Improved large-scale immunomagnetic separation device

Abstract

This invention relates to an apparatus and method for immunomagnetic separation having a flexible, inflatable processing chamber and a magnetic array.

Description

[0001] Cross-application of related applications

[0002] This application claims priority to U.S. Provisional Application No. 62 / 930,917, filed November 5, 2019, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This invention relates to large-scale cell separation, and more particularly to immunomagnetic separation of cells, as well as innovative methods to make such separation faster, more efficient and more economical. Background Technology

[0004] With the emergence of methodologies and new technologies capable of extracting mammalian cells and converting them into active pharmaceutical ingredients that can remove harmful cells from the host, there has been renewed interest in isolating key primitive cell subpopulations from peripheral blood mononuclear cells (PBMCs) for these processes. Large-scale clinical cell isolation typically processes 10 [units of something] per batch. 9 Up to 10 11 T cells are a key subset of cells used in the production of transgenic cells (CAR T cells) in this emerging field of cell and gene therapy, typically comprising 30-45% of leukocyte isolates. To obtain sufficient T cells to begin inoculating these active drugs, 10 10 One PBMC (peripheral blood mononuclear cell) is sufficient. On the other hand, isolating stem cells that constitute 0.5-2% of the original cell population requires more than ten times the number of initial cells. In either case, immunomagnetic separation requires three basic steps: (1) labeling the target cells with magnetic material; (2) separating the cells from the mixture; and (3) recovering the target cells. A potential fourth step may be required, namely, the desired separation of the magnetic material from the target cells.

[0005] The shared separation system 1 described in WO 2016 / 183032 (hereinafter referred to as "'3032") is Figure 1The present invention is shown and includes the following elements: (1) a combined incubation, separation, and thin linear fluid chamber (FC) 2, which pivots at its midpoint to facilitate processing steps in the purification process, such as mixing reagents, performing magnetic separation against gravity, moving buffer and buffer menisci above the collection surface, and using advantageous orientation to fill and empty the chamber; (2) a magnetic array 55, which, when in contact with FC 2, is particularly adapted to perform (a) enhancing the loading of magnetic nanoparticles onto target cells, (b) cell separation, and (c) subsequent purification processing steps of these magnetically immobilized separated cells. As disclosed in '3032, item "(c)" relates to a processing step for removing paracells that are typically entrained during magnetic separation, which, unlike other external field magnetic separations, does not require a cycle of suspension and magnetic separation to remove the products of these paracells. Instead, the entrained paracells are removed from the magnetically held cells by passing the buffer and buffer menisci above. Thus, '3032 discloses in detail a method and operation for efficiently performing immunomagnetic cell separation with a minimum number of steps. System 1 of '3032 can also perform similar separation on other entities. It should be noted that '3032 discloses a system 1 in which the magnetic array 55 and FC 2 can be coupled and decoupled in order to perform various steps employed in the separation process.

[0006] System 1 described in '3032' can be used not only for isolation of large-scale separations but also for separations of smaller quantities, and it can be easily scaled up or down due to the availability of FC 2 with different collection surface areas. The disclosed system 1 is also designed for labeling cells with highly magnetic colloidal nanoparticles that can be separated from the container using an external gradient magnetic field. Cell separation using significantly larger magnetic beads (approximately 1-5 μm) for cell labeling can also be advantageously performed using the method disclosed in '3032, but typically requires additional steps.

[0007] The result of all these considerations is Figure 1The diagram shows a 3 / 4 view of key components of the cell separation system 1 disclosed in '3032'. Item 2 is a collection chamber with thin, rigid sides and ports 3 for introducing or removing fluid. As disclosed in '3032', chamber 2 is fitted in a frame 4 to allow connection to a rotary actuator assembly and a linear actuator assembly bracket. Thus, the plane of the processing chamber 2 is capable of rotation and lateral translation, as indicated by arrows 9 (lateral movement) and 10 (rotational movement). System 1 also shows a magnetic array 5 with an iron backplate 6 that provides yoking and holding magnetic elements 7 in place, wherein the magnetic elements can be arranged in various ways, such as parallel rows of magnetic elements with north and south poles facing the backplate 6. As disclosed in '3032', this arrangement generates a strong magnetic field gradient that attracts the magnetic elements to the planar surface defined by those magnetic pole surfaces. Finite element analysis of various magnetic arrangements is also disclosed, demonstrating that the magnetic field gradient can be well controlled, treating the gradient as a function of distance from the planar surface and magnetic reach and hold. The magnetic array 5 can preferably be fixed in space at a 45° angle, with the magnet 7 facing downwards. Alternatively, the magnet can be fixed to the magnetic array 5, allowing the magnetic array 5 to be in direct contact with the processing chamber 2, enabling the magnetic array and the processing chamber to rotate or translate as a single unit. Clearly, when the magnetic array 5 is in contact with the processing chamber 2, a strong magnetic field gradient is applied to the contents of the processing chamber.

[0008] '3032 discloses a detailed protocol and method for isolating CD3+ T cells from peripheral blood mononuclear cells (PBMCs) using System 1, and the advantages of the rotational and translational properties of the processing chamber 2. In short, the processing chamber 2, in a near-vertical position, is filled with a volume of PBMCs and a second volume of anti-CD3 FF, and mixed by rotational and / or translational oscillation of the processing chamber 2, during which the CD3 T cells are magnetically labeled. Next, the processing chamber 2 is translated into contact with the magnetic array 5, which allows the magnetically labeled cells to reach the upper inner surface of the processing chamber 2, where the magnetically labeled cells are held securely enough that the fluid flowing through them within the processing chamber 2 does not expel them. '3032 discloses that the slow flow of the buffer solution through which the target cells are thus held, and the meniscus, allows for the forced return of paracells entrained during the magnetic separation step to the suspension by this moderate agitation, resulting in a very high level of purity. After two or three such cleaning cycles (or "meniscus scrubs"), the processing chamber 2 can be positioned vertically, the magnetic array 5 can be removed, and the product cells can be suspended in a desired volume of buffer solution using the rotational and lateral translation properties of system 1. Summary of the Invention

[0009] According to the present invention, the applicant has recognized that the collection walls of the processing chamber need to be very thin because a maximum magnetic field gradient needs to be applied to the contents of the processing chamber, since the gradient generated by the planar magnetic array decreases sharply with distance from those planar surfaces. This thinness makes such fragile chambers not only costly due to their difficulty in manufacturing and sterilization, but also necessitates a support system to prevent deformation of the processing chamber during processing steps that require rotation or lateral shaking, such as mixing operations or "meniscus erasure" processes (referring to the process of passing a buffer buffer meniscus through magnetically held target cells to obtain high purity).

[0010] According to the invention, the applicant also recognizes an imaginative solution to these chamber problems: forming narrow, rectangular, rigid-walled chambers from flexible bags (e.g., appropriately sized blood bags). This can be achieved by confining such bags between substantially parallel walls and placing them under moderate pressure, which forces the walls of the flexible bag into close contact with the confining walls and provides a rigid chamber with a wrinkle-free collection surface. Since the magnetic field gradient of this type used herein decreases sharply with distance from the planar magnets, the need for such a flat, wrinkle-free surface should not be overemphasized. Wrinkles in such a collection surface would lead to significant loss of target cells because they are at a reduced magnetic field gradient at their height above the planar magnet array. It should be noted that, in addition to using a synchronous pump to keep the collection processing chamber under pressure to form a rigid chamber, a simple ballast-type auxiliary chamber with an appropriate volume, filled with pressurized air and fitted with a pressure-limiting valve can also be used. Large (2L) blood bags equipped with appropriate pressure-limiting valves have been successfully used.

