced through the filtration line 482, as discussed hereafter. A permeate pump 492, positioned along the waste line 490, is operable to pump the waste removed by the filter to the waste storage unit 472a. In one embodiment, the filter 484 is preferably an elongated hollow fiber filter, but other tangential flow or cross-flow filtration means known in the art (e.g., flat sheet membrane filters) may also be used without departing from a broader aspect of the present invention.

[0160] In one embodiment, the valves of the first fluid assembly 440 and the second fluid assembly 444, as well as the bioreactor line valves (i.e., valves 432, 434, 436, 438), the sterile line valve 462, the interconnection line valve 452, and the filtration line 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 allow a specific flow of fluid through it. 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 performed automatically according to a programmed protocol to enable proper operation of module 200 / device 600. The second controller 210 mounted on the second module 200 / device 600 is intended to be capable of instructing the operation of these valves (linear actuators) and pumps.

[0161] 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 comprises six steps, each containing multiple user-controllable / selectable parameters, and is performed by the controller 210. During the activation phase, two pre-seeding reagents and two post-seeding reagents may be used. The cell input to the activation phase is cells that are ready to be activated. Following activation, the cells may be concentrated and washed to remove any residual reagent components that are undesirable for subsequent process steps. The transduction phase similarly comprises six steps, each containing multiple user-controllable / selectable parameters, and is performed by the controller 210. During the transduction phase, two pre-seeding reagents and two post-seeding reagents may be used. 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 undesirable for subsequent process steps. The amplification phase, in part, comprises three steps (seeding, cell culture, and harvesting), each of which includes multiple user-controllable / selectable parameters and is performed by the controller 210. During the seeding step, the system will add culture medium to the transduction vessel to dilute the contents to the desired cell density for amplification. During the cell culture step, the user can select a sampling frequency and define a feeding strategy used to amplify cells in culture vessels 704, 706. During the harvesting step, harvesting can be performed at a preset time or initiated by the user once the target cell dose is achieved.

[0162] In one embodiment, parameters that can be controlled or selected by the 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 step includes parameters for up to two reagents that can be added to the culture vessel before seeding cells, and parameters for up to two reagents that can be added to the culture vessel after seeding cells. Before transferring reagents to the culture vessel, the user may transfer air or liquid through the system. After the incubation of the reagents, the culture vessel may be rinsed before seeding cells. The input cell volume parameter defines the parameters for adding source cells into the culture vessel. Before adding cells to the culture vessel, the user may manually mix the cells in the source bag. Additionally, the source bag may be rinsed to maximize the transfer of input cells. The incubation parameter defines the parameters during the incubation of cells in culture vessels 704, 706. The user may set the target seeding density and activation volume, as well as sampling-related parameters. Volume reduction parameters define the parameters for enriching cells after activation. Cells are enriched through volume reduction, either by using a hollow fiber filter (HFF) or by scooping up the liquid without disturbing the cells and aspirating the liquid from the culture vessel (i.e., perfusion without adding medium to the inlet so that the volume in the culture vessel decreases) (also known as high-speed perfusion (HSP)).

[0163] The washing parameters define the parameters for washing cells after volume reduction in order to prepare them for transduction. Cells are washed using either a hollow fiber filter (HFF) or high-speed perfusion (HSP). In one embodiment, the HSP washing protocol includes the following process steps: 1) Initial sedimentation phase - 1) The activated vessel remains stable for a fixed amount of time, allowing cells to settle on the vessel membrane; 2) Allows for very slow activated vessel mixing to enhance the homogeneity of the supernatant without disturbing the settling cells; 3) Simultaneously adds medium and removes supernatant while maintaining a stable activated vessel volume; 4) Allows for very slow activated vessel mixing to enhance the homogeneity of the supernatant without disturbing the settling cells, during the washing duration; 5) Simultaneously adds medium and removes supernatant while maintaining a stable activated vessel volume until the washing target duration has elapsed or the washing target medium volume has been consumed; 6) Allows for dilution of the activated vessel contents with medium to the target vessel volume; 7) Allows for very slow activated vessel mixing to enhance the homogeneity of the supernatant without disturbing the settling cells; and 8) Performs low-flow removal of the supernatant to the target activated vessel volume without disturbing the settling cells.

