Disposable kit and culture vessel for biotreatment system

Abstract

A disposable kit for a biological treatment system includes a tray having a spine having a first window and a second window; at least one biological treatment vessel received within the tray; a pinch valve manifold removably received by the ridge within the first window; a first conduit section removably received by the ridge within the second window; and a second conduit section removably received by the ridge within the second window.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application Serial No. 63 / 352,483, filed on June 15, 2022, which is hereby incorporated by reference in its entirety. Technical Field

[0003] Embodiments of the present invention generally relate to bioprocessing systems and methods, and more particularly, to a disposable bioprocessing kit and culture vessel for use in a bioprocessing system. Background Art

[0004] Various medical therapies involve the extraction, cultivation and expansion of cells used in downstream treatment processes. For example, chimeric antigen receptor (CAR) T cell therapy is a cell therapy that redirects the patient's T cells to specifically target and destroy tumor cells. The basic principle of CAR-T cell design involves recombinant receptors that combine antigen binding and T cell activation functions. The general premise of CAR-T cells is to artificially produce T cells for markers found on cancer cells. Scientists can remove T cells from the human body, genetically modify them, and put them back into the patient's body to make them attack cancer cells. CAR-T cells can be derived from the patient's own blood (autologous), or from another healthy donor (allogeneic).

[0005] The first step in the production of CAR-T cells involves removing blood from a patient and isolating white blood cells using apheresis (e.g., leukapheresis). After a sufficient amount of white blood cells have been harvested, the leukapheresis product is enriched for T cells, which involves depleting unwanted cell types. Then, if desired, a specific antibody conjugate or marker can be used to isolate a T cell subpopulation with a specific biomarker from the enriched subpopulation.

[0006] After the separation of target T cells, the cells are activated in an environment in which they can actively proliferate. For example, magnetic beads coated with anti-CD3 / anti-CD28 monoclonal antibodies or cell-based artificial antigen presenting cells (aAPC) can be used to activate cells, and the magnetic beads can be removed from the culture using magnetic separation. Then, CAR gene transduction T cells are used by integrating gamma retrovirus (RV) or lentivirus (LV) vectors. Viral vectors use viral mechanisms to attach to patient cells, and when entering cells, the vector introduces genetic material in the form of RNA. In the case of CAR-T cell therapy, this genetic material encodes CAR. RNA is reverse transcribed into DNA and permanently integrated into the genome of patient cells; thereby allowing CAR expression to be maintained when cells divide and grow in large quantities in bioreactors. Then, CAR is transcribed and translated by patient cells, and CAR is expressed on the cell surface.

[0007] After the T cells are activated and transduced with a viral vector encoding a CAR, the cells are expanded in large quantities in a bioreactor to achieve the desired cell density. After expansion, the cells are harvested, washed, concentrated, and formulated for infusion into the patient.

[0008] Existing systems and methods for manufacturing infusible dosed CAR T cells typically require many complex operations involving a large number of human touch points, which increases the time of the overall manufacturing process and increases the risk of contamination. Although recent efforts to automate the manufacturing process have eliminated some human touch points, these systems may still have problems with high costs, inflexibility, and workflow bottlenecks. In particular, systems that utilize increased automation are very expensive and inflexible because they require consumers to adapt their processes to the specific equipment of the system. WIPO International Publication No. WO 2019 / 106207 (which is hereby incorporated by reference herein) discloses systems and methods for bioprocessing that have successfully addressed many of the shortcomings of the prior art.

[0009] However, in view of the foregoing, there exists a need for bioprocessing systems and methods that improve upon the teachings contained in the '207 publication in terms of overall functionality, flexibility, adaptability, and ease of use. Summary of the invention

[0010] The following summarizes certain embodiments that are comparable in scope to the original claimed subject matter. These embodiments are not intended to limit the scope of the claimed subject matter, but rather, these embodiments are intended only to provide a brief overview of possible embodiments. In fact, the present disclosure may encompass various forms that may be similar or different from the embodiments set forth below.

[0011] In one embodiment, a kit for magnetic cell separation is provided. The kit includes: a first plug manifold having at least four plugs; a separation chamber configured for use with a centrifugal processing chamber of a cell processing device, the separation chamber being in fluid communication with the first plug manifold; a mixing bag configured for use with a heating / cooling mixing chamber of the cell processing device, the mixing bag being in fluid communication with the first plug manifold; a second plug manifold having at least four plugs, the second plug manifold being in fluid communication with the first plug manifold; a magnetic cell separation holder in fluid communication with the second plug manifold, the magnetic cell separation holder being configured for use with a magnetic field generator of the magnetic cell separation device; and a plurality of cell processing bags in fluid communication with the first plug manifold and / or the second plug manifold.

[0012] In another embodiment of the present invention, a method for magnetic cell separation using a disposable kit is provided. The method comprises the following steps: engaging a first plug manifold having at least four plugs with a plug manifold interface of a cell processing device; placing a separation chamber in a centrifugal processing chamber of the cell processing device, the separation chamber being in fluid communication with the first plug manifold; placing a mixing bag in a heating / cooling mixing chamber for cell processing, the mixing bag being in fluid communication with the first plug manifold; engaging a second plug manifold with a plug manifold interface of a magnetic cell separation device; and inserting a magnetic cell separation holder into a slot of the magnetic cell separation device, the magnetic cell separation holder being in fluid communication with the second plug manifold. The magnetic cell separation device is configured to generate a magnetic field for holding beads-bound cells in the magnetic cell separation holder when the magnetic cell separation holder is received in the slot.

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

[0014] In another embodiment, a method for isolating target cells is provided. The method comprises the following steps: 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 through a flow path within the magnetic field multiple times to hold the bead-bound target cells in an area of ​​the flow path within the magnetic field.

[0015] In another embodiment, a device for magnetic cell separation is provided. The device includes: a plug manifold interface located on a base and configured to receive a plug manifold of a cell processing kit; a magnetic field generator located in the base; and a slot formed in the base, the slot being configured to removably receive a magnetic cell separation holder and selectively bring the holder into operative contact 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, the housing including a centrifugal processing chamber, a pump assembly, a plug manifold interface configured to receive a plug manifold of a removable cell processing kit, a heating / cooling mixing chamber, and a magnetic separation module (IM). The IM includes: a base; an IM plug manifold interface on the base, the IM plug manifold interface configured to receive the plug manifold of the removable cell processing kit; a magnetic field generator located in the base; and a notch formed in the base, the notch configured to removably receive a magnetic cell separation holder and selectively operatively contact the holder with the magnetic field generator.

[0017] In another embodiment, a method for magnetically separating cells is provided. The method includes the following steps: inserting a magnetic cell separation holder into a slot of a separation device; moving a magnetic field generator of the separation device from a retracted position to an engaged position, in which the magnetic field generated by the magnetic field generator does not act on the magnetic cell separation holder so as to hold the bead-bound cells in the magnetic cell separation holder, and in which the magnetic field generated by the magnetic field generator is sufficient to hold the bead-bound cells in the magnetic cell separation holder; and flowing a bead-bound cell population into the magnetic cell separation holder to capture the bead-bound cells in the magnetic cell separation holder.

[0018] In yet another embodiment, a method for bioprocessing is provided. The method includes the following steps: providing a bioprocessing system having a first bioreactor vessel and a second bioreactor vessel; activating a cell population in the first bioreactor vessel; genetically modifying the cell population to produce a genetically modified cell population; and expanding the genetically modified cell population in the first bioreactor vessel and the second bioreactor vessel.

[0019] In another embodiment, a method for bioprocessing is provided. The method includes the following steps: providing a bioprocessing system having a first bioreactor vessel and a second bioreactor vessel; activating, genetically modifying, and expanding a first cell population in the first bioreactor vessel; and activating, genetically modifying, and expanding a second cell population in the first bioreactor vessel.

[0020] In another embodiment, a method for bioprocessing is provided. The method includes the following steps: providing a bioprocessing system having a first bioreactor vessel and a second bioreactor vessel; activating a cell population in the first bioreactor vessel; transferring the cell population out of the first bioreactor vessel; genetically modifying the cell population to produce a genetically modified cell population; transferring the genetically modified cell population to at least one of the first bioreactor vessel and the second bioreactor; and amplifying the genetically modified cell population 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; a process drawer that can be received in the housing and can be moved between a closed position and an open position, the process drawer configured to receive at least one culture vessel therein; and a cabinet positioned in a vertical stacking relationship with the housing, the cabinet including at least one vertical storage drawer slidably received in 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 within the tray; a valve manifold mounted to a rear portion of the tray and configured to engage with an array of linear actuators of the bioprocessing apparatus; at least one peristaltic pump tube configured to engage with a peristaltic pump of the bioprocessing apparatus; and a tubing organizer that holds a plurality of tubes fluidically 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 method of bioprocessing is provided. The method includes the following steps: positioning a disposable bioprocessing kit within a process drawer of a bioprocessing apparatus such that a culture vessel of the disposable kit is received atop a rocking assembly of the bioprocessing apparatus; connecting a tubing organizer to a door of a cabinet of the bioprocessing apparatus, the tubing organizer holding a plurality of tubing tails for fluid connection to a plurality of media bags and / or reagent bags installed in the cabinet; and fluidly connecting at least one of the plurality of tubing tails to at least one of the plurality of media bags and / or reagent bags.

[0024] In another embodiment, a rocking mechanism for a bioreactor vessel is provided. The rocking mechanism 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 configured to receive the bioreactor vessel thereon. The motor is controllable to drive the eccentric roller to transmit a force against the underside of the rocking plate to tilt the rocking plate and the bioreactor vessel.

[0025] In another embodiment, a method of bioprocessing is provided. The method includes the steps of receiving a bioreactor vessel atop a wobble plate, and actuating a motor to cause an eccentric roller to exert a force on the underside of the wobble plate to tilt the wobble 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 wobble plate received on top of the fulcrum and configured to pivot thereon; an eccentric roller in contact with the underside of the wobble plate; a motor configured to drive the eccentric roller so that the eccentric roller applies a force on the underside of the wobble plate to pivot the wobble plate about the fulcrum; and a bioreactor vessel received on top of the wobble plate.

[0027] In another embodiment, a method of bioprocessing is provided. The method includes the steps of providing a bioreactor vessel having a gas permeable, liquid impermeable membrane; initiating a gas flow; and passing the gas flow across a bottom surface of the membrane to induce 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 a bottom surface of a 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 and a pair of openings in the tray adjacent to the top of the pair of support legs; at least one bioreactor vessel positioned within the disposable tray at a vertical position corresponding to the vertical position of the pair of openings; and at least one fan configured to circulate atmospheric air from below the bioreactor vessel upward and through a first opening of the pair of openings, across a bottom surface of a gas permeable, liquid impermeable membrane of the bioreactor vessel, through a second opening of the pair of openings, and back below 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 stakes, and a gas permeable, liquid impermeable membrane sandwiched between the base and the lid and held in place by the plurality of heat stakes.

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

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

[0033] In another embodiment, a method for assessing the integrity of a bioprocessing system is provided. The method includes the steps of: pouring a liquid from a first container through a second container to a third container; measuring the mass of the second container during the pouring step; and generating a notification indicating the presence of a leak if the mass of the second container changes by more than a threshold.

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

[0035] In another embodiment, a bioprocessing system is provided. The bioprocessing system includes: a source pump configured to pump a first fluid from a source to a bioprocessing vessel through a first flow line; a process pump configured to circulate fluid out of the bioprocessing vessel through a circulation line and through a filter line; a waste pump configured to pump waste removed by a filter along the filter line to a waste reservoir through a waste line; a first valve configured to separate the bioprocessing vessel from the first flow line, the filter line, and the waste line; and a controller configured to control one of the source pump and the process pump to pressurize at least one of the first flow line and / or the circulation line, and monitor a pressure decay within at least one of the first flow line and / or the circulation line.

[0036] In yet another embodiment, a sensing chamber for a bioprocessing system is provided. The sensing chamber includes a front plate, a rear plate, at least one fluid channel between the front plate and the rear plate, a first port in fluid communication with the fluid channel and allowing fluid to flow into the fluid channel, and a second port in fluid communication with the fluid channel and allowing fluid to flow out of the fluid channel. At least one fluid channel includes multiple sections that allow multiple parameters of the fluid to be sensed using 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 technology, and the second sensing device is configured to sense at least one parameter of the fluid using a second sensing technology. The first sensing technology is different from the second sensing technology.

[0037] In one embodiment, a method for sensing a parameter of a fluid is provided, comprising the steps of: allowing a fluid to flow from a bioprocessing vessel into a fluid channel of a sensing component; electrochemically analyzing the fluid in the fluid channel via contact of the fluid with at least one electrode; 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; and a flow-through sensing chamber having a front plate and a rear plate, a fluid channel between the front plate and the rear plate, a first port in fluid communication with the fluid channel and allowing fluid to flow into the fluid channel, and a second port in fluid communication with the fluid channel and allowing fluid to flow out of the fluid channel. The flow-through sensing chamber is mounted to the tray.

[0039] In another embodiment, a disposable kit for a bioprocessing system is provided. The disposable kit includes: a tray having a ridge, the ridge having a first window and a second window; at least one bioprocessing vessel received in the tray; a pinch valve manifold removably received by the ridge in the first window; a first tubing segment removably received by the ridge in the second window; and a second tubing segment removably received by the ridge in the second window.

[0040] In another embodiment, a culture vessel for a bioprocessing system is provided. The culture vessel includes: a base; a lid connected to the base, the lid having a vent opening; and a gas permeable, liquid impermeable membrane sandwiched between the base and the lid. The lid includes a baffle adjacent to the vent port. The baffle is configured to prevent fluid within the bioprocessing vessel from contacting the vent port. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] The present invention will be better understood by reading the following description of non-limiting embodiments with reference to the accompanying drawings, in which:

[0042] Figure 1is a schematic illustration of a biological processing system according to an embodiment of the present invention.

[0043] Figure 2 is a schematic illustration of a biological treatment system according to another embodiment of the present invention.

[0044] Figure 3 is a schematic illustration of a cell processing and separation system according to an embodiment of the present invention.

[0045] Figure 4 yes Figure 3 Perspective view of the separation module of the cell processing and separation system.

[0046] Figure 5 is a top plan view of the separation module.

[0047] Figure 6 is a perspective view of a stopcock manifold interface of a separation module according to an embodiment of the present invention.

[0048] Figure 7 is an enlarged perspective view of the stopcock manifold interface.

[0049] Figure 8 is another perspective view of the separation module.

[0050] Fig. 9 is a rear perspective view of the separation module.

[0051] Fig.10 It is a front exploded perspective view of the separation module.

[0052] Fig.11 It is a rear exploded perspective view of the separation module.

[0053] Fig.12 is an enlarged perspective view of the bubble sensor assembly of the separation module.

[0054] Fig.13 is a side cross-sectional view of the bubble sensor assembly.

[0055] Fig.14 is a front perspective view of a magnetic field generator assembly of a separation module according to an embodiment of the present invention.

[0056] Fig.15 is another front perspective view of the magnetic field generator assembly.

[0057] Fig.16 is a rear perspective view of the magnetic field generator assembly.

[0058] Fig.17 is a rear perspective view of a portion of a magnetic field generator assembly.

[0059] Fig.18is a simplified front perspective view of a bracket of a magnetic field generator assembly.

[0060] Fig.19 is a simplified rear perspective view of the bracket.

[0061] Fig. 20 is a cross-sectional view of the magnetic field generator assembly in the retracted position.

[0062] Fig.21 is a cross-sectional view of a magnetic field generator assembly with a separator retainer received in a notch of a separator module.

[0063] Fig. 22 is a cross-sectional view of the magnetic field generator assembly in an extended position.

[0064] Fig.23 is a cross-sectional view of a magnetic field generator assembly in an extended position within a breakaway retainer received in a slot.

[0065] Fig.24 is a cross-sectional view of the magnetic field generator assembly in an extended position with the breakaway retainer locked within the notch.

[0066] Fig.25 is a cross-sectional view of the magnetic field generator assembly illustrating a misaligned position of a separation holder.

[0067] Fig.26 is a method for communicating with Figure 4 A perspective view of the separation module used together with the magnetic cell separation holder.

[0068] Fig. 27 yes Fig.26 Exploded perspective view of the magnetic cell separation holder.

[0069] Fig.28 yes Fig.26 Side elevation view of the column of the magnetic cell separation holder.

[0070] Fig.29 yes Fig.28 Exploded view of a column.

[0071] Fig.30 is a perspective view illustrating the insertion of a magnetic cell separation holder into a slot in a separation module.

[0072] Fig.31 According to another embodiment of the present invention, Figure 4 A perspective view of the separation module used together with the magnetic cell separation holder.

[0073] Fig.32 yes Fig.31Perspective view of the magnetic cell separation holder.

[0074] Fig.33 According to aspects of the present disclosure Fig.31 A top plan view of a magnetic cell separation holder illustrating the magnetic field distribution of the magnetic field generator.

[0075] Fig.34 is a simplified perspective view of a magnetic cell separation holder according to another embodiment of the present invention.

[0076] Fig.35 is a simplified perspective view of a magnetic cell separation holder according to yet another embodiment of the present invention.

[0077] Fig.36 is used with Figure 3 Schematic illustration of a disposable kit for washing and concentrating a cell product used in conjunction with a processing device.

[0078] Fig.37A is used with Figure 3 A schematic illustration of a disposable kit for magnetic cell separation used with a processing device and a separation module is shown in Figure 3 Installation on processing equipment and separation modules.

[0079] Fig.37B yes Fig.37A A schematic representation of a disposable kit for magnetic cell separation is shown in Figure 3 Installation on processing equipment and separation modules.

[0080] Fig.37C is a schematic illustration of a disposable kit for magnetic cell separation according to another embodiment of the present invention, the disposable kit being shown mounted on Figure 3 on processing equipment and separation modules.

[0081] Fig.38A It is shown in Figure 3 Utilize on processing equipment and separation modules Fig.37A and Fig.37B Flowchart of the magnetic cell separation workflow / process for the disposable kit.

[0082] Fig.38B It is shown in Figure 3 Utilize on processing equipment and separation modules Fig.37C Flowchart of the magnetic cell separation workflow / process for the disposable kit.

[0083] Fig.39 is used with Figure 3 Schematic illustration of a disposable kit for dosage preparation / formulation used with a processing device and a separation module.

[0084] Fig.40 yes Fig.39 A schematic diagram of a disposable kit for dosage preparation / dispensing is shown in Figure 3 Installation on processing equipment and separation modules.

[0085] Fig.41 It is shown in Figure 3 Utilize on processing equipment and separation modules Fig.39 Flowchart of the workflow / process for the dosing preparation of a single-use kit.

[0086] Fig.42 is a perspective view of a bioprocessing system / apparatus showing process drawers and cabinets in a closed position according to an embodiment of the present invention.

[0087] Fig.43 yes Fig.42 Another perspective view of a bioprocessing apparatus showing the cabinet in an open position.

[0088] Fig.44 yes Fig.42 A perspective view of a cabinet of a bioprocessing apparatus illustrating an extended position of a vertical drawer of the cabinet.

[0089] Fig.45 This is a front elevation view of the cabinet.

[0090] Fig.46 yes Fig.42 A perspective view of a housing and a process drawer of a bioprocessing apparatus illustrating an open position of the process drawer.

[0091] Fig.47 yes Fig.42 Top plan view of a process drawer of a bioprocessing facility.

[0092] Fig.48 is a perspective view of a pair of platform rocker assemblies of a process drawer according to an embodiment of the present invention.

[0093] Fig.49 yes Fig.42 Perspective view of the waste drawer of a biological processing facility.

[0094] Fig.50 is used with Fig.42 A perspective view of a disposable bioprocessing kit for use with a bioprocessing apparatus.

[0095] Fig.51 yes Fig.50 A rear perspective view of a tray of a disposable bioprocessing kit.

[0096] Fig.52 yes Fig.50 Perspective view of the anchor comb of the disposable bioprocessing kit.

[0097] Fig.53 yes Fig.52 Front elevation view of the anchor comb.

[0098] Fig.54 yes Fig.50 A perspective view of the tubing organizer of a disposable bioprocessing kit.

[0099] Fig.55 yes Fig.50 A perspective view of a sampling card of a disposable bioprocessing kit.

[0100] Fig.56 yes Fig.53 Front elevation view of the sampling card.

[0101] Fig.57 is a perspective view illustrating the insertion of trays and culture vessels of a disposable set in a process drawer of a bioprocessing apparatus.

[0102] Fig.58 is a side cross-sectional view illustrating trays and culture vessels of a disposable kit received in a process drawer of a bioprocessing facility.

[0103] Fig.59 is a top view of a process drawer of a bioprocessing apparatus showing various alignment features and sensors of the bioprocessing apparatus.

[0104] Fig.60 is an enlarged front perspective view of a peristaltic pump assembly of a bioprocessing apparatus illustrating alignment and engagement features of the peristaltic pump assembly.

[0105] Fig.61 is an enlarged rear perspective view of a peristaltic pump assembly of a bioprocessing apparatus illustrating alignment and engagement features of the peristaltic pump assembly.

[0106] Fig.62 is an enlarged perspective view of a linear actuator array of a bioprocessing apparatus showing alignment and engagement features of the linear actuator array.

[0107] Fig.63 is a perspective cross-sectional view of a process drawer of a bioprocessing facility.

[0108] Fig.64 yes Fig.50 An exploded perspective view of the culture vessels of the disposable bioprocessing kit.

[0109] Fig.65 yes Fig.64 A bottom plan view of a culture dish.

[0110] Fig.66 yes Fig.42 A perspective view of a portion of a rocking assembly of a bioprocessing apparatus

[0111] Fig.67 yes Fig.66 Another perspective view of the swing assembly.

[0112] Fig.68 yes Fig.66 Another perspective view of the rocking assembly of FIG. 1 , illustrating the engagement of the rocking assembly with the culture vessel.

[0113] Fig.69 It is a graphic Fig.66 Schematic diagram of the operation of the swing assembly.

[0114] Fig.70 yes Fig.42 Cross-sectional view of a process drawer of a bioprocessing facility.

[0115] Fig.71 yes Fig.42 Another cross-sectional view of a process drawer of a bioprocessing apparatus showing the recirculating air flow path.

[0116] Fig.72 yes Fig.42 Another cross-sectional view of a process drawer of a bioprocessing apparatus showing turbulent recirculating air flow at the interface with the culture vessels.

[0117] Fig.73 yes Fig.50 A perspective cross-sectional view of a tray of a disposable bioprocessing kit illustrating the recirculating air flow path.

[0118] Fig.74 is a perspective cross-sectional view of the process drawers and trays of a bioprocessing facility illustrating the recirculating air flow paths.

[0119] Fig.75 is another perspective cross-sectional view of a process drawer and tray of a bioprocessing apparatus illustrating the recirculating air flow path.

[0120] Fig.76 is another perspective cross-sectional view of a process drawer and tray of a bioprocessing apparatus illustrating the recirculating air flow path.

[0121] Fig.77 According to an embodiment of the present invention Fig.42 Rear perspective view of a flow-through sensing chamber of a bioprocessing device.

[0122] Fig.78 yes Fig.77 A front perspective view of a flow-through sensing chamber.

[0123] Fig.79 yes Fig.77 A cross-sectional perspective view of a flow-through sensing chamber.

[0124] Fig.80 yes Fig.77 A perspective view of the back plate of the flow-through sensing chamber.

[0125] Fig.81 yes Fig.50 An enlarged perspective view of the backbone of a disposable bioprocessing kit illustrating the location of the flow-through sensing chamber.

[0126] Fig.82 yes Fig.50 Another enlarged perspective view of the backbone of a disposable bioprocessing kit illustrating the location of the flow-through sensing chamber.

[0127] Fig.83 is a schematic illustration showing the integration of a flow-through sensing chamber with various sensing devices.

[0128] Fig.84 is another schematic illustration showing the integration of a flow-through sensing chamber with various sensing devices.

[0129] Fig.85 is a diagram illustrating an embodiment of the present invention Fig.42 Block diagram of the fluid flow architecture of a bioprocessing device.

[0130] Fig.86 yes Fig.85 Detailed view of a portion of a block diagram illustrating a first fluidic component of a fluid flow architecture.

[0131] Fig.87 yes Fig.85 Detailed view of a portion of a block diagram illustrating a second fluidic component of the fluid flow architecture.

[0132] Fig.88 yes Fig.85 Detailed view of a portion of a block diagram illustrating the sampling components of the fluid flow architecture.

[0133] Fig.89 yes Fig.85 Detailed view of a portion of a block diagram illustrating the filtration flow path of the fluid flow architecture.