[0011] As further disclosed below, the applicant has inventively designed a magnetic component coupled to a narrow, rectangular, rigid-walled processing chamber, in the form of a bag, such as a blood bag, placed between substantially parallel walls, and optionally under moderate pressure. The coupling between the magnetic component and the bag-shaped processing chamber can be permanent, and the gradient of the magnetic component and the bag are perfectly aligned.

[0012] In another aspect of the invention, the invention can provide an apparatus in which i) a narrow, rectangular, rigid-walled processing chamber in the form of a bag (e.g., a blood bag) is positioned between substantially parallel walls, and ii) the magnetic components can be arbitrarily coupled and decoupled, with the precise alignment required for repeatable processing. This configuration presents significant challenges for: (1) the processing chamber is a large, thin-walled, narrow, linear chamber operating under pressure, which must maintain its shape throughout the entire cycle of coupling and decoupling; and (2) the magnetic field gradient generated by the magnetic array drops sharply from its planar surface, making it a critical issue to approach all areas of the collection surface of the processing chamber. In one aspect, a system is disclosed herein that accomplishes the task of coupling and decoupling these critical components with the precision required for repeatable processing. The configuration of the disclosed exemplary system can facilitate automation by employing commonly available actuators. Furthermore, since the separation system can utilize gravity during cell separation via its pivoting capability, gravity can also be used or contribute to the coupling and decoupling of the magnetic array and the processing chamber.

[0013] Reversible coupling between the magnetic components and the processing chamber can be achieved by forming a novel and independent housing for the processing chamber, comprising: a robust cover, preferably transparent, to allow observation of the processing chamber during processing; and a base plate or bottom platform, uniquely constructed to allow close contact between the processing chamber and the magnetic components (i.e., the processing chamber and the magnetic array). This base plate can be obtained by starting with a rigid rectangular plate (preferably aluminum), slightly larger than the back plate of the magnet array, from which a roughly central rectangular region is cut, allowing the magnets of the magnetic array to be inserted integrally into the space, such that the top of each magnet is level with the top surface of the plate or base plate. A support structure is required to hold the bottom pressure surface of the processing chamber in place. This can be achieved by: (1) placing horizontally and parallel thin members in the cut space and in a direction corresponding to the orientation of each magnet in the magnetic array, the thin members being fixed to two opposing surfaces created by the cut, thereby forming a grid-like structure; (2) employing support members that can be fitted between the magnets in the magnetic array with sufficient tolerance so that they can be easily moved in and out of the space; (3) such spaced support members that they can be installed between adjacent magnets or every other magnet when the slotted or grid-like base plate is placed above the magnetic array; and (4) by placing a thin sheet, for example, 1 mm rigid, above the grid-like support structure. Acrylic sheets are used to create a smooth, completely flat bottom confinement wall for the processing chamber. By appropriately selecting the thickness of the rigid bottom plate or base plate and the height of the block magnets used to construct the magnetic arrays, the planar surfaces of these arrays can be made within 0.5 to 1.0 mm of the bottom surface of the processing chamber, thereby effectively allowing the maximum magnetic field gradient to be applied to the contents of the chamber.

[0014] In summary, given the teachings of this disclosure, it is clear that many of the advantages described in '3032, particularly those concerning system automation and elimination of operator tasks, arise from the decoupling of these two main components (the processing chamber and the magnetic array). This method also offers another significant advantage: it eliminates the need for a separate processing station for magnetically labeled target cells prior to their introduction, which is required for the coupling unit. Decoupling the processing chamber and the magnetic array also simplifies product harvesting, which can be achieved simply by removing the product from the processing chamber.

[0015] In one aspect, the invention can provide a system for magnetically separating a target biological entity from a fluid suspension of a target biological entity and a para-biological entity in a processing chamber. The processing chamber may be provided in the form of a blood bag. The system may include a platform configured to receive the processing chamber at its upper surface, the chamber having an opening through which it may be filled with a cell suspension containing a magnetized or magnetizable target biological entity, wherein the processing chamber is a fluid chamber having a collection surface. Furthermore, the system may include a magnetic element mounted on the platform and movable relative to the platform, such that at a first selected position on the platform, the magnetic element is magnetically coupled to the processing chamber to apply a magnetic field to the collection surface, thereby attracting the target biological entity to the collection surface. A chamber control assembly connected to the processing chamber and the magnetic element may also be provided; the chamber control assembly is operable to pivot the separation chamber and the magnetic element about an axis, thereby moving the magnetic element from the first selected position to a second selected position in response to rotation of the platform, wherein the second selected position is further away from the platform than the first selected position. The magnetic element may be movable in a direction perpendicular to the upper surface.

[0016] Furthermore, the platform may include one or more pillars on which magnetic elements are movably mounted to allow the magnetic elements to move from a first selected position to a second selected position on one or more pillars, the second selected position being further away from the platform than the first selected position. The magnetic elements may be configured to move from the first selected position to the second selected position on one or more pillars in response to rotation of the platform. The magnetic elements may include an array of magnets, and the platform may include cavities extending therethrough, the magnetic elements being sized to be mounted within the cavities.

[0017] Furthermore, the system may include a plurality of longitudinal nonmagnetic rods arranged parallel to each other in a spaced-apart manner within the cavity, wherein a plurality of openings are provided between corresponding pairs of longitudinal nonmagnetic rods. Magnetic elements may include an array of longitudinally extending magnets, the magnets being sized to fit within corresponding openings of the plurality of openings when the magnetic element is in a first selected position. The platform may also include a nonmagnetic sheet disposed above and in contact with the plurality of longitudinal nonmagnetic rods to provide a flat surface for engagement with the processing chamber. A cover may be disposed above the upper surface to define a space between the cover and the upper surface for receiving and holding the processing chamber. Additionally, a cam may be provided that contacts the upper surface and the cover, the cam being rotatable to change the distance between the upper surface and the cover, and the cover being movable in a direction perpendicular to the upper surface to change the distance between them.

[0018] In another aspect, the invention provides a system for magnetically separating a target biological entity from a fluid suspension of a target biological entity and a para-biological entity in a processing chamber of bag form. The chamber may have an opening through which it can be filled with a cell suspension containing a magnetized or magnetizable target biological entity, wherein the processing chamber is a fluid chamber having a collection surface. The system may include: a platform having a cavity extending from an upper surface to an opposing lower surface; a plurality of longitudinal nonmagnetic rods arranged parallel to each other in a spaced-apart relationship within the cavity, wherein a plurality of openings are disposed between corresponding pairs of longitudinal nonmagnetic rods; and a plurality of magnets disposed near the upper surface in the plurality of openings within the cavity, such that the magnets can be magnetically coupled to the processing chamber to apply a magnetic field to the collection surface, thereby attracting the target biological entity to the collection surface. The system may further include a nonmagnetic sheet disposed on the upper surface and in contact with the plurality of longitudinal nonmagnetic rods to provide a flat surface for receiving and supporting the processing chamber. Attached Figure Description

[0019] The above overview and the following detailed description of exemplary embodiments of the present invention can be further understood when read in conjunction with the accompanying drawings, wherein:

[0020] Figure 1 A simplified view of some components of the cell separation system disclosed in '3032' is shown schematically;

[0021] Figure 2 A schematic top view of a standard blood bag is shown, which has been modified to optimize its use in the apparatus of the present invention as a processing chamber for the purification of immunomagnetic cells.