[0164] In one embodiment, with respect to the transduction phase, the steps are similar to those described above for the activation phase. In one embodiment, a transport cell parameter is provided, which defines the parameters for transporting activated cells from the activation vessel into the transduction vessel. Before transporting the cells to the culture vessel, the system may mix the cells in the activation vessel. A portion or all of the contents of the activation vessel may be transported to the transduction vessel. Additionally, the activation vessel may be rinsed to maximize the transport of cells.

[0165] Finally, the target seeding general parameters define the parameters for setting the starting conditions for cells during amplification. The cell culture parameters define the feeding strategy used to culture cells during amplification. Users can define the feeding period based on user-configurable parameters. Exemplary feeding strategies include single-shot medium addition (fed-batch) or continuous medium addition (perfusion). The harvest parameters define the parameters that enable cell harvesting. Users can define the volume of cells to harvest and initiate harvesting at a defined time or at any desired time. As will be recognized, these parameter selections and settings can be performed using interface 609 or through an off-board user interface or terminal communicating with the instrument 600 (e.g., through a data port on the back of the instrument 600), but wireless communication means are also possible.

[0166] As shown above, the apparatus 600 and flow architecture 400 also enable sampling of the contents of culture vessels 704, 706, for example, using the sampling tubing tail 748 of the sampling card 722.

[0167] In one embodiment, the sampling sequence includes tilting platform rocker assemblies 640, 642 to mix and homogenize the contents in the culture vessel (the mixing rate depends on the vessel volume), operating a process pump 456 to circulate the vessel contents back from the vessel outlet port (416 or 426) to the vessel inlet port (412 or 422) in the sampling tubing, prompting the user to take a sample, stopping the circulation and mixing, and finally clearing the sampling tubing.

[0168] In one embodiment, the use of two culture vessels 704, 706 within the processing drawer 604 of the apparatus allows parallel processing to be carried out in the manner disclosed below. In one embodiment, all activation steps can be carried out in 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 carried out. In another embodiment, during activation in the first culture vessel 704, the transduction reagent action can be carried out in the second culture vessel 706 before adding the post-activated cells from the first culture vessel 704 to the second culture vessel 706 for the transduction and amplification steps (e.g., adding a pre-seeding reagent to the second culture vessel 706, incubating, and rinsing the culture vessel 706). In yet another embodiment, the activation, transduction, and amplification steps can be carried out in a single culture vessel (e.g., the first or second culture vessels 704, 706).

[0169] Referring to Figure 90, another workflow 1000 activated by the bioprocessing apparatus 600 is illustrated. As shown therein, the workflow or method 1000 includes the steps of performing a series of activation steps 1002 and a series of transduction steps 1004 in a first culture vessel 704, and the steps of amplifying a population of genetically recombinant cells (post-transduction) in a parallel amplification step 1006 using both the first and second culture vessels 704, 706. This involves the step of transferring a fraction of genetically recombinant cells from the first culture vessel 704 to the second culture vessel, so that the parallel amplification 1006 can be performed simultaneously using both culture vessels 704, 706.

[0170] Referring to Figure 91, another workflow 1100 activated by the bioprocessing apparatus 600 is illustrated. As shown therein, the workflow or method 1100 includes carrying out the steps of activation, transduction, and amplification in parallel (but independent) workflows. This includes, for example, the step of carrying out the activation step 1102, transduction step 1104, and amplification step 1106 entirely for a first population of cells in a first culture vessel 704, and the step of carrying out the parallel activation step 1108, transduction step 1110, and amplification step 1112 entirely for a second population of cells in a second culture vessel 706. In one embodiment, the first and second populations of cells can be supplied from a single population of cells that is split between the first and second culture vessels 704, 706 during the input step of the activation phase. In another embodiment, the first and second populations of cells can be different (e.g., come from different sources).