[0134] Fig.90 is a diagram illustrating the use of an embodiment according to the present invention Fig.42 A flow chart of a method for bioprocessing performed by a bioprocessing apparatus.

[0135] Fig.91 is a diagram illustrating the use of an embodiment according to the present invention Fig.42 A flow chart of a method for bioprocessing performed by a bioprocessing apparatus.

[0136] Fig.92 is a diagram illustrating the use of an embodiment according to the present invention Fig.42 A flow chart of a method for bioprocessing performed by a bioprocessing apparatus.

[0137] Fig.93 is a diagram illustrating an embodiment of the present invention Fig.42 Block diagram of the fluid flow architecture of a bioprocessing device.

[0138] Fig.94 is a diagram illustrating another embodiment of the present invention Fig.42 Block diagram of the fluid flow architecture of a bioprocessing device.

[0139] Fig.95 is a diagram illustrating another embodiment according to the present invention Fig.42 Block diagram of the fluid flow architecture of a bioprocessing device.

[0140] Fig.96 is a diagram illustrating another embodiment according to the present invention Fig.42 Block diagram of the fluid flow architecture of a bioprocessing device.

[0141] Fig.97 According to another embodiment of the present invention, Fig.42 A perspective view of a disposable bioprocessing kit for use with a bioprocessing apparatus.

[0142] Fig.98 yes Fig.97 A perspective view of the tray of a disposable bioprocessing kit.

[0143] Fig.99 yes Fig.98 A top plan view of a pallet.

[0144] Fig.100 yes Fig.98 A bottom plan view of a pallet.

[0145] Fig.101 yes Fig.98 A rear plan elevation view of a pallet showing the spine frame of the pallet.

[0146] Fig.102 yes Fig.98 Another rear elevation view of the tray showing the pinch valve manifold and pump tubing segments connected to the spine frame.

[0147] Fig.103 is an elevation view of the culture vessel facing side of the manifold showing the ridges and pinch valves.

[0148] Fig.104 is a rear elevation view of a pinch valve manifold of a culture vessel showing the back plate of the pinch valve manifold.

[0149] Fig.105 is an elevation view of the membrane of the pinch valve manifold.

[0150] Fig.106 is a front elevation view of a pinch valve manifold of a culture vessel showing the fluid plates of the pinch valve manifold.

[0151] Fig.107 is an interior view of a fluid plate showing the fluid flow pathways and fluid ports of the fluid plate.

[0152] Fig.108 is a rear elevation view of the spine showing the pinch valve manifold with the back plate removed.

[0153] Fig.109 yes Fig.97 A perspective view of the left tube organizer card and the right tube organizer card of the disposable kit of FIG.

[0154] Fig.110 is a plan view of the left pipe organizer card.

[0155] Fig.111 is a plan view of the right pipe organizer card.

[0156] Fig.112 is a perspective view of a drip tray of a bioprocessing system showing left and right tube organizer cards attached to the drip tray.

[0157] Fig.113 yes Fig.97 A perspective view of a sampling card of a disposable kit.

[0158] Fig.114 yes Fig.113 A perspective view of the sample manifold of the sampling card.

[0159] Fig.115 yes Fig.97 A perspective view of the base anchor of the disposable kit.

[0160] Fig.116 is another perspective view of the base anchor.

[0161] Fig.117 yes Fig.97 A perspective view of the pivoting anchor of the disposable kit.

[0162] Fig.118 is another perspective view of the pivot anchor.

[0163] Fig.119 yes Fig.97 A perspective view of a disposable bioprocessing kit of FIG. 1 showing the installation of tubing / fluid flow lines.

[0164] Fig.120 yes Fig.97 An exploded top perspective view of a culture vessel of a disposable bioprocessing kit.

[0165] Fig.121 yes Fig.120 Bottom perspective view of a culture dish.

[0166] Fig.122 is a cross-sectional perspective view of a culture vessel showing a vent port with a barbed connector.

[0167] Fig.123 is a cross-sectional perspective view of a culture vessel according to another embodiment of the present invention showing a female gel port. DETAILED DESCRIPTION

[0168] Hereinafter, reference will be made in detail to exemplary embodiments of the present invention, examples of which are illustrated in the accompanying drawings. Wherever possible, the same reference characters are used throughout the drawings to refer to the same or like parts.

[0169] As used herein, the terms "flexible" or "collapsible" refer to structures or materials that are pliable or capable of bending without breaking, and may also refer to compressible or expandable materials. An example of a flexible structure is a bag formed from polyethylene film. The terms "rigid" and "semi-rigid" are used interchangeably herein to describe a "non-collapsible" structure, that is, a structure that does not fold, collapse, or otherwise deform to significantly reduce its elongated dimension under normal forces. Depending on the context, "semi-rigid" may also refer to a structure that is more flexible than a "rigid" element, such as a bendable tube or catheter, but is still a structure that does not collapse longitudinally under normal conditions and forces.

[0170] As used herein, the term "vessel" means a flexible bag, a flexible container, a semi-rigid container, a rigid container, or a flexible or semi-rigid conduit, as the case may be. As used herein, the term "vessel" is intended to cover bioreactor vessels having semi-rigid or rigid walls or partial walls, and other containers or conduits commonly used in biological or biochemical processes, including, for example, cell culture / purification systems, mixing systems, media / buffer preparation systems, and filtration / purification systems, such as chromatography and tangential flow filter systems, and their associated flow paths. As used herein, the term "bag" means a flexible or semi-rigid container or vessel, which is used, for example, as a containment device for various fluids and / or media.

[0171] As used herein, "fluidically coupled" or "fluidly connected" means that components of a system are capable of receiving or transferring fluids between components. The term fluid includes gases, liquids, or combinations thereof. As used herein, "electrically communicating" or "electrically coupled" means that certain components are configured to communicate with each other by direct or indirect signaling by way of direct or indirect electrical connections. As used herein, "operably coupled" refers to a connection that may be direct or indirect. The connection is not necessarily a mechanical attachment.

[0172] As used herein, the term "tray" refers to any object capable of at least temporarily supporting a plurality of components. The tray can be made of a variety of suitable materials. For example, the tray can be made of a cost-effective material suitable for sterilized and single-use disposable products.

[0173] As used herein, the term "functional closed system" refers to a plurality of components that make up a closed fluid path that may have an inlet port and an outlet port to add or remove fluid or air to or from the system without compromising the integrity of the closed fluid path (e.g., to maintain an internally sterile biomedical fluid path), whereby the ports may include, for example, a filter or membrane at each port to maintain sterile integrity when fluid or air is added to or removed from the system. Depending on a given embodiment, the components may include, but are not limited to, one or more conduits, valves (e.g., a multi-port shunt), vessels, receptacles, and ports.

[0174] Embodiments of the present invention provide systems and methods for manufacturing cell immunotherapy with biological samples (e.g., blood, tissue, etc.). In one embodiment, a method includes: genetically modifying a cell population in a bioreactor vessel to produce a genetically modified cell population; and amplifying the genetically modified cell population in the bioreactor vessel without removing the genetically modified cell population from the bioreactor vessel to produce a number of genetically modified cells sufficient for one or more doses used in cell therapy treatment. In certain embodiments, one or more of the methods may include: using magnetic beads or non-magnetic beads to activate cells in the same bioreactor vessel to produce an activated cell population before genetically modifying the cells; and washing the genetically modified cells on the bioreactor vessel to remove unwanted substances.

[0175] refer to Figure 1, a schematic diagram of a bioprocessing system 10 according to an embodiment of the present invention is illustrated. The bioprocessing system 10 is configured for use in the manufacture of cellular immunotherapy (e.g., autologous cellular immunotherapy), wherein, for example, a human blood, fluid, tissue, or cell sample is collected and a cellular therapy is produced from or based on the collected sample. One type of cellular immunotherapy that can be manufactured using the bioprocessing system 10 is a chimeric antigen receptor (CAR) T cell therapy, however, other cellular therapies may also be produced using the present invention or systems of aspects thereof without departing from the broader aspects of the present invention. Figure 1 As shown in the figure, the manufacture of CAR T cell therapy generally starts with collecting the patient's blood and separating lymphocytes by apheresis. Collection / single collection can be performed in a clinical setting, and the single collection product is then sent to a laboratory or manufacturing facility for the production of CAR T cells. In particular, once the single collection product is received for treatment, the desired cell population (e.g., white blood cells) is enriched or separated from the collected blood for the manufacture of cell therapy, and the target cells of interest are separated from the initial cell mixture. Then, the target cells of interest are activated, genetically modified to specifically target and destroy tumor cells, and amplified to achieve the desired cell density. After amplification, cells are harvested and a certain amount is prepared. Then, the preparation is usually cryopreserved and transported to a clinical setting for thawing, preparation, and ultimately infusion into the patient.

[0176] Further references Figure 1 , the bioprocessing system 10 of the present invention includes a plurality of different modules or subsystems, each of which is 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, which is configured to perform the steps of enrichment and separation; a second module 200, which is configured to perform the steps of activation, genetic modification and amplification; and a third module 300, which is configured to perform the steps of harvesting the amplified cell population. In one embodiment, each module 100, 200, 300 is communicatively coupled to a dedicated controller (e.g., communicatively coupled to the first controller 110, the second controller 210 and the third controller 310, respectively). The controllers 110, 210 and 310 are configured to provide substantially automated control of the manufacturing process within each module. Although the first module 100, the second module 200 and the third module 300 are illustrated as including a dedicated controller for controlling the operation of each module, it is contemplated that a master control unit can be used to provide global control of the three modules. Each module 100, 200, 300 is designed to work in conjunction with the other modules to form a single coherent bioprocessing system 10, as discussed in detail below.

[0177] By automating the processes within each module, the consistency of the products from each module can be improved and the costs associated with a large number of manual operations can be reduced. In addition, as discussed in detail below, each module 100, 200, 300 is essentially functionally closed, which helps to ensure patient safety by reducing the risk of external contamination, ensure regulatory compliance, and help avoid the costs associated with open systems. In addition, each module 100, 200, 300 is scalable to support both development at low patient numbers and commercial manufacturing at high patient numbers.

[0178] Further references Figure 1 , the specific way of dividing the process steps in different modules that each provide closed and automated bioprocessing allows efficient use of capital equipment to a degree not seen in the art so far. As will be appreciated, the step of amplifying the cell population to achieve the desired cell density before harvesting and preparation is typically the most time-consuming step in the manufacturing process, while the enrichment and separation steps, harvesting and preparation steps, and activation and genetic modification steps are much less time-consuming. Therefore, attempting to automate the entire cell therapy manufacturing process can also aggravate the bottleneck that hinders the workflow and reduces manufacturing efficiency in the process in addition to being logistically challenging. In particular, in a fully automated process, although the steps of enrichment, separation, activation, and genetic modification of cells can occur quite quickly, the amplification of genetically modified cells occurs very slowly. Therefore, the manufacture of cell therapy with a first sample (e.g., the blood of a first patient) will proceed quickly until the amplification step, which requires a lot of time to achieve the desired cell density for harvesting. In the case of a fully automated system, the entire process / system will be monopolized by the amplification equipment that performs the amplification of cells from the first sample, and the processing of the second sample may not begin until the entire system is vacated for use. In this regard, in the case of a fully automated bioprocessing system, the entire system is essentially offline and unavailable for processing a second sample until the entire cell therapy manufacturing process, from enrichment to harvesting / formulation, is completed for the first sample.

[0179] However, embodiments of the present invention allow for more than one sample (from the same or different patients) to be processed in parallel, providing a more efficient use of capital resources. As alluded to above, this advantage is a direct result of the particular way in which the process steps are divided into the three modules 100, 200, 300. With particular reference to Figure 2In one embodiment, a single first module 100 and / or a single third module 300 can be utilized in combination with multiple second modules (e.g., second modules 200a, 200b, 200c) in the bioprocessing system 12 to provide parallel and asynchronous processing of multiple samples from the same or different patients. For example, the first module 100 can be used to enrich and separate a first single collection product from a first patient to produce a first population of isolated target cells, and the first population of target cells can then be transferred to one of the second modules, such as module 200a, for activation, genetic modification, and amplification under the control of the controller 210a. Once the first population of target cells is transferred out of the first module 100, the first module can be used again to process a second single collection product from, for example, a second patient. Then, the second population of target cells generated in the first module 100 from the sample taken from the second patient can be transferred to another second module, such as second module 200b, for activation, genetic modification, and amplification under the control of the controller 201b.

[0180] Similarly, after the second population of target cells is transferred out of the first module 100, the first module can be used again to process a third single collection product from, for example, a third patient. Then, the third target population of cells generated from a sample taken from a third patient in the first module 100 can be transferred to another second module, such as the second module 200c, for activation, genetic modification, and amplification under the control of the controller 201c. In this regard, for example, the expansion of CAR-T cells for the first patient can occur simultaneously with the expansion of CAR-T cells for the second patient, the third patient, etc.

[0181] The method also allows post-processing to occur asynchronously as desired. In other words, the patient cells may not all grow at the same time. The culture may reach final density at different times, but the plurality of second modules 200 are not coupled, and the third module 300 may be used as desired. In the case of the present invention, although samples may be processed in parallel, they do not have to be carried out in batches.

[0182] Harvesting of the expanded cell populations from the second modules 200a , 200b , and 200c may also be accomplished using a single third module 300 , when each expanded cell population is ready for harvesting.

[0183] Thus, by separating the steps of activation, genetic modification, and expansion (which are the most time consuming and share certain operational requirements and / or require similar culture conditions) into independent, automated, and functionally closed modules, another system equipment for enrichment, separation, harvesting, and formulation is not occupied or offline while expansion of one cell population is performed. As a result, the manufacture of multiple cell therapies can be performed simultaneously, thereby maximizing the utilization of equipment and floor space and improving the efficiency of the overall process and facility. It is contemplated that additional second modules 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 allows for plug-and-play-like functionality, which enables manufacturing facilities to be easily scaled up or down.

[0184] In one embodiment, the first module 100 may be any system or device capable of producing a target population of enriched and separated cells for use in a biological process (such as the manufacture of immunotherapy and regenerative medicine) from a single collection product taken from a patient. The third module 300 may be any system or device capable of harvesting and / or preparing CAR-T cells or other modified cells produced by the second module 200 for infusion into a patient, for use in cellular immunotherapy or regenerative medicine. In certain embodiments, the first module 100 and the third module 300 are configured similarly or identically so that the first module 100 may first be used to enrich and separate cells (which are then transferred to the second module 200 for activation, transduction and amplification (and in some embodiments, harvesting)), and then also for cell harvesting and / or preparation at the end of the process. In this regard, in some embodiments, the same device can be used for front-end cell enrichment and separation steps, as well as back-end harvesting and / or preparation steps.

[0185] Reference now Figure 3, illustrating an exemplary configuration of the first module 100 (and in some embodiments, the third module 300). In one embodiment, the first module 100 (and the third module 300) includes a processing device 102 and a separation module 104. In one embodiment, the processing device 102 and the separation module 104 may be mechanically interconnected to each other, such as via a bracket 105 mounted to the corresponding bottom of the device. The processing device 102 may be, for example, a Sefia S-2000 cell processing instrument available from Cytiva. In one embodiment, the processing device may be configured to be the same or substantially similar to the device 900 disclosed in WIPO International Publication No. WO 2019 / 106207. Therefore, the processing device 102 includes a base 106, which accommodates a centrifugal processing chamber 108, a high dynamic range peristaltic pump assembly 111, a stopcock manifold interface 112, and a heating-cooling-mixing chamber (thermomixer) 114. As indicated below, the stopcock manifold interface 112 is configured to receive a single-use disposable kit specifically configured to perform cell concentration, platelet removal and separation based on density gradient, washing and / or final preparation, and provides a simple and reliable means of docking multiple fluid or gas pipelines together using, for example, Luer fittings. In the base 106 is a motor drivingly connected to a plurality of (in this case, four) output shafts, the output shaft being operable to move the stopcock of the disposable kit between an open position and a closed position under the control of a controller. In one embodiment, the pump assembly 111 is rated to provide a flow rate as low as about 3mL / min and as high as about 150mL / min. The processing device 102 may further include a set of sensors configured to monitor the various parameters of the various fluids of the device 102 itself and the various fluids disposed by the device 102.

[0186] like Figure 3 As further shown in the figure, the processing equipment 102 of the first module 100 and / or the third module 300 also includes a generally T-shaped hanger assembly 116, which extends from the base 106 and includes a plurality of hooks 118 for hanging a plurality of bags, which are used to accommodate or receive the fluid used in the biological treatment operation performed by the first or third module. In one embodiment, there may be six hooks. Each hook may include an integrated weight sensor or weighing sensor (not shown) for monitoring the weight of each vessel / bag. In one embodiment, the bag may be, for example, a sample source bag, a process bag, a separation buffer bag, a washing bag, one or more storage bags, a waste bag after separation, a washing waste bag, a medium bag, a release bag and / or a collection bag, depending on the specific process being performed. The processing equipment 102 also includes a centralized control unit, such as a controller 110, for performing one or more biological treatment operations in an automatic or semi-automatic manner according to (multiple) algorithms stored in a memory.

[0187] Further references Figure 3, and more specifically with reference to Figure 4-11 , showing a separation module 104 of the first module 100 and / or the third module 300. The separation module 104 includes a base / housing 130, a stopcock manifold interface 132 located on the base 130 and configured to receive a stopcock manifold of a single-use disposable cell processing kit, and a vertical aperture or slot 134 in the base configured to removably receive a magnetic cell separation holder 136 of the separation module 104, the purpose of which will be described below. The separation module 104 further includes a support rod 138 having one or more hooks 140 or pegs for hanging a fluid bag or vessel thereon. In one embodiment, the hook 140 may be configured with or connected to a weighing sensor for real-time quality monitoring of the contents of the bag. Although Figure 3 The separation module 104 is illustrated as having two hooks 140, but there may be more or less than two hooks. For example, in one embodiment, the separation module 104 has four hooks 140. In one embodiment, the inner surface of the housing 130 and / or its base structure may be coated or covered with a conductive paint or coating to shield, for example, EMC disturbances. In one embodiment, the housing 130 may be made of plastic, and the base structure supporting the housing may be metal, however in some embodiments, both the base structure and the housing may be formed of plastic or similar non-conductive materials.

[0188] In one embodiment, the separation module 104 includes a drip chamber holder 113 to insert and hold a drip chamber of a disposable bioprocessing kit (e.g., for washing, dosing preparation, formulation, and / or cell separation), as described below. In one embodiment, the drip chamber holder can accommodate different diameters or shapes to accommodate different versions of disposable kit drip chambers (e.g., Fig.36 The drip chamber 380 of the kit 350 shown in FIG. Fig.37A and Fig.37B In one embodiment, the drip chamber holder may include one or more spring plungers to improve the grip of the drip chamber during insertion.

[0189] like Figure 5-7As best shown in the figure, the stopcock manifold interface 132 includes one or more latches or clamps 142, 143 that are selectively deployable to hold the cell processing kit in place on the interface 132, as described below. The interface 132 further includes an array of stopcock pins or splined output shafts 144 that are drivingly connected to at least one stopcock motor 146 housed within the base 130. In one embodiment, there are 6 output shafts that are configured to interface with a corresponding one of the stopcocks of the 6-stopcock manifold of the disposable cell processing kit, however it is contemplated that more or less than 6 stopcock pins may be utilized without departing from the broader aspects of the present invention and depending on the specific 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 to move the stopcock of the disposable cell processing kit received on the interface 132 between an open position and a closed position under the control of the controller, as described below. Notably, Figure 4 The 6-stopcock interface shown in the figure is capable of docking with a 4- or 6-stopcock manifold of a disposable cell processing kit.

[0190] Specific reference Figure 4-6 , Fig.12 and Fig.13 , the separation module 104 may include a plurality of sensors for monitoring various operating parameters of the separation module 104 and parameters or conditions of the flow lines and / or fluids therein. For example, in one embodiment, the separation module 104 may include a line pressure sensor assembly 148 having an interface below which a pressure sensor is positioned and a bubble sensor / detector assembly 150, both of which form part of the stopcock manifold interface 132 for monitoring pressure and the presence of bubbles, respectively, within one or more of the fluid flow lines connected to the module 104. As shown therein, the bubble sensor assembly 150 includes a housing 152 having an upwardly facing channel 154 or passageway therein with which the bubble sensor is associated. The passage 154 is sized and dimensioned to receive a length of tubing, and the cover 156 is selectively movable between an open position and a closed position relative to the housing 152 to capture and retain the length of tubing within the passage 154. In one embodiment, the housing 152 and the cover 156 are formed of a material having poor electrical conductivity, such as anodized aluminum or plastic, so that any electrical current present will pass through the housing 130 of the separation module 104 and not through the bubble sensor 150, which may 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 for cooling the internal components of the housing 130 during operation.

[0191] refer to Fig.10 , Fig.11 and Figure 14-19 , the separation 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 a space therebetween) mounted to a movable bracket 166. Although pairs of magnets 162, 164 are illustrated, it is contemplated that for the same final height, each of the illustrated magnets 162 and 164 may be made of a single long magnet, or a stack of several shorter magnets, without departing from the broader aspects of the present invention. As described in detail below, the bracket 166 is movable between an extended position, in which the magnets 162, 164 are positioned on opposite sides of the slot 134 for generating a magnetic field within the slot 134, and a retracted position, in which the magnets 162, 164 are moved rearward of the slot 134 so as to generate no magnetic field (or only a small or negligible magnetic field) within the slot 134. The carriage 166 is slidably connected to and supported by an upper shaft 168 and a lower shaft 170 received by bushings or bearings 172 in the carriage assembly 166, and is operatively connected to a lead screw 174 received by a center bushing 176 of the carriage 166. The lead screw 174 is rotatable to slidably move the carriage 166 between its extended position and its retracted position, as disclosed in detail below.

[0192] like Fig.14 and Fig.161, the magnetic field generator assembly 160 includes a motor 178 that is drivingly connected to the lead screw 174 via a gear box 180 and a belt 182 (which connects a timing pulley 183 of the gear box 180 to a timing pulley 184 of the lead screw 174). The motor 178 is thus configured to rotate the lead screw 174 to extend or retract the carriage 166 and the magnets 162, 164. As also shown therein, the magnetic field generator assembly 160 further includes a sensor array for detecting movement of the carriage 166, the position of the carriage 166 (and therefore the magnets 162, 164), and the presence of the magnetic separation holder 136 within the slot 134. For example, the magnetic field generator assembly 160 includes a first sensor 186 and a second sensor 188 for detecting and confirming movement of the carriage 166, a third sensor 190 for detecting the presence of the magnetic cell separation holder 136 within the slot 134 in the housing, and a crank sensor 192. In one embodiment, the sensors 186, 188, 190, 192 are inductive proximity sensors, however, other types of sensors known in the art may also be utilized without departing from the broader aspects of the present invention. In conjunction with the detection of the magnetic cell separation holder 136, the magnetic field generator assembly 160 also includes a slidable locking pin 194 having a flange 196 (or washer) configured to engage the rear of the bracket 166 adjacent to its top edge. The locking pin 194 also includes a coil spring 198 configured to bias the locking pin 194 toward the front of the separation module 104 (i.e., toward the notch 134), the purpose of which is described below.

[0193] Now go to Figure 20-25 , the operation of the magnetic field generator assembly 160 and the positioning of its bracket 166 will now be described. Fig. 20 , using the second sensor 188 and the third sensor 190 to perform the detection of the presence or absence of the magnetic cell separation holder 136 within the slot 134. At the beginning of the process, the bracket 166 is in its retracted position, in which the bracket 166 is sensed by the sensor 188 and the sensor 186. In this position, the locking pin 194 is in its retracted position (because the locking pin 194 is prevented from sliding forward due to the engagement of the flange 196 with the rear of the bracket 166). In particular, the bracket 166 overcomes the bias of the spring 198 and holds the locking pin 194 in its retracted position, thereby releasing the slot 134 for the magnetic cell separation holder 136 to be inserted.

[0194] like Fig.21, the magnetic cell separation holder 136 is now inserted. When the motor 178 rotates the lead screw 168, the carriage 166 is driven forward toward the slot 134 and the separation holder 136. Due to the bias of the spring 198 that pushes the locking pin 196 forward, the locking pin 194 and its flange 196 move forward with the carriage 166. As shown therein, when the flange 196 or disk of the locking pin 194 is pushed forward, it is detected by the sensor 190 (and the first sensor and the second sensor 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 separation holder 136 engaged with the slot 134.