[0022] Figure 3A A side view of an inflatable flexible chamber according to an exemplary configuration of the invention is shown schematically. The inflatable flexible chamber is in the form of a deflated flexible blood bag, for example, disposed within a “walled” frame, wherein an inflatable bladder is prepared to pressurize the blood bag to form a processing chamber.

[0023] Figure 3B The display is shown schematically. Figure 3A Flexible blood bags in Figure 3A A side view of the "walled" frame being inflated to form a rigid treatment chamber;

[0024] Figure 4 A top view schematically illustrates an exemplary configuration of a slotted rigid non-magnetic plate according to the invention, which can support... Figure 3B One side of the pressurized inflatable blood bag processing chamber, in which slots in a non-magnetic plate allow a magnet array to make close contact with the processing chamber;

[0025] Figure 5A A top view schematically illustrates an exemplary configuration of a planar magnetic array according to the present invention, which can "interleave" the magnets of the planar magnetic array to... Figure 4 In the non-magnetic plate;

[0026] Figure 5B schematically shown Figure 5A A side view of a planar magnet array, depicting magnets located on a backplate;

[0027] Figure 6A schematically shown Figures 5A-5B A side view of a planar magnet array, which is related to... Figure 4 The support plates are mechanically linked, and a cover plate is added above the support plates to form a frame for holding an inflatable flexible chamber (such as a flexible blood bag);

[0028] Figure 6B schematically shown Figure 6A A partial sectional view of the system, the view relative to Figure 6A The direction shown is rotated 180° around its axis of rotation, which causes the magnet to move through the gap in the slotted non-magnetic plate;

[0029] Figure 7 An exploded perspective view schematically illustrates an exemplary configuration of a pivotable magnetic separation device according to the present invention, which includes... Figure 4-6B The planar magnetic array and processing chamber, wherein pivoting / rotating action is used to engage or disengage the magnets of the array as they rotate under the influence of gravity, and a portion thereof containing a pivotable magnetic separation device that houses an inflatable flexible separation processing chamber is shown disassembled.

[0030] Figure 8 schematically shown Figure 7 The device is shown in a cross-sectional view along section line 8-8 when it is not disassembled (i.e., when the frame supports the separation processing chamber and is in close contact with the planar magnetic array).

[0031] Figure 9 An exemplary configuration of the cam mechanism according to the invention is schematically shown, which can change the depth of the separation processing chamber; and

[0032] Figure 10 The illustration schematically shows how to use the pivoting of the exemplary system of the present invention to bring the processing chamber into or out of contact with the magnetic array in order to perform the procedural steps of immunomagnetic separation. Detailed Implementation

[0033] Referring now to the accompanying drawings, in which like elements are numbered the same throughout, an exemplary manner according to the invention to realize the many advantages of the separation system having magnetic array 55 and the flexible, inflatable processing chamber 16 (e.g., a blood bag) is to permanently fix these components together, such as... Figure 2 , Figure 3A , Figure 3B As shown. Another exemplary method is to make the processing chamber / bag 16 and the separation system having the magnet array 55 movable relative to each other, for example... Figure 2 , 6A As shown in Figure 6B.

[0034] First, move to chamber 16. Figure 2 An exemplary flexible inflatable processing chamber 16 according to the present invention is depicted, which can be adapted to... Figure 3A and Figure 6AThe improved blood bag 16 used in the configuration is provided. The improved blood bag 16 may include an inlet port 17 and an outlet port 18 (but may also be used in other locations) on opposite sides of the bag for connection to an inlet pump and an outlet pump, respectively, to pump fluid (solution or gas) into or out of the bag 16 independently or simultaneously. It should be noted that the corners 19, 20 of the bag 16 are fused to block flow toward the corners of the bag, where the absence of good fluid flow may impede processing. Diverters 21 and 22 may be designed to facilitate push flow through the bag 16. A significant advantage of using blood bags or similar materials as processing chambers 16 in clinical cell separation is that they are compatible with blood products, sterile, familiar to the user, and inexpensive single-use products.

[0035] Reference Figure 3A The illustration shows a system 100 according to the invention, comprising a flexible, inflatable processing chamber 16 disposed between parallel retaining walls, illustrated as a deflated blood bag, one retaining wall being 23' and the other retaining wall shown as a magnetic array 55. The inflatable processing chamber 16 is pressurized to form a rigid processing chamber, its walls being formed flush with the retaining walls 23' and the magnetic array 55 within the frame 23, as shown. Figure 3B As shown. Under moderate hydraulic or pneumatic pressure (less than 1.0 psi), blood bag 16 can remain rigid throughout the processing steps, and the flexible wall remains wrinkle-free.

[0036] like Figure 3A As shown, as a possible method of pressurizing the blood bag 16, the ballast airbag 25 can be kept closed by a clamp 26. As described above, one wall of the frame may include a magnetic array 55 of magnets 57, for example, on which a thin (approximately 0.5 to 1 mm thick) rigid non-magnetic sheet 52 is placed, such as... Acrylic sheets, rigid non-magnetic sheets, are used to form a smooth, flat surface. The opposing wall 23' can be easily removed, allowing the flexible, inflatable treatment chamber 16 to be inserted and held in place within the frame 23 once pressurized.

[0037] Figure 3B schematically shown Figure 3A The device is in place, but the flexible, inflatable processing chamber 16 is inflated within the frame 23. Opening the clamp 26 allows air from the air bladder 25 to inflate and pressurize the blood bag 16, forming a rigid, inflatable chamber 16. With the blood bag 16 pressurized, appropriate arrangements of the walls 23', the rigid non-magnetic sheet 52, the magnetic array 55, and the blood bag 16 can be achieved to transmit a magnetic field to the processing chamber / bag 16, for example, to perform a magnetic cell separation process. A synchronous pump linked to a pressure gauge with feedback can be used to maintain the rigidity and constant pressure of the inflatable flexible chamber 16 throughout the process, which typically lasts 50 to 160 minutes, or even longer if additional steps are included.

[0038] For a separation device 100 constructed by permanently coupling a processing chamber 16 to a magnetic array 55, positive cell selection using indirect magnetic labeling can be performed as follows: (1) a cell suspension can be mixed with labeled monoclonal antibodies (mAbs) and incubated in a suitable external system coupled to system 100; (2) unbound mAbs can then be removed by centrifugation if desired; (3) ordinary capture magnetic nanoparticles can then be added to the mAb-labeled cells and incubated; (4) the mixture from step (3) can then be pumped into a pressurized processing chamber 16 in which separation can be performed immediately; and (5) non-target cells entrained during magnetic separation can then be removed from the magnetically collected cells by performing 1-3 meniscus erasure cycles on the separated magnetically held cells (e.g., as disclosed in '3032'); and (6) finally, the processing chamber is removed from the magnetic field gradient to recover the cells (in the case of positive selection).

[0039] To make it easier to Figure 3A , Figure 3B The system 100 is used for magnetic processing, such as immunomagnetic separation, which requires a method to bring the magnet 57 of the magnetic array 55 into close contact with at least one wall of the modified blood bag 16.