[0171] Now looking at Figure 92, we see another workflow 1200 activated by the bioprocessing apparatus 600. As shown there, the workflow or method 1200 includes the steps of performing an activation step 1202 for a population of cells in a first culture vessel 704, and then, in step 1204, transferring the activated population of cells completely out of the bioprocessing apparatus 600 from the first culture vessel 704 for off-board transduction. After transduction out of the module / apparatus 600, the cell volume is transferred into the second culture vessel 706 of the bioprocessing apparatus 600 for a post-transduction volume reduction and post-transduction washing step 1206 in the second culture vessel 706. As shown there, the amplification step 1208 is also performed in the second culture vessel 706.

[0172] In one embodiment, the bioprocessing apparatus 600, disposable bioprocessing kit 700, and flow architecture of the present invention allow washing (e.g., using a hollow fiber filter) to be performed both after activation and after transduction.

[0173] In connection with the use of the bioprocessing apparatus 600 for performing activation, transduction, and amplification of cell populations in the manner described above, a common requirement is that all disposable devices used in cell culture and bioprocessing are sterile during their operating period, functionally closed, and fully reliable in order to ensure batch quality and product safety. Accordingly, embodiments of the present invention also provide leak-tightness verification and blockage detection checks to be performed on the disposable bioprocessing kit 700 (including culture vessels 704, 706 and associated tubing) before use. Figure 93 illustrates the flow architecture 1300 used by the apparatus 600 and disposable kit 700 according to embodiments of the present invention. The flow architecture 1300 is generally similar to the flow architecture 400 disclosed above.

[0174] As shown therein, the flow architecture / system 1300 includes multiple pneumatic interfaces (for example, two pneumatic interfaces 1302, 1304, or four pneumatic interfaces 1302, 1304, 1306, 1308) that allow air to be drawn into the system 1300. The pneumatic interfaces 1302, 1304, 1306, 1308 allow for leak-tight connections with respect to sterile air filters 1310, 1312, 1314, 1316 associated with each interface (which form part of the disposable kit 700). System 1300 further includes three-way valves 1322 and 1323 in addition to the three-way valve 1320, the three-way valve 1322 enabling switching of the airflow path so that the kit passes through sterile air filters 1310, 1312 to the ambient atmosphere outside the kit in process drawer 604, or to connect to pressure monitoring sensor 1324. The system 1300 further includes two peristaltic pumps 1326, 1328 (e.g., process pump 456 and source pump 454 of peristaltic pump assembly 641) intended to act as pressurizing means and pinch valves during leak-tightness verification processes and as liquid management means during normal operation as disclosed above, a set of up to 20 pinch valves 1330 (#1 to #20) (e.g., formed by valve manifold 712 and linear actuator array 643), and one peristaltic pump 1332 (e.g., waste pump 492 of peristaltic pump assembly 641) intended to act as a pinch valve during leak-tightness verification and as liquid management means during normal operation.

[0175] Air can be drawn into the system via a system peristaltic pump through a pneumatic interface that allows for selective connection of the flow path to the atmosphere via a sterile air filter. In one embodiment, there are two main uses of this interface: (1) to allow pressurization of a portion of the disposable kit 700 during kit integrity checks, as discussed below, and (2) to draw sterile air to clear fluid from the line during various automated workflows.