[0195] like Fig. 22 , the carriage 166 is then driven by the motor 178 and lead screw 168 to its forward-most position until the opposing magnets 162, 164 are aligned with opposite sides of the slot 134. As shown therein, the locking pin 194 is prevented from further forward movement due to its seated engagement with the inserted separator retainer 136 (i.e., it contacts the seat in the separator retainer 136), and thus the flange 196 continues to be detected by the sensor 190. However, in this position, the carriage 166 is in front of and away from the sensors 186, 188, and thus the presence of the carriage 166 is not detected by these sensors. Thus, as will be appreciated, detection of flange 196 by sensor 190 indicates that separator retainer 136 is received in slot 134 , and failure of first or second sensor 186 , 188 to detect bracket 166 indicates that bracket 166 and its magnets 162 , 164 are in a forward working position in which a magnetic field may be generated within slot 134 .

[0196] Now go to Fig.23 , when the bracket 166 and magnets 162, 164 move forward toward the extended position, but the separator retainer 136 is not received in the slot 134 in the housing 130, the locking pin 194 is free to move forward with the bracket 166 under the bias of the spring 198 (i.e., the forward movement of the locking pin 194 does not contact the seat in the separator retainer 136). The locking pin 136 thus slides forward until its end bottoms out and reaches the end of its range of motion. In this position, the distal end of the locking pin 194 blocks the slot 134, thereby preventing the insertion of the separator retainer 136, and the flange 196 is in front of the sensor 190, so that the flange 196 is not detected by the sensor 190, thereby indicating that the separator retainer 136 is not present. Fig.23 As shown in , the absence of the separator retainer 136 can be detected even when the tray is not in its forward-most position (ie, sensor 186 detects the presence of the tray 166, but sensor 188 does not).

[0197] refer to Fig.24 , and as indicated above, if the separator retainer 136 is properly inserted into the slot 134, the locking pin 194 moves forward with the bracket 166 until the locking pin 194 is seated in a recess or seat in the separator retainer 136. In this position, the locking pin 194 prevents the separator retainer 136 from being removed from the slot 134. However, if Fig.25 , if the separator retainer 136 is not properly positioned in the slot 134, the seat 199 in the separator retainer 136 is misaligned with the distal end of the locking pin 194. This misalignment prevents the locking pin 194 from entering the recess / seat 199. Therefore, the locking pin 194 is prevented from traveling forward far enough to align the flange 196 with the sensor 190. In this position, the sensor 188 does not detect the bracket 166, indicating that the bracket 166 has moved forward. However, because the sensor 190 does not detect the flange 196 of the locking pin 194 in this position of the bracket 166, it indicates that the separator retainer 136 is not properly received in the slot 134. Once in Fig.24 In the position shown in the figure, wherein the locking pin 194 holds the separation holder 136 in place within the slot 134, and wherein the magnets 162, 164 are aligned with opposite sides of the slot 134, a magnetic field can be generated to capture the bead-bound cells within the separation holder 136 in a manner known in the art and discussed in more detail below.

[0198] Reference again Fig. 9 , Fig.11 , Fig.15 and Fig.16In one embodiment, the separation module 104 further includes a manual crank 171 operatively connected to the linear screw 174. The crank 171 is operable to manually move the bracket 166 and the magnets 162, 164 to the retracted position in an emergency or when electrical power is lost. The crank 171 has a pivotable handle that remains closed when not in use but can be deployed when needed. A ball stop screwed into the handle maintains the handle in the closed position. In one embodiment, the crank 171 may include a pawl and ratchet mechanism so that when the crank is closed, the pin separates the pawl from the ratchet due to the force of the spring. In this position, the crank rotates freely because the pawl and the ratchet are not in contact. In order to open the crank 171, the operator must deploy a crank arm lever, which presses the pawl against the ratchet by the force of the spring and by the retreat of the pin. Since the pawl and ratchet are now in contact, the crank 171 can be rotated in a clockwise direction to engage the lead screw 174, which corresponds to rearward movement of the carriage 166. As mentioned above, a sensor 192 is provided to detect the open position of the crank 171. In one embodiment, the crank 171 is configured such that rotation in the opposite direction is prevented, making manual forward movement of the carriage 166 impossible (thereby preventing inadvertent or accidental activation of the magnetic circuit).

[0199] Return to reference Fig. 9 , the rear of the separation module 104 may include a connector 151 for connecting to an electrical power supply source for providing power to the separation module 104, a switch 153 for turning the separation module 104 on and off, a communication connector 155 for communicatively connecting the separation module 104 to the controller, and a plurality of openings 157, through which an internal fan 159 may exhaust air to keep the separation module 104 at an optimal operating temperature. In one embodiment, the communication connector 155 may be a USB connector, but other wired or wireless communication devices known in the art may also be used. In one embodiment, the separation module 104 is communicatively coupled to the processing device 102 and controlled by its controller 110. In this regard, all information obtained by the various sensors of the separation module 104 (e.g., about the magnetic field generator assembly 160, the position and state of the cell processing kit on the interface 132, and / or the parameters of the fluid passing through the various flow lines, etc.) is transmitted to the controller of the processing device 102, where the information is analyzed and then used by the controller to control the operation of the separation module 104, generate alarms, etc. Therefore, the separation module 104 does not need to be equipped with a separate processor and memory, which increase cost and complexity.

[0200] In combination with the operation of the separation module 104, Figure 4, the front of the separation module 104 may include an array of indicator lights for communicating the status / position of the magnetic field generator assembly to the operator. For example, the indicator lights 161 may include a green indicator light indicating that the bracket 166 and magnets 162, 164 are in their retracted positions, a flashing yellow indicator light indicating that the bracket 166 is moving, and a solid yellow indicator light indicating that the bracket and magnet are in their extended positions for magnetically holding the bead-bound cells passing through the separation holder 136. In another embodiment, the front of the separation module 104 may alternatively or additionally include pictograms, including, for example, a first pictogram indicating that the separation holder 136 can be inserted into the slot 134 in the separation module 104 when illuminated, a second pictogram indicating that the application / process has been successfully completed and the magnetic circuit is disconnected (and the separation holder 136 can be removed from the separation module 104) when illuminated, and a third pictogram in the form of a lock or other icon indicating that the separation holder 136 is properly locked in place when illuminated.

[0201] As discussed in detail below, separation module 104 provides a bioprocessing function that will be performed in a single easy-to-use system to expand the array. These processes may include, for example, enrichment and magnetic separation of cells, washing, and dosing preparation including cell harvesting and final preparation. As known in the art, cell selection or separation based on magnetic particles involves the targeted binding of antibodies or ligands to magnetic particles (e.g., beads) via cell surface molecules to separate certain cells from a cell mixture. Once combined, the cells connected to the magnetic particles can be separated from unbound cell populations. For example, a cell mixture including bound cells and unbound cells can pass through a separation column positioned in a magnetic field generator (e.g., the magnetic field generator assembly 160 of separation module 104), which captures magnetic particles and therefore captures associated bound cells. Unbound cells pass through a column without being captured. In an embodiment, magnetic cell separation holder 136 and / or separation module 104 may be specifically configured to be used for using various magnetic separation bead types (including, for example, Miltenyi beads, Dynabeads, and StemCellEasySep beads) to carry out cell enrichment and separation. An exemplary configuration of the separator retainer 136 is provided below.

[0202] As indicated above, the magnetic cell separation 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 separation holder 136 can be specifically designed for use with nano-sized magnetic beads (such as, for example, Miltenyi beads). Figure 26-29In one embodiment, the magnetic cell separation holder 136 of the separation module 104 may include a body portion 274 that receives and holds a vertical post 280 therein and a handle 276 connected to the body portion 174 to allow a user to easily manipulate (e.g., install and remove the separation holder 136 into and from a slot 134 in the separation module 104). In one embodiment, the body portion 274 and the handle portion 276 are integral and may be formed by molded halves 277, 278 of the clamping post 280. Fig.26 As best shown in FIG. 1 , 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. Fig.28 and Fig.29 In one exemplary embodiment, the column shell can be a billet extruded aluminum shell that is 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 opposite ends of the column 280, each end cap 282 including a female glue port for directly docking to a certain length of PVC tubing 284, 286, an O-ring (for forming a fluid seal) and a heat-sealed mesh (useful in the process of retaining beads before adding sealant). In one embodiment, the column is filled with a magnetic retaining element and a sealant. In one embodiment, the magnetic retaining element is an array of ferromagnetic balls or beads. The sealant utilized can be a biocompatible epoxy resin. To apply the sealant, the column is filled with ferromagnetic balls or beads, the sealant is added to completely wet the beads, and then the excess sealant is removed via centrifugation and the sealant is cured.

[0203] like Fig.26 and Fig. 27 104, a first length of PVC tubing 284 enters the upper end of the column 280 vertically from above and forms an inlet flow path for the bead-bound cells to enter the column 280 when the separation holder 136 is received in the slot 134 of the separation module 104. A second length of PVC tubing 286 exits the lower end of the column 280, thereby providing an outlet path for the 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 of PVC tubing 286 is directed through the handle 276, where the second length of PVC tubing 286 vertically exits the separation holder 136. Although not shown, the first length of tubing 284 and the second length of tubing 286 include connectors for integrating the column 280 with the flow path of a magnetic cell separation kit or cartridge received on the interface 132 of the separation module, as described below. Fig.30The magnetic cell separation holder 136 is shown installed into a slot 134 in the separation module (ie, by sliding the magnetic cell separation holder 136 into the slot 134 from above). Removal of the magnetic cell separation holder 136 is performed by sliding the holder upwards within the slot 134.

[0204] Now go to Figure 31-35 , various other exemplary configurations of magnetic cell separation holders 136 for use with separation module 104 are illustrated. As disclosed above, some magnetic cell separation techniques may incorporate nanometer-sized particles (e.g., beads having a diameter of about 50 nm or less), while other techniques may use larger particles (e.g., beads having a diameter of about 2 μm or more). For example, smaller particles may be desirable because smaller particle sizes may avoid receptor activation on target cells. In addition, downstream steps may skip particle removal because nanometer-sized particles may have little effect on downstream processing or cell function. However, smaller nanometer-sized magnetic particles may be separated using a magnetic cell separation procedure that involves using a magnetic field gradient enhancer to amplify the applied magnetic field gradient. In contrast, larger particles have a higher magnetic moment. Therefore, the separation of some larger particles may not involve a magnetic field gradient enhancer. However, in the case of larger particles, the separation column in the magnetic field generator may reach capacity before capturing a sufficient number of bead-bound cells. In particular, the beads bind cells to accumulate on the inside of the passage to the point where the additional beads bind cells to be captured are no longer located in a region with a sufficiently high gradient to overcome the viscous resistance that pushes them through the passage. Therefore, the use of larger particles may require multiple capture and elution cycles to obtain a desired yield, which increases the complexity of cell separation techniques based on magnetic particles. As disclosed below, some configurations of magnetic cell separation holders can avoid the need to perform multiple cycles, that is, by passing the cell mixture through a nonlinear flow path in a magnetic field, passing the cell mixture through a magnetic field or circulating in a magnetic field, and / or passing through the magnetic field multiple times. As used herein, nonlinear means not on a straight line through a magnetic field. For example, the flow path may be spiral or helical, or may have one or more curves or profiles in a magnetic field.

[0205] like Figure 31-33As shown in , the magnetic cell separation holder 250 for use with the separation module 104 is shown as being coupled to the magnetic field generator assembly 160 (i.e., received between the magnets 162, 164 of the magnetic field generator assembly 160). As mentioned above, the magnetic field generator 160 is configured to generate a magnetic field within the slot 134 (also referred to herein as the receiving area 134). The receiving area 134 and the magnetic field have a major axis (defining the longitudinal extent of the magnetic field) and a minor axis, whereby the gradient and field strength are substantially constant along a line parallel to the major axis (and may decrease at the end of the major axis). Observe the cross-sectional area perpendicular to the major axis (see, e.g. Fig.32 ), the gradient is essentially constant along lines extending into and out of the page.

[0206] The magnetic cell separation holder 250 is configured to be removably coupled to the magnetic field generator 160, for example, to be received within the receiving area / recess 134 of the magnetic field generator 160. Fig.31 and Fig.32 As shown in FIG. 1 , in one embodiment, the magnetic cell separation holder 250 includes a body 252 that can be formed of any suitable non-magnetic material configured to accommodate cell separation and is coupled to the magnetic field generator 160. In one embodiment, the body 252 is generally rectangular in shape, having a longitudinal axis (in the middle) of the magnetic field that is greater than the width or thickness of the body. Fig.31 The body 252 has a longitudinal extent (in the vertical direction) and includes a plurality of channels or grooves 254 extending along the body 252 for receiving and retaining the tube 256. The tube 256, for its part, can be configured to retain cells bound to the magnetic particles under a magnetic field and allow unbound cells to pass through, as is known in the art. For example, the magnetic particles can be Dynabeads or SCT beads, however, other magnetic particle / bead types can be utilized without departing from the broader aspects of the present invention.

[0207] The tube 256 is guided along the body 252 and / or through the body 252 via the groove 254, and defines a flow path for the flow of a fluid (e.g., a cell mixture). The groove 254 and the tube 256 are positioned so that when the magnetic cell separation holder 250 is coupled to the magnetic field generator 160 (i.e., received in the notch 134), the flow path defined by the tube 256 is positioned within the magnetic field. In addition, the groove 254 and therefore the tube 356 and the flow path defined thereby are configured so that when the magnetic cell separation holder 250 is coupled to the magnetic field generator 160, the direction of fluid flow in the flow channel at a first position within the magnetic field (i.e., through the tube) is different from the direction of fluid flow in the flow channel at a second position within the magnetic field, as discussed below.

[0208] For example, Figure 31-33As shown in FIG. 1 , in one embodiment, the body 252 may include eight substantially vertical channels or grooves 254, with each longitudinal corner of the body 252 adjacent to two channels or grooves 254. The tube 256 is guided through the grooves 254 in a manner such that a plurality of serially connected and fluidly interconnected loops are formed. In the case where the body includes eight grooves 254, four serially connected loops are formed by guiding the tube 256 through the grooves 254. It is contemplated that the body 252 may be formed with more or less than eight grooves to accommodate more or less than four loops as desired. Positioning the tube 256 into a loop provides an increased residence time of the cell mixture in the magnetic field (the total time that the cell mixture passes through the high gradient magnetic field) without reducing the flow rate (by reducing the flow rate or expanding the cross-sectional area of ​​the flow path), thereby enabling better capture of bead-bound cells compared to a single vertical path through the magnetic field (with the same flow rate and the same longitudinal length of the magnetic field generator).

[0209] Fig.32More clearly show the tube loop of magnetic cell separation holder 250. As shown therein, a plurality of tube loops each include a first portion 258 extending substantially linearly along the longitudinal extent of the main body 252, wherein the longitudinal extent of the main body 252 is aligned with the long axis of the magnetic field and magnets 162, 164, and the magnetic field has a substantially constant gradient along the line parallel to the long axis of the magnet and therefore parallel to (and ultimately colinear with) the pipeline path extending along the longitudinal axis of the holder. The tube loop further includes a second portion 260 extending from the first portion 258 and forming a first return bend, a third portion 262 extending substantially linearly and parallel to the first portion 258, and a fourth portion 264 extending from the second portion and forming a second return bend. As indicated above, the loops are connected in series to each other so that the fourth portion / bend 264 of the first loop in the plurality of loops is fluidly connected to the first portion 258 of the second loop to provide a fluid interconnection of the first loop with the second loop for circulation between loops in the magnetic field. For example, the fluid in one of the loops first passes through the substantially vertical first portion 258, enters the first return bend 260, and then enters the substantially vertical third portion 262. Then, the fluid enters the fourth portion / bend 264 and enters the next 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 portion 258 and the third portion 262 are substantially parallel but respectively in opposite directions. Although not shown, the tube 256 has an inlet end for connecting to a source (e.g., a process bag) and for receiving a cell mixture therefrom, and an outlet end for selectively connecting to a waste bag and / or a collection bag. Multiple loops of the tube 256 are in the middle of the inlet end and the outlet end. In certain embodiments, the flow path may have an even number of lengths (e.g., vertical portions) so that the inlet and outlet are on the same end of the magnetic cell separation holder 250. In other embodiments, the flow path may have an odd number of vertical sections such that the inlet and outlet are located on opposite ends of the magnetic cell separation holder 250 .

[0210] In one embodiment, the magnetic cell separation holder 250 may include a handle 266 or finger grip portion that enables a user to grasp the magnetic cell separation holder 250 to position it within the receiving area 134 or remove it from the receiving area 134. Fig.31As best shown in FIG. 1 , the magnetic cell separation holder 250 is inserted between the magnetic field plates 162, 164 of the magnetic field generator 160. For example, the position of the groove 254 and therefore the longitudinal lanes 258, 262 of the tube 256 within the magnetic field generator 160 can cover the position with the highest magnetic field strength in the magnetic field. In another example, the position of the groove 254 and therefore the vertical lanes 258, 262 of the tube 256 within the magnetic field generator 160 can cover the position with the highest magnetic field gradient in the magnetic field while meeting the magnetic field strength requirements of the magnetic particles. Fig.33 The position of the vertical track of the tube 256 within the magnetic field is illustrated when the magnetic cell separation holder 250 is received in the slot 134 between the magnets 162, 164. As illustrated therein, the position of the body 252 and the groove 254 of the magnetic cell separation holder 250 and the magnets 162, 164 are configured and sized such that when the magnetic cell separation holder 302 is coupled to the magnetic field generator 350, the vertical track of the tube 256 is positioned within the high gradient region 268 of the magnetic field generator 160.

[0211] Now go to Fig.34 In one embodiment, the magnetic cell separation holder 300 may include a ferromagnetic core 270 that extends substantially the entire length of the magnetic field and is surrounded by the tube 256. In one embodiment, the ferromagnetic core 270 may be an integral part of the body 252 of the separation holder 250, or it may be an additional component. The use of the ferromagnetic core 270 allows a higher gradient to be generated over a longer length than a system without a ferromagnetic core. In particular, the ferromagnetic core produces additional parallel high gradient regions along the longitudinal axis of the magnetic plate, thereby allowing a longer length of pipeline to be guided within the same magnetic field volume than without a ferromagnetic core. In one embodiment, the ferromagnetic core 270 may be formed by various ferromagnetic materials (such as, for example, iron).

[0212] Fig.35 is a simplified illustration of another configuration for a magnetic cell separation holder according to another embodiment of the present invention. As illustrated therein, the tube 256 is not formed into a plurality of longitudinal loops, but the tube 256 is wound or wrapped in a substantially spiral or helical configuration. As shown, the plurality of loops 272 extend in a direction substantially perpendicular to the longitudinal direction, so that the flow through each loop 272 is substantially perpendicular to the longitudinal direction (e.g., horizontally rather than vertically) within the magnetic field. Similar to Figure 31-31 , the multiple loops 272 within the magnetic field provide a longer travel length for the fluid within the flow path of the tube 256 than a single rod extending linearly through the magnetic field. In one embodiment, the horizontal or spiral loops 272 of the tube 256 may surround the ferromagnetic core 278.

[0213] Although Figure 31-35The tubes 306 are shown arranged in substantially vertical and horizontally extending loops (i.e., parallel or perpendicular to the longitudinal direction / long axis of the magnetic plate and receiving area), respectively, but it is contemplated that the tubes may be arranged in any configuration that provides a flow path of increased length / distance within the magnetic field generated by the magnetic field generator compared to a single linear path through the magnetic field. This may be achieved by using multiple loops of any orientation / direction (such that the cell mixture passes through the magnetic field multiple times) and / or by using a single or multiple non-linear paths through the magnetic field (e.g., the tubing has curved or arcuate portions within the magnetic field).

[0214] In one embodiment, the tubing may be arranged to form multiple loops, where the tubing is looped around the outside of the receiving area 134 (i.e., outside the magnetic field) so that all flow within the magnetic field area flows in the same direction (e.g., from top to bottom or from bottom to top). In addition, it is contemplated that all tubes in the magnetic field may extend in the same direction, and the system may include a manifold at the top and a manifold at the bottom to allow parallel flow.

[0215] Further, it is contemplated that the magnetic separation holder may be configured with a flow path / tube, which turns into multiple paths through a magnetic field and reconverges. In addition, in one embodiment, the main body 252 of the magnetic cell separation holder 250 may be configured as a fluid device (i.e., without the need for a separate tube 256) with (multiple) integral flow paths. In particular, it is contemplated that the flow path and / or the ferromagnetic core may be made entirely of metal. This will allow people to further utilize the higher gradient region of the magnetic field. In yet another embodiment, it is contemplated that the flow path may be injection molded into an insert. It is contemplated that people may insert a mold with a metal frame to add more gradient regions. Similarly, it is contemplated that people may manufacture / print the flow path by suitable non-ferrous materials (e.g., plastics, etc.) additive manufacturing.

[0216] Although it has been disclosed above that the magnetic field generator may consist of two opposing magnetic plates forming a permanent magnet, the invention is not so limited in this respect. In particular, it is contemplated that the magnetic field generator may be an electromagnet that generates a field substantially similar to the field generated by the permanent magnet.

[0217] As disclosed above, the processing device 102 and the separation module 104 are intended to be used in combination with each other to perform various functions, schemes and / or workflows associated with the separation, harvesting and final preparation of cell products in an automatic or semi-automatic manner. In particular, the processing device 102 and the separation module 104 can be controlled according to an instruction set executed by a controller (e.g., controller 110 or 310) of the processing device 102 and stored in a memory of the processing device 102, so as to perform various operations associated with these processes in order with minimal or no human intervention. In one embodiment, the processing device 102 is configured and operable to perform any of the schemes set forth in WIPO International Publication No. WO 2019 / 106207 and performed by the device 900 disclosed therein, wherein the separation module 104 provides additional functions and possible workflows, as described below. In fact, as disclosed above and described in more detail below, the processing device 102 and the separation module provide, for example, fluid management, centrifugation, temperature control, cell separation, cell washing, cell concentration, cell preparation and preparation.

[0218] In conjunction with the processing device 102 and the separation module 104, embodiments of the present invention provide various single-use disposable / consumable kits designed to be used with the processing device 102 and / or the separation module 104 to assist in performing processes and / or workflows associated with the separation, harvesting, and final formulation of cell products. Fig.36 , a disposable washing kit 350 is shown for use with processing apparatus 102. Washing kit 350 is a single-use disposable kit that is utilized in conjunction with apparatus 102 to wash and concentrate fresh or thawed input product after an optional temperature-controlled initial dilution. Fig.36 As shown in , kit 350 includes a cassette or manifold 352 having four stopcocks 354, 356, 358, 360, an input line 362 fluidly connected to the stopcock 354, a final product / collection container or bag 364 fluidly connected to the stopcock 356 via line 366, a wash solution line 368 and a resuspension solution line 370 fluidly connected to the stopcock 358 via line 372, and a waste container or bag 374 fluidly connected to the stopcock 360 via line 376. As shown therein, the input line 362, the wash solution line 368, and the resuspension solution line 370 may be equipped with end caps 378 that maintain the sterility of the lines during transport and storage and may be removed or cut off just prior to use so that bags containing fresh / thawed input product, wash solution, and resuspension solution, respectively, may be connected to the lines via any means known in the art, such as sterile welding, etc.

[0219] like Fig.36As further shown in the figure, the input line 362 includes an in-line drip chamber 380 with an integral filter. The kit 350 further includes a separation chamber 382 fluidly connected to the box 352 via a flow line 384, and a branch pipe tail 386 fluidly connected to the box 352 via the line 384. The pipe tail 386 and the second pipe tail connected to the stopcock 358 of the box are equipped with a hydrophobic filter 388. In one embodiment, the hydrophobic filter is a 2 micron hydrophobic filter. In one embodiment, the kit 250 can be sterilized by means known in the art (such as, for example, ethylene oxide sterilization) and sealed in a blister package for transportation to the end user and for storage.

[0220] In use, the manifold 352 is mounted on the stopcock manifold interface 112 of the processing device 102 so that the corresponding motor output shaft of the interface 112 engages with a corresponding one of the four stopcocks 354, 356, 358, 360 for controlling the position of the stopcock. The separation chamber 382 is received within the centrifugal processing chamber 108. The input line 362 is connected to a bag containing the input product to be washed, the wash solution line 372 is connected to a bag containing the wash solution, and the resuspension solution line 370 is connected to a bag containing the resuspension solution, and these bags are suspended from the hook 118 of the processing device 102 along with the waste bag 374 and the collection bag 364. The washing and optional concentration process is then performed according to a pre-programmed instruction set stored in a memory and utilized by the controller 110 of the processing device 102. In one embodiment, the washing process utilizing the processing device 102 and the disposable kit 350 optionally includes initially diluting the input product, concentrating the input product (so as to reduce its volume), washing the input product with a washing solution for one or more washing cycles, and then resuspending the input product and collecting the resuspended input product in a collection bag.