[0040] In this regard, and in another aspect, the present invention can provide a support structure 30 that provides a retaining wall for the processing chamber / bag 16, and innovatively achieves... Figure 4 The requirements are shown. Figure 4 A large, rigid rectangular nonmagnetic base plate 31, such as a 7-8 mm thick aluminum plate, is shown, having a cavity 32 disposed therein. The size of the cavity 32 can be slightly larger than the size of the processing chamber / bag 16 if a magnetic field gradient is to be applied to all parts of the bag 16. A plurality of nonmagnetic rods 33, such as 1.5 × 12 mm flat aluminum rods, are spanned across the cavity 32. The rods 33 can be held in the cavity 32 by slots disposed within the nonmagnetic plate 31 around the outer periphery of the cavity 32, with the corresponding ends of the rods 33 placed in the slots, such as... Figure 4 As shown.

[0041] The spacing 34 between adjacent rods 33 should be large enough that one or more block magnets 57 of the planar magnetic array 55 can be smoothly inserted into the spacing, such as... Figure 6A , Figure 6B As shown. Holes 36 can be provided at the four corners of the base plate 31 to secure the protruding supports 65 above the base plate 31, thereby holding the cover 42 of the enclosed processing chamber / bag 16 in place, as shown. Figure 4 , Figure 7As shown. Hole 36 can also be used to fix the protruding support 43 at each of the four corners below the base plate 31, as shown. Figure 7 As shown. Support 43 can be used to suspend the magnetic array 55, as described below. Figure 7 A more comprehensive description. In Figure 4 and Figure 5A In the middle, the central line 35 describes the line around which the substrate system 30 will pivot to form the direction required for the cell purification process.

[0042] In another aspect, the inventors recognized that by permanently coupling the processing chamber 16 to the magnetic array 55, Figure 3A , Figure 3B System 100 has some processing limitations. For example, if the processing chamber 16 and the magnetic array 55 can be easily decoupled, certain processes of contacting and decoupling the magnet 57 from the chamber 16 can be performed as follows: In the decoupled state between the array 55 and the chamber 16, a cell mixture can be introduced into the processing chamber 16 (and then appropriately surrounded and pressurized), followed by labeling with a monoclonal antibody (mAh). The system 100 can then be shaken left and right around a pivot point for mixing and incubation; next, ordinary capture magnetic material is introduced into the processing chamber 16, which is shaken again for mixing and incubation. It should be noted that the last step cannot be performed in the coupled state between the array 55 and the chamber 16 because, after the introduction of the magnetic material, and likely before the system is fully mixed, the large amount of magnetic material added will immediately separate. Furthermore, after the introduction, mixing, and incubation of the magnetic reagent with the target cells, there may be steps involving coupling and decoupling between the processing chamber 16 and the magnetic array 55. For example, intermittent coupling (20 to 30 seconds) of the processing chamber 16 and the magnetic array 55 can be employed to enhance the movement of magnetic nanoparticles by altering the magnetic field gradient, while larger cells remain relatively stationary. This could potentially cause any unbound magnetic nanoparticles to find a target cell to bind to, thereby enhancing the magnetic loading of the target cell.

[0043] In the positive selection case, after removing used cells from processing chamber 16 and while chamber 16 and magnetic array 55 remain engaged, buffer washing with magnetically held cells can be performed, followed by meniscus erasure to obtain highly purified cell products. By holding magnetically separated cells (targeted and entrained) in place and placing the buffer and buffer meniscus above them, entrained paracells during magnetic separation can be easily removed, allowing the entrained cells to move into the liquid phase, thus producing highly purified cells, which can be easily obtained by decoupling processing chamber 16 and magnetic array 55 and emptying chamber 16. In addition to all the advantages described above, a system with a decoupleable magnetic array 55 and chamber 16 avoids the additional step of removing processing chamber 16 from the enclosed space required by the coupling unit.

[0044] Forming the support structure for the processing chamber wall in contact with the magnetic array 55 presents a challenge, not only because the wall is thin, preferably less than 1.0 mm, but also because the processing chamber 16 is pressurized. For such a chamber 16, confined to a depth of 8-10 mm, adaptation to 2×10 7 At the optimized separation concentration of 1×10⁶ cells / mL 10 Individual cells are magnetically separated, and the thin-walled collection surface area can be approximately 500 cm². 2 At an internal pressure of 0.8 PSI, there is a load of approximately 65 pounds on this surface. Deformation may occur on this surface when the magnetic array 55 is not pressed against the wall, and during coupling and decoupling of the array 55 and the collection chamber 16. Holding the pressurized collection chamber wall in place and preventing deformation of this wall could lead to uneven collection of target cells. Preventing uneven collection of target cells and / or chamber wall deformation presents a significant challenge. Furthermore, a device that automatically couples and decouples the array 55 and the chamber 16 in a relatively simple manner would be most advantageous.

[0045] It is important to note that the surface area of ​​the collection surface calculated above coincides with another important consideration of the system of the present invention. Extensive experimental data has shown that only when the number of cell layers collected is less than about 6 (possibly 7 layers) can the paracellular cells entrained during magnetic separation be effectively removed by meniscus erasure. In this case, it is readily apparent by alternative considerations that 10 layers... 10 Separating 40-50% of the total cells requires approximately 520 cm⁻¹. 2 The collection area must meet this condition. Therefore, the collection surface needs to be large enough to accommodate the collection of the desired cells in fewer than 7 cell layers.

[0046] In response to the advantages of the system recognized above, which allows for the coupling and decoupling of array 55 and chamber 16, in another aspect of the invention, means is provided for reversibly coupling the flexible, inflatable processing chamber 16 to the magnetic array 55. For example, in an exemplary configuration, the means of the invention may include suspending the magnetic array 55 below a base plate 31 via cylindrical support pillars 43, with corresponding sleeve bearings 44 mounted at the four corners of the base plate 31, such as... Figure 6A-8 As shown. The length of the support 43 should be long enough so that when the magnetic array 55 is in its bottom position, no magnetic field gradient is applied to the processing chamber / bag 16, as shown. Figure 6A , Figure 7 As shown.

[0047] In other words, when the magnetic array 55 is suspended below the base plate 31 on the support column 43 and the sleeve bearing 44, gravity will cause the magnetic array 55 to be in its lowest position on the cylindrical support column, that is, completely detached from the processing chamber / bag 16, as... Figure 6A , Figure 7 As shown. On the other hand, if device 40 is flipped upside down, gravity will cause the magnetic array 55 to slide downwards and into the slotted grid at the bottom of the housing, thereby engaging. Thus, one embodiment of the coupling and decoupling described thus can be gravity-driven, where the task can be accomplished simply by rotating the system, and is advantageous for magnetic separation schemes (such as those disclosed in '3032').

[0048] Clearly, if system 40 is mounted on a pivot fixed to magnetic array 55, engagement of the magnetic field gradient will occur in the opposite direction to that described above. While gravity can meet all requirements, a simple actuator can be used where fully automatic engagement / disengagement is desired in any direction. Gravity-assisted engagement can also be part of an automated system, as it requires actuators with less energy. Furthermore, providing a locking position for the system can be advantageous. For example, if rotating the system 180° results in magnetic array 55 being on top, a locking mechanism that holds the two components in place can be used to maintain their engagement regardless of the system's orientation.