[0176] Figure 94 illustrates another flow architecture / system 1400, which 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 similar reference numbers specify similar parts. As shown therein, system 1440 has four pneumatic interfaces 1302, 1304, 1306, and 1308, one of which (pneumatic interface 1306) is connected to a three-way valve 1318, which switches between atmosphere and pressure sensor. The advantage of flow architecture / system 1400 is that the culture vessel can be pressurized independently of the rest of disposable kit 700 (before initiating bioprocessing operations). This makes it possible to check the culture vessel at one pressure and the rest of the kit at another pressure (potentially higher than what the culture vessel can withstand). Furthermore, this allows the rest of Kit 700 and its flow line to be tested under negative pressure, which is typically avoided in culture vessels as it can cause membranes to be removed or displaced.

[0177] Figure 95 illustrates another flow architecture / system 1402, which can be used by apparatus 600 and disposable kit 700 instead of architecture 1300 or 1400, according to another embodiment of the present invention. Flow architecture / system 1402 is similar to flow architecture / system 1400, where similar reference numbers specify similar parts. However, as shown therein, flow architecture 1402 in Figure 95 omits the hollow fiber filter (HFF) and waste pump. Flow architecture 1402 in Figure 95 is operable in a similar manner to that described above in relation to flow architecture 1400 in Figure 94.

[0178] Figure 96 illustrates yet another flow architecture / system 1410, which can be used by the apparatus 600 and disposable kit 700 instead of architectures 1300, 1400, or 1402, according to another embodiment of the present invention. Flow architecture / system 1410 is similar to flow architecture / system 1402, where similar reference numbers designate similar 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 Figure 95). In particular, it is recognized that utilizing two or more pressure sensors can provide certain advantages depending on the particular architecture and application (in contrast to the single pressure sensor used in the architecture of Figure 95). In some embodiments, 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 and 1412 may have the same or similar pressure ranges.

[0179] In one embodiment, the flow architecture / system 1410 may further include an accumulator 1414. The accumulator 1414 functions as a volumetric buffer and can be constructed as a storage section or as the length of a tubing. Regardless of the specific construction or configuration, the accumulator 1414 has a volume greater than or equal to the total volume of the fluid flow paths between / from the sterile air filter 1316 and the second pressure sensor 1412. In use, in the event of clogging, the presence and location of the accumulator 1414 ensure that the volume of fluid accumulates within the accumulator 1414 without contacting the sterile air filter.

[0180] In many of the components, systems, devices, and architectures disclosed above, the use (or adoption) of sterile air filters has been referred to. In some embodiments, one or more (or all) of these sterile air filters may be hydrophobic, allowing them to be exposed to (or in contact with) a fluid, while still maintaining their integrity and function as intended. In further other embodiments, depending on the specific system or architectural layout and application, non-hydrophobic filters may be used, with or without accumulators or similar devices.

[0181] In one embodiment, the leak-tightness verification described above is performed independently on three differentiated segments of the disposable culture kit. In one embodiment, the first segment includes the entire disposable kit (i.e., the 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 first segment is tested in two phases (pressurization phase and pressure drop monitoring phase). In one embodiment, the second segment includes two culture vessels, 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 second segment is tested in two phases (pressurization phase and pressure drop monitoring phase). In one embodiment, the third segment includes the entire disposable kit (i.e., the entire fluid flow path), excluding the T / U loop (sensor bypass) and the tubing segment between the source pump 1328 / 454 and the tubing tails 1334a-d. Testing of the third segment is performed in three phases: pressurization phase, pressure reduction monitoring, and pressure release phase.

[0182] As described above, the leak-tightness verification and blockage detection method disclosed above enables the end user to perform an automated integrity test on the entire disposable kit 700 before initiating bioprocessing operations. This enables the end user to detect possible leaks in the disposable kit and / or blocked / clogged lines (which would negatively impact the ability to perform automated workflows and, ultimately, batch quality).