[0221] During the initial dilution step, parameters such as temperature, post-dilution mixing, dilution mixing time, and dilution mixing rate may be entered or retrieved from memory, and the initial dilution is performed using wash solution from a bag connected to the wash solution line 372. During the concentration / volume reduction step, parameters such as prime (or not prime) flow lines with input product, input bag flushing during the last volume reduction cycle, input bag flushing volume, and input bag manual mixing (during the middle of the input bag flushing step) may be selected and / or entered and / or enabled or disabled. In addition, the number of wash cycles performed during the wash phase may be entered and selected. Finally, during the resuspension phase, a prompt indicating the user to switch the wash and resuspension fixtures after the wash phase may be enabled or disabled, and the volume of the final product at the end of the resuspension phase may be entered and / or selected. In one embodiment, the wash process performed using the processing device 102 and the kit 350 may be used to wash and concentrate the input product before and / or after activation, transduction, and amplification.

[0222] In one embodiment, the processing device 102, the separation module 104, and the kit 350 allow for the use of an algorithm to control the filling of the resuspension medium into the separation chamber 382 during resuspension to achieve an accurate, small final product volume in order to avoid volume overshoot. A method of resuspending an intermediate volume to achieve a desired final volume is performed simultaneously with the flushing of the separation chamber of the cell product and includes: at a first step, extracting the contents of the intermediate volume of the separation chamber into the final bag line; at a second step, calculating the number of flush cycles and the associated fill volume required to reach the final volume; at a third step, filling the separation chamber 382 until a final target of a flush cycle volume of 10 mL is achieved; at a fourth step, incrementally filling 1 mL volume increments with a 2 second pause between increments until the target flush cycle volume is reached; at a fifth step, extracting the flush volume toward the final / collection bag; and repeating steps three to five until the number of flush cycles is completed and the final volume of the output bag is reached. During the extraction of the flush volume toward the final bag, air inhalation during the filling step is taken into account, and the fill volume of the next flush cycle is subsequently adjusted to ensure that the total final volume effectively reaches the target value. It is contemplated that the general process steps disclosed above may be modified as desired such that, for example, an initial filling of the separation chamber is performed to any desired volume, and the separation chamber is then incrementally filled with any desired smaller incremental volumes, with pauses of selected durations occurring between the smaller volume increments (i.e., the volumes and pause durations specified above may be modified as desired).

[0223] refer to Fig.37A and Fig.37B , showing a single-use disposable magnetic cell separation kit 800 for use with the processing device 102 and the separation module 104 ( Fig.37B104, respectively). Under the control of the controller 110 of the processing device 100, the magnetic cell separation kit 800 and the associated protocols implemented by using such a kit allow for initial dilution, volume reduction, washing, incubation, post-incubation washing, magnetic separation, and final resuspension of a cell population, as disclosed below. In one embodiment, the magnetic cell separation kit 800 includes a cassette or manifold 802 having four stopcocks 804, 806, 808, 810. The kit 800 also includes a line 811 fluidly connected to the first stopcock 804 and configured for fluid connection to an accessory on the magnetic cell separation holder 136, a line 812 fluidly connected to the second stopcock 806 and configured for fluid connection to a second accessory on the magnetic cell separation holder 136, a collection bag 813 fluidly connected to the fourth stopcock 810 via a line 814, a tubing tail 815 fluidly connected to the third stopcock 808 for fluid connection to a resuspension buffer bag (not shown) containing a suspension medium for resuspending the positive fraction of the cells after bead separation, and a tubing tail 816 fluidly connected to the third stopcock 808 for fluid connection to a bag (not shown) containing a release buffer for releasing cells from the magnetic beads in the magnetic cell separation holder 136. Fig.37A and Fig.37B As further shown in FIG. 8 , the magnetic cell separation kit 800 further includes a pair of tubing tails 817, 818, which are fluidically connected to the second stopcock 804 and the fourth stopcock 810, respectively, and are equipped with a hydrophobic filter of the type described above. The kit 800 also includes a negative component bag 820 fluidically connected to the first stopcock 804 via a pipeline 819. Fig.37A As shown in FIG. 8 , the manifold 802 is configured for mounting on the manifold interface 132 of the separation module 104 .

[0224] Further references Fig.37A and Fig.37B, the kit 800 further includes a second manifold 821 having four stopcocks 822, 823, 824, 825 and configured to be received on the manifold interface 112 of the processing device 102. The kit 800 additionally includes a final collection / transfer bag 826 fluidly connected to the second stopcock 823, a process bag / incubation bag 827 also fluidly connected to the second stopcock 823, a line 828 fluidly connected to the first stopcock 822 and having an inline drip chamber 829 with a 200 micron filter. The line 828 additionally includes a branch line 830 having a tubing tail and a branch line 831 having a sampling pillow. As illustrated, the kit 800 further includes a line 832 fluidly connected to the first stopcock 822 for connecting to a platelet-free buffer bag for platelet depletion. The line 832 includes a branch line 833 having a filter. The kit 800 also includes a pipeline 834, which is fluidically connected to the fourth stopcock 825 and has a branch pipeline 835, which has a filter. The pipeline 834 is configured to be fluidically connected to a bag containing a separation buffer, which is used to perform a wash cycle during the incubation of beads and an optional wash cycle after incubation for removing excess beads. As illustrated, the kit 800 includes a waste bag 836 fluidly connected to the fourth stopcock 825 and a spare bag 837 (which is not used during the separation process) fluidly connected to the third stopcock 824. As illustrated, some of the pipelines are equipped with a sampling pillow 838 and / or a filter 839. Further, the kit 800 includes a separation chamber 840, which is configured to be received in the centrifugal chamber 108 of the processing device 102. The line 841 interconnects the manifold 802 on the separation module 104 with the manifold 821 on the processing device 102 for fluid flow therebetween and includes a section of the peristaltic pump tubing 842 configured to engage the peristaltic pump assembly 111 of the processing device 102 and a drip chamber 843. The kit 800 additionally includes an additional tubing tail having a sterile air filter 844. A line 845 is also fluidly connected to the third stopcock 824, the opposite end of which is configured for fluid connection to a bottom port in a process bag 846, which also forms part of the disposable kit 800. In an embodiment, the kit 800 can be sterilized by means known in the art, such as, for example, ethylene oxide sterilization, and sealed in a blister pack for transport to an end user and for storage.

[0225] Now go to Fig.37C, shows a single-use disposable magnetic cell separation kit 1800 for use with a processing device 102 and a separation module 104 according to another embodiment of the present invention. Under the control of the controller 110 of the processing device 100, the magnetic cell separation kit 1800 and the associated protocols implemented by using such a kit allow for initial dilution, volume reduction, washing, incubation, post-incubation washing, magnetic separation, and final resuspension of a cell population, as disclosed below. In one embodiment, the magnetic cell separation kit 1800 is substantially similar to Fig.37A and Fig.37B 800, and includes a box or manifold 1802 having four stopcocks 1804, 1806, 1808, 1810. The kit 1800 also includes a line 1811 fluidly connected to the first stopcock 1804 and connected (or configured for fluid connection) to one side of the magnetic cell separation holder / column 136, a line 1812 fluidly connected to the second stopcock 1806 and connected (or configured for fluid connection) to the opposite side of the magnetic cell separation holder / column 136, a transfer bag 1813 fluidly connected to the fourth stopcock 1810 via a line 1814, a line tail 1815 fluidly connected to the third stopcock 1808 for fluid connection to a resuspension buffer bag (not shown) containing a suspension medium for resuspending the positive components of the cells after bead separation, and a line 1816 fluidly connecting the line 1812 to the third stopcock 1808. Line 1816 may include a drip chamber filter 1817 (e.g., a 40 micron drip chamber filter) such that cells flow from the source bag through the drip chamber filter 1817, through the magnetic cell separation holder / column 136, and to the negative component bag 1820. Fig.37C As further shown in FIG. 1 , the magnetic cell separation kit 1800 further includes a tubing tail 1818 fluidly connected to the fourth stopcock 1810 and equipped with a hydrophobic filter of the type described above. The kit 1800 also includes a line 1819 fluidly connected to the first stopcock 1804 for fluid connection to a negative component bag 1820. Fig.37A and Fig.37B In the embodiment of FIG. 1 , the manifold 1802 is configured to be mounted on the manifold interface 132 of the separation module 104 .

[0226] Further references Fig.37CThe kit 1800 further includes a second manifold 1821 having four stopcocks 1822, 1123, 1824, 1825 and configured to be received on the manifold interface 112 of the processing device 102. The kit 1800 additionally includes a line fluidly connected to the first stopcock 1822 for connecting to a first transfer bag 1832, which is a supplement to the transfer bag (i.e., the second transfer bag 1813), and the kit 1800 includes a final collection / transfer bag 1826 also fluidly connected to the first stopcock 1822. Fig.37C As further shown in the figure, lines 1827 and 1828 are fluidly connected to the second stopcock 1823 and are configured to be fluidly connected to a source bag (not shown) and a process / incubation bag (not shown), respectively. As shown, line 1827 may have an online drip chamber. As shown, the kit 1800 further includes lines 1833 and 1834, which are fluidly connected to the fourth stopcock 1825 for connecting to a platelet-free buffer bag (not shown) for platelet depletion and a separation buffer bag (not shown) for performing a wash cycle during bead incubation and an optional post-incubation wash cycle for removing excess beads. Waste bag 1836 is fluidly connected to the third stopcock 1824. The pipeline 1841 interconnects the manifold 1802 on the separation module 104 with the manifold 1821 on the processing device 102 for fluid flow therebetween, and includes a section of the peristaltic pump conduit 1842 configured to engage the peristaltic pump assembly 111 of the processing device 102 and a drip chamber 1843. In one embodiment, the pipeline 1841 is equipped with a large drip chamber 1844. The kit 1800 additionally includes an additional pipe tail with a sterile air filter 844. In one embodiment, the kit 1800 can be sterilized by means known in the art (such as, for example, ethylene oxide sterilization) and sealed in a blister package for transportation to an end user and for storage. In contrast to the kit 800, the kit 1800 includes a magnetic cell separation holder / column 136 already attached to it / integrated with it.

[0227] Now go to Fig.38A , diagram for use Fig.37A and Fig.37B 850 of an exemplary scheme for magnetically separating cells using a magnetic cell separation kit 800, a processing device 102, and a separation module 104. As indicated above, when used in conjunction with the processing device 102 and the separation module 104, the magnetic cell separation kit 800 allows for initial dilution, volume reduction, washing, incubation, post-incubation washing, magnetic separation, and final resuspension of a cell population. In one embodiment, Fig.38AThe illustrated protocol 850 performs an optional initial dilution of the apheresis product, concentrates the cells, depletes platelets, separates CD3+ cells (for example) using magnetic beads in the separation holder 136, and resuspends the cells in a preselected solution for downstream use (for example, for activation, transduction, and amplification, and ultimately for formulation and dosing preparation). As shown therein, at step 852, magnetic cell separation beads (e.g., Miltenyi beads, Dynabeads, and StemCell EasySep beads) are inserted into the process bag 846 before starting. Kit testing can be performed at step 854. Initial dilution is performed in a further step. Volume reduction is then performed at step 856, after which the cells are transferred to a process bag positioned with the thermal mixing chamber 114 of the processing device 102 at step 858. The cells and beads are then incubated in the thermal mixing chamber 114 at step 860, and a post-incubation wash is performed at step 862 to remove excess beads. In one embodiment, the process bag containing the cells and beads is a three-dimensional process bag, which provides improved thermal control due to the flat bottom surface of the bag (while the top surface remains flexible). Another advantage of using a three-dimensional process bag is improved fluid transfer (e.g., during bag-to-bag separation and flushing steps, the bottom and top surfaces of the bag remain separated and less likely to trap or hold cells). During the incubation step, control of the volume used for incubation (to target-specific cell density), control of temperature, and control of movement of the mixed carrier are achieved.

[0228] After incubation and washing, magnetic separation of the bead-bound cells is then performed at step 864 by inserting the magnetic cell separation holder 136 into the slot 134 in the separation module 104 and applying a magnetic field to hold the bead-bound cells in the column or flow path of the magnetic cell separation holder (as the case may be). Flushing and separation are performed at step 866, after which the target cells are collected using a resuspension buffer at step 868. In one embodiment, step 868 may include replacing the separation buffer with a medium, performing an elution cycle in 3 steps: (1) separating the main amount of cells from the column and collecting the volume, (2) performing an elution cycle with fresh medium and collecting the volume, and (3) flushing the pipe / bag and collecting the volume. An optional volume reduction step may also be performed before the target cells are resuspended. In one embodiment, an air plug may be used to help remove the bead-bound cells from the separation holder / column, as more specifically disclosed in WIPO International Publication No. WO 2019 / 106207.

[0229] In one embodiment, the processing device 102, the separation module 104 and the magnetic cell separation kit 800 can be used to circulate the bead-bound cell population back and forth through the magnetic field to separate / capture the bead-bound cells (rather than passing through the magnetic field once). For example, the bead-bound cell population after incubation can be pumped (via pump 111) from the first bag through the magnetic cell separation holder 136 in the magnetic field positioned in the slot 134 in the separation module 104 to the second bag. When the cell mixture passes through the magnetic field generated by the magnetic field generator, the bead-bound cells are held / captured in the above-described manner in the fluid path between the relative plates extending through the magnetic cell separation holder 136 and positioned in the magnetic field generator, while the unbound cell population, the uncaptured bead-bound cells and the other contents of the cell mixture pass through the magnetic field generator and enter the second bag on the other side of the magnetic field generator. Then the pump 111 of the processing device 102 is reversed to pump the cell mixture from the second bag through the magnetic cell separation holder 136 and back to the first bag. When the cell mixture passes through the magnetic field generated by the magnetic field generator again, additional bead-bound cells are held / captured in the portion of the fluid path of the magnetic cell separation holder 136 positioned within the magnetic field. This process (bag-to-bag circulation / transfer of the cell mixture) can be repeated until a sufficient number of bead-bound cells are held in the fluid path (or its magnetic cell separation holder). As will be appreciated, the transfer of the cell mixture back and forth between the first bag and the second bag causes the cell mixture to pass through the magnetic field multiple times, thereby increasing the capture efficiency of the system.

[0230] As described above, the circulation of the cell mixture back and forth between bags on opposite sides of the magnetic field generator has essentially the same effect as increasing the distance traveled by the cell mixture within the magnetic field by utilizing multiple loops, channels, or by using a non-linear flow path through the magnetic field (as described above in conjunction with Figure 31-35 In particular, by circulating the cell mixture back and forth, the total "distance" that the cell mixture travels within the magnetic field is increased compared to a single linear passage through the magnetic field. This ensures that bead-bound cells that are not retained during the first passage through the magnetic field can be captured in a subsequent pass before collection. Therefore, in conjunction with the above, it is contemplated that the fluid path in the region of the magnetic field generator (e.g., the flow path within the magnetic cell separation holder) may take the form of any of the embodiments described above. For example, the flow path within the magnetic field may include multiple loops, tracks, spirals, contours, turns, etc. to increase the dwelling distance within the magnetic field. In particular, it is contemplated that Figure 31-35 The flow path configuration shown in can be used in conjunction with the separation sequence (bag-to-bag cycle) described above. In other embodiments, a straight path through a magnetic field can be utilized to capture bead-bound cells.

[0231] As also mentioned above, the kit 800 implements and allows the collection of both the positive and negative components caused by the separation (in the collection bag 826 and the negative component bag 820, respectively). In particular, the negative component can be collected in the negative component bag 820 for other potential uses, rather than allowing the negative component to flow to waste. Although the embodiments disclosed above discuss the use of magnetic separation to collect bead-bound cells, the kit 800 additionally allows negative selection, whereby the desired cell population is not labeled with magnetic beads, while other cells are labeled with such beads, so that the undesirable cell population is captured in the magnetic cell separation holder, and the desired unlabeled cell population is allowed to pass through the separation holder and is collected after the bead-bound cell population is captured in the magnetic cell separation holder.

[0232] refer to Fig.38B , diagram for use Fig.37C 1850, an exemplary scheme 1850 of magnetic cell separation kit 1800, processing device 102 and separation module 104 for magnetic separation of cells. Scheme 1850 is substantially similar to scheme 1800, however, it includes additional separation steps to maximize bead-bound cell capture, as disclosed below. As shown therein, at step 1852, before starting, magnetic cell separation beads (e.g., Miltenyi beads, Dynabeads and StemCellEasySep beads) are inserted into a process bag connected to pipeline 1828. Then, the thermal mixing chamber 114 (i.e., thermal mixer) can be configured for scheme 1850. Kit testing can be performed at step 1854. Initial dilution is performed in a further step 1855. Volume reduction is then performed at step 1856, after which the cells are transferred to a process bag positioned together with the thermal mixing chamber 114 of the processing device 102 at step 1858. The cells and beads are then incubated in the thermomixing chamber 114 at step 1860 , and a post-incubation wash is performed at step 1862 to remove excess beads.

[0233] After incubation and washing, magnetic separation of the bead-bound cells is then performed in a two-step process. In a first separation or batch capture step 1864, the bead-bound cells are passed through the magnetic cell separation holder / column 136 at step 1866, and the bead-bound cells are eluted at step 1868 to collect the target cells. In a second separation or batch capture step 1870, the fluid / medium is passed through the magnetic cell separation holder / column 136 a second time at step 1872, and the fluid / medium is eluted at step 1874 to collect the target cells that were not captured during the first batch capture step 1864.

[0234] Particularly, in the first separation stage or batch capture step 1864, magnetic column 136 is connected, and by the part of separation washing solution from separation buffer bag (not shown) transfer through pipeline 1834 and directly transfer to negative component bag 1820, and transfer from pipeline 1834 through pipeline 1812, through magnetic column 136 and through pipeline 1811 (and finally arrive negative component bag 1820) to pre-charge. Next, the part of the content of the source bag is transferred through pipeline 1828, through manifold 1821, through manifold 1802 via pipeline 1841, through pipeline 1816 and drip chamber filter 1817, through magnetic column 136 and arrive negative component bag 1820. Therefore, the part in the cell is captured inside column 136, and the supernatant is inside the negative component bag. The separation washing solution from the separation buffer bag (not shown) is used to rinse column 136, and is transferred to the negative component bag after passing through the column. Next, the remainder of the content of the source bag is transferred through pipeline 1828, through manifold 1821, through manifold 1802 via pipeline 1841, through pipeline 1816 and drip chamber filter 1817, through magnetic column 136 and arrive negative component bag 1820.Therefore, additional cells are captured in post 136 insides, and supernatant is in negative component bag inside.Separation washing solution from separation buffer bag (not shown) is used to rinse post 136 again, and is transferred in the negative component bag after passing post.At this moment, all cells should be captured in post 136 insides.This has finished step 1866 described above.

[0235] During the elution step 1868, the magnet of the column 136 is turned off, and a first primary rinse is performed by transferring a separation wash solution from a separation buffer bag (not shown) through line 1841 and to the second transfer bag 1813 to be stored. The contents of the second transfer bag 1813 then leave the bag 1813, pass through the manifold 1802, pass through the separation column 136, pass through line 1841, pass through the manifold 1821, and reach the first transfer bag 1832 to elute the column 136. Thus, the elution of the column 136 is performed using the stored separation wash solution from the second transfer bag 1813 to the first transfer bag 1823. During this elution step, a subset of cells is transferred from the column 136 to the first transfer bag 1832, however, some of the cells may remain inside the column 136 after elution. The separation chamber 840 is then filled with eluted cells from the first transfer bag 1832 via the manifold 1821 (so that the cells are separated between the separation chamber 840 and the column 136).

[0236] A second rinse is then performed by transferring the separation wash solution from a separation buffer bag (not shown) through line 1841, through manifold 1802 to line 1812, and through lines 1811 and 1814 to a second transfer bag 1813 to be stored again. This step elutes the cells from column 136 and transfers them to second transfer bag 1813. The contents of second transfer bag 1813 (including the eluted cells) then leave bag 1813, pass through manifold 1802, through line 1841, and reach first transfer bag 1832. The contents of first transfer bag 1832 are then circulated back to second transfer bag 1813 by passing such contents through line 1841, through line 1812, through column 136, through line 1811, and finally to line 1814. The eluted cells are thus transferred from first transfer bag 1832 to second transfer bag 1813. In one embodiment, this second flush can be performed any desired number of times to ensure that substantially all of the cells are eluted. All of the eluted product is then transferred from the second transfer bag 1813 to the first transfer bag 1832. The cells from the separation chamber 840 from the first flush are also transferred to the first transfer bag 1832 so that all of the eluted cells are inside the first transfer bag 1832. This completes the first separation stage or batch capture step 1864.

[0237] In the second separation stage or batch capture step 1870, the contents of the first transfer bag 1832 pass through line 1841, through the manifold 1802 and line 1816 and the drip chamber filter 1817, through the magnetic column 136 and reach the negative component bag 1820. Next, a portion of the separation wash solution from the separation buffer bag (not shown) passes through line 1834, through the manifold 1802 and line 1816 and the drip chamber filter 1817, through the magnetic column 136 and reach the negative component bag 1820. The separation chamber 840 is then filled with the separation wash solution from the separation buffer bag. The solution is then transferred from the separation chamber 840 to the first transfer bag 1832. Next, the solution from the first transfer bag 1832 passes through line 1841, through the manifold 1802 and line 1816 and the drip chamber filter 1817, through the magnetic column 136 and reach the negative component bag 1820. The separation buffer solution is then replaced with the resuspension medium by passing the resuspension solution from a resuspension buffer bag (not shown) through line 1815, through manifold 1802, through line 1841, through manifold 1821, and to first transfer bag 1832 (i.e., a portion of the resuspension solution is transferred from the resuspension buffer bag to first transfer bag 1832). The resuspension solution from first transfer bag 1832 is then transferred through column 136 and to negative component bag 1820. At this point, the bead-bound cells are captured inside magnetic column 136.

[0238] During the elution step 1874, the magnet of the column 136 is turned off, and the resuspension solution from the resuspension buffer bag passes through the manifold 1802, through the line 1811, through the magnetic column 136, through the line 1812, through the manifold 1802 again, through the line 1841, through the manifold 1821, and to the final collection bag 1826. This first portion of the cells from the collection bag 1826 is then transferred to the separation column 840. Next, a portion of the resuspension solution from the resuspension bag is transferred through the line 1841 and transferred to the final collection bag 1826. Another rinse is then performed by passing the resuspension solution through the line 1841, back through the column 1836, and to the second transfer bag 1813 to rinse the remaining cells out of the column 136. The contents of the second transfer bag 1813 are then transferred to the final collection bag 1826. The contents of the final collection bag 1826 are then circulated back to the second transfer bag through the column. The eluted cells are thus transferred from the final collection bag 1826 to the second transfer bag 1813. In one embodiment, this second rinse may be performed any desired number of times to ensure that substantially all of the cells are eluted. All of the eluted product is then transferred from the second transfer bag 1813 to the final collection bag 1826. The eluted cells are then transferred from the final collection bag 1826 to the separation chamber 840 to mix with the previously eluted cells. Next, the resuspension solution from the resuspension buffer bag (not shown) is transferred to the first transfer bag 1832, and the separation chamber 840 is filled by transferring the resuspension solution from the first transfer bag 1832 to the separation chamber 840. This ends the elution step 1874, and all of the eluted cells are contained within the separation chamber 840.

[0239] Final product formulation may then be performed via centrifugation in the separation chamber 840 and extraction of the supernatant from the separation chamber 840 to a waste bag 1836. The cells within the separation chamber 840 are then transferred to a final collection bag 1826. Finally, the resuspension solution from the first transfer bag 1832 is transferred to the separation chamber 840 and then extracted to the final collection bag 1826.

[0240] Thus, the protocol 1850 concentrates cells from the apheresis product, depletes platelets (platelet-free phase), separates CD3+ cells using magnetic beads, and resuspends the cells in a preselected solution. As disclosed above, after the first separation and collection of the separated cell product, the separation step is repeated (i.e., the cell product is separated a second time through the column), which ensures that the target cell elution and collection are maximized.