[0049] All embodiments disclosed in '3032 can be readily implemented by means of the following steps: (1) using an emptied pressurized processing chamber, in decoupling mode, reagents and cells are pumped into the system at an appropriate angle (close to 45°); (2) in the absence of a magnetic field, reagents are mixed and agitated with the target cells by pivoting the processing chamber; (3) magnetic separation of the target cells can be performed against gravity, which occurs at a wide range of favorable angles that hold the units together; (4) processing steps such as meniscus erasure of magnetic cells can be performed with magnet engagement, and if coupling is gravity-driven, pivoting through those angles of magnet engagement; and (5) decoupling the units so that the product can be obtained. A system capable of arbitrarily placing the contents of the processing chamber in a gradient magnetic field also provides another option for the system, namely, suspending magnetically collected cells in the presence of a washing buffer when the magnet is detached, and pivoting through those corresponding angles, followed by reorienting the processing chamber for a second or third suspension and magnetic separation. Therefore, step 4 provides another method for removing the entrained paracellular cells.

[0050] Furthermore, a method for altering the depth of the processing chamber is disclosed, which can be used to reduce processing reagents after performing magnetic separation. This possibility allows for the integration of numerous reagent and time-saving methods into the separation protocol. For example, for the separation of fully single-harvest products, a typical volume for separation in this system would be approximately 330 mL, and this separation can be performed in a processing chamber at a depth of 7 mm. For positive separation, magnetically collected cells require immersion in clean buffer followed by at least two cycles of meniscus wiping, which requires at least 660 mL of buffer. By reducing the depth of the processing chamber to approximately 3 mm after magnetic separation, and by reducing the required buffer volume by more than half before adding washing buffer, the time spent pumping buffer into and out of the processing chamber is reduced. All of this is significant from both an economic and cell viability perspective, as shorter processing time necessarily translates to higher viability.

[0051] The terms flexible bag, collection or processing chamber, and blood bag with port are used interchangeably. An external magnetic field gradient is a magnetic field gradient formed in free space using magnetic pole pieces, the polarity of the pole pieces, a special arrangement of the pole pieces, and power; all of these can be used to create very different gradient field spectra in space. '3032 discloses in great detail the analysis of the planar magnetic array used in this paper.

[0052] Exemplary Applications

[0053] Many magnetic nanoparticles can be used for immunomagnetic separation using the device of the present invention. However, highly magnetic colloidal nanoparticles (HMNPs) in the 140 nm size range (such as those of Liberti et al. (US5,698,271; US ​​6,120,856), which are colloidal and highly magnetic (approximately 84% magnetic mass)) are ideal because they can magnetically label cells by diffusion forces, and cells thus labeled with HMNPs can be separated in external magnetic devices with gradients slightly higher than 4-6 kGauss / cm. Magnetic nanoparticles of this size (150 nm) are advantageous because the magnetic aggregation of these labeled materials or entities can be well controlled, and in fact, as we have found, they can be collected in individual monolayers in a uniform magnetic field gradient, which is readily generated in a radial gradient quadrupole magnetic device.

[0054] We further found that when target cells are magnetically collected in a monolayer or near-monolayer, it is unnecessary to perform the cycle of target cell suspension and magnetic collection as is conventional in the art to remove entrained paracells. Instead, target cells collected in a fairly homogeneous layer can have their entrained paracells removed simply by passing a buffer meniscus over it, while those collected cells are magnetically held in place. We term this process of purifying and separating cells “meniscus erasure.” Therefore, it seems reasonable to suggest that surface tension can be used to remove any non-target cells that may be weakly retained for simple secondary purification processes. Evidence supporting the view that “meniscus erasure” is a very gentle process is our finding that the process has no negative impact on cell viability. Of course, this leads to higher target cell yields.

[0055] Based on these fundamental and other findings, several design principles can be incorporated into the device of this invention: (1) the distance that the target cells need to travel to the collection wall should be as small as possible to minimize the entrainment of adjacent cells (based on experimental data showing that the greater the distance, the greater the entrainment); (2) principle "(1)", combined with adaptation to a large number of cells (10 9 -10 11 The need for cell separation requires collecting cells in a chamber with a small depth (less than 15 mm within the magnetic field gradient) on a surface area sufficient to relatively uniformly stratify the cells into 6-7 monolayers; (3) the advantage of stratified cell collection is that it eliminates or minimizes any accumulation of collected target cells, thus allowing the use of "meniscus erasure," which essentially dictates that a flat surface large enough to stratify the cell layers into 6-7 monolayers is used for magnetic collection, since the cylindrical surface (quadrupole separator) must be very high to accommodate the required processable volume.

[0056] To collect target cells in the layers, it is necessary to develop a planar magnetic field gradient capable of forming a uniform surface area (large enough to separate cells into up to seven monolayers). We have demonstrated that by forming such a gradient, target cells can be collected in near-monolayers. To facilitate the passage of the buffer meniscus through the large area of ​​magnetically held cells, a thin, linear collection processing chamber was created, which pivots at its midpoint, allowing agitation fluid and air bubbles to flow through the cells, thereby literally erasing paracellular cells. Furthermore, the pivoting capability of this chamber is used to perform various steps of immunomagnetic separation in an optimal manner. For example, reagents can be added to the processing chamber and mixed by left-right shaking, the processing chamber can be tilted to an optimal angle for filling or emptying, and magnetic separation can be performed against gravity, which we have demonstrated results in a reduction of entrained paracellular cells.

[0057] Go to Figure 5A , Figure 5B , Figure 5A , Figure 5B The diagram schematically illustrates block magnets 57 optimally spaced on the magnetic backplate 56 to generate a strong and substantially uniform gradient force on the plane defined by the tops of the block magnets 57 over the area defined by those block magnets 57. It should be noted that this area will be comparable to (slightly smaller than) the cavity 32 of the base plate system 30. Small holes 38 drilled into the magnetic array 55 hold the supports 59 (…). Figure 9 If the bag originates from a blood bag with a hole or slit at the top for hanging (as with many blood bags), the support will keep the processing chamber / bag 16 secure. Drill holes 39 at the four corners of the magnetic array 55 house bearings (not shown) that will allow the magnetic array 55 to be connected to the base plate system 30, as described below. Line 37 indicates the centerline of the magnetic array 5. Figure 5B A side view of the magnet array 55 is described, showing how the magnets 57 are spaced apart on the magnetic backplate 56.

[0058] Figure 6A An exemplary configuration of the functional automated separation system 40 is schematically shown, with a cross-sectional view illustrating how a planar magnetic array 55 is connected to the underside of a base plate 31 via a support 43 and a sleeve bearing 44, allowing the magnetic array 55 to slide upwards so that paired magnets 57 of the magnetic array 55 can pass through an open space 34 in the base plate 31. The processing chamber / bag 16 is depicted under pressure. For simplicity... Figure 6A , Figure 6B The accompanying drawings do not show a thin (0.5-1.0 mm) rigid plastic sheet located below the processing chamber / bag 16 and above the rod 33. (The rigid plastic sheet is shown and described in later views, for example...) Figure 7 (The rigid plate 52 in the middle.) We have shown that this rigid plate 52 placed on these support rods 33 ( Figure 7 This provides a perfectly flat surface for the processing chamber / bag 16, preventing deformation even under pressures up to 4 psi. Figure 6A middle, The thick acrylic sheet 42 (optionally allowing visualization of the contents of the processing chamber / bag 16) provides a top wall for confining the processing chamber / bag 16 during processing. The sheet 42 can be held in place by a wing nut 46 and mounted on a support 65 fixed to the drilled hole 36 of FIG. 3. The cover sheet 42 can be spring-loaded by placing a spring 51 between the wing nut 46 and the sheet 42.