[0183] As described above, mammalian cell culture processes may require significantly complex fluid transfer management operations that must be performed in a precise and safe manner. Therefore, the ability to detect leakage events is a critical function that should be continuously performed to trigger alarms if batch viability could be potentially compromised. In light of the above, embodiments of the present invention also intend to verify leak-tightness and detect blockages in the disposable kit 700 using real-time monitoring of the mass involved in the bioprocess. In most (if not all) of the bioprocessing operations disclosed herein, four situations are conventionally always present or occurring: (1) a fluid is held in 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 recirculated from a container or vessel and returned to the same container or vessel. Therefore, multiple leak and / or blockage detection processes can be carried out as disclosed below, insofar as the source container, intermediate container, and / or destination container include 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 the same container to the same container in a leak-tight manner (e.g., using peristaltic pump assembly 641), and a control unit (e.g., controller 210) for monitoring variations in the mass of each container. Load cells can include, as disclosed above, for example, bed plates supporting various containers (e.g., culture vessels 704, 706, waste bags, etc.), or pegs or hooks having integrated load cells (e.g., hooks 620 on vertical storage drawers 614, 616 of cabinet 608 for suspending culture bags, reagent bags, and other bags).As disclosed below, the controller (for example, controller 210) is configured to monitor the mass fluctuations of each container, activate pumping means for transferring fluid between containers, run the mass equilibrium equation, and generate an alarm or warning if the solution to the mass equilibrium equation does not indicate the absence of leak-tightness or blockage.

[0184] 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 (for example, 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, and that there is no leakage. However, if the change in mass exceeds the threshold, this indicates that fluid has leaked from the container, and the controller 210 issues an alarm to the user.

[0185] In another embodiment, a method for detecting leakage or blockage involves the steps of 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 the apparatus 600 is controlled by the controller 210 and pumps 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 in it) is first determined. Then, the volume of fluid from the first container is transferred to the second container. Next, 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 fluid volume in the first container and the mass of the transferred fluid volume 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 leak detection process without requiring that the entire fluid volume be transferred between containers. In particular, in some embodiments, the controller 210 is configured to verify whether (and remains below) the transfer flow rate plus a specific leak rate detection threshold, and whether (and remains above) the destination container mass volume absolute variation is the transfer flow rate minus a specific leak rate detection threshold. If not, a leak alarm will be triggered by the controller 210.

[0186] In yet another embodiment of leak-tightness verification using real-time mass balance monitoring, the objective is to maintain a constant mass in an intermediate (e.g., a third) container. Therefore, simultaneous action of two pumps in the peristaltic pump assembly 641 is required, where the transfer of liquid from the source to the intermediate container must be controlled based on the source container fluctuation with respect to a specific flow setpoint, and the transfer of liquid from the intermediate to the destination container must be controlled based on the destination container fluctuation with respect to a specific flow setpoint. The control unit 210 is configured to verify whether (and remains below) the absolute source container mass volume fluctuation plus a specific leak rate detection threshold, whether (and remains below) the absolute intermediate container volume fluctuation below a specific leak rate detection threshold, and whether (and remains above) the absolute destination container mass volume fluctuation above the transfer flow rate minus a specific leak rate detection threshold. If any of these conditions are not met, the controller 210 is then configured to generate an alarm.

[0187] In yet another embodiment, leak-tightness verification is performed by the controller 210 by controlling the pump to recirculate the fluid from the first container, out of the first container, and back into the first container. Thus, the fluid pumping is performed 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 (and remains below) a specific leak rate detection threshold. Otherwise, a leak alarm will be triggered by the controller 210. The variation for this process is when the sample volume is drawn out of the recirculation loop. In this case, the controller 210 verifies whether the absolute mass-volume variation of the first container remains below the specific leak rate detection threshold plus the sampling flow rate.

[0188] Accordingly, embodiments of the present invention utilize real-time mass balance calculations to check leak-tightness and / or detect blockages in Kit 700 before using Kit 700 in a bioprocess operation. However, the methods disclosed herein are not limited to determining leaks before using Kit 700 in a bioprocess, but can also be used during the bioprocess for real-time leak verification or blockage detection. Thus, it may be possible to take corrective action in relation to any blockages or leaks detected in order to save or recover a batch.