[0241] Reference now Fig.39, showing a single-use disposable dosing preparation kit 500 for use with the processing device 102 and the separation module 104. As described below, the dosing preparation kit 500 and the associated protocols implemented by using such a kit allow for automation of volume splitting, dilution, mixing, cryopreparation, and dosing of cell products under the control of the controller 110 of the processing device 100. The dosing 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 the stopcock 504 via peristaltic pump tubing 518, a plurality of tubing lines 520, 522, 524 fluidly connected to the cassette 502 for fluid connection (e.g., aseptic welding) to one or more media bags (not shown), a final formulation / collection bag 526 fluidly connected to the stopcock 508 via line 528, and a bag 530 (for containing an initial / intermediate product from which a final dosing / preparation is produced) fluidly connected to the stopcock 508 via line 532. In one embodiment, the process bag 516 is a three-dimensional process bag. As further shown therein, the kit 500 further includes a waste bag 534 fluidly connected to the stopcock 514 via a line 536, and a plurality of freezer bag connection lines 538, 540, 542, 544 fluidly connected to the stopcocks 510, 512, 514 (for connection to a plurality of freezer bags using sterile welding or other connection means). Finally, the line 518 is equipped with a pair of hydrophobic filters 546, 547 on opposite sides of the peristaltic pump tubing section, and the kit 500 further includes an air inlet line 548 fluidly connected to the stopcock 510 and having a hydrophobic filter 549. In one embodiment, the hydrophobic filter is a 2 micron hydrophobic filter. In one embodiment, the kit 500 can be sterilized by means known in the art (such as, for example, ethylene oxide sterilization) and sealed in a blister package for transportation to an end user and for storage.

[0242] Fig.40The integration / installation of the dosing preparation kit 500 on the processing device 102 and the separation module 104 is shown. As shown therein, on the separation module side, the stopcock manifold / box 502 is installed on the stopcock manifold interface 132 of the separation module 104 so that the corresponding motor output shaft 144 of the motor 146 engages with a corresponding one of the six stopcocks 504, 506, 508, 510, 512, 514 for controlling the position of the stopcock. The waste bag 534 is suspended on one of the hooks 140 on the rod 138 of the separation module, and one or more of the pipelines 538, 540, 542, 544 are aseptically welded (or connected via other means) to the corresponding frozen bag, which is then suspended on one or more of the hooks 140 on the rod 138 of the separation module 104. Finally, the tail section of tubing with the air filter 547 is connected to the line pressure sensor 148 of the separation module 104 , and the portion of the line connecting the 3D process bag 516 to the stopcock manifold 502 engages with the bubble sensor assembly 150 of the separation module 104 .

[0243] On the processing equipment 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 equipment 102 (the single hook 118 has an integrated load cell or weight sensor for sensing the weight of the (multiple) bags suspended thereon). The media bag (not shown) is aseptically welded (or connected via other means) to the media lines 520, 522, 524 and suspended from another hook 118 of the hanger assembly 116 of the processing equipment 102 (the other hook 118 also has an integrated load cell or weight sensor for sensing the weight of the (multiple) bags suspended thereon). The 3D process bag 516 of the kit is placed inside the hot mixing chamber 114 of the processing equipment 102. Finally, the section of the 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 equipment 102, and the tail of the tubing with the air filter 546 is connected to the pressure sensor (not shown) of the processing equipment 102.

[0244] Go to Fig.41, illustrates a method 550 for preparing a dose of a cell product using a processing device 102, a separation module 104, and a dose preparation kit 500. As indicated above, the dosing regimen is executed in an automated manner by the controller 110 of the processing module 102, both of which are controlled by a data connection between the processing module 102 and the separation module 104. The method 550 includes testing and priming the kit 500 at step 552, which, in one embodiment, may include venting air from the 3D process bag 516, the freezing bag, and the freezing bag lines 538, 540, 542, 544 to minimize air in the bag at the end of the process, priming the 3D mixing bag 534 (to equalize the amount of air inside the 3D process bag 534), priming the media bag lines 520, 522, 524, and calibrating the pump 111 by flowing media from the media bag connected to the line 520 (to calibrate the pump speed using the precise weight pulled from the bag on the load cell / hook). Next, at step 554, the initial product in the bag 530 is separated. In one embodiment, this involves transferring all of the input product from the bag 530 to the process bag 516 positioned in the hot mixing chamber 114 of the processing device 102, and mixing the input product in the hot mixing chamber 114 for a preselected or preset duration. Then the preset or preselected volume of product is transferred from the process bag 516 to the formulation bag 526. The remaining volume of product is transferred back from the process bag 516 to the initial input bag 530. In one embodiment, at step 556, the 3D process bag 516 is then flushed with the medium from the media bag connected to the pipeline 520, and the flushing volume is pumped to the input bag 530. In one embodiment, the flushing volume and flushing mixing time can be selected by the user. As further shown therein, at step 558, the formulation preparation is then performed. In one embodiment, this includes transferring a predetermined volume of medium from the media bag connected to the pipeline 520 to the process bag 516 in the hot mixer 114, and transferring such medium to the formulation bag 526.

[0245] If selected / desired, freezer bag preparation and dosing may be performed at steps 560 and 562, respectively, to prepare additional bags (which may be freezer bags for cryopreservation purposes). In such a case, at step 560, a selected volume of the separated product from the input bag 530 is then transferred to the process bag 516 within the thermomixer 114 (wherein excess separated product is retained in the input bag 530). A predetermined volume of media from the media bag connected to line 524 and / or the media bag connected to line 522 is pumped to the process bag 516 within the thermomixer 114, where it is then temperature regulated at a predetermined / preselected temperature (for a period of time calculated by the controller 110 as required to regulate to the target temperature). Next, media from the media bag connected to line 520 is transferred to the process bag 516 within the thermomixer (after prompting), and the volume within the process bag 516 is mixed with such media. As expected, frozen bag dosing is performed by transferring the preselected 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. Accurate control of the transferred volume is achieved by ensuring accurate peristaltic pump flow rates and controlling flow timing. The peristaltic pump flow rate set point is calibrated during the initial pre-filling step to account for possible deviations from the nominal baseline of the peristaltic pump pipeline 518 and / or peristaltic pump 111. Therefore, this scheme allows the preparation / production of one preparation bag 526 and up to four frozen bags (connected to lines 538, 540, 542, and 544, respectively). Therefore, 1 to 5 dosing / bags of up to four components (initial product plus three media) of user-selected volumes are achieved by such a system and method of the present invention.

[0246] Now go to Figure 42-45, illustrating an exemplary embodiment of a second module 600 (also referred to herein as a bioprocessing device 600) for activation, transduction, and amplification of cells (e.g., cells enriched and isolated using the first module 100). The second module 600 can be, for example, a device / system configured to perform the workflow and methods described above in conjunction with the second module 200, and can be configured to operate similarly to the module 200 disclosed in WIPO International Publication No. WO 2019 / 106207. As shown therein, in one embodiment, the second module 600 includes a housing 602, a latchable process drawer 604 slidably received in the housing 602, and a latchable waste bag drawer 606 located below the process drawer 604 and also slidably received in the housing 602. Both the process drawer 604 and the waste bag drawer 606 can be moved between a closed position and an open position for insertion and removal of various components of the second module 600, as disclosed below. As discussed 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 portion of the housing 602 includes a power connection port or cable, one or more communication ports (e.g., RJ45 and RS485 ports), at least one inlet for receiving a supply of carbon dioxide, air oxygen and / or nitrogen, etc., one or more outlet / exhaust ports, and / or multiple (e.g., three) USB ports. The drawer 604 may also include a status indicator 605, multiple USB or other ports 607 for data transmission, and an input terminal 609.

[0247] The second module 600 also includes a cabinet 608 positioned in a vertical stacked relationship relative to the housing 602 (e.g., mounted on top of the housing 602). The cabinet 608 includes a pair of latchable doors 610, 612 hingedly mounted about a vertical axis, which 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 interlocking mechanism (e.g., a pneumatic latch or pin) that is used to maintain the doors 610, 612 in a closed position while bioprocessing operations are performed. In one embodiment, the cabinet 608 further includes a plurality of vertically oriented storage drawers 614, 616 that are slidably received within the cabinet 608. Although in Fig.43 and Fig.44 Two vertical storage drawers 614, 616 are shown in the figure, but there may be more or less than two drawers. In one embodiment, the storage drawers 614, 616 are slidably mounted on upper and / or lower tracks within the cabinet 608, thereby allowing the drawers 614, 616 to be easily moved between the stowed position and the extended position (in Fig.43 and Fig.44614, 616 are movable between the cabinet 608 and the doors 610, 612, in the stowed position, in which the drawers are received within the cabinet 608 and the doors 610, 612 are closeable, and in the extended position, the drawers 614, 616 extend from the cabinet 608, thereby allowing easy access to components and accessories mounted to the left and right vertical sides of the drawers 614, 616.

[0248] like Fig.45 As best shown in FIG. 1 , the interior surfaces of the doors 610, 612 include mechanisms (e.g., a specific array of pegs or pins 618) for releasably connecting the tube organizer cards and / or the 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 the sampling cards of the disposable kit, while the right door 612 may include an array of pegs for holding the tube organizer cards of the disposable kit. In one embodiment, both the tube organizer cards and the sampling cards may be mounted to the right door 612. Fig.43 and Fig.45 , one or both of the vertical storage drawers 614,616 may include a hook 620 for receiving a medium, a reagent and / or other fluid / solution bag on one or each of its faces, and the medium, the reagent and / or other fluid / solution bag are used to be used in the various biological treatment operations performed by the equipment 600. The hook 620 may be operatively connected to a load cell or integrated with it, to monitor the weight of the (multiple) bag connected thereto. In one embodiment, the first vertical drawer 614 is configured to receive one or more media bags 622, and the second vertical drawer 616 is configured to receive one or more reagent bags 624. In this respect, the first vertical drawer 614 may be referred to as a media tray or a compartment, and the second vertical drawer 616 may be referred to as a reagent tray or a compartment. The first vertical drawer 614 is equipped with a media drip tray 626 on its opposite side for catching leaks or drips from the media bag hanging on the hook 620, while the second vertical drawer 616 is equipped with a reagent drip tray 628 on its opposite side for catching leaks or drips from the reagent bag 624 hanging on the hook 620. In one embodiment, the drip trays 626, 628 are removable from the drawers 614, 616, respectively.

[0249] In one embodiment, one or more of the vertical drawers 614, 616 may be housed within a refrigerated compartment forming part of the cabinet 608 for maintaining a fluid or solution contained in one of the bags 622, 624 at a predetermined temperature. Similar to the housing 604, the cabinet 608 may also include a status indicator light 634. Although Figure 42-45 The waste bag drawer 606 is illustrated as part of the lower housing 602, but it is contemplated that the waste bag drawer may alternatively be housed within the cabinet 608 (e.g., as a horizontally oriented drawer or as a vertically mounted drawer). Fig.43 and Fig.46, the process drawer 604 includes an upward facing notch 630 configured to receive an anchor comb 632 that facilitates guiding tubing from the cabinet 608 into the process drawer 604. In one embodiment, the entire apparatus 600 is sized and dimensioned so as to be supported by a table or desk and to provide a user with easy access to the process drawer 604 and the cabinet 608. Control of the apparatus 600 and its functions is performed by an onboard controller (e.g., controller 210), as disclosed below.

[0250] Now go to Fig.46 and Fig.47 , which illustrates a detailed view of the process drawer 604. Fig.46 and Fig.47 As best shown in , the process drawer 604 includes a first interior space 636 configured to receive a disposable bioprocessing kit and a second interior space 638 positioned to the rear of the first interior space 436, and the functional components of the device 606 are installed in the second interior space 638. For example, in one embodiment, the second interior space 638 accommodates a peristaltic pump assembly 641, an array of pinch valves or a linear actuator array 643 (for controlling fluid flow through an array of fluid flow lines), and other components and devices necessary to perform the functions of the device 600. In one embodiment, the peristaltic pump assembly 641 and other components and devices may be configured as disclosed in WIPO International Publication No. WO 2019 / 106207. As Fig.47 As shown in FIG. 6 , a first platform rocker assembly 640 and a second platform rocker assembly 642 are mounted within the first interior space 636 and are configured to support a culture vessel (also referred to herein as a bioreactor vessel) of a disposable bioprocessing kit thereon in a manner disclosed below. The platform rocker assemblies 640, 642 each have a cover 644 through which a plurality of culture vessel support or mounting posts 646 extend for supporting the culture vessels of the disposable kit. In one embodiment, each platform rocker assembly 640, 642 includes four support posts 646, such as Fig.48 640,642. As also shown therein, a sensor assembly 648 associated with each platform rocker assembly 640,642 is provided to detect the presence of a culture vessel and / or measure the temperature in the culture vessel. In other embodiments, the sensor assembly 648 can be used to measure various additional parameters (e.g., temperature, carbon dioxide concentration, oxygen concentration, etc.) of the culture received in the culture vessel atop each platform rocker assembly 640,642 and / or to determine whether the culture vessel is properly positioned and seated on the rocker assembly. As discussed below, each platform rocker assembly 640,642 includes a plurality of load cells 658,660,662 for sensing the weight / quality of the culture vessel supported by the mounting post 646.

[0251] Reference again Fig.47 , the process drawer 604 includes a plurality of features that are configured to contain leaks and prevent or discourage any fluid from collecting within the process drawer 604. For example, the process drawer 604 includes a sealing element 650 that forms a fluid-tight seal between each platform rocker assembly 640, 642 and the bottom of the process drawer 604 (which extends around the periphery of each rocker assembly) and between the rocker assemblies 640, 642 themselves. In addition, each culture vessel support column 646 is equipped with a sealing element in the form of a flexible bellows 652 that forms a seal between the support column 646 and the cover 444. The sealing element 650 and the bellows 652 prevent any fluid from entering the space below the cover 644 of the platform rocker assembly 640, 642. Further, the bottom of the process drawer 604 is formed with a peripheral channel 654 that collects fluid that has overflowed or leaked. The drain hole 656 in the channel 654 provides a means of egress for the fluid collected in the channel 654 of the process drawer 604. The drain hole 656 is in fluid communication with the waste drawer 606 below the process drawer 604 so that any fluid that overflows or leaks into the process drawer 604 is drained directly into the waste drawer 606 to prevent damage to the electromechanical devices in the process drawer 604.

[0252] Fig.49 The configuration of the waste drawer 606 is shown, as shown therein, the waste drawer 606 includes a plurality of load cells 664. In one embodiment, there are four load cells positioned adjacent to the four corners of the waste drawer 606. As indicated above, the waste drawer is slidably received in the housing 602 below the process drawer 604 and is configured to receive a waste bag. In one embodiment, the conduit connected to the waste bag is guided from the process drawer along a groove behind the front panel of the process drawer to leave the process drawer and then freely guided downward to the waste drawer. In addition, as indicated above, the waste drawer 606 is configured to directly receive fluid that has leaked into the process drawer via the drain hole 656 in the process drawer 604.

[0253] Reference now Fig.50, a single-use disposable bioprocessing kit 700 for use with a bioprocessing apparatus 600 is illustrated. The bioprocessing kit 700 includes a generally rectangular tray 702 sized and dimensioned to be received in a first interior space 636 of a process drawer 604, and a pair of culture vessels 704, 706 received within the tray 702. The tray 702 has a pair of openings or windows below the culture vessels 704, 706, and supports the culture vessels 704, 706 in a raised position such that when the tray 702 is positioned within the first interior space 636 of the process drawer 604, the culture vessels 704, 706 are lifted from the tray 702 when engaged with the support columns 646 of the platform rocker assemblies 640, 642. As shown in FIG. Fig.50 and Fig.51 , the tray 702 includes a pair of legs 708, 710 at the front and rear thereof that support the tray 702 on the bottom of the process drawer 604. The legs 708, 710 are hollow and form a low point for the tray 702. Thus, in the event of a leak or spill within the tray 702 (as compared to the process drawer 604), the fluid will collect and be contained in the bottom of the legs 708, 710.

[0254] Further references Fig.50 and Fig.51 , the tray 702 further includes a first window 709 and a second window 711 in the rear of the tray 702, within which are positioned a valve manifold 712 and up to three segments 714, 716, 718 of peristaltic pump tubing for engagement with a peristaltic pump assembly 641 mounted in a process drawer 604 at the rear of the tray 702. The valve manifold 712 may be, for example, a fluid vessel as disclosed in U.S. Patent Application Publication No. 2020 / 0238282, configured to interface with a plurality of linear actuators having plungers of a linear actuator array 643, which is also mounted in the process drawer 604 at the rear of the tray 702. Alternatively, the valve manifold 712 may be formed by a plurality of fluid flow lines configured to be acted upon by a plurality of pinch valves of a pinch valve array, as disclosed in WIPO International Publication No. WO 2019 / 106207. Valve manifold 712 fluidly interconnects culture vessels 704, 706, media and reagent bags in cabinet 608, waste bags in waste drawer 606, and sampling lines to form a fluidic network or architecture as disclosed in the '207 publication or similar to the fluidic network or architecture disclosed in the '207 publication. Fig.50 The connections of the various tubes to the valve manifold 712 are shown.

[0255] Therefore, if Fig.50As further illustrated in FIG. 6 , the disposable kit 700 further includes a tubing organizer card 720 holding a plurality of tubing tails 726 that are fluidly connected to the valve manifold 712 and configured for connection to various media and reagent bags contained within the cabinet 608, and a sampling card 722 holding a plurality of sampling tubing tails that are also fluidly connected to the valve manifold 712. Finally, the disposable kit 700 also includes an anchor comb 632 that is received in a notch 630 in the process drawer 604 and facilitates guiding tubing from the cabinet 608 (e.g., from the tubing organizer 720 and sampling card 722) into the process drawer 604 and into the valve manifold 712. As discussed below, the anchor comb 632, tubing organizer 720, and sampling card 722 provide a means of organizing all tubing tails during and after installing the kit 700 and connecting various media, reagents, and other bags / containers. In one embodiment, the above combination Fig.50 The disposable kit 700 of all elements described may be sterilized by means known in the art, such as, for example, ethylene oxide sterilization or gamma sterilization, and sealed in a blister pack for shipping to an end user and for storage.

[0256] like Fig.52 and Fig.53 , the anchor comb 632 includes a body portion 730 having a passage 732 therethrough. Within the passage 732 are a plurality of tubing retaining elements 734 for retaining and maintaining a length of tubing in an organized manner. As disclosed above, during installation, the anchor comb 632 is received within the slot 630 of the process drawer 604 and facilitates directing various channels of tubing from the cabinet 608 into the process drawer 604, where the various channels are fluidly connected to the valve manifold 712.

[0257] refer to Fig.54, a detailed view of a duct organizer 720 according to an embodiment of the present invention is illustrated. The duct organizer 720 includes a generally rigid plate body 736 and a plurality of duct retaining channels 738, which are molded into or otherwise connected to the rigid plate body 738 and are configured to receive and retain a corresponding plurality of duct tails 726 therein. In one embodiment, the channels 738 extend upward from the lower right corner of the plate body 736 along its right hand side, turn back on themselves and extend generally downward at a certain angle toward the lower right corner of the plate body 738, turn back on themselves again and extend upward from the lower left corner of the plate body 736 along its left hand side. Therefore, the duct tails 726 received in these channels 738 follow the same tortuous path. Thus, this serpentine configuration of the channel 738 maximizes the length of tubing tails 726 that can be held by the tubing organizer, thereby allowing a considerable degree of play to facilitate connection of the tubing tails 726 to the various bags and / or containers contained within the cabinet 608 of the bioprocessing facility 600. Thus, the tubing organizer 720 maintains the tubing tails 726 in an organized and easily accessible manner, which helps to minimize setup time.

[0258] like Fig.54 As also shown in FIG. 1 , the plate body 736 includes features that allow the duct organizer 720 to be removably mounted or suspended on the inside of the door 612 of the cabinet 608, such as Fig.43 612 is shown in. Such features may include, for example, installation and / or positioning apertures 740, through which bolts 618 or hooks on door 612 are received. In use, once pipeline organizer 720 is attached to the inner face of door 612, the user can easily grasp the end of the pipeline tail 726 extending into the gap of plate body 736 or the release area 742, and remove it from its seated position with its corresponding channel 738. Then, by means of aseptic technology, such as, for example, aseptic pipe welding, pipeline tail 726 can be connected to a media bag, a reagent bag or other vessel contained in cabinet 608. This process can be repeated until all fluid connections are formed between the bag contained in cabinet 608 and the valve manifold 712 contained in drawer 604.

[0259] refer to Fig.55 and Fig.56, illustrating a detailed view of a sampling card 722 according to an embodiment of the present invention. As shown therein, the sampling card / device 722 includes a main body portion 744 having a manifold 746 and a plurality of sampling pipe tails 748 fluidly connected to the manifold 746. The sampling card 722 also includes a supply 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 pipe organizer 720, the main body portion 744 of the sampling card 722 includes features that allow the sampling card 722 to be removably mounted or suspended on the inside of the door 612 of the cabinet 608. Such features may include, for example, mounting and / or locating apertures 754 through which a latch 618 or hook on the door 612 is received. In use, once the sampling card 722 is attached to the inner face of the door 612, the user can draw a sample from one of the culture vessels 704, 706 using one of the easily accessible sampling tubing tails 748 on the sampling card 722. Thus, samples can be easily drawn during a bioprocessing operation without opening the process drawer 604 and without having to pause the operation.

[0260] Now go to Figures 57-63 , illustrates the installation and seating of the tray 702 within the process drawer 604 of the bioprocessing apparatus 600. As shown therein, by opening the process drawer 604 and lowering the tray 702 from above into the process drawer 604, the tray 702 is received within the first interior space 636 of the process drawer 604, such that the culture vessels 704, 706 of the disposable set 700 are in a front-to-back relationship within the process drawer 604. In this position, the valve manifold 712 is positioned directly in front of and aligned with the linear actuator array 643, and the three segments 714, 716, 718 of the peristaltic pump tubing are positioned directly in front of and aligned with the peristaltic pump assembly 641. As indicated above, when the tray 702 is lowered into the process drawer 604, the culture vessels 704, 706 are received on the support / mounting columns 646 of the corresponding platform rocker assemblies 640, so that the culture vessels 704, 706 are lifted from their seated engagement with the tray 702 and instead supported by the support columns 646.

[0261] like Figure 59-62 As best shown in FIG. 1 , the tray 702 and process drawer 604 of the disposable kit 700 have a number of cooperating features that facilitate proper positioning of the tray 702 within the process drawer 604 and allow verification of proper positioning. Fig.59As shown in , the tray 702 and the process drawer 604 include a plurality of engagement features / surfaces 756 that cooperate with each other when the tray 702 is properly positioned within the process drawer 604. The process drawer 604 includes a plurality of sensors 758 associated with the engagement features 756 of the drawer in the second interior space 636, which can detect when the cooperating engagement features 756 on the tray 702 and the process drawer 604 engage with each other, thereby indicating proper positioning of the tray 702. In one embodiment, the engagement features 756 associated with the tray 702 are located on the trunk of the tray, such as Fig.59 , and corresponding engagement features 756 associated with the process drawer 604 (and sensor 758) are positioned adjacent to the linear actuator array 643 and the peristaltic pump assembly 741, respectively. In one embodiment, the engagement features 756 associated with the process drawer 604 are pins of the sensor 758. In addition to detecting proper alignment and positioning of the tray 702 within the process drawer 604 as indicated above, the platform rocker assemblies 640, 642 include sensors 648 configured to detect proper positioning of the culture vessels 704, 706.

[0262] 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 pivoting pump shoe 762 that facilitate proper engagement of the peristaltic pump assembly 641 with the backbone of the tray 602. These features also minimize tolerance stack-up issues regarding engagement and actuation of the peristaltic pump assembly 741 and liner actuator array 742 with the segments 714, 716, 718 of peristaltic pump tubing and the valve manifold 712, respectively.

[0263] In one embodiment, the peristaltic pump assembly 641 and the solenoid actuators of the valve manifold 712 are configured to move toward and physically engage corresponding features of the disposable set when the disposable set is positioned in the process drawer and the drawer is closed. Fig.59 , module 600 includes a motorized engagement mechanism that physically moves the assembly comprising the peristaltic pump assembly 641 and the solenoid array 643 toward corresponding features in the disposable kit 700 (segments 714, 716, 718 of peristaltic pump tubing and valve manifold 712), wherein a fixed distance of movement is limited by features that prevent further movement. Disengagement simply involves operating the motorized engagement mechanism in reverse.

[0264] Reference now Fig.64 and Fig.65, showing the configuration of the bioreactor / culture vessels 704, 706 of the disposable bioprocessing kit 700. For ease of illustration, only the culture vessel 704 is shown (the 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 of polycarbonate, however other materials known in the art may also be utilized without departing from the broader aspects of the present invention. Fig.64 As shown in, lid 766 comprises a plurality of support bearings 772 or gussets that strengthen lid 766 and provide increased strength and durability. Lid 766 also comprises an inlet 774 and an outlet 776 to which pipeline can be connected. As illustrated therein, inlet 774 and outlet 776 are molded into the lid so that pipeline at least initially extends vertically from lid 776. This configuration of ports 774, 776 is convenient to be provided with, because pipeline can be more easily connected to culture vessel 704 from above. As further illustrated, vent port 777 is provided in the top of lid 766. In one embodiment, lid 766 comprises rounded corners (e.g., corner 778), which eliminates / prevents any stagnant zone.