[0059] It should be noted that Figure 6A The separation system 40 is described as vertical, with the magnetic array 55 suspended from the base plate 31. In this case, gravity places the magnetic array 55 in its lowest position, i.e., the sleeve 44 is mounted on the stop 45 on the support 43. If the separation system 40 is now pivoted 180° about the pivot point 41, the system 40 will... Figure 6B As shown. Figure 6B This illustrates how gravity will cause the magnetic array 55 to slide downwards, allowing the magnet 57 to move within the space 34 of the plate 31. By appropriately selecting the height of the magnet 57, the thickness of the base plate 31, and the position of the sleeve bearing 44, the magnet 57 can be positioned even closest to the surface of the base plate 31, near the processing chamber / bag 16. Thus, simply by pivoting the separation system 40, we have created a system for engaging and disengaging two key components of the system: the magnetic array 55 and the processing chamber / bag 16. It is equally evident that for… Figure 6A As shown, when tilted nearly 90° in either direction, the magnetic array 55 and the processing chamber / bag 16 can remain detached. This allows the separation system 40 to be used for mixing reagents in the absence of a magnetic field gradient. Similarly, in Figure 6B In this orientation, the magnetic array 55 and the processing chamber / bag 16 are in contact over a wide range of angles, where gravity holds them together. This capability allows the separation system 40 to be used, for example, in a meniscus erasure process, as disclosed in '3032'.

[0060] A locking mechanism can be added to hold the magnetic array 55 and the processing chamber / bag 16 together or apart, thereby extending the utility of the separation system 40. For example, if the separation system 40 is used as described, in order to perform magnetic separation, the system 40 must be inverted (e.g., Figure 6B As described above, this is used to engage the processing chamber / bag 16 and the magnetic field gradient. While separation against gravity has proven beneficial, there may be situations where this is not the case or not beneficial. By employing a locking mechanism against the effects of gravity, the separation system 40 can be locked in place. Figure 6AThe locking mechanism can be positioned and rotated 180° to utilize gravity for separation or other operations. Another advantage of the locking mechanism is the way the control units can be combined or separated. For example, without... Figure 6A In the case of a locking mechanism that starts at a specific orientation, when the unit rotates and reaches the orientation where gravity causes movement, torsional forces exist on the support and sleeve bearings, which can wear down these components. A simpler method is to rotate the device 180° in the locked state and then release the lock. This method minimizes wear on the components and allows for precise control of the timing of applying or removing the magnetic field.

[0061] Figure 7 The rendition 50 of the separation system 40 is shown, mounted on a rotatable shaft 54 ​​and located within a support structure 53 that allows 360° rotation. This rendition shows the components of system 50 in an exploded view, creating a space in which a flexible container for a rigid processing chamber 16 is placed. A prominent feature of the base plate 31 is the grid-like structure of the support rods 33 supporting the pressurized processing chamber / bag 16, clearly showing the individual support rods 33 and the open spaces 34 between them. A thin, non-magnetic rigid sheet 52, preferably less than 1.5 mm thick, covers the rods 33 of the base plate 31, providing a smooth and flat surface to the sides of the processing chamber / bag 16 for compression under pressure. Compression springs 51 can be positioned above the cover 42 on four support pillars 65 fixed to the base plate 31. When the processing chamber / bag 16 is pressurized, the springs 51 should match and exert a slightly greater force to resist the upward pressure on the cover 42. To fix the depth of the flexible inflatable treatment chamber 16, multiple sets of thick-walled cylinders (not shown) with a height of 3-12 mm can be placed on the corner support 65 for optimal separation, which sets a distance limit on the distance from the lower side of the cover 42 to the top surface of the corner support 65.

[0062] Figure 8 It shows Figure 7 The system 50 is a cross-sectional view taken along section line 8-8. Magnets 57 are interleaved with each other and flush with the top of support rods 33 in a way that shows that this exemplary design allows the gradient field of the planar magnetic array 55 to apply its force to the processing chamber 16 without constraint.

[0063] The design and concept disclosed herein enable another practical application. It may be desirable to be able to vary the depth of the collection chamber during the required operation of the described separation scheme. To illustrate this advantage, consider a typical single-collection product (approximately 7 × 10⁻⁶) on this system. 9 The total number of nucleated cells (TNC) will be separated in a final volume of 350 mL (2 × 10⁻⁶ cells at separation time). 7(cells / mL). For this separation, a surface area of ​​440 cm² was used. 2 The blood bags were appropriately modified to a fixed depth of 8 mm. The maximum filling or emptying rate of the system was 60 mL / min, as cells would be expelled at a higher rate by shear force. This took 6 minutes. Furthermore, after the agitation meniscus erasure procedure, the system was allowed to stand for 6–8 minutes to allow any target cells expelled during the process to be recollected. Therefore, for 3 meniscus erasure cycles, 6 filling / emptying cycles were required, plus two standing phases, for a total of 60 minutes. Additionally, during these cycles, 3 × 350 mL of buffer waste was generated, which became part of the hazardous waste.

[0064] On the other hand, if the processing chamber depth is set to 8 mm during the initial separation (to accommodate the total number of cells being processed and to achieve the optimal cell concentration during separation), and then reduced to 3 or 4 mm in subsequent processing steps, this not only reduces the total chamber volume, but we also found in the simulation system that the smaller depth actually provides more effective meniscus erasure for those subsequent purification steps.

[0065] Therefore, for the aforementioned depth variations, the volume of each filling / draining step is reduced to 131 mL, thereby reducing the time requirement for these steps by more than half. Furthermore, the "resting" time during recollection is also reduced because the expelled cells are closer to the collection surface, where the gradient at a depth of 3 mm is almost twice that at a depth of 8 mm. The aforementioned 56-minute processing steps can be reduced to an estimated 20 minutes, which is highly significant for yield.

[0066] Figure 9 A mechanism for changing the depth of the processing chamber is shown, which employs an elliptical cam 60 attached to a lever 61. By selecting the size of the ellipse, for example 8 × 4 mm, the lever 61 can be moved from an initial 8 mm position to a 4 mm position or any desired scale. Four such cams 60 and levers 61 can be positioned at each corner of the chamber housing unit. Figure 9 Also shown is a support 59 that extends through the magnetic array 55 and the base plate 31 to hold the processing chamber / bag 16 in the proper position within the separation system 40, if the bag originates from a blood bag with a hole or slit at the top for hanging, as is the case with many blood bags.

[0067] Several methods can be used to reduce the depth at the aforementioned base plate 31 and on the inserted inflatable blood bag 16. First, consider that if a flexible bag (such as blood bag 16) is inserted into the cross-section of system 40, it will be subjected to a pressure of approximately 0.7 psi. Therefore, as mentioned above, for a large bag, the cap 42 can exert a total force of approximately 60 lbs. This force needs to be counteracted by the compression spring 51 that holds the cap 42 in place. A significantly stronger spring would be required to expel fluid from the collection chamber. A better approach might be to adjust the cam 60 to its smaller size and use an outflow pump to perform the emptying of most of the processing chamber, thus enabling a reduction in depth. Nevertheless, with this simple mechanism, the filling and emptying cycles of these processes can be reduced, as can the time required for target cells that might be forced to remain suspended during meniscus erasure to return to the collection surface.