[0189] In the embodiments disclosed above, the first source bag / container may be a culture medium bag, the second destination bag / container may be a waste bag, and the third intermediate bag / container may be a culture vessel or bioreactor vessel. However, the present invention is not intended to be limited in this respect, and various bags / vessels can be used as the first, second, and third containers, insofar as fluid can be transferred between and / or through such containers. Furthermore, although the mass balancing process for verifying leak-tightness and detecting blockages has been described as being carried out on the bioprocessing apparatus 600 and the disposable bioprocessing kit 700, the present invention is not intended to be limited in this respect. In particular, it is intended that the mass balancing technique can be carried out on various systems and devices, including the processing apparatus 102 and isolation module (and disposable kits therefor) disclosed above in relation to the first and third modules 100, 300.

[0190] As used herein, any element or step described in the singular and preceded by the phrase "a" or "an" should be understood not to exclude any plurality of such element or step unless such exclusion is expressly stated. Furthermore, any reference to "one embodiment" of the present invention is not intended to be interpreted as excluding the existence of additional embodiments that also incorporate the described features. Moreover, unless the opposite is expressly stated, any embodiment that "comprising," "including," or "having" an element or plurality of elements having a particular characteristic may include additional such elements that do not possess that characteristic.

[0191] This written description, using examples, discloses several embodiments (including the best mode) of the invention and enables a person skilled in the art to practice embodiments of the invention (including fabricating and using any device or system, and carrying out any incorporated method). The patentable scope of the invention is defined by the claims and may include other examples conceivable by a person skilled in the art. Such other examples are intended to be within 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 that differ only slightly from the literal language of the claims. [Explanation of Symbols]