[0265] Further references Fig.64 The membrane 768 may be formed of a suitable gas permeable material (e.g., silicone and / or polystyrene) or a porous material having a pore size that does not allow water or microorganisms to pass through, although other materials known in the art may also be utilized without departing from the broader aspects of the present invention. The membrane 768 includes a plurality of positioning / retaining holes 780 along the periphery of the membrane 768, the purpose of which will be described below. For its part, the gasket 770 may be formed of various materials known in the art (such as, for example, silicone) and includes a corresponding plurality of positioning / retaining holes 782, which are positioned along the periphery of the gasket 770 and aligned with the holes 780 in the membrane 768.

[0266] In one embodiment, the cover 766 and the base 764 are connected to each other via heat stakes along the perimeter of the cover 766 and the base 764. In one embodiment, heat stakes 781 extend through each of the positioning / retaining holes 780, 782 of the membrane 768 and the gasket 770, respectively, and are used to anchor the membrane 768 and the gasket 770 between the base 764 and the cover 766. In one embodiment, the cover 766 can be configured with heat stake pins 784 extending downwardly from its underside so that during assembly, the heat stake pins 784 extend through the corresponding positioning / retaining holes 780, 782 of the membrane 768 and the gasket 770, respectively, and are received in corresponding holes 786 in the perimeter of the base 764 and heat staked to the base 764. In one embodiment, about 20 to about 40 heat stakes are used to join the cover 766 to the base 764, and more preferably approximately 34 heat stakes are used. While the embodiments described herein utilize heat staking to connect the cover to the base, it is contemplated that other connection means may be utilized, such as fasteners, snap-fit ​​connections, etc., without departing from the broader aspects of the present invention.

[0267] In one embodiment, the upper surface of the base 764 has a textured surface that allows air flow and eliminates the need for a mesh (which has been customary on prior designs). Fig.65 , the flange region 788 of the base 764 includes a plurality of ribs 790 that provide increased rigidity and strength and a more robust interconnection with the lid 766 (which in addition provides a more reliable and robust anchoring of the membrane 768 and the gasket 770). The corners of the underside of the base 764 each include pin recesses 791, 792, 793, 794 that are configured to receive therein the mounting / supporting posts 646 of the platform rocker assembly 640 or 642 that supports the culture vessel 704. In one embodiment, one of the pin recesses (e.g., recess 794) is oval in shape, which provides improved positional tolerance when mounting the culture vessel 704 atop the platform rocker assembly 640. The base 764 is further provided with an IR sensor window 796 for measuring the temperature of the gas or fluid(s) within the culture vessel 704 using a sensor positioned below the culture vessel 704 within the process drawer 604, and a sensor recess 798 which is utilized by the sensor 648 of the platform rocker assembly 640 or 642 to determine whether the culture vessel 704 is present within the process drawer and / or properly positioned therein. Finally, as Fig.65 As shown in FIG, the base 764 includes an array of small openings 799 that provide fluid communication between the atmosphere within the process drawer 604 and the underside of the membrane 768 for gas transfer during bioprocessing. In one embodiment, there are hundreds of small openings 799 in the base 764.

[0268] As indicated 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 can be utilized to provide mixing of fluids within the culture vessels 704, 706 to support bioprocessing operations therein, including the mechanisms disclosed in WIPO International Publication No. WO 2019 / 106207. Figures 66-68 The configuration of the platform rocker assembly 640, 642 according to another embodiment of the present invention is illustrated (for simplicity and ease of understanding, the rocker assembly 640 is depicted). As shown therein, the platform rocker assembly 640 includes a base 870, a fulcrum 872 received on the base 870 defining a central pivot axis 873, a motor 874 mounted to the base 870 and having an eccentric roller 876 driven by the motor 874, a rocking plate 878 received atop the fulcrum 872 and in contact with the eccentric roller 876 and pivotable about the fulcrum 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 fulcrum 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 other embodiments, instead of a circular roller moving along an eccentric path, a cam-shaped roller may be used.

[0269] like Fig.67 and Fig.68 , the rocking plate 878 includes four support posts 646 received by pin recesses 791, 792, 793, 794 in the base 764 of the culture vessel 704. The motor 874 is controllable (e.g., under the control of the controller 210 of the second module 200 (i.e., the apparatus 600)) to drive the eccentric roller 876 to transfer force against or remove force from the underside of the rocking plate 878 depending on the position of the eccentric roller 876 to tilt the rocking plate 878 and the culture vessel 704 received on the rocking plate 878 upward and / or downward. Although the motor 874 may be capable of being controlled by a main controller, the platform rocker assemblies 640, 642 may alternatively have a dedicated controller positioned on the base plate 872 below the rocking plate 878. Due to the force (or lack thereof) from the eccentric roller 876, the rocking plate 878 and the culture vessel 704 supported thereon pivot about the fulcrum axis 873 of the fulcrum 872.

[0270] In one embodiment, each of the support columns 646 may be configured with 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) load cells 882 extending through the rocker plate 878 and engaging the underside of the culture vessel 704 for measuring the mass of the culture vessel 704. Fig.67 As further shown in FIG. 8 , the rocking plate 878 may be equipped with a tilt sensor 884 configured to measure the degree of tilt of the rocking plate 878 (and therefore the culture vessel 704 ) for use by the controller when performing the rocking / mixing process.

[0271] As indicated above, when motor 874 is actuated, eccentric circular roller 876 transmits a force against the bottom surface of wobble plate 878, thereby moving wobble plate 878 upward or downward, depending on the direction of rotation of motor 874. When in constant operation, the circular profile of eccentric circular roller 876 imparts a continuous sinusoidal wobble profile to the contents of culture vessel 704. This wobble motion is Fig.69 878. The monitoring of the rocking plate 878 using the tilt sensor 884 allows closed-loop control of the tilt angle, homing and discharge operations, and the detection of fault event conditions. The use of the support column 646 to support the culture vessel 704 on the rocking plate 878 allows the entire bottom of the culture vessel 704 to remain unobstructed, which allows better ventilation, heat transfer and other functions, as discussed below. 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. As will be appreciated, in particular, mammalian cells are highly sensitive to the shearing forces induced by the small-scale eddy currents on the highly turbulent fluid state. Therefore, the strong vibration, impact or other mechanical stimulation that causes excessive turbulence, foam formation or overflow is potentially harmful. Therefore, the continuous sinusoidal swing profile of the platform rocker assembly 640, 642 minimizes the existence of such small-scale eddy currents by removing any high-frequency mechanical stimulation, thereby providing safer and gentler mixing conditions, which is particularly beneficial to mammalian cell culture.

[0272] As indicated above, ventilation and heat transfer by the pedestal 764 and film 768 of culture vessels 704,706 are important for various bioprocessing operations.Typically, some cell cultures (for example mammalian cell cultures) must be surrounded by a sterile, homogeneous incubation atmosphere for cell growth at a suitable temperature and CO2 concentration.The mode of providing such physicochemical conditions depends on application, cell type specificity and the degree to which they are adapted to suspend or adhere to growth.In some cases, process may require cell growth on the monolayer on the top of a gas permeable membrane.In this case, heat and mass transfer occur by the passive diffusion of the local gradient of the direct region at the transmembrane both sides.Embodiments of the present invention optimize such phenomenon by the gas permeable membrane 768 on the bottom of inducing culture vessels 704,706 and the turbulent flow interaction between the incubation atmosphere recirculation flow.

[0273] Figure 70-72A cross-sectional view of a portion of a process drawer 604 of a bioprocessing apparatus 600 is presented, wherein the tray 702 and culture vessels 704, 706 of a disposable bioprocessing kit 700 are positioned therein. As disclosed above, the process drawer 604 forms an incubation chamber 902, within which the tray 702 and culture vessels 704, 706 are positioned. As shown therein, the culture vessels 704, 706 are supported by support columns 646 of platform rocker assemblies 640, 642. Within the process drawer 604 is a heating element / device 904 (e.g., positioned above and below each culture vessel). For example, a heater 904 may be positioned below each culture vessel 704, 706, and adjacent to the top of the process drawer 604, for heating the incubation chamber 902 and the culture vessels 704, 706. The process drawer 604 also includes a pair of fans or blowers 906, 908 adjacent to its front and rear walls within the cover 644 of the rocker assembly 640, 642. As further shown therein, the cover 644 may include a pair of opposing shutters or air passages 910, 912, near which the blowers 906, 908 are positioned, thereby allowing air to leave the space adjacent to the rear of the process drawer 604 within the cover 644 (defining the incubation atmosphere recirculation chamber 915) and re-enter the recirculation chamber 915 from the front of the process drawer 604. As discussed below, a temperature sensor 914 and a carbon dioxide sensor 916 are also positioned in at least one position along the recirculation air flow path for measuring the temperature of the recirculation air flow and the carbon dioxide concentration of the recirculation air flow. As further shown therein, a supply source 918 of carbon dioxide is selectively fluidly connected to the process drawer 604 (e.g., via a carbon dioxide inlet port on the rear of the housing 602 of the bioprocessing device 600) and a valve 920. The process drawer 604 also includes a gas port 922 that allows fluid communication between the interior of the process drawer 604 and ambient air (thereby eliminating the need to have a dedicated, separate oxygen supply source). The components described above form a system 900 for liquid-to-atmosphere direct mass transfer of the bioprocess system 600, the operation of which will be described below.

[0274] Further references Fig.70 , the temperature sensor 914 and the carbon dioxide sensor 916 are electrically connected or otherwise communicated with a controller (e.g., the main controller 210 of the apparatus 600, although a dedicated controller for executing the recirculated air flow process is also contemplated) for receiving information about the temperature and carbon dioxide concentration of the recirculated air flow. The controller 210 is also electrically connected or otherwise communicated with the valve 920, fans 906, 908, and heater 904 for controlling their operation in response to the sensor readings and specified set points.

[0275] Reference now Fig.71, the controller 210 is operable to control the fans 906, 908 to generate the recirculating air flow 924. As discussed below, the tray 702 and the process drawer 604 each include various ducting features 926 that ensure that the recirculating air flow 924 exits the recirculating chamber 915 through the louvers 912 adjacent to the rear 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 of the process drawer 604, and re-enters the recirculating chamber 915 through the louvers 910. In this regard, the fan 908 pushes the recirculating air flow 924 outward from the recirculating chamber 915, while the fan 906 draws the recirculating air flow 924 into the recirculating chamber 915.

[0276] refer to Fig.72 , a fan 908 pushes the incubation atmosphere through the incubation atmosphere recirculation chamber 915, and a duct feature 926 directs the recirculating air flow 924 across the bottom of the culture vessels 704, 706. In doing so, the duct feature and the configuration of the underside of the base 764 of the culture vessels 704, 706 induce the formation of local 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.

[0277] Figures 73-76 The ducting features of the system 900 are more clearly illustrated, which allow the recirculating air flow 924 to be directed from the recirculating chamber 915, across the bottom of the culture vessels 704, 706 and back into the recirculating chamber 915 under the influence from the fans 906, 908. As shown therein, the interior facing sides of the legs 708, 710 of the tray 702 are formed with indentations or recessed areas 930, which allow the recirculating air 924 exiting / entering the recirculating chamber 915 to travel upward or downward along the interior surfaces of the legs 708, 710, as the case may be. Figures 73-76 Specifically illustrated is how the recirculation air 924 present in the recirculation chamber 915 is directed upward by the recessed areas 930 of the legs 710 of the tray 702. Thus, the recessed areas 930 of the legs 708, 710 and the outer surface of the recirculation chamber form a vertical air path for the recirculation air flow 924. As the air exiting the louvers 912 travels upward within the recessed areas 930 of the legs 710, the air is obstructed at the point where the legs 710 intersect the bottom of the tray 702. Fig.73 and Fig.74, the tray 702 includes a pair of lateral vent openings 930 located at a height that generally corresponds to the vertical height of the bottom of the culture vessels 704, 706. Thus, the vent openings 932 redirect the recirculating air flow 924 laterally through such openings 932 and toward the culture vessels 704, 706, where it interacts with the bottom geometry of the culture vessels 704, 706 and their corresponding gas permeable membranes, thereby causing the formation of local turbulence 928. The recirculating air flow 924 moves across the bottom of the culture vessels 704, 706, where it enters the opposing vent openings, travels downward within the recessed area 930 of the legs 708, and re-enters the recirculating chamber 915 through the louvers 910.

[0278] As disclosed above, the formation of local turbulence 928 in the recirculating air stream 924 helps to 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 stream 924 and the control actions provided by the control unit 210, the temperature sensor 914, the carbon dioxide sensor 916, the heater 904 and the carbon dioxide control valve 920 allow the volume inside the incubation chamber 902 to be homogenized. Therefore, the system 900 provides heat and mass transfer optimization. As will be appreciated, the constant availability of oxygen only tens of microns away from the cell monolayer supports higher cell concentrations and minimizes the physicochemical gradients across the surface of the membrane 768 of the culture vessels 704, 706.

[0279] As described above, the apparatus 600 includes a plurality of sensors and monitoring devices for monitoring the bioprocessing operation when performing the bioprocessing operation, including monitoring various parameters of the cell culture in the culture vessels 704, 706. This may include, for example, using the sampling conduit tail 748 of the sampling card 722 and / or using sensors to periodically draw samples from the culture vessels 704, 706 to sense various parameters of the culture in the vessel. For example, the sensor assembly 648 accommodates an IR sensor for temperature measurement and for detecting the presence of the culture vessel in the process drawer. The window 796 in the base 764 of the culture vessel 704, 706 enables the temperature of the membrane in the culture vessel to be measured based on IR, and thus measures the temperature of the liquid in the culture vessel.

[0280] refer to Figures 77-84 In one embodiment, the apparatus 600 may further include a flow-through sensing chamber 950 (also referred to herein as a flow-through sensing apparatus 950), which may be used to measure or monitor various parameters of a fluid within the apparatus 600 (e.g., a culture(s) within the culture vessels 704, 706) using a variety of different sensing / measuring devices and without extracting any fluid from the system. Figures 77-80As best shown in FIG. 1 , the flow-through sensing chamber 950 includes a first plate 952, a second plate 954 connected to the first plate 952 in a facing relationship, and a fluid channel 956 intermediate the first plate 952 and the second plate 954. In one embodiment, the fluid channel 956 is formed by a release 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 height of the fluid channel 956 may be between about 0.1 mm and about 1 mm. The chamber 950 further includes a first port 958 in fluid communication with the fluid channel 956 for facilitating the flow of fluid into the chamber 950 and its fluid channel 956, and a second port 960 in fluid communication with the fluid channel 956 for facilitating the flow of fluid out of the chamber 950 and its fluid channel 956. In one embodiment, the ports 958, 960 are in fluid communication with opposite ends of the fluid channel 956.

[0281] like Fig.78 , in one embodiment, the plates 952, 954 may have features that facilitate alignment and coupling of the plates to one another. For example, one of the plates (e.g., plate 952) may have a pair of notches 957 that receive corresponding tabs 959 of the other of the plates (e.g., plate 954). As discussed below, the back plate / first plate 952 includes a plurality of mounting and locating holes 961 extending therethrough that facilitate mounting the chamber 950 to the tray 702 of the disposable kit 700. In one embodiment, the first / back plate 952 and the second / front plate 954 are generally rectangular in shape, are transparent, and are made of biocompatible plastic, glass, or a combination of plastic and glass, however the invention is not intended to be so limited in this regard.

[0282] like Fig.78 and Fig.79 As best shown in FIG. 1 , the fluid channel 956 includes a plurality of segments or sensing locations 962, 964, 966 that allow or facilitate interrogation of the fluid within the fluid channel 956 or monitoring of the fluid using a plurality of sensing devices and techniques. In one embodiment, the fluid within the fluid channel 956 may be interrogated using a variety of different sensing devices associated with a respective one of the plurality of sensing locations 962, 964, 966. In one embodiment, the segment 966 has one or more sensors 968 located within the fluid channel 956 and configured to maintain continuous contact with the fluid passing through the fluid channel 956. Fig.77 As shown in FIG. 9A , the second plate 954 includes a plurality of electrodes 970 that extend into the fluid channel 956 and are accessible from a laterally extending flange 972 of the second plate 954. In one embodiment, the electrodes 970 are gold plated electrodes.

[0283] As indicated above, the flow-through sensing chamber 950 allows the fluid within the fluid channel 956 to be interrogated using a variety of different sensing devices for measuring a variety of different parameters of the fluid. For example, in one embodiment, the sensing location 962 can be configured as a reflective light interrogation segment that is configured with a gold-coated mirror 974 behind the fluid channel 956 that reflects light emitted by the sensing device. Thus, the sensing location 962 can be suitable for various techniques for monitoring / sensing biological variables, such as, for example, optical density sensing, turbidity measurement, digital holographic microscopy, light dynamic scattering, and / or optical interferometry, etc. In one embodiment, the sensing location 964 can be configured as a transmission and backscattered light interrogation segment that allows the fluid within the fluid channel 956 to be interrogated using a transmission or backscattered light sensing instrument. For its part, the sensing location 966 can be configured as a fluorescence sensor interrogation segment having various sensors 968 in contact with the fluid within the fluid channel 956, allowing monitoring or sensing of various parameters of the fluid, such as, for example, dissolved oxygen, pH, carbon dioxide, analytes, etc. Electrode 970 faces rearward (opposite ports 958, 960) and is configured to be contacted by a spring-biased pin of one or more measuring devices suitable for various electrochemical measurement techniques such as, for example, electrical impedance spectroscopy, amperometry, amperometry and / or polarography, etc.

[0284] Fig.81 and Fig.82 The positioning of the flow-through sensing chamber 950 on the backbone of the tray 702 of the disposable bioprocessing kit 700 is illustrated. As indicated above, the chamber 950 can be connected to the tray 702 by receiving the snap pins 976 located on the backbone of the tray 700 within the corresponding mounting apertures 961 of the chamber 950. As shown therein, in one embodiment, the chamber 950 can be mounted to the tray 700 between the valve manifold 712 and the peristaltic pump tubing segments 714, 716, 718. Although the pins 976 are illustrated as being used to mount the chamber 950 to the tray 700, it is contemplated that other means of connection such as, for example, clamping, clamping, fasteners, snap fits, press fits, etc. can also be utilized without departing from the broader aspects of the present invention. In one embodiment, the chamber 950 can form part of the disposable bioprocessing kit 700.

[0285] Fig.83 and Fig.84 A schematic diagram of a flow-through sensing chamber 950 and various sensing instruments / devices for monitoring various parameters of the fluid within a fluid channel 956 is presented. Fig.83 As shown in FIG. 6 , for example, a first electrochemical sensing instrument 978 and a second electrochemical sensing instrument 980 onboard the device 600 may interface with the electrode 970 via a spring biased pin 982. Fig.84As shown in , the reflected light instrument 984 can be positioned and configured to interrogate the fluid within the first sensing location, the first fluorescence instrument 986 and the second fluorescence instrument 988 can be positioned and configured to interrogate the fluid within the second sensing location 964, and the transmitted / backscattered light instrument 990 is positioned and configured to interrogate the fluid within the third sensing location 966.

[0286] Thus, embodiments of the present invention provide an online sensing chamber 950 that provides various optical and electrical measurements of fluid within the fluid channel 956 of the chamber 950, thereby avoiding any need to directly interrogate either of the culture vessels 704, 706. In use, when it is desired to monitor or measure various parameters of the culture within either of the culture vessels 704, 706, the peristaltic pump assembly 641 is used to pump the fluid through the sensing chamber 950, where it can be interrogated by a set of sensor instruments / devices. That is, the chamber 950 disclosed herein facilitates the use of electrochemical and optical sensing technologies on a single fluid channel, thereby allowing multi-parameter monitoring of physicochemical growth conditions, cell type metabolic activity (lactate, glucose, etc.), and viable cell density and total cell count measurements of the cell culture within the culture vessels 704, 706.

[0287] The components of the bioprocessing apparatus 600 (also referred to as the second module 200) have been disclosed in detail, with reference to Figures 85-89 An embodiment of a fluid flow architecture or system 200 within an apparatus 600 is shown. As disclosed above and described in more detail below, the configuration of the bioprocessing apparatus 600 and kit 700, together with the fluid flow architecture 200 provided thereby, allows for cell activation, genetic modification and amplification of cell products, and ancillary or related protocols, workflows, and methods in an automated and functionally closed manner. In one embodiment, the flow architecture or system 400 may be as described in WIPO International Publication No. WO 2019 / 106207. Figure 3-7 , however other configurations are possible. Fig.85, the system 400 includes a first bioreactor vessel (e.g., culture vessel 704) and a second bioreactor vessel 420 (e.g., culture vessel 706). The first bioreactor vessel includes at least a first port 412 and a first bioreactor line 414 in fluid communication with the first port 412, and a second port 416 and a second bioreactor line 418 in fluid communication with the second port 416. Similarly, the second bioreactor vessel includes at least a first port 422 and a first bioreactor line 424 in fluid communication with the first port 422, and a second port 426 and a second bioreactor line 428 in fluid communication with the second port 426. The first bioreactor vessel 410 and the second bioreactor vessel 420 together 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 more than two bioreactor vessels.

[0288] As discussed below, the first bioreactor line 414 and the second bioreactor line 418 of the first bioreactor vessel 410 and the first bioreactor line 424 and the second bioreactor line 428 of the second bioreactor vessel 420 each include a corresponding valve for controlling the flow of fluid therethrough. In particular, the first bioreactor line 414 of the first bioreactor vessel 410 includes a first bioreactor line valve 432, while the second bioreactor line 418 of the first bioreactor vessel 410 includes a second bioreactor line valve 424. Similarly, the first bioreactor line 424 of the second bioreactor vessel 420 includes a first bioreactor line valve 436, while the second bioreactor line 428 of the second bioreactor vessel 420 includes a second bioreactor line valve 438.

[0289] Further references Fig.85 The system 400 also includes a first fluid assembly 440 having a first fluid assembly pipeline 442, a second fluid assembly 444 having a second fluid assembly pipeline 446, and a sampling assembly 448. An interconnection pipeline 450 having an interconnection pipeline valve 452 provides fluid communication between the first fluid assembly 440 and the second fluid assembly 444. Fig.85As shown in , the interconnecting pipeline 450 also provides fluid communication between the second bioreactor line 418 and the first bioreactor line 414 of the first bioreactor vessel 410, thereby allowing the fluid to circulate along the first circulation loop of the first bioreactor vessel. Similarly, the interconnecting pipeline also provides fluid communication between the second bioreactor line 428 and the first bioreactor line 424 of the second bioreactor vessel 420, thereby allowing the fluid to circulate along the second circulation loop of the second bioreactor vessel. In addition, as discussed below, the interconnecting pipeline 450 further provides 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, thereby allowing the contents of the first bioreactor vessel 410 to be transferred to the second bioreactor vessel 420. Fig.85 As illustrated in FIG. 1 , in one embodiment, an interconnect 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 .

[0290] As Fig.85 As shown, the first fluid assembly 440 and the second fluid assembly 450 are arranged along the interconnection line 450. In addition, 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 through the first bioreactor line 414 of the first bioreactor vessel and the first bioreactor line 424 of the second bioreactor vessel 420, respectively. The second fluid assembly 444 is in fluid communication with the second port 416 of the first bioreactor vessel 410 and the second port 426 of the second bioreactor vessel 420 via the interconnection line 450.

[0291] A first pump 454 of a peristaltic pump assembly 641 capable of providing bidirectional fluid flow is disposed along the first fluid assembly line 442, and a second pump or circulation line pump 456 of a peristaltic pump assembly 641 capable of providing bidirectional fluid flow is disposed along the interconnecting line 450, the functions and purposes of which will be discussed below. Fig.85 As also shown, the sterile air source 458 is connected to the interconnection line 450 via 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. Fig.85A sterile air source 458 is shown connected to the interconnect line 450, but in other embodiments, the sterile air source may be connected to the first fluid component 440, the second fluid component 444, or a fluid flow path between the second bioreactor line valve and the first bioreactor line valve of the first bioreactor or the second bioreactor without departing from the broader aspects of the present invention.