[0068] Figure 10 A schematic diagram is shown of immunomagnetic separation performed oriented on system 50, which uses gravity to engage or disengage chamber complex 62, which includes an inflatable processable chamber 16. Acrylic thick sheet 42 and thin rigid non-magnetic sheet 52, wherein magnetic array 55 is used for different steps of the scheme. Figure 10 In the first frame, parallel rectangles show the separated chamber complex 62 and planar magnetic array 55, as the magnetic array 55 is held in place in its lower configuration by gravity. In this configuration, the separation unit is at approximately -45°, and the single-absorption product, mAb, and ferrofluid are added sequentially, with the system agitated between - / +45° for mixing and incubation (upper middle frame). In the upper right frame, the system is rotated so that the magnetic array 55 is on top and now engaged with the chamber complex 62, and the antigravity separation of the magnetically labeled entities occurs (ideally). In the same orientation, the product can be recovered (negative option) or the used PBMC can be pumped into the waste. Furthermore, in the same configuration, buffer can be added to the collection chamber, and the system is agitated because the magnetic array 55 remains engaged with the chamber complex 62, and the buffer for the magnetically held target cells can be meniscus wiped as the buffer and meniscus pass over the target cells, as shown in the lower left frame. In this way, entrained paracellular cells are very effectively forced back into the suspension. After multiple erasure cycles, the required amount of buffer solution can be added, and the orientation can be reversed so that the magnetic array 55 is at the bottom (bottom right screen), and the cells can be suspended and recovered, as shown in the last screen.

[0069] The following examples illustrate the utility of the above disclosure and describe an innovation that can be used to prepare tumor-free T cells or subsets thereof:

[0070] Example I. Negative selection of CD3+ cells using gravity-driven magnetic field gradients and the engagement / disengagement of processing chambers.

[0071] Frozen cells were thawed at room temperature (RT), centrifuged, and the particles were resuspended in RPMI medium containing 10% fetal bovine serum. The suspended cells were centrifuged and resuspended twice more in the same buffer, and finally centrifuged a fourth time in cell separation buffer to achieve a cell count of 1 × 10⁻⁶. 8 Cells / mL. Pump 18 mL of suspension into a processing chamber / bag 16 of approximately 3 × 6.75”, which has an inlet port 17 and an outlet port 18 at opposite ends, see [link to relevant documentation]. Figure 2 The bag (processing chamber) is positioned within frame 23 between wall 23' and magnetic array 55, as follows. Figure 3A As shown, and in Figure 10 As shown in the upper left image. The flexible, inflatable processing chamber 16 is set to a depth of 8 mm and the bag is pressurized with 0.5 psi of compressed filtered air before the cell mixture is pumped into the bottom port. The top port 18 is attached to a similar pressurized ballast bladder 25 and also has a safety valve set at 0.5 psi. Thus, air is expelled from the system as the cell mixture enters the bag. Next, 18 mL of a mouse monoclonal proprietary mixture (all IgG1 cells and targeting all cells except CD3+ cells) is pumped into the processing chamber and as shown in the top left image. Figure 10 The second screen shows shaking to mix the reagents. Two shaking cycles (5 oscillations each) are used, followed by static incubation for 14 minutes. At this time, 36 mL of 20 μg / mL rat anti-mouse IgG1 ferrofluid is pumped into the treatment chamber, and the treatment chamber is shaken as above. After incubation for 10 minutes, 18 mL of separation buffer is added to the treatment chamber so that the treatment chamber contains 90 mL of suspension. As described above, the treatment chamber is shaken to mix the contents, and the system is rotated 180° to engage the magnetic array 55 with the chamber complex 62. Figure 10 (Top right image), thereby applying a strong upward pull to the magnetically labeled cells in the processing chamber, causing them to reach the top surface of the processing chamber.

[0072] Fifteen minutes later, the non-magnetic fraction (negatively selected cells – fraction I) was collected and analyzed by flow cytometry. Next, with the magnetic array 55 still on top of and engaged with the chamber complex 62, the treatment chamber was filled with 80 mL of cell buffer, and the system was rotated 180°. This disengaged the magnetic array 55 from the treatment chamber / bag 16, and the system was shaken, causing the buffer and large air bubbles to pass through the cells, resulting in the movement of the collected non-target cells into the suspension. Figure 10(Bottom right image). This requires three shaking cycles, each with five oscillations. The system is then rotated again so that the magnet array 55 is on top and engaged with the processing chamber 16, allowing for a second separation and recovery of non-magnetic cells (fraction II). The table below shows the analysis of these negative components. By flow analysis, the original product was 60.13% CD3+.

[0073] Components Purity % as determined by flow analysis Yield% Combined output Component I 98.53 52 Component II 97.60 13 65％

[0074] Notably, in this process, the agglutinated product is incubated with a mixture of mAh, and unbound antibodies do not need to be removed before the addition of a standard capture ferromagnetic fluid (rat anti-mouse IgG1 FF). This is a significant advantage, likely unique to nanoparticles in this size range, as unbound mAh is typically removed before the introduction of a standard capture material. This capability is likely due to the high binding capacity of these FFs and their size. We have found that these colloidal nanoparticles for magnetic cell separation exhibit very slow aggregation in the 135–150 nm range compared to micrometer-sized particles when some lectin is added.

[0075] Example II. Negative selection of CD3+ cells from a single-absorption product containing circulating tumor cells (CTCs).

[0076] Currently, CAR T-cell therapy is most successful in the treatment of B-cell carcinoma. Obviously, the apheresis products from such patients are likely to contain cancerous B cells. However, in the negative selection for CD3+ cells, these tumor cells are removed along with normal B cells, which are then specifically targeted by mAh in an appropriate mixture. Currently, significant efforts are being made to treat solid tumors using CAR T technology. When preparing CD3+ formulations for manufacturing CAR T cells for these patients, it is crucial to ensure that no tumor cells are left to contaminate the negative components. Because solid tumors originate from the epithelium and there is extensive experience in the isolation and identification of circulating tumor cells (CTCs), as described in U.S. Patents US 7,332,288B2 and 6,645,731B2 by Terstappen et al., we hypothesize that adding an anti-epithelial antibody to the incubation mAh mixture may be advantageous. For this example, an anti-epithelial mAh (clone VU1D9) is added to the incubation mixture.

[0077] To evaluate our system's ability to clear CTCs and eliminate non-CD3+ cells, negative selection for CD3+ cells was performed by incorporating a colon cancer cell line (Colo 205) into the apheresis product prepared above. These cells were then processed using CellTracker. TMFluorescent staining was performed using red CMTPX dye (Thermo-Fisher). Based on the fact that metastatic cancer patients often have at least 200 CTC / mL in their blood, 3 × 10⁻⁶ CTC / mL was used in the original product. 6 200 Colo 205 cells were incorporated into each total nuclear cell line. An IgG1-type anti-epithelial cell mAh (clone VU1D9) was added to a proprietary mAh mixture optimized for removing all cells except CD3-negative cells at a concentration of 0.5 μg / mL mixture / cell incubation suspension. Separation was performed as described above. The results (yield / purity) of CD3+ cells in the supernatant were almost identical to the data above.