[0192] 10 Bioprocessing Systems 12 Bioprocessing Systems 100 First module 102 Processing Unit 104 Isolation Module 105 Bracket 106 Base 108 Centrifugal Processing Chamber 110 First controller 111 High Dynamic Range Peristaltic Pump Assembly 112 Stopcock Manifold Interface 113 Drip Chamber Holder 114 Heating-cooling-mixing chamber (thermal mixer) 116 Hanger Assembly 118 hooks 130 Base / Housing 132 Stopcock Manifold Interface 134 slots 136 Magnetic Cell Isolation Holder 138 Support pole 140 hooks 142 Latches, Clamps 143 Latch, Clamp 144 Output shaft 146 Stopcock Motor 148 Line pressure sensor assembly 150 Bubble Sensor Assembly 151 Connector 152 Housing 153 switches 154 channels 155 Communication Connector 156 Cover 157 Opening 159 Internal fan 160 Magnetic Field Generator Assembly 161 Indicator Light 162 Permanent Magnets 164 Permanent Magnets 166 Carriage 168 Upper shaft 170 Lower shaft 171 Crank 172 Bushings, bearings 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 Pins 196 Flange 198 Coil Springs 199 Seat section, recess 200 Second module 200a, 200b, 200c: Second module 210 Second controller, control unit 210a, 210b, 210c controllers 250 Magnetic Cell Isolation Holder 252 Main body 254 channels, race 256 Tubes 258 Part 1 260 Part 2 262 Third part 264 Part 4 266 Handle 268 High gradient region 270 ferromagnetic cores 272 loops 274 Main body part 276 Handle 277 Half body 278 Half body 280 columns 280a, 280d Sample Acquisition Device 282 End Cap 284 PVC tubing of first length 286 PVC tubing of second length 300 Third Module 310 The third controller 350 Cleaning Kit 352 Cassette, Manifold 354 Stopcock 356 Stopcock 358 Stopcock 360 Stopcock 362 Input line 364 Final product / collection container or bag 366 Line 368 Cleaning solution line 370 Resuspension solution line 372 Line 374 Waste container or bag 376 Line 378 End cap 380 In-line drip chamber 382 Separation chamber 384 Line 386 Tubing tail 388 Hydrophobic filter 400 System 410 First bioreactor vessel 412 First port 414 First bioreactor line 416 Second port 418 Second bioreactor line<**********>420 Second bioreactor vessel 422 First port 424 First bioreactor line 426 Second port 428 Second bioreactor line 430 Bioreactor array 432 First bioreactor line valve 434 Second bioreactor line valve 436 First bioreactor line valve 438 Second bioreactor line valve 440 First fluid assembly 442 First fluid assembly line 444 Second fluid assembly 446 Second fluid assembly line 448 Sampling Assembly 448 Sampling Assembly 450 interconnection lines 452 Interconnection Line Valve 454 First pump, peristaltic pump, source pump 456 Second pump, circulation line pump, process pump 458 Sterile air source 460 sterile air source lines 462 Line Valve 464a~f Tubing Tail 466a~f First storage section 468a~f Tubing Tail Bulb 470a~d Tubing Tail 472a~d Second storage section 474a~e Tubing Tail Valve 476a~476d Sampling lines 478a~d Sample Line Valve 482 Filtration Line 484 filters 486 Upstream filtration line valve 488 Downstream filtration line valve 490 Waste line 492 Waste pump 500 Dosage Preparation Kit 502 Stopcock Manifold 504 Stopcock 506 Stopcock 508 Stopcock 510 Stopcock 512 Stopcock 514 Stopcock 516 Process Bags 518 Peristaltic pump tubing 520 culture medium lines 522 culture medium line 524 culture medium lines 526 Final Blend / Collection Bag 528 Line 530 Initial Product Bag 532 Line 534 Waste Bag 536 Line 538 Freezing Bag Connection Line 540 Freezing Bag Connection Line 542 Freezing Bag Connection Line 544 Freezing Bag Connection Line<00​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​ 644 cover 646 Support Posts 648 sensors 650 Seal Element 652 Bellows 654 Peripheral Channels 656 Drain hole 658 load cell 660 load cell 662 load cell 664 load cell 700 Bioprocessing Kit 702 Tray 704 Culture Vessel 706 Culture Vessel 708 Legs 709 First Window 710 Legs 711 Second Window 712 Valve Manifold 714 Peristaltic pump tubing segment 716 Peristaltic pump tubing segment 718 Peristaltic pump tubing segment 720 Tubing Organizer Cards 722 Sampling Card 726 Tubing Tail 730 Main body part 732 aisle 734 Tubing retaining element 736 Plate body 738 Tubing Retention Channel 740 Mounting and / or positioning aperture 742 Clearance, relaxation area 744 Main body part 746 Manifold 748 Sampling Tubing Tail 750 Feedline 752 Return Line 754 Mounting and / or positioning aperture 756 Engagement Features / Surface 758 Sensors 760 Engagement structure 762 Pivot pump shoe 764 base 766 Lid 768 Membrane 770 Gasket 772 Enhanced Support 774 Entrance Port 776 Exit Port 777 Ventport 778 Corner 780 Location / Retention Hole 781 Heat Staking Section 782 Location / Retention Hole 784 Heat Staking Pins 786 Hole 788 Flange area 790 Rib 791 Pinwell 792 Pinwell 793 Pinwell 794 Pinwell 796 IR sensor window 798 Sensorwell 799 opening 800 Magnetic Cell Isolation Kit 802 Cassette, Manifold 804 First stopcock 806 Second stopcock 808 Third stopcock 810 Fourth stopcock Line 811 Line 812 813 Collection Bag Line 814 815 Tubing Tail 816 Tubing Tail 817 Tubing Tail 818 Tubing Tail Line 819 820 Negative Fraction Bags 821 Second Manifold 822 First stopcock 823 Second stopcock 824 Third stopcock 825 Fourth stopcock 826 Final Collection / Transfer Bag 827 Process Bags / Incubation Bags 828 Line 829 Inline Drip Chamber 830 Branch Line 831 Branch Line Line 832 833 Branch Line Line 834 835 Branch Line 836 Waste Bags 837 Spare bag 838 Sampling Pillow 839 Filter 840 Separation Chamber Line 841 842 Peristaltic pump tubing 843 Drip Chamber 844 Sterile air filter 845 line 846 Process Bags 870 Base 872 Fulcrum 873 Fulcrum axis line 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 Louvers, air passages 912 Louvers, air passages 914 Temperature Sensor 915 Recirculation Chamber 916 Carbon Dioxide Sensor 918 Carbon Dioxide Supply Unit 920 Carbon Dioxide Control Valve 922 Gasport 924 Recirculating Air Flow 926 Ducting Features 928 Dwelling turbulence 930 Recessed area 932 Vent opening 950 Flow-through sensing chamber 952 First Plate 954 Second plate 956 Fluid Channels 957 Notch 958 First port 959 tabs 960 Second port 961 Mounting and positioning holes, mounting aperture 962 Sensing Locations 964 Sensing Locations 966 Sensing Locations 968 Sensor 970 electrode 972 Flange 974 Miller 976 Snap Pin 978 First electrochemical sensing instrument 980 Second electrochemical sensing instrument 982 pins 984 Reflective light equipment 986 First fluorescent device 988 Second fluorescent device 990 Transmitted light / backscattered light instrument 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