[0292] Now refer to Figure 86 to Figure 88 , showing a detailed view of the first fluid component 440, the second fluid component 444 and the sampling component 448. Fig.86 , the first fluid assembly 440 includes a plurality of pipe tails 464a-f, each of which is configured for selective / removable connection to one of the plurality of first reservoirs 466a-f. Each pipe tail 464a-f of the first fluid assembly 440 includes a pipe tail valve 468a-f for selectively controlling the flow of fluid to or from a corresponding one of the plurality of first reservoirs 466a-f of the first fluid assembly 440. Although Fig.86 Specifically, the first fluid assembly 440 is shown to include six fluid reservoirs, but more or fewer reservoirs may be utilized as desired to provide input or collection of various process fluids. As described below, it is contemplated that each pipe tail 464a-f may be individually connected to a reservoir 466a-f at a desired time during operation of the fluid assembly 440.

[0293] Specific reference Fig.87 , the second fluid assembly 444 includes a plurality of pipe tails 470a-d, each of which is configured for selective / removable connection to one of the plurality of second reservoirs 472a-d. Each pipe tail 470a-d of the second fluid assembly 444 includes a pipe tail valve 474a-e, which is used to selectively control the flow of fluid to or from a corresponding one of the plurality of second reservoirs 472a-d of the first fluid assembly 444. Although Fig.87The second fluid assembly 444 is specifically shown to include four fluid reservoirs, but more or fewer reservoirs may be used as desired to provide input or collection of various processing fluids. In one embodiment, as discussed below, at least one of the second reservoirs (e.g., second reservoir 472d) is a collection reservoir for collecting amplified cell populations housed in a cabinet 608 of the device 600. In one embodiment, the second reservoir 472a is a waste reservoir or bag housed in a waste drawer 606 of the device 600, the purpose of which is discussed below. Although it is contemplated that the waste bag may be housed in the drawer 606 of the device 600, it is also contemplated that the waste bag may be housed in the media cabinet 608. For example, in one embodiment, the waste bag may be suspended on a hook in the media cabinet. The hook may be associated with a pressure sensor to achieve both waste quality measurement, leak detection, and blockage detection. In such an arrangement in which the waste bag is housed in the cabinet 608, the waste drawer 606 may be omitted.

[0294] In one embodiment, the first reservoir 466a-f and the second reservoir 472a-d are single-use / disposable flexible bags housed within the cabinet 608 of the apparatus 600 and fluidly connected to the manifold 712 via the tubing tails of the tubing organizer 720. In one embodiment, the bags are substantially two-dimensional bags having opposing panels that are welded or secured together around their perimeters and support connecting conduits for connection to their respective tails, as is known in the art.

[0295] In one embodiment, the reservoir / bag can be connected to the pipe tail of the first pipe assembly and the second pipe assembly using a sterile welding device. In one embodiment, the welding device can be positioned next to the device 600, and the welding device is used to weld one of the pipe tails to the tail of the pipe on the bag (while maintaining sterility). Therefore, the operator can provide the bag when the bag is needed (for example, by grabbing the pipe tail from the pipe organizer 720 and inserting its free end into the welding device, placing the free end of the bag tube adjacent to the end of the pipe tail, cutting the tube with a new razor blade, and heating the cut end when the razor is pulled away, while the two pipe ends are pressed together while still molten, so that they are resolidified together). Conversely, the bag can be removed by heat sealing the line from the bag and cutting at the heat seal to separate the two closed lines. Therefore, when desired, the reservoir / bag can be connected separately, and the present invention does not need to connect all reservoirs / bags at the beginning of the scheme, because the operator will be able to access the appropriate pipe tail during the whole process, and connect the reservoir / bag in time for its use. Indeed, while it is possible for all reservoirs / bags to be pre-connected, the present invention does not require pre-connection, and one benefit of the second module 200 is that it allows the operator to access the fluid components / lines during operation so that used bags can be connected in a sterile manner, and disconnected so that other bags can be connected aseptically during the protocol, as discussed below.

[0296] like Fig.88 As illustrated in , the sampling assembly 448 includes one or more sampling lines, such as sampling lines 476a-476d (which may be the sampling pipe tail 748 of the sampling card 722), that are fluidly connected to the interconnected line 450. Each of the sampling lines 476a-476d may include a sampling line valve 478a-d, which can be selectively actuated to allow fluid to flow from the interconnected line 450 through the sampling lines 476a-476d. As also shown therein, 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 interconnected line 450. The sample collection device can take the form of any sampling device known in the art, such as, for example, a syringe, a dip tube, a bag, etc. Although Fig.88The sampling assembly 448 is shown connected to the interconnecting pipeline, but in other embodiments, the sampling assembly may be fluidly coupled to the first fluid assembly 440, the second fluid assembly 444, the fluid flow path between the second bioreactor line valve 434 and the first bioreactor line valve 432 of the first bioreactor vessel 410, and / or the fluid flow path between the second bioreactor line valve 438 and the first bioreactor line valve 436 of the second bioreactor vessel 420. The sampling assembly 448 desirably provides fully functionally closed fluid sampling at one or more points in the system 400.

[0297] Return to reference Fig.85 In one embodiment, the system 400 may also include a filter line 482 that is connected at two points along the interconnect line 450 and defines a filtration loop along the interconnect line 450. A filter 484 is positioned along the filter line 482 for removing permeate waste from the fluid passing through the filter line 482. As shown therein, the filter line 482 includes an upstream filter line valve 486 and a downstream filter line valve 488 positioned on the upstream and downstream sides of the filter 484, respectively. The waste line 490 provides fluid communication between the filter 484 and the second fluid component 444, and in particular with the pipe tail 470a of the second fluid component 444 that is connected to the waste reservoir 472a. In this regard, the waste line 490 transports waste removed from the fluid passing through the filter line 482 by the filter 484 to the waste reservoir 472a. As shown in FIG. Fig.85 , the filter line 482 surrounds the interconnect line valve 452 so that the fluid flow through the interconnect line 450 can be forced through the filter line 482, as discussed below. An osmotic pump 492 positioned along the waste line 490 is operable to pump waste removed by the filter to the waste reservoir 472a. In one embodiment, the filter 484 is desirably an elongated hollow fiber filter, however, other tangential flow or cross flow filtration devices known in the art, such as, for example, flat sheet membrane filters, may also be utilized without departing from the broader aspects of the present invention.

[0298] In one embodiment, the valves of the first fluid assembly 440 and the second fluid assembly 444 and the bioreactor line valves (i.e., valves 432, 434, 436, 438, sterile line valve 462, interconnect line valve 452, and filter line valves 486, 488) are formed by the engagement of one of the linear actuators of the linear actuator array 643 with the valve manifold 712 to block or allow a specific flow of fluid therethrough. In one embodiment, the operation of the valves and pumps disclosed above (i.e., the linear actuators of the linear actuator array 643 and the three peristaltic pumps 454, 456, 492 of the peristaltic pump assembly 641) is automatically performed according to a programming scheme to achieve proper operation of the module 200 / device 600. It is contemplated that the second controller 210 onboard the second module 200 / device 600 can direct the operation of these valves (linear actuators) and pumps.

[0299] As indicated above, the bioprocessing device 600 is configured to perform activation, transduction and amplification phases of cell processing in combination with the disposable bioprocessing kit 700. In one embodiment, the activation phase comprises six steps, each of which includes multiple user-controllable / selectable parameters and is performed by the controller 210. During the activation phase, two pre-inoculation reagents and two post-inoculation reagents can be used. The cell input of the activation phase is a cell ready to undergo activation. After activation, it is possible to concentrate and wash the cells to remove any residual reagent components that are not desired in subsequent process steps. The transduction phase also comprises six steps, each of which includes multiple user-controllable / selectable parameters and is performed by the controller 210. During the transduction phase, two pre-inoculation reagents and two post-inoculation reagents can be used. The cell input of the transduction phase is a cell that has been activated in a previous phase. After transduction, it is possible to concentrate and wash the cells to remove any residual reagent components that are not desired in subsequent process steps. As such, the amplification phase comprises three steps (inoculation, 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 media to the transduction vessel to dilute the contents to the desired cell density for expansion. During the cell culture step, the user can select the sampling frequency and define the feeding strategy for the cells in the expansion culture vessels 704, 706. During the harvesting step, harvesting can be performed at preset time points or initiated by the user once the target cell dosing is achieved.

[0300] In one embodiment, the parameters that can be controlled or selected by the user include reagent parameters before and after inoculation, input cell volume, incubation, volume reduction, washing, target inoculation and cell culture. The reagent step before or after inoculation includes parameters for up to two reagents that can be added to the culture vessel before inoculating cells and for up to two reagents that can be added to the culture vessel after inoculating cells. Before the reagent is transferred to the culture vessel, the user can transfer air or liquid through the system. After the reagent incubation, the culture vessel can be rinsed before the inoculation of cells. The input cell volume parameter defines the parameters for adding source cells to the culture vessel. Before adding cells to the culture vessel, the user can manually mix the cells in the source bag. In addition, the source bag can be rinsed to maximize the transfer of input cells. The incubation parameter defines the parameters during the cell incubation in the culture vessel 704, 706. The user can set the target inoculation density and the volume for activation, as well as sampling related parameters. The volume reduction parameter defines the parameters for concentrating cells after activation. Cells are concentrated using hollow fiber filters (HFF) or via volume reduction by skimming and aspirating the liquid out of the culture vessel without disturbing the cells (i.e., perfusing without adding media to the inlet so that the volume within the culture vessel is reduced), also known as high speed perfusion (HSP).

[0301] Wash Parameters define the parameters used to wash the cells after volume reduction in order to make them ready for transduction. Cells are washed using either hollow fiber filters (HFF) or high speed perfusion (HSP). In one embodiment, the HSP wash protocol includes the following process steps: 1) an initial sedimentation phase - the activation vessel is maintained stable for a fixed amount of time to allow cells to settle on the vessel membrane; 2) very slow activation vessel mixing is optionally implemented to enhance homogeneity of the supernatant without disturbing cell sedimentation; 3) simultaneous addition of medium and removal of supernatant while maintaining the activation vessel volume stable; 4) in the middle of the wash duration, very slow activation vessel mixing is optionally implemented to enhance homogeneity of the supernatant without disturbing cell sedimentation; 5) simultaneous addition of medium and removal of supernatant while maintaining the activation vessel volume stable until the wash target duration has passed or the wash target medium volume has been consumed; 6) optionally diluting the activation vessel contents with medium to a target vessel volume; 7) optionally implementing very slow activation vessel mixing to enhance homogeneity of the supernatant without disturbing cell sedimentation; and 8) low flow rate removal of supernatant without disturbing cell sedimentation to a target activation vessel volume.

[0302] In one embodiment, for the transduction phase, the steps are similar to the description of the activation phase provided above. In one embodiment, transfer cell parameters are provided that define the parameters for transferring activated cells from the activation vessel to the transduction vessel. The system can mix the cells in the activation vessel before transferring the cells to the culture vessel. Some or all of the contents of the activation vessel can be transferred to the transduction vessel. Additionally, the activation vessel can be rinsed to maximize the transfer of cells.

[0303] Finally, the target inoculation universal parameter defines the parameters for setting the initial conditions for the cells during the expansion. The cell culture parameter defines the supply strategy for culturing cells during the expansion. The user can define the supply cycle based on the user-configurable parameters. The exemplary supply strategy includes a single-shot medium addition (supply batch) or a continuous medium addition (perfusion). The harvest parameter defines the parameters for realizing cell harvesting. The user can define the cell volume to be harvested, and start harvesting at a defined time point or when desired. As will be appreciated, the selection and setting of these parameters can be performed using interface 609 or by a non-airborne user interface or terminal (e.g., by a data port on the back of the device 600) communicating with the device 600, but wireless communication means are also possible.

[0304] As indicated above, the apparatus 600 and flow architecture 400 also allow for sampling of the contents of the culture vessel(s) 704 , 706 using, for example, a sampling conduit tail 748 of a sampling card 722 .

[0305] In one embodiment, the sampling sequence includes tilting the platform rocker assemblies 640, 642 to mix and homogenize the contents of the culture vessel (the mixing speed depends on the vessel volume), actuating the process pump 456 to circulate the vessel contents from the vessel outlet port (416 or 426) in the sampling line and back to the vessel inlet port (412 or 422), prompting the user to take a sample, stopping circulation and mixing, and finally clearing the sampling line.

[0306] In one embodiment, the use of two culture vessels 704, 706 within the process drawer 604 of the device allows parallel processing to be performed in the manner disclosed below. In one embodiment, all activation steps can be performed in the first culture vessel 704, after which the cells are transferred to the second culture vessel 706, and transduction and amplification of the cells are performed in the second culture vessel 706. In another embodiment, during activation in the first culture vessel 704, before adding the activated cells from the first culture vessel 704 to the second culture vessel 706 for transduction and amplification steps, transduction reagent actions (e.g., adding (multiple) pre-inoculation reagents to the second culture vessel 706, incubating and rinsing the culture vessel 706) can be performed in the second culture vessel 706. In another embodiment, the activation, transduction and amplification steps can be performed in a single culture vessel (e.g., the first culture vessel 704 or the second culture vessel 706).

[0307] refer to Fig.90 , illustrates another workflow 1000 implemented by the bioprocessing device 600. As shown therein, the workflow or method 1000 includes performing a series of activation steps 1002 and a series of transduction steps 1004 in a first culture vessel 704, and amplifying the genetically modified cell population (after transduction) in a parallel expansion step 1006 using both the first culture vessel 704 and the second culture vessel 706. This involves transferring a small portion of the genetically modified cells from the first culture vessel 704 to the second culture vessel so that the parallel expansion 1006 can be performed using both culture vessels 704, 706 at the same time.

[0308] refer to Fig.91 , another workflow 1100 implemented by the bioprocessing device 600 is illustrated. As shown therein, the workflow or method 1100 includes the steps of performing activation, conversion and amplification in parallel but independent workflows. This includes, for example, performing activation steps 1102, transduction steps 1104 and amplification steps 1106 for the first cell population completely within the first culture vessel 704, and performing parallel activation steps 1108, transduction steps 1110 and amplification steps 1112 for the second cell population completely within the second culture vessel 706. In one embodiment, the first cell population and the second cell population may be derived from a single cell population separated between the first culture vessel 704 and the second culture vessel 706 during the input phase of the activation phase. In another embodiment, the first cell population and the second cell population may be different (e.g., from different sources).

[0309] Now go to Fig.92, illustrates yet another workflow 1200 implemented by the bioprocessing device 600. As shown therein, the workflow or method 1200 includes: performing an activation step 1202 in a first culture vessel 704 for a cell population, and then transferring the activated cell population from the first culture vessel 704 completely out of the bioprocessing device 600 for off-board transduction at step 1204. After transduction outside the module / device 600, the cell volume is transferred to a second culture vessel 706 of the bioprocessing device 600 for post-transduction volume reduction and post-transduction wash steps 1206 in the second culture vessel 706. As also shown therein, an amplification step 1208 is also performed in the second culture vessel 706.

[0310] In one embodiment, the bioprocessing apparatus 600, disposable bioprocessing kit 700 and flow architecture of the present invention allow washing to be performed both after activation and after transduction, for example using hollow fiber filters.

[0311] With regard to the use of the bioprocessing apparatus 600 to perform activation, transduction and expansion of a cell population in the manner described above, a common requirement for all disposable devices used in cell culture and bioprocesses is to be sterile during their operating period, but also functionally closed and fully reliable, in order to ensure batch quality and product safety. Therefore, embodiments of the present invention also provide for performing leak-free verification and blockage detection checks on the disposable bioprocessing kit 700 (including culture vessels 704, 706 and associated tubing) prior to use. Go to Fig.93 , illustrates a flow architecture 1300 employed by the apparatus 600 and the disposable kit 700 according to an embodiment of the present invention. The flow architecture 1300 is generally similar to the flow architecture 400 disclosed above.

[0312] As shown therein, the flow architecture / system 1300 includes a plurality of pneumatic interfaces (e.g., two pneumatic interfaces 1302, 1304 or four pneumatic interfaces 1302, 1304, 1306, 1308) that allow air to be drawn into the system 1300. The pneumatic interfaces 1302, 1304, 1306, 1308 allow for leak-free connections relative to sterile air filters 1310, 1312, 1314, 1316 associated with each interface and forming part of the disposable kit 700. In addition to the three-way valve 1320, the system 1300 further includes a three-way valve 1322 and a three-way valve 1323, the three-way valve 1322 allowing switching of the air flow path to connect the kit through the sterile air filters 1310, 1312 to the ambient atmosphere outside the kit within the process drawer 604 or to the pressure monitoring sensor 1324. The system 1300 further includes: two peristaltic pumps 1326, 1328 (e.g., the process pump 456 and the source pump 454 of the peristaltic pump assembly 641), which are intended to act as pressurizing devices and pinch valves during the leak-proof verification process and as liquid management devices during normal operation, as disclosed above, a group of up to twenty pinch valves 1330 (#1 to #20) (e.g., formed by the valve manifold 712 and the linear actuator array 643); and one peristaltic pump 1332 (e.g., the waste pump 492 of the peristaltic pump assembly 641), which is intended to act as a pinch valve during the leak-proof verification and as a liquid management device during normal operation.

[0313] Air can be drawn into the system via a peristaltic pump through a pneumatic interface that enables the flow path to be selectively connected to the atmosphere via a sterile air filter. In one embodiment, this interface has two primary purposes: (1) to allow for pressurization of portions of the disposable kit 700 during a kit integrity check, as discussed below, and (2) to draw in sterile air to purge fluid from the lines during various automated workflows.

[0314] Fig.94Another flow architecture / system 1400 that can be adopted by the device 600 and the disposable kit 700 instead of the architecture 1300 according to another embodiment of the present invention is illustrated. The flow architecture / system 1400 is similar to the flow architecture / system 1300, wherein the same reference numerals indicate the same components. As shown therein, the system 1440 has four pneumatic interfaces 1302, 1304, 1306, 1308, one of which (pneumatic interface 1306) is connected to a three-way valve 1318 that switches between the atmosphere and the pressure sensor. The advantage of the flow architecture / system 1400 is that the culture vessel can be pressurized independently of the rest of the disposable kit 700 (before starting the bioprocessing operation). This allows the culture vessel to be checked at one pressure, and the rest of the kit to be checked at another pressure (which may be higher than the pressure that the culture vessel can withstand). This also enables the rest of the kit 700 and its flow lines to be tested under negative pressure, which is typically avoided in the culture vessel because the membrane can be removed or displaced.

[0315] Fig.95 Another flow architecture / system 1402 that can be employed by the apparatus 600 and disposable kit 700 in place of architecture 1300 or 1400 according to another embodiment of the present invention is illustrated. The flow architecture / system 1402 is similar to the flow architecture / system 1400, wherein like reference numerals designate like components. However, as shown therein, Fig.95 The flow architecture 1402 omits the hollow fiber filter (HFF) and the waste pump. Fig.95 The flow architecture 1402 can be combined with the above Fig.94 The flow architecture 1400 operates in a similar manner to that described above.

[0316] Fig.96 Yet another flow architecture / system 1410 that may be employed by the apparatus 600 and disposable kit 700 in lieu of architectures 1300, 1400, or 1402 according to another embodiment of the present invention is illustrated. The flow architecture / system 1410 is similar to the flow architecture / system 1402, wherein like reference numerals designate like components. However, as shown therein, there is an additional pressure sensor 1412 (in lieu of) fluidly connected to the three-way valve 1320. Fig.95 In particular, it has been recognized that utilizing more than one pressure sensor may provide certain advantages (compared to Fig.95In one embodiment, the first pressure sensor 1324 and the second pressure sensor 1412 may have different pressure ranges suitable for their specific uses. However, it should be appreciated that in some embodiments, the first pressure sensor 1324 and the second pressure sensor 1410 may have the same or similar pressure ranges.

[0317] In one embodiment, flow framework / system 1410 may further include accumulator 1414.Accumulator 1414 is used as volume buffer, and can be configured as reservoir or certain length of pipeline.No matter how specific construction or configuration, the volume that accumulator 1414 has is greater than or equal to the total volume of the fluid flow path between sterile air filter 1316 and the second pressure sensor 1412 / from sterile air filter 1316 and the second pressure sensor 1412.In use, in the case of having obstruction, the existence and position of accumulator 1414 ensure that the fluid of this volume will accumulate in accumulator 1414 and do not contact sterile air filter.In one embodiment, accumulator can be such as via snap-fit ​​connection and be installed to the tray of disposable kit 700.As illustrated, accumulator 1414 is positioned along the ventilation line from pinch valve manifold, to prevent sterile air filter from wetting under empty head.

[0318] In many of the components, systems, devices, and architectures disclosed above, reference has been made to the use or adoption of sterile air filters. In one embodiment, one or more or all of these sterile air filters may be hydrophobic so that they may be exposed to or contact fluids and still maintain their integrity and functionality as expected. In yet other embodiments, non-hydrophobic filters with or without accumulators or similar devices may be employed, depending on specific system or architecture layouts and applications.

[0319] In one embodiment, the non-leakage verification mentioned above is performed independently on three different sections of the disposable culture kit. In one embodiment, the first section includes the entire disposable kit (that is, the whole of its fluid flow path) except the pipeline segment between the source pump 1328 / 454 and the pipeline tail 1334a-d (for example, the pipeline tail of the pipeline organizer 720). The test of the first section is carried out in two stages, i.e., the pressurization stage and the pressure decay monitoring stage. In one embodiment, the second section includes two culture vessels and the pipeline from the inlet port until the supply pump, and the pipeline segment between the source pump 1328 / 454 and the pipeline tail 1334a-d. The test of the second section is carried out in two stages, i.e., the pressurization stage and the pressure decay monitoring stage. In one embodiment, the third section includes the entire disposable kit (that is, the whole of its fluid flow path) except the pipeline segment between the T / U loop (sensor bypass) and the source pump 1328 / 454 and the pipeline tail 1334a-d. The test of the third section was conducted in three phases, namely the pressurization phase, pressure decay monitoring, and pressure release phase.

[0320] As indicated above, the leak-free validation and plugging detection methods disclosed above enable an end user to run an automated integrity test on the entire disposable set 700 prior to commencing a bioprocessing operation. This enables the end user to detect possible leaks and / or plugged / pinched lines within the disposable set, which would negatively impact the ability to conduct an automated workflow and ultimately the quality of the batch.

[0321] As indicated above, mammalian cell culture processes can require significantly complex liquid transfer management operations that must be performed in an accurate and safe manner. Therefore, the ability to detect leak events is a critical function that should be performed continuously so that an alarm is sounded if the viability of a batch may potentially be compromised. In view of the foregoing, embodiments of the present invention also contemplate using real-time monitoring of the qualities involved in the bioprocess to verify the leak-free nature of the disposable kit 700 and detect blockages therein. In most, if not all, of the bioprocessing operations disclosed herein, four situations typically always exist or occur: (1) the fluid is maintained within a closed container, (2) the fluid is transferred from a source container to a destination container, (3) the fluid is perfused from a source container to a destination container through an intermediate container, and / or (4) the fluid is recirculated from a container or vessel and returned to the same container or vessel. Thus, as long as the source, intermediate, and / or destination containers include a device / mechanism for measuring the mass of such containers (e.g., one or more load cells associated with each container, as disclosed above), a device for pumping fluid from one container to another or circulating to and from the same container in a leak-free manner (e.g., using a peristaltic pump assembly 641), and a control unit (e.g., controller 210) for monitoring changes in the mass of each container, a plurality of leak and / or blockage detection processes may be performed, as disclosed below. The load cells may include, for example, a base plate supporting various containers (e.g., culture vessels 704, 706, waste bags, etc.) or a peg or hook with an integrated load cell (e.g., a hook 620 on a vertical storage drawer 614, 616 of a cabinet 608 for hanging media, reagents, and other bags), as disclosed above. As disclosed below, a controller (e.g., controller 210) is configured to monitor changes in the mass of each container, actuate a pumping device to transfer fluid between the containers, execute a mass balance equation, and generate an alarm or warning if the solution to the mass balance equation does not indicate leak-free or the absence of a blockage.

[0322] In one embodiment, no pumping action occurs, and the control unit 210 merely verifies that the mass of the first container remains substantially constant (e.g., within a predetermined or preset change threshold for a predetermined duration). If the mass changes by less than a predetermined threshold amount, this indicates that the volume of the fluid within the first container remains constant, thereby indicating 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 generates a warning to the user.