[0078] To test the effectiveness of removing epithelial cells during negative selection, CD3+ cells recovered during negative selection were subjected to a reaction at a concentration of 3 × 10⁶ cells. 6 CTC detection was performed on replicate 5 mL aliquots at a concentration of 10,000 cells / mL. It should be noted that if Colo 205 cells were not removed, a maximum of 10,000 cells could be detected [(200 / 3×10⁻⁶ cells / mL).] 6 )×(3×10 7 [×5 = 10,000 tumor cells]. However, in reality, only 50-60% is expected. Add 8 μg / mL of ferrofluid conjugated with VU1D9 anti-Epcam and biotin BSA to a replicate 5 mL sample, mix and incubate for 20 min, then add 0.8 μg / mL of streptavidin, mix and incubate for 5 min. (The purpose of this last step is to allow streptavidin to bind unbound FF to FF already bound to Colo205 cells, thereby increasing their magnetic loading and significantly enhancing their magnetic separation ability.) After separation in a quadrupole magnetic separator, discard the supernatant, remove the separation tubes from the magnetic device, and carefully rinse the walls of these tubes with 2.0 mL of buffer to allow any collected cells to be transferred from the tube side to a 2 mL volume for subsequent separation. Repeat this volume reduction process while retaining the magnetically collected cells until the sample volume is 200 μL. The sample is then coated onto a polylysine-coated slide and the cells are counted using a fluorescence microscope. In the control incorporation assay, 55% of incorporated cells were captured. No fluorescent cells were detected in the negative CD3+ cell assay. Given that the above-described protocol for CTC detection can only detect samples of 5 cells / mL, adding anti-Epcam monoclonal antibody to our mAh mixture is clearly an effective means of removing such cells from these formulations. This application could be very important because the starting cells are expanded during CAR T cell production, and the potential for expanding CTCs is likely present.

[0079] The above disclosure illustrates how gravity and the force of a compressive spring can be used to automate a process that would otherwise require many steps. The mating of the cell collection chamber 16 with the planar magnetic array 55 can be achieved using various mechanical / electronic components, which would require considerable technical work and rather complex fabrication. The concept disclosed herein eliminates this need. These examples not only demonstrate the practicality of this device concept but also prove their application in the preparation of initial materials for CAR T cells and in other cell applications requiring high purity.

[0080] In addition to immunomagnetic cell separation using the device of the present invention, the ability of such devices to intermittently apply magnetic field gradients to the contents of the separation / processing chamber may be advantageous for another important requirement in the manufacture of cell therapy constructs. For example, in co-pending application WO 2018 / 022694A1, it has been demonstrated that positively selected T cells magnetically labeled with multivalent common trapping agents (such as streptavidin ferrofluidic nanoparticles) (Liberti et al., US 5,698,271, US 6,120,856) can subsequently be activated and expanded by simply adding a biotinylated anti-CD28 antibody. In the case of positively selected CD4+ cells, these nanoparticles are linked to the CD4 epitope via a specific antibody, and activation / amplification requires the addition of two antibodies, namely biotinylated anti-CD3 and biotinylated anti-CD28. In the step of adding the latter antibody to the common capture and separation of cells, the application of intermittent magnetic field gradients has been shown to result in significantly greater expansion. Therefore, the device of the present invention is ideal for this application. In the absence of a magnetic field gradient, purified positive isolated cells are suspended in a collection chamber, and an activator is added and mixed by applying shaking and intermittent magnetic field gradients to the contents, either by coupling the collection chamber and the magnetic array via an actuator or by using gravity to cause the coupling or decoupling.

[0081] Conclusion: The above specific description is intended to illustrate and demonstrate the invention, and should not be construed as limiting the scope of the invention, which will be defined by the words and equivalents of the appended claims.

[0082] The foregoing specification cites numerous patent and non-patent publications and patent applications, the full disclosure of each of which is incorporated herein by reference.

[0083] Although certain embodiments of the invention have been described and / or illustrated above, various other embodiments will be apparent to those skilled in the art from the foregoing description. Therefore, the invention is not limited to the specific embodiments described and / or illustrated, but is capable of considerable changes and modifications without departing from the scope of the appended claims.

Claims

1. A system for magnetically separating a target biological entity from a fluid suspension of a target biological entity and a para-biological entity in a processing chamber, the system comprising: A platform having a cavity extending therethrough from an upper surface to an opposite lower surface and configured to receive the processing chamber at the upper surface of the platform, the chamber having an opening through which the processing chamber can be filled with a cell suspension having a magnetized or magnetizable target biological entity or para-biological entity, wherein the processing chamber is a fluid chamber having a collection surface. A magnetic element is mounted on the platform within the cavity and is movable within the cavity in a direction perpendicular to the upper surface of the platform, such that the magnetic element can be moved to a position close to the upper surface of the platform to magnetically couple to the processing chamber to apply a magnetic field to the collection surface, thereby attracting magnetized target biological entities or para-biological entities to the collection surface, and the magnetic element can be moved to a position spaced away from the upper surface of the platform to non-magnetically couple to the processing chamber; as well as A chamber control assembly, connected to the processing chamber, platform, and magnetic element, operable to pivot the processing chamber, platform, and magnetic element as a single unit about an axis 360°, such that the processing chamber can be positioned above or below the platform.

2. The system according to claim 1, wherein, The platform includes one or more pillars, and the magnetic element is movably mounted on the pillars to allow the magnetic element to move toward or away from the upper surface of the platform by a distance.

3. The system according to claim 2, wherein, The magnetic element is configured to move on one or more pillars in response to the rotation of the processing chamber, platform and magnetic element as a single unit about an axis.

4. The system according to claim 1, wherein, The magnetic element is configured to move in response to the rotation of the processing chamber, platform, and magnetic element as a single unit about an axis.

5. The system according to claim 1, wherein, The magnetic element comprises an array of magnets.

6. The system according to claim 1, wherein, The magnetic element is sized to be installed inside the cavity.

7. The system according to claim 1, comprising a plurality of longitudinal non-magnetic rods arranged parallel to each other in the cavity at intervals.

8. The system according to claim 7, wherein, Multiple openings are provided between corresponding pairs of longitudinal non-magnetic rods, and wherein the magnetic element comprises an array of longitudinally extending magnets, the magnets being sized to fit into corresponding openings of the multiple openings when the magnetic element is positioned near the upper surface of the platform.

9. The system according to claim 7, wherein, The platform includes a nonmagnetic sheet disposed above and in contact with the plurality of longitudinal nonmagnetic rods to provide a flat surface for engagement with the processing chamber.

10. The system of claim 1, further comprising a cover disposed above the upper surface to define a space between the cover and the upper surface for receiving and holding the processing chamber.

11. The system of claim 10, further comprising a cam in contact with the upper surface and the cover, the cam being rotatable to change the distance between the upper surface and the cover.

12. A system for magnetically separating a target biological entity from a fluid suspension of a target biological entity and a para-biological entity in a processing chamber, the chamber having an opening through which the chamber can be filled with a cell suspension having a magnetized or magnetizable target biological entity or para-biological entity, wherein the processing chamber is a fluid chamber having a collection surface, the system comprising: A platform having a cavity extending therethrough from an upper surface to an opposite lower surface; Multiple magnets are disposed in the cavity and movable within the cavity relative to the upper surface of the platform, such that the multiple magnets can be moved to a position close to the upper surface, thereby magnetically coupling the multiple magnets to the processing chamber to apply a magnetic field to the collection surface, thereby attracting magnetized target biological entities or para-biological entities to the collection surface, and the multiple magnets can be moved to a position spaced away from the upper surface of the platform so as to non-magnetically couple to the processing chamber; as well as A chamber control assembly, connected to the processing chamber, platform, and multiple magnets, is operable to pivot the processing chamber, platform, and multiple magnets as a single unit around an axis, allowing the processing chamber to be positioned above or below the platform. The platform includes one or more pillars, on which a plurality of magnets are movably mounted to allow the magnetic elements to move toward or away from the upper surface of the platform by a distance.

13. The system of claim 12, comprising a nonmagnetic sheet disposed on the upper surface and in contact with the plurality of longitudinal nonmagnetic rods to provide a flat surface for receiving and supporting the processing chamber.

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