Claims

1. A base and, A motor mounted on the aforementioned base, the motor having an eccentric roller driven by the motor, A rocking plate in contact with the eccentric roller, configured to receive a bioreactor vessel on it, In a rocking mechanism for a bioreactor vessel, The motor drives the eccentric roller, transmits force to the lower side of the oscillating plate, and is capable of controlling the tilting of the oscillating plate and the bioreactor vessel. The rocking plate includes a plurality of mounting pins for holding the bioreactor vessel on the rocking plate, The plurality of mounting pins maintain the bioreactor vessel in a vertical relationship, spaced apart from the rocking plate, and the rocking mechanism allows the bottom surface of the bioreactor vessel to be accessible for heat transfer and / or ventilation.

2. The oscillation mechanism according to claim 1, wherein the plurality of mounting pins are at least four mounting pins configured to engage with the bioreactor vessel adjacent to the corners of the bioreactor vessel.

3. The rocking mechanism according to claim 1, further comprising a tilt sensor connected to the rocking plate, the tilt sensor configured to measure the tilt angle of the rocking plate.

4. The rocking mechanism according to claim 1, further comprising at least one load cell associated with the base, which allows for the measurement of the mass of the bioreactor vessel received on the rocking plate.

5. The rocking mechanism according to claim 1, further comprising a pivot point that defines the pivot axis of the rocking plate, the pivot point on which the rocking plate is supported.

6. The rocking mechanism according to claim 1, further comprising a compression spring configured to maintain contact between the rocking plate and the eccentric roller.

7. The rocking mechanism according to claim 1, wherein the eccentric roller is a circular roller configured to move along an eccentric path.

8. The rocking mechanism according to claim 1, further comprising a load cell associated with at least one of the mounting pins.

9. A bioprocessing system, Bass and, The pivot point attached to the aforementioned base, A rocking plate received on the aforementioned pivot point, and configured to pivot on the aforementioned pivot point, The eccentric roller in contact with the lower side of the oscillating plate, A motor that drives the eccentric roller and causes the eccentric roller to exert a force on the lower side of the oscillating plate, the motor configured to pivot the oscillating plate around the pivot point, A bioreactor vessel is received on the aforementioned rocking plate, A plurality of mounting pins extending from the rocking plate and supporting the bioreactor vessel in a vertical relationship spaced apart with respect to the rocking plate, the plurality of mounting pins enabling the bottom surface of the bioreactor vessel to be accessible for heat transfer and / or ventilation, A bioprocessing system comprising [the specified element].

Images