[0323] In another embodiment, the method for detecting leakage or blockage includes monitoring the mass of the first source container and the second destination container, and transferring the fluid from the first container to the second container. For example, the pump of the device 600 is controlled by the controller 210 to pump the fluid from the first container to the second container, while monitoring the mass of each container using an associated load cell. In particular, in such an embodiment, the mass of the first container (and therefore the mass of a certain volume of fluid therein) is first determined. Then a certain volume of fluid from the first container is transferred to the second container. Next, the mass of the second container (and therefore the mass of a certain volume of fluid in the second container) is determined. Then, the controller 210 compares the original mass of a certain volume of fluid in the first container with the mass of a certain volume of fluid in the second container, and if there is no leakage or blockage, they should be approximately equal. If the difference between the original mass of a certain volume of fluid in the first container and the mass of a certain volume of fluid transferred in the second container exceeds a threshold, the controller 210 generates a notification or warning. In one embodiment, the controller can also perform the above-mentioned leak detection process without transferring the entire volume of fluid between containers. In particular, in one embodiment, the controller 210 is configured to verify whether the source container mass volume absolute change is below / remains below the transfer flow rate plus a specified leak rate detection threshold, and whether the destination container mass volume absolute change is above or equal to / remains above or equal to the transfer flow rate minus the specified leak rate detection threshold. If not, a leak alarm will be triggered by the controller 210.

[0324] In yet another embodiment of leak-free verification using real-time mass balance monitoring, the goal is to keep the mass of an intermediate (e.g., third) container constant. Therefore, simultaneous action of the two pumps of the peristaltic pump assembly 641 is necessary, wherein the liquid transfer from the source container to the intermediate container must be controlled based on the source container change relative to a specified flow set point, and the liquid transfer from the intermediate container to the destination container must be controlled based on the destination container change relative to a specified flow set point. The control unit 210 is configured to verify whether the source container mass volume absolute change is lower than / remains lower than the transfer flow rate plus a specified leak rate detection threshold, whether the intermediate container volume absolute change is lower than / remains lower than the specified leak rate detection threshold, and whether the destination container mass volume absolute change is higher than or equal to / remains higher than or equal to the transfer flow rate minus the specified leak rate detection threshold. If any of these conditions do not exist, the controller 210 is configured to generate a warning.

[0325] In yet another embodiment, controller 210 makes fluid recirculate from the first container, leaves the first container and returns to the first container to perform non-leakage verification by controlling pump.Therefore, the pumping of fluid occurs in an open loop, and control unit 210 is configured to verify that the mass volume absolute change of the first container is lower than / remains lower than the leakage rate detection threshold value of appointment.If not, then the leakage alarm will be triggered by controller 210.The modification of this process is whether the sample volume is sucked out from the recirculation loop.In this case, controller 210 verifies whether the mass volume absolute change of the first container remains lower than the leakage rate detection threshold value of appointment and adds the sampling flow rate.

[0326] Thus, embodiments of the present invention utilize real-time mass balance calculations to check for leak-free and / or detect blockages in the kit 700 prior to utilizing the kit 700 in a bioprocess operation. However, the methods disclosed herein are not limited to determining leaks prior to using the kit 700 in a bioprocess, but may also be used for real-time leak verification or blockage detection during a bioprocess. Thus, it may be possible to take remedial action with respect to any blockage or leak detected in order to save or salvage the batch.

[0327] In the embodiments disclosed above, the first source bag / container may be a media bag, the second destination bag / container may be a waste bag, and the third intermediate bag / container may be a culture vessel or a bioreactor vessel. However, the present invention is not intended to be so limited in this regard, and various bags / containers may be used as the first, second, and third containers, as long as the fluid can be transferred between and / or through such containers. In addition, although the mass balance process for verifying leak-freeness and for detecting blockages has been described as being performed on the bioprocessing device 600 and the disposable bioprocessing kit 700, the present invention is not intended to be so limited in this regard. In particular, it is contemplated that the mass balance technology can be performed on various systems and devices, including the processing equipment 102 and separation modules (and disposable kits therefor) disclosed above in conjunction with the first module 100 and the third module 300.

[0328] As disclosed above, the bioprocessing system 10 of the present invention includes a first module 100, a second module 200, and a third module 300 (which may be configured the same as or similar to the first module 100), which are configured to perform a plurality of discrete automated bioprocessing operations, including enrichment and separation, activation, genetic modification and amplification, and harvesting. In one embodiment, the first module 100 (including the processing device 102 and the separation module 104) can be used to separate target cells (e.g., using Fig.37A and Fig.37BThe target cells are then transferred from the first module 100 to the second module 200 for activation, transduction, and expansion of the transduced target cells. Optionally, the present invention contemplates that the first module 100 (including the processing device 102 and the separation module 104) can be used to perform the split and dosing preparation operation, the freezing preparation operation, and the washing operation (e.g., using the kit 350 without using the second module 200 for any further bioprocessing operation (e.g., cell activation, transduction, or expansion)).

[0329] The processing device 102 of the first module 100 can also be used downstream as a stand-alone device (i.e., without requiring or using the separation module 104) to perform harvesting and washing of the expanded cell population (using Fig.36 and kit 350 described above). For such operations, a different tubing set, such as a standard Sefia disposable tubing set, may be used and operated by processing device 102 alone.

[0330] In yet another embodiment, the first module 100 (including the processing equipment 102 and the separation module 104) can also be used downstream to perform the separation and dosing preparation operation and the freezing preparation operation as part of the preparation process. In this case, the first module 100 can be referred to as the third module 300, however, as noted above, it can be configured to be the same or substantially similar to the first module 100 and include a processing equipment (e.g., the processing equipment 102, such as the Sefia S-2000 cell processing instrument, and the separation module 104).

[0331] In this way, the first module 100 or its processing equipment 102 can be used on both sides of the second module 200, that is, before the activation of cells using the second module 200, and after the expansion using the second module 200, to perform automated cell separation, separation and dosing preparation, cryopreservation and / or harvesting and washing as desired. The versatility of the first module 100 is thus demonstrated because it can perform multiple functions in series with different second modules 200. That is, when the second module 200 is processing the target cell population, each module 100 is free to start processing cells intended for or taken from other second modules 200 operating in the production facility. Therefore, a cell therapy manufacturer can utilize the first module 100 or the third module 300 at the same time without limiting its manufacturing capabilities or requiring additional equipment, which would not be fully utilized if the additional equipment was dedicated to a single second module 200.

[0332] As discussed above, in one embodiment, the processing device 102 of the first module 100 may be connected to Fig.36The washing kit 350 is used together to provide automated dilution, washing and concentration of cell products in both the upstream and downstream of, for example, a typical CAR-T manufacturing process. Therefore, the kit 350 and the first module 100 can utilize temperature management to process thawed cell products during the dilution phase or during the washing harvest. For example, the washing kit 350 (in combination with the first module 100) can be used upstream of the second module 200 to process fresh or thawed cell materials (e.g., PBMC, T cells). The thawed PBMC and T cells will be the output stored from the separation step (e.g., cryopreserved backup materials). Downstream of the second module 200, the washing kit 350 (in combination with the first module 100) can be applied to, for example, harvesting after T cell amplification. In addition, the kit 350 and the module 100 can be used to wash cell materials.

[0333] Thus, as described above, the module 100 and kit 350 and associated software (used upstream or downstream of the second module 200) are configured to provide primary dilution (i.e., optional dilution of the initial input source material with a wash solution), settling and initial product wash (i.e., volume reduction and wash with a user selected number of wash cycles), supernatant extraction (i.e., removal of supernatant from the wash), cell resuspension (i.e., post-wash resuspension of cells in a wash solution or a dedicated resuspension solution), and final product resuspension, which includes extraction of the cells into a final product bag. In one embodiment, the initial dilution may be skipped entirely or limited to a limited number of process steps. It may simply provide a volume transfer, or a volume transfer with temperature preconditioning and / or automated mixing. Additionally, wash cycles may be skipped entirely or configured with a limited number of wash cycles.

[0334] refer to Fig.97 , illustrates another configuration of a single-use disposable bioprocessing kit 1500 for use with the bioprocessing apparatus 600. The bioprocessing kit 1500 is generally similar in configuration and function to the Fig.50 As will be apparent from a comparison of the corresponding figures, the bioprocessing kit 1500 includes a generally rectangular tray 1502 sized and dimensioned to be received in the first interior space 636 of the process drawer 604, and a pair of culture vessels 1504, 1506 (also referred to as bioprocessing vessels or bioreactor vessels) received in the tray 1502. Fig.99 and Fig.100As best shown in the figures, the tray 1502 has a pair of openings or windows 1508, 1510 in the floor of the tray 1502 beneath the culture vessels 1504, 1506, and the tray 1502 supports the culture vessels 1504, 1506 in a raised position such that when the tray 1502 is positioned within the first interior space 636 of the process drawer 604, the culture vessels 1504, 1506 are lifted from the tray when engaged with the support columns 646 of the platform rocker assemblies 640, 642.

[0335] like Fig.98 As shown in FIG. 1 , the tray 1502 includes a ridge 1512 at one end thereof, the purpose of which will be described below. The tray 1502 is also equipped with a pair of handles 1514, 1516 at its opposite front and rear ends for facilitating positioning and removal of the tray 1502 within the process drawer 604. In one embodiment, the handles 1514, 1516 may be removably connected to the tray 1502, such as by a snap fit or other means known in the art. Fig.99 As best shown in FIG. 1 , the tray 1502 includes longitudinally extending walls 1518 that define duct passages 1520 between such walls 1518 and the side walls of the tray 1502 through which ducts are guided and contained. Fig.99 and Fig.100 , the bottom plate of the tray 1502 includes a plurality of through openings 1522 for the tilt platform pins / posts (allowing the tilt platform pins to engage the culture vessels 1504, 1506), and a plurality of positioning protrusions 1524, which cooperate with corresponding features on the underside of the culture vessels 1504, 1506 to ensure proper positioning of the culture vessels 1504, 1506 within the tray 1502 on the openings 1508, 1510. A plurality of tube guide clips 1526 are provided adjacent to the opposite lateral sides of the tray 1502, which allow the tubing (e.g., tubing from / to the culture vessels) to be neatly stored, guided and organized. The tray 1502 further includes a pair of snap-fit ​​connectors 1528 at the ends of its spine for receiving the spine 1512, and an air duct cover 1530.

[0336] Now go to Fig.101, showing the spine 1512 of the tray 1502. The spine 1512 includes a generally rectangular first opening 1532 for accommodating a valve manifold 1534 (which may be the same or similar to the valve manifold 712), a second opening 1536 for the pump tubing segments 714, 716 (to be engaged by the peristaltic pump), and a third opening 1538 intended to serve as a gripping area for facilitating disengagement of the spine 1512 and / or the valve manifold 1534. Side guides 1540 with tactile feedback elements assist in the insertion of the tray 1502, provide a rough registration with the pinch valve hardware at the rear of the tray 1502, and provide tactile feedback that the tray 1502 is properly positioned within the process drawer. In one embodiment, the side guides provide an angled or funnel shape to facilitate and guide the connection of the spine to the tray. The first opening 1532 and the second opening 1536 have a plurality of snap-fit ​​recesses 1542, 1544, respectively, which allow for removable and secure connection of the valve manifold 1534 and the pump conduit (e.g., pump conduit connectors 714, 716). Although snap-fit ​​connections are contemplated, other connection means known in the art may also be utilized without departing from the broader aspects of the present invention. As illustrated, the tray 1502 includes only two conduit segments, rather than three (e.g., Fig.81 ), because there is no separate waste pump (through Fig.96 ), no tubing segments for the waste pump are required. In one embodiment, the first opening 1532 can include chamfered corners 1546 or other error-proofing elements to ensure that the valve manifold 1534 is properly oriented within the first opening 1532. That is, the chamfered corners or other mechanisms ensure that the pinch valve manifold 1534 can only be connected to the spine 1512 in a single orientation of the pinch valve manifold 1534.

[0337] Space 1548 is provided for mating a biosensor (if utilized). The spine 1512 further includes a protective flange 1550 extending horizontally at the top of the spine 1512. An opening 1552 allows for a snap-fit ​​connection of the handle 1516.

[0338] Fig.102 The valve manifold 1534 is shown connected to the spine 1512 in the first opening 1532 and the pump conduit connectors 714, 716 are connected to the spine 1512 in the second opening 1536, and Fig.103Also shown is a valve manifold 1534 connected to the spine 1512. In an embodiment, the tubing connectors / tubing segments 714, 716 each include connectors, such as a rod block connector 1554 and a right angle connector 1556 at opposite ends thereof (where the right angle connector 1556 has means for interconnecting with snap fit recesses 1544). The valve manifold 1534 is similar to the valve manifold 712 disclosed above, and can be, for example, a fluid vessel configured to interface with a plurality of linear actuators of the linear actuator array 643. The valve manifold 1534 is fluidly interconnected with the culture vessels 1504, 1506, the media bags and reagent bags in the cabinet 608, the waste bags in the waste drawer 606, and the sampling lines to form a fluid network or architecture as disclosed above.

[0339] Figures 104-107 More particularly, the configuration of the valve manifold 1534 according to an embodiment of the present invention is shown. The valve manifold 1534 includes: a non-fluid contacting back plate 1558 having a plurality of valve bowls 1560, in which the plungers of the linear actuators of the actuator array 643 are used to allow or restrict / prevent fluid flow through the flow path of the valve manifold 1534; a membrane 1562, which creates a fluid seal and valve function when the plunger is actuated; and a fluid plate 1564, which contains fluid paths, valve bowls, sealing ridges, and interface points (e.g., glue ports with various conduits). The membrane 1562 is sandwiched between the back plate 1558 and the fluid plate 1564. As shown therein, the back plate 1558 and the fluid plate 1564 include a plurality of vent interfaces 1566 and vent ports 1568, respectively, and a plurality of alignment openings 1570 for receiving alignment pins in a process drawer, which ensure proper alignment and positioning of the tray 1502 in the process drawer. The valve manifold 1534 further includes a plurality of snap-fit ​​connectors 1572 configured to engage with the snap-fit ​​recesses 1542 on the spine 1512 to retain the valve manifold 1534 within the opening 1532 in the spine 1512. Fig.104 and Fig.106 As shown in , the valve manifold 1534 includes a chamfered corner 1574 that corresponds to the chamfered corner 1546 in the opening 1532 to facilitate proper orientation of the valve manifold 1534 relative to the ridge 1512. Fig.104 As shown in FIG, back plate 1558 includes a plurality of ports 1576 that serve as interface ports for leak testing of manifold 1534. In one embodiment, the face of fluid plate 1564 includes a plurality of fluid port identifiers and vent port identifiers (e.g., numbers or letters identifying the various ports to facilitate proper architectural assembly).

[0340] Fig.107The fluid paths within the valve manifold 1534 formed by the fluid plate 1564 are best illustrated. In one embodiment, port 1578 is fluidly connected to the tubing tail bundle, port 1580 is fluidly connected to the pump (via pump tubing segments 712, 714), port 1582 is fluidly connected to the culture vessels 1504, 1506, port 1584 leads to the vent filter, ports 1586 are fluidly connected to each other, and ports 1588 are fluidly connected to each other. In one embodiment, the pinch valve manifold 1534 has four fluid subnetworks, three represented by three sets of dark / bold lines, and the fourth represented by no dark / bold line. Fig.108 The fluid plate 1564 and membrane 1562 are shown mounted to the spine 1512 (with the back plate 1558 removed).

[0341] Return to reference Fig.97 In addition to the tray 1502 and culture vessels 1504, 1506, the disposable kit 1500 also includes additional left and right tube tail cards / organizers 1590, 1592 and a sample card / organizer 1594, respectively. Figures 109-112 The configuration of pipeline tail card 1590,1592 is more particularly shown.Card 1590,1592 is used for the purpose identical with pipeline organizer 720, that is, organize and hold fluid and be connected to multiple pipeline tails of valve manifold 1534 (and be configured to be used for being connected to various media bags and reagent bags contained in cabinet 608, as described above).As shown therein, left tail card 1590 and right tail card 1592 both comprise multiple tails and wrap mandrel 1596, pipeline tail wraps mandrel 1596 around described multiple tails, and multiple clips 1598, to fix (multiple) pipeline tails extending across the bottom of card.In one embodiment, left tail card 1590 comprises three tails and wraps mandrel 1596, and right tail card 1592 comprises six tails and wraps mandrel 1596, yet under the situation of not departing from the broader aspects of the present invention, more or less mandrels can be adopted. The tubing cards 1590, 1592 further include a corresponding tail bundle entry chute 1600 at one corner of the card 1590 and a plurality of clips 1602 to secure the tail end when not in use. In one embodiment, the cards 1590, 1592 may also include a plurality of bottom snap clips 1604. Each card 1590, 1592 further includes a plurality of hanging clips 1606 for mounting the tubing card 1590, 1592 as desired (e.g., on a drip tray at the bottom of the cabinet 608). Either card may additionally include a plurality of swivel clips 1608 for guiding and retaining tubing. Fig.112 A left tubing card 1590 and a right tubing card 1592 are shown attached to opposite sides of a drip tray 1610 (which may be the same as or similar to drip trays 626 or 628).

[0342] Fig.113 and 114 15 shows a configuration of a sample card 1594 according to an embodiment of the present invention. As shown therein, the sample card 1594 is generally rectangular in shape and has a plurality of keyhole openings 1612 for hanging the sample card 1594 on hooks or latches on the inside of a door 610 or 612 of a cabinet 608. The face side of the sample card 1594 also includes pairs of opposing recessed holes 1614 that receive sample manifolds 1616. A plurality of swivel hooks / clips 1618 for a dual tail bundle are provided along the left side of the card, and the upper end of the card includes a half-moon shaped tail bundle track 1620. In an embodiment, the rear side of the card 1594 may be provided with a waffle or rib for strengthening and stiffening the card and preventing or discouraging warping.

[0343] refer to Fig.114 The sample manifold 1616 includes a pair of inlet / outlet ports 1622, 1624, a pair of opposing end caps 1626 defining a manifold 1628 therebetween, and a plurality of sample tail ports 1630 in fluid communication with the manifold 1628. A handle 1632 in the form of a flange is provided to facilitate insertion, positioning, and removal of the manifold 1616, and to facilitate connection of tubing to the sample tail ports 1630.

[0344] Turn again Fig.97 , and refer to Figures 115-118 The disposable kit 1500 further includes a base anchor 1634 and a pivot anchor 1636. Fig.115 and Fig.116 As shown in FIG. 1 , the base anchor 1634 includes a passage 1638 for receiving a tube bundle therethrough, notches or guides 1640 on opposite sides of the anchor 1634, a first set of clips 1642, a second set of clips 1644, and a foolproofing element 1646. Fig.117 and Fig.118 As shown in FIG. , the pivot anchor 1636 includes a flange raceway 1648 , a plurality of tube wrapped recesses 1650 along the raceway, and an error proofing element 1652 . Fig.119 The disposable kit 1500 is shown with tubing installed and the locations of the base anchor 1634 and the pivot anchor 1636 are shown.

[0345] Fig.120 and Fig.121 The configuration of culture vessel 1504 is shown (culture container 1506 is the same). Culture vessels 1504, 1506 are substantially similar to culture vessels 704, 706 and include a base 1656, a lid 1658 connected to the base 1656, a gas permeable, liquid impermeable membrane 1660 sandwiched between the base 1656 and the lid 1658, and a gasket 1662 sandwiched between the membrane 1660 and the lid 1658. Fig.120 As best shown in FIG. 1 , the opposite end of the cover has a glue port 1664 for connecting inlet and outlet pipes, as described above in conjunction with Fig.64 and Fig.65 The vent port 1666 is disposed at the top center of the cover 1658. Fig.120 and Fig.121 As also shown in FIG. 1 , a sensor flag 1668 is disposed in a receptacle in the base 1656, the sensor flag 1668 being configured to be detected by a sensor associated with the process drawer for ensuring that the culture vessels 1504, 1506 are properly positioned and seated. In one embodiment, the sensor flag 1668 may be connected to the base within the receptacle via a snap-fit ​​connection. Fig.122 and Fig.123 As shown in FIG. 1 , it is contemplated that various types of ventilation ports may be used for connection to ventilation conduits. For example, Fig.122 The barbed vent port 1670 is shown, and Fig.123 The female gel port vent port 1672 is shown.

[0346] Further references Fig.122 In one embodiment, no matter how the specific configuration is, the culture vessel 1504 may include an annular wall or baffle 1674, which surrounds the vent port in the lid 1658 of the culture vessel 1504 and extends downward from the lid into the culture vessel. Although the baffle is illustrated as being annular in shape, other shapes, such as square or rectangular, are also envisioned. In addition, it is contemplated that instead of having a continuous baffle, a pair of relative linear baffles may be provided on the opposite side of the vent port in the top of the culture vessel. Baffle 1674 is configured to prevent and / or prevent the fluid or medium in the culture vessel from leaving the culture container through the vent line (and therefore prevent and / or prevent the fluid or medium from entering the vent line and contacting the air filter positioned therein). In particular, during the swinging of the culture vessel 1504, the medium in the vessel moves end to end in a wave-like motion. This swinging action can cause the fluid to contact the lid of the culture vessel adjacent to the vent port. However, the presence of baffle 1674 prevents this fluid wave from contacting the lid in the area of ​​the vent port, thereby preventing and / or blocking such fluid from entering the vent line and / or contacting the filter. It is further contemplated that the baffles described herein may be present on any of the culture or bioprocessing vessels described herein, and are not limited to Fig.122 The embodiment shown in .

[0347] As indicated above, disposable kit 1500 may be used with device 600 (instead of or in place of kit 500 ).

[0348] As used herein, an element or step that is narrated in the singular and begins with the word "one" or "a kind of" should be understood as not excluding a plurality of said elements or steps, unless such exclusion is explicitly stated. In addition, 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 narrated features. In addition, unless explicitly stated to the contrary, an embodiment that "comprises", "includes" or "has" an element or a plurality of elements with a particular property may include additional such elements that do not have that property.

[0349] This written description uses examples to disclose several embodiments of the invention (including the best mode), and also to enable one of ordinary skill in the art to practice the embodiments of the invention (including making and using any devices or systems and performing any incorporated methods). The patentable scope of the invention is defined by the claims, and may include other examples that occur to one of ordinary skill in the art. Such other examples are intended to be within the scope of the claims if they have structural elements that are not different from the literal language of the claims, or if they include equivalent structural elements that are not substantially different from the literal language of the claims.

Claims

1. A disposable kit for a bioprocessing system, comprising: a tray having a spine with a first window and a second window; at least one bioprocessing vessel received within the tray; a pinch valve manifold removably received by the spine within the first window; a first conduit segment removably received by the spine within the second window; as well as A second conduit segment is removably received by the spine within the second window.

2. The disposable kit according to claim 1, further comprising: A tubing organizer card having multiple mandrels for holding lengths of tubing in a coiled configuration.

3. The disposable kit according to claim 2, wherein: The tube organizer card has a plurality of clips for mounting the tube organizer card to a drip tray of the bioprocessing system.

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

5. The disposable kit according to claim 1, further comprising: A sample card has a sample manifold removably received thereon, the sample manifold having a plurality of ports for fluid connection to sample tubes.

6. The disposable kit according to claim 5, wherein: The sample card includes a semicircular raceway for guiding sample tubes to the sample manifold.

7. The disposable kit according to claim 2, further comprising: A tubing passage extends within the tray and is defined between the inner and outer walls of the tray for accommodating tubing extending from the tubing organizer card to the pinch valve manifold.

8. The disposable kit according to claim 1, further comprising: A pair of handles are configured to facilitate positioning the tray within a process drawer of the bioprocessing system.

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

10. The disposable kit of claim 1, wherein: The tray is configured to be received in a temperature controlled process drawer of the bioprocessing system.

11. The disposable kit of claim 1, wherein: The pinch valve manifold is a fluidic vessel having four fluidic sub-networks.

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

13. The disposable kit of claim 1, wherein: The pinch valve manifold includes elements configured to allow the pinch valve manifold to be connected to the spine in only a single orientation.

14. The disposable kit of claim 1, wherein: The at least one bioprocessing vessel comprises a base and a lid, a vent port in the lid, and a baffle adjacent to the vent port; The baffle is configured to prevent fluid in the bioprocessing vessel from contacting the vent port.

15. The disposable kit of claim 14, wherein: The baffle is annular in shape.

16. The disposable kit of claim 14, wherein: The vent port includes one of a barbed connector and a female gel port.

17. A culture vessel for a biological processing system, comprising: Pedestal; a cover connected to the base, the cover having a vent opening; as well as a gas permeable, liquid impermeable membrane sandwiched between the base and the cover; wherein the cover includes a baffle adjacent to the vent port; and Wherein, the baffle is configured to prevent the fluid in the bioprocessing vessel from contacting the ventilation port.

18. The culture vessel according to claim 17, wherein: The baffle is annular in shape.

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